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BCL2 inhibition by ABT-199 in T-cell acute lymphoblastic leukemia (T-ALL) Pieter Van Vlierberghe Bioluminescent Cell-based Assay Seminar Day Monday 31 march 2014 UCL De Duve institute Brussels, Belgium Short Bio sketch PhD 2003-2008 Pediatric Oncology Erasmus Medical Center Rotterdam, The Netherlands Postdoc 2008-2013 Institute for Cancer Genetics Columbia University Medical Center New York, USA 2 Short Biosketch Turner BioSystems Modulus® II Microplate Mul9mode Reader 3 Short Bio sketch Odysseus type II grant 2013-2017 Center for Medical Genetics Ghent University CellTiter-Glo® Luminescent Cell Viability GloMax®-Multi Microplate Reader Caspase-Glo® 3/7 Apoptosis Assay BCA protein assays Luciferase assays miRNA-mRNA regulation 4 From normal development to malignant transformation Early T-‐cell precursor ALL (ETP-‐ALL) Coustan-‐Smith et al., Lancet Oncology, 2009 Immature ETP-‐ALL 5 Genetic heterogeneity in human ETP-ALL ETP JAK/STAT signaling Myeloid development Chromatin remodeling Non-ETP IL7R JAK1 JAK3 ETV6 EZH2 Genetic heterogeneity provides challenges for the implementation of a uniform targeted therapy in ETP-ALL Zhang et al., Nature 2012 6 Unique features of human ETP-ALL ETP Non-ETP Human ETP-ALLs have a unique gene expression signature 7 Therapeu1c targets in human ETP-‐ALL gene expression profile ETP Non-ETP 8 High BCL2 expression in human ETP-ALL ETP-ALL Other genetic subtypes human T-ALL BCL-2 Fold change > 2; p<0.05 9 High BCL2 expression a reflection of T cell differentiation? 10 High BCL2 expression a reflection of T cell differentiation? BCL-2 11 BCL2 inhibition as a therapeutic strategy ABT-263 (Navitoclax) : ABT-199 : BCL-2, BCL-xL BCL-2 only Davids & Letai, Cancer Cell 2013 Davids & Letai, Journal of Clinical Oncology 2013 12 ABT-199 as a new therapeutic strategy in human cancer January 2013 Clinical Phase I Trials in relapsed or treatment-resistant CLL Tumor Lysis Syndrome Reports on anti-tumoral effect in: acute myeloid leukemia multiple myeloma estrogen receptor-positive breast cancer 13 Protein CCRF-CEM PEER KARPAS45 JURKAT CUTTL 1 KOPTK 1 PF382 DND41 TALL-1 ALL-SIL LOUCY mRNA expression ABT-199 expression in human T-ALL cell lines BCL-‐2 expression BCL-2 β-actin 14 ABT-199 and cell viability in human T-ALL cell lines CellTiter-Glo® Luminescent Cell Viability GloMax®-Multi Microplate Reader 15 ABT-199 IC50 in human T-ALL cell lines CellTiter-Glo® Luminescent Cell Viability GloMax®-Multi Microplate Reader 16 Correlation ABT-199 IC50 and BCL2 expression CellTiter-Glo® Luminescent Cell Viability GloMax®-Multi Microplate Reader 17 ABT-199 induces apoptosis Caspase-Glo® 3/7 Apoptosis Assay GloMax®-Multi Microplate Reader 18 In vivo xenograft model human T-ALL Luciferase positive LOUCY cells Engraftment of cells (6 weeks) Measure luminescence on different time points Oral gavage 100 mg ABT-199/kg or vehicle 19 In vivo xenograft model human T-ALL 20 Genetic subtypes of human T-ALL ETP-ALL Other genetic subtypes human T-ALL 21 In vitro ABT-199 sensitivity primary T-ALL samples Hypothesis The cell of origin and cooperative genetic defects define ABT-199 sensitivity in T-ALL CellTiter-Glo® Luminescent Cell Viability