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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