Abstract: Multiple myeloma is a biologically heterogenous plasma-cell disorder. In this study, we aimed at dissecting the functional impact on transcriptome of gene mutations, copy-number abnormalities (CNA), and chromosomal rearrangements (CR). Moreover, we applied a geno-transcriptomic approach to identify specific biomarkers for personalized treatments. Experimental Design: We analyzed 514 newly diagnosed patients from the IA12 release of the CoMMpass study, accounting for mutations in multiple myeloma driver genes, structural variants, copy-number segments, and raw-transcript counts. We performed an in silico drug sensitivity screen (DSS), interrogating the Cancer Dependency Map (DepMap) dataset after anchoring cell lines to primary tumor samples using the Celligner algorithm. Results: Immunoglobulin translocations, hyperdiploidy and chr(1q)gain/amps were associated with the highest number of deregulated genes. Other CNAs and specific gene mutations had a lower but very distinct impact affecting specific pathways. Many recurrent genes showed a hotspot (HS)-specific effect. The clinical relevance of double-hit multiple myeloma found strong biological bases in our analysis. Biallelic deletions of tumor suppressors and chr(1q)-amplifications showed the greatest impact on gene expression, deregulating pathways related to cell cycle, proliferation, and expression of immunotherapy targets. Moreover, our in silico DSS showed that not only t(11;14) but also chr(1q)gain/amps and CYLD inactivation predicted differential expression of transcripts of the BCL2 axis and response to venetoclax. Conclusions: The multiple myeloma genomic architecture and transcriptome have a strict connection, led by CNAs and CRs. Gene mutations impacted especially with HS-mutations of oncogenes and biallelic tumor suppressor gene inactivation. Finally, a comprehensive geno-transcriptomic analysis allows the identification of specific deregulated pathways and candidate biomarkers for personalized treatments in multiple myeloma.

Abstract: Long noncoding RNA (lncRNAs) are key epigenetic factors that drive the origin and progression of human cancers via mechanisms that are largely unknown. We have previuosly reported the clinical significance of a lncRNA signature in multiple myeloma (MM) as independent risk predictor for clinical outcome; and recently identified a lncRNA RROL (RNA Regulator of Lipogenesis) with impact on MM cell proliferation. Here we describe a unique regulatory function for RROL in the control of gene networks involved in the de novo lipogenesis (DNL) pathway, ultimately impacting MM cell growth and survival. Based on growth and survival impact of RROL depletion, we performed integrated transcriptomic analysis of RNA-seq data after RROL depletion in MM cell lines and CD138+ patient MM cells, and identified a set of significantly modulated metabolic genes including the acetyl Co-A Carboxylase 1 (ACC1) gene, encoding a rate-limiting enzyme of the DNL pathway. This metabolic pathway converts nutrients into fatty acids serving for energy storage or biosynthesis of membranes and signaling molecules. Consistent with the transcriptional control of ACC1, we have observed that RROL inhibition in cell lines and primary MM cells significantly decreased the incorporation of C14-radiolabeled glucose into de novo synthesized lipids. Importantly, supplementation with exogenous palmitate, the main downstream product of DNL pathway, rescued the growth inhibitory effect of RROL depletion on MM cells, further confirming the importance of the DNL pathway in the oncogenic activity of RROL in MM. To understand the molecular mechanism through which RROL regulates ACC1 expression and its metabolic axis, we evaluated the RROL interactome in MM cells. RNA-Protein Pull Down (RPPD) and in vivo RNA yeast three-hybrid (Y3H) assays led to the identification of MYC as relevant direct partner of RROL. These results were further validated by qRT-PCR analysis of MYC-bound RNA obtained through RNA immunoprecipitation (RIP) assay. Using experimental model of conditional MYC KD (P493-6), we found that RROL exerts regulatory activity on ACC1 only in the presence of MYC. Mapping of MYC genomic occupancy by ChIP-seq and gene expression after MYC KD in MM cells revealed that ACC1 is a direct transcriptional target of MYC in cells expressing RROL. These data indicate that RROLand MYC cooperate in the transcriptional control of ACC1. Moreover, we found that RROL itself is transcriptionally regulated by MYC, suggesting the existence