Targeting MM at the Nexus between Cell Cycle and Transcriptional Regulation Via CDK7 Inhibition

Deregulated transcription and cell cycle control are hallmarks of cancer that are especially frequent in multiple myeloma (MM). Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription; and targeting of cell cycle CDKs has been long pursued as an attractive therapeutic strategy. Among CDKs, CDK7 presents a unique therapeutic opportunity as it functions as a CDK activating kinase (CAK), licensing the activity of cell cycle CDKs, and also serves as a core component of the general transcription factor TFIIH. Here we elucidated the biological role of CDK7 and its transcriptional regulatory landscape in MM, using genetic as well chemical approaches, including tools for CDK7 rapid protein degradation (dTAG) and the selective covalent inhibitor YKL-5-124 that targets a cysteine residue (C312) located outside of the kinase domain. We have observed that CDK7 inhibition via YKL-5-124 robustly inhibited the phosphorylation of the CDK1, 2 and 4 activation loops in a representative panel of MM cell lines at concentrations as low as 50 nM. This reduction was not observed in MM cells expressing a resistant mutation in the reactive cysteine (C312S). Consistent with decrease of CAK activity, we observed G1 arrest and S phase loss after CDK7 inhibition, which was also associated with a rapid and transient loss of Ser2 and Ser5 phosphorylation of the RNA Pol2 C-terminal domain. To understand the effect of CDK7 inhibition on MM cell growth and viability, we evaluated activity of YKL-5-124 across a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. Longer exposure to YKL-5-124 caused apoptotic cell death in MM cells; however treatment with an inactive analog or in cells expressing the C312S mutation failed to inhibit MM cell proliferation, confirming that the antiproliferative potency of YKL-5-124 resides in its unique characteristic to covalently bind to C312 domain. Importantly, CDK7 inhibition impaired primary MM cells proliferation alone and when cultured in the presence of BM microenvironment. Selective pharmacological degradation of endogenously tagged CDK7 confirmed impact of CDK7 inhibition on MM cell proliferation via inhibition of CDK7 transcriptional and cell cycle activities. To complement the pharmacological studies, we have established MM cells to express inducible CRISPR/Cas9 constructs encoding 4 independent small guide RNAs targeting CDK7, resulting in the reduction of the abundance of CDK7 protein by 20-60% which was sufficient to inhibit MM cell viability over time, phenocopying pharmacologic inhibition of CDK7. These results support the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data suggest significant role for CDK7 in the CDK-pRB-E2F pathway in MM, which was strengthened by the observation of a positive correlation between expression of CDK7 and expression of E2F target genes in primary MM cells (n=409). Finally, we have evaluated the in vivo effect of CDK7 inhibition in several murine models of human MM. In the localized subcutaneous model, and the disseminated MM model where treatment with YKL-5-124 decreased tumor burden and improved survival. The effect of CDK7 inhibition explored in an aggressive, genetically engineered model of Myc-dependent MM, revealed evidence of response by decline in measurement of monotypic serum immunoglobulins. In conclusion, our study demonstrates that CDK7 contributes to the 'transcriptional addiction' and the cell cycle deregulation frequently observed in MM and represents an attractive molecular vulnerability to be exploited therapeutically. Disclosures Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy. Fulciniti:NIH: Research Funding.

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Disruption of the m-SWI/SNF Complex Mediated By Recurrent Non-Coding Mutations in BCL7A Induces Tumor Cell Proliferation in Multiple Myeloma

Blood ◽

10.1182/blood-2020-142178 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 40-40

Author(s):

