Primary Multiple Myeloma Cells Research Articles

Combined inhibition of Wee1 and Chk1 as a therapeutic strategy in multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents that have significantly improved clinical outcome, most patients relapse and develop drug resistance. MM is characterized by genomic instability and a high level of replicative stress. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways. In this study, we reported that high CHK1 and WEE1 expression is associated with poor outcome in independent cohorts of MM patients treated with high dose melphalan chemotherapy or anti-CD38 immunotherapy. Combined targeting of Chk1 and Wee1 demonstrates synergistic toxicities on MM cells and was associated with higher DNA double-strand break induction, as evidenced by an increased percentage of γH2AX positive cells subsequently leading to apoptosis. The therapeutic interest of Chk1/Wee1 inhibitors' combination was validated on primary MM cells of patients. The toxicity was specific of MM cells since normal bone marrow cells were not significantly affected. Using deconvolution approach, MM patients with high CHK1 expression exhibited a significant lower percentage of NK cells whereas patients with high WEE1 expression displayed a significant higher percentage of regulatory T cells in the bone marrow. These data emphasize that MM cell adaptation to replicative stress through Wee1 and Chk1 upregulation may decrease the activation of the cell-intrinsic innate immune response. Our study suggests that association of Chk1 and Wee1 inhibitors may represent a promising therapeutic approach in high-risk MM patients characterized by high CHK1 and WEE1 expression.

Liposome-Encapsulated Melphalan Exhibits Potent Antimyeloma Activity and Reduced Toxicity.

Multiple myeloma (MM), a plasma cell cancer in bone marrow, remains an incurable disease. Melphalan, an alkylating agent, is a conventional anticancer drug that is still widely used for MM treatment in clinics. However, melphalan-induced organ toxicity and side effects are common. In this study, we loaded melphalan into a liposomal capsule and constituted liposomal melphalan (liposomal MEL). Liposomal MEL particles were approximately 120 nm in size and stable in vitro. The liposomal particles could be effectively taken up by MM cells. In vitro cytotoxicity assays using MM cell lines and primary MM cells showed that liposomal MEL exhibited similar anti-MM activity compared to an equivalent amount of free melphalan (free MEL) compound. In animal models, liposomal particles had bone marrow enrichment and prolonged half-life in vivo. Liposomal MEL exposure resulted in less liver and colon organ toxicity than exposure to an equivalent amount of free MEL-treated mice. Importantly, liposomal MEL had potent anti-MM activity in vivo in a human MM xenograft mouse model. Overall, our findings suggested that liposome-encapsulated melphalan was an effective drug modification of the melphalan compound and showed promise in MM treatment.

SMG1, a nonsense-mediated mRNA decay (NMD) regulator, as a candidate therapeutic target in multiple myeloma.

Early data suggested that CC-115, a clinical molecule, already known to inhibit the mammalian target of rapamycin kinase (TORK) and DNA-dependent protein kinase (DNA-PK) may have additional targets beyond TORK and DNA-PK. Therefore, we aimed to identify such target(s) and investigate a potential therapeutic applicability. Functional profiling of 141 cancer cell lines revealed inhibition of kinase suppressor of morphogenesis in genitalia 1 (SMG1), a key regulator of the RNA degradation mechanism nonsense-mediated mRNA decay (NMD), as an additional target of CC-115. CC-115 treatment showed a dose-dependent increase of SMG1-mediated NMD transcripts. A subset of cell lines, including multiple myeloma (MM) cell lines sensitive to the endoplasmic reticulum stress-inducing compound thapsigargin, were highly susceptible to SMG1 inhibition. CC-115 caused the induction of UPR transcripts and cell death by mitochondrial apoptosis, requiring the presence of BAX/BAK and caspase activity. Superior antitumor activity of CC-115 over TORK inhibitors in primary human MM cells and three xenograft mouse models appeared to be via inhibition of SMG1. Our data support further development of SMG1 inhibitors as possible therapeutics in MM.

Long Noncoding RNA LINC01410 Interacts with the Minichromosome Maintenance (MCM) Complex to Promote Tumor Cell Growth in Multiple Myeloma

The human genome contains thousands of loci that produce long non-protein coding RNAs (lncRNAs) that play critical roles in healthy cell function as well as tumorigenesis. We have recently described the lncRNA landscape in newly diagnosed multiple myeloma (MM), identified their role as independent risk predictors for clinical outcome, and validated the functional contribution of specific lncRNAs to MM cell growth and viability. Although these examples suggest the significance of lncRNAs in MM pathobiology, the vast majority of lncRNA genes have not been functionally tested. To identify lncRNAs that promote MM cell growth, we performed a large-scale cell viability screen of lncRNAs using CRISPR interference (CRISPRi) to inhibit gene expression. To find lncRNA targets, we first interrogated our large RNA-seq dataset from 360 newly diagnosed MM patients and identified 913 lncRNA transcripts expressed in primary MM cells. We designed the CRISPRi library to target the transcription start site of these 913 lncRNA genes, each with 7 sgRNAs. We screened 3 MM cell lines (H929, KMS12BM, KMS11) and identified 32 lncRNA loci that modified cell growth upon CRISPRi targeting, with 7 lncRNAs impacting all three cell lines tested. We then evaluated the differential expression of these lncRNAs in primary MM cells compared to normal plasma cells (PC). We observed that LINC01410 was significantly overexpressed in MM patients compared to normal PC, especially in MM patients with t(4;14) translocations, and was an independent risk predictor that highly correlated with overall survival in newly diagnosed MM patients. We next knocked down LINC01410 using gapmeR antisense oligonucleotides (ASO) in a panel of MM cell lines and observed time-dependent inhibition of MM cell viability, including in the t(4;14) cell lines. As LINC01410 appears to localize to both the cytosol and nucleus, we also utilized siRNAs to specifically target the cytosolic isoform and observed a robust reduction of cell growth, suggesting that both cellular localizations are important for the activity of LINC01410. Transcriptomic analysis indicated a set of genes modulated in response to LINC01410 depletion, with an enrichment in genes involved in the cell cycle and DNA replication. An RNA-protein pull down (RPPD) assay identified three out of six MCM complex subunits (MCM4, MCM5, MCM7) as relevant protein interactors of LINC01410 and was confirmed using RNA-immunoprecipitation (RIP) assay. The MCM complex is a DNA helicase essential for genomic DNA replication, and we hypothesize that LINC01410 affects cell proliferation by interacting with this complex. We are now investigating this functional interaction by looking at the impact of LINC01410 modulation on the assembly of the complex, by using integrated mass spectrometry analysis, and on its ability to load onto chromatin by using chromatin immunoprecipitation sequencing (ChIP-Seq). In conclusion, we report the identification of LINC01410 as a novel lncRNA dependancy that supports MM cell growth via interacting with the MCM complex and controlling the cell cycle/DNA replication axis. Promising developments in the field of lncRNA inhibitors may allow forthcoming clinical applications.

