Multiple Myeloma Cell Survival Research Articles

Co-operation of MCL-1 and BCL-XL anti-apoptotic proteins in stromal protection of MM cells from carfilzomib mediated cytotoxicity.

BCL-2 family proteins are important for tumour cell survival and drug resistance in multiple myeloma (MM). Although proteasome inhibitors are effective anti-myeloma drugs, some patients are resistant and almost all eventually relapse. We examined the function of BCL-2 family proteins in stromal-mediated resistance to carfilzomib-induced cytotoxicity in MM cells. Co-cultures employing HS5 stromal cells were used to model the interaction with stroma. MM cells were exposed to CFZ in a 1-hour pulse method. The expression of BCL-2 family proteins was assessed by flow cytometry and WB. Pro-survival proteins: MCL-1, BCL-2 and BCL-XL were inhibited using S63845, ABT-199 and A-1331852 respectively. Changes in BIM binding partners were examined by immunoprecipitation and WB. CFZ induced dose-dependent cell death of MM cells, primarily mediated by apoptosis. Culture of MM cells on HS-5 stromal cells resulted in reduced cytotoxicity to CFZ in a cell contact-dependent manner, upregulated expression of MCL-1 and increased dependency on BCL-XL. Inhibiting BCL-XL or MCL-1 with BH-3 mimetics abrogated stromal-mediated protection only at high doses, which may not be achievable in vivo. However, combining BH-3 mimetics at sub-therapeutic doses, which alone were without effect, significantly enhanced CFZ-mediated cytotoxicity even in the presence of stroma. Furthermore, MCL-1 inhibition led to enhanced binding between BCL-XL and BIM, while blocking BCL-XL increased MCL-1/BIM complex formation, indicating the cooperative role of these proteins. Stromal interactions alter the dependence on BCL-2 family members, providing a rationale for dual inhibition to abrogate the protective effect of stroma and restore sensitivity to CFZ.

#43. Long-chain acyl-CoA synthetases support multiple myeloma cell proliferation, survival and mitochondrial function

#43. Long-chain acyl-CoA synthetases support multiple myeloma cell proliferation, survival and mitochondrial function

Synergy between BRD9- and IKZF3-Targeting as a Therapeutic Strategy for Multiple Myeloma.

Progress in the treatment of multiple myeloma (MM) has resulted in improvement in the survival rate. However, there is still a need for more efficacious and tolerated therapies. We and others have shown that bromodomain-containing protein 9 (BRD9), a member of the non-canonical SWI/SNF chromatin remodeling complex, plays a role in MM cell survival, and targeting BRD9 selectively blocks MM cell proliferation and synergizes with IMiDs. We found that synergy in vitro is associated with the downregulation of MYC and Ikaros proteins, including IKZF3, and overexpression of IKZF3 or MYC could partially reverse synergy. RNA-seq analysis revealed synergy to be associated with the suppression of pathways associated with MYC and E2F target genes and pathways, including cell cycle, cell division, and DNA replication. Stimulated pathways included cell adhesion and immune and inflammatory response. Importantly, combining IMiD treatment and BRD9 targeting, which leads to the downregulation of MYC protein and upregulation of CRBN protein, was able to override IMiD resistance of cells exposed to iberdomide in long-term culture. Taken together, our results support the notion that combination therapy based on agents targeting BRD9 and IKZF3, two established dependencies in MM, represents a promising novel therapeutic strategy for MM and IMiD-resistant disease.

Protective Effects of High-Density Lipoprotein on Cancer Risk: Focus on Multiple Myeloma.

