Normal Bone Marrow Plasma Cells Research Articles

IRF2 Regulates De Novo Active Chromatin Regions and Participates in the Development and Pathogenesis of Multiple Myeloma

Multiple Myeloma (MM) is the second most common hematologic neoplasia, and it remains largely incurable due to the genetic heterogeneity of the patients. We have recently characterized the epigenome of plasma cells from patients with MM, describing the de novo activation of the chromatin as a unifying factor underlying the pathobiology of the MM. Precisely, we identified 1556 de novo active regions, showing the H3K27ac chromatin activation mark, in MM in comparison to different subpopulations of normal B cells. Moreover, those de novo active regions where enriched in Transcription Factor Binding Sites (TFBS). In order to defined the potential role of these regions we have performed a CRISPR-Cas9 screening using guides against 56 TF that presented TFBS in de novo active regions and that are expressed in MM. A CRISPR-Cas9 library containing 10 guides for each gene was transduced in two MM cell lines (MM.1R and KMS-11) and the presences of guides were analyzed at days +1, +14 and +21. Out of the 22 TFs shown to be essential for MM survival, there were 3 members of the IRF TF family (IRF1, IRF2 and IRF5). Single knockdowns identified IRF2 and the previously described IRF4 as the best candidates. To characterize the signalling pathways involved in IRF family regulation of MM, the chromatin regions where IRF1, IRF2 and IRF4 are bound were mapped using CUT&RUN in MM.1R and KMS-11 cell lines. Interestingly, the three TF were located in 573 out of the 1556 de novo active regions. Notably, IRF2 was present in the 86.9% of those regions, and in 40% of the cases, the binding occurred independently of IRF1 or IRF4 ( Fig. 1). Additionally, the genomic distribution demonstrated that the regions where IRF2 binds alone were enriched in promoters compared to those regions where IRF1 and IRF4 also bound, being these enriched mostly in the gene body. Gene Ontology studies showed that IRF2, as well as IRF1/4 were involved in regulation of cell death in response to oxidative stress, while only IRF2 autonomously was involved in the regulation of pathways relevant for MM such as the kinase dysregulation, cell migration and osteoblast regulation. Furthermore, using our previous RNA-seq data, we observed that the majority of the genes regulated uniquely by IRF2 presented an overexpression in MM in comparison to different subpopulations of normal B cells. This overexpression was already identified in plasma cells from MGUS (Monoclonal Gammopathy of Undetermined Significance) patients and SMM (Smouldering MM) stages in contrast to normal bone marrow plasma cells. Finally, we examined the impact of IRF2 and IRF4 expression on the outcome of MM using RNA-seq data from 664 patients from MMRF (MM Research Foundation) CoMMpass (IA18) database. We observed that patients with higher expression of IRF2 presented significantly poorer Progression Free Survival (PFS) and Overall Survival (OS) in univariate analysis. Interestingly, in multivariate analyses taking into account the classic high-risk genetic factors for MM, the expression of IRF2 together with del13q and amp1q resulted in a significantly lower PFS (global p < 0.001) and the expression of IRF2 together with amp1q and del17p resulted in statistically significant worse OS (global p < 0.001). On a multivariate model, MM patients were stratified according to the number of events ( IRF2 expression, del13q and amp1q for PFS and IRF2 expression, amp1q and del17p for OS) into four risk groups both for PFS and OS ( Fig. 2). In conclusion, our study demonstrate that IRF2 plays a significant role in the pathogenesis of MM by participating in the regulation of the de novo active chromatin regions, specifically regions related to kinase pathway. Additionally, IRF2 represents a robust biomarker that in combination with classical genetic markers improves stratification of MM patients. These results establish IRF2 as a promising candidate for new therapeutic strategies in patients with MM.

S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma.

Multiple myeloma (MM) is the second most prevalent hematologic malignancy and is incurable because of the inevitable development of drug resistance. Methionine adenosyltransferase 2α (MAT2A) is the primary producer of the methyl donor S-adenosylmethionine (SAM) and several studies have documented MAT2A deregulation in different solid cancers. As the role of MAT2A in MM has not been investigated yet, the aim of this study was to clarify the potential role and underlying molecular mechanisms of MAT2A in MM, exploring new therapeutic options to overcome drug resistance. By analyzing publicly available gene expression profiling data, MAT2A was found to be more highly expressed in patient-derived myeloma cells than in normal bone marrow plasma cells. The expression of MAT2A correlated with an unfavorable prognosis in relapsed patients. MAT2A inhibition in MM cells led to a reduction in intracellular SAM levels, which resulted in impaired cell viability and proliferation, and induction of apoptosis. Further mechanistic investigation demonstrated that MAT2A inhibition inactivated the mTOR-4EBP1 pathway, accompanied by a decrease in protein synthesis. MAT2A targeting in vivo with the small molecule compound FIDAS-5 was able to significantly reduce tumor burden in the 5TGM1 model. Finally, we found that MAT2A inhibition can synergistically enhance the anti-MM effect of the standard-of-care agent bortezomib on both MM cell lines and primary human CD138+ MM cells. In summary, we demonstrate that MAT2A inhibition reduces MM cell proliferation and survival by inhibiting mTOR-mediated protein synthesis. Moreover, our findings suggest that the MAT2A inhibitor FIDAS-5 could be a novel compound to improve bortezomib-based treatment of MM.

The Frequency of Alternative Splicing in Multiple Myeloma Is Determined By the Non-Homologous End Joining Pathway

