Selective inhibition of CDK4/CDK6 sensitizes bone marrow myeloma cells for killing by proteasome inhibitors carfilzomib and PR-047 through cell cycle-dependent expression of pro-apoptotic Noxa and Bim.

Abstract Abstract 2854 Poster Board II-830 Targeting the cell cycle in combination with cytotoxic killing is a rational approach to cancer therapy. Progression in multiple myeloma (MM) stems from both loss of apoptotic control in the bone marrow (BM) microenvironment and dysregulation of the cyclindependent kinases (CDK)4 and CDK6, which precedes uncontrolled proliferation of myeloma cells in vivo in particular during relapse. This reinforces the critical importance of targeting CDK4/CDK6 in MM. Through selective and reversible inhibition of CDK4/CDK6 with PD 0332991, the only known CDK4/6-specific inhibitor, we have recently developed a novel strategy to sensitize primary myeloma cells for cytotoxic killing by diverse cytotoxic drugs. These include carfilzomib (PR-171), an irreversible selective inhibitor of the chymotrypsin-like activity of the proteasome, and PR-047, an orally bioavailable analog of carfilzomib. We showed that induction of prolonged early G1 arrest following inhibition of CDK4/CDK6 markedly enhances cytotoxic killing of primary BM myeloma cells by either carfilzomib or PR-047 despite protection by BM stromal cells. The enhancement of cytotoxic killing is further augmented during synchronous S phase entry upon removal of PD 0332991 subsequent to induction of prolonged G1 arrest in myeloma cell lines. In both cases, the enhancement in carfilzomib (or PR-047) mediated killing is not associated with cell cycle regulation of the proteasome activity. It is caspase-dependent, requiring only a brief (one hour) exposure to the proteasome inhibitor at concentrations as low as 30 nM. This killing is mediated by synergistic and rapid induction of mitochondrial membrane depolarization and activation of downstream caspase-9. Further, it is apparently initiated by cell cycle-dependent expression of the pro-apoptotic BH3-only proteins, which neutralize the anti-apoptotic Bcl-2 family proteins upstream of mitochondrial depolarization. Bim is upregulated during early G1 arrest to neutralize the anti-apoptotic MCL-1 and Bcl-2. By contrast, Noxa is silenced in G1 but dramatically upregulated in S phase, in particular when combined with carfilzomib. Importantly, targeting CDK4/CDK6 with PD 0332991 in combination with either carfilzomib or PR-047 leads to complete eradication of myeloma cells ex vivo, in contrast to the combination of PD 0332991 with other proteasome inhibitors. Selective inhibition of CDK4/CDK6 in combination with carfilzomib (or PR-047), therefore, not only halts tumor cell proliferation but also potently induces synergistic killing that is likely to profoundly inhibit cell cycle reentry and self-renewal in MM. PD 0332991 is a small molecule with bio-availability and proven tumor suppressing activity in both human myeloma xenograft and immunocompetent mouse myeloma models. It is well tolerated in humans as indicated by the ongoing Phase I/II clinical trials in myeloma and previous phase I trials in mantle cell lymphoma and solid tumors. Evidence from Phase 2 trials of carfilzomib indicates that it is also well tolerated, in fact, the peripheral neuropathy that is commonly observed with the proteasome inhibitor bortezomib appears to be less severe and possibly less frequent. Mechanism-based targeting of CDK4/6 in combination with selective proteasome inhibitors, like carfilzomib and PR-047, thus represents a new and promising therapeutic strategy for multiple myeloma and potentially other hematopoietic malignancies. Disclosures: Off Label Use: PD 0332991 is going to be used as a CDK4/6-specific inhibitor. Parlati:Proteolix, Inc.: Employment, Equity Ownership. Aujay:Proteolix, Inc.: Employment, Equity Ownership. Niesvizky:Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Seattle Genetics, Inc: Research Funding; Proteolix: Research Funding, data monitoring committee.

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Induction of sequential G1 arrest and synchronous S phase entry by reversible CDK4/CDK6 inhibition sensitizes myeloma cells for cytotoxic killing through loss of IRF-4.

Blood ◽

10.1182/blood.v114.22.299.299 ◽

2009 ◽

Vol 114 (22) ◽

pp. 299-299

Author(s):

Xiangao Huang ◽

Maurizio Di Liberto ◽

Scott Ely ◽

David S Jayabalan ◽

Isan Chen ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Research Funding ◽

