Search

Histone deacetylase inhibitors (HDACis) are antitumor agents with distinct efficacy in hematologic tumors. Pracinostat is a pan-HDACi with promising early clinical activity. However, similar to other HDACis, its activity as a single agent is limited. Diffuse large B-cell lymphoma (DLBCL) includes distinct molecular subsets or metabolically defined subtypes that rely in different ways on the B-cell receptor signaling pathway, oxidative phosphorylation, and glycolysis for their survival. The antitumor activity of pracinostat has not been determined in lymphomas. We performed preclinical in vitro activity screening of 60 lymphoma cell lines that included 25 DLBCLs. DLBCL cells belonging to distinct metabolic subtypes were treated with HDACis for 6 hours or 14 days followed by transcriptional profiling. DLBCL xenograft models enabled assessment of the in vivo antilymphoma activity of pracinostat. Combination treatments with pracinostat plus 10 other antilymphoma agents were performed. Western blot was used to assess acetylation levels of histone and nonhistone proteins after HDACi treatment. Robust antiproliferative activity was observed across all lymphoma histotypes represented. Focusing on DLBCL, we identified a low-sensitivity subset that almost exclusively consists of the oxidative phosphorylation (OxPhos)-DLBCL metabolic subtype. OxPhos-DLBCL cells also showed poorer sensitivity to other HDACis, including vorinostat. Transcriptomic analysis revealed fewer modulated transcripts but an enrichment of antioxidant pathway genes after HDACi treatment of OxPhos-DLBCLs compared with high-sensitivity B-cell receptor (BCR)–DLBCLs. Pharmacologic inhibition of antioxidant production rescued sensitivity of OxPhos-DLBCLs to pracinostat whereas BCR-DLBCLs were unaffected. Our study provides novel insights into the antilymphoma activity of pracinostat and identifies a differential response of DLBCL metabolic subtypes to HDACis.

Several functional parameters from baseline (18)F-fluorodeoxyglucose positron emission tomography (PET)/computed tomography have been proposed as promising biomarkers of treatment efficacy in diffuse large B-cell lymphoma (DLBCL). We tested their ability to predict outcome in 2 cohorts of DLBCL patients receiving conventional immunochemotherapy (rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine sulfate, and prednisone [R-CHOP] regimen), either every 14 (R-CHOP14) or 21 days (R-CHOP21). Baseline PET analysis was performed in 141 patients with DLBCL treated with R-CHOP14 in the prospective SAKK38/07 study (NCT00544219) of the Swiss Group for Clinical Cancer Research (testing set). Reproducibility was examined in a validation set of 113 patients treated with R-CHOP21. In the SAKK38/07 cohort, progression-free survival (PFS) at 5 years was 83% for patients with low metabolic tumor volume (MTV) and 59% for those with high MTV (hazard ratio [HR], 3.4; 95% confidence interval [CI], 1.6-7.0; P = .0005), whereas overall survival (OS) was 91% and 64%, respectively (HR, 4.4; 95% CI, 1.9-10; P = .0001). MTV was the most powerful predictor of outcome also in the validation set. Elevated metabolic heterogeneity (MH) significantly predicted poorer outcomes in the subgroups of patients with elevated MTV. A model integrating MTV and MH identified high-risk patients with shorter PFS (testing set: HR, 5.6; 95% CI, 1.8-17; P < .0001; validation set: HR, 5.6; 95% CI, 1.7-18; P = .0002) and shorter OS (testing set: HR, 9.5; 95% CI, 1.7-52; P < .0001; validation set: HR, 7.6; 95% CI, 2.0-28; P = .0003). This finding was confirmed by an unsupervised regression tree analysis indicating that prognostic models based on MTV and MH may allow early identification of refractory patients who might benefit from treatment intensification. This trial was registered at www.clinicaltrials.gov as #NCT00544219.

Classic hairy cell leukemia (HCL) is a tumor of mature clonal B cells with unique genetic, morphologic, and phenotypic features. DNA methylation profiling has provided a new tier of investigation to gain insight into the origin and behavior of B-cell malignancies; however, the methylation profile of HCL has not been specifically investigated. DNA methylation profiling was analyzed with the Infinium HumanMethylation27 array in 41 mature B-cell tumors, including 11 HCL, 7 splenic marginal zone lymphomas (SMZLs), and chronic lymphocytic leukemia with an unmutated (n = 7) or mutated (n = 6) immunoglobulin gene heavy chain variable (IGHV) region or using IGHV3-21 (n = 10). Methylation profiles of nontumor B-cell subsets and gene expression profiling data were obtained from public databases. HCL had a methylation signature distinct from each B-cell tumor entity, including the closest entity, SMZL. Comparison with normal B-cell subsets revealed the strongest similarity with postgerminal center (GC) B cells and a clear separation from pre-GC and GC cellular programs. Comparison of the integrated analysis with post-GC B cells revealed significant hypomethylation and overexpression of BCR–TLR–NF-κB and BRAF-MAPK signaling pathways and cell adhesion, as well as hypermethylation and underexpression of cell-differentiation markers and methylated genes in cancer, suggesting regulation of the transformed hairy cells through specific components of the B-cell receptor and the BRAF signaling pathways. Our data identify a specific methylation profile of HCL, which may help to distinguish it from other mature B-cell tumors.

