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Targeting autophagy in chronic myeloid leukemia through inhibition of the core autophagy protein ATG4B Porter, Vanessa Lynn 2018

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 Targeting Autophagy in Chronic Myeloid Leukemia through Inhibition of the Core Autophagy Protein ATG4B by  Vanessa Lynn Porter  BSc, Trinity Western University 2015  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  February 2018  © Vanessa Lynn Porter, 2018     ii  Abstract Treatment of chronic myeloid leukemia (CML) targets the BCR-ABL1 fusion oncoprotein that characterizes its pathogenesis using tyrosine kinase inhibitors (TKIs); however, drug resistance and relapse can occur when BCR-ABL1-independant survival pathways such as autophagy are activated. Our lab found that the key autophagy enzyme ATG4B is upregulated in CML stem/progenitor cells from patients that clinically do not respond to TKIs vs. patients that do. Knockdown of ATG4B was found to suppress autophagy and sensitize CML cells to TKIs. This study investigates if combined suppression of BCR-ABL1 and ATG4B by novel ABL1 and ATG4B inhibitors in autophagy-inducing conditions may present a novel therapeutic approach to overcome TKI-resistance in CML. I found that inhibition of ATG4B by DB2 significantly inhibits growth and induces apoptosis in CML cell lines alone and in combination with TKIs when autophagy is induced during serum deprivation. There also is a decrease in colony forming cells after DB2+TKI treatment compared to TKIs alone in non-responding CML cells (p<0.05) but is well tolerated in normal bone marrow. Inhibition of ATG4B by DB inhibitors alters autophagy as seen by an accumulation of autophagosomes detected by western blots and florescent tracking. When DB2 is combined with TKI treatment in an aggressive mouse leukemia model, there are no significant differences in the leukemia burden between TKI treatment alone and the combination. However, another model shows that when ATG4B knockdown is combined with 70% caloric restriction, LC3B accumulates in the leukemic cells, and the mice have decreased engraftment and prolonged survival compared to the fed scrambled control mice, suggesting activation of autophagy is important to potentiate loss of ATG4B. This     iii  study presents ATG4B inhibition as a specific means to target autophagy and circumvent drug resistance in CML.     iv  Lay Summary Treatment for CML relies on targeted therapies to disable the fusion protein, BCR-ABL1, which characterizes CML as a disease. However, BCR-ABL1 does not always act independently in persisting cell survival, which is why therapeutic resistance and disease relapse often occurs. One critical pathway involved in many cancers, including CML, is the cellular recycling process known as autophagy. Several key autophagy genes are expressed differently in leukemia stem cells. The most substantial change is the increased presence of the core autophagy protein ATG4B. I used novel ATG4B inhibitors in combination with TKIs within immortalized CML cell lines, patient samples, and multiple mouse models. The small molecule inhibitors and genetic knockdown of ATG4B caused autophagy to be impaired and CML cells to be sensitized to TKI treatment. Many other cancer types have identified ATG4B as a cancerous target, so this study will provide insight into the feasibility of targeting ATG4B in treatment.       v  Preface I, Vanessa Porter, performed and designed the experiments within this thesis under the supervision of Dr. Xiaoyan Jiang and with the assistance of Dr. Min Chen and Dr. Katharina Rothe. I performed the cell line biological assays after slightly altering conditions previously optimized by Dr. Katharina Rothe. I also performed all the primary cell assays except for the normal bone marrow colony-forming cell assay testing DB1 toxicity, which was performed by Dr. Katharina Rothe. Dr. Min Chen greatly assisted me in the in vivo experiments using a mouse model of human leukemia and performed the irradiation and intravenous injection on her own, and assisted me for the luciferase imaging and some post-mortem analyses. The ATG4B inhibitors used in this study were discovered and modified by Dr. Robert Young’s laboratory at Simon Fraser University and given to us through our collaboration.  The primary cell samples used in this study were obtained and used according to procedures approved by the University of British Columbia Clinical Research Ethics Board under certificate number H12-02372. All animal handling was done in the Animal Resource Centre of the British Columbia Cancer Research Center using Animal Care Committee (University of British Columbia) approved procedures under certificate A15-0062.       vi  Table of Contents  Abstract .......................................................................................................................................... ii	Lay Summary ............................................................................................................................... iv	Preface .............................................................................................................................................v	Table of Contents ......................................................................................................................... vi	List of Tables .................................................................................................................................. x	List of Figures ............................................................................................................................... xi	List of Abbreviations ................................................................................................................. xiii	Acknowledgements .................................................................................................................... xvi	Chapter 1: Introduction ................................................................................................................1	1.1	 Chronic Myeloid Leukemia ................................................................................................... 1	1.1.1	 The Clinical and Biological Features of CML ............................................................ 1	1.1.2	 CML Treatment: Clinical Advancements ................................................................... 3	1.1.3	 CML Treatment: IM Resistance ................................................................................. 5	1.1.4	 CML Treatment: Additional Treatment Options ........................................................ 8	1.2	 Autophagy .............................................................................................................................. 9	1.2.1	 History of Autophagy ................................................................................................. 9	1.2.2	 Players in the Autophagy Process ............................................................................. 10	1.2.3	 Regulation of Autophagy .......................................................................................... 15	1.2.4	 Autophagy and Cancer Including CML .................................................................... 16	1.3	 The Core Autophagy Protease ATG4B ............................................................................... 18	    vii  1.3.1	 The Human ATG4 Homologues ............................................................................... 18	1.3.2	 ATG4B in Cancer including CML ........................................................................... 20	1.3.3	 Discovery of Novel ATG4B Inhibitors ..................................................................... 22	1.4	 Summary .............................................................................................................................. 23	1.5	 Hypothesis............................................................................................................................ 23	1.6	 Thesis Objectives ................................................................................................................. 23	Chapter 2: Materials and Methods ............................................................................................25	2.1	 Cell Culture Materials .......................................................................................................... 25	2.1.1	 Cell Lines .................................................................................................................. 25	2.1.2	 Primary Human Cells ................................................................................................ 25	2.1.3	 Inhibitors ................................................................................................................... 26	2.2	 In vitro Assays ..................................................................................................................... 27	2.2.1	 Cell Starvation Assays .............................................................................................. 27	2.2.2	 Viability Assay .......................................................................................................... 27	2.2.3	 Apoptosis Assay ....................................................................................................... 27	2.2.4	 Colony-Forming Cell (CFC) Assay .......................................................................... 28	2.3	 Transfections and Transductions ......................................................................................... 28	2.3.1	 Lipofectamine Transfection of a mCherry-EGFP-LC3 Vector ................................ 28	2.3.2	 Lentiviral Transfection and Virus Harvest ............................................................... 29	2.3.3	 Knockdown of ATG4B in BV173YFP/Luc Cells ......................................................... 30	2.4	 Molecular Techniques .......................................................................................................... 30	2.4.1	 Cell Lysis and Protein Quantification ....................................................................... 30	    viii  2.4.2	 Western Blotting ....................................................................................................... 31	2.4.3	 LC3B Puncta Monitoring through Immunofluorescence ......................................... 31	2.4.4	 LC3B Puncta Monitoring through Endogenous Fluorescence ................................. 32	2.4.5	 RNA Extraction and Quantitative Real-Time PCR (qRT-PCR) ............................... 33	2.5	 In vivo Assays ...................................................................................................................... 33	2.5.1	 Transplantation of BV173YFP/Luc Cells into Immunodefficient Mice with DB2+DA Treatment .............................................................................................................................. 33	2.5.2	 Transplantation of BV173YFP/Luc + shATG4B into Immunodefficient Mice with Caloric Restriction ................................................................................................................ 34	Chapter 3: Results ........................................................................................................................36	3.1	 The Viability Effects of Novel ATG4B Inhibitors from the DB Series in CML Cell Lines…… ...................................................................................................................................... 36	3.2	 Inhibition of ATG4B by DB Inhibitors in Combination with TKIs is Sensitized by Serum Starvation ...................................................................................................................................... 38	3.3	 DB2 is Selective to Primary CML Cells Without Targeting Normal Bone Marrow ........... 43	3.4	 DB Series Inhibitors Alter Activated Autophagy in CML Cells ......................................... 45	3.5	 DB2 + DA Combination Does Not Reduce Leukemia Burden In Vivo .............................. 51	3.6	 ATG4B Knockdown in BV173YFP/Luc cells ......................................................................... 54	3.7	 Caloric Restriction in vivo May Potentiate ATG4B Knockdown ........................................ 56	Chapter 4: Discussion ..................................................................................................................61	4.1	 Discussion ............................................................................................................................ 61	4.2	 Conclusions and Future Directions ...................................................................................... 70	    ix  References .....................................................................................................................................74	Appendix A ...................................................................................................................................86	Appendix B ...................................................................................................................................87	     x  List of Tables Table 3.1: Overview of the “DB” series of ATG4B inhibitors ..................................................... 36     xi  List of Figures Figure 1.1: Mechanisms of disease pathogenesis in CML ............................................................. 7 Figure 1.2: The players of the autophagy process ........................................................................ 11 Figure 1.3: The roles of ATG4B in the processing of the ATG8 homologues LC3 and GABARAP ................................................................................................................................... 14 Figure 1.4: Crystal structure of ATG4B ....................................................................................... 20 Figure 3.1: IC50s of  four novel ATG4B inhibitors: DB1, DB2, DB3, and DB4 ......................... 37 Figure 3.2: The biological effects of the DB series inhibitors in combination with IM in K562 cells after 72 hours of treatment .................................................................................................... 39 Figure 3.3: The biological effects of the DB series inhibitors in combination with IM in IMR cells after 72 hours of treatment .................................................................................................... 40 Figure 3.4: The biological effects of the DB series inhibitors in combination with IM in BV173 cells after 72 hours of treatment .................................................................................................... 41 Figure 3.5: The colony forming abilities of healthy bone marrow samples after treatment with DB inhibitors in combination with TKIs ...................................................................................... 43 Figure 3.6: The biological effects of DB2 combined with the TKI NL in CML NR samples. .... 44 Figure 3.7: Changes in autophagy after treatment with the DB inhibitors and CQ ...................... 47 Figure 3.8: LC3B immunofluorescence in CD34+ CML cells after DB2 and CQ treatment ....... 48 Figure 3.9: Dose-dependent effects on LC3-I and LC3-II after DB2 treatment ........................... 51 Figure 3.10: Analysis of leukemia progression in mice two weeks following oral gavage treatment. ...................................................................................................................................... 52     xii  Figure 3.11: The fluorescence of LC3B in BV173YFP/Luc (YFP+) engrafted cells in the mice bone marrow two weeks after treatment.. .............................................................................................. 