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Sensitization of diffuse large B-cell lymphoma to chemotherapy by targeting autophagy

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Sensitization of diffuse large B-cell lymphoma to chemotherapy by targeting autophagy

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Content 1

Sensitization of Diffuse Large B-Cell Lymphoma to Chemotherapy
by Targeting Autophagy

By Livia Taitano

Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the
Requirements for the Degree
MASTER OF SCIENCE
Molecular Microbiology and Immunology

Advisor: Dr. Shou-Jiang Gao, PhD

August 2018

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ACKNOWLEGMENTS

I would like to express my thanks and appreciation to my adviser Dr. Shou-Jiang Gao for his
encouragement and guidance towards research and writing this thesis. Your advice on both
research and my career has been very important to me.

I would also like to thank my other thesis committee members, Dr. Chengyu Liang and Dr.
Weiming Yuan, for their guiding comments and suggestions. My best regards to all member of
Dr. Gao’s lab for being a supportive team.

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TABLE OF CONTENTS
LIST OF FIGURES…………………………………………………………………………….….4
ABSTRACT……………………………………………………………………………………… 5
BACKGROUND………………………………………………………………………………..... 6
Diffuse Large B-Cell Lymphoma…………………………………………………..…...…….. 6
Doxorubicin ………………………………………………………………………...…...……. 7
Autophagy……………………………………………………………………....…..…...……. 9
Autophagy Inhibitor: Chloroquine………………………………………………….…..……. 11
RESULTS……………………………………………………………………………..….….…. 14
Chloroquine synergizes with Doxorubicin to inhibit cell proliferation of DLBCL cells ...… 14
Chloroquine synergizes with Doxorubicin to increase induced apoptosis…....…………...…. 17
Doxorubicin induces autophagic flux in DLBCL cells…………………………….……..…. 20
Western Blot Analysis of P62/SQSTM1 and LC3…………………………………........ 21
Immunofluorescence Staining of P62/SQSTM1 and LC3…………………..……..…… 25
DISCUSSION………………………………………………………………………….….…… 31
FUTURE DIRECTIONS……………………………………………………………….…..…... 35
MATERIALS AND METHODS…………………………………………………………..…… 36
Annexin V staining………………………………………………………………….….……. 36
Cell Culture………………………………………………………………………….….……. 36
Immunofluorescence Assay………………………………………………………….….…… 36
Western Blot analysis……………………………………………………………….….……. 37
Statistical Analysis………………………………………………………………………...… 37
REFERENCES………………………………………………………………………….………. 38

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LIST OF FIGURES
Figure 1. Anatomy of Autophagy………………...………………………………………………10
Figure 2. Growth Curves: DLBCL treated with Doxorubicin and Chloroquine .............................14
Figure 3. Annexin V- DAPI staining for Apoptosis……………………………………...……17,18
Figure 4. Western Blot Analysis of P62 and LC3………………………………………………...21
Figure 5. OCI-LY1 Cells Immunofluorescence Staining for P62 and LC3………….…………....25
Figure 6. RIVA Cells Immunofluorescence Staining for P62 and LC3...………………………...27
Figure 7. U2932 Cells Immunofluorescence Staining for P62 andLC3 …….……………....……29

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ABSTRACT
Diffuse large B-cell lymphoma (DLBCL) is a malignant cancer originating from B-cells.
It is the most common B-cell tumor in adults and is categorized as one of the most aggressive
non-Hodgkin lymphomas accounting for 30% to 40% of all newly diagnosed cases of NHL. The
most common treatment for DLBCL is R-CHOP, which is a combination therapy including
rituximab (a monoclonal antibody); three chemotherapy agents: cyclophosphamide,
Doxorubicin, and vincristine; and prednisone (a steroid). The five-year overall survival rate for
older adults is ~60%. Autophagy is a cell “self-eating” process, a form of cell recycling.
Autophagy degrades damaged proteins and organelles for cell use and is a survival mechanism in
the case starvation. Inhibition of autophagy in tumor cells has been shown to enhance the
efficacy of anticancer drugs. Doxorubicin is the main cytotoxic ingredient in CHOP/R-CHOP.
The goal of this project is to determine if inhibition of autophagy can sensitize DLBCL to
Doxorubicin treatment. This project has demonstrated that autophagy inhibitor Chloroquine
synergizes with Doxorubicin to inhibit cell proliferation and induce apoptosis in DLBCL cell
lines.

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BACKGROUND
Diffuse Large B-Cell Lymphoma
Diffuse large B-cell lymphoma (DLBCL) is a malignant cancer originating from B-cells.
(1) It is the most common B-cell tumor in adults and is categorized as one of the most aggressive
non-Hodgkin lymphomas. (1) It accounts for 30% to 40% of all newly diagnosed cases of NHL.
Most people diagnosed with DLBCL are 65 or over, but it can occur at any age, including in
children. (1,3)
DLBCL can arise from both normal B cells or from a malignant transformation of other
types of lymphoma or leukemia. (1-5) This disease displays marked clinical heterogeneity.
Several distinct subtypes of DLBCL have been identified through gene expression profiling,
each subtype having a distinct clinical presentation and prognosis. (3,4) The usual treatment for
each of these is still chemotherapy, often in combination with an antibody targeted at the tumor
cells. The most common treatment is R-CHOP, which is a combination therapy including
rituximab (a monoclonal antibody); three chemotherapy agents: cyclophosphamide,
Doxorubicin, and vincristine; and prednisone (a steroid). The five-year overall survival rate for
older adults is ~60%. A third of cases are refractory or relapse after initial response. (3,4)
DLBCL gene expression profiling (GEP) studies have identified distinct molecular
subtypes. (2,3) Most DLBCL cases can be divided into three cell-of-origin (CCO) molecular
subtypes: germinal center B-cell-like (GCB), activated B-cell-like (ABC), or the intermediate
subtype, primary mediastinal B-cell lymphoma (PMBL). (2) The GCB subtype is the most
common. About 50 percent of DLBCL cases fall into this subtype, mostly in children and
younger patient cases. The ABC subtype accounts for approximately 30 percent of cases, usually
in patients of advanced age. (5) The PMBL subtype accounts for about 10 percent of all DLBCL
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cases and is commonly found in younger female patients. A few remaining cases of DLBCL do
not fall into any of these categories and are referred to as “unclassifiable.” (5)
The GCB subtype has the best prognosis with ~66% of treated patients surviving more
than five years. The ABC subtype exhibits an inferior outcome following R-CHOP (3-year
progression-free survival [PFS] of ∼40% vs 75%, P < .001) in comparison with the GCB
subtype. (4) A majority of refractory and/or relapse cases are ABC subtype. (3)

