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The bone marrow (BM) microenvironment actively promotes multiple myeloma pathogenesis, and therapies targeting both cancer cells and the niche are highly effective. We were interested in identifying novel signaling pathways supporting multiple myeloma–BM cross-talk. Mutations in the transmembrane receptor Roundabout 1 (ROBO1) were recently identified in patients with multiple myeloma; however, their functional consequences are uncertain. Through protein structure–function studies, we discovered that ROBO1 is necessary for multiple myeloma adhesion to BM stromal and endothelial cells and that ROBO1 knockout (KO) compromises BM homing and engraftment in a disseminated mouse model. ROBO1 KO significantly decreases multiple myeloma proliferation in vitro and intra- and extramedullary tumor growth in vivo. Mechanistically, the ROBO1 C-terminus is cleaved in a ligand-independent fashion and is sufficient to promote multiple myeloma proliferation. Vice versa, mutants lacking the cytoplasmic domain, including the human-derived G674* truncation, act dominantly negative. Interactomic and RNA-sequencing studies suggest that ROBO1 may be involved in RNA processing, supporting further studies.Significance:ROBO1 is highly expressed in t(4;14) multiple myeloma and supports homing and dissemination to the BM niche. ROBO1 knockout causes reduced tumor growth in intramedullary and extramedullary myeloma animal models, while the ROBO1 C-terminus is cleaved in multiple fragments and it is necessary and sufficient to sustain myeloma proliferation.

Supplementary Figure 1 showing ROBO1 expression in primary MM cells versus normal donor bone marrow plasma cells via gene expression profiling and immunohistochemistry. Supplementary Figure 2 showing distribution of ROBO1 expression in the CoMMpass database; ROBO1 expression in CoMMpass database according to cytogenetics; ROBO1 expression in IFM170 database according to cytogenetics; ROBO1 expression in t(4;14) cell lines WT or KO for MMSET. Supplementary Figure 3 showing ROBO1 protein down regulation after ROBO1 knock down via shRNA. Supplementary Figure 4 showing effects of shRNA4- and shRNA-5 mediated ROBO1 KD on cell viability across a panel of MM cell lines with western confirming proper protein down regulation. Supplementary Figure 5 showing successful ROBO1 KO across several KMS11 monoclones, but not H929 monoclones. Supplementary Figure 6 showing expression of ROBO1 FL construct in polyclonally and monoclonally-transduced OPM2 cells. Supplementary Figure 7 showing expression of FL ROBO1 addback in distinct KMS11 ROBO1 KO monoclones. Supplementary Figure 8 showing radiographic images of WT versus KO ROBO1 mu-SCID mice. Supplementary Figure 9 showing adhesion defect in ROBO1 KO KMS11 cells that is fully rescued by FL ROBO1 addback. Supplementary Figure 10 showing Kaplan-Meier survival curve of disseminated mouse model injected with ROBO1 KO versus FL OPM2 cells. Supplementary Figure 11 showing cleaved cytosolic ROBO1 fragments in 293T cells transduced with FL ROBO1 tagged with C-LAP tag and in WT MM.1S cells. Supplementary Figure 12 showing interacting partners of ROBO1 cytosolic domain as deconvoluted via STRING database. Supplementary Figure 13 showing differentially expressed genes in ROBO1 WT versus KO plasmacytoma represented via Volcano plot. Supplementary Figure 14 showing gene set enrichment analysis of differentially expressed genes in ROBO1 WT versus KO plasmacytoma. Supplementary Figure 15 showing schema of patient-derived ROBO1 mutation and expression of G674 truncation in WT and KO ROBO1 OPM2 cells. Supplementary Table 1 showing clinical annotation of newly diagnosed MM parents used in Supplementary figure 1.

