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A novel oncogene AHI-1 interacts with BCR-ABL and JAK2 and mediates cellular resistance to tyrosine kinase… DeGeer, Donna 2010

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A Novel Oncogene AHI-1 Interacts With BCR-ABL and JAK2 and Mediates Cellular Resistance to Tyrosine Kinase Inhibitors in CML by Donna DeGeer B.Sc., McMaster University 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES  (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  May 2010  © Donna DeGeer, 2010  i  ABSTRACT Chronic myeloid leukemia is a myeloproliferative disorder characterized by the presence of the Philadelphia chromosome, encoding a unique fusion gene BCR-ABL. The current first line treatment for patients diagnosed with CML involves administration of the ABL kinase inhibitor imatinib mesylate (IM). However, early relapses and acquired drug resistance remain a current impediment to successful treatment for many patients. This suggests the necessity for alternate treatment options which may include combination therapy targeting multiple vital proteins involved in the malignancy of the leukemia. AHI-1 (Abelson helper integration site 1) is a recently discovered oncogene that is highly deregulated in murine lymphomas and leukemias. AHI-1 displays a significant pattern of overexpression in a Philadelphia chromosome positive cell line K562 cells. To investigate AHI-1’s involvement in CML, AHI-1 was either stably overexpressed or suppressed in K562 cells. Interestingly, an increase in cellular proliferation and colony formation and a decrease in apoptosis were observed in the presence of IM when AHI-1 was overexpressed, while suppression of AHI-1 had the opposite effects. Phosphorylation and total protein expression levels of several proteins known to be involved in BCR-ABL signalling were quantified. Interestingly, elevated phosphorylation and total gene/protein expression levels of several of these proteins were observed when AHI-1 was overexpresessed, in particular NFκB and JAK2/STAT5 displayed increased expression. Due to the strong effects AHI-1 had on the JAK2/STAT5 signalling cascade, we then inhibited JAK2 activity using a new JAK2 inhibitor, TG101209. AHI-1 overexpression led to a reduction in the cellular response to the inhibitor while suppression of AHI-1 caused an increase in sensitivity in viability, apoptosis, and colony forming cell assays. Finally, a combination of IM and TG101209 was examined in the same K562 cells lines. Results from suggest that using a combination treatment approach was  ii  more effective at inhibiting cellular viability and colony formation than either treatment alone. These findings together suggest that AHI-1 may play an important role in mediating cellular resistance to IM and TG101209 and activates several BCR-ABL signalling pathways, and that it may be a vital target in eradicating the malignant leukemic cells arising in CML. A  iii  TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii TABLE OF CONTENTS ............................................................................................................... iv LIST OF TABLES ......................................................................................................................... vi LIST OF FIGURES ...................................................................................................................... vii ABBREVIATIONS ..................................................................................................................... viii ACKNOWLEDGMENTS ............................................................................................................. ix DEDICATION .................................................................................................................................x CHAPTER 1: BACKGROUND ......................................................................................................1 1.1 INTRODUCTION TO CML ...........................................................................................................1 1.2 DISEASE PROGRESSION AND CURRENT TREATMENT .................................................................3 1.3 AUTOREGULATION OF C-ABL AND CONSTITUTIVE ACTIVATION OF BCR-ABL ............................5 1.4 BCR-ABL SIGNALLING ...........................................................................................................7 1.5 ABELSON HELPER INTEGRATION SITE 1 (AHI-1) GENE..........................................................11 1.6 AHI-1 INVOLVEMENT IN CML ...............................................................................................14 1.7 A NOVEL JAK2 INHIBITOR TG101209 ..................................................................................17 1.8 EXPERIMENTAL OUTLINE .......................................................................................................19 CHAPTER 2: MATERIALS AND METHODS ...........................................................................20 2.1 CELL LINES ............................................................................................................................20 2.2 CELL CULTURE ......................................................................................................................20 2.3 DETERMINATION OF AN IC50 VALUE FOR TG101209 ............................................................21 2.4 CELLULAR VIABILITY ASSAY .................................................................................................21 2.5 APOPTOSIS ASSAY .................................................................................................................21 2.6 COLONY FORMING CELL ASSAY .............................................................................................22 2.7 RNA EXTRACTION .................................................................................................................23 2.8 CDNA SYNTHESIS .................................................................................................................23 2.9 QUANTITATIVE REAL TIME PCR ............................................................................................24 2.10 PROTEIN EXTRACTION AND QUANTIFICATION ......................................................................24 2.11 WESTERN BLOT ANALYSIS ...................................................................................................25 2.12 STATISTICAL ANALYSIS .......................................................................................................26 iv  CHAPTER 3: RESULTS ...............................................................................................................28 3.1 CONFIRMATION OF AHI-1 EXPRESSION IN K562 CELLS .........................................................28 3.2 RESPONSE OF AHI-1 TRANSDUCED CELLS TO IM TREATMENT ................................................30 3.3 DETERMINATION OF AN IC50 VALUE FOR TG101209 ............................................................34 3.4 RESPONSE OF AHI-1 TRANSDUCED CELLS AND IM RESISTANT CELLS TO TG101209 TREATMENT ...........................................................................................................................37 3.5 RESPONSE OF AHI-1 TRANSDUCED CELLS AND IM RESISTANT CELLS TO COMBINED IM AND TG101209 TREATMENT .............................................................................................41 3.6 DIFFERENTIALLY EXPRESSED BCR-ABL SIGNALLING GENES IN AHI-1 TRANSDUCED CMLCELLS ............................................................................................................................46 3.7 DIFFERENTIALLY EXPRESSED BCR-ABL SIGNALLING PROTEINS IN AHI-1 TRANSDUCED CML CELLS ...........................................................................................................................49 CHAPTER 4: DISCUSSION .........................................................................................................55 CHAPTER 5: CONCLUSION ......................................................................................................66 REFERENCES ..............................................................................................................................68 APPENDICES ...............................................................................................................................73 TABLE A1 LIST OF PRIMER SEQUENCES .......................................................................................73 TABLE A2 PRIMARY AND SECONDARY ANTIBODY CONDITIONS...................................................74 TABLE A3 RAW DATA FOR UNTREATED SAMPLES FOR VIABILITY, ANNEXIN V-PE, AND CFC .......75 TABLE A4 SUMMARY OF P VALUES FOR Q-RT-PCR AND WESTERN BLOTS .................................76 FIGURE A1 FACS PLOTS FOR IMATINIB, TG101209, AND COMBINATION TREATMENTS ..............78 FIGURE A2 ADDITIONAL DOSES TESTED FOR COMBINATION TREATMENT....................................84 TABLE A5 COMPARISON OF THE SH4 + AHI-1 DOUBLE INFECTED CELL LINE WITH PARENTAL K562CELLS ................................................................................................................86  v  LIST OF TABLES TABLE A1 LIST OF PRIMER SEQUENCES .......................................................................................73 TABLE A2 PRIMARY AND SECONDARY ANTIBODY CONDITIONS...................................................74 TABLE A3 RAW DATA FOR UNTREATED  SAMPLES FOR VIABILITY, ANNEXIN V-PE, AND CFC .......75  TABLE A4 SUMMARY OF P VALUES FOR Q-RT-PCR AND WESTERN BLOTS .................................76 TABLE A5 COMPARISON OF THE SH4 + AHI-1 DOUBLE INFECTED CELL LINE WITH PARENTAL K562CELLS ................................................................................................................86  vi  LIST OF FIGURES FIGURE 1: ACQUISITION OF THE PHILADELPHIA CHROMOSOME IN CML .......................................2 FIGURE 2: OVERVIEW OF BCR-ABL SIGNALING ........................................................................10 FIGURE 3: ABELSON HELPER INTEGRATION SITE I GENE ............................................................13 FIGURE 4: AHI-1 MEDIATED ACTIVATION OF BCR-ABL AND JAK2/STAT5 IN CML ..............16 FIGURE 5: STRUCTURE AND FUNCTION OF A JAK2 INHIBITOR TG101209 ..................................18 FIGURE 6: CONFIRMATION OF AHI-1 EXPRESSION IN K562 CELLS..............................................29 FIGURE 7: RESPONSE OF AHI-1 CELLS TO IM TREATMENT ........................................................32 FIGURE 8: IC50 VALUE DETERMINATION FOR TG101209 IN K562 CELLS ..................................35 FIGURE 9:CELLULAR RESPONSE OF AHI-1 TRANSDUCED CELLS AND IM RESISTANT CELLS TO TG101209 ...................................................................................................................39 FIGURE 10: CELLULAR RESPONSE OF AHI-1 TRANSDUCED CELLS AND IM RESISTANT CELLS TO COMBINATION THEARAPY ........................................................................................43 FIGURE 11: CHANGES IN BCR-ABL SIGNALING GENE TRANSCRIPTS .........................................47 FIGURE 12: WESTERN BLOTS OF BCR-ABL SIGNALING PROTEINS ............................................52 FIGURE A1: FACS PLOTS FOR IMATINIB, TG101209, AND COMBINATION TREATMENT .............78 FIGURE A2: ADDITIONAL DOSES TESTED FOR COMBINATION TREATMENT................................84  vii  LIST OF ABBREVIATIONS Ahi-1/AHI-1 = Abelson helper integration site-1 ALL = Acute lymphocytic leukemia A-MuLV = Abelson-murine leukemia virus AP= Accelerated phase BCR = Breakpoint cluster region BP= Blast crisis phase CFC= Colony forming cell CML = Chronic myeloid leukemia CP= Chronic phase CTCL = Cutaneous T-cell lymphoma ERK = Extracellular signal regulated kinase FACS = Fluorescence-activated cell sorting GAPDH = Glyceraldehydes 3-phosphate dehydrogenase IKK = I kappa B kinase IL = Interleukin IM = Imatinib (BCR-ABL inhibitor) JAK = Janus kinase NFB = Nuclear factor kappa B MAPK = Mitogen activated protein kinase MHC = Major histocompatibility complex ORF = Open reading frame PEST = Sequence containing proline, glutamic acid, serine, threonine Ph+ = Philadelphia chromosome positive PI3K = Phosphoinositide-3-kinase PXXP = Proline rich sequence qRT-PCR = Quantitative real rime polymerase chain reaction RNAi = RNA interference RT-PCR = Reverse transcriptase polymerase chain reaction SH2 = Src homology 2 SH3 = Src homology 3 shRNA = Short hairpin RNA SRC = Sarcoma SS = Sezary syndrome STAT = Signal transducer and activator of transcription TBST = Tris-buffered saline tween-20 TG = TargeGene (TG101209) WD40 = Tryptophan-aspartic acid 40  viii  ACKNOWLEDGEMENTS I would first like to thank my supervisor Dr. Xiaoyan Jiang for giving me the opportunity to work on this project, and for her assistance and academic and personal support for the duration of this project. I would also like to thank my committee members Dr. Gregg Morin and Dr. Sandra Dunn for their suggestions and guidance. I would like to give a special thanks to Kathleen Newmarch for assisting me this past summer with completion of several biological assays. I would like to acknowledge Kyi Min Saw, Leon Zhou, Margaret Hale, and Helena Wang for their technical support and assisting me with trouble shooting. Finally I would like to recognize Sharmin Esmailzedah, Erin Kennah, Min Chen, and several other students of the Eaves lab for their feedback and for the discussions that have contributed to my understanding of my results and project.  I am very grateful for having the opportunity to work in such a  supportive environment and have found the experience to be highly educational and personally rewarding.  ix  DEDICATION I would like to dedicate my thesis to my family and friends, but especially my parents Lynda and Dave who have supported me during some challenging but rewarding experiences over the past two and a half years. Also I would like to give a special dedication to my beloved late nana, Olive Snelgrove Hutley, for inspiring me to pursue research in the exciting field of CML and to always follow my heart.  