GloMax®-Multi Microplate Reader 22 In vitro ABT-199 sensitivity human T-ALL ETP JAK/STAT signaling Non-ETP IL7R JAK1 JAK3 23 JAK3 mutant murine T-ALL JAK3 mutant Harvest HSC from BM Tail vein injec9on Monitor leukemia development JAK3 mutant T-ALL tumors Control thymus BCL-2 actin Prof. Jan Cools, KU Leuven 24 Conclusions ABT-199 is a promising new drug for human cancer Our pre-clinical data suggest ABT-199 as a promising new drug for specific subtypes of pediatric T-ALL 25 An unbiased high-‐throughput microRNA library screen iden1fies novel microRNA regulators of key oncogenes and tumor suppressor genes Pieter Van Vlierberghe Bioluminescent Cell-based Assay Seminar Day Monday 31 march 2014 UCL De Duve institute Brussels, Belgium microRNAs § small, non-coding RNA molecules § post-transcriptional regulation of gene expression miRISC mRNA 3’ 5’ 5’ microRNA 3’ 3’ untranslated region (3’UTR) microRNAs § small, non-coding RNA molecules § post-transcriptional regulation of gene expression miRISC mRNA 3’ 5’ 5’ microRNA 3’ mRNA degradation translational inhibition microRNAs § small, non-coding RNA molecules § post-transcriptional regulation of gene expression miRISC mRNA 3’ 5’ 5’ microRNA 3’ § microRNA function? microRNA targets! § microRNA target prediction algorithms! 3’UTR microRNA library screen 3’UTR reporter assay HEK-‐293T cells reporter construct reporter gene 3’UTR microRNA library screen miRNA mimic 470 microRNAs 3’UTR 5’ 3’ 12 cancer genes 3’ 5’ promotor > 15.000 co-‐transfec1ons 5640 interac1ons tested interac1on iden1fica1on reporter gene ac1vity microRNA interactomes benchmark data set 3’UTR microRNA library screen 3’UTR microRNA library screen 3’UTR reporter assay HEK-‐293T cells reporter construct reporter gene miRNA mimic 470 microRNAs 3’UTR 5’ 3’ 12 cancer genes 3’ 5’ promotor > 15.000 co-‐transfec1ons 5640 interac1ons tested interac1on iden1fica1on reporter gene ac1vity microRNA interac1on score rn − medianrn Δ(with ) MAD rn = log rexp rnorm microRNA interactomes benchmark data set 3’UTR library screen NOTCH1 PHF6 RB1 MYC FBXW7 EZH2 470 miRNAs (miRbase 9.2) MYB Cancer gene microRNA interactomes miRNA-‐FBXW7 interactome miRNA-‐NOTCH1 interactome 44 interac1ons iden1fied 21 interac1ons iden1ed 4 confirmed (already described in literature) 4 confirmed (already described in literature) 4 0 novel interac1ons (not yet described) 16 novel interac1ons (not yet described) Cancer gene microRNA interactomes rescue experiments! 120% 100% 80% miR-‐204 interactome 60% 40% 20% 0% reporter gene ac1vity scrambled miRNA wild-‐type scrambled miRNA miR-204 mutated binding site(s) 140% 120% 100% 80% 60% interactome 40% 20% 0% scrambled miRNA miR-‐223 miR-204 miR-223 scrambled miRNA miR-223 Acknowledgements Center for Medical Gene9cs (Ghent) Sofie Peirs Frank Speleman Filip Ma_hijssens Béatrice Lintermans Evelien Van den Eeckhoudt Department of Pediatric Hemato-‐ Oncology (Ghent) Tim Lammens Barbara De Moerloose Yves Benoit Department of Clinical chemistry, microbiology and immunology (Ghent) Tom Taghon Inge Van de Walle VIB Inflamma9on Research Center (Ghent) Steven Goossens Lab Jules Meijerink (Ro_erdam) Jules Meijerink Kirsten Canté-‐Barre_ Lab Jean Soulier (Paris) Jean Soulier 35
