Early G1 Arrest Research Articles

Adaptation to ex vivo culture reduces human hematopoietic stem cell activity independently of the cell cycle

AbstractLoss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols that rely on culture. However, the kinetics and mechanisms through which this occurs remain incompletely characterized. In this study, through time-resolved single-cell RNA sequencing, matched in vivo functional analysis, and the use of a reversible in vitro system of early G1 arrest, we defined the sequence of transcriptional and functional events that occur during the first ex vivo division of human LT-HSCs. We demonstrated that the sharpest loss in LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment, limit the global variability in gene expression, and transiently upregulate gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programs in culture. However, contrary to the current assumptions, we demonstrated that the loss of HSC function ex vivo is independent of cell cycle progression. Finally, we showed that targeting LT-HSC adaptation to culture by inhibiting the early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrated that controlling early LT-HSC adaptation to ex vivo culture, for example, via JAK inhibition, is critically important to improve HSC gene therapy and expansion protocols.

Synergistic Activity of the HSP90 Inhibitor Ganetespib With Lapatinib Reverses Acquired Lapatinib Resistance in HER2-Positive Breast Cancer Cells.

Lapatinib is an FDA-approved EGFR and HER2 tyrosine kinase inhibitor for the treatment of HER2-positive breast cancer patients. However, its therapeutic efficacy is limited by primary or acquired resistance. In the present study, we established breast cancers cells with acquired lapatinib resistance and investigated the antitumor activity of the second-generation HSP90 inhibitor ganetespib in association with lapatinib in lapatinib-sensitive and -resistant cells. The combination treatment showed synergistic inhibition of HER and the downstream PI3K/Akt and Ras/MEK/ERK pathways, in addition to enhancing induction of early apoptotic cell death and G1 arrest in both parent and lapatinib-resistant cells in vitro. The joint administration of ganetespib and lapatinib depleted the aberrant nuclear transcription factor STAT3, a mediator of the cell cycle and apoptosis-related pathways that is probably involved in the lapatinib resistance of HER2-positive breast cancer cells. In conjunctive with the augmented inhibition of tumor growth observed in both SKBR3 and SKBR3-L xenografts compared to monotherapy, our data provide a sound preclinical basis for combination treatment with lapatinib and ganetespib for refractory HER2-positive breast cancer.

Iso-suillin-induced DNA damage leading to cell cycle arrest and apoptosis arised from p53 phosphorylation in A549 cells

Extensive investigations have revealed that iso-suillin, a secondary metabolite isolated from Suillus flavus, could induce cell cycle arrest and apoptosis in human chronic myeloid leukemia K562 cells, human hepatocellular carcinoma SMMC-7721 cell line, and human small cell lung cancer H446 cell line in vitro.In the present study, human lung cancer A549 cells were used to reveal the mechanism of iso-suillin's effects on lung adenocarcinoma, which were detected both in vitro and in vivo.Results showed that iso-suillin potently inhibited A549 cell proliferation through an early G1 arrest. Iso-suillin also induced A549 cell apoptosis in vitro. Phosphorylation of p53 at serines 15 and 20 may be one of the pivotal factors for cell cycle arrest and apoptosis after treatment of iso-suillin in A549 cells.Moreover, in an A549 xenograft model, tumor growth and progression could be inhibited by iso-suillin. Body weight change and some vital organs toxicity was also roughly examined, no significant toxic effects of iso-suillin were shown (at a dose of 5 mg/kg for each administration).The in vitro and in vivo anti-tumor effects implied that iso-suillin may act as a tumor growth inhibitor, and its induction of p53 phosphorylation is pivotal for cell cycle arrest and apoptosis in A549 cells.

Composite Copy Number Variation in CDK4, CDKN2A and RB1 Predisposes Mantle Cell Lymphoma to Expansion of PD1+ Tumor Cells and Resistance to CDK4/6 Inhibitor Therapy as Revealed by Integrative Longitudinal scRNA-seq

Composite Copy Number Variation in CDK4, CDKN2A and RB1 Predisposes Mantle Cell Lymphoma to Expansion of PD1+ Tumor Cells and Resistance to CDK4/6 Inhibitor Therapy as Revealed by Integrative Longitudinal scRNA-seq

Disrupting GRINL1A Association with Cereblon Accelerates IKZF1/3 Degradation and IMiD Killing of Mantle Cell Lymphoma Cells By CDK4/6 Inhibition