INTRODUCTION: EMZL is a heterogeneous disease with variable risk for relapse and progression. Based on age ≥70 years, stage III-IV and elevated LDH, Thieblemont et al (Blood. 2017) developed the MALT-IPI to identify high-risk patients. In this index, disease characteristics (stage and LDH) account for 66% while a disease nonspecific characteristic (age) for 33% of the index score. We reported (Am J Hematol. 2019) that EMZL with multiple mucosal sites (MMS) at diagnosis is characterized by shorter survival and increased incidence of higher grade transformation. To better recognize disease-attributable high-risk patients, we developed a new EMZL prognosis score chiefly based on patient's disease characteristics. METHODS: The revised (R)-MALT-IPI was developed using a retrospective data set of 405 EMZL patients treated at the University of Miami (UM) from 1995 to 2017. Cox proportional hazards regression analysis was conducted to evaluate the effect of the potential prognostic variables on progression-free survival (PFS) and overall survival (OS) and to develop the new index R-MALTI-IPI based on PFS. Model validation was performed in two independent cohorts of EMZL patients from the University of Iowa/Mayo Clinic Molecular Epidemiology Resource (MER) database (n=297) and the IELSG-19 study (n=400) used for the development of MALT-IPI. Performance of various prognostic indices was compared using AIC statistics, and concordance c-statistics by Harrell (CH) and by Gonen and Heller (CGH). RESULTS: Among the candidate variables tested in univariable analysis, the following were statistically significant predictors of shorter PFS: age >60, age ≥70, anemia (Hb1, number of nodal sites >4, and presence of MMS at diagnosis, defined as EMZL with ≥2 different extranodal sites excluding spleen and bone marrow. A stepwise Cox regression analysis yielded a multivariable model with four independent predictors of shorter PFS: age >60 (HR=1.53, p=0.010), elevated LDH (HR=1.73, p=0.004), stage III-IV (HR=2.03, p=0.0003) and presence of MMS (HR=2.78, p60, elevated LDH, stage III-IV, and 2 points for MMS. The R-MALT-IPI defined 4 risk groups: low-risk (score 0 (35%), reference group), low-medium risk (score 1 (39%), HR=1.91, p=0.005), medium-high risk (score 2 (13%), HR=3.77, p For validation, we analyzed R-MALT-IPI index performance in independent Iowa/Mayo Clinic MER and IELSG-19 cohorts. Both R-MALT-IPI and MALT-IPI were useful in distinguishing PFS and OS in all the cohorts. In the UM training cohort, the concordance c-statistics' values for the two indices were similar: for PFS, CH=0.6893 and CGH=0.6611 for R-MALT-IPI, and CH=0.6551 and CGH=0.6367 for MALT-IPI; for OS, CH=0.7017 and CGH=0.6813 for R-MALT-IPI, and CH=0.7029 and CGH=0.67715 for MALT-IPI. In the validation cohorts, the concordance c-statistics' values for the two indices were also similar, but slightly lower than in the UM cohort for PFS. When comparing medium-high to high-risk R-MALT-IPI groups, there was a reduction of 4 years in median PFS in the UM cohort, and reduction in median EFS of 5.6 years in the MER cohort, an important difference between these risk groups identified by the R-MALT-IPI index. CONCLUSIONS: R-MALT-IPI is a new index for EMZL centered principally on disease characteristics. Overall, there is a similar prediction of PFS (EFS) by R-MALT-IPI and MALT-IPI indexes; however, R-MALT-IPI better recognizes a high-risk group accounting for 13% of EMZL patients with short median PFS and thus obviates the waiting period needed to recognize patients with shorter EFS24. Collaborative studies addressing best treatment approach for these high-risk EMZL patients are eagerly needed. Disclosures Alderuccio: Agios: Other: Immediate family member; Foundation Medicine: Other: Immediate family member; OncLive: Consultancy; Targeted Oncology: Honoraria; Puma Biotechnology: Other: Immediate family member; Inovio Pharmaceuticals: Other: Immediate family member. Thieblemont:Cellectis: Membership on an entity's Board of Directors or advisory committees; Kyte: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Roche: Honoraria, Research Funding; Gilead: Honoraria; Novartis: Honoraria. Cerhan:Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding. Zucca:Kite, A Gilead Company: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grant, Research Funding; AstraZenaca: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Merck: Research Funding; Celltrion Helathcare: Membership on an entity's Board of Directors or advisory committees; Abbvie: Other: Travel Grant. Lossos:NIH: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Janssen Scientific: Membership on an entity's Board of Directors or advisory committees.

The PIM kinases are highly expressed in activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). Oncogenic cooperation between PIMs and MYC has been demonstrated. Transgenic mice co-expressing Em-PIM and Em-MYC showed accelerated lymphomagenesis. Conversely, knockdown of PIMs dramatically decreased cMYC levels and lowered tumor incidence. Based on these preclinical data, a treatment strategy aiming at disrupting the oncogenic cooperation between PIMs and MYC may improve the outcome of DLBCL. Therefore, we treated a panel of DLBCL cell lines with increasing dose of the clinically relevant pan-PIM inhibitor (PIMi) AZD1208 (from 0.1 to 10μM) for 48 hours (Hrs), which resulted in a dose-dependent growth inhibition with a stronger efficacy in ABC DLBCL cell lines. (Figure 1A)The analysis of a CRISPR loss-of-function screening in three ABC (LY3, TMD8, HBL1) and three GCB (SUDHL-4, Pfeiffer, BJAB) DLBCL cell lines (Reddy et al, 2017) showed that PIM2 silencing led to significantly decreased viability irrespective of cell-of-origin (Figure 1B), suggesting that this oncogene is essential for cell proliferation in DLBCLs. To identify the genes through which PIMs drive the lymphoma phenotype we performed gene expression profiling using 4 ABC DLBCL cell lines (RIVA, TMD8, SUDHL-2, U2932) treated with either DMSO or AZD1208 at 1μM for 4, 8 and 12 Hrs. We observed induction of 3,439 genes whereas 2,473 genes were downregulated. (Figure 1C) Gene pathway analysis showed that AZD1208 led to downregulation of genes regulated by MYC, including its known downstream p53 and NFKB target genes. On the other hand, AZD1208 treatment broadly induced MHC class II and antigen presentation genes as well as PI3K/AKT, cell cycle and glutaminase genes. (Figure 1D) Using a high-throughput screening approach, we found that the inhibitors of cell cycle (such as the BCL2 inhibitor venetoclax/ABT199 and the PLK4 inhibitor CFI-400945) and of glutaminase (CB839) enhanced the antiproliferative effect of AZD1208, whereas combinations with the PI3K/AKT/mTOR inhibitors had negligible synergistic effect. (Figure 1E) In conclusion, our study revealed previously unknown mechanisms of action of PIM inhibitors and provides a framework for future combination strategies. Disclosures Younes: Xynomics: Consultancy; Biopath: Consultancy; Genentech: Research Funding; AstraZeneca: Research Funding; Syndax: Research Funding; BMS: Research Funding; HCM: Consultancy; Celgene: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Takeda: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Curis: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Abbvie: Honoraria; Pharmacyclics: Research Funding. Bertoni:Nordic Nanovector ASA: Research Funding; Acerta: Research Funding; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants; Jazz Pharmaceuticals: Other: travel grants.

