Response Of Multiple Myeloma Cells Research Articles

TRIM44, a Novel Prognostic Marker, Supports the Survival of Proteasome-Resistant Multiple Myeloma Cells.

TRIM44, a tripartite motif (TRIM) family member, is pivotal in linking the ubiquitin-proteasome system (UPS) to autophagy in multiple myeloma (MM). However, its prognostic impact and therapeutic potential remain underexplored. Here, we report that TRIM44 overexpression is associated with poor prognosis in a Multiple Myeloma Research Foundation (MMRF) cohort of 858 patients, persisting across primary and recurrent MM cases. TRIM44 expression notably increases in advanced MM stages, indicating its potential role in disease progression. Single-cell RNA sequencing across MM stages showed significant TRIM44 upregulation in smoldering MM (SMM) and MM compared to normal bone marrow, especially in patients with t(4;14) cytogenetic abnormalities. This analysis further identified high TRIM44 expression as predictive of lower responsiveness to proteasome inhibitor (PI) treatments, underscoring its critical function in the unfolded protein response (UPR) in TRIM44-high MM cells. Our findings also demonstrate that TRIM44 facilitates SQSTM1 oligomerization under oxidative stress, essential for its phosphorylation and subsequent autophagic degradation. This process supports the survival of PI-resistant MM cells by activating the NRF2 pathway, which is crucial for oxidative stress response and, potentially, other chemotherapy-induced stressors. Additionally, TRIM44 counters the TRIM21-mediated suppression of the antioxidant response, enhancing MM cell survival under oxidative stress. Collectively, our discoveries highlight TRIM44's significant role in MM progression and resistance to therapy, suggesting its potential value as a therapeutic target.

Liquid Overlay and Collagen-Based Three-Dimensional Models for In Vitro Investigation of Multiple Myeloma.

Multiple myeloma (MM) clones reside in the bone marrow (BM), which plays a role in its survival and development. The interactions between MM and their neighboring mesenchymal stromal cells (MSCs) have been shown to promote MM growth and drug resistance. However, those interactions are often missing or misrepresented in traditional two-dimensional (2D) culture models. Application of novel three-dimensional (3D) models might recapitulate the BM niche more precisely, which will offer new insights into MM progression and survival. Here, we aimed to establish two 3D models, based on MSC spheroids and collagen droplets incorporating both MM cells and MSCs with the goal of replicating the native myeloma context of the BM niche. This approach revealed that although MSCs can spontaneously assemble spheroids with altered metabolic traits, MSC spheroid culture does not support the integration of MM cells. On the contrary, collagen-droplet culture supported the growth of both cell types. In collagen, MSC proliferation was reduced, with the correlating decrease in ATP production and Ki-67 expression, which might resemble invivo conditions, rather than 2D abundance of nutrients and space. MSCs and MMs were distributed homogenously throughout the collagen droplet, with an apparent CXCL12 expression in MSCs. In addition, the response of MM cells to bortezomib was substantially reduced in collagen, indicating the importance of 3D culture in the investigation of myeloma cell behavior, as drug resistance is one of the most pertinent issues in cancer therapy.

Targeting Mutant KRAS with Novel Mutant-Specific Pharmacological Inhibitors: Activity Against Myeloma and Other Lymphoid Malignant Cells and Molecular Mechanisms of Resistance

Oncogenic RAS mutations are frequent genomic drivers in hematologic neoplasias such as multiple myeloma (MM) but are traditionally considered non-“druggable”. Novel specific inhibitors of G12C and G12D KRAS mutants are clinically active in solid tumors, but their impact on KRAS-mutant lymphoid malignancies has not been explored. Here, we examined the activity of these inhibitors against G12C/D KRAS-mutant cells from MM, B-ALL and T-ALL; the functional sequelae of pharmacological KRAS inhibition; and the potential mechanisms of resistance to these agents. We assessed the activity of novel G12C- (MRTX1257) and G12D- (MRTX1133) specific pharmacological KRAS inhibitors against 2 MM lines with G12C (KHM1B, XG7) and 2 with G12D (KARPAS620, KP6) KRAS mutations; as well as the G12D mutant KOPN-8 (B-ALL) and CCRF-CEM (T-ALL) lines. KRAS inhibitors were tested at concentration ranges achievable in patients with solid tumors. Potent and mutant-specific activity was achieved with both of these KRAS inhibitors in their respective lines, with IC50 in the range of 5-150 nM after 3-7d treatment. This level of in vitro activity was similar to KRAS-mutant lines (e.g., in our in vitro studies and also in the literature) from solid tumors that are clinically responsive to these agents. Furthermore, the KRAS inhibitors effectively abrogated downstream MAPK and AKT pathway activation (e.g. phosphorylation of ERK or AKT by immunoblotting). MRTX1133 led to potent induction of cell death of KARPAS620, KP6 and KOPN8 cells (flow cytometry for Annexin V/PI staining) while MRTX1257 induced cell death in KHM1B and had a predominantly cytostatic effect on XG7 cells. Combinations of MRTX1257 with clinically established anti-MM drugs (incl. melphalan, bortezomib, pomalidomide, trametinib) caused in some cases supra-additive effects, but - more importantly - showed no antagonism with any combination. RNA sequencing revealed that KRAS inhibitor treatment downregulated RAS effector genes (e.g., ETV4, ETV5), and negative regulators of RAS signaling (e.g., DUSP6, SPRY4 reflecting a RAS-driven negative feedback loop controlling expression of these genes), and molecules involved in cell cycle regulation (e.g., D-type cyclin), anti-apoptosis, and DNA replication/repair. Conversely, KRAS inhibition upregulated genes involved in cell cycle arrest and stress responses. Importantly, we performed genome-scale CRISPR activation studies in 4 MM lines with G12C/G12D mutant KRAS to identify genes whose gain-of-function (GOF) enhances or suppresses MM cell response to the respective KRAS inhibitors. Top “hits” from these screens were validated with individual sgRNAs. CRISPR activation of mutant KRAS itself promotes MM cell escape from treatment, likely due to stoichiometric reasons and decreased target saturation at a given drug concentration. Notably, CRISPR activation of positive regulators of RAS-MAPK signaling (e.g., SHOC2), and upstream surface receptors (e.g., EGFR) were among top genes mediating KRAS inhibitor resistance. Beyond KRAS itself, these top “hit” genes are typically not recurrently mutated, amplified, differentially expressed (e.g., compared with normal plasma cells) in MM and are not associated with “high-risk” clinical behavior. Some (e.g., EGFR) of the genes identified from these CRISPR studies are not typically expressed in MM cells at diagnosis, thus these results outline how ectopic expression (e.g., EGFR) or overexpression of these “hit” genes could represent candidate biomarkers for decreased response or early relapse in future clinical studies of KRAS inhibitors in KRAS-mutant MM. Ongoing studies are addressing how these genomic perturbations may affect the in vivo response of KRAS-mutant cells to KRAS inhibitors alone or in combinations with established therapies. Our study documents that novel KRAS G12C and G12D inhibitors exhibit potent and mutant-specific activity against MM and other lymphoid malignant cells at dose levels that are clinically achievable in solid tumor patients. This study also provides functional evidence about genes and molecular pathways that regulate MM cell sensitivity vs. resistance to KRAS inhibitors. These results create a framework for ongoing and future efforts to translate the use of KRAS inhibitors into clinical studies for MM and other lymphoid malignancies.

