Targeting the Cell Cycle in Lymphoma Therapy Selina

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

CDK Inhibitors Non-Selective CDK 4/CDK 6 inhibitors Targeting multiple CDKs, some are transcription factors • Flavopiridol CDK 9 • Daniciclib CDK 7 Selective CDK 4/CDK 6 inhibitors • PD 0332991 (palbociclib) Lymphoma, Myeloma Solid tumors-Breast Cancer (ER+/HER-) –Breakthrough therapy, combination with letrozole, Phase 3 • LEE 011 Breast Cancer –combination with letrozole and BYL 719, Phase 3 Melanoma • LY 2835219 Non-Small Cell Lung Carcinoma Breast Cancer

The Cell Cycle Positive Negative Go Cyclin D + CDK 4/6 M G 1 G 2 p. S-Rb-E 2 F Cyclin E + CDK 2 S p. ST-Rb CDK: Cyclin-Dependent Kinase p 18 INK 4 c (CDKN 2 C) E 2 F release p 16 p 15 p 18 p 19 p 21 p 27 p 57 mid-G 1 checkpoint

Targeting CDK 4/6 with PD 0332991 (palbociclib) • • • Selective CDK 4/CDK 6 inhibitor (IC 50 11 n. M) Orally bioavailable pyridopyrimidine Competing with ATP for binding to the kinase site of CDK 4/CDK 6 Induces early G 1 arrest Reversible Low in toxicity • Selectively and potently inhibits CDK 4/6 phosphorylation of Rb in primary human myeloma cells (IC 50 60 n. M ) • Inhibits tumor growth in the NOD-SCID human myeloma xenograft models and the immune-competent mouse 5 T models Fry et al. , 2004, Mol Cancer Ther Baughn et al. , 2006, Cancer Research Menu et al. , 2008, Cancer Research

Targeting CDK 4/CDK 6 in combination therapy CDK 4/6 Inhibitor Partner agent (low dose, selective ) Weill-Cornell Mantle cell lymphoma Multiple Myeloma Mantle cell lymphoma Phase I single agent Phase I/II PD-bortezomib-Dex Phase I PD 0332991 -bortezomib 2014 Mantle cell lymphoma Phase I PD 0332991 -Ibrutinib Multiple myeloma Phase I PD 0332991 -Lenalidomide-Dex

Prolonged early G 1 arrest (p. G 1) Hypothesis Go Cyclin D + CDK 4/6 M G 1 G 2 PD 0332991 p. S-Rb S Prolonged inhibition of CDK 4/6 Prolonged early G 1 arrest (p. G 1) Expression of only genes programmed for early G 1 Sensitizing tumor cells to cytotoxic killing

p. G 1 Hypothesis Go Cyclin D + CDK 4/6 M G 1 G 2 PD 0332991 Reversible p. S-Rb S Release of prolonged early G 1 block Cell cycle synchronization incomplete restoration of scheduled gene expression Further sensitizing tumor cells to cytotoxic killing

Targeting CDK 4/CDK 6 in Recurrent MCL Single agent PD 0332991 Phase I study • Inhibition of CDK 4/CDK 6 by PD 0332991 leads to prolonged G 1 arrest (p. G 1) 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 ★ ★ p. G 1 -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

Inhibition of CDK 4/6 induces early G 1 arrest in MCL cells of both responders and non-responders initially. Can we identify genes that differentiate sensitivity from resistance to targeting CDK 4 in combination with bortezomib? M. Di Liberto, D. Chiron, C. Mason, P. Martin, J. Leonard, S. Ely, unpublished

Prolonged early G 1 arrest (p. G 1) Hypothesis Go Cyclin D + CDK 4/6 M G 1 G 2 PD 0332991 p. S-Rb S Prolonged inhibition of CDK 4/6 Prolonged early G 1 arrest (p. G 1) Expression of only genes programmed for early G 1 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 Lymph node biopsy CD 5+CD 19+ isolation WES (50 ng DNA) CNV SNVs WTS (100 -100 ng RNA) m. RNA abundance Alternative splicing