Chandraditya Chakraborty ◽

Eugenio Morelli ◽

Srikanth Talluri ◽

Sanika Derebail ◽

Yan Xu ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Ectopic Expression ◽

Luciferase Reporter ◽

Rna Seq ◽

Loss Of Function ◽

Private Company ◽

Advisory Committees ◽

Cell Growth And Viability

Multiple myeloma (MM) is a biologically heterogeneous plasma cell malignancy, however, the mechanisms underlying this complexity are incompletely understood. By deep (70x) whole-genome sequencing (WGS) of 312 primary MM patients, we observed mutations in about 62% of patients in the 5' untranslated region and intron 1 of the 12q24.3 region which cytogenetically marks the BCL7A gene. Integration of WGS with RNA-seq data suggested a widespread loss of BCL7A expression in MM cells as compared to normal plasma cells (PC). To understand the effect of noncoding mutations in BCL7A promoter we performed in-vitro luciferase reporter assay in MM cell lines (H929, MM1S and KK1) devoid of BCL7A mutation in 5'UTR and found that the normalized luciferase reporter activity indicated that the mutation in the promoter significantly reduced BCL7A promoter activity. Next, we recapitulated the loss of BCL7A observed in MM patients, in a panel of MM cell lines using shRNA based genetic interference and observed the appearance of a more proliferative phenotype. The effects of BCL7A loss in MM cells were further confirmed using CRISPR-Cas9 system. BCL7A-KO (knock-out) cells had higher proliferative rate compared to wild type (WT) cells and lentiviral add back of BCL7A plasmid reversed this effect. This phenomenon was validated in a in vivo model, where we found that mice bearing BCL7A KO MM cells developed tumors faster than mice injected with control or add-back cells, suggesting that BCL7A loss increases tumorigenesis in vivo. On the other hand, ectopic expression of BCL7A significantly reduced cell viability and colony formation over time, inducing apoptotic cell death and impacting genes involved in chromatin and chromosome organization, DNA repair and cell cycle. Interestingly, BCL7A is an important member of the m-SWI/SNF chromatin remodeling complex. Using comparative mass spectrometry analysis, we observed that BCL7A expression is essential for the formation of a functional canonical m-SWI/SNF complex in MM cells, controlling the incorporation of active sub-units into the complex. These data suggest that loss-of-function mutations in the BCL7A region may disrupt the formation of SWI/SNF complex activating a transcriptional program that leads to MM cell growth and viability. We therefore evaluated the transcriptomic changes in MM cells upon BCL7A perturbation in a panel of MM cell lines by whole genome RNA-seq. Integrated analysis of modulated genes identified a set of 23 genes significantly upregulated in the presence of BCL7A depletion and downregulated in presence of BCL7A ectopic expression. To investigate whether these genes are involved in the phenotypic and functional effects observed in MM after BCL7A depletion, we performed LOF (loss-of-function) studies (si-RNA screen) using these genes in scrambled and BCL7A KD (knock-down) MM cells. Among others, we observed that MM cells are highly sensitive to the inhibition of RPS3A (V-Fos transformation effector protein) only in the context of BCL7A loss. Our RNA-seq data revealed that RPS3A is highly expressed in primary MM cells, and its expression significantly correlates with low expression of BCL7A in MM patients. Importantly, we observed that RPS3A binds to the core m-SWI/SNF complex proteins; SMARCC2 and SMARCB1 in the absence of BCL7A. RPS3A gene encodes a ribosomal protein that is a component of the 40S subunit and its overexpression transforms NIH3T3 mouse fibroblasts and induces tumor formation in nude mice and also used as an expression marker in squamous cell carcinoma. In conclusion, we here report biological consequences of a frequent non-coding mutation of BCL7A in MM and loss-of-function mutations in the BCL7A region may disrupt the formation of SWI/SNF complex activating a transcriptional process that leads to MM cell growth and viability. Altogether this study shed lights on a new pathogenic mechanism that may drive MM growth with potential translational implication, that may be applicable to other hematological diseases. Disclosures Anderson: Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Fulciniti:NIH: Research Funding. Munshi:BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company; Janssen: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.

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Activity of Carfilzomib (CFZ) in Acute Myeloid Leukemia (AML) As a Single Agent and in Novel Combinations

Blood ◽

10.1182/blood-2020-141459 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 6-7

Author(s):

Mao Yu Peng ◽

Yasmin Abaza ◽

Martina Mcdermott ◽

Monica Mead ◽

Dennis J. Slamon ◽

...

Keyword(s):

Cell Cycle ◽

Synergistic Effect ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Fatty Acid Synthase ◽