Introduction: Alternative Splicing (AS) plays a pivotal role in regulating cellular processes in both normal and malignant cells. Previous studies indicated mutations in the spliceosome complex, such as SF3B1, can cause aberrant AS in acute myeloid leukemia and multiple myeloma (MM) patients. However, there is also a known interaction between AS and the DNA damage response in other cancer types, which we investigate here. Materials and Methods: RNA-sequencing data of newly diagnosed MM patients (n=598) from the MMRF CoMMpass dataset were utilized to generate whole transcriptome AS profiles. For each patient, RNA-seq data was aligned to Hg38 reference genome using STAR while expression level was measured by Salmon. AS profiles were established from their alignment and expression profile using SUPPA2. Splicing level was defined by Percentage of Spliced-In (PSI). UMAP plots were generated for all patients using top 20,000 AS events ranked by variation in the sample set. High-quality differential AS events between groups of patients were obtained from the following thresholds: significant splicing level difference (|PSI|>10% and P <0.05 from T-test), sufficient expression level (TPM>1), not similarly spliced (|PSI|>10% and P-value<0.05 from T-test) in normal bone marrow plasma cells (N=5). Patient samples were annotated for cytogenetic, mutation, copy number and structural variation status. Pathway analysis was conducted for differentially expressed genes (DEGs). Patients' overall survival and progression-free survival was displayed with Kaplan-Meier curves. Survival difference was measured by log-rank test and cox-regression (when other well-known risk factors were incorporated). Results: We examined differential AS by defining and comparing high and low activity of key myeloma KEGG pathways including the spliceosome, proteasome, DNA repair and ubiquitination pathways. The DNA repair pathway generated significantly more (P<0.05) differential AS events (N=1,197) than the other pathways. To examine this further, we performed the analysis for the three main DNA repair mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) pathways. The number of differential AS events (N=2,999) between NHEJ-high (N=121) and -low (N=36) samples was significantly more than for the other DNA repair pathways (P<0.05). NHEJ-high samples were significantly enriched for TP53 mutations (15% vs. 6%, P=2x10-5, Fisher's exact test), biallelic-TP53 events (10% vs. 4%, P=3x10-5), and biallelic-TENT5C (21% vs. 10%, P=1x10-9) events (Figure 1). Additionally, expression of LIG4 and XRCC4, encoding DNA IV ligase complex, a key component of the NHEJ pathway, correlated highly (, Spearman correlation) with the number of differential AS events. The NHEJ-high group also harbored significantly more structural abnormalities than the NHEJ-low group (mean 87 vs. 19, P=6x10-4, Mann-Whitney U test), indicating their different status of DNA stability that was highly associated with this pathway. Survival analysis indicated that patients in the NHEJ-high group showed a significant worse progression-free survival (PFS) compared to others (Logrank statistic=7.3, P=0.007). In a multivariate analysis including other high-risk factors such as MGP-Double-Hit, t(4;14), t(14;16), TP53 mutation, 17p del, 1q gain/amp, and ISS=III, the NHEJ-high group retained significance (Hazard Ratio=1.8, P=1.6x10-5). To incorporate AS events we further applied a backward elimination approach to automatically select an optimal prognostic model from top 200 highly variant AS events ranked by variation and 7 well-known risk factors along with high NHEJ activity in PFS for the entire population. This model contained 16 significant risk factors (P<0.05, Multivariate cox regression) and 12 of them were AS events. NHEJ-high samples were significantly enriched (P<0.05, Fisher exact test) for eight of the AS events that were associated with poor prognosis. Conclusion: AS frequency is associated with the NHEJ pathway in MM. NHEJ activity is also associated with DNA instability through increased number of structural rearrangements and is a high-risk factor. Figure 1. High expression of the NHEJ pathways is associated with TP53 and TENT5C abnormalities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Altered Expression of CCR10 Indicates Malignant Transformation of Bone Marrow Plasma Cells

Introduction. Phenotypic analysis of tumor cells by flow cytometry is now essential for diagnosis and treatment of hematological malignancies, including multiple myeloma (MM). Surface molecules such as CD19, CD56, CD27 and CD45 have been used to distinguish normal bone marrow (BM) plasma cells from malignant plasma cells (MM cells). Molecules that are relatively specific to plasma cells (CD38 and SLAMF7) are considered targets for antibody-based therapies in MM. Among the vast numbers of surface molecules, chemokine receptors have been attracting much attention as the regulators of migration and tissue localization of plasma cells and MM cells. CXCR4 (C-X-C chemokine receptor type 4) has been reported to guide the movement of plasma cells and myeloma cells to the BM, where its ligand CXCL12 (C-X-C motif chemokine 12) is abundantly produced. CCR10 (C-C motif chemokine receptor 10) is another chemokine receptor, whose ligand is CCL27 (C-C motif chemokine ligand 27) and CCL28, also known to be involved in cell homing and migration. CCR10 has been reported to be highly expressed on mucosal derived plasma cells, however its expression in MM cells and its role within the BM microenvironment has not been well studied. In this study, we analyzed the expression of CCR10 on BM lymphocytes from patients of plasma cell disorders in order to understand its significance in the BM microenvironment.Materials and methods. We analyzed a public available gene expression dataset (GSE 22886) in order to study CCR10 gene expression in human plasma cells. To examine CCR10 gene expression in myeloma cells, we utilized the gene expression data set of multiple cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE: http://www.broadinstitute.org/ccle). Multi-color flow cytometry was performed to study CCR10 expression on bone marrow lymphocytes. BM plasma cells were determined as CD38++ CD138+ or CD38++ SLAMF7+. Monoclonal plasma cells (myeloma clones) were determined as CD19- CD56- or CD19- CD56+ cells and polyclonal plasma cells (normal plasma cells) as CD19+ CD56- cells. CCR10 expression of BM plasma cells, B cells, CD4 T cells, CD8 T cells and NK cells of MGUS, newly diagnosed MM, relapsed-refractory MM patients were analyzed.Results. Gene expression dataset analysis showed that, among the normal leukocyte subsets, plasma cells had the highest CCR10 gene expression compared to other leukocytes (p<0.001). Myeloma cell lines showed the highest expression of CCR10 gene expression among all the other cancer cell lines, indicating the specificity of CCR10 expression in MM cells. Multi-color flow cytometry showed significantly high expression of CCR10 on BM plasma cells compared to other lymphocytes in the bone marrow of MGUS patients (p<0.0001). Within the MGUS BM plasma cells, monoclonal plasma cells had significantly lower CCR10 expression compared to polyclonal plasma cells (p<0.05). Additionally, monoclonal plasma cells from relapsed-refractory MM patients had significantly lower CCR10 expression compared to those from MGUS and newly diagnosed MM patients. These data indicate a role of CCR10 decrease in malignant plasma cells leading to MM progression. We further analyzed other surface molecules that may be related to CCR10 on monoclonal plasma cells. CCR10 expression did not correlate with CD56 expression, while CD45- monoclonal plasma cells tend to have lower CCR10 expression compared to the CD45+ counterparts in newly diagnosed MM patients (p<0.001).Conclusions. Gene expression dataset analysis and multi-color flow cytometry have showed that CCR10 is highly expressed in normal plasma cells. However, we show here for the first time that plasma cells start to decrease CCR10 expression at malignant transformation and during disease progression. Since, CCL27, the ligand of CCR10, is known to induce chemotaxis to plasma cells, loss of CCR10 may lead to migration or metastasis of MM cells to different sites. Further research is now on-going to elucidate the significance and mechanism of altered CCR10 expression in MM cells. This study may lead to a better understanding of MM biology and to the development of a novel strategy to target malignant plasma cells. DisclosuresNo relevant conflicts of interest to declare.