Lipid metabolism is intrinsically linked to tumorigenesis. And one of the most important characteristics of cancer is the modification of lipid metabolism and its correlation with oncogenic signaling pathways within the tumors. Because lipids function as signaling molecules, membrane structures, and energy sources, lipids are essential to the development of cancer. Above all, the proper immune response of tumor cells depends on the control of lipid metabolism. Changes in metabolism can modify systems that regulate carcinogenesis, such as inflammation, oxidative stress, and angiogenesis. The dependence of various malignancies on lipid metabolism varies. This review delves into the modifications to lipid metabolism that take place in cancer, specifically focusing on multiple myeloma. The review illustrates how changes in different lipid pathways impact the growth, survival, and drug-responsiveness of multiple myeloma cells, in addition to their interactions with other cells within the tumor microenvironment. The phenotype of malignant plasma cells can be affected by lipid vulnerabilities, and these findings offer a new avenue for understanding this process. Additionally, they identify novel druggable pathways that have a major bearing on multiple myeloma care.

Research Progress on the Mechanism of PI3K/AKT/mTOR Signaling Pathway in Multiple Myeloma --Review

PI3K/AKT/mTOR signaling pathway is of great significance in the development and prognosis of tumors, and is closely related to the pathogenesis of multiple myeloma (MM). PI3K/AKT/mTOR signaling pathway can participate in the regulation of MM through multichannel and multitarget, such as regulating the tumor microenvironment of MM cells survival, affecting tumor development and migration, regulating the proliferation, apoptosis and autophagy of MM cells. It have shown that after the PI3K/AKT/mTOR signaling pathway is inhibited, the apoptosis and autophagy of MM cells are activated, which promote the death of MM cells and inhibit the metastasis and recurrence of MM cells. Therefore, indepth study of the mechanism of PI3K/AKT/mTOR signaling pathway in MM is helpful to elucidate the pathogenesis and prognosis of MM.

CRIP1 involves the pathogenesis of multiple myeloma via dual-regulation of proteasome and autophagy

Multiple myeloma (MM) is an incurable hematological malignancy of the plasma cells. The maintenance of protein homeostasis is critical for MM cell survival. Elevated levels of paraproteins in MM cells are cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as a backbone agent and successfully improved the outcome of patients; however, the increasing activity of autophagy suppresses the sensitivity to PIs treatment. The transcription levels of CRIP1 were explored in plasma cells obtained from healthy donors, patients with newly diagnosed multiple myeloma (NDMM), and relapsed/refractory multiple myeloma (RRMM) using Gene expression omnibus datasets. Doxycycline-inducible CRIP1-shRNA and CRIP1 overexpressed MM cell lines were constructed to explore the role of CRIP1 in MM pathogenesis. Proliferation, invasion, migration, proteasome activity and autophagy were examined in MM cells with different CRIP1 levels. Co-immunoprecipitation (Co-IP) with Tandem affinity purification/Mass spectrum (TAP/MS) was performed to identify the binding proteins of CRIP1. The mouse xenograft model was used to determine the role of CRIP1 in the proliferation and drug-resistance of MM cells. High CRIP1 expression was associated with unfavorable clinical outcomes in patients with MM and served as a biomarker for RRMM with shorter overall survival. Invitro and invivo studies showed that CRIP1 plays a critical role in protein homeostasis via the dual regulation of the activities of proteasome and autophagy in MM cells. A combined analysis of RNA-seq, Co-IP and TAP/MS demonstrated that CRIP1 promotes proteasome inhibitors resistance in MM cells by simultaneously binding to de-ubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promoted proteasome activity and autophagosome maturation by facilitating the dequbiquitination and stabilization of PA200. Our findings clarified the pivotal roles of the CRIP1/USP7/PA200 complex in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the pathogenesis of MM. A full list of funding sources can be found in the acknowledgements section.

S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma.