Doxorubicin
Doxorubicin (DOXO) is a common anthracycline chemotherapy drug used in the
treatment of DLBCL. Doxorubicin is the main cytotoxic ingredient in CHOP/R-CHOP.
Anthracyclines are active clinical agents that have multiple mechanisms of cytotoxicity. The
major limitation of anthracyclines’ therapeutic potential is the cardiotoxic effects. (7)
Doxorubicin ’s major anticancer effects occur through the inhibition of topoisomerase II and
generation of DNA double-strand breaks. (7) Subsequent TOP2A poison-mediated cytotoxicity
is postulated to involve the mismatch repair genes MSH2 and MLH1because loss of DNA
mismatch repair function results in resistance to Doxorubicin. (7,9) Doxorubicin activates the
DNA damage response (DDR) pathway in cancer cells, leading to p53 activation and apoptosis.
(9) The second cytotoxic mechanism of Doxorubicin, usually discussed in the context of
cardiotoxicity but also occurring in chemotherapy treated cancer cells, is oxidative stress caused
by reactive oxygen species (ROS) originating from damaged mitochondria. (7,8) Re-oxidation of
the DOXO-semiquinone radical back to Doxorubicin leads to the formation of ROS such as
hydrogen peroxide, causing oxidative stress, which can be deactivated by glutathione peroxidase,
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catalase and superoxide dismutase (25). Damaged organelles and reactive oxygen species,
associated with oxidative stress, can induce autophagy. (8)
Mai, etc. al. found that Doxorubicin has subtype-specific mechanisms of cytotoxicity in
DLBCLs, resulting from the differences in the subcellular distribution pattern. (10) They found
that, in a cell line model of ABC-DLBCL, Doxorubicin is often enriched in the cytoplasm and is
often excluded from the nucleus. As a result, Doxorubicin induced cytotoxicity in ABC-DLBCLs
is more often dependent on oxidative stress, rather than DNA damage response. They
corroborated these findings by gene signature analysis, demonstrating that basal oxidative stress
status predicts treatment outcome among patients with ABC DLBCL, but not patients with GCB-
DLBCL. (10)
To investigate the cause of inefficient DDR activation by Doxorubicin in ABC-DLBCL
cells, Mai, etc. al. examined the subcellular distribution of Doxorubicin. (10) These analyses
showed that in GCB-DLBCL cell lines, at least 60% of the DOXO signals were concentrated in
the nucleus. Among these, SUDHL5 had the lowest amount of nuclear DOXO (60%), correlating
with a weaker DDR response relative to other GCB lines. Among the 6 ABC-DLBCL cell lines
tested, only 2 had 60% or more DOXO signals in the nucleus. The 3 cell lines showing the least
concentration of nuclear DOXO (Ly3, Ly10, and Riva) were also the ones that were most
resistant to DOXO-induced DDR. (10) In comparison, the 3 cell lines showing 50% to 90%
nuclear DOXO signals had moderate (SUDHL2and HBL1) to strong (U2932) DDR activation.
Therefore, inefficient DDR activation by Doxorubicin in ABC-DLBCL cells is likely caused by
inadequate nuclear accumulation of the drug. (10)

