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Abstract Anti-CD38 monoclonal antibodies like Daratumumab (Dara) are effective in multiple myeloma (MM); however, drug resistance ultimately occurs and the mechanisms behind this are poorly understood. Here, we identify, via two in vitro genome-wide CRISPR screens probing Daratumumab resistance, KDM6A as an important regulator of sensitivity to Daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). Loss of KDM6A leads to increased levels of H3K27me3 on the promoter of CD38, resulting in a marked downregulation in CD38 expression, which may cause resistance to Daratumumab-mediated ADCC. Re-introducing CD38 does not reverse Daratumumab-mediated ADCC fully, which suggests that additional KDM6A targets, including CD48 which is also downregulated upon KDM6A loss, contribute to Daratumumab-mediated ADCC. Inhibition of H3K27me3 with an EZH2 inhibitor resulted in CD38 and CD48 upregulation and restored sensitivity to Daratumumab. These findings suggest KDM6A loss as a mechanism of Daratumumab resistance and lay down the proof of principle for the therapeutic application of EZH2 inhibitors, one of which is already FDA-approved, in improving MM responsiveness to Daratumumab.

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterised by aberrant production of immunoglobulins requiring survival mechanisms to adapt to proteotoxic stress. We here show that glutamyl-prolyl-tRNA synthetase (GluProRS) inhibition constitutes a novel therapeutic target. Genomic data suggest that GluProRS promotes disease progression and is associated with poor prognosis, while downregulation in MM cells triggers apoptosis. We developed NCP26, a novel ATP-competitive ProRS inhibitor that demonstrates significant anti-tumour activity in multiple in vitro and in vivo systems and overcomes metabolic adaptation observed with other inhibitor chemotypes. We demonstrate a complex phenotypic response involving protein quality control mechanisms that centers around the ribosome as an integrating hub. Using systems approaches, we identified multiple downregulated proline-rich motif-containing proteins as downstream effectors. These include CD138, transcription factors such as MYC, and transcription factor 3 (TCF3), which we establish as a novel determinant in MM pathobiology through functional and genomic validation. Our preclinical data therefore provide evidence that blockade of prolyl-aminoacylation evokes a complex pro-apoptotic response beyond the canonical integrated stress response and establish a framework for its evaluation in a clinical setting.

Abstract We here defined the impacts of γ-secretase inhibitors (GSIs) on T-cell-dependent BCMA-specific multiple myeloma (MM) cell lysis and immunomodulatory effects induced by bispecific antibodies (BisAbs). GSIs-induced membrane BCMA (mBCMA) accumulation reached near maximum within 4 h and sustained over 42h-study period on MM cell lines and patient MM cells. GSIs, i.e., 2 nM LY-411575 or 1 μM DAPT, robustly increased mBCMA densities on CD138+ but not CD3+ patient cells, concomitantly with minimum soluble/shed BCMA (sBCMA) in 1 day-culture supernatants. In ex vivo MM-T-cell co-cultures, GSIs overcame sBCMA-inhibited MM cell lysis and further enhanced autologous patient MM cell lysis induced by BCMAxCD3 BisAbs, accompanied by significantly enhanced cytolytic markers (CD107a, IFNγ, IL2, and TNFα) in patient T cells. In longer 7 day-co-cultures, LY-411575 minimally affected BCMAxCD3 BisAb (PL33)-induced transient expression of checkpoint (PD1, TIGIT, TIM3, LAG3) and co-stimulatory (41BB, CD28) proteins, as well as time-dependent increases in % effector memory/central memory subsets and CD8/CD4 ratios in patient T cells. Importantly, LY41157 rapidly cleared sBCMA from circulation of MM-bearing NSG mice reconstituted with human T cells and significantly enhanced anti-MM efficacy of PL33 with prolonged host survival. Taken together, these results further support ongoing combination BCMA-targeting immunotherapies with GSI clinical studies to improve patient outcome.

Existing clinical models cannot fully capture smoldering multiple myeloma (SMM) heterogeneity. Here, integration of 42 genetic alterations from 214 SMM patients using an unsupervised binary matrix factorization clustering approach results in the identification of 6 distinct molecular and clinical subtypes.

Abstract Interaction of plasmacytoid dendritic cells (pDCs) with multiple myeloma (MM) cells, T- or NK-effector cells in the bone marrow (BM) microenvironment induces tumor cell growth, as well as inhibits innate and adaptive immune responses. Defining pDC-MM interaction-triggered immunosuppressive mechanism(s) will enable design of interventional therapies to augment anti-MM immunity. In the present study, we show that pDC-MM interactions induce metabolic enzyme Ecto-5' Nucleotidase/CD73 in both pDCs and MM cells. Gene expression database from MM patients showed that CD73 levels inversely correlate with overall survival. Using our pDC-MM coculture models, we found that blockade of CD73 with anti-CD73 Abs: decreases adenosine levels; activates MM patient pDCs; triggers cytotoxic T lymphocytes (CTL) activity against autologous patient MM cells. Combination of anti-CD73 Abs and an immune-stimulating agent TLR-7 agonist enhances autologous MM-specific CD8 + CTL activity. Taken together, our preclinical data suggest that the therapeutic targeting of CD73, alone or in combination with TLR-7 agonist, represents a promising novel strategy to restore host anti-MM immunity.

