Myeloma Cell Lines Research Articles

Gain or Amplification of Chromosome 1q Mediate Multiple Myeloma Resistant to Xpo-1 Inhibitor Selinexor Via Transcription Factor ETV3

Background: Gain or amplification of chromosome 1q, which characterized as a poor prognosis indicater, is one of the most frequent cytogenetic abnormalities in multiple myeloma (MM), occurring in nearly 40% of newly diagnosed patients. Though several genes, such as IL6R and CKS1B, have been reported to related to its severity, there still remains a mist for us to explore. So, we further explore the potential mechanism that may illustrate the malignant phenotype of gain or amplification of chromosome 1q in multiple myeloma, and the ETS transcription factors ETV3 may make sense. Objective：Explore the functions ETV3 played in the malignant phenotype of gain or amplification of chromosome 1q in multiple myeloma. Methods：Dataset GSE2658 from GEO database was used to analyze the correlation of ETV3 expression and survival of newly diagnosed multiple myeloma (NDMM). Dataset GSE4581 from GEO database was used to investigate the relationship between the number of 1q copies and expression of ETV3. Next, ETV3 expression on different myeloma cell lines was detected by RT-PCR,WB and high ETV3 expression cell lines were selected for further study. Knocking down of ETV3 on these cell lines were performed and CCK-8 was used to detected the proliferation ability. XPO-1 inhibitor selinexor was used with different concentrations (0-300nM) to treat these cells for 24h and 48h, and apoptosis was detected by flow cytometry. Further, RNAseq was performed to explore the related changed genes that might be regulated by ETV3. Result s : Survival analysis showed MM patients with higher ETV3 expression exhibit worse prognosis according to Dataset GSE2658 (P mini=0.046). As for Dataset GSE4581, 414 patients were included, among which 186 patients carried more than 2 copies of 1q21. We found that along with the increasing of 1q21+ copies, expression of ETV3 increased (p<0.01). The results of RT-PCR and West-blots showed that all the MM cell lines expressed different level of ETV3, among which H929 and AMO1 were extremely high. Hence, we knocked down ETV3 on these two cell lines (H929 Sh-ETV3 and AMO1 Sh-ETV3) in subsequent experiments. Results showed that after knocking down of ETV3, the proliferation rate of myeloma cells was reduced (p<0.05). Besides, myeloma cells knocked down of ETV3 were more sensitive to selinexor (p<0.01). Based on the above results, we analyzed the genes changed after knocking down of ETV3 by RNAseq, results indicated several genes such as PKN1 might play a part in this process. Conclusion s : MM patients carrying with higher 1q21+ copies expressed higher level of ETV3 genes, which indicated a worse prognosis. Knocking down ETV3 on myeloma cells reduced their proliferation ability and enhanced their sensitivity to selinexor. PKN1 might involved in this process. Key words: ETV3, multiple myeloma, Gain or amplification of chromosome 1q, selinexor

Regulation of BCMA By the Ubiquitin Proteasome System Enables Optimization of BCMA-Targeting Therapies in Multiple Myeloma

Background: The exclusive expression of the B-cell maturation antigen (BCMA), in normal and malignant plasma cells, currently makes it a prominent target for immune cell therapies in multiple myeloma (MM) including chimeric antigen receptor T-cell (CART) and bi-specific T cell engaging antibody (BiTE) therapies. Despite initial high response rates, most patients eventually relapse highlighting the need to enhance therapy efficacy. Although loss of BCMA-antigen expression marks one reason for therapy failure, little is known about the mechanistic means of antigen escape. The ubiquitin proteasome system (UPS) controls abundance and protein degradation of about 80-90% of all cellular proteins. Protein degradation is initiated by substrate ubiquitination, which is orchestrated by the sequential action of enzymes whereby only E3-ligases possesses substrate specificity. Deubiquitinating enzymes (DUBs) can reverse substrate ubiquitination. Against this background, we raised the question whether BCMA antigen presentation, expression, and activity is regulated by the UPS and whether the UPS can be utilized to enhance BCMA directed CART therapies in MM. Methods: Protein turnover and regulation by the UPS was assayed using cycloheximide and proteasomal inhibitors (PI) followed by immunoblotting. Direct substrate ubiquitylation was confirmed by ubiquitylation assays based on immunoprecipitation and image-based analysis. Changes in antigen surface presentation were determined using FACS analysis. Changes in cellular signaling and downstream effects were assessed by immunoblotting and quantitative PCR (qPCR). Primary CD138 + cells were obtained from bone marrow aspirations followed by MACS ® cell separation. To assess the effect of PI on CART killing capacity, a classic co-culture was set up in vitro. Using a proximity-based protein purification method (TurboID), UPS-related interactors of BCMA were identified. Results: For the first time, we show that BCMA is a short-lived protein with a half-life of approximately 30 min in MM cells. Degradation of BCMA is abrogated upon PI treatment. Inhibition of γ-secretase, an established protease of the extracellular domain of BCMA does not impair BCMA turnover. Furthermore, we show that BCMA is indeed ubiquitylated in MM cells and that ubiquitylation is induced by the ligand APRIL. These results support the hypothesis that the UPS regulates BCMA abundance. In both MM cell line models and primary CD138 + MM cells of PI refractory/ relapsed MM (RRMM) patients, inhibition of the proteasome leads to a significant increase of BCMA on the cell surface without promoting oncogenic NF-κB signaling in MM cells. Subsequently, we show that the increase in BCMA surface expression upon PI treatment substantially increases the efficacy of BCMA directed CART cells in co-cultures with MM cells and primary CD138 + MM patient cells. Specific members of the UPS (E3 ligases and DUBs) identified from our proteome wide BCMA interactome analysis are currently being validated. Conclusion: Here we show, for the first time, that BCMA is ubiquitylated and degraded by the ubiquitin proteasome system in multiple myeloma. The increase in BCMA expression upon proteasomal inhibition enhanced efficacy of BCMA-targeting CART cells. These insights provide a rationale for combining BCMA CART cells with PI in patients with BCMA CART relapsed/refractory disease and persistence. The identification of BCMA-specific E3-ligases and DUBs may represent novel therapeutic targets to specifically prevent BCMA degradation and antigen escape.

IRF2 Regulates De Novo Active Chromatin Regions and Participates in the Development and Pathogenesis of Multiple Myeloma

Multiple Myeloma (MM) is the second most common hematologic neoplasia, and it remains largely incurable due to the genetic heterogeneity of the patients. We have recently characterized the epigenome of plasma cells from patients with MM, describing the de novo activation of the chromatin as a unifying factor underlying the pathobiology of the MM. Precisely, we identified 1556 de novo active regions, showing the H3K27ac chromatin activation mark, in MM in comparison to different subpopulations of normal B cells. Moreover, those de novo active regions where enriched in Transcription Factor Binding Sites (TFBS). In order to defined the potential role of these regions we have performed a CRISPR-Cas9 screening using guides against 56 TF that presented TFBS in de novo active regions and that are expressed in MM. A CRISPR-Cas9 library containing 10 guides for each gene was transduced in two MM cell lines (MM.1R and KMS-11) and the presences of guides were analyzed at days +1, +14 and +21. Out of the 22 TFs shown to be essential for MM survival, there were 3 members of the IRF TF family (IRF1, IRF2 and IRF5). Single knockdowns identified IRF2 and the previously described IRF4 as the best candidates. To characterize the signalling pathways involved in IRF family regulation of MM, the chromatin regions where IRF1, IRF2 and IRF4 are bound were mapped using CUT&RUN in MM.1R and KMS-11 cell lines. Interestingly, the three TF were located in 573 out of the 1556 de novo active regions. Notably, IRF2 was present in the 86.9% of those regions, and in 40% of the cases, the binding occurred independently of IRF1 or IRF4 ( Fig. 1). Additionally, the genomic distribution demonstrated that the regions where IRF2 binds alone were enriched in promoters compared to those regions where IRF1 and IRF4 also bound, being these enriched mostly in the gene body. Gene Ontology studies showed that IRF2, as well as IRF1/4 were involved in regulation of cell death in response to oxidative stress, while only IRF2 autonomously was involved in the regulation of pathways relevant for MM such as the kinase dysregulation, cell migration and osteoblast regulation. Furthermore, using our previous RNA-seq data, we observed that the majority of the genes regulated uniquely by IRF2 presented an overexpression in MM in comparison to different subpopulations of normal B cells. This overexpression was already identified in plasma cells from MGUS (Monoclonal Gammopathy of Undetermined Significance) patients and SMM (Smouldering MM) stages in contrast to normal bone marrow plasma cells. Finally, we examined the impact of IRF2 and IRF4 expression on the outcome of MM using RNA-seq data from 664 patients from MMRF (MM Research Foundation) CoMMpass (IA18) database. We observed that patients with higher expression of IRF2 presented significantly poorer Progression Free Survival (PFS) and Overall Survival (OS) in univariate analysis. Interestingly, in multivariate analyses taking into account the classic high-risk genetic factors for MM, the expression of IRF2 together with del13q and amp1q resulted in a significantly lower PFS (global p < 0.001) and the expression of IRF2 together with amp1q and del17p resulted in statistically significant worse OS (global p < 0.001). On a multivariate model, MM patients were stratified according to the number of events ( IRF2 expression, del13q and amp1q for PFS and IRF2 expression, amp1q and del17p for OS) into four risk groups both for PFS and OS ( Fig. 2). In conclusion, our study demonstrate that IRF2 plays a significant role in the pathogenesis of MM by participating in the regulation of the de novo active chromatin regions, specifically regions related to kinase pathway. Additionally, IRF2 represents a robust biomarker that in combination with classical genetic markers improves stratification of MM patients. These results establish IRF2 as a promising candidate for new therapeutic strategies in patients with MM.