Cell Cycle Progression ◽

Specific Inhibitor ◽

S Phase ◽

G1 Arrest ◽

Myeloma Cells ◽

Cycle Dependent ◽

Phase Entry

Abstract Abstract 299 Dysregulation of the cell cycle is a hallmark of cancer. However, targeting the cell cycle in cancer therapy has only been modestly successful since broad-spectrum cyclindependent kinase (CDK) inhibitors lack specificity and are highly toxic. The critical importance of controlling CDK4/CDK6 in cancer treatment is further exemplified by recent evidence of prominent CDK4/CDK6 dysregulation in human cancers, including breast cancer, metastatic lung adenocarcinoma, glioblastoma, mantle cell lymphoma and multiple myeloma (MM). To advance mechanism-based targeting of the cell cycle in cancer, we have developed a novel strategy that both inhibits cell cycle progression and enhances cytotoxic killing in tumor cells using PD 0332991(PD), the only known CDK4/CDK6-specific inhibitor that is also reversible, potent and orally bioavailable. We demonstrated by BrdU pulse-labeling that inhibition of CDK4/CDK6 with PD in primary bone marrow (BM) myeloma cells and human myeloma cell lines (HMCL) (IC50 60nM) leads to a complete early G1 arrest in the absence of apoptosis and upon release of the G1 block, synchronous cell cycle progression to S phase. Furthermore, prolonged early G1 arrest enhances cytotoxic killing of MM cells by diverse clinically relevant drugs at low dose, including bortezomib, carfilzomib (PR-171) and dexamethasone, and this is dramatically augmented during synchronous S phase entry. The enhancement of cytotoxic killing in either G1 arrest or synchronous S phase entry is sustained in the presence of BM stromal cells. This killing is caspase-dependent and triggered by the loss of mitochondrial outer membrane potential and activation of the intrinsic apoptosis pathway. Time course studies of cell cycle-specific gene expression by expression profiling, quantitative real time RT-PCR and immunoblotting further revealed that the expression of IRF-4, essential for normal plasma cell differentiation and myeloma cell survival, is strictly cell cycle-dependent: elevated in G1 and markedly declined in S phase. The IRF-4 protein is also markedly reduced (50%) by bortezomib treatment, resulting in a combined 5-fold reduction in S phase. This suggests that differential enhancement of cytotoxic killing in G1 arrest and S phase is mediated by cell cycle-dependent IRF-4 expression. Indeed, shRNA interference confirms that by antagonizing mitochondrial depolarization, IRF-4 is required to protect myeloma cells from cell cycle-dependent enhancement of bortezomib killing. By timely administration and discontinuation of PD treatment, we have further demonstrated in a human MM 1.S. xenograft myeloma model that it is feasible to induce sequential G1 arrest and synchronous S phase in vivo. This leads to synergistic tumor suppression through amplification of bortezomib killing of myeloma cells, but not normal BM cells. As PD is orally bio-available, specific and low in toxicity, our novel strategy has been implemented in the first phase I/II multi-center clinical trial targeting CDK4/CDK6 with PD in combination with bortezomib and dexamethasone in MM. Preliminary bone marrow immunohistochemistry demonstrates PD preferentially and completely inhibits CDK4/CDK6-specific phosphorylaton of Rb and DNA replication in tumor cells, but not other bone marrow cells in all patients. One patient achieved VGPR (12.5%) while 1 patient each achieved MR and SD respectively for an ORR 25% (Niesvizky et al, submitted). Collectively, our preclinical and clinical data indicate, for the fist time, that selective targeting of CDK4/CDK6 in combination therapy is a promising mechanism-based therapy for MM and potentially other cancers. Disclosures: Off Label Use: PD 0332991 is going to be used as a CDK4/6-specific inhibitor.. Chen:Pfizer, Inc.: Employment, Equity Ownership. Wilner:Pfizer, Inc.: Employment, Equity Ownership. Niesvizky:Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Seattle Genetics, Inc: Research Funding; Proteolix: Research Funding, data monitoring committee. Chen-Kiang:Pfizer Inc.: Research Funding.

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Synergistic Loss of IRF4 and Induction of IRF7 Sensitizes Primary Myeloma Cells to IMiD Killing by IFNβ in Prolonged Early G1 Arrest Induced by CDK4/CDK6 Inhibition

Blood ◽

10.1182/blood.v120.21.572.572 ◽

2012 ◽

Vol 120 (21) ◽

pp. 572-572

Author(s):

Xiangao Huang ◽

David Jayabalan ◽

Maurizio Di Liberto ◽

Jackson D Harvey ◽

Anna C. Schinzel ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Research Funding ◽

S Phase ◽

G1 Arrest ◽

Combination Therapies ◽

Employment Equity ◽

Myeloma Cells ◽

Sequential Combination ◽

Equity Ownership

Abstract Abstract 572 The immunomodulatory drugs (IMiD) lenalidomide (Len) and pomalidomide (Pom) are effective therapies for multiple myeloma (MM), improving both disease-free and overall survival in relapsed or refractory MM with a favorable toxicity profile. However, MM remains incurable due to the eventual development of drug resistance, and the mechanism of IMiD action is not well understood. Developing novel mechanism-based combination therapies and defining the mechanism of IMiD action are thus timely and necessary. By inducing prolonged early G1 arrest (pG1) through inhibition of CDK4/CDK6 with a highly specific, potent and reversible inhibitor, PD 0332991, we have now developed a novel sequential combination therapy that both inhibits proliferation of MM cells and sensitizes them to IMiD killing. Our rationales are as follows: 1) cell cycle dysregulation underlies unrestrained proliferation of MM cells in relapse, as in other cancers; 2) dysregulation of CDK4 or CDK6, which drives cell cycle progression through early G1, precedes the increase in proliferation in MM progression; 3) inhibition of CDK4/CDK6 by PD 0332991 arrests the cell cycle in early G1 in all Rb-positive primary bone marrow myeloma cells (BMMM)s, ex vivo and in MM patients in a phase I/II clinical trial; 4) pG1 sensitizes MM cells to killing by diverse clinically relevant agents in pG1 and in subsequent synchronous S phase entry after the release of early G1 block. Our replication kinetics data show that Len induces a dose-dependent late G1 arrest by 48 hours in MM cell lines, but apoptosis and reduction of viable cells is not evident until 72 hours, and appears independent of late G1 arrest. However, killing by Len or Pom is markedly accelerated and enhanced in pG1 induced by PD 0332991 for 24 hours (twice the time needed to induce G1 arrest in MM cells). Importantly, acceleration of early G1 arrest by PD 0332991 sensitizes BMMMs to killing by Len (16/20 cases) and by Pom (3/4 cases) despite protection by bone marrow stromal cells. Thus, IMiDs preferentially target MM cells arrested in early G1, in contrast to most cytotoxic agents, which primarily target tumor cells in S phase, thereby providing a strong rationale for improving IMiD therapy by prior induction of pG1. Whole transcriptome sequencing (WTS, RNA-Seq) and q-PCR analyses of BMMMs further revealed that correlating with Len killing, genes of the interferon (IFN) signaling pathway are coordinately and prominently induced by Len, and by Len and pG1 in synergy, but not by pG1 alone. These data provide the first direct evidence for induction of IFN by IMiD and enhancement by pG1 in BMMMs, suggesting a pivotal role for IFN in mediating IMiD killing in synergy with pG1 in MM. pG1 halts scheduled gene expression in early G1 and prevents the expression of genes programmed for other cell cycle phases, as we have demonstrated by WTS in conjunction with q-PCR and immunoblot analyses. Synergistic induction of IFN may stem from the imbalance in gene expression in pG1 and its interplay with IMiD signaling. Indeed, pG1 activates the synthesis of IRF4, an essential survival factor of MM cells, but markedly amplifies the loss of IRF4 protein induced by Len or Pom through transcriptional and posttranscriptional mechanisms. This leads to induction of IRF7, a primary and direct target of IRF4 repression, and IFNb, which is activated by IRF7. The essential roles of IRF4 and IRF7 in mediating IMiD killing and pG1 sensitization by IFNb signaling have been further confirmed by shRNA silencing in representative MM cell lines that have been characterized by WTS and shown to recapitulate pG1 sensitization of Len and Pom killing. In summary, we have developed a novel sequential combination therapy that both inhibits proliferation and enhances IMiD killing of MM cells by induction of pG1 through selective CDK4/CDK6 inhibition. This therapy combines oral compounds with excellent toxicity profiles and acts in pG1; thus, it may serve as a maintenance therapy to both control tumor expansion and prevent self-renewal. This study presents the first WTS-validated therapeutic strategy in MM, and demonstrates, for the first time, that the IRF4-IRF7-IFNb pathway mediates IMiD killing and pG1 amplifies it. Further investigation may uncover novel molecular therapeutic targets and biomarkers for genome-based patient stratification for cell cycle-based IMiD combination therapies. Disclosures: Huang: Celgene, Corp: Research Funding. Off Label Use: PD 0332991 is a CDK4/CDK6 selective inhibitor Lenalidomide is an Immunomodulatory drug. Mark:Millenium Inc.: Speakers Bureau; Celgene Corp: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Hussein:Celgene, Corp: Employment, Equity Ownership. Randolph:Pfizer, Inc.: Employment, Equity Ownership. Niesvizky:Onyx, Millemium, Celgene. Speakers bureau: Millenium and Celgene: Consultancy, Research Funding. Chen-Kiang:Bristol-Myers Squibb: Consultancy; Pfizer, Inc.: Research Funding.

