Expression In Multiple Myeloma Cells Research Articles

Analysis of COVID-19-Associated Proteins Identifies TANK-Binding Kinase 1 As an Immunotherapeutic Target in Multiple Myeloma

Background and Rationale. Patients with multiple myeloma (MM) are at a higher risk of viral infection due to immune deficiency, as recently highlighted by the severity of COVID-19 in MM patients. However, the mechanism(s) underlying the lack of anti-viral immune response in MM are unclear. We have previously shown that there are more plasmacytoid dendritic cells (pDCs) in the bone marrow of MM patients than in the bone marrow of healthy individuals and that these dysfunctional patient pDCs suppress T and NK cell-mediated immunity. Importantly, pDCs play a key role in both recognizing viral nucleic acid and initiating anti-viral immunity via type 1 interferon (IFN) response signaling. Therefore, we analyzed the immune-pathway proteins implicated in COVID-19-host interactions [COVID-19 protein interaction map, Gordon et al, Nature 2020; 583, 459-568] to assess whether pDC-MM interactions modulate these pathways to confer immune suppression and susceptibility to COVID-19. Results. We first assessed whether the expression of human genes involved in the COVID-19-host interaction changes when ex vivo MM cells are co-cultured with patient pDCs (compared to MM cells alone). We found that 41 of these genes were differentially expressed in MM cells when exposed to pDCs (log2Fold change: ± 3.5-fold; p < 0.00001), and this expression varied widely (median (log10): -0.13 to 4.5; adj p < 0.001). Three of these genes are linked to innate immunity and are essential for IFN production in MM cells - TBK1, IRF3 (Interferon regulatory factor 3), and RAE1 (interferon-inducible mRNA nuclear export factor). Importantly, pDCs downregulated the expression of TBK1 (Log2FC: -0.5) and RAE1 (Log2FC: -0.3) and upregulated the expression of IRF3 (Log2FC: 1.5) in MM cells after coculture (compared to MM cells alone, p < 0.0001). Concordantly, analysis of publicly available gene profiling datasets on relapsed MM patients showed low levels of TBK1 and RAE1 and higher levels of IRF3 (n = 50) (Log2FC: TBK1: -0.208; RAE1: -0.286; IRF3: 0.273; vs normal; p < 0.05). Of note, TBK1 expression was positively correlated with prognosis (n = 350, p = 0.026). We found that most of the 41 genes were also expressed in pDCs (NCBI GEO Datasets based on pDC cell line CAL1 and human peripheral blood pDCs). In unstimulated pDCs, TBK1 expression was significantly lower than IRF3 and RAE1 expression (2-3-fold, p < 0.05). Yet treating pDCs with CpG-ODN type-A, which induces IFN-α expression, increased both TBK1 (adj P = 0.004) and RAE1 (adj P = 0.043) expression, without significantly altering IRF3 expression. Finally, COVID-19 relies on the host ubiquitin-proteasome system for propagation, and we found that targeting ubiquitin receptor Rpn13 with the specific inhibitor RA190 restored TBK1 expression in MM cell lines, indicating the potential clinical use of targeting Rpn13 in restoring anti-viral immune responses in MM. Conclusion. Taken together, we show that TBK1 is downregulated in pDCs, and pDCs interacting with MM cells further decrease TBK1 expression in MM cells, thereby attenuating IFN-mediated anti-viral immune response signaling. Our findings suggest that: 1) low levels of TBK1 may confer increased susceptibility of MM patients to COVID-19 and 2) targeting TBK1 may restore the anti-viral immune response and reduce COVID-19 severity in MM.

Suppression of Multiple Myeloma Via Inhibition of EFNB2 Reverse Signaling

Multiple myeloma (MM) cells display a reciprocal relationship with neighboring niche cells in the bone marrow (BM), particularly vascular endothelial cells (BM ECs). This relationship drives disease initiation, progression, and therapy resistance, but the mechanisms underlying this interaction remain incompletely understood. Here, we show that silencing ephrin B2 (EFNB2) blocked MM cell proliferation and survival in vitro and abrogated MM engraftment and repopulation in vivo. EFNB2 expression is also increased in MM patients with adverse-risk cytogenetics and decreased survival, suggesting an important role for EFNB2 in MM disease progression. To identify novel regulators of MM pathogenesis, we performed transcriptome analyses of ECs from multiple human tissues and analyzed their capacity to promote MM growth. The EPH receptor, EPHB1, was overexpressed in human ECs that support MM expansion in culture. Silencing EPHB1 expression in ECs using siRNA or a lentiviral shRNA abrogated MM cell expansion compared to EC co-cultures treated with non-targeting shRNA and blocked the expansion of MM cells capable of in vivo repopulation in immune deficient mice. Additionally, transgenic Vk*MYC mice, which develop indolent murine MM through activation-induced deaminase-dependent MYC activation in germinal B cells, displayed increased expression of EPHB1 and EPHB4 in BM ECs compared to wild type BM ECs. We found that expression of EFNB2, an EPHB1/4 binding partner, is upregulated in 3 human MM cell lines and in primary MM cell populations from patients, whereas other EPHB1/4 binding partners were either not detected or expressed at a low level. shRNA-mediated inhibition of EFNB2 in MM cells blocked MM cell cycling, leading to a near 70-fold reduction in MM cell growth at day 12 of culture, and blunted MM cell repopulation when transplanted into NSG mice. In order to determine the therapeutic potential of targeting EFNB2 in MM in vivo, we transplanted irradiated NSG mice with MM cells and subsequently administered a single chain variable fragment (scFv) targeting EFNB2 on days +4, +6, +10 and +12. Mice treated with the EFNB2 scFv displayed significantly decreased engraftment of MM cells in vivo compared to control mice. MM reconstitution in vivo. Silencing EFNB2 expression in MM cells downregulated several pathways including those involved in cellular movement, cell death and survival, cellular assembly, cell cycle and DNA repair. Several STAT5 target genes were also downregulated in MM cells treated with shRNA-EFNB2, supporting the notion that STAT5 signaling is activated by ephrin B proteins (Bong et al. Proc Natl Acad Sci. 2007;104(44):17305-17310). Consistent with these findings, EFNB2 overexpression via dCas9-VPR increased MM cell survival, proliferation and STAT5 phosphorylation. Conversely, silencing EFNB2 decreased STAT5 activation, increased MM apoptosis, and decreased MM proliferation. Importantly, STAT5 inhibition in MM cells overexpressing EFNB2 blocked EFNB2-mediated MM cell proliferation, suggesting that EFNB2-mediated MM growth is dependent on STAT5. Overexpressing a mutant form of human EFNB2 with GFP replacing the intracellular cytoplasmic PDZ signaling domain did not result in cell growth comparable to overexpressing the full-length EFNB2. Silencing EPHB4, the main EFNB2 binding partner expressed on MM cells, resulted in significant impairment of growth in wild-type MM cells but not those with full-length EFNB2 overexpression. Updated downstream signaling and in vivo data of these will be presented at the meeting. Interrogation of MM clinical databases, including the Total Therapy studies (Zhan F. et al. Blood. 2006;108(6):2020-2028) and the CoMMpass data (Multiple Myeloma Research Foundation, https://research.themmrf.org/, accessed on 20 October 2021, n = 773 patients), revealed that EFNB2 expression is increased in MM patients with adverse risk cytogenetics, including t(4;14) and gain of chromosome 1q, while lower EFNB2 expression is associated with the more favorable, "standard risk" disease category, hyperdiploid MM. Increased EFNB2 expression is also associated with advanced stage disease and decreased survival in MM patients. Our analyses suggest that EFNB2 signaling represents an important new mechanistic target in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ruxolitinib Enhances Anti-MM Function of Cytotoxic T-Cells By Upregulating IFNγ and Gzmb