Multiple myeloma (MM) is the second most prevalent hematologic malignancy and is incurable because of the inevitable development of drug resistance. Methionine adenosyltransferase 2α (MAT2A) is the primary producer of the methyl donor S-adenosylmethionine (SAM) and several studies have documented MAT2A deregulation in different solid cancers. As the role of MAT2A in MM has not been investigated yet, the aim of this study was to clarify the potential role and underlying molecular mechanisms of MAT2A in MM, exploring new therapeutic options to overcome drug resistance. By analyzing publicly available gene expression profiling data, MAT2A was found to be more highly expressed in patient-derived myeloma cells than in normal bone marrow plasma cells. The expression of MAT2A correlated with an unfavorable prognosis in relapsed patients. MAT2A inhibition in MM cells led to a reduction in intracellular SAM levels, which resulted in impaired cell viability and proliferation, and induction of apoptosis. Further mechanistic investigation demonstrated that MAT2A inhibition inactivated the mTOR-4EBP1 pathway, accompanied by a decrease in protein synthesis. MAT2A targeting in vivo with the small molecule compound FIDAS-5 was able to significantly reduce tumor burden in the 5TGM1 model. Finally, we found that MAT2A inhibition can synergistically enhance the anti-MM effect of the standard-of-care agent bortezomib on both MM cell lines and primary human CD138+ MM cells. In summary, we demonstrate that MAT2A inhibition reduces MM cell proliferation and survival by inhibiting mTOR-mediated protein synthesis. Moreover, our findings suggest that the MAT2A inhibitor FIDAS-5 could be a novel compound to improve bortezomib-based treatment of MM.

Cells of the Osteoblast Lineage Cross-Talk with Myeloid-Derived Suppressor Cells to Promote Multiple Myeloma Growth

Multiple myeloma (MM) is a plasma cell malignancy that thrives in the bone marrow (BM) and is highly influenced by the BM tumor microenvironment. Osteolytic bone disease is a hallmark of MM and is partly due to osteoblast (OB) inhibition. MM cells suppress osteoblast maturation and function via inhibition of the critical Runt-related transcription factor 2 (Runx2) in immature OBs. However, immature cells of the osteoblast lineage (e.g. pre-osteoblasts) remain present and even increased in the MM bone microenvironment and their contribution to the progression of MM has not been well understood. We previously developed a syngeneic mouse model of MM in which Runx2 is specifically deleted in the immature OBs of C57BL6/KaLwRij mice (OB-Runx2 -/- mice), and demonstrated that OB-Runx2 deficiency creates a highly inflammatory BM microenvironment that is responsible for MM cell bone-homing to new bone sites. We therefore hypothesize that pre-osteoblasts play a role as promoters of MM cell survival and proliferation. To determine the direct effect of pre-osteoblasts on MM cells, we established co-cultures of pre-osteoblasts with MM cells. 5TGM1 murine MM cells were cocultured with MC3T3 murine pre-osteoblasts and newborn mouse calvarial pre-osteoblasts respectively. Within 48hrs of co-culture, each of pre-osteoblast populations slightly stimulated proliferation of 5TGM1 MM cells. These data suggest that pre-osteoblasts do not directly affect myeloma growth in vitro. Therefore, we next focus our studies on the role of pre-osteoblasts in MM progression in vivo. Firstly, we used OB-Runx2 deficient mouse model to assess the impact of pre-osteoblast accumulation on MM progression in vivo. 2x10 5 5TGM1-Luciferase MM cells were injected into the right tibia of OB-Runx2 -/- mice and control mice. Bioluminescence imaging and serum IgG2bκ (a soluble marker of 5TGM1 MM cells) ELISA demonstrated that, compared with controls, OB-Runx2 -/- mice had a much larger tumor burden. Following, to more thoroughly evaluate the role of pre-osteoblasts in early stage of MM cell engraftment in bones, we used MC3T3 murine pre-osteoblasts to modulate pre-osteoblast populations at the sites of MM engraftment. 2x10 5 5TGM1-Luciferase MM cells were injected directly into the right tibia of NSG mice with or without equal numbers of MC3T3 pre-osteoblasts. Tumors grew significantly larger when co-injected with 5TGM1 MM cells and MC3T3 pre-osteoblasts than injected with 5TGM1 MM cells alone, which was confirmed by luciferin imaging and serum IgG2bκ ELISA. These data suggest that pre-osteoblasts provide critical support for MM progression in vivo. Given that pre-osteoblasts have no direct effect on the growth of MM cells in vitro, we hypothesized that the pre-osteoblastic niche may regulate MM cells via other intermediary cells in BM. We previously showed that OB-Runx2 deficiency induces an immunosuppressive BM microenvironment that is marked by an increase of myeloid-derived suppressor cells (MDSCs). To investigate whether pre-osteoblasts support MM cell survival and proliferation through the functional crosstalk with MDSCs, 5TGM1 MM cells were co-cultured with MC3T3 pre-osteoblasts and mouse calvarial pre-osteoblasts respectively in the presence or absence of MDSCs. Interestingly, pre-osteoblasts significantly promoted the proliferation of MM cells under the existence of MDSCs. To extend these findings in vivo, we next determined whether MDSC depletion can alleviate the promoting effect of pre-osteoblasts on MM progression. Gemcitabine (GEM) is an FDA-approved anti-cancer agent and MDSC inhibitor used in the treatment of solid tumors but not commonly used in MM therapy. We again injected 5TGM1-Luciferase MM cells into the right tibia of NSG mice with or without co-injection of MC3T3 pre-osteoblasts. We treated tumor-bearing mice with PBS or GEM (i.p. 30 mg/kg/week) for 4 weeks. Bioluminescence imaging and serum IgG2bκ ELISA demonstrated that GEM could overcome the proliferative effect of pre-osteoblasts on MM cells in mouse model, and this effect was independent of NK, B and T cells. Taken together, using in vivo and in vitro models, we show that pre-osteoblasts have a positive regulatory impact on MM cells through communication with MDSCs. Ongoing mechanistic studies aim to delineate how pre-osteoblasts modulate the functions of MDSCs to promote MM progression.

