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Experimental Medicine Program Student Research Day 2014-06-25

Targeting Treatment Naïve CML Stem/Progenitor Cells from Imatinib-Nonresponders by Combination Treatment with JAK2 (BMS-911543) and ABL Inhibitors. Leon ( Hanyang Lin) , Min Chen, Katharina Rothe, Matthew V. Lorenzi, Adrian Woolfson and Xiaoyan Jiang

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Experimental Medicine Program Student Research Day 2014-06-25

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  1. Targeting Treatment Naïve CML Stem/Progenitor Cells from Imatinib-Nonresponders by Combination Treatment with JAK2 (BMS-911543) and ABL Inhibitors Leon (Hanyang Lin), Min Chen, Katharina Rothe, Matthew V. Lorenzi, Adrian Woolfson and Xiaoyan Jiang Terry Fox Laboratory, BC Cancer Agency Department of Medicine, UBC Vancouver, Canada Discovery Medicine Oncology, Bristol-Myers Squibb, Princeton, United States Experimental Medicine Program Student Research Day 2014-06-25

  2. Chronic Myeloid Leukemia (CML) Chronic Phase (CP) Accelerated Phase (AP) Blast Phase (BP) 3-5 years 0.1-1 years Adapted from Ren R, Nature Reviews, 2005

  3. Chronic Myeloid Leukemia (CML) p210BCR-ABL BCR ABL Y177 Y1294 Tyrosine Kinase DNA BD Actin BD SH2 NLS SH3 S/T kinase Rho-GEF PI3K JAK2 Ras AKT STAT5 MAPK Increased proliferation Decreased apoptosis Massive accumulation of myeloid cells in circulation (Major characteristic of CML) Sattler et al, Cytokine Growth Factor Rev 8:63, 1997

  4. Challenges in CML Treatment: CML Stem Cells Are Insensitive to TKIs IM IM IM • Major problems associated with TKI therapy: • - Early relapse, acquired drug resistance • Ineffectiveness in eliminating CML stem cells Current 1st line: Imatinib Alternatives:Dasatinib, Nilotinib Druker BJ Blood , 2008

  5. AHI-1-BCR-ABL-JAK2 Complex Regulates TKI Response/resistance of CD34+ CML Cells • Current Challenges: • TKI Resistance of CML Stem Cells BCR-ABL-dependent mechanisms: Increased BCR-ABL expression BCR-ABL mutations Increased drug efflux and reduced drug influx Genomic instability Ctrl AHI-1sh4 Imatinib 5 µM 80 60 % inhibition relative to untreated cells BCR-ABL-independent mechanisms: Not addicted to BCR-ABL for survival Protected by BM microenvironment Activation of pro-survival pathways: Autophagy, Wnt//β-catenin, etc 40 20 0 Non-responders Responders Blast crisis Chomel et al, Oncotarget, 2011 Jiang et al, J Natl Cancer Inst, 2007; Zhou et al, J Exp Med, 2008 Corbin et al, JCI, 2011; Hamilton et al, Blood 2012

  6. Hypothesis and Objectives Relapse Stem cells are insensitive to TKIs TKIs TKIs JAK2 inhibitor (BMS-911543) JAK2 BCR-ABL AHI-1 Combined suppression of BCR-ABL and JAK2 activities to destabilize this protein complex may be more effective at eliminating CML stem cells

  7. Combination Treatment with ABL and JAK2 Inhibitors Is More Effective at Reducing pSTAT5 Levels in CML Cells 1 2 3 4 5 6 7 8 K562 P < 0.05 pSTAT5 STAT5 GAPDH 2 hours after drug treatment Relative pSTAT5 levels 1 K562 DMSO Ctrl 2 K562 1.0 µM BMS 3 K562 0.05 µm IM 4 K562 IM + BMS 5 K562R DMSO Ctrl 6 K562R 1.0 µM BMS 7 K562R 0.05 µm IM 8 K562R IM + BMS K562R (IM-resistant cells) P < 0.05 P < 0.05 n=3

  8. Combination Treatment Results in a Significant Reduction in Colony Formation of CML Cells Total CFC P < 0.05 P < 0.05 CML cell lines (600 cells) No. of CFC per 200 input cells Methylcellulose ± inhibitors No drug No drug No drug No drug BMS-911543 BMS-911543 BMS-911543 BMS-911543 BMS-911543 + IM BMS-911543 + IM BMS-911543 + IM BMS-911543 + IM IM IM IM IM 12 days 37ºc P < 0.05 P < 0.05 200 cells 200 cells No. of CFC per 200 input cells Small colonies: <50 cells Medium colonies: 50-300 cells Large colonies: >300 cells