Effect of TRIM31 Gene Silencing on the Proliferation and Apoptosis of U266 Cells and Its Mechanism

To investigate the effect of the tripartite motif containing 31 (TRIM31) gene silencing on the proliferation and apoptosis of multiple myeloma cells and its possible mechanism. The normal bone marrow plasma cells (nPCs) were selected as control, and the mRNA and protein expression levels of TRIM31 in human multiple myeloma cell lines (U266, RPMI-8226, NCI-H929 and KMS-11) were detected by RT-qPCR and Western blot. Recombinant lentivirol vector containing shRNA-TRIM31 and its negative control were used to infect U266 cells respectively, and the mRNA expression level of TRIM31 in infected cells was detected by RT-qPCR. Then cell proliferation, colony forming and apoptosis were analyzed by CCK-8, soft agar assay, and flow cytometry, respectively. The protein expression levels of TRIM31, cleaved-caspase-3, BCL-2, Bax, p-Akt (Ser473), Akt and PI3K (p110α) were evaluated by Western blot. In addition, the PI3K/Akt signaling pathway-specific inhibitor LY294002 and TRIM31-shRNA lentivirus were used to interfere with U266 cells, and the cell proliferation, apoptosis, and protein expression of p-Akt (Ser473) and Akt were detected by CCK-8, flow cytometry and Western blot, respectively. Compared with nPCs, the expression levels of TRIM31 mRNA and protein in U266, RPMI-8226, NCI-H929 and KMS-11 cells were significantly increased (P<0.001), especially in U266 cells. After lentivirus infection, the levels of TRIM31 mRNA and protein in U266 cells were significantly decreased (P<0.001). TRIM31 silencing significantly inhibited the proliferation of U266 cells (P<0.05), attenuated the ability of cell cloning, improved cell apoptosis, up-regulated the protein expressions of cleaved-caspase-3 and Bas as well as down-regulated expressions of BCL-2, p-Akt (Ser473) and PI3K (p110α). There was no significant effect on Akt protein. Intervention of LY294002 significantly enhanced the inhibition on cell proliferation and the promotion on apoptosis mediated by TRIM31 gene silencing in U266 cells. TRIM31 gene silencing can inhibit U266 cell proliferation and promote its apoptosis, which may be closely related to inhibition of PI3K/Akt signaling pathway.

LncRNA transcription factor 7 is related to deteriorating clinical features and poor prognosis in multiple myeloma, and its knockdown suppresses disease progression by regulating the miR-203-mediated Jagged1-Notch1 signaling pathway.

Multiple myeloma (MM) remains a challenge to treat, and its precise pathogenic mechanisms have not been fully clarified. The present study aimed to evaluate the relation between long non-coding RNA transcription factor 7 (lnc-TCF7) and clinical features, as well as the prognosis of patients with MM, and to determine the effects of lnc-TCF7-knockdown on the regulation (and regulatory mechanisms) of MM progression. lnc-TCF7 expression was detected in the bone marrow plasma cells of 86 patients with MM and 30 healthy controls. In patients with MM, the clinical data were collected, and event-free survival (EFS) and overall survival (OS) analyses were conducted. In vitro, lnc-TCF7 expression was detected in MM cell lines and normal bone marrow plasma cells. Using Roswell Park Memorial Institute 8226 cells, functional experiments were conducted following lnc-TCF7 short hairpin (sh)RNA transfection, and compensation experiments were performed after lnc-TCF7 shRNA transfection alone and in combination with a microRNA (miR)-203 inhibitor. lnc-TCF7 expression was increased in patients with MM compared with the healthy controls and was positively related to β-2-microglobulin expression and International Staging System stage, while negatively associated with complete response, EFS and OS. In vitro, lnc-TCF7 was upregulated in MM cells compared with normal bone marrow plasma cells, and its knockdown suppressed MM cell proliferation while promoting apoptosis. Compensation experiments showed that miR-203 inhibition promoted MM progression by regulating the Jagged1-Notch1 signaling pathway in lnc-TCF7-knockdown cells. In conclusion, increased lnc-TCF7 expression was related to deteriorating clinical features and prognosis, and lnc-TCF7-knockdown inhibited disease progression by regulating the miR-203-mediated Jagged1-Notch1 signaling pathway activation in MM.

Characterization of complete lncRNAs transcriptome reveals the functional and clinical impact of lncRNAs in multiple myeloma

Multiple myeloma (MM) is an incurable disease, whose clinical heterogeneity makes its management challenging, highlighting the need for biological features to guide improved therapies. Deregulation of specific long non-coding RNAs (lncRNAs) has been shown in MM, nevertheless, the complete lncRNA transcriptome has not yet been elucidated. In this work, we identified 40,511 novel lncRNAs in MM samples. lncRNAs accounted for 82% of the MM transcriptome and were more heterogeneously expressed than coding genes. A total of 10,351 overexpressed and 9,535 downregulated lncRNAs were identified in MM patients when compared with normal bone-marrow plasma cells. Transcriptional dynamics study of lncRNAs in the context of normal B-cell maturation revealed 989 lncRNAs with exclusive expression in MM, among which 89 showed de novo epigenomic activation. Knockdown studies on one of these lncRNAs, SMILO (specific myeloma intergenic long non-coding RNA), resulted in reduced proliferation and induction of apoptosis of MM cells, and activation of the interferon pathway. We also showed that the expression of lncRNAs, together with clinical and genetic risk alterations, stratified MM patients into several progression-free survival and overall survival groups. In summary, our global analysis of the lncRNAs transcriptome reveals the presence of specific lncRNAs associated with the biological and clinical behavior of the disease.

Frequent methylation of the tumour suppressor miR‐1258 targeting PDL1: implication in multiple myeloma‐specific cytotoxicity and prognostification

miR-1258 is localised to the first intron of ZNF385B at chromosome 2q31.3. miR-1258 promoter methylation was studied in 147 samples including 10 normal buffy coat, eight normal bone marrow plasma cells, 16 human myeloma cell lines (HMCLs), 20 MGUS, 63 diagnostic myeloma, and 30 relapsed myeloma samples by methylation-specific PCR. In myeloma lines, miR-1258 methylation, verified by pyrosequencing, was detected in 62·5% HMCLs but not normal controls, and expression of miR-1258 correlated with that of ZNF385B. 5-Aza-2'-deoxycytidine resulted in promoter demethylation and ZNF385B/miR-1258 re-expression. Luciferase assay confirmed programmed cell death ligand-1 (PDL1) as a direct target of miR-1258. Over-expression of miR-1258 in completely methylated myeloma cells led to reduced cellular proliferation and enhanced apoptosis, hence a tumour suppressor role, in addition to repression of PDL1. In primary samples, miR-1258 methylation, with lower expression of miR-1258, was detected in 49·2% diagnostic myeloma, imparting an inferior PFS (P=0·034) in addition to 50·0% relapsed myeloma but not MGUS. Therefore, miR-1258 is a tumour suppressor miRNA co-regulated with its host gene, and frequently hypermethylated in active myeloma instead of MGUS, hence acquired during myeloma progression. Methylation-mediated miR-1258 silencing led to overexpression of PDL1 and inferior PFS, implicating miR-1258 in the modulation of myeloma-specific cytotoxicity.

