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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 80-80
Author(s):  
David Chiron ◽  
Peter Martin ◽  
Maurizio Di Liberto ◽  
Xiangao Huang ◽  
Priyanka Vijay ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Clinical Response ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Selective Inhibition ◽  
G1 Arrest ◽  
Cyclin D ◽  
Mantle Cell ◽  
Whole Transcriptome

Abstract 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.

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

Blood ◽  
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.

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 ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3738-3738 ◽  
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.

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

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 500-500
Author(s):  
Xiangao Huang ◽  
David Jayabalan ◽  
Maurizio Di Liberto ◽  
Zhengming Chen ◽  
Anna C Schinzel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Clinical Response ◽  
Research Funding ◽  
Ex Vivo ◽  
Cell Cycle Control ◽  
Selective Inhibition ◽  
Hematologic Malignancies ◽  
G1 Arrest ◽  
Myeloma Cells

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

Targeting the Cyclin D - CDK4/6 - Rb Axis in Mantle Cell Lymphoma with the Novel Translation Inhibitor Silvestrol,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3498-3498
Author(s):  
Lapo Alinari ◽  
Ryan B. Edwards ◽  
Courtney J. Prince ◽  
William H. Towns ◽  
Rajeswaran Mani ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Tumor Cells ◽  
Cell Lymphoma ◽  
S Phase ◽  
Cyclin D ◽  
Mantle Cell ◽  
Phase Entry

Abstract Abstract 3498 During cell cycle progression, D class cyclins activate cyclin dependent kinases (CDK) 4 and 6 to phosphorylate and inactivate Rb, allowing E2F-1 mediated transcription of additional cell cycle genes including cyclin E to drive S phase entry. This critical pathway is nearly universally dysregulated in cancer, providing tumor cells a strong growth advantage and escape from normal mitotic control. Substantial research is being directed toward targeting this pathway in many cancer types, with some preliminary successes being achieved with pharmacologic inhibitors of CDK4/6. However the development of alternative strategies to block this pathway could potentially provide broad therapeutic benefit. A prime example of a tumor with a disrupted cyclin D axis is Mantle Cell Lymphoma (MCL), in which the t(11;14) translocation places CCND1, the gene for cyclin D1, under the control of an immunoglobulin promoter. This results in sustained cyclin D1 expression in tumor cells and concomitant Rb inactivation, S phase entry and cell division. MCL is a relatively uncommon subset of Non-Hodgkin Lymphoma, but accounts for a disproportionate number of deaths. Treatments are limited and relapse is nearly universal; thus, new treatment strategies are essential for this disease. Silvestrol is a structurally unique, plant-derived cyclopenta[b]benzofuran with potent in vitro and in vivo anti-tumor activity in several model systems including B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). Silvestrol inhibits the initiation step of translation by preventing assembly of eIF4A and capped mRNA into the eIF4F complex, leading to selective loss of short half-life proteins such as Mcl-1 and cyclin D1. We therefore hypothesized that silvestrol, through the depletion of cyclin D1, would demonstrate efficacy in MCL. Silvestrol showed low nanomolar IC50 values in the JeKo-1 (13 nM), Mino (17 nM) and SP-53 (43 nM) MCL cell lines at 48 hr (MTS assay; cell death confirmed by propidium iodide flow cytometry). This potency was similar in primary MCL tumor cells. Longer exposure times substantially improved the cytotoxicity of silvestrol assessed at 48 hr (approximately 50% effect achieved with a 16 hr exposure vs. 80% effect with a 24 hr exposure), suggesting that the cellular impacts of this agent increase with exposure time. Cyclins D1 and D3 were dramatically reduced in MCL cell lines with just 10 nM silvestrol at 16 hr (cyclin D2 was undetectable in these cells), with subsequent loss of Rb phosphorylation as well as cyclin E mRNA and protein, culminating in G1 cell cycle arrest. Similar to what we previously showed in CLL and ALL cells, silvestrol treatment under these conditions also caused loss of Mcl-1 protein with concurrent mitochondrial depolarization, although the exact mechanism of silvestrol-mediated cytotoxicity in these cells is still under investigation. In an aggressive xenograft mouse model of MCL, silvestrol produced a highly significant improvement in survival [median survival of vehicle vs. silvestrol treated mice (1.5 mg/kg every 48 hr) = 27 vs. 38 days; P<0.0001] without detectable toxicity. Together, these data demonstrate that the translation inhibitor silvestrol has promising in vitro and in vivo activity in MCL preclinical models. Furthermore, as the cyclin D/CDK/Rb axis is disrupted in most tumor types, this strategy may be broadly effective in other cancers as well. Disclosures: No relevant conflicts of interest to declare.

