The role of endoplasmic reticulum protein GRP78 in normal hematopoeises and PTEN-null leukemogenesis

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THE ROLE OF ENDOPLASMIC RETICULUM PROTEIN GRP78 IN NORMAL
HEMATOPOIESIS AND PTEN-NULL LEUKEMOGENESIS

by
Shiuan Wey

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR, AND CELLULAR BIOLOGY)

August 2011

Copyright 2011 Shiuan Wey

ii

Dedication

To my parents, Kwo-Dong Wey and Sheue-Ching Hong
and to my husband, Hsien-Chun Lo for their support along the way.

iii

Acknowledgements
First and foremost, I would like to thank my advisor, Dr. Amy Lee for her
wonderful guidance, persistent encouragement and tremendous amount of support during
my graduate study at USC. Amy definitely is an exceptional mentor, demonstrating great
dedication, courage and enthusiasm toward research but not compromising time spent on
the guidance of her students. I would like to thank her for making the Lee lab such an
academically enriched and pleasurable environment to learn and work in.
I am so grateful to my excellent committee members throughout the years, Dr.
Michael Kahn for the many helpful discussions, insightful suggestions and supportive
encouragement; Dr. Darryl Shibata for the helpful discussions, insightful ideas and much
assistance on the pathological aspects for my thesis project. I would also like to thank Dr.
Parkash Gill for the wonderful collaboration and helpful discussions, Dr. William Carroll
(New York University Cancer Institute, NY) and Dr. Deepa Bhojwani (St. Jude
Children’s Research Hospital, TN) for the wonderful collaboration, Dr. Valtor Longo and
his lab for the use of the hematology analyzer, Dr. Yvonne Lin and her lab for the helpful
discussions and Dr. Allen Yang for providing us the clinical patient samples and cell line.
I am also grateful to my former undergraduate mentor, Dr. Alex Hon-Tsen Yu (National
Taiwan University, Taiwan) for the continuous support and concern throughout the years.
I would like to thank all the former and current members of the Lee lab for
making the lab such a warm and enjoyable atmosphere to be in every day. Former
members: Min Ni, Risheng Ye, Miao Wang, Hui Zhou, Dezheng Dong, Yong Fu, Peter
Baumeister, Jianze Li, Changhui Mao. Current members: Biquan Luo, Kyle Pfaffenbach,
iv

Wan-Ting Chen, Genyuan Zhu, Yi Zhang, Chun-Chih Tseng, Jieli Shen, Michelle Pong,
Kate Ott and John Johnson. I am so grateful for their great help, discussions and
wonderful friendship that I am sure will go on for many years to come.
I am grateful for the generous technical support and advice from the USC Norris
Comprehensive Cancer Center core facilities: Lora from the Flow Cytometry Core
Facility, Alex and Moli from the Translational Pathology Core, Ernie and Tony from the
Cell and Tissue Imaging Core, and Diane from the Immune Monitoring Core.
Last but not least, I would like to thank my husband, my parents and loving
family members (Chi, Cindy, Leonard, Julia). They have always been there by my side to
share my joy and excitement and have given me the encouragement, strength and
inspiration to keep me going through the hard times.

v

Table of Contents

Dedication ii
Acknowledgements iii
List of Tables vii
List of Figures viii
Abstract xi

Chapter 1: Overview and Introduction 1
1.1 ER stress and ER chaperones 1
1.2 GRP78 and its role in cancer 4
1.3 ER stress proteins and signaling associated to hematopoiesis
and immunity
7

Chapter 2: Inducible knockout of GRP78 in the hematopoietic system
suppresses Pten-null leukemogenesis and AKT oncogenic signaling

12
2.1 Introduction 12
2.2 Materials and methods 15
2.3 Results 22
2.3.1 GRP78 expression is elevated in patient leukemic
blasts, leukemia cell lines and early relapse in childhood
leukemia
22
2.3.2 Creation of mouse models with conditional biallelic
deletion of Pten and Grp78 in the hematopoietic system
25
2.3.3 Grp78 heterozygosity suppresses Pten null-mediated
MPD characteristics and blast cell expansion
28
2.3.4 Pten null Grp78 heterozygous mice show MPD
characteristics after longer latency period
32
2.3.5 Knockdown of GRP78 inhibits AKT/S6K activation in
bone marrow and leukemia cells
34
2.3.6 Knockdown of GRP78 in human leukemic cells
enhances AraC-induced apoptosis
40
2.3.7 Targeting cell surface GRP78 with monoclonal
antibody suppresses Pten null-induced leukemogenesis
43
2.4 Discussion 48

vi

Chapter 3: Inducible knockout of GRP78 reveals its role in hematopoietic
stem cell survival and cellular homeostasis

55
3.1 Introduction 55
3.2 Materials and methods 58
3.3 Results 64
3.3.1 Creation of mouse model with conditional deletion of
Grp78 in the hematopoietic system
64
3.3.2 GRP78 deficiency reduces primitive cell pool through
increased cell death
66
3.3.3 Deletion of GRP78 in the hematopoietic system leads to
lymphopenia and altered hematopoiesis
69
3.3.4 GRP78-null induced alteration of hematopoiesis is cell
autonomous
73
3.3.5 Knockout of GRP78 in bone marrow cells activates
UPR signaling pathways
79
3.3.6 GRP78 deficient mice exhibit retarded growth and
develop cardiomyopathy and lung congestion
81
3.3.7 GRP78 deficient mice exhibit differential expression of
cytokines and chemokines
87
3.4 Discussion 91

Chapter 4: Conclusions and perspectives 99

Bibliography 106

vii

List of Tables
Table 2.1: Primer sequences for mouse genotyping

16
Table 3.1: Primer sequences for RT-PCR and real-time PCR

63
Table 3.2: Differential cytokine and chemokine expression in serum of GRP78
deficient mice

90

viii

List of Figures
Figure 2.1: Elevated Grp78 expression in leukemia and Grp78 expression
inversely correlates with time to relapse in childhood ALL

24
Figure 2.2: The breeding scheme for the double conditional knockout mice

25
Figure 2.3: Grp78 heterozygosity impedes PTEN null-induced leukemogenesis

27
Figure 2.4: PTEN and GRP78 were knockdown in the peripheral blood and
bone marrow of mutant mice

28
Figure 2.5: Grp78 heterozygosity suppresses PTEN null-mediated MPD
characteristics

30
Figure 2.6: Grp78 heterozygosity suppresses PTEN null-mediated blast cell
expansion and prolongs survival

31
Figure 2.7: Pten null Grp78 heterozygous mice show MPD characteristics after
longer latency period

33
Figure 2.8: Knockdown of GRP78 suppresses AKT signaling

36
Figure 2.9: Knockdown of GRP78 suppresses ER stress induced AKT
activation

37
Figure 2.10: Knockdown of GRP94 does not suppress AKT activation by serum
stimulation

38
Figure 2.11: GRP78 knockdown suppresses PI(3,4,5)P3 production at the cell
periphery

39
Figure 2.12: Knockdown of GRP78 sensitizes human leukemia cells to AraC
and overexpression confers resistance

42
Figure 2.13: Knockdown of GRP78 sensitizes human leukemia cells to AraC

43
Figure 2.14: Targeting cell surface GRP78 with monoclonal antibody
suppresses Pten null-induced leukemogenesis

47

Figure 3.1: Grp78 is expressed in multiple blood cell lineages

64
Figure 3.2: The breeding scheme for the Grp78 conditional knockout mice

65
ix

Figure 3.3: GRP78 was knockdown in the bone marrow of mutant mice

65
Figure 3.4: GRP78 deficiency in the bone marrow reduced HSC-enriched
population

66
Figure 3.5: GRP78 deficiency does not change bone marrow morphology and
total cellularity

67
Figure 3.6: GRP78 deficiency leads to decreased common lymphoid and
myeloid progenitor cells

68
Figure 3.7: GRP78 deficient LSK cells displayed increased cell death

69
Figure 3.8: GRP78 deficiency leads to lymphopenia

70
Figure 3.9: GRP78 deficiency leads to decreased lymphocytes and increased
monocytes, granulocytes in the peripheral blood

71
Figure 3.10: GRP78 deficiency leads to altered lymphoid and myeloid
differentiation in the bone marrow

72
Figure 3.11: Reduction of HSC-enriched population is intrinsic to GRP78
deficiency in BM cells

74
Figure 3.12: GRP78 deficient BM cells transplanted in WT microenvironment
leads to decreased common lymphoid progenitor cells, common myeloid
progenitor cells and megakaryocyte-erythroid progenitor cells

75
Figure 3.13: GRP78 deficient BM cells transplanted in WT microenvironment
leads to decreased lymphocytes and monocytes in the peripheral blood

76
Figure 3.14: GRP78 deficient BM cells transplanted in WT microenvironment
leads to altered lymphoid and myeloid differentiation in the bone marrow

77
Figure 3.15: GRP78 deficient BM cells transplanted in WT microenvironment
leads to altered lymphoid and myeloid differentiation in the spleen

78
Figure 3.16: Knockout of GRP78 in bone marrow cells activates UPR signaling
pathways

81
Figure 3.17: GRP78 conditional knockout mice exhibit body weight drop and
retarded growth rate

82
Figure 3.18: GRP78 conditional knockout mice exhibit lower food
consumption, blood glucose level and less accumulated fat

83
x

Figure 3.19: A minority of GRP78 conditional knockout mice succumb to early
fatality resulting from myocardial infarction

84
Figure 3.20: GRP78 conditional knockout mice exhibited signs of
cardiomyopathy and lung congestion

85
Figure 3.21: GRP78 expression level in the heart and lung of cKO mice was
similar to WT level

86
Figure 3.22: WT recipient transplanted with GRP78 deficient BM cells
exhibited normal growth rate yet moderately enlarged heart

87
Figure 3.23: Summary diagram of alteration of hematopoiesis, UPR signaling
and apoptosis in Grp78 conditional knockout in the hematopoietic system.

92

xi

Abstract
The endoplasmic reticulum (ER) is an intracellular organelle for protein folding,
lipid synthesis and Ca
2+
storage. It is also responsible for the transportation for most of
the secretory and transmembrane proteins. When the protein load exceeds the ER folding
capacity, the ER undergoes stress and activates a set of signaling cascades that is termed
the unfolded protein response (UPR). The multifunctional GRP78 is the major ER
molecular chaperone with protein folding abilities and the master regulator of the UPR,
and recently has been shown that a subfraction of it is localized on the cell surface acting
as a co-receptor for various signaling pathway activation.
Traditionally GRP78 is regarded as protective against hypoxia and nutrient
starvation prevalent in the microenvironment of solid tumors, thus, its role in the
development of hematologic malignancies remains to be determined. In this thesis,
elevated GRP78 expression was detected in leukemic blasts of adult patients, leukemia
cell lines and inversely correlates with time to relapse in childhood acute lymphocytic
leukemia. To directly elucidate the requirement of GRP78 in leukemogenesis, we created
a biallelic conditional knockout mouse model of GRP78 and PTEN in the hematopoietic
system. Strikingly, heterozygous knockdown of GRP78 in PTEN null mice is sufficient
to restore the hematopoietic stem cell (HSC) population back to the normal percentage
and suppress leukemic blast cell expansion. AKT/mTOR activation in PTEN null bone
marrow cells is potently inhibited by Grp78 heterozygosity, corresponding with
suppression of the PI3K/AKT pathway by GRP78 knockdown in leukemia cell lines.
This is the first demonstration that GRP78 is a critical effector of leukemia progression,
xii

at least in part through control of oncogenic AKT signaling. Furthermore, overexpression
of GRP78 renders human leukemic cells more resistant to AraC-induced apoptosis
whereas knockdown of GRP78 sensitizes them, suggesting GRP78 is a novel potent
therapeutic target for leukemia.
Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is
regulated by both intrinsic gene expression and interactions with extrinsic factors in the
microenvironment. GRP78 has been shown to be critical for the maintenance of cellular
homeostasis and prevention of apoptosis. Homozygous knockout mice of GRP78 are
embryonic lethal at E3.5, indicating GRP78 is essential for embryonic cell growth and
pluripotent cell survival. However, this has not been investigated in adult hematopoietic
stem cells. Here we generated a conditional knockout mouse model that acutely deletes
GRP78 in the hematopoietic system. GRP78 deficiency results in a significant reduction
of HSCs, progenitor and lymphoid cell populations yet an increase in myeloid lineage
granulocytes and monocytes in cKO mice. The GRP78-null induced reduction of the
HSC pool can be attributed to enhanced apoptosis. In agreement, GRP78 deficient BM
cells exhibited activated UPR signaling in all three branches and induced expression of
pro-apoptotic CHOP and caspase activation. Transplanting Grp78 KO hematopoietic
cells into WT microenvironment also yielded a decrease in HSCs, suggesting that GRP78
in hematopoietic cells is required for HSC survival and homeostasis. In addition, a
multiplex cytokine assay reveals alteration in several cytokine serum levels in cKO mice,
suggesting GRP78 plays a pleiotropic role and is essential in regulating hematopoietic
system homeostasis and maintenance.

1

Chapter 1
Overview and Introduction

1.1 ER stress and ER chaperones
The endoplasmic reticulum (ER) is an essential compartment for the synthesis and
modification of secretory and membrane proteins. It is also crucial calcium storage. Upon
physiological and pathophysiological situations of ER perturbations, such as nutrient
deprivation, disruption of calcium homeostasis, inhibition of protein glycosylation of
disulfide bond formation, hypoxia, viral or bacterial infection and even plasma cell
differentiation and pancreatic β-cell function and survival may lead to the accumulation
of misfolded proteins in the ER and therefore result in ER stress. ER stress is defined
when the protein loading exceeds the ER folding capacity. When this occurs, the cells
trigger an evolutionarily conserved signaling pathway, the unfolded protein response
(UPR) which activates a cascade of signaling molecules to enhance the ER folding
capacity by induction of molecular chaperones and enzymes and degradation of
misfolded proteins to help the cells better cope with the stress.
There are three major branches in the UPR signaling pathway which are mediated
by PKR-like ER kinase (PERK), the activating transcription factor 6 (ATF6) and inositol-
requiring kinase 1 (IRE1 α). GRP78, the master regulator of UPR, directly interacts with
all three sensors and maintains them in an inactive form in non-stressed situations (J Wu
& R J Kaufman, 2006). During ER stress conditions, GRP78 is titrated away by
accumulated proteins, and therefore GPR78 is released from the UPR sensors, which
2

allows the activation and transduction of UPR signals across the ER membrane to the
cytosol and the nucleus.
PERK is an ER transmembrane protein, which has both a lumenal and cytosolic
domain and has protein kinase activity. The important function of PERK is attenuation of
global protein synthesis through phosphorylation of eIF2 α during UPR activation.
Phosphorylation of eIF2 α inhibits the assembly of 80s ribosome and therefore inhibits the
synthesis of proteins. However, certain mRNAs that contain regulatory sequences in the
open reading frame in 5’-untranslated regions require the phosphorylation of eIF2 α for
translation. The transcription factor ATF4 is such an example. Translational upregulation
of ATF4 can induce the expression of UPR target genes that promote ER folding capacity
and adaptation to stress.
IRE1 α is transmembrane Ser/Thr protein kinase which also has site specific
endoribonuclease (RNase) activity. In response to ER stress, IRE1 α dimerizes and
autophosphorylates, and thereby activating its RNase activity to cleave a 26 base intron
from mRNA encoding X-box-binding protein 1 (XBP1), resulting in a translational
frameshift and translation of a spliced form of XBP1 (XBP1-s) which is a more stable
and potent transcription factor of target genes including DnaJ, p58, ERdj4, EDEM, and
PDI, all involved in protein folding and ER-associated degradation (ERAD) (Calfon et al.,
2002; A.-hwee Lee, Iwakoshi, & Glimcher, 2003).
ATF6, a basic leucine zipper (bZIP) transcription factor, when released from
GRP78 upon ER stress, translocates from the ER to the Golgi apparatus, where it is
cleaved by S1P and S2P proteases to generate the active nuclear form of ATF6 (p50).
3

This regulated intramembrane proteolysis is a process by which bZIP-containing
transcription factors traffic from the ER to the Golgi where they are cleaved and therefore
release their functional isoforms. Cleaved ATF6 and spliced XBP1 act in parallel to
mainly induce transcription of genes encoding ER chaperones and enzymes that promote
protein folding and maturation.
Therefore, the UPR activation results in attenuation of general protein translation,
transcriptional induction of ER chaperones and enzymes to increase ER folding capacity
and initiate ERAD to eliminate ER misfolded protein aggregates which in all is an
adaptive response.
However, under severe and prolonged ER stress conditions where the cells fail to
resolve the ER stress and cannot restore ER homeostasis, the UPR activates unique
pathways that lead to cell death through apoptosis (J Wu & R J Kaufman, 2006). This
protects the organism by removing the stressed cells that have severe accumulation of
misfolded proteins. ER stress induced apoptosis is mediated largely by CHOP (C/EBP
homologous protein), a transcription factor that is downstream of PERK-eIF2 α-ATF4
pathway. It inhibits the anti-apoptotic B-cell leukemia/lymphoma 2 (Bcl-2) and induces
numerous pro-apoptotic factors such as DNA damage-inducible gene 34 (GADD34)
which promote protein synthesis and oxidative stress in cells leading to cell death.
The homeostasis of the ER is mainly maintained by the protein folding machinery
consisting of molecular chaperones and folding enzymes (Min Ni & Amy S Lee, 2007).
The 78 kD glucose regulated protein, GRP78, also known as immunoglobulin heavy
chain binding protein (BiP) is a major chaperone involved in multiple steps of protein
4

processing. GRP78 can form a large multiprotein complex with other ER chaperones,
GRP94, PDI (protein disulfide isomerase), ERp72, GRP170/ORP150, UGGT (UDP-
glucose glycoprotein-glucosyltransferase), CaBP1 (calcium binding protein 1),
cyclophilin B and SDF-L1 (Meunier, Usherwood, Chung, & Linda M Hendershot, 2002).
Among them, the multifunctional GRP78 is the best characterized not only as a molecular
chaperone with protein folding abilities, but also the master regulator of the unfolded
protein response (UPR), and recently been shown that a subfraction of it is localized on
the cell surface acting as a co-receptor for various signaling pathway activation (Min Ni,
Yi Zhang, & Amy S Lee, 2011; Pfaffenbach & Amy S Lee, 2010). Another chaperoning
system consists of calnexin, calreticulin and GRP58/ERp57 which recognize the nascent
peptide with monoglucosylated N-linked glycans (Min Ni & Amy S Lee, 2007).

1.2 GRP78 and its role in cancer
The metabolic environment of tumors is often acidic, hypoxic, and nutrient
deprived, having reduced amounts of both amino acids and glucose. This can be due to
both poor vascularization and rapid growth of tumor cells, and the intrinsic property of
cancer cells with elevated glucose metabolism and higher glycosylation rates. When cells
undergo glucose or oxygen deprivation, they activate the UPR. Evidence has shown that
the microenvironment of tumors resembles physiological ER stress and hence the UPR is
often activated in tumor cells for cell survival (Feldman, Chauhan, & Koong, 2005; J Li
& A S Lee, 2006). Emerging studies have shown that the UPR is an important
mechanism required for cancer cells to maintain survival, malignancy and therapy
5

resistance. The UPR is an adaptive survival mechanism for cancer cells to survive in the
unfriendly microenvironment.
Because of its antiapoptotic property, induction of GRP78 has been reported as a
prosurvival factor for cells undergoing ER stress. It has been well documented that
GRP78 is highly elevated in a variety of cancer cell lines, solid tumors, and human
cancer biopsies, associating to malignancy and metastasis (Daneshmand et al., 2007; D.
Dong et al., 2008). The elevation of GRP78 transcription under various stress conditions
suggests the involvement of Grp78 in an effective defense mechanism for enhanced cell
survival. Grp78 has been shown to directly interact with apoptotic pathway intermediates,
block caspase activation, and eventually lead to apoptosis inhibition and increased cell
survival (Pootrakul et al., 2006; Reddy et al., 2003). As tumor progression requires
proliferation as well as inhibition of tumor cell death, the inherent roles and antiapoptotic
properties of Grp78 indicate a potential role in cancer progression. GRP78 is also
required for ER integrity and stress-induced autophagy, which may be important for
cancer cell survival (J Li et al., 2008). In agreement to this, the expression of GRP78 is
markedly higher in primary tumors compared with that in benign tissues. This has been
documented in various cancers, including breast cancer (Fernandez et al., 2000; Gazit, J.
Lu, & a S. Lee, 1999), hepatocellular carcinoma (Shuda et al., 2003), lung cancer
(Uramoto et al., 2005; Q. Wang et al., 2005) and prostate cancer (Daneshmand et al.,
2007; Pootrakul et al., 2006).
Tumor resistance to therapy is a major challenge in cancer treatment. Thus, the
need of discovering predictive factors for chemoresistance is crucial for screening and
improving adjuvant therapies for cancer patients to avoid unnecessary side effects to
6

treatments which may turn out to be ineffective or unnecessary. Emerging evidence is
suggesting that GRP levels may be exploited as a prognostic as well as diagnostic marker
for chemoresponsiveness. GRP78 expression level was reported to be such a novel
predictor of breast cancer patient’s responsiveness to adriamycin-based chemotherapy (E.
Lee et al., 2006). By performing a retrospective cohort study of 127 stage 2 and 3 breast
cancer patients treated with adriamycin-based chemotherapy, tumor specimens of the
patients were analyzed for GRP78 expression against the respective drug resistance status,
designated by the time to recurrence (TTR). They found that ~65% of the subjects
expressed high levels of GRP78 which was in concert with previous breast cancer studies
(Fernandez et al., 2000), and revealed a significant association between GRP78
expression level and resistance to adriamycin-based chemotherapy.
In the case of prostate cancer, it has been proposed that due to its antiapoptotic
and prosurvival properties GRP78 may be involved in the resistance of prostate cancer
cells to chemotherapy or castration-resistance (Pootrakul et al., 2006). In support of this
notion, GRP78 expression is upregulated during the transition from localized prostate
cancer to metastatic castration resistance based on in vitro evidence and patient cohort
studies. Using established cell line models, GRP78 expression is elevated in both
castration resistant LNCaP-derived cell line, C42B and androgen deprived LNCaP cells
compared to LNCaP cells grown in androgen-rich media (Pootrakul et al., 2006).
Corresponding with development of castration resistance, high level of GRP78
expression in prostate tumors is associated with greater relative risk or recurrence and
overall survival. It was shown that prostate cancer patients with strong GRP78
immunoreactivity in the primary tumor have a higher risk for clinical recurrence and
7

death compared to those with weak GRP78 expression (Daneshmand et al., 2007).
GRP78 protein expression is significantly higher in malignant prostate cancer tissue than
in benign prostatic tissue. The expression intensity is also significantly associated with
patient survival and clinical recurrence, demonstrating that GRP78 expression levels may
provide prognostic information in tumors (Daneshmand et al., 2007; Pootrakul et al.,
2006). This suggests that GRP78 may be a potential target for molecular therapy.