53 Figure 3.12: Knockdown of ATG4B using shRNA in BV173YFP/Luc cells in vitro.. .................... 54 Figure 3.13: Analysis of shATG4B mice vs. SHC mice with and without CR.. .......................... 57 Figure 3.14: The fluorescence of LC3B in BV173YFP/Luc (YFP+) engrafted cells in the mice bone marrow seven weeks after injection.. ............................................................................................ 58 Figure 3.15: Analysis of weights and survival of the SHC and shATG4B mice post-injection.. . 59                    xiii  List of Abbreviations ABL = Abelson  AKT = Protein Kinase B AMPK = AMP-activated protein kinase  APC = Allophycocyanin ATG = Autophagy-related B-ALL = B cell Acute Lymphoblastic Leukemia BafA = Baflomycin A1 BCR = Breakpoint Cluster Region BECN1 = Beclin-1 BME = Beta-Mercaptoethanol CAMKK2 = calmodulin-dependent protein kinase kinase 2 CCR = Complete Cytogenetic Response CFC = Colony Forming Cell CHR = Complete Hematological Response CL3 = Containment Level 3 CML = Chronic Myeloid Leukemia CML-AP = CML-Accelerated Phase CML-BP = CML-Blast Crisis Phase CML-CP = CML-Chronic Phase CMR = Complete Molecular Response CQ = Chloroquine CR = Caloric Restriction DA = Dasatinib DB1 = DB1-043 DB2 = DB2-082 DB3 = DB2-113 DB4 = DB2-114 DMEM = Dulbecco's Modified Eagle Medium DMSO = Dimethylsulfoxide DNA = Deoxyribonucleic Acid EPO = Erythropoietin  ERK = Extracellular Signal-Regulated Kinase FACS = Fluorescence-Activated Cell Sorting FBS = Fetal Bovine Serum FIP200 = FAK Family Kinase-Interacting Protein FISH = Fluorescent in situ Hybridization FOXO = Forkhead Box O GABARAP = Gamma-Aminobutyric Acid Receptor-Associated Protein Precursor  GFP = Green Fluorescent Protein GM-CSF = Granulocyte/Macrophage-Colony Stimulating Factor     xiv  G-SCF = Granulocyte-Macrophage Progenitor HCQ = Hydrochloroquine HSC = Hematopoetic Stem Cell IC50 = 50% Inhibitory Concentration IL = Interleukin IM = Imatinib JAK = Janus Kinase LC3 = MAP1-LC3 LIR = LC3-Interacting Region LSC = Leukemic Stem Cell LTC-IC = Long-Term Culture Initiating Cell  Luc = Luciferase MDM = Iscove’s Modified Dulbecco’s Medium MEK = (MAPKK) Mitogen-Activated Protein Kinase Kinase miRNA = microRNA MMR = Major Molecular Response mTORC = Mammalian Target of Rapamycin Complex NBM = Normal Bone Marrow NL = Nilotinib NSG = NOD/SCID-Interleukin 2 Receptor Gamma-Chain-Deficient Mice NR = CML Non-Responder P/S = Pennicillin/Streptomycin PDAC = Pancreatic Ductal Adenocarcinoma  PDGFR = Platelet-derived Growth Factor Receptor PE = Phosphatidyethanolamine PEI = Polyethyleimine PFS = Progression-free Survival Ph = Philidelphia Chromosome PI = Propidium Iodide PI3K = Phospho-Inositide-3-Kinase PI3P = phosphatidylinositol-3-phosphate PKA = cAMP-dependant protein kinase A PO = Ponatinib PtdIns3K = ATG14-containing class III phosphatidylinositol 3-kinase qRT-PCR = Quantitative Real-Time Polymerase Chain Reaction RAF = Raf proto-Oncogene RAS = Rat Sarcoma Viral Oncogene RFP = Red Fluorescent Protein rIFN-a = Recombinant Interferon-alpha RPMI = Rosewell Park Memorial Institute RT = Room Temperature SCF = Stem Cell Factor     xv  SEM = Standard Error of Mean SFM = Serum-free Medium SHC = shRNA empty scrambled vector shRNA = small hairpin RNA SRC = Sarcoma-family Kinase STAT = Signal Transducer and Activator of Transcription TBST = Tris-Buffered Saline Tween 20 TKI = Tyrosine Kinase Inhibitor TMZ = Tomololamide UBL = ubiquitin-like  ULK = UNC-51-like Autophagy Activating Kinase  VPS = PI3K Vacuolar Protein Sorting YFP = Yellow Fluorescent Protein       xvi  Acknowledgements I believe collaboration is the greatest driver of science, and I know there have been many people that have helped make this research project possible.  I would like to thank my supervisor Dr. Xiaoyan Jiang for always listening to my ideas and driving me to explore them as proper research questions. You have supported and pushed me towards great opportunities that have helped me grow immensely as a scientist. I have enjoyed your lab very much and I thank you for everything you have done for me as a mentor! I also would like to thank my supervisory committee Drs. Sharon Gorski and Gregg Morin. Your ideas and discussions during my committee meetings provided me with invaluable insights into your areas of expertise.  I am so lucky to have ended up with a lab full of such an engaging and wonderful people that support me in both my project and otherwise. Kat, I feel so lucky to have worked under you as I continued this project that we share. Your scientific mind is brilliant and you are a natural-born teacher through and through. Min, I could not have done any of my mouse work without your help and expertise.  Everyone else in the lab, past and present, namely Josephine, Kelly, Ryan, Sujie, Will, Leon, Young, Jason, and Andrew, you guys are great colleagues but most importantly you are great friends.  Furthermore, I would like to thank everyone in TFL, BCCRC, and the ATG4B team that has given me advice and support. I feel very lucky to be surrounded by so many intelligent minds that all have such unique and inquisitive perspectives. My research would not be possible without the teams at Stem Cell Assay, Flow Core, and the Animal Resource Centre. I would like to thank Dr. Robert Young’s lab for providing all the inhibitors I study in this project. Thank you     xvii  especially to Dr. Peter Clark who has been an immense help teaching me about the chemistry side of things and going over data with me.   I also would like to thank the BC Cancer Agency, the Leukemia and Lymphoma Society of Canada, Mitacs Accelerate, ESH, Medical Genetics, the Canadian Institutes of Health Research, and the University of British Columbia for providing me with financial support throughout my endeavors.   Lastly, my utmost warmest thanks to my parents, my brother Stephen, my best friend Shaila, my roommates Ann and Hilary, along with the rest of my family and friends for providing me with much needed emotional support through all these endless years of school!                  xviii           For all the scientists, past and present, who make an ocean of difference  one grain of sand at a time  1  Chapter 1: Introduction  1.1 Chronic Myeloid Leukemia 1.1.1 The Clinical and Biological Features of CML Chronic myeloid leukemia (CML) is a clonal myloproliferative malignancy that affects 1-2 new individuals per 100,000 people per year, accounting for 15-20% of diagnosed leukemia cases in North America.1 Diagnosis of CML typically involves the expansion of the myeloid cell lineage and, in 95% of patients, the presence of the shortened 22q chromosome.1 This “minute” chromosome first described by Norwell and Hungerford2 was the first chromosomal abnormality associated with a specific malignancy. It was fittingly given the name “Philadelphia chromosome” (Ph) after the city of its discovery. The Philadelphia chromosome now is known to be caused by a reciprocal t(9:22)(q34;q11.2) event in a hematopoietic stem cell that results in derivative 9q+ and 22q- chromosomes.3 The translocation event fuses the Breakpoint Cluster Region (BCR) gene on chromosome 9 with the Abelson Tyrosine Kinase 1 (ABL1) proto-oncogene on chromosome 22 to form the BCR-ABL1 fusion gene on chromosome 22q- (Fig. 1.1).4,5 The exact break points of the translocation event are variable and have different modes of molecular and clinical pathogenesis.6-8 For instance, expression of p210 form of BCR-ABL1 in the hematopoietic stem cells of transgenic mice or transplantation models causes a CML-like disease whereas the p190 form causes an aggressive B cell lymphoblastic leukemia; those same isoforms are also the most common in their human disease counterparts.9-11 These studies have concluded that BCR-ABL1 expression in the correct cell type can efficiently cause a CML-like myeloproliferative disease, confirming it alone is necessary and sufficient to produce CML.11,12    2  The molecular pathogenesis of CML relies on the BCR-ABL1 fusion protein that characterizes the disease. ABL1 is a non-receptor tyrosine kinase expressed in most human tissues to shuttle signals from the cell surface to the nucleus mainly for cytoskeletal rearrangements.13,14 BCR contains several modular domains important for interacting with signaling partners.15 This fusion constitutively activates the tyrosine kinase activities of the ABL domain through trans-autophosphorylation in BCR-ABL1 dimers and tetramers and also adds regulatory domains from the BCR domain that allows the tyrosine kinase to interact with new signaling partners.15 Transgenic expression of BCR-ABL1 containing a point mutation that inactivates its kinase activity in mice is insufficient to cause disease, confirming that upregulation of tyrosine kinase activity BCR-ABL1 is essential for its pathogenesis.15 BCR-ABL1 drives proliferation, growth factor independence, and apoptosis evasion through the activation of oncogenic pathways such as PI3K/AKT, JAK/STAT, and RAS/RAF/MEK/ERK.15-18 These pathways and more maintain the leukemic CD34+ stem/progenitor population, particularly CML stem cells in the bone marrow, and drives massive proliferation of the myeloid linage in the peripheral blood, leading to disease. CML is an aging-related disease with the median age of diagnosis at 65 years, and accordingly the prevalence of disease in the United States has increased from 25-30,000 in the year 2000 to 80-100,000 in 2015 due to the aging population.19 There are no known major geographical or environmental associations with the disease but there is a sex-bias of men having a greater prevalence of CML than women.19 The CML disease course is triphasic beginning in the relatively benign chronic phase (CML-CP) and then progressing through the accelerated phase (CML-AP) and then ending in the fatal blast crisis (CML-BP) phase.20 Most patients (90-  3  95%) initially present in the CML-CP of disease and may be asymptomatic (50%) or have non-specific symptoms such as fatigue, anemia, night sweats, and/or mild abdominal discomfort due to splenomegaly.19,20 Clinical presentation of CML-CP is an increased number of Ph+ granulocytes in the peripheral blood and bone marrow with <15% blast cells.20 The patient may display signs of anemia or splenomegaly due to an abolishment of the blood cells during therapy and the engraftment of leukemic cells in the spleen, respectively. With sufficient therapy, most patients will stay in the CML-CP and their disease will be maintained over several years. However, some will progress into CML-AP defined by >15% blasts in the peripheral blood and bone marrow, >20% basophils in the peripheral blood, and platelet counts <100,000/µl.20 The most fatal outcome of CML is when patients enter CML-BP and the leukemic cells become more primitive and destructive. These patients present >30% blasts in the peripheral blood or bone marrow, blast clusters in the bone marrow, and signs of extramedullary disease.20 Few patients, regardless of stage, become fully cured of disease and must remain on therapy for the duration of their lives.  1.1.2 CML Treatment: Clinical Advancements Treatment for CML aims to elicit three types of disease response: hematological, cytogenic, and molecular. The clinical effects of treatment can cause either a complete or partial hematological response. To achieve a complete hematological response (CHR), the peripheral white blood cell counts must normalize to < 10 x 109 L-1, platelet counts to <450 x 99 L-1, and there must be no signs of immature blast cells or splenomegaly.21 After the Ph chromosome was discovered and could be monitored through fluorescent in-situ hybridization (FISH) of metaphase chromosomes, the cytological response to treatment was also used to measure   4  treatment response. The different cytogenic responses consist of complete (CCR, no Ph+ metaphases), major (0-35% Ph+), and minor (36-90% Ph+).21 After the advancement of reverse-transcriptase polymerase chain reaction (RT-PCR), a more sensitive measurement now used clinically in the molecular detection of BCR-ABL1 transcripts in the peripheral blood. For example, patients have a major molecular response (MMR) when the BCR-ABL1 transcripts have a 3-log reduction from the pre-treatment numbers after 3 months of tyrosine kinase inhibitor (TKI) therapy.21-23 A complete molecular response (CMR) accordingly has no BCR-ABL1 transcripts detectable in the threshold of RT-PCR. Currently in the clinic, CML patients are checked for their hematological response every 2 weeks until CHR is achieved and then every 3 months, cytogenic response every 6 months until CCR in achieved and then every 12-18 months, and molecular response every 3 months and then still monitored routinely even after CMR is achieved.20,21  Before the discovery of TKI therapy, CML was first treated with cytotoxic drugs such as busulfan and hydroxyurea to reduce the granulocyte expansion and prevent disease manifestations; however, most CML patients would progress to CML-BP and therefore CML was an ultimately fatal diagnosis.24,25 The next treatment option that became available in the 1980s was recombinant interferon-alpha (rIFN-α), a cytokine released by the immune system typically to control the immune response to pathogens.24,26 The rIFN-α therapy specifically targeted immune cells unlike the previous non-specific chemotherapy treatment, and accordingly many patients reached CHR and the annual death rate of CML reduced by 30% in phase II clinical trials.27 Even so, the rIFN-α treatment was not curative and the severe adverse effects of the drug were intolerable for many patients.26    5  Researchers and clinicians believed targeting the BCR-ABL1 oncoprotein would be revolutionary for this disease, and this was proven true by Druker et al.28 The first TKI therapy targeting BCR-ABL1 in CML was Imatinib Mesylate (IM). This 2-phenylaminopyridine-based compound competitively inhibits the ATP-binding site in the inactive form of ABL to block the conformational switch to the active form.29 Without ABL’s tyrosine kinase activity, BCR-ABL1 is rendered non-functional and Ph+ leukemic cells are pushed towards apoptosis.28 IM treatment does inhibit a few other tyrosine kinases in addition to BCR-ABL1 such as the platelet-derived growth factor receptor (PDGFR), ARG, and c-KIT, but it does not target the closely related SRC family kinases.30 Phase I clinical trials with IM showed that oral doses up to 400 mg were rapidly absorbed, had a half-life in the peripheral blood of 13-16 hours, and had low toxicity and side effects especially compared to the highly toxic rIFN-α.31 The first phase III clinical trial with IM induced a CHR in 95% of patients and CMR in 87% of patients over 18 months, compared to 55% and 35%, respectably, in the rIFN-α treated group.32 These revolutionary results fast-tracked IM into becoming the first-line treatment for CML patients just one year after its introduction into the clinic and still is used as the first-line therapy today.31,33  1.1.3 CML Treatment: IM Resistance Cancer cells are constantly changing due to the genomic instability that comes with rapid division, therefore when monotherapy is applied to these cells it promotes selection of leukemic clones that are resistant to IM therapy. The most common mechanism of resistance is the accumulation of BCR-ABL1 mutations in the tyrosine kinase domain that alter IM’s binding ability to BCR-ABL1 (Fig. 1.1).34,35 The point mutations typically reside within the ABL ATP-binding site to alter IM’s binding pocket, and the most difficult to target is the gatekeeper   6  mutation T315I.36,37 Another set of mutations alter domains that affect the conformational switch of BCR-ABL1 to favour the active form which IM cannot bind to.36 Patients may also develop resistance through amplification of the BCR-ABL1 gene, which was observed experimentally in CML cell lines that were cultured in gradually increasing concentrations of IM as well as in CML patients.35,38,39 Cancer cells also evade therapy by varying expression of influx/efflux proteins that shuttle small molecules through the cell, and in CML the efflux pumps P-glycoprotein and BRCP are known to target IM and mutations in their respective genes have been described.40-42  As CML progresses, the leukemic cells begin to accumulate mutations in pathways that are independent from BCR-ABL1 (Fig. 1.1). The first recognized BCR-ABL1-independent mechanism of IM resistance was overexpression of the SRC family kinase Lyn in IM-resistant CML cell lines, and dual suppression of BCR-ABL1 and Lyn activities was effective in suppressing cell proliferation.43,44 Since then, molecular and cellular changes that promote stem cell maintenance45,46, stress response47,48, genomic instability49,50, niche interactions51, and down-stream signaling partners52,53 have also been identified to cause IM-resistance in CML. Activation of these additional pathways is beneficial for the CML cells to become less addicted to BCR-ABL1 and therefore less targetable during treatment. Dual inhibition of activated survival pathways along with BCR-ABL1 is necessary to block the leukemic growth once additional mutations have arisen.    