Lenalidomide (Len) and Pomalidomide (Pom) are immunomodulatory drugs (IMiDs) used for the treatment of multiple myeloma (MM) and have efficacy in other hematologic malignancies. Although as single agents, IMiDs rarely achieve complete remission despite a high overall response rate and durability, they potentiate the clinical response to diverse partners including Rituximab in mantle cell lymphoma (MCL). In MM, the expression of cereblon (CRBN), a substrate receptor of the CRL4CRBN E3 ligase, is required for IMiD's anti-myeloma activity. Binding of IMiDs to CRBN promotes the recruitment of transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) to CRL4CRBN, for ubiquitination-mediated degradation. This leads to loss of IRF4 and ultimately apoptosis. Crystal structure studies have further identified five putative inhibitors of CRBN (GRINL1A, MBOAT7, OTUD7B, C6orf141 and MEIS2). But no information for their endogenous expression or biological function is available, and the mechanism that mediates IMiD sensitivity in MCL is unknown. To determine the mechanism of IMiD action and if any of the putative CRBN inhibitors acts endogenously in MCL, we first demonstrated by whole transcriptome sequencing (WTS), that only POLR2M (encoding GRINL1A) and MBOAT7 mRNAs were expressed along with IKZF1 and IKZF3 in primary human MCL cells and MCL cell lines. Single-cell RNA sequencing (scRNA-seq) analysis of peripheral mononuclear cells (PBMCs) from treatment-naïve MCL patients further demonstrated that POLR2M, but not MBOAT7, was co-expressed with CRBN and IKZF1 in MCL cells, T cells and NK cells. These data suggest that GRINL1A may modulate the CRBN activity in MCL cells. To address this, we found that Len effectively kills the CCMCL1 MCL cell line, despite c-Myc overexpression and loss of p16INK4a, but not the JEKO-1 cell line, which harbors a deletion in TP53. However, Pom kills both MCL cell lines in a time-dependent manner via apoptosis, as indicated by PARP cleavage. Moreover, Pom killing was accelerated and markedly augmented by prior induction of prolonged early G1 arrest (pG1) through selective inhibition of CDK4/6 with palbociclib. GRINL1A and CRBN proteins were coordinately elevated by Pom within 6 hours, unless the MCL cells were already arrested in G1 by palbociclib pretreatment. By contrast, the MBOAT7 protein level remained unchanged by Pom and even decreased in pG1. This implies that GRINL1A and CRBN proteins may be stabilized by association with each other selectively, and this association is opposed by induction of pG1. Indeed, IP-Western blotting confirmed that GRINL1A and CRBN were in the same immune complex in both CCMCL1 and JEKO-1 cells while MBOAT7 was not. Importantly, induction of pG1 reduced the association of GRINL1A with CRBN, greater by one hour of exposure to Pom and more prominently in CCMCL1 cells than in JEKO-1 cells. Consistent with augmented Pom killing by cooperative reduction of the GRINL1A-CRBN complex in pG1, IKZF1 and IKZF3 were completely degraded by 6 hours of Pom exposure in pG1, but significantly less so by Pom alone. Thus, GRINL1A selectively associates with CRBN in MCL cells, and rapid disruption of this association through induction of G1 arrest by CDK4/6 inhibition accelerates Pom killing via IKZF1/3 degradation. In summary, we have demonstrated for the first time that 1) two putative CRBN inhibitors POLR2M and MBOAT7 are expressed in primary human MCL cells by whole transcriptome sequencing; 2) POLR2M, but not MBOAT7, is co-expressed with CRBN and IKZF1 in primary human MCL cells, T cells and NK cells by scRNA-seq; 3) Induction of prolonged early G1 arrest by CDK4/6 inhibition augments Pom killing, even in Len-resistant MCL cells; and 4) GRINL1A, but not MBAOT7, associates with CRBN in MCL cells and induction of G1 arrest cooperates with Pom to reduce this complex, concurrent with accelerated loss of IKZF1/3 and apoptosis despite overexpression of c-Myc, deletion of p16INK4a or TP53 in MCL cells. Our findings shed light on the mechanism of IMiD action in MCL and identify GRINL1A as the first endogenous inhibitor for the CRL4CRBN E3 ligase in MCL cells. As immunomodulatory drugs, Pom and Len act on immune cells in the tumor-microenvironment as well, especially NK cells and T cells. Co-expression of POLR2M with CRBN and IKZF1 in human PBMCs, as we discovered, provides further new insights into controlling the endogenous CRBN inhibitor in IMiD therapy. Disclosures No relevant conflicts of interest to declare.

Cooperative Repression of IRF4 and Induction of IRF7 By CDK4/6 Inhibition Sensitizes Mantle Cell Lymphoma Cells to IMiD-Mediated Apoptosis Independent of p16 Deletion or c-Myc Overexpression

Lenalidomide (Len) and pomalidomide (Pom) are standard of care therapies for multiple myeloma (MM) and also exhibit clinical efficacy in some lymphomas such as diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). As single agents, each rarely achieves complete remission despite a high overall response rate and durability; however, they potentiate the clinical response to diverse partners including Rituximab in MCL. As immunomodulatory drugs (IMiDs), Len and Pom target multiple cell types in vivo and have been shown to promote the expansion of NK cells in MCL. Len in combination with BTK inhibitors apparently leads to synthetic lethality in the activated B cell-like DLBCL cell lines, but the mechanism that mediates IMiD sensitivity in MCL cells remains largely unknown.We found that Len markedly enhanced ACP196 (second generation BTK inhibitor) killing of the CCMCL1 MCL cell line, despite c-Myc overexpression and deletion of p16INK4a, but not the JEKO-1 cells, which harbor TP53 deletion. Therefore Len appears to enhance killing by BTK inhibition but does not confer sensitivity to resistant MCL cells. Next, we investigated how inhibition of CDK4 might sensitize MCL cells to Len or Pom killing, given that dysregulation of cyclin D1 and CDK4 (CDK6 is silenced in MCL) underlies uncontrolled proliferation and disease progression, and inhibition of CDK4 is clinically efficacious in MCL. Indeed, priming of CCMCL1 cells by prior induction of G1 arrest with the selective CDK4/6 inhibitor palbociclib markedly sensitized them to Len-induced apoptosis, but only marginally so in JEKO-1 cells. Kinetic studies revealed that Len reduced the IRF4 protein markedly in CCMCL1 cells arrested in G1 by CDK4 inhibition, but marginally so in cycling CCMCL1 cells and not at all in Len-resistant JEKO-1 or Maver-1 cells. Reduction of IRF4 protein by Len in prolonged G1 arrest was concomitant with induction of IRF7 protein and tightly associated with apoptosis, as evidenced by PARP and caspase-3 cleavage. Moreover, inhibition of CDK4 conferred sensitivity to Pom in the Len-resistant JEKO-1 cells, and it was concurrent with reduction of IRF4 and induction of IRF7.The triple combination of Len, Ibrutinib, and Rituximab in MCL has been shown to be clinically active in relapsed MCL. To explore the underpinning, we found by RNA-seq that CD20 mRNA expression was downregulated in normal primary human peripheral B cells when they were activated into the cell cycle and in primary MCL cells, suggesting an inverse correlation between cell cycling and CD20 mRNA expression. However, surface CD20 expression was not collinear with CD20 mRNA expression and there was no evidence for upregulation of CD20 by BTK inhibition. On this basis, upregulation of the rituximab target CD20 is unlikely to be the primary mechanism for improved clinical response to this triple combination in MCL.In summary, our data suggest cooperative repression of IRF4 and induction of IRF7 as a central mechanism that underlies enhancement of Len and Pom killing by inhibition of BTK or CDK4, independent of deletion of the physiologic CDK4/6 inhibitor p16INK4a or overexpression of c-Myc. Furthermore, it appears that induction of prolonged early G1 arrest by Palbociclib confers Pom sensitivity independent of p53, implicating a potential to combine Palbociclilb with Pom for treatment of MCL with p53 loss or mutation. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2300: CDK4/6 inhibition triggers dissociation of MEIS2 from CRL4CRBN E3 ligase to induce interferon response and reprogram myeloma for IMiD vulnerability

