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Infection remains the leading cause of morbidity and mortality in patients with multiple myeloma because of the cumulative effect of disease, treatment, and host-related factors. Given that infectious risk is cumulative through the course of the disease, preventing infections is paramount. Optimal preventive strategies include vaccination against common pathogens, antimicrobial prophylaxis, infection control measures, and immunoglobulin replacement in a small subset of patients; however, there are no universally accepted guidelines for infection prevention. This Review provides a consensus statement from a panel of 36 experts with global representation, which was convened by The International Myeloma Society to review existing literature and current guidelines, address issues associated with the risk of infection and prevention of infectious complications in multiple myeloma in the context of emerging therapies, and offer recommendations for preventing these complications.

Summary Background Indatuximab ravtansine (BT062) is an antibody-drug conjugate that binds to CD138 and synergistically enhances the antitumor activity of lenalidomide in preclinical models of multiple myeloma. This phase 1/2a study was done to determine the safety, activity, and pharmacokinetics of indatuximab ravtansine in combination with immunomodulatory drugs in patients with relapsed or refractory multiple myeloma. Methods This open-label, phase 1/2a study took place at nine hospital sites in the USA. Eligible patients were aged 18 years or older, had relapsed or refractory multiple myeloma, and ECOG performance status or Zubrod score of 2 or below. Patients who received indatuximab ravtansine with lenalidomide and dexamethasone (indatuximab ravtansine plus lenalidomide) had failure of at least one previous therapy. Patients treated with indatuximab ravtansine with pomalidomide and dexamethasone (indatuximab ravtansine plus pomalidomide) had failure of at least two previous therapies (including lenalidomide and bortezomib) and had progressive disease on or within 60 days of completion of their last treatment. In phase 1, patients received indatuximab ravtansine intravenously on days 1, 8, and 15 of each 28-day cycle in escalating dose levels of 80 mg/m2, 100 mg/m2, and 120 mg/m2, with lenalidomide (25 mg; days 1 to 21 every 28 days orally) and dexamethasone (20–40 mg; days 1, 8, 15, and 22 every 28 days). In phase 2, the recommended phase 2 dose of indatuximab ravtansine was given to an expanded cohort of patients in combination with lenalidomide and dexamethasone. The protocol was amended to allow additional patients to be treated with indatuximab ravtansine plus pomalidomide (4 mg; days 1 to 21 every 28 days orally) and dexamethasone, in a more heavily pretreated patient population than in the indatuximab ravtansine plus lenalidomide group. The phase 1 primary endpoint was to determine the dose-limiting toxicities and the maximum tolerated dose (recommended phase 2 dose) of indatuximab ravtansine, and the phase 2 primary endpoint was to describe the objective response rate (ORR; partial response or better) and clinical benefit response (ORR plus minor response). All patients were analysed for safety and all patients with post-treatment response assessments were analysed for activity. This study is registered with ClinicalTrials.gov , number NCT01638936 , and is complete. Findings 64 (86%) of 74 screened patients were enrolled between July 3, 2012, and June 30, 2015. 47 (73%) patients received indatuximab ravtansine plus lenalidomide (median follow-up 24·2 months [IQR 19·9–45·4]) and 17 (27%) received indatuximab ravtansine plus pomalidomide (24·1 months [17·7–36·7]). The maximum tolerated dose of indatuximab ravtansine plus lenalidomide was 100 mg/m2, and defined as the recommended phase 2 dose for indatuximab ravtansine plus pomalidomide. An objective response for indatuximab ravtansine plus lenalidomide was observed in 33 (71·7%) of 46 patients and in 12 (70·6%) of 17 patients in the indatuximab ravtansine plus pomalidomide group. The clinical benefit response for indatuximab ravtansine plus lenalidomide was 85% (39 of 46 patients) and for indatuximab ravtansine plus pomalidomide it was 88% (15 of 17 patients). The most common grade 3–4 adverse events in both groups were neutropenia (14 [22%] of 64 patients), anaemia (10 [16%]), and thrombocytopenia (seven [11%]). Treatment-emergent adverse events (TEAEs) that led to discontinuation occurred in 35 (55%) of the 64 patients. Five (8%) patients with a TEAE had a fatal outcome; none was reported as related to indatuximab ravtansine. Interpretation Indatuximab ravtansine in combination with immunomodulatory drugs shows preliminary antitumor activity, is tolerated, and could be further evaluated in patients with relapsed or refractory multiple myeloma. Funding Biotest AG.

Background Indatuximab ravtansine (BT062) is an antibody-drug conjugate that binds to CD138, which is overexpressed on multiple myeloma (MM) cells. Patients and Methods We report from 2 clinical studies of patients with relapsed and/or refractory MM previously treated with an immunomodulatory drug and a proteasome inhibitor. Single- and multi-dosing schedules were investigated to define dose-limiting toxicities, maximum tolerated dose (MTD), recommended phase II dose, and to describe safety, efficacy, and pharmacokinetics. Results In the first-in-human study, indatuximab ravtansine was administered to 32 patients on day 1 of each 21-day cycle. The MTD was 160 mg/m2. In the phase I/IIa study, indatuximab ravtansine was administered to 35 patients on days 1, 8, and 15 of each 28-day cycle, and the MTD/recommended phase II dose was 140 mg/m2. Most (88%) adverse events were grade 1 or 2, the most common being diarrhea and fatigue. There was rapid clearance of indatuximab ravtansine and no relevant accumulation. Over 75% of heavily pretreated patients achieved stable disease or better. With the multi-dose schedule, minor and partial responses occurred in 14.7% of patients, the median time to progression was 3 months, and the median overall survival was 26.7 months. Conclusion Our data support further investigation of indatuximab ravtansine as part of a combination regimen for relapsed and/or refractory MM.

OBJECTIVE: To understand the experience of patients with relapsed and refractory multiple myeloma (RRMM) receiving idecabtagene vicleucel (ide-cel), a B-cell maturation antigen-directed chimeric antigen receptor T cell therapy, in the pivotal, phase 2 KarMMa trial. METHODS: Optional semi-structured interviews before leukapheresis (pre-treatment) captured expectations and after ide-cel infusion (1, 2, and 3 months post-treatment), assessed treatment experience, ide-cel advantages/disadvantages, and health and well-being. In a mixed-method analysis, treatment experiences were categorized by clinical response status, health and well-being, and self-reported recovery after infusion. RESULTS: Pre-treatment interviews indicated unmet treatment needs. In post-treatment interviews, most patients reported the positives of ide-cel outweighed negatives (69%, n = 27/39). Most common advantages of ide-cel were efficacy (18-64%), favorable side-effect profile (46-68%), and recovery time (13-18%); most common disadvantages were related to side effects (13-20%). When analyzed by clinical response, patients most often had stringent complete or very good partial response and improved health and well-being with mild or severe recovery from the infusion (27/58, 47%). Most patients with minimal clinical response reported mild infusion recovery (5/6, 83%). CONCLUSIONS: Patient interviews before ide-cel treatment showed unmet needs in triple-class exposed RRMM. Post-treatment experiences generally favored ide-cel versus previously received treatments. ispartof: LEUKEMIA RESEARCH vol:120 ispartof: location:England status: published