Whole Transcriptome Sequencing Reveals Recurrent NOTCH1 Mutations in Mantle Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 436-436 ◽  
Author(s):  
Robert Kridel ◽  
Barbara Meissner ◽  
Sanja Rogic ◽  
Merrill Boyle ◽  
Adele Telenius ◽  
...  
Keyword(s):  
Overall Survival ◽  
Mantle Cell Lymphoma ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
The Other ◽  
Mantle Cell ◽  
Whole Transcriptome ◽  
Notch1 Mutations

Abstract Abstract 436 Background: Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma that is characterized by the hallmark t(11;14)(q13;q32) translocation, as well as a high number of secondary chromosomal alterations. Further, a small number of genes such as TP53, ATM and CCND1 have been reported to be recurrently mutated in MCL, but do not fully explain the biology and do not adequately account for the wide spectrum of clinical manifestations, response to treatment and prognosis. The aim of this study was to discover new somatic mutations that could contribute to our understanding of the pathogenesis of MCL. Methods: In our discovery cohort, we sequenced the transcriptomes of 18 clinical samples (11 diagnostic and 7 progression biopsies) and 2 mantle cell lymphoma-derived cell lines (Mino and Jeko-1). For this purpose, whole transcriptome shotgun sequencing was performed on RNA extracted from fresh frozen tissue. We assembled an extension cohort of 103 diagnostic patient samples and 4 additional cell lines (Rec-1, Z-138, Maver-1, JVM-2), and performed Sanger sequencing of NOTCH1 exons 26, 27 and 34 on genomic DNA. We further exposed the 6 cell lines to 1 μM of the γ-secretase inhibitor XXI (compound E) for 7 days and measured cellular proliferation with an EdU incorporation assay. Survival analysis was carried out in the 113 patients with diagnostic biopsies and available outcome data. Results: NOTCH1 mutations were found in 14 out of 121 patient samples (11.6%) and in 2 out of 6 cell lines, Mino and Rec-1 (33.3%). The majority of these mutations (12 out of 14) lie in exon 34 that encodes the PEST domain of NOTCH1 and consist of either small frameshift-causing indels (10 cases) or nonsense mutations (2 cases). These mutations are predicted to cause truncations of the C-terminal PEST domain. To gain further insight into functional relevance, we treated 6 cell lines with compound E, an inhibitor of the γ-secretase complex that plays a critical role in the release of the intracellular domain of NOTCH1 after ligand-induced activation. In Rec-1, that harbours a NOTCH1 mutation, we observed a significant decrease in proliferation (mean percentage of cells in culture incorporating EdU decreasing from 47.5% to 1.4%, p<.001). No effect of compound E was observed in Mino, the other cell line with a NOTCH1 mutation, nor in the 4 cell lines that are wild type for NOTCH1. Outcome correlation analysis showed that NOTCH1 mutations are associated with poor overall survival (1.56 versus 3.86 years respectively, p=.001), but not with significantly shortened progression-free survival (0.88 versus 1.73 years respectively, p=.07). Discussion: We have identified recurrent mutations in NOTCH1 in a subset of patients with MCL (11.6%). The frequency and the pattern of mutations are strikingly similar to what has recently been reported in chronic lymphocytic leukemia, the other major CD5 positive B-cell malignancy (Nature, 2011 Jun 5, 475:101–105 and J Exp Med, 2011 Jul 4, 208:1389–1401). NOTCH1 mutations are associated with adverse prognosis as evidenced by shortened overall survival. This latter finding, however, should ideally be validated in a larger and uniformly treated cohort. Finally, the sensitivity of the Rec-1 cell line to compound E suggests that NOTCH1 mutations could serve as the target for tailored therapy in mantle cell lymphoma. Disclosures: Sehn: Roche/Genentech: Consultancy, Honoraria, Research Funding. Connors:Roche: Research Funding.