The Transmembrane Receptor Roundabout 1 (ROBO1) Is Necessary for Multiple Myeloma Proliferation and Homing to the Bone Marrow Niche

Introduction Multiple myeloma (MM) is characterized by dissemination and accumulation of plasma cells in the bone marrow (BM), which promotes tumor cell growth and therapy resistance. ROBO1 is a conserved transmembrane receptor of the Ig superfamily with no intrinsic catalytic activity, and its role in MM pathogenesis is unknown. Material and Methods We first analyzed ROBO1 expression via western blot and/or immunohistochemistry (IHC). Gene expression profiling in a cohort of 170 newly diagnosed MM patients (IFM170) was used to compare ROBO1 expression across primary MM and BM stroma cells (BMSC), and normal BM plasma cells (PC). We used short hairpin RNA (shRNA) for stable ROBO1 knock down (KD) and CRISPR-Cas9 for ROBO1 knock out (KO). For protein structure-function and rescue studies, ROBO1 KO MM cells were transduced with a lentiviral vector expressing either full-length (FL) or truncated ROBO1 mutants devoid of extracellular (Cyt) or intracellular domain (DeltaCyt), including patient-derived truncating mutations, with a C-terminus triple FLAG tag. FLAG immunoprecipitation (IP) followed by mass spectrometry or western blotting and immunofluorescence (IF) were used to identify ROBO1 interacting partners and ROBO1 cellular localization. We used a hydrogel encapsulation system to study proliferation in a 3D system. To study extramedullary and intramedullary MM growth in vivo, WT and ROBO1 KO OPM2 were injected either subcutaneously (plasmacytoma model) or intra-medullary in femoral bones of donor mice which were then implanted subcutaneously in recipient SCID mice (µ-SCID model). PET-CT was used to assess tumor volume. Mouse tumors were retrieved for IHC and RNA extraction followed by RNA sequencing. To study dissemination and homing, KO and FL addback OPM2 cells were injected intravenously in SCID mice. Femurs and plasmacytoma were retrieved at endpoint for IHC. Results ROBO1 is highly expressed in human MM cell lines and primary MM cells with highest expression in cells carrying the high risk t(4;14) cytogenetic and low/absent expression in normal PC. Of human cancer cell lines, ROBO1 expression was limited to late B cell lineage; and ROBO1 KD was selectively cytotoxic against MM, but not other hematologic cancers. ROBO1 KO significantly decreases proliferation in a 3D culture system and tumor growth in extramedullary (mean tumor volume KO versus WT plasmacytoma: 457 versus 1323 mm3, p value= 0.02) and intramedullary (mean tumor volume KO versus WT: 823 versus 2684 mm3, p value= 0.001) murine models of human MM. ROBO1 KO MM cells show decreased adhesion to BM endothelial and BMSC, which is fully rescued by FL ROBO1 addback. To address whether ROBO1 loss alters dissemination/homing of MM cells in vivo, we injected mice intravenously with ROBO KO or FL addback OPM2 cells. While ROBO1 KO resulted in a modest, non-statistically significant prolongation in mouse OS (90 versus 75 days, respectively, p value 0.2), the pattern of disease was strikingly different. As expected, ROBO1 FL mice developed hindlimb paralysis with extensive BM infiltration with MM. Importantly, ROBO1 KO mice demonstrated reduced BM infiltration and developed solitary plasmacytoma. We next showed that ROBO1 C-terminus domain is necessary and sufficient to rescue ROBO1 KO proliferative defect while expression of ROBO1 truncations, including patient-derived frameshift mutations, acted as dominant negative. IP showed avid interaction of ROBO1 with ABL1. Interestingly, we showed that the cytosolic domain of ROBO1 undergoes cleavage and translocates to the nucleus, where its function is now being studied. Conclusions We show that ROBO1 is necessary for MM homing to the BM niche and for MM growth within and outside the BM space. ROBO1 cytosolic domain undergoes proteolytic cleavage and translocates to the nucleus and is necessary and sufficient to rescue ROBO1 KO defective proliferation. Based on our data, we propose a dual model for ROBO1 in MM: the full transmembrane receptor is involved in regulating adhesion, dissemination and homing of MM cells within the BM niche; the cleaved intracellular C-terminus domain participates in transcriptional regulation, promoting MM proliferation. These data suggest that ROBO1 C-terminus may be a novel molecular target in MM. Disclosures Roccaro: Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding; European Hematology Association: Research Funding; AstraZeneca: Research Funding; Transcan2-ERANET: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding. Ghobrial:Takeda: Consultancy; Sanofi: Consultancy; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Janssen: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

AL Amyloidosis — Pathogenesis and Prognosis Are Determined By the Amyloidogenic Potential of the Light Chain and the Molecular Characteristics of Malignant Plasma Cells

