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.

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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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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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.

Blood ◽

10.1182/blood.v114.22.2854.2854 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2854-2854

Author(s):

Maurizio Di Liberto ◽

Xiangao Huang ◽

Rediet Zewdu ◽

Francesco Parlati ◽

Monette Aujay ◽

...

Keyword(s):

Cell Cycle ◽

Research Funding ◽

Proteasome Inhibitors ◽

Specific Inhibitor ◽

Selective Inhibition ◽

G1 Arrest ◽

Employment Equity ◽

Myeloma Cells ◽

Equity Ownership ◽

Cycle Dependent

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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Bim Is Required to Sensitize Mantle Cell Lymphoma Cells for Killing by Bortezomib, but Not Ara-C, by Selective Inhibition of CDK4/CDK6

Blood ◽

10.1182/blood.v118.21.1655.1655 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1655-1655

Author(s):

Xiangao Huang ◽

Maurizio Di Liberto ◽

Jamieson Bretz ◽

David Chiron ◽

Peter Martin ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Research Funding ◽

Cell Cycle Progression ◽

Phase Synchronization ◽

Selective Inhibition ◽

S Phase ◽

G1 Arrest ◽

Cycle Progression ◽

Mantle Cell

Abstract Abstract 1655 Mantle cell lymphoma (MCL) is characterized by aberrant cyclin D1 expression due to the t (11: 14) translocation. In conjunction with elevation of CDK4/CDK6, this promotes cell cycle progression through G1 and unrestrained cell proliferation. As MCL remains incurable despite initial response to therapy, mechanism- and genome-based therapies that both control the cell cycle and enhance cytotoxic killing are urgently needed. We have recently developed such a regimen by inhibition of CDK4/CDK6 with PD 0332991 (PD), a selective inhibitor of CDK4 and CDK6 that is also potent, reversible and orally bioavailable. We demonstrate that 1) inhibition of CDK4/CDK6 with PD leads to early G1 arrest; 2) upon release of the G1 block, synchronous cell cycle progression to S phase occurs; and 3) S phase synchronization following prolonged early G1 arrest (pG1-S) sensitizes MCL cells to killing by diverse clinically relevant agents at reduced doses, including proteasome inhibitors bortezomib and carfilzomib, and the nucleoside analog Ara-C (cytarabine), both in vitro and in a mouse model of MCL. These findings implicate a unified mechanism for cell cycle sensitization of cytotoxic killing. To elucidate the underpinning mechanism, we show that sensitization to cytotoxic killing by CDK4/CDK6 inhibition requires an intact Rb, the substrate of CDK4/CDK6, but is independent of p53. Gene expression profiling and quantitative RNA and protein analyses further demonstrate that prolonged inhibition of CDK4/CDK6 with PD halts the gene expression program in early G1 and depletes the expression of genes programmed for other phases of the cell cycle, such as cyclin A (G1/S), thymidine kinase (S), CDK1 and cyclin B (G2/M) and selective metabolic genes. Removal of PD restores the CDK4/CDK6 activities and the expression of scheduled cell cycle genes but leaves many others in the pG1 state. This leads to S phase synchronization with impaired metabolism. Accordingly, the magnitude of bortezomib and Ara-C killing in pG1-S greatly exceeds the enrichment of S phase cells. Selective inhibition of CDK4/CDK6, therefore, sensitizes MCL cells for cytotoxic killing in S phase synchronization through induction of a persistent metabolic imbalance in prior pG1. pG1 alone induces caspase activation moderately in MCL cells, but markedly augments apoptosis induced by either bortezomib or Ara-C in pG1-S. This enhancement of apoptosis is apparently mediated by an alteration of the ratios of pro-apoptotic BH3-only proteins (Bim, Noxa and Puma) to anti-apoptotic proteins (Mcl-1, Bcl-2 and Bcl-xL), which lowers the threshold for caspase-9 activation. Importantly, Bim is selectively required to sensitize MCL cells for killing by bortezomib, but not Ara-C, at low doses as indicated in studies of Bim-deficient MCL cell lines. Corroborating these findings, loss of one allele of Bim attenuates the enhancement of bortezomib killing in pG1-S in untransformed primary mouse B cells after activation by BCR and CD40 signaling. Thus, the synergistic actions of PD-bortezomib and PD-AraC in MCL therapy are distinguishable by the requirement for Bim. Furthermore, we found that the three Bim isoforms are expressed at variable levels but undetected in 30% of primary MCL tumor cells, consistent with the reported mutations and bi-allelic deletion of Bim (BCL2L11) in MCL. RNA-Seq analysis of samples from patients enrolled in a phase I study of PD in combination with bortezomib in MCL further reveals that the mutation burden in BCL2L11 is ∼3-fold higher in a clinically non-responder compared with a responder. Collectively, our data demonstrate that by halting scheduled gene expression in prolonged early G1 arrest, selective and reversible inhibition of CDK4/CDK6 provides a mechanism-based strategy to sensitize MCL cells for cytotoxic killing by bortezomib, Ara-C, and potentially other emerging agents. By lowering the threshold for caspase activation, Bim is selectively required for sensitization to killing by low dose bortezomib, but not Ara-C, and may serve as a biomarker for genome-based selection of cytotoxic partners in therapeutic targeting of CDK4/CDK6 in MCL. Disclosures: Martin: Millennium Pharmaceuticals, Inc.: Research Funding, Speakers Bureau. Smith:Pfizer: Research Funding; Millenium: Research Funding. Leonard:Pfizer, Inc.: Consultancy; Millenium: Consultancy; Johnson and Johnson: Consultancy; Onyx: Consultancy. Chen-Kiang:Pfizer, Inc.: Research Funding.