Supplementary Methods

Multiple myeloma (MM) is a disease of clonally differentiated plasma cells. MM is almost always preceded by precursor conditions, monoclonal gammopathy of unknown significance (MGUS), and smoldering MM (SMM) through largely unknown molecular events. Genetic alterations of the malignant plasma cells play a critical role in patient clinical outcomes. Del(17p), t(4;14), and additional chromosomal alterations such as del(1p32), gain(1q) and MYC translocations are involved in active MM evolution. Interestingly, these genetic alterations appear strikingly similar in transformed plasma cell (PC) clones from MGUS, SMM, and MM stages. Recent studies show that effectors of the innate and adaptive immune response show marked dysfunction and skewing towards a tolerant environment that favors disease progression. The MM myeloid compartment is characterized by myeloid-derived suppressor cells (MDSCs), dendritic cells as well as M2-like phenotype macrophages that promote immune evasion. Major deregulations are found in the lymphoid compartment as well, with skewing towards immune tolerant Th17 and Treg and inhibition of CD8+ cytotoxic and CD4+ activated effector T cells. In summary, this review will provide an overview of the complex cross-talk between MM plasma cells and immune cells in the microenvironment and the molecular mechanisms promoting progression from precursor states to full-blown myeloma.

Orthostatic hypotension (OH) is a well-recognized phenomenon occurring in multiple myeloma (MM) patients undergoing autologous stem cell transplant (ASCT), and is associated with significant morbidity and mortality. A retrospective analysis of patients admitted for first ASCT between June 2012 and April 2014 found that 161/222 (73%) patients were diagnosed with OH during the course of ASCT, including 51 patients who were found to have OH on the day of first orthostatic vitals check. Excluding these 51 patients, 110/171 (64%) patients developed OH during the peri-transplant period, at a median of 7 days post ASCT (95% CI: 6.5–8.5). OH did not significantly impact length of hospitalization, progression free and overall survival. Multivariable analysis revealed four risk factors (i.e. ≥0.5% weight loss/day, white race, gabapentin, antihypertensives) and two protective factors (i.e. antihistamine, proton pump inhibitor) associated with the development of peri-transplant OH.

Multiple myeloma (MM) is a cancer of terminally differentiated plasma cells. MM remains incurable, but overall survival of patients has progressively increased over the past two decades largely due to novel agents such as proteasome inhibitors (PI) and the immunomodulatory agents. While these therapies are highly effective, MM patients can be de novo resistant and acquired resistance with prolonged treatment is inevitable. There is growing interest in early, accurate identification of responsive versus non-responsive patients, however limited sample availability and need for rapid assays are limiting factors. Here, we test dry mass and volume as label-free biomarkers to monitor early response of MM cells to treatment with bortezomib, doxorubicin, and ultraviolet light. For the dry mass measurement, we use two types of phase-sensitive optical microscopy techniques: digital holographic tomography and computationally-enhanced quantitative phase microscopy. We show that human MM cell lines (RPMI8226, MM.1S, KMS20, and AMO1) increase dry mass upon bortezomib treatment. This dry mass increase after bortezomib treatment occurs as early as 1 hour for sensitive cells and 4 hours for all tested cells. We further confirm this observation using primary multiple myeloma cells derived from patients and show that a correlation exists between increase in dry mass and sensitivity to bortezomib, supporting the use of dry mass as a biomarker. The volume measurement using Coulter counter shows a more complex behavior; RPMI8226 cells increase the volume at an early stage of apoptosis, but MM.1S cells show the volume decrease typically observed with apoptotic cells. Altogether, this cell study presents complex kinetics of dry mass and volume at an early stage of apoptosis, which may serve as a basis for the detection and treatment of MM cells.

Multiple myeloma (MM) is a highly heterogenous disease that exists along a continuous disease spectrum starting with premalignant conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) that inevitably precede MM. Over the past two decades, significant progress has been made in the genetic characterization and risk stratification of precursor plasma cell disorders. Indeed, the clinical introduction of highly effective and well-tolerated drugs begs the question: would earlier therapeutic intervention with novel therapies in MGUS and SMM patients alter natural history, providing a potential curative option? In this review, we discuss the epidemiology of MGUS and SMM and current models for risk stratification that predict MGUS and SMM progression to MM. We further discuss genetic heterogeneity and clonal evolution in MM and the interplay between tumor cells and the bone marrow (BM) microenvironment. Finally, we provide an overview of the current recommendations for the management of MGUS and SMM and discuss the open controversies in the field in light of promising results from early intervention clinical trials.