INTRODUCTION. Systemic light chain amyloidosis (AL) is caused by accumulation of plasma cells producing misfolded monoclonal light chains depositing as amyloid fibrils in different organs, most frequently heart and kidney.AIM of our study is first assessing the molecular characteristics of malignant plasma cells from AL-patients in relation to those from MGUS, asymptomatic, and symptomatic myeloma: Are these plasma cells different, does this difference explain amyloidogenicity? Does AL correspond to a certain developmental stage during evolution of symptomatic myeloma? Secondly, to what extent is prognosis determined by amyloid-deposition (organotropism, amount, amyloidogenicity) vs. number and molecular characteristics of malignant plasma cells?PATIENTS & METHODS. Consecutive patients (n=3023) with AL (n=582), MGUS (n=306), asymptomatic (n=444, AMM), or previously untreated, therapy-requiring multiple myeloma (n=1691, MM) were included. CD138-purified plasma cell samples were subjected to iFISH (n=582/306/444/1691), 1297 to gene expression profiling using Affymetrix U133 2.0 plus arrays (n=196/64/272/765), 712 to RNA- (n=124/52/38/489), and 258 to whole exome sequencing (n=115/53/39/51). Samples of normal bone marrow plasma cells, memory B-cells, and polyclonal plasmablasts were used as comparators. The CoMMpass-cohort (n=647) was used as comparator for the mutational spectrum of myeloma.RESULTS.Prognosis. By AL-factors. Expectedly, organ involvement, i.e. heart only vs. kidney only vs. heart+kidney vs. other (overall survival (OS), P=.001), the amount of free light chains (dFLC ≥18 mg/dL, HR=2.56, P=.01), and the cardiac European Mayo IIIB score (I/II/IIIA/IIIB, median OS 110/55/16/3 months, HR=1/1.94/3.73/7.90, P<.001) strongly determine prognosis (Fig. 1A). By malignant plasma cell factors. High proliferation rate (HR=3.58, P=.001) and expression-based risk factors for MM (GEP70 high, HR=2.38, P=.005; Rs-score high HR=4.63, P<.001) identify patients with very adverse prognosis (Fig. 1A). Tumor load, e.g. plasma cell infiltration >10%/>30% (HR=1.31/1.81, P=.01, P=.002) and M-protein ≥ 30g/l (HR=3.01, P=.005), are likewise prognostic (Fig. 1A). In multivariate analysis, all tested AL-specific (European Mayo IIIB score) and malignant plasma cell factors (proliferation or GEP70 and plasma cell infiltration) are independent. Molecular characteristics.iFISH. As MM (96.2%) and AMM (92.8%) AL-patients (93.1%) carry at least one recurrent myeloma typical aberration. The mean number of progression-associated aberrations in AL (n=0.98) fits between MGUS (n=0.85) and AMM (n=1.45) with significant difference compared to AMM (P<.001) unlike to MGUS. Main differences in frequency are found for t(11;14) and hyperdiploidy with a comparable pattern of non-etiologic aberrations. Gene expression (GEP and RNA-seq). Aberrant plasma cells in AL amyloidosis show the least difference with AMM, followed by MGUS and MM. In principal component analysis, AL overlaps with AMM and MGUS, independent of presence or absence of heart involvement (Fig. 1B). Pairwise assessment of similarity using a multivariate generalization of the squared Pearson correlation coefficient shows closest similarity to AMM and MM followed by MGUS, with comparable differences to normal plasma cells, polyclonal plasmablasts, and memory B-cells. Significantly more AL-patients present with higher proliferation rate vs MGUS (P<.001) and AMM (P<.02). AL and MM differ significantly regarding distinct molecular entities as determined by GEP (e.g. TC-classification; Fig. 1C). Mutation spectrum in AL amyloidosis vs. MM. From the 20 most frequently synonymously mutated non-Ig transcripts (CoMMpass-cohort), 16 could likewise be detected in AL amyloidosis, i.e. KRAS, NRAS, IGLL5, DIS3, FAM46C, MUC16, BRAF, TRAF3, PCLO, RYR2, FATA4, CSMD3, TP53, DNAH5, RYR2A, and FLG. CCND1 mutations were significantly more frequent in AL and AMM compared to MM (P=.02).DISCUSSION & CONCLUSION. Pathogenesis and prognosis of AL amyloidosis are explained both by AL-specific and malignant plasma cell characteristics. Aberrant plasma cells in AL amyloidosis show the same aberration- and expression pattern and a “molecular age” between MGUS and AMM, most closely resembling the latter. AL amyloidosis is thus mostly a rather early plasma cell dyscrasia with an unstable and toxic immunoglobulin light chain. [Display omitted] DisclosuresSeckinger:Celgene: Research Funding; EngMab: Research Funding; Sanofi: Research Funding. Hose:Celgene: Honoraria, Research Funding; Sanofi: Research Funding; EngMab: Research Funding.

Role of RACK1 on cell proliferation, adhesion, and bortezomib-induced apoptosis in multiple myeloma

Receptor for activated C kinase 1 (RACK1), a scaffold protein, plays a crucial role in the progression of various cancers. However, the biological function and underlying mechanism of RACK1 in multiple myeloma (MM) cells remain unclear. The present study aimed to explore the function of RACK1 on the cell proliferation, adhesion, and bortezomib-induced apoptosis in MM. We found that RACK1 was significantly overexpressed in myeloma cell lines and primary myeloma cells compared with normal bone marrow plasma cells. Moreover, immunofluorescence revealed that RACK1 was primarily expressed in the cytoplasm of MM cells. Knockdown of RACK1 impaired growth of MM cells, blocked entry into the S-phase of the cell cycle, and resulted in reduced cell adhesion rates. More importantly, knockdown of RACK1 decreased the proliferation of MM cells by activating P-P38 and P-ERK in the MAPK/ERK signaling pathway. We also found that altered expression of RACK1 is associated with bortezomib-mediated MM cell apoptosis. In summary, these results may provide a possible target for therapy in MM.

CD38 as Immunotherapeutic Target in Light Chain Amyloidosis and Multiple Myeloma-Association With Molecular Entities, Risk, Survival, and Mechanisms of Upfront Resistance.

Monoclonal antibodies against the cell surface antigen CD38, e.g., isatuximab, daratumumab, or Mor202, have entered the therapeutic armamentarium in multiple myeloma due to single agent overall response rates of 29 vs. 36 vs. 31%, effectivity in combination regimen, e.g., with lenalidomide or bortezomib plus dexamethasone, and tolerable side effects. Despite clinical use, many questions remain. In this manuscript, we address three of these: first, upfront CD38 target-expression in AL-amyloidosis, monoclonal gammopathy of unknown significance (MGUS), asymptomatic, symptomatic, and relapsed multiple myeloma. Second, relation of CD38-expression to survival, disease stages, molecular entities, and high-risk definitions. Third, alternative splicing or lack of CD38-expression as potential mechanisms of upfront resistance. We assessed CD138-purified plasma cell samples from 196 AL-amyloidosis, 62 MGUS, 259 asymptomatic, 764 symptomatic, and 90 relapsed myeloma patients, including longitudinal pairs of asymptomatic/symptomatic (n = 34) and symptomatic/relapsed myeloma (n = 57) regarding interphase fluorescence in situ hybridization (n = 1,380), CD38-expression by gene expression profiling (n = 1,371), RNA-sequencing (n = 593), and flow cytometry (n = 800). Samples of normal bone marrow plasma cells (n = 10), memory B-cells (n = 9), polyclonal plasmablastic cells (n = 9), and human myeloma cell lines (n = 54) were used as comparators. CD38 was expressed in all malignant plasma cell samples, but significantly lower compared to normal plasma cells with small but significant downregulation in longitudinal sample pairs. Higher CD38 expression was associated with the presence of t(4;14) and high-risk according to the UAMS70-gene score, lower expression was associated with del17p13 and hyperdiploidy in symptomatic myeloma as well as t(11;14) in asymptomatic myeloma. Higher CD38-expression was associated with slower progression to symptomatic and relapsed myeloma and better overall survival in the latter two entities. CD38 expression, t(4;14), del17p13, and gain of 1q21 are independently prognostic in multivariate analysis. By contrast, high CD38-expression is associated with adverse survival in AL-amyloidosis. Regarding mechanisms of upfront anti-CD38-treatment resistance, lack of CD38-expression and alternative splicing of receptor binding-sites could be excluded. Here, of the two protein coding CD38-transcripts CD38-001 (eight-exon, full length) and CD38-005 (truncated), CD38-001 conveyed >97% of reads spanning the respective CD38 splice junction.