Abstract Inhibition of CDK4/6 has emerged as an effective therapy in human cancer. Palbociclib, the first selective CDK4/6 inhibitor (CDK4/6i), is FDA-approved for treatment of metastatic breast cancer, and abemaciclib has been fast tracked for approval as a front line breast cancer treatment. Recent evidence in mouse models of breast cancer further indicates that CDK4/6 inhibition triggers anti-tumor immunity, but the precise tumor intrinsic mechanism that mediates CDK4/6 inhibition for a clinical response remains to be defined. To address this, we inhibited CDK4/6 with palbociclib in sequential combination with immunomodulary drugs (IMiD)s in primary bone marrow myeloma cells (BMMCs), using normal bone marrow plasma cells as a control. IMiDs, lenalidomide (Len) and pomalidomide (Pom), are standard of care for multiple myeloma (MM) and lymphoma. Each rarely achieves complete remission despite a high overall response rate and durability. However, each markedly potentiates the clinical efficacy of diverse partners by unknown mechanisms. Cereblon (CRBN), a substrate receptor of the CUL4-ROC1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase, is requited for IMiD's anti-tumor activity. Binding of Len or Pom to CRBN accelerates the recruitment of transcription factors IKZF1/3 to CRL4CRBN for ubiquitination and degradation in MM cells. One of the targets of IKZF1/3 is IRF4, which is required for survival of MM cells and reduced after exposure to Len or Pom. Moreover, MEIS2, a homeobox transcription factor was identified to be an substrate of CRBN in crystal structure and by biochemical screen, implicating a role for MEIS2 in modulates IMiD's anti-tumor activity. However, evidence for MEIS2 expression in myeloma cells is lacking. Previously, we have shown that prolonged early G1 arrest (pG1) induced by sustained CDK4/6 inhibition reprogrammed cancer cells for vulnerability to diverse clinically relevant agents ex vivo and in animal models. To address if CDK4 inhibition reprograms MM cells for IMiD vulnerability, first we show that the intrinsic IMiD sensitivity in primary BMMCs ex vivo is a major determinant for the clinical response to IMiD therapy in myeloma patients. Second, we discovered that MEIS2 is aberrantly expressed and associated with CRBN in BMMCs. Third, MEIS2 is regulated by the cell cycle in primary MM cells and required for the survival of MM cells in part by maintaining IRF4 expression. Finally, CDK4/6 inhibition liberates CRBN from association with MEIS2 in pG1. This rapidly accelerates Len-mediated ubiquitination and degradation of IKZF1/3, loss of IRF4, de-repression of IRF7 and induction of interferon (IFN) responsive genes that culminated in TRAIL-mediated apoptosis. Dissociation of MEIS2 from CRBN thus represents a noel mechanism by which CDK4/6 inhibition reprograms myeloma for vulnerability to IMiDs through induction anti-tumor IFN response. Citation Format: Xiangao Huang, David Jayabalan, Maurizio Di Liberto, Zhengming Chen, Anna Schinzel, Scott A. Ely, Adriana Rossi, Morton Coleman, Joseph M. Lane, William C. Hahn, Tomer M. Mark, Ruben Niesvizky, Selina Chen-Kiang. CDK4/6 inhibition triggers dissociation of MEIS2 from CRL4CRBN E3 ligase to induce interferon response and reprogram myeloma for IMiD vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2300.

Abstract 1523: Repression of IRF4 and CYTIP unleashes anti-tumor interferon response in CDK4 inhibitor therapy in mantle cell lymphoma

Abstract Inhibition of CDK4/6 has emerged as an effective cancer therapy, but the underlying mechanism remains obscure. We have addressed this question in mantle cell lymphoma (MCL), a Non-Hodgkin's B cell lymphoma that remains incurable due to the development of drug resistance. As dysregulation of CDK4 (CDK6 is silenced) and cyclin D1 underlies unrestrained proliferation of MCL cells, targeting CDK4 also represents a rational approach to MCL therapy. Previously we have demonstrated in preclinical studies that sustained inhibition of CDK4 with palbociclib led to prolonged early G1 arrest (pG1) that both prevented proliferation of MCL cells and reprogrammed them for killing by diverse agents. On this basis, we hypothesized that CDK4 inhibition may prolong and deepen the clinical response to its partner drug in combination therapy. Indeed, in a phase I clinical trial of palbociclib plus ibrutinib (BTK inhibitor) in recurrent MCL (n=28), complete response was observed in 43% of patients as compared with 23% in response to ibrutinib alone. Moreover, the responses were rapid and durable; only 4 of the 18 responding-patients have progressed in the 3.5 years since the trial opened. To address how CDK4 inhibition enhances therapy vulnerability, in a separate phase I clinical trial palbociclib was administered to MCL patients for 12 days to induce pG1; bortezomib was given in pG1 and again after synchronous S phase reentry (pG1-S). At the optimal dose combination only one of 6 patients progressed and complete remission in one patient continues into its 6th year. Longitudinal integrative RNA and exome-sequencing (WTS/WES) of MCL cells isolated from lymph node biopsies at baseline, in pG1 (day 8) and in pG1-S (day 21) of individual patients showed that inhibition of CDK4 induced pG1 in all patients initially, regardless of the subsequent clinical response. However, induction of pG1 led to a striking difference in cellular gene expression in pG1 (day 8/day 1) in MCL cells of responders (R) and non-responders (NR). There were 2041 genes upregulated and 1483 genes downregulated 1.5-fold or greater in R (N=4, EdgeR, FDR 0.05), but only 39 genes upregulated and 401 genes dowregulated in NR (N=3). Moreover, 5 genes were down regulated in R and upregulated in NR. Gene set enrichment analysis together with loss and gain of function studies revealed that among them, loss of IRF4 in R led to de-repression of IRF7 and induction of the interferon response. This in turn resulted in TRAIL mediated apoptosis due to repression of CYTIP downstream of IRF4 repression. In summary, by longitudinal functional genomics of purified MCL cells and functional studies, we have discovered that CDK4 inhibition reprograms MCL for therapy vulnerability through induction of anti-tumor IFN response, and identified CYTIP and IRF4 as biomarkers that discriminate sensitivity from resistance to therapeutic targeting of CDK4. Citation Format: Maurizio Di Liberto, Peter Martin, Xiangao Huang, Priyanka Vijay, David Chiron, Scott Ely, Christopher Mason, Olivier Elemento, John Leonard, Selina Chen-Kiang. Repression of IRF4 and CYTIP unleashes anti-tumor interferon response in CDK4 inhibitor therapy in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1523.

