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

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

DD Domain Y177 S/T kinase Rho-GEF Y1294 SH3 SH2 BCR ABL Tyrosine Kinase NLS DNA BD Actin BD p210 BCR/ABL Ras MAPK PI3K AKT JAK2 STAT5 Increased proliferation Decreased apoptosis Massive accumulation of myeloid cells in circulation (Major characteristic of CML) The First Line of CML Treatment: Tyrosine Kinase Inhibitors (TKIs) Imatinib BCR-ABL kinase domain Current 1 st line: Imatinib Alternatives: Dasatinib, Nilotinib Druker et al, Blood 112:4808, 2008

Abelson Helper Integration Site 1 (AHI-1) and CML lin - lin - lin Zhou et al, J Exp Med 205:2657, 2008 Jiang et al, Blood 102:2976, 2004 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. AHI-1 STAT5 Stem cell proliferation, survival & maintenance IL-3R IL-3 Autocrine loop Stem cell genomic instability BCR-ABL JAK2 P SH3 WD40 N P TKI -response protein degradation Chen et al, J Natl Cancer Inst 105:405, 2013

Drug Screen: Experimental Design HoechstYFPMerge Prestwick compound library: small molecules -100% marketed drugs -known bioavailability and safety in humans AHI-1-YFP + CML cells 48 H K562-AHI-1 + IM 24 H Pres library (10  M) Pres library Fixation HCS protocol adjustment HCS Scan Data analysis Min Chen & Kaiji Hu Relative transcript levels AHI-1 K562K562 AHI-1

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

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.

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

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

Inhibition of PP2A in Combination With IM Leads To Apoptosis of K562 and K562 IM-Resistant Cells LB1.0 5uM LB1.2 5uM K562-IMR IM 5uM H48H β-Actin C. Caspase 8 C. Caspase 3 P<0.05

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

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

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

G2/M Arrest Leads to Mitotic Catastrophe Untreated – 40X 5uM LB1.2 – 40X DAPIAnti-α-tubulinMerge

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

Phosho-β-Catenin (Y86) β-Catenin (total) 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 LB H Phosho-β-Catenin (T41,S45) GAPDH 48H Imatinib BCR-ABL β-catenin transcriptional activation repressed β-catenin degradation T41 S45 Y654 β-catenin pp Y86 PP2A PP2A inhibitors BCR-ABL T41 S45Y654 β-catenin Y86 PP2A pp β-catenin transcriptional activation β-catenin stability IP: β-Catenin (total) WB: B-Catenin (total) IP: β-Catenin (total) WB: PP2A-C 35kDa 40kDa +CIPIgG IP 100kDa 70kDa +C IP: β-Catenin (total) WB: BCR-ABL 100kDa 130kDa +CIPIgG 170kDa Potential mechanism β-Catenin Phosphorylation

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.

Acknowledgments Dr Xiaoyan Jiang Will Liu Min Chen Katharina Rothe Kevin Lin Leon Lin Rachel Huang Sharmin Esmailzadeh Kyi Min Saw Helena Wang Josephine Leung Elianne Abramovich Dr. Catherine Pallen Dr. Sandra Dunn Committee members