The Brd-Inhibitor OTX015 Shows Pre-Clinical Activity in Anaplastic Large T-Cell Lymphoma (ALCL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4872-4872 ◽  
Author(s):  
Michela Boi ◽  
Paola Bonetti ◽  
Maurilio Ponzoni ◽  
Maria Grazia Tibiletti ◽  
Anastasios Stathis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Cell Lines ◽  
Proliferative Activity ◽  
Research Funding ◽  
Cell Lymphoma ◽  
G1 Arrest ◽  
Apparent Difference ◽  
Mrna Levels ◽  
Rt Pcr

Abstract Abstract 4872 Background: ALCL, is clinically/biologically heterogeneous disease, including ALK+ and ALK- systemic forms. Despite the progresses in understanding the molecular pathogenesis of ALCL, the therapy is still based on chemotherapy, thus the identification of new treatment modalities is needed. Bromodomain-containing proteins are components of transcription factors complexes and determinants of epigenetic memory. Inhibitors of BRD2/3/4, members of the Bromodomain and Extraterminal (BET) family, have recently shown antitumor activity in different hematological malignancies models. Here, we report anti-proliferative activity of OTX015, a novel selective orally bioavailable BRD2/3/4 inhibitor, in a panel of ALCL cell lines. Material and Methods: Eight established human cell lines derived from ALK+ and ALK- anaplastic large cell lymphoma (ALCL) were treated with increasing doses of OTX015 (OncoEthix SA) and MTT assays were performed after 72h exposure. For cell cycle analysis, cells were treated and stained with Click-iT Edu Flow Cytometry Assay Kits (Invitrogen) and 7-AAD and analyzed for DNA content using a FACScan flow cytometer. Results were analyzed with FlowJo 7.6.3 software. RNA was extracted using the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit according to the manufacturer's instructions. RT-PCR was performed on using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For senescence detection, cells were stained using a b-Galactosidase Staining Kit (Calbiochem). Results: We assessed OTX-015 anti-proliferative activity in eight ALCL cell lines. The majority (5/8) of the cell lines were sensitive, with IC50 between 36 and 546 nM. There was no apparent difference between ALK+(6) and ALK- (2) cell lines. Cell cycle analyses revealed G1 arrest and a concomitant decrease of the S phase after 24h OTX015 exposure in 4/4 ALCL cell lines, without an increase in cell death, suggesting a cytostatic effect of OTX015. An increase in the percentage of senescent cells after treatment with the BRD-inhibitor was observed in the most sensitive ALK+ALCL cell line. To understand the mechanism of action of OTX015, we assessed MYC mRNA levels before and after treatment. We observed that OTX015 suppressed the transcription of MYCgene and some of its downstream target genes (such as NCL and CAD) in 4/4 ALCL cell lines, with less efficacy in the most resistant one. Conclusion: OTX015 is a new potent BRD-inhibitor with evident anti-proliferative activity in several ALCL cell lines. The down-regulation of MYC gene, followed by cell cycle G1 arrest and increase of cellular senescence, was observed after OTX015 treatment, appearing one of the possible mechanisms of action of the compound. The compound appears worth of further investigation as a new promising therapeutic agent in ALCL and in other mature T-cell tumors. Disclosures: Bonetti: OncoEthix SA: Research Funding. Cvitkovic:OncoEthix SA: Membership on an entity's Board of Directors or advisory committees. Inghirami:OncoEthix SA: Research Funding. Bertoni:OncoEthix SA: Research Funding.