Introduction: Multiple myeloma (MM) tumor cells evade host immunity through the interaction of checkpoint proteins on immune cells which creates an immunosuppressive milieu in the bone marrow (BM) microenvironment. The JAK/STAT signaling pathway in MM plays a pivotal role in mediating the effects of many different molecules, including interleukins, interferons (IFNs), colony-stimulating factors, growth factors, and cytokines. Our previous study demonstrated that the JAK1/2 inhibitor ruxolitinib (RUX) reverses checkpoint inhibition by downregulating PD-L1, PD-L2, CXCL12, CXCR4, MUC1, and CD44 expression in MM cells. We have reported that RUX overcomes resistance of MM patients to lenalidomide and glucocorticosteroids in an ongoing clinical trial. In this study, we further examined IFNγ and granzyme B (GZMB) expression in MM treated with RUX alone or in combination with anti-BCMA/CD3 bispecific antibodies. IFNγ is a key moderator of cell-mediated immunity that enhances anti-tumor effects of CD8 T cells and GZMB induces cancer cell death by cytotoxic T lymphocytes and natural killer cells. Method: Two fresh bone marrow (BM) aspirates from MM patients in complete remission (CR) were collected following informed consent. BM mononuclear cells (MCs) were isolated using density gradient centrifugation with Histopaque-1077 (Sigma, St. Louis) according to a standard protocol. MM tumor cells were isolated from the human myeloma xenograft LAG- λ1 after it was established in SCID mice. LAG- λ1 MM cells were co-cultured with BMMCs and treated with or without RUX alone or in combination with anti-BCMA/CD3 bispecific antibody for 48 hours. Following incubation, supernatant was collected for ELISA, and the cells were washed three times using PBS in preparation for flow cytometric analysis. Supernatants of culture medium from each experimental group were collected, and protein levels determined according to the IFN-gamma (R&D Systems™) and Granzyme B (R&D Systems™) ELISA protocols, without dilution. Each data was the average of duplicate samples. Results: IFNγ and GZMB levels were markedly increased when LAG- λ1 MM cells were co-cultured with BMMCs without any treatment. The BMMCs maintain the normal secretory activities of IFNγ and GZMB. IFNγ and GZMB levels in the culture supernatants were increased in BM treated with RUX alone. The level of IFNγ and GZMB were further increased in BMMCs co-cultured with LAG--λ1 MM cells that were treated with RUX. We further examined BMMCs co-cultured with LAG- λ1 MM cells treated with RUX in combination with anti-BCMA/CD3 bispecific antibody. The results showed that the levels of IFNγ and GZMB in the supernatants was markedly increased. We also determined the amount of CD8+ T-cells in BMMCs co-cultured with LAG--λ1 MM cells treated with or without RUX using flow cytometric analysis. The results showed that CD8+ T-cells were increased when BMMCs co-cultured with LAG- λ1 MM cells treated with 1, 5, and 15 µM. However, CD56+ cells were only slightly increased in BMMCs co-cultured with LAG- λ1 MM cells that were treated with RUX. Summary Immune checkpoint blockade therapy has become a novel therapeutic approach to treat cancer patients. We previously showed RUX reverses checkpoint inhibition by downregulating PD-L1 in MMBM. In this study, we further demonstrated RUX upregulates IFNγ and GZMB and CD8+ T-cells in MMBM. RUX may enhance anti-MM effects of immune-based therapies including anti-BCMA/CD3 bispecific antibody treatment as shown in these preclinical studies.

HDAC Inhibition Involves CD26 Induction on Multiple Myeloma Cells Via the c-Myc/Sp1-Mediated Prompter Activation and Overcomes Therapeutic Resistance By Humanized Antibody

Background: CD26, a 110-kDa transmembrane glycoprotein, which is expressed on several tumor cells including malignant lymphoma has been implicated in tumorigenesis, whereas, little is known about its roles in plasma cell malignancies. Recently, we have identified that CD26 is uniformly and intensely expressed in osteoclasts, while its expression in plasma cells of patients with multiple myeloma (MM) reveals heterogenous due to its cellular diversity or immune escape, leading to a marked heterogeneity of response to humanized anti-CD26 monoclonal antibody (mAb) therapy in MM. Decreased expression levels of CD26 in MM cells are one of the mechanisms underlying innate and acquired resistance to CD26mAb therapy in MM. Aim: Here, we clarify the potential impact of epigenetic modification by HDAC inhibition (HDACi) with isoform selective- or broad- inhibitors on the regulation of CD26 expression in MM cells and elucidate its mechanisms, thereby affecting the performance of CD26mAb in refractory MM. Methods and Results: Immunostaining of bone marrow tissues of MM showed that CD26pos/CD138pos plasma cells were detected in several patients but not in others. First, we investigated the impact of HDACi on CD26 expression of MM cells. Although the cell surface expression of CD26 was relatively low or not detected on 5 MM cell lines (KMS26, KMS27, KMS28, KMS11, RPMI8226), cultured alone, the increased expression in CD26 levels of MM cells was detectable within 24 hr of the treatment with HDAC1i; FK228, HDAC3i; BG45, MS-275, RG2833 or HDAC6i; nexturastat A, tubastatin A, ACY-1215 as well as broad HDACi; LBH-589, SAHA by flow cytometry. Its expression increased further during the exposure to each HDACi and its maximum increase was observed at 72 hr of the treatment. Then, subsequent removal of HDACi for 48 hrs resulted in a decline of CD26 expression on MM cells to reduced or baseline levels. In addition, the levels of CD26mRNA were concomitantly augmented, which was paralleled with an increase in the induction of CD26 protein and its DPPIV enzymatic activity in MM cells. We also examined the effect of each HDACi on the viability of CD26neg MM cells in the absence or presence of CD26mAb.CD26mAb alone did not induce lysis of CD26neg MM cell lines at any doses, whereas, combining each HDACi plus CD26mAb at 10 μg/ml synergistically facilitated lysis of CD26neg MM cells via direct effects as well as NK cell- mediated ADCC by CD26mAb. Of note, HDACi plus CD26mAb in combination readily augmented lysis of CD26neg cell populations, refractory to HDACi or CD26mAb alone. Next, gene expression profiles in KMS11, KMS27 and RPMI8226, left untreated or treated with LBH589 or RG2833 revealed that Myc is one of the overlapped genes, altered in its expression. The 5'-flanking region of human CD26 gene, representing the promoter activity contains potential binding sites of several transcriptional factors including Sp1 and c-Myc. So, further to elucidate the mechanisms by which CD26 expression is induced in MM cells by HDACi, we examined the epigenetic status in histone and non-histone at the CD26 promoter of MM cells with the treatment of HDACi. First, ChIP-qPCR demonstrated that the acetylation in histone 3 lysine 27 is increased at the CD26 promoter of 5 MM cell lines after exposure to each HDACi, suggesting parts of mechanisms in the CD26 promoter activation. Moreover, the expression level of c-Myc was time-dependently decreased in KMS11, KMS27 and RPMI8226 with the treatment of LBH-589 or RG2833 both at mRNA and protein levels and was significantly down-regulated after 12 to 24 hr of exposure. On the other hand, the acetylation of c-Myc on K323 was significantly increased after 12 to 24 hr of exposure to each HDACi. ChIP-qPCR revealed that the recovery % IP/INPUT showing the binding of c-Myc to the CD26 promoter was exposure time-dependently reduced in KMS11, KMS27 and RPMI8226 by the exposure to LBH589 or RG2833. Namely, in the absence of HDACi, c-Myc was present on the CD26 promoter via binding to Sp1 on the proximal G-C box and repressed CD26 promoter in MM cells, whereas, in the presence of HDACi, c-Myc was highly detached from Sp1 on the CD26 promoter after 12 to 24 hr with its increased acetylation, leading to activate CD26 promoter and initiate CD26 transcription in MM cells. Conclusion: HDACi not only shows anti-MM activity itself but sensitizes MM cells with CD26 antigen loss to CD26mAb via c-Myc/Sp1-mediated CD26 induction and augments its cytotoxicity.