CRIP1 Involves the Pathogenesis of Multiple Myeloma Via Dual-Regulation of Proteasome and Autophagy

Multiple myeloma (MM) is an incurable hematological malignancy of plasma cells. The maintenance of protein homeostasis is critical for the survival of MM cells. Elevated paraprotein in MM cells would be cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as the backbone agent and successfully improved the outcome of patients, but the increasing activity of autophagy suppresses the sensitivity to PIs treatment. In the present study, we elucidated that CRIP1 was a critical player in protein homeostasis via the dual regulation of proteasome and autophagy in MM. A combination analysis of RNA-seq, Co-IP and Tandem affinity purification/mass spectrometry identified that high-level CRIP1 represents a biomarker for relapsed/refractory MM with shorter overall-survival of patients. CRIP1 plays a critical role in MM progression and PIs resistance by binding to deubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promotes proteasome activity and autophagosome maturation in MM by stabilizing PA200. Therefore, our findings clarified the pivotal roles of CRIP1/USP7/PA200 complex formation in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the progression of MM.

Targeting RBM39-MEK5 Axis Synergizes with Bortezomib to Inhibit Multiple Myeloma Malignancy

Multiple myeloma (MM) is a type of haematological malignancy characterised by the accumulation and expansion of malignant plasma cells in the bone marrow (BM). Despite recent advances in chemotherapy, MM remains an incurable disease for most patients and therefore requires novel therapies. Abnormal alternative splicing is a hallmark of cancer and is associated with poor survival and an ultra-high-risk population in MM (Bauer et. al. Haematologica 2021). Targeting splicing is a promising therapeutic strategy in MM. Our previous study showed that RBM39 is highly expressed in primary MM cells and cell lines and correlates with poor overall survival (Tong et. al. Haematologica 2020). To investigate the underlying role of RBM39 in MM, we knocked down RBM39 in MM cell lines. We found that knockdown of RBM39 inhibited MM cell proliferation, induced apoptosis and cell cycle arrest at the G2/M phase, and inhibited subcutaneous tumour growth. Indisulam has been reported to induce RBM39 degradation via the proteasomal pathway by acting as a molecular glue to link RBM39 to the E3 ubiquitin ligase DCAF15 (Han et. al. Science 2017). In our study, Indisulam treatment in MM cells was shown to degrade RBM39 via DCAF15. Indisulam could induce apoptosis and G2/M phase arrest in MM cell lines and inhibite tumour growth in vivo. It also significantly decreased MM cell viability in a co-culture system with MM BM-derived mesenchymal stromal cells. Indisulam was also able to inhibit primary CD138+ cells isolated from newly diagnosed MM patients. To explore the mechanistic role of RBM39 in promoting MM malignancy, we performed RNA immunoprecipitation (RIP)-Seq and RNA sequencing in RBM39 knockdown MM cells to investigate the alternative splicing targets of RBM39. KEGG pathway analysis of differentially expressed and alternatively spliced genes revealed the enrichment of the MAPK pathway. We then identified MEK5, a non-redundant member of the MAPK family, as a potential target of RBM39, which is highly expressed in MM. Full-length MEK5 contributes to increased proliferation and survival of MM cells. We further demonstrated that MEK5 is bound and alternatively spliced by RBM39 and that splicing of full-length MEK5 is dependent on RBM39. Given the importance of the RBM39-MEK5 axis in MM, we also found that inhibition of RBM39 with Indisulam or MEK5 with the inhibitor BIX02189 increased the cytotoxicity of bortezomib in vivo and in vitro. In conclusion, we have demonstrated that the RBM39-MEK5 axis, in which RBM39 maintains splicing of full-length MEK5, is essential for MM cell survival and proliferation. Mechanistically, shRNA-mediated knockdown or Indisulam-mediated degradation of RBM39 caused extensive altered splicing, including mis-splicing of MEK5. Targeting RBM39 or MEK5 could not only inhibit MM malignancy, but also enhance the cytotoxicity of bortezomib for MM.

A Functional Genomic Screen to Identify Novel Genes Involved in Multiple Myeloma Tumour Development