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Autophagy
The term ‘autophagy’ is derived from the Greek meaning ‘eating of self’. It was first
coined by Christian de Duve over 40 years ago. There are three main defined types of autophagy:
macro-autophagy, micro-autophagy, and chaperone-mediated autophagy, all of which promote
proteolytic degradation of cytosolic components at the lysosome. (6,11) Macro-autophagy
delivers cytoplasmic cargo to the lysosome through a double membrane-bound vesicle, referred
to as an autophagosome, which fuses with the lysosome to form an autolysosome. In micro-
autophagy, cytosolic components are directly taken up by the lysosome itself through
invagination of the lysosomal membrane. Both macro-and micro-autophagy can engulf large
structures through both selective and non-selective mechanisms. In chaperone-mediated
autophagy (CMA), targeted proteins are translocated across the lysosomal membrane in a
complex with chaperone proteins (such as Hsc-70) that are recognized by the lysosomal
membrane receptor lysosomal-associated membrane protein 2A (LAMP-2A), resulting in their
unfolding and degradation.(11) Autophagy is similar to a cellular ‘recycling factory’ that also
promotes energy efficiency through ATP generation and mediates damage control by removing
non-functional proteins and organelles. (6) The basic mechanisms of Autophagy are described in
Figure 1.
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Figure 1: Anatomy of autophagy. Autophagy occurs through a multistep process including 4 control
points: 1. initiation, 2. nucleation, 3. maturation, and 4. delivery and degradation of AV contents. These
steps occur irrespective of whether autophagy has been induced through stress and/or ubiquitinated
substrate accumulation, or through starvation. During initiation, nascent AV membranes derived from
multiple potential sources (including isolated membranes, ER, or mitochondria outer membranes) form a
cup-like structure onto which autophagosomal machinery, including LC3, dynamically associates. As the
cup-like structure enlarges, it sequesters substrate, which includes ubiquitinated proteins or organelles in
the case of stress and/or substrate-induced autophagy, and soluble cytoplasm in the case of starvation-
induced autophagy. The double membrane comprising the nascent AV then closes to form the mature
AV, which then targets and fuses with the lysosome. In the lysosome, hydrolytic enzymes digest the
contents and inner membrane of the AV, with autophagic machinery (i.e., LC3) recycled through the
cytoplasm for recruitment to other nascent autophagosomes. (11)
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There is significant evidence that indicates that autophagy can confer cancer cells to
stress tolerance, which serves to maintain tumor cell survival (12,14). Knockdown of essential
autophagy genes in tumor cells has been shown to enhance the induction of cell death (11). In
vivo models, have shown that exposure to metabolic stress impairs survival in autophagy-
deficient cells compared with autophagy-proficient cells (13,23). Cytotoxic and metabolic
stresses, including hypoxia and nutrient deprivation, can activate autophagy for recycling of ATP
and to maintain cellular biosynthesis and survival. (8,13,24) Inhibition of autophagy in tumor
cells has been shown to enhance the efficacy of anticancer drugs, supporting its role in
cytoprotection. (12,21)
Cross-talk between autophagy and apoptosis exists at many levels because both pathways
share mediators, ranging from the core machinery to upstream regulators (11). Recent findings
suggest that p62/SQSTM1 mediates a link between autophagy and the extrinsic apoptotic
pathway (15). P62/SQSTM 1 was shown to bind caspase-8 to enable its aggregation and
activation as well as to enhance TRAIL-mediated apoptosis. TRAIL is a cytokine with activity
against multiple tumor types in which it induces apoptosis. (15)
Our laboratory has previously shown that inhibition of autophagy either by using specific
inhibitors or by knocking down key genes in the autophagy pathway leads to cell proliferation
arrest and apoptosis in DLBCL cells. (26,27) These results indicate that targeting autophagy may
be a possible therapeutic strategy in the treatment of DLBCL.

Autophagy inhibitors
Of the known autophagy inhibitors, only Chloroquine (CQ) and Hydroxychloroquine
(HCQ) have been thoroughly evaluated in humans. They are commonly used in treatments as
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antimalarial drugs and in autoimmune disorders (18). Chloroquine and Hydroxychloroquine are
lysosomotropic drugs that prevent the acidification of lysosomes, whose digestive hydrolases
depend on low pH (19).
The ability of autophagy inhibition to enhance chemo sensitivity and tumor regression
has been shown in animal models. The addition of Chloroquine, in a colon cancer xenograft
model, to vorinostat treatment was shown to significantly reduce tumor burden and to increase
apoptosis (16). Chloroquine enhanced the therapeutic efficacy of the Src inhibitor saracatinib in a
prostate cancer xenograft mouse model (12,17). Saracatinib decreased tumor growth by 26%
compared with control-treated mice, and Chloroquine plus saracatinib further inhibited tumor
growth by 64% (12,17). This combination also led to at least a 2-fold increase in the number of
apoptotic tumor cells in the group treated with saracatinib plus Chloroquine, suggesting that
suppression of autophagy drives cells into apoptosis (12,17). In a Myc-induced murine
lymphoma model, inhibition of autophagy by Chloroquine enhanced cyclophosphamide-induced
tumor cell death and it delayed the time-to-tumor recurrence (20,12). Phase I/II clinical trials
evaluating this combination are ongoing in patients with relapsed/refractory multiple myeloma.
Phase I clinical trial in dogs with spontaneous occurring lymphoma with a combination
Doxorubicin and Chloroquine or hydroxychloroquine has shown promising results with an
overall positive response rate and improved progression-free interval (22). These studies indicate
that autophagy inhibition can enhance the antitumor efficacy of chemotherapeutic agents that use
diverse cellular mechanisms of action.
Since chemotherapy often induces autophagy in cancer cells as a result of stress, which
might lead to resistance, targeting autophagy might sensitize the cancer cells to chemotherapy.
The aim of this project is to determine if inhibition of autophagy can sensitize DLBCL to
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Doxorubicin. The efficacy of autophagy inhibitor Tenovin-6 alone in the cell proliferation arrest
and cell death of DLBCL cells was determined by Dr. Yuan. (26) This project demonstrated that
Chloroquine in addition to Doxorubicin inhibited cell proliferation and survival in DLBCL cell
lines.

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RESULTS
Chloroquine Synergizes with Doxorubicin to inhibit cell proliferation of DLBCL cells
Figure 2: Growth Curves of DLBCL cell lines OCI-LY1 (GCB), RIVA (ABC), and U2932 (ABC). A.
LY1, RIVA, and U2932 cells treated with different concentrations of Doxorubicin (nM) and 25
chloroquine for 3 days. B. LY1, RIVA, and U2932 cells treated with different concentrations of
Doxorubicin (nM) and 50 uM Chloroquine for 3 days.

