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Therapeutic resistance in acute lymphoblastic leukemia: cIAP2 inhibition sensitizes B cell acute lymphoblastic leukemia to anti-CD19 chimeric antigen receptor T cell
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Therapeutic resistance in acute lymphoblastic leukemia: cIAP2 inhibition sensitizes B cell acute lymphoblastic leukemia to anti-CD19 chimeric antigen receptor T cell
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Content
Therapeutic resistance in acute lymphoblastic leukemia:
cIAP2 inhibition sensitizes B cell acute lymphoblastic leukemia to
anti-CD19 Chimeric Antigen Receptor T cell
by
Hye Na Kim
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
(CANCER BIOLOGY AND GENOMICS)
December 2021
Copyright 2021 Hye Na Kim
ii
Dedication
This work is wholeheartedly dedicated to my parents, siblings and husband who have supported
me throughout my journey to pursue my PhD. Without their encouragements, it would have not
been possible to finish what I have done.
iii
Acknowledgement
I take this opportunity to express my sincere gratitude towards everyone who have helped me
during my PhD.
First of all, I would like to thank my mentor Dr. Yong-Mi Kim for her guidance and support
throughout the entire time I have spent in her laboratory. She inspired me to pursue PhD and
challenged me to become a critical thinker to become a better scientist. Her mentorship and
endless support pushed and brought my work to a higher level.
I also would like to acknowledge my previous and current colleagues. Especially I would like to
thank Enzi Jiang, Yongsheng Ruan, Heather Ogana, Zesheng Wan and Samantha Hurwitz for
helping me with experiments done in this thesis. But more importantly, they all have made my
time in the lab enjoyable. Also, I would like thank collaborators Dr. Heisterkamp, Dr. Bhojwani, Dr.
Oberley, Dr. Moghimi, Annie Luong and Justin Le for providing materials, clinical insights,
comments, and feedback for this thesis.
I would like to deeply thank my committee members, Dr. Stallcup, Dr. Abdel-Azim, and Dr. Parekh
for being wonderful supporters. It is my honor to be one of the last students for Dr. Stallcup before
his retirement, as not only his scientific knowledge but his kindness and supports aspired me. I
sincerely thank Dr. Parekh and Dr. Abdel-Azim, for providing insightful feedbacks. Their clinical
remarks have enabled this thesis to be clinically achievable so that it can benefit people in need
in the future.
Lastly, I would like to thank my family and my friends. My father has always motivated me to
challenge myself and achieve higher goals. My mom inspired me to become a cancer scientist so
that I can contribute my work to help cancer patients. I express my deepest respect to my mom
for never giving up and fighting the battle to conquer her disease. My brother and sister are always
my best friends who encourages me in times of hardship. Lastly, I would like to thank my husband
for his steadfast support and love.
iv
Table of contents
Dedication ................................................................................................................................... ii
Acknowledgements .................................................................................................................... iii
List of Tables ............................................................................................................................. vii
List of Figures ........................................................................................................................... viii
Abstract ...................................................................................................................................... ix
Chapter 1: Adhesion Molecules in Chemotherapy Resistance of Leukemia .............................. 1
1.1 Acute lymphoblastic leukemia ........................................................................................ 1
1.2 Minimal residual disease (MRD) and cell adhesion mediated drug resistance
(CAM-DR) in leukemia ................................................................................................... 2
1.3 Leukemia and leukemia stem cells (LSCs) .................................................................... 2
1.4 Cell adhesion molecules (CAMs) in leukemia ................................................................ 3
1.5 Cadherins in CAM-DR in leukemia ............................................................................... 5
1.5.1 VE Cadherin ........................................................................................................ 6
1.5.2 N-Cadherin .......................................................................................................... 7
1.5.3 E-Cadherin .......................................................................................................... 7
1.5.4 Targeting cadherins in leukemia ........................................................................ 8
1.6 Selectins in CAM-DR in leukemia .................................................................................. 9
1.6.1 Targeting selectin in cancer .............................................................................. 11
1.7 Integrins in CAM-DR in leukemia ................................................................................ 12
1.7.1 Integrin 1 (CD49a) .......................................................................................... 13
1.7.2 Integrin 2 (CD49b) ........................................................................................... 14
1.7.3 Integrin 3 (CD49c) ........................................................................................... 14
1.7.4 Integrin 4 (CD49d) .......................................................................................... 15
1.7.5 Integrin 5 (CD49e) ........................................................................................... 16
1.7.6 Integrin 6 (CD49f) ........................................................................................... 17
1.7.7 Integrin 7 (ITGA7) ........................................................................................... 18
1.7.8 Integrin 9 (ITGA9) ........................................................................................... 19
1.7.9 Integrin L (CD11a, LFA-1) ............................................................................... 19
1.7.10 Integrin M (CD11b) .......................................................................................... 20
1.7.11 Integrin V (CD51, VNRA, MSK8) .................................................................... 20
1.7.12 Integrin β1 (CD29) ............................................................................................ 21
1.7.13 Integrin β3 ......................................................................................................... 22
1.7.14 Integrin β7 ........................................................................................................ 22
1.7.15 Targeting integrins ........................................................................................... 23
1.8 Conclusion .................................................................................................................. 24
Chapter 2: PI3Kdelta and BRD4 as targets in Chemotherapy Resistance of Leukemia .......... 32
2.1 Introduction ................................................................................................................. 32
2.2 Material and methods .................................................................................................. 34
2.2.1 Patient samples and cell culture ...................................................................... 34
2.2.2 Starvation and activation assay for detection of phosphorylated-AKT at
serine 473 residue ............................................................................................. 34
2.2.3 Western blot ...................................................................................................... 34
2.2.4 Chromatin immuno-precipitation (chIP) ............................................................. 35
v
2.2.5 RNA extraction and qPCR ................................................................................ 35
2.2.6 Apoptosis analysis with Annexin V and DAPI staining ..................................... 36
2.2.7 Cell cycle analysis ............................................................................................ 36
2.2.8 Cell proliferation assay ..................................................................................... 36
2.2.9 Flow cytometry .................................................................................................. 37
2.2.10 Cell adhesion assay ......................................................................................... 37
2.2.11 Animal studies ................................................................................................... 37
2.2.12 Data analysis and statistics ............................................................................... 38
2.3 Results ......................................................................................................................... 38
2.3.1 PI3K𝛅 and BRD4 expression in B-ALL .............................................................. 38
2.3.2 SF2535 downregulates c-Myc and p-AKT in B-ALL .......................................... 38
2.3.3 SF2535 induces apoptosis in B-ALL cells through changes in the intrinsic
apoptotic pathway ............................................................................................. 40
2.3.4 SF2535 causes cell cycle changes and suppresses cell counts in B-ALL ........ 40
2.3.5 SF2535 decreases surface integrin expression ................................................ 41
2.3.6 SF2535 decreases peripheral leukemic burden in mouse model ..................... 42
2.4 Discussion .................................................................................................................... 43
2.5 Conclusion ................................................................................................................... 45
Chapter 3: cIAP2 inhibition in anti-CD19 CAR T cell therapy resistant Leukemia .................... 52
3.1 Introduction .................................................................................................................. 52
3.2 Methods ...................................................................................................................... 54
3.2.1 Patient derived bone marrow aspirate or peripheral blood sample
processing and culturing .................................................................................. 54
3.2.2 Anti-CD19 CAR T production and transduction of healthy donor PBMC
derived T cells ................................................................................................... 54
3.2.3 Western blot analysis of target protein in primary. B-ALL ................................ 55
3.2.4 Annexin V/ 7AAD staining for accessing viability of primary B-ALL cells ......... 56
3.2.5 Phospho-flow cytometry for staining phosphorylated p65 in primary
B-ALL cells ....................................................................................................... 56
3.2.6 BIRC3 overexpression and knockdown in primary B-ALL cells via
lentivirus transduction ...................................................................................... 57
3.2.7 CRISPR/Cas9 mediated knockout of BIRC3 gene in primary B-ALL ............... 57
3.2.8 RT-qPCR ........................................................................................................... 58
3.2.9 Homing of BIRC3 KD cells into bone marrow .................................................. 58
3.2.10 In vivo treatment of BIRC3 KD cells with anti-CD19 CAR T cells .................... 59
3.3 Results ......................................................................................................................... 60
3.3.1 Primary B-ALL cells express cIAP2 after 24hours of anti-CD19 CAR T cell
exposure .......................................................................................................... 60
3.3.2 TNF-ɑ secreted by anti-CD19 CAR T induces cIAP2 expression in
B-ALL cells ........................................................................................................ 62
3.3.3 Overexpression of cIAP2 in primary B-ALL delays anti-CD19 CAR T
mediated apoptosis in vitro ............................................................................... 63
3.3.4 BIRC3 overexpression in primary B-ALL increases p65 in the cytoplasm
and PD-L1 expression on the surface after anti-CD19 CAR T treatment ......... 63
3.3.5 shRNA vector transduced knockdown and CRISPR/Cas9 mediated
knockout of BIRC3 sensitizes primary B-ALL cells toward anti-CD19 CAR
T therapy ........................................................................................................... 64
3.3.6 SMAC mimetic LCL161 mediated inhibition of cIAP1 and cIAP2
sensitizes B-ALL cells towards anti-CD19 CAR T treatment ........................... 65
vi
3.3.7 Evaluation of cIAP2 inhibition in combination with anti-CD19 CAR T
in vivo ............................................................................................................... 67
3.4 Discussion .................................................................................................................... 67
References .............................................................................................................................. 83
Appendices ............................................................................................................................. 100
Appendix A: Supplementary information for chapter 2 ..................................................... 100
Appendix B: Supplementary information for chapter 3 ..................................................... 110
vii
List of tables
Table 1.1. Description of cadherin inhibitors ............................................................................. 25
Table 1.2. Description of selectin inhibitors .............................................................................. 25
Table 1.3. Clinical trials for selectin inhibitors in leukemia ....................................................... 26
Table 1.4. Description of integrin inhibitors .............................................................................. 26
viii
List of figures
Figure 1.1 BM microenvironment ............................................................................................. 28
Figure 1.2 Cadherin and adherens junction. ............................................................................ 29
Figure 1.3. Dimerization of integrins in leukemia. .................................................................... 30
Figure 1.4. Integrin signaling in leukemia ................................................................................ 31
Figure 2.1. SF2535 downregulates c-Myc and p-AKT .............................................................. 46
Figure 2.2. SF2535 induces apoptotic effects in B-ALL cells .................................................. 48
Figure 2.3. SF2535 prolongs G0+G1 phase arrest and attenuates S phase ........................... 49
Figure 2.4. SF2535 affects adhesion molecules ...................................................................... 50
Figure 3.1. anti-CD19 CAR T treatment induces overexpression of cIAP2 and increases
phosphorylation of p65 at serine 536 in B-ALL cells ............................................ 71
Figure 3.2. TNF-ɑ induces cIAP2 expression in B-ALL cells ................................................... 73
Figure 3.3. Overexpression of BIRC3 protects primary B-ALL from anti-CD19 CAR T ............ 75
Figure 3.4. BIRC3 overexpression in B-ALL increases p65 and surface PD-L1 expression
upon anti-CD19 CAR T treatment ........................................................................ 77
Figure 3.5. BIRC3 knockdown and knockout sensitizes B-ALL cells toward anti-CD19 CAR
T therapy .............................................................................................................. 78
Figure 3.6. SMAC mimetics AT406 and LCL161 sensitizes B-ALL cells toward anti-CD19
CAR T cells by downregulating both cIAP1 and cIAP2, but not XIAP ................... 81
Figure 3.7. Evaluating BIRC3KD combined with anti-CD19 CAR T in vivo ............................. 83
Figure 3.8. Graphical abstract of cIAP2 mediated anti-CD19 CAR T resistance in B-ALL ...... 84
ix
Abstract
Although 5-year survival rate of pediatric leukemia patients has exceeded 85%, relapse
and refractory disease remains a problem. Cell adhesion mediated drug resistance (CAM-DR) in
leukemia has been proposed for a long time. Interaction between leukemia cells surrounding
microenvironment elicits pro-survival intracellular signaling in B-ALL, thus providing drug
resistance.
In chapter 1, we highlighted some of surface molecules that are known to provide
chemotherapy-resistance in major types of leukemia, ALL, AML, ALL and AML. Especially we
focused on cadherins, selectins and integrins, and how subtypes of each surface molecule
provide resistance to therapy in vitro, in vivo and in patients. We also introduced how surface
molecule inhibitors are being translated into clinic.
In chapter 2, we preclinically evaluated a signaling pathway, that is found downstream of
integrin signaling and is implicated as a critical survival signal in drug resistance of leukemia, the
pAKT pathway. Here, we evaluated BRD4 and PI3K inhibition in primary B-ALL with small
molecule inhibitor SF2535.The PI3K/Akt pathway¾and in particular PI3Kδ¾is known for its role
in drug resistant B-cell acute lymphoblastic leukemia (B-ALL), and it is often upregulated in
refractory or relapsed B-ALL. Myc proteins are transcription factors responsible for transcribing
pro-proliferative genes, and c-Myc is often overexpressed in cancers. However, c-Myc is difficult
to target directly. Since BRD4 is responsible for c-Myc transcription in B-ALL, we study the effect
of BRD4/PI3Kδ dual inhibition to downregulate c-Myc and AKT signal. Our studies show
BRD4/PI3Kδ dual inhibition induces apoptosis in B-ALL by downregulating c-Myc and
phosphorylation of AKT. SF2535 also decreases anti-apoptotic protein BCL-2 and increases
cleavage of caspase-3, -7 and PARP. SF2535 treatment induces cell cycle arrest and decreases
cell count in primary B-ALL model in vitro. Interestingly, dual inhibition of BRD4/PI3Kδ by SF2535
decreases the mean fluorescence intensity (MFI) of surface integrin α4, α5, α6, and β1 while
x
increasing MFI of CXCR4, indicating that SF2535 may work through inside-out signaling of
integrins.
In the last chapter 3, we began to study the role of adhesion to stromal cells in the context
of immunotherapy resistance as an extension of our work in chemotherapy resistance. Here, we
came to focus on the cIAP2 mediated anti-CD19 CAR T resistance. Chimeric antigen receptor T
(CAR T) therapy has shown promising result in pediatric patients with relapsed leukemia, yet
recurrence of the disease after CAR T therapy represents an obstacle to overcome in the
immunotherapy of B-ALL. We identified upregulation of cIAP2 protein in primary and immortalized
B-ALL cells after anti-CD19 CAR T treatment, which can serve as a biological target to increase
anti-CD19 CAR T treatment efficacy. Increased cIAP2 in primary B-ALL correlated with increased
phosphorylation of p65. In primary B-ALL, overexpression of BIRC3 (cIAP2) delayed apoptosis
after 24hours of anti-CD19 CAR T treatment in vitro. Furthermore, BIRC3 overexpressed primary
B-ALL presented higher percentage of PD-L1 on their surface after CAR T treatment. To
determine whether inhibition of cIAP2 sensitizes B-ALL cells toward CAR T therapy, we have
generated BIRC3 knocked-down cells with shRNA lentivirus as well as CRISPR/Cas9 mediated
BIRC3 knocked-out primary B-ALL. Both KD and KO cells decreased cIAP2 protein level and
were more sensitive to CAR T therapy in first 24hours of treatment. Taken together, we suggest
cIAP2 as a biological target that can increase CAR T efficacy in treating patients with
relapsed/refractory leukemia.
Through our studies, we highlight the importance of surface molecules in drug resistance
and anti-apoptotic proteins in immunotherapy. Deeper understanding of drug-resistance in
leukemia is needed for eradication of the disease.
1
Chapter 1. Adhesion Molecules in Chemotherapy Resistance of
Leukemia
1.1 Acute lymphoblastic leukemia
Leukemia is a type of cancer that starts in the bone marrow and causes rapid expansion of
abnormal blood cells. Leukemia can be categorized based on the lineage and developmental
stage affected. Major types of leukemia are acute lymphoblastic leukemia (ALL), acute
myelogenous leukemia (AML), chronic lymphoblastic leukemia (CLL) and chronic myelogenous
leukemia (CML). Acute leukemia arises when bone marrow produces immature white blood
cells that cannot function properly. Usually, acute leukemia is faster than chronic leukemia. On
the other hand, chronic leukemia involves more mature cells, and they progress slowly.
Lymphoblastic (or lymphocytic) leukemia develops when cells that forms T- B- or NK cells are
affected. Myelogenous leukemia derives from granulocyte and monocyte forming cells.
Leukemia happens frequently in younger patients compared to older patients. In fact, leukemia
is the most common cancer in pediatric patient under age of 15 [1]. Almost 3 out of 4 patients
have ALL, and rest are AML patients. Large fraction of ALL patients represents B cell ALL (B-
ALL). Improved diagnostic tools and therapeutic drugs have achieved high survival rate of
pediatric leukemia patients. 5-year survival rate of pediatric leukemia patients has exceeded 85-
90% [2]. Leukemia incidence in older patients is lower than pediatric patients, yet prognosis is
much poorer.
2
1.2 Minimal residual disease (MRD) and cell adhesion mediated drug
resistance (CAM-DR) in leukemia
Despite the improved overall survival of leukemia patients, relapsed and refractory leukemia
remains a problem. Chemoresistant minimal residual disease (MRD) contributes to the recurrence
of the disease. Patients with relapsed leukemia in the bone marrow (BM) have worse outcomes
than patients with relapses in the central nervous system or testis [3], suggesting the contribution
of the BM to the progression and aggressiveness of the disease. Indeed, the BM
microenvironment is known to govern leukemia quiescence [4-6], proliferation [7], drug resistance
[8], and leukemogenesis [9]. Leukemia cells communicate with BM through surface molecules
called cell adhesion molecules (CAMs). The CAM-mediated interaction of leukemia cells with the
surrounding microenvironment contributes to drug resistance, which is called cell adhesion-
mediated drug resistance (CAM-DR). Surface molecule overexpression and CAM-DR have been
addressed in many subtypes of leukemia, including B- and T-acute lymphoblastic leukemia (B-
and T-ALL) and acute myeloid leukemia (AML). CAM-DR is one mechanism through which
leukemia cells obtain chemoresistance, and resistant clones will result in the recurrence of the
disease. Due to aberrant expression, CAMs serve not only as a prognostic tool for detecting MRD
in leukemia but can also be targeted to sensitize drug-resistant cells to chemotherapy [10-13].
CAM inhibition in leukemia is being actively evaluated in preclinical and clinical studies. In this
review, we will focus on the major groups of CAMs—cadherin, selectin, and integrin—and their
role in drug resistance in leukemia
1.3 Leukemia and leukemia stem cells (LSCs)
Leukemia is a type of cancers that affects a patient's blood and bone marrow. Leukemia
can be categorized into different subtypes depending on the progression of the disease (acute
and chronic) or a lineage and developmental stage of cells (myeloid or lymphoblastic). Four main
3
subtypes of leukemia that will be discussed in this review are: acute lymphoblastic leukemia (ALL)
[14-16], acute myeloid leukemia (AML) [17, 18], chronic lymphocytic leukemia (CLL) [19, 20], and
chronic myeloid leukemia (CML) [21, 22].