The incorporation of novel agents and monoclonal antibody-based therapies into the treatment of multiple myeloma (MM) has significantly improved long-term patient survival. However, the disease is still largely incurable, with high-risk patients suffering shorter survival times, partly due to weakened immune systems. Bispecific molecules, including bispecific antibodies (BisAbs) and bispecific T-cell engagers (BiTEs), encourage immune cells to lyse MM cells by simultaneously binding antigens on MM cells and immune effector cells, bringing those cells into close proximity. BisAbs that target B-cell maturation antigen (BCMA) and GPRC5D have shown impressive clinical activity, and the results of early-phase clinical trials targeting FcRH5 in patients with relapsed/refractory MM (RRMM) are also promising. Furthermore, the safety profile of these agents is favorable, including mainly low-grade cytokine release syndrome (CRS). These off-the-shelf bispecific molecules will likely become an essential part of the MM treatment paradigm. Here, we summarize and highlight various bispecific immunotherapies under development in MM treatment, as well as the utility of combining them with current standard-of-care treatments and new strategies. With the advancement of novel combination treatment approaches, these bispecific molecules may lead the way to a cure for MM.

Subodh Kumar et al. identify a gene signature correlated with genomic instability and poor survival in esophageal adenocarcinoma (EAC), using a combination of integrative genomic analysis of patient data and laboratory validation in cell line models and mice. They find that inhibitors of some of the identified proteins, including TTK, could be used to reduce genomic evolution as well as inhibit growth of EAC cells.

Relapse following BCMA targeted CAR T-cell therapy is frequently observed in patients with multiple myeloma (MM). Here, by single cell transcriptome profiling on serially collected bone marrow samples, the authors report biallelic loss of BCMA as the mechanism of resistance underlying both relapse and lack of response to a second CAR T infusion in a patient with MM.

Abstract: Chimeric antigen receptor (CAR) T cells (CARTs) have shown tremendous potential for the treatment of certain B-cell malignancies, including patients with relapsed/refractory multiple myeloma (MM). Targeting the B-cell maturation antigen (BCMA) has produced the most promising results for CART therapy of MM to date, but not all remissions are sustained. Emergence of BCMA escape variants has been reported under the selective pressure of monospecific anti-BCMA CART treatment. Thus, there is a clinical need for continuous improvement of CART therapies for MM. Here, we show that a novel trimeric APRIL (a proliferation-inducing ligand)–based CAR efficiently targets both BCMA+ and BCMA− MM. Modeled after the natural ligand-receptor pair, APRIL-based CARs allow for bispecific targeting of the MM-associated antigens BCMA and transmembrane activator and CAML interactor (TACI). However, natural ligands as CAR antigen-binding domains may require further engineering to promote optimal binding and multimerization to adequately trigger T-cell activation. We found that using a trimeric rather than a monomeric APRIL format as the antigen-binding domain enhanced binding to BCMA and TACI and CART activity against MM in vitro and in vivo. Dual-specific, trimeric APRIL-based CAR are a promising therapeutic approach for MM with potential for preventing and treating BCMA escape.

Abstract: Rapid remission by induction therapy has long been recognized as an important predictor for long-time survival in acute leukemia. However, the impact of response kinetics on multiple myeloma (MM) seems to be different and remains unexplored. The relationship between response kinetics and outcome were assessed in 626 patients with newly diagnosed MM who were included in a prospective, nonrandomized clinical trial (BDH 2008/02). Patients were assigned to either immunomodulatory drug- or proteasome inhibitor–based therapy. The response depth, time to best response (TBR) and duration of best response (DBR) were collected. Depth of response was associated with superior outcomes, consistent with findings from other studies. However, the early responders (defined as TBR ≤3 months) showed significantly worse survival compared with late responders. We found that patients with rapid complete remission experienced inferior survivals comparable to those attaining a gradual partial remission. Moreover, 4 distinct response kinetics patterns were identified. Patients with gradual and sustained remission (“U-valley” pattern) experienced superior outcomes, whereas poor outcomes were observed in rapid and transient responders (“roller coaster” pattern) (median overall survival, 126 vs 30 months). The effects of response patterns on survival were confirmed in patients at different stages of disease and cytogenetic risk, including transplant-eligible patients and those attaining different extents of response depth. Collectively, our data indicated that slow and gradual response is a favorable prognostic factor in MM. In addition to response depth, the kinetic pattern of response is a simple and powerful predictor for survival even in the era of novel agents.

Isatuximab is a monoclonal antibody targeting the transmembrane receptor and ectoenzyme CD38, a protein highly expressed on hematological malignant cells, including those in multiple myeloma (MM). Upon binding to CD38-expressing MM cells, isatuximab is thought to induce tumor cell killing via fragment crystallizable (Fc)-dependent mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), as well as via direct Fc-independent mechanisms. Here, these mechanisms of action were investigated in MM and diffuse large B-cell lymphoma (DLBCL) cell lines, as well as in peripheral blood mononuclear cells derived from healthy donors, and in MM patient-derived samples. Our findings show that isatuximab-mediated cytotoxicity occurred primarily via ADCC and ADCP in MM cell lines and via ADCC and apoptosis in DLBCL cell lines expressing high levels of CD38. We identified the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) pathway and MM cell-secreted transforming growth factor-beta (TGF-β) as tumor cell-related features that could suppress CD38-mediated ADCC. Furthermore, we established that isatuximab can directly activate natural killer (NK) cells and promote NK cell-mediated cytotoxicity via crosslinking of CD38 and CD16. Finally, isatuximab-induced CDC was observed in cell lines with high CD38 receptor density (>250,000 molecules/cell) and limited expression of inhibitory complement regulatory proteins (CD46, CD55, and CD59;

Recognition of intronic polyadenylation (IpA) signals can lead to expression of truncated proteins lacking C terminal domains. Analysis of 3ʹ -seq and RNA-seq shows that IpA is widespread in circulating immune cells, while multiple myeloma cells show loss of IpA isoforms that are normally expressed in plasma cells, impacting key genes in the disease.