Characterization of a New Metabolic Score Correlated with CD38 Cell-Surface Expression and Response to Daratumumab Treatment in Multiple Myeloma

Multiple myeloma (MM) is the second most common hematological malignancy characterized by the uncontrolled accumulation of tumor plasma cells within the bone marrow. During the last 10 years, the development of new therapeutics, including the immune based therapies, significantly improved the life quality and survival of patients. However, the high molecular and clinical heterogeneity of this disease often leads to the development of resistance and relapses. Thus, MM unfortunately remains without definitive treatment in the majority of cases. Although few studies described the mechanisms of resistance to therapies developed by myeloma cells, a better understanding and new strategies to overcome the drug resistance mechanisms linked to Daratumumab remains of major interest for patients care. Among the factors that could be involved in myeloma cell resistance to anti-CD38 immunotherapies, we focused on the mitochondrial metabolism in MM, already described as a significant factor influencing response to treatments in several cancers. First, we defined a metabolic score using RNAseq data from different MM models, calculated with the gene expression of 112 genes involved either in the electron transport chain (Oxphos) or glycolysis. Each gene expression value was standardized and the metabolic score was built using the following equation: score= Σ(Glycolysis gene standardized expression) - Σ(Oxphos gene standardized expression). In our unique panel of 40 Human myeloma cell lines (HMCLs), high metabolic score significantly correlated with the functional metabolic profiles assessed by a Seahorse XFe96 analyzer included high mitochondrial and glycolysis activities on 25 HMCLs (r=0.6, p<0.05). Moreover, we tested the potential prognostic value of this metabolic score using cohorts of newly diagnosed MM patients treated by high dose of chemotherapy and autologous hematopoietic stem cell transplantation. In the MMRF CoMMpass cohort (674 patients), 32% of the patients with high metabolic score, defined using maxstat algorithm, were associated with a poor outcome (score>0.07; p<0.0005). These results were validated in an independent cohort of MM's patient (85 patients, Montpellier cohort), with a significant poor outcome associated with a high score (p<0.0005). Furthermore, we calculated the metabolic score in a cohort of MM's patients at diagnosis, who were then treated with anti-CD38 MoAb Daratumumab after relapse (Montpellier cohort N=42). Interestingly, we observed a significant difference between the mean score of patients with progressive or stable disease (n=13, mean score of 0.3380) and the mean score of patients with very good partial response or complete response (n=22, mean score of -0.0703), p<0.005). To validate these observations, the potential prognostic value was tested in that cohort: patients with high metabolic score were associated with a poor outcome (score>0.08; p<0.0005) suggesting a potential role of metabolism in the response to Dara treatment. We next questioned the possible link between the CD38 expression level at the surface of MM cells and their metabolic profile. For that purpose, we determined the CD38 surface antigen binding capacity (SABC) on 23 HMCLs representatives of MM heterogeneity. We observed a significant anti-correlation between the CD38 expression and the basal oxygen consumption rates (OCR) (r=-0.5, p<0.05) quantified with seahorse. Currently, correlations between CD38 expression measured on primary samples from patients with MM (MFI values) and their respective metabolic score are ongoing. However, these data suggest a link between mitochondrial activity, CD38 cell-surface expression and response to anti-CD38 MoAb. Altogether, our data demonstrated metabolism deregulation is associated with a prognostic value in newly diagnosed MM patients. Furthermore, we also reported a link between MM cell metabolism, CD38 expression and response to anti-CD38 MoAb treatment.

The synthetic oleanane triterpenoid CDDO-2P-Im binds GRP78/BiP to induce unfolded protein response-mediated apoptosis in myeloma.

Synthetic oleanane triterpenoids (SOTs) are small molecules with broad anticancer properties. A recently developed SOT, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-4(-pyridin-2-yl)-1H-imidazole (CDDO-2P-Im or '2P-Im'), exhibits enhanced activity and improved pharmacokinetics over CDDO-Im, a previous generation SOT. However, the mechanisms leading to these properties are not defined. Here, we show the synergy of 2P-Im and the proteasome inhibitor ixazomib in human multiple myeloma (MM) cells and 2P-Im activity in a murine model of plasmacytoma. RNA sequencing and quantitative reverse transcription PCR revealed the upregulation of the unfolded protein response (UPR) in MM cells upon 2P-lm treatment, implicating the activation of the UPR as a key step in 2P-Im-induced apoptosis. Supporting this hypothesis, the deletion of genes encoding either protein kinase R-like endoplasmic reticulum kinase (PERK) or DNA damage-inducible transcript 3 protein (DDIT3; also known as CHOP) impaired the MM response to 2P-Im, as did treatment with ISRIB, integrated stress response inhibitor, which inhibits UPR signaling downstream of PERK. Finally, both drug affinity responsive target stability and thermal shift assays demonstrated direct binding of 2P-Im to endoplasmic reticulum chaperone BiP (GRP78/BiP), a stress-inducible key signaling molecule of the UPR. These data reveal GRP78/BiP as a novel target of SOTs, and specifically of 2P-Im, and suggest the potential broader utility of this class of small molecules as modulators of the UPR.