Leukemic cells have been described to interact with and remodel BM to support leukemic
cell expansion and survival [23]. LSCs are capable of self-renewal and thus able to maintain
survival in optimized in vitro co-culture systems and in immunocompromised mice [24]. CAMs
play an important role in the interaction between LSCs and the hematopoietic niche [25, 26].
Firstly, it has been shown that the N-cadherin
+
CD34⁺CD38⁻LSCs population has a critical role in
the development of AML [27, 28]. Moreover, the downregulation of E-cadherin suppressed the
adhesion of AML cells to BM-derived MSCs and enhanced the anti-leukemic effect of cytarabine
[29]. Secondly, in an AML mouse model, LSCs adhered to the vascular niche which protected
LSCs from chemotherapy through E-selectin/E-selectin ligands and this effect was ameliorated
by GMI-1271 [30]. Thirdly, VLA-4 is one of the most prominent integrins involved in LSCs [26].
Recently a study showed that inhibition of Kindlin-3-mediated VLA-4 adhesion mobilized LSCs in
the BM and prolonged survival of mice with CML [31]. Furthermore, the integrin αVβ3 was
expressed in particular on CD34
+
cells in AML with NPM1 mutation [32].
1.4 Cell adhesion molecules (CAMs) in leukemia
BM is a complex tissue with various components. Mesenchymal stromal cells (MSC) [33,
34], endothelial cells [6, 35], osteoblasts [36], adipocytes [37], neurons [38], Schwann cells [39],
and megakaryocytes [40] comprise the endosteal and vascular BM niches. Soluble factors such
as chemokines [41], the exosome [42, 43], or microRNA [44] facilitate crosstalk between cells in
the BM [45, 46] Extracellular matrix (ECM) proteins deposited from cells provide the BM
architecture and determine the stiffness of tissue, which affects the cell proliferation and
chemosensitivity of leukemia [47]. The BM microenvironment has been studied for its role in
4
leukemia support and drug resistance [48, 49]. A previous study examined BCR-ABL positive
(Ph
+
) (Tom-1, Nalm-27 and Sup-B15) and BCR-ABL negative (Ph
−
) cell lines (REH and Nalm-6)
cultured on primary bone marrow stromal cells (BMSC) or osteoblasts (HOB) divided into three
populations by relative distance to the supportive layer–S (suspended), phase bright (PB), and
phase dim (PD). Out of the three populations, the PD leukemic population planted under the
BMSC layer demonstrated increased quiescence, resistance to cytarabine (Ara-C), methotrexate
(MTX), and vincristine (VCR), and increased glycolysis [50]. This result shows the importance of
crosstalk between leukemia and the surrounding microenvironment. One of the most well-known
mechanisms of the BM–leukemia cell interaction is the CXCR4/CXCL12 axis [4, 51], yet there are
many more surface molecules that are directly associated with adhesion and interaction [52-54].
Cell adhesion molecules (CAMs) are cell surface proteins that are specialized for adhesion to
other types of cells or the ECM. This review will primarily focus on leukemia-relevant adhesion
receptors from four major families of CAMs−cadherin, immunoglobulin superfamily CAM (IgCAM),
selectin and integrin [55].
CAMs are single-pass transmembrane proteins with extracellular, transmembrane, and
intracellular structures. The extracellular domain of CAMs recognizes specific ligands or counter-
receptors, and the intracellular (cytoplasmic) domain translates external stimuli into intracellular
signaling, while the transmembrane domain stabilizes the structure of the molecule.
While selectins act as a monomer, cadherins form a homodimer and integrins must form
a heterodimer to be fully functional. Ca
2+
is required for stabilization of the extracellular domain
[56-58], as well as in selectins for proper binding to ligands [59]. Integrins are dependent and
regulated by other divalent cations as well [60, 61]−Ca
2+
binding maintains the folded and inactive
conformation of integrins while the heterodimer travels from the Golgi to the cell surface, and
integrin undergoes conformational changes upon replacement of Ca
2+
with Mg
2+
or Mn
2+
[60-62].
BM is abundant in binding partners for CAMs as each component of BM discussed above
express diverse ligands and secret ECM [63-70] (Figure 1.1). Fibronectin, collagen and laminin
5
secreted in the BM will interact with cellular surface molecules [71, 72]. Leukemia-induced BM
remodeling can interrupt homeostasis and shift equilibrium towards leukemia progression by
overexpressing binding partners for CAMs [73-76]. At the same time, leukemia cells can
aberrantly express CAMs to facilitate surface molecule-mediated interaction with the BM
microenvironment, thereby inducing cell adhesion-mediated drug resistance (CAM-DR). Recently,
the mitochondrial transfer from mesenchymal cells to leukemia has been shown to promote drug
resistance in T-ALL, indicating the diverse aspects that BM can modulate to provide leukemia
protection [77].
1.5 Cadherins in CAM-DR in leukemia
Cadherins are a type of CAM that participates in forming adherent junctions between
adjacent cells. Cadherins can be subdivided into several different groups including Type I and
Type II classical cadherins [78], desmosomal cadherins [79], proto-cadherins [80], seven-pass
transmembrane cadherins, and FAT and Dachsous cadherins [81]. The extracellular domain
contains cadherin extracellular repeats that exert homotypic Ca
2+
dependent adhesion, while the
intracellular domain binds p120-catenin and β or γ-catenin [82-84]. Furthermore, β-catenin will
interact with ɑ-catenin, which binds actin filaments [85-87]. Adherens junction complexes formed
between two cells connect epithelial and endothelial cells [88] (Figure 1.2). E-cadherin, N-
cadherin, and P-cadherin are classified as Type I, while VE-cadherins are Type II classical
cadherins, which have been well-studied in the context of cancer biology. In metastatic epithelial
tumors, the downregulation of E-cadherin compensated by the expression of other cadherins,
such as N-cadherin, is a hallmark of the epithelial–mesenchymal transition (EMT). This “cadherin
switching” enables tumor cells to acquire a metastatic phenotype that is different from the parental
population. Indeed, E-cadherin is considered a tumor suppressor as it inhibits transformation by
blocking β-catenin signaling [89]. Therefore, dysfunctional or decreased expression of E-cadherin
is associated with cancer progression and metastasis. However, cadherin switching is a late event
6
in tumorigenesis and is context-dependent (e.g., exposure to certain soluble factors or interaction
with specific ECM proteins) [90, 91].
1.5.1 VE cadherin
Although leukemia does not necessarily undergo EMT, E-cadherin expression is reduced
by hypermethylation of the E-cadherin gene promoter [92, 93], while VE-cadherin and N-cadherin
expression contributes to chemoresistance in BCR-ABL
+
(Ph
+
) ALL and CML [94, 95]. VE-
cadherin expression along with PECAM-1 expression in ALL enhances the adhesion of leukemia
cells to human brain-derived microvasculature endothelial cells (HBMECs) and their migration
through the HBMEC monolayer, suggesting a role of VE-cadherin in the central nervous system
(CNS) infiltrating leukemia [96]. Furthermore, stromal cells upregulate VE-cadherin expression in
BCR-ABL
+
leukemia cell lines(K562 and SUP-B15) and increase resistance to imatinib by
stabilizing β-catenin [97]. β-catenin is an important component in cadherin-mediated adhesion as
it bridges the cytoplasmic tail of cadherin to the actin cytoskeleton and stabilizes the adherent
junction. Since β-catenin binds to transcription factors to initiate transcription, cadherin adhesion
is often associated with the activation of Wnt/β-catenin intracellular signaling pathways [98]. A
subpopulation of the Ph
+
B-ALL cell line SUP-B15 presents leukemia stem cells (LSCs) and
expresses stem cell markers (CD34, CD38, and c-Kit) and endothelial antigens (Flk-1 and
PECAM-1); moreover, LSCs express VE-cadherin after a long-term co-culture on stromal cells
[99]. The overexpression of VE-cadherin on Ph
+
/VE-cadherin
+
LSC populations stabilizes β-
catenin, maintaining β-catenin as constitutively active and thus promoting self-renewal
independent of Wnt signaling. The same group later showed that VE-cadherin regulates apoptosis
in Ph
+
ALL [100].
7
1.5.2 N-Cadherin
Apart from VE-cadherin, N-cadherin is also associated with LSCs. In AML patients treated
with a HAD regimen of homoharringtoninetcytosine (HHT), cytarabine (Ara-C), and daunorubicin
(DNR), the N-cadherin and Tie2 expressing CD34
+
/CD38
−
/CD123
+
LSC population presented
higher expansion than AML patients who did not receive chemotherapy, indicating that the
expression of N-cadherin and Tie2 on AML cells provides a survival benefit against the therapy
[101]. In addition, the N-cadherin expressing cell line KG-1 and AML patient-derived bone marrow
mononuclear cells (BMMNCs) were able to form more colonies compared to an N-cadherin
negative control [28]. A higher percentage of N-cadherin
+
cells were shown to remain at the G0/G1
phases compared to N-cadherin
−
cells and showed higher engraftment in NOD/SCID mice
compared to the negative control. Indeed, N-cadherin
+
cell bearing mice had a significantly shorter
survival than the N-cadherin
−
engrafted mice. This suggests a role of N-cadherin in maintaining
the stem cell-like properties and survival of LSCs, which results in relapse. Mesenchymal stromal
cell (MSC)-N-cadherin adhesion in CML LSC also provides tyrosine kinase imatinib resistance by
stabilizing N-cadherin/β-catenin complex formation and the nuclear translocation of β-catenin in
concert with the activation of exogenous Wnt/β-catenin signaling [94]. When the N-cadherin-
mediated adhesion of CML cells to MSCs was interrupted with anti-N-cadherin short cyclic HAV
peptide (NCDH), the CML cells gained sensitivity toward imatinib treatment.
1.5.3 E cadherin
Another study supported E-cadherin as an important mediator for AML pathogenesis,
indicated by stalled differentiation accompanied with high proliferation [102]. In this study,
carbohydrate-binding protein lectin LecB induced differentiation of the AML cell line THP-1 and
increased apoptosis of cells in a dose and time dependent manner. LecB-induced differentiation
was mediated by increased autophagy and decreased cellular b-catenin levels, the balance of
8
which is a crucial factor for regulating the differentiation of AML cells. In differentiating cells, LecB
was in proximity to the membrane E-cadherin and further promoted the co-localization of E-
cadherin and b-catenin. Interestingly, fewer LecB treated THP-1 cells were suspended in the
supernatant, suggesting greater adhesion to the cell culture plate, but the direct association of E-
cadherin and adhesion was not investigated in this study.
Drugs developed to interrupt the interactions between malignant cells and the
microenvironment decreased cadherin expression on malignant cells as a secondary effect
without directly targeting cadherin. Recently, an adenosine analogue, Cordycepin, was proposed
as an anti-leukemia therapeutic adjuvant. Cordycepin’s anti-leukemic effect in U937 and K562
cells was achieved through the reduced attachment of leukemia cells to MSC by decreasing N-
cadherin expression on leukemia cells and vascular cell adhesion molecule-1 (VCAM-1) in MSCs
[103]. Targeting bone marrow endothelial cells (BMECs) with combretastatin, a microtubule
assembly inhibitor, increased AML cell dislodgement from BMECs [104]. Combretastatin
decreased the expression of VCAM-1 and VE-cadherin on BMECs, and dislodged AML cells
shifted G0/G1 to G2/M in their cell cycle. A combination treatment of combretastatin and cytotoxic
chemotherapy increased induction apoptosis in AML cells. Taken together, cadherins play an
important role in leukemia drug resistance. Particularly, chemotherapy-resistant leukemic stem
cell (LSC) populations utilize cadherin, which further stabilizes β-catenin and thus activates the
expression of genes important for self-renewal.
1.5.4 Targeting cadherins in leukemia
Cadherin inhibitors were developed based on compelling preclinical data and were
translated into clinical trials. A cyclic pentapeptide ADH-1 against N-cadherin is the most studied
cadherin inhibitor in cancer models. Thus far, this pentapeptide has been tested as a single agent
or in combination with conventional chemotherapy in patients with N-cadherin expressing solid
9
tumors [105-109]. Due to N-cadherin’s role in tumor metastasis, drug resistance and bone marrow
homing, N-cadherin has been proposed as a potential target to treat hematological malignancies
in patients [110] (Table1.1).
FX06 is a naturally occurring peptide derived from the Bb15-42 sequence of human fibrin that
is cleaved and released from the parental fibrin and competitively binds to VE-cadherin [111].
FX06 was evaluated in myocardial infarction, yet it failed to show significant baseline benefits
compared to a placebo-treated group, although the necrotic core zone significantly decreased
[112]. FX06 has not been tested in any cancer to date.
Celecoxib is a nonsteroidal anti-inflammatory drug that inhibits prostaglandin-endoperoxide
synthase 2, also known as COX-2. Interestingly, the effects of celecoxib were studied to treat
calcific aortic valve disease (CAVD) because of their cadherin-11 binding properties [113, 114].
Celecoxib was shown to promote anoikis by downregulating E-cadherin in osteosarcoma cell line
MG-63 by decreasing PI3K/Akt [115]. However, E-cadherin downregulation is a characteristic of
EMT in invasive tumors when accompanied with N-cadherin expression; thus celecoxib has been
proposed to induce EMT in ovarian cancer [116]. Despite different views on celecoxib, there have
been more than 100 related clinical trials in the U.S. for cancer patients, including two trials on
leukemia and hematological malignancies [117]. Celecoxib showed the inhibition of proliferation
and survival by downregulating β-catenin in Ph
+
CML [118], restoring imatinib sensitivity in
imatinib-resistant CML [119], and exerting an anti-tumor effect in the HL-60 AML cell line [120],
yet its relationship with cadherin-11 is not yet specified. As Wnt signaling has been shown to be
an aberrantly upregulated pathway in leukemia [121] and is involved with chemoresistance, this
warrants further exploration of celecoxib as a viable therapy to reverse CAM-DR in leukemia.
1.6 Selectins in CAM-DR in leukemia
Selectins (CD62) are single-chain transmembrane glycoproteins that mediate calcium-
dependent carbohydrate-binding. There are three major types of selectins: L-selectins are majorly
10
expressed on leukocytes, E-selectins are expressed on endothelial cells, and P-selectins are
expressed on activated platelets [122, 123]. Selectins share common structures: (1) Calcium-
dependent lectin domain, (2) an epidermal growth factor (EGF)-like domain, (3) a variably-sized
repeated region, (4) a transmembrane domain, and (5) a cytoplasmic domain [124]. The main
function of selectins is to facilitate leukocyte tethering and rolling along endothelial cells, which is
an initial step of the transmigration of leukocytes through the endothelial barrier. Briefly, free
floating cells expressing selectin ligands, such as P-selectin glycoprotein ligand-1 (PSGL-1), bind
to P-selectin expressing endothelial cells. Upon engagement, the leukocyte movement will slow
down and the cells remain in proximity to the vessel wall, while integrin-ICAM/VCAM-1
interactions and other cytokine-mediated tight adhesions between leukocytes and endothelial
cells strengthen the binding for transmigration. As a result, leukocytes can travel to distant sites
of inflammation, and hematopoietic stem cells (HSCs) can home into the bone marrow [125].
Because the endothelium binding of leukocytes is a prerequisite of metastasis, selectins
are well-known to be involved in cancer progression [126, 127]. E-selectin expressed on the
endothelium is the primary source of binding partners for leukocytes, and T-ALL cells were not
able to adhere to interleukin-2 activated human umbilical vein endothelial cells (HUVEC) upon E-
selectin inhibition with a monoclonal antibody [128]. E-selectin expression in the BM vascular
niche has been proposed to be regulated by Runt-related (RUNX) transcription factor, and RUNX
silencing was shown to downregulate E-selectin expression and lead a subsequent decrease in
AML engraftment in the BM in mice [129].
Leukemia cells interact with E-selectin through various ligands such as CD43, CD44, and
PSGL-1. Particularly, myeloblasts favor PSGL-1 for interactions with endothelial selectins, while
lymphoblasts express less PSGL-1. PSGL-1 expressed on the surface of the leukemia cells can
bind to P-selectin along with E-selectin on endothelial cells [130]. In contrast, lymphoblasts mainly
use CD43 and/or CD44 to bind to endothelial selectins [131]. Therefore, even though specific
ligands are preferentially used in different cells, this does not mean that other molecules are less
11
crucial in conferring CAM-DR. Indeed, myeloblasts use PSGL-1, CD44, and CD43 to various
extents during E-selectin binding. Therefore, different patients show different profiles of E-selectin
ligand expression levels. Interestingly, nilotinib treatment upregulated E-selectin, ICAM-1, and
VCAM-1 expression on human endothelial cells [132], which may result in the increased
adherence of leukemia cells to E-selectin and the evasion of chemotherapy-induced cytotoxicity.
In fact, a high baseline level of soluble E-selectin along with VEGF, PAI-1, and low initial soluble
ICAM-1 were proposed as prognostic factors for poor outcomes in pediatric ALL [133]. Leukemia
itself can express selectins on the surface to promote migration and progression. Human BCR-
ABL1 (p210) retrovirus transduced murine leukemia expressed integrin subunit alpha-6 and L-
selectin, which were used to metastasize into the central nervous system, predominantly in
meninges [134].
1.6.1 Targeting selectin in cancer
Currently, selectin inhibition is actively being translated into leukemia treatments (Table 1.2).
GMI-1271 (Uproleselan), an E-selectin antagonist, is intended to inhibit E-selectin expression on
endothelial cells so that E-selectin-mediated drug resistance in leukemia can be prevented.
Preclinical investigations of GMI-1271 in multiple myeloma (MM) showed the sensitization of E-
selectin ligand rich in MM toward bortezomib [135]. GMI-1359, a dual E-selectin and CXCR4
inhibitor, significantly decreased bone metastasis, synergized the docetaxel effect in prostate
cancer cells [136], and sensitized MM toward carfilzomib [137]. Currently, GMI-1271 is being
investigated for its safety and efficacy in AML patients (Table 1.3). Crizanlizumab (Adakveo) is a
monoclonal antibody against P-selectin, which is expressed on the surface of the activated
endothelium and platelets. Crizanlizumab is used to reduce vaso-occlusive crises (VOC) in adult
and pediatric patients with sickle cell disease (SCD). Clinical trials are open for dose confirmation
and safety in both adult and pediatric SCD patients to evaluate the safety and efficacy on SCD-
12
related complications along with a combination study of myelofibrosis with ruxolitinib. YSPSL
(rPSGL-lg), a P-selectin glycoprotein ligand IgG fusion protein, binds to P-selectin and was
evaluated in ischemia-reperfusion injury, liver disease, and kidney functions but has not been
tested in cancer [138].
GMI-1271 is currently the only selectin inhibitor in clinical trials and is being investigated for
its safety and efficacy in AML patients (Table 3). There is increasing evidence in support of the
importance of targeting the BM microenvironment due to its role in therapy resistance [139-141].
In these clinical trials, GMI-1271 will be administered in combination with existing chemotherapies,
which highlights how targeting both the microenvironment and leukemia cells may be necessary
in order to ameliorate CAM-DR.