7   Figure 1.1: Mechanisms of disease pathogenesis and TKI resistance in CML. Disease is initiated in CML after a translocation event in a hematopoietic stem cell (HSC) that creates the fusion gene BCR-ABL1. The signalling activities of BCR-ABL1 drive a mass expansion of mature myeloid cells in the peripheral blood and bone marrow. During CML-CP, these cells are reliant on BCR-ABL1 for survival so TKI therapy can effectively abolish much of this expansion. The selective pressure of therapy can cause the leukemic cells to accumulate mutations and other molecular changes that are both BCR-ABL1-dependant and BCR-ABL1-independent and ultimately lead to TKI resistance. These resistant clones become more primitive and destructive causing an acute disease of infiltrating blast cells that can be fatal.  One of the main contributing factors to IM-resistance and disease relapse in CML is the persistence of the leukemic stem cells (LSCs) during and after treatment (Fig. 1.1).54-56 These cells are the long-term quiescent population that resides in the protective bone marrow niche.57 Although IM therapy had groundbreaking success to overall survival of chronic phase CML, most patients will not be cured of CML and will have to remain on IM therapy for the duration   8  of their lives.58,59 Patients that display CMR with no detectable BCR-ABL1 transcripts often still relapse after discontinuation of TKI therapy.60,61 Past studies have shown that primitive CD34+CD38- LSC subpopulations expressing very low levels of BCR-ABL1 are more elusive to therapy and can persist longer than those with higher BCR-ABL levels detected in more mature cells.54,62 These LSCs persist often because of pathways independent of BCR-ABL1 that promote G1 arrest (e.g. PI3K/AKT/FOXO),63 self-renewal (e.g. hedgehog signaling),64,65 niche interactions (e.g. Wnt signaling, adhesion proteins),66-68 and starvation resistance (e.g. autophagy)46.  Targeting these non-dividing cells within their niche proves nearly impossible for TKIs, and still is the largest challenge in CML treatment. The only curative treatment of CML remains to be allogenic stem cell transplant, in which the entire bone marrow of a patient is irradiated to kill all of the host’s long-term stem cells and then healthy donor cells are transplanted in their place.69 However, this procedure is not a suitable first-line therapy because of the rarity of finding matching donors and the high chance of graft rejection.70,71 Additionally, because CML is most commonly diagnosed in older adults the procedure itself often is too high-risk to perform.70 1.1.4 CML Treatment: Additional Treatment Options Since the introduction of IM, several new TKIs became available to treat patients that were resistant or intolerant to IM. The first second-generation inhibitor developed was another phenylaminopyramidine-based compound Nilotinib (NL), which acts similarly to IM as a competitive inhibitor of the ATP binding site, but with a higher affinity to the ABL kinase domain and accordingly more in vivo potency and selectivity.72 In addition, NL was found to be effective against 32/33 point mutations in the ATP binding site that cause IM-resistance except   9  the gatekeeper T315I mutation.73,74 Also similar to IM, NL inhibits the tyrosine kinases PDGFR, ARG, and c-KIT without inhibiting the SRC family kinases.30 The first phase II clinical trial showed the rates of MMR in the NL treated group were twice that of the IM treated group after 12 months of treatment (NL=44% vs. IM=22%).75 Meanwhile, a non-phenylpyramidine-based SRC-family kinases inhibitor, Dasatinib (DA), became clinically available to treat IM-resistant patients.76,77 DA has more in vitro and in vivo potency than both IM and NL but it targets a larger number of other tyrosine kinases, which contributes to an added toxicity.30,78 Although potent, DA also cannot bind to the T315I mutants and therefore a third generation inhibitor, Ponatinib (PO), was developed.79 Due to its adverse side effects PO is clinically used as a last line therapy and will not be given to patients unless they have had no response to two other TKIs and/or have a confirmed T315I mutation.80 Even so, studies have shown that some compound mutations and the T315M (requires two subsequent amino acid changes) in the ATP binding site can confer resistance to PO as well.81  1.2 Autophagy 1.2.1 History of Autophagy Macroautophagy, hereafter autophagy, is a catabolic cell recycling process that degrades cellular components to increase nutrient availability and eliminate toxic waste. This process was first discovered 60 years ago when mitochondria were observed inside membrane-bound vesicles of mouse kidneys, and these vesicles were later discovered to contain lysosomal hydrolases.82 This process was coined “autophagy” by Christian de Duve at the 1963 Ciba Foundation symposium on lysosomes, meaning “self-eating” to describe the digestion of cellular components.83 Researchers began to unravel the functionality of the process when they observed   10  increased autophagy in starved/amino acid deprived animals, and reduced autophagy in animals supplied with sufficient amino acids in their diet.84,85 The general opinion of the field in that time was that autophagy served as the cellular garbage disposal mechanism to replenish missing nutrients but its true importance was not elucidated for several more decades. The players of the process were famously characterized in 1992 by Dr. Yoshinori Ohsumi’s group, for which he was awarded the 2016 Nobel Prize.86 They conducted a mutagenesis screen in yeast for genes that are important for properly functioning autophagy when yeast was grown on medium devoid of nutrients. In the past 25 years since the initial characterization of autophagy, research in its roles in homeostasis, immunity, ageing, cancer, and other diseases have been rapidly progressing as researchers discovered autophagy is much more than just the cellular “garbage-disposal”.83 1.2.2 Players in the Autophagy Process  Unlike other autophagy mechanisms, macroautophagy is able to sequester degradation targets in an independent double-membrane vesicle and then fuse with a lysosome for degradation. The process is separated into four main parts: induction, nucleation, elongation, and fusion/completion (Fig. 1.2). All the players in this process were first characterized in yeast and then homologous proteins were found in mammalian cells. For the purposes of this review, the mammalian homologues will be described.    11   Figure 1.2: The players of the autophagy process. The process is initiated after the ULK complex is released from the repressive mTORC1 complex. The ULK complex then can phosphorylate the PtdIns3K complex to aid in homing elongation factors to the phagophore. The ATG12-ATG5-ATG16L1 and LC3-II complexes are required for elongation of the phagophore into a mature autophagosome. The autophagosome then fuses with the lysosome and hydrolases are released into the vesicle to degrade the sequestered cargo.   The initiation complex of autophagy is comprised of the ULK1/2-ATG13-FIP200 proteins, which remains stable in the cell regardless of nutrient availability.87 This complex actively initiates autophagy unless it is inhibited through phosphorylation of ULK1/2 and ATG13 by mTORC1 - which is upregulated by a high nutrient density in the cell as well as many other processes.87 Upon cell starvation or mTOR inhibition, the initiation complex is released and it activates nucleation through phosphorylation of BECN1 within the ATG14-containing   12  class III phosphatidylinositol 3-kinase (PtdIns3K) complex.88 The PtdIns3K complex localizes at the site of phagophore generation which can be the endoplasmic reticulum, the plasma membrane, the golgi apparatus, the mitochondria, or other double membrane structures. Within this complex are PIK3C3, PIK3R4, PtdIns3K/VPS34, and BECN1, with BECN1 being the most crucial for regulation and induction of autophagy.89,90 VPS34 deposits lipid phosphatidylinositol (PI) on the surface of the phagophore that gets phosphorylated into phosphatidylinositol-3-phosphate (PI3P) and serves as a docking site for proteins bringing degradation targets into the autophagosome.91 Next, the elongation step requires the conjugation of two ubiquitin-like (UBL) proteins to the membrane of the phagophore for it to mature into an autophagosome.92 The first system forms the ATG12-ATG5-ATG16L1 dimeric complex that becomes associated with the membrane via attachment of ATG16L1.92 The complex is brought together through the irreversible conjugation of the UBL protein ATG12 to ATG5, catalyzed by the E1-like activating enzyme ATG7 and the E2-like conjugating enzyme ATG10.93-95 ATG16L1 binds to ATG5 and then ATG16L1 dimerizes with another ATG12-ATG5-ATG16L1 complex and then attaches to the phagophore membrane.96 The second UBL conjugation system attaches lipidated mammalian ATG8 homologues to the autophagosome membrane (Fig. 1.3); these homologues are divided up into the MAP1-LC3 (LC3) and GABARAP subfamilies with the LC3 system being the most understood.92,97  The translated LC3 protein first is cleaved by ATG4B into LC3-I, which has an exposed C-terminus glycine residue necessary for subsequent conjugation.98 The E1-like activating enzyme ATG7 then transfers LC3-I to the E2-like conjugating enzyme ATG3.99 The ATG12-ATG5-ATGL1 complex recruits the lipid phosphatidyethanolamine (PE) from lipid   13  membranes and catalyzes the lipidation of LC3-I to PE and the attachment of PE to the membrane to form LC3-II.100 Once the autophagosome is fully formed, ATG4B cleaves off the PE domain from LC3-II to release LC3-I back into the cytosol to be reused in subsequent rounds of the process. In the final step of autophagy, the autophagosome must close off from the cytosol and fuse with the lysosome to degrade its contents. The completed autophagosome is trafficked through the cell to the lysosomes by microtubules, and then fuses to form an autophagolysosome.101 The acidic hydrolases within the lysosome degrade the macromolecules engulfed by the autophagosome into their smaller components such as amino acids, nucleotides, fatty acids, carbohydrates, and more.102 These molecules get released back into the cytosol to be reused in metabolic processes.    14   Figure 1.3: The roles of ATG4B in the processing of the ATG8 homologues LC3 and GABARAP. ATG4B first facilitates the cleavage of the translated product pro-LC3/GABARAP into LC3-I/GABARAP-I, which have a revealed C-terminus glycine residue necessary for subsequent conjugation. The E1-like enzyme ATG7 binds the C-terminus of LC3-I/GABARAP-I and facilitates the transfer of it to the E2-like enzyme ATG3. The conjugation of LC3-I/GABARAP-I to a phosphotidylethanolamine (PE) domain requires the ATG16L1-ATG5-ATG12 E3-like complex to interact with ATG3 and liposomes containing PE to help catalyze the lipidation of LC3-I/GABARAP-I. The lipidated LC3-II/GABARAP-II is attached to the autophagosome membrane during this step and remains there until completion of autophagy. Once autophagy is complete, ATG4B cleaves off the PE domain to release LC3-I/GABARAP-I back into the cytosol.     15  1.2.3 Regulation of Autophagy Autophagy is a tightly-regulated catabolic response to many types of cellular stresses such as nutrient starvation, hypoxia, growth factor deprivation, and ER stress.103 Various pathways are responsible for sensing the levels of these stressors within the cell and then inhibiting or inducing the autophagic response. The carbon and nitrogen content of the cell is sensed by cAMP-dependant protein kinase A (PKA) and mTOR pathways, and these two mediators block autophagy in nutrient-rich conditions.104,105 The energy-sensing kinase AMP-activated protein kinase (AMPK) upregulates autophagy through ULK1 phosphorylation during low-energy conditions, as determined by the AMP/ATP ratio.88 ER stress activates autophagy when the cytosolic Ca2+ concentrations increase and the calmodulin-dependent protein kinase kinase 2 (CAMKK2) activates AMPK, or when the unfolded proteins in the ER accumulate and induce autophagy.106 Hypoxia also positively regulates autophagy through inhibition of mTORC1.107 Several other pathways that become activated by extracellular signals such as growth factors and insulin are down-regulated in their absence and result in an upregulation of autophagy.108  These stressors can be applied to the cell experimentally to upregulate autophagy and study its effects in different models. Autophagy can be induced in vitro through manipulation of the cellular medium or through chemical induction. Common methods are depriving the cells of serum, glucose, or glutamine to decrease the nutrient and growth factor content within the cell.109 Rapamycin can chemically induce autophagy through inhibition of mTOR, although this method affects many additional pathways and has indirect toxicity.110 Autophagy has also been discovered to be upregulated in mammals after fasting and even in-between meals.111,112 After 48   16  hours of fasting in mice, there is increased LC3B puncta in the hepatocytes and all leukocytes, while 48 hours of fasting in human volunteers increases the LC3B puncta only in neutrophils and not in any other leukocytes.112 Autophagy inhibitors are also commonly used in experimental studies. However, the most characterized inhibitors such as chloroquine (CQ) and bafilomycin A1 (BafA) target lysosomal degradation and not autophagosome development, and therefore are not specific to macroautophagy.109 New inhibitors that are specific to macroautophagy would be useful when researching its roles.  