And-Gate CAR T-Cells to Improve Tumour Specificity and Targeting of Low-Expression Antigens in Multiple Myeloma

Whilst chimeric antigen receptor (CAR) T cells have shown remarkable success, further novel targets are required. However, our previous cell surface proteomics work in multiple myeloma (MM) showed that target choice is highly limited by an absence of truly tumour-specific antigens, and often very low on-tumour expression of target proteins in some primary samples. One approach to improve tumour specificity is to use combinatorial antigen recognition, or “AND-gates”, whereby a CAR T cell must recognise two different antigens for full activation and target cell elimination. It has also been reported that this approach might overcome low target expression. We therefore decided to explore the potential for AND-gate targeting in MM and identify a suitable target pairing. To determine a comprehensive list of viable AND-gate combinations for MM, we first integrated our MM proteomics data with a normal tissue proteomics dataset. From 777 extracellular proteins expressed in primary MM, we identified a total of 287,890 possible pairwise combinations. We next excluded any pairs with overlapping expression in vital, healthy tissues, reducing potential combinations to 664. Additional filtering steps to remove difficult-to-target proteins and combinations where either target was expressed on T cells - thus avoiding T cell fratricide - resulted in a final list of 92 AND-gate targets. From our prioritised candidates, we selected TNFRSF8 and TMPRSS11E as an exemplar pairing. By mass spectrometry, both TNFRSF8 and TMPRSS11E were expressed across all 8 profiled primary MM samples at comparable levels to existing CAR T cell targets, such as GPRC5D, and had no predicted overlapping expression in normal tissue. To test our AND-gate combination, we designed a ‘split-CAR’ system in which TNFRSF8 was targeted by a 1 st generation CAR with an intracellular signalling domain, but without a co-stimulatory receptor (CAR-only construct), and TMPRSS11E was targeted by a construct with a chimeric co-stimulatory receptor (CCR), but lacking an intracellular signalling domain (CCR-only construct). Constructs were initially validated in a Jurkat cell line model of T cell activation. Jurkats were transformed with CCR-only, CAR-only, or both constructs, as well as with full 2 nd generation CARs targeting TNFRSF8 or TMPRSS11E. These cells were co-cultured with the TNFRSF8+TMPRSS11E+ human MM cell line XG1 or the TNFRSF8+TMPRSS11E- erythroleukemic cell line K562. As anticipated, neither the CCR-only nor the CAR-only Jurkats were activated, with no production of IFNγ or IL2 ( Fig. 1A). Whilst Jurkats expressing the full 2 nd generation TMPRSS11E CAR were activated in co-culture with XG1, Jurkats expressing the TNFRSF8 2 nd generation CAR were not, despite expression of TNFRSF8 on XG1. Jurkats co-expressing both components of the split-CAR were markedly activated when co-cultured with TNFRSF8+TMPRSS11E+ XG1 cells (but not with TNFRSF8+TMPRSS11E- K562 cells), as demonstrated by a large increase in expression of CD69, IFNγ, and IL2 ( Fig. 1A). Thus, the split-CAR system showed a very high degree of specificity, requiring both antigens to be co-expressed on the target cell. Moreover, it was activated even when full 2 nd generation CARs against TNFRSF8 were not. Next, we sought to investigate the cytolytic activity of our split-CAR constructs in donor T cells. In a cytotoxicity assay, T cells expressing the CAR-only or CCR-only construct did not exhibit significant killing of XG1 cells ( Fig. 1B). In parallel with the Jurkat reporter assays, only 2 nd generation TMPRSS11E CAR T cells, and not 2 nd generation TNFRSF8 CAR T cells, were cytotoxic to XG1. Finally, donor T cells expressing both components of the split-CAR demonstrated potent killing of dual-positive XG1 cells ( Fig. 1B), but not single-positive K562 cells. We have thus developed an analytical pipeline for the discovery of AND-gate CAR T cell targets for MM, also applicable to other tumours. This approach led to the development of an extremely potent, but highly selective, split-CAR targeting TNFRSF8 and TMPRSS11E. Importantly, our split-CAR targeted very low levels of TNFRSF8 antigen that were insufficient to trigger activation of a 2 nd generation TNFRSF8 CAR-T. In summary, our work suggests that AND-gate targeting can increase not just specificity, but potency, even against very low expression tumour targets in MM. Link to preprint https://doi.org/10.1101/2023.04.04.535580

Unraveling Diverse Mechanisms of Complex Structural Variant Interactions through Multiomic Data in Multiple Myeloma

Introduction: Structural variants (SVs) play a role in proliferation of plasma cells that characterizes multiple myeloma (MM). Several hallmark SVs have been identified in MM, including translocations involving the immunoglobulin heavy chain locus and several partner genes, as well as deletions and duplications affecting key genes such as TP53, RB1, and MYC. Complex SVs such as templated insertions, chromothripsis, and chromoplexy are associated with disease progression but have not yet systematically been studied. Methods: Paired short-read (Illumina, average depth 75x), long-read HiFi (PacBio, 16x), optical genome mapping (OGM; Bionano Genomics, 341x) and Micro-C (Cantata Bio, 45x) data were sequenced from 8 relapsed MM patient-derived xenograft samples and 2 cell lines, covering t(11;14), t(4;14), and hyperdiploid subgroups. Reads were aligned to the hg38 reference genome while complex SVs (chromothripsis, chromoplexy and templated insertion) were systematically identified and accurately reconstructed by combining short-read (Manta), HiFi (pbsv), OGM, and Micro-C data (EagleC). Chromosome organization including loops, topologically associated domains (TADs) and A/B compartments were called from Micro-C data (Mustache and Juicer). Haplotype phasing was conducted using small variants detected from HiFi data. De novo assembly of the genome was conducted using HiFi and Micro-C data and compared to the reference genome. Reconstructed derivative chromosome interactions due to complex SVs were further identified by aligning Micro-C reads to the newly assembled genomes. Previously identified super-enhancers from H3K27Ac ChIP-seq from MM cell lines were mapped onto these SVs and expression of target genes assessed by RNA-seq. Results: 1,340 SVs were found in 10 samples using OGM of which 828 were identified in short-read WGS data. 16 complex SVs including chromothripsis, chromoplexy and templated insertions were detected in 10 samples, and we describe two (Fig.1) in detail here. A complex templated insertion consisting of a t(2;6;8) was found in a t(11;14) PDX sample with copy number gains at breakpoints which could not be resolved from short-read data alone. OGM data revealed a single contig consisting of DNA from chromosomes 2, 6, and 8. Micro-C data revealed interactions between these chromosomes, specifically the gained regions on chromosomes 2 and 8 showed more interactions with chromosome 6 than with other chromosomes, indicating insertions into chromosome 6. As a result, both MYC at chromosome 8 and GALM at chromosome 2 were inserted into the TXNDC5 locus, containing a known MM super-enhancer, leading to the over-expression of both MYC and GALM (log 2FC=1.7 and 1.0) in this sample. A de novo genome assembly indicated neo-TAD formation encompassing the super-enhancer and both genes. In coMMpass dataset, high expression of GALM and MYC are significantly associated with inferior overall (p=0.002 and 0.05, logrank test) and progression-free survival (p=0.01 and 0.09). Another complex templated insertion consisting of a t(3;4;16) was identified in a hyperdiploid PDX sample, again with copy number gains at breakpoints. Although the short-read data indicated an assembly similar to the previous templated insertion SV, the OGM and Micro-C data indicated a 3-way chromosome swap where the short arm of chromosome 3 was translocated to the short arm of chromosome 4, the short arm of chromosome 4 was translocated to the long arm of chromosome 16, and the long arm of chromosome 16 was translocated to the short arm of chromosome 3. Separate contigs for the t(4;16), t(3;16) and t(3;4) were confirmed by OGM data and only 2-way chromosomal interactions were seen in the Micro-C data. This complex event led to numerous oncogenes juxtaposed to super-enhancers, which caused over-expression of target genes such as ACD and RANBP10 (log 2FC=1.0 and 0.4), both of which were on chromosome 16 and are significantly associated with inferior overall (p=0.02 and p=0.004) and progression-free survival (p=0.003 and p=0.005). Conclusion: By utilizing multiomic techniques we were able to reconstruct complex SV events, showing different mechanisms of generating templated insertion events and identifying their biological consequences. We demonstrated how these complex events could potentially facilitate tumor proliferation via engaging the super-enhancers and regulating the expression of key oncogenes.