PIK3IP1 Inhibition of PI3K in G1 Arrest Induced By CDK4 Inhibition Reprograms MCL for Ibrutinib Therapy

Inhibition of CDK4/6 has emerged as a promising therapy for diverse human cancers. Mantle cell lymphoma (MCL), in which aberrant cyclin D1 expression and CDK4 activity underlie unrestrained proliferation of tumor cells, remains incurable. Targeting Bruton tyrosine kinase (BTK) with ibrutinibin recurrent MCLhas unprecedented single agent activity, with a completion remission (CR) rate of 21%. However, progression on ibrutinib is virtually universal in MCL and is associated with aggressive proliferation of tumor cells and a dismal clinical outcome.By longitudinal integrated RNA- and exome-seq analysis, we previously discovered a mutation in the ibrutinib binding site (BTKC481S) in MCL that was specific to relapse from a durable response, but absent in both primary resistance and resistance following a transient response. Irrespective of mechanism, ibrutinib resistance was associated with enhanced PI3K activation as determined by Western blotting of MCL cells isolated from serial biopsies. Induction of prolonged early G1 arrest (pG1) by selective inhibition of CDK4 with palbociclib sensitized to PI3K inhibition independent of this BTK mutation and to ibrutinib when the wild type BTK was retained. In both cases, synergistic killing appears to occur via cooperative inactivation of PI3K. Collectively, our data suggest that dual inhibition of CDK4 and BTK or PI3K restrain the expansion of resistant clones or even eliminate them to deepen or prolong the clinical response to targeting BTK.To test this hypothesis, we initiated a phase I clinical trial of palbociclib plus ibrutinib in recurrent MCL patients in August of 2014. All 5 dose levels included ibrutinib daily and palbociclib administered on days 1-21 of each 28-day cycle (Figure 1). Based on an intent-to-treat analysis of 18 patients, 12 (67%) patients have responded to treatment including 8 (44%) complete responses (CR). The median time to CR was 3 cycles and no responding patients have progressed on study. The therapy was well tolerated (see ASH abstract by Martin et al for clinical information). RNA- and exome-seq were performed to assess the genomic alternations and gene expression on purified MCL cells from samples before therapy, on progression and on treatment from PR patients. Initial analysis did not detect non-synonymous mutation in CDK4, CCND1, or genes in the BCR or PI3K signaling pathway. Hemizygous deletion of Rb, p53, ATM and the INK4A/B locus and amplification of PIK3CA and PIK3CB occurred in 30-50 % of patients before therapy. These copy number variations were predominantly associated with primary resistance (4 subjects) but did not preclude CR or PR. By contrast, expression of PIK3IP1, a putative PI3K inhibitor, was markedly reduced on progression in all primary resistant patients, but not in the PR patient. Function studies demonstrated that PIK3IP1 was required for pG1 sensitization to BTK and PI3K inhibition in MCL cells in vitro. Moreover, PIK3IP1 was induced in pG1 together PI3K inhibition in MCL cells in patients responding to palbociclib alone in a separate clinical trial (Martin, DiLiberto et al, unpublished), suggesting that PIK3IP1 mediates pG1 reprogramming.In summary, the high CR rate and durability support our hypothesis that induction of prolonged early G1 arrest by CDK4 inhibition reprograms MCL cells for deepened and durable targeting of BTK in MCL. Our data further suggest inhibition of PI3K by PIK3IP1 as a potential mechanism to overcome ibrutinib resistance. A phase II multi-center clinical trial is planned to further elucidate the genes and mechanisms that discriminate sensitivity from resistance to dual targeting of CDK4 and BTK. [Display omitted] DisclosuresBlum:Pharmacyclics: Research Funding. Ruan:Pharmacyclics: Research Funding, Speakers Bureau. Martin:Janssen: Consultancy, Honoraria, Other: travel, accommodations, expenses; Celgene: Consultancy, Honoraria; Gilead: Consultancy, Other: travel, accommodations, expenses; Novartis: Consultancy; Acerta: Consultancy; Teva: Research Funding.

Abstract IA08: Reprogramming human cancer cells in CDK4/6 inhibitor therapy

Abstract CDK4 and CDK6 drive cell cycle progression through early G1 and are frequently deregulated in human cancer. Selective inhibition of CDK4/6 with palbociclib (PD 0332991) has demonstrated exciting clinical efficacy in diverse human cancers. However, the mechanism that discriminates sensitivity from resistance to targeting CDK4/6 remains obscure. Mantle cell lymphoma (MCL) is an incurable non-Hodgkin's lymphoma where deregulated CDK4 activity and cyclin D1 expression underlies unrestrained proliferation and disease progression. In a phase I clinical trial in recurrent MCL, we demonstrated that palbociclib alone produced a durable clinical response with a favorable toxicity profile. To investigate the mechanism for targeting CDK4/6, we have now shown in primary cancer cells that 1) inhibition of CDK4/6 leads to early G1 arrest that requires Rb, the CDK4/6 substrate; 2) prolonged early G1 arrest (pG1) sensitizes Rb-proficient cancer cells to killing by diverse clinically-relevant agents; and 3) pG1 sensitization stems from continuous expression of genes scheduled for early G1 only. This leads to an imbalance in genes expression, which is exacerbated in synchronous progression to S phase (pG1-S) after palbociclib withdrawal, due to incomplete restoration of cell cycle-coupled gene expression. In a hypothesis-driven phase Ib clinical trial, targeting CDK4 with palbociclib in sequential combination with the proteasome inhibitor bortezoimb was well tolerated (n=16). It exhibited a durable palbociclib dose-dependent clinical activity, including one complete remission for over 3 years with only one progression at the optimal dose combination (n=6). Longitudinal integrative whole transcriptome and whole exome sequencing of tumor cells isolated from serial lymph node biopsies demonstrated that palbociclib initially induced pG1 in MCL cells of all patients, regardless of copy number variation or mutation (ATM, p53). Cell cycle control by palbociclib is thus initially intact in MCL, but is insufficient to predict the clinical response. As predicted, only genes programmed for early G1 were expressed pG1, concurrent PI3K inactivation in primary MCL cells. However, <1% of the 1400 genes suppressed (not programmed) in pG1 in clinically-responding patients were activated in non-responding patients. These genes were critical for redox homeostasis, suggesting that PI3K inactivation and redox stress mediates pG1 sensitization. This study represents the first investigation of genes that discriminate sensitivity from resistance in targeting CDK4/CDK6 in human cancer, through integrative longitudinal analysis of whole exome and whole transcriptome sequencing in concert with protein expression analysis and functional studies. Selective inhibition of CDK4 induces pG1 in all MCL patients, which apparently reprograms MCL for clinical response to bortezomib through PI3K inactivation and suppression of genes for redox homeostasis. Citation Format: Selina Chen-Kiang, Maurizio Di Liberto, Priyanka Vijay, David Chiron, Xiangao Huang, Scott Ely, Olivier Elemento, Christopher Mason, Lewis Cantley, John P. Leonard, Peter Martin. Reprogramming human cancer cells in CDK4/6 inhibitor therapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr IA08.