1.2.4 Autophagy and Cancer Including CML Autophagy has context-specific roles in cancer as both tumour suppressive and tumour promoting depending on the stage of cancer and the cell type. For non-cancerous cells, autophagy reduces the aggregation of potentially harmful damaged proteins and organelles that can cause irregular behaviors or induce damage to the cell. Key tumour suppressing roles of autophagy are maintaining a metabolic homeostasis113, limiting inflammation in the tissue114, clearing harmful mitochondrial waste115, defending against pathogens114, and aiding stem cell maintenance116. Loss of proficient autophagy often occurs as normal cells transform into malignant cells and alter their normal homeostasis.117,118 For instance, monoallelic deletion of BECN1 is found in breast, ovarian, and prostate cancers and heterozygous deletion of BECN1 in mice causes spontaneous heptacellular carcinoma as they age.119,120  As cancer progresses, the cells go through metabolic reprogramming where they begin to upregulate pathways normally active during stress response, including autophagy. This is the point where autophagy transforms into a tumour promoting pathway because it allows the cell to live through environments it usually could not.117 The upregulation of autophagy allows the cell   17  to be more resistant in stressful conditions including hypoxia,121 starvation,122 therapy (reviewed later), and the epithelial-mesenchymal transition123. Importantly, much like in normal cells autophagy maintains the cancer stem cell population that can repopulate a cancer following treatment.124,125 There has been increased autophagy reported after cancer treatments both in preclinical and clinical studies. Notable examples include cisplatin for ovarian cancer,126 5-fluorouracil for colon cancer,127 epirubicin for breast cancer,128,129 radiation and tomozolomide (TMZ) for glioma,130  and importantly, imatinib for chronic myeloid leukemia131.  Autophagy inhibitors such as chloroquine have been tested in combination with standard anti-cancer therapies or other therapies and have shown promising results in preclinical testing and have led to over 50 clinical trials.132 However, CQ/HCQ therapy has had mixed results regarding tolerated dose and efficacy depending on the tumour type and concurrent therapy. For instance, a recent phase I/II glioblastoma clinical trial investigating HCQ in combination with radiation and TMZ found no significant survival improvements with the addition of HCQ unlike a previous study, and the therapy had dose-limiting toxicity due to myelosuppression.133 By contrast, a very interesting trial in pre-operative pancreatic ductal adenocarcinoma (PDAC) showed that after patients were treated with HCQ and gemcitabine, the patients whose LC3-II protein levels increased by more than 51% in peripheral blood mononuclear cells had an increased progression-free survival (PFS) compared to those who’s LC3-II levels stayed low (PFS; 15 months compared with 6.9 months).134 The effects of autophagy inhibitors in cancer are still quite unknown although evidence has shown that it is worth exploring.  The connection between autophagy and CML was first recognized by an increased autophagic response following treatment with IM or other non-targeted drugs in CML cell lines   18  and primary patient samples.131,135 This response was attributed to ROS generation, ER stress response, and a dose-dependent increase in gene and protein expression of key autophagy players.131,136-138 The increase in autophagy induces CML cells to become senescent rather than apoptotic, causing them to be more elusive to treatment.139 Pharmacological or genetic suppression of autophagy in combination with IM increases apoptosis and depletes the CML stem/progenitor population in IM-resistant cells.131,135,140 In contrast, activation of autophagy through mTORC1 inhibition or oncolytic expression of BCLN1 has also been shown to also have therapeutic benefits in CML in recent reports.141,142 This may be because the BCR-ABL1 protein is suggested to be targeted by autophagic vesicles following IM treatment resulting in decreased protein levels.143 The autophagic response in CML is quite evident but its therapeutic implications have not been completely elucidated.  1.3 The Core Autophagy Protease ATG4B 1.3.1 The Human ATG4 Homologues An important step in the autophagy process is the attachment of lipidated ATG8/LC3 to the autophagosome membrane, and the initiating step of this process is the cleavage of pro-LC3 into LC3-I by ATG4. There are four human homologues of the yeast ATG4 protein (ATG4A-D) and each has subtle differences in their protease functions towards the 6 human homologues of ATG8: LC3A-C, GATE-16, GABARAP, and GABARAPL.144 ATG4B has the broadest spectrum of ATG8 homologue targets as it effectively cleaved all 6 ATG8 targets more efficiently than any other ATG4 homologue.144 ATG4A had the next largest spectrum and is especially effective at targeting GATE-16 and weakly targets GAPARAP but cannot cleave   19  LC3A-C.144 ATG4C and ATG4D have the lowest efficacy in cleaving ATG8 homologues, with no activity towards GABARAP and weak activities towards LC3 and GATE16.144   ATG4B is a cysteine protease with activity specific to the ATG8 human homologue families.145 The initial cleavage of pro-LC3 into LC3-I by ATG4B is necessary for the subsequent conjugation of LC3 to the autophagosome membrane, and the delipidation step allows the LC3-I species to be recycled for faster autophagy turnover.146 ATG4B interacts with the LC3 and GABARAP subfamilies via their C-terminal LC3-interacting region (LIR) that is important for both cleavage and stabilization of the unlipidated forms of LC3 and GABARAP (Fig. 1.4).147 Expression of an inactive mutant of ATG4B in human cells halts autophagy and causes an accumulation of the degradation marker SQSTM1/p62 due to the defects in autophagosome closure.148 Without ATG4B, unlipidated LC3-I and GABARAP-I become unstable in the cell and are targeted by the proteasome for degradation, therefore causing the LC3-II/LC3-I ratio to increase.147 Mice harboring an atg4b knockout mutation show lowered levels of both the lipidated (LC3-II) and unlipidated (LC3-I) forms of LC3 and GABARAP subfamilies throughout a wide range of tissues due to the deficient processing.149 These mice are phenotypically viable but they display abnormal inner ear pathologies that manifest in behavioral changes and balance problems.149    20   Fig. 1.4: Crystal structure of ATG4B. (A) Regulatory regions on ATG4B, with red indicating positive regulation and blue indicating negative regulation. (B) The complex of ATG4B with substrate LC3 and non-substrate LC3. Reprinted with permission from Springer Nature150.   1.3.2 ATG4B in Cancer including CML Several studies have implicated ATG4B as having an impact on cancer progression. Transcript and protein levels of ATG4B have been reported to be raised in cancer tissues   21  compared to normal controls in prostate cancer,151 breast cancer,152 and lung cancer.153 Reciprocally, the miRNA responsible for targeting ATG4B transcripts, miR34a, is downregulated in these cancers and has shown increased DNA methylation on its promoter leading to its silencing.151,154 Knockdown or knockout of ATG4B slows tumour growth in breast cancer and osteosarcoma in vivo and sensitizes cells to treatment or serum starvation in vitro.152,153 The success seen using chloroquine in cancer therapy has prompted researchers to search for an autophagy-specific target for therapy, and ATG4B has been considered a suitable target. Pre-clinical research has begun to discover small molecules that inhibit ATG4B activities and have then used these inhibitors either alone or in combination with other cancer therapies. A benzotropolone derivative confirmed to inhibit ATG4B blocked autophagy in LC3B-GFP mice that were nutrient restricted and slowed tumour growth when combined with oxiplatin in colorectal tumour xenografts.155 In Saos-2 osteosarcoma, small molecule inhibition of ATG4B alone reduces cancer cell viability and tumour growth.153 Conversely, there is a context-specific response to ATG4B in cancer much like autophagy.  Expression of the enzymatically inactivated form ATG4B (C74A) in prostate cancer could either amplify the effects of therapy or contribute to chemoresistance depending on the subtype of tumour and the accompanying treatment.156 Additionally, a small molecule that was discovered to be an ATG4B agonist induced autophagy in TNBC cell lines and had anti-proliferative effects in vitro.157   Our lab has recently shown that several key autophagy genes are differentially expressed in CML stem/progenitor cells compared to normal bone marrow, most substantially was ATG4B and its deregulating miRNA miR-34a.154 Importantly, ATG4B gene and protein expression was even higher in the IM-nonresponders compared to IM-responders, suggesting that this enzyme   22  could be a biomarker for treatment response. Upon stable knockdown of ATG4B, autophagy becomes suppressed and primitive CML cells are sensitized to TKI therapy. This evidence agrees with past literature in other cancers that suggests ATG4B may be a suitable target in the autophagy survival pathway.  1.3.3 Discovery of Novel ATG4B Inhibitors Our lab is in collaboration with the medicinal chemistry team in Dr. Robert Young’s lab at Simon Fraser University who provide us with novel ATG4B inhibitors. My project focuses on inhibitors that were found through an in vitro assay that uses a fluorescently active substrate that binds ATG4B and becomes cleaved to emit a change in fluorescent emission.158 A high-throughput screening of over 200,000 compounds yielded nine positive hits that were confirmed by structure-activity relationship studies (unpublished data). The top candidate compound, DB1-043 (DB1), has been chemically modified into four different analogs which in microscale thermophoresis assays show consistent Kd values for binding to ATG4B. The analogue with the best in vivo pharmacokinetics and in vitro potency and selectivity is DB2-082 (DB2). This compound has an in vitro IC50 to ATG4B of 2.1µΜ and does not readily bind ATG4A and other cysteine proteases (IC50 > 100uM). In mouse studies, DB2 shows good absorption, good bioavailability, and has a half-life of approximately 6 hours (unpublished data). The binding site of DB2 on ATG4B has not been elucidated yet but the drug binding occurs independent of the substrate concentration, suggesting a non-competitive inhibition model. My study mainly focuses on the inhibitor DB2, but also investigates its analogues DB1, DB3, and DB4, in the CML model.    23  1.4 Summary In progressed disease, CML cells acquire mutations and other molecular changes in BCR-ABL1-independent survival mechanisms to circumvent TKI treatments. Autophagy has been characterized as a mechanism used by cancer cells to provide a renewal of cellular molecules that allows them to persist during treatment. In CML, the addition of autophagy inhibitors to TKI therapy showed promising combination effects. One particular autophagy enzyme, ATG4B, was overexpressed in CML stem/progenitor cells compared to normal bone marrow, and importantly even more so in IM-nonresponders. Thus, inhibition of this key autophagy protein in combination with TKIs may provide a possible treatment strategy to specifically inhibit autophagy and sensitize the cells to standard therapy. 1.5 Hypothesis I hypothesize that combined suppression of BCR-ABL1 and ATG4B activities by new ABL and ATG4B inhibitors in autophagy-inducing conditions can have therapeutic benefits over TKI treatment alone through an inhibition of autophagy in CML models.  1.6 Thesis Objectives 1. Investigate the ability of new ATG4B inhibitors alone or in combination with ABL inhibitors to inhibit the growth of CML cell lines and CD34+ CML stem/progenitor cells from IM-nonresponders in vitro; 2. Investigate the impact on autophagy following ATG4B inhibition by small molecule inhibitors in CML cells by Western blot analysis and advanced autophagy assays;       24  3. Optimize in vivo assay conditions for investigation of the efficacy of ATG4B small molecule inhibition/genetic knockdown, alone or in combination with TKIs, to block leukemia development.   25  Chapter 2: Materials and Methods 2.1 Cell Culture Materials 2.1.1 Cell Lines The human Ph+ cell lines used in this study were K562, IM-Resistant K562 (IMR), and BV173. K562 cells were derived from a 52-year-old CML-BP patient harboring a p210 BCR-ABL1 fusion protein with no BCR-ABL1 kinase mutations. IMR cells were derived from the K562 cell line by culturing the cells in increasing concentrations of IM until resistant clones arose that did not have BCR-ABL1 mutations in the kinase domain (provided by Dr. A. Turhan, University of Poitiers, France). BV173 cells were derived from a 47-year-old B cell acute lymphoblastic leukemia patient harboring a p190 BCR-ABL1 fusion protein without BCR-ABL1 kinase mutations. The cell lines were maintained in Rosewell Park Memorial Institute (RPMI) culture medium containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), and 1% β-mercaptomethanol (BME) and incubated at 37°C and 5% CO2. Stock vials containing 1x106 cells were cryologically frozen at -135°C until use.  2.1.2 Primary Human Cells Patient samples were obtained from heparin-anticoagulated peripheral blood (PB) or bone marrow (BM) of CML-CP patients at the time of diagnosis. Normal bone marrow cells (NBM) were obtained from healthy adults as a voluntary donation. Informed consent was obtained in accordance with the Declaration of Helsinki, and the procedures were approved by the Research Ethics Board at the University of British Columbia (certificate H12-02372). The mononuclear cells were isolated from the samples using Ficoll-Paque density gradient separation and the stem/progenitor population was enriched using the EasySep™ Human CD34 Positive Selection   26  Kit (STEMCELL Technologies). The purity of the stem/progenitor population was verified by staining the cells for the CD34 surface marker with allophycocyanin (APC)-labeled anti-human CD34 antibody (BD Biosciences) and then checking the CD34+ expression with a fluorescent-activated cell sorter (FACS) to confirm the purity was >85%. Stock vials were cryologically frozen at -135°C until use. IM non-responding patients were exclusively used in this study. To be characterized as a responder, patients had to have achieved a >3-log reduction in BCR-ABL1 transcript levels at 12 months of treatment (MMR) and show no Ph+ cells after cytogenetic analysis at 6 months of treatment (CCR).22 IM non-responders therefore do not meet these criteria and still have detectable transcripts and Ph+ cells. As these samples were taken at diagnosis, the IM response was determined on the retrospective samples after treatment.  2.1.3 Inhibitors Cytotoxic pharmaceutical drugs targeting ATG4B, BCR-ABL1, and lysosomes were used in this study. The ATG4B inhibitors DB1-043 (DB1), DB2-082 (DB2), DB1-113 (DB3), and DB1-114 (DB4) were provided by Dr. R. Young (Simon Fraser University, Burnaby) as 40mM stock solutions dissolved in DMSO. The inhibitors were aliquoted into 10mM stock solutions in DMSO and stored at -20°C. When making the working solutions, the first 1:10 dilution was done in a 40% DMSO / 60% medium solution. All subsequent dilutions were done in 100% medium. The inhibitor CQ was in an aqueous stock solution of 50mM and frozen at -20°C. The stock was thawed and diluted in aqueous culture medium to make the working solutions. The TKIs IM and NL were obtained from Novartis (Novartis, Basel, Switzerland) and DA was provided by Bristol-Myers Squibb (Princeton, USA). The powder form of the TKIs were reconstituted into   27  10mM stock solutions to store at -20°C. For IM, the working solution was diluted 1:10 into 100% aqueous medium for all dilutions. For DA, the first 1:10 dilution was done in a 40% DMSO / 60% medium solution and then all subsequent dilutions were in 100% medium. Lastly, NL was first diluted 1:10 dilution in a 60% DMSO / 40% medium solution and then all subsequent dilutions were in 100% medium.  