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Induction of Early G1-Arrest by CDK4/CDK6 Inhibition Sensitizes Mantle Cell Lymphoma Cells to Selective PI3Kδ Inhibition by GS-1101 Through Enhancing the Magnitude and Duration of p-AKT Inhibition

Blood ◽

10.1182/blood.v120.21.791.791 ◽

2012 ◽

Vol 120 (21) ◽

pp. 791-791

Author(s):

David Chiron ◽

Peter Martin ◽

Maurizio Di Liberto ◽

Xiangao Huang ◽

Scott A Ely ◽

...

Keyword(s):

Cell Cycle ◽

Board Of Directors ◽

Research Funding ◽

Human Cancer ◽

Single Agent ◽

Specific Inhibitor ◽

Selective Inhibition ◽

G1 Arrest ◽

Advisory Committees ◽

Indolent Lymphomas

Abstract Abstract 791 The phosphatidylinositol-3-kinase (PI3K) signaling pathway is constitutively activated at a high frequency in human cancer. As the first PI3K-specific inhibitor, GS-1101 (CAL-101) selectively targets p110δ (PI3Kδ). It has emerged as a promising single-agent therapy for chronic lymphocytic leukemia and indolent lymphomas. For aggressive non-Hodgkin lymphoma such as mantle cell lymphoma (MCL), efficacy has been observed but the extent and duration of tumor control has been modest, encouraging development of mechanism-based combination therapy. Since cell cycle dysregulation is frequently amplified in relapse/refractory diseases, we hypothesize that targeting the cell cycle may sensitize non-indolent lymphomas to inhibition of PI3Kδ by GS-1101, and test this hypothesis in MCL based on the following: 1) dysregulated cyclin D1 and CDK4 expression is a primary cause for unrestrained cell cycle progression and proliferation in MCL; 2) by induction of prolonged early G1 arrest (pG1) through selective inhibition of CDK4/CDK6 with PD 0332991 we have recently developed a novel strategy that both inhibits proliferation of tumor cells and sensitizes them to cytotoxic killing; and 3) induction of pG1 by PD 0332991 demonstrated encouraging clinical activity and an excellent toxicity profile in a phase I single-agent study in MCL. To test this hypothesis, we first demonstrate by whole transcriptome sequencing (WTS, RNA-Seq) that PI3Kδ is the predominant PI3K catalytic subunit expressed, and that only few non-synonymous single-nucleotide variants are present in the coding sequences of genes in the PI3K-AKT pathway in primary MCL tumor cells (N=10), including the analyzed PI3K subunits, AKT1, PTEN and PDK1. Moreover, despite a multitude of genetic abnormalities, mutations in the coding regions of core G1-cell cycle genes, including cyclin D1, CDK4, and CDK4/6 inhibitors CDKN2C (p18INK4c) and CDKN2D (p19INK4d) are also rare in primary MCL cells. Analysis of protein expression by immunoblotting has confirmed the WTS analysis and further demonstrated that AKT is constitutively phosphorylated on serine 473 by mTORC2 (p-AKT) downstream of PI3K in primary MCL cells. These findings reinforce the rationale for combining selective inhibition of PI3Kδ with selective inhibition of CDK4/CDK6 in targeting MCL. GS-1101 treatment does not result in cell cycle arrest in proliferating MCL cell lines (N=6), including Jeko-1 and MAVER-1 cells, which recapitulate the expression of PI3K and G1 cell cycle genes in primary MCL cells based on WTS and immunoblot analyses. GS-1101 transiently reduces p-AKT in proliferating MCL cells, confirming that MCL cells are intrinsically responsive to GS-1101 but also implying a potential mechanism for resistance. Prior induction of pG1 by selective inhibition of CDK4/CDK6 with PD 0332991 reduces p-AKT, amplifies and sustains the loss of p-AKT, and enhances apoptosis in response to GS-1101. Finally, validating the G1 cell cycle-dependence of GS-1101 killing, all primary MCL cells tested are responsive to PI3Kδ inhibition by GS-1101 when they are arrested in early G1 ex vivo in stromal co-culture. This loss of viability is accelerated at a reduced GS-1101 concentration when G1 arrest is accelerated by PD 0332991, despite the presence of cytokines and growth factors that are known to activate PI3K. This study presents the first sequential combination of selective inhibition of CDK4/CDK6 with a selective partner, the PI3Kδ-specific inhibitor GS-1101, in primary human cancer cells, and the first WTS-validated therapeutic strategy that leads to sensitization of MCL cells by cell cycle control and PI3K inhibition. Our data demonstrate, for the first time, that the magnitude and duration of GS-1101 killing is G1 cell cycle-dependent, and suggest a strategy to sensitize proliferating lymphoma cells to selective PI3Kδ inhibition by induction of early G1-arrest through CDK4/CDK6-specific inhibition. Disclosures: Off Label Use: PD 0332991 is a CDK4/CDK6 selective inhibitor GS-1101 is a PI3K-delta specific inhibitor. Martin:Cephalon: Consultancy; Celgene: Consultancy; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Huang:Celgene, Corp: Research Funding. Lannutti:Gilead Sciences Inc: Employment. Leonard:Gilead/Calistoga: Consultancy, Honoraria. Mason:HESI Advisory Board: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MorganStanley: Consultancy; Shriner's Hospital: Consultancy; Illumina, Inc.: Ownership interest (inc stock options) in a publicly traded company, Ownership interest (inc stock options) in a publicly traded company Other; PerkinElmer: Consultancy. Chen-Kiang:Bristol Myers Squibb: Consultancy; Pfizer: Research Funding.