Single Agent ◽

Therapeutic Strategies ◽

Private Company ◽

Advisory Committees

Background:Recent advances in targeted therapy have expanded the available therapeutic optionsfor patients with AML. However, many patients still have suboptimal outcomes, particularly in the relapsed/refractory setting, underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs), such as bortezomib, exhibit antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis. CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib making it an agent of interest in AML therapy. Here we assessed the activity of CFZ as a single agent and in novel combinations with Ara-C and/or other agents targeting potential vulnerabilities in AML cell lines. Methods:20 AML cell lines were treated with a single dose of CFZ for 7 days, proliferation inhibition was measured using an IC50 cutoff for CFZ of 10 nM. 2 sensitive (ML2 and MV411) and 2 resistant (AML193 and NOMO1) cell lines were selected for further analysis. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM and Ara-C 200 nM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA). Cell lines were aligned according to CFZ IC50. Several proteins involved in various physiological pathways exhibited a potential correlation with CFZ sensitivity. Combination treatments with CFZ and agents targeting these pathways were carried out in selected cell lines. Results:Single-agent CFZ induced apoptosis with apoptotic rates >85% in sensitive cell lines and only 10% in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive, but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis as the major mechanism of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ was combined with Ara-C. RPPA revealed that AML cell lines with higher expression of autophagy-related proteins (Atgs) were more resistant to CFZ treatment. Combining autophagy inhibitor hydroxychloroquine (HCQ) or ROC-325 with CFZ produced a synergistic effect to induce apoptosis in several CFZresistant cell lines. RPPA also revealed that lower basal levels of fatty acid synthase (FASN), a key enzyme involved in lipogenesis, correlated with CFZ sensitivity and CFZ resistant lines tendedto have higher basal FASN levels. The combination of CFZ with a FASN inhibitor resulted in a significant synergistic apoptosis-inducing effect that was observed in the AML lines tested. Conclusion:CFZ demonstrated single agent activity in the nanomolar range in human AML cell lines. The addition of standard-of -care chemotherapy to CFZ did not show antagonism. Combining CFZ with agents targeting autophagy or lipid-metabolism showed synergistic effect in apoptosis. These results suggest a role for CFZ in combination therapeutic strategies for AML patients. Disclosures Mcdermott: TORL Biotherapeutics:Current equity holder in private company;1200 Pharma:Current equity holder in private company.Slamon:TORL Biotherapeutics:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;1200 Pharma:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;Novartis:Consultancy, Research Funding;Eli Lilly:Consultancy;Bayer:Consultancy, Research Funding;Pfizer:Consultancy, Other: stock, Research Funding;Syndax:Research Funding;Aileron:Research Funding;Genetech:Research Funding;Biomarin:Membership on an entity's Board of Directors or advisory committees;Seattle Genetics:Other: Stock;Amgen:Other: Stock.Larson:BMS, Bioline, Celgene, Juno, Janssen:Research Funding;TORL Biotherapeutics:Current equity holder in private company.

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Pre-Clinical Validation of a Novel Erk1/2 and CDK4/6 Inhibitor Combination in Multiple Myeloma (MM)

Blood ◽

10.1182/blood-2020-142596 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 22-23

Author(s):

Sophia Adamia ◽

Shruti Bhatt ◽

Yu-Tzu Tai ◽

Kenneth Wen ◽

Catherine A Nicholas ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Board Of Directors ◽

Cell Lines ◽

Synergistic Effects ◽

Single Agent ◽

Cycle Phase ◽

G1 Arrest ◽

Synergistic Activity ◽

Advisory Committees

Whole-genome sequencing analysis of newly diagnosed and relapsed multiple myeloma (MM) samples identified recurrent mutations in genes involved in the MAPK pathway, highlighting the potential of RAS/RAF/MEK/ERK signaling as a therapeutic target. Genomic studies identified translocations that involve IGH and set of partner genes MMSET, FGFR3, and CCND1 as primary events in MM. CDK4/CDK6 is overexpressed in MM, and CDK6 overexpression correlates with poor OS, suggesting that CDK4/6 are promising targets for MM therapy. Recent studies demonstrated synergistic activity of combined novel ERK1/2i inhibitor LY3214996 and CDK4/6i LY2835219 in solid tumors, but analogous studies have not been done in MM. Here we used preclinical models of MM to investigate inhibiting Erk1/2, CDK4/6, or both using ERK1/2i, CDK4/6i, or combination therapy. MM cell lines, RAS mutated or wild type (WT), were sensitive to ERK1/2i at IC50<0.5uM, and CDK4/6i at IC50<3uM. Synergistic effects of the Erk1/2i and CDK4/6i were noted in both RAS mutated and WT MM cell lines when ERK1/2i combined with CDK4/6i. Combination of ERK1/2i+CDK4/6i resulted in dose-dependent G0/G1 arrest in RAS mutated and WT MM cells. Similar effects were seen in RAS mutated cells treated with ERK1/2i or CDK4/6i as a single agent. ERK1/2i + CDK4/6i treatment triggered modest early apoptosis in RAS mutated MM cells, while in RAS WT MM cells this effect was more evident. Using dynamic BH3 profiling assay, we found that short-term treatment of MM cell with ERK1/2i and CDK4/6i led to increased overall mitochondrial priming in response to promiscuous BIM peptide in all MM cell lines. Even single agent treatment with ERK1/2i and CDK4/6i was able to enhance priming of RAS mutated or WT cells. Thus, ERK1/2i and CDK4/6i may activate mitochondrial apoptotic signaling in MM cells alone or in combination, consistent with observed synergistic cytotoxicity. HD PBMC and ARH77 cells were tested as controls. These cells were resistant to ERK1/2i and CDK4/6i at a broad range of concentrations, suggesting a favorable therapeutic index. The clinical potential of CDK4/6i+ERK1/2i was supported by an in vivo study demonstrating a significant (P=0.0004) decrease of the MM burden in CDK4/6i+ERK1/2i treated mice, without adverse effects. Proliferation and apoptosis studies of PCs from MM patient BM samples in the presence and absence of autologous BMSC/BMSCI-CM suggest potent and strong synergistic effects of ERK1/2i+CDK4/6i in MM and may allow successful use in clinic. To address the underlying mechanism of the synergism between Erk1/2i and CDK4/6i, we evaluated their cellular and transcriptional activity in MM cells. Gene expression profiling showed significant downregulation of RAS and CDK4/6 signaling pathway genes in MM cells as a result of ERK1/2i and CDK4/6i treatment at specific concentration ratios (3:1/1:3). Further evaluation of functional effects of ERK1/2i and CDK4/6i, alone or in combination, demonstrated that the synergistic effect of these inhibitors in MM cells is achieved through inhibition of p-S6, downregulation of c-myc, and correlate with ERK1/2i+CDK4/6i induced cell arrest in the G1 cell cycle phase. We noted increased ERK1/2 phosphorylation, which generally results in compensatory activation of parallel signaling pathways or in the loss of negative feedback. Regardless, ERK1/2i+CDK4/6i retained the inhibitory activity of the downstream signaling network, as demonstrated by the inhibition of cytoplasmic (p-RSK1) and nuclear (c-myc) targets of ERK at protein and mRNA levels. Treatment with ERK1/2i+CDK4/6i significantly decreased the levels of p-Rb and E2F1, downstream targets of CDK4/6. Recent studies shown that, in addition to cell cycle regulation, CDK4 and CDK6 induce tumorigenesis through regulation of inflammatory cytokines that are induced via NFκB pathway activation. CDK4/6i functional effects on MM cells cannot be limited to cell cycle arrest, CDK4/6i might also inhibit cytokines, which are produced in MM cells by NFκB activation. Overall, we shown that ERK1/2i+CDK4/6i induced cell proliferation and led to the key target molecule (p-c-myc, p-RSK, p-S6, p-RB, and E2F1) downregulations suggesting on-target activity of these inhibitors in MM cells. Importantly, our studies demonstrate strong synergistic anti-MM activity with ERK1/2+CDK4/6 therapy, providing a preclinical framework for clinical trials to improve patient outcome in MM. Disclosures Letai: Novartis: Research Funding; AbbVie: Consultancy; AstraZeneca: Consultancy; Zentalis: Membership on an entity's Board of Directors or advisory committees; Flash Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dialectic: Membership on an entity's Board of Directors or advisory committees; Chugai: Other: Lecture Fees. Anderson:Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics..