Anti-cancer activity of Biochanin A against multiple myeloma by targeting the CD38 and cancer stem-like cells

Multiple myeloma (MM) is a malignant neoplasm of B-cells characterised by the uncontrolled proliferation of plasma cells in the bone marrow. Despite the advancements in MM treatment, it has a poor prognosis with a median survival of 3–5 years. CD38 has been one of the key therapeutic targets for MM, due to its high expression in MM cells conferring its role in immune evasion and cancer progression. Therefore, novel compounds targeting CD38 with low toxicity are warranted for improved MM therapy. In this study we aimed to investigate the anticancer activity of Biochanin A (BCA), an isoflavone, against Multiple myeloma. BCA treatment induced apoptosis in MM cells and showed reduced cytokine expression levels. In-addition, BCA significantly reduced the CD38 population and cancer stem-cell markers in dose dependent manner. Consistently, we observed BCA treatment significantly reduced the stemness markers and invasion capability of cells. Furthermore, BCA treatment significantly attenuated the tumour growth in U266 induced tumour model in NOD/SCID mice. Mechanistic studies revealed that BCA anti-cancer activities are exerted by modulating the NF-κB and MAPK signalling pathways. Overall, this study enlightens the application of BCA as a novel treatment modality for multiple myeloma with superior efficacy and reduced toxicity.

GAS6/TAM signaling pathway controls MICA expression in multiple myeloma cells.

NKG2D ligands play a relevant role in Natural Killer (NK) cell -mediated immune surveillance of multiple myeloma (MM). Different levels of regulation control the expression of these molecules at cell surface. A number of oncogenic proteins and miRNAs act as negative regulators of NKG2D ligand transcription and translation, but the molecular mechanisms sustaining their basal expression in MM cells remain poorly understood. Here, we evaluated the role of the growth arrest specific 6 (GAS6)/TAM signaling pathway in the regulation of NKG2D ligand expression and MM recognition by NK cells. Our data showed that GAS6 as well as MERTK and AXL depletion in MM cells results in MICA downregulation and inhibition of NKG2D-mediated NK cell degranulation. Noteworthy, GAS6 derived from bone marrow stromal cells (BMSCs) also increases MICA expression at both protein and mRNA level in human MM cell lines and in primary malignant plasma cells. NF-kB activation is required for these regulatory mechanisms since deletion of a site responsive for this transcription factor compromises the induction of mica promoter by BMSCs. Accordingly, knockdown of GAS6 reduces the capability of BMSCs to activate NF-kB pathway as well as to enhance MICA expression in MM cells. Taken together, these results shed light on molecular mechanism underlying NKG2D ligand regulation and identify GAS6 protein as a novel autocrine and paracrine regulator of basal expression of MICA in human MM cells.

Abstract 5352: Genome-wide CRISPR-cas9 screening identifies KDM6A as a modulator of CD38 expression in multiple myeloma: Therapeutic implications

Abstract Daratumumab (Dara) is the first-in-class monoclonal antibody targeting CD38 which triggers both direct and immune mediated cytotoxicity in preclinical in vitro and in vivo models of multiple myeloma (MM) in the bone marrow (BM) milieu. Importantly, Dara combination therapies can achieve high frequency and extent of response in both relapsed/refractory and newly diagnosed MM. However, relapse of MM is commonly observed due, at least in part, to downregulation of CD38 on MM cells. Here we utilize genome-wide CRISPR-cas9 knockout (KO) screening to identify novel regulators mediating CD38 expression and Dara sensitivity in MM cells. We identified 46 different sgRNAs to be positively correlated with MM cytotoxicity triggered by Dara and NK cells treatment. We further performed second round screening using the cell population with lowest 5% CD38 expression after CRISPR lentiviral library transduction. Importantly, KDM6A was the top ranked of the overlapping genes from these two screenings. Next, we individually KO of KDM6A in MM cells and found that both mRNA and protein levels of CD38 were significantly decreased, as well as the cell surface CD38 expression detected by flow cytometry. Re-introduction of KDM6A into KDM6A KO cells restored the expression level of CD38 and Dara-induced cytotoxicity, indicating that KDM6A positively regulates CD38 expression in MM cells. Since KDM6A regulates demethylation of lysine 27 at histone H3 (H3K27) to activate gene expression, we next examined whether CD38 expression was altered by methylation status of H3K27. From our ChIP-seq and ChIP Q-PCR data, we identified that H3K27me3 level was highly enriched on the promotor area of CD38 in KDM6A KO cells compared to control cells. These data indicate that KDM6A modulates CD38 expression by regulating H3K27me3 level on the CD38 promotor, thereby mediating sensitivity of MM cells to Dara-induced cytotoxicity. H3K27me3 is regulated by the balance of demethylase (KDM6A) and methyltransferase (EZH2). However, downregulating H3K27me3 on the CD38 promoter by enhancing KDM6A activity is challenging. Therefore, we next explored whether EZH2 inhibitor can restore CD38 expression and overcome Dara resistance in KDM6A KO cells. Indeed, H3K27me3 level on the CD38 promotor area was significantly decreased by EZH2 inhibitor. Moreover, EZH2 inhibitor treatment upregulated both CD38 mRNA and protein levels in KDM6A KO cells. Importantly, EZH2 inhibitor also restored sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity. Taken together, our studies demonstrate that KDM6A regulates H3K27me3 level on CD38 promotor area and CD38 expression in MM. Moreover, we validate that combination treatment with EZH2 inhibitor and Dara can overcome Dara resistance in preclinical MM models, providing the rationale for combination clinical trials to overcome Dara resistance and improve patient outcome. Citation Format: Jiye Liu, Lijie Xing, Teru Hideshima, Kenneth Carl Anderson. Genome-wide CRISPR-cas9 screening identifies KDM6A as a modulator of CD38 expression in multiple myeloma: Therapeutic implications [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 5352.

Targeting an MDM2/MYC Axis to Overcome Drug Resistance in Multiple Myeloma.