2.2 In vitro Assays  2.2.1 Cell Starvation Assays To activate autophagy, cell lines were cultured in RPMI medium with a reduced concentration of FBS through the duration of various assays. The lowest concentrations of FBS in which the cells could still proliferate was selected for each cell line. K562 and IMR cells were cultured in 1% FBS medium and BV173 cells were cultured in 2% FBS medium.  2.2.2 Viability Assay CML cell lines were seeded at an appropriate density depending on the cell line (1x105 cells/ml for K562 and IMR, 2x105 cells/ml for BV173) in a 12-well plate containing 1ml of RPMI medium supplemented with 1% P/S, 1% BME, and varying amounts of FBS. Different drug combinations were added to the plate at the appropriate concentrations at the start of the assay. The cell densities were counted after 72 hours of treatment using trypan blue dye with a Neubauer hemocytometer.  2.2.3 Apoptosis Assay A portion of the CML cell line cell suspension that was plated for the viability assay was taken to analyze in an apoptosis assay after 72 hours of treatment. The cells were prepared using an Annexin V Apoptosis Detection Kit APC (eBioscience), which stained the cells with Annexin   28  V APC and Propidium Iodide (PI). This kit differentiates cell populations that are undergoing early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptosis from viable cells (Annexin V-/PI-). These populations were collected on a FACSCalibur (BD Bioscience) and analyzed using FlowJo® 10 software.  2.2.4 Colony-Forming Cell (CFC) Assay One bottle of methylcellulose medium (80ml) (STEMCELL Technologies) was supplemented with growth factors (300 U EPO, 2µg IL-6, 2µg IL-3, 2µg GM-CSF, and 2µg G-CSF), topped up to 100ml with Iscove’s Modified Dulbecco’s Medium (MDM) (STEMCELL Technologies), and then partitioned into 3ml aliquots. 2000-3000 CD34+ CML or NBM cells ±	experimental treatments were seeded and mixed thoroughly in the 3ml aliquots. Using a 3ml syringe with a blunt end needle, 1.2ml of the cell suspension was plated into duplicate CFC plates. The plates were incubated with adjacent water plates for humidity at 37°C and 5% CO2. After 12-14 days of incubation, each plate was enumerated according to colony type (erythroid, granulopoietic, and mixed) and expressed as a percent of the DMSO control. 2.3 Transfections and Transductions 2.3.1 Lipofectamine Transfection of a mCherry-EGFP-LC3 Vector One day before the transfection, 1x105 K562 cells were seeded into 500µl of complete RPMI medium and left overnight. The next day, two lipofectamine mixtures were prepared: 75µl Opti-MEM® medium (Thermo Fisher Scientific) + 4.5µl Lipofectamine® 3000 (Invitrogen) and 75µl of Opti-MEM + 6µl p3000 buffer (Invitrogen) and 1µg of mCherry-EGFP-LC3B vector (provided by Dr. Sharon Gorski, BC Cancer, Vancouver). The DNA mixture was added into the Lipofectamine® mixture and incubated at room temperature (RT) for 10 minutes to conjugate.   29  The cells were transfected with 50µl of the Lipofectamine®-DNA mix per well of cells and then incubated for 48 hours at 37°C and 5% CO2.  2.3.2 Lentiviral Transfection and Virus Harvest  The fetal kidney cell line 293T was used as hosts to produce lentivirus in a polyethyleimine (PEI) transfection. The 293T cells were seeded at 6x106 cells per 10cm culture plate (require 7 plates per vector) in 7ml Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS and 1% glutamine to be incubated overnight at 37°C and 5% CO2. The next day, the culture medium was replaced with 4.5ml fresh DMEM medium 2-4 hours before the transfection. For each plate, a solution was made containing 6µg desired construct, 3.9µg packaging construct ΔR, 1.5µg packaging construct REV, and 2.1µg vesicular stomatitis virus glycoprotein envelope construct, all in 250µl Opti-MEM® medium. Another solution was made with 40µg PEI in 250µl Opti-MEM® medium. The two mixtures and the 293T cells were brought downstairs to containment level 3 (CL3) where the PEI mix was added dropwise to the construct mix, vortexed, and incubated for 20 minutes at RT. The PEI-DNA conjugate was added to each plate and the transfection was left in CL3 for 48 hours at 37°C and 5% CO2. Produced virus in the supernatant of the 293T cell cultures was harvested and filtered through a 0.45µm low protein-binding filter (Pall Corporation) to remove cellular debris. The virus was then collected by ultrcentrifugation at 2.5x103 x g for 90 minutes at 4°C. Collected virus was resuspended in 100µl of Iscove’s medium containing 5% DNAse under slow rotation for 1 hour at RT. The virus aliquots were frozen at -80°C in CL3.    30  2.3.3 Knockdown of ATG4B in BV173YFP/Luc Cells BV173YFP/Luc were seeded at 1x106 cells/ml in RPMI only medium and then brought into CL3 for the infection. Concentrated lentivirus of the empty scrambled vector (SHC) or the ATG4B shRNA vector (shATG4B) was added to the cells at a concentration of 10µl per 1ml cell suspension along with protamine sulphate at a concentration of 12.5µl per 1ml cell suspension. The cells were incubated in CL3 overnight (16 hours) at 37°C and 5% CO2 for a maximum transduction efficiency. Infected cells were harvested and washed 3X PBS to remove all virus from the supernatant. The cells then were safe to be brought out of CL3 and were plated into complete RPMI medium for 24 hours. Antibiotic selection was applied to the cells using 0.5µg/ml pyromycin for at least 48 hours before using the cells in subsequent assays.  2.4 Molecular Techniques 2.4.1 Cell Lysis and Protein Quantification Washed cell pellets were lysed in lysis buffer (90% phosphorylation solubilizing buffer (PSB), 10% NP-40 Alternative Protein Grade Detergent (Calbiochem), 0.1% sodium dodecyl sulphate (SDS, Sigma Aldridge), 0.5% 200 mM phenylmethylsulfonyl fluoride (PMSF, Sigma Aldridge), 0.5% protease inhibitor cocktail (PIC, Sigma Aldridge) for 40-60 minutes at 4°C. The cell lysate was centrifuged at 13 000 x g at 4°C for 20 minutes. The supernatant containing the protein contents of the cells was kept and the pellet was discarded. The absorbances of the lysates were measures using a Bradford reagent (Bio-Rad) and measured at an absorbance of 570 nm on a spectrophotometer. The absorbances were compared to a Bovine Serum Albumin (BSA) standard curve to determine protein concentrations.    31  2.4.2 Western Blotting The samples used for western blotting were prepared with 20-30 µg of protein lysate, 4x loading dye, and ddH2O, and then heated for 10 minutes at 90°C to denature the proteins. Samples were loaded onto a 12% sodium dodecyl sulfate polyacrylamide-gel with 1.0 mm wells and run on an electrophoresis machine (SDS-PAGE) along with a PageRuler Pre-stained Protein Ladder (Fermentas) at 100V for 15 minutes, and then 150V until the loading dye reached the end of the gel. The separated proteins were transferred from the gel onto a polyvinylidene difluoride (PVDF) membrane (Millipore) at 100V for 1 hour while being kept cool with ice packs. The membrane was blocked with 5% skim milk powder in Tris-buffered saline Tween 20 (TBST) for 1 hour while rotating at RT. Membranes were washed with TBST and the primary antibody was added to the membrane in 2% BSA overnight while rotating at 4°C. The primary was washed off with 3x10 minute rotating washes with TBST and then the corresponding secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology) was added in 2% skim milk for 1 hour rotating at RT. The membrane was washed again for 3x10 minutes and the secondary on the membrane with reacted with a chemiluminescence enhancing reagent. Protein levels were visualized by exposing the membrane on KODAK BioMax Xar autogradiography film and processing in a film developer. Primary antibodies used in this study include anti-LC3B (Cell Signaling, 1:1000 dilution), anti-p62 (Cell Signaling, 1:1000 dilution), anti-ATG4B (Cell Signaling, 1:1000 dilution), and anti-β-Actin (Applied Biological Materials, 1:3000 dilution). 2.4.3 LC3B Puncta Monitoring through Immunofluorescence  Poly-L-lycine coated microscope slides were prepared with a 0.5-0.8cm diameter circle drawn with a liquid blocker PAP pen (Daido Sangyo Co. Ltd.). Cell lines and CD34+ cells (0.5-  32  1x105) were transferred into the PAP circle in 100µl of Dulbecco’s Phosphate Buffered Saline (PBS, STEMCELL Technologies) and were left to settle for 10 minutes at 37°C. The cells were then fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton-X for 5 minutes, and blocked with 3% BSA for 1 hour. The LC3B primary antibody (Cell Signalling) was added to the PAP circle at 1:200 dilution in a PBS staining solution containing 0.1% gelatin and 0.2% goat serum overnight at 4°C. The slides were washed in PBS and then stained with the secondary antibody (anti–rabbit IgG FITC-conjugate, Invitrogen) at 1:1000 dilution in PBS staining solution for 1 hour in the dark. The cells were washed with PBS and ddH2O (last wash) before ProLong® Gold antifade reagent with DAPI (Life Technologies) was added to the slide and a cover slip was mounted on with nail polish. The slides were kept in the dark at 4°C until being imaged on Leica SP5II Inverted LSCM on the 60x objective (immersion oil).  2.4.4 LC3B Puncta Monitoring through Endogenous Fluorescence Poly-L-lycine coated microscope slides were prepared with a 0.5-0.8cm diameter circle drawn with a liquid blocker PAP pen. Cell lines (0.5-1x105) transfected with the mCherry-EGFP-LC3B vector were transferred into the PAP circle in 100µl of PBS and were left to settle for 10 minutes at 37°C. The cells were then fixed with 4% paraformaldehyde for 10 minutes and then washed with PBS and ddH2O (last wash) before ProLong® Gold antifade reagent with DAPI was added to the slide and a cover slip was mounted on with nail polish. The slides were kept in the dark at 4°C until being imaged on Leica SP5II Inverted LSCM on the 60x objective (immersion oil).    33  2.4.5 RNA Extraction and Quantitative Real-Time PCR (qRT-PCR) RNA was extracted from cell suspensions using TRIzol (Life Technologies) according to the company SOP. The RNA was resuspended in RNase-free ddH2O (Life Technologies) and a nanodrop ND-100 spectrophotometer (Thermo Fisher Scientific) was used to determine the concentrations. 300-500ng RNA was reverse-transcribed into cDNA using the SuperScript® VILOTM Master Mix (Life Technologies) as per the company SOP. Each well in a 96-well plate was prepared for qRT-PCR with 6µl of a 2X SYBR® Green PCR Master Mix (Life Technologies), 1µl of 20 µM gene specific primers, 1µl cDNA and brought to 12µl with RNase-free ddH2O. The plate was centrifuged down to remove droplets and bubble and then run on the 7500 Real Time PCR System (Applied Biosystems).  2.5 In vivo Assays  2.5.1 Transplantation of BV173YFP/Luc Cells into Immunodefficient Mice with DB2+DA Treatment 8-10 week old female NOD/SCID-interleukin 2 receptor γ-chain-deficient (NSG) mice were sub-lethally cesium irradiated overnight (315 cGy). The following day, the mice were injected intravenously with 2.5x106 BV173 cells expressing yellow fluorescent protein (YFP) and luciferase (BV173YFP/Luc). The mice’s body weight and activity levels were monitored every other day throughout the duration of the experiment. Two weeks after injection, the mice were split into the following treatment groups for two weeks of oral gavage: vehicle (propylene glycol), 15 mg/kg DA, 50 mg/kg DB2, 100 mg/kg DB2, 50 mg/kg DB2 + 15 mg/kg DA, and 100 mg/kg DB2 + 15 mg/kg DA. Due to incompatibilities between DA and DB2, the combination mice received the DB2 dose 1 hour before receiving DA. The engraftment of BV173YFP/Luc cells   34  were imaged in vivo at 2, 6, and 8 weeks post-injection using the Xenogen IVIS® 50 Bioluminescence Imaging System and the Live Imaging Software Version 3.0 after intraperitoneally injection with 3mg D-Luciferin. The engraftment of BV173YFP/Luc cells was measured post-mortem from harvested spleen, liver, bone marrow, and peripheral blood by measuring the endogenous YFP expression in viable cells (PI-) on a FACSCalibur (BD Bioscience) machine and analyzing on FlowJo® 10 software. All animal handling was done in the Animal Resource Centre of the British Columbia Cancer Research Center using Animal Care Committee (University of British Columbia) approved procedures. 2.5.2 Transplantation of BV173YFP/Luc + shATG4B into Immunodefficient Mice with Caloric Restriction 15-16 week old male NSG mice were sub-lethally cesium irradiated overnight (315 cGy). The following day, the mice were injected intravenously with 1.4x106 BV173YFP/Luc cells that were expressing either a scrambled control vector (SCR) or ATG4B shRNA (shATG4B) vector to knockdown ATG4B expression. The day after injection, both SCR and shATG4B mice were split into a normally fed group and a 70% caloric restriction (CR) group. The CR mice were housed singly and fed 3g of Caloric Restriction 2020 (70%, Vit, Min, ENVIGO) once per day. The mice were monitored for body weight and activity daily though the duration of the experiment. The engraftment of BV173YFP/Luc cells was imaged in vivo at 2, 4, and 6 weeks post-injection using the Xenogen IVIS® 50 Bioluminescence Imaging System and the Live Imaging Software Version 3.0 after intraperitoneally injection with 3mg of D-Luciferin. The engraftment of BV173YFP/Luc cells was measured post-mortem from harvested spleen, liver, bone marrow, and peripheral blood by measuring the endogenous YFP expression in viable cells (PI-) on a   35  FACSCalibur machine and analyzing on FlowJo® 10 software. All animal handling was done in the Animal Resource Centre of the British Columbia Cancer Research Center using Animal Care Committee (University of British Columbia) approved procedures.  .    36  Chapter 3: Results   3.1 The Viability Effects of Novel ATG4B Inhibitors from the DB Series in CML Cell Lines Four novel ATG4B inhibitors were evaluated in the Ph+ cell lines K562 and BV173 to assay their effects on proliferation (Fig. 3.1). The IC50s of the drugs were determined by the reduced viability throughout increasing drug concentrations. Dr. Young’s lab also assessed the in vitro IC50s of the inhibitors towards ATG4B and other cysteine proteases in cell-free assays158 (unpublished data). The in vitro and in vivo potency of these inhibitors is reviewed in Table 3.1. The overall least potent drug in vivo with an IC50 of 10µM was the parental compound DB1. After the chemical changes in the benzyl group, the potency increased in the subsequent iterations of the drug (Table 3.1). The most potent compound in cell-free assays, DB3, was the second least potent compound within the Ph+ cell lines (K562 IC50 = 6.2µM), and DB2 was slightly more potent (K562 IC50 = 4.3µM). The compound DB4 was the most potent (K562 IC50 = 1.6µM) in Ph+ cell lines, but had a greater potency in cells than it did in vitro (Table 3.1). Between the two cell lines, BV173 cells were slightly more sensitive than K562 cells to all ATG4B inhibitors. For both K562 and BV173, once these cells were put into low-serum medium to induce starvation conditions, the IC50 reduced, showing ATG4B inhibition was potentiated by starvation/stress conditions (in starved K562, DB1 = 7.5µM, DB2 = 2.3 µM, DB3 = 2.6µM, DB4 =0.52µM).       