Transcript Huang, Di Liberto, Niesvizky, Chen-Kiang, unpublished

Integrative whole transcriptome sequencing
in myeloma and MCL therapy
Selina Chen-Kiang
Professor
Weill Cornell Medical College
October 24 , 2013
Disclosures: None
The Cell Cycle
Positive
Go
Cyclin D + CDK4/6
M
pS-Rb-E2F
G1
G2
Cyclin E + CDK2
S
pST-Rb
CDK: Cyclin-dependent Kinase
CKI: Cyclin-dependent kinase inhibitor
E2F release
Negative
p16
p15
p18
p19
p21
p27
p57
mid-G1
checkpoint
The Weill-Cornell Medical College Team
Goal
• To develop superior, mechanism-based combination
cancer therapy using novel reagents
• To identify biomarkers
• To develop functional genomics for patient and therapy
stratification
Approach
• Bench to bedside and back
• Integrating novel reagents with basic research, clinical
application, pathology and functional genomics
Targeting CDK4/CDK6 with PD 0332991 (palbociclib)
• Highly potent and specific CDK4/6 inhibitor (IC50 11-60 nM)
• Induces complete early G1 arrest
• Reversible
• Orally bioavailable pyridopyrimidine
• Low in toxicity
• Inhibits tumor growth in the NOD-SCID
human myeloma xenograft models and the immune-competent
mouse 5T models
• Prolonged G1 arrest (pG1) and synchronization into S-phase (pG1-S)
sensitize myeloma cells to killing by proteasome inhibitors
Fry et al., 2004, Mol Cancer Ther
Baughn et al., 2006, Cancer Res
Menu et al., 2008, Cancer Res
Huang et al., 2012, Blood
Targeting CDK4/CDK6 in combination therapy
PD 0332991
Partner agent (low dose, selective )
Weill-Cornell
Mantle cell lymphoma
Multiple Myeloma
Mantle cell lymphoma
Acute myeloid Leukemia
Mantle cell lymphoma
Multiple myeloma
Advanced Solid tumors
Breast cancer
Metastatic liposarcoma
Glioblastoma
Phase I single agent study
Phase I/II PD-bortezomib-Dex
Phase I PD-bortezomib
Phase I PD-AraC
(2/2013)
Phase I PD-Ibrutinib
(2013)
Phase I PD-Lenalidomide
(2013)
Phase I single agent (2006, completed)
Phase I/II –letrozole front line (2009-completed)
Phase I single agent (9/2010—completed)
Phase I single agent (10/2010--
Non-small cell lung carcinoma Phase I single agent ( 2/2011--
Targeting the Cell Cycle in Multiple Myeloma
The second most common hematopoietic cancer
•
Incurable
•
Dysregulation in apoptosis and cell cycle control
•
CDK4/CDK6-specific phosphorylation of Rb and cell proliferation
increase with disease progression in MM (Ely et al., 2005)
MM cells
•
Hypothesis
Go
Cyclin D + CDK4/6
M
G1
G2
PD 0332991
Reversible
pS-Rb
S
Release of prolonged early G1 block
Cell cycle synchronization
incomplete restoration of scheduled gene expression
Further sensitizing tumor cells to cytotoxic killing
Inhibition of
CDK4/CDK6
Early G1 genes
Late G2, S,
G2/M
genes
Huang, Di Liberto et al, Blood, 2012
Hypothesis
Go
Cyclin D + CDK4/6
M
G1
G2
PD 0332991
Reversible
pS-Rb
S
Release of prolonged early G1 block
Cell cycle synchronization
incomplete restoration of scheduled gene expression
Further sensitizing tumor cells to cytotoxic killing
Targeting CDK4/CDK6 in Mantle Cell Lymphoma
First in human PD 0332991 single-agent clinical trial
— pG1 (prolonged inhibition of CDK4/CDK6)
• Inhibition of CDK4/CDK6 by PD 0332991 leads to prolonged
G1 arrest (pG1) and increased tumor-specific cell death in
MCL (n=17)
• PD (125 mg/d orally 21 of 28 d) is generally well tolerated with
neutropenia, fatigue and diarrhea as most common
adverse events
• 1 complete response, 2 partial response, 5 SD > 1 year
Leonard, Vallabhajosula, Martin,
Chen-Kiang et al, Blood 2012
Does prolonged early G1 arrest (pG1) induced by
selective CDK4/CDK6 inhibition reprogram myeloma cells
for IMiD killing?