The bone marrow (BM) microenvironment actively promotes multiple myeloma pathogenesis, and therapies targeting both cancer cells and the niche are highly effective. We were interested in identifying novel signaling pathways supporting multiple myeloma–BM cross-talk. Mutations in the transmembrane receptor Roundabout 1 (ROBO1) were recently identified in patients with multiple myeloma; however, their functional consequences are uncertain. Through protein structure–function studies, we discovered that ROBO1 is necessary for multiple myeloma adhesion to BM stromal and endothelial cells and that ROBO1 knockout (KO) compromises BM homing and engraftment in a disseminated mouse model. ROBO1 KO significantly decreases multiple myeloma proliferation in vitro and intra- and extramedullary tumor growth in vivo. Mechanistically, the ROBO1 C-terminus is cleaved in a ligand-independent fashion and is sufficient to promote multiple myeloma proliferation. Vice versa, mutants lacking the cytoplasmic domain, including the human-derived G674* truncation, act dominantly negative. Interactomic and RNA-sequencing studies suggest that ROBO1 may be involved in RNA processing, supporting further studies. Significance: ROBO1 is highly expressed in t(4;14) multiple myeloma and supports homing and dissemination to the BM niche. ROBO1 knockout causes reduced tumor growth in intramedullary and extramedullary myeloma animal models, while the ROBO1 C-terminus is cleaved in multiple fragments and it is necessary and sufficient to sustain myeloma proliferation.

AL amyloidosis (AL) is an incurable plasma cell disorder. The solely pathogenic mechanism in AL is deposition of immunoglobulin free light chains (FLC) organized in fibrils in target organs. Surprisingly, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, which are the specific target of botulinum neurotoxin (BoNT), are involved in the docking and fusion of secretory vesicles. We hypothesized that certain BoNT serotypes may block FLC secretion and trigger cytotoxicity triggering a terminal UPR. Gene expression profiling in a cohort of 170 newly diagnosed multiple myeloma patients (IFM170) was used to interrogate SNAREs expression in malignant plasma cells. We developed tetracycline inducible, bicistronic vectors expressing distinct BoNT serotypes (BoNT/A-F), T2A and GFP. Lentivirally transduced cells would express BoNT in a 1:1 stochiometric ratio with GFP, upon doxycycline (dox) administration. We transduced AL cell lines stably expressing rRTA with Tet-On lentivirus expressing 7 distinct BoNTs and performed two sets of experiments. First, we performed time-course viability assays on polyclonally transduced cells and compared relative proportion of GFP+ cells over time. Then, we single-cell sorted transduced cells, triggered BoNT expression and assessed GFP kinetic and apoptosis at 24, 48 and 72 hours post dox. SNAREs cleavage following induction of BoNT expression was evaluated via WB in GFP+ clones. To assess if BoNT cytotoxicity correlated with cessation of FLC secretion, we performed a secretion assay in monoclones expressing distinct BoNTs. IFM170 GEP and AL/MM cell lines analysis showed VAMP2, VAMP3 and SNAP23 as the top expressed SNAREs. By using polyclonally transduced cells, we show that GFP+ (transduced) cells are rapidly depleted over time after dox, across all serotypes, except BoNT/B, consistent with cytotoxic effect. We noted an association between SNAP23 and VAMP3 cleavage and BoNT toxicity, suggesting that dual targeting of SNAP23/VAMP3 may be necessary to mediate BoNT cytotoxicity. We next show that only BoNTs causing early cytotoxicity (A, D, E and F) significantly inhibited FLC secretion and induced UPR activation, presumably through FLC retention. Indeed, cytotoxic BoNTs, activated PERK pathway with eIF2a phosphorylation (p-eIF2a); ATF4, CHOP and GADD34 upregulation. Importantly preliminary data in CHOP KO AL cells show rescue of BoNT_induced death, confirming that a terminal UPR is the mechanism of cytotoxicity of BoNT in AL. We show that in all seven BoNT serotypes examined, except for BoNT/B, simultaneous cleavage of SNAP23 and VAMP3 predicts BoNT cytotoxicity and correlates with cessation of FLC secretion and terminal UPR. Overall, our preliminary data provide proof of concept that targeting FLC secretion is feasible and of therapeutic efficacy in preclinical AL models, suggesting potential clinical translatability of this innovative approach. Citation Format: Maria Moscvin, Peter Gregor Czarnecki, Tianzeng Chen, Annamaria Gullá, Kenneth Carl Anderson, Giada Bianchi. Targeting protein secretion as a novel therapeutic strategy in AL amyloidosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 141.