Background Multiple myeloma (MM) is characterized by an immunosuppressive microenvironment that enables tumor development. One of the mechanisms of immune evasion used by MM cells is the inhibition of NK cell effector functions; thus, the restoration of NK cell antitumor activity represents a key goal for new immunotherapeutic approaches, increasing tumor cell recognition, avoiding tumor escape and potentially enhancing the effect of other drugs. Methods Here we investigate the potential of NKTR-255, a novel polymer-conjugated human IL-15 to engage the IL-15 pathway and overcome the inhibitory status observed in NK cells from MM patients. For this purpose, we have analyzed ex vivo and in vivo effects of NKTR-255 on phenotypic features, effector functions and cytotoxicity of NK cells against MM cells. Results We observed that incubation with NKTR-255 was able to tilt the balance towards an activated phenotype in NK cells isolated from peripheral blood mononuclear cells of MM patients, with increased expression of activating receptors (NKG2D, NKp46, NKp30, DNAM-1, CD69, TRAIL) on the surface of treated NK cells. This resulted in an enhanced degranulation, cytokine release and anti-tumor cytotoxicity when the NK cells were exposed to both MM cell lines and primary MM cells. For a more accurate assessment of the effect of NKTR-255 on NK cell activity in an autologous setting in the presence of the bone marrow (BM) milieu, we cultured whole BM samples from non-treated newly-diagnosed MM patients with increasing doses of NKTR-255 for 5 days. NK cells experienced a dose-dependent induction of proliferation and activation (as shown by increased expression of CD69 and NKG2D), which translated in a reduced viability of CD138+ MM cells in the presence of NKTR-255. We further evaluated the in vivo effect of NKTR-255 in fully humanized immunocompetent mice subcutaneously engrafted with H929 MM cells. Compared to placebo, weekly injection of the mice with NKTR-255 increased the number of circulating NK cells in peripheral blood and delayed tumor growth. Finally, we also tested in vitro and in vivo efficacy of a combination of NKTR-255 with daratumumab. We observed a more efficient antibody-dependent cellular cytotoxicity against MM cells in vitro and decreased tumor growth in vivo, where NKTR-255 rescued CD38+ NK cell levels from depletion by daratumumab. Conclusions Taken together, these results support the restoration and expansion of NK cell activity in MM with NKTR-255, providing rationale for its clinical use as a novel immunotherapeutic approach for MM patients alone or in combination with monoclonal antibodies or other immunomodulatory drugs.

Background Multiple Myeloma (MM) is a disease driven by numerous genetic and epigenetic alterations, however true drivers of the disease have yet to be identified. Methods To identify new dependencies and actionable therapeutic targets in MM we integrated gene expression and genetic dependency (CRISPR KO). Results We found that many of the specific and potent dependencies in MM are transcription factors, especially those establishing plasma cell identity. Among others, the POU2AF1 gene, which encodes the OCA-B/BOB-1, a B cell transcriptional coactivator protein, represented the most striking dependency in MM. Although BOB-1 is expressed throughout B-cell development, we found it to be highly expressed in CD138+ plasma cells from patients with precursor conditions (MGUS and SMM) as well and established MM compared to normal plasma cells (NPC). Loss-of-function studies using shRNA, siRNAs as well as antisense GapMers specific for BOB-1 confirmed significant impact on MM cell viability. Transcriptomic analysis by RNA-sequencing revealed a small set of genes commonly modulated in MM cell lines upon BOB-1 depletion, including the XBP1- BHLHA15 axis involved in lipid synthesis and unfolded protein response (UPR). Interestingly, among the genes most significantly upregulated by BOB-1 depletion was heme oxygenase 1 (HMOX1), that was affected via the NRF2/Keap1 pathway. We observed that HMOX1 expression is significantly lower in MM cells from patients compared to normal plasma cells and correlates with poor clinical outcome, suggesting important role in MM. Moreover, we found that siRNA depletion of HMOX1 reverted the inhibition of MM cell growth caused by BOB-1 KD, confirming significant role for HMOX1 in the BOB-1 addiction observed in MM cells. Next, we performed gene set enrichment analysis (GSEA) and observed ribosome biogenesis pathways and mRNA translation and elongation processes, along with WNT and senescence pathways, to be significantly enriched among genes modulated by BOB-1 depletion in MM cells. Since high protein load is a feature of MM, we evaluated the role of BOB-1 in the translational efficiency of MM cells. In MM cell lines, BOB-1 knockdown decreased de novo protein synthesis, while its overexpression significantly enhances protein synthesis compared to control cells. As MM is characterized by excess production of monoclonal immunoglobulins, we evaluated impact of BOB-1 perturbation on intracellular kappa and lambda light chains production. We observed changes in the intracellular abundance of the light chains with BOB-1 modulation in all MM cell lines tested. As a result, BOB-1 depletion was associated with induction of resistance to proteasome inhibition. Conclusion In conclusion, we report BOB1 as a specific dependency in MM cells with potential role on modulating the protein load/capacity balance in MM cells and therefore the sensitivity to proteasome inhibition.

Background Multiple myeloma (MM) is an incurable hematologic malignancy requiring long-term, continuous therapy. Despite its chronic and unrelenting course, studies examining quality of life (QOL), psychological distress, and prognostic awareness by line of therapy are lacking. Methods We conducted a cross-sectional, multi-site study of patients undergoing treatment for MM (excluding maintenance therapy) between 6/2020-1/2021. To capture the full spectrum of treatment, we conducted purposeful sampling and recruited patients to 3 cohorts based on lines of therapy: 1) newly diagnosed receiving first-line therapy; 2) 2-3 lines; and 3) ≥ 4 lines. Patients completed validated questionnaires to assess their QOL, symptom burden, fatigue, psychological distress, and perceptions of prognosis. We used multivariate linear regression models to examine the association between lines of therapy, QOL, psychological distress, with patient’s perception of their prognosis. Results We enrolled 180 patients with MM (newly diagnosed (n=60), 2-3 lines (n=60), and ≥4 lines of therapy (n=60)). QOL, symptom burden, mood, and fatigue scores did not differ by lines of therapy. The rate of clinically significant depression, anxiety, and PTSD symptoms were 23.9% (43/180), 23.9% (43/180), and 24.4% (44/180), respectively. Overall, 84% (147/175) of patients reported that it is “extremely” or “very” important to know about their prognosis, and the majority (66.1%, 117/177) stated that they had received adequate information regarding their prognosis. Most patients, 84.7% (149/176) reported that their oncologist told them their cancer was incurable but only 30.6% (53/173) acknowledged that they were terminally ill and only 42.0% (73/174) reported that they thought their cancer was incurable. Patients receiving 2-3 lines of therapy were more likely to acknowledge their terminal illness (36.7% vs. 19.6%, p=0.045) and that their MM was incurable (90.0% vs. 75.9%, p=0.047) compared to those receiving first-line therapy. QOL and psychological distress were not associated with patient’s perception that their MM was incurable. However, patients who acknowledged their terminal illness reported higher depression (B=1.52, P = 0.009), anxiety (B=1.52, P=0.0037), symptom burden (B=7.42, P=0.007), and lower QOL (B=-14.78, p=0.001). Conclusions MM patients undergoing treatment experience impaired QOL and elevated psychological distress across the disease continuum, irrespective of their line of therapy. Although the majority reported that their oncologist has told them that their cancer is incurable, a substantial proportion still have significant misperceptions of their prognosis. Interventions are needed to improve patients’ QOL, reduce their psychological distress, and enhance patients’ perceptions of their prognosis to facilitate informed decision-making.