Simple SummaryWe show that high expression of MDM2 is associated with poor prognosis and enhanced drug resistance in human myeloma cell lines (HMCLs). Inhibition of MDM2 by RNAi or by the MDM2/XIAP dual inhibitor MX69 significantly increased the sensitivity of resistant HMCLs and primary MM samples to bortezomib and other anti-myeloma drugs, demonstrating that MDM2 can modulate drug response. We uncovered a novel oncogenic regulatory loop between MDM2 and c-Myc that mediated MM drug resistance. MDM2 inhibition resulted in a remarkable induction of apoptosis and suppression of relapsed MM cell growth. Mechanistically, MDM2 stabilized c-Myc mRNA to upregulate its expression, while c-Myc transcriptionally inducedMDM2. Targeting this regulatory loop with MX69 re-sensitized chemo-resistant MM cells to anti-myeloma agents, irrespective of TP53 tumor suppressor status, and prolonged survival in xenograft MM mouse model. These results establish a rationale for therapeutic targeting of the MDM2/c-Myc axis to improve the clinical outcome of patients with refractory/relapsed MM.Background: MDM2 is elevated in multiple myeloma (MM). Although traditionally, MDM2 negatively regulates p53, a growing body of research suggests that MDM2 plays several p53-independent roles in cancer pathogenesis as a regulator of oncogene mRNA stability and translation. Yet, the molecular mechanisms underlying MDM2 overexpression and its role in drug resistance in MM remain undefined. Methods: Both myeloma cell lines and primary MM samples were employed. Cell viability, cell cycle and apoptosis assays, siRNA transfection, quantitative real-time PCR, immunoblotting, co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), soft agar colony formation and migration assay, pulse-chase assay, UV cross-linking, gel-shift assay, RNA-protein binding assays, MEME-analysis for discovering c-Myc DNA binding motifs studies, reporter gene constructs procedure, gene transfection and reporter assay, MM xenograft mouse model studies, and statistical analysis were applied in this study. Results: We show that MDM2 is associated with poor prognosis. Importantly, its upregulation in primary MM samples and human myeloma cell lines (HMCLs) drives drug resistance. Inhibition of MDM2 by RNAi, or by the MDM2/XIAP dual inhibitor MX69, significantly enhanced the sensitivity of resistant HMCLs and primary MM samples to bortezomib and other anti-myeloma drugs, demonstrating that MDM2 can modulate drug response. MDM2 inhibition resulted in a remarkable suppression of relapsed MM cell growth, colony formation, migration and induction of apoptosis through p53-dependent and -independent pathways. Mechanistically, MDM2 was found to reciprocally regulate c-Myc in MM; MDM2 binds to AREs on c-Myc 3′UTR to increase c-Myc mRNA stability and translation, while MDM2 is a direct transcriptional target of c-Myc. MDM2 inhibition rendered c-Myc mRNA unstable, and reduced c-Myc protein expression in MM cells. Importantly, in vivo delivery of MX69 in combination with bortezomib led to significant regression of tumors and prolonged survival in an MM xenograft model. Conclusion: Our findings provide a rationale for the therapeutic targeting of MDM2/c-Myc axis to improve clinical outcome of patients with refractory/relapsed MM.

Extracellular vesicles mediate the communication between multiple myeloma and bone marrow microenvironment in a NOTCH dependent way.

Multiple myeloma (MM) is an incurable hematologic neoplasm, whose poor prognosis is deeply affected by the propensity of tumor cells to localize in the bone marrow (BM) and induce the protumorigenic activity of normal BM cells, leading to events associated with tumor progression, including tumor angiogenesis, osteoclastogenesis, and the spread of osteolytic bone lesions. The interplay between MM cells and the BM niche does not only rely on direct cell-cell interaction, but a crucial role is also played by MM-derived extracellular vesicles (MM-EV). Here, we demonstrated that the oncogenic NOTCH receptors are part of MM-EV cargo and play a key role in EV protumorigenic ability. We used in vitro and in vivo models to investigate the role of EV-derived NOTCH2 in stimulating the protumorigenic behavior of endothelial cells and osteoclast progenitors. Importantly, MM-EV can transfer NOTCH2 between distant cells and increase NOTCH signaling in target cells. MM-EV stimulation increases endothelial cell angiogenic ability and osteoclast differentiation in a NOTCH2-dependent way. Indeed, interfering with NOTCH2 expression in MM cells may decrease the amount of NOTCH2 also in MM-EV and affect their angiogenic and osteoclastogenic potential. Finally, we demonstrated that the pharmacologic blockade of NOTCH activation by γ-secretase inhibitors may hamper the biological effect of EV derived by MM cell lines and by the BM of MM patients. These results provide the first evidence that targeting the NOTCH pathway may be a valid therapeutic strategy to hamper the protumorigenic role of EV in MM as well as other tumors.

Imaging-Based Screening of Deubiquitinating Proteases Identifies Otubain-1 as a Stabilizer of c-MYC.

Simple SummaryThe transcription factor c-MYC drives growth and proliferation in most cancers. The c-MYC protein is short-lived and rapidly degraded. However, deubiquitinating enzymes can counteract its degradation and increase the levels of this oncogenic protein. We altered the expression of 41 individual deubiquitinases and measured the abundance of c-MYC. We discovered that the deubiquitinating enzyme OTUB1 slows the degradation of the transcription factor c-MYC and increases its abundance in the nucleus. Further, patients with high expression of OTUB1 show poor clinical outcomes in the c-MYC-dependent cancer multiple myeloma. Finally, elevating OTUB1 expression in multiple myeloma cells stimulates more aggressive growth. Thus, inhibition of OTUB1 may blunt c-MYC activity, making it a promising target for cancer treatment.The ubiquitin–proteasome pathway precisely controls the turnover of transcription factors in the nucleus, playing an important role in maintaining appropriate quantities of these regulatory proteins. The transcription factor c-MYC is essential for normal development and is a critical cancer driver. Despite being highly expressed in several tissues and malignancies, the c-MYC protein is also continuously targeted by the ubiquitin–proteasome pathway, which can either facilitate or inhibit c-MYC degradation. Deubiquitinating proteases can remove ubiquitin chains from target proteins and rescue them from proteasomal digestion. This study sought to determine novel elements of the ubiquitin–proteasome pathway that regulate c-MYC levels. We performed an overexpression screen with 41 human proteases to identify which deubiquitinases stabilize c-MYC. We discovered that the highly expressed Otubain-1 (OTUB1) protease increases c-MYC protein levels. Confirming its role in enhancing c-MYC activity, we found that elevated OTUB1 correlates with inferior clinical outcomes in the c-MYC-dependent cancer multiple myeloma, and overexpression of OTUB1 accelerates the growth of myeloma cells. In summary, our study identifies OTUB1 as a novel amplifier of the proto-oncogene c-MYC.

Aberrant ENPP2 expression promotes tumor progression in multiple myeloma

Ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) has been recently linked to tumor development. However, its role in modulating multiple myeloma (MM) disease progression remains unclear. Here, we demonstrated that CD138+ cells isolated from MM patients presented with higher expression of ENPP2 compared with CD138- cells. Treatment of MM cells with IL-6 resulted in ENPP2 upregulation. ENPP2 overexpression promoted proliferation, inhibited apoptosis, increased lysophosphatidic acid (LPA) generation, and upregulated osteoclastogenesis mediator expression in MM cells. In contrast, ENPP2 inhibition induced apoptosis, suppressed proliferation and survival, decreased LPA generation and downregulated osteoclastogenesis mediator expression. In an MM xenograft mouse model, ENPP2 knockdown significantly reduced MM tumor burden by inhibiting cell proliferation and inducing apoptosis. Furthermore, ENPP2 knockdown decreased the levels of LPA, osteoclastogenesis mediators in sera of mice with MM. Our findings revealed the tumor-promoting role of ENPP2 in MM, thus providing new molecular evidence for targeting the ENPP2-LPA axis in MM therapy.