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Lenalidomide Targets Myeloma Cells Preferentially During Prolonged Early G1 Arrest but Not Synchronization Into S Phase by Selective and Reversible Inhibition of CDK4/CDK6 through Loss of IRF-4

Blood ◽

10.1182/blood.v116.21.449.449 ◽

2010 ◽

Vol 116 (21) ◽

pp. 449-449 ◽

Cited By ~ 1

Author(s):

Xiangao Huang ◽

Maurizio Di Liberto ◽

David Jayabalan ◽

Mohamad Hussein ◽

Sophia Randolph ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Proteasome Inhibitor ◽

Research Funding ◽

Phase 1 ◽

Proteasome Inhibitors ◽

S Phase ◽

G1 Arrest ◽

Myeloma Cells ◽

Sequential Combination

Abstract Abstract 449 Dysregulation of cyclin-dependent kinase (CDK)4 and CDK6 is common in human cancer and precedes unrestrained proliferation of tumor cells in multiple myeloma (MM) patients, especially during refractory relapse. MM remains incurable due to the eventual development of drug resistance despite initial response to two main lines of therapy with proteasome inhibitors and immunomodulatory drugs. Therapeutic strategies that both control the cell cycle and enhance cytotoxic killing are thus urgently needed in MM. Although targeting the cell cycle in cancer therapy has only been modestly successful because broad-spectrum CDK inhibitors lack specificity and are highly toxic, we have recently developed such a therapy by selective inhibition of CDK4/CDK6 in sequential combination with proteasome inhibitors. Using PD 0332991, the only known selective inhibitor of CDK4/CDK6 that is also potent, reversible and bioavailable, we have demonstrated that 1) induction of prolonged early G1 arrest by inhibition of CDK4/CDK6 markedly enhances the killing of primary BM myeloma cells by proteasome inhibitors despite stromal protection, and 2) release from the G1 block upon PD 0332991 withdraw leads to synchronous progression to S phase, which further augments cytotoxic killing of MM cells. To optimize targeting CDK4/CDK6 in MM, we have investigated lenalidomide as an alternative cytotoxic partner by first determining its cell cycle targeting specificity, taking advantage of the exceptional precision and efficiency with which PD 0332991 induces early G1 arrest and cell cycle synchronization. We show by simultaneous analyses of BrdU pulse labeling and DNA content per cell that lenalidomide preferentially targets MM cells following prolonged early G1 arrest by PD 0332991 pretreatment for 24 hours (twice the time needed to induce G1 arrest in MM cells), but not those synchronized into S phase after release from the G1 block. This is distinct from proteasome inhibitors (bortezomib, carfilzomib and ONX-0921), which preferentially target MM cells synchronized into S phase over those arrested in G1. MM cells in G2/M appear to be less sensitive to both proteasome inhibitors and lenalidomide. However, these cells are rendered sensitive to these compounds upon cell cycle reentry through inhibition of CDK4/CDK6 and induction of early G1 arrest. Time course studies of DNA replication further reveal that lenalidomide alone (3 uM, daily) induces G1 arrest by 48 hours, which precedes evidence of apoptosis and reduction of viable cells at 72 hours. While the magnitude of G1 arrest induced by lenalidomide is dose-dependent (1-50 uM), the timing of cytotoxic killing does not vary. Prior induction of prolonged early G1 arrest by PD 0332991 (24 hours) enhances (> 5-fold) and also accelerates lenalidomide killing by at least 24 hours, leading to eradication of some MM cell lines. This acceleration and enhancement of lenalidomide killing appears to be mediated by synergistic reduction of IRF-4, as we have found in cell cycle enhancement of proteasome inhibitor killing. Most importantly, acceleration of early G1 arrest by inhibition of CDK4/CDK6 in primary bone marrow myeloma cells enhances lenalidomide killing in the presence of bone marrow stromal cells. Thus, the immunomodulatory compound lenalidomide induces G1 arrest and is cytotoxic for myeloma cells directly and preferentially in G1, in contrast to proteasome inhibitors, which preferentially target MM cells in S phase. Induction of prolonged early G1 arrest accelerates and enhances subsequent lenalidomide killing, which appears to be mediated by loss of IRF-4 in common with cell cycle enhancement of proteasome inhibitor killing. To implement targeting CDK4/CDK6 in combination therapy, a multi-center phase 1/2 clinical trial targeting CDK4/6 with PD 0332991 in sequential combination with bortezomib and dexamethasone in relapsed refractory MM is in progress. Data from the phase 1 portion indicate that PD 0332991 is well tolerated, and directly and completely inhibits CDK4/CDK6 and the cell cycle in tumor cells in MM patients with promising clinical efficacy. Given the known clinical efficacy of lenalidomide in MM, our findings suggest lenalidomide as an attractive cytotoxic partner for targeting CDK4/CDK6 in sequential combination therapy to both control tumor expansion and enhance tumor killing in the treatment of MM. Disclosures: Off Label Use: PD 0332991 is a cell cycle CDK4/CDK6 inhibitor. Hussein:Celgene: Employment. Randolph:pfizer: Employment, Equity Ownership. Niesvizky: Celgene: Consultancy, Research Funding, Speakers Bureau; Millennium: Consultancy, Research Funding, Speakers Bureau; Onyx: Consultancy, Research Funding, Speakers Bureau.

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A Phase I Trial of PD 0332991, a Novel, Orally-Bioavailable CDK4/6-Specific Inhibitor Administered in Combination with Bortezomib and Dexamethasone to Patients with Relapsed and Refractory Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.1877.1877 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1877-1877 ◽

Cited By ~ 2

Author(s):

Ruben Niesvizky ◽

Scott Ely ◽

David S Jayabalan ◽

Megan C. Manco ◽

Seema Singhal ◽

...