Introduction Long noncoding RNAs (lncRNAs) are profoundly dysregulated in multiple myeloma (MM), yet their tumor-promoting features remain to be defined. Here, we have coupled extensive transcriptomic profiling of patient samples (n=360) with a genome-wide CRISPR interference (CRISPRi) viability screen (4 cell lines) to generate a map of lncRNA "dependencies" in MM; moreover, we describe the molecular and functional role – as well as its therapeutic potential – of the screen top hit RNA Regulator of Lipogenesis (RROL), a nuclear-retained lncRNA generated by alternative splicing of MIR17HG. Methods Loss-of-function (LOF) studies using antisense oligonucleotides (ASO) confirmed the RROL dependency in a large panel of MM cell lines and CD138+ patient cells, in vitro and in vivo in animal models. This effect was not antagonized in DROSHA KO MM cells or by ectopic expression of MIR17HG-derived miR-17-92, indicating a microRNA-independent role of RROL. Results Transcriptomic analysis after RROL depletion indicated a significant impact on the de novo lipogenesis (DNL) gene networks. RROL occupancy at DNL gene loci was detected by chromatin isolation by RNA precipitation followed by qRT-PCR (ChIRP-qPCR) and confirmed by DUAL RNA FISH analysis. At functional level, we demonstrated that RROL depletion reduces DNL in MM cells using an unbiased lipidomic profiling as well as by measuring the incorporation of C14-radiolabeled glucose into lipids. Importantly, we also proved that exogenous palmitate, the downstream product of DNL pathway, can significantly rescue the growth inhibitory effect of RROL depletion in MM cells; thus implicating a role for DNL in the growth promoting effect of this lncRNA. RNA-Protein Pull Down (RPPD) and in vivo RNA yeast three-hybrid (Y3H) assays led to identify c-MYC (MYC) as a relevant protein interactor of RROL; a finding validated by RIP-qPCR. Coupling RNA FISH with immunofluorescence, we co-detected RROL and MYC at the ACC1 promoter. Importantly, neither RROL or MYC could occupy ACC1 promoter and exert regulatory control in the absence of the other factor. Moreover, using in vitro (Co-IP/MS) and in vivo (BioID) assays, we identified WDR82 as an RROL-dependent MYC partner implicated in the transcriptional control of ACC1 expression. Finally, to therapeutically antagonize RROL, we have screened >100 ASOs and provided the basis to develop a first-in-class RROL inhibitor. This inhibitor has shown a very strong anti-MM activity in vitro (IC50 Conclusion 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 ongoing development of RROL inhibitors may allow clinical application of this unique targeted therapy in MM.

Background Efforts are required to improve potency and durability of CD38- and BCMA-based immunotherapies in human multiple myeloma (MM). Methods We here delineated the molecular and cellular mechanisms underlying novel immunomodulatory effects triggered by BCMA pyrrolobenzodiazepine (PBD) antibody drug conjugate MEDI2228 which may augment efficacy of these immunotherapies. RNA sequencing followed by gene set enrichment analysis showed that MEDI2228 significantly enriched IFN I-signaling and induced type I interferon (IFN I)-stimulated genes (ISGs) in MM cell lines. The most MEDI2228-enhanced IFN-driven genes include chemokines/cytokines and receptors (i.e., CXCL9/10, CCL4L1/2, CCL22, CCL1/3/4/5, CCL3L1/3, TNFSF9/10, CSF2 (GM-CSF), CCR7), RSAD2, CASP1, ISGs (i.e., XAF1, TRIMs, IFITs, ISG15, GBP2/3, OAS1/2/L, RNASEL, MX1/2, FAS), RUNX3, GZMB, IKBKE, IRF1/6/7/9, STAT1/2/4/6, MB21D1(CGAS), TMEM173 (STING), IFIT1/2/3/5, and SOCS1/2/3. Results Regardless of genetic heterogeneity and resistance to current anti-MM therapies, MEDI2228 induced dose- and time-dependent DNA damage-ATM/ATR-CHK1/2 pathways, activation of cGAS-STING-TBK1-IRF3 and STAT1-IRF1-signaling cascades, as well as increased CD38 expression. It overcame CD38 downregulation triggered by IL6 and bone marrow stromal cell culture supernatant (BMSC-sup), via activation of STAT1-IRF1 without phosphorylation of STAT3 in immunomodulatory drugs (IMiDs)- and bortezomib-resistant MM cell lines. In contrast, MEDI2228 did not change CD38 expression and survival in BCMA-negative NK effector cells. Significantly, MEDI2228 with anti-CD38 monoclonal antibody Daratumumab (Dara) synergistically induced NK-mediated lysis of MM cell lines and autologous resistant patient MM cells, even in the presence of BMSC-sup and BMSCs. In parallel, MEDI2228 increased membrane expression of NKG2D ligands (NKG2DLs), i.e., MICA/B and ULBPs, in all MM cells tested, including Dara-resistant patient cells, correlating with enhanced MM cell susceptibility to NK cell killing. Since MEDI2228, but not its MMAF-ADC homolog M3 even used at >1-log higher concentrations enhanced surface expression of CD38 and NKG2DLs on MM cells, the potent DDR-mediated immunomodulation triggered by MEDI2228 vs M3 is critical in rendering MM cells more susceptible to Dara-induced NK cell killing. Importantly, M2 still activated STAT1/IRF1 signaling to induce CD38 and MICA/B expression in MM tumors grown in mice. All MM1S tumor-bearing NSG mice reconsituted with human NK cells became tumor-free following a single low dose M2 with Dara treatment, with 100% host survival. Conclusions Taken together, our data showed that MEDI2228 restored MM sensitivity to CD38 targeting by Dara without depleting NK cells and potentiated immunogenic cell death of MM cells. These results therefore provide the mechanistic rationale for clinical evaluation of combination CD38- and BCMA-directed immunotherapies to further improve patient outcome in MM.