1.7 Integrins in CAM-DR in leukemia
Integrins are calcium independent type-I transmembrane proteins with a shared structure
of the extracellular domain, the transmembrane domain, and the cytoplasmic domain. Composed
of 18 alpha (a) and 8 beta (b) subunits, integrins participate in cell–cell or cell–ECM adhesion. To
date, 24 integrin heterodimers with different combinations of a and b subunits are known and can
interact with their ligands in arginine–glycine–aspartic acid (RGD) sequence dependent and
independent manners [142, 143] (Figure 1.3). Figure 1.3 does not represent a comprehensive list
of integrin heterodimers but rather includes integrins described by currently available publications
in the field of leukemia, which are reviewed in this review. During “inside-out” signaling, binding
of talin to the cytoplasmic tail of the β subunit increases integrins’ affinity toward their ligands by
undergoing conformational changes from low-affinity to high-affinity state [144, 145]. On the other
hand, binding of a ligand or a counter-receptor to a specific domain on the α subunit or the αβ
heterodimer results in spatial separation of cytoplasmic tails of α and β subunits. This event allows
adaptor proteins such as talin and vinculin to engage with the β tail and associate with the
13
cytoskeleton and form a protein complex called focal adhesion and integrin clustering [146-148]
(Figure 1.4). Integrins can be internalized and recycled, thus controlling availability of integrin
heterodimers on the plasma membrane [143, 149]. These processes will translate external stimuli
and environmental cues into intracellular signaling and mediate adhesion, cell spreading,
migration, proliferation and survival in cells. Integrin-dependent adhesion to ECM can convert
mechanical forces into biochemical signals, allowing cells to recognize biophysical properties of
given BM microenvironment [150]. Few studies analyze redundancy between integrins in CAM-
DR, and how it might affect integrin targeting. Future studies will need to address this gap of
knowledge. Here, we summarized integrins by name.
1.7.1 Integrin a 1 (CD49a)
Integrin alpha 1 subunit forms a heterodimer with the integrin beta 1 subunit to form a1b1,
which binds to collagen and laminin [151, 152]. a1b1 is also called very late antigen 1 (VLA-1)
because it is expressed on the surface of long-term activated T cells [153]. VLA-1 mediates the
adhesion of intraepithelial lymphocytes (IELs), such as the CD8
+
T cells found in the intestinal
epithelium, to collagen [154]. a1 was also suggested to be a potential marker for stromal cells and
is expressed in more than 80% of human derived non-transformed bone marrow stroma cells. In
this study, only a1 expressing stroma precursors was able to give rise to colony-forming unit-
fibroblasts (CFU-F) compared to the a1 negative subgroup of the stromal population, suggesting
a1 as a marker for stromal precursor cells [155]. Embryonic fibroblasts derived from a1 deleted
mice were not able to spread or migrate to either collagen IV or laminin, suggesting their
importance in cell spreading and migration [156]. However, the role of VLA-1 in CAM-DR in
leukemia is still unknown.
14
1.7.2 Integrin a 2 (CD49b)
Integrin alpha 2 forms a heterodimer with the beta 1 subunit to form a VLA-2 molecule that
binds to collagen and laminin [157]. A real-time quantitative polymerase chain reaction study on
134 de novo AML patients revealed higher ITGA2 expression in AML patients compared to the
33 normal controls [158]. Moreover, ITGA2
high
patients had significantly lower complete remission
(CR) rates and a shorter overall survival compared to the ITGA2
low
groups. ITGA2 expression
decreased significantly in the patients who achieved CR but increased again in relapsed patients,
suggesting that ITGA2 is a marker for a poor prognosis in AML. The a2b1 mediated adhesion of
the T-ALL cell lines Jurkat and HSB-2 and the primary T-ALL blasts toward collagen I decreased
doxorubicin induced apoptosis [159]. a2b1 mediated adhesion activates the MAPK/ERK pathway,
which inhibits the doxorubicin-induced activation of c-Jun N-terminal kinase (JNK) and maintains
the pro-survival protein Bcl-2 family member Mcl-1. The same group extended a2b1-collagen
mediated doxorubicin resistance in the AML cell lines HL-60 and U937 [160]. In AML, collagen
binding through a2b1 inhibited the activation of the pro-apoptotic protein Rac1, thereby preventing
Rac1 induced DNA damage.
1.7.3 Integrin a 3 (CD49c)
VLA-3 interacts with ligands in both an RGD-dependent and RGD-independent manner.
VLA-3 mediated adhesion to fibronectin requires RGD-motif recognition, whereas binding to
collagen and laminin does not require an RGD-motif in the ligands [161]. VLA-3 binding to
fibronectin increased in the presence of the Mg
2+
and Mn
2+
−divalent cations required for integrin
activation, whereas binding to collagen and laminin was less affected. Integrin-a3 was identified
as a marker for long-term repopulating hematopoietic stem cells (LT-HSCs) that expand from
15
CD34+ human cord blood cells and retain their self-renewal ability with a long-term engraftment
pattern compared to short term HSCs (SC-HSCs) [162]. Furthermore, ITGA3 knockdown with
short hairpin RNA against ITGA3 did not affect the stemness of the cells but decreased the long-
term reconstitution ability in NSG mice. However, the specific role of VLA-3 in CAM-DR in
leukemia is still unclear.
1.7.4 Integrin a 4 (CD49d)
Integrin α4 binds with either the b1 or b7 subunit to form α4β1 or a4b7 heterodimer. Integrin α4 exerts
physiological effects including cell adhesion and migration, while triggering intracellular signaling,
thereby indicating the promotion of leukemia cell drug resistance and survival. Integrin-a4
knockout mice are embryonically lethal [163]. There are specific relationships between integrin
α4, epigenetics, metabolism, and cell surface markers. Histone deacetylase inhibitor treatment
may downregulate VLA-4 for various AML cell lines, primary patient samples, and normal
hematopoietic stem cells [164]. The expression of G9a, a histone methyltransferase related to
gene silencing, correlates with integrin α4 expression in pediatric B- and T-ALL. Furthermore, G9a
depletion or inhibition with BIX01294 was shown to abrogate the ability of ALL cell migration
towards the endothelial monolayer [165]. Moreover, it was recently reported that tetraspanin
(CD9)
+
B-ALL is associated with a poor prognosis. In this study, CD9 physically interacted with
VLA-4 and mediated the affinity to VCAM-1. CD9 inhibition interrupted the leukemia–stroma
interactions and sensitized B-ALL cells to chemotherapy [166]. CD98 has been shown to interact
with the cytoplasmic domains of β1 and β3 and mediate the adhesive signaling of integrin
α4/VCAM-1 in AML [167]. The redox modulation of adjacent thiols in VLA-4 by AS101, an IL-1β
converting enzyme, restored the chemosensitivity of AML cells by decreasing PI3K/Akt/Bcl2
signaling [168]. It has been demonstrated that integrin α4 and α5 are involved in Jurkat T-ALL
adhesion-independent chemoresistance [169]. Our studies also showed that both deletion and
16
inhibition with natalizumab of integrin α4 sensitize primary B-ALL cells to chemotherapy [170].
Furthermore, the CD49d antisense drug ATL1102 efficiently downregulated the CD49d mRNA
level of B-ALL in vitro [171]. Anti–VLA-4 antibodies (SG/73, SG/17) were shown to increase
chemosensitivity in human AML cells and eradicate minimal residual disease (MRD) in
experimental mice when combined with chemotherapy [8]. Integrin α4 has been shown to be a
prognostic marker of poor overall survival in B-ALL [170]. Interestingly, a report from the Children's
Oncology Group found that high VLA-4 expression is associated with a better clinical outcome in
pediatric AML and is an independent predictor of relapse [172]. Similar results were found in a
study of the Southwest Oncology Group trials [173].
1.7.5 Integrin a 5 (CD49e)
The integrin α5 subunit can dimerize with integrin β1 to form a5b1 (VLA-5), which binds to the
RGD sequence in fibronectin [174]. Both murine and CD34
+
human HSCs were shown to bind to
a recombinant peptide of the VLA-5 binding RGD-motif of fibronectin in vitro [175]. The
preincubation of B6.Hbb
d
/Hbb
d
, Gpi-1
a
/Gpi-1
a
mice-derived BM cells incubated with the
fibronectin binding domain including the peptide CH-296 decreased the engraftment of BM cells
in recipient mice, and the intravenous injection of the CH-296 peptide caused an increase in the
percentage of progenitor cells in the spleen, suggesting the importance of VLA-5 in HSC
engraftment in the BM. VLA-5 has been suggested as a therapeutic target in leukemia. A subset
of ALL includes an alteration in the IKAROS gene, which is correlated with a poor prognosis. The
exon 5 deletion of Ikzf1 in pre-B cells arrests the cells in an “adherent phase”, where survival and
proliferation depend on stable adhesion to the stroma with increased Erk1-2 MAPK activity [176].
The expression of the dominant-negative Ikaros isoform IK6 in the T-ALL (Jurkat) and B-ALL cell
lines (RS4;11, Nalm6) lifted the transcription suppression of FUT4, which fucosylates α5β1 on
leukemia cells and tightens the adhesion of ALL cells to fibronectin in the ECM. This increased
17
adhesion was achieved via activation of the FAK/Akt pathway upon Lewis X (Le
X
, CD15 or SSEA-
1) modification of α5β1 [177]. In U937 and blasts from AML patients, a4b1 and a5b1 mediated the
adhesion of cells to fibronectin, and the addition of the Wnt antagonist sFRP induced resistance
to daunorubicin 16407823 [178]. Both adhesion and the Wnt pathway contribute to
chemoresistance in AML and require the activation of glycogen synthase kinase 3b (GSK3b).
Upon serum starvation of AML U937, VLA-5 binding to fibronectin regulates specific pro-survival
functions through the activation of GSK3b [179]. VLA-5 inhibition with an anti-integrin a5 antibody
sufficiently decreased adhesion of the Ph
+
ALL cell line SUP-B15 to fibronectin, while a
combination of VLA-5 inhibition with imatinib synergistically increased apoptosis in SUP-B15 cells
in vitro [180]. Furthermore, the inhibition of VLA-5 with disintegrin, an antibody, or knocking down
integrin-a5 impaired the engraftment of SUP-B15 cells in immunodeficient mice. A combination of
integrin-a5 inhibition with the FAK inhibitor TAE226 prolonged the survival of SUP-B15 engrafted
mice, suggesting that the inhibition of VLA-5 combined with conventional chemotherapy may
improve the outcome for Ph
+
ALL patients.
1.7.6 Integrin a 6 (CD49f)
Integrin a6 dimerizes with b1 to form VLA-6 [181] or with b4 to form a6b4, which is also known
as TSP-180 [182]. a6 has been suggested to be a biomarker for minimal residual disease since it
is expressed on pre-B-ALL at diagnosis, and the signal is preserved or expressed with a higher
intensity after therapy [12, 183]. a6 was found to be expressed significantly more strongly not only
in relapsed B-ALL but also in ecotropic viral integration site-1 positive(EVI1
+
) AML cases. In this
study, the drug sensitivity of EVI1 AML cells was restored after the inhibition of integrin a6 [184].
Functionally, a6 is suggested to play an important role in the chemoresistance and metastasis of
leukemia cells. EVI1
+
AML cell lines and primary cells were able to bind to laminin better than cell
lines with low EVI1. This adhesion is specifically mediated by ITGA6 and ITGB4 expression on
18
EVI1
+
AML cells, and small-hairpin RNA against EVI1 decreased the expression of ITGA6 and
ITGB4. Moreover, the inhibition of ITGA6 or ITGB4 with neutralizing antibodies restored
chemosensitivity against Ara-C in EVI
+
AML cells. In another study, a6 on the surface of ALL was
shown to facilitate the invasion of ALL cells into the central nervous system by binding to laminin
during the process of migration toward the cerebrospinal fluid [185].
Since a high expression of integrin α6 was found on day 29 of an MRD test on B-ALL in the
Children’s Oncology Group (COG) P9906 clinical trial, we proposed the drug resistance role of
integrin α6. Firstly, we showed that the integrin α6 blockade de-adhered the B-ALL cell from
laminin-1 and OP9 stromal cells. Secondly, P5G10, an anti-integrin α6 antibody, in combination
with chemotherapy, prolonged the survival of B-ALL xenograft mice. Thirdly, integrin α6 deletion
induced apoptosis of B-ALL cells involving Src signaling [186]. Recently, it has been shown that
the inhibition of integrin-a6 is correlated with decreased cell surface deformability using single-
beam acoustic tweezers, while no changes in inhibition were shown for integrin α4 [187].
1.7.7 Integrin a 7 (ITGA7)
Integrin-a7 binds with b1, which is expressed on skeletal and cardiac muscle [188-191].
ITGA7 was more significantly increased in AML patients with granulocytic sarcoma (GS)
compared to patients with GS. Furthermore, integrin-a7 mediated the phosphorylation of ERK in
the surface integrin-a7 expressing AML cell lines PL21 and THP1, thus promoting the proliferation
of these cells [192]. ITGA7 also has been suggested to be a biomarker for AML patients as ITGA7
expression in AML patients was significantly increased compared to the control and correlated
with a poorer prognosis. Patients with either high ITGA7 mRNA or protein expression had shorter
event-free survival (EFS) and overall survival (OS) compared to low ITAG7 patients [193].
19
1.7.8 Integrin a 9 (ITGA9)
Integrin-a9 dimerizes with the b1 subunit to form an a9b1 that is distributed in the airway
epithelium, squamous epithelium, smooth and skeletal muscle, and hepatocytes [194]. a9b1
recognizes tenascin-C [195], osteopontin [196], and VCAM-1 [197]. While a9b1 shares nearly 40%
of its amino-acid sequence homology with integrin-a4, both have distinct functions. Integrin-a9
knockout mice develop bilateral chylothorax and die 6 to 12 days after birth due to respiratory
failure [198]. The roles of integrin-a9 in the context of leukemia have not been elucidated. Recently,
the dual inhibition of a4b1 and a9b1 with BOP ((N-(benzenesulfonyl)-L-prolyl-L-O-(1-
pyrrolidinylcarbonyl)tyrosine, a small molecule antagonist against integrin a4b1 and a9b1)
demonstrated the successful HSC mobilization potential from the bone marrow to the peripheral
blood. While a single dose of BOP increased the mobilization of hCD45
+
CD34
+
cells by about 2
fold compared to the saline control, a combination of BOP with the CXCR4 inhibitor AMD3100
increased the mobilization of HSC by three fold [199]. This result suggests that a9b1,
concomitantly with a4b1, is involved in the integrin mediated adhesion of HSCs in the bone marrow.
Indeed, CD34
+
CD133
+
hematopoietic stem and progenitor cells (HSPCs) expressed a9
transcripts and a9b1 on the surface. Integrin-a9 mediates the adhesion of CD34
+
cells to
osteoblasts, and the addition of functional blocking antibody against a9b1 and Y9A2 significantly
decreased the proliferation and differentiation of CD34
+
HSPC cells [200].
1.7.9 Integrin a L (CD11a, LFA-1)
Integrin-aL dimerized with a b2 subunit is called lymphocyte function-associated antigen-1
(LFA-1). In an early study using T cells derived from leukocyte adhesion deficient (LAD) patients
with genetic defects in b2 showed a decreased expression of LFA-1 and LFA-1 LAD derived T
cells still bound to endothelial cells similar to normal T cells via complementary binding through
20
VLA-4, but their transmigration through the endothelial layer of LAD derived T cells was
significantly decreased [201]. In hematological malignancies, T-cell neoplasms, including T-ALL,
almost always express LFA-1, while LFA-1 expression in lymphoma and B-cell neoplasms,
including T-ALL, CLL, HCL, and SLL, vary between patients [202, 203]. LFA-1 on the T-ALL cell
line (Sup T1 and Jurkat) and primary T-ALL were shown to play a critical role in binding T-ALL
cells to the BM stroma (HS-5 and patient derived BM) and regulating the survival of T-ALL cells
[204].
1.7.10 Integrin a M (CD11b)
The integrin-aMb2 heterodimer is called Mac-1 and is known to bind to fibrinogen [205],
platelet factor 4 [206], and ICAM-1 [207]. The expression of Mac-1 was suggested to be a
biomarker for a poor prognosis [208]. The Mac-1 mediated adhesion of U937 and HL-60 cells to
plastic was suggested to elicit a survival benefit in leukemia cells treated with phorbol ester, and
these Mac-1 mediated adherent cells are susceptible to undergo anoikis when forced to be de-
adhered, suggesting adhesion dependent survival.
1.7.11 Integrin a V (CD51, VNRA, MSK8)
Integrin-aV can dimerize with b1, b3, b5, b6, and b8. Heterodimers including aV can bind to
fibronectin [209] and vitronectin [210, 211]. In AML, aVb3 is suggested to cooperate with the
fibroblast growth factor receptor (fgf-R) to increase proliferation, especially the subset of AML that
has Hox-overexpression induced by MLL fusion protein [212]. In this study, the MLL-ELL
transduction of primary murine bone marrow cells increased the expression of b3 integrin via
HoxA10, and the aVb3-mediated adhesion of cells to vitronectin increased Syk, Pak1, and Fak1.
aVb3 activity was reversed through the b-catenin and Cdx4 dependent decrease in ITGB3
promoter activity upon fgf-R inhibition in these cells.
21
1.7.12 Integrin β1 (CD29)
Integrin b1 is the most common beta subunit heterodimer partner for integrin alpha
subunits [213]. In cancer, upregulated expression of β1 is indicative of a poor prognosis [214] and
plays roles in chemoresistance by binding to ligands and eliciting downstream signaling events.
Berrazouane et al. reported that b1 promotes chemoresistance in T-ALL primary blasts via ABC
transporter-mediated doxorubicin efflux and the downstream activation of PYK2 [215]. Integrin
a2b1 binds to collagen and upregulates ABCC1 via the ERK/MAPK pathways to modulate efflux
[216]. Similarly, collagen-binding b1 integrins contribute to doxorubicin resistance in AML by
reducing DNA damage through Rac1 inhibition [217].
β1 also has roles in apoptosis inhibition. Estrugo et al. demonstrated that leukemia cell lines
HL60 and Jurkat adhere to b1 integrin ligands fibronectin, laminin, or collagen-1 and are protected
from radiation, Ara-C, and FasL-induced apoptosis [218]. These b1 integrin-mediated cell-matrix
interactions inhibit procaspase-8 activation via the PI3K/AKT pathway. Additionally, b1 integrin
ligation to fibronectin impairs both procaspase-3 and procaspase-9 activation associated with the
intrinsic apoptotic pathway.
The tetraspanin superfamily is known to be associated with the activation, ligand binding,
and inside-out signaling of β1 integrins and can promote cancer cell survival [219, 220]. When b1
is expressed with tetraspanin CD82, chemoresistance is promoted by increasing PKCα activation
and the downstream clustering of b1 integrin, leading to AML cell survival via the activation of p38
MAPK for DNA damage repair [221]. In summary, integrin b1 is implicated in the chemoresistance
of leukemia via chemotherapy efflux, intracellular signaling, and apoptosis inhibition.