Sec61 blockade therapy overrides resistance to proteasome inhibitors and immunomodulatory drugs in multiple myeloma.

Multiple Myeloma (MM) is an incurable neoplasm of mature B cells and the second most prevalent hematological malignancy worldwide. While combinations of proteasome inhibitors like bortezomib (Bz) and immunomodulators (IMiDs) like lenalinomide (Len) are generally effective in newly diagnosed patients, some do not respond to this first-line therapy, and all others will eventually become drug resistant. We previously reported that inhibiting the Sec61 translocon with mycolactone synergizes with Bz to induce terminal unfolded protein response in MM cells, irrespective of their resistance to proteasome inhibition. Here, we examined how Sec61 blockade interferes with IMiD action and whether it overrides resistance to Len. With this aim, we knocked out the IMiD target CRBN in the MM1S cell line and a Bz-resistant subclone to generate Len- and Len/Bz-resistant daughters, respectively. Both the Len- and Len/Bz-resistant clones were susceptible to mycolactone toxicity, especially the doubly resistant one. Notably, the synergy between mycolactone and Bz was maintained in these two clones, and mycolactone also synergized with Len in the two Len-susceptible ones. Further, mycolactone enhanced the therapeutic efficacy of the Bz/Len combination in both mice engrafted with parental or double drug resistant MM1S. Together, these data consolidate the interest of Sec61 blockers as new anti-MM agents and reveal their potential for treatment of refractory or relapsed MM.

The Pre-Existing T Cell Landscape Is Associated with Response to High Dose Melphalan and Autologous Stem Cell Transplant in Multiple Myeloma

Introduction: High dose melphalan and autologous stem cell transplant (ASCT) remain a cornerstone of therapy for transplant-eligible newly diagnosed multiple myeloma (MM) patients. The bone marrow microenvironment may affect the response of MM cells to high dose melphalan and ASCT. The changes in the immune landscape and associations with response to ASCT remain largely unknown. Here we investigate the role of the microenvironment in response to high dose melphalan using scRNA and TCR sequencing in paired pre- and post-ASCT bone marrow samples from the Indiana Myeloma Registry. Methods: Paired bone marrow (BM) aspirates were collected from MM patients (n=40) after induction therapy but before ASCT and again after ASCT but prior to maintenance therapy (range 58-142 days post-ASCT). All patients had measurable disease after induction therapy. After ASCT, patients were evaluated based on their response to ASCT and divided into those who achieved a stringent complete response (n=17) and were negative for measurable residual disease (MRD) at 10-6 by flow cytometry (responders), or those who achieved a partial response or less (n=23) (non-responders). BM mononuclear cells were isolated by Ficoll and CD138 magnetic bead selection into CD138+ve and CD138-ve subsets. Paired pre- and post-ASCT CD138-ve samples underwent scRNA and TCR sequencing (10X Genomics), resulting in 488,479 high quality cells. Samples were independently normalized, cells integrated, and clusters annotated for cell type. Significant gene expression differences between clusters were calculated. Results: Significant differences in the proportions of different immune cells in pre- vs. post-ASCT samples were identified with increases in B cells (2.6% vs 19.6%; P<.001) and B-cell progenitors (1.5% vs 8.9%; P<0.001) and decreases in CD4 T cells (24.8% vs 10.2%; P<0.001) and monocytes/myeloid cells (27.8% vs 21.0%; P=0.005) in post-ASCT samples. The overall proportions of CD8 T cells remained stable (27.1% vs 25.1%; P=0.2), but there were significant differences in the proportion of cells within the CD8 T cell clusters, including an increase in effector CD8 T cells (8.6% vs 10.7%; P=0.01) that expressed IFNG, KLRG1, TBX21, and PRF1 as well as a decrease in second cluster of CD8 T cells (2.3% vs 0.6%; P<0.01) expressing IL7R and TCF7. When comparing responder and non-responder pre-ASCT samples, we found a higher percentage of a CD4 T cell subset (10.0% vs 7.6%; P=0.04) in responders, which expressed SELL, CCR7, and ICOS indicating they are likely non-effector memory cells or naïve cells. There was also a higher proportion of myeloid dendritic cells (1.47% vs 1.08%; P=0.05) and granulocyte progenitors (0.98% vs 0.62%; P=0.04) in responders in post-ASCT samples. TCR sequencing revealed expanded CD8 T cells but not CD4 T cells, suggesting the CD4 T cell subset of interest may have a modulating effect of MM cells response to high dose melphalan rather than a specific anti-myeloma immune response. Conclusions: We comprehensively describe the immune microenvironment in the BM of MM patients around ASCT and identify a pre-existing CD4 T cell subset that is associated with depth of response to ASCT. Further studies are required to better define the role of this population in modulating the response to melphalan. Prospective follow up of patients is ongoing to assess which clusters are associated with progression-free and overall survival.

Looking into Endoplasmic Reticulum Stress: The Key to Drug-Resistance of Multiple Myeloma?

Multiple myeloma (MM) is the second most common hematologic malignancy, resulting from the clonal proliferation of malignant plasma cells within the bone marrow. Despite significant advances that have been made with novel drugs over the past two decades, MM patients often develop therapy resistance, especially to bortezomib, the first-in-class proteasome inhibitor that was approved for treatment of MM. As highly secretory monoclonal protein-producing cells, MM cells are characterized by uploaded endoplasmic reticulum stress (ERS), and rely heavily on the ERS response for survival. Great efforts have been made to illustrate how MM cells adapt to therapeutic stresses through modulating the ERS response. In this review, we summarize current knowledge on the mechanisms by which ERS response pathways influence MM cell fate and response to treatment. Moreover, based on promising results obtained in preclinical studies, we discuss the prospect of applying ERS modulators to overcome drug resistance in MM.

Comprehensive characterization of the epigenetic landscape in Multiple Myeloma.