Background We have previously reported the identification of FEN1 a driver of genomic instability in myeloma (MM) as well as esophageal adenocarcinoma (EAC) cells. We demonstrated that FEN1 is overexpressed in MM cell lines and in clinical datasets of MM and several other cancers including EAC, and FEN1-knockdown inhibited spontaneous DNA breaks, homologous recombination (HR) activity as well as genomic instability in MM cells. We now show that FEN1-overexpression in non-cancerous (normal fibroblasts and bone marrow/stroma HS5) cells increases DNA breaks and genomic instability, as assessed by micronucleus assay. Methods In order to further evaluate the impact on genomic instability, control and FEN1-overexpressing cells were cultured for three weeks and new genomic changes acquired in cultured relative to “day 0” cells (representing baseline genome), were identified using single nucleotide polymorphism (SNP) arrays. Results Overall, the acquisition of amplification and deletion events were increased by ~ 3-fold in FEN1-overexpressing relative to control cells. Evaluation by RNA sequencing in two different non-cancerous cell types showed that FEN1-overexpression was associated with upregulation of several interconnected pathways including DNA double strand break repair, cell cycle, mitotic G2 M phases, TP53, homologous DNA pairing and strand exchange. Top downregulated pathways included several metabolic pathways and an apoptosis pathway. These data demonstrate a significant role and the impact of FEN1 on DNA repair and genome maintenance, especially HR. We next identified FEN1-interacting proteins from two different MM cell lines (MM1S, RPMI8226) by mass spectrometry. Forty-one proteins interacted with FEN1 in both MM cell lines including two helicases (ASCC3, RUVBL2) and several proteins involved in DNA damage response and recruitment. To investigate the functional relevance of FEN1-ASCC3 interaction, FEN1 was overexpressed in non-cancerous (stromal HS5) cells and ASCC3 was suppressed in control as well as FEN1-overexpressed cells, and impact on different parameters of genome stability (HR activity, micronuclei) and growth (cell viability and DNA replication) monitored. FEN1-overexpression increased HR activity, whereas ASCC3-knockdown inhibited spontaneous as well as FEN1-induced HR activity. Consistent with these data, FEN1-overexpression also increased genomic instability, whereas ASCC3-knockdown inhibited spontaneous as well as FEN1-induced genomic instability as assessed by micronucleus assay. Importantly, FEN1-overexpression also increased DNA replication (as assessed by BrdU-labelling), whereas ASCC3-knockdown inhibited spontaneous as well as FEN1-induced DNA replication in these cells. Conclusion These data suggest that helicase activity of ASCC3 is coupled to endonuclease activity of FEN1 to cause genomic instability and cancer cell proliferation, and is currently being investigated in MM and other cancer models to develop translational application.

Background Patients (pts) with RRMM who were exposed to immunomodulatory agents, proteasome inhibitors (PIs), and anti-CD38 monoclonal antibodies (mAbs) have poor outcomes with subsequent treatments. Ide-cel, a BCMA-directed CAR T-cell therapy, has shown frequent, deep, and durable responses in triple-class exposed pts with RRMM in the pivotal KarMMa study (NCT03361748; Munshi NC et al. N Engl J Med 2021). Here, we report updated results from this trial. Methods Eligible pts had received ≥3 prior regimens (including an immunomodulatory agent, a PI, and an anti-CD38 mAb) and had disease refractory to their last regimen per IMWG criteria. After 3 days of lymphodepletion (cyclophosphamide 300 mg/m2 + fludarabine 30 mg/m2), pts received 150–450×106 CAR+ T cells (target dose levels). Primary endpoint was overall response rate (ORR); key secondary endpoint was complete response (CR) rate (CR + stringent CR). Other secondary endpoints included duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Results In total, 128/140 enrolled pts received ide-cel. Data are presented for the treated pts. Median age was 61 y. Pts had received a median of 6 (range, 3–16) prior regimens, with 84% being triple-class refractory and 26% penta-refractory (lenalidomide, pomalidomide, bortezomib, carfilzomib, and daratumumab). Most pts (88%) had received bridging therapy. At data cutoff (Dec 21, 2020), the median follow-up among surviving pts was 24.8 mo. In total, 94/128 pts (73%) achieved an overall response, with a CR rate of 33%; median DOR and PFS were 10.9 mo and 8.6 mo, respectively. In pts who achieved ≥CR, median DOR was 21.5 mo and median PFS was 22.4 mo. Outcomes improved at higher doses, with ORR of 81%, CR rate of 39%, median DOR of 11.3 mo, and median PFS of 12.2 mo at 450×106 CAR+ T cells; in pts who achieved ≥CR at this dose, median DOR was not reached. Responses were observed in all subgroups, including difficult-to-treat subsets (eg, high tumor burden [ORR, 71%], extramedullary disease [70%], and R-ISS stage III disease [48%]). Median OS was 24.8 mo; the estimated 24-mo OS rate was 51%. The most common any-grade toxicities were neutropenia (91%) and cytokine release syndrome (CRS; 84%). CRS was mostly grade 1/2; 5 pts (4%) had grade 3, 1 pt each had grade 4 and 5 events (both at 300×106). Investigator-identified neurotoxicity was reported in 23 pts (18%); 5 (4%) had grade 3 and 0 had grade ≥4 events. Tocilizumab was used in 67 and 3 pts, and steroids were used in 19 and 10 pts with CRS and neurotoxicity, respectively. Conclusion Updated results from the KarMMa trial continue to demonstrate durable and deep responses with ide-cel in heavily pretreated, triple-class–exposed pts with RRMM. Efficacy and safety data are consistent with prior reports and support a favorable clinical benefit–risk profile for ide-cel across the target dose levels.

The heterogeneity and specification of pro-epicardial cells (pro-EpiCs) to fibroblast and smooth muscle cell (SMC) are unknown. We applied single-cell RNA sequencing (scRNA-seq) paired with RNAScope, bioinformatics, and lineage tracing in an unbiased manner to identify the previously uncharacterized molecular heterogeneity of the pro-EpiCs isolated from pro-epicardium (PE). We found that pro-EpiCs labeled by Tbx18 Cre/+ are heterogeneous, with three clusters displaying differential expression profiles and distinct spatial locations. Cluster 1 are mesothelial cells, and Cluster 2 express SMC markers. Surprisingly, Cluster 3 express Isl1 and markers of pacemaker progenitor cells (PMPC) but not marker of atrial cardiomyocytes. Our studies conclude that pro-EpiCs are heterogeneous and SMC-like cells are specified in the PE, while fibroblasts are not specified in PE but epicardium. We identified a region in PE that contains PMPCs, which translocate through the inflow tract to the sinoatrial node. The expression profile of Cluster 3 cells unifies previous studies regarding the origins and markers of PMPCs.