•
•
•
•
•
•
Enhances IMiD clinical efficiency at lower doses
Mechanism of IMiD killing
Biomarker for IMiD killing
Maintenance therapy
Control of MM stem cell renewal
Control of second primary malignancy
pG1 enhances and accelerate lenalidomide killing
of primary myeloma cells in stromal co-culture
Huang, Di Liberto, Niesvizky, Chen-Kiang, unpublished
pG1 sensitizes primary myeloma cells to IMiD killing
independent of prior treatments or cycling status, but
dependent on Rb
Lenalidomide: 17/21
Pomalidomide: 3/4
Huang, Di Liberto, Jayabalan, Niesvizky, Chen-Kiang, unpublished
Inhibition of CDK4/CDK6 (complete early G1) overrides
cell cycle regulation by lenalidomide (incomplete late G1)
Go
Cyclin D
+ CDK4/6
(Rb-deficient)
M
G1
G2
S
Cyclin E/A
+ CDK2
Huang, Di Liberto Chen-Kiang, unpublished
Induction of pG1 by CDK4/CDK6 inhibition Enhances
Lenalidomide and Pomalidomide Killing
(pG1)
Huang, Di Liberto, Chen-Kiang, unpublished
Synergistic increase in CRBN
and loss of IRF4 by IMiDs and pG1
•
IMiDs reduces IRF4 in myeloma cells (Li et al., 2011; Lopez Girona et al., 2012
•
CRBN (cereblon) is required for the anti-myeloma activity of IMiDs (Zhu et al., 2011)
Huang, Di Liberto, Chen-Kiang, unpublished
Analysis of RNA-Sequencing
(RNA-Seq) Data
Genomics and Bioinformatics
Discovery of biomarkers by
Whole Transcriptome Sequencing (WTS, RNA-Seq)
RNA abundance, variant, indel
50x50 paired-end RNA sequencing on a HiSeq2000,
76 million reads per sample
Use the Burrow-Wheeler Aligner to align reads to the
genome ( Building 37)
SAMtools and Genome Analysis Toolkit to call nonreference variants.
X. Huang, D. Chiron,
M. DiLibiberto, C. Mason
Predict gene fusions,
polyA sites
Algorithms: Rmake,
Alexa, SnowShoes
RNA-Seq
Sequencing Data
R-make: Distributed, Parallel
Alignment on HPC nodes
reads
References
5
Adapters
Find ncRNAs and
new TARs
Algorithms: Rmake,
Aceview
rRNA
miRBase
4
RefSeq
hg19
Alignments
& QC
1
annotations
bigwigs
BAM files
3
2
Counts and differential
expression by gene, exon,
allele, splice isoform, &
transcript
Algorithms: TopHat, Rmake,
Cufflinks, BayesASE
Genetic variation:
(SNVs, indels,
CNVs)
Algorithms: Rmake,
BWA/SAMtools
GATK, HGVS,
VarScan
High reproducibility with low input
10ng
100ng
1000ng
B
Raw Reads per Gene (10ng)
A
R2=0.95
Raw Reads per gene (1000ng)
Genomics and Bioinformatics
Lenalidomide induced interferon responses
in primary myeloma cells
Huang, Di Liberto, Chen-Kiang, unpublished
IRF7 mediates lenalidomide killing and pG1 sensitization
•
IRF7 is a direct target of IRF4 in ABC DLBCL revealed by ChIP-Seq (Yang et al., 2012)
TRAIL
Huang, Di Liberto, Chen-Kiang, unpublished
IRF7 is also inhibited by FoxO, which protects myeloma
cells IMiD killing
Huang, Di Liberto, Chen-Kiang, unpublished
pG1 Sensitizes MCL Cells to IMiD Killing
through synergistic reduction of IRF4
Di Liberto, Chen-Kiang, unpublished
Summary
• Lenalidomide and Pomolidomide induce incomplete late G1 arrest
independent of Rb in myeloma cells
• Induction of pG1 (prolonged early G1 arrest exceeding the
scheduled arrest time) by PD 0332991 overrides late G1 arrest
induced by IMiDs.