22
1.7.13 Integrin β3
It has been suggested that integrin b3 may have functional redundancies with b1 integrins
[222]. In AML, b3-mediated signaling is required for leukemogenesis and leukemia survival [223],
in part through SYK activation. To date, the function of b3 in the chemoresistance in leukemia has
not been studied. In hepatocellular carcinoma, the upregulation of Galectin-1, which elevates
aVb3expression, was found to activate the PI3K/AKT pathway and is correlated with a poor
sorafenib response [224]. The antagonism of b3 with cilengitide in M2 macrophages led to the
promotion of tumor cells, and the loss of integrin b3 signaling promoted an immunosuppressive
tumor environment [225]. Clearly, b3 signaling is important in drug response and cancer
progression, which may be grounds for similar studies on leukemia.
1.7.14 Integrin β7
Integrin b7 is present on lymphocytes as a subunit of the a4b7 heterodimer and mediates
binding to fibronectin, VCAM-1 [226], and mucosal addressin cell adhesion molecule 1 (MAdCAM-
1) [227]. a4b7 is less well studied in the context of leukemogenesis and drug resistance and is
mainly involved with lymphocyte homing and trafficking. In hematopoietic progenitor cells, a4b7
and MAdCAM-1 contribute to the recruitment of cells into the bone marrow following
transplantation, and the inhibition of MAdCAM-1 significantly reduces homing [228]. For blood
cancers, it has been suggested that the expression of a4b7 plays a role in the leukemic evolution
of T cell lymphoblastic lymphomas and the dissemination of lymphoma cells to VCAM-1-positive
vascular spaces [229]. In T cell leukemias with gastrointestinal involvement, it was found that the
expression of a4b7 is linked with homing to MAdCAM-1 on endothelial cells in the intestinal
mucosa [230]. In summary, while a4b7 may not be involved with leukemogenesis, its roles in
lymphocyte homing have effects on the progression of leukemia in different organs.
23
1.7.15 Targeting integrins
The preclinical evaluation of integrin inhibition has suggested promising results for the
sensitization of leukemia cells to chemotherapy. Knocking out ITGA4 restored the sensitivity of
BCR-ABL
+
murine leukemia toward nilotinib (NTB), and a blockade of integrin alpha 4 with a
monoclonal antibody sensitized primary B-ALL engrafted xenograft mice to chemotherapy [170].
Since the b1 (CD29) subunit dimerizes with many different a units, blockade of b1 is an attractive
target for leukemia therapy. In T-ALL, the b1 blockade with b1 specific antibody AIIB2 inhibited
cell-matrix interactions and decreased the Matrigel effect on T-ALL colony formation. Furthermore,
AIIB2 in combination with doxorubicin significantly prolonged the survival of CEM xenograft
mouse models [215]. OS2966, a humanized monoclonal antibody, will be used in a phase I clinical
trial for glioblastoma and may also have efficacy in the treatment of hematological malignancies
by targeting multiple integrins on leukemia cells and the surrounding microenvironment [231]. As
for other integrins, targeting the active form of the integrin β7 subunit, specifically the MMG49
epitope in the N-terminal region of active β7, showed multiple anti-myeloma effects in vivo without
damaging normal hematopoietic cells [232]. The efficacy of chimeric antigen receptor (CAR) T
cells against αvβ3 in melanoma and αvβ6 in ovarian, breast, and pancreatic xenograft mice
models has also been evaluated [233, 234] for integrin targeting CAR T therapy in hematological
malignancies.
Despite the compelling in vitro and in vivo anti-tumor effects of integrin blockades in tumor
models, the preclinical evaluation of integrin targeting has not yet been successfully translated
into a clinical platform. Several clinical trials evaluating integrin inhibition in solid tumors were
terminated due to infusion-related reactions and non-significant anti-tumor activity (NCT00915278)
[235], insufficient clinical data (NCT00684996), or low enrollment (NCT00675428). A phase II trial
of abituzumab (EMD 525797) targeting αv in combination with cetuximab and FOLFIRI in
metastatic colorectal cancer is expected to be completed by August 2021 (NCT03688230).
24
Integrin targeting is useful for the detection of cancers, and many clinical trials target integrin
in the CT/PET imaging of cancer patients. A novel radiotracer 99mTc-RWY detecting integrin
alpha 6 is in an early phase I clinical trial for SPECT imaging in breast cancer (NCT04289532),
and the safety of the radiotracer and potential clinical applications are being evaluated. Likewise,
many other types of integrin tracing molecules are being evaluated for their efficacy in imaging
cancer patients (NCT04285996, 02807945, 02749019).
Although the inhibition of integrin has not yet been successfully translated into a clinical trial
for leukemia, integrins remain a valid target for cancer therapy, as they can serve as a targetable
biomarker. Targeting the active form of the integrin β7 subunit, specifically the MMG49 epitope in
the N-terminal region of active β7, showed multiple anti-myeloma effects in vivo without damaging
normal hematopoietic cells [232]. The efficacy of CAR T cells against αvβ3 in melanoma and αvβ6
in ovarian, breast, and pancreatic xenograft mice models has also been evaluated [233, 234] for
integrin targeting CAR T therapy in hematological malignancies.
In summary, integrin blockades must be further investigated using preclinical systems that
can accurately recapitulate the biological environments in patients, thus allowing the integrin
blockade to exert anti-tumor effects [236]. To overcome this challenge, integrins can serve as a
good target for tumor imaging in patients or for immunotherapy, including CAR T therapy. As
discussed in this section, integrin blockades have been shown to increase chemosensitivity of
leukemia cells and provide support for further studies of integrins as a viable target to abolish
CAM-DR in leukemia.
1.8 Conclusion
Despite advances in cancer therapy and the increased overall survival rate of cancer
patients, the eradication of leukemia still remains a challenge. Pediatric ALL has a good prognosis
overall, yet relapse and refractory disease remain a problem. AML has a worse prognosis, and
there is an unmet need for the improvement of patient outcomes. The impact of the
25
microenvironment on cancer cell progression and drug resistance has been often neglected, yet
it is apparent that leukemia cells actively communicate and interact with the surrounding
microenvironment for their survival. Therefore, disrupting the interactions between leukemia cells
and the surrounding cells or ECM protein may lead to apoptosis or sensitization toward
chemotherapy. Cadherins, selectins, and integrins are known cell adhesion molecules that are
involved in CAM-DR in leukemia. Their aberrant expression and association in CAM-DR in
different types of leukemia have been studied. Furthermore, a preclinical evaluation of the efficacy
of the CAM blockade was performed on subtypes of leukemia and showed promising results with
an anti-leukemic effect. The FDA has granted a Breakthrough Therapy designation and Fast
Track designation for the E-selectin inhibitor Uproleselan, which shows both the urgency of finding
an effective drug for leukemia treatment and the importance of microenvironment–leukemia
interactions in leukemia treatment. The translation of more CAM inhibitors into a clinical platform
will advance leukemia therapy and eradication of the disease.
Table 1.1. Description of cadherin inhibitors
Drug name Description Indication References
ADH-1 N-cadherin inhibitor Solid tumors [106-108]
FX06 Competitive inhibitor of
fibrin E1 binding to VE-
cadherin
Cardiac reperfusion injury
and myocardial infarction
[112, 237]
Celecoxib COX-2 inhibitor Evaluated for anti-cancer
effects by binding to
cadherin-11 and
regulating E-cadherin
expression
[238-240]
Table 1.2. Description of selectin inhibitors.
Drug name Description indication References
GMI-1271
(Uproleselan)
E-selectin inhibitor Small molecule inhibitor
against E-selectin on
endothelial cells to treat
AML and potentially other
hematologic cancers
[30]
26
GMI-1359 E-selectin/ CXCR4
dual inhibitor
Targeting E-selectin and
CXCR4 to reduce tumor
metastasis to bone
marrow
[136, 137]
Crizanlizumab
(Adakveo)
Monoclonal
antibody against P-
selectin
Reduction of vaso-
occlusive crises in sickle
cell disease patients
[241]
YSPSL (rPSGL-lg) Recombinant P-
selectin
glycoprotein ligand
IgG fusion protein
Myocardial infarction, red
blood cell disorders,
anemia, transplant,
ischemic-reperfusion
injury
[242, 243]
Table 1.3. Clinical trials for selectin inhibitors in leukemia.
Drug Target Condition
or disease
Phase Intervention
/treatment
References
GMI-1271 E-selectin AML I/II Evaluation of GMI-
1271 treatment
combined with
mitoxantrone,
etoposide, cytarabine
and idarubicin in AML
patients
NCT023062
91
GMI-1271 E-selectin Relapsed/
refractory
AML
III Efficacy of
uproleselan (GMI-
1271) in combination
with mitoxantrone,
etoposide and
cytarabine (MEC) or
fludarabine,
cytarabine and
idarubicin (FAI) in
relapsed/refractory
AML patients
NCT036164
70
GMI-1271 E-selectin AML (adults
60 years
and older)
II/III Evaluation of
uproleselan
combined with
cytarabine or
daunorubicin in older
AML patients
receiving intensive
induction
chemotherapy.
NCT037013
08
Table 1.4. Description of integrin inhibitors
Drug name Description Indication References
Natalizumab
(Tysabri®)
Monoclonal antibody
against α4
Multiple sclerosis and Crohn’s
disease
[170]
27
Vedolizumab
(Entivio)
Monoclonal antibody
against α4β7
severe ulcerative colitis or Crohn's
disease
[244, 245]
Volociximab Monoclonal antibody
against integrin α5β1
Solid tumors including kidney,
lung, ovarian cancer, melanoma,
and pancreatic cancer
[246-249]
ATN-161 non-RGD based peptide
targeting α5β1 and ανβ3
Solid tumors including prostate,
colon, and hepatocellular cancers
[250-252]
Intetumumab
(CNTO95)
human αν monoclonal
antibody
Inhibition of tumor growth [231, 253]
Etaracizumab
(MEDI-522)
Monoclonal antibody
against ανβ3
Psoriasis, kidney cancer [254, 255]
Abituzumab
(EMD525797)
Monoclonal antibody
against ανβ6
Metastatic prostate cancer [256, 257]
Cilengitide
(EMD 121974)
first anti-angiogenic
small molecule targeting
the integrins αvβ3, αvβ5
and α5β1
Inhibition of endothelial cell–cell
and cell–ECM interactions and
angiogenesis
[258-261]
GLPG0187 a small molecule
inhibitor for ανβ1, ανβ3,
ανβ5, ανβ6 and α5β1
Solid tumors including high-grade
gliomas and colorectal carcinoma
[262-264]
OS2966 Humanized monoclonal
antibody against β1
integrins
Glioblastoma, meningioma, ALL,
and AML
[231, 265]
28
Figure 1.1 BM microenvironment BM includes many types of cellular and non-cellular
components. Cellular components express ligands or counter receptors, such as VCAM-1 or
ICAM, that will bind to CAMs. Cells can also secret extracellular matrix (ECM) proteins that will
bind to CAMs.
29
Figure 1.2 Cadherin and adherens junction. Upon engagement in homotropic manner,
cytoplasmic tail of cadherins will bind to actin through p120, b-catenin and a-catenin protein
complex. Cadherin mediated protein complex formation is observed at adherens junction where
adjacent cells are connected to each other.
30
Figure 1.3. Dimerization of integrins in leukemia. Dimerization of a and b integrins forms a
functional heterodimer unit. This figure does not represent a comprehensive list of integrin
heterodimers but rather includes integrins described by currently available publications in the field
of leukemia, which are reviewed in this thesis
31
Figure 1.4. Integrin signaling in leukemia Talin binding to cytoplasmic tail of b-subunit activates
integrin heterodimer and increases affinity of the complex towards ligands. Activation of integrin
is followed by conformational change of the heterodimer and separation of cytoplasmic tails of
each subunit, allowing recruitment of proteins. Recruited proteins, such as kindlin, paxillin, FAK
and Src forms a protein complex that initiates integrin mediated intracellular signaling that results
in cell adhesion, migration, survival and mechanotransduction of leukemia cells.
32
Chapter 2. PI3Kdelta and BRD4 as targets in Chemotherapy Resistance
of Leukemia
2.1 Introduction
Despite a high five-year survival rate, relapsed and refractory B-cell acute lymphoblastic
leukemia (B-ALL) remains a problem in children [14] the and the prognosis for adult B-ALL
patients is poor [266]. During treatment, leukemia cells interact with the bone marrow (BM)
microenvironment and obtain a survival benefit, known as cell adhesion-mediated drug resistance
(CAM-DR) [45]. This drug resistance in B-ALL can be achieved by increased pro-survival
intracellular signaling as a result of adhesion to the BM microenvironment. The PI3K-AKT
pathway has been identified as one of the most significant pro-survival pathways in CAM-DR and
leukemia cell-BM stromal cell contact has been shown to upregulate phosphorylated AKT in B-
ALL [267]. Despite great interest in inhibition of the AKT pathway via targeting PI3K isoforms in
leukemia, a clinically available drug for B-ALL treatment remains elusive [267-269].
In addition, PI3K inhibition facilitates degradation of the transcription factor MYC through
the GSK-3β-dependent MYC phosphorylation pathway [270]. Emerging reports have indicated
oncogenic protein c-Myc plays a critical role in survival, proliferation, and drug resistance in both
B and T-ALL [271-274]. However, direct targeting of Myc has been a challenge due to its
"undruggable" protein structure [275]. Currently, targeting c-Myc transcription by interfering with
chromatin-dependent signal transduction to RNA polymerase by BRD4 inhibition has shown great
promise [275, 276]. BRD4 is a member of the bromodomain and extraterminal domain (BET)
family of proteins which binds to acetylated lysine residues at promoter and enhancer regions,
including regions for the MYC gene [277]. BRD4 has been proposed to be a critical chromatin
regulator that maintains disease progression in acute myeloid leukemia (AML) [278]. As a result,
suppression of BRD4 with shRNA or JQ1, a bromodomain inhibitor, caused anti-leukemic effects
both in vitro and in vivo. An increasing number of studies show promising results of BET protein
33
inhibition with preclinical inhibitors, such as JQ1 in AML cell lines, ex vivo patient samples or
mouse models [279, 280]. BET inhibition also has been shown to be efficient against primary
childhood B-ALL by decreasing c-Myc protein stability, suppressing progression at DNA
replication forks, and sensitizing primary B-ALL towards dexamethasone both in vitro and in vivo
[281]. There are a few BET inhibitors that have been used in clinical trials, including OTX015 (MK-
8628), an analog of JQ1, in a Phase 1 trial for AML [282]. In this dose-escalation study, three
patients achieved complete remission and two additional patients had partial blast clearance [282].
Previous studies have shown that concomitant inhibition of PI3K and BRD4 by SF2523
blocks MYC expression and activation, promotes MYC degradation, and markedly inhibits
neuroblastoma cell growth and metastasis [283]. Taken together, PI3K and BRD4 inhibition
causes downregulation of c-Myc owing to promotion of c-Myc degradation and attenuation of c-
Myc transcription. Therefore, it is a rationale for synthesis of a dual targeting PI3K and BRD4
inhibitor [284, 285].
Herein, we evaluated SF2535, a novel small molecule inhibitor of PI3Kδ and BRD4, in B-ALL. We
have reported the chemical structures of SF2535, which is a derivative of SF2523 [283]. Both
SF2535 and SF2523 were found from a discovery of the 5-morpholino-7H-thieno[3,2-b]pyran-7-
one (TP-scaffold) system, which was the foundation of a new compound class of potential PI3K
inhibitors with improved potency. As BRD4 bromodomains (BDs) are targets of TP-scaffold
inhibitors, both SF2535 and SF2523 bind to BRD4 BD1 to a similar extent according to
displacement and NMR titration experiments [283]. Unlike SF2523, which is a highly selective
and potent inhibitor of PI3K, particularly of the PI3Kα isoform, SF2535 specifically targets PI3Kδ.
Since the PI3Kδ isoform is expressed selectively in hematopoietic cells and PI3Kδ signaling is
active in many B-cell leukemias and lymphomas [286], SF2535 is evaluated here in B-ALL.
34
2.2 Material and methods
2.2.1. Patient samples and cell culture
Bone marrow samples were obtained from B-ALL patients after informed signed consent from
patients in compliance with the Institutional Review Board regulations of Children’s Hospital Los
Angeles. Primary B-ALL blasts from bone marrow aspirates were isolated by Ficoll (GE
Healthcare) gradient centrifugation and co-cultured with irradiated OP9 stroma cells (ATCC) in
MEM-alpha supplemented with 20% fetal bovine serum (FBS, Invitrogen), 100U/ml penicillin and
100µg/ml streptomycin at 37℃ and 5% CO2. Patient sample information is listed in Supplementary
Table S2.1.
2.2.2. Starvation and activation assay for detection of
phosphorylated-AKT at serine 473 residue
B-ALL cells were serum-deprived by washing twice with Dulbecco’s Phosphate-Buffered Saline
(DPBS, Invitrogen) and cultured in MEM-alpha media at 37℃ and 5% CO2 overnight. Following
another wash with DPBS, B-ALL cells were treated with vehicle control DMSO or SF2535 for 30
minutes. Subsequently, FBS was added to a final concentration of 20% to all cells except for the
no-activation control groups. Whole cell lysates were isolated after 1 hour for use in Western blot
analysis for phosphorylated-Akt
Ser473
(p-AKT
S473
) detection.
2.2.3. Western blot
B-ALL cells were harvested and lysed in M-PER buffer (Invitrogen) containing 1% protease
inhibitor cocktail (VWR). Protein concentration was determined by Bradford protein assay.
Proteins were separated by 4-12% Bis-Tris protein gels (Invitrogen) and transferred to PVDF
membranes (Invitrogen). The antibodies (Abs) used are listed in Supplementary Table S2.2.
35
2.2.4. Chromatin Immuno-precipitation (ChIP)
Cells were treated with DMSO or SF2535 5µM for 18 hours and were then harvested and
processed using ChIP kit according to the manufacturer instructions (Abcam). In brief, the cells
were fixed with 1.1% Formaldehyde, quenched by 10% glycine, and lysed. The lysates were
sonicated in order to shear DNA to form optimal DNA fragments with optimal size of 200-1000bp.
A portion of the diluted chromatin was set aside for the INPUT. Diluted chromatins were incubated
with anti-BRD4 (1:50, CST), anti-histone H3 (4µg, Abcam) as positive control, or no antibody as
negative control overnight with rotation at 4°C.The antibody binding beads were added and
washed according to the manufacturer’s instructions. The samples were treated with DNA-
purifying slurry and Proteinase K to purify DNA. Samples were subjected to qPCR using the c-
MYC promoter primers (F: 5’-GAGCAGCAGAGAAAGGGAGA-3’, R: 5’-
CAGCCGAGCACTCTAGCTCT-3’). Fold enrichment was analyzed as described previously
described [287].
2.2.5. RNA Extraction and qPCR
Cells were treated with DMSO or 5µM SF2535 for 6 hours. Total RNA was extracted using the
Qiagen RNeasy kit (Qiagen) and cDNA was produced by the SuperScript III First-Strand
Synthesis System (Invitrogen). cDNA was amplified by specific c-Myc primers (F 5’-
CTTCTCTCCGTCCTCGGATTCT-3’; R 5’-GAAGGTGATCCAGACTCTGACCTT-3’) and GAPDH
primers (F 5’-GTTGCCATCAATGACCCCTTCATTG-3’; R 5’-
GCTTCACCACCTTCTTGATGTCATC-3’) with PowerUp SYBR Green Master Mix (Applied
Biosystems) using a ABI 7900HT qPCR machine. Relative expression levels of c-Myc were
normalized to GAPDH expression and calculated as described previously [287].