Background: Human multiple myeloma (MM) cell lines (HMCLs) have been widely used to understand the molecular processes that drive MM biology. Epigenetic modifications are involved in MM development, progression, and drug resistance. A comprehensive characterization of the epigenetic landscape of MM would advance our understanding of MM pathophysiology and may attempt to identify new therapeutic targets.Methods: We performed chromatin immunoprecipitation sequencing to analyze histone mark changes (H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3 and H3K36me3) on 16 HMCLs.Results: Differential analysis of histone modification profiles highlighted links between histone modifications and cytogenetic abnormalities or recurrent mutations. Using histone modifications associated to enhancer regions, we identified super-enhancers (SE) associated with genes involved in MM biology. We also identified promoters of genes enriched in H3K9me3 and H3K27me3 repressive marks associated to potential tumor suppressor functions. The prognostic value of genes associated with repressive domains and SE was used to build two distinct scores identifying high-risk MM patients in two independent cohorts (CoMMpass cohort; n = 674 and Montpellier cohort; n = 69). Finally, we explored H3K4me3 marks comparing drug-resistant and -sensitive HMCLs to identify regions involved in drug resistance. From these data, we developed epigenetic biomarkers based on the H3K4me3 modification predicting MM cell response to lenalidomide and histone deacetylase inhibitors (HDACi).Conclusions: The epigenetic landscape of MM cells represents a unique resource for future biological studies. Furthermore, risk-scores based on SE and repressive regions together with epigenetic biomarkers of drug response could represent new tools for precision medicine in MM.

Functional Genomic Landscape of Natural Killer Cell Evasion in Multiple Myeloma

Natural killer (NK) cell-based therapies are considered promising future approaches for multiple myeloma (MM) treatment, but immune evasion mechanisms are poorly understood. To determine the mechanisms regulating MM cell response to NK cells, we performed genome-wide (GW) and targeted CRISPR screens in MM cell lines. To further investigate the transcriptional impact of genes identified as regulators of sensitivity to NK cells, we performed additional pooled CRISPR screens with a single-cell (sc) transcriptome readout using the CROP-seq platform and integrated these findings with data from phenotypic assessment of NK cell response of pooled “DNA-barcoded” cell line including 15 MM cell lines (PRISM platform) and molecular profiling of MM patient samples.Our loss-of-function (LOF) and gain-of-function (GOF) GW-CRISPR screens were performed in the MM1.S, LP1, KMS11 MM lines treated with ex vivo expanded NK cells vs untreated control. The top LOF hits were validated using a focused library of ~600 genes. As expected, LOF of class I HLA /antigen presentation machinery genes, transcriptional regulators of HLA and IFNg pathway genes sensitized MM cells to NK cells, confirming that these pathways represent prominent suppressors of NK cell killing. Moreover, LOF of death receptors or downstream effectors was associated with NK resistance; while LOF of the negative regulators of death receptor signaling (e.g. CFLAR, and XIAP) sensitized to NK cell killing.To mechanistically dissect the transcriptional impact of genes identified as regulators of sensitivity to NK cells, we performed scRNAseq using the CROP-seq platform. Pools of MM1.S and LP1 expressing single-guide (sg)RNAs targeting 31 select hits were co-cultured with NK cells for 24 h or left untreated, followed by scRNA-seq and sgRNA detection, differential gene-expression analysis and patient data correlation.The single cell transcriptional profiling of each CRISPR-based LOF for genes of interest documented that disruption of TRAF2, NFKBIA or NFKBIB enhanced NF-kB signaling and was associated with increased expression of the death receptor FAS, a key mediator of NK cell sensitivity (Figure), and changes in expression of BIRC3, CD70, and CXCL10(the latter may further increase immune reactivity through recruitment of T and NK cells). Interestingly, in our PRISM data, high transcriptional NF-kB signatures and/or presence of TRAF3 mutations (an activator of NF-kB signaling) correlated with decreased NK cell response. Among the individual genes most highly correlated with NK cell resistance was CFLAR, a known NF-kB target gene, and negative regulator of death receptor-mediated apoptosis. In contrast, PTEN and NLRC5 mutations were associated with enhanced NK cell sensitivity in several MM lines tested, consistent with their depletion in the GW LOF CRISPR screens and the HLA I regulatory function of NLRC5.We correlated our findings with patient-derived data from the CoMMpass study. Mutations of NF-kB negative regulators TRAF2 and NFKBIA were associated with increased expression of NF-kB target genes, consistent with our CROP-seq data. NFKBIA mutations were also linked to reduced HLA-E expression in both MM patient and CROP-seq data, suggesting a potential explanation for the NK-sensitizing effect of NFKBIA disruption. NLRC5 mutations were associated with lower HLA-E expression consistent with CROP-seq data, suggesting that, although rare, NLRC5mutations may represent a more NK-sensitive MM subset. TRAF3 alterations occurred both in a distinct TRAF3-altered cluster and in a subset of CoMMpass patients with WHSC1 translocations, consistent with the observations in our PRISM data set. CFLAR expression was enriched in patients with TRAF3 alterations belonging to both of these groups. The aggregate of molecular data from MM patient samples and PRISM or CRISPR-based functional studies preclinically raise the possibility that TRAF3 and WHSC1/t(4;14) alterations may contribute, at least in part through CFLAR, to decreased NK cell response in MM cells.Our data illuminate the complex mechanisms of response to NK cells in MM, highlighting the different effects of distinct molecularly defined subgroups of MM tumor cells with increased susceptibility to NK cell treatment, underlining the potential of such studies as a blueprint for identification of biomarkers individualized use of NK cell-based therapies in MM. [Display omitted] DisclosuresMustjoki: Novartis: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Janpix: Research Funding. Mitsiades: H3 Biomedicine: Research Funding; FIMECS: Consultancy, Honoraria; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Nurix: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; BMS: Research Funding; Fate Therapeutics: Consultancy, Honoraria; Novartis: Research Funding; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Arch Oncology: Research Funding; Abbvie: Research Funding; Ionis Pharmaceuticals: Consultancy, Honoraria.