Abstract B cell lymphoma and multiple myeloma (MM) are the most common hematological malignancies which benefit from therapeutic monoclonal antibodies (mAbs)-based immunotherapies. Despite significant improvement on patient outcome following the use of novel therapies for the past decades, curative treatment is unavailable for the majority of patients. For example, the 5-year survival of MM is currently less than 50%. In the 1980s, interferon-α was used as monotherapy in newly diagnosed or previously treated MM with an overall response rate of 15–20%. Noticeably, a small subset of patients who responded to long-term interferon-α further achieved sustained complete remission. Since 1990, interferon-α-containing regimens have been used as a central maintenance strategy for patients with MM. However, the systemic administration of interferon-α was ultimately limited by its pronounced toxicity. To address this, the selective mAb-mediated delivery of interferon-α has been developed to enhance specific killing of MM and B-cell malignant cells. As such, targeted interferon-α therapy may improve therapeutic window and sustain responses, while further overcoming suppressive microenvironment. This review aims to reinforce the role of interferon-α by consolidating our current understanding of targeting interferon-α with tumor-specific mAbs for B cell lymphoma and myeloma.

Several histone modifiers have been implicated in the survival of multiple myeloma cells. Here, the authors reveal a role for the histone demethylase KDM3A in the survival of this haematologic cancer, and show that mechanistically KDM3A removes H3K9 methylation from the promoters of KLF2 and IRF4, genes essential for myeloma cell survival.

The approval of the first two monoclonal antibodies targeting CD38 (daratumumab) and SLAMF7 (elotuzumab) in late 2015 for treating relapsed and refractory multiple myeloma (RRMM) was a critical advance for immunotherapies for multiple myeloma (MM). Importantly, the outcome of patients continues to improve with the incorporation of this new class of agents with current MM therapies. However, both antigens are also expressed on other normal tissues including hematopoietic lineages and immune effector cells, which may limit their long-term clinical use. B cell maturation antigen (BCMA), a transmembrane glycoprotein in the tumor necrosis factor receptor superfamily 17 (TNFRSF17), is expressed at significantly higher levels in all patient MM cells but not on other normal tissues except normal plasma cells. Importantly, it is an antigen targeted by chimeric antigen receptor (CAR) T-cells, which have already shown significant clinical activities in patients with RRMM who have undergone at least three prior treatments, including a proteasome inhibitor and an immunomodulatory agent. Moreover, the first anti-BCMA antibody–drug conjugate also has achieved significant clinical responses in patients who failed at least three prior lines of therapy, including an anti-CD38 antibody, a proteasome inhibitor, and an immunomodulatory agent. Both BCMA targeting immunotherapies were granted breakthrough status for patients with RRMM by FDA in Nov 2017. Other promising BCMA-based immunotherapeutic macromolecules including bispecific T-cell engagers, bispecific molecules, bispecific or trispecific antibodies, as well as improved forms of next generation CAR T cells, also demonstrate high anti-MM activity in preclinical and even early clinical studies. Here, we focus on the biology of this promising MM target antigen and then highlight preclinical and clinical data of current BCMA-targeted immunotherapies with various mechanisms of action. These crucial studies will enhance selective anti-MM response, transform the treatment paradigm, and extend disease-free survival in MM.

Immunomodulatory drugs and monoclonal antibody-based immunotherapies have significantly improved the prognosis of the patients with multiple myeloma (MM) in the recent years. These new classes of reagents target malignant plasma cells (PCs) and further modulate the immune microenvironment, which prolongs anti-MM responses and may prevent tumor occurrence. Since MM remains an incurable cancer for most patients, there continues to be a need to identify new tumor target molecules and investigate alternative cellular approaches using gene therapeutic strategies and novel treatment mechanisms. Osteoclasts (OCs), as critical multi-nucleated large cells responsible for bone destruction in >80% MM patients, have become an attractive cellular target for the development of novel MM immunotherapies. In MM, OCs are induced and activated by malignant PCs in a reciprocal manner, leading to osteolytic bone disease commonly associated with this malignancy. Significantly, bidirectional interactions between OCs and MM cells create a positive feedback loop to promote MM cell progression, increase angiogenesis, and inhibit immune surveillance via both cell–cell contact and abnormal production of multiple cytokines/chemokines. Most recently, hyper-activated OCs have been associated with activation of programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway, which impairs T cell proliferation and cytotoxicity against MM cells. Importantly, therapeutic anti-CD38 monoclonal antibodies and checkpoint inhibitors can alleviate OC-induced immune suppression. Furthermore, a proliferation-inducing ligand, abundantly secreted by OCs and OC precursors, significantly upregulates PD-L1 expression on MM cells, in addition to directly promoting MM cell proliferation and survival. Coupled with increased PD-L1 expression in other immune-suppressive cells, i.e., myeloid-derived suppressor cells and tumor-associated macrophages, these results strongly suggest that OCs contribute to the immunosuppressive MM BM microenvironment. Based on these findings and ongoing osteoimmunology studies, therapeutic interventions targeting OC number and function are under development to diminish both MM bone disease and related immune suppression. In this review, we discuss the classical and novel roles of OCs in the patho-immunology of MM. We also describe novel therapeutic strategies simultaneously targeting OCs and MM interactions, including PD-1/PD-L1 axis, to overcome the immune-suppressive microenvironment and improve patient outcome.

