ALBERT

Description: Multiple myeloma (MM) is a plasma cell dyscrasia characterized by bone marrow infiltration of clonal plasma cells. The recent literature has clearly demonstrated clonal heterogeneity in terms of both the genomic and transcriptomic signature of the tumor. Of note, novel studies have also highlighted the importance of the functional cross-talk between the tumor clone and the surrounding bone marrow milieu, as a relevant player of MM pathogenesis. These findings have certainly enhanced our understanding of the underlying mechanisms supporting MM pathogenesis and disease progression. Within the specific field of small non-coding RNA-research, recent studies have provided evidence for considering microRNAs as a crucial regulator of MM biology and, in this context, circulating microRNAs have been shown to potentially contribute to prognostic stratification of MM patients. The present review will summarize the most recent studies within the specific topic of microRNAs and circulating microRNAs in MM.

Description: MicroRNAs (miRNAs), short non-coding RNAs which tune gene expression at post-transcriptional level, are recently emerging as key players in pathogenesis, progression and drug-resistance of multiple myeloma (MM). In this disease, they can act either with tumor-promoting or tumor-suppressing functions, depending on the nature of target mRNAs. Nowadays, effective strategies are available both to replace or to inhibit the expression of deregulated miRNAs, thus prompting the design of miRNA-based therapeutic strategies. We have recently demonstrated that miR-125b has tumor suppressor activity in MM and that enforced expression of synthetic miR-125b-5p mimics induces significant anti-MM activity in vitro and in vivo by targeting cell addiction to IRF4/cMyc pro-survival signaling. Moreover, we uncovered a functional feedback loop between cMyc and miR-125b in MM cells, whereas cMyc directly suppresses miR-125b transcription which, in turn, negatively regulates cMyc expression by targeting IRF4 mRNA. In the present study, we investigated the therapeutic potential of synthetic miR-125b-5p mimics combined with cMyc targeting agents, including the 10058-F4 small molecule inhibitor of cMyc-Max heterodimerization and the BET-bromodomain inhibitor JQ1, which is reported to inhibit cMyc transcription. At this aim, 3 MM cell lines (NCI-H929, SK-MM-1 and RPMI-8226) transfected with either miR-125b-5p mimics or scrambled oligonucleotides (miR-NC) were exposed to 10058-F4 (ranging from 10 to 100 μM) or JQ1 (ranging from 0,1 to 2μM) or DMSO. Effects on cell proliferation were then evaluated by CCK-8 assay at 24h, 48h and 72h time points, while the occurrence of apoptotic cell death was assessed by Annexin V flow-cytometry assay. Importantly, we found that enforced expression of miR-125b-5p mimics significantly and synergistically (synergistic index, SI 〉1) increases growth-inhibitory and pro-apoptotic activities of both 10058-F4 and JQ1. Similar results were observed in SK-MM-1 cells co-transfected with miR-125b-5p and cMyc siRNAs, while cMyc-defective U266 cells were not sensitized to either 10058-F4 nor JQ1 upon transfection with miR-125b-5p mimics. Furthermore, combinatorial treatments with JQ1 and miR-125b-5p mimics resulted in a stronger downregulation of cMyc protein, as compared to single molecules alone. Indeed, these results confirmed that impairment of cMyc activity/expression mediates the anti-MM synergistic effects between 10058-F4 or JQ1 and overexpression of miR-125b-5p by synthetic mimics. In conclusion, our data demonstrate a cMyc-mediated synergistic anti-MM activity of synthetic miR-125b-5p mimics with 10058-F4 or JQ1 cMyc targeting agents, providing the rationale for a more advanced preclinical investigations for the design of early clinical trials. Disclosures No relevant conflicts of interest to declare.