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Pre-Clinical Evaluation of a Small Molecule Inhibitor of CDK1 as Potential Therapy for MYC Expressing Mutiple Myeloma Tumors

Blood ◽

10.1182/blood.v112.11.3667.3667 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3667-3667 ◽

Cited By ~ 1

Author(s):

Seda Zeng ◽

Zhi Hua Li ◽

Marta Chesi ◽

Chungyee Leung-Hagesteijn ◽

Sheng-ben Liang ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Cell Lines ◽

Cell Cycle Progression ◽

Myeloma Cell ◽

S Phase ◽

Causative Role ◽

Cyclin D ◽

Cycle Progression ◽

Myeloma Cells

Abstract Through the recent elucidation of molecular and cellular processes in multiple myeloma (MM) pathogenesis that effect well-defined proliferative and survival pathways, a number of molecular targets for anti-cancer agents have emerged. This includes proteins involved in cell cycle regulation. A recent model of early MM pathogenesis proposes that at least one of the cyclin D genes is dysregulated in all myeloma tumors facilitating activation of cyclin dependent kinase (CDK)4 (or CDK6), and transition from G1 to S phase. It is hypothesized that this renders myeloma cells more responsive to bone marrow (BM) derived proliferative stimuli including IL-6 that upregulates MYC expression in MM cells and cooperates with cyclin D to promote transit to S-phase. In addition, recent studies have established a causative role of MYC dysreguation in progression from monoclonal gammopathy to myeloma. Thus an approach targeting deregulated cell cycle progression and MYC may prove effective MM therapy. Purvalanol is a potent and selective inhibitor of CDK1, an important regulator of cell cycle progression and has been reported to induce MYC-dependent apoptosis. Activity of purvalanol against a panel of 14 standardized, annotated myeloma cell lines was measured in a 48 hour MTT viability assay. IC50-s of all 14 cell lines ranged between 5 uM to 7.5 uM. Western blot and real-time PCR analysis revealed variability of MYC protein and mRNA levels between the 14 myeloma cell lines. We compared potential therapeutic activity of purvalanol against 3 myeloma cell lines (U266, H929, KMS12PE) with low, intermediate, and high levels of c-MYC expression, respectively. All three cell lines demonstrated G2-M growth arrest however marked apoptosis as determined by PI/annexin V staining was observed only in the cell lines with the highest level of MYC expression. Exposure of MM patient-derived BM mononuclear cells to purvalanol preferentially induced apoptosis of CD138+ MM cells. In contrast, purvalanol was minimally cytotoxic to the non-myeloma cell fraction and to non-transformed fibroblast cell lines (MRC5, IMR90 and W138) and failed to inhibit normal bone marrow-derived CD34 colony formation. Recent studies have demonstrated that the BM microenvironment offers protection of myeloma cells from chemotherapeutic agents by common mechanisms. Myeloma cell lines were cultured in 3 different conditions to mimic the tumor’s protective microenvironment. Soluble factors produced by the BM, IL6 and IGF-1 induced a modest degree of resistance to purvalanol while co-culture on BM stroma cells was completely protective. Studies evaluating the in vivo efficacy of purvalanol in a novel Vk*myc transgenic mouse model that spontaneously develops myeloma with a low proliferative index are ongoing and will be presented. The CDK1 inhibitor, purvalanol demonstrates broad single agent activity against myeloma cells with enhanced activity against MYC overexpressing cells. However, the strong protective effect of the BM microenvironment suggest that combination with agents that can reverse cell-adhesion mediated drug resistance may prove beneficial in optimizing the efficacy of this class of drugs for the treatment of MM.