x  CHAPTER 1: BACKGROUND 1.1 Introduction to CML CML is a clonal, multilineage, disorder arising from the neoplastic reciprocal translocation between the long arms of chromosomes 9 and 22 t(9;22)(q34;q11), producing the abnormally short Philadelphia chromosome (Figure 1a)1-6. CML occurs with an incidence rate of approximately 1-2 new cases per 100,000 each year. This characteristic translocation is thought to occur in a more primitive hematopoietic stem cell compartment characterized by the CD34+CD38- phenotype, and results in the production of the oncoprotein BCR-ABL2,3,5,7(Figure 1b), which is the initial malignant event triggering the onset of CML. Following this initial event, other mutations occur within all cell compartments at all stages of differentiation. CML is an interesting and unique form of leukemia in that, unlike most types of leukemia, it is characterized by one recurring genetic malignancy that is both necessary and sufficient for propagation of the disease and survival of the cancer cells. In fact, because CML is dependent on the presence of the oncogene, it has been called a leukemia with an “oncogene addiction”8. It has been one of the most extensively studied leukemias and is often used as a model for studying other less well defined malignancies, and is the first leukemia to be successfully treated with targeted molecular therapy2. Although BCR-ABL is well documented as the trigger for onset of the disorder, the accumulation of secondary mutations within the cell is required for progression of the disease9.  1  Figure 1: Acquisition of the Philadelphia Chromosome in CML. a) Reciprocal translocation between the long arms of chromosomes 9 and 22 producing the Philadelphia Chromosome, encoding BCR-ABL. b) Acquisition of the BCR-ABL oncoprotein within the stem cell compartment and development of additional mutations within other cellular compartments (Legend:  HSC=hematopoietic stem cell, CMP=common myeloid progenitor,  CLP= common lymphoid progenitor, GMP= granulocyte/macrophage progenitor, MEP= megakaryocyte/erythrocyte progenitor). a) c-ABL Chromosome 9 Chromosome 22 c-BCR Philadelphia Chromosome  b) BCR-ABL  Additional mutations  HSC CLP  CMP  GMP  Granulocytes Monocytes  Additional mutations  MEP  Platelets Erythroid  Natural Killer  T Cells  B Cells  2  1.2 Disease Progression and Current Treatment Patients diagnosed with CML progress through three stages of the disease; the chronic phase (CP), accelerated phase (AP), and finally the terminal blast crisis phase (BP)9. During the chronic phase, which lasts approximately 3 to 4 years, patients are generally asymptomatic, with the exception of an over-proliferation of differentiated myeloid cells, in particular the granulocytes and monocytes1. As patients progress through the three stages, an accumulation of more primitive like myeloid and lymphoid blast cells accumulate, which are unable to differentiate into the various lineages, resulting in a severe deficiency of healthy mature differentiated cells. Blast crisis is characterized by the presence of at least 30% blast cells in the peripheral blood or bone marrow9. Progression to the terminal blast crisis phase is characterized by the acquisition and accumulation of additional genetic mutations. Interestingly, as patients progress through the stages of the disorder, the levels of BCR-ABL increase in cells at all stages of differentiation, in particular in the stem/progenitor CD34+ cell compartment. The only current therapy that has been successful at completely eradiating the leukemia is through allogenic stem cell transplantation. However, this treatment option is rarely feasible due to a lack of suitable stem cell donors, and the age of the patients being treated. However, due to the discovery of one unique CML aberration, current treatment options have focused on inhibition of the BCR-ABL kinase activity10. The first line treatment for patients diagnosed with CML is the oral administration of the tyrosine kinase inhibitor IM (Glivec or STI571). IM acts a potent and specific inhibitor for the ABL kinase through binding to the ATP site of the kinase domain, and also binds to cKIT, platelet derived growth factor receptor (PDGFR), and the ABL homolog ARG11. Patients treated in the early phase of the disease respond remarkably well to the treatment, with approximately between 80 to 90% displaying a complete cytogenetic  3  response to treatment. Although promising, this treatment option is not without its drawbacks. Patients who are initially treated during the later phases of the disease do not respond as effectively to the treatment (approximately 40% in accelerated phase and 20% in blast crisis), and even those that do initially respond will often suffer relapses, most commonly due to the acquisition of point mutations within the kinase domain of ABL, resulting in an inability of IM to bind to its site. Furthermore, sensitive Q-RT-PCR reveals that patients treated with the inhibitor still show a very small molecular response, as indicated by the continued presence of BCR-ABL transcripts, suggesting that treatment involving ABL inhibition is not entirely effective at eradicating these leukemic BCR-ABL positive stem cells. This indicates the necessity of long term therapy or the need to target other critical players involved.  4  1.3 Autoregulation of C-ABL and Constitutive Activation of BCR-ABL Normally, the ubiquitously expressed c-ABL kinase encoded by its gene on chromosome 9 is able to shuttle between the nuclear and cytoplasmic fractions of the cell12-15. Alternative splicing of the first two exons in the ABL1 transcript produces two isoforms (1a and 1b), one (1b isoform) of which undergoes myristolylation at its amino terminus 10. C-ABL is a non-receptor tyrosine kinase involved in signal transduction from cell surface growth factors and adhesion receptors, and acts to regulate cytoskeletal structure, cell differentiation, cell division, apoptosis, and cell response to stress16-28. C-ABL and its homolog ARG resemble the SRC and TEC family tyrosine kinases, and contain several signalling domains including a catalytic domain, a variable N terminal region (cap region), SH3, an SH2 domain, activation loop, and several proline rich sequences within the C terminus. Furthermore, c-ABL contains three nuclear localization and one nuclear export signals allowing it to shuttle between the two cellular compartments in response to specific cellular signals10. C-ABL undergoes strict autoregulation thought to be controlled by the SH3 and SH2 domains. Regulation differs slightly between the two isoforms due to the presence of the myristoylated region of 1b. Generally, binding of the SH3 domain to a linker segment between the SH2 and catalytic domains, and binding of the SH2 domain with the carboxy terminus of the kinase domain creates a folding of the kinase into an inactive state preventing binding to downstream proteins. In order for the SH2 domain to bind, the kinase domain must be in an open conformation. A specific conformational change, located at the base of the activation loop, must occur within the kinase domain to create the docking site for SH2. In the case of the 1b isoform, binding of the myristoyl group to the kinase domain is essential for maintaining the inactive form, whereas in the 1a isoform interactions between the SH3 and SH2 are crucial, however, the exact mechanisms of control remains to be determined. Upon fusion  5  with BCR from chromosome 22, the coiled coil domain at the N terminus of BCR overcomes this autoregulation leading to tetramerization and dimerization causing autophosphorylation of BCR-ABL thus resulting in a constitutively activated non receptor tyrosine kinase26. Additionally, new domains are created that recruit several adaptor proteins that can bind to the oncoprotein. Rather that maintaining the capacity to shuttle between the two compartments, BCR-ABL relocates to the cytoplamic compartment where it activates several altered signal transduction cascades involved in cellular proliferation, survival, and growth factor independence. The most common form of BCR-ABL exists as a p210kD protein, however, two other forms of p185kD and p230kD have been reported29.  6  1.4 BCR-ABL Signalling As mentioned previously, the malignant BCR-ABL protein contains several signalling domains involved in the activation of downstream signalling cascades including the RAS/ERK(MAPK), PI3K/AKT, NF-kB, and JAK2/STAT5 pathways (Figure 2) -30 Combined, these cascades are involved in cellular resistance to apoptosis, increase in cellular survival, resistance to tyrosine kinase inhibitors, reduction in cell adhesion, reduction in cellular response to cell cycle check mechanisms, reduction in the ability of the cells to repair damaged DNA, and growth factor independence. Activation of the RAS pathway depends on the GRB-2/Gab-2 adaptor complex binding to the SH2 domain of BCR-ABL31-33. Upon this binding, BCR-ABL phosphorylates the SHC adaptor protein, which in turn activates the GTP/GDP exchange factor SOS (son of sevenless). This allows for the exchange of GDP bound to Ras in its inactive form with GTP causing the activation of the protein. Ras then activates several kinases including the MEK1/2 kinase, which in turn leads to the activation of ERK1/2 (MAPK 1/2). Studies have demonstrated that down regulation of this pathway leads to an increase in cellular response to cell cycle control mechanism, slows proliferation of the cells, and increases cellular response to apoptosis. Strict control of this pathway is therefore essential to maintain proper control of cell division and survival. The GRB-2/Gab-2 adaptor complex is also involved in activation of the PI3K pathway31. The p85 subunit of PI3K binds to this complex and is phosphorylated by BCR-ABL34-36. Activated PI3K can then phosphorylate the major serine threonine kinase AKT, which leads to inactivation of the proapoptic protein BAD. Cross talk exists between this pathway and the NF  7  κB pathway as AKT is known to phosphorylate the NF-κB1 family member (p50 subunit) leading to its activation37. The NF-κB signalling pathway is a complex set of interactions consisting of several family members. Briefly five members exist including NF-κB1 (p105/p50), NF-κB2 (p100/p52), RelA (p65), cREL, and RELB, each with different cellular functions37. The NF-κB proteins are generally involved in controlling transcription of genes involved in immunity and the inflammatory response including, but not limited to, IL-2, IL-6, GM-CSF, and class I MHC. The active form of NF-κB consists of homo and hetero dimers with the most abundant form consisting of NF-κB1 (p105/p50) bound to RelA (p65). Simply described, the precursor NFκB1 (p105 subunit) is cleaved to form the p50 subunit which is activated by AKT, while the RelA or p65 subunit is phosphorylated by protein kinase A38-43. The two phosphorylated proteins then form a heterodimer which can then translocate to the nucleus and act as a nuclear transcription factor (Figure 2b). To add complexity to this pathway, activity of the NF-κB family members, (particularly that of NF-κB1 which has been the most extensively studied family member) depends on phosphorylation of its binding partner IκB (Inhibitor of kappa B) which is required for the release of NF-κB40. Phosphorylation is mediated by the IKKα and β kinases (Inhibitor of kappa B kinase), particularly by the IKKα kinase. Dysfunction in the regulation of the NF-κB proteins has now been widely linked to the development of several leukemias and lymphomas. The final pathway of interest includes the Janus Kinases (JAKs) and Signal Transducers and Activators of Transcription (STAT) proteins. Four family members of the JAK family exist including JAK1, JAK2, JAK3, and TYK2, while seven exist for the STAT family; STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6 arising from several gene duplication events44. JAK2 activity has been widely described in terms of its involvement in haematopoiesis  8  and specifically overactivation or mutation of JAK2 has been linked to hematologic malignancies including Ph+ CML45, and is known to preferentially activate the STAT3, STAT5a and STAT5b transcription factors in these malignancies46. Once phosphorylated the STAT proteins form homo and heterodimers and are able to translocate to the nucleus where they regulate several genes involved in the immune response, cellular growth, and cellular differentiation.  9  Figure 2: Overview of BCR-ABL Signalling. a) BCR-ABL signalling cascades that are active in CML. Emphasis is on the JAK/STAT, PI3K/AKT, and Ras/MAPK signalling pathways. b) Simplified overview of NF-κB activation.  a)  b)  PI3K  IKKα/β  NFkB 105  PKA  AKT IkB  NF kB p65  NFkB (50)  NFkB (50) NF kB p65  Nuclear Transcription Factor  10  1.5 Abelson Helper Integration Site 1 (AHI-1) Gene Ahi-1/AHI-1 is a recently identified oncogene initially identified in the v-abl induced murine pre-B cell lymphomas (MuLV)47. The Abelson murine virus is a defective, replicationincompetent retrovirus that requires the assistance of a helper virus which integrates at the Ahi-1 locus via proviral intentional mutagenesis.  Ahi-1/AHI-1 involvement in leukemogenesis is  indicated by the presence of several mutations within the Ahi-1 gene in murine leukemias and lymphomas, leading to overexpression of the full length as well as expression of truncated forms of the protein11,48,49. Recently, mutations in human AHI-1 have been found to be associated with Joubert syndrome, an autosomal recessive brain disorder. Abnormal cerebellar development and axonal decussation occur in individuals with point mutations in AHI-1 that generate stop codons, amino acid substitutions, or splicing errors within AHI-1 protein50,51. Ahi-1/AHI-1 contains several domains indicating its potential function as a signalling protein. Some of the major sequences include an SH3 domain, seven WD 40 domains, two PEST motifs, several phosphorylation sites, and several SH3 binding sites47. Additionally the human form of AHI-1 contains an N terminus coiled coil domain not found on the murine protein (Figure 3). AHI-1 consists of three major isoforms found in normal human blood. Isoform I contain all domains mentioned previously. Isoform III resembles Isoform I containing all major domains, although shorter than isoform I, it contains additional coding sequences not present in either isoform I or II. Isoform II completely lacks the C terminus, suggesting that it may have a different function in the cell than the other two (Figure 3). AHI-1 expression is normally under strict cellular control with expression level being the highest in the most primitive hematopoietic stem cells. Its expression is downregulated through the process of differentiation50.  