R-ISS Stage-Dependent Single-Cell Sequencing Analysis Uncovers Oncogenes and Potential Immunotherapeutic Targets in Multiple Myeloma

Multiple myeloma (MM) accounts for ~10% of all haematologic malignancies. Little is known about high intratumour heterogeneities in patients stratified by the Revised International Staging System (R-ISS). Herein, we constructed a single-cell transcriptome atlas to compare differential expression patterns among stages. We found that a novel cytotoxic plasma cell (PC) population exhibited with NKG7 positive was obviously enriched in stage II patients. Additionally, a malignant plasma cell population with significantly elevated expression of MKI67 and PCNA was associated with unfavourable prognosis and Epstein-Barr virus (EBV) infection in our collected samples. Moreover, Ribonucleotide Reductase Regulatory Subunit M2 (RRM2) was found and verified to promote proliferation of MM cell lines, suggesting RRM2 may serve as a detrimental marker in MM. The percentages of CD8+ T cells and NKT cells decreased along with R-ISS stages, reflecting the plasticity of the tumour immune microenvironment. Importantly, their crosstalks with myeloid cells and PC identified several potential immunotargets such as SIRPA-CD47, and CD74-MIF, respectively. Collectively, this study provided an R-ISS-related single-cell MM atlas and revealed the clinical significance of novel PC clusters, as well as potential immunotargets in MM progression.

Targeting IGF1R/Insr Pathway with Approved ALK-Inhibitors Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma

Background: The treatment of multiple myeloma (MM) has advanced rapidly with the discovery of proteasome inhibitors (PIs) bortezomib (BTZ) and carfilzomib (CFZ). PIs disrupt the equilibrium between production and disposal of excess and/or misfolded proteins in MM cells, leading to apoptosis. However, despite this success, essentially all patients with MM eventually develop resistance to PIs. Therefore, identifying strategies to overcome PIs resistance is clinically important. ALK inhibitors exhibit anti-MM activity ex vivo. Previously, the ALK inhibitor ceritinib has been shown to inhibit IGF1R/InsR signaling. Signaling through the IGF1/IGF1R axis contributes to acquired resistance to BTZ, and PI activity is reduced in the presence of IGF1, suggesting that IGF1R inhibitors may enhance the cytotoxic effect of PIs. To date, IGF1R/InsR inhibitors have not been successful as single agents in clinical trials and no formally approved drugs are available. Importantly, the drug repurposing approach offers a promising strategy for drug development, particularly for MM. Therefore, we aimed to identify i) whether ALK inhibitors are cytotoxic in MM by targeting the IGF1R/InsR pathway, and ii) whether they can be combined with PIs to overcome PI resistance in vitro and in vivo. Methods: A set of PI-naïve and PI-resistant cells was used in this study. The cytotoxicity of drugs was determined by CCK8 assay in cell lines and by CellTiter-Glo assay in primary cells. Genome-wide CRISPR/Cas9-based loss-of-function screening using the Brunello library was performed in the AMO-1 cell line to identify the mechanism of action of ceritinib. Kinase inhibitor selectivity data were retrieved from ChEMBL, v30. Western blotting was performed to assess the levels of total and phosphorylated proteins. RNA sequencing was used to determine the number of transcripts in PI-sensitive and PI-resistant cells, and RNA-seq data from patients included in the CoMMpass study were analyzed. Unbiased LC-MS/MS was performed to determine the effects of ceritinib and carfilzomib on intracellular metabolites. An in vivo mouse model based on orthotopic injection of AMO-BTZ cells into the femur of NSG mice was used to determine the effect of the drug combination in vivo. Results: Initially, seven ALK inhibitors were tested in PI-naïve and PI-adapted MM cell lines. Based on IC 50 values, the most effective ALK inhibitors to induce cytotoxicity in MM were ceritinib > brigatinib > entrectinib. The combination of ceritinib, brigatinib, and entrectinib showed synergistic cytotoxicity with PI BTZ and CFZ and overcame PI-resistance in four different sets of PI-adapted cells. The strongest synergistic cytotoxicity was observed between ceritinib and CFZ in CFZ-adapted cells. CRISPR/Cas9-screening identified genes involved in the negative regulation of mTORC signaling (DDIT4, NPRL2/3, TSC1/2) and transcription factor FOXO1 as the major resistance candidates to ceritinib. Subsequently, ceritinib treatment significantly inhibited mTORC signaling and impaired purine-pyrimidine and amino acid production pathways, suggesting a metabolic and proliferation shut-down and amino acid starvation due to targeting of the upstream receptor tyrosine kinases (RTK), which are essential for cell viability and thus not identified by CRISPR screening. Further search in the ChemBL database identified InsR and IGF1R, as RTK that was potently inhibited by ceritinib. PI-naïve and PI-resistant cell lines as well as MM patients were negative for ALK, but positive for InsR and IGF1R expression. Accordingly, co-treatment of PI-resistant cells with a combination of InsR/IGF1R inhibitors and CFZ induced strong synergistic cytotoxicity, resembling the synergistic cytotoxicity observed for ceritinib and CFZ, whereas InsR/IGF1R inhibitors did not show any synergistic cytotoxicity with ceritinib, suggesting targeting the same pathway. Importantly, the combination of ceritinib and CFZ was superior to CFZ in an orthotopic mouse model bearing PI-resistant MM cells and showed strong synergistic cytotoxicity in primary cells from MM patients progressing under or after PI-containing therapy. Conclusion: Ceritinib, an FDA-approved drug, overcomes PI resistance in MM by targeting InsR/IGF1R signaling, which is essential for PI resistance in MM. Therefore, ceritinib represents a promising, potential option for the treatment of PI-resistant MM.

CREB1 Promotes Immune Escape of Multiple Myeloma Cells By Inducing HLA-E

Introduction: Multiple Myeloma (MM) is a disease of clonal plasma cells, which accumulate in the bone marrow (BM) and rely on the support of the microenvironment to survive. Immune escape is a common resistance mechanism employed by tumor cells to avoid cytotoxicity by effector immune cells. HLA-E, a non-classical major histocompatibility complex (MHC) class I molecule, inhibits the function of specific subtypes of Natural Killer (NK) and T cells, leading to tumor immune escape. HLA-E expression correlates with worse progression-free survival in newly diagnosed patients with MM. Therefore, we decided to better elucidate the function and regulation of HLA-E expression in MM. Results: We observed that HLA-E mRNA expression increases in the transformation from normal plasma cells to MM. Since INF-gamma induces HLA-E in MM cells and CREB1 itself promotes the production of IFN-gamma, we hypothesize that CREB1 could regulate HLA-E expression in MM. We first analyzed RNA-sequencing data from patients with MM in the CoMMpass database: gene set enrichment analysis (GSEA) showed increased expression of several pathways related to interferon signaling in patients with high CREB1 expression. We then specifically looked at differences in HLA-E and we confirmed that patients with high CREB1 expression had statistically significant higher HLA-E levels compared with patients with low CREB1 expression. CHIP-sequencing assays in MM cell lines demonstrated that CREB1 can directly influence HLA-E expression by binding to HLA-E promoter. To further prove these data, we evaluated HLA-E levels in gain-of and loss-of function models of CREB1. We observed an increase of HLA-E levels by overexpression of wild type CREB1 and reduction of HLA-E levels by CREB1 silencing or pharmacological inhibition of CREB1 with 666-15 (CREBi). We then investigated the relationship between CREB1 and STAT1. Indeed, IFN-gamma signals through the STAT1-JAK pathway. Interestingly, while treatment with IFN-gamma strongly induces HLA-E without affecting CREB1 levels or its function, CREBi + INF-gamma reduces HLA-E, STAT1, and phospho-STAT1 levels. Conversely, immunomodulatory drugs (lenalidomide-LEN and pomalidomide-POM) and HDAC inhibitor, Panobinostat, promote the phosphorylation of STAT1, resulting in the transcription of IFN-target genes, including HLA-E. The increase in HLA-E was then reverted by CREB1 inhibition. Since HLA-E impairs NK cell function, we treated MM cells in the presence of NK cells with CREBi alone or in combination with immunomodulatory drugs (IMiDs). Treatment with CREBi or CREBi + IMiDs potentiated the killing effects of NK cells. Animal studies are ongoing to confirm these data in vivo. Conclusion: In conclusion, our study defines the role of CREB1 in modulating HLA-E expression; CREB1 inhibition improves NK cell-mediated cytotoxicity in MM, representing a novel strategy to tackle immune escape.