• pG1 reprogram myeloma and MCL cells for IMiD killing, in part
through synergistic reduction of the IRF4 protein.
• Lenalidomide and Pomolidomide induce interferon responses in
primary myeloma cells through de-repression of IRF7 transcription
• pG1 enhances IRF7 transcription and interferon production induced
by IMiDs, leading to TRAIL-mediated killing.
A Phase 1 Open-Label Study of PD 0332991 in
Combination with Lenalidomide and Dexamethasone in
Patients with Relapsed or Refractory Multiple Myeloma
BMBx
Cycle 1 and
Cycle 2
Day
1
BMBx
Cycle 1
8
15
21 22
Dex
Dex
Dex
Dex
20mg PO
20mg PO
20mg PO
20mg PO
28
Lenalidomide PO once daily
PD 0332991 (at predetermined
dose) PO once daily
(PD 0332991 will be given for days 1-14 followed by a 14 day rest period for a 28-day cycle, Lenalidomide will be given
predetermined dose level on days 8-21, and Dexamethasone is given once weekly on Days 8, 15, 22; bone marrow
tes ng will be performed Cycle 1, Days 1 and 8; Cycle 2, Day 1 only).
Mark, Niesvizky, Di Liberto, Chen-Kiang
Targeting CDK4/CDK6 in combination therapy
PD 0332991
Partner agent (low dose, selective )
Weill-Cornell
Mantle cell lymphoma
Multiple Myeloma
Mantle cell lymphoma
Acute myeloid Leukemia
Mantle cell lymphoma
Multiple myeloma
Advanced Solid tumors
Breast cancer
Metastatic liposarcoma
Glioblastoma
Phase I single agent study
Phase I/II PD-bortezomib-Dex
Phase I PD-bortezomib
Phase I PD-AraC
(2/2013)
Phase I PD-Ibrutinib
(2013)
Phase I PD-Lenalidomide
(2013)
Phase I single agent (2006, completed)
Phase I/II –letrozole front line (2009-completed)
Phase I single agent (9/2010—completed)
Phase I single agent (10/2010--
Non-small cell lung carcinoma Phase I single agent ( 2/2011--
Targeting CDK4/CDK6 in hematologic malignancies
• Both inhibits the cell cycle in tumor cells and
reprograms them to cytotoxic killing
pG1
• forces an imbalance in gene expression,
• Increases redox stress,
• Mechanism-based combination therapy
pG1 iMiDs, GS-1101, ibrutinib
pG1-S bortezomib, carfilzomib, Ara C
• Genome based patient and therapy stratification
Acknowledgements
Weill Cornell Medical College
Xiangao Huang
Maurizio Di Liberto
David Chiron
Adriana Rossi
David Jayabalan
Selina Chen-Kiang
Ruben Niesvizky
Tomer Mark
Pfizer
Sophia Randolph
Broad Institute
Anna Schinzel
Bill Hahn
Peter Martin
John Leonard
Scott Ely
Chris Mason
Olivier Elemento
Celgene
Mohamad Hussein
Patients
Lymphoma Research Foundation
Leukemia and Lymphoma Society
Starr Cancer Consortium
NIH/NCI