Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor...
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Transcript of Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor...
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
G1
S
G2
M
p21p27p57
Negative
Go
Positive
Cyclin D + CDK4/6
pS-Rb-E2F
Cyclin E + CDK2
pST-Rb E2F release
p16p15p18p19
CDK: Cyclin-dependent Kinase
CKI: Cyclin-dependent kinase inhibitor
mid-G1 checkpoint
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
The Weill-Cornell Medical College Team
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 TherBaughn et al., 2006, Cancer ResMenu et al., 2008, Cancer ResHuang et al., 2012, Blood
Targeting CDK4/CDK6 in combination therapy
Partner agent (low dose, selective )
PD 0332991
Weill-CornellMantle cell lymphoma Phase I single agent studyMultiple Myeloma Phase I/II PD-bortezomib-Dex Mantle cell lymphoma Phase I PD-bortezomib Acute myeloid Leukemia Phase I PD-AraC (2/2013)Mantle cell lymphoma Phase I PD-Ibrutinib (2013)Multiple myeloma Phase I PD-Lenalidomide (2013)
Advanced Solid tumors Phase I single agent (2006, completed) Breast cancer Phase I/II –letrozole front line (2009-completed)Metastatic liposarcoma Phase I single agent (9/2010—completed)Glioblastoma 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
ce
lls
Release of prolonged early G1 block
Cell cycle synchronization incomplete restoration of scheduled gene expression
Further sensitizing tumor cells to cytotoxic killing
Cyclin D + CDK4/6 PD 0332991Reversible
G1
S
G2
M
Go
pS-Rb
Hypothesis
Inhibition of CDK4/CDK6
Early G1 genes
Late G2, S, G2/M genes
Huang, Di Liberto et al, Blood, 2012
Release of prolonged early G1 block
Cell cycle synchronization incomplete restoration of scheduled gene expression
Further sensitizing tumor cells to cytotoxic killing
Cyclin D + CDK4/6 PD 0332991Reversible
G1
S
G2
M
Go
pS-Rb
Hypothesis
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)
(Rb-deficient)
G1
S
G2
M
GoCyclin D+ CDK4/6
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 non-reference variants.
X. Huang, D. Chiron,M. DiLibiberto, C. Mason
Genetic variation: (SNVs, indels, CNVs)Algorithms: Rmake, BWA/SAMtoolsGATK, HGVS,VarScan
2
Counts and differential expression by gene, exon, allele, splice isoform, & transcriptAlgorithms: TopHat, Rmake, Cufflinks, BayesASE3
Predict gene fusions, polyA sitesAlgorithms: Rmake, Alexa, SnowShoes
5
reads
Alignments & QC
BAM files
annotations
bigw
igs
R-make: Distributed, Parallel Alignment on HPC nodes
References
hg19hg19
RefSeqRefSeq
miRBasemiRBase
rRNArRNA
AdaptersAdapters
Find ncRNAs and new TARsAlgorithms: Rmake, Aceview
4
1
RNA-SeqSequencing Data
RNA-SeqSequencing Data
10ng
100ng
1000ng
A B
Raw Reads per gene (1000ng)
Raw
Rea
ds p
er G
ene
(10
ng)
R2=0.95
Genomics and Bioinformatics
High reproducibility with low input
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 Killingthrough 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
Mark, Niesvizky, Di Liberto, Chen-Kiang
Targeting CDK4/CDK6 in combination therapy
Partner agent (low dose, selective )
PD 0332991
Weill-CornellMantle cell lymphoma Phase I single agent studyMultiple Myeloma Phase I/II PD-bortezomib-Dex Mantle cell lymphoma Phase I PD-bortezomib Acute myeloid Leukemia Phase I PD-AraC (2/2013)Mantle cell lymphoma Phase I PD-Ibrutinib (2013)Multiple myeloma Phase I PD-Lenalidomide (2013)
Advanced Solid tumors Phase I single agent (2006, completed) Breast cancer Phase I/II –letrozole front line (2009-completed)Metastatic liposarcoma Phase I single agent (9/2010—completed)Glioblastoma 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
Selina Chen-Kiang
Weill-Cornell Medical College
Weill Cornell Medical College
Xiangao HuangMaurizio Di LibertoDavid ChironAdriana RossiDavid JayabalanSelina Chen-Kiang
Ruben Niesvizky Tomer Mark
Peter MartinJohn Leonard
Scott ElyChris MasonOlivier Elemento
PfizerSophia Randolph
Patients
CelgeneMohamad Hussein
Acknowledgements
Lymphoma Research FoundationLeukemia and Lymphoma Society Starr Cancer ConsortiumNIH/NCI
Broad InstituteAnna Schinzel Bill Hahn