Background Peripheral neuropathy (PN) is an important toxicity that limits the use of bortezomib (Btz). Attempts to reduce PN have included its subcutaneous (SC) administration. Patients and Methods We retrospectively analyzed 307 patients with newly diagnosed multiple myeloma from a single Chinese center, receiving Btz-based regimens administered either via SC injection (SC group, n = 167) or intravenous (IV) infusion (IV group, n = 140). The efficacy and safety of Btz administration via SC and IV were then compared. Results Most baseline characteristics were similar between these 2 groups. A lower frequency of adverse events, especially grade ≥ 3 PN (P = .002), was observed in the SC group compared with the IV group. The estimated median Btz dosage when PN developed was higher (20.8 mg/m2 vs. 15.6 mg/m2), and fewer patients reduced or discontinued Btz owing to adverse events in the SC group compared with the IV group. The overall response rate (≥ partial response [PR]) was comparable (94.8% vs. 96.2%). However, patients in the IV group required fewer cycles to achieve PR, whereas a larger proportion of patients in the IV group achieved ≥ very good PR. After a median follow-up of 23 months (range, 1-84 months), no significant difference in median progression-free survival (not arrived vs. 33.0 ± 2.735 months) and overall survival (not arrived vs. 56.0 months) was noted. Conclusion SC Btz is associated with better tolerance; however, IV administration achieves a faster and deeper response in Chinese patients with newly-diagnosed multiple myeloma.

The Blood and Marrow Transplant Clinical Trials Network (BMT CTN) Myeloma Intergroup Workshop on Minimal Residual Disease and Immune Profiling was convened on December 1, 2016 at the American Society of Hematology (ASH) meeting to discuss the emerging data and technologies for minimal residual disease assessment and immune profiling in myeloma. Particular emphasis was placed on developing strategies to incorporate these techniques into clinical trial design. This document reviews the literature, summarizes the topics discussed in the workshop and provides recommendations for integration of these techniques into future clinical trial design.

Castleman disease (CD) is a rare and heterogenous group of disorders sharing in common an abnormal lymph node pathology. CD comprises distinct subtypes with different prognoses. Unicentric CD and multicentric CD are featured by specific systemic manifestations and may be associated with Kaposi sarcoma, non-Hodgkin and Hodgkin lymphoma, and POEMS syndrome. Multicentric CD is classically associated with systemic symptoms and poorer prognosis. In this article, the authors review how to diagnose the disease, keeping in context the clinical findings, biochemical changes and complications associated with CD.

Background With the deluge of available genomic data in recent years, it is becoming evident that MM cells are characterized by cell cycle dysregulation, epigenetic heterogeneity, and perturbation of the transcriptional landscape. We here elucidated the biological role of CDK7 and explored the functional consequence of its inhibition in MM using chemical and genetic approaches, including a recently reported selective CDK7 covalent inhibitor YKL-5-124, and engineered systems for rapid CDK7 protein degradation (dTAG). As previously shown with non-selective inhibitors, CDK7 inhibition via YKL-5-124 was active against a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. The efficacy of YKL-5-124 was confirmed in vivo in several murine MM models. Selective pharmacological degradation of endogenously tagged CDK7 and inducible KO/KD cell systems confirmed impact of CDK7 inhibition on MM cell proliferation supporting the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data support the notion of CDK7 as a central hub in the oncogenic CDK-pRb-E2F pathway in MM cells, with its expression and activity positively correlated with E2F transcriptional output in patient cells. Conclusion Importantly, dual inhibition with low doses of YKL-5-124 and BRD4 inhibitor JQ1 respectively, displayed superior activity against a panel of MM cell lines and primary MM cells compared to single perturbation alone by both converging on a subset of key SE-associated dependencies as well as impacting distinct oncogenic expression programs. Isobologram analysis revealed strong synergism with a combination index (CI)

Background Primary plasma cell leukemia (pPCL) is an aggressive subtype of multiple myeloma that is characterized by ≥20% circulating tumor cells (CTCs), whereas CTC levels in newly diagnosed multiple myeloma (NDMM) are Methods Transcriptomic data were generated of CD138-enriched bone marrow (BM) plasma cells from both NDMM patients enrolled in the Cassiopeia (NCT02541383) and HO143 (EudraCT 2016-002600-90) trials and pPCL patients from the EMN12/HO129 (EudraCT 2013-005157-75) trial. NDMM CTC levels were determined with the EuroFlow NGF protocol. HR FISH was defined as the presence of either t(4;14), t(14;16) or del17p.154 NDMM and 29 pPCL patients were divided into a discovery and validation cohort to construct and test a classifier for PCL-like disease. Subsequently, data from 8 additional NDMM cohorts were used to assess the association of PCL-like status with progression-free survival (PFS) and overall survival (OS) in Cox proportional hazards (PH) models including conventional HR markers. Results Baseline CTC levels were determined in 297 NDMM and 51 pPCL patients. CTCs could be detected in 87% of NDMM patients, with a limit of detection Conclusions (1) pPCL cannot only be identified clinically, but also molecularly. (2) PCL-like status is a novel marker for HR disease in NDMM that identifies patients with a tumor transcriptome similar to pPCL and has independent prognostic value in the context of conventional HR markers. (3) PCL-like status could help detect NDMM patients with early stage or borderline pPCL.

Background Resistance to immune approaches poses a major challenge to effective immunotherapy and long-term clinical outcome in multiple myeloma (MM). Here we identified loss-of-function of gamma-aminobutyric acid receptor-associated protein (GABARAP) as a tumor-intrinsic mechanism of resistance to bortezomib (BTZ)-induced immunogenic cell death (ICD), the immunogenic consequence of apoptosis resulting in specific anti-MM immunity via T-cell priming by dendritic cells (DCs). Methods We found that BTZ induces ICD in human and murine MM cell lines that is dependent on the exposure of calreticulin (CALR), which drives DCs-mediated phagocytosis of MM cells. DCs-phagocytosis is followed by a specific T cell activation, with a significant increase of CD4+ effector memory (EM), total CD8+, CD8+ EM, and CD8+ terminally differentiated EM cells. Isolated T cells after co-cultures showed the presence of MM specific CTLs that were able to efficiently induce lysis of MM cells. Our results were validated using primary cells from MM patients. Moreover, induction of a protective immune response was confirmed in vivo. Specifically, treatment of 5TGM1 tumors with BTZ induced a tumor regression in a syngeneic model; and injection of live 5TGM1WT two weeks after regression did not result in tumor development, consistent with induction of immunological memory as confirmed by ELISPOT of mouse splenocytes. We identified a specific ICD signature induced by BTZ in mice; and we found that increased expression of the human orthologs of this signature was positively correlated with OS (p=0.01) in patients enrolled in the IFM/DFCI 2009 study. Notably, these functional immunologic sequelae were abrogated after BTZ treatment of CALRKO MM cells both in vitro and in vivo, confirming the obligate role of CALR exposure in the ICD process. Results By interrogating the IFM/DFCI dataset and focusing on genes involved in ICD processes that were correlated with MM patients clinical outcome, we found that low levels of GABARAP (chr17p13.1), an autophagy regulator and putative CALR binding partner, negatively impact MM patient clinical outcome (EFS, p=0.0032); even excluding HR patients with 17p deletion (EFS, p=0.018). Interestingly, KMS11 cells carrying monoallelic deletion of GABARAP were resistant to induction of ICD by BTZ; and sensitivity was restored after overexpression of the gene. Moreover, GABARAPKO in 3 ICD-sensitive cell lines abrogated the induction of ICD by BTZ; and add-back experiments by pre-treatment with recombinant CALR or GABARAP overexpression in KO clones restored ICD. Finally, CyTOF confirmed that treatment of GABARAPKO cells with BTZ failed to activate an efficient T cell response. Conclusions our study demonstrates that loss-of-function of GABARAP, particularly in HR patients with 17p deletion, contributes to tumor immune evasion and ICD resistance. These studies provide the framework for novel combination treatments to restore anti-MM immunity and improve patient outcome in HR MM.