OAB-019: CRISPR screens with single-cell transcriptome readout reveal potential mechanisms of response to natural killer cell treatment in multiple myeloma

<h3>Background</h3> Natural killer (NK) cell-based therapy is a promising approach to improve treatment responses in multiple myeloma (MM), which remains incurable. However, genetic determinants and mechanisms of sensitivity or resistance to NK cells in MM are incompletely understood. Here, we investigated the transcriptional impact of genes identified as regulators of sensitivity to NK cells in genome-wide CRISPR screens and integrated the findings with patient-derived data. <h3>Methods</h3> To systematically dissect the mechanisms by which select genes identified in our previous genome-wide studies influence MM cell response to NK cells, we performed pooled CRISPR screens with a single-cell (sc) transcriptome readout using the CROP-seq platform. Based on genome-wide screen ranking and biological relevance, 31 genes were selected. Pools of MM1.S and LP1 expressing sgRNAs targeting select hits were co-cultured with NK cells for 24 h or left untreated, followed by scRNA-seq and sgRNA detection and assignment to cells, followed by differential gene-expression analysis and patient data correlation. <h3>Results</h3> The analysis of the transcriptomic responses in MM cells upon NK cell exposure revealed that engagement by NK cells resulted in activation of IFNy-JAK-STAT signaling and upregulation of HLA class I genes, including the inhibitory NKG2A ligand HLA-E, as well as HLA class II. We then evaluated the impact of each sgRNA perturbation on gene expression in each condition, compared to the untreated control. Silencing of IFNy signaling mediators (JAK1, JAK2, STAT1, IFNGR2) prevented the induction of the IFNy response upon NK cell exposure, providing a mechanism for the observed sensitization to NK cells via reduced expression of HLA I genes. Disruption of TRAF2, NFKBIA, or NFKBIB, which also sensitized MM1.S to NK cells, induced NF-kB signaling. The induced NF-kB signature included increased expression of the death receptor FAS, pivotal for susceptibility to NK cells in the MM1.S CRISPR screen. We then correlated the findings with patient data from the CoMMpass dataset. Mutations in the NF-kB negative regulators TRAF2 and NFKBIA were associated with increased expression of NF-kB target genes also identified experimentally by CROP-seq. Also, NFKBIA mutations were linked to reduced HLA-E expression in both MM patients and CROP-seq, suggesting a potential explanation for the NK-sensitizing effect of NFKBIA disruption. Interestingly, patients with NLRC5 mutations had lower HLA-E expression consistent with CROP-seq data, indicating that although rare, NLRC5 mutations may predispose MM cells to higher NK-sensitivity. <h3>Conclusions</h3> Our data shed light on mechanisms of response to NK cells in MM and identify potential therapeutic targets for combination treatment. Furthermore, patient data correlation highlights subgroups that might have an increased susceptibility to NK cell treatment, highlighting the potential of such studies in the identification of predictive biomarkers.

Eosinophils Upregulate PD-L1 and PD-L2 Expression to Enhance the Immunosuppressive Microenvironment in Multiple Myeloma

Multiple myeloma is a clonal proliferation of malignant plasma cells and in the vast majority of patients remains incurable. The biology of the malignant plasma cells (PCs) in multiple myeloma (MM) is highly influenced by the bone marrow (BM) microenvironment in which they reside. Specifically, BM stromal cells (SCs) have been shown to interact with MM cells to promote their growth, survival and resistance to conventional chemotherapy. In contrast, it remains unclear how the BM microenvironment modulates the response of MM cells to novel agents, such as proteasome inhibitors or responses to innate immune cells. Moreover, it remains unclear which specific cell types within the BM modulate the therapeutic response of MM cells.MM cell lines and patient tumor cells co-cultured with BM and BM-derived eosinophils demonstrated increased growth, proliferation and survival in the presence of the proteasome inhibitors bortezomib and carfilzomib. BM eosinophils have been reported to support the survival of plasma cells by secreting APRIL (a pr oliferation- i nducing l igand), among other growth factors, and contribute to the long-term maintenance of malignant plasma cells. Results indicated that addition of APRIL to MM cultures increased cell proliferation.Targeting immune checkpoints such as programmed cell death protein 1 (PD-1), programmed cell death 1 ligand 1 (PD-L1) and PD-L2 has achieved noteworthy benefit in multiple cancers by blocking immuno-inhibitory signals and enabling patients to produce an effective antitumor response. Programmed death-ligand 1 (PD-L1) and PD-L2 are ligands for the receptor PD-1 within a negative costimulatory network that leads to a T cell exhaustion phenotype, preventing appropriate cellular response to antigens. Treatment with patient BM, BM-derived eosinophils or APRIL led to a substantial upregulation of PD-L1 and PD-L2 on the surface of MM cells (Fig. 1A, B). Furthermore, pre-treatment of MM cells with eosinophils or APRIL suppressed anti-tumor immunity by cytotoxic T lymphocytes (CTLs). shRNA-mediated knockdown of PD-L1 and PD-L2 on MM cells enhanced the cytotoxic effect of CTLs (Fig. 1C). Treatment with the FDA-approved proteasome inhibitors bortezomib, carfilzomib and ixazomib also dramatically and rapidly upregulated PD-L1 and PD-l2 levels on MM cells. Treatment of MM cells with proteasome inhibitors also compromised the anti-myeloma effect of CD8+ T cells. Chemical compound screening was then performed and revealed that small molecule kinase inhibitors that suppressed the basal and drug-induced expression of PD-L1 and PD-L2 on MM cells. Novel small molecule kinase inhibitors that suppressed PD-L1 and PD-L2 on MM cells also triggered CTL activation with antimyeloma benefit.Conclusions: BM-derived eosinophils enhanced immuno-suppressive microenvironmental effects and promoted resistance of MM cells to all proteasome inhibitors tested. APRIL, secreted by eosinophils, promoted MM growth and upregulated the checkpoint proteins PD-L1 and PD-L2 on MM cells. Thus, standard-of-care antimyeloma agents such as proteasome inhibitors as well as the BM-secreted cytokine APRIL suppressed antimyeloma immunity. Antibody-mediated blockade of the PD-L1/PD-L2~PD-1 axis restores antitumor immunity in cancer patients with therapeutic benefit, but exhibits numerous adverse side effects. The development of novel small molecules that suppress checkpoint proteins on MM cells may overcome the drawbacks associated with monoclonal-based checkpoint inhibitors to provide greater specificity, increased tumor response rates and fine-tune therapy while managing adverse events. Small molecules also provide increased oral bioavailability and higher stability at ambient temperature facilitating purification during clinical production. Kinase inhibitors that suppress PD-L1 and PD-L2 to promote antimyeloma immune effects may also limit tumor progression in immunocompetent murine models of myeloma. [Display omitted] DisclosuresMalek:Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau.