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

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

Mapping of the Multiple Myeloma Transcriptional Core Regulatory Circuitry Reveals TCF3 As a Novel Dependency and an Oncogenic Collaborator of MYC

Multiple Myeloma (MM) is a complex plasma cell malignancy driven by numerous genetic and epigenetic alterations that are acquired over time. The events controlling and modifying transcriptomic changes that drive MM cell growth and progression remains undefined. To reveal the epigenetic circuitry governing myeloma cells, we performed a comprehensive analysis integrating data obtained from Multiplexed Indexed T7 Chromatin IP (Mint-ChIP), Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq), and RNA-seq in 10 primary MM cells as well as 3 MM cell lines to identify genome-wide the master transcription factors (TFs), the enhancer elements they occupy, and the genes they regulate. Using these data, we have identified myeloma-specific core regulatory circuitry which includes several well-established regulators of MM such as IKZF, E2F, MYC and IRF family of genes. For example, our data show elevated MYC at numerous tissue specific enhancers in myeloma cells, including those that regulate lineage specifying transcription factors such as IRF4 and TCF3 (aka E2A). When translocated to the immunoglobulin enhancer, MYC in turn is regulated by these lineage transcription factors thus integrating MYC into the interconnected transcriptional core regulatory circuitry of MM (Figure 1a,b).We propose that this oncogenic “re-wiring” accounts for the observed addiction of MM cells to lineage factors such as IRF4 and in this work, we implicate the B-cell factor TCF3 as a novel multiple myeloma dependency. Using myeloma cell lines and primary samples, we observed elevated enhancer activity at TCF3 in primary CD138+ cells from myeloma patients compared to normal plasma cells (NPCs) (Figure 1c). As a result, TCF3 expression is significantly upregulated in our large cohort of MM patients (n=370) compared to normal bone marrow plasma cells (n=18). As MYC proteins can only bind pre-established and acetylated regions of active chromatin, we hypothesize that enhancer specifying lineage transcription factors such as TCF3 may cooperate with MYC to alter tissue specific gene expression programs. We show that TCF3 is regulated by a large proximal enhancer that is bound by MYC, and is highly sensitive to chemical perturbation of enhancer co-activators such as BRD4. As a helix-loop-helix transcription factor that similar to MYC binds short (CANNTG) E-box sequences, we computationally predict co-occupancy of MYC and TCF3 at ~80% of all enhancers that form the multiple myeloma transcriptional core regulatory circuitry. To evaluate the functional role of TCF3 in myeloma cells, we established TCF3 knock down myeloma cell lines and followed the cell growth over time. Stable knockdown of TCF3 preferentially blocks proliferation of IgH MYC translocated cell lines (such as MM1.S cells) versus non-translocated lines (such as U266 cells). Finally, high expression of TCF3 correlates with poor clinical outcome in myeloma patients. Together these data suggest TCF3 acts as an oncogenic collaborator with deregulated MYC and implicates transcriptional control of lineage as a dependency in multiple myeloma.Figure 1: Transcriptional core regulatory circuitry of multiple myeloma: A) ChIP-Seq tracks of IRF4, MYC, BRD4, and H3K27ac occupancy at the IRF4, IgH enhancer, and TCF3 loci respectively. B) Schematic of transcription factor to enhancer connectivity of the partial multiple myeloma transcriptional core regulatory circuitry highlighting interactions between IRF4, MYC, and TCF3 (computationally predicted based on TCF3 motif data). C) ChIP-Seq tracks of H3K27ac occupancy at the TCF3 locus in patient multiple myeloma (top, n=3) or normal plasma cells (bottom, n=2). [Display omitted] DisclosuresBradner:Acetylon: Other: Scientific Founder; Novartis: Employment. Anderson:Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees.

Deregulated expression of long non-coding RNA UCA1 in multiple myeloma.

Long non-coding RNAs (lncRNAs) are RNA transcripts longer than 200 nucleotides that are not translated into proteins. They are involved in pathogenesis of many diseases including cancer and have a potential to serve as diagnostic and prognostic markers. We aimed to investigate lncRNA expression profiles in bone marrow plasma cells (BMPCs) of newly diagnosed multiple myeloma (MM) patients in comparison to normal BMPCs of healthy donors (HD) in a three-phase biomarker study. Expression profile of 83 lncRNA was performed by RT2 lncRNA PCR Array (Qiagen), followed by quantitative real-time PCR using specific TaqMan non-coding RNA assays analyzing 84 newly diagnosed MM patients and 25 HD. Our analysis revealed dysregulation of two lncRNAs; NEAT1 (sensitivity of 55.0% and specificity of 79.0%) and UCA1 (sensitivity of 85.0% and specificity of 94.7%). UCA1 levels correlated with albumin and monoclonal immunoglobulin serum levels, cytogenetic aberrations, and survival of MM patients. Our study suggests a possible prognostic impact of UCA1 expression levels on MM patients.

Targeting EZH2 in Multiple Myeloma Could be Promising for a Subgroup of MM Patients in Combination with IMiDs

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. DisclosuresGoldschmidt: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.

Cooperative Supression of MEIS2 Mediates Dexamethasone Enhancement of Lenalidomide Killing in Myeloma Cells