To determine effects of Doxorubicin and Chloroquine alone and in combination on cell
proliferation, we treated OCI-LY1 (GCB), RIVA (ABC), and U2932 (ABC) cell lines with
different concentrations of Doxorubicin (based on cell line) and either 25uMChloroquine (Fig 2.
A) and 50uMChloroquine (Fig 2. B) for 3 days.
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Treatment with Doxorubicin significantly inhibited the proliferation of OCI-LY1 cells.
Treatment with Chloroquine alone also significantly inhibited the proliferation of OCI-LY1 cells.
However, OCI-LY1 cells, treated with 200uM and 500uM Doxorubicin, showed little to no
synergistic effect when in combination with 25uM Chloroquine. (Fig 2. A) OCI-LY1 cells
showed a little more synergistic effect when in combination with 50uM Chloroquine. (Fig 2. B)
Therefore, single treatment of Chloroquine or Doxorubicin alone already had significant
inhibition on cell proliferation but overall there was no significant difference between single
treatment Doxorubicin and combination treatment, in OCI-LY1 cells.
Doxorubicin treatment significantly inhibited the proliferation of RIVA cells. Treatment
with Chloroquine also significantly inhibited the proliferation RIVA cells. Dual treatment with
25uM Chloroquine and 100nM Doxorubicin had some synergistic effects. (Fig 2. A). Treatment
with 100nM and 200nM Doxorubicin combined with 50uM Chloroquine had strong synergistic
effects compared to single treatment with Doxorubicin or Chloroquine alone. (Fig 2. B)
Therefore, although single treatment of Chloroquine or Doxorubicin alone already had
significant inhibition on cell proliferation, there was a significant difference between single
treatment Doxorubicin and combination treatment. The synergistic effects of Chloroquine and
Doxorubicin combination treatment are dosage dependent in RIVA cells.
In U2932 cells, cell proliferation was significantly inhibited by treatment with
Doxorubicin alone. Chloroquine treatment also significantly inhibited the proliferation of U2932
cells. Treatment with Doxorubicin in combination with 25uM Chloroquine showed some
synergistic effects compared to single treatments. (Fig 2. A) There were stronger synergistic
effects in cells treated with 100nM Doxorubicin in combination with 50uM Chloroquine (Fig 2.
B). There was little to no synergistic effect when treated with combination 50nM Doxorubicin
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and 50uM Chloroquine compared to 50uM Chloroquine alone. Dosage may have been too low to
show much effect. The synergistic effects appear to be dosage dependent.
Overall, both Chloroquine and Doxorubicin can induce arrest of cell proliferation of
DLBCL cell lines but the synergistic effects of these two agents vary according to cell lines.
There were synergistic cytotoxic effects with the addition of Chloroquine to Doxorubicin
treatment. The inhibition of cell proliferation was stronger in cells treated with Doxorubicin in
combination with Chloroquine at 50uM concentration. This synergistic effect was stronger in
ABC-type cell lines, RIVA and U2932, than in GCB-type, LY1. In OCI-LY1 cells, there was no
significant difference between cells treated with Doxorubicin only and Doxorubicin with
Chloroquine. These results indicated that the cell cytotoxic effects of Chloroquine in addition to
Doxorubicin on DLBCL cells increased in a dosage dependent manner, but this effect was cell
line dependent. These results also indicate that there is some genotype correlation.

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Chloroquine synergizes with Doxorubicin to increase apoptosis

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Figure 3: Annexin V staining for Apoptosis. A. Representative results of Annexin V- DAPI staining in
OCI-LY1 cells treated with 100nM Doxorubicin and 50uM Chloroquine for 48hrs. B. RIVA and U2932
cell lines representative results of Annexin V- DAPI staining after treatment with 500nM Doxorubicin
and 50uM Chloroquine for 48hrs. C. Percentages of Annexin V+ cells in LY1, RIVA, and U2932 cell
lines treated for 24 hrs. and 48 hrs.

In OCI-LY1 cells, there was significant increase in the percent of Annexin V+ cells with
treatment of 100nM Doxorubicin at 24 and 48hrs. There was also significant increase in the
percent of Annexin V+ cells with treatment of 50uM Chloroquine at 24 and 48hrs. In OCI-LY1
cells treated with combination of Doxorubicin and Chloroquine, there was significant increase in
Annexin V+ cells compared to individual treatment of Doxorubicin or Chloroquine at both 24
and 48hrs (Fig 3. A&C). Percentages of both early and late apoptotic cells increased after dual
treatment in comparison to single treatments (Fig 3. A). The majority of Annexin V+ cells
detected were in the early apoptosis stage. While there was synergistic increase in the percent of
Annexin V+ when treated with combination Doxorubicin and Chloroquine, there was not a
significantly change at 48hr compared to 24hrs.
In RIVA cells, the percent of Annexin V+ cells increased significantly when treated with
500nM Doxorubicin. Chloroquine alone also significantly increased the percent of Annexin V+
cells. (Fig 3. B&C) Dual treatment with 500nM Doxorubicin and 50uM Chloroquine further
significantly increased the percent Annexin V+ cells in compared to individual Doxorubicin or
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Chloroquine treatment at 24 and 48hrs. (Fig 3. C) The majority of Annexin V+ cells detected
were in the early apoptosis stage. There was not a significantly change in the percent of Annexin
V+ at 48hr compared to 24hrs when treated with combination Doxorubicin and Chloroquine.
Treatment with Doxorubicin significantly increased the percent of Annexin V+ cells in
U2932 cells. Treatment with Chloroquine alone also significantly increased the percent of
Annexin V+ cells in U2932 cells 24 and 48hrs. (Fig 3. B&C) Dual treatment with 500nM
Doxorubicin and 50uM Chloroquine significantly increased Annexin V+ cells, a majority of
which were in the early apoptosis stage, in comparison to both untreated and single treatments at
24 and 48hrs. (Fig 3. C). The percent of Annexin V+ at 48hr compared to 24hrs, when treated
with combination Doxorubicin and Chloroquine, did not significantly change.
In conclusion, Doxorubicin and Chloroquine can induce apoptosis in DLBCL cell lines
but the synergistic effects are not time dependent. In DLBCL cells treated with combination
Doxorubicin and Chloroquine, the percent of Annexin V+ at 48hr compared to 24hrs did not
have much difference. This result may be caused by the DLBCL cells being in a state of cell
cycle arrest. There was stronger cytotoxic effect in OCI-LY1 cells, treated with Doxorubicin
alone and in combination. This is consistent with previous research that GCB-type cells are more
susceptible to chemotherapy treatment (4). Overall, these results indicated that Chloroquine
synergized with Doxorubicin to induce apoptosis in DLBCL cells.