Description: Although estrogens have been shown to regulate normal B cell proliferation and differentiation, the impact of estrogen receptor (ER) signaling on B cell malignancies, including Waldenström Macroglobulinemia (WM), remains unexplored. To address this issue, we have analyzed, through preclinical WM models, the druggability either of the classical estrogen receptor ERβ, an emerging target for acute leukemias and certain types of lymphomas, and of the novel, more recently deorphanized G-protein coupled ER GPER, whose therapeutic potential in solid tumors has recently emerged. Both ERβ and GPER were found expressed at mRNA and protein level in a panel of WM and IgM-secreting lymphoma cell lines. However, while treatment with ERβ agonists did not elicit any effect, selective GPER activation significantly reduced WM cell viability. On this basis, we investigated GPER expression pattern and therapeutic potential in WM. By interrogating public microarray datasets, we found GPER transcript significantly upregulated in WM as compared to normal B cells; moreover, immunohistochemical analysis showed elevated GPER expression in lymph node biopsies from newly-diagnosed WM patients as compared to healthy lymph nodes. Treatment with the selective GPER agonist G-1 ( (±) -1-[(3aR*,4S*,9bS*)-4-(6-Bromo-1,3-benzodioxol-5-yl) -3a,4,5,9b-tetrahydro-3H cyclopenta [c]quinolin-8-yl] ethanone) led to reduced viability, clonogenicity and migration of WM and IgM-secreting lymphoma cell lines (IC50 at 48h ranging between 2.5 and 5.0 mM), including MYD88 and CXCR4 mutated cell lines, and even in the presence of bone marrow-derived stromal cells, while it did not affect healthy CD19+ B cell viability; conversely, GPER antagonists G-36 and G-15 slightly enhanced cell proliferation. The growth inhibitory activity of G-1 was associated with accumulation in G2/M cell cycle phase and induction of apoptosis, the latter phenomenon assessed by Annexin V/7AAD staining, analysis of mitochondrial membrane depolarization and cleavage of caspase 3, 7 and 9; hybridization of an antibody-array also highlighted G-1-induced down-regulation of anti-apoptotic proteins, including survivin and Bcl2. A significant effect of G-1 was also observed in primary tumor cells from refractory WM patients. Moreover, G-1 synergistically enhanced bortezomib and ibrutinib cytotoxicity in vitro. Importantly, intraperitoneal injection of G-1 (2mg/kg) significantly reduced the growth of BCWM-1 xenografts in NOD/SCID mice. To gain further insight into the consequences of selective GPER activation, we performed gene expression profile and GSEA analysis of WM cell lines treated with G-1. Of note, the p53 signaling pathway was strongly induced upon GPER activation, and upregulation of p53 and of its target genes or microRNAs (p21CIP1, Bax, Bad, PUMA and miR-34a) was confirmed both in G-1 treated WM cell lines and primary WM cells; moreover, G-1 combination with bortezomib or ibrutinib led to stronger increase in p53 and p21CIP1 levels as compared to single agent treatment. Finally, p53 knock-down partially reversed G-1-dependent anti-WM effects, thus suggesting p53 as downstream mediator of GPER. In summary, we report a significant anti-tumor activity upon selective GPER activation in WM cells, with apoptosis induction and a potent synergistic anti-WM activity in combination with ibrutinib and bortezomib. Altogether, these preclinical data suggest GPER agonists as a potential therapeutic option in WM. Disclosures Munshi: OncoPep: Other: Board of director.

Description: The human fibroblast growth factor receptor (FGF-R) family plays an essential role in a wide range of cellular processes, such as cell growth, proliferation, differentiation, migration and survival. It has been reported that FGF-Rs are expressed in hematopoietic cells; and FGF/FGFR signaling deregulation is largely involved in hematologic malignancies, including Waldenström macroglobulinemia (WM). WM is still an incurable disease, and patients succumb due to disease progression. Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We aimed to investigate the role of FGF/FGF-R system in FGF-dependent WM cell lines by using an anti-pan FGF trap molecule (NSC12), responsible for FGF/FGF-R blocking. We first interrogated the GSE9656 dataset in order to confirm the expression of FGFs and FGF-Rs in WM cells, demonstrating an enrichment of several FGF- and FGF-R-isoforms in primary WM patients' derived tumor cells compared to the normal cellular counterpart (P

Description: Besides the well described function of RNA to produce proteins, a large volume of transcribed product has non-coding function. A recent analysis of RNA repertoire has identified a family of non-coding transcripts with sequence longer than 200 nucleotides, the long intergenic non-coding RNAs (lincRNAs). Although lincRNAs have been considered to provide regulatory functions, their precise role in cellular biology remains unclear. Using our RNA-seq data from 360 newly-diagnosed patients and 18 normal plasma cells, we have recently described the landscape of lincRNAs in multiple myeloma (MM) and reported their role as independent risk predictors for survival outcome. We have now studied the functional role of a lincRNA, the miR-17-92 primary precursor linc-MIR17HG, present in our lincRNA profile and highly correlated with overall survival in MM. We observe that inhibition of linc-MIR17HG by antisense LNA GapmeRs (n=2) leads to apoptosis in 12 genotypically distinct MM cell lines as well as in 13 primary patient MM cells. These effects are not fully rescued by expression of miR-17-92 microRNAs, suggesting a distinct biological function for linc-MIR17HG in MM. We therefore performed gene expression profile in 2 MM cell lines (AMO1 and NCI-H929) and in 2 primary patient MM cells after short-term suppression of linc-MIR17HG; and, at these early time points (18-36h), we found significant downregulation of a subset of genes (FC〉2; p0.4; p