Multiple myeloma (MM) is an incurable malignancy characterised by uncontrolled proliferation of plasma cells (PCs) in the bone marrow (BM). MM is a genetically heterogeneous disease with each patient's PCs harbouring unique genetic mutations, however the development of MM tumours is not only dependent on the underlying genetics but also on the selective pressures applied by the BM microenvironment. Hence, we hypothesise that identifying the dependencies which promote MM cell outgrowth in the BM microenvironment will allow for the identification of new drug targets. This project aims to use an in vivo functional screen, combining CRISPR-Cas9 gene editing, using a single guide RNA (sgRNA) library targeting thousands of genes, with a murine model of MM, to identify novel genes involved in MM tumour development. The Bassik human apoptosis and cancer CRISPR knockout library (Addgene #101926) was used to transduce MM cells with 31,324 unique sgRNAs targeting 3,015 genes and 1,500 control regions. The human MM cell line OPM2 was transduced with the Cas9 transgene (FuCas9GFP), followed by the sgRNA expression vector (pMCB320) generating OPM2-Cas9-sgRNA cells. These cells were then injected (5x10 5 cells/mouse, n=8 mice + 1 tumour-naive control) into the tibiae of immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ (NSG) mice. Four weeks post-injection, the primary tumour within the injected tibia was isolated and sgRNA frequencies were assessed via next generation sequencing and compared with that of the initial library and the injected cells using the MAGeCK algorithm. To identify genes that play roles in MM tumour formation in patients, we assessed the expression of our identified genes in CD138-selected BM PCs from newly diagnosed MM patients (n=155) when compared with normal PCs from healthy donors (n=5) in microarray dataset E-MTAB-363 (ArrayExpress). Furthermore, to identify potential pathway involvement of our identified genes we performed gene set enrichment analysis (GSEA) using the University of California San Diego and Broad Institute developed GSEA software, comparing to Hallmark Gene Sets. Comparison of the in vitro cultured cells with the initial library identified 34 genes with undetectable (p<0.05) sgRNAs post in vitro culture, suggesting these genes play key roles in the survival of OPM2 cells. This list contained a number of genes known to control MM cell survival, such as IRF4 and CCND2. Additionally, sgRNAs targeting a further 115 genes were significantly depleted (p<0.05) in vitro and were completely undetectable in vivo, suggesting that these are implicated in cell proliferation and tumour formation in vivo. This gene list contained some already known MM drivers such as MYC, KRAS and DIS3. Furthermore, we identified 28 genes where CRISPR knockout had no significant effect in vitro but were critical dependencies for tumour development, with sgRNAs being undetectable (p<0.05) in vivo. This list contained genes known to be important for MM and BM stromal cell interaction such as ALCAM and SPP1. Notably, 25% of the genes identified as critical dependencies for OPM2 cells in vitro and/or in vivo are significantly upregulated >2-fold (p<0.05; limma) in MM patient PCs when compared with normal PCs (E-MTAB-363), suggesting these may play a role in tumour development in patients. Gene set enrichment analysis revealed that the genes that were essential in vitro were predominantly involved in cell survival related pathways such as cell cycle regulation (G2M checkpoint) and DNA replication/repair; however, the uniquely in vivo genes are predominantly involved in cell metabolism pathways such as MTORC1 signalling, oxidative phosphorylation and glycolysis. Overall, this study has successfully used a functional genetic screen to identify novel gene dependencies required for in vitro MM cell growth and in vivo MM tumour development. A secondary genetic screen will be performed in vivo to validate and refine this list of candidate genes. These validated genes will be further investigated using patient derived data sets to determine if they represent common vulnerabilities in patients and in multiple MM cell lines to investigate their specific mechanistic and pathway involvement. Identification and understanding the driving dependencies which promote MM cell outgrowth in the BM microenvironment will allow for the identification and development of new drug targets to improve patient outcomes.

