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

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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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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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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Induction of Metabolic Impairment In Prolonged Early G1 Arrest Induced by CDK4/CDK6 Inhibition Sensitizes Myeloma Cells for Proteasome Inhibitor Killing During Subsequent S Phase Synchronization

Blood ◽

10.1182/blood.v116.21.2989.2989 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2989-2989 ◽

Cited By ~ 1

Author(s):

Maurizio Di Liberto ◽

Xiangao Huang ◽

Jamieson Bretz ◽

Scott A Ely ◽

Rediet Zewdu ◽

...

Keyword(s):

Cell Cycle ◽

Combination Therapy ◽

Proteasome Inhibitor ◽

Research Funding ◽

Phase Synchronization ◽

Phase 1 ◽

S Phase ◽

G1 Arrest ◽

Tumor Killing ◽

Sequential Combination

Abstract Abstract 2989 Sequential drug combination is a rational approach to maximize tumor killing and minimize side effects in cancer therapy. However, this is rarely achieved because the mechanism of drug action is often incompletely understood and the cell cycle specificity of individual drugs unknown. 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. This highlights the critical importance of targeting CDK4/CDK6 in MM. We have now developed, for the first time, a novel therapeutic strategy to selectively inhibit CDK4/CDK6 in sequential combination with clinically relevant cytotoxic drugs for maximal tumor killing at reduced doses in MM. CDK4 and CDK6 promote reentry and progression of the cell cycle through G1. PD 0332991, the only known CDK4/CDK6-specific inhibitor, is potent, reversible and bioavailable. We showed that inhibition of CDK4/CDK6 with PD 0332991 induces early G1 arrest and upon release from the G1 block, synchronous progression to S phase and G2/M with exceptional precision and efficiency in MM cells in vitro and in animal models. This provides a unique means to determine the cell cycle targeting specificity of individual compounds for optimal combination. Simultaneous analysis of BrdU pulse-labeling (30 minutes) and DNA content per cell reveals that bortezomib, a reversible proteasome inhibitor; carfilzomib (PR-171), an irreversible selective inhibitor of the proteasom; and its oral analog ONX-0912 (PR-047) all preferentially target MM cells synchronized into S phase over those arrested in G1, but not cells in G2/M. On this basis, killing of myeloma cells by proteasome inhibitors is markedly enhanced in prolonged G1 arrest induced by PD 0332991 and further augmented during synchronous entry into and progression through S phase upon release from the G1 block, in vitro and in vivo in the native bone marrow niches. Induction of early G1 arrest by PD 0332991 requires Rb, the