Interestingly, this mirrors what is observed for BCR-ABL  11  expression levels in CML cells suggesting a possibly cooperative role between the two. AHI-1 is highly expressed in several human leukemia and lymphoma cell lines (Figure 3b). In particular, AHI-1 is very highly expressed in patients with Sezary syndrome (CTCL) and CML, as well as in a human cell line derived from a patient in blast crisis CML (K562 cells)50,51. Previous studies involving stable infection of murine lymphoma BaF3 cell line with full length BCR-ABL, Ahi-1, or both has led to a deeper understanding of the cooperative activities between the two oncoproteins52. Overexpression of BCR-ABL or Ahi-1 alone in this growth factor dependent cell line led to an increase in cellular proliferation and growth factor independence. Furthermore, overexpression of BCR-ABL and Ahi-1 significantly enhanced these perturbations supporting an oncogenic cooperative mechanism involved in leukemogenesis. Further in vivo studies involving injection of parental BaF3, Ahi-1 or BCR-ABLtransduced BaF3, and Ahi-1 and BCR-ABL co-transduced BaF3 cells into NOD/SCID-β2 immunodeficient mice further support the oncogenic activity of Ahi-152. Mice injected with the parental BaF3 did not display any disease 120 days following injection, while those injected with Ahi-1 overexpressing and BCR-ABL expressing BaF3 cells developed a lethal leukemia within 70 and 41 days respectively. Injection of the co-transduced cells caused a lethal leukemia within 26 days of injection, further supporting a cooperative role between Ahi-1 and BCR-ABL.  12  Figure 3: Abelson Helper Integration Site 1 Gene. a) Three human AHI-1 isoforms that exist in normal human blood cells. Images show all important protein domains involved in signalling functions. b) Elevated levels of AHI-1 transcript levels in human leukemia (orange) and lymphoma (blue) cells lines as well as in the CML cell line K562 as compared to normal human bone marrow (red).  a) Chromosome 6  Human AHI-1 Y PEST  I  Y  PEST  C  N  PXXP  Coiled-Coil  WD40  PXXP  SH3  Y  II N  C PXXP  PXXP  Y  Y  III N  C  PXXP  PXXP  Adapted by Permission from Macmillan Publishers Ltd: Leukemia 20, 1593-1601 ©2006  b)  Jiang et al., Blood 102: 2976. 2004  13  1.6 AHI-1 Involvement in CML Studies involving K562 cells and primary patient blood samples have revealed a striking correlation between cellular resistance to IM and AHI-1 expression levels52. Blood samples from CML patients who had not been treated with IM, were obtained from patients in the chronic phase and blast crisis phase. Some of these patients following treatment proved to be nonresponders to the IM therapy.  Analysis of AHI-1 transcripts within the lin-CD34+  stem/progenitor cell compartment revealed that the non-responders displayed a significantly higher expression level of AHI-1 than the responders, and that those in blast crisis also expressed significantly higher AHI-1 transcript levels compared to patients in the chronic phase. Interestingly, when AHI-1 was suppressed in these primary samples, using a mediated retroviral approach, and a colony forming cell assay was performed, the transduced cells displayed a significant reduction in the number and size of colonies formed indicating an increased response to IM. In vitro studies involving the K562 cells support the previous results found in primary samples.  K562 cells with overexpression of AHI-1 demonstrated a significant decrease in  cellular sensitivity to IM treatments in comparison to the parental K562 cells, as determined by a colony forming cell assay, while cells with suppression of AHI-1 demonstrated the opposite effect (Figure 4a)52. Additionally, when these cell lines were treated with IM, and protein expression levels of cellular and phosphorylated BCR-ABL was analyzed, the results indicated that AHI-1 overexpression significantly increased the amount of total and phosphorylated BCRABL expressed in the cells. While suppression of AHI-1 led to a significant reduction of total and phosphorylated BCR-ABL (Figure 4b).  Several other downstream signalling cascades  demonstrated the similar effects, with the most striking differences observed in JAK2 and 14  STAT5. Overexpression of AHI-1 not only led to an increase in the levels of phosphorylated JAK2 and STAT5, but in total levels of these proteins (Figure 4b). Due to the strong effects on the BCR-ABL signalling cascade, co-immunoprecipitation experiments were performed to determine if AHI-1 might interact with any of these signalling proteins. The results revealed a direct interaction between BCR-ABL and AHI-1, as well as between AHI-1 and phosphorylated BCR-ABL, and AHI-1 with JAK2 and phosphorylated JAK2 (Figure 4c)52. These results suggest a potential mechanism of AHI-1 action in which AHI-1 forms a stable complex with BCR-ABL and JAK2, and through the activation of STAT5 can lead to an increase in cellular survival/proliferation and resistance to imatinib (Figure 4d).  15  Figure 4: AHI-1-Mediated Activation of BCR-ABL and JAK2/STAT5 in CML. a) K562 response to IM treatments with AHI-1 overexpression (AHI-1) and AHI-1 suppression (AHI/Sh4) as determined by a colony forming cell (CFC) assay. Significant differences are observed when AHI-1 is suppressed at a dose of 1µM as compared to parental cells, and at all doses when AHI-1 is overexpressed as compared to parental cells. b) AHI-1 manipulation strongly affects the total levels of BCR-ABL, JAK2, and STAT5, as well as the levels of these phosphorylated proteins. c) AHI-1 directly interacts with BCR-ABL and JAK2 in the K562 cells. d) Model of AHI-1--BCR-ABL-JAK2 complex regulation of constitutive activation of BCR-ABL and JAK2-STAT5 pathway, resulting in increased proliferation and reduced TKI response of CML stem/progenitor cells. a)  c)  b)  d)  Zhou et al, JEM, 2008  16  1.7 A Novel JAK2 Inhibitor TG101209 As mentioned previously, AHI-1 displayed significant effects on JAK2/STAT5 signalling, thus an approach in which JAK2 is inhibited and effects are observed in K562 cells with and without AHI-1 overexpression or suppression is of interest. JAK/STAT signalling is a complex pathway involving several family members of both proteins. Due to the fact that AHI-1 exerts its effects most strongly on JAK2/STAT5 signalling specifically, we were interested to inhibit this pathway52. This signalling pathway involves an autocrine loop which is stimulated when interleukin-3 (IL-3) binds to its receptor, which results in the homodimerization of JAK2 proteins that can cross phosphorylate each other, which upon phosphorylation can then phosphorylate and activate the transcription factor STAT5. Inhibition of JAK2 activity in theory would thus result in a decrease not only in the activity level of STAT5, but also JAK2 due to the cross phosphorylation of this protein53. Several JAK inhibitors are currently available, including the popular AG490, however, several of these inhibitors are non-specific for the JAK2 kinase. One inhibitor, TG101209, currently in preclinical trials (Figure 5a), is another inhibitor that is highly specific for the mutant V617F JAK2 involved in myeloproliferative disorders, but also inhibits the wild type JAK2 in K562 cells54.  Studies involving determination of the  phosphorylated and total protein levels of STAT5 and JAK2 have demonstrated strong and specific effects on JAK2/STAT5 signalling with the use of this inhibitor (Figure 5), and thus is an appropriate choice for further investigation as indicated by a reduction in the phosphorylated levels of these proteins while total protein levels remain unaffected54-55.  17  Figure 5: Structure and Function of a JAK2 Inhibitor TG101209. a) Molecular structure of TG101209. b) Image of TG101209 binding to the ATP pocket of the JAK2 kinase domain.  a)  b)  Pardanani et al., Leukemia (8): 1658-68. 2007  18  1.8 EXPERIMENTAL OUTLINE Based on previous studies using the same K562 model system, the AHI-1 oncoprotein was found to strongly affect both BCR-ABL and JAK2/STAT5 signalling52. When AHI-1 is overexpressed there is a significant increase in the expression of total BCR-ABL, JAK2, and STAT5, thus I predicted that AHI-1 will also affect other key BCR-ABL signalling cascades, and that overexpression of AHI-1 will lead to increased signalling of other key downstream oncogenes that are activated by BCR-ABL including AKT, ERK, and NF-кB. To test this hypothesis, several Q-RT-PCR and western blot analyses were performed to determine the RNA and protein expression of these other key factors. In the same previous experiments overexpression of AHI-1 led to a significant reduction in cellular sensitivity to imatinib while suppression of AHI-1 led to a significant increase in cellular sensitivity to imatinib as determined by a CFC assay. I predicted that overexpression and suppression of AHI-1 will also affect cellular viability and apoptosis, and to test this hypothesis, both a viability assay and an annexin V stain were performed. Because AHI-1 led to an increase in the level of JAK2/STAT5 signalling, I also predicted that treatment of the AHI-1 overexpressing cells with a novel JAK2 inhibitor TG101209 will display heightened resistance as compared to parental cells. This was determined using a viability assay, annexin V stain, and a CFC assay of the K562 cells in the presence of varying doses of TG101209. Finally, I predicted that the AHI-1 overexpressing cells and IM resistant cells would respond better to a combination therapy approach in which both BCR-ABL and JAK2 are inhibited. The response of these cells was determined using a viability assay, annexin V stain, and a CFC assay using various doses of TG101209 combined with a 1 µM dose of imatinib.  19  CHAPTER 2: MATERIALS AND METHODS 2.1 Cell Lines As described previously, K562 cells were infected with a lentiviral vector (EF1-IRES-YFP) containing the full length human AHI-1 cDNA, to produce an AHI-1 overexpressing K562 cell line (AHI-1 lenti clone B5)52. To generate a K562 AHI-1 suppressing cell line, the cells were infected with a retroviral construct (RPG) containing the H1 promoter encoding a small hairpin RNA against AHI-1 as previously reported52. The oligonucleotides encoding the AHI-1 siRNA that inhibited AHI-1 expression (AHI-SH4) were: 5’-GATCCCCGTGATGATCCCGACACTATTTCAAGAGAATAGTGTCGGGATCATCACTTTTTA-3’and 51  5’-GCTTAAAAAGTGATGATCCCGACACTATTCTCTTGAAATAGTGTCGGGATCATCACGGG-3’  .  Finally, a K562 cell line that was generated to be highly resistant to IM was obtained from Dr. Ali Turhan from The University of Poitiers, Poitiers, France and was used (IM resistant) in the study. Verification of cells transduced with each construct was performed using FACS analysis and screening for GFP or YFP expression.  2.2 Cell Culture All K562 cells were cultured in RPMI media containing 10% heat inactivated fetal bovine serum, 100U/mL penicillin, 0.1mg/ml streptomycin, and 1 x 104  mercaptoethanol (StemCell Technologies, Vancouver, BC) at 37c. Cells were maintained in 25cm2 rectangular canted neck cell culture flasks with vented caps (Corning Inc, Lowell, MA) and passaged every two to three days.  20  2.3 Determination of an IC50 Value for TG101209 Parental K562 cells were cultured in 6 well dishes containing 2 ml of RPMI media in duplicate containing either 0.001, 0.01, 0.1, 1, 5, or 10 M of TG101209 (TargeGen, Inc, San Diego, CA) and cells were counted using the Beckman coulter vi cell analyzer at 24 and 48 hours. Cell viability was plotted using Microsoft excel, and IC50 value was extrapolated from both the 24 and 48 hour graphs. Furthermore, western blot analysis (procedure to be described later), was performed using the same doses of the inhibitor and protein expression analysis for pSTAT5 (Y694), total STAT5, pJAK2(Y1007/1008), and total JAK2 (Appendix Table 2).  2.4 Cellular Viability Assay 2 x 105 K562 cells from the parental K562, K562 SH4 bulk, K562 AHI-1 lenti clone B5, and IM Resistant cells were plated in a six well dish containing 2 ml of RPMI media in duplicate and treated with either IM (Novartis, Basel, Switzerland) alone, TG101209 alone, or a combination of the two inhibitors. IM treatment included doses of 1, 5, or 10 M, and cells were counted at 24 and 48 hours using haemocytometer counts of trypan blue excluding cells, or with the Beckman Coulter vi-cell analyzer. TG101209 treatment included doses of 0.5, 5, or 10 M, and cells were counted using the Beckman Coulter vi-cell analyzer. Combination treatment included manipulation of TG101209 concentration with 1 M of IM. Doses of TG101209 included 0.1, 0.5, 1, 2.5, and 3.5 M. Cells were counted at 24 and 48 hours using the Beckman Coulter vi cell analyzer.  2.5 Apoptosis Analysis The apoptosis analysis was performed using an apoptosis detection kit (BD pharmingen TM, San Jose, CA) following the manufacturer’s instructions. 2 x 105 cells were plated in 6 well dishes 21  and treated with IM alone, TG101209 alone, or a combination of the two inhibitors. Treatment doses of each inhibitor or combination of inhibitors was the same as used in the viability assay. Cells were cultured for either 24 or 48 hours with the inhibitor. 5 x 104 cells were washed with PBS, and resuspended in 50 L of 1X binding buffer and incubated in 2.5 L of 7-AAD (7 amino-actinomycin D) and 2.5 L of PE-conjugated Annexin V antibody at room temperature in the dark for 15 minutes. An additional 200 L of binding buffer was then added for FACS analysis. Unstained negative controls, Annexin V-PE stained, and 7AAD stained controls were used for each cell line analyzed. Parameters for each cell line were determined using negative controls. Early apoptotic cells were defined as Annexin V positive cells, late apoptotic cells were defined as annexin V and 7AAD double positive cells.  2.6 Colony Forming Cell Assay  CFC assays were performed on all K562 cell lines. 80 mL of methylcellulose H4230 (Stem Cell Technologies, Vancouver, B.C)  was mixed with 20 mL Iscove’s Media (Stem Cell  Technologies, Vancouver, B.C) and divided into 3 mL aliquots, and 3 L of penicillin/streptomycin was added. IM, TG101209, or a combination of treatments was added to each aliquot. IM treatments again included doses of 1, 5, or 10 M, TG101209 treatment included doses of 0.5, 1, 2.5, 3.5, or 5 M, and combination treatments included 1 M of IM with 0.5, 1, 2.5, 3.5 M of TG101209. 500 cells were added to each aliquot, and samples were vortexed, and allowed to incubate for 5 minutes for the bubbles to disperse. 3 mL syringes with blunt end needles were then used to plate 1.3 mL of media to tissue culture dishes in duplicate as described in the manufacturer’s instructions (Stem Cell Technologies, Vancouver, and B.C).  