37    Table 3.1: Overview of the “DB” Series of ATG4B Inhibitors Compound Structure In vitro IC50 to ATG4B K562 Fed/Starved IC50 BV173 Fed/Starved IC50 DB1    2.9µM  10µM / 7.5µM 10µM /  7.1µM DB2    2.1µM 4.3µM /  2.3µM 3.2µM /  1.7µM DB3    0.67µM 6.2µM /  2.6µM 4.6µM /  1.7µM DB4    2.5µM 1.6µM /  0.52µM 1.1µM /  0.45µM     38   Figure 3.1: IC50s of four novel ATG4B inhibitors: DB1, DB2, DB3, and DB4. (A) IC50s of the four drugs under normal (10% FBS) and serum starved (1% FBS) conditions when treated for 72 hours in K562 cells. The IC50s (fed and serum starved, respectively) for DB1 were 10µM and 7.5µM, DB2 were 4.3µM and 2.3µM, DB3 were 6.2µM and 2.6µM and DB4 were 1.6µM and 0.52µM. (B) IC50s of the four drugs under normal (10% FBS) and serum starved (2% FBS) conditions when treated for 72 hours in BV173 cells. The IC50s (fed and serum starved, respectively) for DB1 were 10µM and 7.1µM, DB2 were 3.2µM and 1.7µM, DB3 were 4.6µM and 1.7µM and DB4 were 1.1µM and 0.45µM. N=3 for fed IC50s, and N=1 for starved IC50s.   3.2 Inhibition of ATG4B by DB Inhibitors in Combination with TKIs is Sensitized by Serum Starvation The abilities of the new inhibitors to decrease viability and induce apoptosis in combination with IM in three Ph+ cell lines was tested under normal and starvation conditions after 72 hours of treatment (Fig. 3.2-3.4). For each of the inhibitors, the approximate starvation IC50 of the inhibitors was used in both fed and starved conditions (DB1: 10µM, DB2: 2µM, DB3:   39  2µM, DB4: 1µM). K562 cells had a moderate decrease in viability but no increased apoptosis under fed conditions when the DB small molecule inhibitors were combined with IM (Fig. 3.2A). However, once starved there was a dramatic decrease in viable cells and an increase in apoptotic cells in the single and combination DB treated cells, except for the drug DB1 (Fig. 3.2B). For the inhibitor DB2, after it was combined with IM the fraction of cells undergoing apoptosis significantly increased from 42% in IM to 92% in IM+DB2 (p<0.01). The viable and apoptotic cells were also measured in IM-resistant K562 (IMR) cells after treatment (Fig. 3.3). These cells were derived from K562 cells that were grown in increasing concentrations of IM until they developed resistance without any ABL1 kinase mutations. The IMR cells had a decrease in viability in the single DB treatments and a further reduction when they were combined with IM and starved (p<0.05). However, the amount of apoptotic cells only increased significantly from 12% with IM treatment to 21% in the starved IM+DB2 combination (p<0.05; Fig. 3.3B) but not in any of the other combinations of IM with the DB inhibitors. BV173 cells had the most drastic differences between the fed and starved conditions with starvation decreasing viability after IM+DB2 treatment to 2% and increasing apoptosis to 90% (p<0.001; Fig. 3.4). Correlating with IC50 results, these cells were more sensitive than K562 to all DB inhibitors as seen by a reduction in viability and an induction in apoptosis in the single DB treatments (Fig. 3.2 & 3.4). The results from all three cell lines showed the DB inhibitors were mainly potentiated by starvation, and significantly more so when combined with IM treatment.    40   Figure 3.2: The biological effects of the DB series inhibitors in combination with IM in K562 cells after 72 hours of treatment. (A) The percent viable (trypan blue negative) and apoptotic (Annexin V+) cells after treatment in fed (10% FBS) conditions. (B) The percent viable (trypan blue negative) and apoptotic (Annexin V+) cells after treatment in starved (1% FBS) conditions. N=3; *p<0.05, **p<0.01, ***p<0.001; error bars = standard error of mean (SEM).     41   Figure 3.3: The biological effects of the DB series inhibitors in combination with IM in IMR cells after 72 hours of treatment (A) The percent viable (trypan blue negative) and apoptotic (Annexin V+) cells after treatment in fed (10% FBS) conditions. (B) The percent viable (trypan blue negative) and apoptotic (Annexin V+) cells after treatment in starved (1% FBS) conditions. N=3; *p<0.05, **p<0.01, ***p<0.001; error bars = SEM.    42   Figure 3.4: The biological effects of the DB series inhibitors in combination with IM in BV173 cells after 72 hours of treatment. (A) The percent viable (trypan blue negative) and apoptotic (Annexin V+) cells after treatment in fed (10% FBS) conditions. (B) The percent viable (trypan blue negative) and apoptotic (Annexin V+) cells after treatment in starved (2% FBS) conditions. N=3; *p<0.05, **p<0.01, ***p<0.001; error bars = SEM.      43  3.3  DB2 is Selective to Primary CML Cells Without Targeting Normal Bone Marrow The therapeutic window of the DB inhibitors was tested by comparing the colony forming abilities of CD34+ CML stem/progenitor cells to CD34+ normal bone marrow (NBM) cells using a CFC assay. The DB inhibitors along had no toxicity on CD34+ NBM cells and there was a decrease in the combination from the TKIs (IM & DA) alone by about 20% for the parental DB1 inhibitor at the working concentration of 10µM (Fig. 3.5). The newer generation (DB2, DB3, DB4) inhibitors had less toxicity in their TKI combinations with >60% CFCs up to 5µM for DB2, 5µM for DB3, and 2µM for DB4 (Fig. 3.5). Using this information and biochemical data received from Dr. Robert Young’s lab (unpublished data), I focused on one of the least toxic and most effective drugs, DB2, and analyzed its effectiveness in CD34+ CML primary cells obtained from IM-nonresponders (NR). The combination effects when NL was combined with the DB2 were 2-fold less than NL alone, which decreased the number of colonies from 45% in the NL treated cells to 22% in the combination (Fig. 3.6). Comparing to the NBM results, there was a therapeutic window in the combinations suggesting this treatment could be selective towards primitive CML cells. To sensitize these cells to DB2 treatment, different methods of starvation and stress induction were tested. Prior to plating in a CFC assay, CD34+ CML cells from the same patient samples were first cultured in serum-free medium (SFM) either containing 4 added growth factors (GFs) or no added GFs for 16 hours to stimulate autophagy. The cells that were pre-cultured in SFM with no added GFs have a reduction in colony formation in the DB2 only condition, which reduced CFCs from 76% in the no pre-culture CFC assay to 55%; however, there is no change in the DB2+NL combination (Fig. 3.6). Also, when the cells were pre-cultured in SFM + 4GFs there was no change in DB2 only condition and an increase in   44  the DB2+NL combination when compared to the no pre-culture CFC assay (Fig. 3.6). These results indicated that there may be a potential therapeutic window in the DB2+TKI combination, which could target the CML cells but spare the patient’s NBM.   Figure 3.5: The colony forming abilities of healthy bone marrow samples after treatment with the four DB inhibitors and in combination with the TKIs DA (100nM) and NL (5µM). The NBM samples were analyzed for GEMM, CFU-GM, and BFU-E colonies and each category of colonies was compared to the total number of colonies in the untreated control. N=3; error bars = SEM; GEMM = mixed progenitor colony, CFU-GM = granulocyte/macrophage progenitor colony, BFU-E = primitive erythroid progenitor colony.   45   Figure 3.6: The biological effects of DB2 combined with the TKI NL in CML NR samples. The colony-forming abilities of DB2 (5µM) combined with NL (5µM) after no pre-culture, an overnight pre-culture with no added growth factors, and an overnight pre-culture with 4 growth factors. The CML cells samples were analyzed for GEMM, CFU-GM, and BFU-E colonies and each category of colonies was compared to the total number of colonies in the untreated control. No culture n=3; pre-culture n=2; *p<0.05; error bars = SEM; GEMM = mixed progenitor colony, CFU-GM = granulocyte/macrophage progenitor colony, BFU-E = primitive erythroid progenitor colony.  3.4 DB Series Inhibitors Alter Activated Autophagy in CML Cells The changes in autophagy flux before and after drug treatment can be tracked by the protein expression of autophagy markers and the formation of autophagosomes visualized under a confocal microscope. Measurement of markers of autophagy induction and completion, LC3B and p62, respectively, is the standard method for determining autophagy levels. I compared the DB inhibitors to the known autophagy inhibitor CQ after 6 hours of treatment and starvation to see what kind of changes in autophagy occur after inhibiting ATG4B (Fig. 3.7A). Quantification was performed on Image J software and averaged from two repeated Western blots. The DB inhibitors had a different LC3B response than CQ after treatment. In BV173 cells, three out of the four inhibitors (DB1, DB2, DB3) caused the levels of LC3-I to decrease much more than after CQ treatment, and caused a LC3-II accumulation similar to CQ. Thus, the LC3-II/LC3-I   46  ratio rose more after ATG4B inhibition than CQ treatment (Fig. 3.7A). However, the decrease in LC3-I levels was not present in K562 cells, but there was an accumulation of LC3-II which also caused the LC3-II/LC3-I ratio to increase (Fig. 3.7A). After 6 hours, there were no significant changes in p62 levels when compared to the starvation control (quantification not shown). Additionally, changes in ATG4B protein levels were also investigated but no significant changes were seen in any of the conditions (quantification not shown). The inhibitor DB4 did not have much change in LC3-II/LC3-I compared to the starvation control in both K562 and BV173 cells, suggesting it may not target ATG4B/autophagy as specifically as DB1, DB2, and DB3. Additionally, the mCherry-EGFP-LC3B vector was transiently expressed in K562 cells to image LC3B puncta (Fig. 3.7B). This vector tags LC3B with both mCherry and EGFP in order to distinguish early and late autophagosome development. The marker EGFP is easily degraded in acidic environments, whereas mCherry is stable, so after the lysosome fusion GFP degrades and the LC3B puncta appear red instead of yellow. Following serum starvation, the LC3B puncta in the untreated cells shifted from yellow to a mix of yellow and red; this was indicative of activated autophagy. These red-only puncta were not present in the CQ, DB2, and DB3 treated cells. There were few red-only LC3B puncta detectable in the DB4 treated cells and less accumulation of yellow puncta. The LC3B puncta were quantified by counting the number of red and yellow puncta within each cell and averaged over 25 cells (Fig. 3.7B). Additionally, immunofluorescence was used to image LC3B puncta in CD34+ CML cells after 16 hours of DB2, CQ, and DB2+CQ treatment (Fig. 3.8). There was significantly increased fluorescence in CQ and DB2 treated cells compared to the no treatment control. However, when DB2 and CQ were combined there was no increase in LC3B signal compared to either DB2 or CQ on their   47  own, probably because both drugs increased LC3B signal close to saturation. These results indicated that the new DB inhibitor series affected autophagy in CML cells, except for DB4, and was consistent with observations of blocked autophagy.  DB2 was analyzed and quantified for dose-dependent LC3-II accumulation after 6 hours and 24 hours of treatment in K562 and BV173 cells (Fig. 3.9). There was a dose-dependent increase in the LC3-II/LC3-I ratio in both fed and serum starved conditions, although the cells required a higher concentration of DB2 to see this accumulation in the fed conditions in both cell lines. In K562 cells, the higher concentrations of DB2 were able to maintain high LC3-II/LC3-I ratios at both 6 hours and 24 hours in the starved conditions especially (Fig. 3.9). The BV173 cells behaved differently to K562 cells with more dynamic changes of LC3B. After 6 hours, the BV173 cells had a very evident dose-dependent LC3-I degradation and LC3-II accumulation both in fed and serum starved conditions (Fig. 3.9A). After 24 hours, LC3-I was still degraded in a dose-dependent manner; however, there was a dose-dependent increase of LC3-II until the highest concentration, which then decreased compared to the lower doses (Fig. 3.9B). These results indicated that there may be more dependence on LC3-I stability by ATG4B in BV173 cells than K562 cells, and that the changes in LC3-I and LC3-II were occurring due to DB2 in a dose-dependent manner.     48   Figure 3.7: Changes in autophagy after treatment with the DB inhibitors and CQ. (A) Western blot analysis of the autophagy proteins ATG4B, p62, and LC3B in K562 and BV173 cells after 6 hours of treatment with DB1 (10µM), DB2 (4µM), DB3 (5µM), DB4 (2µM), and CQ (10µM) in serum starvation conditions. Quantification of the LC3-II/LC3-I ratio is also shown. N=2 (B) Visualization of LC3B puncta in K562 cells transduced with the mCherry-EGFP-LC3B vector after 6 hours of treatment on a confocal microscope. Quantification of the red and yellow puncta also shown; 25 cells per condition were quantified. St = starved; error bars = SEM.     49   Figure 3.8: LC3B immunofluorescence in CD34+ CML Cells after DB2 and CQ Treatment. Primary CML cells were treated for 16 hours in SFM with no added GFs and then stained for LC3B. LC3B puncta was visualized on a confocal microscope. N = 30 cells quantified on ImageJ software; *p<0.05, **p<0.01, ***p<0.001, N.S. = no significance; error bars = SEM.                         50   Figure 3.9: Dose-dependent effects on LC3-I and LC3-II after DB2 treatment. (A) Western blotting of LC3 6 hours and (B) 24 hours after DB2 treatment in K562 and BV173 cells. Increasing doses was tested in both fed (10% FBS) and serum starved (1% FBS in K562 and 2% in BV173) conditions. The LC3-II/LC3-II quantification is also shown. N=2; *p<0.05, **p<0.01; error bars = SEM.   51  3.5 DB2 + DA Combination Does Not Reduce Leukemia Burden In Vivo After confirming the efficacy of the DB inhibitors in vitro, the abilities of one inhibitor, DB2, to block leukemia development of an aggressive xenograft leukemia model was tested in vivo. With the help of Dr. Min Chen, we injected 2.5 million BV173YFP/Luc cells into the tail vein of female NSG mice. These cells contained YFP so they could be simply analyzed by flow cytometry and luciferase so that the leukemia development could be tracked in the mouse by bioluminescence imaging. Two weeks after injection, the mice were checked for uniform engraftment (Fig. 3.10A), then oral gavage treatment was started with the propylene glycol vehicle control, DA (15mg/kg), DB2 (50 mg/kg), DB2 (100 mg/kg), DA + DB2 (50mg/kg), and DA + DB2 (100mg/kg) once daily for two weeks. Two weeks after treatment, only the DA + DB2 (100mg/kg) combination showed a slight decrease in luciferase signal compared to mice treated with DA alone, but DA + DB2 (50mg/kg) did not show any reduction (Fig. 3.10A). There were no obvious changes in mice treated with DB2 alone compared to the vehicle. One mouse per group was sacrificed for further analyses of BV173YFP/Luc cell engraftment in bone marrow, spleen, liver, and peripheral blood, as well as organ sizes, BCR-ABL1 transcripts, and LC3B immunofluorescence imaging. The spleen and liver sizes were enlarged in diseased mice compared to the no injection control mouse (Fig. 3.10B). However, the combination treated mice did not have a reduction in spleen and liver sizes than the mouse singly treated with DA. Similar to the imaging, the BV173YFP/Luc engraftment levels in the DA + DB2 (100mg/kg) mouse were slightly lower than DA in all organs (Fig. 3.10C). BCR-ABL1 transcript levels were also lower in the DA + DB2 (100mg/kg) mouse than the DA mouse in the spleen but not the bone marrow (Appendix A). To investigate any impact on autophagy, I analyzed the relative fluorescence after   52  staining for LC3B in YFP+ engrafted cells in the bone marrow. DA, DB2 (100mg/kg), and DA + DB2 (100mg/kg) treated mice all had 2.5-fold increased LC3B puncta than the vehicle but there were no differences to each other (p<0.001; Fig. 3.11). The final analyses on surviving mice was performed 4 weeks after treatment on DA, DA + DB2 (50mg/kg), and DA + DB2 (100mg/kg) mice (Appendix B). There were no significant changes in engraftment, circulating leukemic cells, or median survival between DA and the two combinations. This mouse model showed early reduction in leukemia development in the DA+ DB2 (100mg/kg) combination but this reduction did not last long-term, and did not significantly increase the survival of these mice (Fig. 3.10D).    