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

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

The Novel CDK4/6-Inhibitor Abemaciclib Induces Early G1-Arrest in MCL Cell Lines, Sensitizes Cells to Cytarabine Treatment and Is Additive with Ibrutinib

Introduction: Mantle cell lymphoma (MCL) is characterized by t(11;14) resulting in a constitutive cyclin D1 overexpression. The cyclin D1-CDK4/6 complex inactivates Rb through phosphorylation, leading to G1/S-phase transition. Therefore, inhibition of CDK4/6 is an efficient and rational approach to overcome cell cycle dysregulation in MCL. We evaluated the efficiency of the novel CDK4/6 inhibitor abemaciclib in various MCL cell lines and in primary MCL cells in combination with cytarabine (AraC) and ibrutinib.Material & Methods: MCL cell lines (Granta 519, JeKo-1, Maver-1, Mino) and primary MCL cells were exposed to abemaciclib alone and combined with AraC or ibrutinib. Cells were pretreated with abemaciclib and exposed to AraC or ibrutinib with or without consecutive wash-out of the CDK4/6 inhibitor. Proliferation and viability were measured by tryptan blue staining and Cell Titer Glo assay. Flow cytometry was used for cell-cycle (PI-staining) and apoptosis analysis (Annexin V PE/7AAD-staining). Western Blot analysis showed protein expression and phosphorylation status of various downstream proteins.Results: Abemaciclib inhibited cell proliferation by induction of early G1-arrest. Western Blot analysis revealed reduced phosphorylation of Rb on serine 795 without changes in CDK 4 and cyclin D1 expression, in line with reversible cell cycle arrest. IC50-values of sensitive cell lines (JeKo-1, Maver-1, Mino) were <30 nM after 72 h. We observed an almost complete and reversible G1-arrest in all sensitive cell lines by FACS analysis (JeKo-1: G1-phase +51,7 %; S/G2-phase -51,7 % at 31,25 nM after 24 h; G1-phase +35,4 %; S/G2-phase -34,8 % after 72 h), whereas cell viability was not reduced.Wash-out of abemaciclib after 24 h resulted in synchronized S-phase entry in all sensitive cell lines (e.g. Mino: G1-phase -20,4 %; S-phase +30,5 %). The sequential combination of abemaciclib followed by AraC showed strong synergy in Mino cells (CI=0,22 for 31,25 nM abemaciclib and 3,33 µM cytarabine). In contrast, simultaneous exposure to abemaciclib had a protective effect against AraC treatment in all sensitive cell lines, due to an ongoing G1-arrest (Mino: CI=-0,19 for 31,25 nM abemaciclib and 3,33 µM AraC).In primary MCL cells, 31,25 nM of abemaciclib had no impact on cell death. Moreover, no sensitization to AraC was observed as all cells were resting in G0-phase.The combination of abemaciclib induced G1 arrest and ibrutinib had additive or synergistic effects in sensitive cell lines (JeKo-1, Mino and Maver).Conclusion: The novel CDK4/6 inhibitor abemaciclib causes reversible G1 cell cycle arrest without loss of viability at low nanomolar doses. Rationale drug combinations exploiting the sequential effect may achieve major benefits, but drug interactions are complex: Pretreatment with abemaciclib sensitizes MCL cell line cells to AraC whereas simultaneous application protects them from AraC treatment. Further analyses explore the interaction with other targeted approaches (inhibitors of the B-cell receptor pathway) to better understand the underlying molecular mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3095: Longitudinal integrative whole transcriptome and exome sequencing identifies genes that reprogram lymphoma cells for clinical response to CDK4/6 inhibition in combination therapy

Abstract CDK4 and CDK6 drive cell cycle progression through early G1 and are frequently deregulated in human cancer. The selective inhibitor palbociclib (PD 0332991) demonstrated exciting clinical efficacy in diverse human cancers, notably in metastatic breast cancer where it more than doubled the progression free survival of patients treated with letrozole. In mantle cell lymphoma (MCL), deregulated CDK4 and cyclin D1 expression underlies unrestrained proliferation and disease progression. In a phase I clinical trial in recurrent MCL, we showed that palbociclib produced a durable clinical response. However, the mechanism that discriminates sensitivity from resistance to targeting CDK4/6 remains obscure. To address this question, we demonstrated that 1) inhibition of CDK4/6 leads to early G1 arrest that requires Rb, the CDK4/6 substrate; 2) prolonged early G1 arrest (pG1) sensitizes Rb+ cancer cells to killing by diverse clinically-relevant agents; and 3) pG1 sensitization stems from continuous expression of genes scheduled for early G1 only. This sensitization is exacerbated in synchronous progression to S phase (pG1-S) after palbociclib withdrawal, due to incomplete restoration of cell cycle-coupled gene expression. In a hypothesis-driven phase Ib clinical trial we further showed that palbociclib induces pG1 and sensitizes MCL cells to the proteasome inhibitor bortezomib. At the optimal dose of palbociclib, only one in 6 patients progressed, and 4 patients had a durable responses including a complete remission for &amp;gt;800 days. Longitudinal integrative whole transcriptome and whole exome sequencing of tumor cells from serial lymph node biopsies demonstrated that cell cycle control by palbociclib is initially intact in Rb+ MCL but is insufficient to predict clinical response. Palbociclib induced pG1 in all patients initially regardless of the subsequent clinical response or patient-specific copy number variation or mutation (ATM, p53). As expected, pG1 maintained the expression of cell cycle genes programmed for early G1 and suppressed those scheduled for other phases, and this was reversible upon release of the early G1 block. However, expressing only genes scheduled for early G1 led to an imbalance in gene expression. Of the 1483 genes suppressed in pG1 compared with the baseline in Responders (Rs) (N = 4), 5 were up-regulated in Non-Responders (NRs) (N = 3); 11 of the 2041 genes up-regulated in pG1 in R were suppressed in NR. These oppositely expressed genes are involved in redox stress and metabolism based on functional analysis, suggesting a role for cell cycle-coupled metabolic imbalance in differential clinical response to targeting CDK4/6. Further validation of these candidate genes in the context of clinical response should advance the mechanism for therapeutic targeting of CDK4/6 as well as genome-based therapy and patient stratification. Citation Format: Maurizio Di Liberto, Peter Martin, David Chiron, Priyanka Vijay, Xiangao Huang, Pedro Blecua, Scott Ely, Olivier Elemento, John P. Leonard, Christopher E. Mason, Selina Chen-Kiang. Longitudinal integrative whole transcriptome and exome sequencing identifies genes that reprogram lymphoma cells for clinical response to CDK4/6 inhibition in combination therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3095. doi:10.1158/1538-7445.AM2015-3095