22  Plates were incubated at 37c for 10 days. Colonies were then counted, and an average between the two dishes was obtained. Each CFC assay was performed with two biological replicates.  2.7 RNA Extraction  Total RNA was extracted from cells using the absolutely RNA microprep kit (Stratagene, La Jolla, CA, USA).  Briefly, 1 x 106 cells were pelleted and washed in sterile Dulbecco’s  Phosphate Buffered Saline (PBS) solution (StemCell Technologies, Vancouver, BC). Cells were resuspended in lysis buffer with -mercaptoethanol. Purification of RNA was performed following the procedure described for tissue culture cells grown in suspension.  The first  extraction of RNA was performed by adding 40L of elution buffer; followed by a second extraction using 30L of elution buffer to yield a total volume of 70L. RNA was quantified using the nanodrop ND-100 spectrophotometer and measuring optical density at 260nm and 280 nm.  2.8 cDNA Synthesis cDNA was synthesized using the protocol for the first-strand cDNA synthesis provided with the SuperScriptTM III Reverse Transcriptase (Invitrogen, Burlington, ON). cDNA was synthesized from 100-250 ng of total RNA and 3g/L of random primers (Invitrogen, Burlington, ON) in 20 L reaction volumes.  23  2.9 Quantitative Real Time PCR  Samples were prepared in 96 well dishes and plated in duplicate or triplicate. Three biological samples were analyzed for each gene. 25L reaction volumes were obtained by combining 12.5L Fast SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), 1L of cDNA, 10.5L of RNAse free PCR water, and 1L of a 20M gene specific primer (Invitrogen, Burlington, ON,Appendix Table 1). Gene expression was quantified using the ABI7500 real time PCR system (Applied Biosystems, Foster City, CA), using a thermal profile consisting of 50C for 2 minutes, 95c for 10 minutes, and 50 cycles of 95c for 15 seconds followed by 1 minute at 60c. Fluorescence was measured using SYBR green as the reporter dye and ROX as a passive reference. All primer efficiency analysis was performed using serial dilutions of each primer set (1X, 1/10X, 1/100X, and 1/1000X) in RNA extracted from K562 cells. Samples were analyzed using the ABI7500 real time PCR system software (Applied Biosystems, Foster City, CA). All measurements were normalized to an endogenous control glyceraldehydes-3phosphate-dehydrogenase (GAPDH), and all gene expression levels are quantified relative to parental K562 cells.  2.10 Protein Extraction and Quantification  Cells were pelleted and washed in PBS and placed at -70c for a minimum of 10 minutes to freeze dry. Protein was extracted from the various cell lines by incubating the pellet in protein lysis buffer consisting of a ratio of 1 ml of protein solubilization buffer (PSB), 50 L of NP-40 Alternative Protein Grade Detergent (Calbiochem, Gibbstown, NJ), 50 L of protease inhibition complex (PIC,Sigma-Aldrich, Oakville, ON), and 5 L of phenylmethyl sulfonyl fluoride  24  (PMSF,Sigma-Aldrich, Oakville, ON). Cells were diluted at a concentration of 1 X 106 cells per 100 L of lysis buffer and incubated for 1 hour at 4c.  Protein lysate was then separated via  centrifugation at 12000 rpm for 10 minutes at 4c, and the supernatent was then collected and stored at -70c. Protein concentration was quantified using the Bradford Assay. Bio Rad Protein Assay Dye Reagent (Mississauga, ON) was diluted at a ratio of 1:4 times dye in super Q water. Protein was diluted at a ratio of 1:20 by adding 5 L of protein lysate to 95 L of PSB in a 96 well plate, and samples were analyzed in duplicate. Standard protein concentration was obtained using purified bovine serum albumin (BSA,Bio-Rad Laboratories, Mississauga, ON) using concentrations ranging from 33.3-1000ng/L. 200 L of Bio-Rad dye was added to each well, and samples were left to incubate at room temperature for 10 minutes.  Absorbance was  measured at 630 nm using the Elx808TM Absorbance Microplate Reader (BioTekk Instruments, Winooski, VT).  2.11 Western Blot Analysis Between 20 and 40 g of protein lysate was added to 2.5-5 L of NuPAGE LDS sample buffer (4X) and 1-2 L NuPAGE reducing agent (10X) and up to 13 L of deionized super Q water for a final sample volume of 10-20L. Samples were heated at 70c for 10 minutes and loaded with 7L of the PageRulerTM prestained protein ladder (Fermentas, Burlington, ON) on NuPAGE Novex 4-12% Novex bis-tris gels (Invitrogen, Burlington, ON).  Gels were run for 45-60  minutes using the Xcell SurelockTM Mini-cell and NuPAGE MOPS SDS running buffer (Invitrogen, Burlington, ON) at 200 V. Transfer of the protein from the gel to Immobilin-PPVDG 0.45 m membrane (Milliporte, Billerica, MA) was performed using NuPAGE Transfer buffer (Invitrogen, Burlington, ON). Transfer time ranged between 60-90 minutes depending on 25  protein size at 30-33 V. Membranes were blocked in Tris Buffered Saline Tween-20 (TBST) with 5% skim milk for 1 hour, washed twice in TBST for 5 minutes. Primary antibodies were diluted at a concentration of 1:500 or 1:1000 in TBST, and 2-5% Bovine Serine Albumin (BSA). Membranes were incubated with primary antibody overnight at 4C.  Primary antibodies  included polyclonal N terminal anti AHI-1 (IMGENEX, San Diego, CA), anti JAK2, anti phospho JAK2 (Y1007/1008), anti STAT5a/b, anti phospho STAT5a/b (Y694), anti phospho SRC, anti AKT, anti phospho AKT (S473), anti NF-B p65, anti NF-B p105/p50, anti phospho NF-B (S536), anti IKK, anti IKK, and anti GAPDH (Cell Signalling Technology, Beverly, MA), anti ERK, and anti phospho ERK (Santa Cruz Biotechnology, Santa Cruz, California). The following day, membranes were washed 3 times in TBST for 10 minutes, and incubated in secondary antibody conjugated to HRP for 1 hour at room temperature, and the membrane was then washed an additional 3 times in TBST for 10 minutes. Secondary antibody concentration ranged from 1:2500 to 1:10000, diluted in 1XTBST and 2-5% BSA. The Appendix Table 2 provided detailed primary and secondary antibody conditions. Western Lightning Western Blot Chemiluminescence Reagent Plus and KODAKTM and BioMax XAR autoradiography film (PerkinElmer, Life and Analytical Sciences, Waltham, MA) was used to image the membrane. Protein expression was quantified using the Image Quant 5.2 software (Molecular Dynamics, Sunnyvale, CA). Protein expression was normalized to GAPDH levels, and expression is determined relative to parental K562 cells.  2.12 Statistical Analysis Errors bars are displayed as standard deviation and differences observed between the various cell lines were analyzed using Students t-test for paired samples with unequal variance. Differences were determined significant at a value of P≤ 0.05.  All viability, annexin V, CFC, and Q-RT26  PCR experiments consisted of two or three technical replicates performed during one experiment and two or three separate biological experiments. Western blots consisted of two or three separate biological replicates. Number of replicates provided indicates the number of separate biological replicates performed.  27  CHAPTER 3: RESULTS 3.1 Confirmation of AHI-1 Expression in K562 Cells AHI-1 expression level was analyzed using both q-RT-PCR and western blotting. RNA was extracted from parental K562, K562 SH4 bulk (with suppression of AHI-1), K562 AHI-1 lenti clone B5 (with overexpression of AHI-1), and K562 IM resistant cells. cDNA synthesis was performed followed by qRT-PCR, and technical replicates of each sample in triplicate were performed. Three separate biological replicates obtained from samples at different time points were analyzed. AHI-1 expression was determined using a primer set specific for the N terminus of AHI-1, and was normalized relative to GAPDH. 5.5-fold overexpression (p=0.01) and 4-fold suppression of AHI-1 (P=0.05) was confirmed in the AHI-1 lenti clone B5 and SH4 bulk cell lines respectively. Interestingly, overexpression of AHI-1 was also detected in the IM resistant cell line (Figure 6a) by approximately 4-fold (P=0.04). Furthermore, AHI-1 expression was quantified via western blotting using two different antibodies that bind to the N terminus of the protein, and one that binds to the C terminus. Again, 2.5-fold overexpression of AHI-1 was observed in the AHI-1 lenti clone B5 sample (P=0.006) and 7-fold suppression in the SH4 bulk (P=0.02) sample. Overexpression of the AHI-1 protein was also observed in the IM resistant cell line by 2-fold (P=0.05) (Figure 6b). A fifth K562 cell line containing both the retroviral SH4 RNA and lentiviral cDNA full length AHI-1 construct was also used to control for possible vector effects, and results from this vector are shown in Table A5d.  28  Figure 6: Confirmation of AHI-1 Expression in K562 Cells a) Using primers specific for the N terminus of AHI-1, suppression was observed in the AHI-1 SH4 bulk cell line and overexpression in the AHI-1 lenti clone B5 cell line. AHI-1 overexpression was also observed in the IM resistant cell line (n=3). b) Western blot analysis of AHI-1 expression normalized to GAPDH confirmed PCR results with significant suppression in the AHI-1 SH4 bulk cell line, overexpression in the AHI-1 lenti clone B5 cell line, and in the IM resistant cell line (n=3). * Indicate significant difference in expression relative to parental at P≤ 0.05.  a) RNA expression of AHI-1 8 7 6 5 4 3 2 1 0  P=0.01 P=0.04  SH4 bulk = AHI-1 suppressed K562 cells AHI-1 lenti = AHI-1 overexpressing K562 cells IM Resistant = Imatinib resistant K562 cells  P=0.05  b) Protein expression of AHI-1 3 2.5 2 1.5 1  P=0.006  P=0.05  P=0.02  0.5 0  29  3.2 Response of AHI-1-Transduced Cells to IM Treatment Biological response to IM treatments in AHI-1-transduced K562 cells was measured using a viability assay, an apoptosis assay, and a colony forming cell assay. For the viability assay, 2 x 105 cells were cultured in 6 well dishes with 1, 5, or 10 µM of IM and cells were counted at 24 and 48 hours. Significant differences in viability were observed at both 24 and 48 hours when AHI-1 was suppressed and overexpressed. Suppression of AHI-1 led to a significant increase in cellular sensitivity as compared to parental cells at 1 and 5 µM at 24 hours (P=0.05 and 0.05) and at 1 µM at 48 hours (P=0.03). AHI-1 overexpressing cells displayed a significant reduction in sensitivity to IM treatments at concentrations of 5 μM at 48 hours as compared to parental cells (P=0.02). As expected the IM resistant cells displayed significantly increased viability as compared to the parental cells at 48 hours with P values of 0.04 at 1µM, 0.008 at 5 µM and 0.05 at 10 µM (Figure 7a and Table A3). Results were confirmed using an apoptosis assay in cells treated with 1, 2.5, or 5 µM of IM for either 24 or 48 hours. Significant increases in sensitivity of the AHI-1 suppressed cells were observed at concentrations of 2.5 and 5 µM of IM treatment at 48 hours (P= 0.05 and 0.05), while significant decreases in sensitivity were observed in the AHI-1 overexpressing cells at 1, 2.5, and 5µM of IM at 24 hours (P=0.03, 0.05, and 0.005). Again, as expected, the IM resistant cells displayed a significant reduction in sensitivity to IM at all treatment doses at both 24 and 48 hours as compared to the parental cells (Figure 7b and Table A3). Final confirmation using a CFC assay validated the sensitivity of the AHI-1 suppressed cells showing an increase in sensitivity at 1 µM of IM (P= 0.05) as compared to the parental cells and the AHI-1 overexpressing cells displaying a decreases in sensitivity at 1 µM (P=0.05) as compared to the parental cells (Figure 7c). The IM resistant cells displayed a reduction in  30  cellular sensitivity at both 1 and 5 µM of treatment with P values of 0.03 and 0.03. In addition to colony number, morphology of the colony was different (Figure 7d) with the AHI-1 overexpressing cells and IM resistant cells producing larger colonies than the parental or AHI-1 suppressed cells.  Raw data is displayed in Table A3 and illustrates that there are small  differences in terms of viability, apoptosis, and colony forming ability across the cell lines even in the absence of treatment. The fifth K562 cell line containing both suppression and overexpression of AHI-1 was also included and data is shown in Table A5.  31  Figure 7: Response of AHI-1-Transduced Cells to IM Treatment a) Cell viability of IM treatment at 24 and 48 hours. Results are expressed as the total number of viable cells relative to no treatment (n=3). Significant differences are observed when AHI-1 is suppressed at 24 hours at 1 and 5 µM (P=0.05 and 0.05) and at 48 hours with 1 µM (P=0.03) and when AHI-1 is overexpressed at 48 hours at 5 µM (0.02). b) Annexin V-PE staining at 24 and 48 hours (n=3). A significant increase in sensitivity is apparent when AHI-1 is suppressed at 48 hours at 2.5 and 5 µM (P=0.05 and 0.05) and a decrease in sensitivity when AHI-1 is overexpressed at 24 hours at 1, 2.5, and 5 µM (P=0.03, 0.05, and 0.005). Percentage of apoptotic cells is expressed relative to no treatment within each group. c) CFC assay of IM-treated cells (n=2). Results are expressed as percent of colony formation relative to no treatment within each group. Suppression of AHI-1 led to a significant increase in sensitivity at 1 μM (P= 0.05), while overexpression led to a reduction in sensitivity at 1 μM (P=0.05). d) Images of colonies formed from the four cell lines at 1 and 5 μM of IM treatment demonstrating differences in morphology between the four cell lines. *Indicate significant differences as compared to parental K562 cells P≤0.05. a)  Cell Viability 24 Hours Imatinib Parental 120 100  SH4 bulk AHI -1 lenti  Cell Viability 48 Hours Imatinib 120 100  IM Resistant 80  80  60  60  40  40  20  20  0  0  32  b) Annexin V-PE 24 hours Imatinib 25  60  Parental SH4 bulk  20  Annexin V-PE 48 Hours Imatinib  50  AHI -1 lenti IM Resistant  40  15 30 10 20 5  10  0  0  c) CFC Imatinib 100 90  Parental  80  SH4 bulk  70 60 50  AHI-1 lenti IM Resistant  40 30 20 10 0  d) 1 µM Imatinib  5 µM Imatinib  Parental K562  K562 SH4 bulk  K562 AHI-1 lenti clone B5  K562 IM Resistant 33  3.3 Determination of IC 50 Value for TG101209  In order to test the efficiency of the JAK2 inhibitor TG101209 a viability assay was performed on the parental K562 cells cultured in various doses of the inhibitor (0.001, 0.01, 0.1, 1, 5, and 10 μM). Viability was determined at 24 and 48 hour time points, and an IC50 value was then determined from the resulting graph. At 24 hours the IC 50 value was determined to be 5 µM, and at 48 hours was 0.5 μM (Figure 8a). Furthermore, validation of inhibition of JAK2 activity was determined via western blot analysis. K562 cells were cultured in the same doses of TG101209 for a period of 4 hours, lysate was extracted, and protein expression analyzed. Results from this analysis showed at least a 25% reduction in the level of phosphorylation of JAK2 (Y1007/1008) was observed at a concentration of 0.1 μM or higher of the inhibitor, while total JAK2 protein levels remained the same (Figure 8b). Interestingly, a 50% reduction in the amount of phosphorylated STAT5 (Y694) was also observed at a dose of 5M, while total STAT5 protein expression remained unaffected (Figure 8b).  