Description: Long noncoding RNAs (lncRNA) are major regulators of chromatin dynamics and gene expression. We have recently performed deep RNA sequencing of CD138+ cells from 360 uniformly-treated, newly-diagnosed multiple myeloma (MM) patients (IFM/DFCI 2009) and described the lncRNA landscape and their role as independent risk predictors for clinical outcome in MM. Moreover, we have identified one of these lncRNAs - lnc-17-92 - as an independent risk predictor highly correlating with EFS and OS in newly-diagnosed MM providing rationale to define its molecular role in MM. Lnc-17-92 is generated at MIR17HG gene locus and is known for being involved in the biogenesis of miR-17-92 cluster of microRNAs. We here establish, for the first time, role of this transcript as a lncRNA with microRNA-independent function and molecular and biological implications in MM. Having confirmed its expression in MM cell lines and primary MM cells, we have utilized antisense oligonucleotides (n=3) to suppress lnc-17-92 expression in large panel of human MM cell lines (HMCLs) (n=12) and primary patient MM cells (n=13). Lnc-17-92 inhibition impaired MM cell proliferation leading to apoptotic cell death. This inhibitory effect was not rescued by ectopic expression of miR-17-92 microRNAs, confirming independent activity of lnc-17-92 on MM cell growth and viability. The microRNA-independent role of lnc-17-92 in transcriptional control was further confirmed using DROSHAKOcells. Analysis of transcriptomic changes after lnc-17-92 modulation in HMCLs and primary MM cells identified bona fide transcriptional targets of lnc-17-92. Using two independent MM RNA-seq datasets, we observed high correlation (R〉 0.4) between lnc-17-92 expression and the expression of 12 of the transcriptional targets identified above. Interestingly, these genes were significantly enriched within metabolic pathways, suggesting an unexplored role for lnc-17-92 in MM cell metabolism. Further analysis using an RNAi-based loss-of-function screening in 3 HMCLs revealed Acetyl-CoA Carboxylase Alpha (ACC1) as a novel myeloma vulnerability. ACC1 encodes the limiting enzyme in the de novo lipogenesis pathway. Analysis of incorporation of C14-radiolabeled glucose into lipids in MM cells revealed that inhibition of ACC1 or lnc-17-92 strongly inhibited de novo lipogenesis in HMCLs and in primary MM cells. We have used ACC1 conditional KD MM cells expressing IPTG-inducible ACC1 shRNAs and confirmed significant role of ACC1 in MM cell growth and survival, both in vitro and in vivo in SCID mice model. Importantly, supplementation of palmitate, the main downstream product of ACC1 activity, significantly reverses the growth inhibitory effect of either ACC1 or lnc-17-92 suppression in MM cells. These data suggest an important role for lipogenesis pathway on lnc-17-92-promoted MM cell growth. We have further investigated mechanism by which lnc-17-92 may exert its transcriptional control. Protein-RNA pulldown assay established MYC as interacting partner of lnc-17-92. This interaction was confirmed by immunoprecipitation of MYC-bound RNA followed by qRT-PCR with specific primers for detection of lnc-17-92. ChIP-seq analysis revealed a direct binding of MYC at regulatory regions of ACC1 in MM.1S cells; these data were corroborated by the decreased ACC1 expression observed in MYC KD MM cells. Taken together, these data suggest that lnc-17-92 may function as a scaffold between MYC and the E-box motifs present on ACC1 intronic sequences, facilitating MYC binding and its transcriptional activity on ACC1. Finally, for translational application, we have pre-clinically investigated ND-646, a clinically applicable small molecule inhibitor of ACC1. Analysis of incorporation of C14-radiolabeled glucose into lipids confirmed its effect on lipogenesis in MM, which was associated with a significant in vitro growth inhibitory activity in large panel of HMCLs and primary patient MM cells. In vivo studies in murine model of human MM, using this oral agent, are ongoing and will be presented. In conclusion, we here report for the first time the microRNA-independent role of lnc-17-92 in MM pathobiology with direct impact on transcriptional control of lipogenesis. The availability of oral inhibitor of this pathway may allow the clinical application of this unique targeted therapy in MM. Disclosures Anderson: Janssen: Other: Advisory Board; Gilead Sciences: Other: Advisory Board; OncoPep: Other: Scientific founder ; Sanofi-Aventis: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder . Munshi:Abbvie: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; Oncopep: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Abbvie: Consultancy.

