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![Page 1: Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor Weill Cornell Medical College October 24, 2013 Disclosures:](https://reader031.fdocuments.us/reader031/viewer/2022032523/56649d835503460f94a68ba6/html5/thumbnails/1.jpg)
Integrative whole transcriptome sequencing in myeloma and MCL therapy
Selina Chen-Kiang
Professor
Weill Cornell Medical College October 24 , 2013
Disclosures: None
![Page 2: Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor Weill Cornell Medical College October 24, 2013 Disclosures:](https://reader031.fdocuments.us/reader031/viewer/2022032523/56649d835503460f94a68ba6/html5/thumbnails/2.jpg)
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
![Page 3: Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor Weill Cornell Medical College October 24, 2013 Disclosures:](https://reader031.fdocuments.us/reader031/viewer/2022032523/56649d835503460f94a68ba6/html5/thumbnails/3.jpg)
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
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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
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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--
![Page 6: Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor Weill Cornell Medical College October 24, 2013 Disclosures:](https://reader031.fdocuments.us/reader031/viewer/2022032523/56649d835503460f94a68ba6/html5/thumbnails/6.jpg)
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
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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
![Page 8: Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor Weill Cornell Medical College October 24, 2013 Disclosures:](https://reader031.fdocuments.us/reader031/viewer/2022032523/56649d835503460f94a68ba6/html5/thumbnails/8.jpg)
Inhibition of CDK4/CDK6
Early G1 genes
Late G2, S, G2/M genes
Huang, Di Liberto et al, Blood, 2012
![Page 9: Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor Weill Cornell Medical College October 24, 2013 Disclosures:](https://reader031.fdocuments.us/reader031/viewer/2022032523/56649d835503460f94a68ba6/html5/thumbnails/9.jpg)
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
![Page 10: Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor Weill Cornell Medical College October 24, 2013 Disclosures:](https://reader031.fdocuments.us/reader031/viewer/2022032523/56649d835503460f94a68ba6/html5/thumbnails/10.jpg)
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
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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
![Page 12: Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor Weill Cornell Medical College October 24, 2013 Disclosures:](https://reader031.fdocuments.us/reader031/viewer/2022032523/56649d835503460f94a68ba6/html5/thumbnails/12.jpg)
pG1 enhances and accelerate lenalidomide killing of primary myeloma cells in stromal co-culture
Huang, Di Liberto, Niesvizky, Chen-Kiang, unpublished
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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
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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
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Induction of pG1 by CDK4/CDK6 inhibition Enhances
Lenalidomide and Pomalidomide Killing
(pG1)
Huang, Di Liberto, Chen-Kiang, unpublished
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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
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Analysis of RNA-Sequencing (RNA-Seq) Data
Genomics and Bioinformatics
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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
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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
![Page 20: Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor Weill Cornell Medical College October 24, 2013 Disclosures:](https://reader031.fdocuments.us/reader031/viewer/2022032523/56649d835503460f94a68ba6/html5/thumbnails/20.jpg)
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
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Lenalidomide induced interferon responses in primary myeloma cells
Huang, Di Liberto, Chen-Kiang, unpublished
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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
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IRF7 is also inhibited by FoxO, which protects myeloma cells IMiD killing
Huang, Di Liberto, Chen-Kiang, unpublished
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pG1 Sensitizes MCL Cells to IMiD Killingthrough synergistic reduction of IRF4
Di Liberto, Chen-Kiang, unpublished
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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.
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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
![Page 27: Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor Weill Cornell Medical College October 24, 2013 Disclosures:](https://reader031.fdocuments.us/reader031/viewer/2022032523/56649d835503460f94a68ba6/html5/thumbnails/27.jpg)
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--
![Page 28: Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor Weill Cornell Medical College October 24, 2013 Disclosures:](https://reader031.fdocuments.us/reader031/viewer/2022032523/56649d835503460f94a68ba6/html5/thumbnails/28.jpg)
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
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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