1.3 ER stress proteins and signaling associated to hematopoiesis and
immunity
In addition to the UPR, other signaling pathways emerge from the ER to the
mitochondria and nucleus forming a complex interconnected signaling network. A
growing body of evidence suggests the UPR and inflammation are interconnected
through various mechanisms, including the production of reactive oxygen species (ROS),
the release of calcium from the ER, the activation of the transcription factor nuclear
factor- κB (NF- κB) and the JUN N-terminal kinase (JNK), and the induction of the acute-
phase response (K. Zhang & Randal J Kaufman, 2008).
Protein folding in the correct conformation is a process that requires the formation
of disulphide bonds. Two ER resident enzymes: protein disulphide isomerase (PDI) and
ER oxidoreductin 1 (ERO1) are involved in disulfide bond formation. The process
requires the transfer of electrons, in which PDI accepts electrons, oxidizing cysteine
residues and forming disulfide bonds, in which the electrons are then passed on to oxygen
8

as the final electron recipient and therefore leading to the production of ROS. Hence,
increased protein folding load in the ER can result in ROS accumulation which might
initiate an inflammatory response.
In addition, NF- κB is a key transcriptional regulator that plays a central role in the
onset of inflammation (Rius et al., 2008). Under normal condition, NF- κB remains
inactive by direct interaction with its inhibitor I κB. The activation of NF- κB is initiated
by phosphorylation of I κB, which leads to degradation of I κB and thereby exposing the
nuclear-localization signal of NF- κB where it is then active and released to translocate
into the nucleus to activate a number of inflammatory genes. Studies have shown that ER
stress and activated UPR has been shown to activate NF- κB by two means. Upon ER
stress, PERK is activated and phosphorylates eIF2 α, leading to a global protein
attenuation. As the half life of I κB is much shorter than NF- κB, translational attenuation
results in an accumulation of free NF- κB and thereby translocating to the nucleus for
transcriptional activation (Deng et al., 2004). Alternatively, activated IRE1 α
autophosphorylates resulting in a conformational change in its cytosolic domain and
thereby can interact a form a complex with tumor-necrosis factor- α-receptor-associated
factor 2 (TRAF2) (F Urano, X. Wang, & Bertolotti, 2000). The IRE1 α-TRAF2 complex
can recruit I κB kinase (IKK) which phosphorylates I κB and leads to degradation of I κB
and nuclear translocation of NF- κB (P. Hu, Han, Couvillon, Randal J Kaufman, & Exton,
2006). Furthermore, the IRE1 α-TRAF2 complex can also recruit the protein kinase JNK,
leading to its activation. Activated JNK induces the expression of inflammatory genes by
phosphorylating the transcription factor activator protein 1 (AP1) (R. J. Davis, 2000).
9

This suggests activated IRE1 α can activate both NF- κB and JNK signaling and therefore
may be an important link between ER stress and the inflammatory response.
Furthermore, an accumulation of misfolded proteins in the ER can cause calcium
leakage from the ER possibly from inositol-triphosphate receptors (Deniaud et al., 2007).
The released calcium from the ER can lead to depolarization of the mitochondria inner
membrane, disrupting electron transport and increasing ROS production (Görlach,
Klappa, & Kietzmann, 2006). Through calcium release, ROS production and misfolded
proteins activating JNK and NF- κB can lead to inflammatory responses and even cell
death (Malhotra & Randal J Kaufman, 2007).
The acute phase response is the systemic inflammatory component of innate
immunity (Yoo & Desiderio, 2003). CREBH is mainly expressed in hepatocytes and
mediates the acute phase response in liver and was shown to be activated by ER stress (K.
Zhang et al., 2006). Its expression is highly induced by pro-inflammatory cytokines, such
as interleukin 6 (IL6), interleukin 1 β (IL1 β) and TNF α. CREBH, similar to ATF6, is a
bZIP-containing transcription factor that resides in the ER membrane. Upon ER stress,
CREBH translocates to the Golgi apparatus where it undergoes proteolytic cleavage by
S1P and S2P, releasing it cytosolic fragment which can then form homodimers or
heterodimers with activated ATF6 and translocate to the nucleus to synergistically
activate the transcription of genes encoding serum amyloid P component and C-reactive
protein, which mediate the acute phase response (K. Zhang et al., 2006).
In addition to the inflammatory response, several other UPR signaling pathways
were shown to be important for immune cell signaling and homeostasis. XBP-1 has been
10

shown to be essential for plasma cell differentiation and UPR induced XBP-1 splicing
was required to restore immunoglobulin production in XBP-1
-/-
B cells, hence suggesting
XBP-1 to be the link between UPR and plasma cell differentiation (Iwakoshi et al., 2003).
XBP-1 was identified to be essential for the survival and development of dendritic cells
(DCs). XBP-1 was found constitutively activated in immature DCs and XBP-1 deficient
chimeric mice exhibited severe reduction of all four subsets of DCs and secreted smaller
amount of IFN γ (Iwakoshi, Pypaert, & Glimcher, 2007). Toll-like receptor (TLR)
signaling in macrophages has been found to activate the IRE1/XBP-1 axis and this is
crucial for host defense. TLR-activated XBP-1 was required for optimal and sustained
production of pro-inflammatory cytokines in macrophages (Martinon, X. Chen, A.-H.
Lee, & Glimcher, 2010).
Many ER chaperones have been shown to be involved and/or required for the
proper folding of immunity-related proteins. GRP78, also known as immunoglobulin
heavy chain binding protein (BiP) is the best characterized. Initially, GRP78 was found
associated with the heavy chain of immunoglobulins in pre-B cells, B cells and at
increased levels in plasma cells (Munro & Pelham, 1986). It was then demonstrated that
GRP78 directly associates to the nascent chains of immunoglobulin, preventing their
premature escape and therefore assuring their proper folding (Bole, L M Hendershot, &
Kearney, 1986). GRP78 (BiP) has been shown to have immunomodulatory properties. It
has been reported that GRP78 can stimulate the release of Th2 cytokines, especially IL-
10 and IL-4 and therefore affect the development of regulatory T cells (Corrigall,
Vittecoq, & Panayi, 2009) and has been demonstrated to coimmunoprecipitate with IL-6
and regulate its secretion from astrocytes (Qian, Zheng, Weber, & Tiffany-Castiglioni,
11

2007). Another ER chaperone protein, GRP94 has also been shown to play an important
role in various immune functions (Jockheck-Clark et al., 2010). GRP94 is required for the
expression of a large number of integrins, as well as Toll-like receptors and regulates
innate immunity of macrophages (Randow & Seed, 2001; Y. Yang et al., 2007), and early
T and B lymphopoiesis (B. Liu & Z. Li, 2008; Staron et al., 2010). Recently, GRP94 has
been identified as a novel cell intrinsic factor required to maintain the interaction of
hematopoietic stem cells (HSCs) with their niche, in which acute elimination of GRP94
in the hematopoietic system resulted in expansion of HSCs attributed to the impaired
interaction of HSCs and the niche, resulting in enhanced HSC mobilization and
compromised homing and lodging ability of primitive hematopoietic cells (Biquan Luo et
al., 2011). In addition, two other ER chaperones, Calnexin and Calreticulin have been
shown to complex with CD1b heavy chains to prevent from degradation and proper
expression (Hüttinger, Staffler, Majdic, & Stockinger, 1999). Furthermore, calnexin has
also been shown to play a role in the proper folding and assembly of MHC class I and
class II molecules (Diedrich, Bangia, Pan, & Cresswell, 2001; Schreiber et al., 1994).
ERdj3 is a DnaJ protein and co-chaperone for GRP78. ERdj3 initially binds to unfolded
protein substrates including unfolded light chains and inhibits protein aggregation until
GRP78 joins the complex (Ying Shen & Linda M Hendershot, 2005).

12

Chapter 2
Inducible knockout of GRP78 in the hematopoietic system suppresses
Pten-null leukemogenesis and AKT oncogenic signaling

2.1 Introduction
One of the most frequently mutated tumor suppressor genes in human cancer is
PTEN (phosphatase and tension homolog deleted on chromosome 10), which encodes for
a non-redundant, plasma-membrane lipid phosphatase that antagonizes the
phosphatidylinositol-3-kinase (PI3K) signaling pathway.(Maehama & Dixon, 1998;
Sansal & Sellers, 2004) Upon loss of PTEN, the PI3K/AKT signaling pathway is
activated, leading to promotion of cell survival, proliferation and angiogenesis, as well as
activation of the mTOR and S6 kinases, resulting in enhanced protein translation
commonly observed in cancers.(Salmena, Carracedo, & Pandolfi, 2008) PTEN also has a
role in the maintenance of the hematopoietic stem cells (HSCs), as shown by ablation of
PTEN function in adult HSCs through crossing of polyinosine-polycytidine (pIpC)-
inducible Mx-1-Cre transgenic mouse line (Kiihn & Aguet, 1995) with a PTEN flox/flox
(Pten
f/f
) mouse line.(Lesche et al., 2002) Induced Cre-expression in postnatal mice
exhausted normal HSCs and promoted excessive proliferation of leukemogenic stem cells,
resulting in the development of myeloproliferative disorders (MPD) and eventually
leukemia.(H. Yilmaz et al., 2006; Jiwang Zhang et al., 2006) The latter study further
showed that the mTOR inhibitor rapamycin effectively suppressed growth of the
leukemogenic stem cells and prevented exhaustion of normal HSCs. Furthermore, recent
13

studies showed that PTEN is down-regulated by BCR-ABL in leukemic stem cells and
PI3K and AKT play critical roles in BCR-ABL induced leukemia in mice.(Kharas et al.,
2008; Peng et al., 2009) Thus, inhibition of the PTEN/AKT/mTOR pathway not only can
suppress solid tumor growth, but could also represent a novel therapeutic strategy against
stem cell disorders that are leukemogenic in nature.
GRP78, also referred to as BiP or HSPA5, is a member of the HSP70 protein
family.(Min Ni & Amy S Lee, 2007) Traditionally it is regarded as a major endoplasmic
reticulum (ER) chaperone facilitating protein folding, protein quality control, calcium
binding and regulation of transmembrane ER inducers.(M. Wang et al., 2009) However,
evidence is emerging that a subfraction of GRP78 can localize to the cell surface under
pathophysiologic conditions, such as in cancer cells, and serves as a co-receptor for
growth and pro-survival signaling mediated by PI3K/AKT.(Uma Kant Misra, Deedwania,
& Salvatore Vincent Pizzo, 2006; Min Ni, Yi Zhang, & Amy S Lee, 2011; Yi Zhang, R.
Liu, Min Ni, Gill, & Amy S Lee, 2010) Strikingly, Pten null prostate tumorigenesis and
AKT activation are potently blocked by targeted knockout of GRP78 in the prostate
epithelium.(Fu et al., 2008) Furthermore, in a variety of cell culture systems, specific
knockdown of GRP78 results in inhibition of AKT activation.
11,14
While GRP78 is
established to protect cancer cells against the adverse hypoxic and nutrient-deprived
microenvironment of solid tumors, (A S Lee, 2007; Pfaffenbach & Amy S Lee, 2010) its
role in initiation and progression of hematologic cancers is not known. We report here
that GRP78 expression was elevated in leukemic blasts of adult patients, leukemia cell
lines and inversely correlates with time to relapse in childhood acute lymphocytic
leukemia. Through creation of a biallelic conditional knockout mouse model of GRP78
14

and PTEN in the hematopoietic system, we demonstrate here that GRP78 deficiency
potently suppresses leukemogenesis and AKT/mTOR signaling in PTEN null bone
marrow cells. Furthermore, we showed that manipulation of GRP78 expression level can
alter the sensitivity of human leukemic cells to drug-induced apoptosis, suggesting that
GRP78 is a novel potent therapeutic target for leukemia.

2.2 Materials and methods
Clinical specimens
Leukemic blasts from bone marrow and peripheral blood samples were generously
provided by Dr. Allen Yang (University of Southern California Keck School of
Medicine) and the University of Southern California Translational Pathology Core. They
were obtained from diagnosed leukemia patients in USC Norris Comprehensive Cancer
Center according to institutional guidelines. The normal peripheral blood mononuclear
cells were purchased from ALLCELLS (catalog #PB003F). The ethical use of the human
tissues for research was approved by the USC Institutional Review Board.
RT-PCR
Total RNA from peripheral blood or bone marrow samples of normal individuals or
leukemia patients were extracted by Trizol reagent (Invitrogen), followed by DNase I
treatment. First-strand cDNA was synthesized with the Superscript II reverse
15

transcriptase (Invitrogen) with oligo d(T) primer according to manufacturer’s instructions.
The primers used for human Grp78 and β-actin are listed in Table 3.1.
Mice
The construction of the Pten
f/f
Grp78
f/f
mice was previously described.(Fu et al.,
2008) The transgenic Mx1-Cre mouse in C57BL/6 background was purchased from the
Jackson Laboratory (catalog #003556). The breeding of the Pten
f/f
Grp78
f/f
or Grp78
f/f
mice with the Mx1-Cre mice to generate the mice cohorts is described in Figure 2.2.
All protocols for animal use were reviewed and approved by the USC Institutional
Animal Care and Use Committee.
Genotyping
For genotyping of the Grp78 floxed allele or knockout (-) alleles, primer sets PF3
and PR3 or PF3 and PTR were used respectively. (S. Luo, C. Mao, Brenda Lee, & Amy S
Lee, 2006) Genotyping for the Cre transgene, primers Cre109 and Cre457 were used.
Reactions for Grp78 floxed, wildtype, knockout alleles and Cre were run under the
following conditions: 94°C for 5 min, followed by 46 cycles of 94°C for 20 sec, 62°C for
30 sec, and 72°C for 30 sec, ended with 72°C for 7 min. For genotyping of the Pten
floxed allele in mice cohorts, primer set Pten-1 and Pten-2 were used. Reactions were
under the following condition: 94°C for 3 min, followed by 46 cycles of 94°C for 30 sec,
62°C for 1 min, and 72°C for 1’10, ended with 72°C for 10 min. For PCR detection of
Pten knockout allele, primer set Pten-F, Pten-R1, Pten-Rko was used. Reactions were
16

under the following condition: 94°C for 3 min, followed by 40 cycles of 94°C for 45 sec,
60°C for 30 sec, and 72°C for 1 min, ended with 72°C for 10 min.
Gene Primer name Sequence
Grp78 PF3 5’-GATTTGAACTCAGGACCTTCGGAAGAGCAG-3’
PR3 5’-GCAATAGCAGCTGCTGTACTGTGAGGATGA-3’
PTR 5’-TTGTTAGGGGTCGTTCACCTAGA-3’
Cre Cre109 5’-AAGAACCTGATGGACATGTTCAGGGA-3’
Cre457 5’-ACGAACCTGGTCGAAATCAGTGCGTTC-3’
Pten Pten-1 5’-AAGCAAGCACTCTGCGAAACTGA -3’
Pten-2 5’-GATTGTCATCTTCACTTAGCCATTGGT -3’
Pten-F 5'-TCCCAGAGTTCATACCAGGA-3'
Pten-R1 5'-AATCTGTGCATGAAGGGAAC-3'
Pten-Rko 5'-GCAATGGCCAGTACTAGTGAAC-3'
Table 2.1: Primer sequences for mouse genotyping
Complete blood count
Peripheral blood was collected via tail bleeding and analyzed using an auto
hematology analyzer BC-2800 vet (Mindray) according to manufacturer’s instructions.
Flow cytometry
Bone marrow cells were flushed from long bones (tibias and femurs) with
Dulbecco’s phosphate-buffered saline without calcium and magnesium (DPBS), and then
filtered through nylon screen (70 µm, BD) to obtain single cell suspension. Bone marrow
cells were resuspended in DPBS with 0.5% BSA and 0.1% sodium azide. 3x106 cells
were used to stain for HSC percentage, using LSK markers (Lin-Sca-1+c-Kit+) and
3.5x10
6
cells used for immunophenotyping for leukemic blast cells. Cells were incubated
with florescence conjugated antibodies for Lin markers [B220 (RA3-6B2/FITC), CD4
(RM4-5/FITC), CD8 (53-6.7/FITC), Gr-1 (RB6-8C5/FITC), Mac-1 (WT.5/FITC), TER-
17

119 (FITC)], c-Kit (2B8/APC-H7) and Sca-1 (D7/PE-cy7), which were all purchased
from BD Pharmingen. To identify leukemic blast cells, anti-CD45 (30-F11/APC) from
eBioscience was used. After washing, cells were resuspended in PBS with 0.1% sodium
azide and 2 µl 7-AAD was added to exclude dead cells. All FACS analyses were
performed on LSR II flow cytometer.
Cell cycle analysis
1x10
7
whole BM cells were incubated with 10 µg/ml Hoechst 33342 (Sigma-
Aldrich) at 37°C for 45 minutes, then stained with primitive hematopoietic cell antibodies
(Lin, Sca-1 and c-Kit) as described above. The stained cells were resuspended in 10%
buffered formalin and incubated at 4°C overnight. To stain for RNA content, pyronin Y
(Polysciences Inc., Warrington, PA) was added to the cells at a final concentration of
0.75 µg/ml and incubated at 4°C for 30 minutes. Cell cycle status was examined using a
LSR II flow cytometer.
LSK apoptosis assay
1×10
6
whole BM cells were stained with primitive hematopoietic cell antibodies (Lin,
c-Kit and Sca-1) as described above. The stained cells were resuspended in 150 μL
1×Annexin V binding buffer (BD Pharmingen) and incubated with Annexin V and
7AAD (both from BD Pharmingen) for 15 min. Cell apoptosis was examined within an
hour using a BD LSR II flow cytometer.