MiR-218 contributes to drug resistance in multiple myeloma via targeting LRRC28.

Multiple myeloma (MM) is a malignant neoplasm featured by obvious drug resistance and poor prognosis. MicroRNAs (miRNAs) are a class of small noncoding RNAs with crucial roles in many biological processes including cancer initiation and progression. The current study aims to investigate the pathogenic role and molecular mechanism of miRNAs in MM drug resistance. In the present study, The expression profile of miRNAs in MM samples was analyzed by microarray and real-time polymerase chain reaction. Protein expressions were detected by Western blot analysis. Cell apoptosis was detected by the Annexin V staining assay. The interaction between miRNA and the targeting mRNA was assessed using Dual luciferase reporter assay. Herein, we show that expression profile of miRNAs is deregulated in MM. miR-218, one of the most aberrational miRNAs in MM, is significantly decreased in MM cells compared to peripheral blood mononuclear cell (PBMC). Genetic manipulation reveals miR-218 control the response of MM cells to anticancer drug bortezomib (BTZ). Overexpression of miR-218 causes a significant aberrant genes expression including leucine rich repeat containing 28 (LRRC28). Mechanistic study shows that miR-218 control the drug response through mediating the expression of LRRC28 in MM cells. Overexpression of LRRC28 significantly reserves miR-218-mediated cell response to BTZ. Taken together, miR-218 is decreased in MM that contributes to BTZ resistance via targeting LRRC28, which might be used as a novel therapeutic target for multiple myeloma.

Mechanisms Regulating Myeloma Cell Responsiveness to Antisense Oligonucleotides Targeting IRF4

Functional genomics studies from our group and others, including CRISPR screens, have documented that interferon regulatory factor 4 (IRF4) is a critical transcription factor (TF) for multiple myeloma (MM) cells in preclinical in vitro and in vivo models; and one of the top most pronounced and recurrent dependencies for MM cells vs. other neoplasias. IRF4 lacks known ligand-binding pocket(s) amenable to selective inhibition by small-molecule pharmacological agents. IRF4 is thus considered, as many TFs, as "undruggable". Recent progress in antisense oligonucleotides (ASOs) and their in vivo properties motivated us to systematically evaluate the response of MM cells to anti-IRF4 ASOs, to obtain direct insights into their potential therapeutic applications in MM and also as functional probes into IRF4 biology. We observed that a panel of anti-IRF4 ASOs (but not control ASO) exhibited dose- and time-dependent activity against genotypically diverse MM cell lines (sub-uM IC50s for most MM lines vs. &amp;gt;10uM against non-MM cells lacking IRF4, e.g. HS5 bone marrow stromal cells [BMSCs]). Major in vitro anti-MM activity could be observed within 3-5 days of treatment; and was preceded by significant decrease in intracellular IRF4 protein levels. Importantly, 24-hour exposure to IRF4 ASO (followed by washout and subsequent culture in ASO-free media for up to 6 days) can induce similar anti-MM activity as continuous in vitro exposure over 6 days. Combinations of IRF4-ASO with several established (e.g. proteasome inhibitor, thalidomide derivative, glucocorticoids) or investigational (e.g. venetoclax) agents for MM result led to enhanced or even synergistic effects in a panel of 6 MM cell lines. For some of these MM cell lines, their in vitro response to IRF4 ASO had statistically significant attenuation in co-cultures with BMSCs: this effect was partially recapitulated in MM cell monocultures supplemented by conditioned media from BMSCs or (to a lesser extent) rhIL6 treatment. To obtain insights into cell-autonomous and nonautonomous mechanisms regulating MM cell responses to IRF4 ASOs, we examined the transcriptional profiles of MM.1S cells exposed to ASOs; and also performed genome-scale CRISPR studies to define genes whose editing (loss-of-function, LOF) or activation (gain-of-function, GOF) alter the MM cell response to the IRF4 ASOs. The transcriptional signature of MM cell treatment with IRF4 ASO (vs. control ASO) comprised distinct clusters of downregulated genes with preferential expression in normal or malignant plasma cells vs. other lineages; CRISPR-validated roles as dependencies for MM cells in vitro (preferentially essential for MM vs. non-MM tumors; or pan-essential); and/or proximity to large areas of chromatin accessibility (defined by H3K27Ac genome-wide ChIP-Seq or ATAC-Seq). Importantly, several genes downregulated by IRF4 ASO treatment are upregulated in MM cells cocultured with BMSCs, possibly explaining at least in part the impact of co-cultures on anti-MM activity of ASO. In our integrated genome-scale CRISPR studies (and validation of many genes with individual sgRNAs), MM cell response to IRF4 ASOs was attenuated by LOF of endonuclease RNase H1 (RNASEH1; which degrades the target RNA when it establishes intracellular hybrids with the ASOs); LOF of diverse genes involved in ASO endocytosis (e.g. IGF2R, PICALM, SH3GL1, RAB5C) or regulation of chondroitin/heparan sulfate (SLC35B2, B3GAT3, B4GALT7, ALG5 and ALG6); GOF of IRF4 itself (likely reflecting a "stoichiometric" effect, e.g. induction of higher IRF4 mRNA levels may require higher concentrations of ASO to maintain anti-MM effect); and GOF of exocytosis-related genes(e.g. CLU, QPCT). Importantly, LOF or GOF of individual genes typically associated with high-risk MM was not associated with decreased MM cell response to the IRF4 ASOs: therefore, retaining intracellular accumulation of IRF4 ASO and productive knockdown of IRF4 mRNA remains a primary driver of anti-MM activity of ASOs even in the context of biologically aggressive MM cells with "high-risk" features. Our results provide comprehensive integrated assessment of the molecular and functional landscapes associated with dysregulation of IRF4; have direct implications for our mechanistic understanding of the role of IRF4 in MM biology; and provide a framework for IRF4 targeting by ASOs or potentially other therapeutic approaches. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Luo:Ionis Pharmaceuticals, Inc.: Current Employment. Kim:Ionis Pharmaceuticals, Inc.: Current Employment. MacLeod:Ionis Pharmaceuticals, Inc.: Current Employment. Mitsiades:Arch Oncology: Research Funding; Sanofi: Research Funding; FIMECS: Consultancy, Honoraria; Karyopharm: Research Funding; Abbvie: Research Funding; Takeda: Other: employment of a relative; TEVA: Research Funding; Janssen/Johnson &amp; Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; EMD Serono: Research Funding; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria.