substrate of CDK4 and CDK6, but not p53. Importantly, the increase in carfilzomib, ONX-0912 or bortezomib mediated killing after S phase synchronization significantly surpasses the enrichment of S phase cells. It is in fact proportional to the time of prior G1 arrest. Kinetics analyses of global gene expression patterns, specific RNA and protein levels and functional shRNA interference show that prolonging early G1 arrest leads to time-dependent uncoupling of gene expression from the cell cycle. PD 0332991 withdraw rapidly restores the CDK4 and CDK6 catalytic activity and scheduled expression of cell cycle genes, hence synchronous progression to S phase and mitosis. This includes upregulation of cyclin A synthesis and Skp2 mediated ubiquitin-proteasome degradation of p27 for S phase entry, mini chromosome maintenance(MCM)s and thymidine kinase for DNA replication, and genes critical for G2/M checkpoint control and mitosis. However, it fails to fully reverse the metabolic impairment (altered glucose, nucleotide and ATP metabolism) induced in prolonged early G1 arrest. This culminates in the loss of IRF-4 required for myeloma survival and selective gain of pro-apoptotic Bim function in G1 arrest and Noxa in S phase in synergy with carfilzomib and bortezomib, which lowers the threshold for activation of the intrinsic apoptosis pathway. Selective inhibition of CDK4/CDK6 in sequential combination therapy thus not only halts tumor cell proliferation but also potently induces synergistic tumor killing. This sequential combination therapy has been implemented in a multi-center phase 1/2 clinical trial targeting CDK4/6 with PD 0332991 in combination with bortezomib and dexamethasone in relapsed refractory MM. Phase 1 data 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. Evidence from phase 2 trials of carfilzomib indicates that it is also well tolerated. The peripheral neuropathy commonly observed with bortezomib appears to be less severe and less frequent. Selective combination with carfilzomib or the oral agent ONX-0912 thus represents a promising alternative to refine targeting CDK4/6 in sequential combination therapy for multiple myeloma and potentially other cancers. Disclosures: Off Label Use: PD 0332991 is a cell cycle CDK4/CDK6 inhibitor Carfilzomib is a proteasome inhibitor. Kirk:Onyx: Employment, Equity Ownership. Randolph:Pfizer: Employment, Equity Ownership. Niesvizky:Celgene: Consultancy, Research Funding, Speakers Bureau; Onyx: Consultancy, Research Funding, Speakers Bureau; Millennium: 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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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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Mutation Burden in CDKN2C, CDK1 and E2F2 Is Associated with Differential Response to Targeting CDK4/CDK6 in Combination with Bortezomib in Mantle Cell Lymphoma,