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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 791-791
Author(s):  
David Chiron ◽  
Peter Martin ◽  
Maurizio Di Liberto ◽  
Xiangao Huang ◽  
Scott A Ely ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Board Of Directors ◽  
Research Funding ◽  
Human Cancer ◽  
Single Agent ◽  
Specific Inhibitor ◽  
Selective Inhibition ◽  
G1 Arrest ◽  
Advisory Committees ◽  
Indolent Lymphomas

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

scholarly journals Abemaciclib-Based Combinational Therapies to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-34
Author(s):  
Yuxuan Che ◽  
Yang Liu ◽  
Lingzhi Li ◽  
Holly Hill ◽  
Joseph McIntosh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Therapeutic Resistance ◽  
Cycle Arrest ◽  
Mantle Cell ◽  
Ic50 Values

Introduction The past decades witnessed dramatic improvement of overall survival rate of mantle cell lymphoma (MCL) patients by constant efforts in developing novel therapeutic strategies that include ibrutinib and venetoclax. Nevertheless, resistance is still a major challenge in refractory/relapsed MCL patients. Chromosomal translocation t(11:14)(q13:q32) of the cyclin D1 (CCND1) gene is the hallmark of MCL, which leads to overexpression of cyclin D1. This overexpression promotes aberrant cell cycle progression by activating CDK4/6. Abemaciclib is a selective CDK4/6 inhibitor used as a clinical treatment of breast cancer and has been shown to be effective in preclinical human MCL xenograft models. It has also been used in a phase II clinical trial as a single agent among refractory/relapsed MCL patients with an objective response rate of 35.7%. In this preclinical study, we aim to evaluate the benefit of a combinational therapeutic strategy using abemaciclib with other molecular targeting agents among MCL patients with therapeutic resistance. Methods Cytotoxic efficacy of abemaciclib as a single agent and in combination with other drugs on different MCL cell lines and primary lymphoma cells from MCL patients with or without resistance was used as a key criterion for screening beneficial therapeutic strategies. Cell apoptosis and cell cycle arrest assays were conducted to further evaluate those effective combinations. Western blot was performed to investigate the mechanism of action of the combinations. Finally, the efficacy of abemaciclib alone or in combination were assessed in ibrutinib-resistant or venetoclax-resistant MCL PDX models in vivo. Results Our preliminary data showed that all MCL cell lines involved in this study were highly sensitive to abemaciclib treatment with IC50 values ranging from 50 nM to 1 µM. Further investigation of abemaciclib cytotoxicity on ibrutinib and/or venetoclax resistant MCL cell lines showed effective inhibition with a higher IC50 values ranging from 5 µM to 10 µM. More importantly, abemaciclib had potent efficacy on cells from primary MCL patients as well as from patients with acquired ibrutinib resistance. Our recent findings revealed that the addition of PI3K inhibitor TGR-1202 significantly enhanced cytotoxicity of abemaciclib in both sensitive and resistant MCL cell lines. Abemaciclib significantly inhibited phosphorylation of Rb1, the active form of the protein, in 4 different MCL cell lines. The active Rb1 maintains the cell in the G1 phase, preventing progression through the cell cycle and acting as a growth suppressor. The result suggests that CDK4/6 inhibition with abemaciclib disrupts CDK4/6 suppressive activity towards pRb-E2F and induce cell cycle arrest in the MCL cells. Interestingly, abemaciclib somehow interrupted phosphorylation of Chk1, which is continuously phosphorylated and hence activated in the MCL cell lines. Inhibiting activation of Chk1 by abemaciclib may induce cell death via unmonitored and accumulated DNA damage. The efficacy of abemaciclib in combination with Bcl-2 or BTK inhibitors in MCL cell lines and isolated cells from MCL patients are ongoing. These data suggest that abemaciclib in combination with other therapeutic drugs could be beneficial in targeting therapeutic resistant MCL cells. Conclusions Abemaciclib showed impressive therapeutic potency on both MCL cell lines and isolated primary cells from MCL patients, which is likely due to the predominant contribution of cyclin D1-CDK4/6 pathway to malignancy. Other agents, such as PI3K inhibitors, can sensitize abemaciclib in therapeutic resistant MCL cells. Thus, an abemaciclib based multi-drug combinational strategy may be a promising therapy for refractory/relapsed MCL patients in the near future. Disclosures Wang: Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Oncternal: Consultancy, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; MoreHealth: Consultancy; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