of a feed-forward regulatory loop in which MYC enhances the expression of RROL that, in turn, drives MYC transcriptional activity to ACC1. We hypothesized that RROL may shape the protein interacting network of MYC to confer specificity for ACC1 promoter. To this end, we performed mass spectrometry analysis of MYC interactome in three MM cell lines in the presence or in the absence of RROL and identified the transcriptional modulator WDR82 as RROL-dependent MYC partner. Interestingly, WDR82 directly interacts with RROL as assessed in the RPPD and RNA Y3H assays, and transcriptionally regulates ACC1 in RROL-dependent manner. These data indicate that RROL catalyzes the interaction of MYC with the transcriptional modulator WDR82 to form a transcriptional ternary complex regulating ACC1 expression. To therapeutically antagonize the RROL lipogenic signaling we have pre-clinically tested small molecule inhibitors of ACC1 (ACC1i). We have observed significant anti-MM activity of ACC1i in vitro in a large panel of MM cell lines and primary MM cells from patients; and in vivo in mouse models of human MM including the localized subcutaneous model and the disseminated model that establish a more aggressive systemic disease. Importantly, we have now developed clinically applicable ASOs and small molecule-like compounds to directly target RROL in MM cells. These studies are ongoing and will be presented. In conclusion, we here report a unique regulatory function of a novel lncRNA supporting MM cell growth via its control of the lipogenic metabolic axis. The availability of oral inhibitors of ACC1 as well as the ongoing development of RROL inhibitors may allow clinical application of this unique targeted therapy in MM. Disclosures Fulciniti: NIH: Research Funding. Chauhan:Oncopeptide AB: Consultancy; consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy.

Abstract: Recent investigations have improved our understanding of the molecular aberrations supporting Waldenström Macroglobulinemia (WM) biology; however, whether the immune microenvironment contributes to WM pathogenesis remains unanswered. We first showed how a transgenic murine model of human-like lymphoplasmacytic lymphoma/WM exhibits an increased number of regulatory T (Treg) cells with respect to control mice. These findings were translated into the WM clinical setting, where the transcriptomic profiling of WM patients'-derived regulatory T cells (Tregs) unveiled a peculiar WM-devoted mRNA signature, with significant enrichment for NF-kB-mediated TNF-a signaling-, MAPK-, PI3K/AKT-related genes; paralleled by different Treg functional phenotype. We demonstrated a significantly higher Treg-induction, -expansion and -proliferation triggered by WM cells as compared to their normal cellular counterpart; with a more profound effect within the context of CXCR4C1013G-mutated WM cells. By investigating the B-to-T cell cross-talk at single-cell level, we identified the CD40/CD40-ligand as a potentially relevant axis supporting WM cell-Treg cell interaction. Our findings demonstrate the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.

Abstract: The human fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) axis deregulation is largely involved in supporting the pathogenesis of hematologic malignancies, including Waldenström macroglobulinemia (WM). WM is still an incurable disease, and patients succumb because of disease progression. Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We aimed to investigate the role of FGF/FGFR system blockade in WM by using a pan-FGF trap molecule (NSC12). Wide-transcriptome profiling confirmed inhibition of FGFR signaling in NSC12-treated WM cells; unveiling a significant inhibition of MYD88 was also confirmed at the protein level. Importantly, the NSC12-dependent silencing of MYD88 was functionally active, as it led to inhibition of MYD88-driven pathways, such as BTK and SYK, as well as the MYD88-downstream target HCK. Of note, both canonical and noncanonical NF-κB cascades were downregulated in WM cells upon NSC12 treatment. Functional sequelae exerted by NSC12 in WM cells were studied, demonstrating significant inhibition of WM cell growth, induction of WM cell apoptosis, halting MAPK, JAK/STAT3, and PI3K-Akt pathways. Importantly, NSC12 exerted an anti-WM effect even in the presence of bone marrow microenvironment, both in vitro and in vivo. Our studies provide the evidence for using NSC12 as a specific FGF/FGFR system inhibitor, thus representing a novel therapeutic strategy in WM.