Abstract 4031: CDK4/6 inhibition amplifies IMiD killing of myeloma cells by accelerating CRL4CRBN-dependent degradation of IKZF1 and IKZF3 in opposition to MEIS2

Abstract The immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) are effective therapies for hematologic malignancies, especially multiple myeloma (MM). Cereblon (CRBN) is required for IMiD's anti-myeloma activity. Emerging evidence has established that CRBN is a substrate receptor of the CUL4-ROC1-DDB1-CRBN (CRL4CRBN) E3 ligase complex and that the homeobox transcription factor MEIS2 is an endogenous substrate of CRL4CRBN. IMiDs block MEIS2 from binding to CRBN, thereby facilitating the recruitment of transcription factors IKZF1 and IKZF3 to CRL4CRBN for ubiquination-proteasome degradation. IKZF1 and IKZF3 appear to be required for MM survival, in part by transcriptional regulation of IRF4. These findings, obtained in heterologous systems and MM cell lines, suggest that modulation of the CRL4CRBN E3 ligase is a critical early event in IMiD killing of MM cells. We investigated its physiologic relevance in primary bone marrow myeloma cells (BMMCs) in co-culture with HS-5 bone marrow stromal cells. We showed that IMiDs preferentially kill BMMC in G1 arrest and that induction of prolonged early G1 arrest (pG1) by selective inhibition of CDK4/CDK6 with palbociclib (PD 0332991) markedly enhanced Len/Pom killing of BMMCs (14/21), unless the BMMCs were refractory or extremely sensitive to Len (n = 7). Importantly, the IMiD sensitivity in BMMCs ex vivo correlates with subsequent clinical responses to IMiD-based therapies in vivo in 7/7 cases characterized. This novel finding suggests that although IMiDs target multiple cell types, the intrinsic IMiD sensitivity is a major determinant for the clinical response to IMiDs in MM. Whole transcriptome sequencing and gene set enrichment analysis further revealed that Len induces the interferon response signature genes in BMMCs, and this is enhanced by palbocicib. While CRBN is ubiquitously expressed, IKZF3 and IRF4 (mRNA and protein) and the IKZF1 protein were barely detectable in normal bone marrow plasma cells and prominently increased in BMMCs. Primary myeloma cells thus appear to be addicted to IKZF1/IKZF3-IRF4 signaling for survival, and IMIDs induce CRL4CRBN-mediated degradation of IKZF1 and IKZF3 to antagonize this survival pathway. Accordingly, palbociclib enhancement of Len killing was associated with rapid acceleration of IKZF1/IKZF3 degradation, within one hour of Len treatment in BMMCs. Palbociclib acts in opposition of MEIS2, because MEIS2 knockdown mimicked palbociclib sensitization and MEIS2 reconstitution antagonized palbociclib sensitization. In summary, we have demonstrated that induction of pG1 by CDK4/6 inhibition sensitizes primary myeloma cells to IMiD killing through rapid acceleration of CRL4CRBN -dependent degradation of IKZF1 and IKZF3 in opposition to MEIS2. These findings are being validated in a clinical trial targeting CDK4/6 with palbociclib in combination with Len in MM. Note: This abstract was not presented at the meeting. Citation Format: Xiangao Huang, David Jayabalan, Maurizio Di Liberto, Tomer M. Mark, Zhengming Chen, Adriana Rossi, Anna C. Schinzel, William C. Hahn, Mohamad A. Hussein, Rubin Niesvizky, Selina Chen-Kiang. CDK4/6 inhibition amplifies IMiD killing of myeloma cells by accelerating CRL4CRBN-dependent degradation of IKZF1 and IKZF3 in opposition to MEIS2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4031. doi:10.1158/1538-7445.AM2015-4031

CDK4/6 Inhibitor PD 0332991 Sensitizes Acute Myeloid Leukemia to Cytarabine-Mediated Cytotoxicity.

Cyclin-dependent kinase (CDK)4 and CDK6 are frequently overexpressed or hyperactivated in human cancers. Targeting CDK4/CDK6 in combination with cytotoxic killing therefore represents a rational approach to cancer therapy. By selective inhibition of CDK4/CDK6 with PD 0332991, which leads to early G1 arrest and synchronous S-phase entry upon release of the G1 block, we have developed a novel strategy to prime acute myeloid leukemia (AML) cells for cytotoxic killing by cytarabine (Ara-C). This sensitization is achieved in part through enrichment of S-phase cells, which maximizes the AML populations for Ara-C incorporation into replicating DNA to elicit DNA damage. Moreover, PD 0332991 triggered apoptosis of AML cells through inhibition of the homeobox (HOX)A9 oncogene expression, reducing the transcription of its target PIM1. Reduced PIM1 synthesis attenuates PIM1-mediated phosphorylation of the proapoptotic BAD and activates BAD-dependent apoptosis. In vivo, timely inhibition of CDK4/CDK6 by PD 0332991 and release profoundly suppresses tumor growth in response to reduced doses of Ara-C in a xenograft AML model. Collectively, these data suggest selective and reversible inhibition of CDK4/CDK6 as an effective means to enhance Ara-C killing of AML cells at reduced doses, which has implications for the treatment of elderly AML patients who are unable to tolerate high-dose Ara-C therapy.

Inhibition of human cytomegalovirus replication by artemisinins: effects mediated through cell cycle modulation.

Artemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G0/G1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G1/S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition.

Sensitizing shRNA Screen for Molecular Targets in CDK4/CDK6-Based Combination Therapy in Multiple Myeloma