Novel Combinatorial Therapeutic Options in NEAT1-Depleted Multiple Myeloma Cells By the Integration of in Vitro and in silico Drug Screening Approaches

Multiple myeloma (MM) is an incurable plasma cells malignancy characterized by high genomic instability and clinical heterogeneity. Transcriptional landscape in MM is characterized by a marked de-regulation of long noncoding RNAs (lncRNAs) that influence disease progression and therapy resistance. We have shown that the lncRNA NEAT1 is overexpressed in MM patients and demonstrated that NEAT1 silencing negatively impact MM cells viability, both in vitro and in vivo suggesting that it may result in higher sensitivity to standard MM treatments. Herein, we adopted in vitro high throughput (HTS) and in silico drug screening approaches to explore the NEAT1 role in chemo-resistance scenario and identify novel putative therapeutic combination strategies. For the HTS approach, a library of 320 selective inhibitors covering 123 targets involved in proliferative and survival pathways, was screened in NEAT1-KD AMO-1 MM cells and their relative scramble condition. Silencing of total NEAT1 was achieved through the gymnotic delivery of LNA-gapmeRs. Synergy score, calculated with Bliss analysis, identified 19 compounds having a synergistic effect, in combination with NEAT1 KD, leading to a decreased MM cells proliferation, compared to single treatments. These inhibitors span different cellular targets, grouped into five categories: cell cycle checkpoint, transcriptional regulators, protein tyrosine kinases, receptors channels, epigenetics regulators. Among the top scoring candidates, Aurora kinase A inhibitors were the most promising compounds able to individually exert a synergistic activity with total NEAT1-KD in vitro. Additionally, transcriptomic analysis was performed by bulk RNA sequencing in NEAT1-KD AMO-1 cell line. Libraries with optimal quality and quantity were run on NextSeq 500. Differential gene expression analysis identified 752 down-regulated genes and 957 up-regulated genes. This signature was used to query the Connectivity Map (CMap) database. Compounds mimicking NEAT1 KD transcriptional signature were selected with the highest connectivity score (> 90). Among the top scoring molecules, Aurora kinase A (AURKA) inhibitors were identified as able to mimic NEAT1 KD transcriptional signature. Both approaches identified AURKA inhibitors as promising compounds able to affect NEAT1 functions. Based on this evidence, we started the validation of two different AURKA inhibitors on a panel of MM cell lines. Cell proliferation was assessed after 96 hours of NEAT1 silencing and 72 hours of drug treatment with Trypan-exclusion method and with live-cell imaging and analysis by taking advantage of Incucyte S3 TM technology. The synergy score was calculated with CompuSyn software. WB was used to assess the drugs on-target activity and qRT-PCR was used to validate the efficiency of NEAT1 KD. These data, suggest that NEAT1 KD and treatment with AURKA inhibitors can have similar biological outcome and can affect similar molecular pathways on MM cells. Overall, our results indicate that NEAT1 silencing combined with Aurora kinase A inhibition could represent a promising therapeutic option for MM patients, supporting the need to further characterize the molecular mechanisms underlying the synergistic effect observed in MM cells.

The Role of Macrophage Migration Inhibitory Factor (MIF) in Regulation of T-Cell Activity in Multiple Myeloma

Background: Multiple myeloma (MM) is the second most common hematological malignancy associated with the overproduction of light chain or monoclonal immunoglobulin. MM remains incurable, with high relapses and refractory disease rates after first-line treatment. Hence, novel therapeutic approaches are needed to improve immune responses to prevent relapses or drug resistance after initial therapies. Immune responses are shaped by several factors, including mutations, pro- and anti-inflammatory cytokines and chemokines, and activation of immune checkpoints in the tumor microenvironment (TME) (Gonzalez et al., 2018). Macrophage migration inhibitory factor (MIF) as a soluble pro-inflammatory cytokine has become one of the most puzzling regulators of innate and adaptive immune responses. In myeloma, MIF was found to be significantly higher in MM cells from relapsed patients than those responding to the treatment. MIF has been implicated in drug resistance and suggested as a biomarker for predicting MM patient responses to proteasome inhibitors (Wang et al., 2020). We previously reported that MIF is highly expressed in MM and regulates CD84 (SLAMF5) expression in MM TME. We showed that MIF secreted from MM cells regulates PD-L1 expression through CD84 on CD14 + monocytic cells in the TME. Inhibition of MIF significantly reduced CD84 and PD-L1 expressions on human CD14 + cells and on myeloid-derived suppressor cells of the mice bearing MM (Lewinsky and Gunes et al., 2021). However, whether MIF directly regulates T-cell activity in MM TME has yet to be explored. Methods and Results: The current study reports that MIF may play a pivotal role in T-cell exhaustion in MM. Ouranalyses in published MM transcriptomic databases show that MM patients with low MIF had significantly longer overall and post-progression survival, indicating the therapeutic potential of MIF in MM. We also noted upregulated MIF gene expression in relapsed or refractory MM (R/RMM) patients (n=2) compared to that in newly diagnosed MM patients (n=7) and healthy individuals (n=9) in our and publicly available MM single-cell RNA sequencing datasets. We detected a profound MIF expression in MM cells and myeloid cell fractions, including monocytes, macrophages, and dendritic cells from the patients' bone marrow (BM) specimens. We previously reported that human MM cell lines highly express MIF. In this study, we assessed the impact of MIF secreted by MM cells on the immune cells, specifically CD8 + T cells. Our results showed that inhibition of MIF reduced the elevated T-cell exhaustion markers, PD1 and CTLA-4, on CD8 + T cells in healthy donors (HDs) peripheral blood mononuclear cells (PBMCs) following the coculturing with MM1.S cells (n=3 *P < 0.05 ). We also found that MIF inhibition induced the T-cell activation marker, LAMP1, on CD8 + T cells in this coculturing setting (n=3, **P < 0.01). Furthermore, we noted MIF inhibition reduced PD1 expression on CD8 + T cells in HDs PBMCs (n=3, **P < 0.01), suggesting MIF may regulate the exhaustion profile of CD8 + T cells. MIF can conduct paracrine or autocrine signalings by binding to its receptors, including CD74 and CD44, and lead to intracellular signaling cascades. Hence, we assessed CD74 gene expression as the counterpart of MIF in R/RMM, MM, and healthy patients. We noted significantly upregulated CD74 expression in R/RMM patients compared to newly diagnosed MM patients and healthy individuals. We also noted higher CD74 expression in CD8 + T cells than in CD4 + T cells in R/RMM patients, suggesting that MIF/CD74 axis may be a potential target to enhance CD8 + T cell-mediated immunity in R/RMM. To further explore the impact of MIF inhibition on MM progression, we performed a pilot in vivo study by inoculating murine 5TGM1 cells into two groups of syngeneic C57BL/KaLwRijHsd mice. After two weeks, the mice were injected with either sterile PBS as a control or a MIF inhibitor. The results of this pilot study showed that MIF inhibition significantly decreased MM cells in BM and PB of myeloma-bearing mice and reduced T-cell exhaustion markers on CD8 + T cells compared to that in the control mice (n=3-4, *P < 0.05, **P < 0.01, ***P < 0.001). Conclusion: Overall, our preliminary data indicate that MIF may have a crucial role in T-cell activity by regulating T-cell exhaustion markers in MM. Further studies are needed to explore the therapeutic potential of MIF to reduce T-cell exhaustion to improve anti-tumor immunity in MM.

