ALBERT

Beschreibung: Multiple Myeloma is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow.Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio 〉 2 and FDR (false discovery rate) 〈 5%) were identified, including the EZH2 histone methyltransferase. EZH2, the enzymatic subunit of Polycomb Repressive Complex 2, is a histone methyltransferases able to repress gene expression by catalyzing H3K27me3 histone mark. EZH2 overexpression has been associated with numerous hematological malignancies, including MM. We thus studied EZH2 role in MM physiopathology and drug resistance. EZH2 expression was analyzed in normal bone marrow plasma cells (BMPCs; N=5), primary myeloma cells from newly diagnosed patients (MMCs; N=206) and human myeloma cell lines (HMCLs; N=40) using Affymetrix microarrays. EZH2 gene is significantly overexpressed in MMCs of patients (median 574, range 105 - 4562) compared to normal BMPCs (median = 432; range: 314 - 563) (P 〈 0.01). The expression is even higher in HMCLs (median 4481, range 581 - 8455) compared to primary MMCs or BMPCs (P 〈 0.001). High EZH2 expression is associated with a poor prognosis in 3 independent cohorts of newly diagnosed patients (Heidelberg-Montpellier cohort - N=206, UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N =158). Furthermore, GSEA analysis of patients with high EZH2 expression highlighted a significant enrichment of genes involved in cell cycle, downregulated in mature plasma cells vs plasmablasts, and EZH2 targets. Specific EZH2 inhibition by EPZ-6438 EZH2 inhibitor induced a significant decrease of global H3K27me3 in all the HMCLs tested (P 〈 0.01) and inhibited MM cell growth in 5 out of the 6 HMCLs tested. The inhibitory effect of EZH2 inhibitor on MM cell growth appeared at day 6 suggesting that it is mediated by epigenetic reprogramming. To confirm that EZH2 is also required for the survival of primary MMCs from patients, primary MM cells (n = 17 patients) co-cultured with their bone marrow microenvironment and recombinant IL-6 were treated with EPZ-6438. As identified in HMCLs, EZH2 inhibition significantly reduced the median number of viable myeloma cells by 35% (P = 0.004) from a subset of patients (n=9) while the other group (n=8) was resistant. Of interest, EPZ-6438 induced a significant global H3K27me3 decrease in both groups of patient. RNA sequencing of 6 HMCLs treated with EPZ-6438 combined with H3K27me3 ChIP analyses allowed us to create an EZ GEP-based score able to predict HMCLs and primary MM cells sensitivity to EZH2 inhibitors. We also observed a synergy between EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment. More interestingly, pretreatment of myeloma cells with EPZ-6438 significantly re-sensitize drug-resistant MM cells to Lenalidomide. Investigating the effect of EPZ-6438/Lenalidomide combination in MMC, we identified that IKZF1, IRF4 and MYC protein levels were significantly more inhibited by the combination treatment (65.5%, 63.9% and 14.8% respectively) compared with Lenalidomide (51.5%, 43% and 2.2%) or EPZ-6438 (45.2%, 38.7% and 6.2%) alone. Clinical trials are ongoing with EZH2 inhibitors in lymphoma and could be promising for a subgroup of MM patients in combination with IMiDs. Furthermore, the EZ score enables identification of MM patients with an adverse prognosis and who could benefit from treatment with EZH2 inhibitors. Disclosures Goldschmidt: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, 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, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

Beschreibung: Redox homeostasis is fundamental for normal cellular functioning and is maintained by the net physiologic balance between production and removal of ROS. Redox signaling has been shown to coordinate the global signal transduction pattern critical for the control of key cellular processes including differentiation and tumorogenesis. In the present study, we review the systematic expression of ROS-producing and antioxidant genes and we draw up a full redox portrait of B cells all the way throughout B- cells expansion and differentiation and in malignant plasma cells. Our data firmly established that B-cells terminal differentiation entails drastic reshaping of the redox profile and underscore the stark transcriptional divide between B cells and plasma cells compartments with a major contribution of the Thioredoxin-Glutathione system in latter one. Furthermore, we advanced significant evidence that the most aggressive Multiple Myeloma cells (MMCs) witness increased expression of ROS producing genes and actively upregulated multiple antioxidant systems, which likely afforded high cytoprotective capacities and compromise chemotherapy efficacy. More importantly, we describe a transcriptional redox signature that enables the demarcation of good versus bad prognosis of MM patients and hold promise for robust molecular signature that could predict responsiveness. Finally, our data pinpointed the Thioredoxin-Glutathione system as actionable targets to circumvent drug resistance. Using Human Myeloma cell lines and primary cultures of MMCs, we validated Thioredoxin-Glutathione system as new critical, key functional node in the oncogenic network of MMCs whose depletion kill tumor cells and improve the response rate. We provided herein a preclinical data, which warrants further clinical investigation for the potential use of redox-modulators as a novel MM treatment strategy. Disclosures No relevant conflicts of interest to declare.

Beschreibung: The contribution focuses on the development of microresonant sensor solution integrated in microfluidic platform for the haemostasis assessment at realistic rheological flow conditions similar to the one in blood vessels. A multi-parameter sensor performs real time analysis of interactions between immobilized collagen and platelets. The detection and characterization of such interactions at controlled flow rates provide information to evaluate the dynamic of each step of primary haemostasis. The microresonant sensor concept was developed and is described in the contribution.