36
2.2.6. Apoptosis analysis with Annexin V and DAPI staining
Following 24 hours or 72 hours treatment of DMSO or SF2535, B-ALL cells were resuspended in
1X Annexin V binding buffer (Becton Dickinson) at a concentration of 1×10
6
cells per mL. 2.5μl
Annexin V PE (BioLegend) and 2.5μl DAPI (50μg/mL, Invitrogen) were added to 100μl of the cell
suspension. After 15 min incubation at room temperature in the dark, B-ALL cells were analyzed
by flow cytometry using BD FACS Canto II.
2.2.7. Cell cycle analysis
B-ALL cells were treated with DMSO or SF2535 (0.2µM 1µM, or 5µM) for 24h. Subsequently,
cells were stained with CytoPhase™ Violet (BioLegend) at 5µM and incubated for 90 minutes at
37℃ and 5% CO2 and analyzed on a BD FACSCanto II flow cytometer. Furthermore, BrdU
incorporation assay (Phase-Flow™ BrdU cell proliferation kit FITC-conjugated, BioLegend)
according to the protocol of the manufacturer was performed as confirmation of the results. In
brief, BrdU solution was added to cell suspension at 0.5μL/mL. Following 1.5-hour incubation, B-
ALL cells were harvested and washed. Buffer A was added for 20 minutes at 4℃ to fix cells. Then
after cell permeabilization and repeat fixation of cells, cells were treated with DNAse and
incubated 1 hour at 37℃. Lastly, 5μL of anti-BrdU antibody was added to each tube for 15 minutes
at room temperature in the dark. Cells were resuspended with PBS containing DAPI (1μg/mL)
prior to acquiring on a flow cytometer.
2.2.8. Cell proliferation assay
1×10
6
B-ALL cells were seeded per condition in triplicates on irradiated OP9 stromal cells as
previously described [186]. B-ALL cells were treated with DMSO or SF2535 (0.2µM 1µM, or
37
5µM) for 24 hours and 72 hours. Cell numbers were counted by Trypan blue exclusion on a
hemocytometer under an inverted phase-contrast microscope.
2.2.9. Flow cytometry
B-ALL cells were treated with the indicated concentration of SF2535 for 24h. Subsequently, B-
ALL cells were resuspended in 100µl PBS containing FACS antibodies or the respective isotype
controls (information can be found in Supplementary Table S2.3). Following incubation at 4°C for
30 min, B-ALL cells were washed by 1ml PBS and resuspended in PBS containing DAPI (1μg/mL)
then analyzed with a BD FACSCanto II flow cytometer. Flow cytometry data was analyzed with
FlowJo 7.0 software (FlowJo LLC).
2.2.10. Cell adhesion assay
2.5 × 10
4
/well irradiated OP9 cells were seeded onto a tissue culture 96-well plates and cultured
overnight. Simultaneously, B-ALL cells were treated with different concentrations of SF2535 or
DMSO for 24 hours. Then live B-ALL cells were harvested, washed once with DPBS, and
resuspended at the final concentration of 0.2 × 10
6
/200µL with culture medium. B-ALL cells were
dispended onto an irradiated OP9 96-well plate with 200 µL in each well and allowed to adhere
for 2 hours at 37°C. Non-adhering cells were removed and remaining cells on OP9 were gently
washed with 100 µL of DPBS. Adherent cells and supernatant cells were counted by Trypan blue
exclusion on a hemocytometer.
2.2.11. Animal studies
Primary relapsed B-ALL cells (LAX56) were intravenously injected into NOD.Cg-
Prkdc
scid
Il2rg
tm1Wjl
/SzJ (NSG, The Jackson Laboratory) mice (1×10
6
cells/mouse). After 3 weeks
of engraftment, SF2535 (30 mg/kg, dissolved in 20% dimethylacetamide (DMA) and 80% Captisol
38
(20% w/v in water for 2mg/ml)) (n=6) or vehicle (n=6) was administered once by intraperitoneal
(i.p.) injection. After 24 hours, mice were sacrificed and bone marrow, spleen, and peripheral
blood were harvested, and red blood cells were lysed by RBC lysis buffer (Invitrogen). The animal
study was performed in compliance with a research protocol approved by the Institutional Animal
Care and Use Committee (IACUC), the Saban Research Institute of Children's Hospital Los
Angeles.
2.2.12. Data analysis and statistics
All statistical analyses were performed using GraphPad Prism 5. The mean was chosen as a
center value for all graphs. 95% confidence interval (95% CI), a standard deviation of the mean
was used as measures of spread as indicated in figure legends and the Results section. Statistical
analysis was performed using paired Student’s t-test or one-way ANOVA followed by Tukey's
multiple comparison tests for statistical analyses as appropriate. A p-value of <0.05 was
considered statistically significant.
2.3 Results
2.3.1. PI3Kδ and BRD4 expression in B-ALL
Expression of PI3Kδ and BRD4 was determined in fifteen primary B-ALL and three B-ALL cell
lines representing various cytogenetics (Supplementary table 2.1). Most of the primary B-ALL and
cell lines expressed similar levels of PI3Kδ despite their difference in cytogenetics, while BRD4
levels were variable (Figure 2.1a).
2.3.2 SF2535 downregulates c-Myc and p-AKT in B-ALL
To determine the effective concentration of SF2535 in primary B-ALL cases, we treated three
primary B-ALL (LAX56, LAX7R, and TXL3) cells with increasing concentrations of SF2535 for 48
39
hours. SF2535 dose-dependently induced apoptosis in all B-ALL cells, and the calculated EC50
values of SF2535 were 2.4μM (95% CI, 1.990μM - 2.935μM), 1.5μM (95% CI, 1.389μM -
1.633μM), and 3.2 μM (95% CI, 2.718μM – 3.670μM) in LAX56, LAX7R and TXL3, respectively
(Supplementary figure S2.1). Based on these values, three different doses of SF2535, 0.2μM,
1μM, and 5μM, were chosen for subsequent studies. As c-Myc transcription is mediated by BRD4
binding to the promoter region [275], we evaluated the specific effect of SF2535 on BRD4 binding
on the c-Myc promoter by chromatin immunoprecipitation (ChIP). In all three cases, SF2535
decreased BRD4 binding to the c-Myc promoter site compared to DMSO (LAX56 P<0.0001,
LAX7R P<0.0001, TXL3 P<0.0001) (Figure 2.1b). Subsequently, mRNA transcript levels of c-Myc
expression were significantly decreased upon SF2535 treatment in all three cases (LAX56
P=0.0020, LAX7R P=0.0002, TXL3 P=0.0009) (Figure 2.1c). Finally, SF2535 prominently
downregulated c-Myc protein expression in a dose-dependent manner, which was determined by
Western blot (Figure 2.1d, Supplementary figure S2.2a). Decrease in c-Myc protein expression
could be restored by the proteasome inhibitor MG132, which shows c-Myc degradation in B-ALL
occurs through the ubiquitin-proteasome pathway [288] (Figure 2.1e, Supplementary figure
S2.2b). SF2535 also decreased phosphorylated AKT in LAX56, LAX7R and TXL3, demonstrating
the on-target effect of SF2535 on PI3Kδ. Following serum starvation of leukemia cells, B-ALL
cells were treated with DMSO (vehicle control) or 0.2μM, 1μM, or 5μM of SF2535 for 30 minutes
prior to 1 hour serum-induced activation. Levels of p-AKT
S473
decreased in a dose-dependent
manner from SF2535 in LAX56, LAX7R, and TXL3 B-ALL cells (Figure 2.1f, Supplementary figure
S2.2c, S2.2d).
40
2.3.3 SF2535 induces apoptosis in B-ALL cells through changes
in the intrinsic apoptotic pathway
To determine the apoptotic effect of SF2535 in primary B-ALL, LAX56, LAX7R, and TXL3 cells
were treated with DMSO or 0.2μM, 1μM, or 5μM SF2535 for 24 hours and 72 hours (Figure 2.2).
The percentages of apoptotic cells were significantly increased after 24 hours with 5μM SF2535
compared to DMSO control for LAX56 (51.48±4.51% vs 31.28±4.94%, P<0.0001), LAX7R
(53.86±14.02% vs 19.98±8.51%, P<0.0001), and TXL3 (39.51±10.49% vs 20.90±4.05%,
P<0.0001) (Figure 2.2 a-c), and were also significantly increased after 72 hours of treatment
(Figure 2.2 d-f) in four independent experiments performed. We further analyzed whether SF2535
affects components of the intrinsic apoptotic pathway, including caspase-3, caspase-7, PARP
and the anti-apoptotic component BCL-2. 5μM SF2535 significantly increased cleaved PARP,
caspase-3, and caspase-7 in LAX56, LAX7R, and TXL3. Moreover, SF2535 5μM markedly
decreased BCL-2 after 72 hours in both LAX7R and TXL3, yet not in LAX56 (Figure 2.2 G-I).
2.3.4 SF2535 causes cell cycle changes and suppresses cell
counts in B-ALL
In order to determine if the decrease in proliferation was due to cell cycle arrest, we performed
cell cycle analysis in SF2535-treated B-ALL cells. LAX56, LAX7R and TXL3 cells were treated
with DMSO control or with 0.2µM, 1µM or 5µM of SF2535. After 24 hours, percentage of cells in
G0+G1 phase increased while the percentage of cells in S phase decreased in all SF2535-treated
groups except for TXL3 treated with 0.2µM of SF2535 (Figure 2.3). In LAX56 cells (Figure 2.3a),
0.2µM, 1µM and 5µM SF2535 when compared to DMSO prolonged G0+G1 phase (P=0.278,
P=0.006, P<0.001, respectively) and arrested S phase (P=0.044, P=0.011, P<0.001, respectively).
Similarly, significant prolonged G0+G1 phase was found in LAX7R (Figure 2.3b) and TXL3 (Figure
41
2.3c). The summarized results of mean and standard deviation of two independent triplicate
experiments and representative flow cytometry figures are depicted in Figure S3. In addition, BrdU
incorporation assays were performed, and consistent results were shown (Figure S4). According
to the apoptotic effect and S phase cell cycle arrest of SF2535, we performed cell count assays
to access the potential proliferation effect of SF2535 in B-ALL cells. We treated B-ALL cells with
DMSO or 0.2μM, 1μM, or 5μM SF2535 for 24 and 72 hours. Both 1μM and 5μM SF2535
significantly inhibited cell proliferation after 72 hours of treatment of LAX56 (Figure 2.3d), LAX7R
(Figure 2.3e), and TXL3 (Figure 2.3f). For instance, at 72 hours, 1μM and 5μM of SF2535 reduced
the number of viable cells compared to (2.74±0.33) × 10
6
in DMSO to (1.49±0.49) × 10
6
in SF2535
1μM (P<0.0001) and (0.29±0.17) × 10
6
in SF2535 5μM (P<0.0001), in LAX56.
2.3.5 SF2535 decreases surface integrin expression
Our previous studies have shown that cell adhesion-mediated drug resistance (CAM-DR) plays a
crucial role in relapsed and refractory B-ALL [170, 289]. Previously, we have shown inhibition of
PI3Kd with idelalisib in B-ALL inhibited homing of cells into the bone marrow [290]. Decrease in
homing may be due to the inability of cells to adhere to surrounding microenvironment upon PI3Kd
inhibition. In order to determine if blockade of PI3Kd affects expression level of surface adhesion
in B-ALL cells, we assessed integrin α4, α5, α6, β1, and CXCR4 expression in SF2535 treated
B-ALL. LAX56, LAX7R and TXL3 were treated with DMSO control or with 0.2µM, 1µM or 5µM of
SF2535. After 24 hours, cells were stained with anti-integrin α4, α5, α6, β1, and CXCR4
antibodies and their mean fluorescence intensity (MFI) was assessed by flow cytometry. To
exclude dead cells which can interfere with flow cytometry data analysis, viable cells were strictly
gated and a representative gating strategy of 5µM SF2535 treated LAX56 cells is shown in
supplementary figure S2.5a. As a result, histograms of integrin α4, α5, α6, β1, and CXCR4
showed relatively small changes in expression levels of integrin subunits and CXCR4 between
42
DMSO and SF2535-treated groups (Figure 2.4a-c). Moreover, MFI of integrin α4, α5, α6, and β1
significantly decreased in SF2535 treated groups while MFI of CXCR4 increased in LAX56,
LAX7R and TXL3 after SF2535 treatment (Figure 2.4d-r, Supplementary figure S2.5). This result
shows dual inhibition of PI3Kd and BRD4 decreases integrin expression on the cell surface that
is important for adhesion of leukemia cells to the microenvironment. However, cells may
compensate for the loss of integrin subunits by expressing other surface molecules implicated in
adhesion, such as CXCR4. In order to further evaluate the physiological and biological relevance
of integrin expression effect, we performed cell adhesion assays. Since SF2535 induces
apoptosis of B-ALL, we pre-treated B-ALL with SF2535 for 24 hours and harvested live cells.
Harvested cells were washed and plated at 0.2 × 10
6
/200µL onto 96 well tissue culture plates
seeded with irradiated OP9 stromal cells, which has multiple integrin ligands [170, 291], for 2
hours allowing B-ALL cells to adhere. We observed that 5µM SF2535 significantly inhibited
adhesion of all three B-ALL lines to stromal cells (Figure S6).
2.3.6 SF2535 decreases peripheral leukemic burden in mouse
model
Finally, we evaluated in vivo efficacy of the drug in a leukemia-engrafted mouse model. In order
to determine the in vivo effects of SF2535, NSG mice were first injected with 1×10
6
LAX56 cells
per mouse. After 3 weeks, the engrafted mice were treated either with the vehicle control (n=6)
or SF2535 (30mg/kg, n=6) (Supplementary Figure S2.7a). Continuous treatment with 30 mg/kg
SF2535 was not well-tolerated by the animals, therefore, SF2535 was administered once to the
mice and the early effects of the drug on B-ALL cells were evaluated. After 24 hours post-injection
of SF2535, bone marrow (BM), spleen cells (SPC), and peripheral blood (PB) were collected and
analyzed for human CD45
+
CD19
+
via flow cytometry (Supplementary figure S2.7b-d). Leukemia
burden, shown as percentage of human CD45
+
CD19
+
, in PB was significantly decreased in
43
SF2535-treated mice (P=0.0202) (Supplementary figure S2.7d) yet there was no decrease of
human leukemia in BM or SPC (Supplementary figure S2.7B, c). This result shows the dose and
timing of SF2535 administration is sufficient to induce apoptosis of circulating leukemia cells in
the peripheral blood, but not enough to induce apoptosis of B-ALL cells that are residing in the
bone marrow or spleen.
2.4 Discussion
PI3K has been targeted by copanlisib and duvelisib which were approved by the FDA for use in
CLL and follicular lymphoma [292, 293]. It has been demonstrated that PI3K also plays a crucial
role in ALL [267, 290]. Our findings show that PI3K is broadly expressed in B-ALL and the key
downstream signal p-AKT is markedly downregulated by dual inhibition of PI3Kd-BRD4 by
SF2535 (Figure 2.1f). In addition, it is well established that c-Myc plays a major role in mature B-
ALL and Burkitt lymphoma [294, 295], however, there are few studies that explore the role of c-
Myc in other types of B-ALL. Ott et al. reported that BET bromodomain inhibition using JQ1 targets
both c-Myc and IL7R in high-risk CRLF2-rerranged and other B-ALL [296]. Moreover, oncogenic
Myc is also a difficult target for cancer therapy, and alternative approaches have been taken to
indirectly target Myc by blocking pathway events upstream of c-Myc [275]. Our previous study
showed feasibility of targeting Myc with a dual-activity PI3K-BRD4 inhibitor [283]. In our present
study, we have demonstrated that c-Myc is expressed in B-ALL, and c-Myc was markedly
downregulated by inhibition of its promoter site by SF2535 (Figure 2.1b-d). SF2535 also led to a
decrease in p-AKT
S473
levels upon inhibition of PI3Kd. Our data show that SF2535 led to
downregulation of both p-AKT and c-Myc in B-ALL.
Inhibition of c-Myc has been shown to result in apoptosis in T-ALL [296, 297]. Our data also
indicate that dual inhibition of PI3Kd and BRD4 results in apoptosis or primary B-ALL cells using
SF2535 (Figure 2.2a-f). Furthermore, we demonstrated that SF2535-induced apoptosis occurs
44
through the intrinsic pathway via increasing cleavage of PARP, caspase-3 and caspase-7 and
decreasing BCL-2 [298] (Figure 2.2g-i). Inhibition of PI3Kd and BRD4 not only induced apoptosis,
but also caused cell cycle arrest and decreased proliferation (Figure 2.3). A potential mechanism
is that BET bromodomain inhibition affects key regulators of the cell cycle such as cyclin D1
expression [283].
Recently, we have suggested that BRD4 regulates the immunosuppressive myeloid tumor
microenvironment which can be blocked by PI3K/BRD4 inhibitors using SF2523 [287]. The bone
marrow environment has been shown to promote CAM-DR in ALL[299]. Our previous studies
have identified the integrin α4 and α6 as an adhesion molecule that plays a critical role in B-ALL
through CAM-DR [170, 186]. Our results indicate dual inhibitors of PI3Kδ and BRD4 using SF2535
affected the expression of adhesion molecules including integrin α4, α5, α6, β1, while CXCR4
was increased (Figure 2.4). This finding suggests a relationship between integrins and PI3Kδ
through outside-in signaling [289] and would warrant further mechanistic studies. It has been
shown that integrin α6 and β1 are regulated by the c-Myc oncogene in colorectal cancer cells
[300, 301] and a murine hematopoietic cell line [302]. Yao et al. recently showed that use of a
PI3Kδ inhibitor resulted in a significant reduction of leukemia metastasis to the central nervous
system due to decreased integrin α6 expression despite minimally decreased bone marrow
disease burden [185]. It is possible that CXCR4 expression compensates for the downregulation
of integrins, which requires further investigation. Our preliminary in vivo results show that dual
inhibition of PI3Kδ and BRD4 led to a reduction of leukemia cell numbers in the peripheral blood
of leukemia bearing mice. Mice tolerated 10mg/kg SF2535 for continuous treatment up to 4 weeks,
yet the low dosage of SF2535 was not effective enough to prolong the survival of leukemia
engrafted mice (data not shown), while higher doses were not well tolerated in mice (data not
shown).
45
2.5 Conclusions
Taken together, these results reveal that SF2535 efficaciously induces apoptosis through
downregulating c-Myc and p-AKT pathways in primary B-ALL providing a rationale for further
preclinical evaluation of PI3Kδ and BRD4 inhibition in B-ALL.
46
47
Figure 2.1. SF2535 downregulates c-Myc and p-AKT.