EZH2 Targeting Induces CD38 Upregulation and Response to Anti-CD38 Antibodies in Multiple Myeloma

Multiple myeloma (MM) is the second most common hematological malignancy characterized by the accumulation of tumor plasma cells within the bone marrow. MM is characterized by high molecular and clinical heterogeneity. During the last 10 years new therapeutic classes including targeted immunotherapies significantly improved the overall survival of MM patients. However, drug resistance and relapse remain major challenges in MM. In MM, marked reduction of CD38 expression via exocytosis, endocytosis, degradation of the antigen-antibody complex and trogocytosis has been associated with resistance to CD38 targeted immunotherapies. Our group previously described a link and a negative correlation between CD38 and EZH2 (Enhancer of Zeste homolog 2) expression during normal B to plasma cell differentiation in association with a transcriptional control of CD38 expression involving the polycomb PRC2 complex (Herviou et al. Leukemia. 2019). Here we identified a significant negative correlation between CD38 expression and EZH2 expression in 2 independent cohorts of MM patients (p<0.05; Montpellier cohort n=198; Compass cohort n=631). Thus, we hypothesized that PRC2 targeting using EZH2 inhibitor could result in CD38 re-expression to overcome daratumumab resistance. We first characterized the membrane CD38 expression by calculating the Antibody Binding Capacity in our unique panel of 40 Human Myeloma Cell lines (HMCL) representative of MM heterogeneity. We observed a heterogeneous expression of the membrane CD38 expression on MM cell lines with cell lines demonstrating no membrane expression and cell lines demonstrating high membrane expression. Next, we selected 3 HMCLs with low (JJN3), intermediate (XG2), and high (XG7) CD38 expression, and treated these cell lines with sublethal concentration of EZH2 inhibitor (EPZ-6438) for 9 days. CD38 membrane expression was characterized at Day 3, Day 6 and Day 9. Interestingly, EZP-6438 treatment induced upregulation of cell surface CD38 expression on all the HMCLs tested with the highest effect measured at day 9. Similar re-expression pattern was observed by western blot at these timing in the JJN3 CD38low cell line. Remarkably, ChIPseq analyses underlined a significant association of H3K27me3 mark at CD38 promoter in CD38neg and CD38low cell lines compared to CD38highcells. In addition, we investigated the efficiency of CD38 monoclonal antibodies (Daratumumab and Isatuximab) lysis through ADCC (Antibody dependent cellular cytotoxicity) assay after 9 days of EPZ-6438 pretreatment. Using JJN3 CD38low HMCL, EPZ-6438 pretreatment significantly improved the ADCC lysis induced by low doses of Daratumumab and Isatuximab treatment, in presence of purified Natural Killer (NK) from healthy donor, together with CD38 re-expression (p<0.05; n=3). Finally, we searched to validate these results using primary MM cells from patients at relapse after Daratumumab treatment. EPZ-6438 pretreatment resulted in a significant CD38 membrane expression upregulation with a 2 fold difference compared to the control condition together with improved ADCC toxicity of anti-CD38 antibodies in vitro. Altogether we demonstrated that treatment of MM cells with EZH2 inhibitor leads to significant upregulation of membrane CD38 expression in cell lines and primary MM cells from patients. CD38 re-expression was linked to an improvement of Daratumumab and Isatuximab ADCC efficiency. Thus, EZH2 targeting may be of therapeutic interest to overcome resistance to anti-CD38 targeted immunotherapies in Multiple Myeloma.

Preclinical Activity of Allogeneic CS1-Specific CAR T-Cells (UCARTCS1) in Multiple Myeloma