A Novel Indirubin-3-Monoxime Derivative Functions As Regulator of Proteostasis Via Targeting TRIM28 in Multiple Myeloma

Introduction: Multiple myeloma (MM) remains an incurable plasma cell malignancy. Agents disturbing the proteostasis play indispensable roles in MM treatment. Our previous studies have shown that the indirubin-3-monoxime (I3MO) works as a novel proteasome inhibitor that effectively inhibits MM cell growth. In this study, we aim to investigate the improvement of I3MO combined with histone deacetylase 6 (HDAC6) inhibitors, and clarified the molecular mechanisms. Methods: We constructed a compound 8b combined with I3MO and HADC6 inhibitor. The anti-MM effects of compound 8b were investigated in vivo and in vitro studies. Western blots, immunofluorescence, and proteasome activity assay were utilized to investigate the effects of 8b on MM cell proteasome activity, aggresome and autophagosome formation. RNA-seq and Dual-luciferase reporter gene assay were performed to identify the downstream targets of 8b treatment. We constructed MM cell lines with TRIM28 overexpression or shRNA knockdown. We further explored the function of TRIM28, one of the targets of 8b treatment, by ChIP-seq and IP-MS. Results: The novel I3MO derivative 8b more efficiently suppressed myeloma cell growth in vitro and in vivo. Compound 8b not decreased proteasome activity but inhibited the formation of aggresomes and autophagosomes. Interesting, we found that compound 8b efficiently inhibited TRIM28 promoter activity and repressed TRIM28 transcription level further. GEO data analysis demonstrated a positive correlation between TRIM28 and the expression of proteasome subunits as well as autophagy-related genes in MM. Further, we utilized ChIP-seq to confirm that TRIM28 could bind to the promoter region of multiple proteasome subunits-encoding genes, including PSMB1. Our data indicated that TRIM28 knockdown not only decreased the proteasome activity, but inhibited the formation of autophagosomes. Knockdown TRIM28 induced a higher sensitivity to BTZ treatment in MM cells. Furthermore, IP-MS with Co-IP analysis showed that TRIM28 interacts with 14-3-3ζ. TRIM28 acts as an E3 ligase in mediating the ubiquitination and degradation of 14-3-3ζ, a well-known negative regulator of autophagy. 14-3-3ζ degradation induced by TRIM28 activates autophagy in MM cells. These data suggested that TRIM28 suppressed by compound 8b would involve in proteostasis by regulation of proteasome activity and autophagy. Conclusions: Our study identified a novel compound 8b, I3MO combined with HDAC6i, efficiently suppressed MM cells survival. Additionally, we for the first time provided novel important insights for the roles of TRIM28 in MM pathobiology by regulation of proteasome activity and autophagy.

Targeting HUWE1 in Multiple Myeloma Leads to Impaired Replicative Stress Response and Enhanced Sensitivity to Bortezomib

Introduction Despite continuous advances in therapies over the last few decades, Multiple Myeloma (MM) remains an incurable malignancy with almost all patients developing relapsed/refractory disease. This emphasizes the need for novel approaches that can improve the efficacy of current standard of care treatments. Previous work from our lab and others has identified dysregulation of the E3 ligase HUWE1 in MM and highlighted it's potential as a therapeutic target. Through its interaction with a diverse set of substrates, HUWE1 is implicated in many key cellular processes including stress responses and DNA replication and repair pathways and is being increasingly recognised as a major regulator of MYC activity. In this study, we uncover a novel role for HUWE1 in DNA replication and repair and assess the combination of HUWE1 and proteasome inhibition as a potential therapeutic strategy in MM. Methods MM cell lines were transfected with SMARTvector Inducible Human HUWE1 shRNA or a non-targeting control (NTC) shRNA (Dharmacon, USA). Replicative stress was induced by treatment with 2 mM Hydroxyurea (HU) and assessed using immunofluorescence staining and iPOND (isolation of proteins on nascent DNA) analysis. Viability was assessed using CellTiter® Glo and combination indices (CI) calculated using CompuSyn software. Commercially available HUWE1 inhibitor BI8622 (MedChemExpress) and novel HUWE1 inhibitors synthesised in house were used throughout the study. Results HUWE1 knockdown and/or inhibition in MM cell lines led to an accumulation of cells in S phase, consistent with previous studies indicating that HUWE1 is required for effective DNA replication. Using proteomic profiling and co-immunoprecipitation, we identified and validated Replication Protein A, 70 kDa (RPA70) as a novel substrate of HUWE1. RPA70 is a subunit of the heterotrimeric RPA complex which plays a critical role in the recruitment of DNA repair proteins. RPA70 is the first subunit to translocate to sites of DNA damage where it binds to DNA and recruits the other subunits (RPA32 and RPA14) forming the complex, thus allowing for initiation of DNA repair. We observed significantly less K63-linked ubiquitination of RPA70 (p=0.00213) in the absence of HUWE1, with an associated decrease in both phosphorylation (p=0.0064) and localisation of RPA subunit RPA32 to DNA following treatment with hydroxyurea (HU) to induce replicative stress. Subsequent iPOND analysis determined that this is associated with reduced recruitment of repair proteins, including RAD51, to DNA. Moreover, induced replicative stress in HUWE1 depleted/inhibited cells resulted in significantly higher levels of DNA damage at 6hrs (p=0.00421) and 24hrs (p=0.0219) post HU treatment. A recent study demonstrated that HUWE1 ubiquitination of MYC promotes the formation of MYC multimers that protect stalled replication forks to limit the formation of double strand breaks (DSB) (Nature 2022;612:148-155). In line with this we also observe decreased ubiquitination of MYC in the HUWE1 knockdown cell line compared to the NTC following HU treatment. Finally, we assessed the efficacy of combining HUWE1 inhibitors with bortezomib which is known to impair DSB repair. Dual inhibition of HUWE1 and proteasome activity resulted in synergistic effects (CI values < 1) and a corresponding increase in DNA damage compared to HUWE1 inhibitor or bortezomib alone as measured by levels of ƴH2AX. Conclusion We have identified RPA as a novel substrate for HUWE1 and demonstrate that targeting HUWE1 results in increased replication stress and a dampened DNA repair capacity, underpinned by reduced recruitment of repair proteins. This work outlines a clear role for HUWE1 in genome stability in MM cells and highlights that HUWE1 inhibitors represent a novel anti-myeloma strategy that acts in synergy with bortezomib to exacerbate DNA damage.

CRISPR-Based Gene-Editing Screens Revealed Modulators of Dexamethasone Sensitivity in Multiple Myeloma

Background: Multiple myeloma (MM) is an incurable plasma cell malignancy that accounts for 10% of hematologic malignancies. Despite improved prognosis due to advances in treatment, most patients ultimately develop therapy resistance and relapse. The glucocorticoid dexamethasone is a potent anti-myeloma agent used in combination with proteasome inhibitors and Immunomodulatory drugs (IMIDs) as frontline treatment. Dexamethasone binds to the glucocorticoid receptor (GR), which induces apoptosis by driving an apoptotic transcriptional program. The mechanisms of Dexamethasone resistance in MM remain poorly understood. Aim: To define cellular processes that modulate dexamethasone sensitivity in MM. Methods: We performed loss-of-function CRISPR screens in the t(4;14), NSD2 overexpressing, MM cell lines KMS11 and LP1 using the genome-wide Brunello sgRNA library. Results: The screens identified genes whose loss altered MM cell sensitivity to dexamethasone, most being cell type-specific suggesting heterogeneity in mechanisms influencing dexamethasone cytotoxicity across MM patients. In KMS11 cells, disruption of the IL10/JAK/STAT signaling pathway and glycosaminoglycans/heparan sulfate synthesis pathway increases sensitivity to dexamethasone. By contrast, loss of chromatin remodeling factors including the polycomb group member EPC1 and the SWI/SNF complex components BRD7 and BRD9, altered dexamethasone response exclusively in LP1 cells. Perturbations resulting in dexamethasone resistance in both cell lines include the GR and its co-chaperone PTGES3 in addition to members of the NFKB inhibitor family (NFKBIA, NFKBIB). By contrast, disruption of the druggable GR co-chaperone FKBP5 or the histone deacetylase HDAC8 increased sensitivity to dexamethasone. In addition, the GID/CTLH E3 ubiquitin ligase complex was also identified as a general determinant of dexamethasone resistance in MM cells as disruption of several constituents of this complex resulted in increased dexamethasone sensitivity in both cell lines. The mechanism by which the GID/CTLH complex confers dexamethasone tolerance in MM cells is currently under investigation. Conclusions: Multiple cellular processes contribute to modulating the cytotoxic effect of dexamethasone in MM. Such processes often regulate expression, stability, or localization of the GR. NFKB signaling and the GID/CTLH E3 ubiquitin ligase activity are key determinants of dexamethasone resistance. Targeting these processes can increase the therapeutic effect of dexamethasone in MM.