Abstract: The human fibroblast growth factor/fibroblast growth factor-receptor (FGF/FGFR) axis deregulation is largely involved in supporting the pathogenesis of hematologic malignancies, including Waldenstrom's Macroglobulinemia (WM). Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We investigated the role of FGF/FGFR system blockade in WM by using a pan-FGF trap molecule, NSC12, a small molecule identified using pharmacophore modeling of the interaction of a minimal PTX3-derived FGF-binding pentapeptide with FGF2. By interrogating the transcriptome signature of patients' BM-derived CD19-positive cells (GEO9656, GEO6691), we found a significant enrichment of FGF/FGFR-driven signaling cascades, including PI3K-AKT, MAPK and STAT3 pathways (FDR & lt;0.25; P & lt;0.05); coupled with higher expression of FGFs (FGF2, FGF7, FGF12, FGF18; P & lt;0.05) in WM cells as compared to their normal cellular counterpart. FGFRs are also shown to be overexpressed in WM. We performed transcriptome profiling of NSC12-treated WM cells, confirming the blockade of the FGFR-signaling blockade; and, importantly, we discovered the efficacious silencing of MYD88 and MYD88-dopwnstream target HCK in WM cells. These findings were confirmed at protein level, showing inhibition of MYD88-driven pathways, such as BTK-, and SYK-phosphorylation. As a further demonstration of the functional impact of NSC12-dependent targeting of MYD88, we could confirm the inhibition of both canonical and non-canonical NF-kB in NSC12-treated WM cells, as assessed at nuclear protein level by WB and by using the NFkB activity assay. In addition, the NSC12-dependent inhibition of MYD88 resulted in silencing of the MAPK-ERK signaling cascade, thus leading to NSC12-induced Myc-silencing in WM cells. We next confirmed the efficacy of NSC12 in silencing bone marrow stromal cell (BMSC)-induced FGFR3 phosphorylation; paralleled by inhibition of of pro-survival pathways, including pAKT, the AKT-downstream pGSK3β; p-ERK; and p-STAT3. Functional sequelae of the FGF/FGFR blockade in WM cells were studied, demonstrating inhibition of WM cell growth, induction of apoptosis, enhanced ER stress and initiation of UPR. Of note, anti-WM activity of NSC12 was also documented using primary bone marrow-derived CD19+ cells isolated from patients with WM. In contrast, NSC12 did not show cytotoxicity on PBMC-derived CD19+ cells isolated from healthy donors. The anti-WM activity exerted by NSC12 was confirmed also within the context of the supportive bone marrow milieu, as shown both in vitro and in vivo. BCWM.1, MWCL1 cells were cultured with NSC12 in the presence or absence of primary WM BMSCs: adherence of WM cells to BMSCs triggered a significant increase in the proliferation, which was inhibited by NSC12in a dose-dependent manner (P & lt;0.05). Using a disseminated humanized WM model, BCWM.1 M-Cherry-Luc tumor-bearing mice were treated with either NSC12 or vehicle control. NSC12-treated mice presented with a significant inhibition of WM tumor growth as demonstrated by bioluminescence imaging analysis. NSC12-dependent reduction of WM tumor cell BM infiltration was confirmed by IHC performed on harvested femurs, in comparison to vehicle-treated mice. Importantly, we assessed MYD88-mRNA expression using harvested bone marrow femurs; and found a significant reduction of MYD88- and MYD88-downstream target HCK in NSC12-treated mice was confirmed ex vivo (P & lt;0.05). Overall, our studies are reporting on the use of NSC12, as a novel potential therapeutic strategy to specifically halt the FGF/FGFR axis in WM; and demonstrate how the observed anti-WM activity exerted by NSC12 may be driven, at least in part, by inhibition of MYD88. Disclosures Motta: Roche: Honoraria; Janssen: Honoraria. Rossi:Alexion: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Advisory board; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Honoraria; Amgen: Honoraria; Sanofi: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Roccaro:Amgen: Other; AstraZeneca: Research Funding; Celgene: Other; Janssen: Other; Italian Association for Cancer Research (AIRC): Research Funding; Transcan2-ERANET: Research Funding; European Hematology Association: Research Funding.