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Enhancing the Immune Surveillance in Multiple Myeloma Via CDK4/6 Inhibition

Blood ◽

10.1182/blood-2020-143327 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 33-34

Author(s):

Yan Xu ◽

Yao Yao ◽

Woojun Daniel Park ◽

Sanika Derebail ◽

Chandraditya Chakraborty ◽

...

Keyword(s):

Cell Cycle ◽

Nk Cells ◽

Board Of Directors ◽

Cell Lines ◽

Nk Cell ◽

Single Agent ◽

Cyclin D ◽

Private Company ◽

Advisory Committees

Deregulation of cyclin D genes is a uniform event in multiple myeloma (MM) and represent a striking addiction as observed in pan-cancer genome-wide CRISPR screening data. However, early stage Cyclin D and other cell cycle kinases inhibitors have shown a lack of single agent activity suggesting that targeting of cell cycle regulation is insufficient to produce a durable response in MM. Recent evidence recognizes the Cyclin D and CDK4 activities within the immune tumor microenvironment, supporting a previously unrecognized immunomodulatory functions of CDK4/6. This is particularly important in MM, a highly heterogeneous disease that resides in a complex ecosystem comprising of immune, endothelial, and stromal cells. We here evaluated the tumor intrinsic and extrinsic effects of CDK4/6 inhibition in MM with the goal to define rationally designed combination strategies to effectively impact MM growth. We have evaluated MM cell sensitivity to CDK4/6 inhibitors (both Palbociclib and Abemaciclib) in a panel of 32 MM cell lines and primary MM patients' samples. As expected, both inhibitors were mostly cytostatic with G0/G1 cell cycle arrest and significant impact on the pRB-E2F axis both in vitro as well as in vivo. In luminescence subcutaneous SCID models engrafted with H929 or MM1S MM cells expressing an E2F-driven luciferase reporter, treatment with low dose Palbociclib or Abemaciclib caused regression of bulky tumors, evidenced by a ~40% reduction in tumor volume at the 14-day end-point and a decreased E2F1 reporter activity. We next studied genome-wide transcriptional response to treatment using RNA-seq analysis in two MM cell lines using multiple doses and duration (24 and 72 hours) to evaluate the effect of short and long exposure to the drug. Gene set enrichment analysis (GSEA) of RNA-seq data confirmed significant downregulation of proliferation and E2F target genes at 24 hours post treatment. Interestingly, after longer exposure to both drugs we observed modulation of signatures for Ag presentation (including upregulation of HLA-A,B, and C; B2MG), innate immune response (ICAM-1 and 2; IL-8 and several CCLs) and interferon inducible genes IFN response (IRF1, IRF9, STAT1, STAT2, STAT4, OSA1, OSA2, MX1). To confirm genomic data and evaluate if CDK4/6i promotes the induction of the senescence-associated secretory phenotype (SASP) program in MM cells, we performed cytokine profile to assess the secretion of 174 soluble factors in the MM cell supernatant upon CDK4/6i. We confirmed a significant increase of chemokines involved in NK cell recruitment (CCL2, CCL4, CCL5), as well as cytokines that promote NK cell proliferation and activation. Moreover, we found that intercellular adhesion molecule-1 (ICAM-1) and the NKG2D ligands ULBP2 and MICA, required for activation of NK cell cytotoxicity and tumor cell targeting, were induced after CDK4/6i in MM cells. Although not secretory per se, these NK cell ligands are part of the transcriptional module linked to the SASP. Overall, these data suggest that, in addition to a more stable cell cycle arrest, CDK4/6i may promote MM cell immune surveillance through induction of the SASP program. To further confirm the immune effects, we tested freshly isolated NK cells from MM patients and healthy donors using in vitro MM-NK cell coculture assay and observed enhanced degranulation and cytokine production (intracellular IFN-γ and TNFα) by NK cells in response to MM cells. We finally investigated the potential of combining CDK4/6i with daratumumab in a standard 4-h ADCC assays using NK cells from MM patients as effector and MM cell lines as target. Daratumumab-mediated ADCC against MM cells was significantly augmented in the combination compared to single agent. In conclusion, we here report a novel anti-MM activity of CDK4/6i which is beyond the previously reported growth arrest. The observed ability to directly modulate the immune system along with decrease in the proliferative potential of MM cells may provide opportunities to develop unique combination approaches in MM. Disclosures Anderson: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.. Fulciniti:NIH: Research Funding. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Legend: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company.