Introduction: The substantial activity of chimeric antigen receptor (CAR) T-cells in multiple myeloma (MM) has recently been shown by the success of BCMA-CAR T-cell therapy. Nevertheless the difficulties in (timely) generation of CART cells for each patient, the limited availability of manufacturing slots, and the frequent BCMAlow MM relapses after therapy urge the investigators to search for i) alternative, "off the shelf", available sources of CAR T-cells and ii) alternative target antigens than BCMA. UCARTCS1 cells are "off the shelf" allogeneic CAR T-cells derived from healthy donors targeting CS1 (SLAMF7), which is a highly attractive target, due to its overexpression on MM cells. Furthermore, the genes coding for the TCRα chain of the TCRab receptor and for CS1 are disrupted in UCARTCS1 cells using Cellectis' TALEN® gene-editing technology. These modifications aim to minimize the potential risk for allo-TCR-mediated graft-versus-host disease (GvHD) upon infusion in patients and diminish CS1-mediated fratricide. In this study, we evaluated the preclinical activity of UCARTCS1 in MM cell lines, in bone marrow (BM) samples obtained from MM patients and in a MM mouse model. We also investigated the potential impact of previous therapy and tumor characteristics on the in vitro efficacy of UCARTCS1. Methods: Luciferase-transduced MM cell lines with different expression levels of CS1 were incubated with UCARTCS1 cells or control (non-transduced CS1/TCRαβ double knock-out) T-cells at different E:T ratios for 24 hours. MM cell lysis was assessed by bioluminescence. Anti-MM activity of UCARTCS1 was also evaluated in 25 BM samples obtained from newly diagnosed (n=10; NDMM), daratumumab-naïve relapsed/refractory patients (n=10; RRMM; median of 3 prior therapies) and from daratumumab-refractory patients (n=5; DRMM; median of 3 prior therapies) in 24-hour flow cytometry-based cytotoxicity assays. The results were correlated to CS1 expression levels of MM cells, Treg frequency, and several other patient characteristics such as treatment history. Results: UCARTCS1 induced strong CS1 CAR-mediated and dose-dependent lysis of different MM cell lines expressing variable levels of CS1 (MM1s, L363, UM9, U266). UCARTCS1 cells also effectively lysed primary MM cells in a dose-dependent and CAR-mediated manner, with a mean maximal lysis of 96.9% (Figure 1). In these BM samples, UCARTCS1 cells also targeted CS1+ non-malignant hematopoietic cells, such as CD4+ T-cells, CD8+ T-cells, B cells, and NK cells, while sparing the CS1low fraction in each cell subset. However, the lysis of normal cells was less efficient than MM cell lysis, which could be explained by substantially lower CS1 expression on normal cells compared to MM cells (Figure 2). CS1 expression on MM cells and UCARTCS1 cytotoxic activity were both comparable in the different patient subgroups. In addition, baseline tumor and immune characteristics, such as the expression level of CS1 on MM cells, the tumor load (MM cell percentage in BM), or frequency of Treg cells in the BM did not affect CAR T-cell mediated cytotoxicity. Finally, UCARTCS1 exhibited durable dose-dependent anti-MM activity in mouse xenograft models after i.v. injection of CAR T-cells compared to mice treated with CAR- control T-cells. Conclusion: UCARTCS1 has potent anti-MM activity against MM cell lines and primary MM cells, as well as in a MM xenograft model. There was no difference in activity between heavily pretreated and newly diagnosed patients, indicating that mechanisms of resistance to prior therapies do not result in reduced susceptibility to UCARTCS1-mediated lysis in vitro. UCARTCS1 was also effective in samples from patients with relatively low CS1 expression, with high tumor burden, or with high Treg numbers. These data support the ongoing phase 1 clinical trial with UCARTCS1 in heavily pretreated MM patients (NCT04142619). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In Situ Characterization of Chromatin Landscape in Progression of Smoldering Precursor Stage to Multiple Myeloma

Introduction Multiple myeloma (MM) is a fatal malignancy of plasma cells and is preceded by a precursor stage, smoldering myeloma (SMM). There is a 50% risk of progression from SMM to overt MM within five years of diagnosis. Most SMM patients do not receive treatment until progression to overt MM and early intervention is beyond the current standard of care. Large sequencing studies have revealed that SMM patients with similar genetic backgrounds experience varying rates of disease progression, overall survival, and treatment response. These findings indicate an unmet need to better define the molecular requirements of disease progression. We hypothesize that altered chromatin states in pre-myeloma cells act as priming events for clonal expansion and enable the transition from pre-myeloma conditions to MM. Characterization of the pre-myeloma epigenome at single-cell resolution may offer insights into the dynamic reprogramming that occurs during myeloma development and provide new biomarkers of disease progression. Methods Bone marrow (BM) aspirates were sampled from 26 patients and healthy donors at different stages of the disease. BM samples were enriched for plasma cells using CD138 microbeads and subjected to single-cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq, 10x Genomics). This cohort comprised five healthy donors, seven low risk (LR), five intermediate risk (IR), and three high risk (HR) SMM patients as well as six newly diagnosed (ND) MM patients based on the 2/20/20 risk stratification model. Batch effects, data denoising, and differential accessibility testing were performed using deep generative modeling via PeakVI. CytoTRACE was used to model cell plasticity and HOMER was used to infer enriched transcription factor footprints in differential accessibility regions (DARs). g:Profiler was used for gene ontology analyses. Results We analyzed >81,000 scATAC-seq profiles. To contextualize tumor cell emergence from normal plasma cells (NPCs), we supplemented our dataset with ~1,000 cells from a public dataset of relapsed refractory MM (RRMM) patients and healthy donors (Fredeet al., Nat Cell Biol, 2021). We identified ~17,000 LRSMM vs. NDMM DARs, ~24,000 HRSMM vs. LRSMM DARs and ~5,000 HRSMM vs. IRSMM DARs. Comparing cells from SMM and MM donors with NPCs revealed few or zero significant DARs, suggesting that intrasample heterogeneity must be properly resolved to accurately describe detailed molecular annotations of each cell group. We thus performed unsupervised clustering of all NPC, SMM, and MM cells to resolve 11 distinct clusters. We defined three NDMM clusters, three NPC/LRSMM clusters, and individual RRMM, NDMM/ HRSMM, HRSMM, IRSMM/LRSMM, NDMM/LRSMM clusters. Comparison of these clusters revealed ~13,000 LRSMM vs. NPCs DARs and ~1,600 IRSMM/LRSMM vs. NPC DARs. Gene ontology analysis of genes neighboring DARs (<50 kb) displayed signatures associated with the P38-MAPK, JNK, and WNT signaling pathways as well as differentiation and development, consistent with previous observations of de-differentiation and increased epigenomic plasticity in MM. BATF, POU3F3, SMAD2, IRF4, JUNB, MYC, FOSL1, and SP1 binding sites were enriched in DARs. We ordered these cell groups along an axis of disease progression, and work to build a deep, generative time-resolved model of MM emergence from pre-myeloma is ongoing. Conclusions We report the largest single-cell chromatin profiling experiment on primary MM cells, focusing on phenotypically early traits of SMM. We characterize distinct sub-groups of SMM patient BM cells and their corresponding regions of accessible chromatin. We are expanding this study through sample-matched WGS to delineate independent genetic and epigenomic contributions to MM progression. scATAC-seq analyses of the matched CD138-negative immune cell fractions are underway alongside analyses of single-cell transcriptome data for both CD138 positive and negative fractions. We anticipate that a deeper understanding of the chromatin states associated with the establishment of overt myeloma will lead to novel therapeutic targets and offer improved predictive prognostics for 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