Keyword(s):

Cell Cycle ◽

Animal Models ◽

Dose Escalation ◽

Research Funding ◽

Specific Inhibitor ◽

S Phase ◽

Stage Ii ◽

G1 Arrest ◽

Myeloma Cells ◽

Orally Bioavailable

Abstract Abstract 1877 Poster Board I-902 Background: PD 0332991 (PD) a potent orally bioavailable small molecule, is the only known CDK4/CDK6-specific inhibitor. Through selective inhibition of CDK4/6, PD induces G1 cell cycle arrest thereby preventing DNA replication. As PD acts reversibly, it can induce synchronous G1-S progression upon its discontinuation. It has been shown that PD potently induces G1 arrest in primary human bone marrow (BM) myeloma cells/lines in the presence of BM stroma and prevents tumor growth in animal models (IC50, ∼60 nM) (Baughn et al 2006). Moreover, it has been shown that induction of prolonged G1 arrest and synchronous S phase entry by PD profoundly enhances bortezomib (B) and dexamethasone (D) killing of primary BM myeloma cells in vitro and in animal models (Huang et al, unpublished). The synergistic anti-myeloma effect provides a compelling rationale for evaluating two methods of targeting CDK4/6 using PD in combination with B and D: a “concurrent regimen” aims at enhancing B killing during prolonged G1 arrest and a “sequential regimen” to enhance B killing during both G1 arrest and synchronous progression to S phase. Methods: Patients (pts) who had relapsed and refractory myeloma after at least 2 previous treatments and who were Rb positive were eligible. This phase 1 dose escalation study is being conducted to assess the safety, tolerability and pharmacokinetics of the combination including dose limiting toxicities (DLTs). PD was given orally once a day for 3 weeks (21 days) followed by one week (7 days) off (Schedule A), starting on Day 1 of each cycle. B was given during G1 arrest by IV bolus together with 20 mg PO of D on Days 8, 11, 15 and 18 of each cycle (up to a maximum of 10 cycles). The study design followed a 3+3 dose-escalation scheme, with planned doses of PD/B starting from 100 mg/1.0 mg/m2 and escalating up to a maximum dose of 125 mg/1.3 mg/m2, respectively. Alternatively, in case of toxicity, de-escalation was planned to a minimum dose of 50 mg/0.7 mg/m2 for PD/B, respectively. Results: Nine patients were enrolled in Schedule A. Pt characteristics included 90% > SD stage II, 50% > ISS stage II with median β2 M 4.0 (range 1.6–10.5), median serum albumin 3.8 (2.2–4.6), median Hgb 10.5 (8.1–14.4) and median calcium 9.9 (8.7–10.7) values at screening. The median number of prior therapies was 6 (2–10) with 7/9 pts showing progression to their latest prior therapy. One patient achieved VGPR (12.5%) while 1 patient each achieved MR and SD respectively for an ORR 25%. The VGPR and MR were achieved with the lowest dose combination (75 mg/0.7 mg/m2 PD/B). Six pts had progression of disease while on therapy. The most commonly reported adverse events were >Grade 3 reversible uncomplicated cytopenias. PD was absorbed relatively slowly with a median Tmax of 4 hours (range 2–8 hours). PD plasma exposures (normalized to the 100 mg dose level) ranged from 345–1128 ng.hr/mL for AUC 0–12 and from 36–111 ng/mL for Cmax and were consistent with those observed in prior solid tumor studies. Immunohistochemistry of BM on Day 8 (prior to initiation of BD) in 7/8 pts demonstrated preferential and complete inhibition of CDK4/6-specific phosphorylation of Rb and Ki67 in tumor cells. Follow up BMs after 21 days, showed G1-S cell cycle progression upon PD withdrawal, confirming PDs synchronization effect. Conclusions: Targeting CDK4/6 with PD to induce prolonged G1 arrest in combination with B/D represents the first mechanism-based targeting of the cell cycle in cancer, and it appears to be effective in MM. Pts are being actively accrued to Schedule B consisting of sequential PD-B/D (12 days of PD followed by B/D as in Schedule A) to assess the safety of this novel schedule and the efficacy following cell cycle synchronization. Disclosures: Niesvizky: Celgene: Consultancy, Research Funding, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Research Funding, Speakers Bureau; Proteolix: Consultancy, Research Funding. Courtney:Pfizer: Employment. DuFresne:Pfizer: Employment. Wilner:Pfizer: Employment. Chen:Pfizer: Employment. Mark:Celgene: Speakers Bureau; Millenium: Speakers Bureau. Coleman:Bristol-Myers Squibb Research & Development: Consultancy.

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Inhibition of CDK4/CDK6 Sensitizes Myeloma to IMiD By Reducing the MEIS2 to Cereblon Ratio That Accelerates IKZF1 and IKZF3 Degradation

Blood ◽

10.1182/blood.v126.23.500.500 ◽

2015 ◽

Vol 126 (23) ◽

pp. 500-500

Author(s):

Xiangao Huang ◽

David Jayabalan ◽

Maurizio Di Liberto ◽

Zhengming Chen ◽

Anna C Schinzel ◽

...

Keyword(s):