Background . PI3Kδ is expressed in B-cells and has a central role in the B-cell receptor signaling in B-cell derived malignancies. Idelalisib was the first-in-class PI3Kδ inhibitors and several second-generation compounds are undergoing clinical investigation as single agents and in combinations. To identify modalities to overcome the resistance that develops to this class of agents, we have developed two idelalisib-resistant models derived from splenic marginal zone lymphoma (SMZL) cell lines. Materials and Methods. Cells were kept under idelalisib (IC90) until acquisition of resistance (RES) or with no drug (parental, PAR). Stable resistance was confirmed by MTT assay after 2-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out. Cells underwent transcriptome and miRNA profiling by RNA-Seq, whole exome sequencing (WES), lipidomics profiling, pharmacological screening (348 compounds), and FACS analysis. Cytokines and growth factor secretion was performed by ELISA. Results. Two RES models were obtained from VL51 and Karpas1718 with 7-10 fold times higher IC50s than PAR counterparts. In both models, conditioned media from RES cells transferred the resistance in the PAR cells. While WES did not identify somatic mutations associated with resistance, RNA-Seq and lipidomics analyses showed that the two cell lines had developed resistance activating different modalities. The VL51 RES model showed an enrichment in BCR-TLR-NFkB (TLR4, CD19, SYK), IL6-STAT3 (IL6, CD44), chemokines (CXCL10, CXCR4, CXCR3) and PDGFR (PDGFRA, PRKCE) signatures, paired with increased p-AKT and p-BTK levels, decreased cardiolipins and sphingomyelins levels, and increased levels of specific triacylglycerols and glycerophosphocholines. In particular, there was an over-expression of surface expression of PDGFRA and secretion of IL6 in the medium. Silencing of both IL6and PDGFRA by siRNAs reverted the resistance, while the silencing of the individual genes had only a partial effect. These data were paired with the acquired sensitivity to the PDGFR inhibitor masitinib, identified in the pharmacologic screening. In the Karpas1718 model, we observed an increased p-AKT activity with an enrichment for B-cell activation signatures (RAG1, RAG2, TCL1A), proliferation (E2F2, MKI67), ERBB signaling (HBEGF, NRG2, ERRB4), increased levels of some triacylglycerols and repressed levels for specific glycerophosphocholines. HBEGF secretion was confirmed by ELISA. The addition of recombinant HBEGF to the medium induced resistance in the PAR cells. Combination with the pan ERBB inhibitor lapatinib was beneficial in the K1718 RES. Recombinant HBEGF also induced resistance to the BTK inhibitor ibrutinib in the PAR cells and in the mantle cell lymphoma SP-53 cell line. Specific members of the let-7 family of miRNAs were repressed in the RES lines derived from both cell lines, indicating the involvement of miRNA deregulation in the mechanism of resistance. Indeed, let-7 members are known to directly target IL6-STAT3 and cytokine signaling cascade, as well PI3K-AKT network. In solid tumors, let-7 members are also expressed at low levels in tumors with constitutive active ERBB signaling, in accordance with the activation of ERBB pathway and p-AKT we observed in our Karpas1718model. Experiments with a LIN28B inhibitor are now on-going. Finally, we validated the findings across a panel of 34 B-cell lymphoma cell lines, in which IL6, PDGFRA, HBEGF and LIN28 expression levels were negatively correlated with idelalisib sensitivity, while the latter was positively correlated with let-7 levels (P Conclusions. We developed two distinct models derived from MZL of secondary resistance to the PI3Kδ inhibitor idelalisib. We identified treatments that might overcome resistance to idelalisib and are worth of further investigations. The two models, driven by different biologic processes, will allow the evaluation of further alternative therapeutic approaches. Disclosures Stathis: PharmaMar: Other: Renumeration; ADC Therapeutics: Other: Institutional research funding; Abbvie: Other: Renumeration; Bayer: Other: Institutional research funding; Novartis: Other: Institutional research funding; MEI-Pharma: Other: Institutional research funding; Roche: Other: Institutional research funding; Pfizer: Other: Institutional research funding; Merck: Other: Institutional research funding. Stuessi:Gilead: Speakers Bureau. Zucca:Gilead: Honoraria, Other: travel grant. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board. Bertoni:Nordic Nanovector ASA: Research Funding; Acerta: Research Funding; Jazz Pharmaceuticals: Other: travel grants; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants.

Introduction. The majority of SMZLs display an indolent course, however the disease is still incurable and a significant proportion of patients (~25-30%) experience poor outcomes surviving 5 years, and for whom tumor material collected before initiation of medical therapy was available. Mutation analysis was performed by CAPP-seq targeted deep next generation sequencing of tumor genomic DNA. A stringent bioinformatic pipeline was applied to suppress the background noise allowing to call variants with a sensitivity of 5x10-2 in FFPE derived DNA. Copy number variations (CNVs) were identified by using the sequencing reads-based GATK4-CNV algorithm. IGHV rearrangements were obtained by using LymphoTrack® IGH FR1 Assay Panel kit. Molecular clusters were identified by an iterative algorithm that maximizes genetic distinctiveness of subgroups by reassigning patients between clusters that are created a priori based on the co-occurrence of genetic lesions. Relative survival, defined as the ratio between actuarial survival observed in the SMZL cohort and expected survival of the general population matched to patients by geographical origin, sex, age and calendar year of diagnosis, was calculated using the Ederer II method. Results. The analysis included 303 patients with a SMZL diagnosis confirmed on spleen histology. The sample size allowed to identify 30% differences in survival for molecular subgroups comprising at least 5% of cases with a statistical power between 80-100%. Median follow-up was 9.2 years. At 10 years, 85% of patients were alive, consistent with the known indolent behavior of this lymphoma. Genes recurrently affected by non-synonymous somatic mutations in >10% of SMZL included KLF2 (24%), NOTCH2 (19%), KMT2D (15%), TNFAIP3 (13%), EP300 and TP53 (10%). Deletion 7q was documented in 25% of cases and IGHV1-2*04 usage in 32%. By cluster analysis, three major molecular subgroups were identified, each of them characterized by a NOTCH pathway mutated gene (Fig. 1A). The first cluster was defined by NOTCH2 and/or KLF2 mutations and was enriched in TNFAIP3 mutations and IGHV1-2*04 gene usage (Fig. 1A). The second cluster was defined by SPEN mutations, and was enriched in KMT2D and other epigenetic gene mutations (Fig. 1A). The third cluster was enriched in NOTCH1 mutations (Fig. 1A). By relative survival analysis, the NOTCH2/KLF2 cluster showed a lower survival compared to the matched general population, indicating a significant impact of the disease on patients' expected survival (Fig. 1B). Conclusions. The large sample size and inclusion of SMZL confirmed by spleen histopathology review allowed for precise estimation of the prevalence of KLF2 and NOTCH2 mutations in this lymphoma. Three molecular clusters were identified in SMZL, each of them containing a NOTCH pathway gene, supporting the relevance of NOTCH signaling in the pathogenesis of SMZL. Patients belonging to the NOTCH2/KLF2 cluster had a lower relative survival compared to the matched general population. Disclosures Traverse-Glehen: Astra Zeneca: Other: Travel; Takeda: Research Funding. Gomes da Silva:Roche: Other: Institution's payment for consultancy, Travelling support; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Celgene: Other: Travelling support; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Institution's payment for consultancy, Travelling support; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Research Funding. Ladetto:Celgene: Honoraria; Jannsen: Honoraria; Acerta: Honoraria; Abbvie: Honoraria; Sandoz: Honoraria; Roche: Honoraria. Rambaldi:Pfizer: Consultancy; Novartis: Consultancy; Omeros: Consultancy; Italfarmaco: Consultancy; Amgen Inc.: Consultancy; Roche: Consultancy; Celgene: Consultancy. Vitolo:Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Gilead: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Zinzani:MSD: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Speakers Bureau. Gaidano:Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Morphosys: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Salles:Servier: Honoraria; Abbvie: Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Amgen: Honoraria; BMS: Honoraria, Other: Advisory Board; Celgene: Honoraria, Other: Advisory Board, Research Funding; Acerta: Honoraria; Janssen: Honoraria, Other: Advisory Board; Merck: Honoraria; Pfizer: Honoraria; Morphosys: Honoraria; Gilead: Honoraria, Other: Advisory Board; Epizyme: Honoraria; Servier: Honoraria, Other: Advisory Board; Takeda: Honoraria. Zucca:Celltrion: Consultancy; AstraZeneca: Consultancy.