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Activation of the ERK Pathway Drives Acquired Resistance to Venetoclax in MM Cell Models

Blood ◽

10.1182/blood-2020-142655 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 21-22

Author(s):

Chandraditya Chakraborty ◽

Yan Xu ◽

Yao Yao ◽

Eugenio Morelli ◽

Anil Aktas-Samur ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Acquired Resistance ◽

Gene Set Enrichment Analysis ◽

Erk Pathway ◽

Rna Seq ◽

Intrinsic Resistance ◽

Private Company ◽

Advisory Committees ◽

High Doses

Multiple myeloma (MM) is a hematological malignancy characterized by various genetic abnormalities including translocations involving the IgH gene at 14q32. Amongst these, t(11;14) is one of the most common translocations. Recent clinical data suggests a significant impact of Venetoclax, a small molecule inhibitor of BCL2, in this subgroup of MM patients, representing the first example of personalized medicine in MM and opening a wide range of research aiming at elucidating its mechanism of action. However, despite the initial positive response to the drug, a significant proportion of patients eventually develop resistance and relapse. To delineate the mechanisms that contribute to the development of an acquired drug-tolerant/resistance phenotype, we modeled the response to Venetoclax in 2 MM cell lines (KMS27 and KMS-12PE with IC50 of 35.47nM and 3.64nM, respectively). Whereas the vast majority of cells plated into 96-well plates were killed within a few days of exposure to a high dose of drug concentration, we detected a small fraction of viable, largely quiescent cells, which were expanded by culturing them in high doses of Venetoclax. We successfully generated 4 independent clones from each cell line, that were single cell-cloned with continued growth in the presence of high doses of Venetoclax. These clones labelled as drug-tolerant expanded persisters (DTEP) were investigated for the mechanisms driving drug tolerance and resistance against Venetoclax. First, we observed that altered expression of apoptotic regulators were associated with Venetoclax resistance in DTEP cells. We indeed observe a significant increase in the anti-apoptotic proteins MCL1 and BCL-XL in DTEP clones, which translated in our observation of improved sensitivity to MCL1 and BCL-xL inhibitors (S63845 and A-1155463 respectively). We performed both whole genome sequencing (WGS) and RNA-seq to evaluate if DTEP cells undergo transcriptional adaptation via genomic or epigenomic regulation and transcriptional reprograming during development of acquired drug resistance. While, WGS analysis didn't show any significant differences between parental and resistant clones, transcriptomic analysis showed both shared and unique transcriptome signatures in the DTEP clones. Gene set enrichment analysis (GSEA) of the common significantly modulated genes in the resistant clones revealed that the genes belonging to the PKA-ERK-CREB pathway were significantly upregulated in resistant clones, while apoptotic genes were downregulated compared to parental cells. Western blot analysis confirmed activation of ERK and the downstream target cAMP response element-binding (CREB) gene in resistant clones; and importantly treatment with the ERK inhibitor U0126 rescued the resistance to Venetoclax, providing a synergistic activity in resistant clones but not in parental cells, with decreased cell viability and increased apoptotic cell death. To evaluate if the ERK pathway was also associated with intrinsic resistance to Venetoclax, we assessed a panel of 24 MM cell lines and then calculated Pearson correlation coefficients between the measured drug activity and individual gene expression levels (by RNA-seq) across all cell lines and subjected the resulting rank-ordered gene list to GSEA. This analysis showed that mechanisms driving the DTEP phenotype are different from those associated with the intrinsic resistance to Venetoclax. RNA processing and splicing pathways were strongly enriched, with high expression of these genes correlating with increased sensitivity. Moreover, among the genes correlated with a resistant phenotype, we observed that the gene G0S2 was significantly downregulated in the resistant cell lines. G0S2 is a tumor suppressor gene that binds and inhibits BCL2. Interestingly, we observed that while G0S2 is downregulated in MM compared to normal plasma cells, t(11:14) patients have a higher expression. We are now in the process of validating G0S2 in MM and its contribution to Venetoclax sensitivity in MM. In conclusion, we here provide evidences of molecular mechanisms of acquired resistance to Venetoclax with activation of the ERK pathway as one of the prime targets. Combining Venetoclax with ERK inhibitor may therefore prevent or overcome the acquired resistance to Venetoclax observed in MM patients. Disclosures Fulciniti: NIH: Research Funding. Munshi:C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Janssen: Consultancy. Anderson:Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