Abstract: Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

Abstract: e16507 Background: The phase II BONSAI trial ( n = 25 ; NCT 03354884) met the primary endpoint demonstrating activity of cabozantinib in untreated metastatic collecting ducts carcinoma (mCDC), a rare and biologically poorly characterized disease. Here we report on molecular analyses of baseline tissue samples. Methods: Formalin-fixed paraffin-embedded (FFPE) samples from 18 mCDC patients enrolled in BONSAI were sequenced by TruSeq RNA Exome kit (Illumina). The data were mapped and quantified using STAR and htseq, respectively. Globaltest and edgeR packages in R were used to assess the correlation between transcriptional profiles and survival data. Nineteen samples underwent DNA Sequencing with the Oncomine Comprehensive Assay Plus (ThermoFisher Scientific). The reads were aligned to the human genome reference (hg19) and analyzed with Opencravat and IonReporter software. Germline variants were excluded based on 1000 Genomes, GnomAd and Exac databases, and Clinical annotation of somatic variants was performed using ClinVar. Results: The global expression levels of thirty-one genes have been found significantly associated with overall survival (OS). The natural grouping of the 18 tumor samples based on the 31-gene signature identified a main group of 11 cases, showing global higher expression levels in 22 out of the 31 genes. This group displayed overall a significant higher OS rate in comparison to the remaining 7 patients, carrying opposite expression trend and mostly undergoing to disease progression. The identified signature was enriched in biological processes like cell junction, cytoskeleton organization and methylation. FOS oncogene was among the 9 genes negatively associated to OS, showing common higher expression in the poorer survival rate group. Furthermore, a 22-gene signature was found significantly associated with progression-free survival (PFS), involving mainly genes associated to cell cycle regulation or recognized as components of Golgi apparatus. Only three genes were globally up-regulated in the group of 9 patients characterized by shorter PFS. Finally, heterogeneous pattern of somatic mutations was identified in 19 tumor samples, with at least 8 genes recurrently affected in more than three patients. Notably, mutated genes were mostly involved in DNA repair and chromatin modification processes. Conclusions: Our findings for the first time define specific molecular signatures that differentiate therapy-specific outcomes in first-line mCDC, warranting further investigation of their involvement in the tumor biology. Clinical trial information: NCT 03354884.

Abstract: Introduction Multiple Myeloma (MM) is characterized by hyperdiploidy (HD) or immunoglobulin gene (IgH) rearrangements as initiating events. Clonal heterogeneity is a hallmark of its biology as highlighted by Next Generation Sequencing. In this context, data on the impact of peculiar mutations, copy number aberrations (CNAs), and chromosomal rearrangements (CRs) at the transcriptomic level are still scanty. In this study, we aimed to dissect the transcriptional deregulation promoted by the most recurrent genomic drivers. Based on this geno-trascriptomic link, we also aimed to identify biomarkers that could suggest personalized treatments. Methods We analyzed 517 newly diagnosed patients from the IA12 release of the CoMMpass study, focusing on mutations in MM driver genes, structural variants, copy number segments and raw transcript counts. RNAseq data was processed with the VOOM/LIMMA pipeline. To perform an in-silico drug sensitivity screen, we anchored cell lines to patients samples using the Celligner algorithm and interrogated the DepMap dataset. Results We first analyzed the global impact of genetic aberrations on the transcriptome. Chr(1q)amp/gain, followed by IgH translocations and HD showed the highest number of deregulated transcripts. Individual mutations had much less impact, with the exception of NRAS and chr(13q) genes (DIS3, TGDS, RB1). Next, we investigated differential influence between hotspots (HS) vs nonHS mutations within driver genes. KRAS and NRAS, showed little changes between nonHS and wild type (WT), as the transcriptome was mostly impacted by HS mutations. IRF4 K123 showed a specific transcriptional profile, while nonHS mutations still carried functional relevance although on different genes. For BRAF, the kinase dead D594 