TCL1 oncogene is overexpressed in aggressive form of human chronic lymphocytic leukemia (CLL) and its dysregulation in mouse B cells causes a CD5-positive leukemia similar to the aggressive form of human CLLs. To identify oncogenes that cooperate with Tcl1, we performed genetic screen in Eμ−TCL1 mice using Sleeping Beauty transposon-mediated mutagenesis. Analysis of transposon common insertion sites identified 7 genes activated by transposon insertions. Overexpression of these genes in mouse CLL was confirmed by real time reverse transcription-polymerase chain reaction. Interestingly, the main known function of 4 of 7 genes (Nfkb1, Tab2, Map3K14, and Nfkbid) is participation in or activation of the nuclear factor-kB (NF-kB) pathway. In addition, activation of the NF-kB is 1 of main functions of Akt2, also identified in the screen. These findings demonstrate cooperation of Tcl1 and the NF-kB pathway in the pathogenesis of aggressive CLL. Identification cooperating cancer genes will result in the development of combinatorial therapies to treat CLL.

Background.Diffuse large B cell lymphoma (DLBCL) represents the most common lymphoma subtype (30%-40% lymphomas). Gene expression profiling (GEP) identifies two main subtypes of DLBCL defined by their postulated cell of origin (COO). Activated B cell-like DLBCL (ABC DLBCL) is characterized by activation of B-cell receptor signaling and the nuclear factor kB pathway: these patients respond worse to standard R-CHOP but better to novel agents (ibrutinib or lenalidomide) than patients with DLBCL of the germinal-center-type (GCB DLBCL). We have previously reported an upregulation of the transcriptional factor ETS1 in up to 25% of DLBCL (Bonetti, Testoni et al. Blood, 2013). ETS1 is involved in many biologic processes, including B cell differentiation. ETS1 undergoes a series of post-translational modifications, and, in particular, ETS1 phosphorylation at Threonine 38 (p-ETS1) is a marker for ETS1 activation. Here, we present the impact of p-ETS1 in DLBCL cellular models and in a large series of clinical specimens. Patients and Methods. Levels of p-ETS1 (ab59179, Abcam) and, as controls, of ETS1 (C-20, Santa Cruz Biotechnology), ERK (93, Santa Cruz Biotechnology), p-ERK-Tyr204 (p-ERK; 7383, Santa Cruz Biotechnology) and IRF4 (4964, Cell Signaling Technology) were analyzed in cell lines derived from ABC (n.=7), GCB (n.=8) and type 3 (n.=4) DLBCL. p-ETS1 was examined by immunohistochemistry on clinical specimens: cases were defined as positive if > 10% of the neoplastic cells nuclei were p-ETS1 positive. Results.p-ETS1 was detected in ABC, not in GCB DLBCL cell lines (100% vs 0%, P Conclusions. Our data identify ETS1 phosphorylation at threonine 38 as i) associated with the ABC DLBCL phenotype; ii) associated with poor PFS in GCB DLBCL; iii) downstream to BCR signaling and amenable to pharmacological interventions with BTK and MEK inhibitors. Disclosures Rossi: Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria.

Background IMGN529 is an antibody drug conjugate (ADC) consisting of an anti-CD37 antibody with direct anti-tumor activity conjugated via a thioether linker to the cytotoxic maytansinoid antimicrotubule agent DM1. IMGN529 has shown pre-clinical (Deckert et al, Blood 2013) and clinical activity in lymphoma (Stathis et al, ASH 2014; NCT01534715). Here, we assessed the anti-tumor activity of IMGN529 on a large panel of B cell and T cell human lymphomas to identify potential biomarkers of response. Methods Fifty-four lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), n.=27; mantle cell lymphoma (MCL), n.=10; anaplastic large T-cell lymphoma, n.=5; marginal zone lymphomas, n=6, others, n=6] were exposed to increasing doses of IMGN529 or to the unconjugated DM1 for 72h. Cell proliferation was measured using the MTT. Apoptosis induction was defined by at least 1.5-fold increase in caspase 3/7 signal activation with respect to controls using the Promega ApoTox-Glo Triplex Assay. CD37 surface expression was assessed by cytofluorimetry. Gene expression profiling (GEP) was done with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES > |2|, P |1.2|; P< 0.05; top 200 up and top 200 down). Results. The IMGN529 median IC50 in the 54 cell lines was 780pM (95%C.I., 263pm-11.45nM). Activity was stronger (P We then compared the baseline gene expression profiling of DLBCL cell lines that were highly sensitive to IMGN529 (IC50< 800pM; "S") versus less sensitive/resistant DLBCL cell lines (IC50>10nM, "R"), separately for germinal center B cell type (GCB) (S, n=11; R, n=8) and for activated B cell like (ABC) (S, n=4; R, n=3). In both DLBCL groups, MYC targets, genes involved in unfolded protein response, glycolysis and DNA repair were enriched in transcripts more expressed in R than S cell lines. Transcripts associated with low sensitivity included CD44, VIM, ANXA2, BCL2, ANXA2P1, HSP90B1, NFKBIZ, CDK6, BIRC5 in GCB and HSPA1B, HSP90AA1, CADM1, CD86, TUBB2A, TUBG1, NOTCH1 in ABC cell lines. HEBP1, PHB, PSME3, RNU6-15, RPL13 were more expressed in both GCB and ABC R. Genes involved in PI3K/AKT/mTOR, hypoxia, INF-gamma, TNFA signaling via NFKB and in complement were more expressed in S than in R cell lines. Genes associated with sensitivity to IMGN529 comprised: CD37 (IMGN529 target), CD79A, CHI3L2, FAM117B, LPAR5, NFATC1, PTPN22, RBM38, SGPP1, SLC6A16 in both GCB and ABC cell lines; BASP1, CXCR5, BIK, LY86, TLR10, CD86, LCK, CD22, PTPN22, BCL6, PIK3IP1, CDKN2A in GCB; AFF3, PIM1, MGMT, PDE4B, NFKBIE, SYK, FOXO1in ABC. Conclusions. IMGN529 showed a very strong anti-tumoral activity in pre-clinical lymphoma models. High expression of CD37 and mostly genes involved in BCR signalling were associated with sensitivity to IMGN529. Conversely, the presence of MYC translocation, a high expression of MYC targets and of genes known to be involved in drug resistance (BCL2, BIRC5, CDK6, heat-shock proteins, annexins, proteasome and tubulin components) appeared to negatively affect the response to the ADC but also represent therapeutic targets for novel combinations to be explored. Disclosures Rossi: Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Sloss:Immunogen Inc: Employment.