34  Figure 8: IC 50 Value Determination for TG101209 in K562 Cells. a) Determination of an IC 50 value in the K562 cells was performed using a cell viability assay in which cells were plated in 0, 0.001, 0.01, 0.1, 1, 5, or 10 μM of TG101209 (n=4). Viability was determined at both 24 and 48 hours and IC 50 values were determined to be 5 and 0.5 µM at these time points respectively. b) Western blot analysis confirming inhibition of JAK2 activity, indicate a reduction in the levels of phosphorylated JAK2 (Y1007/1008) and phosphorylated STAT5 (Y694), while total levels of these proteins remain unaffected (n=2).  a)  to e v it a le r la v iv r u s t n e c re P  24 hours K562 9 0 2 1 0 1 G T o n  100 80 60 40 20 0  Concentration of TG101209 (uM)  48 hours K562 100 80 60 40 20 0  Concentration of TG101209 (uM)  35  b)  STAT5 Expression in TG101209  n o i s s e r p x e n i e t o r P  9 0 1.4 2 1 1.2 0 1 G 1 T 0.8 o 0.6 n o t 0.4 e 0.2 iv t 0 la e r  n io s s e r p x e n i e t o r P  9 0 2 1 1.2 0 1 1 G 0.8 T 0.6 o 0.4 n 0.2 o t 0 e v it a l e r  pSTAT 5 (Y694) Expression in TG101209 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0  JAK2 Expression in TG101209  pJAK2 (Y1007/1008) Expression in TG101209 1.2 1 0.8 0.6 0.4 0.2 0  Concentration TG BL-1 uM  Concentration TG101209 (uM)  36  3.4 Response of AHI-1-Transduced Cells and IM Resistant Cells to TG101209 Treatment  Biological response of K562 cells to a JAK2 inhibitor TG101209 was assayed using viability, apoptosis, and CFC assays. In a viability assay, 2 x 105 cells were plated in 0.5, 5, or 10 μM of TG101209, viability was determined at 24 and 48 hour time points. In terms of total cell viability, the only significant differences observed were between the parental K562 and the AHI-1 lenti clone B5 cells at 48 hours at all doses tested 0.5, 5, and 10 µM of TG (P=0.03, 0.03, and 0.01 respectively) (Figure 9a). The IM resistant cells displayed a similar response to JAK2 inhibition as the parental cells. An analysis of the percentage of apoptotic cells was also determined at 24 and 48 hours times points, and results from this assay indicated that suppression of AHI-1 resulted in a significant increase in cellular sensitivity at 24 hours at a 5 µM dose of TG101209 as compared to parental K562 cells (P=0.02), while both the AHI-1 overexpressing and IM resistant cells appeared to be less sensitive to TG at a dose of 10µM (P=0.03 and 0.05 respectively) (Figure 9b). Finally, in a CFC assay in which colonies were counted at a 10 day time point, significant differences between the response of the AHI-1 overexpressing or suppressed cells and the parental cells indicated significant differences only at a 0.5 µM dose of the treatment with the AHI-1 lenti cells displaying an decrease in cellular sensitivity (P=0.02) and the AHI-1 suppressed cells displaying an increase in cellular sensitivity (P=0.04) (Figure 9c). Results from the CFC assay also suggested that there were no significant differences in how the parental K562 and IM resistant cells responded to treatment. Differences in the morphology of the colonies were also noted at these concentrations with the AHI-1 suppressed cells producing smaller colonies than the AHI-1 overexpressing cells (Figure 9d). Raw data is displayed in Table A3 and illustrates 37  that there are small differences in terms of viability, apoptosis, and colony forming ability across the cell lines even in the absence of treatment The fifth K562 cell line containing both suppression and overexpression of AHI-1 was also included and data is shown in Table A5.  38  Figure 9: Cellular Response of AHI-1-Transduced Cells and IM Resistant Cells to TG101209 a) Viability assays revealed a significant differences between AHI-1 lenti clone B5 and parental K562 cells at 48 hours at all doses (P=0.03, 0.03, and 0.01) (n=3). b) Annexin V PE staining confirmed significant differences between the AHI-1 overexpressing and parental cells at 24 hours at 10 µM (P=0.05) and the AHI-1 suppressed cells and parental cells at 24 hours at 5 µM (P=0.03) (n=3). c) The number of colonies formed after 10 days significantly differed between the AHI-1 lenti clone B5 cells and the parental cells at 0.5 µM (P=0.02) and between the SH4 bulk cells and parental cells at 0.5 µM (P=0.04) (n=2). d) Colony morphology also differed at these doses. * indicate significant difference as compared to parental K562 at P≤0.05.  Percent viable cells relative to no treatment  a) Cell Viability 48 hours TG101209  Cell Viability 24 hours TG101209 Parental SH4 bulk  120 100 80 60 40 20 0  AHI-1 lenti IM Resistant  80 60  40  * *  *  20 0  b) 60 50  Annexin V-PE 24 hours TG101209 Parental SH4 bulk AHI -1 lenti  40 30 20 10 0  IM Resistant  90  Annexin V-PE 48 hours TG101209  80 70 60 50 40 30 20 10 0  39  c)  CFC TG101209 120 100  Parental SH4 bulk  80 60  AHI-1 lenti IM Resistant  40 20 0  d)  0.5 µM TG101209  1 µM TG101209  Parental K562  K562 SH4 bulk  K562 AHI-1 lenti clone B5  K562 IM Resistant  40  3.5 Response of AHI-1-Transduced Cells and IM Resistant Cells to Combined IM and TG101209 Treatment The final set of biological assays involved using a combination of IM treatments with TG101209 to determine if a rescue in the response of the AHI-1 overexpressing cell line and IM resistant cell line that were previously resistant to relative low doses of IM treatments could be obtained. To do this, a low 1 M dose of IM was used for the parental K562, K562 SH4 bulk, K562 AHI-1 lenti clone B5, and IM resistant cell lines. Various doses of TG101209 were combined with IM including 0.1, 0.5, 1, 2.5, 3.5, and 5 M (Figure A2). Again, cellular response was determined using a viability assay, an apoptosis assay, and a colony forming cell assay. The viability assay began with 2 X 105 cells in 6 well plates, and total cell counts of viable cells was determined at a 24 hour time points (Figures 10a, A2). Both the parental K562 and the K562 SH4 bulk cell lines responded equally to or better to a combination of treatments at all doses as compared to either treatment alone with the most significant differences occurring when 1 µM of IM is combined with 5µM of TG101209 (Figure10a). However, although the AHI-1 lenti clone B5 and IM resistant cell lines also appear to respond better to combination treatment as compared to IM alone, significant improvements in response to combination was not significantly different than using TG10209 alone. An apoptosis analysis of the same four cell lines was assayed using the Annexin V-PE kit described in methods and materials. Cells were again cultured in 1 M of IM combined with either 0.1, 0.5, 1, 2,5, or 3.5 M of TG101209 (Figure A2). The most significant effects of combination therapy was observed at a dose of 3.5 M of TG101209 combined with 1 M of IM, but the combined effects were only more significant than either treatment alone for the SH4 bulk cells (Figure 10b). Again, although there appear to be some additive effects of combination  41  treatment as compared to single treatments for the other cell lines, these effects were not significantly different than using TG101209 alone for the remaining three cell lines. Finally, a colony forming cell assay was performed in which approximately 250 cells were plated in tissue culture dishes in duplicate using a combination of 1 M of IM and 0.1, 0.5, 1, or 2.5 M of TG101209 (Figure A2). Colonies were counted at 10 days, and only those consisting of 30 cells or more were considered a colony. At a dose of 0.5 µM of TG101209 and 1 µM of IM, using a combination of treatments was more effective at inhibiting colony formation then each treatment alone for all four cell lines (Figure 10c). Not only was colony number affected by AHI-1 expression levels, but the morphology of the colonies appeared quite different, with the K562 AHI-1 lenti clone B5 cells producing larger colonies than the K562 SH4 cells (Figure 10d). Raw data is displayed in Table A3 and illustrates that there are small differences in terms of viability, apoptosis, and colony forming ability across the cell lines even in the absence of treatment.  42  Figure 10: Cellular Response of AHI-1-Transduced Cells and IM Resistant Cells to Combination Therapy a) In a viability assay, combination therapy was significantly more effective than single treatment for parental and SH4 cells, and more effective for the AHI-1 and IM resistant cells although values are not significant (n=2). b) Combination therapy significantly caused an increase in the cellular response of the SH4 bulk cells by Annexin V-PE staining (n=2). c) Combination therapy was significantly more effective than either treatment alone for all cell lines by performing a CFC assay (n=2). d) Colonies formed from the different cell lines. a)  120  11 uM µM IM IM 100 80 60  55uM µMTG TG 11 uM µM IM IM ++ 5 uM TG 5 µMTG  40 20 0  43  b)  Annexin V PE Imatinib and TG101209 70  µMIM IM 11uM  60 50  3.5uM µMTG TG 3.5  40 30  3.5TG µM++IM 3.5 1 µMTG  20 10 0  c)  120 100  P=0.0009  40 20  µMIM IM 11uM  P=0.03  80 60  P=0.04  P=0.05  0.5uM µM 0.5 TG TG 11uM µMIM IM++ 0.5 0.5uM µM TG TG  0  44  d) 0.1 µM TG101209 + 1 uM IM  0.5 µM TG101209 + 1 uM IM  1 µM TG101209 + 1 uM IM  Parental K562  K562 SH4 bulk  K562 AHI-1 lenti clone B5  K562 IM Resistant  45  3.6 Differentially Expressed BCR-ABL Signalling Genes in AHI-1-Transduced CML Cells Due to previous observations in which AHI-1 expression modulated the expression level of some BCR-ABL signalling proteins, several other candidate genes were selected for Q-RTPCR study. Representative genes from highly active signalling cascades in CML including BCR-ABL, ERK (MAPK), SRC, AKT, JAK2, STAT5, NF-kB, and IKKα/β were selected. Parental K562, K562/SH4 bulk, K562/AHI-1 lenti clone B5, and K562 IM resistant cells were used. Expression levels of selected genes were carefully analyzed by including technical replicates in triplicate, and repetition of each PCR reaction three times. All gene expression data were normalized to GAPDH and expressed relative to the parental K562 cells. Of the selected genes, overexpression of AHI-1 is associated with a significant increase in the gene expression of ERK (P=0.001), AKT (P=0.04), JAK2 (P=0.05), STAT5 (P=0.03), NF-кB (P=0.02), and IKKα/β/γ (P=0.02), while suppression of AHI-1 only led a significant decrease in gene expression of STAT5 (P=0.03) and NF-кB (P=0.05) as compared to parental K562 cells (Figure 11 and Table A4). Interestingly, the same genes that were overexpressed in the AHI-1 lenti clone B5 cell line were also found to be overexpressed within the IM resistant cells also displaying significant patterns of overexpression of ERK (P=0.004), JAK2 (P=0.005), STAT5 (P=0.05), NF-кB (P=0.05), and IKKα/β/γ (P=0.003). BCR-ABL and SRC gene expression were not significantly affected by AHI-1 expression levels (Figure 14). P values are summarized in Table A4. The fifth K562 cell line containing both suppression and overexpression of AHI-1 was also included and data is shown in Figure A2.  46  Figure 11: Changes in BCR-ABL Signalling Gene Transcripts Q-RT-PCR analysis of the levels of BCR-ABL signalling genes relative to GAPDH in AHI-1 transduced K562 cells and IM resistant cells (n=3). Values shown are the mean ± SEM of triplicate measurements. *Indicates significant expression difference from parental K562 cells at P≤0.05  SH4 bulk = AHI-1 suppressed K562 cells AHI-1 lenti = AHI-1 overexpressing K562 cells IM Resistant = IM resistant K562 cells  3 2.5 2 1.5 1 0.5 0  BCR-ABL 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0  JAK2 12 10 8 6 4 2 0  P=0.005  * P=0.05  *  7 6 5 4 3 2 1 0  AKT P=0.04 *  STAT5 P=0.05 P=0.03 * * P=0.03  *  47  IKK a/b/y 8 7 6 5 4 3 2 1 0  NF kB  P=0.02 P=0.003  *  *  7 6 5 4 3 2 1 0  SRC 1.4 1.2 1 0.8 0.6 0.4 0.2 0  P=0.03 P=0.05  *  *  P=0.05  *  ERK 9 8 7 6 5 4 3 2 1 0  P=0.002 * P=0.004  *  48  3.7 Differentially Expressed BCR-ABL Signalling Proteins in AHI-1-Transduced CML Cells Typically a protein is considered the functional unit of the cell, thus in addition to studying which genes are affected by AHI-1 expression, it was important to determine if any of these changes could be detected at the protein level. The same set of BCR-ABL signalling proteins was assayed via western blotting; JAK2, STAT5, AKT, ERK, NF-Bp105/p50, NF-B p65, SRC, IKK, and IKK. In addition to total protein levels, it was of interest to analyze expression levels of several phosphorylated proteins, as this would lead to a deeper understanding of the mechanism of AHI-1 control over these signalling pathways. Again, the same four cell lines were studied; parental K562, K562 SH4 bulk, K562 AHI-1 lenti clone B5, and K562 IM resistant cells. Experiments were performed on two biological replicates except for IKKα/β and pNF-κB (S536) which have only been performed once. The first signalling cascade that was studied was the JAK2/STAT5 proteins. Surprisingly, results (Figure 12a) were not consistent with what had previously been observed in this pathway (Figure 4b), and is explained in further detail in the discussion section. Analysis of total JAK2 and total STAT5 revealed that no significant differences were observed across the four cell lines for JAK2, but significant differences were observed for total STAT5 when AHI-1 is overexpressed as compared to the parental cells (P=0.004) (Figure 12a). Additionally, levels of phosphorylated JAK2 (Y1007/1008), and phosphorylated STAT5 (Y694) were assayed, and no significant difference were observed between the AHI-1 overexpressing and AHI-1 suppressed cells for either phosphorylated protein Table A4 Members of the NF-B pathway including NF-Bp150/p50 and NF-Bp65 subunits were assayed, as well as phosphorylated levels of the p65 subunit (Figure 12b), and the upstream  49  proteins IKK and IKK were analyzed (Figure 12c). Total protein levels of NF-Bp105/p50 subunits were expressed 3-fold higher in the AHI-1 overexpressed cells as compared to the parental cells displaying significant increases in expression (P=0.02 and P=0.004). IKK was strongly affected by both AHI-1 suppression and overexpression with the AHI-1 overexpressing cells displaying a 2.5-fold increase in the expression as compared to parental cells and AHI-1 suppression causing a 6-fold decrease in expression, while IKK was only affected by AHI-1 suppression displaying a 3-fold decrease in expression relative to parental cells (Figure 12c). Total level of NF-B p65 was not significantly affected by AHI-1 expressing cells, however, expression levels of the phosphorylated form indicated by NFB (s536) was strongly affected by both overexpression and suppression of AHI-1 displaying a 2.5-fold increase and a 2-fold decrease when AHI-1 is overexpressed and suppressed respectively (Figure 12b). Upon analysis of AKT and ERK, protein expression levels revealed that neither of these proteins is significantly affected by AHI-1 expression. Analysis of phosphorylated AKT (S473) confirmed that this signalling cascade remains equally active in all cell lines studied (Figure 12d). Analysis of phosphorylated ERK revealed more interesting results (Figure 12e). ERK is expressed in two isoforms one is 38kD (Y202) and one is 43 kD (Y204). Significant effects were observed for the p38 isoform with the AHI-1 overexpressing cells displaying a 3.5-fold increase expression relative to the parental cells (P=0.02) and although changes were observed for the p43 subunit when AHI-1 was overexpressed but these changes were not significant. (Figure 12e). Finally, phosphorylated levels of the SRC protein, one which shares significant homology to the BCR-ABL tyrosine kinase, was analyzed. SRC also exists as two isoforms one is 69kD subunit and one is a 71kD. While it initially appeared that both subunits were strongly  50  affected by AHI-1 suppression and overexpression, these differences were not significant (Figure 12f). The fifth K562 cell line containing both suppression and overexpression of AHI-1 was also included and data is shown in Figure A2.  