Targeting Iron Homeostasis As a Therapeutic Strategy in Multiple Myeloma

Multiple myeloma (MM) is a hematological cancer characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow (BM). Despite the survival improvement provided by current treatments, the majority of patients relapse and eventually become resistant to all treatments. Drug resistance and relapse are the main current challenges in the treatment of MM and development of new therapeutic interventions is a pressing need (Pinto et al., 2020 Cancers). Abnormal iron homeostasis is implicated in tumorigenesis and the progression of several cancers. Ironomycin is a small synthetic derivative of salinomycin that induces DNA damage and non-apoptotic cell death by sequestering iron in lysosomes (Mai et al., 2017 Nat Chem). Preclinical studies in Acute Myeloid Leukemia (Garciaz et al., 2022 Cancer Discovery) and Diffuse Large B-Cell Lymphoma (Devin et al., 2022 Can Res) have revealed the therapeutic interest of ironomycin in treating hematological malignancies. Here, we have evaluated the effect of ironomycin on MM cells, alone and in combination with standard anti-myeloma treatments. A list of 63 genes related to iron metabolism in cancer was defined. Using Maxstat R function, we showed that 6 genes out of the 63 investigated have a prognostic value in a cohort of newly diagnosed MM patients (n=345). Based on these prognostic genes, we created a gene expression profile (GEP)-based risk score as the sum of the beta coefficients weighted by ±1 according to the patient signal above or below the probe set Maxstat value as previously reported (Herviou et al., 2018 Clinical Epigenetics). This "Iron Score” (IS) was significantly associated with high-risk myeloma (OS and EFS) in 3 independent cohorts of newly diagnosed MM patients (TT2 cohort (N=345), HM cohort (N=206) and TT3 cohort (N=158)), as well as in a cohort of 188 patients at relapse treated with bortezomib monotherapy (Mulligan cohort N=188). GSEA analysis revealed that high-risk MM patients defined by iron score are significantly enriched in genes involved in cell cycle, TFRC targets and hypoxia (p < 0.001). We analyzed the therapeutic interest of ironomycin in 19 human myeloma cell lines (HMCLs). Ironomycin inhibits MM cell growth at nanomolar concentrations compared to other iron chelators. Ironomycin caused a significant cell growth inhibition mediated by cell cycle arrest and triggering of apoptosis, without significant ferroptosis induction. Moreover, cell proliferation arrest correlated with increased DNA damage and caspases activation, and a metabolic swift towards a glycolytic or quiescent metabolism. Iron could also play a role in transcriptional regulation of cancer cells mediated by epigenetic reprogramming. Iron dependent modifications of histone or DNA methylation have been shown to play a significant role in solid cancers. Ironomycin also induced a downregulation of major oncogenes of MM cells including MMSET, c-MYC and CCND1. Furthermore, ironomycin treatment resulted in an epigenetic dysregulation with H3K27ac, H3K4me3 and H3K9me3 downregulation. With major importance, we validated the therapeutic interest of ironomycin in primary MM cells from patients (n=5) without significant toxicity for non-tumor cells from the microenvironment. Furthermore, ironomycin presented a low toxicity on hematopoietic progenitors (CFU assays, n=5) compared to conventional treatment. We tested the therapeutic interest to combine ironomycin with conventional drugs used in MM treatment. The combination of ironomycin with IMiDs (lenalidomide and pomalidomide) had a synergistic effect on cell growth arrest and the activation of apoptotic mechanisms, associated with a marked reduction of several targets of IMiDs including Cereblon (CRBN), MYC, IRF4, IKZF1 and IKZF3. Our data demonstrate that targeting iron homeostasis using ironomycin could be of therapeutic interest in high-risk MM defined by the IS, especially in combination with IMiDs.

The Multi-Kinases Inhibitor ARQ531 Enhanches Anti-MM Activity of Imids By Dual Deregulation of MAP- and Cyclin-Dependent Kinases Activities