Background On average 10% of SMM patients progress to symptomatic MM per year with in first 5 years of diagnosis. However, a subset of SMM patients re-classified as high risk patients on the basis of risk markers which identify risk of progression within 2 years. Although recent studies have evaluated the high-risk SMM, genomic background of SMM patients who do not progress to MM after long-term follow-up (>=5 years) has not been described. Methods Here, we evaluated transcriptomic and genomic changes enriched in non-progressor (NP) (no progression after 5 years of follow-up) precursor conditions (N=31) with those progressed within short period time (N=71) and compared them with changes observed in newly diagnosed MM (N=192). Additionally, using transcriptome, epigenome and whole genome profiling we also studied additional unique samples from 18 patients at their precursor stage as well as when progressed to MM. Results Overall, we have observed significantly lower mutational load for NP SMM (median SNV 5460 vs. 7018, p 90% accuracy and >0.80 area under the curve on ROC using ten-fold cross validation. This indicated that not only the load but also the patterns of mutations (type, location, frequency) are different between two conditions. We also found that NP samples have significantly lower heterogeneity (p 80%) with AUC score 0.80. Our transcriptomic analysis measured the distance between progressor and NP SMM as well as MM and found that NP SMM are less similar to MM which is closer to progressor SMM. Epigenomic analysis yield that 75 SEs regions were differentially utilized between precursor and symptomatic MM stage. The targeted genes included BMP6, PRDM1, STAT1 and RAB21 and possibly regulating genes related to oncogenic KRAS activities. Conclusions In conclusion, our results now provide the basis to develop genomic definition of SMM as well as risk driving features.

Background Immunotherapies in MM targeting BCMA, have shown remarkable clinical benefits. However, two recent reports highlighted that biallelic loss of BCMA cause resistance to anti-BCMA therapy. In both studies BCMA locus was deleted bringing in focus importance of del16p. We have evaluated 2883 MM patients at diagnosis and relapse to understand frequency of BCMA targeting events and characteristics of MM patients with BCMA deletion. Methods We observed del16p in 8.58% (7.6% to14.6% in individual studies) of newly-diagnosed patients (n=2458).Frequency of 16p loss in both HMM and NHMM were similar, suggesting its independence from MM subtypes. Overall CN loss was significantly higher in patients with BCMA loss compared to rest of the MM patients. High risk deletion events such as del1p and del17p were more likely to be observed in patients with loss of BCMA locus (OR [95% CI] 19.3(13.1-25.8), FDR = 1.5e-65; and 8.8(6.3-12.1), FDR = 5.5E-39, respectively)]. MM patients with loss of BCMA locus have increased mutational load (8202 with 95% HDI 6921 and 9535) compared to those without BCMA locus loss (6975 with 95% HDI 6626 – 7343); probability of difference greater than 0 was 96.8% and difference of the means were 1222 [95% CI 112-2589]. To understand the risk profile of patients with loss of BCMA locus, we next focused on the observation that BCMA loss frequently co-occurs with other deletions. Results We observed that when BCMA and TP53 or BCMA and del1p loss are present in the same patient, they are likely to have same clonality. These data suggested a possibility of co-occurrence of these events in same cell. To further investigate this observation, we used single cell DNA sequencing data from patients with sub clonal and clonal BCMA locus loss. Interestingly, almost all cells with BCMA loss also had p53 loss, while not all p53 loss cells had BCMA loss suggesting that the chronology of this copy number alternation may suggest first p53 loss followed by BCMA loss. Our data from a patient with BCMA targeting therapy also indicated that BCMA loss tend to co-occur with TP53 deletions (OR=5.67 [95% CI 4.12-7.84], p value Conclusions Our data from large scale copy number profiles showed that even without treatment pressure, monoallelic BCMA deletions are frequent events. Moreover, patients with these events show increased genomic loss. Such behavior potentially make these cells vulnerable for biallelic loss of other genes. Our results highlight that by looking at mRNA or protein expressions at bulk sample would not directly indicate the presence or absence of cells with target loss and therefore evaluating single cell level data are necessary. These results suggest the need to study del16p in patients being targeted for BCMA-directed therapy and its association with del17p raises question about the role of BCMA targeted therapy in high-risk myeloma.

Background Mitochondria are enticing potential targets against cancer, owing to their role as signaling hubs orchestrating key homeostatic functions. Of special interest is ClpP, a resident mitochondrial protease suggested to maintain OXPHOS by degrading damaged protein components and regulating the assembly of mito-ribosomes. While the exact role of ClpP in mammals remains unclear, its manipulation has been shown to induce leukemic cell death. Prompted by its distinctive expression in malignant plasma cells (PCs), we investigated the role of ClpP in maintaining mitochondrial and cellular homeostasis in multiple myeloma (MM) cells and tested it as a possible anti-myeloma target. Methods We analyzed the expression of ClpP mRNA in public and proprietary datasets of normal and malignant PCs and MM cell lines. We performed stable shRNA-mediated knockdown of ClpP (ClpPkd) in MM cell lines and analyzed its sequelae combining electron microscopy, Seahorse and ATP assays, transcriptomics, proteomics, and metabolomics. A proteolytically inactive ClpP mutant (ClpPmut) was expressed to entrap ClpP substrates for subsequent mass spectrometry identification and wet validation. Results ClpP mRNA was significantly higher in bone marrow-purified malignant vs. normal PCs, and MM cells were the highest ClpP-expressing human cancer cell lines. Attesting to a crucial role in myeloma, ClpPkd MM cell lines disappeared from culture due to rapid onset of cell cycle arrest and apoptosis. Intriguingly, toxicity in MM proved independent of the currently acknowledged ClpP-controlled mitochondrial functions, i.e., mito-ribosome assembly and OXPHOS maintenance. Indeed, Seahorse demonstrated different bioenergetics across MM lines, ranging from mixed oxidative/glycolytic to almost exclusively glycolytic. Yet, ClpPkd failed to abate ATP in glycolytic MM lines, but proved equally toxic across all lines, thus unveiling an energy-independent vulnerability. To unbiasedly define the role of ClpP in MM, we undertook a threefold orthogonal approach employing RNA-seq, proteomics, and metabolomics. Their integrated analysis upon ClpPkd revealed an unexpected impact of ClpPkd on protein translation in the cytosol via the processing of nuclear-encoded RNA, coupled with metabolic changes indicative of impaired fatty acid oxidation and pentose phosphate pathway. Finally, mass spectrometry of ClpPmut-entrapped partners identified myeloma-specific mitochondrial substrates, including chaperones and enzymes involved in RNA metabolism and oxidative stress. RNA-seq analyses are further characterizing the pathways impacted by ClpPkd and guiding wet validation experiments. Conclusions Overall, our data strongly suggest that ClpP is vital to MM cells due to a novel non-bioenergetic function, and that its manipulation is lethal via a broad perturbation of mitochondrial and cellular homeostasis.

