Pieter Van Vlierberghe

Bioluminescent Cell-based Assay Seminar Day Monday 31 march 2014 UCL De Duve institute

Brussels, Belgium

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

2  

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

3  

Short Biosketch

Turner  BioSystems  Modulus®  II  Microplate  Mul9mode  Reader  

4  

Short Bio sketch

Odysseus type II grant 2013-2017

Center for Medical Genetics Ghent University

GloMax®-Multi Microplate Reader

CellTiter-Glo® Luminescent Cell Viability

Caspase-Glo® 3/7 Apoptosis Assay

BCA protein assays

Luciferase assays miRNA-mRNA regulation

5  

From normal development to malignant transformation

Immature    ETP-­‐ALL  

Coustan-­‐Smith  et  al.,  Lancet  Oncology,  2009    

Early  T-­‐cell  precursor  ALL  (ETP-­‐ALL)  

6  

Genetic heterogeneity in human ETP-ALL

JAK/STAT signaling

IL7R JAK1 JAK3

Myeloid development ETV6

Chromatin remodeling

EZH2

Genetic heterogeneity provides challenges for the implementation of a uniform targeted therapy in ETP-ALL

ETP Non-ETP

Zhang et al., Nature 2012

Unique features of human ETP-ALL

7  

ETP Non-ETP

Human ETP-ALLs have a unique

gene expression signature

Therapeu1c  targets  in  human  ETP-­‐ALL  gene  expression  profile  

8  

ETP Non-ETP

High BCL2 expression in human ETP-ALL

9  

BCL-2

Fold change > 2; p<0.05

ETP-ALL Other genetic subtypes human T-ALL

High BCL2 expression a reflection of T cell differentiation?

10  

High BCL2 expression a reflection of T cell differentiation?

11  

BCL-2

BCL2 inhibition as a therapeutic strategy

12  Davids & Letai, Journal of Clinical Oncology 2013

Davids & Letai, Cancer Cell 2013

ABT-263 (Navitoclax) : BCL-2, BCL-xL ABT-199 : BCL-2 only

ABT-199 as a new therapeutic strategy in human cancer

13  

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

ABT-199 expression in human T-ALL cell lines

14  

mRN

A  ex

pres

sion

P

rote

in

LOU

CY

ALL

-SIL

TALL

-1

DN

D41

C

UTT

L1 K

OP

TK1 P

F382

JUR

KAT

K

AR

PAS

45

PE

ER

CC

RF-

CE

M

BCL-2

β-actin

BCL-­‐2  expression  

ABT-199 and cell viability in human T-ALL cell lines

15  CellTiter-Glo® Luminescent Cell Viability

GloMax®-Multi Microplate Reader

ABT-199 IC50 in human T-ALL cell lines

16  CellTiter-Glo® Luminescent Cell Viability

GloMax®-Multi Microplate Reader

Correlation ABT-199 IC50 and BCL2 expression

17  CellTiter-Glo® Luminescent Cell Viability

GloMax®-Multi Microplate Reader

ABT-199 induces apoptosis

18  Caspase-Glo® 3/7 Apoptosis Assay GloMax®-Multi Microplate Reader

In vivo xenograft model human T-ALL

19  

Luciferase positive LOUCY cells

Engraftment of cells (6 weeks)

Oral gavage 100 mg ABT-199/kg

or vehicle Measure luminescence on

different time points

In vivo xenograft model human T-ALL

20  

Genetic subtypes of human T-ALL

21  

ETP-ALL Other genetic subtypes human T-ALL

In vitro ABT-199 sensitivity primary T-ALL samples

22  

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

In vitro ABT-199 sensitivity human T-ALL

23  

JAK/STAT signaling

IL7R JAK1 JAK3

ETP Non-ETP

JAK3 mutant murine T-ALL

24  

Harvest  HSC  from  BM  

JAK3  mutant  

Tail  vein  injec9on  

Monitor  leukemia  development  

Control thymus JAK3 mutant T-ALL tumors

BCL-2

actin

Prof. Jan Cools, KU Leuven

Conclusions  

25  

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

Pieter Van Vlierberghe

Bioluminescent Cell-based Assay Seminar Day Monday 31 march 2014 UCL De Duve institute