Fatty Acid Binding Proteins As a Novel Therapeutic Target in Multiple Myeloma

The fatty acid binding protein (FABP) family is instrumental in fatty acid oxidation, lipid shuttling and signal transduction, but the roles of FABPs in multiple myeloma (MM) have not been explored previously, despite these being safely targetable and holding great translational promise. Using the Broad's Cancer Dependency Map (DepMap) Crispr screening tool, we found that MM, and in fact almost every cancer, shows dependency on FABP5. In patient microarray datasets, we found that increased FABP5 expression in MM cells correlates with poor overall survival (OS) and shorter relapse free survival (Zhan et al., 2006., Mulligan et al., 2006., Carrasco et al., 2006). Interestingly, MM cells from “proliferative” (most aggressive) patient subgroups have the highest expression of FABP5 when comparing the molecular MM subtypes (Zhan et al. 2006). We also found that MM cells from relapsed patients have increased FABP5 expression versus newly-diagnosed patients (Chng et al., 2007). Finally, we analyzed 779 patients in the MMRF CoMMpass dataset and confirmed worse progression-free survival (PFS) and OS in patients with higher than mean FABP5 expression levels in their tumor cells (log-rank-value for high vs. low expression <0.0001 for both PFS and OS) (Fig. 1). Overall, our data strongly suggest that elevated FABP5 is a novel risk factor for poor MM prognosis and that targeting the FABP family holds great promise as a new therapeutic avenue.We next demonstrated that FABP5 is the most highly expressed FABP family member in myeloma cell lines and patient samples, and that inhibition of FABP proteins with two inhibitors, BMS309403 and SBFI-26, negatively impacts MM cell cycle and cell number, and induces apoptosis in human (MM.1S, RPMI-8226, H929, and OPM2) and mouse (5TGM1 and Vk*Myc) MM cells in vitro in a dose- and time-dependent manner. Next, using the MM.1S/SCID-beige xenograft and the syngeneic 5TGM1-TK/C57BL/KaLwRij (KaLwRij) models, we found that pharmacological FABP inhibition improves survival and decreases tumor burden. In both models, mice were tail vein inoculated with MM cells (5x10^6 MM.1S or 1x10^6 5TGM1-TK) and started on intraperitoneal treatments the following day. SCID-beige mice received 5 mg/kg BMS309403, 1 mg/kg SBFI-26, both, or vehicle 3x/week; KaLwRij mice received 5 mg/kg BMS309403 or vehicle 3x/week. Mice were analyzed biweekly with live bioluminescent imaging of whole mice starting 10-12 days post inoculation. In the SCID-beige model, all treatments reduced tumor burden starting at day 21 and led to significantly increased survival, compared to the vehicle. Similar trends were also seen in the KaLwRij group for BMS309403 treatments; SBFI-26 and combinations are now being explored in this model.After demonstrating strong evidence that FABP inhibition reduced tumor growth in vitro and in vivo, we performed RNA-sequencing of human MM.1S cells treated with BMS309403, SBFI-26, both inhibitors, or neither, at 50 μM for 24 hours and analyzed gene and pathway alternations using dbString and Ingenuity pathway analyses with cutoffs of FC≤|1.2|. RNA-sequencing pathway analysis revealed that single or combination treatment decreased IRE1-mediated unfolded protein response, endoplasmic reticulum (ER) unfolded protein response, and chromatin silencing. At the individual gene level, major oncogenic genes demonstrated decreased expression including c-myc, KRAS and XBP1. Western blot confirmed that Myc protein was decreased with FABP inhibition in MM cells, illustrating a novel role for FABP proteins in tumor cell survival and proliferation. To confirm that FABP inhibition has negative effects on MM cells, Myc signaling and ER stress, and to rule out inhibitor off-target effects, we also generated FABP5 knockout MM.1S cell pools using Crispr/Cas9, which we are analyzing with RealTime-Glo and RNA-sequencing.In conclusion, FABP inhibition slowed tumor growth and induced tumor cell death in vitro and in two mouse models. RNA-sequencing revealed two potential, novel mediators of FABP action in MM cells: Myc pathway inhibition and enhanced ER stress. Dysfunction in these two pathways via FABP inhibitors potentially triggered cell cycle arrest, decreased proliferation, and increased apoptosis in MM cells. Combined with clinical data from CoMMpass and other datasets, this suggests that FABP inhibition in MM is an untapped therapeutic avenue that should be further explored and developed. [Display omitted] DisclosuresMarinac: GRAIL Inc: Research Funding; JBF Legal: Consultancy.

Identification of Novel Targets Based on Splicing Alterations for Undruggable RAS/CDK Signaling Cascade in Multiple Myeloma

Background: RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis. Targeting these pathways represents a novel therapeutic strategy in MM. Our ongoing studies (>420 patients) demonstrate that aberrantly spliced transcript expressions can predict MM patient survival outcomes better than gene expression alone, indicating a significant role of splicing mechanism in MM pathophysiology. These studies also identified intron retentions as the predominant recurrent alterations (~32% of spliced genes were retained introns) in MM. We evaluated splicing alterations associated with pathway-level responses after RAS/CDK inhibition in order to identify and validate novel molecular targets.Methods/results: MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to inhibit RAS and CDK pathways. Our studies demonstrated strong synergistic (IC<0.5) MM cytotoxicity triggered by this combination treatment, which triggered dose-dependent manner G0/G1 phase growth arrest. We assessed early death cascade in MM cells after Ei+Ci treatment, and demonstrated significant priming to selective peptides BIM, BAD, and MS1 or HRK, suggesting dependency on BCL2 and MCL1 or on BCL-XL proteins. Our studies showed that Ei+Ci treatment induced inhibition of key target molecules in Erk1/2 and CDK4/6 signaling including c-myc, p-RSK, p-S6, p-RB, and E2F1, suggesting on-target activity of Ei and Ci. Patient MM cells co-cultured with or without autologous BM stromal cells remain equally sensitive to Ei+Ci, suggesting that this combination can overcome the protective effects of the MM BM milieu. Moreover, our in vivo study demonstrated a significant (P=0.0004) MM burden decrease in Ei+Ci-treated mice. We evaluated the effect of Ei+Ci treatment on target gene expression in BM cells isolated from flushed femurs of treated animals with Ei, Ci or Ei+Ci, and observed downregulation of Erk1/2-CDK4/6-dependent gene signature. Therefore, we suggest that these inhibitors selectively target Erk1/2, CDK4/6 and their downstream substrates both in vitro/vivo.We next evaluated aberrantly spliced transcript expression in MM cells, with/without Erk1/2 knockdown (KD) or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. This observation was further supported by principal component analyses: upregulation in response to Erk1/2 KD and downregulation due to treatment with Ei+Ci were considered spliced gene-signatures linked to RAS/CDK modulation. Gene/pathway enrichment analyses of these genes showed their involvement in cell proliferation and regulation of epigenetic networks in MM. Importantly, these analyses suggest that overexpression of RAVER1/SNRPB core splicing regulator genes are associated with RAS/CDK pathway regulation. These genes encode subunits of U1/2/4/5 spliceosome complex and are involved in intron retention processes, a marker of malignant transformation. We compared signature-gene expressions from 558 MM patient samples to the signature-genes in plasma cells from normal donors and observed significant (p<2e-11) upregulation of genes with progression from MGUS to sMM, and, also to overt MM . SNRPB overexpression is associated with shorter overall patient survival (p<0.01), while RAVER1 is linked with poor outcomes. SNRPB proteins are also overexpressed in MM cells. Our studies evaluating SNRPB effects on RNA splicing showed both upregulation of transcripts with full intron retention and transcripts with cryptic stop codons utilizing intronic sequences causing their partial retention. We evaluated RAVER1 and SNRPB expression in BM cells from animals treated with Ei and Ci alone or in combination. We observed significant downregulation of RAVER1/SNRPB (p=0.001) in BM samples obtained from animals treated with Ei+Ci. We observed decreased intron retention events in genes in treated samples, consistent with our in vitro analyses in MM cell lines and patient samples. Thus, RAVER1/SNRPB overexpression contributes to the aberrant transcriptome splicing associated with RAS/CDK cascade in MM.Conclusions: Our studies 1) show an association between RNA processing and RAS-CDK pathways in MM, 2) identify a core splicing protein, SNRPB/RAVER1, as a novel target for modulating this cascade, and 3) suggest that targeting spliceosome complexes represents a promising therapy in MM. DisclosuresLetai: Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board. Anderson: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: 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.