Background Tumor-associated macrophages, derived from peripheral blood monocytes, support malignant plasma cell proliferation in the bone marrow (BM). Since peripheral blood absolute monocyte count (AMC) could reflect the BM microenvironment, we sought to evaluate the prognostic significance of AMC in multiple myeloma (MM). Methods We used nationwide Veterans Affairs electronic health records to include treatment-naive MM patients diagnosed between 2000 and 2019 without concomitant aplastic anemia, myelodysplastic syndrome, myeloproliferative neoplasm, and acute or chronic leukemia. We obtained AMC (×10-3) closest to and within 90 days prior to each timepoint: at diagnosis and every 3 months from diagnosis up to 2.5 years. Patients were stratified by AMC: low (1.25). Our selection criteria excluded any treatment-related change in AMC. Overall survival (OS) was evaluated using Kaplan-Meier estimator and logrank tests. Cox models were used for multivariable analysis. Results We identified and analyzed 10,822 patients (median age 70 years; interquartile range [IQR] 63-77) with a median follow up of 2.9 years (IQR 1.3-5.3). At diagnosis, 25.3% of patients presented with abnormal AMC. Patients with low, severely elevated, elevated, and normal AMC at diagnosis had median OS of 2.3, 2.7, 3.1, and 3.6 years (p Conclusion Abnormal AMC in MM at diagnosis or follow up is significantly associated with inferior OS, independent of known prognostic factors. Survival was also inferior for patients who had normal AMC at diagnosis and developed abnormal AMC during follow up, possibly suggesting changes in the BM microenvironment. Overall, AMC is a readily available metric that could be included in the risk stratification of MM at diagnosis and beyond.

Background Multiple myeloma (MM) is an incurable hematologic malignancy requiring long-term, continuous therapy. Although caregivers of MM patients play a critical role in supporting their loved ones throughout the illness course, studies examining caregiver quality of life (QOL), psychological distress, and prognostic awareness are lacking. Methods We conducted a cross-sectional, multisite study of patients undergoing treatment with MM (excluding maintenance therapy) and their caregivers between 6/2020-3/2021. Eligible caregivers were identified by the patient as the primary caregiver and enrolled to 1 of 3 cohorts based on lines of therapy: 1) newly diagnosed receiving first-line therapy; 2) 2-3 lines; 3) ≥4 lines of therapy. Caregivers completed validated questionnaires to assess their QOL, psychological distress, and perceptions of prognosis. Patients also completed validated questions to assess their perception of prognosis. We used descriptive statistics to describe caregiver QOL, psychological distress, and perception of prognosis. We then descriptively compared psychological distress and perception of prognosis between patient and caregiver dyads. Results We enrolled 127 caregivers of MM patients (newly diagnosed (n=43), 2-3 (n=40), and ≥ 4 lines of therapy (n=44)). Median caregiver age was 61.8 years (range 24.0-81.9); 71.7% (91/127) were female. Caregiver QOL and psychological distress did not differ by lines of therapy. The rate of clinically significant depression, anxiety, and PTSD symptoms were 15.8% (20/127), 44.1% (56/127), and 24.4% (31/127), respectively. When examining dyads, caregivers reported higher rates of clinically significant anxiety symptoms (44.4% vs. 22.5%) compared to MM patients. Caregivers reported less clinically significant depression symptoms (15.3% vs. 24.2%) and similar rates of clinically significant PTSD symptoms (24.2% vs 25.0%). Overall, 89.6% (112/125) of caregivers reported that it is ‘extremely’ or ‘very’ important to know about the patient’s prognosis and 55.6% (70/126) stated that they had received adequate information regarding the patient’s prognosis. Most caregivers (84.2%, 101/120), reported that the oncologist told them the patient’s cancer was incurable. In contrast, only 53.6% (59/110) of caregivers reported that they thought the patient’s cancer was incurable and 37.9% (58/114) acknowledged that the patient is terminally ill. When examining dyads, caregivers were more likely to report that the patient is terminally ill (50.1% vs. 30.1%). There was no difference in caregivers’ and patients’ report that the oncologist said MM is incurable (83.3% vs. 84.2%). Conclusions Caregivers of patients undergoing treatment for MM experience substantial psychological distress across the disease continuum. Interventions are needed to improve caregivers’ QOL, to reduce their psychological distress and to enhance caregiver perceptions of the patient’s prognosis to facilitate informed decision-making.

Introduction CAR T cells have demonstrated unique potency for tumor cytoreduction and durable response in hematological malignancies. However, disease relapse remains a concern due to the emergence of antigen negative variants, tolerization of CAR T cell populations and lack of T cell persistence. We postulated that vaccination with a personalized cancer vaccine, that we developed, in which patient derived tumor cells are fused with autologous dendritic cells would enhance CAR T cell efficacy through the expansion of T cell clonal populations and the vaccine mediated enhancement of T cell activation and persistence. In the present study, we examined the potential synergy between CAR T cells targeting CD19 and syngeneic DC/tumor fusions. Methods/Results CD19 CAR T cells and DC/tumor fusions were studied in the context of a murine A20 lymphoma model, and their combination effectively lysed A20 cells in a CTL assay in a higher percentage than CAR T cells alone (20% vs 34%). Moreover, we examined the interaction of vaccine and CAR T cells ex vivo using the IncuCyte S3 Live-Cell Analysis System which allows for live cell visualization of lysis of A20 cells over time. We studied the impact of combining vaccine educated and CAR T cells as well as an individual T cell population that underwent sequential vaccine mediated stimulation followed by transduction with the CD19 CAR. Even in this assay, coculture with either combined vaccine educated and CAR T cells or sequentially vaccine educated, and transduced T cells demonstrated the highest levels of cytotoxicity that was maintained over time. Enhanced lysis by combined vaccine stimulation and CAR T cells was similarly demonstrated in another tumor cell line, 5TGM1, a multiple myeloma cell line transduced to express CD19. On the contrary wild type 5TGM1 cells were recognized by the DC/tumor fusion stimulated cells in contrast to CAR T cells alone (40% vs. 8%). Finally, we examined the capacity of vaccine educated T cells in conjunction with CAR T cells to target A20 cells in an immunocompetent murine model. Mice were challenged with 1 × 106 A20 Mcherry-Luc and after lymphoma engraftment animals were treated with 3 × 106 T cells consisting of CAR T cells, vaccine educated T cells or the combination. Serial bioluminescence imaging demonstrated greatest reduction in tumor burden using combined CAR T and vaccine educated T cells with 4/5 animals still disease free at day 13 after tumor challenge. Conclusions Combined therapy with T cells stimulated by DC/tumor fusions and CAR T cells exhibited potent lysis of murine lymphoma and myeloma cells compared to CAR T cells or vaccine educated T cells alone in in vitro and immunocompetent murine models. These findings suggest potent synergy between these modalities that may overcome recognized pathways of resistance including the broadening of the tumor specific response and vaccine mediated activation of CAR T cell populations.