Active STAT3 Prevents Selinexor-Induced Caspase-Independent Apoptosis in Multiple Myeloma

Multiple myeloma (MM) is an acquired malignant plasma cell disorder that typically develops late in life with a median age at diagnosis of 69 years and results in approximately 12,000 deaths per year. Currently, the standard treatment strategy for MM is a combination of 3 drugs with different mechanisms: dexamethasone, a proteasome inhibitor, and an immunomodulatory agent. Many MM patients acquire a “double-hit” of their TP53 on Chromosome 17p region after multple rounds of treatment and become resistant to these conventional therapeutics. Thus, novel effective treatments for high-risk and relapsed and refractory (RR)-MM are urgently needed to overcome drug-resistance, especially in TP53 null patients. In many cancers, nuclear exporter protein XPO1 overexpression is induced by c-Myc and/or loss of p53. Previous studies have demonstrated that many RR-MM patients show increased expression/activity of XPO1, which is associated with aberrant cytosolic localization of its protein and RNA cargoes. Selective XPO1 inhibitor Selinexor (Sel) has been approved by FDA to treat relapsed and refractory multiple myeloma (RR-MM) in combination with a bortezomib + DEX regimen. Addition of Sel to this regiment significantly improves the overall response rate (~75% vs. ~60%) and progress free survival (13.9 months vs 9.5 months), regardless of risk factors including TP53 status. However, only ~50% of RR-MM patients benefit from Sel treatment. In addition, Sel has significant dose dependent side effects. It is important to determine the mechanism of Sel-resistance in order to identify biomarker(s) to predict Sel-resistance and develop novel effective combination therapies. . In this study, we compared the response of 10 human MM cell lines to Sel treatment and identified Sel sensitive and resistant cell lines. Based on EC50, we divided the cell lines into Sel-sensitive group with EC 50 values between 80.4 nM to 211.8 nM, and Sel-resistant groups with EC 50 values >741.4 nM. We found that Sel sensitivity was notably independent of TP53 mutation status, as well as other genetic mutations and chromosomal translocations. In resistant cells, high dose of Sel induces an off-target Caspase-mediated apoptosis because it could not be influenced by XPO1-knockdown but could be prevented using a pan-caspase inhibitor Z-VAD. In sensitive cell lines, low dose of Sel induces a Caspase-independent type of cell death because it could not prevented by pre-treated with Z-VAD. To investigate which programmed cell death pathways Sel works through, sensitive cells were treated using with Sel + inhibitors for necroptosis, ferroptosis, pyroptosis, and parthanatos. We found only parthanatos inhibitor could significantly prevent Sel-induced cell death. The parthanatos death pathway functions via DNA-damage/poly (ADP-ribose) polymerase-1 (PARP-1) activation. While proper PARP-1 levels are required for DNA damage repair, over-activation results in the release of apoptosis-inducing factor (AIF) from mitochondria to cytoplasm which then trans-localize to the nucleus where it functions with macrophage migration inhibitory factor (MIF) to induce DNA fragmentation and cell death. We show via Western blotting and immunofluorescence imaging that upon Sel treatment, AIF shifts from the cytosol into the nucleus. Sel treatment was further assessed in combination with with Dex, where it showed a low synergistic effect with Dex in the sensitive cells but not in resistant cells, specifically when used sequentially. When assessing mechanisms of Sel resistance, phosphorylated STAT3 was uniquely observed in the resistant cell lines. To determine if STAT3 mediates resistance, resistant cell lines were pre-treated with a STAT3-PROTAC (SD36). Our study suggested that p-STAT3 predicts Sel-resistance, and Sel + STAT3 inhibitor combination is a novel effective regimen for MM, especially for these Sel-resistant cases.

Secondary Genetic Events Impact the Expression of Key Immunotargets on the Surface of Multiple Myeloma Cells

Background: Multiple myeloma (MM) is characterized by complex genomic aberrations including primary translocations of the immunoglobin heavy chain (IGH) locus and secondary mutations involving key oncogenic drivers. This temporal complexity represents a constant challenge for the clinical management of MM, but at the same time may provide new avenues for immunotherapy-based approaches including CAR-T cells and T-cell engaging antibodies. In this study, we investigated the impact of secondary genetic events on the surface expression of common targets for immunotherapy in MM. Methods: Cell models harboring two hotspot activating mutations in KRAS (G12A, A146T) and TP53 (R282W) were created using the MM cell lines OPM-2 and AMO1 via CRISPR-Cas9 technology. Direct stochastic optical reconstruction microscopy ( dSTORM) was used for high-resolution receptor density estimation and epitope quantification of common immunotherapeutic targets such as CD38, BCMA and SLAMF7. Results: dSTORM imaging of cell line models carrying patient-derived secondary genetic alterations revealed a distinct pattern linked to each mutation respectively. In KRAS G12A, we observed a reduction in BCMA density by 2-fold as compared to wild type cells (5.9 ± 0.3 vs. 10.8 ± 1.2 localization clusters/µm 2, p=0005). This reduction in BCMA surface expression was accompanied by a decrease in other key immunotargets, including SLAMF7 (6.0 ± 0.8 vs 11.8 ± 0.8 localization clusters/µm 2, p <0,0001) and CD38 (12.2 ± 1.5 vs 17.9 ± 1.6 localization clusters/µm 2, p=0,012). In KRAS A146, CD38 density increased, whereas mean BCMA and SLAMF7 localizations were consistently reduced as compared to wild type control. To ensure that BCMA downregulation is a consistent feature in genomic high-risk disease, we next simulated a TP53 double-hit situation by introducing an inactivating hotspot mutation R282W in a TP53 deletion background. Surfaceome profiling by dSTORM revealed a minor increase in CD38 levels for TP53R282W/Del as compared to wild type control (51.5 ± 5.7 vs. 36.4 ± 4.2 localization clusters/µm 2, p=0.06). Interestingly, the expression level of BCMA, known to be comparably low on WT tumor cells, further decreased in TP53 double-hit cells (0.6 ± 0.1 vs 1.1 ± 0.1 localization clusters/µm 2, p=0.002), whereas no major differences in the cluster density of SLAMF7 were observed. This leaves room for speculation whether these effects on biallelic TP53 inactivated tumor cells may underlie reduced susceptibility towards BCMA-targeting therapy. Of note, we also observed altered cell morphology in the TP53 double-hit model, characterized by elongated stress fibers enriched with CD138, potentially interfering with receptor accessibility of surface receptors (Figure 1). Recently, secondary genetic events have been reported in BCMA and GPRC5D following targeted immunotherapy inducing acquired drug resistance. We created cell models representing homo- and heterozygous genetic alterations in BCMA and GPRC5D, studies to assess their effects on the surfaceome are ongoing and will be presented at the meeting. Summary: Our data suggest that surface epitope expression in MM cells seems to be less agnostic to secondary genomic events than expected. These findings may provide a direct link between genetic high-risk disease and inferior efficacy of anti-BCMA immunotherapies in the context of e.g. TP53 double-hit MM.

Elucidating the Role of Tolerogenic Dendritic Cells in Immune Tolerance and Disease Progression in Multiple Myeloma

Introduction: Previous research has demonstrated that programmed death-ligand 1 (PD-L1) in multiple myeloma (MM) cells not only suppresses anti-tumor immune responses but also enhances malignant potential through the PD-L1-PD-1 interaction [Ishibashi M, et al. Cancer Immunol Res. 2016]. However, it has not yet been shown that monotherapy and combination therapies utilizing PD-1/PD-L1 antibodies are sufficiently effective for MM. One reason for this is the absence of myeloma-specific cytotoxic T lymphocytes induction in the MM tumor microenvironment. In certain cancers, tumor-infiltrating dendritic cells (DCs) undergo conversion into tolerogenic DCs, which are crucial for inducing and maintaining T-cell tolerance, although this remains unclear in MM. Thus, this study aimed to investigate the function and induction mechanisms of tolerogenic DCs in the MM microenvironment. Materials and Methods: (1) Flow cytometry (FCM) was employed to assess the two conventional DC (cDC; lineage negative, HLA-DR +CD11c high) subsets, cDC1 (CD141 + cDC) and cDC2 (CD1c + cDC), in bone marrow (BM) samples obtained from MM patients (n=17) and healthy controls (n=13). (2) CD14 + monocytes were cultured with IL-4 and GM-CSF to generate monocyte-derived DCs (moDCs). Simultaneously, moDCs were co-cultured with MM cell lines using a trans-well system. The characteristics and functions of moDCs were analyzed using real-time PCR, RNA-sequencing (RNA-seq), western blotting, ELISA, and FCM after lipopolysaccharide stimulation. Results: The frequency of cDC1 subsets was significantly lower in MM patients compared to controls, while the major cDC2 subset did not differ. Similarly, when moDCs were co-cultured with MM cells, moDC numbers were markedly reduced. These co-cultured moDCs exhibited downregulated expression of activation markers CD80, CD86, and CD83, along with decreased phosphorylation of ERK and S6K. Conversely, these moDCs upregulated the expression of immunosuppressive factors IL-10, ARG1, and NOS2 through increased transcription factor C/EBPβ. Moreover, these moDCs inhibited T-cell proliferation, activation, and interferon-γ production in comparison to control moDCs, indicating the presence of immature and tolerogenic phenotypes. However, these phenotypes were not observed in moDCs co-cultured with soluble factors secreted from MM and stromal cells. Treatment with AZD3965 improved the phenotype of moDCs co-cultured with MM cells, reversing their suppressive effect on T-cell proliferation. AZD3965 targets monocarboxylate transporter 1 (MCT1) in MM cells, thereby suppressing extracellular lactate accumulation and acidic pH in the MM microenvironment. Furthermore, RNA-seq analysis comparing moDCs co-cultured with and without MM cells revealed downregulation of gene sets associated with activated DCs in moDCs co-cultured with MM. Notably, moDCs co-cultured with MM showed high enrichment of gene sets related to lysosome biogenesis and NOD-like receptor signaling. This suggests that inflammasome activation in these moDCs leads to caspase-1 activation, resulting in increased levels of mature IL-1β and IL-18 in their cell culture supernatants. Additionally, co-culturing CD38-positive MM cells with CD203a/CD73-positive HS-5 stromal cells and NAD + resulted in the generation of adenosine in the cell culture supernatant. Adenosine promotes the immaturation of mature DCs; however, inflammasome activation does not occur in these DCs. Conclusions: Patients with MM exhibit a reduced frequency of cDC1 subsets, which are crucial for cross-presentation to CD8 + T cells. moDCs in acid pH conditions derived from MM cells display an immature phenotype and impaired function due to decreased DC activation signals. Furthermore, inflammasome activation promotes the secretion of IL-1β and IL-18, contributing to pro-tumorigenic inflammation in the MM microenvironment. Moreover, adenosine generated via the CD38/CD203a/CD73 adenosinergic pathway promotes the immaturation and dysfunction of moDCs. Thus, the characteristics of tolerogenic DCs may vary depending on the MM microenvironment. These findings highlight the induction of tolerogenic DCs by the metabolic microenvironment in MM, leading to immune tolerance and disease progression.