Introduction: Kinases inhibitors are emerging as promising therapeutic tools for various blood tumors. We previously reported that a novel small tyrosine kinase inhibitor, ARQ531, constrains leukemia cells proliferation and survival by disrupting multiple tumor-addicted programs based on its kinase inhibition profile. As result we pursued its application in pre-clinical models of Multiple Myeloma (MM). Methods: Inhibitory effects of ARQ531 on cell viability were investigated in a panel of MM cell lines and primary tumor cells (collected from NDMM and RRMM patients) by CellTiterGlo luminescent assay over a range of drug concentrations and time. Specific transcriptomic profiling of ARQ531-treated MM cells was performed by RNA-Seq analysis. Molecular mechanism of this small molecule on MM cells was further investigated by qPCR and western blot analyses. The therapeutic efficacy of ARQ531 combination with different anti-MM drugs was also explored and CalcuSyn software was employed to confirm synergistic effects. Mice engrafted with MM cells were used to determine in-vivo anti-MM activity of ARQ531 in combination with lenalidomide. Results: ARQ531 inhibited cell viability of MM cell lines (n=20) with IC50 value of 3±1 µM. Furthermore, inhibitory effects of ARQ531 on cell viability were evaluated in a panel of primary MM cells with different mutational status (including those with RAS mutations), with an IC50s value ranging from 0.9 to 4 µM. Consistent with the effect on cell viability, ARQ531 increased the apoptotic rate of MM cells tested. Moreover, anti-MM effects of ARQ531were not reduced in presence of bone marrow stromal cells (BMSCs), and, more importantly such treatment showed a good therapeutic window. A transcriptome profiling analysis of ARQ 531-treated cells demonstrates wide oncogenic transcriptional programs dysregulation, as well as specific interference with kinases involved in cell cycle progression including MAP Kinases and CDK. Given that these kinases are targetable resistance mechanisms for immunomodulatory drugs (IMiDs) in MM, we combined ARQ531 with lenalidomide, pomalidomide or iberdomide observing synergistic effects. Different gene-editing approaches further supported ARQ531-identified profiling as crucial for observed anti-MM activity of tested drugs. We also confirmed that co-treatment in vivo leads to impaired engraftment and prolonged survival in a MM xenograft mouse model, by disrupting key cell proliferation pathways including c-MYC. Conclusions: Our data show that the small multi-kinases inhibitor ARQ531 has a promising therapeutic activity against MM in preclinical models thus supporting the feasibility of targeting oncogenic driven programs in MM cells with ARQ531 alone or in combinations with IMiDs.

Phase 1 Trial of AUC-Targeted Melphalan in Myeloma Patients Undergoing Autologous Transplant (The MyMel Study)

Standard 200 mg/m2 high dose melphalan (HDM) yields a 500% variation in plasma melphalan exposure, i.e. area under the curve (AUC), leading to unpredictable DNA damage and widely variable clinical responses. Strategies to predict melphalan AUC have been unsuccessful given population PK models (Nath, Br J Clin Pharmacol 2010 & Cho Clin Pharmacol Ther 2017) are ineffective on an individual patient level and only explain ~20% of the variability observed, and test dose predictions have failed to accurately represent melphalan AUC for doses >100 mg/m2. PK directed dose adjustment of HDM would require a 24-hour turnaround for sample collection, shipping, processing and data analysis. Previously we described methods for measuring melphalan PK after the Day -2 HDM dose, allowing for real-time dose adjustments (Sweiss, Br J Clin Pharmacol 2020). Here, we set out to perform the first phase 1 study of AUC-targeted HDM in ASCT (NCT04483206). This multicenter phase 1 trial (Fig 1) is designed to test 7 AUC ranges (13-21 mg*h/L) to determine the maximum tolerated systemic exposure (MTSE). The feasibility portion focuses on the first 2 AUCs (phase A, n=20) in patients with ≥2L therapy, where we measure accuracy of achieving target AUCs of 13.5 and 14.5 mg∗h/L, respectively. Patients receive HDM 100 mg/m2 on day -3 with PK measurement and subsequent dose-adjustment on day -1 to achieve the total target AUC. 7 blood samples are drawn, processed and shipped frozen for LC-MS/MS analysis. PK parameter estimations are determined from noncompartmental analysis (NCA) and per FDA guidance used to adjust doses. Nonlinear mixed effects modeling (NONMEM) is performed in parallel to compare PK estimations. Using observed first-dose HDM clearance (Clobs) from NCA and assuming that Cl is consistent between doses, linear dose-proportional adjustments are performed to achieve the target AUC0-inf. After a planned FDA review of these cohorts, we will begin to dose escalate to determine the MTSE with up to 5 AUC levels (15.5-19.5 mg∗h/L). Bone marrow (BM) MRD status will be assessed at Day 90 as part of the Phase B primary objective. Correlatives include measuring DNA damage in circulating PBMCs post-HDM to correlate with AUC, piloting a DNA fiber combing assay on primary MM cells from patient BM samples to quantify DNA replicative stress, and assessing PROs using NCI PRO-CTCAE v.1.0. We have enrolled 8 patients into the first AUC cohort with 110 blood samples collected. Median age was 69 (56-75) years, with 7 (87.5%) males, and 4 (50%) Blacks. The median day -3 HDM Clobs and AUC0-inf were 22.05 (16.3-38.2) L/hr and 9.53 (6.97-11.82) mg∗h/L, respectively. All shipped blood samples were received in suitable frozen condition, and in 7 (87.5%) of 8 patients, LC-MS/MS run with NCA estimation and dose reporting was done within 12 hours of receiving samples. One patient whose samples were delayed from shipping had the dose reported on day-1 resulting in 5 of 7 day-1 samples collected. Covariates of HDM PK (i.e., FFM, hematocrit) were entered into our developed PK model to further enrich our data prediction. All 8 day -1 doses were adjusted, with a resulting median total BSA-based HDM dose of 139.6 (110.93-192.6) mg/m2. After adjustment, median day -1 Clobs and AUC0-inf were 24.48 (19.3-35.8) L/hr and 3.84 (1.7-7) mg∗h/L, respectively. For accuracy of dose adjustment, 107 (97.3%) samples were evaluable for PK parameter estimation, with 3 concentrations excluded (n=3 patients). One subject whose last 2 day -1 samples were not collected was removed from analysis as it resulted in 19% AUC extrapolation and 33% variance between day -3 and -1 Cl values. Of 7 patients, 6 (85.7%) achieved the target AUC, with less than 10% variance in Cl between doses. (Fig 2). In the one patient, it was assessed that the day -1 Cl deviated by 24% as a result of prolonged time from drug admixture to completion of infusion leading to drug degradation. Estimated melphalan Cl generated from NCA and NONMEM correlated well (r=0.99). This is the first trial in MM implementing a novel personalized dosing approach using drug exposure as the target to define each cohort. We demonstrate feasibility of real-time PK sampling, shipping, processing, NCA PK parameter estimation, and dose adjustment of HDM. So far we report a high rate of accuracy in achieving the target AUC which we hypothesize will deliver reliable DNA damage to MM cells. Enrollment is active and mature data will be presented at the conference. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The serine protease matriptase inhibits migration and proliferation in multiple myeloma cells.