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Doxorubicin induces autophagic flux in DLBCL cells.
To determine the mechanism by which Chloroquine sensitizes DLBCL to Doxorubicin
we examined autophagic flux. This verified that cell survival after chemotherapy treatment with
Doxorubicin is through autophagy. DLBCL cell lines LY1, RIVA and U2932 were treated with
Doxorubicin and Chloroquine and examined by Western blot (Fig 4). SQSTM1/p62 and LC3 are
the most common proteins used as indicators of autophagic flux. P62 recruits and binds to
ubiquinated proteins for degradation in the autolysosome. LC3 exists in two forms: LC3I, which
is found in the cytoplasm and LC3II, which is membrane-bound and is converted from LC3I, to
initiate formation and lengthening of the autophagosome. Traditionally, the degradation of P62
and increase in LC3II/LC3I ratio together indicate an increase in autophagic flux.
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Figure 4: Western Blot Analysis of P62 and LC3II. A. OCI-LY1 cell line treated with 100nM
Doxorubicin and 50uM Chloroquine. The relative expressions of P62/Actin and LC3II/LC3I at 24 and
48hrs are represented in the bar graphs next to the corresponding blot. B. RIVA cell line treated with
500nM Doxorubicin and 50uM Chloroquine for 24 and 48hrs with corresponding relative expression bar
graphs of P62/Actin and LC3II/LC3I. RIVA cells treated with 500nM Doxorubicin and 100uM
Chloroquine for 24 hrs. C. U2932 cells treated with 500nM Doxorubicin and 50uMChloroquine for 24,
48, and 72hrs. U2932 cells treated with 500uM Doxorubicin and 100uMChloroquine for 24.

RIVA cells treated with 500nM Doxorubicin alone showed increase in LC3II/LC3I ratio
and increase in P62 compared to untreated and Chloroquine alone at 24hrs. At 48hrs, LC3II is
decreased compared to untreated, but P62 was still increased. 50uM Chloroquine alone showed
increase in both P62 and LC3II, as expected with autophagic flux being blocked. With dual
treatment of Doxorubicin and Chloroquine, there was increase in LC3II at 24hrs compared to
Doxorubicin or Chloroquine alone. Although, at 48hrs LC3II was decreased compared to
control, but still higher than Chloroquine alone. Dual treatment P62 was increased at both 24 and
48hrs. Treatment with Rapamycin showed little change in LC3II at 24hrs or slightly decreased at
48hrs, but LC3I was also decreased at 24 and 48 hrs. P62 was decreased at both 24 and 48hrs as
expected, compared to untreated. Both P62 and LC3II increase with the addition of Chloroquine.
Therefore, this result is probably due to increased LC3 turnover, which is cell line specific, in the
cell. Based on these results, it is likely that many cells have initiated apoptosis at this point,
which is consistent with the apoptosis data (Fig 3) or is undergoing cell cycle arrest. RIVA cells
treated with 100uM Chloroquine in addition to 500nM Doxorubicin at 24hrs showed increase in
both P62 and LC3II, compared to untreated and single treatments. These results indicate a
synergistic effect with Doxorubicin and Chloroquine in RIVA cell line.
OCI-LY1 cells treated with 100nM Doxorubicin only showed increase in both P62 and
LC3II/LC3I ratio at both 24 and 48hrs, compared to untreated cells. Chloroquine alone showed
increase in both P62 and LC3II, as expected with autophagy being blocked, at 24 and 48hrs.
Treatment with Rapamycin showed little change in LC3II at 24hrs or slightly decreased at 48hrs,
23

but LC3I was also decreased at 24 and 48 hrs. There was increase in the LC3II/LC3I ratio. P62
was decreased at both 24 and 48hrs as expected. P62 increased slightly, as did LC3II/LC3I ratio
increase, with the addition of Chloroquine in comparison to rapamycin alone, as expected.
Combination treatment of 100uM Doxorubicin and 50uM Chloroquine showed increased
LC3II/LC3I, at both 24 and 48hrs, compared to untreated and single treatments. P62, at 24hrs,
was increased compared to untreated and single treatments. At 48hrs, P62 was not much
difference than Doxorubicin treatment alone, and P62 was lower than Chloroquine treatment.
U2932 cells treated with 500nM Doxorubicin alone showed increased P62 at 24 and
48hrs. At 72hrs, P62 was decreased. In U2932 treatment with Doxorubicin showed little change
or decrease in LC3II, but there is also decrease in LC31 expression at 24, 48, and 72hrs.
Chloroquine alone showed increase in both P62 and LC3II/LC3I, as expected with autophagy
being blocked, at 24 and 48hrs. Combination treatment with 500nM Doxorubicin and 50uM
Chloroquine showed LC3II was consistently increased at 24, 48, and 72hrs compared to
untreated and single treatments. P62 levels, compared to untreated or singles treatments, were
increased at 24hrs. P62 appeared have little difference at 48hrs, compared to Chloroquine alone,
or at 72hrs, compared to Doxorubicin alone. U2932 cells, treated with dual treatment of 500nM
Doxorubicin and 100uM Chloroquine for 24hrs, showed increase in P62 in comparison to
untreated and single treatments. LC3II/LC3I was also strongly increased in comparison to
untreated and single treatments, showing synergistic effects with dual Doxorubicin and
Chloroquine treatment. These results indicate increase in autophagic flux.
Overall, these results indicate that there is induction of autophagic flux in DLBCL cells
when treated with Doxorubicin. The synergistic effects with combination treatment of
Doxorubicin and Chloroquine occur in a dosage and time dependent manner. The extent of this
24