Transcriptomic Features Influencing Anti-Myeloma Drug Resistance in Human Multiple Myeloma Cell Lines

Despite increasing number of new drugs, multiple myeloma (MM) remains a significant challenge in hematology. This problem primarily arises from the emergence of drug resistance, which could either result from pre-existing drug-resistant clones, or from the development of secondary genomic and phenotypic drug resistance features upon drug exposure. The use of primary MM patient-derived samples, while considered the gold standard in preclinical MM research, presents challenges due to notoriously low plasma cell yields and short ex vivo lifespan. Moreover, these cells often fail to survive cryopreservation, making them difficult to use in reproducible drug sensitivity screens. To address this issue, the MM research community has established numerous culture-adapted MM cell lines (HMCLs), representative of all cytogenetic MM subtypes and characterized by DNA and RNA sequencing. However, a comprehensive evaluation of their drug sensitivity has been lacking. In this study, we sought to bridge this gap by profiling the sensitivity of over 30 HMCLs to both established and novel drugs relevant to MM treatment. We explored the transcriptomic and genomic features that correlate with drug sensitivity of these cell lines, and investigated whether these features were associated with the outcomes of patients enrolled in the MMRF CoMMpass study. Our initial results suggest potential pathways universally implicated in MM drug resistance, which could be targeted therapeutically. We performed drug sensitivity screening in 96-well plates, testing established drugs such as proteasome inhibitors, immunomodulatory drugs, dexamethasone, and melphalan, alongside novel treatment modalities including the BCL2 inhibitor venetoclax, XPO1 inhibitor selinexor, and histone deacetylase inhibitor panobinostat. Optimal cell density and drug dilutions were ensured to capture the full spectrum of drug response. After 4 days of incubation, cell proliferation was measured using a metabolic MTS assay, and EC50 values were computed using four-parameter logistic regression. Genomic and transcriptomic data were obtained from HMCL66 dataset from the Keats laboratory. Drug sensitivity was analyzed using a log-transformed EC50 matrix, which was used for unsupervised hierarchical clustering of cell lines, revealing two primary clusters: multi-drug resistant (MDR) and multi-drug sensitive (MDS) cell lines. Gene set enrichment analysis revealed that MDR cell lines showed positive enrichment for KEAP1/NFE2L2 (Figure 1A), UPR/PERK pathways, and type I interferon signaling. Negative enrichment in MDR cell lines was observed for TP53-regulated transcription of cell death genes, with the top leading edge genes being key apoptosis inducers such as BAX, BID, APAF1, and FAS. We used the obtained leading edge gene lists to assign respective pathway activation scores to the CoMMpass patients, based on their transcriptomic profile, using the ssGSEA singscore package. High activation score of KEAP1/NFE2L2 was associated with shorter overall survival (HR = 1.57, p = 0.016), irrespective of type of first-line therapy (Figure 1A). Our study represents a significant milestone in characterizing the HMCL drug sensitivity landscape, suggesting that MDR cell lines inherently exhibit transcriptional features associated with high activity of the KEAP1/NFE2L2 and PERK pathways. These pathways have been previously associated with PI and IMiD resistance, but interestingly, our findings also link these signatures with the sensitivity to a variety of other anti-MM drugs. Conversely, MDS cell lines displayed enhanced basal activity of TP53-regulated pro-apoptotic genes, which might increase their apoptotic priming and consequently increase their susceptibility to drug-induced apoptosis. With the rapid evolution of protein-protein interaction inhibitors, future exploration of KEAP1-NRF2 or MDM2-TP53 interaction inhibitors could potentially enhance myeloma cell sensitivity. In the future, our team plans to expand the drug panel used in the screen and provide this enriched dataset as a public resource, thereby facilitating wider access and collaborative advancements in the field. Study supported by National Science Centre Grants PRELUDIUM 2018/31/N/NZ5/03214, ETIUDA 2020/36/T/NZ5/00610 and Polish Stem Cell Bank ExCELLent grant.

Lymphodepletion Enables Successful BCMA CAR-T Cell Engraftment and Tumour Control in the Syngeneic Vk*MYC Model of Aggressive Myeloma

DISCUSSION AND CONCLUSION Chimeric antigen receptor (CAR)-T cells directed against B-cell maturation antigen (BCMA), have yielded impressive results in clinical trials for multiply relapsed/refractory multiple myeloma (MM). However, progression-free survival is short, demonstrating a need for improvements. While numerous advances have been made to improve this treatment modality, these have not always translated to superior clinical outcomes. A major limitation is that novel therapeutics are often not tested in a relevant in vivo model. These novel strategies have been largely tested only in immunodeficient models of MM. To better recapitulate the host-tumour-effector cell interactions, particularly in the TME, we sought to develop a system to study CAR-T cell activity in the Vk*MYC model of MM. Here, we report on the first known successful model of CAR-T cell activity in an immunocompetent model of MM. We sought to identify a BCMA CAR construct that is capable of binding to both human and mouse BCMA. A novel nanobody recognizing BCMA was developed by immunizing llamas with human BCMA and the resulting single-domain antibodies (sdAbs) were subsequently isolated and cloned. These sdAbs were then screened for binding to mouse BCMA. A binder capable of recognizing both human and mouse BCMA was identified and cloned into our murine CAR construct, with CD28 and CD3z signaling domains, herein referred to as sdBCMA2.CD28.CD3z. Murine CAR-T cells were then generated by transducing activated healthy murine splenocytes with a retroviral vector carrying the sdBCMA2.CD28.CD3z construct. These CAR-T cells were then characterized in vitro using standard cytotoxicity, proliferation, and cytokine release assays. To assess activity in an immunocompetent model, female C57BL/6J mice were injected intravenously with Vk12598 Vk*MYC cells. CAR-T cells were later administered intravenously. Mice were bled weekly to quantify M-protein disease burden by serum protein electrophoresis and for phenotypic characterization of circulating cells. When co-cultured with BCMA+ human MM cell lines and with Vk*MYC cell lines, murine sdBCMA2.CD28.CD3z CAR-T cells effectively lysed target cells, proliferated, and released inflammatory cytokines. To assess the in vivo activity of these CAR-T cells, we injected intravenously female C57BL/6J mice with Vk12598 cells, and then administered sdBCMA2.CD28.CD3z CAR-T cells intravenously. While there was some evidence of survival improvement, the median overall survival (mOS) of 35 days compared to 28 days, for CAR-T vs. Mock T-cells, respectively, the difference was non-significant, and the survival benefit was not durable. To address this, we incorporated lymphodepletion by total body irradiation (TBI) followed by CAR-T cell administration the next day. By incorporating lymphodepletion prior to CAR-T cell administration we were able to significantly reduce M-protein surrogates of disease burden, as well as achieve durable remissions in the TBI + CAR-T arm with an mOS not yet reached after experimental day 91, compared to an mOS of 42 days in the TBI + Mock T-cell arm. The addition of TBI also significantly improved the engraftment of CAR-T cells, as assessed by flow cytometry. The TBI + CAR-T arm also had significantly increased IL-6, IL-1b, IL-2, and TNF-a in the serum, compared to the TBI + Mock arm, at 7 days post administration of CAR-T cells. The durable remission achieved using the combination of our novel sdBCMA2.CD28.CD3z CAR-T cells with lymphodepletion is a pivotal advancement in modelling MM CAR-T cell activity in this clinically relevant model system. Our approach represents the first time that CAR-T cells were successfully used to treat the Vk12598 model of MM that faithfully recapitulates relapsed MM. The incorporation of lymphodepletion significantly improves CAR-T cell engraftment, in line with clinical CAR-T cell therapy paradigms. The cytokine profile observed is consistent with the cytokine elevations typically observed after CAR-T cell administration in patients. As our construct is capable of binding to both human and mouse BCMA, we are therefore able to fully characterize our lead assets in a relevant model system. Our strategy will enable further research to dissect host-tumour-effector cell interactions, to better inform the design and characterization of novel CAR-T cell therapies in MM.