Background: Multiple myeloma (MM) is an incurable malignancy of plasma cells. The serine protease matriptase is frequently dysregulated in human carcinomas, which facilitates tumor progression and metastatic dissemination. The importance of matriptase in hematological malignancies is yet to be clarified. In this study, we aimed to characterize the role of matriptase in MM.Materials and Methods: mRNA expression of matriptase and its inhibitors hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2 was studied in primary MM cells from patient samples and human myeloma cell lines (HMCLs). We further investigated the effect of matriptase on migration and proliferation of myeloma cells in vitro. By use of the CoMMpass database, we assessed the clinical relevance of matriptase in MM patients.Results: Matriptase was expressed in 96% of patient samples and all HMCLs tested. Overexpression of matriptase in vitro reduced proliferation, and significantly decreased cytokine-induced migration. Conversely, matriptase knockdown significantly enhanced migration. Mechanistically, overexpression of matriptase inhibited activation of Src kinase.Conclusions: Our findings may suggest a novel role of matriptase as a tumor suppressor in MM pathogenesis.

Inhibition of NF-κB DNA Binding Suppresses Myeloma Growth via Intracellular Redox and Tumor Microenvironment Modulation.

Multiple myeloma is a plasma cell malignancy that is still largely incurable, despite considerable progress in recent years. NF-κB is a well-established therapeutic target in multiple myeloma, but none of the currently available treatment options offer direct, specific pharmacologic targeting of NF-κB transcriptional activity. Thus, we designed a novel direct NF-κB inhibitor (IT848) as a drug candidate with strong potential for clinical translation and conducted comprehensive in vitro and in vivo mechanistic studies in multiple myeloma cell lines, primary multiple myeloma cells, xenograft models, and immunocompetent mouse models of multiple myeloma. Here, we show that IT848 inhibits NF-κB activity through inhibition of DNA binding of all five NF-κB subunits. IT848 treatment of multiple myeloma cell lines and patient samples inhibited proliferation and induced caspase-dependent and independent apoptosis. In addition to direct NF-κB inhibitory effects, IT848 treatment altered the redox homeostasis of multiple myeloma cells through depletion of the reduced glutathione pool, selectively inducing oxidative stress in multiple myeloma but not in healthy cells. Multiple myeloma xenograft studies confirmed the efficacy of IT848 as single agent and in combination with bortezomib. Furthermore, IT848 significantly improved survival when combined with programmed death protein 1 inhibition, and correlative immune studies revealed that this clinical benefit was associated with suppression of regulatory T-cell infiltration of the bone marrow microenvironment. In conclusion, IT848 is a potent direct NF-κB inhibitor and inducer of oxidative stress specifically in tumor cells, displaying significant activity against multiple myeloma cells in vitro and in vivo, both as monotherapy as well as in combination with bortezomib or immune checkpoint blockade.

ATRA works synergistically with the γ-secretase inhibitor crenigacestat to augment BCMA on multiple myeloma and the efficacy of BCMA-CAR T-cells.

B-cell maturation antigen (BCMA) is the lead antigen for CAR T-cell therapy in multiple myeloma (MM). A challenge is inter- and intra-patient heterogeneity in BCMA expression on MM cells and BCMA downmodulation under therapeutic pressure. Accordingly, there is a desire to augment and sustain BCMA expression on MM cells in patients that receive BCMA-CAR T-cell therapy. We used all-trans retinoic acid (ATRA) to augment BCMA expression on MM cells and to increase the efficacy of BCMA-CAR T-cells in pre-clinical models. We show that ATRA treatment leads to an increase in BCMA transcripts by quantitative PCR and an increase in BCMA protein expression by flow cytometry in MM cell lines and primary MM cells. Analyses with super-resolution microscopy confirmed increased BCMA protein expression and revealed an even distribution of non-clustered BCMA molecules on the MM cell membrane after ATRA treatment. The enhanced BCMA expression on MM cells after ATRA treatment led to enhanced cytolysis, cytokine secretion and proliferation of BCMA-CAR T-cells in vitro, and increased efficacy of BCMA-CAR T-cell therapy in a murine xenograft model of MM in vivo (NSG/MM1.S). Combination treatment of MM cells with ATRA and the γ-secretase inhibitor crenigacestat further enhanced BCMA expression and the efficacy of BCMA-CAR T-cell therapy in vitro and in vivo. Taken together, the data show that ATRA treatment leads to enhanced BCMA expression on MM cells and consecutively, enhanced reactivity of BCMA-CAR T-cells. The data support the clinical evaluation of ATRA in combination with BCMA-CAR T-cell therapy and potentially, other BCMA-directed immunotherapies.

All-trans retinoic acid works synergistically with the γ-secretase inhibitor crenigacestat to augment BCMA on multiple myeloma and the efficacy of BCMA-CAR T cells.