Introduction. PQR309 is a novel oral dual PI3K/mTOR inhibitor (Cmiljanovic et al, AACR 2015), being now evaluated as single agent in a phase I study in patients with relapsed or refractory lymphomas (NCT02249429). We previously reported activity of PQR309 as single agent in a large panel of lymphoma cell lines, and early combination data in two diffuse large B-cell lymphoma (DLBCL) cell lines (Tarantelli et al, ASH 2014). Here, we present extended combination data that might enhance the anti-proliferative effect of the single drug. Methods. A panel of DLBCL cell lines derived from activated B-cell like (ABC) or germinal center B (GCB) subtype, with or without BCL2 or MYC deregulation, were used: RIVA (ABC, BCL2 amplification), SU-DHL-2 (ABC), TMD8 (ABC) and U2932 (ABC, BCL2 amplification); DOHH2 (GCB, BCL2 and MYC translocations), SU-DHL-6 (GCB, BCL2 translocation), KARPAS422 (GCB, BCL2 translocation), OCI-LY18 (GCB, BCL2 and MYC translocations), SU-DHL-10 (GCB), OCI-LY1 (GCB, BCL2 translocation). Cell lines were exposed to increasing doses of PQR309 alone or in combination with increasing doses of other agents for 72h and synergism was assessed with the Chou-Talalay combination index (CI): 1.1, no benefit. Results. The combination of PQR309 with the BCL2 inhibitor venetoclax (ABT199) gave positive results in 6/8 cell lines, with strong synergism in three (U2932, median CI=0.1, SU-DHL-6, 0.14, Karpas422, 0.14) and synergism in the remaining three (TMD8, 0.5, RIVA, 0.56, DOHH2, 0.65). No benefit was observed in SU-DHL-2 and OCI-LY18. The combination of PQR309 with the BTK inhibitor ibrutinib was synergistic in 3/4 ABC-DLBCL (RIVA, 0.42; U2932, 0.6; TMD8, 0.57), while it was of no benefit in SU-DHL-2. The combination of PQR309 with the immunomodulator lenalidomide was of benefit in 4/4 ABC-DLBCL: synergistic in three (RIVA, 0.38; U2932, 0.4; TMD8, 0.5) and additive effect SU-DHL-2 (1.01). The combination of PQR309 with the HDAC-inhibitor Panobinostat was synergistic in 5/6 (KARPAS422, 0.21; U2932, 0.65; SU-DHL-6, 0.67; DOHH2, 0.8; SU-DHL-2, 0.83), but not in the TMD8 (1.14). In our models, synergism was observed in only 2/5 cell lines (TMD8, 0.6; DOHH2, 0.89) exposed to both PQR309 and anti-CD20 monoclonal antibody, while not in KARPAS422, SU-DHL-6, or U2932. Finally, since we had previously observed an up-regulation of the PIM1 kinase after exposure to PQR309 (Tarantelli et al, ICML 2015), potentially acting as a mechanism of adaptive resistance, we evaluated the addition of the PIM inhibitor AZD1208 to PQR309 in four DLBCL cell lines. The combination showed synergism in two (OCI-LY1, 0.29; SU-DHL-10, 0.57), an additive effect in TMD8 (0.95) and no benefit in the SU-DHL-2 (1.15). Conclusions. The novel dual PI3K/mTOR inhibitor PQR309 showed synergism when combined with additional targeted agents in different DLBCL models, including some derived from double hit lymphomas. In particular, the combination with the BCL2 inhibitor venetoclax showed very good results, especially in cell lines bearing BCL2 gene deregulation due to chromosomal translocation or genomic amplification, and the combination with a PIM inhibitor might overcome early adaptive resistance to PQR309. These data provide the basis for future pre-clinical and clinical studies. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Stathis:PIQUR Therapeutics AG: Research Funding. Fabbro:PIQUR Therapeutics AG: Employment. Wicki:PIQUR Therapeutics AG: Employment, Research Funding. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees. Bertoni:PIQUR Therapeutics AG: Research Funding; Oncology Therapeutic Development: Research Funding.