Background AL amyloidosis (AL) is an incurable plasma cell (PC) disorder. The solely pathogenic mechanism in AL is deposition of immunoglobulin free light chains (FLC) organized in fibrils in target organs. Surprisingly, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, which are the specific target of botulinum neurotoxin (BoNT), are involved in the docking and fusion of secretory vesicles. We hypothesized that certain BoNT serotypes may block FLC exocytosis, causing retention of FLC-loaded vesicles and triggering a terminal unfolded protein response (UPR). Methods Gene expression profiling in a cohort of 170 newly diagnosed multiple myeloma (MM) patients (IFM170) was used to interrogate SNAREs expression in malignant plasma cells. Western blotting (WB) was used to assess SNARE expression across MM and AL cell lines. We developed tetracycline inducible, lentiviral vectors expressing distinct BoNT serotypes (BoNT/A-F), T2A and GFP. Lentivirally transduced cells would express BoNT in a 1:1 stochiometric ratio with GFP, upon doxycycline (dox) administration, allowing for flow cytometry-based analysis. A vector comprising solely T2A and GFP was used as negative control. We transduced AL cell lines with Tet-On lentivirus expressing 7 distinct BoNTs and performed two sets of experiments. First, we performed time-course viability assays on polyclonally transduced cells and compared relative proportion of GFP+ cells over time. Then, we single-cell sorted transduced cells, triggered BoNT expression and assessed GFP kinetic and apoptosis via AnnexinV/DAPI flow cytometry at 24, 48 and 72 hours post dox. SNAREs cleavage following induction of BoNT expression was evaluated via WB in GFP+ clones. To assess if BoNT cytotoxicity correlated with cessation of FLC secretion, we performed a secretion assay in monoclones expressing distinct BoNTs. Results IFM170 GEP analysis showed VAMP2, VAMP3 and SNAP23 as the top expressed SNAREs. This was further confirmed in AL/MM cell lines. By using polyclonally transduced cells, we show that GFP+ cells are rapidly depleted over time after dox, across all serotypes, except BoNT/B, consistent with cytotoxic effect. Similarly, we observed rapid apoptosis in monoclones expressing any BoNT serotypes, except BoNT/B. We noted an association between SNAP23 and VAMP3 cleavage and BoNT toxicity, suggesting that dual targeting of SNAP23/VAMP3 may be necessary to mediate BoNT cytotoxicity. We next show that only BoNTs causing early cytotoxicity significantly inhibited FLC secretion. Cytotoxic BoNTs, activated PERK pathway with eIF2a phosphorylation (p-eIF2a); CHOP and GADD34 upregulation, presumably through FLC retention. Conclusions We show that cytotoxic BoNTs block FLC secretion, trigger a terminal UPR and induce apoptosis in AL and MM models. We provide proof of concept that targeting FLC secretion has a potential clinical translatability.