Extra-medullary disease (EMD) in multiple myeloma (MM) is associated with poor prognosis and resistance to chemotherapy. However, molecular alterations that lead to EMD have not been well defined. We developed bone marrow (BM)- and EMD-prone MM syngeneic cell lines; identified that epithelial-to-mesenchymal transition (EMT) transcriptional patterns were significantly enriched in both clones compared to parental cells, together with higher levels of CXCR4 protein; and demonstrated that CXCR4 enhanced the acquisition of an EMT-like phenotype in MM cells with a phenotypic conversion for invasion, leading to higher bone metastasis and EMD dissemination in vivo. In contrast, CXCR4 silencing led to inhibited tumor growth and reduced survival. Ulocuplumab, a monoclonal anti-CXCR4 antibody, inhibited MM cell dissemination, supported by suppression of the CXCR4-driven EMT-like phenotype. These results suggest that targeting CXCR4 may act as a regulator of EMD through EMT-like transcriptional modulation, thus representing a potential therapeutic strategy to prevent MM disease progression.

Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM.

2015 was a groundbreaking year for the multiple myeloma community partly due to the breakthrough approval of the first two monoclonal antibodies in the treatment for patients with relapsed and refractory disease. Despite early disappointments, monoclonal antibodies targeting CD38 (daratumumab) and signaling lymphocytic activation molecule F7 (SLAMF7) (elotuzumab) have become available for patients with multiple myeloma in the same year. Specifically, phase 3 clinical trials of combination therapies incorporating daratumumab or elotuzumab indicate both efficacy and a very favorable toxicity profile. These therapeutic monoclonal antibodies for multiple myeloma can kill target cells via antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent phagocytosis, as well as by direct blockade of signaling cascades. In addition, their immunomodulatory effects may simultaneously inhibit the immunosuppressive bone marrow microenvironment and restore the key function of immune effector cells. In this review, we focus on monoclonal antibodies that have shown clinical efficacy or promising preclinical anti-multiple myeloma activities that warrant further clinical development. We summarize mechanisms that account for the in vitro and in vivo anti-myeloma effects of these monoclonal antibodies, as well as relevant preclinical and clinical results. Monoclonal antibody-based immunotherapies have already and will continue to transform the treatment landscape in multiple myeloma.

We analyzed whole genomes of unique paired samples from smoldering multiple myeloma (SMM) patients progressing to multiple myeloma (MM). We report that the genomic landscape, including mutational profile and structural rearrangements at the smoldering stage is very similar to MM. Paired sample analysis shows two different patterns of progression: a "static progression model", where the subclonal architecture is retained as the disease progressed to MM suggesting that progression solely reflects the time needed to accumulate a sufficient disease burden; and a "spontaneous evolution model", where a change in the subclonal composition is observed. We also observe that activation-induced cytidine deaminase plays a major role in shaping the mutational landscape of early subclinical phases, while progression is driven by APOBEC cytidine deaminases. These results provide a unique insight into myelomagenesis with potential implications for the definition of smoldering disease and timing of treatment initiation. [ABSTRACT FROM AUTHOR]

Alternative cleavage and polyadenylation (ApA) is known to alter untranslated region (3ʹUTR) length but can also recognize intronic polyadenylation (IpA) signals to generate transcripts that lose part or all of the coding region. We analyzed 46 3ʹ-seq and RNA-seq profiles from normal human tissues, primary immune cells, and multiple myeloma (MM) samples and created an atlas of 4927 high-confidence IpA events represented in these cell types. IpA isoforms are widely expressed in immune cells, differentially used during B-cell development or in different cellular environments, and can generate truncated proteins lacking C-terminal functional domains. This can mimic ectodomain shedding through loss of transmembrane domains or alter the binding specificity of proteins with DNA-binding or protein–protein interaction domains. MM cells display a striking loss of IpA isoforms expressed in plasma cells, associated with shorter progression-free survival and impacting key genes in MM biology and response to lenalidomide. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) has proven clinically susceptible tomodulation of pathways of protein homeostasis. Blockade of proteasomal degradation of polyubiquitinated misfolded proteins by the proteasome inhibitor bortezomib (BTZ) achieves responses and prolongs survival in MM, but long-term treatment with BTZ leads to drug-resistant relapse in most patients. In a proof-of-concept study, we previously demonstrated that blocking aggresomal breakdown of polyubiquitinated misfolded proteins with the histone deacetylase 6 (HDAC6) inhibitor tubacin enhances BTZ-induced cytotoxicity in MM cells in vitro. However, these foundational studies were limited by the pharmacologic liabilities of tubacin as a chemical probe with only in vitro utility. Emerging from a focused library synthesis, a potent, selective, and bioavailable HDAC6 inhibitor, WT161, was created to study the mechanism of action of HDAC6 inhibition in MM alone and in combination with BTZ. WT161 in combination with BTZ triggers significant accumulation of polyubiquitinated proteins and cell stress, followed by caspase activation and apoptosis. More importantly, this combination treatment was effective in BTZ-resistant cells and in the presence of bone marrow stromal cells, which have been shown to mediate MM cell drug resistance. The activity of WT161 was confirmed in our human MM cell xenograft mouse model and established the framework for clinical trials of the combination treatment to improve patient outcomes in MM. [ABSTRACT FROM AUTHOR]