OTX015 (MK-8628) has shown anti-lymphoma activity in both the preclinical and clinical settings (Boi et al, CCR 2015; Stathis et al, EORT-NCI-AACR 2014). Here we report in vivo data on OTX015 in combination with four targeted compounds in DLBCL. Methods. NOD-Scid (NOD.CB17-Prkdcscid/NCrHsd) mice were subcutaneously engrafted with the activated B-cell like DLBCL cell line SUDHL2 (15 x106 cells) and then divided into 10 groups (6 mice each). Treatment with OTX015 was started 3 days after the graft, and treatments with other drugs were initiated when mice developed palpable tumors (100 mm3). Tumor size was measured two times per week using digital calipers. Tumor volumes were calculated using the equation V = [length x width2]/2 (width and length are the shortest and the longest diameters of each tumor, respectively). Tumor specimens were collected at the end of treatment and necrosis was semi-quantitatively defined on the total amount of neoplastic tissue. Results. Xenografts of SU-DHL-2 were treated with control or OTX015 (50 mg/kg once daily; QDx7/w x5w), the BTK-inhibitor ibrutinib (5 mg/kg; QDx2/w x5w), the mTOR-inhibitor everolimus (1 mg/kg, Qdx2/w x5w), the HDAC-inhibitor vorinostat (15 mg/kg; QDx2/w x5w), the anti-CD20 monoclonal antibody rituximab (3 mg/kg; QDx1/w x5w) as single agents or in OTX015-containing combinations. No weight loss was reported. OTX015, in accordance previously published data (Boi et al, CCR 2015), as single agent delayed tumor growth. Almost complete eradication of tumors was observed in mice treated with OTX015 combinations (p Conclusions. OTX015-containing combinations with everolimus, ibrutinib, vorinostat and rituximab showed very promising in vivo activity in an ABC-DLBCL model, providing the rationale for future clinical studies. Figure 1. In vivo treatment of ABC-DLBCL SU-DHL-2 xenografts with MK-8228 as single agent and in combination with other targeted drugs. Figure 1. In vivo treatment of ABC-DLBCL SU-DHL-2 xenografts with MK-8228 as single agent and in combination with other targeted drugs. Disclosures Riveiro: Oncology Therapeutic Development: Employment. Cvitkovic:Oncology Therapeutic Development: Employment, Membership on an entity's Board of Directors or advisory committees. Bertoni:PIQUR Therapeutics AG: Research Funding; Oncology Therapeutic Development: Research Funding.

Introduction: Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of plasma cells (PCs) in the bone marrow (BM). Over 50% of patients die within 5 years of diagnosis. The transition from normal PCs to active MM, via premalignant condition (monoclonal gammopathy of undetermined significance; MGUS), consists of many oncogenic events, including upregulation of cyclin D1 and IRF4 genes, as well as activating mutations in NRAS, and KRAS. Despite recent advances in oncogenomics, further studies are needed to identify critical players in MM pathogenesis that could be targeted for pharmacological intervention to improve outcome. Recently, aberrations in epigenetic modifications, including DNA modifications and miRNA expression have been shown to play a crucial role in development and progression of MM. miRNAs are short non-coding RNAs that bind to complementary sequences on target messenger RNA (mRNAs) and downregulate their expression by inhibiting mRNA translation, or inducing its degradation. miRNA analysis, may lead to improved understanding of MM biology and classification, by establishing associations between gene expression changes and MM molecular and clinical features. To assess whether miRNA deregulation plays a critical role in the development of MM we performed small RNA next generation sequencing in the PCs isolated from 3 patients at the MGUS stage and after that they developed active disease, but still untreated. miRNA deregulation was also validated in an independent set of newly diagnosed MM patients (n.34) compared to non-cancer age matched donors (n.6). Mechanisms of transcriptional regulation and biological roles of the differentially expressed miRNAs were also analyzed. Methods: 1x106 CD138+ frozen PCs (purity>90%) from different 3 donors before and after the disease development were used for the analysis. RNA was extracted with RNA-DNA-protein kit (Norgen Biotek) and 0.8µg of total RNA was used to generate the cDNA libraries using TruSeq Small RNA Library Preparation Kit. The obtained cDNA libraries were sequenced on an ILLUMINA system through the OSU Genomic Shared Resource (GSR). Myeloma cells (MM.1S) were treated with pan-HDACi for 24 hours and total miRNA expression was analyzed by nCounter microRNA array (NanoString Technologies, Inc.). miRNA deregulation upon use of several pan-HDAC'i in clinical use (LBH589, SAHA and AR-42) were validated in 4 different cell lines and in the MM cells of newly diagnosed MM patients. Chromatin immunoprecipitation, silencing RNA for specific histone deacetylase enzymes (HDACI, HDAC2, HDAC3, and HDAC6), western blot analysis, luciferase assays, stem loop RT-PCR, q-RT-PCR and cell proliferation assays were also performed. Results: We found that several miRNAs are commonly deregulated during disease transition. Some of these miRNAs, including miR-223, miR-221, miR-222, miR-92a and miR-93 (p Conclusions: Collectively, we believe, that these findings support the key role of miRNA aberrant expression in PC transformation. Their role in regulating the expression of IRF4 during myeloma development and lead us to speculate that this explains why IRF4 and c-Myc mRNA levels are higher in newly diagnosed MM patients, without obvious chromosomal abnormalities, compared to MGUS patients. Understanding of the molecular bases of how c-Myc expression can be regulated by a specific histone deacetylase via modulation of miRNA levels will have important impact not only on myeloma therapy, but also other hematopoietic malignancies involving c-Myc deregulation. Disclosures No relevant conflicts of interest to declare.