Dexamethasone (Dex) enhances the clinical response to most therapies in multiple myeloma, the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide in particular. As a steroid, Dex may modulate the therapeutic response by acting on multiple cell types in vivo. Whether and how Dex enhances the tumor intrinsic clinical response to IMiDs is not well understood. To address this important question, we focus on the role of Dex in modulating the proximal events in IMiD signaling that lead to the killing of myeloma cells.Cereblon (CRBN), a substrate receptor of the CRL4CRBN E3 ligase, is required for IMiD's anti-myeloma activity. MEIS2 is a homeobox transcription factor of the mammalian myeloid ecotropic insertion site (MEIS) family, which plays a pivotal role in development and leukemogenesis. Recent crystal structure studies in conjunction with biochemical screen have identified MEIS2 as an endogenous substrate of CRBN. IMiDs apparently bind CRBN and block MEIS2 from binding to CRBN, thereby facilitating the recruitment of transcription factors IKZF1 and IKZF3 to CRL4CRBN for degradation. This then leads to loss of IRF4, a target of IKZF1/3, necessary for myeloma survival. This model posits that MEIS2 inhibits the function of CRBN in myeloma cells. However, as all the studies of MEIS2 were performed heterologous cell lines, evidence that supports a function of MEIS2 in myeloma cells is lacking.We now showed by RNA-seq and protein analysis that MEIS2 was absent in normal bone marrow plasma cells (BMPC), but highly expressed in freshly isolated primary bone marrow myeloma cells (BMMC)s at both RNA and protein levels. We further showed that MEIS2 was regulated by the cell cycle: reduced in G1 arrest and restored in S phase in human myeloma cell lines (HMCL)s as well as primary BMMCs. Depletion of MEIS2 markedly enhanced lenalidomide killing, and overexpression of MEI2 attenuated lenalidomide killing of HMCL MM1.S. These results provide the first evidence that MEIS2 is aberrantly expressed in primary myeloma cells and acts within myeloma cells to inhibit IMiD killing.To investigate if Dex enhances the myeloma intrinsic response to IMiDs, we first determined that lenalidomide and pomolidomide induced late G1 arrest within 24 hours, as evidenced by the repression of cyclin A, the increases in p21 and p27 proteins, and the reduction of myeloma cells in S phase by BrdU labeling. Kinetic studies further indicate that induction of G1 arrest precedes apoptosis, detectable at 72 hours in MM1.S and CAG cells. These data suggest that IMiDs preferentially kill myeloma cells arrested in G1. Dex killed CAG and MM1.S cells marginally at 10 nM and killing was not greater at 100 nM. However, concurrent addition of Dex enhanced lenalidomide killing, more prominently in CAG cells then in MM1.S cells. Addition of Dex at these concentrations to CAG cells after 48 hours of exposure to lenalidomide leads to synergistic and greater killing in 24 hours in a dose-dependent manner. These data indicate that Dex enhances lenalidomide killing, preferentially in G1 arrest.To investigate the mechanism by which Dex enhances lenalidomide killing, we found that Dex did not regulate MEIS2 or CRBN protein expression in CAG cells, in line with its inability to significantly kill myeloma cells. Sequential addition of lenalidomide and Dex led to a > 2-fold reduction in the MEIS2 protein, which corresponds to a > 2-fold reduction in the MEIS2/CRBN ratio and synergistic killing. Concurrent addition of Dex and lenalidomide also resulted in a 2-fold reduction of the MEIS2 protein and the MEIS2/CRBN ratio, but it required 72 hours of the presence of Dex instead of 24 hours.In summary, our study demonstrates, for the first time, that MEIS2 is expressed in primary myeloma cells and inhibits IMiDs killing. Dex enhances IMiD killing of myeloma cells through cooperative suppression of MEIS2 at proximal IMiD signaling. These findings suggest a novel mechanism by which Dex enhances the anti-myeloma activity of IMiD, which has important implication for myeloma therapy. DisclosuresRossi:Celgene: Consultancy, Speakers Bureau; Onyx: Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Janssen: Speakers Bureau. Niesvizky:Celgene: Consultancy, Research Funding, Speakers Bureau; Onyx: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Research Funding, Speakers Bureau.

RAS Association Domain Family Member 4 (RASSF4): A New Potent Tumor Suppressor in Multiple Myeloma

Multiple myeloma (MM) is a genetic highly heterogeneous plasma cell malignancy. In MM, the RAS/MAPK pathway is the most frequently mutated pathway, leading to aberrant MEK/ERK and PI3K/Akt signaling and thus stimulating MM cell growth and survival. Increasing evidence indicates that next to their classical oncogenic effects, RAS proteins can also induce growth inhibitory effects and apoptosis. These tumor suppressive effects are mainly mediated through the Ras-Association Domain Family (RASSF), a group of 10 RAS effector proteins (RASSF1-10) and numerous isoforms. Based on the localization of the Ras-association (RA) domain this family is further subdivided into two groups. RASSF1 to 6 harbor a C-terminal RA domain and are termed the classical group, while RASSF7-10 contain a N-terminal RA domain and are referred to as non-classical RASSF proteins. The classical RASSF members also carry a SARAH domain on the C-terminus, allowing the RASSF proteins to bind each other and the mammalian sterile 20-like kinases MST and MST-2. Currently, no data about the biological role of RASSF proteins in the pathogenesis of MM is available.Here, we first investigated the expression of the classical RASSF proteins RASSF1-6 in MM using publically available gene expression profiling data of 2 independent newly diagnosed patient cohorts (namely the Heidelberg-Montpellier and the UAMS-TT2 cohort) and 1 cohort of relapsed patients (Mulligan-cohort). We report that RASSF4 was the only classical RASSF protein that was consistently found downregulated in MM cells compared to normal bone marrow plasma cells, correlating with a bad prognosis in all cohorts. Treatment with epigenetic modulating agents including histone deacetylase inhibitors (HDACi) and DNA methyltransferases inhibitors significantly increased RASSF4 expression both in vitro and in vivo, indicating that RASSF4 downregulation in MM is due to epigenetic silencing. Forced RASSF4 expression induced a strong G2-phase arrest and caspase-3 mediated apoptosis in human MM cell lines and strongly reduced the viability of primary CD138+ MM cells. Moreover, RASSF overexpression significantly reduced in vivotumor growth. In addition, we showed RASSF4-MST1 binding and the activation of the downstream signaling pathways JNK/Jun, p38 and p53. RNA sequencing and gene set enrichment analysis following forced RASSF4 expression furthermore revealed downregulation of genes involved in protein metabolism, the unfolded protein response and translation. RASSF4 overexpression also sensitized MM cells to the proteasome inhibitor bortezomib, the specific MEK1/2 inhibitor trametinib and the ROS inducer Prima-1Met. Consequently, combining trametinib with HDACi, e.g. panobinostat, resulted in very strong synergistic anti-MM effects.In conclusion, we identified RASSF4 as a new potent tumor suppressor in MM that is epigenetically silenced in malignant plasma cells and provide a rationale for testing novel therapeutic strategies enhancing RASSF4 expression (using HDACi or gene therapy) in combination with MEK/ERK inhibitors and bortezomib. DisclosuresHose:Takeda: Other: Travel grant; EngMab: Research Funding; Sanofi: Research Funding.