18

Cell culture and transfection
The human leukemia NB4 cells (generously provided by Dr. Allen Yang,
University of Southern California Keck School of Medicine) and HL60 cells were
maintained in RPMI medium supplemented with 10% FBS and 1%
penicillin/streptomycin. For knockdown of GRP78, the siRNA against Grp78 is 5’-
ggagcgcauugauacuagatt-3’, the control siRNA is 5’-aaggagacguauagcaacggu-3’. For
knockdown of Grp94, the siRNA against Grp94 is 5’-aucugggacaagcgaguuuuu-3’.
Transfection of siRNA was described.(Fu et al., 2008) Forty-eight hours after siRNA
transfection, the cells were treated with 300 nM Tg (Thapsigargin) or AraC (24 hours)
prior to harvesting. For serum stimulation experiments, 24 hours after siRNA transfection,
HL60 cells were serum starved for 16 hours, followed by 10% serum stimulation prior to
harvesting. For overexpression of GRP78, NB4 cells at 60% confluency were transfected
with either pcDNA or Flag-tagged GRP78 expression vector [pcDNA-F-GRP78)] as
previously described.(Yi Zhang et al., 2010) Transfection was performed using BioT
(Bioland Scientific) following manufacturer’s instructions. Forty-eight hours after
transfection, the cells were treated with AraC as indicated for 24 hours prior to harvesting.
Western blot analysis
Whole cell lysates were prepared from Ficoll-Paque isolated peripheral blood
mononuclear cells of normal control and leukemia patients, leukemia cell lines, single-
cell suspensions of bone marrow cells or splenocytes from the indicated mice. Cells were
lysed in RIPA buffer supplemented with competent protease-inhibitor mixture (Roche)
and phosphatase-inhibitor mixture (Roche) and 25 µg cell lysate was subjected to SDS-
19

PAGE. The immunoblot membranes were incubated with primary antibodies at 4°C
overnight and the protein signals were detected with ECL reagent (Roche) or Supersignal
chemiluminescence reagent (PIERCE) after reacting with HRP-conjugated secondary
antibody. The primary antibodies used are as follows. Monoclonal mouse anti-GRP78
(1:2000) and monoclonal mouse anti-caspase-7 (1:4000) are from BD Pharmingen.
Mouse anti-PTEN (26H9, 1:1000), rabbit anti-AKT (1:1000), rabbit anti-p-AKT (Ser473,
1:1000), rabbit anti-p-AKT (Thr308, 1:1000), rabbit anti-S6K (1:1000), rabbit anti-p-S6K
(Thr389, 1:1000), rabbit anti-p-PI3K p85 (Tyr458 1:1000), mouse anti-p44/42 MAPK
(ERK1/2) (1:1000), mouse anti p-p44/42 MAPK (ERK1/2) (Thr202/Tyr204, 1:1000),
rabbit anti-GSK3 β (1:1000), rabbit anti-p-GSK3 β (Ser9 1:1000), rabbit anti-p38 MAPK
(1:1000), rabbit anti-p-p38 MAPK (Thr180/Tyr182 1:1000) are from Cell Signaling.
Mouse anti-PARP (1:500) is from Santa Cruz Biotechnology. Mouse anti- β-actin
(1:5000) is from Sigma.
Microscopy and immunofluorescence analysis
For immunofluorescence staining procedures, HL60 cells were transfected with
sictrl or siGrp78 as previously described in methods, followed by 12 hour serum
starvation and then were either untreated or stimulated with 10% FBS for 3 minutes.
Serum starved untreated or stimulated cells were fixed by adding an equal volume of 4%
paraformaldehyde to cells in media for 20 minutes at room temperature (RT). After two
washes with TBS (25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 7.4), cells were rinsed
with distilled H
2
O and applied to lysine coated slides (Snowcoat X-tra™, Surgipath
Medical Instruments). Cells were post-fixed with 4% paraformaldehyde for 15 minutes at
20

RT. After three washes with TBS, cells were permeabilized for 15 minutes at RT with
0.5% saponin (47036, Sigma). After three washes with TBS, cells were blocked with
10% goat serum in TBS for 30 minutes at 37℃. This was followed by incubation with
mouse anti-PI(3,4,5)P3 IgG antibody (Z-P345b, Echelon Biosciences) diluted in TBS for
60 minutes at 37℃. Cells were rinsed three times with TBS containing 1% goat serum
and incubated with Alexa Fluor 488 goat anti-mouse antibody (Invitrogen) for 30 minutes
at 37℃. After three final washes with distilled H
2
O, cells were mounted with Vectashield
antifade medium with DAPI (H-1200, Vector Laboratories). Samples were stored at 4 ℃
in dark until use.
Confocal images were obtained by Zeiss LSM 510 laser scanning confocal
microscope under 40 × or 100 × magnification. For the fluorescence quantification,
monocolor confocal microscope images under 100 × magnification with thickness of
500nm were acquired below the saturation level of the camera. All images were taken
using constant settings. Digital images (n ≥30 for each group) were imported into
McMaster Biophotonics Facility (MBF)-ImageJ software. Pixel intensities at the cell
perimeter around the leading edge were collected using a 4 µm tangent line and averaged
by the software. The edge intensities from the serum-stimulated cells were averaged and
normalized to the edge intensities of non-stimulated cells.
Trypan blue exclusion assay
10,000 cells/well were plated into a 96-well plate and treated with either PBS 5 µM or 10
µM AraC for 24 hours. Trypan blue solution (0.4%, Sigma) was added into the medium
21

at 1:1 dilution and incubated for 5 minutes. The numbers of trypan blue positive cells and
total cells were counted using a hemocytometer.
Annexin V/7-AAD apoptosis detection
For apoptosis detection, cells were collected by centrifugation at 1,500 rpm for 5
minutes at 4°C and washed by PBS once. The cells were resuspended at the concentration
of around 1-1.5x10
6
cells/ml in the binding buffer (10 mM Hepes/NaOH (pH 7.4), 140
mM NaCl, 2.5 mM CaCl2), and then incubated with 5 µl PE-Annexin V and 5 µl 7-
amino actinomycin (7-AAD) for 15 minutes at room temperature. Cell apoptosis was
analyzed on LSR II flow cytometer.
Microarray analysis for clinical specimens
Expression values for Grp78 mRNA were acquired from a previously published
genome-wide study of 60 patients with relapsed childhood acute lymphoblastic leukemia
(ALL) on the Affymetrix (Santa Clara, CA) U133A microarray platform. (Bhojwani et al.,
2006) Gene expression data is publically available through Gene Expression Omnibus
(http://www.ncbi.nlm.gov/geo); series accession number GSE3912. Bone marrow blasts
were obtained from patients enrolled on the Children’s Oncology Group protocol
AALL01P2 for first medullary relapse.(Raetz et al., 2008) Thirty-seven patients relapsed
early (<36 months from initial diagnosis) and 23 patients relapsed late ( ≥36 months). The
Affymetrix probeset 211936_at represents the gene HSPA5 (also known as GRP78).
Normalization was performed by the method of “Robust Multiarray Analysis” (Irizarry et
al., 2003) and the normalized probeset intensity values were used for subsequent analyses.
22

Microarray analysis for adult tissues and cell lines
The Grp78 mRNA expression values were downloaded from the Amazonia
microarray database, which can be accessed on the website:
http://amazonia.transcriptome.eu/expression.php?geneId=Hs.716396. The data presented
in Figure 2.1C was from studies performed with normal adult tissues and human
leukemia cell lines.
Statistical analyses
Kaplan-Maier survival curves were constructed using GraphPad (Prism5
software), and log-rank analysis was used to analyze the results. For bar graphs, the
unpaired 2-tailed Student’s t-test was used to compute P values, and the error bars reflect
standard error (s.e.) (*, P<0.05, **, P<0.01, ***, P<0.001)

2.3 Results
2.3.1 GRP78 expression is elevated in patient leukemic blasts, leukemia cell lines
and early relapse in childhood leukemia
Patient bone marrow or peripheral blood samples were collected from patients
diagnosed with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute
lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL). Mononuclear
cells were purified and expression of GRP78 was first determined by RT-PCR. Examples
of the RT-PCR results are shown in Figure 2.1A and a summary of an expanded study is
shown below. These results reveal that normal controls expressed low basal level of
23

Grp78 transcripts, which was generally elevated in all four types of patient samples. In
agreement with the mRNA results, compared to normal control, GRP78 protein
expression was also generally elevated in mononuclear cells from leukemia patients
(Figure 2.1B). Additionally, analysis of multiple microarray databases further revealed
that Grp78 mRNA was expressed at low level in whole blood, moderate level in bone
marrow but higher level (by about 5-fold) in myelogenous leukemia cell lines K562 and
HL60 (Figure 2.1C). Thus, increase in GRP78 expression associates with leukemogenesis.
In addition to promoting solid tumor progression, GRP78 is known to confer
resistance to anti-cancer agents in solid tumors, however, its role in therapeutic resistance
in leukemia is not known.(A S Lee, 2007) The outcome for relapsed leukemia is
extremely poor. Duration of first remission is an important prognostic factor following
retrieval therapy and early relapse portends a poor response. Previously we performed a
genome-wide microarray analysis of bone marrow blasts obtained from 60 patients with
relapsed childhood ALL (Bhojwani et al., 2006). Our study revealed that leukemic blasts
at relapse demonstrated higher expression of cell cycle regulators and apoptosis inhibitors
compared to their diagnostic counterparts. Differential expression of these genes was
more pronounced in early versus late relapse. The median value of Grp78 expression was
456 in patients with relapsed ALL. Among the 37 patients who relapsed early (<36
months from initial diagnosis) and 23 patients who relapsed late ( ≥36 months), Grp78
level was significantly higher in patients who relapsed early vs. late (mean of 779 vs.
450; p=0.008, Figure 2.1D). These results demonstrate that Grp78 expression inversely
correlates with time to relapse in childhood ALL.
24

Figure 2.1: Elevated Grp78 expression in leukemia and Grp78 expression inversely correlates with
time to relapse in childhood ALL. (A) Grp78 mRNA levels measured by RT-PCR from peripheral blood
or bone marrow (asterisk) samples of the indicated types of leukemia patients, with -actin levels as
control. Below is a summary of Grp78 transcript level in an expanded study after normalization against -
actin. (B) GRP78 protein expression in Ficoll-Paque isolated peripheral blood mononuclear cells of normal
control and leukemia patients (AML, CML, ALL, CLL). Below is the quantitation of the relative GRP78
level normalized against -actin. (C) Relative Grp78 mRNA levels in the indicated tissues and cell lines
from Amazonia microarray database, with the level of Grp78 mRNA in blood set as 1. The data are
presented as mean ± s.e. (D) Grp78 expression level determined from microarray database of relapse ALL
patients (n=60) was plotted against time to relapse. The horizontal bar represents median GRP78
expression (456) of all ALL patients and the vertical bar at the 36 month time point separates patients with
early relapse (left) and late relapse (right).

25

2.3.2 Creation of mouse models with conditional biallelic deletion of Pten and Grp78
in the hematopoietic system
The association between elevated Grp78 expression and leukemia suggests that
GRP78 may be a major effector for leukemogenesis and subsequent therapeutic
resistance. To test directly the requirement of GRP78 for leukemogenesis, we created a
new mouse model containing the Pten floxed allele, the Grp78 floxed allele and the
Mx-1-Cre transgene.

Figure 2.2: The breeding scheme for the double conditional knockout mice. The generation of parental
Pten
f/f
Grp78
f/f
was described previously.
15
Pten
+/+
Grp78
+/+
Mx1-Cre was commercially purchased from the
Jackson Laboratory. The genotypes indicated with the gray shade were used in this study. The genetic
background of the parental mouse strain is indicated below within the square brackets. The numbers below
the genotypes indicate the expected probability of the indicated genotype among the offspring.
Pten and Grp78 were deleted in the hematopoietic system from 6-8 week old Pten,
Grp78 floxed mice carrying the Mx-1-Cre transgene by administrating pIpC every other
day for a total of 7 doses to induce Cre expression.(Kiihn & Aguet, 1995) The mice were
analyzed 6 days post pIpC injection. Littermates without the Cre transgene
26

(Pten
f/f
Grp78
+/+
and Pten
f/f
Grp78
f/+
) are phenotypically equivalent to animals with a WT
Pten allele; they served as wild-type (WT) normal controls and were also injected with
pIpC. Since mice with homozygous deletion of both Pten and Grp78 (cPten
f/f
Grp78
f/f
)
died during the pIpC administration interval (data not shown), this study focused on Pten
null, Grp78 heterozygous (cPten
f/f
Grp78
f/+
) mice. Partial reduction of GRP78 in
heterozygotes is a valuable model mimicking anti-GRP78 therapeutic agents that achieve
partial suppression of GRP78 in human diseases. The status of Pten and Grp78 deletion
was validated by PCR in isolated bone marrow cells (Figure 2.3A), splenocytes (Figure
2.3B) and peripheral blood (Figure 2.4A). Western blot analysis of bone marrow cells
(Figure 2.3C) and splenocytes (Figure 2.3D) confirmed that PTEN expression was
completely ablated in the cPten
f/f
and cPten
f/f
Grp78
f/+
mice and GPR78 expression was
reduced in the cPten
f/f
Grp78
f/+
mice. As reported previously,(H. Yilmaz et al., 2006;
Jiwang Zhang et al., 2006) cPten
f/f
mice developed MPD which includes hunched posture,
enlarged lymph nodes in the axillary areas and an increased number of immature
progenitor cells in peripheral blood (Figure 2.2E). Strikingly, despite loss of PTEN,
cPten
f/f
Grp78
f/+
mice resembled WT normal controls with normal posture and no
apparent increase in immature progenitor cells in the peripheral blood (Figure2.3E).
27

Figure 2.3: Grp78 heterozygosity impedes PTEN null-induced leukemogenesis. (A-B) Representative
PCR genotyping results from WT (P
f/f
78
f/+
), PTEN null (cP
f/f
) and PTEN null, Grp78 heterozygous mice
(cP
f/f
78
f/+
) bone marrow (A) and splenocytes (B) 6 days post completion of pIpC treatment. (C-D) Western
blot results for detection of GRP78 and PTEN protein level in the bone marrow (C) and splenocytes (D)
performed in duplicates. (E) (Upper panel) Hunched posture of PTEN null mice compared to normal
posture of wild-type and cP
f/f
78
f/+
mice. (Lower panel) Detection of immature progenitor cells (dark
arrows) in the blood smear of PTEN null

mice. A magnified image of one of these cells is shown in the
upper right corner. Scale bar represents 30 µm.

cGrp78
f/+
mice were also used to serve as internal controls. This was to determine
if there were any effects of only acutely knocking out one allele of Grp78 with both the
Pten alleles intact. Both PCR genotyping and western blot analysis of the bone marrow
cells (Figure 2.4B-C) showed efficient knockdown of GRP78 both at the DNA and
28

protein level. These mice were also analyzed at the 6 day timepoint post completion of
the pIpC treatment.

Figure 2.4: PTEN and GRP78 were knockdown in the peripheral blood and bone marrow of mutant
mice. (A) Representative PCR genotyping results from cPten
f/f
, cPten
f/f
Grp78
f/+
, Pten
f/f
Grp78
f/f
and
Pten
f/f
Grp78
f/+
peripheral blood cells 6 days post completion of pIpC treatment. (B) Representative PCR
genotyping results from WT (78
f/+
) and Grp78 heterozygous mice (c78
f/+
) bone marrow 6 days post
completion of pIpC treatment. (C) Western blot results for detection of GRP78 protein level in the bone
marrow performed in duplicates.

2.3.3 Grp78 heterozygosity suppresses Pten null-mediated MPD characteristics and
blast cell expansion
Compared to WT controls, the cPten
f/f
mice showed substantially enlarged spleen,
liver and thymus (Figure 2.5A), and H&E staining of the paraffin tissue sections showed
effaced splenic architecture and myeloid cell infiltration in the liver (Figure 2.5B). The
cPten
f/f
Grp78
f/+
mice exhibited intermediate enlargement of spleen and liver size but not
the thymus, close to normal splenic architecture and absence of the myeloid cell
29

infiltration in the liver (Figure 2.5A-C). In agreement with previous reports, PTEN null
mice showed significant decrease in bone marrow cells and hematopoietic stem cells
(HSCs) as measured by the percentage of Lin
-
Sca-1
+
c-Kit
+
cells (referred to as ‘LSK’
cells), which include both long-term and short-term HSCs.(Guo et al., 2008) Grp78
heterozygosity reversed these trends such that in the cPten
f/f
Grp78
f/+
mice, bone marrow
cell numbers were partially restored and the percent of HSCs in the bone marrow was
close to normal level (Figure 2.5C). By examining the cell cycle profiles of the HSCs
(LSK cells), we further determined that the increase in proliferation in cPten
f/f
HSC was
suppressed in cPten
f/f
Grp78
f/+
HSC (Figure 2.5D), while apoptosis was not affected
(Figure 2.5E). For comparison, we also analyzed cGrp78
f/+
mice which did not exhibit
any obvious abnormality compared to littermate WT mice, exhibiting normal organ size
and morphology, no loss in total bone marrow cell number or HSC population (Figure
2.5A-C). Collectively, these results provide proof-of-principle that anti-GRP78 agents
that partially suppress GRP78 can potentially suppress development of PTEN null-
mediated myeloproliferative disease without affecting normal organ function.
30

Figure 2.5: Grp78 heterozygosity suppresses PTEN null-mediated MPD characteristics. All analyses
were performed 6 days post completion of pIpC treatment. (A) Organ size and morphology from mice of
the indicated genotypes. (B) H&E staining of paraffin sections of spleen (upper) and liver (lower) of the
indicated genotypes. The scale bar represents 200 µm. (C) Quantitation of the spleen weight (n=8), total
bone marrow (BM) cell number (n=8), and % of LSK cells in bone marrow (n=5) for each indicated
genotypes. (D) Quantitation of cell cycle distribution of LSK cells from WT (n=3) cP
f/f
(n=5) and cP
f/f
78
f/+

(n=5). (E) Flow cytometric analysis of apoptotic LSK cells using Annexin V and 7-AAD. All data are
presented as mean ± standard error (s.e.) (*P<0.05, **P<0.01 and ***P<0.001 Student’s t-test).
To further assess whether Grp78 heterozygosity affects leukemic blast cell expansion,
we analyzed for leukemic blast cell presence using side scatter pattern and CD45 staining
in bone marrow cell suspensions.(Guo et al., 2008) Dramatic increase of blast cell
frequency was observed in PTEN null mice as previously described.(H. Yilmaz et al.,
2006) In contrast, despite Pten deletion, the percent of blast cells in the bone marrow in
cPten
f/f
Grp78
f/+
mice was similar to normal WT level (Figure 2.6A and B). This implies
31

that partial suppression of one cellular moiety, GRP78, is sufficient to suppress PTEN
null-induced leukemic blast expansion. In agreement, cPten
f/f
Grp78
f/+
mice showed a
statistically significantly prolonged life span than the cPten
f/f
mice (P<0.05) (Figure 2.6C).

Figure 2.6: Grp78 heterozygosity suppresses PTEN null-mediated blast cell expansion and prolongs
survival. (A) Representative results of leukemic blast cells identification. P1 population in green represents
neutrophil region. P2 population in red represents leukemic blast cell region. (B) Quantitation of the
leukemic blast cell percentages of WT (n=7), cP
f/f
(n=7) and cP
f/f
78
f/+
(n=10) shown in (A). The data are
presented as mean ± s.e (**P<0.01, Student’s t-test). (C) Kaplan-Meier survival curve of WT (n=10),
PTEN null (cP
f/f
, n=14), PTEN null, Grp78 heterozygous mice (cP
f/f
78
f/+
, n=21) and Grp78 heterozygous
mice (c78
f/+
, n=5).

32

2.3.4 Pten null Grp78 heterozygous mice show MPD characteristics after longer
latency period
In analyzing these two groups of mice at a later time point (21 days) post injection,
we observed that compared to 6 days post injection, the spleen and the thymus of the
cPten
f/f
Grp78
f/+
mice were further enlarged with evident signs of effacement of the
splenic architecture and myeloid cell infiltration in the liver (Figure 2.7A and B). There
was a further decrease in bone marrow cells and HSC (LSK cells) population (Figure
2.7C). The percent of blast cells in the bone marrow was increased by 4-fold,
corresponding with the trend of increase in white blood cells, specifically monocytes and
granulocytes in blood count analysis, while the other organs remained phenotypically
normal (Figure 2.7D). These data strongly suggest that the cPten
f/f
Grp78
f/+
mutant mice
showed delayed development of leukemia and this could be a contributing cause for their
eventual death between 3 to 14 weeks. The underlying reason for the leukemia
development at the later timepoint in cPten
f/f
Grp78
f/+
mutant mice awaits further
investigation. One possible explanation is that the incomplete deletion efficiency of Mx1-
Cre in the primitive stem cell pool, where there may be a minority of cells that escaped
the knockdown of GRP78 in cPten
f/f
Grp78
f/+
BM, in which these cells with only Pten
deletion and intact GRP78 have greater survival and proliferation advantage, and
therefore in the long run, expand and initiate leukemogenesis phenotype in
cPten
f/f
Grp78
f/+
mutant mice. This may be a possibility as western blot results using BM
cells of cPten
f/f
Grp78
f/+
mice at 3-4 week timepoint exhibit an increase in GRP78 level
compared to that at 6 days post deletion (data not shown).
33

Figure 2.7: Pten null Grp78 heterozygous mice show MPD characteristics after longer latency period.
All analyses were performed 21 days post completion of pIpC treatment. (A) Organ size and morphology
from mice of the indicated genotypes. (B) H&E staining of paraffin sections of spleen (upper) and liver
(lower) of the indicated genotypes. The scale bar represents 200 m. (C) Quantitation of the spleen weight
(n=6), total bone marrow (BM) cell number (n=6), and % of LSK cells in bone marrow (n=5) and % blast
cells in bone marrow (n=6) for each indicated genotypes. (D) Complete blood count with tail peripheral
blood from WT (n=7), cP
f/f
(n=7) and cP
f/f
78
f/+
(n=7) mice. Peripheral blood was collected via tail bleeding
and analyzed using an auto hematology analyzer BC-2800 vet (Mindray) according to manufacturer’s
instructions. All data are presented as mean ± standard error (s.e.) (*P<0.05, **P<0.01 and ***P<0.001
Student’s t-test).