effect was also cell line dependent. The results showed consistent increase of autophagic flux in
DLBCL cell lines at 24hrs, but at 48hrs it varied. It is possible that Doxorubicin was too
cytotoxic at 48hrs, and the cells activated Cell Cycle arrest or the Apoptosis pathway. P62
expression was increased with Doxorubicin treatment compared to autophagy inducer
Rapamycin in all cell lines. It could be possible that Doxorubicin has some inhibitory effect on
autophagy. Therefore, further study is needed to elucidate what other pathways Doxorubicin may
be affecting in DLBCL cells. Immunofluorescence staining of LY1, RIVA, and U2932 was done
to further verify the assertions of increased autophagic flux.

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Figure 5: Immunofluorescence staining for LC3 and P62 in OCI-LY1 cells. OCI-LY1 cells treated with
100nM Doxorubicin and 50uMChloroquine for 24hrs.

OCI-LY1 cells treated with 100nM Doxorubicin alone showed increased speckling in
LC3(green) and P62(red) compared to untreated cells. Treatment with 50uM Chloroquine
increased LC3intensity and speckling, increased P62 intensity, and P62-LC3 localization
(yellow), as expected with autophagy inhibitor. 25nM Rapamycin treatment showed little change
in P62 and LC3. Rapamycin in combination with 50uM Chloroquine showed increase in LC3
intensity and speckling, increase in P62 intensity, and P62-LC3 localization (yellow). Similar to
Rapamycin and Chloroquine combination, Dual treatment of 100nM Doxorubicin and 50uM
Chloroquine showed increase in LC3 intensity and speckling, increase in P62 intensity, and P62-
LC3 localization (yellow). These results are consistent with the previous western blot (Fig 4. A)
indicate that autophagic flux was increased, in OCI-LY1 cells, with treatment of Doxorubicin.
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Figure 6: Immunofluorescence Staining of P62 and LC3 in RIVA cells. RIVA cells were treated with
500nM Doxorubicin and 50uMChloroquine for 24 and 48hrs.

RIVA cells treated with 500nM Doxorubicin alone showed increase in LC3(green)
speckling and P62(red) accumulation at both 24 and 48hrs compared to untreated cells.
Treatment with 50uM Chloroquine increased LC3intensity and speckling, increased P62
intensity, and P62-LC3 localization (yellow), as expected with autophagy inhibition. 25nM
Rapamycin treatment showed slight increase LC3 and P62 at 24 and 48hrs. Rapamycin in
combination with 50uM Chloroquine showed increase in LC3 intensity and speckling, increase
in P62 intensity, and P62-LC3 localization (yellow). Similar to Rapamycin and Chloroquine
combination, cells treated with 500nM Doxorubicin and 50uM Chloroquine, showed increase in
LC3 intensity and speckling, and increase in P62 intensity and P62-LC3 localization (yellow)
single treatments of Doxorubicin or Chloroquine. These combination effects were stronger at
48hrs. The results indicate increase in autophagic flux. The results are consistent with western
blot results at 24hrs but showed stronger increased autophagic flux at 48hrs compared to western
blot. (Fig 4. B)
28

29

Figure 7: Immunofluorescence Staining of P62 and LC3 in U2932 cells. U2932 cells were treated with
500nM Doxorubicin and 50uMChloroquine for 24 and 48hrs.

U2932 cells treated with 500nM showed increase in LC3(green) speckling and P62(red)
accumulation at both 24 and 48hrs compared to untreated cells. This is different from the western
blot result, but indicates the lack of LC3II may have been a sensitivity issue. Treatment with
50uM Chloroquine increased LC3intensity and speckling, increased P62 intensity, and P62-LC3
localization (yellow), as expected with autophagy inhibitor. 25nM Rapamycin treatment showed
decrease in P62 and increase in LC3 speckling. Rapamycin in combination with 50uM
Chloroquine showed significant increase in LC3 intensity and speckling, increase in P62
intensity, and P62-LC3 localization (yellow). Cells treated with dual treatment, 500nM
Doxorubicin and 50uM Chloroquine, showed significant increase in LC3 intensity and speckling,
and increase in P62 intensity and P62-LC3 localization (yellow) compared to single treatments
of Doxorubicin or Chloroquine. These combination synergistic effects were stronger at 48hrs.
These results indicate increase in autophagic flux, which is consistent with western blot results
(Fig 4. C)
Overall, DLBCL cells lines showed synergistic increase in LC3(green) speckling,
increased P62(red) intensity, and increased P62-LC3 co-localization(yellow) with combination
treatment of Doxorubicin and Chloroquine. The intensity of this synergistic effect is cell line and
phenotype specific, as well as time dependent. These results are consistent with the western blots
at 24hrs (Fig 4) but there was some variance in 48hrs results. The IFA results support that
Doxorubicin does induce autophagic flux in DLBCL cells.