CDK4 and CDK6 are rarely mutated but are overexpressed or hyperactivated at a very high frequency in human cancers. By inhibiting CDK4/CDK6 with an exceptionally selective and reversible inhibitor, palbociclib (PD 0332991), we have developed a novel strategy to reprogram cancer cells for cytotoxic killing through induction of prolonged early G1 arrest (pG1). We have demonstrated that pG1 sensitizes cancer cells expressing Rb, the substrate of CDK4 and CDK6, to cytotoxic killing by forcing an imbalance in gene expression because only genes scheduled for early G1 are expressed. This sensitization is exacerbated after palbociclib withdrawal due to incomplete restoration of gene expression despite S phase synchronization (pG1-S). This study aims to identify genes that mediate pG1-S sensitization to two clinically-relevant agents for myeloma, the proteasome inhibitors carfilzomib and bortezomib, in model cell lines by a sensitizing pool genome-wide shRNA screen, and by validating the hits in a clinical trial of palbociclib in combination with bortezomib and dexamethasone.We ranked the hits based on the enrichment of target shRNAs, and representation in replica of each cell lines and among different human myeloma cell lines (HMCLs) as well as functional analyses. In myeloma cells, cell cycle control by palbociclib was intact in all hits, demonstrating that CDK4 and CDK6 are indispensable for myeloma replication. Among the top ranking 20 candidates, we found that NEDD4L was essential for proteasome inhibitor killing, FTH1 modulated the threshold of killing by diverse agents especially in pG1-S, and IL10RAappeared to be required for pG1-S sensitization to proteasome inhibitors.Moreover, RNA-sequencing analysis of primary myeloma cells from a phase II clinical trial targeting CDK4/CDK6 with palbociclib in combination with bortezomib in myeloma revealed that a higher level of FTH1 expression in myeloma cells in vivo correlated with sensitivity to this therapy, suggesting a role for FTH1 in differential sensitivity to this CDK4/CDK6-based therapy in myeloma. Selective inhibition of CDK4/CDK6 with palbociclib, or another specific inhibitor such as LY2835219 or LEE011, in combination therapy has now achieved unprecedented clinical efficacy in diverse human cancers. Most notably, palbociclib more than doubled the progression free survival of metastatic breast cancer patients when it was combined with letrozole, and has been designated a “breakthrough therapy” by the FDA for breast cancer. Our work provides the first insight into genes that mediate cell cycle sensitization to cytotoxic killing through selective CDK4/CDK6 inhibition. It provides an exciting potential for further investigation in a clinical context, such as the ongoing phase I clinical trial combining palbociclib with the immunomodulatory drug lenalidomide in patients with relapsed/refractory myeloma. DisclosuresHuang:Celgene: Research Funding. Off Label Use: PD 0332991 (palbociclib) is a specific CDK4/CDK6 inhibitor used to stop the cell cycle.. Niesvizky:Onyx: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Millennium: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau. Chen-Kiang:Celgene: Consultancy, Research Funding.

Cell-cycle reprogramming for PI3K inhibition overrides a relapse-specific C481S BTK mutation revealed by longitudinal functional genomics in mantle cell lymphoma.

Despite the unprecedented clinical activity of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in mantle cell lymphoma (MCL), acquired resistance is common. By longitudinal integrative whole-exome and whole-transcriptome sequencing and targeted sequencing, we identified the first relapse-specific C481S mutation at the ibrutinib binding site of BTK in MCL cells at progression following a durable response. This mutation enhanced BTK and AKT activation and tissue-specific proliferation of resistant MCL cells driven by CDK4 activation. It was absent, however, in patients with primary resistance or progression following transient response to ibrutinib, suggesting alternative mechanisms of resistance. Through synergistic induction of PIK3IP1 and inhibition of PI3K-AKT activation, prolonged early G1 arrest induced by PD 0332991 (palbociclib) inhibition of CDK4 sensitized resistant lymphoma cells to ibrutinib killing when BTK was unmutated, and to PI3K inhibitors independent of C481S mutation. These data identify a genomic basis for acquired ibrutinib resistance in MCL and suggest a strategy to override both primary and acquired ibrutinib resistance. We have discovered the first relapse-specific BTK mutation in patients with MCL with acquired resistance, but not primary resistance, to ibrutinib, and demonstrated a rationale for targeting the proliferative resistant MCL cells by inhibiting CDK4 and the cell cycle in combination with ibrutinib in the presence of BTK(WT) or a PI3K inhibitor independent of BTK mutation. As drug resistance remains a major challenge and CDK4 and PI3K are dysregulated at a high frequency in human cancers, targeting CDK4 in genome-based combination therapy represents a novel approach to lymphoma and cancer therapy. Cancer Discov; 4(9); 1022-35. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973.

Integrative Whole Transcriptome and Exome Sequencing Reveals Reprogramming Of Mantle Cell Lymphoma In Prolonged Early G1 Arrest For Clinical Response To Selective Inhibition Of CDK4 In Combination Therapy

CDK4 and CDK6, which drive cell cycle entry and progression through G1 in the presence of cyclin D, are overexpressed at a high frequency in human cancers. Targeting CDK4 with the first selective and potent CDK4/CDK6 inhibitor, palbociclib (PD 0332991), has recently achieved unprecedented clinical efficacy in both hematologic malignancies and solid tumors. Most notably, palbociclib more than tripled the progression free survival of breast cancer patients treated with letrozole. In mantle cell lymphoma (MCL), CDK4 overexpression and aberrant cyclin D1 expression leads to unrestrained cycling and proliferation that underlies disease progression. In the first phase I single-agent palbociclib clinical trial in recurrent MCL, inhibition of CDK4 by palbociclib alone resulted in a durable clinical response with tumor regression in some MCL patients, including one complete response and two partial response.However, the fundamental mechanism for differential clinical response to selective targeting of CDK4/CDK6 remains obscure. To address this question, we have developed a novel strategy that both inhibits proliferation of cancer cells and reprograms them for cytotoxic killing by reversible inhibition of CDK4/CDK6. We have shown that: 1) inhibition of CDK4/6 with palbociclib leads to early G1 arrest that is dependent on Rb, the substrate for CDK4/CDK6; 2) prolonged early G1 arrest (pG1) reprograms cancer cells for killing by diverse agents; 3) pG1 sensitization is exacerbated in synchronous S phase entry (pG1-S) upon palbociclib withdrawal. Further, pG1 sensitization appears to stem from restricted expression of genes scheduled for early G1 only, which is exacerbated in pG1-S due to incomplete restoration of cell cycle-coupled gene expression.To advance targeting CDK4 in MCL, we have implemented this strategy combining palbociclib with bortezomib at a reduced dose (1.0 mg /m2) in a phase I clinical trial (Pa-Btz) in recurrent MCL. palbociclib was administered to MCL patients on days 1-12 of a 21-day cycle to induce pG1; bortezomib was given on days 8 and 11 in pG1 and on days 15 and 18 in pG1-S. Pa-Btz was well tolerated and appeared to have a palbociclib dose-dependent durable clinical activity, with only one of 6 patients progressed at the optimal dose combination.We investigated the genes that mediate pG1 reprogramming by integrative whole exome sequencing (WES) and whole transcriptome sequencing (WTS). The dynamic changes in cell cycle-coupled gene expression were determined within individual patients in primary MCL tumor cells isolated from serial lymph node biopsies at baseline, in pG1 (day 8) and in pG1-S (day 21) in conjunction with immunohistochemistry (IHC). palbociclib inhibited CDK4 and induced pG1 in all patients initially, regardless of the clinical response, mutations in p53 or ATM, cyclin D1 3'UTR deletion, or other patient-specific deletions, amplifications and mutations. No mutations were detected in CDK4, which was expressed in primary MCL cells over CDK6. Induction of pG1 maintained the expression of cell cycle genes programmed for early G1 (CDK4, cyclin D and Rb), and prevented the expression of those scheduled for late G1 (cyclin A), S phase (Ki 67, TK) and G2/M (CDK1, cyclin B), and this was completely reversible upon release of the early G1 block.However, induction of pG1 also led to an imbalance in the expression of other cellular genes due to restricted expression of only genes programmed for early G1. Among the 868 genes that were suppressed in pG1 (not programmed for early G1) in MCL tumor of clinically-responding patients (N=4, EdgeR, FDR 0.05), 9 were conversely activated in the non-responding patients (N=4, EdgeR, FDR 0.05). These genes are involved in redox stress, metabolism and cell migration, suggesting a potential role of cell cycle-coupled metabolic imbalance in differential clinical response to targeting CDK4/6.Thus, selective inhibition of CDK4 led to Rb-dependent pG1 in tumor cells of all MCL patients despite a multitude of genomic aberrations. Integrative WES and WTS analysis of serial tumor biopsies revealed that pG1 reprograms MCL cells by inducing an imbalance in gene expression that is associated with the clinical response to the Pa-Btz therapy. Defining the functions of the candidate genes identified in the context of clinical response should shed light on the mechanism for therapeutic targeting of CDK4/CDK6 and advance genome-based patient stratification. Disclosures:Off Label Use: PD 0332991 is a CDK4/CDK6 selective inhibitor. Martin:Teva: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech: Speakers Bureau; Millennium: Research Funding; Seattle Genetics: Consultancy, Speakers Bureau. Leonard:millennium: Consultancy.