Cell Cycle ◽

Clinical Response ◽

Research Funding ◽

Ex Vivo ◽

Cell Cycle Control ◽

Selective Inhibition ◽

Hematologic Malignancies ◽

G1 Arrest ◽

Myeloma Cells

Abstract Lenalidomide (Len) and pomalidomide (Pom) are immunomodulatory drugs (IMiDs) effective in hematologic malignancies, in combination therapies for multiple myeloma (MM) in particular. Cereblon (CRBN), a component of the CRL4CRBN E3 ligase, is required for IMiD's anti-myeloma activity. Emerging evidence suggests that IMiDs bind CRBN and block an endogenous substrate MEIS2 from binding to CRBN, thereby facilitating the recruitment of transcription factors IKZF1 and IKZF3 to CRL4CRBN and their degradation. This then leads to loss of IRF4 necessary for myeloma survival. The clinical relevance of these novel findings, however, has not been defined. To address this question, we've investigated the mechanism of IMiD action and the functional consequences in freshly isolated primary bone marrow myeloma cells (BMMCs) (n=31) in stromal co-culture ex vivo in the context of the clinical response to Len or Pom in vivo before or after biopsy. We showed by whole transcriptome sequencing, protein analysis and functional assays that 1) BMMCs are addicted to IKZF3-IRF4 for survival; 2) Len-mediated IRF4 loss leads to de-repression of IRF7, induction of interferon (IFN) response genes and TRAIL-mediated apoptosis; and 3) the magnitude of IFN induction is tightly associated with killing of BMMCs by Len. Importantly, the IMiD sensitivity in BMMCs ex vivo correlated with the prior or subsequent clinical response to IMiD-based therapies in individual myeloma patients, suggesting that the clinical response to IMiDs in myeloma is largely intrinsic to myeloma cells. IMiDs have been reported to cause cell cycle arrest. We found that before evidence of killing, Len and Pom induced late G1 arrest by both repressing CCNA2 (encoding cyclin A) mRNA synthesis and elevating p21 and p27 proteins independent of Rb and p53. This result suggests that IMiDs preferentially kill cells in G1 arrest, and that induction of p rolonged early G1 arrest (pG1) beyond the normal G1 transit time by selective inhibition of CDK4/CDK6 with palbociclib (PD 0332991, Ibrance) may sensitize MM cells to IMiD killing, as it does to killing by other agents. Indeed, induction of pG1 by palbociclib overrides cell cycle regulation by Len, and sensitizes BMMCs to Len-mediated apoptosis by augmenting the loss of IRF4 protein and the induction of IRF7, IFNb and TRAIL. Further investigation revealed that induction of pG1 by CDK4/CDK6 inhibition sensitizes primary myeloma cells to IMiD killing by rapid acceleration of Len-mediated loss of IKZF1 and IKZF3 proteins, within one hour of IMiD addition. Loss and gain of function studies demonstrates that MEIS2 opposes pG1 sensitization to Len killing; however, MEIS2 itself is regulated by the cell cycle. Induction of pG1 reduces the ratio of MEIS2 to CRBN by both reducing the MEIS2 protein rapidly and increasing the CRBN protein at a later time in cooperation with Len. In summary, our data provide the first evidence that induction of prolonged early G1 arrest by selective inhibition of CDK4/CDK6 amplifies IMiD killing of primary myeloma cells by both repressing MEIS2 and increasing CRBN protein in cooperation with Len. This leads to a profound reduction in the ratio of MEIS2 to CRBN that accelerates the loss of IKZF1, IKZF3 and IRF4, and enhances IFN and TRAIL induction. Reducing the MEIS2/CRBN ratio thus represents a novel mechanism by which CDK4/CDK6 inhibition sensitizes myeloma to IMiDs, and a means for developing mechanism-based IMiD therapy through cell cycle control. Disclosures Huang: Celgene: Research Funding. Off Label Use: Palbociclib (PD 0332991) is a specific CDK4/CDK6 inhibitor used to stop the cell cycle.. Rossi:Calgene: Speakers Bureau. Pearse:Celegen: Consultancy. Mark:Calgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Niesvizky:Celgene: Consultancy, Speakers Bureau. Chen-Kiang:Celgene: Consultancy.

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Induction of Sustained Early G1 Arrest by Selective Inhibition of CDK4 and CDK6 Primes Myeloma Cells for Synergistic Killing by Proteasome Inhibitors Carfilzomib and PR-047

Blood ◽

10.1182/blood.v112.11.3670.3670 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3670-3670

Author(s):

Xiangao Huang ◽

Kathryn Bailey ◽

Maurizio Di Liberto ◽

Francesco Parlati ◽

Ruben Niesvizky ◽

...

Keyword(s):

Cell Cycle ◽

Phase I ◽

Proteasome Inhibitor ◽

Proteasome Inhibitors ◽

Selective Inhibition ◽

Selective Inhibitor ◽

Cytotoxic Agents ◽

Rational Approach ◽

Phase I Trials ◽

Myeloma Cells

Abstract Targeting the cell cycle in combination with cytotoxic killing is a rational approach to cancer therapy. Dysregulation of the cyclin-dependent kinases CDK4 and CDK6 precedes uncontrolled proliferation of myeloma cells in vivo, in particular during relapse and drug resistance. This finding reinforces the critical importance of targeting CDK4/6 in myeloma, but success with broad-spectrum CDK inhibitors has been modest. Using the only known selective inhibitor of CDK4/6, PD 0332991, we have developed a novel approach to prime chemoresistant myeloma cells for synergistic killing by diverse cytotoxic agents. We show that selective inhibition of CDK4/6 by PD 0332991 leads to sustained cell cycle arrest in early G1 in the absence of apoptosis. However, it markedly augments cytotoxic killing by PR-171 (carfilzomib), a selective inhibitor of the chymotrypsin-like activity of the proteasome, or PR-047, an orally bioavailable analog of carfilzomib. Synergistic killing of myeloma cells arrested in early G1 by carfilzomib (or PR-047) is caspase-dependent, and requires only a brief (one hour) exposure to the proteasome inhibitor at concentrations as low as 60 nM. This effect is mediated by synergistic and rapid induction of mitochondrial membrane depolarization and activation of downstream caspase-9 within 6 hours of removal of carfilzomib or PR-047. As PD 0332991 acts as an ATP-competitive inhibitor of the CDK4/6 kinase domain, inhibition of CDK4/6 and the cell cycle by PD 0332991 is reversible. Importantly, targeting CDK4/6 with PD 0332991 in combination with either carfilzomib or PR-047 leads to complete eradication of myeloma cells ex vivo, in contrast to the combination of PD 0332991 with other proteasome inhibitors. Selective inhibition of CDK4/6 in combination with carfilzomib (or PR-047), therefore, not only halts cell proliferation but also potently induces synergistic killing that is likely to eliminate cell cycle reentry and generation of resistant cells. PD 0332991 is a small molecule with bio-availability and proven tumor suppressing activity in both human myeloma xenograft and immunocompetent mouse myeloma models. It is well tolerated in humans as shown by the ongoing Phase I/II clinical trials in myeloma and previous phase I trials in mantle cell lymphoma and solid tumors. Evidence from Phase I trials of carfilzomib indicates that it is also well tolerated, in fact, the peripheral neuropathy that is commonly observed with proteasome inhibitor bortezomib appears to be less severe and possibly less frequent. Mechanism-based targeting of CDK4/6 in combination with selective proteasome inhibitors, like carfizomib and PR-047, thus represents a new and promising therapeutic strategy for multiple myeloma and potentially other hematopoietic malignancies.