51  Figure 12: Western Blots of BCR-ABL Signalling Proteins a) Western blot analysis of protein expression and phosphorylation of JAK2/STAT5 in AHI-1 transduced K562 cells and IM resistant cells (n=2). b) NF-кB signalling (n=3 for 105/50kD, n=2 for 65kD, n=1 for p65 kD). c) IKKα/β signalling (n=1). d) AKT signalling (n=2). e) ERK signalling (n=2). f) SRC signalling (n=2). *Indicate significant difference as compared to parental at P≤0.05.  SH4 bulk = AHI-1 suppressed K562 cells AHI-1 lenti = AHI-1 overexpressing K562 cells IM Resistant = IM resistant K562 cells a) JAK2 4 3 2 1 0  1.5 1 0.5 0  2  STAT5  1.5 1 0.5 0  P=0.004  *  3 2.5 2 1.5 1 0.5 0  pJAK2 (Y1007/1008)  p STAT5 (Y694)  52  b) NF kB p 50  NF kB p 105 5 4 3 2 1 0  P=0.02  *  NF kB p 65 2.5 2 1.5 1 0.5 0  P=0.004  5 P=0.02 4 * 3 2 1 0  *  P=0.02  *  p NF kB (S536) 3 2.5 2 1.5 1 0.5 0  c)  Ikka  Ikkß 1.2 1 0.8 0.6 0.4 0.2 0  3 2.5 2 1.5 1 0.5 0  d) 1.2 1 0.8 0.6 0.4 0.2 0  AKT  p AKT (s473) 1.2 1 0.8 0.6 0.4 0.2 0  53  e) ERK 2 1.5 1 0.5 0  p 38 p 43  pERK (Y202/Y204) 5 4 3 2 1 0  P=0.02  *  f)  54  CHAPTER 4: DISCUSSION Results from the present study have led to a deeper understanding of the mechanisms of how the AHI-1 oncoprotein may be involved in mediating BCR-ABL signalling in CML. AHI-1 plays a vital role in the regulation of cellular response/resistance to tyrosine kinase inhibitor IM treatment in vitro as demonstrated by overexpression or suppression of AHI-1 in a CML cell line (K562) model system. While biological effects were observed both in liquid and semi solid culture, the most profound effects could be observed from the CFC assay. Results from IM treatments alone indicated a significantly greater response in terms of cellular viability when AHI-1 is suppressed in K562 cells as compared to parental cells at 1 and 5 µM treatment doses at 24 hours and at 1 µM of treatment at 48 hours. However, when AHI-1 is overexpressed there are only significant differences from the parental cells at 48 hours and only at a dose of 5 µM. Similarly, the only significant differences observed between the parental cells and the IM resistant cells occurred at 48 hours but not at 24 hours. This suggests that suppression of AHI-1 strongly affects cellular sensitivity to IM treatments and are likely highly dependent on BCRABL signalling for survival. The fact that the only differences between the parental cells and the AHI-1 overexpressing and IM resistant cells occurs at 48 hours may suggest that these cells may be utilizing compensatory mechanisms allowing for the cells to survive even after 48 hours significantly better than the parental cells. In a recent study using LAMA cells, results indicated that an IM resistant cell line was producing GM-CSF when cells were cultured in the presence of IM or nilotinib, which led to the activation of JAK2/STAT5 signalling, independently of BCRABL, allowing the cells to survive IM treatments56. Perhaps in this case, the parental cells may be capable of independent activation of this pathway as well, but only for a limited amount of time, while the AHI-1 lenti and IM resistant cells are able to sustain these signalling pathways for 55  a longer time period. Or perhaps, another signalling pathway such as NF-κB which is highly active in these two cell lines is compensating for the diminished BCR-ABL activity. An apoptosis assay supported these results, significant differences between the AHI-1 suppressed cells and the parental cells was observed at 48 hours at doses of 2.5 and 5 µM, and between the AHI-1 overexpressing cells and parental cells at 24 hours at all doses tested. Combined with the previous results suggests that AHI-1 overexpression leads to an immediate protective mechanism that prevents apoptosis in the cells at 24 hours, but because apoptosis is comparable to the parental cells at 48 hours as opposed to what is observed in the viability assay, differences observed at 48 hours are likely due to a cellular proliferative advantage, possibly due to the overactivation of the ERK signalling cascade observed within these cells, which is pathway well known for its effects on cellular proliferation. Results from the CFC assay revealed that the only significant difference obtained between the AHI-1 suppressed cells and overexpressed cells as compared to the parental cells was observed at a low dose of 1 μM of IM treatment, again indicating the toxicity of higher doses of this drug. Future studies involving treatment with intermediate doses of IM would be of interest to determine if there is a critical dose which can inhibit the AHI-1 overexpressing cells without conferring toxicity to normal cells. Potential doses of 2.5 μM and 3.5 μM for both the viability and CFC assays would be the next logical step. Interestingly, the behaviour pattern of the AHI-1 overexpressing cells often mirrored the pattern of cellular viability and colony formation observed within the K562 cells that are highly resistant to IM. The IM resistant K562 cell lines that were used do not contain any of the mutations often found within the kinase domain of BCR-ABL, suggesting a mechanism of resistance which may be caused by AHI-1 overexpression. Validation of AHI-1 overexpression both by Q-RT-PCR and western blotting  56  supports this theory showing that AHI-1 is in fact overexpressed in these IM resistant cells both at the transcript and protein level.  As mentioned previously, one potential mechanism of  resistance may be overactivation of several altered BCR-ABL signalling cascades in particular the JAK2/STAT5 cascade that was reported in previous studies52. A hypothesis in which AHI-1 binds to both BCR-ABL and JAK2 and may lead to an increase in cellular resistance due to overactivation of downstream factors, including STAT5 and activation of an autocrine loop involving interleukin-3 produced in primitive CML cells and other signalling pathways activated by BCR-ABL. As a transcription factor, STAT5 is known to be involved in regulating several genes involved in cellular differentiation, proliferation, and cell cycle regulation, therefore inhibition of JAK2 with TG101209 to reduce STAT5 signalling was the next logical step in elucidating resistance mechanisms in this AHI-1 overexpressing cell line. Treatment with TG101209 in a viability assay revealed that all cell lines responded to an increasing dose of the inhibitor, but the only significant difference between cell lines occurred when comparing the AHI-1 overexpressing cells with the parental cells at 48 hours at all doses tested. Inhibitory effects on the AHI-1 suppressed cells using TG101209 were not as strong as IM treatments suggesting that other BCR-ABL dependent or independent signalling cascades in these cells are likely more prevalent than JAK2 signalling. The fact that at 48 hours the AHI-1 overexpressing cells are significantly less sensitive than the parental cells suggests that there are other pathways that compensating for the lack of JAK2 signalling in these cells that may not be as prevalent in the parental cells. Again, the ERK pathway that is highly active in these cells may be a potential cellular proliferation pathway that is leading to an increase in proliferation. An apoptosis assay further suggests that the differences in terms of cellular viability is more a proliferative effect and less of a cell apoptotic effect as there are few differences between  57  the parental cells and the AHI-1 suppressed and overexpressed cells by TG101209 treatment. The only significant differences occurred at 24 hours when AHI-1 is suppressed at a dose of 5 µM, or when AHI-1 is overexpressed at a dose of 10 µM. This also supports the theory that activation of cellular proliferation cascades as opposed to anti apoptotic cascades are likely a factor affected by AHI-1 expression. The majority of toxic effects that could be observed occurred once again in semi solid culture when a small number of cells are plated in the CFC assay, in which a dose as low as 1 μM was apparently toxic to even the AHI-1 overexpressing cells. The most significant differences of the biological effects observed between the AHI-1 overexpressing and suppressed cells as compared to the parental cells were observed at 0.5 µM of treatment. These differences between the colony forming cell assay as opposed to either liquid culture assay suggesting that this is a more sensitive assay to detect the effects of AHI-1. One possibility for the stronger effects observed both with IM and TG101209 treatment in semi solid culture compared to liquid culture is that notable differences, including length of time and number of cells, varies dramatically between these two approaches. CFC assays are typically performed over a period of 10-14 days allowing for prolonged effects of the drug, and only about 200 cells per culture dish as compared to 2 x 105 used in the viability and annexin V assays. Because colonies form from single cells, there is presumably no paracrine signalling required for the initiation of colony formation as opposed to liquid culture in which there is both paracrine and autocrine signalling. Results from this study are consistent with previous studies in the lab involving studies of IM sensitivity in K562 cells, suggesting that use of a CFC assay is a much more sensitive and accurate approach in detecting cellular response to IM than either a viability or apoptosis assay. In order to develop a complete biological understanding of how AHI-1 affects the cells, and whether it exerts its effects more on the differentiated population or more  58  primitive population, further investigation using the same techniques involving primary samples obtained from IM responders and nonresponders with CML is necessary. Primary samples obtained from CML patients would need to be sorted into the CD34+ and CD34- compartments prior to performing the viability and annexin V staining. If, in fact, that treatment with IM causes independent activation of JAK2/STAT5 signalling, then inhibition of both BCR-ABL and JAK2 activities would be the next logical step in determining if a restoration of the response of the AHI-1 overexpressing and IM resistant cells is possible. Studies again involving cell viability, apoptosis, and colony forming ability revealed that at specific doses, a combination of the two inhibitors was significantly better at restoring the response of the IM resistant cells and AHI-1 overexpressing cells to either inhibitor alone. When a dose of 5 μM of TG101209 is combined with 1 μM of IM, both the AHI-1 overexpressing cells and IM resistant cells respond better than either did at 1 μM of IM alone in a viability assay, and although results were not significant when comparing 5 µM of TG101209 alone with a combination treatment for these two cell lines, that might be explained by the fact that this has only been replicated two times, additional experiments may prove significant differences. Analysis of apoptotic cells confirmed that although combination therapy was more effective than single treatments for the AHI-1 overexpressing and IM resistant cells, the differences were not significant, probably due to the low number of replication experiments, or the fact that a specific critical dose is required to observe these effects. Results from the CFC assay were more convincing, when a dose of 0.5 μM of TG101209 combined with 1 μM of IM showed a significantly increased response of all cell lines tested, as compared to either treatment alone. These results suggest that combined inhibition of JAK2 and BCR-ABL is effective at restoring some level of cellular response to treatment when AHI-1 is overexpressed, but additional  59  replicates and further optimization of additional doses of treatment are needed. Further studies involving co-immunoprecipitation of JAK2, AHI-1, and BCR-ABL will be necessary to determine if the interaction between AHI-1, JAK2, and BCR-ABL is disrupted upon treatment with either inhibitor alone or with a combination of inhibitors. These results do however, suggests the potential of JAK2 inhibition in treating patients with CML who have developed resistance to current treatments involving only BCR-ABL inhibition. It is interesting to note that at all doses of combination treatment, the AHI-1 overexpressing cells continued to display a significantly higher level of resistance than the AHI-1 suppressed