Brussels, Belgium

An  unbiased  high-­‐throughput  microRNA  library  screen  iden1fies  novel  microRNA  regulators  of  key  

oncogenes  and  tumor  suppressor  genes  

microRNAs

miRISC

3’

5’

5’

3’

mRNA

microRNA

3’ untranslated region (3’UTR)

§  small, non-coding RNA molecules

§  post-transcriptional regulation of gene expression

microRNAs

miRISC

3’

5’

5’

3’

mRNA

microRNA

§  small, non-coding RNA molecules

§  post-transcriptional regulation of gene expression

mRNA degradation translational inhibition

§  small, non-coding RNA molecules

§  post-transcriptional regulation of gene expression

§  microRNA function? microRNA targets!

§  microRNA target prediction algorithms!

microRNAs

miRISC

3’

5’

5’

3’

mRNA

microRNA

3’UTR microRNA library screen

reporter  construct   miRNA  mimic  

reporter  gene  3’UTR    

promotor  

5’   3’  5’  3’  

HEK-­‐293T  cells  

reporter  gene  ac1vity  

12  cancer  genes  470 microRNAs

benchmark  data  set    microRNA  interactomes  

>  15.000  co-­‐transfec1ons    

5640  interac1ons  tested  

 interac1on  iden1fica1on  

3’UTR  reporter  assay   3’UTR  microRNA  library  screen  

3’UTR microRNA library screen

reporter  construct   miRNA  mimic  

reporter  gene  3’UTR    

promotor  

5’   3’  5’  3’  

HEK-­‐293T  cells  

Δ(rn −medianrn

MAD) rn = log

rexprnorm

reporter  gene  ac1vity  

microRNA  interac1on  score  

with  

12  cancer  genes  470 microRNAs

benchmark  data  set  microRNA  interactomes  

>  15.000  co-­‐transfec1ons    

5640  interac1ons  tested  

 interac1on  iden1fica1on  

3’UTR  reporter  assay   3’UTR  microRNA  library  screen  

3’UTR library screen

EZH2

MYB MYC

NOTCH1 PHF6 FBXW7

RB1

470 miRNAs (miRbase 9.2)

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)                    

Cancer gene microRNA interactomes

0%

20%

40%

60%

80%

100%

120%

140%

scrambled miRNA

miR-223 scrambled miRNA

miR-223

0%

20%

40%

60%

80%

100%

120%

scrambled miRNA

miR-204 scrambled miRNA

miR-204

wild-­‐type   mutated  binding  site(s)  

miR-­‐204  

miR-­‐223  

interactom

e  interactom

e  

repo

rter  gen

e  ac1v

ity  

repo

rter  gen

e  ac1v

ity  

rescue  experiments!  

Acknowledgements  

35  

Center  for  Medical  Gene9cs  (Ghent)  Sofie  Peirs  Frank  Speleman  Filip  Ma_hijssens  Béatrice  Lintermans  Evelien  Van  den  Eeckhoudt  

Lab  Jules  Meijerink  (Ro_erdam)  Jules  Meijerink  Kirsten  Canté-­‐Barre_  

Lab  Jean  Soulier  (Paris)  Jean  Soulier  

VIB  Inflamma9on  Research  Center  (Ghent)  Steven  Goossens  

Department  of  Clinical  chemistry,  microbiology  and  immunology  (Ghent)  Tom  Taghon  Inge  Van  de  Walle  

Department  of  Pediatric  Hemato-­‐Oncology  (Ghent)  Tim  Lammens  Barbara  De  Moerloose  Yves  Benoit