Combined Targeting of Chromatin Modifying Enzymes LSD1, EZH2 and Histone Deacetylases (HDACs) Has Superior Efficacy Against Human Mantle Cell Lymphoma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2429-2429
Author(s):  
Warren Fiskus ◽  
Sunil Sharma ◽  
Rekha Rao ◽  
Ramesh Balusu ◽  
Sreedhar Venkannagari ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Core Protein ◽  
Cell Lymphoma ◽  
Amine Oxidase ◽  
Combined Treatment ◽  
Reversible Inhibitor ◽  
Mantle Cell

Abstract Abstract 2429 PRC (polycomb repressive complex) 2 contains three core protein components, i.e., EZH2, SUZ12 and EED, of which EZH2 has the SET domain with its intrinsic histone methyltransferase activity that mediates the trimethylation (Me3) of lysine (K) 27 on histone (H) 3-a repressive chromatin mark for gene expression. We have previously reported that treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep as well as treatment with the pan-histone deacetylase inhibitor panobinostat (PS, Novartis Pharma) deplete PRC2 complex proteins. LSD1 (KDM1A) is a demethylase of H3K4Me2, and inhibiton of LSD1 leads to increase in H3K4Me3-a permissive mark for gene expression. In the present studies, we determined the chromatin-modifying and cytotoxic effects of LSD1 inhibition alone and in combination with PS or DZNep in cultured (JeKo-1 and Z138C) and primary human Mantle Cell Lymphoma (MCL) cells. Treatment with the non-amine oxidase reversible inhibitor of LSD1 CIT0665 (250 to 1000 nM), or the more potent analogue HCI2509 (20 to 250 nM), dose-dependently increased the levels of H3K4Me2 & Me3, p21 and p27, while decreasing the levels of cyclin D1, which was associated with inhibition of cell proliferation and accumulation of the MCL cells in the G1 phase of the cell cycle. Abrogation of LSD1 by a specific shRNA treatment also induced similar chromatin, cell cycle and growth inhibitory effects. Exposure to CIT0665 or HCI2509 disrupted the binding of LSD1 with the co-repressor CoREST and HDAC1, without affecting the levels of these proteins. As noted above, treatment with PS (10 to 50 nM) dose-dependently depleted the levels of not only EZH2, SUZ12 and the PRC1 complex protein BMI1, but also of LSD1 in MCL cells. PS treatment alone also depleted the levels of AKT, cRAF, CDK4 and cyclin D1, as well as induced cell cycle growth inhibition and apoptosis of MCL cells. Co-treatment with PS enhanced the chromatin modifying effects of CIT0665 or HCI2509. The combination synergistically induced apoptosis of the cultured MCL cells (combination indices, CI <1.0). This was associated with greater induction of p27 and depletion of cyclin D1. Treatment with PS and HCI2509 also synergistically induced loss of viability of primary MCL cells (CI <1.0). We have previously reported that DZNep dose-dependently depleted EZH2, SUZ12 and BMI1 expression, inhibited H3K27Me3 levels, induced p21, p27 and FBXO32 (muscle atrophy F-box protein, also called atrogin-1) levels in cultured and primary MCL cells. Here, we determined the effects of co-treatment with HCI2509 and DZNep in MCL cells. Combined treatment with HCI2509, although not synergistic, enhanced the apoptosis of MCL cells induced by DZNep. Taken together these findings indicate that combined targeted depletion of the level and activity of LSD1 by PS and CIT0665 or HCI2509 along with PS-mediated depletion of PRC2 proteins, BMI and HDACs exerts superior activity against MCL cells. These studies also support the in vivo testing of combined epigenetic therapies in the treatment of MCL. Disclosures: Sharma: Novartis: Research Funding.

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