Here we show that overexpression or activation of B cell maturation antigen (BCMA) by its ligand a proliferation-inducing ligand (APRIL) promotes human multiple myeloma (MM) progression in vivo. BCMA downregulation strongly decreases viability and MM colony formation; conversely, BCMA overexpression augments MM cell growth and survival via induction of AKT, MAPK, and NFκB signaling cascades. Importantly, BCMA promotes in vivo growth of xenografted MM cells harboring p53 mutation in mice. BCMA-overexpressing tumors exhibit significantly increased CD31/microvessel density and VEGF compared with paired control tumors. These tumors also express increased transcripts crucial for osteoclast activation, adhesion, and angiogenesis/metastasis, as well as genes mediating immune inhibition including PD-L1, TGFβ, and IL-10. These target genes are consistently induced by paracrine APRIL binding to BCMA on MM cells, which is blocked by an antagonistic anti-APRIL monoclonal antibody hAPRIL01A (01A). 01A is cytotoxic against MM cells even in the presence of protective bone marrow (BM) myeloid cells including osteoclasts, macrophages, and plasmacytoid dendritic cells. 01A further decreases APRIL-induced adhesion and migration of MM cells via blockade of canonical and non-canonical NFκB pathways. Moreover, 01A prevents in vivo MM cell growth within implanted human bone chips in SCID mice. Finally, the effect of 01A on MM cell viability is enhanced by lenalidomide and bortezomib. Taken together, these data delineate new molecular mechanisms of in vivo MM growth and immunosuppression critically dependent on BCMA and APRIL in the BM microenvironment, further supporting targeting this prominent pathway in MM. [ABSTRACT FROM AUTHOR]

B-cell maturation antigen (BCMA), highly expressed on malignant plasma cells in human multiple myeloma (MM), has not been effectively targeted with therapeutic monoclonal antibodies. We here show that BCMA is universally expressed on the MM cell surface and determine specific anti-MM activity of J6M0-mcMMAF (GSK2857916), a novel humanized and afucosylated antagonistic anti-BCMA antibody-drug conjugate via a noncleavable linker. J6M0-mcMMAF specifically blocks cell growth via G2/M arrest and induces caspase 3-dependent apoptosis in MM cells, alone and in coculture with bone marrow stromal cells or various effector cells. It strongly inhibits colony formation by MM cells while sparing surrounding BCMA-negative normal cells. J6M0-mcMMAF significantly induces effector cell-mediated lysis against allogeneic or autologous patient MM cells, with increased potency and efficacy compared with the wild-type J6M0 without Fc enhancement. The antibody-dependent cell-mediated cytotoxicity and apoptotic activity of J6M0-mcMMAF is further enhanced by lenalidomide. Importantly, J6M0-mcMMAF rapidly eliminates myeloma cells in subcutaneous and disseminated mouse models, and mice remain tumor-free up to 3.5 months. Furthermore, J6M0-mcMMAF recruits macrophages and mediates antibody-dependent cellular phagocytosis of MM cells. Together, these results demonstrate that GSK2857916 has potent and selective anti-MM activities via multiple cytotoxic mechanisms, providing a promising next-generation immunotherapeutic in this cancer. [ABSTRACT FROM AUTHOR]

HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FC&ggr;R binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 lgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the lgG1 analog, evidenced by increased cell surface expression of gran-zyme B-dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FC&ggr;R-dependent mechanisms, and was significantly more effective than the lgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cyno-molgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM [ABSTRACT FROM AUTHOR]

CD38, a type II transmembrane glycoprotein highly expressed in hematological malignancies including multiple myeloma (MM), represents a promising target for mAb-based immunotherapy. In this study, we describe the cytotoxic mechanisms of action of daratumumab, a novel, high-affinity, therapeutic human mAb against a unique CD38 epitope. Daratumumab induced potent Ab-dependent cellular cytotoxicity in CD38-expressing lymphoma- and MM-derived cell lines as well as in patient MM cells, both with autologous and allogeneic effector cells. Daratumumab stood out from other CD38 mAbs in its strong ability to induce complement-dependent cytotoxicity in patient MM cells. Importantly, daratumumab-induced Ab-dependent cellular cytotoxicity and complement-dependent cytotoxicity were not affected by the presence of bone marrow stromal cells, indicating that daratumumab can effectively kill MM tumor cells in a tumor-preserving bone marrow microenvironment. In vivo, daratumumab was highly active and interrupted xenograft tumor growth at low dosing. Collectively, our results show the versatility of daratumumab to effectively kill CD38-expressing tumor cells, including patient MM cells, via diverse cytotoxic mechanisms. These findings support clinical development of daratumumab for the treatment of CD38-positive MM tumors. [ABSTRACT FROM AUTHOR]

The unmet need for improved multiple myeloma (MM) therapy has stimulated clinical development of monoclonal antibodies (mAbs) targeting either MM cells or cells of the bone marrow (BM) microenvironment. In contrast to small-molecule inhibitors, therapeutic mAbs present the potential to specifically target tumor cells and directly induce an immune response to lyse tumor cells. Unique immune-effector mechanisms are only triggered by therapeutic mAbs but not by small molecule targeting agents. Although therapeutic murine mAbs or chimeric mAbs can cause immunogenicity, the advancement of genetic recombination for humanizing rodent mAbs has allowed large-scale production and designation of mAbs with better affinities, efficient selection, decreasing immunogenicity, and improved effector functions. These advancements of antibody engineering technologies have largely overcome the critical obstacle of antibody immunogenicity and enabled the development and subsequent Food and Drug Administration (FDA) approval of therapeutic Abs for cancer and other diseases. [ABSTRACT FROM AUTHOR]

