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Integrative whole transcriptome sequencing in myeloma and MCL therapy

Integrative whole transcriptome sequencing in myeloma and MCL therapy . Selina Chen-Kiang Professor Weill Cornell Medical College October 24 , 2013. Disclosures: None. Positive. Go. p16 p15 p18 p19. Cyclin D + CDK4/6. mid-G1 checkpoint. pS-Rb-E2F. p21 p27 p57.

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Integrative whole transcriptome sequencing in myeloma and MCL therapy

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  1. Integrative whole transcriptome sequencing in myeloma and MCL therapy Selina Chen-Kiang Professor Weill Cornell Medical College October 24 , 2013 Disclosures: None

  2. Positive Go p16 p15 p18 p19 Cyclin D + CDK4/6 mid-G1 checkpoint pS-Rb-E2F p21 p27 p57 Cyclin E + CDK2 pST-Rb E2F release The Cell Cycle Negative M G1 G2 S CDK: Cyclin-dependent Kinase CKI: Cyclin-dependent kinase inhibitor

  3. 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

  4. 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 xenograftmodels 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

  5. Targeting CDK4/CDK6 in combination therapy PD 0332991 Partner agent (low dose, selective ) Weill-Cornell Mantle cell lymphoma Phase I single agent study Multiple 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--

  6. 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

  7. Hypothesis Go PD 0332991 Reversible Cyclin D + CDK4/6 M pS-Rb G1 G2 S Release of prolonged early G1 block Cell cycle synchronization incompleterestoration of scheduled gene expression Further sensitizing tumor cells to cytotoxic killing

  8. Inhibition of CDK4/CDK6 Early G1 genes Late G2, S, G2/M genes Huang, Di Liberto et al, Blood, 2012

  9. Hypothesis Go PD 0332991 Reversible Cyclin D + CDK4/6 M pS-Rb G1 G2 S Release of prolonged early G1 block Cell cycle synchronization incompleterestoration of scheduled gene expression Further sensitizing tumor cells to cytotoxic killing

  10. 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

  11. 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 IMiDkilling • Maintenance therapy • Control of MM stem cell renewal • Control of second primary malignancy

  12. pG1 enhances and accelerate lenalidomide killing of primary myeloma cells in stromal co-culture Huang, Di Liberto, Niesvizky, Chen-Kiang, unpublished

  13. 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

  14. Go CyclinD + CDK4/6 Inhibition of CDK4/CDK6 (complete early G1) overrides cell cycle regulation by lenalidomide (incomplete late G1) (Rb-deficient) M G1 G2 S CyclinE/A + CDK2 Huang, Di LibertoChen-Kiang, unpublished

  15. Induction of pG1 by CDK4/CDK6 inhibition Enhances Lenalidomide and PomalidomideKilling (pG1) Huang, Di Liberto, Chen-Kiang, unpublished

  16. 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

  17. Analysis of RNA-Sequencing (RNA-Seq) Data Genomics and Bioinformatics

  18. 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

  19. RNA-Seq Sequencing Data Predict gene fusions, polyA sites Algorithms: Rmake, Alexa, SnowShoes R-make: Distributed, Parallel Alignment on HPC nodes Find ncRNAs and new TARs Algorithms: Rmake, Aceview reads References 5 Adapters rRNA Counts and differential expression by gene, exon, allele, splice isoform, & transcript Algorithms: TopHat, Rmake, Cufflinks, BayesASE miRBase 4 RefSeq hg19 Alignments & QC 3 Genetic variation: (SNVs, indels, CNVs) Algorithms: Rmake, BWA/SAMtools GATK, HGVS, VarScan bigwigs BAM files 1 2 annotations

  20. High reproducibility with low input A B 10ng R2=0.95 100ng Raw Reads per Gene (10ng) 1000ng Raw Reads per gene (1000ng) Genomics and Bioinformatics

  21. Lenalidomide induced interferon responses in primary myeloma cells Huang, Di Liberto, Chen-Kiang, unpublished

  22. 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

  23. IRF7 is also inhibited by FoxO, which protects myeloma cells IMiDkilling Huang, Di Liberto, Chen-Kiang, unpublished

  24. pG1 Sensitizes MCL Cells to IMiD Killing through synergistic reduction of IRF4 Di Liberto, Chen-Kiang, unpublished

  25. 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.

  26. 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

  27. Targeting CDK4/CDK6 in combination therapy PD 0332991 Partner agent (low dose, selective ) Weill-Cornell Mantle cell lymphoma Phase I single agent study Multiple 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--

  28. 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

  29. Acknowledgements Weill Cornell Medical College Xiangao Huang Maurizio Di Liberto David Chiron Adriana Rossi David Jayabalan Selina Chen-Kiang Ruben Niesvizky TomerMark Peter Martin John Leonard Scott Ely Chris Mason Olivier Elemento Celgene MohamadHussein Pfizer Sophia Randolph Broad Institute Anna Schinzel Bill Hahn Lymphoma Research Foundation Leukemia and Lymphoma Society Starr Cancer Consortium NIH/NCI Patients

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