B-cell maturation antigen (BCMA) is the lead antigen for chimeric antigen receptor (CAR) T-cell therapy in multiple myeloma (MM). A challenge is inter- and intra-patient heterogeneity in BCMA expression on MM cells and BCMA downmodulation under therapeutic pressure. Accordingly, there is a desire to augment and sustain BCMA expression on MM cells in patients that receive BCMA-CAR T-cell therapy. We used all-trans retinoic acid (ATRA) to augment BCMA expression on MM cells and to increase the efficacy of BCMA-CAR T cells in pre-clinical models. We show that ATRA treatment leads to an increase in BCMA transcripts by quantitative reverse transcription polymerase chain reaction and an increase in BCMA protein expression by flow cytometry in MM cell lines and primary MM cells. Analyses with super-resolution microscopy confirmed increased BCMA protein expression and revealed an even distribution of non-clustered BCMA molecules on the MM cell membrane after ATRA treatment. The enhanced BCMA expression on MM cells after ATRA treatment led to enhanced cytolysis, cytokine secretion and proliferation of BCMA-CAR T cells in vitro, and increased efficacy of BCMA-CAR T-cell therapy in a murine xenograft model of MM in vivo (NSG/MM.1S). Combination treatment of MM cells with ATRA and the γ- secretase inhibitor crenigacestat further enhanced BCMA expression and the efficacy of BCMA-CAR T-cell therapy in vitro and in vivo. Taken together, the data show that ATRA treatment leads to enhanced BCMA expression on MM cells and consecutively, enhanced reactivity of BCMA-CAR T cells. The data support the clinical evaluation of ATRA in combination with BCMA-CAR T-cell therapy and potentially, other BCMA-directed immunotherapies.

Paired miRNA- and messenger RNA-sequencing identifies novel miRNA-mRNA interactions in multiple myeloma

Multiple myeloma (MM) is an incurable cancer of terminally differentiated plasma cells that proliferate in the bone marrow. miRNAs are promising biomarkers for risk stratification in MM and several miRNAs are shown to have a function in disease pathogenesis. However, to date, surprisingly few miRNA-mRNA interactions have been described for and functionally validated in MM. In this study, we performed miRNA-seq and mRNA-seq on CD138 + cells isolated from bone marrow aspirates of 86 MM patients to identify novel interactions between sRNAs and mRNAs. We detected 9.8% significantly correlated miRNA-mRNA pairs of which 5.17% were positively correlated and 4.65% were negatively correlated. We found that miRNA-mRNA pairs that were predicted by in silico target-prediction algorithms were more negatively correlated than non-target pairs, indicating functional miRNA targeting and that correlation between miRNAs and mRNAs from patients can be used to identify miRNA-targets. mRNAs for negatively correlated miRNA-mRNA target pairs were associated with gene ontology terms such as autophagy, protein degradation and endoplasmic stress response, reflecting important processes in MM. Targets for two specific miRNAs, miR-125b-5p and miR-365b-3p, were functionally validated in MM cell line transfection experiments followed by RNA-sequencing and qPCR. In summary, we identified functional miRNA-mRNA target pairs by correlating miRNA and mRNA data from primary MM cells. We identified several target pairs that are of potential interest for further studies. The data presented here may serve as a hypothesis-generating knowledge base for other researchers in the miRNA/MM field. We also provide an interactive web application that can be used to exploit the miRNA-target interactions as well as clinical parameters associated to these target-pairs.

Mechanisms underlying synergism between circularized tumor necrosis factor-related apoptosis inducing ligand and bortezomib in bortezomib-sensitive or -resistant myeloma cells.

Mechanisms underlying interactions between a novel, clinically relevant circularized tumor necrosis factor-related apoptosis inducing ligand (TRAIL) agonist, circularly permuted TRAIL (CPT) have been examined in multiple myeloma (MM) cells sensitive or resistant to bortezomib (BTZ). Various MM cell lines for example, U266, including those resistant to bortezomib-resistant U266 cells were exposed to low nanomolar concentrations of bortezomib±CPT and apoptosis monitored. Circularly permuted TRAIL and bortezomib synergistically induced apoptosis in both BTZ-naïve and -resistant cells. The regimen up-regulated DR4 receptor internalization in MM cells, known to modulate both NF-κB and extrinsic apoptotic pathways. CPT/BTZ disrupted the non-canonical NF-κB pathway, reflected by tumor necrosis factor (TNF) receptor associated factors 3 (TRAF3) up-regulation, NF-κB inducing kinase down-regulation, diminished p52 and p50 processing, and B-cell lymphoma-extra large (BCL-XL) down-regulation, but failed to inactivate the canonical NF-κB pathway, reflected by unchanged or increased expression of phospho-p65. The regimen also sharply increased extrinsic apoptotic pathway activation. Cells exhibiting TRAF3 knock-down, dominant-negative Fas-associated protein with death domain, knock-down of caspase-8, BCL-2/BCL-XL, or exposure to a caspase-9 inhibitor displayed markedly reduced CPT/BTZ sensitivity. Concordant results were observed in bortezomib-resistant cells. The regimen was also active in the presence of stromal cells and was relatively sparing toward normal CD34+ hematopoietic cells. Finally, ex vivo results revealed synergism in primary MM primary cells, including those BTZ, and the CPT/BTZ regimen significantly decreased tumor growth in a patient-derived MM xenograft model. These results indicate that the CPT/BTZ regimen acts via the non-canonical NF-κB as well as intrinsic/extrinsic apoptotic pathways to induce cell death in MM cells, and may represent an effective strategy in the setting of bortezomib resistance.