The Combination Of Palbociclib Plus Bortezomib Is Safe and Active In Patients With Previously Treated Mantle Cell Lymphoma: Final Results Of a Phase I Trial

IntroductionMantle cell lymphoma (MCL) is characterized by cell cycle dysregulation due to cyclin D1 and CDK4 overexpression. Palbociclib (PD 0332991) is an orally bioavailable, specific, reversible inhibitor of CDK4/6 that induces prolonged early G1 arrest (pG1) in MCL cells and durable remissions in patients with MCL. Moreover, we have evidence that palbociclib-induced pG1 sensitizes MCL cells to killing by bortezomib and that sensitization is amplified upon withdrawal of palbociclib, when MCL cells synchronously enter S phase (pG1-S). Targeting CDK4 in combination with bortezomib, therefore, is a rational and novel therapeutic combination. We report the final results of a phase I trial of palbociclib plus bortezomib in patients with previously treated MCL. MethodsAdults with previously treated MCL and adequate bone marrow and organ function were received palbociclib orally at doses of 75 mg (dose level 1), 100 mg (dose level 2), or 125 mg (dose levels 3 and 4) for 12 days. Bortezomib was administered by IV or SC injection at 1 mg/m2 (dose levels 1-3) or 1.3 mg/m2 (dose level 4) on days 8, 11, 15, and 18 of each 21-day cycle. Subjects underwent core needle biopsies of tumor tissue pre-treatment, on day 8 (in pG1) and on day 21 (in pG1-S phase) of cycle 1. Subjects were restaged following cycles 2, 5, and 8 and then every 4 cycles. Subjects could remain on the study regimen until progression, unacceptable toxicity, or withdrawal. Dose levels were escalated according to the standard 3+3 schema. Dose limiting toxicity (DLT) was defined as treatment-related grade 3-4 toxicity occurring during cycle 1 or a delay in cycle 2 of > 1 week due to treatment-related grade 4 neutropenia or thrombocytopenia. The primary objective was to estimate the maximum tolerated dose of the combination. Secondary objectives included response rate, duration of response, and evaluation of the pharmacokinetic and pharmacodynamic profiles at multiple time points and across all dose levels. ResultsNineteen subjects were enrolled: 6 in dose level 1, 3 in dose level 2, 7 in dose level 3, and 3 in dose level 4. The median age was 64 years (range 42-81). The median number of prior therapies was 3 (range 1-7). The number of subjects with low, intermediate, and high-risk MIPI scores was 6, 11, and 2, respectively. Two subjects experienced DLT: thrombocytopenia (level 1), neutropenia (level 3). Grade 3-4 hematologic toxicity included neutropenia (63%), thrombocytopenia (53%), lymphopenia (32%), and anemia (11%). Treatment-related grade 3-4 non-hematologic toxicity included zoster (1). Grade 1-2 toxicities occurring in >2 pt included: fatigue (47%), pain (42%), bleeding/bruising (37%), increased creatinine (26%), constipation (26%), rash (21%), nausea/vomiting (21%), sensory neuropathy (21%), dyspnea (21%), hypoalbuminemia (16%), cough (16%), edema (16%), infection (16%), increased AST (16%), hypocalcemia (16%), increased alk phos (16%). Reasons for ultimately stopping treatment include: progression (9), toxicity (6), and non-compliance (1). All 3 patients at dose level 4 required dose delays/reductions during cycle 2 due to toxicity. There appeared to be an association with dose of palbociclib and response, with one responder at each of dose levels 1 and 2, and 4 patients remaining free from progression for 1 year at dose level 3, including one complete response. Only one responding patient progressed on therapy. All patients with serial biopsies achieved pG1 on day 8, with reduction in CDK4/CDK6-specific Rb phosphorylation and Ki67 by immunohistochemistry. The primary MCL tumor cells express cell cycle genes scheduled for early G1 such as cyclin D1 and CDK4, but not genes programmed for other phases of the cell cycle such MKi67, E3F3, CDK1, CCNA2, as determined by RNA-seq. ConclusionDaily palbociclib 125 mg for 12 days can be safely combined with bortezomib 1 mg/m2 twice weekly, while higher doses were limited by myelosuppression. The combination induced durable responses in some patients. Palbociclib induced pG1, even at the lowest dose. However, the initial cell cycle control by palbociclib did not predict clinical response. Rather, pG1 appears to induce an imbalance in gene expression that is associated with response to the combination of palbociclib plus bortezomib. Strategies to control the cell cycle and dissect the underpinning mechanisms appear promising in MCL and warrant further evaluation. Disclosures:Martin:Teva: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech: Speakers Bureau; Millennium: Research Funding; Seattle Genetics: Consultancy, Speakers Bureau. Ruan:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics, Inc.: Membership on an entity's Board of Directors or advisory committees. Leonard:Millennium: Consultancy.