53   Figure 3.10: Analysis of leukemia progression in mice two weeks following oral gavage treatment. (A) Luciferase imaging of the BV173YFP/Luc engraftment throughout the mice before and after treatment. (B) Harvested spleen and liver sizes. (C) FACS analyzed engraftment of BV173YFP/Luc cells in the hematopoietic organs. (D) Survival curve of the different treatment groups.   54    Figure 3.11: The fluorescence of LC3B in BV173YFP/Luc (YFP+) engrafted cells in the mice bone marrow two weeks after treatment. Overall fluorescence of LC3B was averaged from 25 YFP+ cells in ImageJ software. Images were taken on a confocal microscope at 60x magnification. N=30 YFP+ cells; ***p<0.001 (compared to vehicle); error bars = SEM.    3.6 ATG4B Knockdown in BV173YFP/Luc cells In preparation for a subsequent mouse model, knockdown of ATG4B was tested in BV173YFP/Luc cells. The cells had a successful knockdown that reduced ATG4B protein expression 10-fold as shown with a western blot (Fig. 3.12A). Analyzing autophagy, there was increased LC3B puncta in the shATG4B cells when visualized using immunofluorescence (p<0.001; Fig.3.12B). The shATG4B cells proliferated much slower than a scrambled control (SHC) in fed medium conditions (p<0.01) and even more in serum starvation conditions   55  (p<0.001; Fig.3.12C). These results were consistent with a successful and stable ATG4B knockdown in BV173YFP/Luc cells prior to their injection into recipient mice.   Figure 3.12: Knockdown of ATG4B using shRNA in BV173YFP/Luc cells in vitro. (A) Western blot analysis of ATG4B protein expression in shATG4B cells compared to SHC control and quantification of knockdown. N=2. (B) Immunofluorescence of LC3B puncta in knockdown cells compared to the SHC control (35 cells quantified on ImageJ software). (C) Proliferation of shATG4B cells and SHC control cells over 72 hours in complete medium (10% FBS) and starved medium (2% FBS). Significance is compared to the SHC fed control at 72 hours. N=3; *p<0.05, **p<0.01, ***p<0.001; error bars = SEM.     56  3.7 Caloric Restriction in vivo May Potentiate ATG4B Knockdown  It was observed in vitro that starvation was important for potentiating the effects of ATG4B inhibition, and thus I wanted to investigate if starvation could potentiate effects in vivo. For this model, ATG4B was knocked-down with shRNA and compared to a SHC control. I injected 1.4 million BV173YFP/Luc cells stably expressing either SHC or shATG4B into NSG mice. The following day, the mice were separated into a normally fed group, and a group receiving a 70% caloric restriction (CR) diet for both the shATG4B and SHC conditions. The mice were imaged with luciferase at 2, 4, and 6 weeks post-injection to check leukemia progression (Fig. 3.13A). The SHC and SHC+CR mice had comparable luciferase signal at all three time points, with a slight decrease in the SHC+CR group. The shATG4B mice had lower signal intensity than the SHC and SHC+CR groups. The shATG4B+CR mice had very little signal throughout all three time points and the engrafted cells appeared to stay in the bone marrow rather than migrate to the spleen and liver.  After 7 weeks, one mouse per group was sacrificed for engraftment and autophagy analyses. The organ sizes correlated with the imaging results, with the liver and spleen from SHC being the largest, the SHC+CR and shATG4B a bit smaller, and the shATG4B+CR organs were comparable to the no cell control mouse (Fig. 3.13B). Leukemia progression was measured by the proportions of YFP+ cells in the bone marrow and peripheral blood, and the BCR-ABL1 transcript levels in the bone marrow and spleen (Fig. 3.13C; Appendix A). The SHC mouse had unusually low engraftment of BV173YFP/Luc cells when measured by FACS and BCR-ABL1 transcripts, and overall SHC+CR had the highest. The shATG4B+CR mouse consistently had less engrafted cells and fewer BCR-ABL1 transcripts throughout the tissues. The impact of   57  shATG4B and CR on autophagy was also analyzed by staining for LC3B puncta in the bone marrow (Fig. 3.14). The human YFP+ cells in the normally fed shATG4B mouse had more LC3B fluorescence compared to the SHC control (p<0.05).  Once CR was added, the LC3B fluorescence increased 2-fold, which was significantly higher than both shATG4B and SHC+CR mice (p<0.001). Over the time of the experiment, the body weights of the CR mice were 2-3g lower between day 10-30 post-injection (Fig. 3.15A). However, once disease progressed in the normally fed mice, the body weights of the fed and CR groups overlapped. The shATG4B+CR (median = 65 days) mice and the shATG4B (median = 63 days) mice both had significantly longer lifespans than the SHC mice (median = 55 days, Fig.3.15B). There were two mice in the shATG4B+CR group that survived up to 73 days. There was no significance between the shATG4B and shATG4B+CR survivals, showing that CR did not improve the later stage development of leukemia within the mice. This model suggested that ATG4B was important for Ph+ leukemia progression, and that CR may enhance the effects of shATG4B during the early progression of Ph+ leukemia but did not confirm that it can reduce the progression of late-stage disease.     58   Figure 3.13: Analysis of shATG4B mice vs. SHC mice with and without CR. (A) Luciferase imaging of the BV173YFP/Luc engraftment throughout the mice 2, 4, and 6 weeks after injection (B) Spleen and liver sizes 7 weeks after injection (C) FACS analyzed engraftment of BV173YFP/Luc cells in the mouse bone marrow and peripheral blood.   59    Figure 3.14: The fluorescence of LC3B in BV173YFP/Luc (YFP+) engrafted cells in the mice bone marrow seven weeks after injection. Fluorescence of LC3B was averaged from 30 YFP+ cells in ImageJ software. Images were taken on a confocal microscope at 60x magnification. N.S. = not significant. *p<0.05, **p<0.01, ***p<0.001; error bars = SEM.     60   Figure 3.15: Analysis of weights and survival of the SHC and shATG4B mice post-injection. (A) The body weights of the SHC and shATG4B groups with and without CR over their lifespan. (B) Survival curve of the SHC and shATG4B groups with and without CR. **p<0.01.   61  Chapter 4: Discussion 4.1 Discussion Although treatment for CML has improved greatly since the discovery of TKIs, the treatment is not curative and some patients develop resistance through upregulation of additional pro-survival pathways. Autophagy has been implicated to be important in the development of several cancers, including CML.132 TKI treatment has been confirmed in multiple studies to cause an upregulation of autophagy, and our lab discovered that ATG4B within the autophagy pathway is also upregulated after TKI treatment.154 At its basal levels, ATG4B is upregulated in CML patient samples compared to NBM, and importantly even more so in IM-non-responding patients compared to IM-responders.154 Knockdown of ATG4B in CML NR samples impairs the proliferative abilities of the stem/progenitor population and even more so when combined with TKIs.154 This data prompted me to study if novel ATG4B inhibitors could inhibit growth of CML cells as the initial knockdown data suggested. This study aimed to optimize the abilities of these inhibitors to block growth of CML cell lines, primary CD34+ CML cells, and an in vivo leukemic xenograft model, as well as investigate the effects of these inhibitors on autophagy.  I received four different inhibitors from Dr. Robert Young’s lab that had been shown to target ATG4B in cell-free biochemical assays. All of these inhibitors were modified from a compound that was found in a high-throughput screen of over 200,000 inhibitors.158 These inhibitors showed potent growth inhibition of the Ph+ cell lines K562 and BV173 (Fig. 3.1). Upon serum starvation, these inhibitors became more potent against the tested cell lines (Table 3.1). Adding FBS into medium introduces growth factors, amino acids, and other nutrients that caused the cells to accelerate their growth. Many of these nutrients are positive regulators of   62  mTORC1, and therefore are negative regulators of autophagy.105,109 Inhibition of a core autophagy protein like ATG4B may impair autophagy and make the cells more susceptible to apoptosis when they are signaled to activate autophagy. BV173 cells were particularly sensitive to serum starvation and required higher serum content in the medium to stay proliferative. In addition, the IC50s of the DB inhibitors were lower overall in BV173 cells (Table 3.1). This could suggest that these cells are more dependent on autophagy/ATG4B during starvation/stress conditions. K562 and BV173 cells are both Ph+ but they are derived from two different types of leukemias; K562 cells are derived from a CML blast-crisis patient whereas BV173 cells are from a patient with Ph+ B cell acute lymphoblastic leukemia (B-ALL).159,160 Therefore, K562 cells are myeloid blast cells and BV173 cells are lymphoid blast cells. In general, Ph+ B-ALL is far less sensitive to TKI therapy than CML and has a much worse prognosis.161 These cells are more dependent on additional pathways for their survival thus making them less addicted to BCR-ABL1, and from observations in my studies one of these survival pathways could be autophagy.161  Serum starvation susceptibility was observed in viability and apoptosis assays in three different cell lines: K562, IMR, and BV173. There were no significant combination effects when the ATG4B inhibitors were combined with IM in all three cell lines in full serum medium. However, upon serum starvation the viability of these cells after ATG4B inhibition decreased drastically and the induced apoptosis rose (Fig. 3.2-3.4). There was even greater cell killing benefits when the ATG4B inhibitors were combined with IM in low-serum medium. IM also induced autophagy in CML cells through additional pathways (reviewed in Chapter 1), thus driving even more autophagy dependence and therefore drug sensitivity.131 There is a   63  homeostatic balance between apoptosis and autophagy through a mutual inhibition of each other.162 Both catabolic processes are cellular responses to stress, but autophagy allows the cell to adapt whereas apoptosis causes cell death.162 Inhibiting a core autophagy protein may cause a bioenergetics shortage within the cell that pushes it towards apoptosis, as seen in these assays.163 Additionally, combining IM with the ATG4B inhibitors directed the cell towards apoptosis through two different pathways. Since there were no combination effects under fed conditions, it seems the autophagy dependence after TKI treatment alone was not enough to push the cells to apoptosis. IMR, an IM-resistant K562 cell line, was the only cell line that did not induce apoptosis in the combination treatments even though they had low viability. It is possible the cells became more senescent rather than apoptotic and proliferated less than the controls. Autophagy is a very important mechanism for both hematopoietic stem cells and leukemic stem cells, so the toxicity of the inhibitors to NBM was compared to the effectiveness in CML cells to make sure ATG4B inhibition could be a targeted therapy.116,164,165 The stem/progenitor population was analyzed using CFC assays in both CML and NBM cells. This assay utilizes the ability of primitive CD34+ populations to proliferate into colonies of different lineages when plated in semi-solid medium containing the appropriate growth factors. In the combinations with the TKIs NL and DA, most of the toxicity in the NBM was from the TKI treatment rather than the ATG4B inhibitors up to a particular dose depending on the compound; for DB2 this was 5µM (Fig. 3.5). The efficacy of the combination treatment with the second-generation TKI NL and the ATG4B inhibitor DB2 was also investigated using non-toxic doses in the CML NRs. There were significant differences between using NL alone and using it in combination with DB2 in the CFC assay (Fig. 3.6A, p<0.05), which differs from the NBM   64  results thus leaving a therapeutic window. Since the cell lines were potentiated by serum starvation, I tested if overnight growth factor deprivation prior to plating in a CFC in CD34+ CML cells could enhance the effects of the combination. A previous study found that culturing hematopoietic cells in medium devoid of growth factors robustly upregulated autophagy in a FOXO3A-dependant manner.165 This strategy did not increase the effects of the combination, but it did reduce the CFC output in the DB2 only treated cells from 76% to 55% of the control (Fig. 3.6B). Additionally, culturing the cells in SFM containing all essential growth factors had a protective effect against the combination, and there was no reduction between the NL and DB2+NL groups. More work must be done to confirm these observations since only two NR samples were tested so there was no significance, but so far these results suggest that limiting added growth factors could have an effect on the CML cells’ sensitivity towards ATG4B inhibition. Another possible experiment would be to put the cells in a hypoxic environment to mimic the bone marrow niche, which also pushes the cells to be more reliant on autophagy.164  Next, I investigated the effects of the DB inhibitors on autophagy and compared them to the autophagy inhibitor CQ. Western blotting and confocal imaging were used as a means to monitor autophagy at 6 hours and 24 hours after treatment. I used 6 hours to compare the effects of the four ATG4B inhibitors to CQ when combined with serum starvation to activate autophagy in K562 and BV173 cells (Fig. 3.7A). Both K562 and BV173 cells had little change in p62 levels at 6 hours compared to the starvation control. Often, p62 takes 24-48 hours to accumulate after autophagy has been blocked.109 A longer time point might be needed to show p62 changes in these cells. p62 has additional roles in cell signaling that are independent of autophagy, which do not require the ubiquitin associated (UBA) or LIR domains on p62.166 These functions include   65  activation of inflammatory genes controlled by NF-κB, activation of the NRF2-dependent anti-oxidant response, and activation of mTORC1.166,167 p62 is used as an autophagy measure because its LIR sequesters it to the autophagosome during non-specific autophagy thus mediating its degradation168, but its expression may be controlled via other mechanisms. ATG4B levels were also monitored to see if the inhibitors had any influence on the protein stability or production in the cells. After treatment with the DB inhibitors, there were no significant changes in ATG4B levels in K562 and BV173 cells, therefore showing the inhibitors do not influence protein levels of ATG4B. In K562 cells, there was very little change in the LC3-I levels across the different conditions and an accumulation of LC3-II after CQ, DB1, DB2, and DB3 treatment but not DB4 treatment (Fig. 3.7A). These observations may suggest DB4 is not specific to ATG4B and has off-target effects in the cell. BV173 cells showed different results in response to the inhibitors. Similar to K562 cells, the CQ, DB1, DB2, and DB3 treated groups all showed an accumulation of LC3-II compared to the starvation control. However, in BV173 cells there was a reduction of LC3-I levels after DB1, DB2, and DB3 treatment but not CQ treatment (Fig. 3.7A). ATG4B is responsible for cleaving pro-LC3 into LC3-I and also cleaving off LC3-II from the autophagosome membrane and recycling it back into LC3-I (Fig. 1.3).147 Since the third pro-LC3 band was not seen, the loss of LC3-I seen at 6 hours may be due more so to the loss of the recycling function rather than the pro-LC3 cleavage. The autophagy inhibitor CQ inhibits the lysosome activity but does not inhibit the