34

2.3.5 Knockdown of GRP78 inhibits AKT/S6K activation in bone marrow and
leukemia cells
To understand the potential mechanisms underlying the relieved leukemic phenotype
of cPten
f/f
Grp78
f/+
mice compared to PTEN null mice, we examined the PI3K/AKT
signaling pathway, in which PTEN is the major negative regulator. In the bone marrow of
the PTEN null mice, strong activation of the AKT/mTOR pathway was detected with
high levels of phosphorylation of AKT
S473
(p-AKT) and the p70 S6 Kinase (p-S6K),
whereas p-AKT level was considerably lower and p-S6K was barely detectable in the
cPten
f/f
Grp78
f/+
mice (Figure 2.8A). The levels of total AKT and S6K were similar in
either genotype. The relative levels of p-AKT
S473
and p-S6K after normalization with the
total AKT and p-S6K were summarized in Figure 2.8B.
To determine whether the decrease in AKT activation in the bone marrow of the
cPten
f/f
Grp78
f/+
mice is a consequence of suppressed leukemogenesis or due to the
requirement of GRP78 in AKT signaling, we performed GRP78 knockdown experiments
in two human leukemia cell lines (HL60 and NB4) using two different stimuli (serum and
ER stress inducer) for AKT activation. The human acute promyelocytic leukemia cell
line HL60 was transfected with siRNA specific for GRP78 or with random siRNA
serving as negative control. Knockdown of GRP78 expression by siRNA in HL60 cells
suppressed serum induced AKT
S473
phosphorylation throughout the entire course of
treatment with no effect on total AKT level (Figure 2.8C and D). Serum stimulation led
to tyrosine phosphorylation of the p85 regulatory subunit of PI3K, relieving its inhibitory
activity on PI3K.(Cuevas et al., 2001) We observed that knockdown of GRP78
suppressed serum-stimulated PI3K p85 phosphorylation, consistent with lower AKT
S473

35

activation in the same cells (Figure 2.8C). Furthermore, knockdown of GRP78 not only
suppressed serum induced AKT
S473
but also AKT
T308
phosphorylation (Figure 2.8E).
GSK3 β, which is a downstream substrate of AKT was also suppressed due to GRP78
knockdown (Figure 2.8E). In contrast, GRP78 knockdown minimally affected the level of
phosphorylated ERK, which showed high constitutive level and remained elevated
throughout serum treatment (Figure 2.8C), and did not affect serum induced p38 MAPK
phosphorylation (Figure 2.8E).

36

Figure 2.8: Knockdown of GRP78 suppresses AKT signaling. (A) Representative Western blot results
using bone marrow cell lysates (n=2 for each genotype) for detection of the indicated protein levels. (B)
Quantitation of the ratio of p-AKT to total AKT and p-S6K to total S6K in (A). The ratio of one of the WT
levels of p-AKT and p-S6K was set as 1. (C) Western blot results of lysates from HL60 cells transfected
with siRNA against Grp78 (siGrp78) or control siRNA (sictrl), followed by serum starvation for 16 hours,
and then stimulated with 10% serum for the indicated time (h). (D) Quantitation of the ratio of p-AKT to
total AKT in (C). The ratio at the 0 hour time point in cells transfected with sictrl was set as 1. (E) Western
blot results of lysates from HL60 cells transfected with siRNA against Grp78 (siGrp78) and control siRNA
(sictrl), followed by 16 hours serum starvation, and then stimulated with 10% serum for the indicated time
(h). (F) (Left) Representative immunofluorescent images of untreated (0 min) or serum stimulated (3 min)
HL60 cells transfected with either sictrl or siGrp78 and stained with anti-PI(3,4,5)P3 antibody. Scale bar
represents 20 µm. (Right) Quantification of relative edge fluorescence intensity of PI(3,4,5)P3 staining in
serum stimulated siRNA transfected cells normalized to non-stimulated siRNA transfected cells (n>30 cells
per condition). The normalized relative intensity of the 3 minute stimulated sictrl cells was set as 1. All data
are presented as mean ± s.e (*P<0.05, *** P<0.001, Student’s t-test).
37

Similarly, knockdown of GRP78 expression in human acute promyelocytic leukemic
cell line NB4 suppressed ER stress induced AKT
S473
phosphorylation. Previously, it has
been demonstrated that the ER stress inducer thapsigargin (Tg) was able to induce AKT
activation.(Fu et al., 2008; Hosoi, Hyoda, Okuma, Nomura, & Ozawa, 2007) In NB4
cells treated with Tg, Western blot analysis showed that p-AKT levels were reduced at all
treatment time points in siGrp78-treated cells expressing a much lower level of GRP78,
while the total levels of AKT was unaffected (Figure 2.9A). The ratio of p-AKT level to
total AKT level was quantitated and summarized in Figure 2.9B. We further determined
that knockdown of GRP78 has no effect on the level of endogenous PTEN expressed in
NB4 cells (Figure 2.9A), thus, knockdown of GRP78 suppresses AKT activation in
leukemic cells independent of PTEN status.

Figure 2.9: Knockdown of GRP78 suppresses ER stress induced AKT activation. (A) Western blot
results of lysates from NB4 cells transfected with siRNA against Grp78 (siGrp78) or control siRNA (sictrl),
followed by 300 nM Tg treatment for the indicated time (h). (B) Quantitation of the ratio of p-AKT to total
AKT in (A). The ratio at the 0 hour time point in cells transfected with sictrl was set as 1. All data are
presented as mean ± s.e (*P<0.05, *** P<0.001, Student’s t-test).

To determine whether the suppression of AKT activation is specific for Grp78
knockdown or a consequence of ER chaperone disruption, another essential
multifunctional ER chaperone, GRP94 (Biquan Luo et al., 2011; C. Mao et al., 2010),
38

was examined. Our results showed that knockdown of GRP94 expression in HL60 cells
did not suppress serum-induced AKT activation as observed for GRP78 knockdown
(Figure 2.10).

Figure 2.10: Knockdown of GRP94 does not suppress AKT activation by serum stimulation. Western
blot results of cell lysates from HL60 cells transfected with siRNA against Grp94 (siGrp94) and control
siRNA (sictrl), followed by 16 hours serum starvation, and then stimulated with 10% serum for the
indicated time (h).
To assess at which step GRP78 is regulating AKT activation, we measured the effect
of GRP78 knockdown on serum stimulated production of phosphatidylinositol 3,4,5-
triphosphate [PI(3,4,5)P3], indicative of PI3K activity. PI(3,4,5)P3 production at the
leading edge of serum stimulated HL60 cells was measured using a monoclonal anti-
PI(3,4,5)P3 antibody previously established for sensitive detection of PI(3,4,5)P3 levels
in cells (Sharma, DesMarais, Sumners, Shaw, & Narang, 2008; Yip et al., 2007). To
examine the requirement of GRP78 in PI(3,4,5)P3 production, HL60 cells were
transfected with siRNA against Grp78 (siGrp78) or control siRNA (sictrl) followed by 12
hour serum starvation, and then stimulated with serum to activate leading edge
PI(3,4,5)P3 production.
39

Figure 2.11: GRP78 knockdown suppresses PI(3,4,5)P3 production at the cell periphery: (Left)
Representative immunofluorescent images of serum stimulated (3 min) HL60 cells transfected with either
sictrl or siGrp78 and stained with anti-PI(3,4,5)P3 antibody or counterstained with DAPI at 100x
magnification. Scale bar represents 3 µm. (Right) Quantification of relative edge fluorescence intensity of
PI(3,4,5)P3 staining in non-stimulated siRNA transfected cells or serum stimulated (3 min) siRNA
transfected cells (n>30 cells per condition). The relative intensity of the 0 minute non-stimulated sictrl cells
was set as 1. All data are presented as mean ± s.e (*** P<0.001, Student’s t-test).
At 3 minutes of serum stimulation, anti-PI(3,4,5)P3 staining showed clear strong
structure in a narrow band at the cell edge in HL60 cells treated with sictrl (Figure 2.8F,
Figure 2.11). In contrast, GRP78 knockdown resulted in a markedly inhibition of
PI(3,4,5)P3 production, evidenced by a significant decrease of anti-PI(3,4,5)P3 staining
at the cell edge of siGRP78 treated cells compared to sictrl treated cells following serum
stimulation (Figure 2.8F, 2.11). Thus, this suggests GRP78 knockdown suppresses AKT
signaling at the level of PI3K activation.

40

2.3.6 Knockdown of GRP78 in human leukemic cells enhances AraC-induced
apoptosis
The association between elevated Grp78 expression level in early relapse of
childhood ALL implies that overexpression of GRP78 could contribute to
chemoresistance. To test this, human leukemia NB4 cells were transiently transfected
with an expression vector for GRP78, or empty vector as a negative control. The cells
were then treated with AraC, a DNA damaging agent commonly used as a chemotherapy
drug against leukemia, with IC
50
value between 5 and 10 µM in leukemic cells (Alenzi,
El-Nashar, Al-Ghamdi, Abbas, Hamad, El-Saeed, Wyse, & Lotfy, 2010a). To monitor for
AraC-induced onset of apoptosis, we measured the cleavage of caspase-7 (C-7) and
PARP as indicators for their activation and onset of apoptosis. In NB4 cells transfected
with empty vector, thus only expressing endogenous level of GRP78, we observed an
increase in the level of cleaved C-7 and PARP in an AraC-dosage dependent manner
(Figure 2.12A). GRP78 overexpression suppressed C-7 and PARP activation in AraC-
treated cells (Figure 2.12A and B). Using siRNA specifically targeting human
Grp78,(Pyrko, Scho, Hofman, T. C. Chen, & Amy S Lee, 2007; Tsutsumi et al., 2006)
we obtained efficient knockdown of GRP78 in NB4 cells. When treated with AraC, cells
with reduced level of GRP78 showed enhanced C-7 and PARP activation (Figure 2.12C
and D). In the trypan blue exclusion assays, live cells possess intact cell membranes that
exclude the trypan blue dye while dead cells take up the dye. In cells treated with control
siRNA, the percentage of trypan blue positive dead cells at 5 and 10 µM AraC were 18%
and 25%, respectively. GRP78 siRNA knockdown in combination with 5 and 10 µM
AraC treatment resulted in an increase to 30% and 40% trypan blue positive cells,
41

respectively (Figure 2.13). To confirm the protective role of GRP78 in chemoresistance
in other leukemia cell lines, HL60 cells were either transfected with control siRNA or
siRNA against GRP78, and then subjected to treatment with 10 µM AraC. Apoptosis was
monitored by AnnexinV/7-AAD FACS analysis (Figure 2.12E). We observed that
knockdown of GRP78 sensitized the HL60 cells to AraC treatment in a time dependent
manner where the enhanced sensitization was most evident at 24 hour treatment (Figure
2.12F). Collectively, these results showed that suppression of GRP78 expression in
human leukemic cells enhances AraC-induced apoptosis and lowers viability.

42

Figure 2.12: Knockdown of GRP78 sensitizes human leukemia cells to AraC and overexpression
confers resistance. (A) Western blot analysis of NB4 cells transfected with pcDNA or Flag-tagged GRP78
expression vector and then treated with indicated AraC concentration for 24 hours for detection of cleaved
caspase 7 and PARP with β-actin as loading control. (B) Quantitation of cleaved caspase 7 and PARP
normalized to β-actin in (A). The ratio at 0 µM AraC in pcDNA cells was set as 1. (C) Western blot
analysis of lysates from NB4 cells transfected with siGrp78 or sictrl and treated with indicated AraC
concentration for 24 hours for detection of cleaved caspase 7 and cleaved PARP with β-actin as loading
control. (D) Quantitation of cleaved caspase 7 and PARP normalized against β-actin in (C). The ratio at 0
µM AraC in sictrl cells was set as 1. (E) Representative AnnexinV/7-AAD flow cytometric apoptosis
analysis of HL60 cells transfected with control siRNA (sictrl) or siRNA against GRP78 (siGrp78). The
cells were either non-treated (0 h) or treated with 10 µM AraC for 24 hours (24 h). (F) Time course
analysis of apoptotic HL60 cells treated with AraC. HL60 cells were transfected with sictrl or siGrp78,
followed by treatment with 10 µM AraC for the indicated time (h). The percentage of apoptotic cells was
measured by AnnexinV/7-AAD flow cytometry. The level of apoptosis at 0 h in cells treated with sictrl was
set as 1. All data are presented as mean ± s.e (*P<0.05, Student’s t-test).
43

Figure 2.13: Knockdown of GRP78 sensitizes human leukemia cells to AraC. Viability of NB4 cells
transfected with siRNA against Grp78 (siGrp78) or control siRNA (sictrl) followed by the indicated AraC
concentration for 24 hours was measured by trypan blue exclusion assay. The data are presented as mean ±
s.e (** P<0.01, Student’s t-test).

2.3.7 Targeting cell surface GRP78 with monoclonal antibody suppresses Pten null-
induced leukemogenesis
To further investigate the potential of targeting GRP78 as a therapeutic target for
leukemia, a monoclonal antibody that specifically targets cell surface GRP78 (MAb159)
was introduced into Pten-null induced leukemic mice. Simultaneous intraperitoneal
administration of pIpC (20µg/gW) and monoclonal antibody 159 against GRP78 was
given to 6-8 week old Pten
f/f
;Mx1-Cre (cP
f/f
) mice every other day for a total of 7 doses to
induce Cre expression. Co-administration of IgG and pIpC to cP
f/f
mice were used as
positive control. In addition, littermates without carrying the Cre transgene (Pten
f/f
) are
phenotypically equivalent to WT mice and were also injected with pIpC. After the first
seven co-injections of both pIpC and MAb159 (or IgG), two additional treatments of
MAb159 or IgG were administrated every other day after the completion of the pIpC
interval. The mice were analyzed 6 days post pIpC injection. Similar to the previous
44

results of pIpC injected Pten
f/f
;Mx1-cre mice at six days post pIpC completion, the
additional IgG administrated cP
f/f
mice [cP
f/f
(IgG)] exhibited a hunched posture and
enlarged lymph nodes in the axillary areas whereas the MAb159 treated mice
[cP
f/f
(MAb159)] showed normal appearance (Figure 2.14A). In accordance with previous
results, cP
f/f
(IgG) mice exhibited greatly enlarged spleen compared to WT controls
whereas the cP
f/f
(MAb159) mice exhibited intermediate spleen enlargement (Figure
2.14B). More importantly, to further assess whether targeting the cell surface GRP78
with a monoclonal antibody affects leukemic blast cell expansion, we performed
immunophenotyping of leukemic blast cells by using side scatter pattern and CD45
staining of bone marrow cell suspensions. There was drastic increase in the blast cell
frequency of cP
f/f
(IgG) mice. Most surprisingly, despite the Pten deletion, cP
f/f
mice that
were treated with the monoclonal antibody against surface GRP78 (MAb159) resulted in
a substantial decrease in the blast cell population compared to IgG treated cP
f/f
mice
(Figure 2.14B). The peripheral blood count profile of WT, cP
f/f
(IgG), cP
f/f
(MAb159)
mice were also examined (Figure 2.14C). There was an overall increase of white blood
cells in cP
f/f
(IgG) mice, including lymphocytes, monocytes and granulocytes compared to
WT. In agreement to the dramatic decrease in blast cells, the peripheral blood count
profile of cP
f/f
(MAb159) mice were close to normal WT levels.
To validate the targeting of the cell surface GRP78 with MAb159 of the bone
marrow cells, 130 µg of biotinylated MAb159 was injected in all three groups of mice,
WT, cP
f/f
(IgG), cP
f/f
(MAb159), one day prior to sacrifice. The intact bone marrow cells
were collected and analyzed for surface GRP78, measured by the biotinylation level
during FACS analysis. The area enclosed by the curve indicated by the count number
45

represents the total number of cells that has surface GRP78 in which the MAb159 can
bind to. The results show that all three groups of mice have surface GPR78 in the bone
marrow cells. In addition, compared to the IgG treated mice, the MAb159 treated mice
had substantially decreased biotinylated count number, indicating great reduction of cell
surface GRP78 (Figure 2.14D).
To elucidate the possible mechanisms behind the relieved leukemic phenotype of
MAb159-treated compared to IgG-treated PTEN null mice, the PI3K/AKT signaling
pathway in which PTEN majorly suppresses was examined. Utilizing flow cytometric
analysis, isolated bone marrow cells from WT, cP
f/f
(IgG), cP
f/f
(MAb159) mice were
permeabilized and stained with p-AKT (Ser473), followed by fluorescent labeled
secondary antibody incubation, and then analyzed by flow cytometry. As expected, there
was an increase of the p-AKT peak mean fluorescent intensity (MFI) in the cP
f/f
(IgG)
mice compared to the WT, indicating activation of the p-AKT in the bone marrow in the
IgG treated PTEN null mice (Figure 2.14D). Interestingly, compared to the p-AKT MFI
of the IgG treated PTEN null mice, there was a reduction of the p-AKT peak mean
fluorescent intensity of the bone marrow cells of MAb159 treated mice, indicating a
suppression of the cytosolic AKT activation. To further validate the suppression of AKT
activation in MAb159 treated PTEN null mice, bone marrow lysates was collected from
WT, cP
f/f
(IgG), cP
f/f
(MAb159) mice and subjected to western blot analysis. In the bone
marrow of the IgG treated PTEN null mice, activation of the AKT pathway was detected
whereas the p-AKT level was considerably lower in the MAb159 treated PTEN null mice
(Figure 2.14E).
46

This study suggests the importance of cell surface GRP78 in PTEN null induced
leukemogenesis as a single monoclonal antibody targeted specifically against GRP78 can
potently attenuate PTEN-null induced AKT activation and suppress the initiation of
leukemic blast cell expansion. However, there was a greater extent of rescued leukemic
phenotype in the genetically deleted Pten
f/f
;Grp78
f/+
;Mx1-cre mice compared to MAb
treated Pten
f/f
;Mx1-cre mice as evidenced by the HSC percentage in BM in cP
f/f
78
f/+
mice
was restored to normal levels whereas the HSC level in cP
f/f
(MAb159) mice was still
reduced although higher than cP
f/f
(IgG) mice (data not shown).

47

Figure 2.14: Targeting cell surface GRP78 with monoclonal antibody suppresses Pten null-induced
leukemogenesis. (A) Hunched posture of Pten
f/f
;Mx1-Cre mice treated with IgG compared to normal
posture of those treated with monoclonal antibody against GRP78 (MAb159). (B) Quantitation of the
spleen weight and leukemic blast cell percentages of WT (n=4), cP
f/f
treated with IgG (n=5), and cP
f/f

treated with MAb159 (n=8). All data are presented as mean ± standard error (s.e.). (C) Complete blood
count with tail peripheral blood from WT (n=4), cP
f/f
treated with IgG (n=5), and cP
f/f
treated with MAb159
(n=8). Peripheral blood was collected via tail bleeding and analyzed using an auto hematology analyzer
BC-2800 vet (Mindray) according to manufacturer’s instructions. All data are presented as mean ± s.e. (D)
Flow cytometry analysis of total BM cells of WT, cP
f/f
(IgG) and cP
f/f
(MAb159) for staining of
biotinylated cell surface GRP78 level and cytosolic p-AKT level. (E) Representative Western blot results
using bone marrow cell lysates for detection of the indicated protein levels.