cells in all three assays. This suggests that although inhibition of these highly resistant cells can partially lead to restoration of a normal response, there is likely another mechanism of resistance in this cell line, possibly overactivation of other oncogenic BCR-ABL signalling cascades or activation of some of the major drug effluxors (ABCB1 and ABCG2) or down regulation of the major drug influxor (OCT1). Studies involving treatment of the four cell lines with a combination of the two inhibitors and analyzing protein expression levels of some of these other signalling cascades or proteins of interest may lead to a better understanding of the resistance mechanisms that develop when AHI-1 is overexpressed. Results from Q-RT-PCR studies indicate that AHI-1 may play a role in manipulation of gene transcription, but predominately when AHI-1 is overexpressed rather than suppressed. It may be that under normal circumstances AHI-1 has functions that include regulation of gene expression, and when overexpressed these normal functions are perturbed resulting in increased gene expression. Under normal circumstances AHI-1 expression may be regulated within the cells such that only a small amount of AHI-1 is actually involved in gene transcription, and suppression of AHI-1 may leave enough remaining AHI-1 to fulfill this normal function. AHI-1  60  contains several signalling domains, and while there is no known DNA binding domain, there is the potential that AHI-1 may directly interact with transcription factors and could act as a transcription co-factor. Another possibility is that through overactivation of signalling cascades involving known potent transcription factors such as NF-κB and STAT5, indirect effects are observed in terms of activation of several other genes. These genes may undergo strict transcriptional regulation within the parental K562 cells, thus suppression of AHI-1 would cause very little effect because this regulation would be maintained, but overexpression may seriously perturb these regulatory mechanisms. The fact that all genes that are overactive in AHI-1 overexpressing cells are also overactive in the IM resistant cells is not surprising considering that fact that AHI-1 is also significantly overexpressed within these cells at endogenous level. These findings support the hypothesis that AHI-1 may play a role in resistance mechanisms to IM through overactivation of several other genes.  Notably, although significant changes are  observed at the level of the transcript for several genes, some of these changes are not necessarily translated to the protein level, as in the case of AKT and ERK. This is often observed in cells whereby transcription and translation do not necessarily correlate. Several mechanisms of translational control may occur to prevent changes in transcription from affecting cellular function, for example degradation of the transcript via micro RNA57-59. Additionally, once a protein has been formed, cells are capable of quickly degrading that protein to prevent further perturbations in normal function. Also, in the case where protein expression is altered without a corresponding change in gene expression may indicate a mechanism in which normal degradation of that protein is insufficient, rather than alteration of gene expression. Results from protein analysis upon AHI-1 manipulation have revealed different possible mechanisms of its cellular function. Evaluation of the ERK pathway suggests that AHI-1 might  61  play a role in manipulation of activity of this pathways, but not necessarily transcription of this protein, because a difference in terms of phosphorylated ERK is observed while total ERK remains unaffected. Contrary to this, observation of changes within the NF-κB and previous studies indicating changes in the JAK2/STAT5 pathway52 seem to indicate that AHI-1 exerts its effects through transcription, translation, or degradation of the protein, resulting in changes of the total protein levels. In the present study changes within the JAK2/STAT5 pathway were not as obvious.  These differences may be explained by slightly different culture conditions.  Previous studies demonstrated strong effects on this pathway when the cells were cultured in the presence of human interleukin-3 (IL-3), while culture conditions in the present study were not performed in the presence of any growth factors. It is well known that JAK2 signalling and IL-3 signalling are related. Upon stimulation of the IL-3 receptor, JAK2 forms homodimers, which can phosphorylate each other, leading to increased STAT5 activity and creating a positive feedback loop which further causes the autocrine production of IL-3 and increasing activation of both the IL-3 and JAK2 pathways51. The fact that AHI-1 manipulation only affects JAK2 signalling significantly in the presence of IL-3 suggests its potential involvement in this signalling pathway. It may be that AHI-1 is downstream of the IL-3 signalling, and that an initial stimulation of the IL-3 pathway via external administration of this growth factor is necessary for AHI-1 to exert its effects on JAK2 and STAT5. To determine how AHI-1 is related to IL-3 pathway, studies involving Q-RT-PCR and western blotting of the IL-3 gene expression and protein expression when AHI-1 is overexpresssed or suppressed are needed. If AHI-1 is downstream of IL-3, then a difference in IL-3 expression may not be observed. A second study in which parental K562 cells are treated with IL-3, and AHI-1 expression is  62  determined via Q-RT-PCR and western blotting would reveal if AHI-1 is affected by IL-3 signalling. The effects observed within the NF-κB pathway have led to the most interesting results, while total levels of the p105/p50 subunits is strongly affected by AHI-1 overexpression, total levels of the p65 subunit appear unaffected. Analysis of activity of the p65 subunit however, as indicated by levels of phosphorylated NF-κB p65 (s536), suggest that AHI-1 does affect signalling by this protein. As mentioned previously NF-κB signalling is quite complex, and different mechanisms of control exist for the different family members. The p105/p50 subunit is strongly controlled by the IKK family members, in particular IKKα. When the NF-κB inhibitor IκB is phosphorylated by IKKα, it is released from the P50 subunit, allowing the subunit to translocate to the nucleus and control the expression of a wide variety of genes38. Since the total levels of IKKα were strongly affected by AHI-1 overexpression, it is not surprising that NF-B signalling is so strongly affected. In addition to total protein level, an analysis of the phosphorylated levels of the NF-κB p50 subunit and IKKα/β would be interesting. Another future study would involve examination of the total levels of the IκB protein as well as its phosphorylated protein. One might expect an increase in the levels of phosphorylated IκB upon AHI-1 overexpression, indicating a greater level of activity of both the p50 and p65 subunits. Because NF-κB appears to be the most strongly affected protein when AHI-1 is manipulated, future directions involving inhibition of this pathway would be of interest. It might be that in the cells displaying resistance to both BCR-ABL and JAK2 inhibition, NF-κB might be highly expressed. Western blotting for NF-κB of cells treated with IM alone, TG101209 alone, or a combination of these treatments may provide additional evidence that this pathway is involved in resistance mechanisms. It would be interesting to investigate how inhibition of  63  NF-κB alone would affect the AHI-1 overexpressing cells, and if combined inhibition of NF-κB with IM is able to rescue the resistant phenotype observed in these cells. Finally, because AHI-1 appears to regulate the transcription and protein level of several of these genes as opposed to simply altering activation of these pathways, it may be that AHI-1 acts as a co-transcription factor. Studies involving chromatin immunoprecipitation followed by microarray analysis (ChIP-chip analysis) would address this hypothesis. A potential caveat in interpreting the previous studies involves the mechanism of action within the infected K562 cell lines. In some rare cases, the addition of a viral vector into the cells may potentially lead to undesired effects caused by integration of the vector into an undesired location in the genome. When interpreting the results it is important to consider such events. Potentially the differences in terms of cellular viability, apoptosis, colony forming ability, and effects on gene/protein expression is not in fact due to AHI-1 expression, but due to other changes that occur within the cell as a result of viral integration or due to indirect effects altering expression of other genes/proteins. To control for this possibility, a fifth K562 cell line was included in the all experiments that contained both the retroviral SH4 RNA and lentiviral cDNA full length AHI-1 construct to ensure that all effects were in fact due to AHI-1 action. Results from these studies indicated that the biological and molecular characteristics of these cells typically displayed characteristics that were intermediate between the parental K562 cell line and the AHI-1 suppressed cells with AHI-1 protein expression being 65% of parental AHI-1 expression, suggesting that there is a partial rescue of the phenotype of the AHI-1 suppressed cells but not complete enough to restore activity to that observed within the parental cells. Raw data for viability, annexin V, CFC, RNA expression, and protein expression is displayed in  64  Figure A2. This suggests that the effects that were observed could be explained by AHI-1 expression, and not simply due to vector effects. A second potential caveat that must be considered when interpreting the results of the biological assays is that there are differences in terms of cellular proliferation, apoptosis, and colony forming ability across the four different K562 cell lines in the absence of treatment. However, the differences observed in the absence of treatment are small in comparison to the differences observed in the presence of treatment suggesting that these initial differences do not affect the differences in terms of cellular effects across the different cell lines in the presence of imatinib or TG101209.  65  CHAPTER 5: CONCLUSION In conclusion, AHI-1 was determined to play a role in K562 cellular resistance to both BCR-ABL inhibition by IM and JAK2 inhibition by TG101209. Overexpression or suppression of AHI-1 in K562 cells affected cellular viability, colony forming ability and apoptosis in response to either treatment alone or a combination on treatments suggesting its involvement in these processes. Interestingly, AHI-1 overexpressing cells showed reduced proliferation and colony formation when treated with IM and TG101209 in combination compared to either IM or TG101209 alone; suggesting that targeting both BCR-ABL and JAK2 activities may be a potential therapeutic option for IM poor responders. Alteration of AHI-1 expression affects transcript levels of several genes involved in BCRABL signalling pathways including JAK2, STAT5, ERK, AKT, NF-κB, and IKK. Changes in some of these genes were also observed at the protein level including ERK, NF-κB, IKK, and STAT5. These results suggest a mechanism by which AHI-1 is involved in regulation of gene transcription rather than simply altering the activity of these signalling cascades. Possible mechanisms of action may include indirect alteration of gene transcription through activation of nuclear transcription factors or by acting as a transcription co-factor. AHI-1 involvement in cellular resistance to IM can be elucidated from these studies by comparing the pattern of gene and protein expression between the AHI-1 overexpressing cells with the IM resistant cells. Similarities in terms of protein expression between these two cell lines suggests a mechanism in which AHI-1 is involved in resistance through overactivation of several BCR-ABL signalling cascades especially the JAK2/STAT5 and NF-κB pathways. The present study has led to a deeper understanding of the biological and molecular activities of the novel oncogene AHI-1, and how it is involved in leukemic cell transformation, 66  altered BCR-ABL signalling and drug response/resistance of CML cells. 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Mol Cell Biol 1999 Nov; 19(11): 7357-7368.  