Only 1% of the genes that were repressed in (p. G 1, day 8) in clinically responding patients (R) were up-regulated in p. G 1 nonresponding patients (NR). Candidate biomarkers for the PD 0332991 -bortezomib therapy? Differential regulated genes • Glucose homeostasis • Redox homeostasis • Cell migration Chiron, Di Liberto, Mason, Martin, et al, unpublished

Targeting CDK 4 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 CDK 4 induces early G 1 arrest that controls cell cycle gene expression in all MCL patients initially. • MCL cells express CDK 4 but not CDK 6, cyclin D 1 but not D 2 or D 3 • CDK 4 is a stable target- no mutation in CDK 4 detected • A small number of genes are oppositely regulated in p. G 1 (day 8 vs day 0) in responders vs non-responders – candidate biomarkers for targeting CDK 4 in combination with bortezomib. Glucose homeostasis Redox homeostasis Cell migration

p. G 1 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 C 481 S mutation in BTK in MCL --Longitudinal integrative WES and WTS analysis Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

Relapse-specific C 481 S mutation in BTK in MCL --Longitudinal integrative WES and WTS analysis BTK C 481 S mutation undetected before Ibrutinib relapse At least two mechanisms of Ibrutinib relapse - • BTKC 481 S mutation is detected in durable ibrutinib response (>14 or 30 months, 2/2. • However, BTKC 481 S mutation is absent in transient ibrutinib response (< 5 months) or primary resistance (6/6) Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

• BTK is inactivated by ibrutinib in MCL cells of both sensitive and resistant patients in vivo • PI 3 K-AKT is activated in ibrutinib resistance BTK Sensistive Resistant BTKC 481 S Sensitive Relapse Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

Induction of p. G 1 by CDK 4 inhibition reprograms MCL cells for killing by ibrutinib via inhibition of BTK and AKT Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

p. G 1 inhibits NF-k. 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 481 S mutation at relapse from ibrutinib after a durable response in MCL • BTK 481 S 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 • p. G 1 sensitizes resistant MCL cells to Ibrutinib killing via cooperative inactivate BTK and AKT, and inactivation of NF-k. B. Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

p. G 1 reprogramming of MCL cells for PI 3 K inhibition regardless of C 481 S BTK mutation Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

p. G 1 reprograms MCL cells for PI 3 K inhibitor killing PI 3 Kd inhibitor GS-1101 (idelalisib) D. Chiron, M. Di Liberto, et al, Cell Cycle, 2013

Induction of p. G 1 sustains the inactivation of AKT by PI 3 Kd inhibitor in MCL cells D. Chiron, M. Di Liberto, et al, Cell Cycle, 2013

p. G 1 reprogramming for PI 3 K inhibition eradicates ibrutinib-resistant lymphoma cells independent of BTK mutation Cell death Live cells Chiron, Di Liberto, Martin et al, Cancer Discovery, 2014

p. G 1 reprogramming of MCL cells for PI 3 K inhibition independent of C 481 S BTK mutation in MCL

Future Directions • Mechanism of p. G 1 sensitization Cancer metabolism • Mechanism-based combination therapy for MCL Targeting CDK 4 with PD 0332991(palbociclib) in combination with ibrutinib Targeting CDK 4 in combination with PI 3 K inhibitor • Mechanism of resistance • Identification of biomarkers via longitudinal integrative WES/WTS and targeted sequencing

The Team Maurizio Di Liberto Xiangao Huang David Chiron David Jayabalan Selina Chen-Kiang John Leonard Peter Martin Adriana Rossi Ruben Niesvizky Tomer Mark Lewis Cantley Chris Mason Olivier Elemento Jihye Paik Scott Ely Steve Gross Tim Mc. Graw Jeff Sharman Patients NIH/NCI V Foundation Lymphoma Research Foundation Leukemia and Lymphoma Society Starr Cancer Consortium