48

2.4 Discussion
Our investigation into the role of GRP78 in the development of leukemogenesis
and therapeutic resistance revealed several novel observations. GRP78 is widely used as
a marker for the induction of ER stress (A S Lee, 2007). Recent studies suggest that
leukemic cells experience ER stress and therapeutic treatments in clinical use for
leukemia also elicits ER stress (Min Ni, H. Zhou, Wey, Baumeister, & Amy S Lee,
2009a). Here we provide direct evidence that Grp78 mRNA expression is upregulated in
the majority of adult patients with AML, CML, ALL or CLL, leukemia cell lines and in
early relapse of childhood leukemia. GRP78 protein level is also generally elevated in
peripheral blood samples of leukemia patients compared to normal control. These results
are consistent with a recent report that primary B-CLL cells from patients constitutively
express high levels of GRP78 protein and that siRNA knockdown of GRP78 in these
cells increased their spontaneous apoptosis (Rosati et al., 2010). These findings, coupled
with proteomic analysis that GRP78 is differentially expressed in the hematopoietic stem
cell (HSC)-like fractions from the bone marrow of leukemia patients (Min Ni, H. Zhou,
Wey, Baumeister, & Amy S Lee, 2009a), provide the first indication that GRP78 may
play an important role in leukemia and chemoresistance.
In this study, through the creation of a biallelic conditional knockout mouse
model of GRP78 and PTEN in the hematopoietic system, we demonstrated that partial
reduction of a single molecular entity, GRP78, in form of Grp78 heterozygosity, is
sufficient to restore the hematopoietic stem cell population back to the normal percentage
and suppress AKT activation and leukemic blast cell expansion mediated by loss of
49

PTEN. We observed that the increase in proliferation in cP
f/f
HSCs was suppressed in
cP
f/f
78
f/+
HSCs, while apoptosis was not affected. This raises the interesting question of
how might Grp78 heterozygosity restore the HSC population to near normal levels? It is
recently reported that depletion of PTEN null HSCs is caused by increases in expression
of p53 and p16 which promote the HSCs to differentiate/mature and migrate out of the
bone marrow HSC pool and this is dependent on mTOR activation.(J. Y. Lee et al., 2010)
Since Grp78 deficiency reduces mTOR activation, this could explain why HSC depletion
is suppressed, resulting in higher number of HSCs in the cP
f/f
78
f/+
mice versus cP
f/f
mice.
Furthermore, we showed that Grp78 heterozygosity by itself has no apparent effect on
development and survival (D. Dong et al., 2008; S. Luo, C. Mao, Brenda Lee, & Amy S
Lee, 2006) and as analyzed here no effect on total bone marrow cell number or HSC
population. Thus, partial GRP78 expression is sufficient to maintain normal organ
homeostasis whereas tumor progression requires optimal level of GRP78.
How might GRP78 contribute to leukemogenesis? While GRP78 is able to confer
multiple anti-apoptotic effects on cancer cells (Fu et al., 2008), here we propose a key
mechanistic explanation, by which a prominent effector, AKT that is activated by the loss
of PTEN, is compromised by reduction of GRP78 in the hematopoietic system. This is
based on our discovery that PTEN-null mediated AKT/mTOR signaling is potently
suppressed in the bone marrow of the PTEN-null Grp78 heterozygous mice. In cell
culture experiments, knockdown of GRP78 by siRNA reduced PI3K/AKT activation
independent of PTEN status and with minimal effect on the ERK and p38 MAPK
pathways. The requirement of GRP78 for AKT activation may involve multiple
mechanisms. For example, GRP78 as a major ER chaperone protein may be required for
50

the processing and cell surface expression of growth factor receptors mediating AKT
activation in the PTEN null model.(Feige & Scheffler, 1987) Additionally, while GRP78
is traditionally regarded as primarily an ER chaperone, its ability to regulate AKT
signaling in the bone marrow and in human leukemic cells is consistent with the recent
exciting discovery that under pathophysiological conditions such as cancer, a subfraction
of GRP78 relocalizes to the cell surface and acts as co-receptor promoting growth
signaling (Min Ni, Yi Zhang, & Amy S Lee, 2011; M. Wang et al., 2009). It has been
reported that ligation of cell surface GRP78 in human cancer cells with antibodies
directed against its carboxyl domain suppresses PI3K/AKT signaling (Uma K Misra &
Salvatore V Pizzo, 2010). Our observation that knockdown of GRP78 suppresses serum-
stimulated phosphorylation of the p85 regulatory subunit of PI3K suggests that GRP78
regulates AKT activation through functional regulation of PI3K and PI(3,4,5)P3
production suggest that this warrants further investigation. Importantly, ER stress or
overexpression of GRP78 is sufficient to promote cell surface expression of GRP78.(Yi
Zhang, R. Liu, Min Ni, Gill, & Amy S Lee, 2010) Since PTEN mutation and
AKT/mTOR signaling represent a major oncogenic pathway in human cancer, promoting
both proliferative and survival pathways, GRP78 suppression will have wide implications
in blocking both solid and hematological cancer progression.
With regard to the additional possible mechanisms of the relieved leukemic
phenotype of the Pten null, Grp78 heterozygous mice, the potential alteration of the Wnt
signaling pathway and apoptotic regulators, Bcl-2 and caspase 7 is discussed below.
Several signaling pathways such as Wnt/ β-catenin (Reya et al., 2003), Hox
(Argiropoulos & Humphries, 2007) and Bmi-1 (Raaphorst, 2003) have been implicated in
51

the self-renewing properties for both normal and leukemic hematopoiesis. However, the
normal homeostatic mechanisms regulating self-renewal in HSCs are likely disrupted in
leukemic stem cells (Chao, Seita, & I L Weissman, 2008). Upregulation of Wnt signaling
is suggested to be associated to leukemogenesis as abnormal upregulated Wnt/ β-catenin
self-renewal signaling was reported to be significantly higher in the GMP population
(candidate LSC in blast crisis) compared to both CML HSCs and normal GMPs, which
directly contributes to its ability to self-renew in vitro and in vivo (Jamieson et al., 2004).
Studies have shown that hypoxia-induced ER stress inhibits normal Wnt protein
processing and secretion as ER stress causes dissociation between GRP78 and Wnt which
is essential for its correct posttranslational processing (Verras, Papandreou, A. L. Lim, &
Denko, 2008). Hence, whether the prolonged leukemogenesis observed in out Pten null,
Grp78 heterozygous mice may be partially attributed to attenuation of Wnt protein
processing due to GRP78 knockdown awaits further investigation.
Another key feature of cancer cells is unregulated proliferation and escape of
apoptosis. Just like tumor cells, this is the case for acute leukemias and leukemia stem
cells. Down-regulation of apoptosis is generally seen in tumor pathogenesis, it is not
surprising that LSKs have deregulated apoptotic signaling. The anti-apoptotic Bcl-2 has
been shown to have a role in lymphoid malignancies and growing evidence suggesting
that it may play a role in leukemic transformation of myeloid cells. It has been found that
leukemia cells from human AML subtypes express Bcl-2 at significantly higher levels
than normal cellular counterparts and show the highest level of Bcl-2 expression in early
LSK progenitors (Konopleva et al., 2002). A recent study has shown that GRP78
suppresses apoptosis mediated by BIK and NOXA and decreases BCL-2 binding to BIK
52

in breast cancer cells (H. Zhou, Yi Zhang, Fu, Chan, & Amy S Lee, 2011). This invites
the exciting possibility that the relieved leukemic phenotype in our Pten null, Grp78
heterozygous mice may be at least in part due to regulation on the anti-apoptotic BCL-2.
Either up-regulation of anti-apoptotic signals or down-regulation of proapoptotic
signals is an important event in cellular transformation to myeloid leukemias. GRP78 has
been shown to have anti-apoptotic properties, in part by regulating caspase 7 activation
and interacting with Bcl-2 signaling proteins (X. Li, K. Zhang, & Z. Li, 2011; Reddy et
al., 2003). Therefore, knocking down the anti-apoptotic GRP78 in the conditional Pten-
null induced leukemia mouse model may suppress the transformation of leukemia cell
development and survival through its anti-apoptotic properties.
Another interesting observation was the potential feedback regulation between
GRP78 expression level and AKT activation. The immunoblot results using BM cell
lysates of the mice reveal that the overall GRP78 level in the cP
f/f
78
f/+
mice exhibit greater
than 50% reduction compared to WT control, corresponding to suppressed AKT
activation in cP
f/f
78
f/+
BM (Figure 2.8A). In addition, there also seems to be a trend of
increased GRP78 expression level in cP
f/f
BM cells which exhibit enhanced AKT
activation (Figure 2.8A). This suggests the possible feedback regulation of GRP78 level
and AKT activation, in which not only is GRP78 upstream of AKT signaling, but may
also be downstream of AKT activation.
With regard to drug resistance in leukemia, it has been reported that PI3K/AKT is
constitutively active in primary AML cells from patients and blocking PI3K with
inhibitor (LY294002) potentiates the response to AraC (Grandage, Gale, Linch, &
53

Khwaja, 2005). Recently, it is reported that the resistance of B-lineage ALL cells to the
anti-leukemic drug vincristine was suppressed by (-)-epigallocatechin gallate (EGCG),
which inhibits the anti-apoptotic function of GRP78 by targeting to its ATP-binding
domain (Ermakova et al., 2006; Uckun et al., 2011). Our observation that suppression of
GRP78 which inhibits PI3K/AKT signaling sensitizes leukemic cells to AraC induced
apoptosis supports the previous finding and further identifies GRP78 as a novel
therapeutic target against AraC-induced chemoresistance in leukemic cells. How might
GRP78 protect leukemic cells against chemotoxic treatment? One explanation is that this
is mediated by the role of GRP78 as a master regulator of ER homeostasis and apoptosis
(M. Wang et al., 2009). Furthermore, ER stress induces alternative splicing of the Grp78
transcript, leading to the production of a cytosolic isoform of GRP78 (GRP78va) which
also protects HL60 cells from ER stress-induced cell death (Min Ni, H. Zhou, Wey,
Baumeister, & Amy S Lee, 2009b). Therapeutic drugs in clinical treatment or pre-clinical
test for leukemia, such as arsenic trioxide and imatinib mesylate, have been shown to
upregulate ER stress markers and induce leukemic cell death through ER stress-mediated
apoptosis.(Min Ni, H. Zhou, Wey, Baumeister, & Amy S Lee, 2009a) AraC is a
nucleoside analogue and its incorporation into DNA causes localized alterations in the
DNA duplex, resulting in inhibition of DNA polymerase as well as stabilization of
covalent topoisomerase I-DNA complexes contributing to cytotoxicity (Chrencik, Burgin,
Pommier, Stewart, & Redinbo, 2003). Previously, we established that GRP78 protects
cells from apoptosis induced by other DNA damaging agents such as topoisomerase
inhibitors through inhibition of caspase-7, which associates with the ER and can be
activated by ER stress (Rao et al., 2002; Reddy et al., 2003). Here we showed that
54

treatment of leukemic cells with AraC leads to activation of caspase-7. Since GRP78 is
known to complex with caspase-7 and suppress its activation,(Ermakova et al., 2006; Rao
et al., 2002; Reddy et al., 2003) GRP78 level can play an important role in
responsiveness to AraC treatment, however, future investigations are required to address
other potential mechanisms. In summary, here we provide proof-of-principle that partial
reduction of GRP78 can arrest leukemogenesis and sensitize leukemic cells to
chemotherapeutic treatment while having no harmful effect on the hematopoietic system.
Therapeutics targeting GRP78 have recently emerged and have shown anti-tumor activity
in solid tumors (Cells et al., 2009; Kim et al., 2010; Yoneda et al., 2008; D.-H. Yu et al.,
2008). Whether these and other agents capable of suppressing GRP78 expression or
activity can be applied for suppression of leukemogenesis warrants vigorous investigation.
55

Chapter 3
Inducible knockout of GRP78 reveals its role in hematopoietic stem cell
survival and cellular homeostasis
3.1 Introduction
In the adult hematopoietic system, hematopoietic stem cell (HSC) regulation of
survival, self-renewal and differentiation is regulated both by the intrinsic gene
expression and extrinsic cues from the microenvironment (Cmp, 2002; Zon, 2008). HSCs
have the property of pluripotent differentiation and give rise to mature blood cells, but
can also self renewal. Self renewal is a biological process that pluripotent stem cells give
rise to daughter cells that have the same potential to differentiate into multilineages and
to renewal. The regulation of HSCs occurs in a cell autonomous manner but is also
influenced by the environment as evidenced by the importance of stem-cell niches (Zon,
2008).
GRP78, also known as immunoglobulin heavy chain binding protein (BiP), is an
essential endoplasmic reticulum (ER) molecular chaperone protein and master regulator
of ER homeostasis and is involved in several cellular processes. The ER is the checkpoint
for proper folding and modification of proteins involved in the secretory pathway (M.
Wang et al., 2009). Metabolic, infectious reasons can result in ER stress. Upon ER stress,
the unfolded protein response (UPR) in which GRP78 is the master regulator of is
activated to help the cells better cope with the stress. The UPR is mediated by three
sensor molecules, namely, PKR-like ER kinase (PERK), inositol-requiring enzyme 1
(IRE1 α) and activating transcription factor 6 (ATF6), which are associated with GRP78
56

and retained inactive under normal unstressed conditions (Pfaffenbach & Amy S Lee,
2010). Upon activation, PERK phosphorylates eIF2 α, which in turn inhibits general
protein translation and activates C/EBP homologous protein (CHOP). IRE1- α is an
endoribonuclease that upon activation initiates the splicing of the mRNA encoding X-
box-binding protein 1 (XBP-1). Spliced XBP-1 is a potent transcriptional activator that
upregulates the transcription of a subset of UPR related genes involved in protein folding,
maturation and degradation in the ER. Activated ATF6 translocates from the ER to the
Golgi, where it is cleaved by S1P/S2P proteases and generates another active
transcription factor.
GRP78 (BiP) has been shown to have immunomodulatory properties. It has been
reported that GRP78 can stimulate the release of Th2 cytokines, especially IL-10 and IL-
4 and therefore affect the development of regulatory T cells (Corrigall et al., 2009) and
has been demonstrated to coimmunoprecipitate with IL-6 and regulate its secretion from
astrocytes (Qian et al., 2007).
It has been shown that GRP78 is essential for embryonic cell growth and
pluripotent cell survival as Grp78
-/-
embryos die on embryonic day 3.5 via ER stress
activated mechanisms (S. Luo, C. Mao, Brenda Lee, & Amy S Lee, 2006). However, the
role of GRP78 in adult stem cells, namely hematopoietic stem cell survival and
homeostasis is still unclear. Therefore, to understand GRP78 function in hematopoietic
stem cell and normal hematopoiesis, we generated an inducible conditional knockout
mouse model by breeding Grp78
f/f
mice (S. Luo, C. Mao, Brenda Lee, & Amy S Lee,
2006) with Mx1-cre transgenic mice (Kiihn & Aguet, 1995) to produce Grp78
f/f
;Mx1-Cre
mice and therefore would allow acute, inducible and efficient homozygous knockout of
57

GRP78 in the hematopoietic system through polyinosinic-polycytidylic acid (pIpC)
administration. GRP78 deficiency resulted in significant reduction in the primitive HSC-
enriched Lin
-
cKit
+
Sca-1
+
(LSK) cell population. Apoptosis analysis revealed that
GRP78 deficient LSK cells were undergoing enhanced apoptosis. In addition, GRP78
depletion in the hematopoietic system resulted in altered hematopoiesis in which
dramatic lymphopenia and increased myelopoiesis was observed. Through bone marrow
transplantation assays, we further determined that the loss of HSC and altered
hematopoiesis is a cell autonomous effect while not excluding the contribution of the
microenvironment factor. Furthermore, we revealed in GRP78-null bone marrow cells
strong activation of all three branches of UPR signaling in which activated ATF6 (p50),
increased xbp-1 splicing and enhanced p-eIF2 α corresponding to CHOP induction and
caspase activation in response to GRP78 depletion. To our knowledge, this is a novel
demonstration of the GRP78-null induced activation of the universally conserved
protective UPR signaling cascade in hematopoietic bone marrow cells. Collectively, our
study establishes GRP78 as an essential regulatory factor for hematopoietic stem cell
survival and maintenance of hematopoiesis function.

58

3.2 Materials and methods
Mice
Grp78
f/f
mice in a mixed C57BL/6; 129/Sv background were generated as
previously described.(S. Luo, C. Mao, Brenda Lee, & Amy S Lee, 2006) Grp78
f/f
mice
were crossed with the transgenic Mx-1-cre mice on a C57BL/6 background (Jackson
Laboratory) to generate Grp78
f/f
, Mx-1-cre mice. Littermates that did not carry the cre
transgene were used as WT controls. Genotyping was performed by PCR using genomic
DNA extracted from mouse tail biopsies as previously described.(Fu et al., 2008) To
induce the genomic deletion, 6 to 8 week-old mice were injected intraperitoneally with
poly-inositic:poly-cytodylic acid (pI.pC; 25 µg/g mouse body weight) every other day for
14 days. Mice were analyzed 6 days post 7 injections of pIpC induction.
All protocols for animal use were reviewed and approved by the USC Institutional
Animal Care and Use Committee.
Complete blood count
Peripheral blood was collected via tail bleeding and analyzed using an auto
hematology analyzer BC-2800 vet (Mindray) according to manufacturer’s instructions.
RT-PCR
Total RNA from bone marrow samples of Grp78
f/f
;Mx-1-cre or Grp78
f/f
mice
were extracted by Trizol reagent (Invitrogen), followed by DNase I treatment. First-
strand cDNA was synthesized with the Superscript II reverse transcriptase (Invitrogen)
59

with oligo d(T) primer according to manufacturer’s instructions. The primers used for
RT-PCR for xbp-1 and β-actin: are listed in Table 3.1.
Multiplex cytokine assay of serum
Six days post completion of the pIpC administration interval, blood was collected
from retro-orbital bleeding from Grp78
f/f
;Mx-1-cre or Grp78
f/f
mice in Microtainer
(Becton Dickinson, NJ) serum gel collection tubes. Blood was allowed to clot for 30 min
and centrifuged for 5 min at 13,000g. Serum was collected from the top of the tube
followed by analysis (Koh, Gray, Higgins, Hubby, & Kast, 2009). A 23-plex mouse
cytokine assay (Bio-Rad Laboratories) was carried out following manufacturer’s
instructions. Briefly, a 96-well Multiscreen Resist Vacuuum Manifold filter plate
(Millipore) was pre-wetted with Bio-plex assay buffer, and multiplex beads were added
to the wells. Multiplex beads were washed twice with Bio-plex wash buffer and 50 µl of
reconstituted standards or diluted serum samples were added to the wells. Serum samples
were diluted 1:3 with mouse serum diluents (Bio-Rad Laboratories). The filter plate was
incubated with gentle shaking at 300 rpm for 30 min at room temperature and washed
three times with Bio-plex wash buffer. Bio-plex detection antibody was then added to the
wells and incubated with shaking at 300 rpm for 30 min room temperature. The filter
plate was washed three times with Bio-plex wash buffer and 50 µl Streptavidin-PE was
added to each well and incubated with shaking at 10 min in the dark. The filter plate was
washed three times with Bio-plex wash buffer and resuspended in 125 µl of Bio-plex
assay buffer and the beads detected using the Bio-plex HTF system (Bio-Rad
60

Laboratories) and results analyzed using the Bio-plex Manager software (Bio-Rad
Laboratories).
Bone marrow transplantation
Grp78
f/f
;Mx-1-cre or Grp78
f/f
mice were lethally irradiated at 9.5Gy. Within 24
hours, 1x10
6
total bone marrow cells of Grp78
f/f
;Mx-1-cre or Grp78
f/f
mice were
transplanted in the lethally irradiated recipient mice. Intraperitoneal injection of pIpC was
administrated to the bone marrow recipient mice 8 weeks after the transplantation. The
mice were then analyzed 6 days post completion of the 7 injections (25µg/gW) of pIpC
that were given every other day.
Flow cytometry
Bone marrow cells were flushed from long bones (tibias and femurs) with
Dulbecco’s phosphate-buffered saline without calcium and magnesium (DPBS), and then
filtered through nylon screen (70 µm, BD) to obtain single cell suspension. Bone marrow
cells were resuspended in DPBS with 0.5% BSA and 0.1% sodium azide for antibody
staining. To study hematopoietic stem and progenitor cells, the following antibodies from
BD Pharmingen were used: Lineage (Lin; which consists of B220 (RA3-6B2), TER119
(TER119), CD4 (RM4-5), CD8 (53-6.7), Gr-1 (RB6-8C5), and Mac-1 (WT.5)), c-Kit
(2B8), Sca-1 (D7), CD34 (RAM34), IL7R α (SB/199) and Fc γRII/III (2.4G2). After
washing, cells were resuspended in PBS with 0.1% sodium azide and 2 µl 7-AAD was
added to exclude dead cells. All FACS analyses were performed on LSR II flow
cytometer.
61

Fluorescence activated cell sorting
Primitive hematopoietic stem and progenitor cells were purified using a FACS Aria
flow cytometer (Becton Dickinson) based on established cell surface phenotypes.
Cell cycle analysis
1x10
7
whole BM cells were incubated with 10 g/ml Hoechst 33342 (Sigma -
Aldrich) at 37°C for 45 minutes, then stained with primitive hematopoietic cell antibodies
(Lin, Sca-1 and c-Kit) as described above. The stained cells were resuspended in 10%
buffered formalin and incubated at 4°C overnight. To stain for RNA content, pyronin Y
(Polysciences Inc., Warrington, PA) was added to the cells at a final concentration of
0.75 µg/ml and incubated at 4°C for 30 minutes. Cell cycle status was examined using a
LSR II flow cytometer.
Apoptosis assay
1×10
6
whole BM cells were stained with primitive hematopoietic cell antibodies
(Lin, c-Kit and Sca-1) as described above. The stained cells were resuspended in 150 μL
1×Annexin V binding buffer (BD Pharmingen) and incubated with Annexin V and
7AAD (both from BD Pharmingen) for 15 min. Cell apoptosis was examined within an
hour using a BD LSR II flow cytometer.