The long non-coding RNA CRNDE regulates growth of multiple myeloma cells via an effect on IL6 signalling

Multiple myeloma (MM) is a currently incurable malignancy of antibody-secreting plasma cells. Long non-coding RNAs (lncRNAs) have been recognised as an important class of regulatory molecules which are increasingly implicated in tumorigenesis. While recent studies have demonstrated changes in expression of lncRNAs in MM, the functional significance and molecular pathways downstream of these changes remain poorly characterised. In this study, we have performed CRISPR-mediated deletion of the locus encoding the lncRNA Colorectal Neoplasia Differentially Expressed (CRNDE), a known oncogenic lncRNA that is overexpressed in plasma cells of MM patients and is a marker of poor prognosis. We found that CRISPR-mediated deletion of the CRNDE locus in MM cells decreases proliferation and adhesion properties, increases sensitivity to Dexamethasone and reduces tumour growth in an in vivo xenograft model. Transcriptomic profiling in CRNDE-deleted MM cells demonstrated that CRNDE activates expression of a number of genes previously implicated in the aetiology of MM, including IL6R. We further demonstrate that deletion of the CRNDE locus diminishes IL6 signalling and proliferative responses in MM cells. Altogether this study reveals the IL6 signalling pathway as a novel mechanism by which CRNDE impacts upon MM cell growth and disease progression.

Abstract 5864: Genetic screens reveal dependencies and chemotherapy modulators in NSD2-driven hematologic malignancies

Abstract The histone methyltransferase NSD2 contributes to the pathogenesis and chemoresistance of various hematologic cancers. In multiple myeloma (MM), NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance. Further, a gain-of-function mutation of NSD2 promotes oncogenic reprogramming and chemoresistance in acute lymphocytic leukemia (ALL). So far, attempts to develop specific and potent NSD2 inhibitors have not been successful. Here, we utilized genome-wide CRISPR-based loss-of-function genetic screens to explore molecular dependencies and modulators of chemotherapy response in hematologic malignancies with aberrant NSD2 activity. We assessed gene essentiality in KMS11-derived NSD2-high and NSD2-low isogenic MM cells. Differential essentiality analysis revealed multiple genes whose disruption is selectively detrimental in cells with NSD2 overexpression. These include the adenine nucleotide regulator Adenylate Kinase 2 (AK2) and the super elongation complex (SEC) component ELL-Associated Factor 1 (EAF1), which were validated by in vitro competition assays. We further found that NSD2-high cells exhibit higher dependency on the druggable anti-apoptotic factor BCL2 and demonstrated a stronger synergy between BCL2 inhibitors and the glucocorticoid dexamethasone in these cells compared to NSD2-low cells. This finding suggests that BCL2 inhibitors can be used to potentiate dexamethasone response in MM patients with t(4;14) chromosomal translocation, where NSD2 is overexpressed. We also utilized the CRISPR-based genetic approach to investigate cellular processes influencing sensitivity of NSD2-driven MM cells to dexamethasone. Our study identified multiple cellular pathways as modulators of dexamethasone response in MM. For example, we showed that the IL10-JAK-STAT pathway mediates dexamethasone resistance whereas certain components of mRNA degradation function as dexamethasone sensitizers. How these pathways and other uncovered key players alter MM cell response to dexamethasone is still under investigation. We previously demonstrated that an activating NSD2 mutation leads to dexamethasone resistance in ALL. We sought to explore the mechanisms by which mutant NSD2 drives dexamethasone resistance and the cellular processes that can be targeted to restore dexamethasone sensitivity in ALL cells. Genome-wide CRISPR screens in RCH-ACV, a dexamethasone-resistant B-ALL cell line harboring the NSD2 mutation, uncovered multiple modulators of dexamethasone response. Intriguingly, we found that disruption of any component of a transcriptional complex involving LMO2, LDB1 and LYL1 sensitizes RCH-ACV cells to dexamethasone. Targeting this complex could be relevant to overcoming dexamethasone resistance in relapsed pediatric ALL. Citation Format: Amin Sobh, Charlotte Kaestner, Alberto Riva, Jianping Li, Richard Bennett, Jonathan Licht. Genetic screens reveal dependencies and chemotherapy modulators in NSD2-driven hematologic malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5864.

A novel BCMA PBD-ADC with ATM/ATR/WEE1 inhibitors or bortezomib induce synergistic lethality in multiple myeloma.

To target mechanisms critical for multiple myeloma (MM) plasma cell adaptations to genomic instabilities and further sustain MM cell killing, we here specifically trigger DNA damage response (DDR) in MM cells by a novel BCMA antibody drug conjugate (ADC) delivering the DNA cross-linking PBD dimer tesirine, MEDI2228. MEDI2228, more effectively than its anti-tubulin MMAF-ADC homolog, induces cytotoxicity against MM cells regardless of drug resistance, BCMA levels, p53 status, and the protection conferred by bone marrow stromal cells and IL-6. Distinctly, prior to apoptosis, MEDI2228 activates DDRs in MM cells via phosphorylation of ATM/ATR kinases, CHK1/2, CDK1/2, and H2AX, associated with expression of DDR-related genes. Significantly, MEDI2228 synergizes with DDR inhibitors (DDRi s) targeting ATM/ATR/WEE1 checkpoints to induce MM cell lethality. Moreover, suboptimal doses of MEDI2228 and bortezomib (btz) synergistically trigger apoptosis of even drug-resistant MM cells partly via modulation of RAD51 and accumulation of impaired DNA. Such combination further induces superior in vivo efficacy than monotherapy via increased nuclear γH2AX-expressing foci, irreversible DNA damages and tumor cell death, leading to significantly prolonged host survival. These results indicate leveraging MEDI2228 with DDRi s or btz as novel combination strategies, further supporting ongoing clinical development of MEDI2228 in patients with relapsed and refractory MM.