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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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Cooperative Supression of MEIS2 Mediates Dexamethasone Enhancement of Lenalidomide Killing in Myeloma Cells

Blood ◽

10.1182/blood.v128.22.3292.3292 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3292-3292

Author(s):

Sonia Allouch ◽

David S Jayabalan ◽

Adriana C Rossi ◽

Ruben Niesvizky ◽

Xiangao Huang ◽

...

Keyword(s):

Bone Marrow ◽

Clinical Response ◽

Cell Lines ◽

Research Funding ◽

Insertion Site ◽

S Phase ◽

G1 Arrest ◽

Dependent Manner ◽

Myeloma Cells ◽

Fold Reduction

Abstract Dexamethasone (Dex) enhances the clinical response to most therapies in multiple myeloma, the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide in particular. As a steroid, Dex may modulate the therapeutic response by acting on multiple cell types in vivo. Whether and how Dex enhances the tumor intrinsic clinical response to IMiDs is not well understood. To address this important question, we focus on the role of Dex in modulating the proximal events in IMiD signaling that lead to the killing of myeloma cells. Cereblon (CRBN), a substrate receptor of the CRL4CRBN E3 ligase, is required for IMiD's anti-myeloma activity. MEIS2 is a homeobox transcription factor of the mammalian myeloid ecotropic insertion site (MEIS) family, which plays a pivotal role in development and leukemogenesis. Recent crystal structure studies in conjunction with biochemical screen have identified MEIS2 as an endogenous substrate of CRBN. IMiDs apparently bind CRBN and block MEIS2 from binding to CRBN, thereby facilitating the recruitment of transcription factors IKZF1 and IKZF3 to CRL4CRBN for degradation. This then leads to loss of IRF4, a target of IKZF1/3, necessary for myeloma survival. This model posits that MEIS2 inhibits the function of CRBN in myeloma cells. However, as all the studies of MEIS2 were performed heterologous cell lines, evidence that supports a function of MEIS2 in myeloma cells is lacking. We now showed by RNA-seq and protein analysis that MEIS2 was absent in normal bone marrow plasma cells (BMPC), but highly expressed in freshly isolated primary bone marrow myeloma cells (BMMC)s at both RNA and protein levels. We further showed that MEIS2 was regulated by the cell cycle: reduced in G1 arrest and restored in S phase in human myeloma cell lines (HMCL)s as well as primary BMMCs. Depletion of MEIS2 markedly enhanced lenalidomide killing, and overexpression of MEI2 attenuated lenalidomide killing of HMCL MM1.S. These results provide the first evidence that MEIS2 is aberrantly expressed in primary myeloma cells and acts within myeloma cells to inhibit IMiD killing. To investigate if Dex enhances the myeloma intrinsic response to IMiDs, we first determined that lenalidomide and pomolidomide induced late G1 arrest within 24 hours, as evidenced by the repression of cyclin A, the increases in p21 and p27 proteins, and the reduction of myeloma cells in S phase by BrdU labeling. Kinetic studies further indicate that induction of G1 arrest precedes apoptosis, detectable at 72 hours in MM1.S and CAG cells. These data suggest that IMiDs preferentially kill myeloma cells arrested in G1. Dex killed CAG and MM1.S cells marginally at 10 nM and killing was not greater at 100 nM. However, concurrent addition of Dex enhanced lenalidomide killing, more prominently in CAG cells then in MM1.S cells. Addition of Dex at these concentrations to CAG cells after 48 hours of exposure to lenalidomide leads to synergistic and greater killing in 24 hours in a dose-dependent manner. These data indicate that Dex enhances lenalidomide killing, preferentially in G1 arrest. To investigate the mechanism by which Dex enhances lenalidomide killing, we found that Dex did not regulate MEIS2 or CRBN protein expression in CAG cells, in line with its inability to significantly kill myeloma cells. Sequential addition of lenalidomide and Dex led to a > 2-fold reduction in the MEIS2 protein, which corresponds to a > 2-fold reduction in the MEIS2/CRBN ratio and synergistic killing. Concurrent addition of Dex and lenalidomide also resulted in a 2-fold reduction of the MEIS2 protein and the MEIS2/CRBN ratio, but it required 72 hours of the presence of Dex instead of 24 hours. In summary, our study demonstrates, for the first time, that MEIS2 is expressed in primary myeloma cells and inhibits IMiDs killing. Dex enhances IMiD killing of myeloma cells through cooperative suppression of MEIS2 at proximal IMiD signaling. These findings suggest a novel mechanism by which Dex enhances the anti-myeloma activity of IMiD, which has important implication for myeloma therapy. Disclosures Rossi: Celgene: Consultancy, Speakers Bureau; Onyx: Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Janssen: Speakers Bureau. Niesvizky:Celgene: Consultancy, Research Funding, Speakers Bureau; Onyx: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Research Funding, Speakers Bureau.