INTRODUCTION: Cancer pathogenesis is usually characterized by a long evolutionary process where genomic driver events accumulate over time, conferring advantage to distinct subclones, allowing their expansion and progression. METHODS: To investigate the multiple myeloma (MM) evolutionary history, we characterized the mutational processes' landscape and activity over time utilizing a large cohort of 89 whole genomes and 973 exomes. To improve the accuracy of mutational signatures analysis, we analyzed both the 3' and 5' nucleotide context of each mutation and we developed the novel fitting algorithm mmSig, which fits the entire mutational catalogue of each patient with the mutational signatures involved in MM pathogenesis. The contribution of each mutational signature was then corrected based on the cosine similarity between the original 96-mutational profile and the reconstructed profile generated without that signature. To reconstruct the genetic evolutionary history of each patient's cancer, we integrated two approaches. First dividing all mutations into clonal (early) or subclonal (late), then subdivided the clonal mutations into duplicated mutations (present on two alleles and therefore acquired before the duplication) or non-duplicated mutations (detected on a single allele), reflecting either pre-gain and post-gain mutations on the minor allele, or post-gain mutations acquired on one of the duplicated alleles. RESULTS Eight mutational signatures were identified, seven of which showed significant similarity with the most recent mutational signature catalogue (i.e SBS1, SBS2, SBS5, SBS8, SBS9, SBS13 and SBS18). The new mutational signature (named SBS-MM1) was observed only among relapsed patients exposed to alkylating agents (i.e melphalan). The etiology of this specific signature was further confirmed by analyzing recent whole genomes public data from human-induced pluripotent stem cells exposed to melphalan (Kucab et al, Cell 2019). Reconstructing the chronological activity of each mutational signature, we identified four different routes to acquire the full mutational spectrum in MM based on the differential temporal activity of AID (SBS9) and APOBEC (SBS2 and SBS13). Our data indicate that AID activity is not limited to the first contact with the GC, but persists in the majority of patients, behaving similarly to a B-memory cells, capable of re-entering the germinal center upon antigen stimulation to undergo clonal expansion several times before MM diagnosis. Next, we confirmed the clock-like nature (i.e constant mutation rate) of SBS5 in MM and other post-germinal center disorders such as chronic lymphocytic leukemia and B-cell lymphomas. Based on the SBS5 mutation rates and the corrected ratio between duplicated and non-duplicated mutations within large chromosomal gains, we could time the acquisition of the first copy number gain during the life history of each MM patient. Intriguingly, the first MM chromosomal duplication was acquired on average 38 years (ranges 11-64) before sample collection. In 23/27 (85%) cases the first multi gain event occurred before 30 years of age, and in 13/27 (48%) before 20 years reflecting a long and slow process potentially influenced and accelerated by extrinsic and intrinsic factors. DISCUSSION Our analysis provides a glimpse into the early stages of myelomagenesis, where acquisition of the first key drivers precedes cancer diagnosis by decades. Defining the time window when transformation occurs opens up for new avenues of research: to identify causal mechanisms of disease initiation and evolution, to better define the optimal time to start therapy, and ultimately develop early prevention strategies. Disclosures Bolli: CELGENE: Honoraria; JANSSEN: Honoraria; GILEAD: Other: Travel expenses. Corradini:Janssen: Honoraria, Other: Travel Costs; Jazz Pharmaceutics: Honoraria; KiowaKirin: Honoraria; Servier: Honoraria; Takeda: Honoraria, Other: Travel Costs; Kite: Honoraria; Novartis: Honoraria, Other: Travel Costs; Gilead: Honoraria, Other: Travel Costs; Roche: Honoraria; Sanofi: Honoraria; BMS: Other: Travel Costs. Anderson:Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board. Moreau:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Papaemmanuil:Celgene: Research Funding. Avet-Loiseau:takeda: Consultancy, Other: travel fees, lecture fees, Research Funding; celgene: Consultancy, Other: travel fees, lecture fees, Research Funding. Munshi:Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Abbvie: Consultancy. Landgren:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Theradex: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Epigenomic changes have become an important component of cellular regulation and ultimately, of our understanding of oncogenomics in Multiple Myeloma (MM) as well as in other cancers. In recent years, both clinical and preclinical studies have confirmed that MM is vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets. Although Pan-HDAC inhibitors are effective as therapeutic agents, there is increasing emphasis on understanding the biological and molecular roles of individual HDACs. Here we have evaluated the role of HDAC8, a member of Class I HDAC isoenzymes in MM. First, we evaluated the expression of HDAC8 in 172 newly-diagnosed MM patients from the IFM myeloma dataset and observed HDAC8 overexpression as well as its significant correlation with poor survival outcome (p To address the functional role of HDAC8 in MM biology and to evaluate its potency as therapeutic target, we used a lentiviral-shRNA delivery system for HDAC8-knockdown in MM1S and OPM2 myeloma cells. The HDAC8 depletion in HMCLs resulted in significant inhibition of proliferation of MM at 1 week as measured by 3[H]-thymidine assay, and as decrease in colony formation evaluated after 3 weeks post transfection (p In conclusion, our results demonstrate an impact of aberrant epigenome on DNA integrity through connection between HDAC8 and DDR pathway, and provide insights into the effect of HDAC8 on cellular growth and survival with potent therapeutic implications in MM. Disclosures Anderson:Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy.

Multiple myeloma (MM) is an incurable malignancy in majority of patients characterized by clonal proliferation of plasma cells. To date, treatment is established based on general conditions and age of patients. However, MM is a heterogeneous disease, featured by various subtypes and different outcomes. Thus, the understanding of MM biology is currently a major challenge to eventually cure the disease. During the last decade, karyotype studies and gene expression profiling have identified robust prognostic markers as well as a widespread genomic landscape. More recently, studies of epigenetic, transcriptional modifications and next generation sequencing have allowed characterization of critical genes and pathways, clonal heterogeneity and mutational profiles involved in myelomagenesis. Altogether, these findings constitute important tools to develop new targeted and personalized therapies in MM.