Background. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma, accounting for 30%-40% of all cases. Despite a major improvement in the cure rate, a large number of DLBCL patients lack therapeutic options. Aberrant changes in histone modifications, DNA methylation and expression levels of non-coding RNA, including microRNA (miRNA), contribute to DLBCL pathogenesis and represent potential therapeutic targets. OTX015 targets bromodomain and extra-terminal (BET) proteins, which are epigenetic readers contributing to gene transcription. It has shown preclinical activity in hematologic and solid tumor models (Gaudio et al, AACR 2014; Noel et al, EORTC-NCI-AACR 2013) and promising early results in an ongoing phase I study (Herait et al, AACR 2014; NCT01713582). To better understand the mechanism of action of OTX015, we studied molecular changes induced by this compound in DLBCL cell lines. Methods. Total RNA was extracted from 2 DLBCL cell lines, the germinal center B-cell (GCB) type DOHH2 and activated B-cell-like (ABC)-type SU-DHL-2, following treatment with 500 nM OTX015 or DMSO for 4h or 8h. RNA samples were labeled with cyanine-3 dye using the Agilent microRNA Complete Labeling System & Hyb Kit and hybridized to the Agilent Human microRNA microarray v.3. Raw expression values were obtained with Agilent Feature Extraction Software, log-transformed and normalized by the quantile method. Data were filtered to exclude relatively invariant features and those below the detection threshold. Limma (Linear Models for Microarray data analysis) was employed using R/Bioconductor and the filtered dataset. Baseline miRNA profiling was obtained from 22 DLBCL cell lines with the Nanostring nCounter Human v2 miRNA Expression Assay kit. Baseline gene expression profiling (GEP) was obtained in these cell lines with the Illumina HumanHT-12 v4 Expression BeadChip. Selected miRNA changes were validated by real-time PCR. Validated miRNA targets were retrieved using the miRWalk database (Dweep et al, 2011). Gene Set Enrichment Analysis (GSEA) software was used to assess enrichment of miRNA targets in the GEP datasets. Results. miRNA profiling of the GCB and ABC DLBCL cell lines exposed to OTX015 identified four downregulated miRNAs and eight which were upregulated. Among them, the oncomirs miR-92a-1-5p (log2 FC, -2.01; P=0.004) and miR-21-3p (log2 FC, -0.37; P=0.0045) were downregulated, while the tumor suppressor miR-96-5p (log2 FC, 0.39; P=0.041) was upregulated. Interestingly, changes of these miRNAs matched GEP variations of validated target genes (e.g., miR-92a-1-5p: CDKN1A, log2 FC, 0.81, CDKN2A, log2 FC, 0.81; miR-96-5p: MYC, log2 FC, -0.57, MYD88, log2 FC, -0.35). We then evaluated if these three miRNAs play a role in OTX015-sensitivity by obtaining baseline miRNA and GEP profiling data in 22 DLBCL cell lines. Compared to 8 cell lines with lower sensitivity to OTX015 (IC50 >500 nM), the 14 sensitive cell lines (IC50 Conclusions. Changes in the expression levels of biologically relevant miRNAs may contribute to response to OTX015. miR-92a-1-5p, the oncomir which was most strongly downregulated by OTX015, is a member of the MYC target MIR17HG (mir-17-92 cluster), involved in the pathogenesis and chemo-resistance of lymphomas, mainly contributing to PI3K/AKT/mTOR pathway activation. Since the cell cycle transcriptional regulator E2F1 is targeted by mir-17-92, OTX015 may contribute to cell cycle arrest and to downregulation of the E2F1 target gene reported with BRD inhibitors in DLBCL cell lines. miR-21-3p, also downregulated by OTX015, is a well-known oncomir, and forced miR-21-3p expression in transgenic mice results in the development of leukemias and lymphomas. miR-96-5p, upregulated by OTX015, targets oncogenes such as RAS or MYC, and low expression has been reported in mantle cell lymphoma. Interestingly, low miR-96-5p baseline levels were associated with higher sensitivity to OTX015, an observation meriting validation in other tumor models and evaluation in clinical studies. Disclosures Stathis: Oncoethix SA: Consultancy, Research Funding. Riveiro:Oncoethix SA: Consultancy, Research Funding; Oncology Therapeutic Development: Employment. Bertoni:Oncoethix SA: Research Funding.

Introduction: The first FDA-approved deacetylase inhibitor (HDACi), suberoylanilide hydroxamic acid (SAHA, Vorinostat), was shown to be effective in vitro by a number of anti-neoplastic mechanisms. Despite minimal single-agent activity in multiple myeloma (MM), phase 1b studies combining HDACi’s with bortezomib salvaged some relapsed patients and prolonged progression free survival (PFS) from days (Vorinostat) to months (Panobinostat), albeit at the cost of significant side effects including fatigue, nausea, and vomiting. Phase 1/2 studies in combination with lenalidomide have demonstrated tolerability and activity in lenalidomide-refractory patients, but randomized trials are lacking. In MM, the anti-neoplastic mechanism of action for HDACi’s is unknown, but at biologically achievable concentrations, it has been theorized that they sensitize MM cells to other drugs by interfering with cell adhesion mediated drug resistance (CAM-DR), primarily involving the integrins CD44, CD49d (VLA-4), CD54 (ICAM-1), and/or CD184 (CXCR4). AR-42 (ARNO Therapeutics) is a novel orally bioavailable phenylbutyrate-based class I/II HDAC inhibitor that has greater anti-proliferative effects compared to Vorinostat in vitro and in vivo. It has been previously shown that in MM cell lines, AR-42 down-regulates the expression of gp130, and inhibits IL-6 induced activation of STAT3 and downstream targets including BCL-XL and Cyclin-D1, with minimal effects on the PI3K/AKT and MAPK pathways. CD44 is a type I transmembrane glycoprotein, which is directly transcribed by β-catenin, and its role in cell adhesion-mediated drug resistance (CAM-DR) for MM as well as other cancers has been largely investigated. Recent published data have shown that CD44 forms a complex with STAT3 and p300 (acetyltransferase) causing STAT3 activation in a cytokine- and growth factor-independent manner, and that CD44 over-expression is one of the main molecular mechanisms that contributes to Lenalidomide resistance in MM cells. Hypothesis: We hypothesize that CD44 down-regulation, both surface expression on MM cells as well as the soluble form in the blood, is the primary effect of AR-42 at concentrations achievable in humans, explaining its weak single agent effect and its improvement when combined with other therapeutic agents in the clinic. Methods: As part of a single center, dose-escalating, first-in-man phase 1 trial of single agent oral AR-42 administered orally three times weekly in 28-day cycles (3 weeks of treatment followed by a 7-day off treatment period), patients were accrued at 20-70 mg TIW in a standard 3+3 cohort design. Using peripheral blood obtained during cycle 1 of therapy, nCounter® GX Human Immunology assays and nCounter miRNA expression profile was performed to assess differentially expressed genes after AR-42 treatment. Enzyme-linked immunoabsorbent assay (ELISA), qRT-PCR and luciferase assays were also performed.To examine whether AR-42 treatment could sensitize the cells to Lenalidomide in vivo, we used GFP+/Luc+ MM.1S cells engrafted in NOD-SCID mice. Results: AR-42 in relapsed MM showed no confirmed partial responses, but did result in marginal responses in 3 out of 13 MM patients. We found that AR-42 in MM cells modulates the expression of many genes coding for surface receptors including CD44, CD48, CD46 and TRAF5 and affects the expression of several miRNAs. Our data show a decrease of CD44 mRNA expression in the CD138+ MM plasma cells and of the soluble CD-44 in the serum of AR-42 treated patients. We also show that in MM cells CD44 down-regulation upon AR-42 treatment is associated with impairment of STAT-3 signaling pathways and direct targeting of its regulatory RNA binding protein, Insulin grow factor 3 binding protein 3 (IGF2BP3), by miR-9-5p. We found that miR-9-5p is up-regulated in vitro and in the cancer cells of MM patients after AR-42 treatment. Moreover we show that AR-42 in combination with Lenalidomide show synergistic apoptotic effect on MM cells, enhancing Lenalidomide anti-myeloma activity invivo. Conclusions: These findings show that CD44 is a therapeutic target for the HDACi AR-42 in MM patients, providing the rationale to support further clinical investigation of AR-42 in combination with IMiDs in patient cohorts with high pretreatment CD44 expression in the serum and on the surface of MM cells. Disclosures Hofmeister: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Honoraria, Research Funding; ARNO Therapeutics: Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees.