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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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Chemosensitisation by poly(ADP-ribose) polymerase-1 (PARP-1) inhitor 5-aminoisoquinoline (5-AIQ) on various melanoma cell lines

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.12019 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 12019-12019 ◽

Cited By ~ 1

Author(s):

S. Radulovic ◽

S. Bjelogrlic ◽

Z. Todorovic ◽

M. Prostran

Keyword(s):

Cell Cycle ◽

Cytotoxic Activity ◽

Cell Lines ◽

Melanoma Cell ◽

Cell Cycle Progression ◽

Single Agent ◽

S Phase ◽

G1 Arrest ◽

Parp 1 ◽

Melanoma Cell Lines

12019 Background: PARP-1 facilitates DNA strand brakes repair and PARP inhibitors were investigated as enhancers of chemoradiotherapy. We investigated whether 5-AIQ potentates the effect of doxorubicin (DOXO), cisplatin (CDDP) and paclitaxel (Ptx) on human (slow-growing) FemX and murine (fast-growing) B16 melanoma cell lines. Methods: Twenty-four hours after cells were seeded in 96 well plates, cytotoxic drugs and 5-AIQ were added to cell medium. For evaluation of single-agent activity, drugs were applied in concentration ranges as follows: CDDP (0.3–30 μM), DOXO (0.1–3 μM), Ptx (1–100 ηM), 5-AIQ (1–100 μM). 5-AIQ (3μM) was combined with CDDP (0.1, 0.3, 1 μM), DOXO (10, 3, 100 ηM), or Ptx (1, 3, 10 ηM). Incubation lasted for 72 hrs when SRB assay was utilized to determine individual and combine activity (interactions calculated with isobole method). For cell cycle analysis B16 cells were seeded on 6 well plates and treated with each drug alone and combinations, using the same concentrations as those for investigation of combine cytotoxic activity. Cell cycle was determined after 72 hrs, on FACS Calibur with propidium iodide dye. Results: 5-AIQ induced minimal changes in cell viability and cell cycle progression on both cell lines, compared to non-treated control. CDDP revealed high activity against FemX (IC50 = 2.85 μM) and B16 cells (IC50 = 8.84 μM), and G0/G1 arrest. In B16 cells 5-AIQ multiply enhanced CDDP’s activity with strong synergistic interaction and cells slightly driven to S phase. Synergism was also detected on B16 cells treated with combination of DOXO (IC50 = 0.2 μM on B16 and 0.89 μM on FemX) and 5-AIQ when DOXO was applied in low concentrations (10 and 30 ηM), while 5-AIQ did not interfere with cell cycle changes. Cytotoxicity of Ptx (IC50 = 6.16 ηM on B16 and <1 ηM on FemX) was stimulated only at higher concentrations. 5-AIQ stimulated G0/G1 and S phase arrest on B16 cells with Ptx of 3 and 10 ηM, respectively. In FemX cells, most of the interactions of 5-AIQ with CDDP, DOXO, and Ptx revealed as antagonistic. Conclusions: PARP-1 inhibitor 5-AIQ enhances cytotoxic activity of both DNA damaging and agents with different mechanism of action, but the effect varies between cell lines with different proliferation rate. No significant financial relationships to disclose.

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