Blood ◽

10.1182/blood.v118.21.3738.3738 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3738-3738 ◽

Cited By ~ 3

Author(s):

Christopher E. Mason ◽

Maurizio Di Liberto ◽

Xiangao Huang ◽

David Chiron ◽

Jamieson Bretz ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Clinical Study ◽

Research Funding ◽

Cell Lymphoma ◽

S Phase ◽

Cytotoxic Agents ◽

G1 Arrest ◽

Mutation Burden ◽

Mantle Cell

Abstract Abstract 3738 Dysregulation of the cell cycle is a hallmark of mantle cell lymphoma (MCL) in which cyclin D1 expression is constitutive due to the t (11:14) translocation and CDK4 levels are elevated. MCL remains incurable despite initial response to therapy. Our goal was to develop a mechanism- and genome-based therapy to both inhibit lymphoma cell proliferation and sensitize them for cytotoxic killing. We have recently developed such a regimen by inhibition of CDK4/CDK6 with PD 0332991 (PD), the only known selective inhibitor of CDK4 and CDK6 that is also potent, reversible and orally bioavailable, in combination with cytotoxic agents. We demonstrate, for the first time, 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 cytotoxic agents at reduced doses, including proteasome inhibitors bortezomib and carfilzomib, and the nucleoside analog cytarabine, in vitro and in a mouse model of MCL (Huang et al, submitted). In a completed phase I clinical study in MCL, PD potently and preferentially inhibited CDK4/CDK6 in lymphoma cells despite extensive chromosomal abnormalities, with an excellent toxicity profile and promising clinical response (Leonard et al, submitted). To advance targeting CDK4/CDK6 in MCL, we have now combined PD with escalating dose of bortezomib in an ongoing phase I clinical study (PD-B) in MCL. In this proof-of-concept study, PD is administered on days 1–12 of a 21-day cycle; bortezomib is administered first in prolonged G1 arrest concurrent with PD on days 8 and 11, and again after PD withdrawal in pG1-S on days 15 and 18. CD19+ MCL tumor cells were isolated at baseline, on day 8 and day 21 for analysis. To elucidate the mechanisms that underlie the progression of MCL and the differential response to this novel, cell-sensitizing therapy, we preformed 50×50 paired-end RNA-Sequencing on a HiSeq2000, using one lane for each sample of clinically responding and non-responding patients enrolled in this clinical trial. We generated an average of 76 million reads for each sample, then used the Burrow-Wheeler Aligner (BWA) to align the reads to the genome (Build 37), and SAMtools and the Genome Analysis Toolkit (GATK) to call non-reference variants. We focused on examining genes in the cell cycle and apoptotic pathways, and our data show 400 mutations in 16 genes including CDKN2C (p18), CDK1, E2F2, BBC3 (PUMA), BCL2L11(BIM), JUN and TP53, which are specific to each patient and whose expression changes dynamically during treatment. Moreover, we observe that the overall mutation burden is higher in a non-responding patient relative to the responding patient, and that certain genes (CDKN2C, CDK1, E2F2) show a highly significant (p=2.2×10–16) enrichment of mutations at baseline in the non-responder. By inhibiting CDK4/CDK6, p18 (CDKN2C) is essential for homeostatic cell cycle control of B cell activation and plasma cell differentiation in immunity. Conversely, mutations and deletions of CDKN2C are frequent in MCL, suggesting that loss of CDKN2C contributes to cell cycle dysregulation in this disease. Our RNA-Seq data reveal specific mutations in CDKN2C that are associated with compromised clinical response to PD, in line with cooperative inhibition of CDK4/CDK6 by p18 and PD in BCR-activated B cells as we reported previously. Gene expression profiling and quantitative RNA and protein analyses further demonstrate that induction of prolonged G1 arrest by inhibition of CDK4/CDK6 with PD halts gene expression in early G1 and depletes the expression of those programmed for other phases of the cell cycle. This leads to a metabolic imbalance, which is not restored in pG1-S, thereby sensitizing MCL cells to cytotoxic killing. Mutations in E2F2, which promotes G1/S transition, and CDK1, which functions in G2/M, may therefore antagonize cell cycle sensitization to cytotoxic killing by CDK4/CDK6 inhibition. These data provide new mechanistic insight into therapeutic targeting of CDK4/CDK6 in MCL, and suggest novel molecular targets for personalizing and advancing cell cycle-based therapy in MCL. Disclosures: Martin: Millennium Pharmaceuticals, Inc.: Research Funding, Speakers Bureau. Leonard:Pfizer, Inc: Consultancy; Millenium: Consultancy; Johnson and Johnson: Consultancy; Onyx: Consultancy. Chen-Kiang:Pfizer, Inc.: Research Funding.