BCMA-Specific ADC MEDI2228 and Daratumumab Induce Synergistic Myeloma Cytotoxicity via IFN-Driven Immune Responses and Enhanced CD38 Expression.

Efforts are required to improve the potency and durability of CD38- and BCMA-based immunotherapies in human multiple myeloma. We here delineated the molecular and cellular mechanisms underlying novel immunomodulatory effects triggered by BCMA pyrrolobenzodiazepine (PBD) antibody drug conjugate (ADC) MEDI2228 which can augment efficacy of these immunotherapies. MEDI2228-induced transcriptional and protein changes were investigated to define significantly impacted genes and signaling cascades in multiple myeloma cells. Mechanisms whereby MEDI2228 combination therapies can enhance cytotoxicity or overcome drug resistance in multiple myeloma cell lines and patient multiple myeloma cells were defined using in vitro models of tumor in the bone marrow (BM) microenvironment, as well as in human natural killer (NK)-reconstituted NOD/SCID gamma (NSG) mice bearing MM1S tumors. MEDI2228 enriched IFN I signaling and enhanced expression of IFN-stimulated genes in multiple myeloma cell lines following the induction of DNA damage-ATM/ATR-CHK1/2 pathways. It activated cGAS-STING-TBK1-IRF3 and STAT1-IRF1-signaling cascades and increased CD38 expression in multiple myeloma cells but did not increase CD38 expression in BCMA-negative NK effector cells. It overcame CD38 downregulation on multiple myeloma cells triggered by IL6 and patient BM stromal cell-culture supernatant via activation of STAT1-IRF1, even in immunomodulatory drug (IMiD)- and bortezomib-resistant multiple myeloma cells. In vitro and in vivo upregulation of NKG2D ligands and CD38 in MEDI2228-treated multiple myeloma cells was further associated with synergistic daratumumab (Dara) CD38 MoAb-triggered NK-mediated cytotoxicity of both cell lines and autologous drug-resistant patient multiple myeloma cells. These results provide the basis for clinical evaluation of combination MEDI2228 with Dara to further improve patient outcome in multiple myeloma.

New Antibcma-CAR for Multiple Myeloma

B Cell Maturation Antigen (BCMA) has appeared as a promising target to use in CAR immunotherapy for multiple myeloma (MM) patients due to specific BCMA expression in plasma cells. We designed a 2nd generation CAR against BCMA adding 4-1BB as co-stimulatory domain. T cells were successfully transfected with our CAR against BCMA. CAR-T BCMA cells were functional in vitro against 3 different MM cell lines. Cytotoxicity assays were performed at ratios 1:1 and 0.5:1 of CAR-T: MM cell lines. All MM cells were eliminated completely at 72 hours. Moreover, CAR-T BCMA cells were functional in an in vivo NSG-mouse model with MM disease already established (Figure 1A).Even published results are promising, high CAR-T cell doses have been needed to achieve clinical responses, which is associated with high toxicities. Moreover, loss of expression of the target antigen (BCMA in our study) appears in a proportion of patients with relapses, indicating that CAR-T cells will not recognize the tumor cell for any longer. Meanwhile, an ideal CAR treatment would be the one achieving permanent responses, with the lowest CAR cell dose possible, to avoid toxicities associated to the treatment.We previously observed that when Natural Killer (NK) cells and MM cells become in contact there is concomitant transfer of NK cell proteins to the MM cell, and also MM proteins are transferred to NK cells. This transfer is performed in lipid structures, such as vesicles and lipid rafts. We hypothesized that after CAR-T and MM cell contact the same phenomenon could occur, with concomitant transfer of the CAR target antigen. This might explain why there is a loss of expression of the target antigen after CAR treatment. Therefore, we studied trafficking of BCMA between MM cells and CAR-T BCMA cells.First, we analyzed BCMA expression in MM cells by confocal fluorescence microscopy and observed that BCMA is continuously being recycled and released in vesicles to the extracellular milieu. To analyze the trafficking of BCMA in MM cells after CAR-T BCMA exposure, we over-expressed BCMA fused to green fluorescent protein (GFP) in MM cells. MM cells over-expressing BCMA-GFP were co-cultured for 4 hours with CAR-T BCMA cells while time lapse in vitro confocal fluorescence microscopy was performed. BCMA transfer was observed to the extracellular milieu in vesicles and also to CAR-T BCMA cells. Moreover, flow cytometry analysis revealed that after 24h of co-culturing CAR-T BCMA cells with MM cells, a loss of BCMA surface expression in MM cells occurred, which was not observed with control non-transduced T cells (Figure 2B). Moreover, we observed that a proportion of CAR-T BCMA cells acquired BCMA expression.To avoid this BCMA transfer we hypothesized that lipid synthesis inhibitors could be useful. Statins by inhibiting the Ac-CoA pathway, block cholesterol synthesis, and also they have an impact in the prenylation of proteins involved in protein degradation and recycling. Moreover, statins are anti-inflammatory agents and show anti-MM properties, suggesting their potential as co-adjuvants for CAR treatment. We tested and confirmed in vitro and in vivo the anti-MM properties of Fluvastatin. Moreover, 3h pre-treatment of MM cells with Fluvastatin before co-culturing with CAR-T BCMA cells decreased significantly loss of intracellular BCMA expression in MM cells (Figure 3C). Surface BCMA expression was the same, suggesting a decreased BCMA secretion to the extracellular milieu after statin treatment. Last, in a MM NSG-mouse model, pretreatment of mice with Fluvastatin (0.85mg/kg) for 4 days, before infusing CAR-T BCMA cells, did not impact negatively in the CAR-T BCMA cells activity. In conclusion, statins could be a good co-adjuvant to use in CAR immunotherapy treatment to prevent loss of BCMA antigen expression in the remaining MM cells after CAR treatment. Moreover, their anti-inflammatory properties could also improve side effects associated to this therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Altered Expression of CCR10 Indicates Malignant Transformation of Bone Marrow Plasma Cells