A Small Molecule That Selectively Targets BLM Helicase Has a Therapeutic Interest in Multiple Myeloma

Hematological malignancies, including MM, are characterized by genomic instability that is possibly related to underlying defects in DNA repair or genomic stability maintenance. RECQ helicases are at the crossroad between replication, recombination, DNA repair and transcription and could represent potent therapeutic targets for cancer therapy.The Bloom’s syndrome helicase, BLM, has DNA annealing and unwinding activities. Through its interaction with TOPOIIIα, BLM unwinds the short stretches of naked duplex DNA and processes homologous recombination (HR) intermediates containing a double holiday junction. It may suppress hyper-sister chromatid exchange (SCE) by disruption of D-loop recombination intermediates and also might be involved in the suppression of crossing over during homology mediated recombination. This helicase facilitates telomere replication by resolving G4 structures. BLM deregulation is also associated with cancer.We recently identified a significant overexpression of BLM in malignant plasma cells compared to normal bone marrow plasma cells. Since BLM, plays important roles in the maintenance of chromosomal stability, we investigated if BLM could play a role in MM pathophysiology and drug resistance.A high BLM expression in MMCs could predict for shorter overall survival (OS) in four independent cohorts of patients (P = 0.003 in the HM cohort (N=206), P = 0.0002 in the UAMS-TT2 cohort (N=345), P = 0.0008 in TT3 cohort (N=186)) and P = 0.04 in Hovon cohort (N=282). A high BLM expression in MMCs could also predict for shorter event free (EFS) survival in the HM and UAMS-TT2 cohorts. BLM expression was significantly higher in the poor prognosis proliferation MM subgroup (P < 0.05). Moreover, we found that BLM was overexpressed in HMCLs (median 1848, range 246 - 10901) compared to primary MMCs or BMPCs (P< .001).The small molecule, ML216, is a membrane permeable selective inhibitor of BLM helicase. ML216 appears to act at the BLM-nucleic acid substrate binding site, inhibiting DNA binding and blocking BLM's helicase activity.We investigated the interest of the BLM inhibitor, ML216, to eradicate MM cells. The effect of ML216 was tested using 10 different human myeloma cell lines (HMCL). ML216 induced a dose dependent inhibition of cell growth in all investigated HMCL with a median IC50 of 4.1 μM (range: 1.2 - 30 μM). Moreover, our tests on HMCL showed that the BLM inhibitor synergizes with Melphalan and Bortezomib, two drugs currently used in myeloma therapy. Furthermore, ML216 induced a significant apoptosis of primary myeloma cells of patients co-cultured with their bone marrow environment and recombinant IL-6 (n=7). BLM inhibitor significantly reduced the median number of viable myeloma cells by 54%, 60% and 74% at respectively 3, 6 and 10 μM (P = 0.001, P < 0.001 and P<0.001 respectively; n=7). Of interest, the normal non myeloma cells present in the culture were significantly less affected by ML216 when used at 3 and 6 μM. These data demonstrated a significant higher toxicity of ML216 on myeloma cells compared to normal bone marrow cells.Taken together, our results underline the therapeutic interest of BLM inhibitor in MM and especially in patients characterized by high BLM expression and a poor prognosis. DisclosuresNo relevant conflicts of interest to declare.

Arginine Methylation Mediated By PRMT5 Has Significant Biological and Clinical Impact in Multiple Myeloma

Arginine-specific methyltransferases critically regulate cellular homeostasis by dictating the biological outcome of target proteins. Among them, Protein Arginine Methyltransferase 5 (PRMT5) has attracted growing interest due to its role as an enzyme mediating epigenetic regulation of anti-cancer target genes, as well as in methylation of non-histone proteins involved in growth-regulating and survival pathways including p53. However, little is known about its biologic function in multiple myeloma (MM).To first evaluate the clinical significance of PRMT5 in MM pathogenesis, we analyzed RNA-seq data from newly-diagnosed MM patients and identified highly upregulated PRMT5 in 320 patients' CD138+ cells compared to 16 samples of normal bone marrow (BM) plasma cells. Additional analysis of PRMT5 expression in two independent datasets also showed further PRMT5 mRNA upregulation during progression of MM. Immunohistochemical staining also confirmed elevated expression of PRMT5 in BM biopsies from MM patients as compared to healthy individuals and monoclonal gammopathy of undetermined significance (MGUS). Moreover, analysis of the prognostic significance of PRMT5 expression in MM patients enrolled on IFM/DFCI 2009 clinical study showed that high PRMT5 expression was associated with poor prognosis in terms of both event free (p= 0.016) and overall survival (p=0.018). Consistently, we also found upregulated PRMT5 expression at both mRNA and protein levels in MM cell lines (N=11) and patients CD138+ MM cells (N=3) as compared to PBMCs from healthy volunteers, associated with a parallel increase of cellular symmetric arginine di-methylation (SDMA) substrates. Interestingly, genetic depletion of PRMT5 in H929 (p53wt) and KMS11 (p53null) MM cell lines by shRNA decreased SDMA levels, associated with cell growth inhibition in a p53-independent manner. Likewise, pharmacological inhibition of PRMT5 with the small molecule inhibitor EPZ015666 triggered decreased SDMA levels, cell growth, survival, and clonogenicity, as well as induction of caspase-dependent apoptosis in MM cell lines. Moreover, although PRMT5 and SDMA levels were increased in MM cells cultured in the presence of BM stromal cell supernatant, cytotoxic activity of EPZ015666 was maintained. Notably, drug treatment significantly impaired cell proliferation of patient MM cells (n=2) even in the presence of BM mononuclear or stromal cells, without toxicity on normal PBMCs. At the level of gene expression modulation, PRMT5 inhibition was associated with downregulation of NF-kB-dependent transcription, evidenced by both gene set enrichment analysis (GSEA) and Ingenuity Upstream Regulator Analysis. Moreover, analysis of protein levels confirmed reduction of both canonical and non-canonical NF-kB pathways, evidenced by significantly decreased NF-kB DNA binding activity by ELISA. Importantly, Mass Spectrometry analysis identified TRIM21 as a new PRMT5 interactor; and EPZ015666-treated cells showed that PRMT5 methylates TRIM21 evidenced by WB analysis. Since TRIM21 mediates monoubiquitination of IKKbeta, thereby triggering its selective autophagy-mediated degradation, we next analyzed EPZ015666 effects on IKKbeta. Treatment increased both monoubiquitination of IKKbeta and the formation of IKKbeta-TRIM21-pBECLIN1-pULK1 autophagic complexes. Conversely, inhibition of autophagosome formation by 3-methyladenine abrogated the anti-MM activity of EPZ015666 and IKKbeta degradation, indicating that selective autophagic degradation of IKKbeta and inhibition of NF-kB signaling mediates EPZ015666-triggered anti-MM activity. Consistent with this view, confocal microscopy analysis also confirmed co-localization of IKKbeta in the autophagosome after EPZ015666 treatment. Finally, stable silencing of TRIM21 in MM cell lines significantly abrogated the anti-proliferative effect of EPZ015666. Collectively, these data delineate arginine methylation as a new control mechanism of MM cell growth, and demonstrate that inhibiting PRMT5 decreases tumor cell survival via blockade of NF-kB signaling, even in the context of the BM milieu. These data demonstrate the biologic and prognostic significance of PRMT5 in MM pathogenesis, and provide the rationale for novel therapies targeting PRMT5 to improve patient outcome in MM. DisclosuresHideshima:Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Munshi:Takeda: Consultancy; Celgene Corporation: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Consultancy, Equity Ownership. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: 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.