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BCL-2 Inhibition By ABT-199 (Venetoclax/GDC-0199) and p53 Activation By RG7388 (Idasanutlin) Reciprocally Overcome Leukemia Apoptosis Resistance to Either Strategy Alone: Efficacy and Mechanisms

Blood ◽

10.1182/blood.v126.23.673.673 ◽

2015 ◽

Vol 126 (23) ◽

pp. 673-673 ◽

Cited By ~ 2

Author(s):

Rongqing Pan ◽

Vivian Ruvolo ◽

Hong Mu ◽

Zhuanzhen Zheng ◽

Joel Leverson ◽

...

Keyword(s):

Research Funding ◽

Acquired Resistance ◽

Cell Cycle Distribution ◽

G1 Arrest ◽

Apoptosis Resistance ◽

Employment Equity ◽

P53 Activation ◽

Equity Ownership ◽

Mdm2 Inhibitor ◽

Refractory Aml

Abstract Acute myeloid leukemia (AML) is primarily treated with chemotherapy, but the 5-year survival rate has only marginally increased over the past few decades, highlighting the need for novel targeted therapy. We have reported elevated expression of BCL-2 in AML and that BCL-2 inhibition by ABT-199 (ABT, venetoclax) induced on-target apoptosis, which could be predicted by BH3 profiling (Pan, et al., Cancer Discovery, 2014). ABT also showed encouraging clinical activity in relapsed/refractory AML (Konopleva et al., ASH 2014), yet MCL-1-mediated resistance may limit its use as monotherapy. p53 mutations are relatively rare in AML, but its functions are often suppressed by overexpressed MDM2 protein. Since p53 and BCL-2 family proteins are central regulators of apoptosis, we asked whether concurrent BCL-2 inhibition and p53 activation (by MDM2 inhibitor) could overcome resistance to apoptosis and synergistically induce apoptosis in AML cells. The novel MDM2 inhibitor RG7388 (RG, Idasanutlin) robustly activated p53 and induced growth inhibition and apoptosis of AML cells in a p53-dependent manner. p53 activation by RG also synergized with BCL-2 inhibition in killing ABT-sensitive cell lines such as MOLM-13 or MV-4-11 (Fig. 1A). After long-term exposure to escalating doses of ABT, initially sensitive cells upregulated MCL-1 and acquired resistance. Nonetheless, the acquired resistance could be effectively abrogated by RG (Fig. 1B). OCI-AML3 cells express a high basal level of MCL-1, and are inherently resistant to ABT. Concurrent p53 activation and BCL-2 inhibition induced synergistic apoptosis and overcame the inherent ABT resistance (Fig. 1C). Next, we studied the underlying mechanisms. p53 activation by RG increased the expression of PUMA and BAX (but not NOXA, Fig. 1D), which are able to counteract MCL-1. In addition, p53 activation quickly dephosphorylated ERK2 and downregulated MCL-1 (Fig. 1E). Surprisingly, ABT increased ERK2 phosphorylation and upregulated MCL-1 (Fig. 1E). Because active ERK2 phosphorylates and stabilizes MCL-1, these results indicate that the observed changes in MCL-1 levels could be attributed to ERK2 phosphorylation status. Consistently, ERK2 dephosphorylation by MEK inhibitors quickly reduced MCL-1. Most importantly, ABT-induced ERK2 phosphorylation and MCL-1 upregulation could be reversed by p53 activation (Fig. 1F). These mechanistic studies provide insights into how p53 activation overcomes acquired/inherent resistance to BCL-2 inhibition. OCI-AML3 cells are relatively resistant to p53 activation by RG. We used concomitant Annexin V staining, EdU pulsing and PI staining to simultaneously analyze apoptosis induction and cell cycle distribution of live cells (Fig. 1G). p53 activation by RG induced cell accumulation in G1 phase, while little apoptosis occurred (Fig. 1H). Addition of ABT dramatically increased apoptosis, reduced G1-arrested cells, and boosted apoptotic hallmarks like the cleavage of caspase-9, -3 and PARP-1 (Fig. 1H-I). ABT did not affect p21 expression and cell cycle distribution, and p53 activation induced robust expression of p21 and G1 arrest. Furthermore, p21 knockdown significantly decreased G1-arrested cells and increased apoptosis following p53 activation, indicating that p21 upregulation and G1 arrest mediate apoptosis resistance to p53 activation. Nonetheless, addition of ABT effectively shifted cell response from G1 arrest to apoptosis, suggesting BCL-2 inhibition can reciprocally overcome apoptosis resistance to p53 activation. Next, we tested the combination in two AML mouse models. In an OCI-AML3-derived mouse model (with inherent resistance to ABT or RG), ABT or RG prolonged survival by 10 or 19 d, respectively, while the combination prolonged mouse survival by 61 d (Fig. 1J-K). Currently, we are also following the survival of mice in a MOLM-13 acquired resistance model. Early results indicate the tumor burden in combination group is <1/100 of that in control/ABT groups and ~1/20 of that in the RG group at day 14 (Fig. 1K-L). In summary, BCL-2 inhibition by ABT and p53 activation by RG can reciprocally overcome resistance to apoptosis encountered by using either treatment alone in vitro and in vivo. Since both BCL-2 and MDM2 overexpression are associated with poor prognosis in AML, the proposed combination of the two clinical-stage compounds could have considerable clinical impact in relapsed/refractory AML. Disclosures Leverson: AbbVie: Employment, Equity Ownership. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Nichols:Roche Pharma: Employment, Equity Ownership.

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Circulating Multiple Myeloma Cells (CMMCs): A Novel Method for Detection and Molecular Characterization of Peripheral Blood Plasma Cells in Multiple Myeloma Precursor States

Blood ◽

10.1182/blood.v124.21.2031.2031 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2031-2031

Author(s):

Brendan Weiss ◽

Kate Sasser ◽

Chandra Rao ◽

Brad Foulk ◽

Steven Gross ◽

...