This study investigated the cytotoxicity and mechanism of action of AS703026, a novel, selective, orally bioavailable MEK1/2 inhibitor, in human multiple myeloma (MM). AS703026 inhibited growth and survival of MM cells and cytokine-induced osteoclast differentiation more potently (9- to 10-fold) than AZD6244. Inhibition of proliferation induced by AS703026 was mediated by G0-G1 cell cycle arrest and was accompanied by reduction of MAF oncogene expression. AS703026 further induced apoptosis via caspase 3 and Poly ADP ribose polymerase (PARP) cleavage in MM cells, both in the presence or absence of bone marrow stromal cells (BMSCs). Importantly, AS703026 sensitized MM cells to a broad spectrum of conventional (dexamethasone, melphalan), novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti-MM therapies. Significant tumour growth reduction in AS703026- vs. vehicle-treated mice bearing H929 MM xenograft tumours correlated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels in vivo. Moreover, AS703026 (<200 nmol/l) was cytotoxic against the majority of tumour cells tested from patients with relapsed and refractory MM (84%), regardless of mutational status of RAS and BRAF genes. Importantly, BMSC-induced viability of MM patient cells was similarly blocked within the same dose range. Our results therefore support clinical evaluation of AS703026, alone or in combination with other anti-MM agents, to improve patient outcome. [ABSTRACT FROM AUTHOR]

Monoclonal gammopathy of undetermined significance (MGUS) is a common disorder of aging and a precursor lesion to full-blown multiple myeloma (MM). The mechanisms underlying the progression from MGUS to MM are incompletely understood but include the suppression of innate and adaptive antitumor immunity. Here, we demonstrate that NKG2D, an activating receptor on natural killer (NK) cells, CD8+ T lymphocytes, and MHC class I chain-related protein A (MICA), an NKG2D ligand induced in malignant plasma cells through DNA damage, contribute to the pathogenesis of MGUS and MM. MICA expression is increased on plasma cells from MGUS patients compared with normal donors, whereas MM patients display intermediate MICA levels and a high expression of ERp5, a protein disulfide isomerase linked to MICA shedding (sMICA). MM, but not MGUS, patients harbor circulating sMICA, which triggers the down-regulation of NKG2D and impaired lymphocyte cytotoxicity. In contrast, MGUS, but not MM, patients generate high-titer anti-MICA antibodies that antagonize the suppressive effects of sMICA and stimulate dendritic cell cross-presentation of malignant plasma cells. Bortezomib, a proteasome inhibitor with anti-MM clinical efficacy, activates the DNA damage response to augment MICA expression in some MM cells, thereby enhancing their opsonization by anti-MICA antibodies. Together, these findings reveal that the alterations in the NKG2D pathway are associated with the progression from MGUS to MM and raise the possibility that anti-MICA monoclonal antibodies might prove therapeutic for these disorders. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is an invariably fatal form of cancer characterized by clonal proliferation of malignant plasma cells in the bone marrow. The canonical Wnt signaling pathway is activated in MM cells through constitutively active β-catenin, a messenger molecule relevant to growth, survival, and migration of MM cells. The identification of a number of small molecular compounds, such as PKF115-584, which disrupt the interaction of the transcriptionally active β-catenin/TCF protein complex, provides valuable new therapeutic tools to target an alternative pathway in MM independent of the proteasome. Here we evaluated the transcriptional, proteomic, signaling changes, and biological sequelae associated with the inhibition of Wnt signaling in MM by PKF115-584. The compound blocks expression of Wnt target genes and induces cytotoxicity in both patient MM cells and MM cell lines without a significant effect in normal plasma cells. In xenograft models of human MM, PKF115-584 inhibits tumor growth and prolongs survival. Taken together, these data demonstrate the efficacy of disrupting the β-catenin/TCF transcriptional complex to exploit tumor dependence on Wnt signaling as a therapeutic approach in the treatment of MM. [ABSTRACT FROM AUTHOR]

A critical role for vascular endothelial factor (VEGF) has been demonstrated in multiple myeloma (MM) pathogenesis. Here, we characterized the effect of the small-molecule VEGF receptor inhibitor pazopanib on MM cells in the bone marrow milieu. Pazopanib inhibits VEGF-triggered signaling pathways in both tumor and endothelial cells, thereby blocking in vitro MM cell growth, survival, and migration, and inhibits VEGF-induced up-regulation of adhesion molecules on both endothelial and tumor cells, thereby abrogating endothelial cell-MM cell binding and associated cell proliferation. We show that pazopanib is the first-in-class VEGF receptor inhibitor to inhibit in vivo tumor cell growth associated with increased MM cell apoptosis, decreased angiogenesis, and prolonged survival in a mouse xenograft model of human MM. Low-dose pazopanib demonstrates synergistic cytotoxicity with conventional (melphalan) and novel (bortezomib and immunomodulatory drugs) therapies. Finally, gene expression and signaling network analysis show transcriptional changes of several cancer-related genes, in particular c-Myc. Using siRNA, we confirm the role of c-Myc in VEGF production and secretion, as well as angiogenesis. These preclinical studies provide the rationale for clinical evaluation of pazopanib, alone and in combination with conventional and novel therapies, to increase efficacy, overcome drug resistance, reduce toxicity, and improve patient outcome in MM. [ABSTRACT FROM AUTHOR]

The proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for the treatment of multiple myeloma (MM). Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance are poorly understood. However, resistance to bortezomib as a single agent develops in the majority of patients, and activity in other malignancies has been less impressive. To elucidate mechanisms of bortezomib resistance, we compared differential gene expression profiles of bortezomib-resistant SUDHL-4 and bortezomib-sensitive SUDHL-6 diffuse large B-cell lymphoma lines in response to bortezomib. At concentrations that effectively inhibited proteasome activity, bortezomib induced apoptosis in SUDHL-6 cells, but not in SUDHL-4 cells. We showed that overexpression of activating transcription factor 3 (ATF3), ATF4, ATF5, c-Jun, JunD and caspase-3 is associated with sensitivity to bortezomib-induced apoptosis, whereas overexpression of heat shock protein (HSP)27, HSP70, HSP90 and T-cell factor 4 is associated with bortezomib resistance. [ABSTRACT FROM AUTHOR]

Examines the role of caveolin-1 in multiple myeloma cells. Demonstration that the caveolin-1 is expressed in multiple myeloma cells; Effect of cholesterol depletion of by beta-cyclodextrin; Finding that cholesterol depletion by beta-cyclodextrin abrogates both interleukin-6- and insulin-like growth factor-I-triggered multiple myeloma cell survival via negative regulation of caveolin-1.

