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