30

DISCUSSION
New targets for cancer therapy are a critical point of research to improve the treatment
and livelihood of current and future patients. Both Chloroquine and Doxorubicin are FDA
approved drugs and have both been studied for general dosage safety. Previous autophagy and
DLBCL work, done by Dr. Yuan, were starting points to the development of this project. His
work showed the efficacy of an autophagy inhibitor Tenovin-6 alone in the treatment of DLBCL.
(26,27) Since DLBCL is treated by R-CHOP combination treatment it was important to
determine if a combination of an autophagy inhibitor and the chemotherapy agent Doxorubicin
could be effective. Due to Doxorubicin’s cardiotoxic effects, a lower dosage of Doxorubicin is
an important factor to take into consideration for future research.
The results of this project showed both Chloroquine and Doxorubicin can induce arrest of
cell proliferation of DLBCL cell lines but the synergistic effects of these two agents vary
according to cell lines. There were synergistic cytotoxic effects with the combination of
Chloroquine and Doxorubicin treatment. The inhibition of cell proliferation was stronger in cells
treated with Doxorubicin in combination with Chloroquine at 50uM concentration. This
synergistic effect was stronger in ABC-type cell lines, RIVA and U2932, than in GCB-type,
LY1. These results indicated that the cell cytotoxic effects of Chloroquine in addition to
Doxorubicin on DLBCL cells increased in a dosage dependent manner, but this effect was cell
line dependent. These results also indicate that there is some genotype correlation. (Fig 2),
Doxorubicin and Chloroquine alone induce apoptosis in DLBCL cell lines, but the
synergistic effects vary according to cell lines (Fig 3). There were synergistic apoptotic effects
with the combination of Chloroquine and Doxorubicin treatment. DLBCL cells, treated with
combination Doxorubicin and Chloroquine, percentage of Annexin V+ at 48hr compared to
31

24hrs did not have much difference (Fig 3. C). This result may be caused by the DLBCL cells
being in a state of cell cycle arrest. There was stronger cytotoxic effect in OCI-LY1 cells,
treated with Doxorubicin alone and in combination. This is consistent with previous research that
GCB-type cells are more susceptible to chemotherapy treatment (4) and with cell proliferation
data (Fig 2). Overall, these results indicated that Chloroquine synergized with Doxorubicin to
induce apoptosis in DLBCL cells but the effect was cell line dependent.
Doxorubicin was tested for autophagic flux because it was important to characterize that
Doxorubicin was affecting autophagy. This ensured that Chloroquine was targeting the
mechanism by which the DLBCL cells survive Doxorubicin. To determine induction of
autophagic flux P62 and LC3II was examined by western blot. (Fig 4) In all DLBCL cell lines,
there was increase in LC3II/LC3I and P62, with combination treatment therapy of Doxorubicin
and Chloroquine at 24hrs. Compared to single treatment and untreated cells, these results
indicate that Doxorubicin induces autophagic flux. The synergistic effects with combination
treatment of Doxorubicin and Chloroquine occur in a dosage dependent manner. The extent of
this effect was cell line dependent. The results showed consistent increase of autophagic flux in
DLBCL cell lines at 24hrs, but at 48hrs it varied. At 48hrs, Doxorubicin was probably too
cytotoxic, and the cells activated Cell Cycle arrest or the Apoptosis pathway. P62 expression was
increased with Doxorubicin treatment compared to autophagy inducer Rapamycin in all cell
lines. It could be possible that Doxorubicin has some inhibitory effect on autophagy. Therefore,
further study is needed to elucidate what other pathways Doxorubicin may be affecting in
DLBCL cells. To further verify that Doxorubicin increased autophagic flux,
immunofluorescence staining of DLBCL cell lines were done
32

RIVA, U2932 and OCI-LY1 cells lines showed increased LC3 (green) speckling,
P62(red) intensity, and increased P62-LC3 co-localization (yellow) with the combination
treatment of Doxorubicin and Chloroquine compared to single treatment (Figures 5, 6, &7). The
intensity of this synergistic effect is cell line and phenotype specific, as well as time dependent.
These results are consistent with the western blots (Fig 4) at 24hrs, and further support that
Doxorubicin does induce autophagic flux in DLBCL cells. The synergistic effects with
combination treatment of Doxorubicin and Chloroquine occur in a dosage dependent manner but
this effect was cell line dependent.
Western blot and immunofluorescence results at 24hrs were consistent indicating
increased autophagic flux. They were also consistent with the cell proliferation and apoptosis
results. Response varied, at 48hrs, comparing western blots and IFA, but they are still consistent
with cell proliferation and apoptosis data. Based on the results, it is likely that many of the
DLBCL cells have initiated apoptosis or are in cell cycle arrest by the 48hr point.
The increased P62, without the addition of autophagy inhibitor, indicates other pathways
may also be active in the DLBCL cells. According to Suhani et. al, expression of the autophagy
substrate SQSTM1/p62 is restored during prolonged starvation depending on transcriptional
upregulation and autophagy-derived amino acids. Amino acids derived from the autophagy–
lysosome pathway are used for de novo synthesis of SQSTM1 under starvation conditions. (28)
In some cell lines after starvation, autophagy triggered transcriptional upregulation of genes, by
the degradation for nutrients, that signal the cells to increase P62 and LC3II production.
There is some evidence that indicate that the expression level of SQSTM1/p62 does not
always inversely correlate with autophagic activity. (28, 29) It is possible that the mitochondria
DNA and oxidative damage from Doxorubicin and recycling of the mitochondria by autophagy
33

may cause upregulation of P62 and LC3 production. (10, 28) Since Doxorubicin uses multiple
pathways to induce cell death, other pathways may be activated in parallel to autophagy. (7,10)
These alternate pathways may account for the behavior of LC3 and P62 in some DLBCL cell
lines (Fig 4.). Further investigation into the pathways can play an important role in understanding
possible crosstalk between mechanisms activated by autophagy. It is possible that the increased
P62, with Doxorubicin treatment alone, may be a result of P62 playing a role in the mediation of
both autophagy and apoptosis in DLBCL cell lines.
Further research is necessary to delineate the mechanisms and develop the logistics of
using autophagy inhibitors in addition to chemotherapy as a clinical treatment of DLBCL, but so
far, the results are promising. Therefore, the results of this project show that the targeting
autophagy is a valuable therapeutic strategy in overcoming chemoresistance in Diffuse Large B-
Cell Lymphoma.