Beschreibung: Shear bulk acoustic type of resonant biosensors, such as the quartz crystal microbalance (QCM), give access to label-free in-liquid analysis of surface interactions. The general understanding of the sensing principles was inherited from past developments in biofilms measurements and applied to cells while keeping the same basic assumptions. Thus, the biosensor readouts are still quite often described using ‘mass’ related terminology. This contribution aims to show that assessment of cell deposits with acoustic biosensors requires a deep understanding of the sensor transduction mechanism. More specifically, the cell deposits should be considered as a structured viscoelastic load and the sensor response depends on both material and topological parameters of the deposits. This shifts the paradigm of acoustic biosensor away from the classical mass loading perspective. As a proof of the concept, we recorded QCM frequency shifts caused by blood platelet deposits on a collagen surface under different rheological conditions and observed the final deposit shape with atomic force microscopy (AFM). The results vividly demonstrate that the frequency shift is highly impacted by the platelet topology on the bio-interface. We support our findings with numerical simulations of viscoelastic unstructured and structured loads in liquid. Both experimental and theoretical studies underline the complexity behind the frequency shift interpretation when acoustic biosensing is used with cell deposits.

Beschreibung: Multiple myeloma (MM) is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow. Epigenetic modifications, including DNA methylation and histone post-translational changes, are involved in MM development and progression, but also in drug resistance. We have derived a large cohort of patient-derived HMCLs that remain dependent on the addition of exogenous MM bone marrow growth factors, reflecting primary tumor conditions. We have described their molecular diversity by analyzing the gene expression profile and mutational landscape and have showed that HMCL molecular diversity reflects part of the molecular heterogeneity of primary MM cells. However, the epigenetic landscape of HMCLs has never been described. A comprehensive characterization of the epigenetic landscape of HMCLs would advance our understanding of MM pathophysiology and may attempt to identify new therapeutic targets. In our study, we presented the epigenetic landscape of HMCLs. We performed chromatin immunoprecipitation sequencing (ChIP-seq) to analyze changes of the histone marks (H3K9me3 to follow heterochromatin, H3K4me1 and H3K27ac to follow enhancer activity, H3K4me3 and H3K36me3 to follow active transcription and H3K27me3 to follow Polycomb-silenced chromatin) on 16 HMCLs, representative of the molecular heterogeneity of MM. The differential analysis of histone modification profiles of HMCLs highlighted links between histone modifications and cytogenetic abnormalities or recurrent mutations. H3K4me3 and H3K27me3 profile analysis revealed specific clusters of HMCL related to 1q gain and t(4;14) translocation, respectively. These two cytogenetic abnormalities lead to deregulation of epigenetic player expression (e.g. SETDB1 and MMSET) and thus, could alter histone modification profile. Using histone modifications associated to enhancer regions (H3K4me1 and H3K27ac), we identified super-enhancers (SE) associated with genes involved in the biology of MM. 607 to 2510 predicted super-enhancers per HMCL were identified, including MAF, MYC, CCND1, CCND2, TRAF3 or NSD2. These super-enhancers differ from typical enhancers in both size and H3K4me1 and H3K27ac levels. The SE-associated genes identified in HMCLs with a prognostic value in two independent cohorts of newly diagnosed patients (CoMMpass cohort; N = 674 and Montpellier cohort; N = 69 with RNA-seq data) were used to build a score predicting MM patient outcome (Figure 1). Moreover, among the 28 genes that compose the risk-score (BSG, HK2, HNRNPC, HSPA9, IL10, ILF3, LDHB, MDH1, MYBPC2, NCL, NUDC, PARP1, PDIA6, PRPS1, RPL8, RPL13A, RPL27A, RPL35, SF3B2, SLC7A5, SLC25A39, SMARCA4, SPN, STC2, THY1, TNPO2, TPR), public datasets of RNAi and CRISPR/Cas9 screening revealed four genes (YWHAQ, IL10, HK2 and THY1) identified as significant essential myeloma genes, suggesting that they could represent potential therapeutic targets. We also identified promoters of genes characterized by a co-localization of H3K9me3 and H3K27me3 repressive marks in HMCLs. We evaluated the prognostic value of these genes in the CoMMpass and Montpellier cohorts, and selected genes associated with poor outcome when their expression is low in MM cells of patients (ARHGEF5, BIVM, DEF8, GRID2IP, HDAC9, HSPA1L, KDM4C, NLRP2, P4HA3, PAG1, PM20D1, RMND5A, SEMA6A, SFMBT2, THEMIS2, TPRKB, ZFP2 and ZNF5188B) underlining potential new tumor suppressor genes. These potential tumor suppressor genes associated with repressive histone marks were used to build a second risk score splitting MM patients in low- and high-risk groups in CoMMpass and Montpellier cohorts. Finally, we explored H3K4me3 marks comparing drug-resistant and -sensitive HMCLs (N = 16) to identify regions involved in drug resistance. From these data, we developed epigenetic biomarkers based on this H3K4me3 modification predicting lenalidomide and romidepsin HDCAi response. This study provides a comprehensive characterization of the MM epigenetic landscape representing unique resources for future biological studies and could help to identify novel critical epigenetic modifications involved in MM progression and drug resistance. Furthermore, risk-scores based on super enhancers and repressive regions together with epigenetic biomarkers of drug response could represent new tools for precision oncology in MM. Disclosures De Boussac: Diag2Tec: Current Employment. Bruyer:Diag2Tec: Current Employment. Vincent:janssen: Membership on an entity's Board of Directors or advisory committees, Other: Congress support; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Congress support; takeda: Membership on an entity's Board of Directors or advisory committees, Other: Congress support. Moreaux:Diag2Tec: Consultancy.