72  APPENDIX Table A1: List of Primer Sequences Gene AHI-1 (N Terminus) BCR/ABL JAK2 STAT5 NFB IKK SRC AKT ERK  Primer Sequence Forward 5’-GCC GAG ATA GCC GGG TTT ATC-3’ Reverse 5’-TCA GTT CGG TGA ATG TAA ACC CC-3’ Forward 5’-GAT CT ACT GGC CCC TGA AG-3’ Reverse 5’-CAT TCC CCT GAC CAT CAA TAA G-3’ Forward 5’-GGA ATA TCT CGA GGT GCT GAA-3’ Reverse 5’-TCC ATC CGT GCA CAA AAT C-3’ Forward 5’-CAG CAG CTG CAG GGA GAC-3’ Reverse 5’-TTG TCC AAG TCA ATG GCA TC-3’ Forward 5’-GGA TGC ATC TGG GGA TGA G-3’ Reverse 5’-TTA ACG TAG CAG AGG GGA CA-3’ Forward 5’-GTC AAG GAG CTG CAG GAG AT-3’ Reverse 5’-GAT GGC CAA GTG CAG GAA-3’ Forward 5’-GAG CCA GGA TTT GAA CCC AG-3’ Reverse 5’-GGA GTC AGG GGT CTFFC GAA AT-3’ Forward 5’-GGC CCA ACA CCT TCA TCA T-3’ Reverse 5’-GAT GGC GGT TGT CCA CTC-3’ Forward 5’-TGG TCA TGG CCA GAA AGC-3’ Reverse 5’GAG TTG CAC TCA TGC AGA ACC-3’  73  Table A2: Primary and Secondary Antibody Conditions Antibody AHI-1 (N terminus) Imgenex AHI-1 (N terminus) Jouberin AHI-1 (C terminus) Imgenex JAK2 STAT5a/b NF κB p105/p50 NF κB p65 NF κB p65 (s536) pJAK2 (Y1007/1008) pSTAT5a/b (Y694) AKT pAKT (s473) ERK pERK (Y202/Y204) pSRC family kinase IKKα IKKβ GAPDH  Blocking 5% Skim Milk in TBST 5% Skim Milk in TBST 5% Skim Milk in TBST 5% Skim Milk in TBST 5% Skim Milk in TBST 5% Skim Milk in TBST 5% Skim Milk in TBST 5% BSA Milk in TBST 5% Skim Milk in TBST 5% BSA Milk in TBST 5% Skim Milk in TBST 5% BSA Milk in TBST 5% Skim Milk in TBST 5% BSA Milk in TBST 5% BSA in TBST 5% Skim Milk in TBST 5% Skim Milk in TBST 5% Skim Milk in TBST  Primary Antibody 1:500 in TBST 3%BSA 1:1000 in TBST 5% BSA 1:500 in TBST 3% BSA 1:1000 in TBST 3%BSA 1:1000 in TBST 3%BSA 1:1000 in TBST 3%BSA 1:1000 in TBST 3%BSA 1:500 in TBST 3%BSA 1:500 in TBST 3%BSA 1:500 in TBST 3%BSA 1:1000 in TBST 3%BSA 1:500 in TBST 3%BSA 1:1000 in TBST 3%BSA 1:500 in TBST 3%BSA 1:500 in TBST 3%BSA 1:1000 in TBST 3%BSA 1:1000 in TBST 3%BSA 1:1000 in TBST 3%BSA  Secondary Antibody 1:2500 in TBST 3% BSA 1:5000 in TBST 3% BSA 1:5000 in TBST 3% BSA 1:10000 in TBST 3% BSA 1:10000 in TBST 3% BSA 1:10000 in TBST 3% BSA 1:10000 in TBST 3% BSA 1:5000 in TBST 3% BSA 1:5000 in TBST 3% BSA 1:10000 in TBST 3% BSA 1:10000 in TBST 3% BSA 1:5000 in TBST 3% BSA 1:10000 in TBST 3% BSA 1:5000 in TBST 3% BSA 1:5000 in TBST 3% BSA 1:10000 in TBST 3% BSA 1:10000 in TBST 3% BSA 1:10000 in TBST  74  Table A3: Raw data for untreated K562 cells, AHI-1-transduced cells and IM resistant cells for viability, apoptosis, and CFC assays a) Total viable cells, percent apoptotic cells, and colony numbers for each cell line without treatment. b) Colonies formed from each cell line. a) Cell Line  Average Average Viable Cells Viable Cells (x106) (x106) 24 hours 48 hours  K562  4.9  8.3  Average Percent Apoptotic Cells 24 hours 15.87  Average Percent Apoptotic Cells 48 hours 12.75  Average Colony Number 10 days  SH4 bulk  6.8  9.6  10.73  15.67  339  AHI-1 lenti Clone B5  6.0  8.8  16.29  13.87  264  IM Resistant  5.0  9.5  17.72  13.19  315  287  b)  TG101209 Experiment  IM Experiment  Parental K562  K562 SH4 bulk  K562 AHI-1 lenti clone B5  K562 IM Resistant  75  Table A4: Summary of P Values for Q-RT-PCR and Western Blots  Gene/Protein  AHI-1 BCR-ABL JAK2  STAT5  RNA p value P=parental S=SH4 bulk A=AHI-1 lenti clone B5 P-A 0.013493774 P-S 0.045271768 S-A 0.02418263 P-A 0.1036428 P-S 0.601784787 S-A 0.093607033 P-A 0.0457287 P-S 0.14954673 S-A 0.014966179  P-A P-S S-A  0.027377286 0.026706139 0.004964812  P-A P-S S-A  0.018860903 0.05231971 0.00813218  P-A P-S  0.044247831 0.1896542  NF κB  AKT  Protein p value P=parental S=SH4 bulk A=AHI-1 lenti clone B5 P-S 0.021523 P-A 0.005629 S-A 0.000749 Not Done  JAK2 P-S 0.12732 P-A 0.285381 S-A 0.096293 pJAK2 P-S 0.109808 P-A 0.208515 S-A 0.140997 STAT5 P-S 0.004377 P-A 0.148639 S-A 0.012021 pSTAT5 P-S 0.122692 P-A 0.391782 S-A 0.216792 NF κB (P105) P-S 0.019164 P-A 0.005677 S-A 0.003264 NF κB (p50) P-S 0.004343 P-A 0.024476 S-A 0.013691 NF κB (p65) P-S 0.589869 P-A 0.083559 S-A 0.030792 pNF- κB (p65) not replicated AKT P-S 0.135474 76  S-A  0.010793059  ERK  P-A P-S S-A  0.001393834 0.650388956 4.14044E-05  SRC  P-A P-S S-A  0.467215914 0.20654054 0.344348681  IKKα/β  P-A P-S S-A  0.023321047 0.386299823 0.016811783  P-A 0.425629 S-A 0.832207 pAKT P-S 0.200278 P-A 0.117788 S-A 0.195973 ERK 38 P-S 0.993458 P-A 0.936721 S-A 0.937555 ERK 43 P-S 0.10429 P-A 0.810163 S-A 0.8495 pERK p38 P-S 0.45184 P-A 0.02426 S-A 0.023048 pERK p43 P-S 0.158842 P-A 0.147633 S-A 0.103678 pSRC p69 P-S 0.067452 P-A 0.533555 S-A 0.423353 p SRC p71 P-S 0.105647 P-A 0.240822 S-A 0.171542 Not Replicated  77  Figure A1: FACS Plots for IM, TG101209, and Combination Treatments. a) IM at 24 hours.  3  10  2  103  10.1  10  100  10 4  28.4  10  56.4  5.14 101  102 FL2-H: Annexin  103  1.06  101  2.79 101  102 FL2-H: Annexin  103  55.4  10  3  10  2  8.53 101  102 FL2-H: Annexin  103  1  0.78  10  0  104  104  4.7  10  10 2  10 1  83  11.5 101  102 FL2-H: Annexin  103  No Treatment  104  10  2  10  4.34  89.2  5.5 101  102 FL2-H: Annexin  103  102 FL2-H: Annexin  103  8.67  10  2  10  4  10  3  10  2  0.65  10  10  53.5  0  10 4  102  10  3  10  2  104  7.99 101  102 FL2-H: Annexin  103  84.1  10.8 101  102 FL2-H: Annexin  1 µM  103  104  10 0  10  3  10  2  10  20.7 101  102 FL2-H: Annexin  103  7.08  104  29.7  AHI-1 lenti Clone B5 55.3  0  104  100  6.98  84.6  6.98 101  102 FL2-H: Annexin  103  10  4  10  3  10  2  0.92  10  104  7.89 101  102 FL2-H: Annexin  103  1.36  104  9.21  SH4 bulk  81  0  100  10 4  5.57  10 1  100  70.2 100  10 1  100  4.48  Parental K562  0  104  29.9  1.41  0  8.21  101  100  104  3  100  3  104  101  100  10  15 101  101  100  FL3-H: 7AAD  FL3-H: 7AAD  10  3  101  FL3-H: 7AAD  93.5  3  10 0  104  3  100 4  76.8 100  27  10  0.82  10 1  100  104  101  100  10  103  9.1  10 0  2.65  102  10 4  102 FL2-H: Annexin  10 2  104  FL3-H: 7AAD  FL3-H: 7AAD  100  100  5.35 101  10 1  10 0  3  88.5  0  104  10 1  10  101  FL3-H: 7AAD  10  102 FL2-H: Annexin  FL3-H: 7AAD  FL3-H: 7AAD  10 4  3.61 101  3  102  FL3-H: 7AAD  91 100  10  2  101  10 0  4  10  10  10 4  7.09  FL3-H: 7AAD  10 1  3  1.15  FL3-H: 7AAD  10 2  10  104  4.85  FL3-H: 7AAD  3  1.27  FL3-H: 7AAD  104  3.79  FL3-H: 7AAD  FL3-H: 7AAD  10  1.59  FL3-H: 7AAD  10 4  10  3  10  2  8.4 101  102 FL2-H: Annexin  103  1.05  104  5.76  K562 IM resistant  10 1  82.7 100  10.8 101  102 FL2-H: Annexin  103  2.5 µM  104  10 0  79.6 100  13.6 101  102 FL2-H: Annexin  103  104  5 µM  78  b) IM at 48 hours  101  3  10  2  102 FL2-H: Annexin  103  10.2  10  10 4  16.9  10  68.6  3  10  2  4.28 101  102 FL2-H: Annexin  103  0  0.18  10 4  0.98  96.7  2.16 101  102 FL2-H: Annexin  103  0.4  10 4  3  102  101  100  10  3  10  2  51.8  10 4  5.34 101  102 FL2-H: Annexin  103  95.4  2.88 101  102 FL2-H: Annexin  103  No Treatment  104  10 0  10  3  10  2  1.15  10 0  104  FL3-H: 7AAD  12.5 101  102 FL2-H: Annexin  103  15.5  10 4  10  3  10  2  20.3 101  102 FL2-H: Annexin  103  0.45  10  0  10 4  1.9  10  3  10  2  10  94.4  3.3 101  102 FL2-H: Annexin  1 µM  103  104  10 0  Parental K562  0  65 100  10 4  30.7  45.7  8.1 101  102 FL2-H: Annexin  103  9.35 101  102 FL2-H: Annexin  103  15.8  104  30.8  3  AHI-1 lenti Clone B5  10 2  1.88  10 0  104  49 100  10 4  19.5  10  3  10  2  4.46 101  102 FL2-H: Annexin  103  2.05  104  23.3  SH4 bulk  10 1  48.9  29.8 101  102 FL2-H: Annexin  103  0.71  10  0  104  43.6 100  10 4  2.06  10 1  100  2  24.3  10 1  100  104  10  104  10 1  67.1  3  10  100  11.5  10 1  100  75.4  10 1  100  1.33  0  10  1.39  10 1  100  28.5  10 2  10 0  2  10  3  104  10  104  10 1  FL3-H: 7AAD  FL3-H: 7AAD  10  14.4  100  10  100  104  103  10 2  10 0  101  10  102 FL2-H: Annexin  3  104  FL3-H: 7AAD  FL3-H: 7AAD  10  6.42 101  10 1  100  104  87.5 100  101  100  0  104  FL3-H: 7AAD  FL3-H: 7AAD  10  1.32 101  3  101  FL3-H: 7AAD  97.1 100  104  2  FL3-H: 7AAD  100  10  10  10 4  11.6  FL3-H: 7AAD  101  3  0.52  FL3-H: 7AAD  102  10  104  5.73  FL3-H: 7AAD  3  0.33  FL3-H: 7AAD  104  1.41  FL3-H: 7AAD  FL3-H: 7AAD  10  0.21  FL3-H: 7AAD  104  10  3  10  2  31.1 101  102 FL2-H: Annexin  103  1.02  104  1.85  K562 IM resistant  10 1  93.6 100  3.68 101  102 FL2-H: Annexin  2.5 µM  103  104  10 0  94.7 100  2.46 101  102 FL2-H: Annexin  103  104  5 µM  79  c) TG101209 24 hours  10  10 4  0  9.77 10  1  2  10 FL2-H: Annexin  10  3  0.7  10  10  1  10  0  4  10  2  1  0  85  10.6  10 0  10 4  10 1  10 2 FL2-H: Annexin  10 3  10  10  FL3-H: 7AAD  73  16.9 10 1  102 FL2-H: Annexin  10 3  0  2  10 FL2-H: Annexin  10  3  10  2  10 FL2-H: Annexin  10  3  10  1  83  9.56 101  102 FL2-H: Annexin  103  No Treatment  10 4  FL3-H: 7AAD  58.9 0  28.7 10  1  2  10 FL2-H: Annexin  10  3  10  10  68.4  22.4 10 1  10 2 FL2-H: Annexin  10 3  10  1  10  0  83.4 0  7.72 10  1  2  10 FL2-H: Annexin  1 µM  10  3  10  4  10  3  10  4  13.1  AHI-1 lenti Clone B5 51.1  10  10  10  37.8 10  0  51.5 10  1  2  10 FL2-H: Annexin  10  3  2.47  10  10 4  0  30.1 10  1  2  10 FL2-H: Annexin  10  3  10  4  10  2  10  1  10  0  SH4 bulk  2  1  29.1  0  10  9.27  20.7  3  10  0  46.8 10  1  2  10 FL2-H: Annexin  10  3  10  4  4  4.6  12.9  103  10 3  10  2  10 FL2-H: Annexin  3.39  1  10 4  0  1  5.8  8.81  2  6.2  2  10  10 2  10  100  3  36  0  3  4  3  10 4  36.4  10  104 1.83  1  2.66  10  4  1  10  10  1  7.13  2  Parental K562  10 0  10  10  8.04  101  10 0  3  3  10 4  100  2  10 0  10  10  10  6.95  2  10  104  1.21  100  10  2  10 FL2-H: Annexin  3  4  104  0.49  1  5.28  11.7 1  47.9 10  104  10  10  44 0  25.8  10 2  10  10  6.71  79.1  1  4  10  0  2  10 0  10  3  10 4  3  10 0  10  10  FL3-H: 7AAD  FL3-H: 7AAD  10  1  10  10 0  10  10  8.7  1  10 4  1  2.45  10  3  0  2  104 1.41  10 2  10  10  10 4  FL3-H: 7AAD  10  10.9 10  10 3 FL3-H: 7AAD  FL3-H: 7AAD  10  85.1 0  10 4  10 3  10  10  10  3.68  10  10  3  FL3-H: 7AAD  85.6  0  10 2  1.84  7.31  FL3-H: 7AAD  1  10  FL3-H: 7AAD  10  2  0.74  3  10  10 4  10 4  3.64  FL3-H: 7AAD  10  0.36  FL3-H: 7AAD  10  3  FL3-H: 7AAD  FL3-H: 7AAD  10  4  10  4.06  FL3-H: 7AAD  0.57  FL3-H: 7AAD  10 4  10  2  K562 IM resistant  101  62.4 10  0  25.9 10  1  2  10 FL2-H: Annexin  2.5 µM  10  3  10  100 4  62.3 100  20.2 10 1  102 103 FL2-H: Annexin  104  5 µM  80  d) TG101209 48 hours 10 4  10 1  10  2  10 3  0.21  FL3-H: 7AAD 100  10 4  10 10 1  10 2 FL2-H: Annexin  10 3  0.53  10  104  1  81.3 0  12 10  1  2  10 FL2-H: Annexin  10  3  0.59  FL3-H: 7AAD  11.8 10 1  10 2 FL2-H: Annexin  10 3  102  0.65  10  2.7  80 10  0  13.8 10  1  2  10 FL2-H: Annexin  10  3  62.8  4.72 101  102 FL2-H: Annexin  10 3  1.76  10  100  10  87.6 100  9.09 101  102 FL2-H: Annexin  10 3  No Treatment  104  10 4  28.2  10  2  10  1  10  0  10 4  25.5  AHI-1 lenti Clone B5 30.7 10 0  37.9 10 1  10 2 FL2-H: Annexin  10 3  1.52  10 4  14.1  SH4 bulk  10 2  10 1  27.7  0  0  45.1 10  1  2  10 FL2-H: Annexin  10  3  10  18.3  10 0 4  100  66.1 101  10 2 10 3 FL2-H: Annexin  10 4  3.39  FL3-H: 7AAD  10  83.9 0  11.1 10  1  2  10 10 FL2-H: Annexin  1 µM  3  10  10 4  10  3  2  10  10  1  51.2  0  10  0  27.5  13.3  FL3-H: 7AAD  10  10  10  3.17  104  102  1  100  10 3  4  10 4  4.44  102  1  10 2 FL2-H: Annexin  10 4 0.61  103  2  79.3 10 1  10 3  10  104  FL3-H: 7AAD  FL3-H: 7AAD  10  3  8.65  10 3  1.43  10  1  17.4  4  4  10  10 0  1  10  10  11.3  Parental K562  10 0  101  0  2  10 4  103  3  10 4  10 3  2  10  0.75  10  10  10 0  101  84.6  10 2 FL2-H: Annexin  3  4  5.59  53.4 10 1  15.1  100  10  104  10 1  10 0  10  10  0  17.9 10 0  6.34  4  10 3  10 0  2  10  10  25.9  1  10 4  10  10  3.03  10 2  103  3  10 4  10 3  10 0  10  10  86.8  10 2 FL2-H: Annexin  0.35  10 1  10 0  8.22 10 1  104  2.98  10  10 0  89.4  10 0  10 4  FL3-H: 7AAD  10  3  10 2 FL2-H: Annexin  FL3-H: 7AAD  FL3-H: 7AAD  10 4  6.07 10 1  2.71  10 2  10  FL3-H: 7AAD  91.5 10 0  FL3-H: 7AAD  10 2  10 1  10 0  4  10 3  10 3  10 2  104  10  2.32  FL3-H: 7AAD  3  0.08  FL3-H: 7AAD  2.2  FL3-H: 7AAD  FL3-H: 7AAD  10  0.27  FL3-H: 7AAD  10 4  34.1 10  1  2  10 FL2-H: Annexin  2.5 µM  10  3  10  10 4  3  K562 IM resistant  2  1  0  10  26.2 0  43 10  1  2  10 FL2-H: Annexin  10  3  10  4  5 µM  81  e) Combination Treatment TG101209 (Concentration Varies as Indicated) and IM (1µM) For All Treatments at 24 Hours  2  10 1  10 0  3  10  2  10 1  91.5 100  2.61 101  102 FL2-H: Annexin  103  87.9  10 0  104  6.39  100  3  10  2  11.8  10 1  10 0  101  46.5  37.5 101  102 FL2-H: Annexin  103  10  3  10  2  2  10 4  4.83  10  2  83.4  8.68 101  102 FL2-H: Annexin  103  10  3  10  2  10 0  104  102 FL2-H: Annexin  103  78.2 100  16.7 101  102 FL2-H: Annexin  103  10 0  104  78.2 100  16.7 101  102 FL2-H: Annexin  103  104  1 µM TG  104  3.15  2.48  10 4  10  3  10  2  10  3  10  2  4.99  73.6  14 101  102 FL2-H: Annexin  103  104  10 0  10  3  10  2  4.96  16  10 1  61.6 100  20.7 101  102 FL2-H: Annexin  103  0.5 µM TG  1.22  10 4  12.8  10 1  100  12.8  10 4  9.3  0.1 µM TG  10 1  10 0  10 1  0.5 µM TG  10 1  FL3-H: 7AAD  FL3-H: 7AAD  3  2  35.6 101  No Treatment  10  10  3.5 µM TG  3.09  100  10 4  10 0  104  3  4.29  18.3  44.5 100  10 1  10 0  103  FL3-H: 7AAD  10  2  10  0.86  Parental K562  10 0  104  FL3-H: 7AAD  FL3-H: 7AAD  3  102 FL2-H: Annexin  1.59  2.5 µM TG  10  10  10 4  4.29  10 1  100  10 4  3  0.1 µM TG 10 4  FL3-H: 7AAD  FL3-H: 7AAD  10  4.28  10  0.86  10 1  No Treatment 10 4  10 4  4.42  FL3-H: 7AAD  10  10  1.28  FL3-H: 7AAD  3  10 4  4.58  FL3-H: 7AAD  10  1.33  FL3-H: 7AAD  FL3-H: 7AAD  10 4  104  10 0  56.3 100  22.7 101  102 FL2-H: Annexin  103  104  1 µM TG  12  SH4 Bulk 10 1  53.2 100  31.6 101  102 FL2-H: Annexin  2.5 µM TG  103  104  10 0  50.8 100  35.9 101  102 FL2-H: Annexin  103  104  3.5 µM TG  82  2  10 1  10 0  3  10  2  10 1  72.2 100  9.96 101  102 FL2-H: Annexin  103  73.8  10 0  104  100  3  10  2  10 4  6.59  10 1  10 0  53.7  38.1 101  102 FL2-H: Annexin  103  10  3  10  2  2  0.93  10 4  3.47  10  3  10  2  81.7  13.9 101  102 FL2-H: Annexin  103  10  2  10 1  61.2 100  25.1 101  102 FL2-H: Annexin  103  10 0  104  61.1 100  23.2 101  102 FL2-H: Annexin  103  104  1 µM TG  0.5 µM TG  AHI-1 lenti Clone B5  102 FL2-H: Annexin  103  104  10 4  9.79  2.78  69.6 100  10 4  13.6  10  3  10  2  7.61  14.9 101  102 FL2-H: Annexin  103  10 0  104  10  3  10  2  1.76  10 4  9.01  10 1  0.1 µM TG  10 1  10 0  2  43.9 101  5.74  10 0  104  FL3-H: 7AAD  FL3-H: 7AAD  3  10  10.1  44.3 100  No Treatment  10  10 0  104  10 1  100  10 4  2  3  11.4  3.5 µM TG  10 1  10 0  103  FL3-H: 7AAD  10  102 FL2-H: Annexin  1.82  10 0  104  FL3-H: 7AAD  FL3-H: 7AAD  3  14.1 101  2.5 µM TG  10  10  10  4.23  10 1  100  10 4  3  10 4  10.8  0.1 µM TG  FL3-H: 7AAD  FL3-H: 7AAD  10  1.62  10  2.99  10 1  No Treatment  10 4  10 4  5.91  FL3-H: 7AAD  10  10  6.17  FL3-H: 7AAD  3  10 4  3.86  FL3-H: 7AAD  10  14  FL3-H: 7AAD  FL3-H: 7AAD  10 4  10  3  10  2  1.92  11.5  10 1  67.9 100  15.5 101  102 FL2-H: Annexin  103  0.5 µM TG  104  10 0  58.1 100  28.5 101  102 FL2-H: Annexin  103  104  1 µM TG  10.2  K562 IM resistant  10 1  53.8 100  29.8 101  102 FL2-H: Annexin  103  2.5 µM TG  104  10 0  56.4 100  31.7 101  102 FL2-H: Annexin  103  104  3.5 µM TG 83  Figure A2: Additional Doses Tested for Combination Treatment a) Viability assay b) Annexin V-PE staining and c) CFC assay in AHI-1-transduced and IM resistant cells.  a)  b)  84  c)  85  Table A5: Comparison of the SH4 cells + AHI-1 Double Infected Cell Line with Parental Cells (given as percent compared to parental cells). a) Viability and annexin V-PE staining at 24 hours. b) Viability and annexin V-PE staining at 48 hours. c) CFC assay. d) Gene transcript and protein expression. a) Assay (treatment) 24 hours Viability (imatinib) Viability (TG101209) Annexin V-PE (imatinib)  0.5 µM  1 µM  2.5 µM  5µM  10 µM  N/A  70%  N/A  144%  49%  120%  N/A  N/A  70%  79%  N/A  66%  72%  50%  N/A  Annexin V-PE (TG101209)  98%  N/A  N/A  66%  72%  Assay (treatment) 48 hours Viability (imatinib) Viability (TG101209) Annexin V-PE (imatinib)  0.5 µM  1 µM  2.5 µM  5µM  10 µM  N/A  56%  N/A  100%  50%  71%  N/A  N/A  88%  94%  N/A  50%  66%  55%  N/A  Annexin V-PE (TG101209)  72%  N/A  N/A  72%  66%  b)  86  c)  Assay (treatment) 10 days CFC (imatinib) CFC (TG101209)  0.5 µM  1 µM  2.5 µM  5µM  N/A  50%  N/A  90%  68%  No colonies  No colonies  N/A  d) Gene  Transcript  Protein  AHI-1  67%  65%  BCR-ABL  144%  N/A  JAK2  107%  77%  STAT5  36%  80%  IKK  189%  48%(IKKα)/64%(IKKβ)  NF-κB  32%  40%(105kD)/105%(50kD) 125%(65kD)/72%(65kDser536)  ERK  77%  79%(38kD)/66%(43kD)  AKT  146%  107%  SRC  91%  183%(69kD)/232%(71kD)  87  

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