Introduction. Phenotypic analysis of tumor cells by flow cytometry is now essential for diagnosis and treatment of hematological malignancies, including multiple myeloma (MM). Surface molecules such as CD19, CD56, CD27 and CD45 have been used to distinguish normal bone marrow (BM) plasma cells from malignant plasma cells (MM cells). Molecules that are relatively specific to plasma cells (CD38 and SLAMF7) are considered targets for antibody-based therapies in MM. Among the vast numbers of surface molecules, chemokine receptors have been attracting much attention as the regulators of migration and tissue localization of plasma cells and MM cells. CXCR4 (C-X-C chemokine receptor type 4) has been reported to guide the movement of plasma cells and myeloma cells to the BM, where its ligand CXCL12 (C-X-C motif chemokine 12) is abundantly produced. CCR10 (C-C motif chemokine receptor 10) is another chemokine receptor, whose ligand is CCL27 (C-C motif chemokine ligand 27) and CCL28, also known to be involved in cell homing and migration. CCR10 has been reported to be highly expressed on mucosal derived plasma cells, however its expression in MM cells and its role within the BM microenvironment has not been well studied. In this study, we analyzed the expression of CCR10 on BM lymphocytes from patients of plasma cell disorders in order to understand its significance in the BM microenvironment.Materials and methods. We analyzed a public available gene expression dataset (GSE 22886) in order to study CCR10 gene expression in human plasma cells. To examine CCR10 gene expression in myeloma cells, we utilized the gene expression data set of multiple cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE: http://www.broadinstitute.org/ccle). Multi-color flow cytometry was performed to study CCR10 expression on bone marrow lymphocytes. BM plasma cells were determined as CD38++ CD138+ or CD38++ SLAMF7+. Monoclonal plasma cells (myeloma clones) were determined as CD19- CD56- or CD19- CD56+ cells and polyclonal plasma cells (normal plasma cells) as CD19+ CD56- cells. CCR10 expression of BM plasma cells, B cells, CD4 T cells, CD8 T cells and NK cells of MGUS, newly diagnosed MM, relapsed-refractory MM patients were analyzed.Results. Gene expression dataset analysis showed that, among the normal leukocyte subsets, plasma cells had the highest CCR10 gene expression compared to other leukocytes (p<0.001). Myeloma cell lines showed the highest expression of CCR10 gene expression among all the other cancer cell lines, indicating the specificity of CCR10 expression in MM cells. Multi-color flow cytometry showed significantly high expression of CCR10 on BM plasma cells compared to other lymphocytes in the bone marrow of MGUS patients (p<0.0001). Within the MGUS BM plasma cells, monoclonal plasma cells had significantly lower CCR10 expression compared to polyclonal plasma cells (p<0.05). Additionally, monoclonal plasma cells from relapsed-refractory MM patients had significantly lower CCR10 expression compared to those from MGUS and newly diagnosed MM patients. These data indicate a role of CCR10 decrease in malignant plasma cells leading to MM progression. We further analyzed other surface molecules that may be related to CCR10 on monoclonal plasma cells. CCR10 expression did not correlate with CD56 expression, while CD45- monoclonal plasma cells tend to have lower CCR10 expression compared to the CD45+ counterparts in newly diagnosed MM patients (p<0.001).Conclusions. Gene expression dataset analysis and multi-color flow cytometry have showed that CCR10 is highly expressed in normal plasma cells. However, we show here for the first time that plasma cells start to decrease CCR10 expression at malignant transformation and during disease progression. Since, CCL27, the ligand of CCR10, is known to induce chemotaxis to plasma cells, loss of CCR10 may lead to migration or metastasis of MM cells to different sites. Further research is now on-going to elucidate the significance and mechanism of altered CCR10 expression in MM cells. This study may lead to a better understanding of MM biology and to the development of a novel strategy to target malignant plasma cells. DisclosuresNo relevant conflicts of interest to declare.

Targeting CD47 As a Novel Therapeutic Strategy in Multiple Myeloma

Introduction: CD47 is an immune checkpoint coordinating the innate and adaptive immune systems which allows cancer cells to escape the innate immune response by sending inhibitory signals to macrophages and regulates the adaptive immune anti-tumor response. Hence, CD47 has been identified as a novel therapeutic target in cancers, including multiple myeloma (MM) which is localized mainly in the bone marrow (BM). MM cells rely heavily on the interactions with tumor microenvironment (TME) and immune system. Macrophages are enriched in the BM of MM patients compared to healthy controls, and were demonstrated to support MM growth, in opposition to their intrinsic phagocytic properties. We study the new class of immune checkpoint inhibitor such as anti-CD47 monoclonal antibody (mAb) which enhance MM killing. We tested two anti-CD47 mAbs provided; Vx1000R (blocking only) - blocking CD47/SIRPα binding and Vx1004R (dual function) - blocking CD47/SIRPα binding and inducing cell death activity.The objectives of this study were: (1) to test the expression of CD47 (Vx1000R and Vx1004R binding) to MM cells, stromal cells (MSP-1 and HS5) and peripheral blood mononuclear cell (PBMC) sub-populations; (2) to study anti-CD47 mAbs binding in hypoxic conditions; (3) to investigate the effect of anti-CD47 mAbs on MM cell apoptosis in 2D and the 3D tissue engineered BM (3DTEBM; a scaffold incorporating BM plasma from MM patients, MM cells, and accessory cells) to reproduce the TME; (4) to test the cytotoxicity of Vx1004R on PBMC sub-populations in 3D culture; and (5) to examine the effect of Vx1004R on MM cell phagocytosis ex vivo by macrophages.Methods: We performed meta-analysis of CD47 mRNA expression in dataset available online in healthy, MGUS, smoldering MM and MM subjects. We tested the binding of Vx1000R and Vx1004R mAbs on MM.1S, H929, RPMI8226 and U266 cells and stromal cells (MSP-1 and HS5) in normoxia (21% O2) and hypoxia (1% O2), and in the 3DTEBM at 24h and 72h. We also tested Vx1000R binding to PBMC sub-populations (from 12 MM patients) including CD3 (T cells), CD14 (monocytes/macrophages), CD16 (NK cells, eosinophils, and neutrophils), CD19 (B cells), CD123 (pDC and basophils) and CD138 (MM cells).Next, we examined the cytotoxic activity of CD47 mAbs on MM cells alone and in co-culture with the BM negative fraction (CD138-depleted) and stromal cells (MSP-1) in 2D cultures by performing MTT survival assay, and in 3DTEBM cultures by flow cytometry. The cytotoxicity of Vx1004R was studied on PBMCs incorporated into a similar 3D model and treated with Vx1004R; proliferation and apoptosis of different sub-populations of PBMC were assessed via flow cytometry. Lastly, the effect of Vx1004R on MM cell phagocytosis by macrophages (CD14+; green) was tested after 2h of co-culture with MM cells (pre-labeled with DiD+; red) in the presence or absence of Vx1004R. Phagocytosis was determined as double positive (green+ red+) cells by flow cytometry.Results: The meta-analysis results demonstrated that CD47 mRNA expression increases with tumor progression (compared to early stage disease and healthy subjects). We demonstrated that MM.1S, H929, U266 and RPMI8226 cells and stromal cells (MSP-1 and HS5) express high levels of CD47, which does not alter in hypoxia, and that MM cells increase CD47 expression in 3DTEBM at 72h. CD47 (Vx1000R) is expressed in different sub-populations of PBMCs with the highest expression in MM cells. In addition, we have shown that anti-CD47 dual function mAb inhibits proliferation of MM cells, especially when co-cultured with negative fraction and MSP-1 stroma in 3DTEBM cultures. Moreover, we showed that anti-CD47 mAb induces phagocytosis of MM cells ex vivo by macrophages.Conclusions: CD47 is a promising target candidate for MM treatment, since CD47 mRNA increases with MM progression, and CD47 protein increases in MM cells in the presence of TME in the 3DTEBM, suggesting involvement in pathophysiology of MM. Moreover, anti-CD47 mAb enhances killing of MM cells when co-cultured with TME; however, it does not affect apoptosis of PBMC sub-populations in the 3DTEBM. Finally, anti-CD47 mAb induces phagocytosis of MM cells ex vivo by macrophages. These results suggest that anti-CD47 mAbs are applicable for MM treatment. Further studies are warranted to examine the effect of anti-CD47 mAbs as a novel strategy to inhibit the progression and dissemination of MM in vivo, especially when combined with chemotherapy. DisclosuresVij:Takeda: Honoraria, Research Funding; Jazz: Honoraria; Abbvie: Honoraria; Celgene: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria; Bristol-Meyers-Squibb: Honoraria; Konypharma: Honoraria. Capoccia:Tioma: Employment. Manning:Tioma: Employment. Azab:Abbvie: Research Funding; Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Cleave Bioscience: Research Funding; Cellatrix LLC: Other: Founder and owner; Glycomimetics: Research Funding; Tioma: Research Funding.

