Targeting the Cell Cycle in Lymphoma Therapy Selina Chen-Kiang Weill Cornell Medical College

CDK Inhibitors Non-Selective CDK4/CDK6 inhibitors Targeting multiple CDKs, some are transcription factors Flavopiridol CDK9 Daniciclib CDK7 Selective CDK4/CDK6 inhibitors PD (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 3 Melanoma LY Non-Small Cell Lung Carcinoma Breast Cancer

The Cell Cycle G1 S G2 M p21 p27 p57 Negative Go Positive Cyclin D + CDK4/6 pS-Rb-E2F Cyclin E + CDK2 pST-Rb E2F release p16 p15 p18 p19 CDK: Cyclin-Dependent Kinase p18 INK4c (CDKN2C ) mid-G1 checkpoint

Targeting CDK4/6 with PD (palbociclib) Selective CDK4/CDK6 inhibitor (IC 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 (IC 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 Ther Baughn et al., 2006, Cancer Research Menu et al., 2008, Cancer Research

Targeting CDK4/CDK6 in combination therapy Partner agent (low dose, selective ) CDK4/6 Inhibitor Weill-Cornell Mantle cell lymphomaPhase I single agent Multiple Myeloma Phase I/II PD-bortezomib-Dex Mantle cell lymphomaPhase I PD bortezomib 2014 Mantle cell lymphoma Phase I PD Ibrutinib Multiple myelomaPhase I PD Lenalidomide-Dex

Cyclin D + CDK4/6 PD 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 Reversible G1 S G2 M Go pS-Rb pG1 Hypothesis

Targeting CDK4/CDK6 in Recurrent MCL Single agent PD Phase I study Inhibition of CDK4/CDK6 by PD 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, et al Blood 2012

Phase I study of PD bortezomib in patients with recurrent MCL Biopsy ★ ★ ★ pG1 pG1-S Martin, Di Liberto, Leonard, et al, unpublished

Phase I study of PD 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 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 ( ng RNA) mRNA abundance Alternative splicing SNVs 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 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 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 homeostasis Redox homeostasis Cell migration

pG1 reprogramming MCL cells for ibrutinib inhibition of Bruton Tyrosine Kinase (BTK) Ibrutinib is effective in MCL. However, relapse is frequent and associated 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, BTK C481S mutation is absent in transient ibrutinib response (< 5months) or primary resistance (6/6) At least two mechanisms of Ibrutinib relapse - BTK C481S 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 BTK C481S BTK 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-  B 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 BTK 481S mutation at relapse from ibrutinib after a durable response in MCL BTK 481S 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-  B. 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 PI3K  inhibitor GS-1101 (idelalisib)

Induction of pG1 sustains the inactivation of AKT by PI3K  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 sensitization Cancer metabolism Mechanism-based combination therapy for MCL Targeting CDK4 with PD (palbociclib) in combination 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 Ely Chris Mason Steve Gross Olivier Elemento Tim McGraw Jihye Paik Jeff Sharman Patients NIH/NCI V Foundation Lymphoma Research Foundation Leukemia and Lymphoma Society Starr Cancer Consortium The Team