62

Real-time quantitative RT-PCR
To detect Grp78 expression, RNA was extracted from sorted mouse whole BM cells
and reverse-transcription was performed as previously described (Min Ni, H. Zhou, Wey,
Baumeister, & Amy S Lee, 2009a). cDNA samples were analyzed in triplicate with the
SYBR Green Supermix (Quanta Biosciences, Gaithersburg, MD) according to
manufacturer’s instructions. The primers used for Grp78 and 18S RNA are listed in Table
3.1.
Western blot analysis
Whole cell lysates were prepared from single-cell suspensions of bone marrow
cells from the indicated mice. Cells were lysed in RIPA buffer supplemented with
competent protease-inhibitor mixture (Roche) and phosphatase-inhibitor mixture (Roche)
and 25 µg cell lysate was subjected to SDS-PAGE. The immunoblot membranes were
incubated with primary antibodies at 4°C overnight and the protein signals were detected
with ECL reagent (Roche) or Supersignal chemiluminescence reagent (PIERCE) after
reacting with HRP-conjugated secondary antibody. The primary antibodies used are as
follows. Monoclonal mouse anti-GRP78 (1:2000) and monoclonal mouse anti-caspase-7
(1:2000) are from BD Pharmingen. Rabbit anti-p-eIF2 α (Ser51, 1:1000), rabbit anti-
eIF2 α (1:1000) are from Cell Signaling. Mouse anti-ATF6 (1:200) is from Imgenex.
Rabbit anti-Calreticulin (1:2000) is from Stressgen. Mouse anti-CHOP (1:1000) is from
Santa Cruz Biotechnology. Mouse anti- β-actin (1:5000) is from Sigma.

63

Statistical analyses
Kaplan-Maier survival curves were constructed using GraphPad (Prism5
software), and log-rank analysis was used to analyze the results. For bar graphs, the
unpaired 2-tailed Student’s t-test was used to compute P values, and the error bars reflect
standard error (s.e.) (*, P<0.05, **, P<0.01, ***, P<0.001)

Table 3.1: Primer sequences for RT-PCR and real-time PCR
Gene Primer name Sequence
Human
Grp78 h78-1F 5’-CGACCTGGGGACCACCTACT-3’
hBiP-br1 5-GCCACAACTTCGAAGACACCA-3’
β-actin human β-actin F 5'-TCGTGCGTGACATTAAGGAG-3'
human β-actin R 5'-AGCACTGTGTTGGCGTACAG-3'
Mouse
Grp78 Grp78 + 5'-TCTCCACGGCTTCCGATAAT-3'
Grp78 - 5'-GTACCTTTGTCTTCAGCTGTCACTC-3'
18s RNA 18s RNA+ 5'-ACGGCCGGTACAGTGAAAC-3'
18s RNA - 5'-GAGGGAGCTCACCGGG-3'
xbp-1 XBP-1 F 5'-GAACCAGGAGTTAAGAACACG-3'
XBP-1 R 5'-AGGCAACAGTGTCAGAGTCC-3'
β-actin mouse β-actin F 5'-GACGGCCAGGTCATCACTAT-3'
mouse β-actin R 5'-GTACTTGCGCTCAGGAGGAG-3'
64

3.3 Results
3.3.1 Creation of mouse model with conditional deletion of Grp78 in the
hematopoietic system
To identify the expression pattern of GRP78 in wildtype mouse bone marrow
(BM) cells, we performed real-time quantitative PCR analysis with sorted subpopulations
of BM cells. As an essential housekeeping protein, GRP78 was expressed in all six tested
subpopulations including LSK cells (Figure 3.1).

Figure 3.1: Grp78 is expressed in multiple blood cell lineages. Grp78 mRNA expression in WT BM
subpopulations measured by quantitative real-time PCR. The experiments were performed in duplicates;
each replicate contains pooled BM from two WT mice.
To examine the role of GRP78 in the hematopoietic system, Grp78
f/f
mice were
crossed with a polyinosine-polycitidine (pIpC) inducible Mx-1-Cre transgenic mouse line
(Kiihn & Aguet, 1995) that allows GRP78 to be acutely deleted in the hematopoietic
system. The schematic diagram of the mating strategy is depicted in Figure 3.2. In this
study, Grp78 was deleted in the hematopoietic system of 6-8 week old adult Grp78
f/f
;Mx-
1-Cre mice (referred to as cKO) mice upon administration of pIpC every other day for a
65

total of 7 injections to activate Cre expression. Littermates without the Cre transgene
(Grp78
f/f
) are phenotypically equivalent to animals with a WT Grp78 allele served as
wild type (WT) normal controls and were also injected with pIpC. The status of Grp78
deletion was validated by PCR in isolated bone marrow cells (Figure 3.3A). Western blot
analysis of bone marrow cells confirmed that GRP78 expression was almost completely
ablated in the cKO mice (Figure 3.3B).

Figure 3.2: The breeding scheme for the Grp78 conditional knockout mice. The generation of parental
Grp78
f/f
was described previously (S. Luo, C. Mao, Brenda Lee, & Amy S Lee, 2006). Grp78
+/+
Mx1-Cre
was commercially purchased from the Jackson Laboratory. The genotypes indicated with the gray shade
were used in this study. The genetic background of the parental mouse strain is indicated below within the
square brackets. The numbers below the genotypes indicate the expected probability of the indicated
genotype among the offspring.

Figure 3.3: GRP78 was knockdown in the bone marrow of mutant mice. (A) Representative PCR
genotyping results from WT (78
f/f
) and Grp78 null mice (c78
f/f
) bone marrow 6 days post completion of
pIpC treatment. (B) Western blot results for detection of GRP78 protein level in the bone marrow
performed in duplicates.
66

3.3.2 GRP78 deficiency reduces primitive cell pool through increased cell death
Examining the effect of acute depletion of GRP78 in adult primitive
hematopoietic cells, we observed that after 6 days of GRP78 deletion there was a
statistically significant reduction in the HSC-enriched Lin
-
c-Kit
+
Sca-1
+
(LSK) cell
population in the bone marrow (Figure 3.4A and B). Upon further analysis, the reduction
in LSK population in the bone marrow stems from decrease in both the Lin
-
c-Kit
+
Sca-1
+

CD34
-
long term HSC-enriched population (LT-HSC) and Lin
-
c-Kit
+
Sca-1
+
CD34
+

short term HSC-enriched population (ST-HSC) shown in figure 3.4C.

Figure 3.4: GRP78 deficiency in the bone marrow reduced HSC-enriched population. (A)
Representative flow cytometric analysis with BM cells using Lin, c-Kit, Sca-1 and CD34. (B) Quantitation
of flow cytometric analysis of HSC-enriched LSK population in the BM. (n=12 for WT, n=16 for cKO) (C)
Quantitation of flow cytometric analysis of Lin
-
c-Kit
+
Sca-1
+
CD34
-
(LT-HSC) and Lin
-
c-Kit
+
Sca-1
+

CD34
+
(ST-HSC) populations in the BM (n=6 for WT, n=6 for cKO). All data are presented as mean ± s.e
(*P<0.05, **P<0.01, Student’s t-test).
67

In addition, there was no apparent difference in morphology of the tibia bone
marrow section between WT and cKO mice (Figure 3.5A) and the total BM cell number
was not statistically different in the mutant mice (P=0.51) (Figure 3.5B). This suggests
that the total number of primitive hematopoietic cells in cKO mice BM is lower
compared to controls.

Figure 3.5: GRP78 deficiency does not change bone marrow morphology and total cellularity. (A)
(Left) Representative H&E sections of the tibia bone of WT and cKO mice at lower magnification (Scale
bar represents 50 µm). (Right) The selected square area in the bone marrow section is further magnified
(Scale bar represents 20 µm). (B) Total bone marrow cell number from WT (n=11) and cKO (n=16) mice.
All data are presented as mean ± s.e.
The progenitor cell populations in the BM were also examined. We observed that
in the cKO mice, there was a statistically significant decrease in the common lymphoid
progenitor (CLP) enriched Lin
-
c-Kit
lo
Sca-1
lo
IL-7R α
+
cells and a significant increase in
the common myeloid progenitor (CMP) enriched Lin
-
c-Kit
+
Sca-1
-
IL-7R α
-
CD34
+

Fc γII/IIIR
lo
cells, whereas there was a modest decrease in the megakaryocyte-erythroid
progenitor (MEP) enriched Lin
-
c-Kit
+
Sca-1
-
IL-7R α
-
CD34
-
Fc γII/IIIR
lo
cells and the
granulocyte-monocyte progenitor (GMP) enriched Lin
-
c-Kit
+
Sca-1
-
IL-7R α
-
CD34
+

68

Fc γII/IIIR
high
cell population was comparable between the cKO mice and their WT
littermates (Figure 3.6).

Figure 3.6: GRP78 deficiency leads to decreased common lymphoid and myeloid progenitor cells.
Quantitation of flow cytometric analysis of lymphoid and myeloid progenitors including common
lymphoid progenitor (CLP), common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP)
and megakaryocyte-erythroid progenitor (MEP) from WT (n=6) and cKO (n=6) mice. All data are
presented as mean ± s.e (*P<0.05, **P<0.01, Student’s t-test).
To determine whether the decrease of the primitive hematopoietic cells in the BM
of the conditionally deleted Grp78 knockout mice was due to increased cell death or
decreased proliferation, we analyzed the apoptotic and cell cycle profile in LSK cells.
Apoptotic LSK cells were examined by flow cytometric analysis of Annexin V and 7-
AAD staining. Our results showed that there was a significant increase in the percentage
of apoptosis LSK cells in GRP78-null mice compared to WT mice (Figure 3.7A).
Furthermore the cell cycle distribution analyzed by Hoechst/Pyronin Y staining showed
that the decrease of LSK cells was not due to proliferation as there was no significant
difference between cKO and WT mice (Figure 3.7B and C). This suggests that GRP78 is
essential in the maintenance and survival of adult hematopoietic stem cells as GRP78
69

deficiency results in decrease in the primitive hematopoietic stem cells at least in part
through enhanced cell death.

Figure 3.7: GRP78 deficient LSK cells displayed increased cell death. (A) Summary of flow cytometric
analysis of apoptotic LSK cells using Annexin V and 7-AAD (n=5 for WT, n=6 for cKO). (B) Summary of
cell cycle distribution of LSK cells from WT (n=4) and cKO (n=4) mice. (C) Representative flow
cytometric analysis of LSK cell cycle status by Hoechst and Pyronin Y staining. All data are presented as
mean ± s.e (*P<0.05, Student’s t-test).

3.3.3 Deletion of GRP78 in the hematopoietic system leads to lymphopenia and
altered hematopoiesis
We further investigated the acute consequence of GRP78 depletion following
Mx1-Cre induction on the hematopoietic phenotypes of cKO mice compared to WT
control. In respect to altered primitive stem and progenitor populations in the conditional
70

Grp78 knockout mice, we analyzed the effect on the hematopoietic organs and
downstream differentiated mature cell populations both in the BM and the peripheral
blood. Compared to WT controls, the cKO mice showed substantially smaller thymus
corresponding to statistically significant lower thymus cellularity, while the spleen and
liver size was comparable to WT siblings (Figure 3.8A and B). H&E staining of the
paraffin tissue sections showed hypocellularity in the thymus, signs of extramedullary
hematopoiesis in the spleen as evidenced by presence of megakaryocytes while no sign of
obvious pathology was observed in the liver (Figure 3.8C).

Figure 3.8: GRP78 deficiency leads to lymphopenia. (A) Organ size and morphology from mice of the
indicated genotypes. (B) Quantitation of the spleen weight (n=14 for WT, n=20 for cKO) and thymus
cellularity (n=4 for WT, n=4 for cKO). (C) H&E staining of paraffin sections of spleen, liver and thymus.
The scale bar represents 200 µm. All data are presented as mean ± s.e (**P<0.01, Student’s t-test).
Complete blood count analysis demonstrated a dramatic 60% decrease in
lymphocyte number while showing a significant 2-fold increase in monocytes and 2.5-
71

fold increase in granulocytes in cKO mice compared to WT siblings. There were signs of
moderate anemic phenotype in the GRP78-null mice as there was moderate yet
significant decrease of hemoglobulin levels and hematocrit percentage while red blood
cells and thrombocyte counts were relatively normal (Figure 3.9A). Consistent to the
blood count analysis results, examination of blood smears further confirmed the
lymphopenia in peripheral blood (Figure 3.9B).

Figure 3.9: GRP78 deficiency leads to decreased lymphocytes and increased monocytes, granulocytes
in the peripheral blood. (A) Complete blood count with tail peripheral blood from WT (n=12) and cKO
(n=16). All data are presented as mean ± s.e (*P<0.05, **P<0.01, ***P<0.001, Student’s t-test). (B)
Peripheral blood smears of WT and cKO mice. Scale bar represents 20 µm.
72

The substantial drop in lymphoid cells and expansion of myeloid cells was also
evident in the BM of Grp78 knockout mice as there was dramatic decrease in both the
B220
+
and CD3
+
cells and significant increase in Gr-1
+
and Mac-1
+
cells in cKO mice
compared to WT littermates (Figure 3.10A and B).

Figure 3.10: GRP78 deficiency leads to altered lymphoid and myeloid differentiation in the bone
marrow. (A) Representative flow cytometric analysis with BM cells using lineage markers B220 and Gr-1
(top) and CD3 and Mac-1 (bottom). (B) Quantitation of lymphoid and myeloid cells in (A) (n=4 for each
genotype). All data are presented as mean ± s.e (*P<0.05, **P<0.01, ***P<0.001, Student’s t-test).
The decrease in lymphoid cells and increase in myeloid cells was consistent in
both the BM and peripheral blood and this corresponded to the upstream decrease of CLP
and decrease in CMP progenitor populations in the BM, suggesting GRP78 deficiency in
the primitive hematopoietic cells leads to an evident alteration of the hematopoietic
phenotype.
73

3.3.4 GRP78-null induced alteration of hematopoiesis is cell autonomous
Since GRP78 was reported to be critical in embryonic stem cell survival and
maintenance (S. Luo, C. Mao, Brenda Lee, & Amy S Lee, 2006) and is a multifunctional
ER chaperone protein which has been shown to be important in various processes within
the cell (D. Dong et al., 2008; Pfaffenbach & Amy S Lee, 2010), it was intriguing to
investigate whether the loss of HSC-enriched LSK cells and alteration of hematopoiesis
observed in Mx1-Cre-induced Grp78 knockout mice was cell intrinsic or
microenvironment driven.
To directly examine GRP78 in the hematopoietic cell autonomous property and
exclude the possible effects of the knockout in the microenvironment, isolated total bone
marrow cells of cKO mice were transplanted into lethally irradiated WT siblings (WT-
cKO). As control, bone marrow cells of WT mice were also transplanted into lethally
irradiated WT littermates (WT-WT). Two months following transplantation to allow full
BM reconstitution, recipient mice were intraperitoneal injected with pIpC (25 µg/gW)
every other day for a total of 7 injections to induce Mx1-Cre mediated deletion. WT-WT
mice were also administrated with pIpC as control. The mice were analyzed 6 days after
the completion of the pIpC induction interval.
While the total BM cellularity was the same, we observed a significant reduction
of HSC-enriched LSK percentage in the BM upon pIpC induction of the cKO cells
transplanted in a WT microenvironment (WT-cKO) (Figure 3.11). Furthermore, the level
of LSK reduction was similar to that in the cKO (Grp78
f/f
;Mx1-cre) mice, suggesting
GRP78 to be essential in maintaining normal HSC homeostasis and this property is
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intrinsic to the cell. Additionally, consistent to what was observed in cKO mice, there
was no significant difference in spleen weight, whereas significant thymus
hypocellularity was also seen in WT-cKO mice, however, to a lesser extent (Figure 3.11).

Figure 3.11: Reduction of HSC-enriched population is intrinsic to GRP78 deficiency in BM cells. (A)
Quantitation of flow cytometric analysis of LSK percentage in BM. (B) Quantitation of total BM cell
number. (C) Quantitation of spleen weight. (D) Quantitation of thymus cellularity. WT-WT (n=3) and WT-
cKO (n=3) for each analysis. All data are presented as mean ± s.e ( **P<0.01, Student’s t-test).
We next examined the effect on the progenitor populations resulting from GRP78
knockout strictly in the bone marrow cells. In agreement to the results in cKO mice, we
also observed a significant decrease in common lymphoid progenitor (CLP) enriched Lin
-

c-Kit
lo
Sca-1
lo
IL-7R α
+
cells in WT-cKO, however to a lesser extent (Figure 3.12).
Consistent to what was observed in cKO mice, a significant decrease in common myeloid
progenitor (CMP) enriched Lin
-
c-Kit
+
Sca-1
-
IL-7R α
-
CD34
+
Fc γII/IIIR
lo
cells was also
found in WT-cKO mice. In addition, the megakaryocyte-erythroid progenitor (MEP)
enriched Lin
-
c-Kit
+
Sca-1
-
IL-7R α
-
CD34
-
Fc γII/IIIR
lo
cells was also significantly
reduced whereas in the cKO mice, the MEP population was only modestly reduced. The
granulocyte-monocyte progenitor (GMP) enriched Lin
-
c-Kit
+
Sca-1
-
IL-7R α
-
CD34
+

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Fc γII/IIIR
high
cell population was comparable to WT-WT (Figure 3.12) which was similar
to the results in cKO mice.

Figure 3.12: GRP78 deficient BM cells transplanted in WT microenvironment leads to decreased
common lymphoid progenitor cells, common myeloid progenitor cells and megakaryocyte-erythroid
progenitor cells. Quantitation of flow cytometric analysis of lymphoid and myeloid progenitors including
common lymphoid progenitor (CLP), common myeloid progenitor (CMP), granulocyte-monocyte
progenitor (GMP) and megakaryocyte-erythroid progenitor (MEP) from WT-WT (n=3) and WT-cKO (n=3)
mice. All data are presented as mean ± s.e (*P<0.05, Student’s t-test).
We further examined the effect of strict GRP78 BM cell knockout on general
hematopoiesis and therefore analyzed mature cell populations of WT-WT, WT-cKO in
peripheral blood, bone marrow and spleen. Blood count analysis showed a general
reduction of white blood cells and consistent to the results in cKO mice, dramatic
lymphopenia was also observed in WT-cKO mice (P<0.001) (Figure 3.13). However,
interestingly, there was a significant reduction in monocytes in WT-cKO whereas in
contrast, a significant increase in monocytes and granulocytes was observed in cKO mice.
This result is in agreement to the reduction of the common myeloid progenitor observed
in the BM of WT-cKO. In addition, similar to cKO mice, a drop in hematocrit percentage
was seen in WT-cKO (Figure 3.13). This may suggest that the drop in lymphocytes and
76

hematocrit in the peripheral blood of cKO mice was mainly a consequence of GRP78
deficiency in bone marrow cells whereas the increase of monocytes and granulocytes in
cKO mice can be in part due to GRP78 deficiency in the microenvironment. However,
this requires further investigation and validation.

Figure 3.13: GRP78 deficient BM cells transplanted in WT microenvironment leads to decreased
lymphocytes and monocytes in the peripheral blood. Complete blood count with tail peripheral blood
from WT (n=3) and cKO (n=3). All data are presented as mean ± s.e (*P<0.05, ***P<0.001, Student’s
t-test).
Upon analysis of mature cells in the BM, consistent to cKO results, we observed a
significant decrease in B220
+
lymphoid cells and a significant increase in Gr-1
+
and Mac-
1
+
myeloid cells (Figure 3.14A and B). However, only borderline decrease in CD3
+

lymphoid cells was seen in WT-cKO mice while a greatly significant reduction (P<0.001)
was observed in cKO mice. As the thymus is the site where T cells are majorly produced
77

and educated, the moderation of CD3
+
lymphoid cells in WT-cKO compared to the level
in cKO mice is consistent with the observation that the decrease of thymus cellularity in
WT-cKO mice is also mitigated compared to the dramatic decrease in cKO mice.

Figure 3.14: GRP78 deficient BM cells transplanted in WT microenvironment leads to altered
lymphoid and myeloid differentiation in the bone marrow. (A) Representative flow cytometric analysis
with BM cells using lineage markers B220 and Gr-1 (top) and CD3 and Mac-1 (bottom). (B) Quantitation
of lymphoid and myeloid cells in (A) (n=3 for each genotype). All data are presented as mean ± s.e
(**P<0.01, ***P<0.001, Student’s t-test).
Flow cytometric analysis with isolated splenocytes of WT-WT and WT-cKO
revealed that strict knockout of GRP78 in the BM results in significant decrease of
lymphoid cells (namely CD3
+
cells) and increase of myeloid cells (namely both Gr-1
+

and Mac-1
+
cells) (Figure 3.15A and B). This altered lymphoid and myeloid
78

differentiation phenotype in which there is lymphoid cell reduction and myeloid cell
expansion was consistently observed in the BM and spleen of WT-cKO mice.