Protein Kinase CSNK1A Regulates the Multiple Myeloma Plasma Cell-Microenvironment Interplay

Multiple myeloma (MM) is a plasma cell malignancy characterized by a strong dependency on the bone marrow microenvironment, which makes plasma cells resistant to pro-apoptotic stimuli and favours the myeloma-associated bone disease (MMABD). Alteration of Wnt/β-catenin, Hedgehog (Hh) and NF-κB signaling by plasma cell-intrinsic or bone marrow stroma-delivered signals sustains MM cell growth and contributes to bone homeostasis alterations. Protein kinase CK1α (CSNK1A1, casein kinase 1α) belongs to a family of monomeric Ser/Thr kinases that regulate cellular processes important in cancer biology such as membrane biogenesis, transport of molecules, signal transduction, transcription, translation and the DNA damage response. CK1α is also an essential regulator of the function of the Wnt, Hh and NF-κB signalling pathways. It is known that osteogenic differentiation is dependent on the activation of Wnt/β-catenin and that CK1α exerts a negative regulative role on this pathway. Thus, CK1α could play a role in MMABD representing a rational therapeutic target. We recently demonstrated that CK1α supports MM plasma cells growth, in part through the regulation of AKT and β-catenin. CK1α is overexpressed in MM patients compared to healthy controls, and its inhibition/silencing in plasma cells causes apoptosis and cell cycle arrest (Manni et al ., 2017, Oncotarget 8(9):14604). To investigate if this kinase could regulate osteogenesis, we analyzed the generation of osteocytes from stromal cells in conditions of CK1α inactivation in BM stromal cells (BMSC). We found that at variance from malignant plasma cells, CK1α silencing in bone marrow stromal cells did not impact on cell survival but it enhanced the differentiation potential towards bone formation, inducing an osteoblastogenic transcriptional program. CK1α silencing in BMSC, led to the stabilization of β-catenin, increased mRNA expression of the osteoblast markers RUNX2, SPP1 and BGLAP, and it increased calcium deposits of stromal cells pushed to differentiate towards osteoblasts/osteocytes. Moreover, we observed that the immunomodulatory drug lenalidomide, by inducing CK1α degradation in both MM and stromal cells, potentiated the effects of CK1α silencing in BMSC, further supporting osteoblast formation. Previous papers reported that MM cells overexpress RUNX2, leading to a higher expression of RANKL, which in turn could be responsible of the inhibition of osteoblast activity in the bone marrow microenvironment (Giuliani et al., 2005 ; Blood 106, 2472). Moreover, elevated expression of RUNX2 in MM cells enhanced tumour growth and bone disease (Trotter et al., 2015, Blood 125, 3598). To investigate the effects of CK1α on RUNX2 levels in MM cells, we created a model whereby the BM microenvironment dependent INA-6 cell lines were stably transduced with lentiviral shRNA vectors to obtain CK1α silencing by addition of IPTG and then were cultured in the presence of BM stromal cells. At variance with BMSC, CK1α silencing in MM cells determined a reduction of RUNX2 in MM cells with a consequent RANKL-induced increase of RUNX2 in stromal cells. Therefore, CK1α could have a positive regulative role on RUNX2 expression in MM cells participating in the impairment of osteoblastogenesis and yet a negative regulative role still on RUNX2 levels in BMSC. Altogether, our data provide a mechanistic framework for the function of CK1α in BMSC biology and in the MM-BMSC crosstalk and support a dual beneficial therapeutic role of CK1α inhibition in MM, with the potential of targeting not only the malignant plasma cells but also the MMABD. DisclosuresNo relevant conflicts of interest to declare.

Immune Microenvironment Contributes to Dysregulation of Aid/Apobec Expression and Genomic Instability in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy with marked genomic instability. Using whole genome sequencing, we have previously demonstrated that myeloma cells display heterogeneity in mutational spectrum and diverse patterns of clonal evolution. We have also identified AID/APOBEC-driven signature as one of 2 biologically distinct mutational signatures responsible for the majority of observed mutations. Our data show that several members of APOBEC family (including 3A, B, C, and G) are up regulated in patient MM cells and cell lines. Using SNP array and whole genome sequencing, we have also confirmed that elevated expression of APOBECs (3B, C, and G) contributes to genomic instability in MM cells. Since genomic instability and associated clonal evolution could also be driven by pressures from microenvironment, in this study we investigated the role of microenvironment in dysregulation of AID/APOBECs in MM cells.In normal cellular environment, AID and APOBECs are expressed by B and T cells and play a key role in adaptive and innate immunity. However, their expression is tightly regulated in order to prevent nonspecific damage to cellular DNA. We hypothesized that abnormal immune components and soluble factors in the bone marrow microenvironment can induce APOBEC expression in MM cells and can contribute to genomic instability. As we have previously shown that pDCs, MDSCs, Th17cells and B1/B2 cell ratio were significantly elevated in peripheral blood and bone marrow of MM patients, we evaluated the role of these immune cells (iDC, pDC, B, B1, and T cells), and soluble components (IL6, IL-17, IL-27, IFNa, and IFNb) on APOBEC expression and activity in myeloma cells. We used purified immune cells from healthy donors and co-cultured them with MM cells. Following a 3 day co-culture, MM cells were purified and analyzed. APOBEC expression by RT-qPCR. Co-culture with immune cells caused increased expression (range: 2-100 fold) of several APOBEC family genes in myeloma cells. Among different immune cells, iDC and pDC induced the expression of AID, A3A, A3G, and A3H; TLR9 activated B cells induced the expression of AID and A3G; B1 cells induced expression of AID, A3A and A3G; and, IL-17 activated T cells induced the expression of AID, A3A and A3G. Among soluble components of immune microenvironment, interferon a and β induced APOBEC expression (A3A, A3G, and A3H; range: 4-15 fold) in myeloma cells after treatment. Interestingly, none of the interleukins tested showed any impact on APOBEC gene expression in these cells. We then tested if IRF9, a downstream effector of type-1 interferons is involved in the interferon-mediated induction of APOBECs. IRF9-siRNA significantly inhibited interferon-mediated induction of APOBECs (by 50%) in myeloma cells suggesting that interferon mediated up-regulation of APOBECs in MM cells is mediated at least in part by IRF9. We also used a functional assay and demonstrated that increased expression of APOBEC genes was associated with increased APOBEC deaminase activity in MM cells. To investigate the genomic impact of interferon induced APOBEC activity, we treated MM cells with IFN-β and evaluated its impact on abasic sites (that could directly result from APOBEC deaminase activity) and micronuclei assay (a marker of genomic instability). The number of abasic sites and micronuclei were increased by 2 and 4 fold respectively, in cells treated with IFN-β. Overall, these results suggest that BM microenvironment can induce APOBEC expression/activity and associated genomic instability in MM cells. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic effect and mechanism of ibrutinib combined with dexamethasone on multiple myeloma.

Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase and has proven to be an effective agent for B-cell-mediated hematological malignancies, including multiple myeloma (MM). Several clinical trials of ibrutinib treatment combined with dexamethasone (DXMS) for relapsed MM have demonstrated high response rates, however, the mechanism still remains unclear. In this study, we explored the therapeutic effect and mechanism of ibrutinib combined with DXMS on MM in vitro and vivo. The apoptosis of MM cell lines and mononuclear cells from MM patients' bone marrow induced by ibrutinib combined with DXMS was detected by flow cytometry and the expression of apoptosis-related proteins were detected by Western blot. A mice MM model was established to verify the therapeutic effect of ibrutinib combined with DXMS on MM. We found that ibrutinib combined with DXMS increased the apoptosis of MM cell lines through the PI3K/PARP pathway, significantly reduced CD38 expression in MM cells from patients in vitro, and reduced tumor size and increased the survival time in mice model. This study provides a theoretical basis for the treatment of relapsed refractory MM with ibrutinib combined with DXMS, and a potential therapeutic target for MM clinical treatment.