(A) PI3Kδ and BRD4 expression in whole cell lysates in B-ALL. (B) Primary B-ALL LAX56, LAX7R
and TXL3 cells were treated with SF2535 at 5µM. After 18 hours, the cells were harvested for
BRD4 ChIP analysis which was performed at the c-Myc promoter site. Data were combined from
three independent experiments per leukemia. Data was analyzed by paired Student’s t-test,
where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. ctrl (DMSO). (C) qPCR data showing the
effect of SF2535 on c-Myc expression in B-ALL cells. Experiment was performed in triplicate.
Data was analyzed by Student’s t test, where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs.
ctrl (DMSO). (D) LAX56, LAX7R and TXL3 cells were treated with 0.1% DMSO or SF2535 (0.2μM,
1μM, or 5μM) for 48 hours. c-Myc expression of B-ALL cells was analyzed by Western blot. (E)
LAX56, LAX7R, and TXL3 cells were pre-treated with either 0.1%DMSO control or proteasome
inhibitor MG132 (4μM) for 45 min and subsequently treated with SF2535 at 5μM for 6 hours. c-
Myc expression was analyzed by Western blot. (F) LAX56, LAX7R and TXL3 cells were cultured
in MEM-α without serum overnight. Subsequently, cells were treated with 0.1% DMSO control,
SF2535 (0.2μM, 1μM, 5μM) for 30 mins. Cells were activated with 20% FBS for 1 hour. Western
blots of p-AKT
S473
and AKT are shown. β-actin was used as internal control for equal protein
loading for Western blots (D-F). One of two independent experiments per leukemia was
performed for D-F.
48
Figure 2.2. SF2535 induces apoptotic effects in B-ALL cells. LAX56, LAX7R and TXL3 B-ALL
cells were cultured in presence of 0.1% DMSO (white bars) or SF2535 (0.2μM, 1μM, or 5μM) (in
red bars) for 24 (A-C) and 72 hours (D-F) and apoptosis was assessed by percent of Annexin V
+
cells by flow cytometry. Data are pooled from four independent experiments performed in
triplicates. P-values are calculated using one-way ANOVA test and Tukey’s multiple comparison
test. *P<0.05, **P<0.01, ***P<0.001 compared to the DMSO group. LAX56, LAX7R and TXL3 B-
ALL cells were cultured in presence of 0.1% or SF2535 5μM for 24 and 72 hours and proteins
were isolated for Western blot analysis (G-I). β-actin was used as loading control. One of two
experiments is shown.
49
Figure 2.3. SF2535 prolongs G0+G1 phase arrest and attenuates S phase. (A) LAX56, (B)
LAX7R and (C) TXL3 B-ALL cells were treatment with DMSO or SF2535 (0.2μM, 1 μM, or 5μM)
for 24 hours. Cell cycle was assessed via flow cytometry after 24 hours of treatment. White bars
indicate G0+G1 Phase. Green bars indicate G2+M Phase. Red bars indicate the S Phase. P-
value *<0.05: Comparing S phase compared to DMSO S phase. P-value #<0.05: Comparing
G0+G1 phase to DMSO G0+G1 phase. The results are representative of two independent
triplicate experiments. To determine the effect of SF2535 on proliferation and cell numbers,
LAX56 (D), LAX7R (E) and TXL3 (F) B-ALL cells were treated with DMSO or SF2535 (0.2μM,
1μM, or 5μM) for 24 and 72 hours. Numbers of live cells were counted by Trypan blue exclusion
on a hemocytometer. Data of three independent triplicate experiments in triplicates are combined.
P-value *<0.05 compared to DMSO; ns, not significant.
50
Figure 2.4. SF2535 affects adhesion molecules. Representative histograms for integrin α4, α5,
α6, β1, and CXCR4 expression for (A) LAX56, (B) LAX7R and (C) TXL3 cells were treated with
0.1% DMSO (in white bars) or SF2535 (0.2μM, 1μM, 5μM in gradient red bars) for 24 hours. (D-
R) Mean fluorescence intensity (MFI) of integrin α4, α5, α6, β1, and CXCR4 were shown on
indicated y-axis in (D-H) LAX56, (I-M) LAX7R and (N-R) TXL3 B-ALL cells. P-value *<0.05
51
compared to DMSO control. One representative experiment out of at least two independent
experiments is shown.
52
Chapter 3. cIAP2 inhibition in anti-CD19 CAR T cell therapy resistant
Leukemia
3.1 Introduction
B cell acute lymphoblastic leukemia (B-ALL) is one of the common type of cancers in
children [1]. Despite favorable outcome in the patients with over 90% of 5-year survival rate,
relapsed/refractory B-ALL still affects many children and is one of the leading causes of cancer-
related deaths in pediatric cancer patients. Prognosis is worse in adolescents and young adults
[2]. In 2017, anti-CD19 chimeric antigen receptor T (CAR T) cell therapy was approved for treating
pediatric patients with relapsed/refractory leukemia after successful multi-center phase II ELIANA
clinical trial. As the name suggest, CAR T carries engineered receptor against target antigen. For
anti-CD19 CAR T, engineered T cells express receptor against CD19 molecule, which is
abundantly expressed in B cell hematological malignancies. Anti-CD19 CAR is a fusion of
extracellular domain from CD19-specific IgG1 monoclonal antibody (FMC63) and intracellular T
cell receptor (TCR) signaling domain [303]. Antigen binding domain of anti-CD19 CAR T forms a
single-chain variable fragment (scFv) from heavy and light chain of FMC63. Second generation
of CAR T, which FDA-approved CAR T belongs to, includes 4-1BB or CD28 co-stimulatory
domains in addition to CD3ζ intracellular domain. Antigen recognition by CAR is independent of
major histocompatibility complex (MHC) independent. It prevents tumor cells escaping MHC-
restricted T cell recognition through downregulating the expression of MHC complex [304]. Anti-
CD19 CAR T has revolutionized treatment of relapsed/refractory leukemia in pediatric patients.
About 70% of anti-CD19 CAR T recipients will achieve complete remission (CR) yet relapse still
happens in 30-50% of the patients with long-term follow-up [305]. Most of the relapse after CAR
T therapy involves CD19 antigen loss due to alternative splicing or mutation in CD19 gene [306].
IAP2 is one of Inhibitor of apoptosis (IAP) protein family that mediates apoptosis pathway
in cells. IAP family includes XIAP (BIRC4) , cIAP1 (BIRC2) , cIAP2 (BIRC3) , NIAP (BIRC1),
53
Survivin (BIRC5) and Bruce (BIRC6). Altered expression of IAP proteins has been seen in various
types of cancers including have overexpression in various cancer types, including hematological
malignancies [307-310]. Prognostic values of abnormal expression of IAP protein varies from
cancer to cancer, as IAP proteins can be both pro-oncogenic and tumor-suppressive depending
on the type of disease and the situation. In CLL, cIAP2 (BIRC3) was found to be both oncogenic
and suppressive in different studies. Mutation in CLL predicts poorer outcome in patients as they
cannot function as negative regulator of non-canonical NF-κB. On the other hand, it was also
suggested to be pro-oncogenic as higher expression of cIAP2 was found in CLL patients [307].
In lymphoma, BIRC3 and MALT1 gene fusion activates NF-κB pathway and is responsible for
drug resistance and rapid disease progression [311]. In B-ALL, targeting cIAP5 or survivin,
another IAP protein family member, with shRNA and antisense oligonucleotide in combination
with chemotherapy eliminated drug-resistant minimal residual disease in mice engrafted with
primary ALL [312]. cIAP2 has not been elucidated much in B-ALL disease setting. Overall,
targeting IAP proteins has been suggested as therapeutic intervention in many cancer types as
they are often overexpressed in abnormal tissue and are associated with poorer prognosis [313].
cIAP2 (BIRC3) was first identified in bacuroviral gene that controls apoptosis of host cell
[314]. cIAP2 binds to caspase-3 and -7 through conserved BIR domain, thus modulating
apoptosis by inhibiting the activation of caspases [315, 316]. Also, it is known to mediate TNF-ɑ
induced NF-κB activation [317, 318]. During this process, cIAP2 works as an E3 ligase and adds
ubiquitin moiety on RIPK1 in order to convert TNF-ɑ induced apoptotic signal into NF-κB mediated
survival of the cell [319]. Here, we show cIAP2 protein is overexpressed in primary B-ALL after
anti-CD19 CAR T treatment and inhibition of cIAP2 in primary B-ALL sensitizes B-ALL cells
toward anti-CD19 CAR T. Furthermore, we suggest combination therapy of cIAP2 inhibition with
anti-CD19 CAR T to increase efficacy of the treatment in pediatric leukemia patients.
54
3.2 Methods
3.2.1 Patient derived bone marrow aspirate or peripheral blood sample
processing and culturing
Bone marrow or peripheral blood aspirates from healthy donor or leukemia patients were obtained
and processed via density gradient centrifugation to isolate mononuclear cells from the sample.
Isolated leukemia cells were transplanted in NOD.Cg-Prkdc
scid
Il2rg
tm1Wjl
/SzJ (NSG) mice and
engrafted cells were harvested from mice bone marrow and spleen. Cells were passaged in mice
up to three times until primary leukemia cells stably grow in vitro growth medium (MEM-alpha
containing 20% of heat-inactivated fetal bovine serum with 1% of penicillin/Streptomycin). For in
vitro expansion of primary B-ALL cells, cells were cultured on tissue culture plates or tissue culture
plates were coated with irradiated OP9 cells. Leukemia cells were passed once a week and
transferred to new sets of OP9 coated plates or dishes to provide continual support of growth.
3.2.2 Anti-CD19 CAR T production and transduction of healthy donor
PBMC derived T cells
All plasmids required for generating anti-CD19 CAR were provided by Dr. Chintan Parekh from
Children’s Hospital Los Angeles. HEK293 cells were transfected with CAR B-CD19, psPAX2
(packaging) and pMD2.G (envelope) with OPTI-MEM (Gibco) and TransIT 293 reagent (Mirus)
as per manufactures’ protocol. After sodium butyrate induction, virus was harvested from HEM293
plated dishes. Harvested virus was ultracentrifuged to concentrate the lentivirus particle and the
viral titer was determined. After titer was determined, CD3 positive T cells enriched from healthy
donor was transduced to produce anti-CD19 CAR T cells. First, thawed PBMC was cultured
overnight the day before enrichment. The next day, CD3 positive T cells were enriched from
PBMC using Pan T cell isolation kit (Miltenyi), and enriched cells were checked for anti-CD56 and
55
anti-CD3 via flow cytometry to confirm the purity. Enriched cells were activated with CD3/ CD28
Dynabeads (Gibco). The next day, cells were transduced with B-CD19 CAR virus with MOI of 10
on retronectin coated non-tissue culture plate. Second dose of virus as added the next day, and
cells were washed and expanded on day 4 in AIM V medium with 5% of heat-inactivated fetal
bovine serum and 25ng/mL of IL-2. For checking the transduction efficiency, cells were harvested
and de-beaded and stained with biotinylated protein-L for 45 minutes at 4℃ in dark. Protein-L
stained cells were washed and incubated with streptavidin for 30minutes at 4℃ in dark. After
incubation, cells were washed and analyzed via flow cytometry.
3.2.3 Western blot analysis of target protein in primary B-ALL
5x10
6
primary B-ALL cells were cultured in 10cm
2
tissue culture dish coated with OP9 overnight.
Then 0.5x10
6
of NT or anti-CD19 CAR T cells were added to the dish. After 24hours of co-culture
of B-ALL with NT or anti-CD19 CAR T on OP9, B-ALL cells were collected by vigorous pipetting
to collect cells that are potentially staying beneath the feeder layer. Cell viability is confirmed via
7AAD/ Annexin V staining flow cytometry prior sorting. After checking cell viability, counted cells
were bound to anti-CD3 magnetic beads per manufacture’s protocol and CD3 negative B-ALL
cells that are not bound to the beads were collected by negative selection. Negative selection
enabled us to collect alive B-ALL cells, whereas positive sorting with CD19 bead was not capturing
CD19
dim
population and also contained non-specific binding of dead cells to the beads. Purity
check was performed on sorted cells with CD3, CD19 and CD45. After enrichment, cytoplasmic
protein was isolated with Mammalian Protein Extraction Reagent with protease inhibitor. Cell
lysates were measured and used for Western Blot analysis for detecting protein of interest.
56
3.2.4 Annexin V/ 7AAD staining for accessing viability of primary B-ALL
cells.
All of cell viability analysis in this manuscript was done with 7AAD/ Annexin V staining flow
cytometry. From each experiment, suspension cells (B-ALL and T cell) were harvested with
vigorous pipetting to collect cells that are potentially hiding under feeder layer. Collected cells
were stained with anti-CD3, anti-CD19 and anti-CD45 for 30minutes at 4℃ in dark. Stained cells
were washed twice with DPBS and further stained with 7AAD/ Annexin V in Annexin V binding
buffer. For analysis, CD3 negative population was analyzed for 7AAD and Annexin V negativity.
CD19 and CD45 status is checked on the cells, but all CD3 negative cells are included in the
analysis to avoid false interpretation of data.
3.2.5 Phospho-flow cytometry for staining phosphorylated p65 in
primary B-ALL cells
0.5x10
6
of primary B-ALL cells were cultured overnight on 24-well tissue culture plate coated with
non-irradiated OP9 cells. 0.05x10
6
of NT or anti-CD19 CAR T cells were added the next day and
co-cultured for 24 hours. After given time, cells were collected from plates with vigorous pipetting
to make sure to harvest leukemia cells that might be staying under OP9 layer. Collected cells
were counted via Trypan Blue exclusion assay, and 0.1x10
6
of cells were stained with anti-CD3,
anti-CD19 and anti-CD45 flow cytometry antibody for 30minutes at 4℃ in dark. Stained cells were
washed with DPBS and fixed with 100μL of Fixation buffer for 60 minutes at room temperature in
dark. After fixation, 2mL of 1X Permeabilization buffer were added to each tube and centrifuged
at 500xg for 5 minutes at room temperature for two times. After centrifugation, cells were stained
with PE-conjugated phosphorylated p65 in 100μL of 1X Permeabilization buffer for 60 minutes at
room temperature in dark. After 60 minutes, 2mL of Permeabilization buffer was added and tubes
57
were centrifuged at 500xg for 5 minutes at room temperature for two times. Cells were
resuspended in 100μL of flow cytometry buffer (PBS containing 5% of fetal bovine serum).
3.2.6 BIRC3 overexpression and knockdown in primary B-ALL cells via
lentivirus transduction
BIRC3 lentivirus particels for overexpression and appropriate control are purchased from Origene.
Lentivirus carrying overexpression or shRNA vector along with appropriate controls were added
to growth medium (without antibiotics) on 48 well non-tissue culture plate coated with retronectin
in MOI of 10. 0.05x10
6
of primary B-ALL cells were added to virus solution and incubated with
virus for 72 hours. After 72 hours, cells were checked with fluorescence microscope and flow
cytometry for increased GFP signal. Once GFP signal is confirmed, GFP positive cells were sorted
via flow cytometry. Sorted cells were expanded in a larger vessel and followed with puromycin
selection to enrich successfully transduced cells. Puromycin was added every other day to
provide selection pressure until resistant cells proliferates under puromycin pressure.
3.2.7 CRISPR/Cas9 mediated knockout of BIRC3 gene in primary B-ALL
sgRNAs for BIRC3 and appropriate negative control for CRISPR/Cas9 mediated KO were
purchased from Thermofisher. CRISPR/Cas9 mediated knockout of BIRC3 gene in primary B-
ALL was done with Neon nucleofection machine and modified single guide RNA (sgRNA) for
BIRC3 purchased from Thermofisher scientific per company protocol. Briefly, 2x10
6
B-ALL cells
were harvested and washed with DPBS, then resuspended in 100μL of supplied resuspension
buffer to keep 20x10
6
/mL density. Cells were kept at 37℃ with 5% CO2 during Cas9-sgRNA
conjugate preparation process. For Cas9-sgRNA conjugation preparation, 1μg of Cas9 protein
(0.5μL) and 250ng (0.5μL of 100μM stock) of sgRNA were mixed in 5μl of Resuspension buffer
per run. If multiple runs were performed, volume of each component were linearly scaled up to
58
the number of conditions repeated. Cas9-sgRNA complex was incubated at room temperature for
10 minutes. For electroporation, 6μL of cells and 6μL of complex were mixed for each 10μL run.
Cell/complex mixture was transferred to Neon tip and electroporated with 1650V, 10ms for 3
pulses. Electroporated cells were transferred to 1 well of 24 well plates coated with irradiated OP9
with 1mL of growth medium and incubated at 37℃ with 5% CO2. Cells from three runs were kept
in the same well to maintain cell density, and knockout efficiency was verified via RT-qPCR and
western blot once cells expanded.
3.2.8 RT-qPCR
For validation of overexpression or KD/KO of the gene, mRNA was isolated from cells using
RNeasy mini kit from Qiagen per manufacturer’s protocol. cDNA was synthesized from isolated
mRNA using the SuperScript™ III First-Strand Synthesis System (Thermo Fisher) as per
manufacturer’s protocol, and cDNA was used for RT-qPCR. Primer pairs for BIRC3 (Origene) and
the control GAPDH were used for RT-Qpcr with PowerUp SYBR Green Master Mix (Thermo
Fisher). Qpcr reactions were run on a QuantStudio™ 7 Flex Real-Time PCR System (Thermo
Fisher). The relative expression of BIRC3 was calculated relative to GAPDH by the delta-delta Ct
method.
3.2.9 Homing of BIRC3 KD cells into bone marrow
0.05x10
6
of negative control or BIRC3 KD primary B-ALL cells were injected into NSG mice via
intravenous injection. On day 4 after injection of primary B-ALL, mice were euthanized and bone
marrow from each mouse was harvest from both femur and tibia. Processed bone marrow cells
were washed with DPBS and red blood cells were lysed using ACK lysing buffer. Lysed cells were
counted via Trypan Blue exclusion assay, and 10x10
6
of counted cells from each mouse were
stained with anti-mouse CD45, anti-human CD45 and anti-human CD19 for 30minutes at 4℃ in
59
dark. After staining, cells were washed with 2mL of DPBS and resuspended in 500μL of flow
cytometry buffer containing DAPI. Homing of B-ALL cells was assessed by determining the
percentage of anti-human CD45/anti-human CD19 double positive cells out of 1x10
6
DAPI
negative lymphocyte events acquired.
3.2.10 In vivo treatment of BIRC3 KD cells with anti-CD19 CAR T
cells
1 week before setting up an experiment, B-ALL cells were transduced with luciferase lentivirus on
48well non-tissue culture plate coated with retronectin. On day 3 post-transduction, cells were
harvested and checked for bioluminescence signal. Once signal is confirmed, cells were
expanded to non-irradiated OP9 coated tissue culture plate. Expanded cells were collected and
washed with DPBS twice and resuspended in sterile saline. 0.05x10
6
of prepared negative control
or BIRC3 KD primary B-ALL cells were injected into NSG mice via intravenous injection (in 200μL/
mouse). On day 4 after engraftment of B-ALL cells, 3x10
6
of NT or CAR T cells were injected into
mice intravenously. Mice were weighted and bioluminescent imaged periodically to access
progression of disease and to prevent mice from experiencing unnecessary pain. If a mouse loses
more than 15% of initial weight, loses weight rapidly over few days (about 2% of weight, roughly
2g in 20-25g mouse), shows sign of discomfort or is lethargic, the mouse was euthanized in CO2
chamber. Euthanized mouse bone marrow was harvested from both femur and tibia. 0.1x10
6
of
processed bone marrow cells were stained anti-human CD3, CD19, CD45, PD-L1, PD-1, ICAM-
1 and anti-mouse CD45 for accession of human leukemia in the bone marrow. Another 0.1x10
6
of processed bone marrow cells were stained with T cell phenotyping antibodies, anti-CD3, CD8,
CD45RA, CD45RO, LAG3 and TIM3. All cells were stained as mentioned above.