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.

SDC2 Expression Is Increased in Myeloma Cells in Response to Loss of Pro-Survival Surface Proteins, CD28 and CD86

Introduction: While recent advances in multiple myeloma (MM) have improved overall survival, most patients will succumb to late-stage and drug resistant disease. Plasma cell (PC) survival outside of the bone marrow is characteristic of late-stage myeloma and pivotal to disease progression. PCs are typically restricted to the bone marrow microenvironment, as they are dependent on stromal interactions for survival. One such interaction is the engagement of CD28 and its ligand CD86, proteins which are best understood for their roles in T-cell activation by antigen presenting cells. However, myeloma cells can express both CD28 and CD86 on the cell surface. Notably, high CD28 expression is seen in 59% of medullary recurrences, and over 90% of extramedullary relapses. Moreover, high CD86 expression is more common in malignant plasma cells as compared to precursor stages and has a reported role in driving expression of IRF4. We have previously shown that expression of CD28 and CD86 is associated with worse outcomes than that observed in patients with low expression in the MMRF CoMMpass study (NCT01454297). We have also shown that silencing of CD28 or CD86 results in cell death and loss of IRF4 protein expression in human myeloma cell lines (HMCL). We confirmed these studies using CRISPR/Cas9 editing and demonstrated loss of proliferation and induction of cell death in HMCLs. However, the mechanism(s) by which CD28 and CD86 contribute to MM cell survival is not fully understood. Methods: To shed light on the mechanistic basis of CD28 and CD86 in the survival of MM, we used CRISPR/Cas9 editing to disrupt expression from RPMI8226 myeloma cells. We then passaged transduced cells to select for populations that can survive loss of CD28 or CD86. These passaged cells were sorted into populations that either retained CD28 or CD86 expression as transduction controls (High), or populations that underwent CRISPR/Cas9 depletion (Low). Surface expression of CD28 High/Low and CD86 High/Low populations was confirmed via flow cytometry and were then processed for RNA isolation and cDNA library preparation. RNA-sequencing was performed using paired-end sequencing, and data were aligned using the STAR aligner. Differentially expressed genes were determined using edgeR with an FDR ≤ 0.05 and a fold-change ≥ 2.0. Validation of RNA-sequencing results was further assessed via western blot and flow cytometry. Results: Analysis of RNA-sequencing results indicated that 57 genes were differentially expressed between High and Low cells, with 23 genes common to both CD28 and CD86 High/Low comparisons. (FDR<0.05). Notably, our results reveal that loss of CD28 or CD86 is associated with an increase in mRNA expression of Syndecan-2 (SDC2). In addition, our preliminary experiments evaluating SDC2 surface expression via flow cytometry suggest that both CD28 Low and CD86 Low populations have increased expression of SDC2. Additionally, in the RNA-seq analysis we also observed that CD28 Low and CD86 Low cells were also able to sustain mRNA expression of IRF4. This result was later confirmed via western blot, demonstrating CD28 Low and CD86 Low cells sustain protein expression of IRF4. Conclusions: Our results demonstrate that RPMI8226 cells that lack CD28 or CD86 expression have increased mRNA and protein expression of SDC2, a member the heparan sulfate proteoglycan family. Since SDC2 expression has also been associated with a number of cancers including colorectal, lung, and pancreatic cancers, these results provide insight to potential alternative pathways that contribute to myeloma survival independent of CD28 and CD86 signaling. We also confirmed that CD28- and CD86-depleted populations were able to maintain IRF4 mRNA/protein levels. This indicates myeloma cells have mechanisms to sustain IRF4 expression in addition to CD28 and CD86 signaling. These results are also consistent with previous findings illustrating importance of IRF4 in myeloma. Finally, we have developed CRISPR/Cas9-depleted CD28 and CD86 populations in additional HMCLs. We are currently in the process of isolating CD28 High/Low and CD86 High/Low populations for additional analysis and will present these upcoming results.