Presents an immunofluoresence microscopy image of neural progenitor cells expressing tyrosine hydroxylase. Result of straining cells β-tubulin (green), tyrosine hydroxylase (red) and green fibrillary acidic protein (GFAP; blue); Microscope through which the image was acquired; Measurement of scale bar.

An introduction to the journal is presented in which the editor discusses the CD38 expression on multiple myeloma (MM) cells.

Multiple myeloma evolves continuously. Here the authors chronologically reconstruct driver events in multiple myeloma, noting a limited repertoire of initiating driver events that shape the evolutionary trajectory of the disease.

Smoldering MM (SMM) is a premalignant stage of multiple myeloma (MM). Here the authors perform whole genome sequencing of unique paired samples of SMM progressing to MM, and show that the genomic landscape at the SMM stage is very similar to MM, but trajectories of evolution can vary from patient to patient.

The number and activity of osteoclasts (OCs) are strongly enhanced by myeloma cells, leading to significant bone lesions in patients with multiple myeloma (MM). Mechanisms remain elusive as to whether myeloma-supporting OCs also induce suppressive immune bone marrow (BM) microenvironment. Here, we first show that OCs significantly protect MM cells against T-cell–mediated cytotoxicity via direct inhibition of proliferating CD4+ and CD8+ T cells. The immune checkpoint molecules programmed death ligand 1 (PD-L1), Galectin-9, herpesvirus entry mediator (HVEM), and CD200, as well as T-cell metabolism regulators indoleamine 2, 3-dioxygenase (IDO), and CD38 are significantly upregulated during osteoclastogenesis. Importantly, the levels of these molecules, except CD38, are higher in OCs than in MM cells. Anti–PD-L1 monoclonal antibody (mAb) and IDO inhibitor partly overcome OC-inhibited T-cell responses against MM cells, confirming their roles in OC-suppressed MM cell lysis by cytotoxic T cells. In addition, Galectin-9 and a proliferation-induced ligand (APRIL), secreted by OCs, are significantly upregulated during osteoclastogenesis. Galectin-9 specifically induces apoptosis of T cells while sparing monocytes and MM cells. APRIL induces PD-L1 expression in MM cells, providing additional immune inhibition by OCs. Moreover, CD38 is significantly upregulated during osteoclastogenesis. When targeted by an anti-CD38 mAb, suppressive T-cell function by OCs is alleviated, associated with downregulation of HVEM and IDO. Taken together, these results define the expression of multiple immune proteins and cytokines in OCs essential for suppressive MM BM milieu. These results further support the combination of targeting these molecules to improve anti-MM immunity. [ABSTRACT FROM AUTHOR]

Patient-derived multiple myeloma (MM) cells are difficult to establish in culture or propagate in vivo in murine model. Here, we describe a zebrafish xenograft model that permits rapid, reliable growth of human MM cells injected into the perivitelline space of albino zebrafish (Casper) embryos 48 hours postfertilization. MM1S and MM1R MM cell lines and primary CD138+ MM cells were stained with CM-Dil red fluorescent dye and suspended in Matrigel prior to their injection. The cells grew at the site of injection and disseminated throughout the developing embryos and larvae. Tumor size was quantified by fluorescent microscopy, and cell fate was followed for 4 days. All of the cell line xenografts showed responses similar to those previously observed with in vitro assays. CD138+ plasma cell xenografts derived from MM patients also grew and were inhibited by the same drugs patients had responded to clinically. Using this technique, we can assess drug sensitivity or resistance with a small number of MM cells in a short period. This raises the possibility that one might be able to assess drug sensitivity in real time with readily obtainable clinical samples. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) cell lines and primary tumor cells are addicted to the MYC oncoprotein for survival. Little is known, however, about how MYC expression is upregulated in MM cells. The mucin 1 C-terminal subunit (MUC1-C) is an oncogenic transmembrane protein that is aberrantly expressed in MM cell lines and primary tumor samples. The present studies demonstrate that targeting MUC1-C with silencing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 editing or with the GO-203 inhibitor is associated with downregulation of MYC messenger RNA and protein. The results show that MUC1-C occupies the MYC promoter and thereby activates the MYC gene by a β-catenin/transcription factor 4 (TCF4)-mediated mechanism. In this way, MUC1-C(1) increases β-catenin occupancy on the MYC promoter, (2) forms a complex with β-catenin and TCF4, and, in turn, (3) drives MYC transcription. Analysis of MM cells using quantitative real-time reverse transcription polymerase chain MYC reaction arrays further demonstrated that silencing MUC1-C is associated with downregulation of MYC target genes, including, CCND2, hTERT, and GCLC. Analysis of microarray data sets further demonstrated that MUC1 levels positively correlate with MYC expression in MM progression and in primary cells from over 800 MM patients. These findings collectively provide convincing evidence that MUC1-C drives MYC expression in MM. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage response. Here, we show that the NAD+-dependent deacetylase SIRT6 is highly expressed in MM cells, as an adaptive response to genomic stability, and that high SIRT6 levels are associated with adverse prognosis. Mechanistically, SIRT6 interacts with the transcription factor ELK1 and with the ERK signaling-related gene. By binding to their promoters and deacetylating H3K9 at these sites, SIRT6 downregulates the expression of mitogen-activated protein kinase (MAPK) pathway genes, MAPK signaling, and proliferation. In addition, inactivation of ERK2/p90RSK signaling triggered by high SIRT6 levels increases DNA repair via Chk1 and confers resistance to DNA damage. Using genetic and biochemical studies in vitro and in human MM xenograft models, we show that SIRT6 depletion both enhances proliferation and confers sensitization to DNA-damaging agents. Our findings therefore provide insights into the functional interplay between SIRT6 and DNA repair mechanisms, with implications for both tumorigenesis and the treatment of MM. [ABSTRACT FROM AUTHOR]