formation/stability of LC3-I, and therefore in this assay CQ treatment did not reduce LC3-I levels at the short-term time point but the absence of autophagosome degradation led to an accumulation of LC3-II.     66  The inhibitor DB2 was analyzed for a dose-dependent LC3 response after 6 and 24 hours of treatment (Fig. 3.9). Both fed and serum starved conditions were tested to monitor the cells in conditions with and without autophagy activation. There was a more potent response to DB2 treatment when starvation was added, but the fed cells also showed a response with higher concentrations of DB2 such as 5µM and 10µM. After 6 hours, both K562 and BV173 cells had an accumulation of LC3-II in a dose-dependent manner, which was especially evident with added starvation (Fig. 3.9A). At 6 hours, the cell may have had abundant remaining processed LC3-I within the cytosol that still could be processed into LC3-II, and therefore the effects of the inhibitor were largely due to decreased LC3-II recycling. After 24 hours of DB2 treatment, the two cell lines showed different responses in LC3 levels (Fig. 3.9B). In K562 cells, the cells maintained their LC3-II accumulation in the higher concentrations and had a decrease in LC3-I levels when treated with 5µM DB2 in serum starvation. In BV173 cells this decrease in LC3-I was evidently dose-dependent until it reduced to almost no LC3-I expression in both fed and serum starved conditions. However, BV173 cells also had an unexpected decrease in LC3-II levels in the higher DB2 concentrations at 24 hours rather than following a dose-dependent pattern as it had at 6 hours. The LC3-II band seen at the highest DB2 concentrations in BV173 cells may actually be remaining pro-LC3 rather than processed LC3-II since Wang et al. showed that the two forms migrate at a very similar rate in SDS-PAGE.169 Mice knockout studies have also shown that complete loss of ATG4B resulted in decreased overall LC3-I and LC3-II levels across several tissue types due to the inability to form LC3-I for conjugation.149 I hypothesize there is a threshold on the inhibitor’s effect of ATG4B’s activities. Partial inhibition may have lead to a blockade of LC3-II recycling, whereas full inhibition lead to a blockade of both LC3-II   67  recycling and proteolytic cleavage of pro-LC3. Similar effects were seen using a small molecule inhibitor of ATG4B in glioblastoma multiforme.170 A remaining question is how LC3-II becomes degraded after full inhibition of ATG4B, since in Saccharomyces cerevisiae loss of Atg8 recycling from the outer autophagosome membrane impairs the ability for the autophagosome to fuse with the lysosome.98,171 Other degradation processes such as proteolysis may degrade excess accumulating autophagosomes. ATG4B partial knockdown vs. complete knockout studies would be a way to investigate the differences between full and partial ATG4B inhibition in mammalian cells.  Additionally, confocal microscopy was used to visualize LC3B puncta within CML cell lines and primary cells. In K562 cells, the mCherry-EGFP-LC3B vector was used to distinguish early and late autophagosome development to see if ATG4B inhibition can block autophagy (Fig. 3.7B). There was an increase in red-only puncta upon serum starvation; however, the number of red-only puncta per cell was quite low in many of the cells. It is possible that a longer time point such as 8 hours may have more lysosome fusion and therefore more red-only puncta within these cells to be detected. In the CQ, DB2, DB3, and DB4 treated cells, there were little to no red-only puncta within the cells, which was indicative of blocked autophagy. Furthermore, the autophagosome puncta were visualized in primary cells using LC3B immunofluorescence (Fig. 3.8). Treatment with CQ, DB2, and CQ+DB2 had significantly increased LC3B puncta compared to the no treatment control. There was a greater increase in LC3B fluorescence in the DB2 treated cells than CQ, although this was insignificant. I believe this was because the concentration of CQ used (10µM) may not have been enough to saturate CML primary cells’ LC3B signal; using 20µM instead could give a larger accumulation. An additional experiment   68  would be to look at LC3B accumulation following TKI treatment, and a TKI combined with DB2, to see if there is a greater accumulation with TKI-induced autophagy. The top DB series inhibitor DB2 was further tested in combination with the second generation TKI, DA, to block an aggressive xenografted leukemia model in vivo. This model uses BV173 cells that are expressing YFP and luciferase for easy imaging and FACS analyses. This model is quick, aggressive, and induces an engrafted leukemia that ultimately is fatal to the mice. Particularly, this is a Ph+ B-ALL model that can test the effectiveness of new drugs to specifically target the late stage of disease. The mice were treated with a clinically relevant dose of DA (15 mg/kg) and two different doses of DB2 (50mg/kg and 100mg/kg), both singly and in combination. When the mice were analyzed 2 weeks later, there was no significant reduction in the leukemia burden between the DA treated mice and DA+DB2 combinations. There are several possible reasons why this combination was not effective in our model. The first possibility is that the two drugs were not compatible when dosed together. Our collaborators in Dr. R. Young’s lab assayed the pharmacokinetics of the DA+DB2 combination and noted that when the two drugs were given together, there were some toxic effects that affected the mice’s behavior (unpublished data). The two drugs also hindered the absorbance of each other when they were given together. To overcome these potential problems, I administered DB2 to the mice approximately 2 hours before administering DA to avoid incompatibility, but it is possible that this break was not long enough to avoid toxicity and/or absorbance issues (no visible toxicity was observed in the mice). Another possible reason why this model did not work was the ineffectiveness of DB2 and the combination under fed conditions in vitro, and the necessity for starvation. The in vivo environment in the bone marrow and peripheral blood does tend to have fewer nutrients and is   69  more hypoxic than complete medium, but this environment is much less controlled than my in vitro model.172 Other optimizations that could be done to achieve better results could be to optimize the given dose of DB2, extend the length of treatment, and test the autophagy effects immediately after treatment. Any of these limitations could have led to the ineffectiveness of the DA+DB2 combination in vivo. I performed an additional mouse model that aimed to fix several issues that may have caused the previous model to be ineffective. Our collaborators were still working on increasing the in vivo stability of DB2, so instead I stably knocked down ATG4B using shRNA in BV173YFP/Luc cells to genetically reduce ATG4B activity. The BV173YFP/Luc cells with shATG4B had 10-fold less ATG4B protein expression, a 2-fold increase in LC3B puncta, and very slow in vitro proliferation (Fig. 3.12). To address the other issue in my previous mouse model, I compared 70% CR to normally fed mice to mimic starvation conditions in vivo. Other tumour mouse models have utilized CR as a treatment group to enhance the effects of inhibitors that affect metabolic pathways like autophagy. It has been shown that putting mice and humans on low-calorie or fasting diets can upregulate autophagy within the leukocytes in the peripheral blood.112 Other xenograft cancer models that have used CR in combination with anti-cancer drugs or gene knockdowns include CQ for melanoma173, the autophagy inhibitor oblongifolin C for cervical cancer174, and knockdown of the translation regulator eEF2K for neuroblastoma175. These studies show that CR has antitumour effects on its own, but when combined with drugs or gene knockdowns that cause dysfunction in degradation or translation, there is a synergistic effect since the cells no longer can adapt to the environment. In my model, combining shATG4B with CR also reduced the leukemia progression more than shATG4B or CR on its own in early   70  analyses. There was less luciferase signal in the shATG4B+CR group compared to the other groups at 2, 4, and 6 weeks post-injection (Fig. 3.13A). Additionally, 7 weeks post-injection there was a reduction in spleen and liver sizes and less engrafted cells in the bone marrow and peripheral blood (Fig. 3.13B&C). Autophagy was affected in this model as shown by an accumulation of LC3B puncta in the shATG4B + CR mouse bone marrow within the YFP+ human cells (Fig. 3.14). However, this anti-leukemia benefit did not last throughout the end stages of the disease model resulting in no significant differences in survival between shATG4B and shATG4B+CR mice; both lived significantly longer than the normally fed SHC control (p<0.01, 3.15B). In the later stages of leukemia, mice from all groups dropped in body weight and were eating less, which therefore made the CR much less controlled (3.15A). Another possibility is that the BV173YFP/Luc cells lost ATG4B knockdown towards the end of the experiment causing the cells to proliferate again.  4.2 Conclusions and Future Directions This study investigated the use of novel inhibitors from the DB series in combination with TKIs in CML cells in vitro and in vivo, but there still are many future directions this project could take. As previously mentioned, starvation was a primary factor for potentiating the effects of the DB inhibitors in CML cell lines, and possibly for potentiating shATG4B in vivo. There may be a way to also potentiate the effects of the inhibitors in CML primary cells in vitro via growth factor deprivation or mimicking the hypoxic niche to increase the autophagy dependence. One hypothesis I had was that ATG4B was important for maintaining the stem/progenitor population in CML. Within the bone marrow niche, HSCs and LSCs are exposed to low density nutrient gradients and hypoxic conditions that cause them to be inherently reliant on   71  autophagy.164,172 Danet et al. found that expanding the HSC stem cell population (CD34+CD38-) in hypoxic conditions in vitro is beneficial for these most primitive cells.176 Assaying the inhibitors under conditions closely resembling the bone marrow niche may give a better idea of their effectiveness in their natural environment. One of the best methods for mimicking the niche in vitro is a long-term culture initiating cell (LTC-IC) assay.177 The LTC-IC assay studies the properties of LSCs by first co-culturing CML cells with feeder cells for 6 weeks to potentially mimic the stem cell niche, and then plating the cells in a CFC assay. However, the best experiment for assaying the survival of CML LSCs in the bone marrow niche would be to engraft CD34+ primary CML cells into irradiated immunodeficient mice and monitor the long-term engraftment within the bone marrow. This experiment could test the importance of ATG4B in the LSCs through a genetic knockdown or knockout of ATG4B in the cells prior to injection, or administering DB2 treatment through oral gavage after the cells have engrafted.  In my studies, the inhibitor DB2 had different responses to autophagy between the cell lines K562 and BV173, which represent two different Ph+ leukemias. K562 had an accumulation of LC3-II and a slight reduction of LC3-I, whereas BV173 had a drastic decrease of LC3-I and an accumulation of LC3-II that led to a reduction in the higher concentrations after 24 hours. Investigating the dependency of these cell lines on ATG4B vs. other ATG4 homologues by looking at gene and protein expression may help explain the discrepancy between these two cell lines. Moreover, this could be expanded to CML primary cells that have high ATG4B expression vs. low ATG4B expression. Investigating the differences in drug sensitivities and autophagy levels between these two groups may stratify patient samples into those who may benefit from ATG4B therapy vs. those that would not. There is a trend that IM non-responders have higher   72  ATG4B expression than responders, but not all non-responders have high levels of ATG4B.154 It would be interesting to investigate the therapeutic differences between non-responders that upregulated ATG4B and those that did not.  I investigated using CR in vivo to induce starvation conditions and potentiate loss of ATG4B in an engrafted Ph+ human leukemia. Although the preliminary results were promising, there are some limitations with the long-term effectiveness of this strategy. Adding a TKI in combination with shATG4B/DB2 and CR would prolong survival even longer and possibly enhance the combination effects. An even better in vivo model for this experiment would be to make a conditional BCR-ABL1-expressing mouse model12 with a genetic knockout of ATG4B within the hematopoietic cells and engraft the stem cell population into recipient mice, and then test the CR diet. This model is widely used in CML studies because the leukemia more closely resembles CML pathogenesis and the cell types and driver mutations are all controlled.15 However, one caveat would be that the cells used in this model have not had the exposure to TKIs that eventually leads to TKI resistance and dependence on other pathways.  In conclusion, this study investigated and optimized the abilities for DB series ATG4B inhibitors/ATG4B knockdown to be used to target autophagy and sensitize CML cells to TKI therapy. These experiments used four ATG4B inhibitors from the DB series that were discovered through high-throughput screening and structure-activity relationship studies. These inhibitors had cellular IC50s ranging from 1µM to 10µM under normal conditions, but the sensitivity increased when the cells were subjected to serum deprivation to induce autophagy. Combining the DB inhibitors with IM enhanced their effectiveness to inhibit growth and induce apoptosis in three cell lines under serum starvation but not in fed conditions. Following treatment with these   73  DB inhibitors, autophagy appeared to be blocked as indicated by a dose-dependent accumulation of LC3-II shown on a western blot and incomplete fusion of autophagosomes with lysosomes as shown by confocal microscopy. Additionally, combining DB2 with TKIs significantly inhibited growth of CD34+ CML stem/progenitor cells from IM-nonresponders but not normal bone marrow cells. However, administering DB2+DA treatment in an in vivo leukemia model did not have significant benefits over using DA on its own.  Interestingly, ATG4B knockdown mice had less engrafted BV173YFP/Luc than the SHC control mice, and once combined with CR this difference was much greater. It appears that starvation may be an important factor to enhance the effects of targeting ATG4B, both in vitro and in vivo. Increased ATG4B and/or autophagy levels in drug resistant patients have been identified in other cancers, and this study presents strong evidence to support targeting ATG4B in treatment of BCR-ABL+ human leukemia.     74  References  1 Druker, B. J. Translation of the Philadelphia chromosome into therapy for CML. Blood 112, 4808-4817, doi:10.1182/blood-2008-07-077958 (2008). 2 Nowell, P. C. & Hungerford, D. A. 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One mouse sacrificed per group; error bars made from duplicate wells.    87  Appendix B  Appendix B: Late-stage mice analyses performed 4 weeks after oral gavage treatment in surviving mice. (A) Luciferase imaging of surviving mice in the DA (15mg/kg), DA+DB2 (50mg/kg), and DA+DB2 (100mg/kg) groups. (B) FACS analysis of the percentage of YFP+ human cells in the peripheral blood in the surviving mice.   

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