P860: THE DE NOVO DNA METHYLTRANSFERASE DNMT3B PLAYS AN IMPORTANT ROLE IN MM CELL GROWTH, CLONOGENICITY AND DRUG RESPONSE

Background: Multiple myeloma (MM) is an incurable plasma cell malignancy due to the development of drug resistance (DR). In about half of the MM patients, modifications are observed in epigenetic modifiers (epiplayers) at the time of diagnosis and this frequency is further increased at relapse, indicating an important role for epiplayers in MM cell DR. However, so far, only for two epiplayers, MMSET and EZH2, a clear role in MM cell DR development has been established. With the aim to identify new, clinically relevant epiplayers involved in MM progression and relapse, we recently compared the RNASeq data from matched newly diagnosed and relapsed patients from the MMRF CoMMpass study. We found that the epiplayer DNMT3B is significantly increased in relapsed patients, suggesting a possible role for DNMT3B in MM relapse. Aims: The aim of this study is to explore the role of DNMT3B in MM cell biology and drug response. Methods: Publicly available gene expression profiling data of three independent cohorts of newly diagnosed MM patients (GSE4581, E-MTAB-372, MMRF-COMMPASS), one cohort of relapsed patients (GSE9782), BM plasma cells (E-MTAB-372), primary MM cells (E- MTAB-372), and human MM cell lines (HMCLs; E-TABM-1088 and E-TABM-937) was used. DNMT3B specific targeting was achieved by using the DNMT3B specific inhibitor Nanaomycin A and genetic inhibition using doxycycline inducible shRNA against DNMT3B. Viability and apoptosis were assessed using a CellTiter-Glo assay and an AnnexinV/7AAD staining respectively. In addition, BrdU incorporation and cell cycle analysis was evaluated to assess cell proliferation. Clonogenic capacity was evaluated by a colony formation assay. Results: We found that DNMT3B mRNA levels increase during disease progression and high DNMT3B mRNA expression correlates with a worse disease outcome in both newly diagnosed and relapsed patients, indicating a role for DNMT3B in MM progression and DR. In line, Nanaomycin A treatment (up to 72h) led to a significant and dose-dependent decrease in cell viability and proliferation and increase in apoptosis in the XG-2, XG-7 and AMO-1 HMCLs. We also validated the anti-MM activity of Nanaomycin A on human primary MM cells. Since DNMT3B is thought to play a significant role in cancer cell stemness, we next evaluated the effect on clonogenicity. A significant and dose-dependent decrease in the number of colonies was observed when low doses of Nanaomycin A were added to AMO-1 (100 and 200 nM) and XG-2 (30 and 50 nM) cells on the day of plating (day 0), but not when added 7 days after plating (day 7). In contrast, treatment with a high Nanaomycin A concentration (800 nM) significantly reduced the number of colonies at both timepoints, indicating that high Nanaomycin A doses are cytotoxic whereas low doses impair the proliferation and clonogenicity of the MM cells. The anti-clonogenic effect of DNMT3B targeting was confirmed by DNMT3B knockdown in AMO-1 cells. Finally, combining Nanaomycin A (100 nM) with bortezomib (Bz, 4 nM) resulted in a significant decrease in colony formation compared to either Nanaomycin A or Bz treatment alone. Summary/Conclusion: Taken together, our findings indicate that DNMT3B is a novel promising target to overcome or delay relapse in MM. DNMT3B targeting impairs MM cell proliferation and clonogenicity and sensitizes the cells to the proteasome inhibitor Bz. In the near future, the anti-myeloma activity of DNMT3B targeting will be validated in vivo using the 5TMM murine MM models and the underlying mechanisms will be further determined.

Timelapse viability assay to detect division and death of primary multiple myeloma cells in response to drug treatments with single cell resolution.

Heterogeneity among cancer cells and in the tumor microenvironment (TME) is thought to be a significant contributor to the heterogeneity of clinical therapy response observed between patients and can evolve over time. A primary example of this is multiple myeloma (MM), a generally incurable cancer where such heterogeneity contributes to the persistent evolution of drug resistance. However, there is a paucity of functional assays for studying this heterogeneity in patient samples or for assessing the influence of the patient TME on therapy response. Indeed, the population-averaged data provided by traditional drug response assays and the large number of cells required for screening remain significant hurdles to advancement. To address these hurdles, we developed a suite of accessible technologies for quantifying functional drug response to a panel of therapies in ex vivo three-dimensional culture using small quantities of a patient's own cancer and TME components. This suite includes tools for label-free single-cell identification and quantification of both cell division and death events with a standard brightfield microscope, an open-source software package for objective image analysis and feasible data management of multi-day timelapse experiments, and a new approach to fluorescent detection of cell death that is compatible with long-term imaging of primary cells. These new tools and capabilities are used to enable sensitive, objective, functional characterization of primary MM cell therapy response in the presence of TME components, laying the foundation for future studies and efforts to enable predictive assessment drug efficacy for individual patients.

Targeting hypersialylation in multiple myeloma represents a novel approach to enhance NK cell–mediated tumor responses

AbstractAbnormal glycosylation is a hallmark of cancer, and the hypersialylated tumor cell surface facilitates abnormal cell trafficking and drug resistance in several malignancies, including multiple myeloma (MM). Furthermore, hypersialylation has also been implicated in facilitating evasion of natural killer (NK) cell–mediated immunosurveillance but not in MM to date. In this study, we explore the role of hypersialylation in promoting escape from NK cells. We document strong expression of sialic acid-derived ligands for Siglec-7 (Siglec-7L) on primary MM cells and MM cell lines, highlighting the possibility of Siglec-7/Siglec-7L interactions in the tumor microenvironment. Interactomics experiments in MM cell lysates revealed PSGL-1 as the predominant Siglec-7L in MM. We show that desialylation, using both a sialidase and sialyltransferase inhibitor (SIA), strongly enhances NK cell–mediated cytotoxicity against MM cells. Furthermore, MM cell desialylation results in increased detection of CD38, a well-validated target in MM. Desialylation enhanced NK cell cytotoxicity against CD38+ MM cells after treatment with the anti-CD38 monoclonal antibody daratumumab. Additionally, we show that MM cells with low CD38 expression can be treated with all trans-retinoic acid (ATRA), SIA and daratumumab to elicit a potent NK cell cytotoxic response. Finally, we demonstrate that Siglec-7KO potentiates NK cell cytotoxicity against Siglec-7L+ MM cells. Taken together, our work shows that desialylation of MM cells is a promising novel approach to enhance NK cell efficacy against MM, which can be combined with frontline therapies to elicit a potent anti-MM response.