Multiple myeloma (MM) is a plasma cell disorder typically characterized by abundant synthesis of clonal immunoglobulin or free light chains. Although incurable, a deeper understanding of MM pathobiology has fueled major therapeutical advances over the past two decades, significantly improving patient outcomes. Proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies are among the most effective anti-MM drugs, targeting not only the cancerous cells, but also the bone marrow microenvironment. However

Key Points MM cells suffer from proteotoxicity and depend on NRF1, the master regulator of proteasome stress response, for survival.KO of NRF1 or its activating protease DDI2 is cytotoxic in myeloma and overcomes de novo and acquired PI resistance., Visual Abstract, Multiple myeloma (MM) cells suffer from baseline proteotoxicity as the result of an imbalance between the load of misfolded proteins awaiting proteolysis and the capacity of the ubiquitin-proteasome system to degrade them. This intrinsic vulnerability is at the base of MM sensitivity to agents that perturb proteostasis, such as proteasome inhibitors (PIs), the mainstay of modern-day myeloma therapy. De novo and acquired PI resistance are important clinical limitations that adversely affect prognosis. The molecular mechanisms underpinning PI resistance are only partially understood, limiting the development of drugs that can overcome it. The transcription factor NRF1 is activated by the aspartic protease DNA damage inducible 1 homolog 2 (DDI2) upon proteasome insufficiency and governs proteasome biogenesis. In this article, we show that MM cells exhibit baseline NRF1 activation and are dependent upon DDI2 for survival. DDI2 knockout (KO) is cytotoxic for MM cells, both in vitro and in vivo. Protein structure-function studies show that DDI2 KO blocks NRF1 cleavage and nuclear translocation, causing impaired proteasome activity recovery upon irreversible proteasome inhibition and, thereby, increasing sensitivity to PIs. Add-back of wild-type, but not of catalytically dead DDI2, fully rescues these phenotypes. We propose that DDI2 is an unexplored promising molecular target in MM by disrupting the proteasome stress response and exacerbating proteotoxicity.

Multiple myeloma (MM), the second most common hematological malignancy, is an incurable cancer of plasma cells. MM cells diffusely involves the bone marrow (BM) and establish a close interaction with the BM niche that in turn supports MM survival, proliferation, dissemination and drug resistance. In spite of remarkable progress in understanding MM biology and developing drugs targeting MM in the context of the BM niche, acquisition of multi-class drug resistance is almost universally inevitable. Exosomes are small, secreted vesicles that have been shown to mediate bidirectional transfer of proteins, lipids, and nucleic acids between BM microenvironment and MM, supporting MM pathogenesis by promoting angiogenesis, osteolysis, and drug resistance. Exosome content has been shown to differ between MM patients and healthy donors and could potentially serve as both cancer biomarker and target for novel therapies. Furthermore, the natural nanostructure and modifiable surface properties of exosomes make them good candidates for drug delivery or novel immunomodulatory therapy. In this review we will discuss the current knowledge regarding exosome’s role in MM pathogenesis and its potential role as a novel biomarker and therapeutic tool in MM.