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Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.133.133 ◽

2010 ◽

Vol 116 (21) ◽

pp. 133-133 ◽

Cited By ~ 2

Author(s):

Patricia Maiso ◽

AbdelKareem Azab ◽

Yang Liu ◽

Yong Zhang ◽

Feda Azab ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Cell Lines ◽

Mechanism Of Action ◽

Plasma Cells ◽

Bone Marrow Microenvironment ◽

Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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CYC065, a Potent Derivative of Seliciclib Is Active In Multiple Myeloma In Preclinical Studies

Blood ◽

10.1182/blood.v116.21.2999.2999 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2999-2999 ◽

Cited By ~ 1

Author(s):

Samantha Pozzi ◽

Diana Cirstea ◽

Loredana Santo ◽

Doris M Nabikejje ◽

Kishan Patel ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Preliminary Data ◽

Research Funding ◽

Preclinical Studies ◽

Dependent Manner ◽

Advisory Committees

Abstract Abstract 2999 Multiple myeloma (MM) is a treatable but incurable hematological malignancy and novel targeted therapies are under investigation. MM is characterized by dysregulation of the cell cycle, consequent to the overexpression of cyclins and their related kinases, the cyclins dependent kinases (CDK), a group of Ser/Thr proteine kinases. CDKs represent a promising therapeutic target, and inhibitors have been developed for anticancer treatment. We have previously studied seliciclib in the context of MM. CYC065, a second generation CDK inhibitor is the more potent derivative of seliciclib. It is mainly active on CDK 2, 5 and 9, involved in progression of the cell cycle and protein transcription. It has already shown promising results in preclinical studies in breast cancer and acute leukemia. We tested CYC065 in in vitro experiments in MM. Our preliminary data in 7 MM cell lines showed cytotoxicity of CYC065, both in MM cell lines sensitive as well as resistant to conventional chemotherapy, with an IC50 ranging between 0.06 and 2μ M, at 24 and 48h. Tritiated thymidine uptake assay confirmed the antiproliferative effects of CYC065 in MM, and its ability to overcome the growth advantage conferred by co-culture with bone marrow stromal cells derived from MM patients, and cytokines like interleukin 6 (10ng/ml) and insulin like growth factor-1 (50ng/ml). The anti-proliferative effect was evident both at 24 and 48h, starting at concentrations as low as 0.015μ M. The AnnexinV/PI assay in the MM1.s cell line confirmed CYC065's ability to induce apoptosis in a time dependent manner starting at 9 hours of treatment, at a concentration of 0.125 μ M, inducing 82% of apoptosis after 48h of exposure. Cell cycle analysis in the same MM1.s cell line showed an increase of subG1 phase, starting at 9 hours of treatment, at 0.125 μ M of CYC065. Preliminary results of western blot analysis confirmed the apoptotic effect of CYC065 in the MM1s cell line, highlighted by the cleavage of caspase 3, 8, 9 and PARP. The compound was tested in primary CD138+ cells isolated from three refractory MM patients, confirming its efficacy at 0.125 μ M, both at 24 and 48h. Comparative analysis in PBMCs from normal donors, for the evaluation of the drug toxicity is ongoing and will be presented. In conclusion our preliminary data confirm the efficacy of CYC065 in MM cell lines and primary MM cells, at nanomolar concentrations. Ongoing mechanistic and in vivo studies will delineate its role in the now increasing spectrum of CDK inhibitors in MM and better define its potential for clinical development in MM. Disclosures: Green: Cyclacel: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Acetylon: Research Funding.