Keyword(s):

Multiple Myeloma ◽

Molecular Characterization ◽

Research Funding ◽

Plasma Cells ◽

Employment Equity ◽

Myeloma Cells ◽

Novel Method ◽

Equity Ownership ◽

Precursor States

Abstract Background Circulating plasma cells (PCs) have been identified as a prognostic factor in patients with myeloma precursor states (MGUS and SMM) and active multiple myeloma (MM). Enumeration of circulating PCs by available methods is not suitable for widespread use and does not provide molecular characterization. We developed and evaluated a novel method for enumeration and molecular characterization of circulating PCs (circulating multiple myeloma cells, “CMMC”), based on the CELLSEARCH® System (Janssen Diagnostics LLC, Raritan, NJ), an automated technology for the capture, enumeration and characterization of rare cells in the peripheral blood. Methods We are performing a prospective study of patients with MGUS and SMM to evaluate CMMCs as biomarker for progression to active MM. Utilizing the CELLSEARCH® System CMMCs were captured by CD138 ferrofluid magnetic particles and identification was defined as CD38+ and CD19-, CD45-. Nonviable cells were excluded by DAPI. Isolated CMMCs were stored and FISH for t(4:14), t(14;16) and del17 was performed. Results We have enrolled 16 patients, MGUS = 3, SMM = 11, and newly diagnosed MM = 2. The Mayo Risk stratification for MGUS patients was: low risk = 2, low-intermediate = 1. All SMM patients were low risk by Mayo Model incorporating serum free light chains. The median number of bone marrow plasma cells for MGUS patients was 7 (range 7-9) and for SMM patients was 15 (range 10-40). The median CMMCs for MGUS = 6 (range 2-55), median CMMCs for SMM = 31 (5-1918). The two patients with NDMM had 5870 and 5 CMMCs, respectively. A single patient with SMM progressed with a symptomatic solitary lumbar plasmacytoma and had CMMCs of 5 and 3 at baseline and progression, respectively. Abnormalities by FISH were detected in both bone marrow and CMMCs. Accrual is ongoing and additional data will be presented at the meeting. Conclusions The CELLSEARCH® CMMC assay can detect, quantify and provide molecular characterization of circulating PCs in MGUS/SMM/MM; longer prospective follow-up is needed to test the prognostic value of CMMCs. Disclosures Weiss: Janssen: Consultancy, Research Funding. Sasser:Janssen: Employment. Rao:Janssen: Employment, Equity Ownership. Foulk:Janssen: Employment. Gross:Johnson & Johnson: Employment, Equity Ownership. Cohen:Janssen: Membership on an entity's Board of Directors or advisory committees. Vogl:Celgene Corporation: Consultancy; Amgen: Consultancy; Millennium/Takeda: Research Funding; GSK: Research Funding; Acetylon: Research Funding. Stadtmauer:Janssen: Consultancy.

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Selective Inhibition of CDK4 and CDK6 Primes Chemoresistant Myeloma Cell for Cytotoxic Killing through Induction of Cell Cycle-Coupled Mitochondrial Dysfunction and Apoptosis

Blood ◽

10.1182/blood.v112.11.3678.3678 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3678-3678 ◽

Cited By ~ 1

Author(s):

Xiangao Huang ◽

Maurizio Di Liberto ◽

Tracey Louie ◽

David S Jayabalan ◽

Scott Ely ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Mitochondrial Dysfunction ◽

Low Dose ◽

Selective Inhibition ◽

Cytotoxic Agents ◽

G1 Arrest ◽

Myeloma Cells

Abstract Dysregulation of cyclin-dependent kinase (CDK)4 or CDK6 activity by gain of function or loss of inhibition is one of the most frequent aberrations in cancer. This includes multiple myeloma (MM), where overexpression of CDK4 (CDK6) precedes unrestrained proliferation of CD138+ bone marrow myeloma cells in vivo, in particular during aggressive tumor growth and relapse. In complex with the D-type cyclin, CDK4 and CDK6 promote cell cycle entry and progression through G1 by inactivating the retinoblastoma protein Rb and antagonizing the INK4 family of CDK inhibitors, suggesting that inhibition of CDK4/6 is a promising approach for cell cycle control in MM. We have now developed a novel approach to both halt cell proliferation and enhance cytotoxic killing of MM cells by selective inhibition of CDK4/6 in combination with cytotoxic agents. We show that knocking down CDK4 and CDK6 expression by shRNA interference or inhibiting CDK4/6 activity with PD 0332991, the only known CDK4/6-specific small molecule inhibitor, leads to sustained G1 arrest and induction of synchronous cell cycle progression upon removal of PD 0332991. Induction of sustained early G1 arrest is not accompanied by apoptosis. However, it primes MM cells for synergistic killing by low dose cytotoxic agents of diverse modes of action, which is further augmented during synchronous S phase entry. Most importantly, induction of sustained G1 arrest with PD 0332991 primes freshly isolated chemoresistant CD138+ bone marrow myeloma cells for killing by low dose proteasome inhibitors in the presence of bone marrow stromal cells. Synergistic killing by PD 0332991 combined with low dose bortezomib (2–6 nM) in early G1 (referred to as PD-B) is mediated by increased neutralization of Mcl-1 and Bcl-2 in the absence of Noxa, as PD-B augments bortezomib activation of Bim and Mcl-1 transcription while silencing Noxa in early G1. This leads to aggregation of Bak, but not Bax, on the mitochondria, mitochondrial membrane depolarization, preferential release of Smac/DIABLO, but not cytochrome c, from mitochondria, reduction of c-IAP and caspase-9 activation. Apoptosis is further amplified through activation of caspase-8 without inducing TRAIL, FASL and TNF-α, the major ligands that trigger the extrinsic apoptosis pathway. Cytotoxic killing by PD-B is recapitulated in synergistic tumor suppression in animal models. Collectively, our ex vivo and in vivo data demonstrate that PD-B induces synergistic killing of MM cells through cell cycle-coupled regulation of Bcl-2 family genes and induction of mitochondrial dysfunction. As PD 0332991 is orally bio-available, potent and low in toxicity, our approaches have formed the basis for an ongoing, first-inclass Phase I/II clinical trial to selectively target CDK4/6 with PD 0332991 in combination with bortezomib and dexamethasone in multiple myeloma. Selective targeting CDK4 and CDK6 in combination with cytotoxic killing, therefore, provides a new and promising mechanism-based therapeutic strategy for multiple myeloma and potentially other cancers.

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