Identification of Genetic Vulnerabilities and Synthetic-Lethal Targets in NSD2-High Multiple Myeloma

The histone methyltransferase NSD2 is overexpressed in 15-20% of multiple myeloma (MM) patients due to the t(4;14) chromosomal translocation. NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance in MM cells. Despite strong efforts by academia and industry, there remains no tool compound or drug that inhibits NSD2 enzymatic activity. Therefore we explored the molecular and biochemical consequences of NSD2 overexpression in MM cells and investigated genetic vulnerabilities associated with high NSD2 levels as well as synthetic-lethal drug-gene interactions in t(4;14)-positive MM cells. We utilized the well-studied isogenic pair of human MM cells derived from the t(4;14)-positive KMS-11 cell line, where NSD2 is disrupted by knocking out either the translocated overexpressed allele (TKO; NSD2-low) or the wild-type non-translocated allele (NTKO; NSD2-high). Untargeted metabolomic profiling of TKO and NTKO cells revealed that NSD2 overexpression substantially alters nucleotide metabolism. NSD2-high cells exhibited an increase in purine synthesis and a decrease in pyrimidine synthesis. In addition, a genome-wide loss-of-function CRISPR gene editing screen using the 76,000 guide Brunello library uncovered genes differentially essential between NSD2-high and low MM cells. Intriguingly, one of the genes whose disruption is selectively lethal in NSD2-high cells encodes Adenylate Kinase 2 (AK2), an enzyme involved in purine metabolism, indicating that defective nucleotide metabolism associated with increased NSD2 expression introduces genetic vulnerabilities that can be therapeutically exploited. We further explored liabilities that can improve therapeutic outcomes in t(4;14) MM. A genome-wide CRISPR screen was performed in KMS-11 MM cells to identify mechanisms of sensitivity and resistance to dexamethasone, a steroid commonly used in MM treatment. As expected, inactivating the gene encoding the glucocorticoid receptor (GR) resulted in remarkable dexamethasone tolerance. We then identified and validated multiple genes/pathways that can alter response of MM cells to dexamethasone when disrupted. For example, inactivation of interleukin 10 (IL10) signaling by disrupting either subunit of the IL10 receptor or components of the downstream JAK/STAT pathway considerably enhances dexamethasone sensitivity. In addition, disruption of many components of heparan sulfate or glycosaminoglycans synthesis pathways, whose targeting has been previously shown to increase sensitivity to conventional MM chemotherapeutic agents, increases susceptibility to dexamethasone. By contrast, genetic perturbations leading to dexamethasone resistance correspond to GR co-chaperones including FKBP4 and PTGES3 and transcriptional coactivators like the nuclear receptor transactivator 1 (NCOA1). Interestingly, disruption of genes encoding proteins implicated in RNA stability and translation such as the N6-methyladenosine (m6A)-containing RNA binding protein YTHDF2 and the PAN2-PAN3 deadenylase complex results in dexamethasone resistance. How these proteins affect the expression of pro or anti-apoptotic genes in response to dexamethasone is under investigation. Our work reveals insight into novel molecular-based treatment options for t(4;14) MM that are independent on direct NSD2 inhibition which remains unsuccessful. Disclosures No relevant conflicts of interest to declare.

Molecular markers of MM cell sensitivity and resistance to Natural Killer cells: implications for anti-MM immunotherapy

Despite recent therapeutic advances, including monoclonal antibodies and T-cell-based therapies, multiple myeloma (MM) remains incurable. Natural killer (NK) cells are highly cytotoxic in preclinical MM studies; while infusion of ex vivo expanded/activated NK cells in clinical studies has been feasible and safe, without graft-versus-host reactions. However, the molecular markers determining MM cell response vs. resistance to NK cells remain incompletely understood. To address this topic, we performed genome-wide CRISPR/Cas9 loss- (LOF) and gain-of-function (GOF) studies in MM.1S cells exposed to primary NK (pNK) cells and identified genes whose knock-out (KO) or activation led to NK cells resistance or sensitivity.

XRCC1-mediated DNA repair is associated with progression-free survival of multiple myeloma patients after autologous stem cell transplant.

Autologous stem cell transplant (ASCT) with high-dose melphalan (HDM) is the standard treatment for fit multiple myeloma (MM) patients. It is generally believed that some DNA repair proteins impact the activity to repair melphalan-induced DNA damage, thus potentially contributing to the patient's clinical response. However, knowledge of these proteins is limited. In the current study, we investigated the roles of XRCC1, a protein involved in base excision repair and single-strand break repair, in melphalan response in MM cells. Small interfering RNA knockdown of XRCC1 significantly increased the accumulation of melphalan-induced DNA damage in MM cells and sensitized them to melphalan treatment, indicating that genetic variation in XRCC1 may impact response to melphalan treatment. We then evaluated the association between an XRCC1 variant with reduced activity, rs25487 (R399Q), and clinical outcomes of 108 MM patients with melphalan therapy. Our results showed that XRCC1 rs25487 was associated with prolonged progression-free survival (PFS) in MM patients. The adjusted hazard ratio for PFS between patients carrying rs25487 AA/AG and GG was 0.42 (95% confidence interval: 0.25, 0.84, P = .014). Taken together, these results indicate that XRCC1 is involved in the repair of melphalan-induced DNA damage and XRCC1 rs25487 variant with impaired DNA repair function influences the clinical responses of HDM in MM patients.