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Blockade of Nuclear Export Protein CRM1 (chromosomal region maintenance 1, XPO1) by a Novel, Potent and Selective CRM1 Inhibitor KPT-185 Induces Significant Antitumor Activity Against Human Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.2913.2913 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2913-2913

Author(s):

Sun-Young Kong ◽

Yosef Landesman ◽

Jana Jakubikova ◽

Michael A. Sellitto ◽

Antonia Cagnetta ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Proliferation ◽

Board Of Directors ◽

Tumor Suppressors ◽

Chromosomal Region ◽

Advisory Committees ◽

Human Multiple Myeloma ◽

Export Protein ◽

Dose Dependent

Abstract Abstract 2913 CRM1 (chromosomal region maintenance 1, XPO1) is the major export protein regulating degradation of key tumor suppressors by transporting them from nucleus to cytoplasm, thus abrogating their function. Highly expressed CRM1 is associated with poor prognosis in several solid tumors, and inhibition of CRM1 restores function of tumor suppressors such as p53, p21 and FOXO and IκB (cellular antagonist of NF-κB). Furthermore, CRM1 knockdown enhances sensitivity of human multiple myeloma (MM) cell lines to topoisomerase II inhibitor (Cancer Res 2009 :69, 17). In this study, we investigated a novel selective CRM1 inhibitor, KPT-185, in human MM cells. Gene expression profiling analysis using GEO database showed that CRM1 expression is significantly increased in CD138+ cells from MM patients versus monoclonal gammopathy of undetermined significance (MGUS) patients or normal donors (p<0.05). Importantly, KPT-185 inhibits cell proliferation against drug sensitive and resistant MM cell lines with either wild type or mutated p53 status (n=7) in a dose-dependent manner (ED50: 5–100nM), evidenced by potently decreased [3H]-thymidine incorporation. MTT assay also demonstrated a significant reduction in cell viability (ED50: 20–120nM), associated with induction of apoptosis by annexin V/PI analysis. The percentage of apoptotic cells increased in a dose-dependent fashion in MM1.S, H929, U266, and RPMI-8226 cells, accompanied by increased activities of caspase 3/7 and 8. Cell cycle analysis confirmed that KPT185 inhibited cell proliferation, associated with 1.4-fold increased G0/G1 phase and a 4.5-fold decreased S phase at 24hr in MM.1S. Immunoblotting analysis next demonstrated that KPT-185-altered cell cycle was associated with increased p53 and p21, as well as decreased cyclin D1, cyclin E, CDK2, CDK4, and CDK6. KPT185 significantly induced cytotoxicity against CD138+ cells purified from MM patients (n=12) including newly diagnosed, advanced stage and relapsed patients. The median ED50 was 20 nM and 291 nM against patient MM cells, as determined by cell proliferation and viability assay, respectively. To evaluate the effects of the bone marrow (BM) microenvironment on KPT-185 treatment, BM stromal cells (BMSCs) derived from MM patients and HS-5 stromal cell line were co-cultured with MM cells, in the presence of serial dilutions of KPT-185. KPT-185 blocked MM cell growth induced by adhesion to BMSCs (p<0.05), indicating that KPT-185 could overcome BMSC-induced MM cell proliferation. KPT-185 also inhibited VEGF and MIP1 β secretion induced by coculture of BMSCs and IL-6-dependent ANBL6 (p<0.05). When MM1.S and INA6 cells were co-cultured with osteoclasts, KPT185 also demonstrated potent cytotoxicity against MM cells. Finally, treatments with KPT-185 and dexamethasone or lenalidomide synergistically induced cytotoxicity against MM1.S, MM1.R, U266, and H929 cells (CI <1.0). KPT-185 combined with melphalan or bortezomib induced additive or synergistic effects against MM cells. Together, results from these preclinical studies establish CRM1 as a promising novel target in MM and strongly support clinical evaluation of KPT-185, alone or in combination with conventional or novel anti-MM agents, to improve patient outcome in MM. Disclosures: Landesman: Karyopharm Therapeutics: Employment. Senapedis:Karyopharm Therapeutics: Employment. Shacham:Karyopharm Therapeutics Inc.: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. McCauley:Karyopharm Therapeutic Inc.: Employment. Saint-Martin:Karyopharm Therapeutics Inc: Employment. Munshi:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.

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

Blood ◽

10.1182/blood.v120.21.791.791 ◽

2012 ◽

Vol 120 (21) ◽

pp. 791-791

Author(s):

David Chiron ◽

Peter Martin ◽

Maurizio Di Liberto ◽

Xiangao Huang ◽

Scott A Ely ◽

...