60
3.3 Results
3.3.1 Primary B-ALL cells express cIAP2 after 24hours of anti-CD19
CAR T cell exposure
In order to determine if anti-CD19 CAR T treatment induces anti-apoptotic protein in primary B-
ALL, 5 cases of primary B-ALL and 3 B-ALL cell lines were treated with no T cells, non-transduced
T cells (NT) or anti-CD19 CAR T cells for 24hours in the presence of OP9 layer. After 24hours,
cells were harvested, and B-ALL cells were enriched by collecting cells that were not bound to
anti-CD3 magnetic beads. We have specifically used negative sorting technique because it
yielded better separation between T cells and B cell fraction, and did not collect dead cells non-
specifically binding to the magnetic beads. When treated cells were enriched using anti-CD19
magnetic beads, cells captured in the column still included CD3+ population, which possibly
represents CD3 positive T cells forming conjugates with CD19 positive B cells. Also, these cells
included high percentage of dead cells as magnetic beads can bind to dead cells. Cell lysates
extracted from collected cells were used for Western blot analysis to identify BCL-2, cIAP1, cIAP2,
XIAP and Survivin. As a result, primary B-ALL cells significantly increases cIAP2 expression after
they were treated with anti-CD19 CAR T cells (Figure 3.1a-e). Overall, BLC2, cIAP1, XIAP and
Survivin protein expression was not specific to anti-CD19 CAR T exposure and no shared pattern
between cases were observed. In cell lines, cIAP2 expression was relatively weaker compared
to primary B-ALL, yet all of the cases increased cIAP2 expression upon anti-CD19 CAR T
exposure (Figure 3.1 f-h). Using the same cell lysates, we identified if increased cIAP2 correlates
with increased phosphorylation of p65 protein. Phosphorylation of NF-κB p65 protein is known to
be required for nuclear translocation of the protein and the subsequent activation of the signaling
pathway. Western blot analysis has revealed increased phosphorylation of NF-κB p65 at serine
536 residue, but no change in total p65 protein quantity in primary B-ALL. This was not necessarily
true in B-ALL cell lines as NALM6 and SupB15 express high level of phosphorylated p65 without
61
any treatment, thus phosphorylation level does not change upon CAR T treatment. RCH-ACV
showed similar pattern seen in primary B-ALL, where B-ALL alone does not express much
phosphorylated p65 but the phosphorylation increased upon CAR T exposure. We further
identified that chemotherapy does not induce cIAP2 in primary B-ALL, suggesting that cIAP2
overexpression is specific to anti-CD19 CAR T therapy (Supplementary figure 3.1 a-g).
Furthermore, we determined if in vivo CAR T treatment also induces cIAP2 expression. To test
this, we injected 50,000 of primary B-ALL LAX56 and injected anti-CD19 CAR T or NT control on
day 4 post injection of B-ALL. After 48hours of T cell injection, mice were sacrificed and bone
marrow was collected from femur. After processing, each BM sample were stained for anti-CD79a
(brown) as a B-cell marker and for cIAP2 (red). As a result, we observed brown cells in LAX56
treated with NT bone marrow and red cells in LAX56 treated with CAR T bone marrow (Figure
3.1 i, j). This data confirms that anti-CD19 CAR T treatment induces cIAP2 in primary B-ALL in
both in vitro and in vivo. Next, we screened cIAP2 expression in post-chemotherapy relapsed B-
ALL and post-CAR T relapsed B-ALL patient samples. cIAP2 was not observed in any of the post-
chemotherapy relapsed B-ALL patient samples as we expected, yet it was also not expressed in
post-CAR T relapse B-ALL except for one sample out of nine cases (Supplementary figure 3.2).
Taken together, we conclude that anti-CD19 CAR T cell induces cIAP2 expression in primary B-
ALL both in vitro and in vivo, and the expression is limited to small time window where the cells
are exposed to anti-CD19 CAR T. Primary B-ALL cells that express cIAP2 protein following CAR
T exposure also increase phosphorated p65 protein, which may potentially provide survival
benefit.
62
3.3.2 TNF-ɑ secreted by anti-CD19 CAR T induces cIAP2 expression in
B-ALL cells
TNF-α is known to support HSC survival by inducing cIAP2 protein [320]. First, we identified
whether anti-CD19 CAR T co-cultured with primary B-ALL on OP9 produces TNF-α. After 24
hours of co-culture, anti-CD19 CAR T co-cultured with target primary B-ALL LAX56 on OP9
significantly increased TNF-α concentration in medium (Figure 3.2a). We further determined if
TNF-α induces cIAP2 in primary B-ALL and B-ALL cell lines by treating with 2 different doses of
TNF-α, 1ng/mL based on ELISA from Figure 2a and another dose that is ten folds higher than
what is observed. Annexin V/ 7AAD flow cytometry revealed that each cell responds differently to
TNF-α (Figure 3.2b). For example, primary B-ALL LAX7R and BLQ5 were more sensitive to TNF-
α compare to LAX56 and HEM310, and overall cell lines were not responding to TNF-α much
compared to primary B-ALL. Cell lysates from each cell showed cIAP2 is induced upon TNF-α
exposure (Figure 3.2c) and cIAP2 was more expressed in cells that were more sensitive to the
treatment. This is different from cIAP2 expression pattern in primary B-ALL and B-ALL cell lines
upon CAR T exposure, which suggests there might be more factors contributing to cIAP2
expression in addition to TNF-α. Then we chose the TNF-α resistant primary B-ALL and
determined whether TNF-α inhibition with monoclonal antibody Adalimumab diminishes cIAP2
expression. To maximize cIAP2 induction, we treated LAX56 and HEM310 with 0.1% BSA or
higher dose of TNF-α (10ng/mL) and added 2 different doses of Adalimumab to each condition.
Western blot analysis showed 10ng/mL of TNF-α increased cIAP2 expression in both LAX56 and
HEM310, and addition of Adalimumab reversed the expression (Figure 3.2e, g). As a summary,
TNF-α secreted by anti-CD19 CAR T upon co-culture with target B-ALL cells contributes to cIAP2
expression on primary B-ALL cells.
63
3.3.3 Overexpression of cIAP2 in primary B-ALL delays anti-CD19 CAR
T mediated apoptosis in vitro
In order to determine the role of cIAP2 in primary B-ALL, we transduced primary B-ALL LAX56
and HEM310 with lentivirus carrying BIRC3 overexpression vector (Figure 3.3a, 3.3b). Overall,
LAX56 had higher transduction efficiency compared to HEM310 both in mRNA and protein level
(Figure 3.3a-3.3d). Then we treated Empty control and BIRC3 overexpressing primary B-ALL
cells with no T, NT or anti-CD19 CAR T cells to see if cIAP2 overexpression affects survival of B-
ALL cells towards CAR T. In both LAX56 (Figure 3.3e) and HEM310 (Figure 3.3g), BIRC3
overexpressed B-ALL cells were more resistant to anti-CD19 CAR T cells after 24 hours of
treatment. Interestingly, BIRC3 OE-LAX56 cell viability was significantly higher after NT treatment
as well. Resistance toward CAR T in BIRC3 overexpressed cells corresponded to how much of
the actual protein is translated in the cells after transduction (Figure 3.3c, 3.3d). In conclusion,
cIAP2 protein expression in primary B-ALL cells delays anti-CD19 CAR T mediated apoptosis in
vitro.
3.3.4 BIRC3 overexpression in primary B-ALL increases
phosphorylation of p65 in the cytoplasm and PD-L1 expression
on the surface after anti-CD19 CAR T treatment
To further dissect the mechanism how overexpression of cIAP2 confers initial resistance to CAR
T, we used NF-κB proteome array to identify if any of NF-κB related proteins are differentially
expressed between Empty-LAX56 and BIRC3 OE-LAX56. NF-κB proteome array revealed
overexpression of many NF-κB proteins, including phosphorylated p65 and total p65 compared
to the control (Figure 3.4a, 3.4b, supplementary figure 3.3). We further validated if increased p65
induces NF-κB target genes in BIRC3 OE-LAX56. PD-L1, also known as CD274, is one of the
64
genes transcribed upon NF-κB p65 nuclear translocation [320]. PD-L1 is especially important in
context of CAR T therapy as PD-L1 expressed on tumor cells can lead to immune suppression
when it binds to PD-1 receptor expressed on activated T cells. In both LAX56 (Figure 3.4c) and
HEM310 (Figure 3.4d), BIRC3 overexpession led to PD-L1 expression on higher percentage of
cells after 24hours of anti-CD19 CAR T therapy. In general, higher percentage of HEM310
expressed PD-L1 on the surface compared to LAX56, probably because this sample is CAR T
refractory B-ALL which might already have an alternative mechanism to express PD-L1 on the
surface rapidly.
3.3.5 shRNA vector transduced knockdown and CRISPR/Cas9
mediated knockout of BIRC3 sensitizes primary B-ALL cells
toward anti-CD19 CAR T therapy
In order to determine the effect of cIAP2 inhibition in B-ALL against CAR T, we generated BIRC3
knockdown (KD) LAX56 with lentivirus carrying BIRC3 shRNA vector and BIRC3 knockout KO
LAX56 using CRISPR/Cas9. Flow cytometry sorted and puromycin selected BIRC3 KD-LAX56
showed significant decrease of BIRC3 mRNA (Figure 5a) and about 40% reduction in protein
level (Figure 3.5b, 3.5c). When treated with no T cells, NT and anti-CD19 CAR T, BIRC3 KD
LAX56 cells were significantly more sensitive to CAR T therapy than negative control LAX56
transduced with empty vector (Figure 3.5d). BIRC3 KD did not affect the basal viability of B-ALL
cells and did not sensitize cells toward NT cell. CRISPR/Cas9 mediated KO of BIRC3 in LAX56
decreased almost 50% of mRNA (Figure 3.5e). Unlike shRNA LV transduction, CRISPR/Cas9
KO cells were not able to be enriched and this may have caused higher mRNA level in KO cells
reflecting mRNA isolated from both knocked-out and not knocked-out population. However,
CRISPR/Cas9 mediated KO yielded much lower cIAP2 protein level (Figure 3.5f and 3.5g).
Interestingly, BIRC3 KO-LAX56 cells had significantly lower basal viability, which caused
65
significantly reduced viability after NT and CAR T treatment. Phopho-flow revealed that BIRC3
KD impaired phosphorylation of p65 in B-ALL cells when they are treated with anti-CD19 CAR T
(Figure 3.5i). This result suggests NF-κB pathway mediated survival of B-ALL is impaired when
insufficient cIAP2 is available, thereby directing TNF-α/TNFR1 receptor mediated signal towards
apoptosis.
3.3.6 SMAC mimetic LCL161 mediated inhibition of cIAP1 and cIAP2
sensitizes B-ALL cells towards anti-CD19 CAR T treatment
Recently, small-molecule drug screening has revealed SMAC mimetic as a sensitizer that
enhances CAR T cytotoxicity [321]. CRISPR screening picked FADD and TRAIL-R2 receptor as
the important mediators of CAR-T cell induced cytotoxicity, and SMAC mimetics additionally
augments apoptotic signal by sensitizing TNF/TNFR1 initiated signal. In order to verify this in our
primary B-ALL model, we have treated primary B-ALL cells with 3 increasing doses of SMAC
mimetics, AT406 and LCL161 in combination with T cells. First, LAX56 cells were treated with no
T, NT or CAR T cells in combination with three different doses of AT406, 0.1, 1 and 10nM (Figure
3.6a). AT406 alone, even at the lowest concentration, was sufficient to significantly lower the
viability of B-ALL cells. LAX56 cells treated with NT in combination with AT406 also further
decreased viability compared to LAX56 cells treated with NT cells alone. Combination treatment
of anti-CD19 CAR T and AT406 significantly lowered the viability of B-ALL cells compared to anti-
CD19 CAR T monotherapy, yet the lowest concentration of the drug yielded the lowest viability.
Viability of LAX56 recovered as higher concentration (1 and 10nM) of AT406 was combined with
anti-CD19 CAR T. This assay was repeated with another SMAC mimetic, LCL161. In LAX56,
LCL161 alone and LCL161 combined with NT were not enough to induce significant decrease of
the viability (Figure 3.6b). Also, lower concentration of LCL161 (1 and 10nM) did not sensitize B-
ALL cells toward anti-CD19 CAR T, yet the highest concentration of LCL161 at 100nM sensitized
66
LAX56 cells toward anti-CD19 CAR T. AT406 and LCL161 combination assay was repeated in 2
more primary B-ALL cases (BLQ5 and HEM310) and 2 cell lines (NALM6 and RCH-ACV)
(Supplementary figure 3.4), and the results were consistent in most of the cases where
combination of SMAC mimetic with anti-CD19 CAR T sensitized cells toward the therapy. The
only exception was NALM6 and RCH-ACV cell lines that were treated with AT406 in combination
with T cells, where combination of 3 different doses of AT406 and anti-CD19 CAR T did not
produce any synergistic effect (Supplementary figure 3.4 c,d). Based on better efficacy of the drug
and effectiveness in cell lines, we further studied how LCL161 provides synergistic effect in B-
ALL. First, we treated cells and isolated cytoplasmic proteins to identify the on-target effect of the
drug on IAP proteins, cIAP1, cIAP2 and XIAP (Figure 6d). Increased dose of LCL161 in LAX161
successfully downregulated cIAP1 and cIAP2, although cIAP2 was lowly expressed. XIAP
intensity was not changed in LAX56 after LCL161. Combination of LCL161 with NT also
decreased the level of cIAP1 in dose dependent manner, whereas cIAP2 did not decrease. XIAP
protein slightly decreased for 1 and 10nM of LCL161 with NT but bounced back up with the highest
concentration. When LAX56 cells were treated with anti-CD19 CAR T in combination with LCL161,
cIAP1 protein was degraded in dose dependent manner yet addition of LCL161 did not completely
degrade the protein as effectively as in LAX56 alone or with NT. cIAP2 protein level was only
decreased at the highest concentration (Figure 6e). XIAP protein level remained the same with
1nM, but was higher when 10 and 100nM of LCL161 in addition to the anti-CD19 CAR T. This
result shows that decrease of cIAP1 protein alone is not sufficient to sensitize primary B-ALL
toward anti-CD19 CAR T. However, decrease of cIAP2 at the highest concentration of LCL161
provided sensitization of the cells toward CAR T despite increased XIAP.
67
3.3.7 Evaluation of cIAP2 inhibition in combination with anti-CD19 CAR
T in vivo
Next, we evaluated the effect of cIAP2 inhibition in vivo by using BIRC3 KD-LAX56 in NSG mice.
First, we determined whether BIRC3 KD affects homing of the B-ALL cells. On day 0, we injected
50,000 of control or BIRC3 KD cells into NSG mice. After 4 days, we ran flow cytometry on BM
samples from each mouse to detect human B-ALL. As expected, BIRC3 KD did not reduce the
homing capability of the B-ALL possibly because primary B-ALL cells do not express cIAP2
without any stimulation and thus are not actively utilizing the protein for biological functions (Figure
3.7b). After confirming that BIRC3 KD does not affect homing of B-ALL cells by day 4, we injected
the primary B-ALL and treated with NT or anti-CD19 CAR T on day 4. All NT treated mice,
including control and BIRC3KD LAX56, had to be euthanized on day 30 or 32 due to their weight
loss and discomfort they were experiencing from the disease progression. There was no survival
benefit of NT treatment compared to no treatment (Figure 3.7c). Interestingly, BM cells harvested
from no T cell treated group showed increased CD19 median fluorescence intensity (MFI) in
BIRC3 KD-LAX56 cells compared to the control (Figure 3.7d). NT treated LAX56 or BIRC3 KD-
LAX56 mice bone marrow was full of mCD45-hCD3- human leukemia cells and almost no CD3
positive T cells were observed. B-ALL cells in the BM of the mice treated with NT expressed low
level of PD-L1 and still retained CD19 molecule on their surface suggesting non-efficient clearing
of leukemia by T cells in the BM. Anti-CD19 CAR T injection into human leukemia engrafted NSG
mice prolonged survival of these mice compared to NT control.
3.4 Discussion
cIAP2 is inhibitor of apoptosis (IAP) protein that protects cells from undergoing apoptosis and thus
provide survival benefit. Its role has not been much elucidated compared to other IAP proteins
such as cIAP1 or XIAP. In this paper, we studied the role of cIAP2 in primary B-ALL during anti-
68
CD19 CAR T cell therapy. In five primary B-ALL cells, cIAP2 was overexpressed after 24hours of
treatment with anti-CD19 CAR T cells. cIAP2 was also upregulated in three of the B-ALL cell lines,
yet overall signal was stronger in primary B-ALL. This suggests stroma-dependency of primary
B-ALL cells may affect the amount cIAP2 activation, but it has not been verified since we have
not identified which surface molecule is responsible. Other anti-apoptotic proteins Bcl-2, cIAP1
and XIAP were already expressed without any T cell stimulation. Level of each protein was
maintained despite the treatment in most of the cases, except for HEM207. In HEM207, which is
a pre-CAR T sample of HEM310, Bcl-2, cIAP1, XIAP and Survivin level decreased with both NT
and anti-CD19 CAR T. This patient did not respond to CAR T therapy and eventually relapsed
with CD19 positive B-ALL, HEM310. Survivin protein is also maintained similarly throughout the
treatment yet is upregulated in some of the cases (BLQ5, HEM310, NALM6) and downregulated
in other (LAX56, HEM207). We also show TNF-α is secreted by anti-CD19 CAR T cells in the
presence of target B-ALL and TNF-α induces apoptosis in B-ALL. However, some of the cells
(LAX7R, BLQ5) were more sensitive to TNF-α compared to the rest of the primary B-ALL and cell
line. Also, NALM6 did not induce cIAP2 with 2 different doses of TNF-α. NALM6 does not strongly
upregulate cIAP2 in the presence of anti-CD19 CAR T cells either, yet it still overexpresses. This
result suggests there may be more factors besides TNF-α that contribute to the cIAP2
upregulation in B-ALL, which we have not identified yet. Also, TNF-α induced cIAP2
overexpression pattern is different from cIAP2 expression pattern induced by anti-CD19 CAR T,
suggesting there may be more factors regulating the expression of cIAP2 in primary B-ALL
exposed to CAR T cells.
Overexpression of cIAP2 in primary B-ALL LAX56 and HEM310 provided delayed
response toward anti-CD19 CAR T therapy. Resistance toward anti-CD19 CAR T correlated how
much mRNA was present in the cells after transduction, and LAX56 was more resistant to CAR
T compared to HEM310. However, even small increase of cIAP2 in HEM310 was enough to
provide resistance to CAR T, suggesting the importance of the protein in CAR T resistance.