Figure 3.15: GRP78 deficient BM cells transplanted in WT microenvironment leads to altered
lymphoid and myeloid differentiation in the spleen. (A) Representative flow cytometric analysis with
BM cells using lineage markers B220 and Gr-1 (top) and CD3 and Mac-1 (bottom). (B) Quantitation of
lymphoid and myeloid cells in (A) (n=3 for each genotype). All data are presented as mean ± s.e (*P<0.05,
Student’s t-test).

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3.3.5 Knockout of GRP78 in bone marrow cells activates UPR signaling pathways
To investigate the underlying mechanism of the reduction of HSC enriched
population and altered hematopoiesis in GRP78 deficient cKO mice, we examined the
unfolded protein response (UPR) signaling pathway in which GRP78 is the master
regulator (Amy S Lee, 2005). The UPR is activated when cells are undergoing ER stress
in which the protein folding load exceeds the folding capacity. The UPR is an
evolutionarily conserved mechanism to help the cells better deal with the stress by
inhibiting general protein translation or increasing the ER protein folding capacity, but
when severe or prolonged ER stress is present, the activated UPR may push the cells
toward apoptosis.
The western blot results using the bone marrow cells of WT and Grp78 cKO mice
demonstrate that there was efficient deletion of Grp78 as the GRP78 level was greatly
depleted in cKO mice (Figure 3.16A). In turn, strong levels of phosphoylated eIF2 α, one
of the indicators of the PERK branch of UPR signaling is largely activated in the cKO
compared to WT controls while the total eIF2 α level remained the same (Figure 3.16A).
CHOP, which is activated further downstream of p-eIF2 α signaling and considered to
play a pro-apoptotic role, is also greatly activated in the bone marrow cells of cKO mice.
Additionally, activation of the ATF6 branch was observed in cKO mice as evidenced by
the enhanced presence of the activated form of ATF6 (p50). Upon examination of the
third UPR branch, the IRE-1 pathway, we measured the splicing of xbp-1 transcription
factor which is the substrate and indicator of IRE-1 activation. There was a 2.5 fold
increase of spliced over unspliced xbp-1 mRNA level in the bone marrow cells of cKO
mice compared to WT littermates (Figure 3.16B). However, there was no obvious
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upregulation or compensation of other chaperone proteins, such as calreticulin levels
remained the same (Figure 3.16A).
Furthermore, we measured the intermediate precursor of caspase-7 activation
[pro-C7 (I)] and the cleavage of caspase-7 [C7(c)] as indicators for their activation and
onset of apoptosis. In the bone marrow cells of cKO mice, we observed an increase in the
level of intermediate pro-caspase-7 and cleaved caspase-7 (Figure 3.16A). This was in
agreement to the activation of pro-apoptotic CHOP observed in the cKO mice.
Thus, depletion of GRP78 in the bone marrow cells of cKO mice triggers all three
branches of the UPR signaling cascade, the PERK, ATF6 and IRE-1 whereas another ER
chaperone, calreticulin was not affected. The pro-apoptotic CHOP and activation of
caspase-7 was also strongly enhanced in the GRP78-null bone marrow cells. This was the
first demonstration of UPR signaling in bone marrow cells upon GRP78 depletion and
demonstrates that Mx1-Cre-induced deletion of Grp78 results in full activation of UPR
signaling and enhanced apoptosis in the bone marrow.
81

Figure 3.16: Knockout of GRP78 in bone marrow cells activates UPR signaling pathways. (A)
Western blot results using bone marrow cell lysates (n=3 for each genotype) for detection of GRP78,
phosphor-eIF2 α, total eIF2 α, CHOP, ATF6 (p50), ATF6 (p90), Calreticulin, pro-caspase 7 intermediate,
cleaved caspase 7 and β-actin. (B) (Upper panel) RT-PCR results for detection of xbp-1 spliced [xbp-1 (s)],
xbp-1 unspliced [xbp-1 (u)] and β-actin mRNA levels from bone marrow cells of WT and cKO mice (n=3
for each genotype). The PCR image was inverted for better clarity. (Lower panel) Quantitation of the ratio
of xbp-1(s) to xbp-1 (u). The average ratio of xbp-1(s)/xbp-1(u) in WT was set as 1. The data is presented
as mean ± s.e. (*P<0.05, Student’s t-test).

3.3.6 GRP78 deficient mice exhibit retarded growth and develop cardiomyopathy
and lung congestion
As gene inactivation caused by interferon or pIpC inducible Mx1-Cre is greatly
efficient and effective in the hematopoietic system, heterogeneous induced gene deletion
in other organs may also come into play (Kiihn & Aguet, 1995). Therefore, aside from
the major hematopoietic system, other possible organ phenotypes in the Grp78
f/f
;Mx1-cre
cKO mice were also examined and investigated.
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The first significant phenotype was the dramatic drop of body weight and retarded
growth rate of cKO mice throughout and after the course of pIpC administration (Figure
3.17A). Prior to pIpC induced Mx1-Cre expression (Day 0), the initial average body
weight of WT and cKO were comparible (Figure 3.17B). However, five days after the
completion of the seven dose pIpC interval (Day 18), the average weight of the cKO mice
lose on average 15% of their initial body weight and at times exhibit a weak, hunched
posture (data not shown) while WT littermates that also undergone pIpC treatment are
normal and continue to gradually gain weight (Figure 3.17B).

Figure 3.17: GRP78 conditional knockout mice exhibit body weight drop and a retarded growth rate.
(A) Average growth curve of WT (n=13) and cKO (n=27) mice starting the first day of pIpC injection. (B)
Representative images of WT and cKO mice at the prior to pIpC treatment (Day 0) and five days after
completion of pIpC treatment interval (Day18).
Upon closer examination of the retarded growth phenotype in cKO mice, we
found that cKO mice exhibit greatly reduced amounts of both white adipose tissue (WAT)
and brown adipose tissue (BAT) (Figure 3.18A). This may be a consequence of GRP78
knockout in the adipose tissue or lesser food intake or a result of both in the cKO mice.
There was lesser food consumption in cKO compared to WT mice throughout the pIpC
treatment interval (Figure 3.18B) and on average the daily grams of food each mouse
83

intook was lower in cKO mice compared to WT controls (Figure 3.18C). In addition, the
normal feeding blood glucose level in cKO mice was statistically significantly lower than
their WT siblings (Figure 3.18D).

Figure 3.18: GRP78 conditional knockout mice exhibit lower food consumption, blood glucose level
and less accumulated fat. (A) Images of white adipose tissue (WAT) and brown adipose tissue (BAT) of
WT and cKO mice. (B) The average food consumption of WT and cKO mice throughout the pIpC
administration interval. (C) The average daily grams of food consumed by each mouse of WT (n=4) and
cKO (n=8) genotype. (D) The average feeding blood glucose level of WT (n=4) and cKO (n=4) mice. The
data is presented as mean ± s.e. (*P<0.05, Student’s t-test).
In addition, a small percentage of the cKO mice succumbed early on either during
the pIpC administration interval or shortly after (Figure 3.19A). We followed the mice up
to 5 weeks after the first pIpC injection and the majority of the cKO mice survived to this
84

timepoint. Upon autopsy of one of the cKO mice that died suddenly on day 13 revealed
that it had died of myocardial infarction evidenced by blood clot formation in the heart
(Figure 3.19B).

Figure 3.19: A minority of GRP78 conditional knockout mice succumb to early fatality resulting
from myocardial infarction. (A) Kaplan-Meier survival curve of WT (n=17) and cKO (n=20) mice. (B)
H&E staining of the heart paraffin section of cKO mice that died early on.
Therefore, to investigate the possible cause of death, a closer examination of the
heart and the lung which oftentimes correlates to cardio abnormalities were performed.
The average size of the heart in cKO mice was larger than WT control, although not
statistically significant (Figure 3.20A and B) while the lung morphology was similar.
85

H&E staining of the paraffin sections of the heart revealed that signs of cardiomyopathy
in the cKO mice corresponding to pulmonary edema where there is fluid accumulation in
the congested lung suggesting inadequate functioning of the heart (Figure 3.20C).
Figure 3.20: GRP78 conditional knockout mice exhibited signs of cardiomyopathy and lung
congestion. (A) Heart and lung morphology of WT and cKO mice. (B) Quantitation of the heart weight of
WT (n=9) and cKO (n=10) mice. The data is presented as mean ± s.e. (*P<0.05, Student’s t-test). (C) H&E
staining of paraffin sections of the heart. The scale bar in heart represents 20 µm and in the lung represents
50 µm.
To further examine whether GRP78 was knockdowned in the heart and lung of
cKO mice, immunofluorescent staining of the paraffin sections of the heart and lung
using anti-GRP78 antibody. Based on the staining results, there was no significant
decrease of GRP78 in both the lung and heart in the cKO mice compared to WT siblings
(Figure 3.21), suggesting that the cardiomyopathy phenotype may not be a direct result of
GRP78 deficiency in the heart but may be secondary to GRP78 deficiency in the bone
marrow or other tissues.
86

Figure 3.21: GRP78 expression level in the heart and lung of cKO mice was similar to WT level.
Representative immunofluorescent staining of paraffin sections of heart and lung using an anti-GRP78
antibody counterstained with DAPI. The scale bar represents 50 µm.
Furthermore, we also measured and compared the growth rate and heart weight in
WT recipient mice transplanted with either WT bone marrow cells (WT-WT) or GRP78
deficient BM cells (WT-cKO) to tease out the possible effects of GRP78 knockout in the
microenvironment and other organs and examine whether the phenotype observed in
cKO mice can be recapitulated in WT-cKO mice and therefore be the consequence of
GRP78 depletion in the bone marrow. Interestingly, while the cKO mice exhibited
dramatic weight loss throughout the pIpC induction interval, the WT-cKO mice showed
normal growth rate comparable to WT-WT control mice (Figure 3.22A). In addition,
when we measured the heart sizes of the mice, we found that there was a borderline
significant increase in the heart weight of WT-cKO mice compared to WT-WT mice
87

(Figure 3.22B). This suggests that the dramatic weight loss and retarded growth observed
in the cKO mice may be a consequence of GRP78 deficiency in the microenvironment or
other sites aside from the bone marrow cells. In contrast, GRP78 deficiency in the bone
marrow alone may result in the moderate enlargement of the heart. This is very intriguing
but requires further investigation.

Figure 3.22: WT recipient transplanted with GRP78 deficient BM cells exhibited normal growth rate
yet moderately enlarged heart. (A) Average growth curve of WT (n=3) and cKO (n=3) mice starting the
first day of pIpC injection. (B) Quantitation of the heart weight of WT-WT (n=3) and WT-cKO (n=3) mice.
The data is presented as mean ± s.e. (student’s t-test)

3.3.7 GRP78 deficient mice exhibit differential expression in cytokines and
chemokines
Since we observed phenotypic changes in the immune system and outside of the
hematopoietic system in GRP78 cKO mice, suggesting involvement of intracellular
regulation, we therefore investigated the immunomodulatory effects of GRP78 on various
88

cytokine and chemokine production to give us a better understanding of its
immunological function.
Analysis of the differential expression of cytokines and chemokines in the serum
of WT and cKO mice provided us with some interesting candidate molecules that may
give insight to the causation of phenotypes observed in the GRP78-null mice. Out of all
the cytokines/chemokines examined between WT and cKO mice, the most differentially
expressed cytokine found was interleukin 1 alpha (IL-1 α), where the IL-1 α level in cKO
mice was dramatically 4 times lower than in WT siblings (P<0.0001) (Figure 3.23). IL-1 α
is a multifunctional proinflammatory cytokine that can affect nearly every cell type (C. a
Dinarello, 1996). It is synthesized as a 31 kD precursor in the cytosol and can be cleaved
by activation of the calcium-dependent, membrane associated cysteine proteases called
calpains (Kobayashi et al., 1990). In addition to its proinflammatory effects, IL-1 α has
been reported to have synergistic interactions with TNF- α for its requirement in the
expression of CD25 (IL-2 α receptor chain) and maturation of thymocytes (Zuniga-
Pflucker, Di, & Lenardo, 1995). It has also been associated to myocardial infarction
modulatory effect (Turner, Warburton, O'Regan, Ball, & Porter, 2010) and body weight
loss and sickness behavior in rats (Bluthé, Beaudu, Kelley, & Dantzer, 1995).
Another significantly differentially expressed chemokine found was
lipopolysaccharide-inducible CXC chemokine (LIX). We found a significant 5-fold
decrease in LIX chemokine level of cKO mice compared to WT siblings. LIX is usually
associated with cell migration and activation in neutrophils (Chandrasekar et al., 2003),
but also found to have a role in hematopoietic stem cells maintenance which the authors
revealed that LIX was also expressed in the hematopoietic stromal cells and its
89

expression is associated with hematopoietic supportive phenotypes (Choong, Yong, A. C.
L. Tan, Biao Luo, & Lodish, 2004). The dramatic reduction of LIX levels and
corresponding decrease in hematopoietic stem cell population in cKO mice suggests the
potential that the regulation of GRP78-null induced HSC reduction may be in part
through LIX chemokine interaction.
In addition, there was a prominent increase in interleukin-7 (IL-7) serum levels in
cKO mice compared to WT. IL-7 is produced by bone marrow and thymic stromal cells
and is the major lymphopoietic and thymopoietic cytokine. IL-7 has been identified as a
non-redundant cytokine essential for proper T and B cell development (von Freeden-
Jeffry et al., 1995; J. T. Tan et al., 2001) and induces proliferation and differentiation of
immature thymocytes (Murray, Suda, Wrighton, F. Lee, & Zlotnik, 1989), and protects
thymocytes from apoptosis by induction of bcl-2 expression (Akashi, Kondo, von
Freeden-Jeffry, Murray, & I L Weissman, 1997). In the Grp78 knockout mice in our
study, we observed a dramatic loss of lymphocytes in the peripheral blood and bone
marrow and severe loss of thymocytes indicating a significantly compromised
lymphopoiesis. Previous reports have shown that HIV-1-mediated T cell depletion
exhibit increased IL-7 production (Napolitano et al., 2001) and lymphopenic patients had
higher levels of serum IL-7, demonstrating the inverse relationship between serum levels
of IL-7 and absolute lymphocyte count (Bolotin, Annett, Parkman, & K. Weinberg, 1999).
Our results are in agreement to the inverse correlation between serum IL-7 levels and
lymphocyte number as previously reported.
90

Table 3.2: Differential cytokine and chemokine expression in serum of GRP78 deficient mice. Table
summarizing the differential expression of cytokines and chemokines in the serum of WT and cKO mice.
Values are mean ± s.e.

91

3.4 Discussion
Our investigation into the role of GRP78 in hematopoietic stem cell survival and
hematopoietic cellular homeostasis revealed many novel observations. Several studies
reveal that GRP78 has antiapoptotic properties and plays a crucial role in early
embryogenesis, oncogenesis, neurodegenerative diseases and atherosclerosis (M. Wang
et al., 2009). It has been shown that GRP78 is required in embryonic cell growth and
pluripotent cell survival (S. Luo, C. Mao, Brenda Lee, & Amy S Lee, 2006). In this study,
through the creation of a conditional knockout mouse model of GRP78 in the
hematopoietic system, we demonstrated for the first time that GRP78 depletion results in
significant reduction of hematopoietic stem cell population due to enhanced apoptosis in
the primitive cell population, indicating GRP78 is essential for hematopoietic stem cell
survival and maintenance. Furthermore, Mx1-cre induced disruption of GRP78 resulted
in significant reduction of lymphoid progenitor cells, corresponding to a dramatic
decrease in lymphocytes both in bone marrow and in circulation and severe impairment
of thymocytes. In contrast, while a decrease in myeloid progenitor cells was observed in
the cKO mice, there was an expansion of myeloid cells in the bone marrow and increased
levels of monocytes and granuolcytes in the peripheral blood. A diagram of the
alterations in the hematopoietic lineages and signaling pathways of the Grp78 conditional
KO mice is summarized in Figure 3.23. Through BM transplantation assays, we further
determined that the loss of HSC is a HSC cell autonomous effect, whereas the loss of
lymphoid populations was also found but to a smaller extent while in contrast, a moderate
decrease in monocytes was observed in the peripheral blood, suggesting that the potential
role of GRP78 in the microenvironment cannot be undermined. As GRP78 is critical for
92

the maintenance of cellular homeostasis and prevention of apoptosis, we observed full
activation of UPR signaling in the BM cells in Grp78 KO mice and induction of CHOP
and caspase mediated apoptosis.

Figure 3.23: Summary diagram of alteration of hematopoiesis, UPR signaling and apoptosis in Grp78
conditional knockout in the hematopoietic system. GRP78 depletion in the hematopoietic system leads
to altered hematopoiesis, activated UPR signaling and enhanced apoptosis. Red arrows represent an
increase and green arrows represent a decrease.
How does GRP78 depletion result in altered hematopoiesis? The knockdown of
this essential molecular chaperone has the potential to impact immune functions and
immunomodulatory effects at multiple levels.
It has been reported that both ATF6 activation and IRE1 α mediated XBP-1
splicing are required for “full induction of the UPR” (X. Li et al., 2011) and we have
93

found both these two branches and PERK activated in the bone marrow of Grp78
knockout mice. Thus, this suggests that GRP78 knockout in the hematopoietic system
results in full activation of the UPR as we see activated signaling in all three branches,
the PERK, ATF6 and IRE1 α pathways and also induction of pro-apoptotic CHOP,
indicative of more severe ER stress which corresponds to the caspase activation observed
in the cKO bone marrow cells (Figure 3.23). In agreement to persistent or severe ER
stress can switch the cytoprotective functions of the UPR into apoptosis programs (M.
Wang et al., 2009), we have observed that in the GRP78-null HSC-enriched population,
enhanced apoptosis persists.
GRP78, also known as immunoglobulin heavy chain binding protein (BiP),
chaperones immunoglobulin heavy and light chains (Nakaki, Deans, & A S Lee, 1989)
and therefore can regulate their synthesis and efficiency of secretion as it has been shown
that GRP78 level is an important factor for secretion efficiency of selective proteins
(Dorner & Randal J Kaufman, 1994). The ER is the site for the folding of proteins that
are destined to be secreted or membrane bound. As a major ER resident molecular
chaperone protein, GRP78 assists and regulates this process. Therefore, GRP78 may also
contribute to the processing of antigens and their association with major
histocompatibility class II molecules (Bonnerot et al., 1994) and cytokine production and
secretion (Qian et al., 2007).
In our study, we observed the generalized immuno-phenotype of decreased
lymphoid cells and increased myeloid cells in the peripheral blood and bone marrow in
our conditional Grp78 knockout mice. The decrease in lymphoid cells corresponds to the
reduction of upstream common lymphoid progenitor (CLP) enriched population in the
94

bone marrow and a severe loss of thymus cellularity in cKO mice. However, while we
observed a general increase in myeloid cells both in peripheral blood and bone marrow in
the cKO mice, we detected a decrease in their common myeloid progenitor (CMP)
enriched population and no significant difference in the granulocyte-monocyte progenitor
(GMP) enriched population. This suggests that the expansion of myeloid cells may not be
a consequence of increased upstream progenitor populations, but may be a result of
inflammation in the GRP78 deficient mice. A growing body of evidence suggests ER
stress and activated UPR signaling can result in stimulation of the inflammatory response
(K. Zhang & Randal J Kaufman, 2008). Several mechanisms have been proposed, one of
which is the link between UPR signaling pathways and NF-κB and JNK-mediated
inflammation. Upon ER stress, activated IRE1 α autophosphorylates, resulting in a
conformational change in its cytosolic kinase domain, in which it can then bind to
adaptor protein tumour-necrosis factor- α-receptor-associated factor 2 (TRAF2) (F Urano
et al., 2000). The IRE1 α-TRAF2 complex can recruit and activate the protein kinase JNK
which then phosphorylates the transcription factor activator protein 1 (AP1) and induces
expression of inflammatory genes (R. J. Davis, 2000). In addition, the IRE1 α-TRAF2
complex can also recruit and activate I κB kinase (IKK), which phosphorylates inhibitor
of NF- κB (I κB), leading to the degradation of I κB and releasing active NF- κB that
translocates to the nucleus and initiates transcription of inflammatory genes (P. Hu et al.,
2006). Alternatively, ER stress activated PERK-eIF2α mediated global protein
translational attenuation can also activate NF- κB. Since the protein half life of I κB is
much shorter than NF- κB, phosphorylated eIF2 α mediated translational inhibition can
lead to accumulation of free and active NF- κB that can translocate to the nucleus and turn
95

