Targeting the Cell Cycle in Lymphoma Therapy

Selina Chen-KiangWeill Cornell Medical College

CDK Inhibitors

Non-Selective CDK4/CDK6 inhibitorsTargeting multiple CDKs, some are transcription factors • Flavopiridol CDK9• Daniciclib CDK7

Selective CDK4/CDK6 inhibitors • PD 0332991 (palbociclib)

Lymphoma, Myeloma Solid tumors-Breast Cancer (ER+/HER-) –Breakthrough therapy, combination with letrozole, Phase 3

• LEE011 Breast Cancer –combination with letrozole and BYL719,

Phase 3Melanoma

• LY2835219Non-Small Cell Lung CarcinomaBreast Cancer

The Cell Cycle

G1S

G2

M

p21p27p57

Negative Go

Positive

Cyclin D + CDK4/6

pS-Rb-E2F

Cyclin E + CDK2

pST-Rb E2F release

p16p15p18p19

CDK: Cyclin-Dependent Kinasep18INK4c (CDKN2C)

mid-G1 checkpoint

Targeting CDK4/6 with PD 0332991 (palbociclib)

• Selective CDK4/CDK6 inhibitor (IC50 11 nM) • Orally bioavailable pyridopyrimidine• Competing with ATP for binding to the kinase site of CDK4/CDK6 • Induces early G1 arrest • Reversible• Low in toxicity • Selectively and potently inhibits CDK4/6 phosphorylation of Rb in primary human myeloma cells (IC50 60 nM )

• Inhibits tumor growth in the NOD-SCID human myeloma xenograft models and the immune-competent mouse 5T models

Fry et al., 2004, Mol Cancer TherBaughn et al., 2006, Cancer ResearchMenu et al., 2008, Cancer Research

Targeting CDK4/CDK6 in combination therapy

Partner agent (low dose, selective )

CDK4/6 Inhibitor

Weill-Cornell

Mantle cell lymphoma Phase I single agent Multiple Myeloma Phase I/II PD-bortezomib-Dex Mantle cell lymphoma Phase I PD 0332991-bortezomib

2014Mantle cell lymphoma Phase I PD 0332991-Ibrutinib Multiple myeloma Phase I PD 0332991-Lenalidomide-Dex

Cyclin D + CDK4/6

PD 0332991

G1

S

G2

M

Go

pS-Rb

Prolonged early G1 arrest (pG1) Hypothesis

Prolonged inhibition of CDK4/6

Prolonged early G1 arrest (pG1)Expression of only genes programmed for early G1

Sensitizing tumor cells to cytotoxic killing

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

pG1 Hypothesis

Targeting CDK4/CDK6 in Recurrent MCL

Single agent PD 0332991 Phase I study

• Inhibition of CDK4/CDK6 by PD 0332991 leads to prolonged G1 arrest (pG1) and increased tumor-specific cell death in MCL (n=17)

• PD 0332991 (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, et al Blood 2012

Phase I study of PD 0332991 + bortezomib in patients with recurrent MCL

Biopsy ★ ★ ★pG1 pG1-S

Martin, Di Liberto, Leonard, et al, unpublished

Phase I study of PD 0332991 + bortezomib in patients with recurrent MCL

% change in tumor size (by patient)

Martin, Leonard, unpublished

M. Di Liberto, D. Chiron, C. Mason, P. Martin, J. Leonard, S. Ely, unpublished

Inhibition of CDK4/6 induces early G1 arrest in MCL cells of both responders and non-responders initially.

Can we identify genes that differentiate sensitivity from resistance to targeting CDK4 in combination with bortezomib?

Cyclin D + CDK4/6

PD 0332991

G1

S

G2

M

Go

pS-Rb

Prolonged early G1 arrest (pG1) Hypothesis

Prolonged inhibition of CDK4/6

Prolonged early G1 arrest (pG1)Expression of only genes programmed for early G1

Sensitizing tumor cells to cytotoxic killing

Longitudinal Integrative analysis of whole exome-sequencing (WES) and whole transcriptome-sequencing (WTS) of serial biopsies, using cheek swab as a control.

Integrative WES and WTS analysis

CD5+CD19+ isolation

WTS (100-100ng RNA)

mRNA abundance

Alternative splicingSNV

s

Lymph node biopsy

WES (50ng DNA)

CNV

Only 1% of the genes that were repressed in (pG1, day 8) in clinically responding patients (R) were up-regulated in pG1 non-responding patients (NR). Candidate biomarkers for the PD 0332991-bortezomib therapy?