Characterization of Multiple Myeloma Subpopulations Associated with Treatment Resistance By Single-Cell RNA Sequencing

Multiple myeloma (MM) has extensive genomic and transcriptomic heterogeneity with identical variable-diversity-joining (VDJ) region recombination of immunoglobulin loci (called repertoire). Even though new treatment approaches have been developed, most cases of MM remain incurable. Recent evidence suggests that epigenetic cell plasticity and changes in cell state underlie the therapy resistance, but the mechanisms remain largely unknown. We attempted to understand the comprehensive architecture of the entire tumor cell populations using repertoire clonality as a fingerprint. First, we subdivided bone marrow samples of primary MM at different stages (n=8) into 11 fractions and performed bulk RNA sequencing (bulk RNA-seq). Analysis of the repertoire revealed that tumor cells were present at very low frequencies in some minor fractions other than the main fraction (Lineage marker - (Lin -)/CD19 -/CD38 +/CD138 +) as the disease stage progressed. Importantly, MM cells were consistently present in CD138 negative fraction (Lin -/CD19 -/CD38 +/CD138 -) regardless of disease stage. Flow cytometry (FCM) analysis confirmed that CD138negative MM cells were present at a rate of less than 10% across the disease stages. Compared with CD138 positive cells, they were slowly cycling and showed unique morphology with large cytoplasm (n=59). To achieve a selective and comprehensive evaluation of MM population, we performed single-cell RNA sequencing (scRNA-seq) coupled with VDJ targeted sequencing (scVDJ-seq) on Lin -/CD19 -/CD38 +(without CD138 gating) samples from 11 MM patients, including those at diagnosis and in remission with minimal residual disease (MRD). This method allowed us to discriminate MM cells (20,084 cells) clearly from others by clonal repertoire sequence even in the post-treatment samples with extremely small populations of tumor cells. Because mRNA levels of CD138 did not correspond to the differential intensity in FCM, we defined gene signatures of CD138 positive and negative cells respectively by bulk RNA-seq (n=6). As a result of integration of these gene signatures using Gene Set Enrichment Analysis (GSEA), the heterogeneity on the CD138 axis was also unraveled in scRNA-seq data. Interestingly, gene ontology (GO) analysis of bulk RNA-seq data revealed that several terms related to H3K4 methylation were positively enriched in CD138 negative fraction, which included KMT2A, KMT2B, KMT2C and KMT2D. Western blotting confirmed significant reductions in H3K4me1 and H3K4me3 levels in CD138 negative cells. Furthermore, primary MM cell culture indicated the plasticity between CD138 positive and negative cells, which was also suggested by trajectory/velocity analysis of scRNA-seq data. Collectively, it was suggested that epigenetic changes, including those in histone modifications, contribute to the heterogeneity along the CD138 axis. We next compared the size of each cluster before and after treatment in scRNA-seq data to identify the MM populations associated with treatment resistance. Remarkably, we identified unique clusters which tended to remain at high proportions against treatment in the CD138 negative population. They were characterized by low expression of MHC class-I genes and TNFRSF17/BCMA, as well as 198 significantly upregulated genes including MCL1, MALAT1 and NEAT1, which have been previously implicated in treatment resistance of MM. To systematically interrogate their contribution in MM cell survival, we performed CRISPR/Cas9 knockout screening of selected 100 genes, which were differentially up-regulated in the resistant clusters and associated with poor prognosis in a MMRF CoMMpass Study database. GO analysis of indispensable genes reproducibly identified among three MM cell lines revealed enrichment of several pathways related to mRNA splicing. These results suggested that the RNA splicing pathway is a promising therapeutic target to eliminate resistant populations. Indeed, we confirmed that inhibition of a splicing factor by CRISPR/Cas9-mediated knockout and pharmacological protein degradation suppresses MM cell proliferation effectively. Taken together, we achieved a complete view of MM diversity by using repertoire clonality and confirmed heterogeneity along the CD138 axis, which may be characterized by epigenetic plasticity. Our results highlight a novel subpopulation as a candidate therapeutic target for cure.

Functional Characterization of GPRC5D alteration and Its Impact on Talquetamab Resistance in Relapsed/ Refractory Multiple Myeloma

Background T-cell engaging (TCE) antibodies show unprecedented efficacy in heavily pretreated penta-refractory multiple myeloma (MM). Responses however are often not durable as resistance mechanisms can arise despite the initial absence of resistance-conferring mutations. Biallelic loss of GPRC5D on chr.12p is a rare, yet well-established mechanism of acquired resistance to the first-in-class anti-GPRC5D directed TCE talquetamab (Lee et al., 2022). At the same time, monoallelic GPRC5D frameshift and missense mutations are much more common and occur in up to 15% of MM patients (Truger et al., 2021). In this study, we investigated the functional relevance of monoallelic GPRC5D alterations and their impact on talquetamab resistance. Methods GPRC5D alterations were modeled in vitro by using CRISPR-Cas9 editing in the OPM-2 MM cell line. The bioluminescence-based cytotoxic assay was performed using PBMCs as effector cells to measure the resistance to talquetamab. Quantitative PCR (qPCR), immunohistochemistry, bulk whole genome sequencing (WGS), and whole genome bisulfite sequencing (WGBS) were performed for molecular, genomic, and epigenetic characterization in a patient with acquired talquetamab resistance. Results Using CRISPR-Cas9, we generated cell line models with mono- vs. biallelic deletions in the GPRC5D gene. Clones with biallelic alterations (OPM-2 Del/Del) displayed a 96.4%, reduced expression of GPRC5D ( p < .0001), leading to in vitro resistance to talquetamab. GPRC5D expression levels in monoallelic models (OPM-2 Wt/Del) decreased by 57.4% ( p = .0004) compared to the wild type. Sensitivity to talquetamab in these clones was equally impaired, however, less pronounced than in the biallelic knock-outs. To assess the impact of monoallelic GPRC5D deletions in the clinic, we next investigated the case of a heavily pretreated 58-year-old patient with lambda light-chain MM treated at our institution with talquetamab in his 7th line of therapy. qPCR was used to measure GPRC5D expression in CD138+ MM cells from the bone marrow of this patient before and at the time of relapse to talquetamab after 15 months of treatment (best response: CR). At the time of relapse, we observed a 94.0% ( p < .0001) decrease in GPRC5D expression upon qPCR assessment as compared to the pre-treatment time point which was confirmed by immunohistochemistry. WGS revealed a novel monoallelic loss of chr.12p encompassing GPRC5D in talquetamab-resistant CD138+ MM cells. Since monoallelic alterations seem unlikely as the sole drivers of resistance, we hypothesized that epigenetic modifications may have contributed to talquetamab resistance in this patient. To this end, WGBS was performed and uncovered a significant shift towards hypermethylation of two regulatory regions of GPRC5D, most likely resulting in the silencing of the second GPRC5D allele. Time-course experiments are currently ongoing to explore the methylation landscape of our CRISPR-Cas9-generated clones with monoallelic GPRC5D deletions. Data for these validation experiments will be presented at the meeting. Conclusions Our study provides the first evidence of the functional impact of monoallelic GPRC5D alterations resulting in relative resistance to talquetamab in vitro. We further report on epigenetic regulation as a potential second-hit mechanism conferring clinical resistance to anti-GPRC5D-directed TCE therapy. This data suggests that patients with monoallelic GPRC5D deletions may be at particular risk for developing resistance to talquetamab.