Description: Proteasome inhibitors (PIs) have improved the treatment of multiple myeloma (MM) and prolonged patient survival, but several challenges remain to overcome drug-resistance and toxicity. Bone marrow microenvironment (BMM) drives tumor progression and PIs-resistance in MM; and agents that inhibit the interaction between MM and BMM have been shown to re-sensitize MM cells to therapy. However, the synchronized in vivo delivery of BMM-targeting agents with PIs has been a challenge so far. Nanoparticles offer a valuable platform to encapsulate drugs, and if functionalized, they can facilitate specific delivery to tumor, thus improving treatment efficacy and reducing off-target effects. Within the BMM, the endothelium plays a relevant tumor promoting role. By analyzing the expression of an array of markers in normal and in MM-related endothelium, we found high levels of P-selectin expression on MM-activated endothelial cells (ECs) than normal cells and on ECs collected from the BM of either MM patients or MM-bearing mice compared to their respectively healthy BMMNCs. We next sought to develop lipid nanoparticles (LNPs) targeting the MM-related endothelium, loaded with both PI and BMM-targeting agent for synchronized delivery and reversal of the BMM-induced drug resistance. At this aim, we developed targeted LNPs towards P-selectin by decorating their surface with P-selectin-glycoprotein-ligand-1 (PSGL-1). PSGL-1-targeted LNPs showed specific binding to recombinant P-selectin than identically non-targeted particles, and to MM-associated endothelium compared to healthy endothelium, both in vitro and in vivo. To reverse BMM-induced resistance, LNPs were loaded with bortezomib (BTZ) together with a BMM disrupting agent, ROCK-inhibitor (Y-27632) that inhibits the downstream signaling of the RhoA GTPase pathway, known to be instrumental to the interaction of MM cells with BMM. Consequently, we tested the effect of synchronized delivery of BTZ and Y-27632 in the same LNP on MM cell survival in co-culture with the BMM in vitro. While Y-27632-loaded LNPs did not affect cell proliferation, LNPs loaded with both Y-27632 and BTZ enhanced responsiveness of MM cells to BTZ, compared to BTZ-loaded LNPs, thus overcoming the BMM-induced resistance. Mechanistically, we observed more significant inhibition of PI3K and MAPK signaling, decrease of pRb and up-regulation of p21 and induction of pro-apoptotic pathway (caspase-3, caspase-9 and PARP) by drug-loaded LNPs, compared to free drugs. In addition, drug-loaded LNPs were able to decrease adhesion and impair the migration of MM cells to ECs. We also investigated the in vivo efficacy of BTZ/Y-27632-loaded PSGL-1-targeted LNPs in a humanized murine model of MM. The synchronized delivery of both agents using dual drug-loaded PSGL-1-targeted LNPs delayed the MM tumor progression and prolonged survival significantly more than all the controls. The synchronized delivery of both agents using dual drug-loaded PSGL-1-targeted LNPs delayed the MM tumor progression and prolonged survival significantly more than all the controls (vehicle, BTZ and Y-27632 alone or in combination as free drugs, or encapsulated in non-targeted or in PSGL-1-targeted LNPs) demonstrating that both P-selectin targeting and combination of Y-27632 with BTZ reverses the BMM-induced drug resistance and enhances the efficacy of therapy in vivo. Altogether, our data demonstrate the ability of PSGL-1-decorated LNPs to specifically target MM-BMM; to efficiently encapsulate and deliver drugs to tumor tissue; to overcome BMM-induced drug resistance in vitro and in vivo, to reduce tumor growth and prolong overall survival. This study provides the preclinical basis for future clinical trials using MM-BMM-targeted nanomedicine able to enhance the effect of PIs or other drugs for the treatment of MM. Disclosures Roccaro: GILEAD: Research Funding; AMGEN: Other: Advisory Board. Vij:Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Azab:Cellatrix LLC: Equity Ownership, Other: Founder and owner; Targeted Therapeutics LLC: Equity Ownership, Other: Founder and owner; Ach Oncology: Research Funding; Glycomimetics: Research Funding.