Background. Mantle cell lymphoma (MCL), which is characterized by the deregulation of cyclin D1 (CCND1), is one of the most common lymphoma subtypes, accounting for 5-10% of all cases. MCL prognosis is often very poor. Several novel non-chemotherapeutic agents have shown promising activity in MCL, but novel agents and in particular new drug combinations are needed to improve patients' outcome. Aberrant changes in histone modifications, DNA methylation and expression levels of non-coding RNA contribute to MCL pathogenesis and represent potential therapeutic targets. Bromodomain and extra-terminal (BET) proteins are epigenetic readers contributing to gene transcription. OTX015 is a bromodomain (BRD) inhibitor that has shown preclinical activity in hematologic and solid tumor models (Gaudio et al, AACR 2014; Noel et al, EORTC-NCI-AACR 2013) as well as promising early results in an ongoing phase I study (Herait et al, AACR 2014; NCT01713582). Here, we present preclinical evidence of OTX015 activity in combination with other targeted drugs in MCL. Material and methods. MCL cell lines (REC1, MAVER1, UPN1, JeKo1, SP53, Mino, Granta519) were exposed to increasing doses of OTX015 alone or in combination with increasing doses of other drugs, including everolimus, BEZ235, ibrutinib, carfilzomib, pomalidomide, 5-AZA, vorinostat and dexamethasone. The MTT assay was performed after 72h exposure. Real-time PCR and Western blotting were used for RNA and protein expression analyses. Synergy was assessed by the Chou-Talalay combination index (CI) with the Synergy R package: CI Results. OTX015 showed antiproliferative activity as a single agent in 6/7 MCL cell lines with IC50s < 500 nM. Four MCL cell lines were exposed to DMSO or OTX015 (500 nM and IC50) for 4 and 24h to evaluate expression of CCND1 and other members of signaling pathways known to be impacted by BRD inhibitors. No reduction in CCND1 was observed at the RNA or protein levels after OTX015 exposure. However, MYC was downregulated and the transcriptional regulator HEXIM1 and histone-coding genes were upregulated. In light of reported strong synergy between OTX015 and the mTOR inhibitor everolimus in diffuse large B-cell lymphoma, we evaluated OTX015 combined with everolimus or the dual PI3K/mTOR inhibitor BEZ235 in 6 MCL cell lines (REC1, MAVER1, UPN1, JeKo1, SP53, Mino). In addition, combinations of OTX015 with the BTK-inhibitor ibrutinib, the proteasome inhibitor carfilzomib, the immunomodulator pomalidomide, the demethylating agent 5-AZA, the HDAC-inhibitor vorinostat, and the glucocorticoid dexamethasone were assessed in REC1 and MAVER1. Combinations of OTX015 with everolimus, pomalidomide, dexamethasone, and ibrutinib showed the strongest activity (Fig 1). Strong synergy between BEZ235 and OTX015 was only seen in the ibrutinib-resistant cell line MAVER1. Conclusions. OTX015 showed preclinical activity as a single agent in MCL cells. The mechanism of action does not appear to involve CCDN1 down-regulation and gene expression profiling studies will be needed to identify the involved pathways. OTX015 had additive or synergistic activity with several targeted compounds in multiple MCL cell lines, identifying combinations that may merit further investigation in the preclinical and clinical settings. Fig. 1. Chou-Talalay analysis of OTX015 combinations in MCL cell lines. Y-axis: CI 2.25, antagonism. Outliers were excluded. C.I., Combination Index. Fig. 1. Chou-Talalay analysis of OTX015 combinations in MCL cell lines. Y-axis: CI 2.25, antagonism. Outliers were excluded. C.I., Combination Index. Disclosures Stathis: Oncoethix SA: Consultancy, Research Funding. Riveiro:Oncoethix SA: Consultancy, Research Funding; Oncology Therapeutic Development: Employment. Bertoni:Oncoethix SA: Research Funding.

Purpose While international stage (ISS) and the presence of absence of cytogenetic abnormalities on FISH somewhat define the clinical risk of MM patients, additional biomarkers are necessary for more precise risk-based classification. Emerging studies have shown that circulating microRNAs (miRNAs) can be detected in patients with a variety of malignancies, including MM, and they could be non-invasive biomarkers. We measured serum miRNA levels of a large cohort of well-characterized previously untreated MM patients and correlated results with clinical outcome to test their prognostic impact. Methods and Patients To profile the expression of circulating microRNAs in the serum of MM patients, we performed NanoString-nCounter microRNA assays on samples obtained from a discovery cohort of 54 newly diagnosed MM patients enrolled on a randomized GIMEMA phase 3 study comparing Velcade-Melphalan-Prednisone-Thalidomide versus Velcade-Melphalan-Prednisone followed by maintenance with Velcade-Thalidomide. To further analyze the expression of the differentially expressed microRNAs, stem-loop-RT-PCR was performed on a validation cohort of 234 MM patients enrolled in the same trial. The prognostic significance of differentially expressed microRNAs were evaluated in relation to progression-free (PFS) and overall survival (OS) using univariate and multivariate Cox proportional hazards models. The utility of incorporating microRNA expression into a risk score with known risk factors – specifically ISS stage and the presence of del17, t(4;14) or t(14;16) by FISH – was also explored. Results Out of the 800 miRNAs evaluated, only 25 were detectable (≥100 counts) in at least 20% of the patients. The expression of these miRNAs were then measured in a validation set, but only 10 (miRs-92a, 21, 30a, 720, 451, 223, 126, 19b, 25 and miR-16) were validated to be differentially expressed. We found that levels of miR-16 and miR-25, used as continuous variables, had significant impact on OS duration: miR-16 (HR 0.87; p=0.019) and miR-25 (HR 0.81; p=0.0012) where low expression corresponded with worse survival. Based on these observations we generated a microRNA-based risk score which was significantly associated with OS duration (p=0.008). We then integrated this score with ISS stage and presence of high risk features by FISH, generating an integrated-microRNA risk score that was significantly associated with OS (p Conclusions Circulating miR-16 and miR-25 can risk stratify elderly, previously untreated, MM patients beyond ISS-stage and high risk genetic features. The opportunity to isolate circulating miRNAs allows us to sequentially sample cancer patients in a relatively non-invasive manner, opening new avenues of investigation for disease stratification and response to therapy. Disclosures: Bringhen: Onyx: Consultancy.