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Integrated Analysis of Global DNA Methylation and Transcription Reveals a Leukemia Subtype with Extreme Hypermethylation Associated with Silencing of Regulators of Differentiation

Blood ◽

10.1182/blood-2018-99-118521 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2608-2608

Author(s):

Claudia Gebhard ◽

Roger Mulet-Lazaro ◽

Lucia Schwarzfischer ◽

Dagmar Glatz ◽

Margit Nuetzel ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Promoter Methylation ◽

Research Funding ◽

Promoter Hypermethylation ◽

Ctcf Binding ◽

P Value ◽

Global Dna Methylation ◽

Employment Equity ◽

Equity Ownership

Abstract Acute myeloid leukemia (AML) represents a highly heterogeneous myeloid stem cell disorder classified based on various genetic defects. Besides genetic alterations, epigenetic changes are recognized as an additional mechanism contributing to leukemogenesis, but insight into the latter process remains minor. Using a combination of Methyl-CpG-Immunoprecipitation (MCIp-chip) and MALDI-TOF analysis of bisulfite-treated DNA in a cohort of 196 AML patients we previously demonstrated that (cyto)genetically defined AML subtypes, including CBFB-MYH11, AML-ETO, NPM1-mut, CEBPA-mut or IDH1/2-mut subtypes, express specific DNA-methylation profiles (Gebhard et al, Leukemia, 2018). A fraction of AML patients (5/196) displayed a unique abnormal hypermethylation profile that was completely distinct from any other AML subtype. These patients present immature leukemia (FAB M0, M1) with various chromosomal aberrations but very few mutations (e.g. no IDH1/2, KRAS, DNMT3A) that might explain the CpG island methylator phenotype (CIMP) phenotype. The CIMP patients showed high resemblance with a recently reported CEBPA methylated subgroup (Wouters et al, 2007 and Figueroa et al, 2009), which we confirmed by MCIp-chip and MALDI-TOF analysis. To explore the whole range of epigenetic alterations in the CIMP-AML patients we performed in-depth global DNA methylation and gene expression analyses (MCIp-seq and RNA-seq) in 45 AML and 12 CIMP patients from both studies. Principle component analysis and t-distributed stochastic neighbor embedding (t-SNE) revealed that CIMP patients express a unique DNA-methylation and gene-expression signature that separated them from all other AMLs. We could discriminate promoter methylation from non-promoter methylation by selecting MCIp-seq peaks within 3kb around TSS. Promoter hypermethylation was highly associated with repression of genes (PCC = -0.053, p-value = 0.00075). Hypermethylation of non-promoter regions was more strongly associated with upregulation of genes (PCC = 0.046, p-value = 4.613e-06). Interestingly, differentially methylated regions also showed a positive association with myeloid lineage CTCF binding sites (27% vs 18% expected, p-value < 2.2e-16 in a chi-square test of independence). Methylation of CTCF sites causes loss of CTCF binding, which has been reported to disrupt boundaries between so-called topologically associated domains (TADs), allowing enhancers located in a particular TAD to become accessible to genes in adjacent TADs and affect their transcription. Whether this is the case is under investigation. In this study we particularly focused on the role of hypermethylation of promoters in CIMP-AMLs. Promoters of many transcriptional regulators that are involved in the differentiation of myeloid lineages of which several are frequently mutated in AML were hypermethylated and repressed, including CEBPA, CEBPD, IRF8, GATA2, KLF4, MITF or MAFB. Notably, HMGA2, a critical regulator of myeloid progenitor expansion, exhibited the largest degree of CIMP promoter hypermethylation compared to the other AMLs, accompanied by a reduction in gene expression. Moreover, multiple members of the HOXB family and KLF1 (erythroid differentiation) were methylated and repressed as well. In addition, these patients frequently showed hypermethylation of many chromatin factors (e.g. LMNA, CHD7 or TET2). Hypermethylation of the TET2 promoter could result in a loss of maintenance DNA demethylation and therefore successive hypermethylation at CpG islands. We carried out regulome-capture-bisulfite sequencing on CIMP-AMLs compared to other AML samples and normal blood cell controls and confirmed methylation of the same transcription and chromatin factor promoters. We conclude that these leukemias represent very primitive HSCPs which are blocked in differentiation into multiple hematopoietic lineages, due to the absence of regulators of these lineages. Although the underlying cause for the extreme hypermethylation signature is still subject to ongoing studies, the consequence of promoter hypermethylation is silencing of key lineage regulators causing the differentiation arrest in these cells. We argue that these patients may particularly benefit from therapies that revert DNA methylation. Disclosures Ehninger: Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

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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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Biomarkers of Overall Survival and Response to Glasdegib and Intensive or Non-Intensive Chemotherapy in Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-111239 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2733-2733 ◽

Cited By ~ 1

Author(s):

Jorge E. Cortes ◽

Akil Merchant ◽

Catriona Jamieson ◽

Daniel A Pollyea ◽

Michael Heuser ◽

...

Keyword(s):