mutation surprisingly impacted the most in comparison to V600 and WT cases. Next, we explored the effect of bi-allelic genetic events with known prognostic impact. TP53 double-hits were associated with an upregulation of PHF19, a MM poor prognostic marker, and downregulation of SLAMF7, a new immunotherapy target. CYLD and TRAF3 double-hits correlated with NF-κB pathway activation, and the former showed a significant BCL2 upregulation. Bi-allelic events on chr13 exhibited gene-specific consequences: DIS3 inactivation deregulated mostly lncRNAs, while TGDS impacted on genes involved in cell-cycle regulation. Regarding chromosomal gains, only chr(1q)amp ( & gt; 3 copies) showed a gene dosage effect with upregulation of the potential therapeutic targets MCL1 and SLAMF7. Given that the BCL2 axis was perturbated by several genetic alterations, we systematically compared the expression levels of BCL2, NOXA, MCL1 and BCL2L1 in CYLD inactivated, t(11;14) and chr(1q)amp patients. BCL2 levels were higher in the CYLD group, which parallels with the overexpression of the anti-apoptotic gene BCL2L1. NOXA, which promotes MCL1 degradation, was significantly upregulated in t(11;14). Chr(1q)amp patients showed a concomitant MCL1 overexpression and NOXA downregulation. To correlate these results to drug sensitivity, we performed an in-silico screen. We first selected MM and lymphoma cell lines from the DepMap dataset based on a gene expression profile that was most similar to the MM samples, then analyzed candidate drugs. The SKMM2 MM cell line, harboring t(11;14), del(CYLD) e NOXAamp was highly sensitive to Venetoclax. The same was true for the lymphoma ones RI1 and OCI-LY3, both harboring NOXAamp, but negative for t(11;14). On the contrary, the U266 and MOLP8 both with t(11;14) carrying a MCL1amp due to a chr(1q)amp were fully resistant. Of note, these latter resulted sensitive to the pan-BCL2 axis inhibitor Sabutoclax. Conclusions Our study suggests a link between the genomic architecture and transcriptome in MM, where CNAs and CRs had a stronger impact on expression than gene mutations. However, given that not all mutations are identical, HS ones need further testing as they may represent a future treatment target. Moreover, the mutational status is crucial since, while mono-allelic events are often of little transcriptional value, compound heterozygosity carries a huge influence on transcriptomic which provides biological basis for the observed prognostic impact of "double-hit" MM. Finally, we suggest that a comprehensive profiling of the BCL2 pathway may identify biomarkers of sensitivity to BCL2 inhibitors in addition to the t(11;14). Disclosures D'Agostino: GSK: Membership on an entity's Board of Directors or advisory committees. Corradini:Celgene: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Sanofi: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Incyte: Consultancy; Daiichi Sankyo: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Other; BMS: Other; F. Hoffman-La Roche Ltd: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Novartis: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Servier: Consultancy, Honoraria; Kite: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Other: Travel and accommodations paid by for; KiowaKirin: Consultancy, Honoraria. Bolli:Celgene: Honoraria; Janssen: Honoraria.

Abstract: Alterations in KRAS have been identified as the most recurring somatic variants in the multiple myeloma (MM) mutational landscape. Combining DNA and RNA sequencing, we studied 756 patients and observed KRAS as the most frequently mutated gene in patients at diagnosis; in addition, we demonstrated the persistence or de novo occurrence of the KRAS aberration at disease relapse. Small-molecule inhibitors targeting KRAS have been developed; however, they are selective for tumors carrying the KRASG12C mutation. Therefore, there is still a need to develop novel therapeutic approaches to target the KRAS mutational events found in other tumor types, including MM. We used AZD4785, a potent and selective antisense oligonucleotide that selectively targets and downregulates all KRAS isoforms, as a tool to dissect the functional sequelae secondary to KRAS silencing in MM within the context of the bone marrow niche and demonstrated its ability to significantly silence KRAS, leading to inhibition of MM tumor growth, both in vitro and in vivo, and confirming KRAS as a driver and therapeutic target in MM.