34

FUTURE DIRECTIONS
Further research, that would benefit my current research completed, would be to
determine if Doxorubicin with Chloroquine increase cell cycle arrest by performing an
examination of cell cycle progression by BrdU (5-bromo-2′-deoxyuridine) and PI
(propidiumiodide) staining. Determine the mechanisms by which Doxorubicin regulates P62 and
LC3 levels. Also, determine if P62 and LC3 are relatively transcriptional upregulated with the
addition of Doxorubicin both alone and in combination through RT-qPCR. Knockdown of
autophagy linked genes to elucidate Doxorubicin role in Autophagy. Targeting of upstream P62
proteins to determine other possible activation of P62 transcriptional pathways is another
prospective research project worthy of attention. Critical future research would be in animal
studies in NOD/SCID mice to measure tumor reduction efficacy. Treatment with a low dose of
Doxorubicin in combination with Chloroquine, will elucidate some critical questions about the
efficacy of using lower dosages of Doxorubicin with Chloroquine to help mediate the toxic side
effects of chemotherapy. An outline of such a project would include: 10 NOD/SCID mice per
group; 4 Groups: Control, Chloroquine alone, Doxorubicin alone, Doxorubicin +Chloroquine
combination; Doxorubicin treatment: 2mg/kg by I.P. injection, Once every 3 weeks; Chloroquine
treatment: 10mg/kg by I.P. injection, Daily.

35

MATERIALS AND METHODS
Antibodies and Reagents
The chemicals used were Doxorubicin (2252, Tocris Bioscience), Chloroquine diphosphate
(C6628, Sigma, St. Louis, MO), bafilomycin A1 (B1793, Sigma), rapamycin (R8781, Sigma).
The antibodies used were: LC3B (CTB-LC3-1-50, Cosmo Bio, Carlsbad, CA), SQSTM1/p62
(PM045, MBL, New York, NY), and β-actin (sc-8432, Santa Cruz, Dallas, TX)

Annexin V staining
PE-Cyanine 7-conjugated anti-annexin V antibody (25-8103-74, eBioscience, San Diego) was
used to detect apoptosis following the instructions of the manufacturer. Samples were run in a
FACS Canto System (BD Biosciences) and analyzed with FlowJo (Treestar, Ashland, OR).

Cell Culture
RIVA, U2932, and OCI-LY1 cells were cultured in RPMI-1640 media. RPMI-1640 media was
supplemented with 100 μg/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum
(#26140-079, Thermo Fisher Scientific). All cells were maintained in a 5% CO2 atmosphere at
37°C.

Immunofluorescence assay
Cells were treated as described then spun onto coverslips at 300 RCF for 5min. After fixation
with 4% paraformaldehyde for 15 min at 4 C, the cells were washed and then blocked with 3%
BSA in PBS for 30 min at 37°C. The cells were then incubated with anti-LC3B antibody (CTB-
LC3-1-50, Cosmo Bio) and SQSTM1/p62 (PM045, MBL, New York, NY), at a 1:500 dilution
36

for 1 h at 37°C. After incubation for 1 h at 37 °C with a secondary antibody (A-11001,
ThermoFisher Scientific), samples were counterstained with 0.5 μg/mL 4-,6-diamidino-2-
phenylindole (DAPI) in PBS for 5 min, and the slides were mounted in FluorSave Reagent
(#345789, Calbiochem, Billerica, MA). Samples were observed with a laser-scanning confocal
microscopy (Nikon Eclipse C1).

Western Blotting
Cells were lysed in RIPA buffer supplemented with 1% SDS and proteinase inhibitor cocktail
(1:100) (P8340, Sigma). 20 μg of Protein lysate for each sample was resolved by SDS-PAGE
and transferred for 50min at 20V onto a PVDF membrane. After blocking with 5% skim milk,
the membrane was probed with primary and then secondary antibodies. The signal was
developed with the Luminiata Crescendo Western HRP substrate (WBLUR0500, EMD
Millipore, Billerica, MA) and visualized with a UVP BioSpectrumImaging System (UVP, LLC,
Upland, CA). Western blot relative expression quantification was determined using Image J.

Statistical analysis
Performed Two Tailed T-test to test for statistical significance. P < 0.05 was considered
statistically significant. * =P < 0.05 and ** =P < 0.001

37

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Abstract (if available)

Abstract Diffuse Large B-cell Lymphoma (DLBCL) is a malignant cancer originating from B-cells. It is the most common B-cell tumor in adults and is categorized as one of the most aggressive non-Hodgkin lymphomas, accounting for 30% to 40% of all newly diagnosed cases of NHL. The most common treatment for DLBCL is R-CHOP, which is a combination therapy including rituximab (a monoclonal antibody)

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Asset Metadata

Creator Taitano, Livia (author)

Core Title Sensitization of diffuse large B-cell lymphoma to chemotherapy by targeting autophagy

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 08/15/2018

Defense Date 05/23/2018

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

Tag autophagy,chemotherapy,chloroquine,diffuse large B-cell lymphoma,DLBCL,Doxorubicin,OAI-PMH Harvest

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Advisor Gao, Shou-Jiang (committee chair), Liang, Chengyu (committee member), Yuan, Weiming (committee member)

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