BCL2 inhibition by ABT-199 in T-cell acute lymphoblastic leukemia (T-ALL)

Transcription

1 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

2 Short Bio sketch PhD Pediatric Oncology Erasmus Medical Center Rotterdam, The Netherlands Postdoc Institute for Cancer Genetics Columbia University Medical Center New York, USA 2

3 Short Biosketch Turner BioSystems Modulus II Microplate Mul9mode Reader 3

4 Short Bio sketch Odysseus type II grant 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

5 From normal development to malignant transformation Early T- cell precursor ALL (ETP- ALL) Immature ETP- ALL Coustan- Smith et al., Lancet Oncology,

6 Genetic heterogeneity in human ETP-ALL ETP Non-ETP JAK/STAT signaling Myeloid development Chromatin remodeling IL7R JAK1 JAK3 ETV6 EZH2 Genetic heterogeneity provides challenges for the implementation of a uniform targeted therapy in ETP-ALL Zhang et al., Nature

7 Unique features of human ETP-ALL ETP Non-ETP Human ETP-ALLs have a unique gene expression signature 7

8 Therapeu1c targets in human ETP- ALL gene expression profile ETP Non-ETP 8

9 High BCL2 expression in human ETP-ALL ETP-ALL Other genetic subtypes human T-ALL BCL-2 Fold change > 2; p<0.05 9

10 High BCL2 expression a reflection of T cell differentiation? 10

11 High BCL2 expression a reflection of T cell differentiation? BCL-2 11

12 BCL2 inhibition as a therapeutic strategy ABT-263 (Navitoclax) : BCL-2, BCL-xL ABT-199 : BCL-2 only Davids & Letai, Cancer Cell 2013 Davids & Letai, Journal of Clinical Oncology

13 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

14 ABT-199 expression in human T-ALL cell lines BCL- 2 expression Protein LOUCY ALL-SIL TALL-1 DND41 CUTTL 1 KOPTK 1 PF382 JURKAT KARPAS45 PEER CCRF-CEM mrna expression BCL-2 β-actin 14

15 ABT-199 and cell viability in human T-ALL cell lines CellTiter-Glo Luminescent Cell Viability GloMax -Multi Microplate Reader 15

16 ABT-199 IC50 in human T-ALL cell lines CellTiter-Glo Luminescent Cell Viability GloMax -Multi Microplate Reader 16

17 Correlation ABT-199 IC50 and BCL2 expression CellTiter-Glo Luminescent Cell Viability GloMax -Multi Microplate Reader 17

18 ABT-199 induces apoptosis Caspase-Glo 3/7 Apoptosis Assay GloMax -Multi Microplate Reader 18

19 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

20 In vivo xenograft model human T-ALL 20

21 Genetic subtypes of human T-ALL ETP-ALL Other genetic subtypes human T-ALL 21

22 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

23 In vitro ABT-199 sensitivity human T-ALL ETP Non-ETP JAK/STAT signaling IL7R JAK1 JAK3 23

24 JAK3 mutant murine T-ALL JAK3 mutant Harvest HSC from BM Tail vein injec9on Monitor leukemia development Control thymus JAK3 mutant T-ALL tumors BCL-2 actin Prof. Jan Cools, KU Leuven 24

25 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

26 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

27 micrornas small, non-coding RNA molecules post-transcriptional regulation of gene expression mirisc 5 mrna 3 5 microrna 3 3 untranslated region (3 UTR)

28 micrornas small, non-coding RNA molecules post-transcriptional regulation of gene expression mirisc 5 mrna 3 5 microrna 3 mrna degradation translational inhibition

29 micrornas small, non-coding RNA molecules post-transcriptional regulation of gene expression mirisc 5 mrna 3 5 microrna 3 microrna function? microrna targets! microrna target prediction algorithms!

30 3 UTR microrna library screen 3 UTR reporter assay 3 UTR microrna library screen HEK- 293T cells reporter construct 3 UTR reporter gene mirna mimic micrornas 12 cancer genes promotor > co- transfec1ons 5640 interac1ons tested interac1on iden1fica1on reporter gene ac1vity microrna interactomes benchmark data set

31 3 UTR microrna library screen 3 UTR reporter assay 3 UTR microrna library screen HEK- 293T cells reporter construct 3 UTR reporter gene mirna mimic micrornas 12 cancer genes promotor > co- transfec1ons 5640 interac1ons tested interac1on iden1fica1on reporter gene ac1vity microrna interac1on score Δ( r median n r with n MAD ) r n = log r exp r norm microrna interactomes benchmark data set

32 3 UTR library screen NOTCH1 PHF6 FBXW7 RB1 EZH2 MYC 470 mirnas (mirbase 9.2) MYB

33 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) 40 novel interac1ons (not yet described) 16 novel interac1ons (not yet described)

34 Cancer gene microrna interactomes 120% rescue experiments! 100% 80% mir- 204 interactome 60% 40% 20% 0% scrambled mirna mir-204 scrambled mirna mir-204 reporter gene ac1vity 140% 120% 100% 80% wild- type mutated binding site(s) mir- 223 interactome 60% 40% 20% 0% scrambled mirna mir-223 scrambled mirna mir-223

35 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 VIB Inflamma9on Research Center (Ghent) Steven Goossens Lab Jules Meijerink (Ro_erdam) Jules Meijerink Kirsten Canté- Barre_ Lab Jean Soulier (Paris) Jean Soulier Department of Clinical chemistry, microbiology and immunology (Ghent) Tom Taghon Inge Van de Walle 35