Keyword(s):

Cell Cycle ◽

Board Of Directors ◽

Research Funding ◽

Human Cancer ◽

Single Agent ◽

Specific Inhibitor ◽

Selective Inhibition ◽

G1 Arrest ◽

Advisory Committees ◽

Indolent Lymphomas

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

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Functional Defects In Neutrophils Derived From Ezh2 Null Mice

Blood ◽

10.1182/blood.v122.21.1556.1556 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1556-1556

Author(s):

Albert Perez-Ladaga ◽

Bennett Caughey ◽

Huafeng Xie ◽

Stuart H. Orkin ◽

David B. Sykes ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Progenitor Cells ◽

Board Of Directors ◽

Cell Lines ◽

Resting Cells ◽

Surface Markers ◽

Advisory Committees ◽

Spontaneous Cell Death

Abstract Introduction We investigate the role of Ezh2 in neutrophil function using murine progenitor cells differentiated into neutrophils lacking the Ezh2 gene. Ezh2 is the catalytic component of the polycomb repressive complex 2, which methylates lysine 27 of histone H3. It is frequently disrupted in myelodysplastic syndromes (MDS) leading to loss of function (Ernst et al., 2010). Mutations in EZH2 are found in 6% of MDS patients and while not strongly linked to cytopenias or blast proportion, they are independently associated with worse overall survival compared to patients with wildtype EZH2 (Bejar R. et al., 2011 and 2012). We hypothesize that Ezh2 mutations may cause qualitative defects in myeloid cells that impact their function and could contribute to the adverse prognosis observed in EZH2 mutant MDS. Methods Bone marrow from Ezh2 null (Ezh2-/-) and littermate control mice (WT) were transduced with HOXB8 fused to the estrogen receptor ligand-binding domain to produce immortalized myeloid progenitor cells. Removal of estrogen from the media allows these cells differentiate into mature neutrophils (Wang G.G., 2006). Differentiated cells were characterized for surface markers by flow cytometry and for gene expression by PCR of mRNA. Spontaneous cell death was measured by annexin/PI staining. Cell cycle patterns were determined by measuring the red emission of PI. Chemotactic function was assessed by counting cells that migrated across a transwell in presence/absence of the attractant zymosan. For phagocytosis experiments, cells were incubated with Fluoresbrite YG carboxylate beads at 37°C or 4°C. Reactive oxygen species (ROS) generation was measured by the oxidation of dihydrorhodamine 123 into fluorescent rhodamine 123. Results Estrogen withdrawal caused differentiation of both WT and Ezh2-/- lines into cells with mature neutrophil morphology after six days (Figure 1a). Both differentiated lines expressed the neutrophil surface markers CD11b and CD62L and the neutrophil-specific genes lactoferrin and Itgb2l. Ezh2 -/- cells had an increased rate of spontaneous cell death compared to WT in undifferentiated (32.81% vs. 20.33%) and mature cells (32.82% vs. 14.23%). Nevertheless, both progenitor cell lines showed similar cell cycle patterns, demonstrating that Ezh2 absence had no other effect on cell cycle progression. Ezh2 -/- neutrophils failed to migrate towards zymosan (Figure 1b). Expression of Tlr2, which binds zymosan, and other Toll-like receptors (Tlr4/5/9) were similar between the differentiated cell lines. Cells incubated with FITC-zymosan at 37°C showed no fluorescence differences between cell lines, indicating similar adherence. Experiments with neutrophils from an MDS patient with homozygous EZH2 mutations demonstrated a similar migration defect. Additional studies in MDS patient samples are ongoing and will be presented. Phagocytosis was reduced in Ezh2-/-cells. Unstimulated, the number of cells ingesting and adhering YG-beads was significantly greater with WT cells than with Ezh2-/-cells. When activated with fMLP, both lines showed increased adherence of YG-beads but the number of phagocytosing Ezh2-/- cells was reduced. The average number of beads ingested by each cell was lower for Ezh2-/- cells compared to WT (5.95 vs 2.94, p < 0.001) in resting cells, and 9.47 vs. 3.73 in fMLP-activated cells, p < 0.01. The fraction of Ezh2-/- neutrophils generating ROS when stimulated with PMA is 2.4-fold higher than for WT cells. ROS production was greatly reduced in the presence of diphenyleneiodonium (DPI), confirming the role of NADPH oxidase in the generation of ROS. Conclusion Our results indicate impaired function of neutrophils derived from Ezh2-/- mice, demonstrating increased spontaneous cell death, impaired migration, decreased phagocytosis, and overproduction of ROS. Qualitative defects observed in neutrophils deficient for EZH2 may help explain the adverse prognosis associated with these mutations in MDS patients. Disclosures: Bejar: Genoptix: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

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