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Damian Lai SRD 2014

Targeting Drug-Insensitive CML Stem/Progenitor Cells with Combination Treatments of New ABL and PP2A Inhibitors. Damian Lai SRD 2014. CML ( C hronic M yeloid L eukemia). Chronic myeloid leukemia (CML) is a multistep, multilineage myeloproliferative disease.

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Damian Lai SRD 2014

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  1. Targeting Drug-Insensitive CML Stem/Progenitor Cells with Combination Treatments of New ABL and PP2A Inhibitors Damian Lai SRD 2014

  2. CML (Chronic Myeloid Leukemia) • Chronic myeloid leukemia (CML) is a multistep, multilineagemyeloproliferative disease. • Incidence 1/100.000, 20-30% of human leukemia in adults (BCR-ABL) HSC CMP CLP GMP MEP Sattler et al, Cytokine Growth Factor Rev 8:63, 1997 Chronic Phase (Accumulation of granulocytes) T cell B cell RBC G M Platelets Accelerated Phase (Adapted from Ren RB Nat Cancer Rev,2005) Blast Crisis

  3. The First Line of CML Treatment: Tyrosine Kinase Inhibitors (TKIs) p210BCR/ABL BCR ABL Y177 Y1294 DD Domain Tyrosine Kinase DNA BD Actin BD SH2 S/T kinase NLS SH3 Rho-GEF PI3K JAK2 Ras Druker et al, Blood 112:4808, 2008 AKT STAT5 MAPK BCR-ABL kinase domain Increased proliferation Decreased apoptosis Imatinib Massive accumulation of myeloid cells in circulation (Major characteristic of CML) Current 1st line: Imatinib Alternatives: Dasatinib, Nilotinib

  4. Abelson Helper Integration Site 1 (AHI-1) and CML Autocrine loop N IL-3 WD40 P IL-3R SH3 P JAK2 BCR-ABL AHI-1 STAT5 Stem cell genomic instability Stem cell proliferation, survival & maintenance TKI -response protein degradation • AHI-1/Ahi-1 is down-regulated during normal hematopoietic cell differentiation. • AHI-1 is highly deregulated in CML stem/progenitor cells, indicating a possible cooperative activity in leukemia development. lin- lin- lin+ 34+38- 34+38+34- Jiang et al, Blood 102:2976, 2004 Zhou et al, J Exp Med 205:2657, 2008 Chen et al, J Natl Cancer Inst 105:405, 2013

  5. Drug Screen: Experimental Design Pres library (10M) Fixation HCS protocol adjustment HCS Scan Data analysis K562-AHI-1 K562-AHI-1 + IM Pres library 48 H 24 H AHI-1-YFP+ CML cells Prestwick compound library: AHI-1 • -1200 small molecules • -100% marketed drugs • known bioavailability and safety in humans Relative transcript levels Hoechst YFP Merge K562 K562 AHI-1 Min Chen & Kaiji Hu

  6. Cantharidin K562 K562-IMR P<0.001 P<0.001 • Not FDA approved • High toxicity in NBM • Need to find alternative drug with similar mechanism of PP2A inhibition P<0.001 P<0.001

  7. Hypothesis and Objective • Hypothesis • A new PP2A inhibitor, to selectively target and destabilize multiple AHI-1-mediated complexes, including AHI-1-BCR-ABL-β-catenin-PP2A, in combination with new TKIs, is more effective in eliminating leukemic stem cells, a population highly resistant to current TKI monotherapy. • Objective • To investigate role of PP2A in CML and evaluate the therapeutic potential of two PP2A inhibitors, LB100 and LB102, alone and in combination with TKI to inhibit the growth of CML stem and progenitor cells in vitro and in vivo.

  8. New Drugs Specifically Target PP2A and Efficacy is Affected by Expression of AHI-1 p<0.05 p<0.05 p<0.05

  9. Inhibition of PP2A In Combination With IM Leads To Decreased Proliferation of K562 and K562 IM-Resistant Cells P<0.01 P<0.01 P<0.01 P<0.01 P<0.01 P<0.01 P<0.01 P<0.01

  10. Inhibition of PP2A in Combination With IM Leads To Apoptosis of K562 and K562 IM-Resistant Cells P<0.05 P<0.05 K562-IMR 24H 48H IM 5uM - - - - - - + + + + + + LB1.0 5uM - - - - - - + + - + + - LB1.2 5uM - - - - - - + - - + + + C. Caspase 8 C. Caspase 3 β-Actin

  11. Inhibition of PP2A Leads to Mitotic Arrest (G2/M Phase) * * * * G2/M phase arrest is dosage dependent * * * * N=3, *, P < 0.05

  12. Inhibition of PP2A Leads to Mitotic Arrest (G2/M Phase) * * * * * * * * * * * * * * * * N=3, *, P < 0.05

  13. G2/M Arrest Leads to Mitotic Catastrophe Multipolar Cell Division Multipolar Cell Division with Lagging Chromosomes Multipolar Spindles K562: 20uM LB100 – 40X

  14. G2/M Arrest Leads to Mitotic Catastrophe Anti-α-tubulin DAPI Merge Untreated – 40X 5uM LB1.2 – 40X

  15. PP2A Inhibition in Combination with IM Affects AHI-1-BCR-ABL-JAK2-STAT and AKT Pathways • BCR-ABL has been shown to stabilize β-Catenin in CML through tyrosine phosphorylation at y86 and y654 residues • PP2A activity supports Wntsignalling pathway leading to stabilization of β-Catenin • Effect of dual inhibition of BCR-ABL and PP2A may converge on β-Catenin

  16. Potential mechanism β-Catenin Phosphorylation 24H 48H K562 IM Resistant Cells 1: Control 2: 5uM IM 3: 5uM LB100 4: 5uM LB1.2 5: 5uM IM + 5uM LB100 6: 5uM IM + 5uM LB1.2 1 2 3 4 5 6 1 2 3 4 5 6 Imatinib PP2A inhibitors Phosho-β-Catenin (Y86) PP2A Phosho-β-Catenin (T41,S45) BCR-ABL β-Catenin (total) BCR-ABL PP2A GAPDH IP: β-Catenin (total) WB: PP2A-C p p p p S45 Y654 Y86 T41 Y86 Y654 T41 S45 +C IP IgG β-catenin β-catenin 40kDa IP: β-Catenin (total) WB: BCR-ABL 35kDa β-catenin stability β-catenin degradation +C IP IgG +C IP IgG 100kDa IP: β-Catenin (total) WB: B-Catenin (total) 70kDa β-catenin transcriptional activation β-catenin transcriptional activation repressed 170kDa 130kDa 100kDa

  17. Conclusion • Combination of LB100 and LB102 with IM significantly inhibits proliferation and induces apoptosis of IM-resistant cells. • LB100 and LB102 induces G2-M arrest leading to mitotic catastrophe. • Combination of LB100 and LB102 with IM affects the AHI-1-BCR-ABL-JAK2-STAT5 pathway and potentially targets β-catenin for protein degradation. • Inhibition of PP2A leads to mitotic catastrophe. This could occur via the activation of CyclinB/CDK1 complex.

  18. Acknowledgments • DrXiaoyan Jiang • Will Liu • Min Chen • Katharina Rothe • Kevin Lin • Leon Lin • Rachel Huang • SharminEsmailzadeh • Kyi Min Saw • Helena Wang • Josephine Leung • ElianneAbramovich Committee members • Dr. Catherine Pallen • Dr. Sandra Dunn

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