on inflammatory gene transcription (Deng et al., 2004). Since we have observed GRP78
deficiency results in full activation of UPR signaling in the bone marrow cells of cKO
mice, specifically the IRE1 α and PERK-eIF2 α is activated, this may result in ER
stress/UPR signaling mediated activation of inflammation and hence increase of
inflammatory cells (neutrophils, macrophages, monocytes, eosinophils, basophils) which
compose the majority of myeloid cell population. This was in agreement to the increased
serum levels of pro-inflammatory interferon gamma (IFN γ) and interleukin 6 (IL-6) in
the cKO mice. Therefore, in the GRP78 cKO mice, the observed decrease of common
myeloid progenitor population yet increase of myeloid cells found in peripheral blood
and bone marrow may be a consequence of ER stress induced inflammation.
It is worthy to note that Mx1-Cre induced deletion may not result in complete
GRP78 deletion efficiency as evidenced by observation of recovery at later timpoints (2
weeks, 3 weeks post induction) of cKO mice. From the complete blood count analysis,
we observed that the dramatic reduction of lymphocytes found at day 6 started to show a
gradual trend of increase back to normal level at the later timepoints (data not shown). In
agreement, the cKO mice also started to gain weight, trending toward normal growth rate
as weeks go by (data not shown). This may be a consequence of incomplete deletion
efficiency of Grp78 by Mx1-Cre induction, as cells that escaped Grp78 deletion have
greater survival advantage compared to GRP78-null cells, and therefore leading to the
expansion and repopulation of intact GRP78 cells in the long run.
The multiplex cytokine assay provided some clues for the GRP78-null induced
alterations in cytokine levels in cKO mice compared to WT controls. This suggested
some potential links of mechanism to many of the observed phenotypes in the cKO
96

mouse. One of the most distinguishably differentiated expressions is the 4-fold drop in
IL-1 α level in cKO mice. IL-1 α is generally considered a proinflammatory cytokine that
stimulates the activity of genes involved in inflammation and immunity (C. A. Dinarello,
1997). However, its functions are not limited to inflammation, as it has been associated
with bone formation and remodeling (Tani-Ishii, Tsunoda, Teranaka, & Umemoto, 1999),
insulin secretion (Sandberg, Eizirik, & Sandler, 1997), appetite regulation and post-
infarction cardiac remodeling (Turner et al., 2010). IL-1 α is synthesized as a 33 kD
molecule, which is proteolytically processed to the bio-active 17 kD form by Calpain in a
calcium dependent manner (Kobayashi et al., 1990). As GRP78 is a major ER chaperone
regulating cellular Ca
2+
homeostasis, depletion of GRP78 in cKO mice can lead to altered
calcium balance and disrupt the calcium dependent cleavage of IL-1 α by Calpain. Hence,
the dramatic decrease of IL-1 α levels in cKO mice can help interpret the loss of mean
body mass and cardiomyopathy phenotypes in the GRP78-null mice. Another significant
chemokine found to be differentially expressed was LIX. There was a 5-fold reduction of
LIX levels in the cKO mice compared to WT controls. The known functions of LIX are
as a chemoattractant for neutrophils and amplification of inflammatory cascades
(Chandrasekar et al., 2003). In addition, it has been reported to have a supportive role for
hematopoietic stem cell maintenance (Choong et al., 2004). With this raises the intriguing
possibility of whether the downregulation of LIX in the cKO mice may contribute to the
reduction of the HSC population and maintenance. This is exciting and requires further
evaluation. Additionally, the 7-fold increase in interleukin-7 (IL-7) levels found in cKO
mice was also brought to our attention. IL-7 is a non-redundant cytokine essential for
thymopoiesis and lymphocyte proliferation and maturation. The dramatic loss of
97

thymocytes and lymphocytes in peripheral blood and bone marrow indicates a significant
disruption of lymphopoiesis. Furthermore, our results are in agreement to the inverse
relationship between serum levels of IL-7 and lymphocyte number as previously reported
(Napolitano et al., 2001), suggesting the increase of IL-7 production as part of a
homeostatic response to T lymphocyte depletion.
Although we show the significant reduction of HSC-enriched population in Grp78
conditional knockout mice is cell autonomous as cKO bone marrow cells transplanted in
WT microenvironment still exhibit HSC loss, the discrepancies in thymus cellularity and
mature cell percentages between cKO mice and WT-cKO mice illustrate that the stromal
microenvironment may also contribute to the overall phenotypic changes. The growth
and development of hematopoietic cells occur within intimate associations and
interactions between the heterogenous stromal microenvironment which are composed of
adipocytes, smooth muscle cells, macrophages, endothelial and osteogenic cells. This
microenvironment is thought to provide a rich surrounding of molecular signals through
combinations of cytokines and chemokines production that mediate hematopoietic cell
maintenance and function (Hackney et al., 2002). Our multiplex cytokine analysis
revealed that there was differential expression of various cytokines/chemokines in
response to GRP78 knockout. As cytokines/chemokines can be produced not only by
hematopoietic cells, but also stroma cells, and therefore gives rise to the potential effect
of the knockout of GRP78 on the microenvironment. This provides as an alternate
mechanism to explain the discrepancy of phenotype between cKO mice and WT-cKO
mice.
98

As we show that GRP78 knockout in the hematopoietic system results in
significant reduction in HSC percentage in BM, recently another important ER chaperone,
GRP94 has been shown to have the opposite effect on HSC population. The study has
identified the ER chaperone protein, GRP94, as a novel cell intrinsic factor required to
maintain the interaction of hematopoietic stem cells (HSCs) with their niche, in which
acute elimination of GRP94 in the hematopoietic system resulted in expansion of HSCs
attributed to the impaired interaction of HSCs and the niche, resulting in enhanced HSC
mobilization and compromised homing and lodging ability of primitive hematopoietic
cells (Biquan Luo et al., 2011). Despite both GRP78 and GRP94 are essential ER
chaperones, the roles these two chaperones play in the hematopoietic system are diverse.
Our studies show that GPR78 is essential in hematopoietic stem cell survival and
hematopoietic system homeostasis and maintenance, with regulation at both the levels of
cell intrinsic and extrinsic stromal microenvironment, suggesting that GRP78 plays a
pleiotropic role in the hematopoietic system.

99

Chapter 4
Conclusions and Perspectives
Endoplasmic reticulum (ER) homeostasis and stress are involved extensively in a
variety of diseases, such as neurodegenerative disorders, inflammation, cancer,
atherosclerosis, and diabetes mellitus (Min Ni & Amy S Lee, 2007; Yoshida, 2007). The
direct role of GRP78 in adult hematopoietic stem cell, hematopoiesis and hematological
diseases, namely leukemia is still unclear. In this dissertation, we set forth to elucidate the
direct role of GRP78 in the a) Pten-null induced leukemia and b) hematopoietic stem cell
and maintenance. These studies contribute to the understanding of the importance of
GRP78 in hematopoiesis and Pten-null induced leukemogenesis.
In the first part of the dissertation, we investigated the role of GRP78 in
leukemogenesis. As GRP78 has been shown to be upregulated in various sold tumors and
cancer cell lines, for the first time, we have provided evidence that GRP78 mRNA
expression is elevated in the majority of adult patients with AML, CML, ALL and CLL
and myelogenous leukemia cell lines, HL60 and K562. In addition, we also detected
upregulated GRP78 protein level in the bone marrow samples of leukemia patients.
Additionally, we observed an increase in the GRP78 level in early relapse patients of
childhood ALL compared to the late relapse, suggesting an inverse relationship between
GRP78 expression level and time to relapse. This provides the first indication that GRP78
may play an important role in leukemia and chemoresistance.
100

To investigate the direct role of GRP78 in leukemogenesis, we generated an
inducible biallelic conditional knockout mouse model of GRP78 and PTEN in the
hematopoietic system in which we can acutely knockdown both PTEN and GRP78 under
the Mx1-cre induced expression by administrating pIpC (an interferon inducer)
intraperitoneally. We demonstrated that partial reduction of GRP78, in the form of Grp78
heterozygosity, was able to suppress Pten null mediated myeloproliferative disorders as
evidenced by undetectable immature progenitor cells in the peripheral blood, normal
splenic and hepatic morphology, and restoration of the HSC population back to the
normal percentage by decreased Pten-null induced proliferation. More importantly,
Grp78 heterozygosity significantly suppressed Pten-null mediated blast cell expansion
and significantly prolonged the survival compared to Pten-null mice. Pten null Grp78
heterozygous mice show MPD characteristics after longer latency period and indications
suggest the mice eventually succumb to leukemia. Furthermore, we show that Grp78
heterozygosity in the hematopoietic system has no apparent effect on the development
and survival of mice and exhibit normal bone marrow cellularity and HSC population.
Therefore, this suggests partial reduction of GRP78 can suppress leukemogenesis while
having no effect on normal hematopoiesis which renders GRP78 as a potential
therapeutic target for leukemia.
Upon investigation of the mechanism behind the suppressed leukemic phenotype
in Pten null Grp78 heterozygous mice, we analyzed the PI3K/AKT pathway which is the
major signaling pathway that PTEN suppresses. While enhanced activation of AKT/S6K
signaling was found in the bone marrow of Pten null mice, excitingly, we observed
downregulation of AKT/S6K activation in the bone marrow of Pten null Grp78
101

heterozygous mice. In cell culture experiments, we performed GRP78 knockdown with
siRNA in two human leukemia cell lines (HL60 and NB4) using two different stimuli
(serum and ER stress inducer) for AKT activation and demonstrated that knockdown of
GRP78 by siRNA suppressed AKT phosphorylation. Consistently, GRP78 knockdown
not only suppressed serum induced AKT
S473
phosphorylation, but also AKT
T308

activation. In agreement, GRP78 knockdown suppressed GSK3 β activation which is a
downstream signaling substrate of serum induced AKT activation while having minimal
effect on ERK and p38 MAPK signaling. In addition, our results suggest that GRP78
knockdown suppresses AKT signaling at the level of PI3K activation as evidenced by a
significant decrease of PI(3,4,5)P3 levels in siGrp78 induced cells. Furthermore, we
determined that the suppression of AKT activation may be specific for Grp78 knockdown,
and not a consequence of ER chaperone disruption, as we showed that knockdown of
GRP94 expression in HL60 cells did not suppress serum-induced AKT activation.
As we observed an inverse association between elevated Grp78 expression level
and time to relapse in childhood ALL, we tested if GRP78 could contribute to
chemoresistance. Our results revealed that overexpression of GRP78 conferred resistance
to AraC (a common used chemotherapy against leukemia) treatment and knockdown of
GRP78 increased chemo-sensitivity. This was confirmed by western blot detection of
apoptosis markers, trypan blue exclusion assay and Annexin V/7-AAD assays.
Therefore, we demonstrated that partial reduction of GRP78 can arrest
leukemogenesis through suppression of AKT signaling. This is in agreement to a
previous report showing that blockage of the AKT/mTOR signaling pathway by
Rapamycin can suppress and rescue the leukemic phenotype in the Pten
f/f
;Mx1-cre
102

leukemic mice (H. Yilmaz et al., 2006). Therefore, in collaboration with Dr. Parkash’s
laboratory, we investigated the potential therapeutic treatment of a monoclonal antibody
specifically against cell surface GRP78 (MAb159) on Pten null-induced leukemia. We
show that MAb159 treated Pten null mice exhibited a relatively normal whole blood
count profile and attenuated Pten null induced leukemic blast cell expansion. This was at
least in part due to suppression of AKT, suggesting the importance of cell surface GRP78
in AKT signaling activation and Pten null induced leukemogenesis.
Although PTEN mutation and deficiency have been found in T-ALL and BCR-
ABL leukemia (Palomero, Dominguez, & Ferrando, 2008; Peng et al., 2009), PTEN
disruption is not one of the well established contributing factors in leukemia. Therefore, it
would be intriguing to investigate whether knockdown of GRP78 in other leukemic
models exhibit similar rescue and suppressive leukemic phenotype. BCR-ABL is a
common fusion oncoprotein resulting from chromosome translocation and is highly
associated with chronic myeloid leukemia (CML) as 95% of CML patients have this
abnormality. To test if knockdown of GRP78 can also suppress BCR-ABL induced
leukemia, apoptosis and transplantation assays of GRP78 knockdowned BCR-ABL
transformed BM cells can be performed. Isolated BM cells from either GRP78
f/f
;Mx1-Cre
or Grp78
f/+
;Mx1-Cre mice can be transformed by lenti-BCR-ABL. BM cells isolated
from GRP78
f/f
mice will also be transformed by lenti-BCR-ABL and serves as control.
Cultured BCR-ABL transformed BM cells can further be treated with pIpC in the
medium to induce Cre expression and therefore knockdown GRP78. Apoptosis and
proliferation assays can be performed to analyze the effect of GRP78 knockdown in
BCR-ABL transformed cells. For in vivo studies, transformed BCR-ABL BM cells from
103

GRP78
f/f
;Mx1-Cre, Grp78
f/+
;Mx1-Cre, and GRP78
f/f
mice mixed with WT BM cells can
be transplanted in lethally irradiated WT mice to examine the effect of GRP78
knockdown in BCR-ABL induced leukemogenesis. Investigating GRP78 suppression in
another leukemic model is exciting and awaits further examination.
In summary, we observed overexpression of GRP78 in human leukemia patients
and revealed an inverse relationship between GRP78 expression level and time to relapse
in childhood ALL patients. Collectively, we provide proof-of-principle that partial
reduction of GRP78 (in the form of Grp78 heterozygosity or monoclonal antibody
targeted) can arrest leukemogenesis through suppression of AKT signaling and can
sensitize leukemic cells to chemotherapy treatment while having no harmful effect on the
hematopoietic system.
GRP78 has been shown to be critical for the maintenance of cellular homeostasis
and prevention of apoptosis. Homozygous knockout mice of GRP78 are embryonic lethal
at E3.5, indicating GRP78 is essential for embryonic cell growth and pluripotent cell
survival (S. Luo, C. Mao, Brenda Lee, & Amy S Lee, 2006). However, this has not been
investigated in adult hematopoietic stem cells. We generated a conditional knockout
mouse model that results in acute homozygous deletion of GRP78 in the hematopoietic
system. GRP78 deficiency results in a significant reduction of HSC-enriched population
in both the LT-HSC and ST-HSC while minimally affecting bone marrow cellularity and
spleen size and morphology. The GRP78-null induced reduction of HSCs is at least in
part due to enhanced apoptosis in the HSC population and this is cell autonomous.
GRP78 conditional knockout mice exhibited lymphopenia as evidenced by decreased
common lymphoid progenitors, significant reduction of CD3
+
and B220
+
lymphoid cells
104

in bone marrow, decreased lymphocytes in the peripheral blood and severe depletion of
thymocytes. While GRP78 deficiency led to decreased common myeloid progenitor
populations, there was a significant increase in Mac-1
+
and Gr-1
+
myeloid cells in the
bone marrow, corresponding to increased granulocytes and monocytes in the peripheral
blood.
Through transplantation assay of transplanting Grp78 KO bone marrow cells into
a WT microenvironment (WT-cKO) resulted in significant reduction of HSC population
compared to WT bone marrow cells transplanted into WT microenvironment (WT-WT).
This suggests GRP78 in hematopoietic cells is required for HSC survival and
homeostasis. In agreement, GRP78 deficient BM cells transplanted in WT
microenvironment resulted in decrease of both common lymphoid and myeloid
progenitor populations, suggesting the requirement of GRP78 in the primitive cell pool is
cell autonomous. A general reduction in the lymphoid populations was also observed in
WT-cKO mice with decreased lymphoid cells in the bone marrow and peripheral blood
and loss of thymus cellularity. However, while an increase in myeloid cells in the bone
marrow was detected, there was a decrease in monocytes and granulocytes in the
peripheral blood in WT-cKO mice. This suggests the increase of monocytes and
granulocytes found in the peripheral blood of cKO may at least in part be a consequence
of GRP78 knockdown in the microenvironment resulting in cytokine level alteration
induced inflammation. However, this requires further investigation and validation.
As GRP78 is essential in maintaining cellular homeostasis and is the master
regulator of the ER stress induced UPR signaling. In GRP78 deficient bone marrow, we
detected full induction of UPR signaling with activation of PERK, ATF6 and IRE1 α
105

pathways and also induction of proapoptotic CHOP indicative of more severe ER stress
which corresponds to the caspase activation observed. In agreement to persistent or
severe ER stress can switch the cytoprotective functions of the UPR into apoptosis
programs (M. Wang et al., 2009), we have observed that in the GRP78-null HSC-
enriched population, enhanced apoptosis persists.
The knockdown of this essential molecular chaperone has the potential to impact
immune functions and immunomodulatory effects at multiple levels. Our multiplex
cytokine assay revealed differential expression in cytokine serum levels in Grp78
knockout mice compared to WT siblings such as interleukin 1 α (IL-1 α), interleukin 7 (IL-
7) and LIX. This reveals candidate cytokines and chemokines with differential
expression that may contribute to the altered hematopoiesis phenotype observed in cKO
mice. This suggests that not only is GRP78 essential in the intrinsic survival and
maintenance of HSC but is also important for the regulation of hematopoiesis in the
stroma microenvironment.
Collectively, our studies show that GPR78 is not only essential in adult
hematopoietic stem cell survival and hematopoietic system homeostasis and maintenance,
but also hematological diseases, namely leukemogenesis and leukemic chemoresistance
through its antiapoptotic properties, chaperoning functions or regulation of signaling
pathways, suggesting that GRP78 plays a pleiotropic role in the hematopoietic system.
106

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Asset Metadata

Creator Wey, Shiuan (author)

Core Title The role of endoplasmic reticulum protein GRP78 in normal hematopoeises and PTEN-null leukemogenesis

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 07/18/2013

Defense Date 06/23/2011

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag AKT,GRP78,Hematopoiesis,leukemia,OAI-PMH Harvest,PTEN

Language English

Advisor Lee, Amy S. (committee chair), Kahn, Michael (committee member), Shibata, Darryl K. (committee member)

Creator Email shannonwey@gmail.com,shiuan.wey@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c127-629905

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Abstract (if available)

Abstract The endoplasmic reticulum (ER) is an intracellular organelle for protein folding, lipid synthesis and Ca²⁺ storage. It is also responsible for the transportation for most of the secretory and transmembrane proteins. When the protein load exceeds the ER folding capacity, the ER undergoes stress and activates a set of signaling cascades that is termed the unfolded protein response (UPR). The multifunctional GRP78 is the major ER molecular chaperone with protein folding abilities and the master regulator of the UPR, and recently has been shown that a subfraction of it is localized on the cell surface acting as a co-receptor for various signaling pathway activation. ❧ Traditionally GRP78 is regarded as protective against hypoxia and nutrient starvation prevalent in the microenvironment of solid tumors, thus, its role in the development of hematologic malignancies remains to be determined. In this thesis, elevated GRP78 expression was detected in leukemic blasts of adult patients, leukemia cell lines and inversely correlates with time to relapse in childhood acute lymphocytic leukemia. To directly elucidate the requirement of GRP78 in leukemogenesis, we created a biallelic conditional knockout mouse model of GRP78 and PTEN in the hematopoietic system. Strikingly, heterozygous knockdown of GRP78 in PTEN null mice is sufficient to restore the hematopoietic stem cell (HSC) population back to the normal percentage and suppress leukemic blast cell expansion. AKT/mTOR activation in PTEN null bone marrow cells is potently inhibited by Grp78 heterozygosity, corresponding with suppression of the PI3K/AKT pathway by GRP78 knockdown in leukemia cell lines. This is the first demonstration that GRP78 is a critical effector of leukemia progression, at least in part through control of oncogenic AKT signaling. Furthermore, overexpression of GRP78 renders human leukemic cells more resistant to AraC-induced apoptosis whereas knockdown of GRP78 sensitizes them, suggesting GRP78 is a novel potent therapeutic target for leukemia. ❧ Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression and interactions with extrinsic factors in the microenvironment. GRP78 has been shown to be critical for the maintenance of cellular homeostasis and prevention of apoptosis. Homozygous knockout mice of GRP78 are embryonic lethal at E3.5, indicating GRP78 is essential for embryonic cell growth and pluripotent cell survival. However, this has not been investigated in adult hematopoietic stem cells. Here we generated a conditional knockout mouse model that acutely deletes GRP78 in the hematopoietic system. GRP78 deficiency results in a significant reduction of HSCs, progenitor and lymphoid cell populations yet an increase in myeloid lineage granulocytes and monocytes in cKO mice. The GRP78-null induced reduction of the HSC pool can be attributed to enhanced apoptosis. In agreement, GRP78 deficient BM cells exhibited activated UPR signaling in all three branches and induced expression of pro-apoptotic CHOP and caspase activation. Transplanting Grp78 KO hematopoietic cells into WT microenvironment also yielded a decrease in HSCs, suggesting that GRP78 in hematopoietic cells is required for HSC survival and homeostasis. In addition, a multiplex cytokine assay reveals alteration in several cytokine serum levels in cKO mice, suggesting GRP78 plays a pleiotropic role and is essential in regulating hematopoietic system homeostasis and maintenance.