  9. Combination Treatment Is More Effective at Reducing pSTAT5 and pCRKL Levels in • CD34+ CML Cells P<0.05 P<0.05 P<0.05 P<0.01 P-STAT5 relative to untreated cells (%) P<0.05 P<0.01 P<0.05 No drug P-CRKL relative to untreated cells (%) DA BMS DA + BMS IgG Ctrl 48 hrs treatment 72 hrs treatment n=4

  10. Combination Treatment Results in a Significant Increase in Apoptosis of CD34+ CML Cells P<0.05 48 hrs Ctrl BMS BMS + DA BMS + DA BMS BMS BMS + IM BMS + IM IM IM DA DA Apoptotic cells above untreated (%) BMS + NL BMS + NL NL NL P<0.05 PI P<0.05 DA BMS+DA P<0.05 Annexin V 72 hrs n=3

  11. Combination Treatment Is More Effective at Reducing Colony Formation in Treatment-naïve CD34+ CML Cells from Subsequent IM-nonresponders Total CFC CML blood samples P<0.001 Methylcellulose± inhibitors P<0.001 P<0.001 % CFCs relative to untreated cells Select CD34+ cells CD34+ cells 12 days CFU-GM BFU-E P<0.001 P<0.001 P<0.01 P<0.001 P<0.05 % CFCs relative to untreated cells % CFCs relative to untreated cells P<0.01 n=10

  12. Combination Treatment Is More Effective at Eliminating More Primitive CML Cells from Subsequent IM-nonresponders CML blood samples Select CD34+ cells Total LTC-IC derived CFCs relative to untreated cells (%) P<0.05 CD34+ cells P<0.05 On feeders for 6 wks  inhibitors Methylcellulose 12 days 37ºc n=3

  13. BMS-911543 Has Less Toxicity on CD34+ Normal Bone Marrow Cells than CML Cells Total CFC P<0.001 P<0.001 % of CFCs relative to untreated cells P<0.01 n=7

  14. In Vivo Combination Effects Day 55 NSG mice age 7-10 weeks % Survival P<0.001 Sub-lethal cesium irradiation Days after transplantation I.V. injection of BV173 cells Weight (grams) Oral gavage drug treatments Days after transplantation

  15. In Vivo Combination Effects Ctrl Vehicle BMS DA DA+BMS Ctrl Vehicle BMS DA DA+BMS Day 55 Spleen CD19 1.0 mm 0.03 0.19 0.21 0.03 0.02 Liver 1.9 3.15 4.53 1.62 1.47 1.0mm Vehicle BMS DA DA+BMS Ctrl BM 0.07 28.9 33 1.34 0.53 CD19 FSC BCR-ABL transcript levels BM Spleen Liver P<0.01 Fold difference relative to vehicle P<0.05 ND ND ND ND ND ND

  16. In Vivo Combination Effects Day 70 NSG mice age 7-10 weeks % Survival P<0.001 Sub-lethal cesium irradiation Days after transplantation I.V. injection of BV173 cells Weight (grams) Oral gavage drug treatments Days after transplantation

  17. In Vivo Combination Effects Vehicle DA DA+BMS Vehicle DA DA+BMS Day 70 Spleen 0.14 0.11 0.03 CD19 1.0 mm Liver 5.55 4.17 1.78 1.0mm Vehicle DA DA+BMS 34.7 26.3 1.17 BM CD19 FSC BCR-ABL transcript levels BM Spleen Liver P<0.001 P<0.001 P<0.01 Fold difference relative to vehicle

  18. Conclusions TKIs Y177 P Y177 P BMS-911543 JAK2 BCR-ABL AHI-1 STAT5 STAT5 Stem cell proliferation & survival TKI response Stem cell proliferation & survival TKI response JAK2 BCR-ABL • Combination treatment: • More effectively reduces pSTAT5 and pCRKL levels • Increases apoptotic cells and inhibits colony growth in CD34+ CML cells • Prolongs survival of leukemic mice AHI-1 Targeting both BCR-ABL and JAK2 activities may be a more effective therapeutic option for CML patients

  19. Acknowledgments Jiang Lab Members: Dr. Xiaoyan Jiang Dr. Min Chen Katharina Rothe Dr. Kevin Lin Sharmin Esmailzadeh Will Liu Rachel Huang Damian Lai Stem Cell Assay Lab: Karen Lambie Helen Nakamoto Kyi Min Saw Animal Resource Centre FACS Facility CML Working Group: Dr. Connie Eaves Dr. Donna Forrest BMS: Dr. Matthew Lorenzi Dr. Adrian Woolfson Collaborators: Dr. T. Holyoake, Scotland Dr. A.Turhan, France Dr. R. Arlinghaus, Houston

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