Chiron, Di Liberto, Mason, Martin, et al, unpublished

Differential regulated genes

• Glucose homeostasis• Redox homeostasis• Cell migration

Targeting CDK4 in combination with bortezomib in MCL

• At the optimal PD 0332991 concentration and reduced bortzomib 1 CR, 1 PR, 2 near PR (43% reduction), 1 SD, 1 PD.

• Inhibition of CDK4 induces early G1 arrest that controls cell cycle gene expression in all MCL patients initially.

• MCL cells express CDK4 but not CDK6, cyclin D1 but not D2 or D3

• CDK4 is a stable target- no mutation in CDK4 detected

• A small number of genes are oppositely regulated in pG1 (day 8 vs day 0) in responders vs non-responders – candidate biomarkers for targeting CDK4 in combination with bortezomib.

Glucose homeostasisRedox homeostasisCell migration

pG1 reprogramming MCL cells for ibrutinib inhibition of Bruton Tyrosine Kinase (BTK)

Ibrutinib is effective in MCL. However, relapse is frequent andassociated with aggressive proliferation and poor prognosis

Relapse-specific C481S mutation in BTK in MCL--Longitudinal integrative WES and WTS analysis

Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

Relapse-specific C481S mutation in BTK in MCL--Longitudinal integrative WES and WTS analysis

• However, BTKC481S mutation is absent in transient ibrutinib response (< 5months) or primary resistance (6/6)

At least two mechanisms of Ibrutinib relapse -

• BTKC481S mutation is detected in durable ibrutinib response (>14 or 30 months, 2/2.

Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

BTK C481S mutation undetected before Ibrutinib relapse

• BTK is inactivated by ibrutinib in MCL cells of both sensitive and resistant patients in vivo• PI3K-AKT is activated in ibrutinib resistance

BTKC481SBTK

Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

Sensistive Resistant Sensitive Relapse

Induction of pG1 by CDK4 inhibition reprograms MCL cells for killing by ibrutinib via inhibition of BTK and AKT

Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

pG1 inhibits NF-kB activation in BCR signaling

Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

Overriding ibrutinib resistance by cell cycle reprogramming

• Integrative WES/WTS identified a BTK481S mutation at relapse from ibrutinib after a durable response in MCL

• BTK481S mutation is absent in transient ibrutinib response or primary resistance, suggesting addition mechanisms for resistance.

• BTK and AKT are concurrently activated in ibrutinib resistance.

• Ibrutinib inactivates BTK in MCL cells of resistant patients.

• Enhanced proliferation of MCL cells at relapse

• pG1 sensitizes resistant MCL cells to Ibrutinib killing via cooperative inactivate BTK and AKT, and inactivation of NF-kB.

Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

pG1 reprogramming of MCL cells for PI3K inhibition regardless of C481S BTK mutation

Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

pG1 reprograms MCL cells for PI3K inhibitor killing

D. Chiron, M. Di Liberto, et al, Cell Cycle, 2013

PI3Kd inhibitorGS-1101(idelalisib)

Induction of pG1 sustains the inactivation of AKT by PI3Kd inhibitor in MCL cells

D. Chiron, M. Di Liberto, et al, Cell Cycle, 2013

pG1 reprogramming for PI3K inhibition eradicates ibrutinib-resistant lymphoma cells

independent of BTK mutation

Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

Cell death

Live cells

pG1 reprogramming of MCL cells for PI3K inhibition independent of C481S BTK mutation in

MCL

Future Directions-

• Mechanism of pG1 sensitizationCancer metabolism

• Mechanism-based combination therapy for MCLTargeting CDK4 with PD 0332991(palbociclib) incombination with ibrutinib

Targeting CDK4 in combination with PI3K inhibitor

• Mechanism of resistance

• Identification of biomarkers via longitudinal integrative WES/WTS and targeted sequencing

Maurizio Di Liberto Xiangao Huang David Chiron David Jayabalan Selina Chen-Kiang

John Leonard Ruben Niesvizky Peter Martin Tomer Mark Adriana Rossi

Lewis Cantley Scott ElyChris Mason Steve GrossOlivier Elemento Tim McGrawJihye Paik Jeff Sharman

Patients

NIH/NCIV FoundationLymphoma Research FoundationLeukemia and Lymphoma SocietyStarr Cancer Consortium

The Team