Gene Expression ◽

Board Of Directors ◽

Research Funding ◽

Intensive Therapy ◽

Employment Equity ◽

Advisory Committees ◽

Expression Levels ◽

Biomarker Analysis ◽

Cytokine Levels ◽

Equity Ownership

Abstract Background: In a previously reported Phase 2 randomized study of patients with acute myeloid leukemia (AML), addition of the investigational agent glasdegib (PF-04449913) to low-dose cytarabine (LDAC) improved overall survival (OS) when compared with LDAC alone. In a non-randomized study arm, glasdegib together with 7+3 chemotherapy was well tolerated and associated with clinical activity. We used a comprehensive biomarker analysis, evaluating gene expression, circulating cytokine levels, and gene mutations, to identify molecular drivers that predict overall response (OR) and OS. Methods: In this Phase 2 multicenter study (NCT01546038), patients with AML who were suitable for non-intensive therapy were randomized (2:1) to LDAC + glasdegib 100 mg QD or LDAC alone, and patients suitable for intensive therapy were assigned 7+3 plus glasdegib 100 mg QD. Whole blood, serum, and bone marrow aspirate samples were collected at baseline, and used to assess 19 genes for expression analysis, 38 analytes for circulating cytokine levels, and 109 genes for mutation analysis. Gene expression was analyzed using TaqMan Low Density Array Cards (TLDCs), cytokine levels were analyzed using quantitative, multiplexed immunoassays (Myriad RBM), and mutation analysis was performed using the Illumina® MiSeq instrument (San Diego, CA). All correlations were performed either for OS or for OR. For gene expression and cytokine analysis, a cut-off value above or below the median expression level for each treatment arm was used to separate samples into two subgroups (< or ≥ the median value) to explore the relationship of expression levels with OS data. Criteria for significance in the non-intensive cohort required one subgroup to have a p-value of <0.05 in the between-treatment arms comparison and the HR difference between the two subgroups to be ≥2 fold. Responses were defined as patients with a complete remission (CR), CR with incomplete blood count recovery (CRi), morphologic leukemia-free state, partial remission (PR), or PRi. For response correlations, genes or cytokines were considered to be differentially expressed if they had a p-value <0.05 and were differentially expressed by ≥2-fold. Results: Within the non-intensive arm (LDAC + glasdegib, n=68; LDAC alone, n=30), expression levels of several genes correlated with improved OS with glasdegib plus LDAC. Lower levels of expression of FOXM1 and MSI2, and higher expression levels of BCL2 and CCND2 correlated with improved OS with the combination. Additionally, lower levels of the cytokines 6CKINE (CCL21), ICAM-1, MIP-1α, and MMP-3 correlated with improved OS. An analysis of correlations of gene expression and cytokine levels with OR could not be completed due to the low number of responders in the LDAC only group (n=2). In the intensive treatment arm (glasdegib and 7+3, n=59), higher PTCH1 expression correlated with improved OS (p=0.0219, median OS 10.8 versus 39.5 months). In this cohort, lower levels of IL-8 (p=0.0225) and MIP-3β (p=0.0403) correlated with lower OS. Expression levels of no genes or cytokines significantly correlated with OR in this arm. We also examined correlations between gene mutation status and OS in both study arms. In the non-intensive arm (LDAC + glasdegib, n=58; LDAC alone, n=25), no genes mutated in at least 5 patients correlated with OS. In the intensive treatment arm (n=47), mutations in FLT3, TP53, CEP170, NPM1, and ANKRD26 correlated with OS (all p<0.05). Patients in this arm with FLT3 mutations responded better than patients with wild type FLT3 (p=0.0336, median OS of 13.1 months versus unreached for FLT3 mutant). Conclusions: In this biomarker analysis, we found that expression levels of a select number of genes and circulating cytokines implicated in AML correlated with OS in the non-intensive and the intensive arms. The improved response for patients with FLT3 mutations and high PTCH1 expression levels in the intensive arm deserves further investigation. These findings need to be verified in larger controlled studies, which are ongoing. Disclosures Cortes: Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Arog: Research Funding. Pollyea:Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Curis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Heuser:Astellas: Research Funding; Daiichi Sankyo: Research Funding; Sunesis: Research Funding; Tetralogic: Research Funding; Bayer Pharma AG: Consultancy, Research Funding; StemLine Therapeutics: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; BergenBio: Research Funding; Karyopharm: Research Funding. Chan:Pfizer: Employment, Equity Ownership. Wang:Pfizer: Employment, Equity Ownership. Ching:Pfizer Inc: Employment, Equity Ownership. Johnson:Pfizer Inc: Employment, Equity Ownership. O'Brien:Pfizer Inc: Employment, Equity Ownership.

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