Background AL amyloidosis (AL) is an incurable plasma cell (PC) disorder. The solely pathogenic mechanism in AL is deposition of immunoglobulin free light chains (FLC) organized in fibrils in target organs. Surprisingly, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, which are the specific target of botulinum neurotoxin (BoNT), are involved in the docking and fusion of secretory vesicles. We hypothesized that certain BoNT serotypes may block FLC exocytosis, causing retention of FLC-loaded vesicles and triggering a terminal unfolded protein response (UPR). Materials and Methods Gene expression profiling in a cohort of 170 newly diagnosed multiple myeloma (MM) patients (IFM170) was used to interrogate SNAREs expression in malignant plasma cells. Western blotting (WB) was used to assess SNARE expression across MM and AL cell lines. We developed tetracycline inducible, lentiviral vectors expressing distinct BoNT serotypes (BoNT/A-F), T2A and GFP. Lentivirally transduced cells would express BoNT in a 1:1 stochiometric ratio with GFP, upon doxycycline (dox) administration, allowing for flow cytometry-based analysis. A vector comprising solely T2A and GFP was used as negative control. We transduced AL cell lines with Tet-On lentivirus expressing 7 distinct BoNTs and performed two sets of experiments. First, we performed time-course viability assays on polyclonally transduced cells and compared relative proportion of GFP+ cells over time. Then, we single-cell sorted transduced cells, triggered BoNT expression and assessed GFP kinetic and apoptosis via AnnexinV/DAPI flow cytometry at 24, 48 and 72 hours post dox. SNAREs cleavage following induction of BoNT expression was evaluated via WB in GFP+ clones. To assess if BoNT cytotoxicity correlated with cessation of FLC secretion, we performed a secretion assay in monoclones expressing distinct BoNTs. Results IFM170 GEP analysis showed VAMP2, VAMP3 and SNAP23 as the top expressed SNAREs. This was further confirmed in AL/MM cell lines. By using polyclonally transduced cells, we show that GFP+ cells are rapidly depleted over time after dox, across all serotypes, except BoNT/B, consistent with cytotoxic effect. Similarly, we observed rapid apoptosis in monoclones expressing any BoNT serotypes, except BoNT/B. We noted an association between SNAP23 and VAMP3 cleavage and BoNT toxicity, suggesting that dual targeting of SNAP23/VAMP3 may be necessary to mediate BoNT cytotoxicity. We next show that only BoNTs causing early cytotoxicity significantly inhibited FLC secretion. Cytotoxic BoNTs, activated PERK pathway with eIF2a phosphorylation (p-eIF2a); CHOP and GADD34 upregulation, presumably through FLC retention. Conclusions We show that cytotoxic BoNTs block FLC secretion, trigger a terminal UPR and induce apoptosis in AL and MM models. We provide proof of concept that targeting FLC secretion has a potential clinical translatability. Disclosures Czarnecki: Clearview: Consultancy. Anderson: Millenium-Takeda: 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; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Bianchi: Karyopharm: Consultancy, Honoraria; MJH: Honoraria; Jacob D. Fuchsberg Law Firm: Consultancy; Pfizer: Consultancy, Honoraria.

Introduction Vascular endothelial injury related to treatment with proteasome inhibitors (PI) has been previously described. Carfilzomib is an irreversible PI and has been associated with cardiovascular toxicity, suggesting increased risk of endothelial injury. Thrombotic microangiopathy (TMA) has been described in multiple myeloma (MM) patients receiving PIs; more often with carfilzomib. In the pediatric transplant population, increased risk of TMA was related to heterozygous mutation in the alternative complement pathway. The homozygous deletions enable uncontrolled complement activation and was found three times more frequently in TMA population. We hypothesized that MM patients with complement mutation are at increased risk of PI-related TMA. Materials and Methods We identified ten cases of renal TMA in MM patients receiving carfilzomib from two medical institutions in Greece and in the United States. TMA was diagnosed based on either renal biopsy or acute kidney injury, new-onset anemia, thrombocytopenia and increased LDH in the absence of disease progression. We performed targeted sequencing of the twelve genes implicated in TMA: CFH, CFI, MCP, CFB, CFHR5, C3, THBD, DGKE, PLG, ADAMTS13, MMACHC and G6PD genes. Multiplex Ligation-Dependent Probe Amplification (MLPA) was performed for the analysis of the CFH-CFHR5 region. Results Patient characteristics and laboratory values at TMA diagnosis are presented in Table 1. The median age of patients was 68 years (range, 47-73), with slight male predominance (60%). Median laboratory values at diagnosis included hemoglobin 9.35 g/dL, platelet count 26,500 x 10 6/L, LDH 356,5 U/L and creatinine 2.25 mg/dL. Patients were treated with carfilzomib doses ranging 20-70 mg/m 2. Regimens included carfilzomib-dexamethasone (Kd, 7 patients), carfilzomib-lenalidomide-dexamethasone (KRd, 1 patient), carfilzomib-pomalidomide-dexamethasone (KPd, 1 patient), and carfilzomib-daratumumab-dexamethasone (DaraKd, 1 patient). All patients had previously received at least one line of therapy and seven patients had previously undergone autologous stem cell transplant (ASCT), 1-12 years prior to TMA diagnosis. The median time between carfilzomib initiation and TMA diagnosis was 4.5 months (range, 1-60 months). Diagnosis was confirmed with renal biopsy in four cases. Carfilzomib was discontinued in all patients and five patients were treated with plasma exchange (PLEX) while one patient received eculizumab. Seven patients demonstrated clinical improvement and resolution of TMA at 1 year after discontinuation of carfilzomib. Two patients progressed to end stage renal disease (ESRD) requiring intermittent hemodialysis, and one patient developed multiorgan failure. Results of genetic panel are shown in Table 2. Deletions of the CFHR3-CFHR5 region were present in seven cases (70%): two patients carrying a homozygous deletion of CFHR3-CFHR1, four patients with a heterozygous deletion of CFHR3-CFHR1, and one patient with heterozygous deletion of CFHR1-CFHR4. Direct gene sequencing revealed identifiable mutations in CD46 (MCP) and CFHR5 in two distinct patients. The functional correlation and clinical significance are yet to be investigated. Conclusions In our cohort of ten patients of carfilzomib-induced TMA, deletions of CFHR3-CFHR5 occurred frequently (70%). In the setting of carfilzomib use, heterozygous CFHR3-CFHR1 deletion may represent a risk factor for the development of TMA. Our data set the bases for larger studies assessing complement mutation as a predisposing factor for PI-induced TMA. Figure 1 Figure 1. Disclosures Kastritis: Amgen: Consultancy, Honoraria, Research Funding; Genesis Pharma: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding. Dimopoulos: Janssen: Honoraria; BMS: Honoraria; Takeda: Honoraria; Beigene: Honoraria; Amgen: Honoraria. Richardson: AbbVie: Consultancy; Secura Bio: Consultancy; Sanofi: Consultancy; GlaxoSmithKline: Consultancy; Karyopharm: Consultancy, Research Funding; Regeneron: Consultancy; AstraZeneca: Consultancy; Oncopeptides: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy; Celgene/BMS: Consultancy, Research Funding; Protocol Intelligence: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Bianchi: Jacob D. Fuchsberg Law Firm: Consultancy; MJH: Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