69
We suggest delayed CAR T response in BIRC3 overexpressed primary B-ALL is due to
upregulation of NF-κB mediated transcription of pro-survival genes. Especially we determined
p65 (RelA) as one of the NF-κB proteins responsible for this resistance. Activation of p65 is
particularly interesting in context of CAR T therapy because it is known to induce expression of
PD-L1 in the tumor cells. Indeed, we identified significant overexpression of PD-L1 on the surface
of BIRC3 overexpressed B-ALL cells compared to the control. Another PD-1 ligand, PD-L2, was
not observed (data not shown). Overexpressed PD-L1 on leukemia can interact with PD-1 on
activated T cells and in turn suppresses T cell activity. For this reason, combination of PD-1
antibody with CAR T therapy has been studied in hematological malignancies and tumors [322-
325]. However, PD-1 inhibition also has been shown to cause regression of the disease [326],
therefore combination of checkpoint inhibition with CAR T still needs to be studied and optimized
for patient selection and timing of administration of the drugs and CAR T cells. Here we suggest
targeting cIAP2 as an alternative method to inhibit PD-1/PD-L1 interaction by decrease the
expression of PD-L1 on the surface of B-ALL. To achieve this, we have generated BIRC3 KD and
KO primary B-ALL. Knockdown of BIRC3 gene by BIRC3 shRNA containing lentivirus
transduction resulted in significant reduction in BIRC3 mRNA but reduced only 40% of the protein.
However, small reduction of the protein was sufficient to sensitize B-ALL cells toward anti-CD19
CAR T therapy in vitro. BIRC3 KD repressed phosphorylation of p65 upon anti-CD19 CAR T, and
as a result PD-L1 expression on the surface of the cells decreased. Reduction of PD-L1 on the
surface of the leukemia cells will lead to decreased interaction of PD-1/PD-L1, and which can help
maintaining T cell activation for prolonged time and efficient clearing of leukemia. CRISPR/Cas9
mediated KO of BIRC3 in primary B-ALL significantly reduced mRNA in the cells, but not as much
as KD. However, cIAP2 signal was much lower in KO cells compared to KD. We cannot explain
what exactly is causing this deviation, yet we suspect the difference might come from the
enrichment of the cells. KD cells were able to be sorted after transduction based on their GFP
signal, whereas BIRC3 KO cells were not enriched because they do not have any distinguishable
70
target. High purity of KD cells leads to lower mRNA, but maybe shRNA is not effective enough to
prevent small amount of mRNA being translated into protein. In contrast, mRNA of BIRC3 is
higher because cell extracts are from both successfully KO and cells that were not KO. However,
gene knockout leads to complete abortion of transcription of the gene, therefore retaining lower
protein level overall. There are several pre-clinical IAP targeting compounds. These compounds
mimics SMAC (second mitochondria-derived activator of apoptosis, thus called SMAC mimetics)
and antagonizes IAP proteins to promote apoptosis in cells. AT406 and LCL161 are monovalent
SMAC mimetics and we showed LCL161 successfully degraded cIAP1 and decreased cIAP2 in
our primary B-ALL. As a result, combination of AT406 or LCL161 with anti-CD19 CAR T cell has
sensitized primary B-ALL cells toward anti-CD19 CAR T in vitro. We would like to point out that
the lower dose of AT406 was better than the higher dose, whereas LCL161 was only effective at
the highest dose. This explains why our in vivo LCL161 study did not show survival benefit of
mice treated with combination therapy (Supplementary figure 3.5). LCL161 dose used in our in
vivo study was not enough to induce degradation of the protein and did not provide the full effect
in primary B-ALL engrafted mice. Therefore, we suggest further investigation of the drug for
appropriate dose, and we expect maximum efficacy of the treatment when proper amount of
SMAC mimetic is combined with CAR T.
71
Figure 3.1. anti-CD19 CAR T treatment induces overexpression of cIAP2 and increases
phosphorylation of p65 at serine 536 in B-ALL cells 10x10
6
of primary B-ALL (a) LAX7R (b)
72
LAX56 (c) BLQ5 (d) HEM207 (e) HEM310 and cell lines (f) NALM6 (g) RCH-ACV and (h) supB15
were cultured on OP9 overnight. The next day, leukemia cells were treated without any T cells,
with non-transduced (NT) or anti-CD19 CAR T (19) in 1:10 effector to target ratio for 24hours. B-
ALL cells were separated from T cells by negative sorting with anti-CD3 magnetic beads. Cell
lysates from CD3 negative sorted B-ALL cells were used for detection of anti-apoptotic proteins
Bcl-2, cIAP1, cIAP2, XIAP and survivin, and phosphorylated p65 (Ser536) and total p65. Bone
marrow from LAX56 engrafted mice treated with (i) non-transduced T cells (NT) or (j) anti-CD19
CAR T cells were stained for CD79a (brown, 1:150) and cIAP2 (red 1:900). Each slides are
scanned with 40x magnification.
73
74
Figure 3.2. TNF-α induces cIAP2 expression in B-ALL cells (a) TNF-α secreted into the growth
medium by anti-CD19 CAR T was measured using ELISA assay. 5x10
6
of primary B-ALL (b)
LAX7R, LAX56, BLQ5, HEM310 and cell lines NALM6 and RCH-ACV were treated with 1ng/mL
or 10ng/mL of TNF-α for 24 hours and viability was assessed by Annexin V/ 7AAD flow cytometry.
Cell lysates from each conditions from (b) were collected and used for detection of cIAP2 (c). (d)
LAX56 and (f) HEM310 were treated with or without 10ng/mL of TNF-α. TNF-α inhibitor
Adalimumab was added 1μg/mL or 10μg/mL to each group of cells. Viability of cells was assessed
via 7AAD/Annexin V flow cytometry after 24 hours. Cell lysates from (e) LAX56 and (g) HEM310
were collected and used for detection of cIAP2 via Western Blot.
75
Figure 3.3. Overexpression of BIRC3 protects primary B-ALL from anti-CD19 CAR T
76
Quantification of BIRC3 mRNA in Empty and BIRC3 overexpressed (a) LAX56 and (b) HEM310.
Quantification of cIAP2 protein in Empty and BIRC3 overexpressed (c) LAX56 and (d) HEM310.
3x10
6
of Empty and BIRC3 overexpressed LAX56 cells were cultured in 10cm
2
cell culture dish
coated with OP9 cells and treated with 1:10 effector to target ratio. Cells were harvested after 24
hours and viability was assessed by Annexin V/7AAD staining via flow cytometry (e, g).
Representative Annexin V/7AAD dot plots are shown (f, h)
77
Figure 3.4. BIRC3 overexpression in B-ALL increases p65 and surface PD-L1 expression
upon anti-CD19 CAR T treatment 10x10
6
of primary B-ALL Empty-LAX56 and BIRC3 KD-LAX56
were cultured on OP9 overnight. Then the cells were treated with non-transduced (NT) or anti-
CD19 CAR T (19) in 1:10 effector to target ratio for 24hours. Cell lysates from magnetic beads
sorted B-ALL cells were used for phosphorylated p65 and total p65 protein detection via Western
Blot. (a) Empty and BIRC3 overexpressed LAX56 cells were cultured as described above, then
protein lysates were used for NF-κB proteome array. Surface expression of PD-L1 in Empty and
BIRC3 overexpressed (c) LAX56 and (d) HEM310 was assessed by flow cytometry
78
79
Figure 3.5 continued
Figure 3.5. BIRC3 knockdown and knockout sensitizes B-ALL cells toward anti-CD19
CAR T therapy (a) quantification of BIRC3 mRNA in negative control and shRNA-mediated
BIRC3 knockdown LAX56 (b) TNF-α mediated induction of cIAP2 protein in negative control and
BIRC3 knockdown LAX56 and (c) quantification of cIAP2 signal over β-actin. 3x10
6
of negative
control and BIRC3 KD LAX56 cells were cultured in 10cm2 cell culture dish coated with OP9
80
cells and treated with (d) 1:10 effector to target ratio for 24 hours. Cells were harvested and
viability was assessed by Annexin V/ 7AAD staining via flow cytometry. (e) quantification of
BIRC3 mRNA in negative sgRNA control and CRISPR/Cas9 mediated KO LAX56 (f) TNF-α
mediated induction of cIAP2 protein in negative control and BIRC3 KO LAX56 and (g)
quantification of cIAP2 signal over β-actin. (h) 3x10
6
of negative control and BIRC3 KO LAX56
cells were cultured in 10cm2 cell culture dish coated with OP9 cells and treated with 1:10
effector to target ratio for 24 hours and the viability was accessed via Annexin V/ 7AAD
flowcytometry. (i) BIRC3 KD cells and the control cells were cultured as the same condition
mentioned above, then collected and stained for intracellular phosphor-p65 (j) PD-L1 expression
on control and BIRC3 KD LAX56 cells were accessed by flow cytometry
81
Figure 3.6. SMAC mimetics AT406 and LCL161 sensitizes B-ALL cells towards anti-CD19
82
CAR T cells by downregulating both cIAP1 and cIAP2, but not XIAP Primary B-ALL LAX56
was treated with three different concentrations of SMAC mimetic (a) AT406 and (b) LCL161.
Viability of treated cells was accessed by Annexin V/ 7AAD flow cytometry and representative
plots are shown in (c). (d) Cell lysates from LAX56 treated with combination therapy of LCL161
and CAR T was used to verify the on-target effect of the drug on cIAP1, cIAP2 and XIAP
proteins. (e) Individual band intensity was divided by β-actin signal and normalized to 0nM of
LCL161 controls.
83
Figure 3.7. Evaluating BIRC3KD combined with anti-CD19 CAR T in vivo. (a) timeline for
the homing experiment (b) percentage of anti-mouse CD45-/anti-human CD19 positive B-ALL
cells homed to the bone marrow after 4 days of engraftment in NSG mice (c) Kaplan-Meier
curve of the in vivo experiment of BIRC3 KD engrafted mice treated with NT or anti-CD19 CAR
T (d) median fluorescence intensity of anti-human CD19 from leukemia cells recovered from
mice engrafted with Empty-LAX56 or BIRC3-KD LAX56 cells
84
Figure 3.8 Graphical abstract of cIAP2 mediated anti-CD19 CAR T resistance in B-ALL
Upon recognition of CD19 on target B-ALL cell, anti-CD19 CAR T cell initiates intracellular
signaling and secret TNF-ɑ. TNF-ɑ will bind to TNFR on B-ALL and in turn elicits TNF-TNFR
mediated extrinsic apoptotic pathway. However, in the presence of cIAP2, B-ALL cell can switch
the death signal into survival signal. Inhibition of cIAP2 by SMAC mimetics can prevent the
activation of survival promoting NF-κB signaling in B-ALL upon TNF-ɑ exposure.
85
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101
Appendices
Appendix A: Supplementary information for chapter 2
Table S2.1 Characteristics of B-ALL
102
Table S2.2 Antibodies’ information of Western blot (WB) or chromatin immunoprecipitation
(ChIP)
103
Table S2.3 Flow cytometry Antibodies information
104
Supplementary figure S2.1. EC50 curve and value of SF2535. Apoptosis cells (Annexin V +)
percentage of (a, d) LAX56, (b, e) LAX7R and (c,f)TXL3 in DMSO(D), 0.04μM, 0.2μM, 0.5μM,
1μM, 5μM, or 10μM SF2535 for 48h were analyzed for calculation of EC50. The value and 95%
confidence intervals of EC50 of (a-c) were inserted in the curve graphs. (d-f) showed the actual
apoptosis percentage of DMSO and SF2535 in white, gradient red, respectively. Apoptosis assay
was performing in triplicate using flow cytometry.
105
Supplementary figure S2.2. Independent repeated experiments data supports
downregulation of c-Myc and p-AKT by SF2535. (a) and (b) c-Myc expression by Western Blot
for Figure 1d and 1e. (c) Schema of phosphorylated AKT (Ser473) (p-AKT
S473
) detection assay.
(d) p-AKT
S473
expression by Western Blot for Figure 1f.
106
Supplementary figure S2.3. SF2535 changes cell cycles. (A) Representative cell cycle gating
strategy, a sample from TXL3 with 5μM SF2535 treatment. Following treatment of DMSO or
SF2535 (0.2μM, 1μM, 5μM) in (B, E) LAX56, (C, F) LAX7R, and (D, G) TXL3 for 24 hour, cell
cycles were analyzed by flow cytometry. Representative histogram of cytophase were showed in
(B-D), while mean ± SD of two independent experiments showed in (E-G).
107
Supplementary figure S2.4. SF2535 prolongs G0+G1 phase arrest and attenuates S phase.
Following treatment of DMSO or SF2535 (0.2μM, 1μM, 5μM) in (A, B) LAX56, (C, D) LAX7R, and
(E, F)TXL3 for 24 hour, cell cycles were analyzed by flow cytometry. (B, D, F) Representative dot
plots of DMSO control (up) and SF2535 5 μM (down) were showed. Experiments were performed
in triplicates. P-value *<0.05: Comparing S phase compared to DMSO S phase. P-value #<0.05:
Comparing G0+G1 phase to DMSO G0+G1 phase.
108
Supplementary figure S2.5. SF2535 affects adhesion molecules. (A) Representative gating
strategy for assessing adhesion molecules in LAX7R treated with SF2535 5μM. Representative
dot plots for integrin α4, α5, α6, β1, and CXCR4 expression for (B) LAX56, (C) LAX7R and (D)
TXL3 cells are treated with DMSO(in blank bars) or SF2535 (0.2μM, 1μM, 5μM in gradient red
bars) for 24 hours. Relative fold changes of mean fluorescence intensity (MFI) of integrin α4, α5,
α6, β1, and CXCR4 for (E-I) LAX56, (J-N) LAX7R and (O-S) TXL3. P-value *<0.05. The results
109
are combination of at least two independent triplicate experiments.
Supplementary figure S2.6. SF2535 moderately inhibits B-ALL adhesion to OP-9 cells. (A)
Schema of adhesion assays. (B) LAX56, (C) LAX7R, and (D) TXL3 cells were treated with
DMSO (D) or SF2535 for 24 hours. After harvest and wash, 0.2×106 alive cells per well were
placed on pre-seeded OP9 cells plate for 2 hours. Adhesion and supernatant cells were counted
by Trypan Blue exclusion. Percentage (%) of alive adhesion cells were presented. Experiments
were performed in triplicates. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 compared to
DMSO group.
110
Supplementary figure S2.7. SF2535 decreases peripheral leukemia burden in primary B-
ALL engrafted mice. (A) Schematic for time flow for the experiment. After3 weeks of 1×106
cells (LAX56) injection per NSG mouse, mice were treated with vehicle or SF2535 (30mg/kg)
(n=6 each). Bone marrow (BM), spleen (SPC), and peripheral blood (PB) were harvested from
each mouse after 24 hours. Percentages of human CD45 and human CD19 double positive
primary B-ALL cells in (B) BM, (C) SPC, and (D) PB are assessed by flow cytometry after
staining with anti-mouse CD45, anti-human CD45 and anti-human CD19. Ns, not significant. P-
value *<0.05.
111
Appendix B: Supplementary information for chapter 3
Supplementary Table S3.1 Characteristics of primary B-ALL
112
Supplementary Table S3.2 Flow cytometry antibody information
113
Supplementary Table S3.3 Western blot antibody information
114
Supplementary figure 3.1 cIAP2 is not induced with chemotherapy in primary B-ALL
3x10
6
of primary B-ALL (a) LAX7R (b) LAX56 (c) BLQ5 (d) HEM310 and cell lines (e) NALM6
and (f) RCH-ACV were treated with combination of Vincristine and Dexamethasone (VD) for or
Nilotinib (NTB). Viability of cells were accessed with Annexin V/ 7AAD flow cytometry after
24hours of chemotherapy. (g) Cell lysates from each cell were used for detection of cIAP1,
cIAP2, XIAP, Survivin and β-actin.
115
Supplementary figure 3.2. cIAP2 is not retained in post-CAR T relapsed B-ALL 9 post-
chemotherapy refractory/relapse patient samples and 9 post-CAR T refractory/relapse patient
116
samples were screened for their cIAP2 level via Western blot.
Supplementary figure 3.3 NF-κB proteome array of Empty and BIRC3 overexpressed
LAX56 Mean pixel intensity of proteins that were differentially expressed in BIRC3
overexpressed LAX56 compared to Empty control.
117
118
Supplementary 3.4 Combination of AT406/ LCL161 with anti-CD19 CAR T in vitro in
primary B-ALL and cell lines Primary B-ALL BLQ5 and HEM310 and cell lines NALM6 and
RCH-ACV were treated with three increasing doses of (a-d) AT406 or (e-h) LCL161 in
combination with NT or anti-CD19 CAR T cells
Supplementary figure 3.5. Low dose of LCL161 in combination with anti-CD19 CAR T
does not prolong survival of NSG engrafted with primary B-ALL (a) schematics for the
experiment (b) Kaplan-meier survival curve from the experiment (c) Bioluminescent imaging of
primary B-ALL LAX56 engrafted mice before injection of T cells (top row) and after 23 days of B-
ALL injection, which is day 19 post injection of T cells.
Abstract (if available)
Abstract
Although the 5-year survival rate of pediatric leukemia patients has exceeded 85%, relapse and refractory disease remain a problem. Cell adhesion-mediated drug resistance (CAM-DR) in leukemia has been proposed for a long time. Interaction between leukemia cells surrounding microenvironment elicits pro-survival intracellular signaling in B-ALL, thus providing drug resistance. Chimeric antigen receptor T (CAR T) therapy has shown promising results in pediatric patients with relapsed leukemia, yet recurrence of the disease after CAR T therapy represents an obstacle to overcome in the immunotherapy of B-ALL. We identified upregulation of cIAP2 protein in primary and immortalized B-ALL cells after anti-CD19 CAR T treatment, which can serve as a biological target to increase anti-CD19 CAR T treatment efficacy. Increased cIAP2 in primary B-ALL correlated with increased phosphorylation of p65. In primary B-ALL, overexpression of BIRC3 (cIAP2) delayed apoptosis after 24hours of anti-CD19 CAR T treatment in vitro. Furthermore, BIRC3 overexpressed primary B-ALL presented a higher percentage of PD-L1 on their surface after CAR T treatment. To determine whether inhibition of cIAP2 sensitizes B-ALL cells toward CAR T therapy, we have generated BIRC3 knocked-down cells with shRNA lentivirus as well as CRISPR/Cas9 mediated BIRC3 knocked-out primary B-ALL. Both KD and KO cells decreased cIAP2 protein level and were more sensitive to CAR T therapy in the first 24hours of treatment. Taken together, we suggest cIAP2 as a biological target that can increase CAR T efficacy in treating patients with relapsed/refractory leukemia.
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Kim, Hye Na
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Therapeutic resistance in acute lymphoblastic leukemia: cIAP2 inhibition sensitizes B cell acute lymphoblastic leukemia to anti-CD19 chimeric antigen receptor T cell
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Keck School of Medicine
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Cancer Biology and Genomics
Degree Conferral Date
2021-12
Publication Date
11/22/2022
Defense Date
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