Despite recent therapeutic advances that have doubled the median survival time of patients with multiple myeloma, intratumor genetic heterogeneity contributes to disease progression and emergence of drug resistance. miRNAs are noncoding small RNAs that play important roles in the regulation of gene expression and have been implicated in cancer progression and drug resistance. We investigated the role of the miR-221--222 family in dexamethasone-induced drug resistance in multiple myeloma using the isogenic cell lines MM1R and MM1S, which represent models of resistance and sensitivity, respectively. Analysis of array comparative genome hybridization data revealed gain of chromosome X regions at band p11.3, wherein the miR-221--222 resides, in resistant MM1R cells but not in sensitive MM1S cells. DNA copy number gains in MM1R cells were associated with increased miR-221--222 expression and downregulation of p53-upregulated modulator of apoptosis (PUMA) as a likely proapoptotic target. We con?rmed PUMA mRNA as a direct target of miR-221--222 in MM1S and MM1R cells by both gain-of-function and loss-of-function studies. In addition, miR-221--222 treatment rendered MM1S cells resistant to dexamethasone, whereas antimiR-221--222 partially restored the dexamethasone sensitivity of MM1R cells. These studies have uncovered a role for miR-221-- 222 in multiple myeloma drug resistance and suggest a potential therapeutic role for inhibitors of miR-221--222 binding to PUMA mRNA as a means of overcoming dexamethasone resistance in patients. The clinical utility of this approach is predicated on the ability of antisense miR-221--222 to increase survival while reducing tumor burden and is strongly supported by the metastatic propensity of MM1R cells in preclinical mouse xenograft models of multiple myeloma. Moreover, our observation of increased levels of miR-221--222 with decreased PUMA expression in multiple myeloma cells from patients at relapse versus untreated controls suggests an even broader role for miR-221--222 in drug resistance and provides a rationale for the targeting of miR-221--222 as a means of improving patient outcomes. [ABSTRACT FROM AUTHOR]

Clonal proliferation of plasma cells within the bone marrow (BM) affects local cells, such as mesenchymal stromal cells (MSCs), leading to osteolysis and fatality in multiple myeloma (MM). Consequently, there is an urgent need to find better mechanisms of inhibiting myeloma growth and osteolytic lesion development. To meet this need and accelerate clinical translation, better models of myeloma within the BM are required. Herein we have developed a clinically relevant, three-dimensional (3D) myeloma BM coculture model that mimics bone cell/cancer cell interactions within the bone microenvironment. The coculture model and clinical samples were used to investigate myeloma growth, osteogenesis inhibition, and myeloma-induced abnormalities in MM-MSCs. This platform demonstrated myeloma support of capillarylike assembly of endothelial cells and cell adhesion-mediated drug resistance (CAM-DR). Also, distinct normal donor (ND)- and MM-MSC miRNA (miR) signatures were identified and used to uncover osteogenic miRs of interest for osteoblast differentiation. More broadly, our 3D platform provides a simple, clinically relevant tool to model cancer growth within the bone-useful for investigating skeletal cancer biology, screening compounds, and exploring osteogenesis. Our identification and efficacy validation of novel bone anabolic miRs in MM opens more opportunities for novel approaches to cancer therapy via stromal miR modulation. [ABSTRACT FROM AUTHOR]

Recent work has delineated mutational profiles in multiple myeloma and reported a median of 52 mutations per patient, as well as a set of commonly mutated genes across multiple patients. In this study, we have used deep sequencing of RNA from a subset of these patients to evaluate the proportion of expressed mutations. We find that the majority of previously identified mutations occur within genes with very low or no detectable expression. On average, 27% (range, 11% to 47%) of mutated alleles are found to be expressed, and among mutated genes that are expressed, there often is allele-specific expression where either the mutant or wild-type allele is suppressed. Even in the absence of an overall change in gene expression, the presence of differential allelic expression within malignant cells highlights the important contribution of RNA-sequencing in identifying clinically significant mutational changes relevant to our understanding of myeloma biology and also for therapeutic applications. [ABSTRACT FROM AUTHOR]

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family that has been recently linked to tumor development. However, its role in modulating multiple myeloma (MM) biology and disease progression remains unexplored. We first demonstrated that patients with MM present with higher expression of Pyk2 compared with healthy individuals. By using loss-of-function approaches, we found that Pyk2 inhibition led to reduction of MM tumor growth in vivo as well as decreased cell proliferation, cellcycle progression, and adhesion ability in vitro. In turn, overexpression of Pyk2 promoted the malignant phenotype, substantiated by enhanced tumorgrowth and reduced survival. Mechanistically, inhibition of Pyk2 reduced activation of Wnt/β-catenin signaling by destabilizing β-catenin, leading to downregulation of c-Myc and Cyclin D1. Furthermore, treatment of MM cells with the FAK/Pyk2 inhibitor VS-4718 effectively inhibited MM cell growth both in vitro and in vivo. Collectively, our findings describe the tumor-promoting role of Pyk2 in MM, thus providing molecular evidence for a novel tyrosine kinase inhibitor as a new therapeutic option in MM. [ABSTRACT FROM AUTHOR]