IT-901 is a novel and selective NF-κB inhibitor with promising activity in pre-clinical models. Here we show that treatment of chronic lymphocytic leukemia cells (CLL) with IT-901 effectively interrupts NF-κB transcriptional activity. CLL cells exposed to the drug display elevated mitochondrial reactive oxygen species, which damage mitochondria, limit oxidative phosphorylation and ATP production, and activate intrinsic apoptosis. Inhibition of NF-κB signaling in stromal and myeloid cells, both tumor-supportive elements, fails to induce apoptosis, but impairs NF-κB-driven expression of molecules involved in cell-cell contacts and immune responses, essential elements in creating a pro-leukemic niche. The consequence is that accessory cells do not protect CLL cells from IT-901-induced apoptosis. In this context, IT-901 shows synergistic activity with ibrutinib, arguing in favor of combination strategies. IT-901 is also effective in primary cells from patients with Richter syndrome (RS). Its anti-tumor properties are confirmed in xenograft models of CLL and in RS patient-derived xenografts, with documented NF-κB inhibition and significant reduction of tumor burden. Together, these results provide pre-clinical proof of principle for IT-901 as a potential new drug in CLL and RS.

Multiple myeloma (MM) is a currently incurable hematologic tumor with heterogeneous clinical behavior and prognosis. During the last years, survival improved due to a better understanding of MM biology and the development of novel drugs, although it still remains unsatisfactory in many cases: new drugs and treatment strategies are needed. CD38 is uniformly expressed at high levels on MM cells and, to a lesser extent, on the surface of normal hematopoietic and non-hematopoietic cells, making this molecule an interesting target for immunotherapeutic approaches.This review discusses the preclinical and clinical experience on different immunotherapeutic agents targeting CD38 in MM.Monoclonal antibodies (mAbs) targeting CD38 are currently changing the treatment scenario in MM, allowing physicians to reach unprecedented results, especially when anti-CD38 mAbs are used in combination with consolidated MM treatments. Other immunotherapies targeting CD38 - such as conjugated anti-CD38 mAbs, bispecific antibodies stimulating T cells to eliminate CD38+ MM cells, and CD38-specific chimeric antigen receptor T cells - are interesting strategies, currently at earlier developmental stages.