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.

Single-Cell Multi-Ome Analysis Reveals Novel Molecular Mechanisms Underlying Subclonal Response to Survivin Inhibition in Relapsed/Refractory Multiple Myeloma

Multiple myeloma is the second-most common hematological malignancy in the US that remains a challenging disease to cure despite recent advancements in treatment strategies. Drug resistance is a major cause of concern in myeloma chemotherapy. We have created an optimization-regularization-based computational prediction method called secDrug that identified several novel secondary drug (‘secDrug‘) combinations against drug-resistant myeloma (relapsed/refractory multiple myeloma or RRMM). Top among our predicted secDrugs includes the survivin inhibitor, YM155. Survivin is a member of the inhibitor of apoptosis protein (IAP) family that has dual roles in mitosis and apoptosis. YM155 inhibits survivin at the transcriptional level by inhibiting the survivin gene promoter. Several studies have demonstrated the efficacy of YM155 in solid tumors and heme malignancies, including myeloma. However, the precise mechanism of action is not yet known. MM cell lines express high levels of IAPs. We observed high in vitro cytotoxicity of YM155 as a single agent (IC 50 range 0.41 to 12.85nM) as well as in combination with the primary drugs like proteasome inhibitors and Immunomodulatory drugs (IMiDs) against >15 human myeloma cell lines representing innate/refractory and acquired/emerging (relapse) resistance. Next, we explored the molecular mechanisms underlying the efficacy of YM155 using pre-vs-post-treatment tumor whole-transcriptome analysis (RNAseq). The top differentially regulated genes include DDIT3 (CHOP), ATF3, TXNDC5 SEC24D, and DHX15 ( Figure 1A). Ingenuity Pathway Analysis (IPA) predicted that, in addition to regulating apoptosis, survivin inhibition in myeloma significantly upregulated unfolded protein response and downregulated vehicle trafficking and genes associated with immune cell function. Further, we performed single-cell transcriptomics (scRNAseq; 10X Genomics) analysis to explore treatment-induced changes to the subclonal architecture. Our scRNAseq results revealed a distinct shift in clusters representing myeloma single-cell subpopulations following YM155 treatment ( Figure 1B). For example, Cluster 1 (53.11%), characterized by high expression of cell survival markers Ki67 and MYC, was pre-dominant in untreated myeloma cells, while Cluster 4 (52.19%) was the primary subclone in post-treatment tumors. Cluster 3 was shared between the untreated and treated groups. Detailed distinctions between each single-cell cluster will be presented. Furthermore, our scRNAseq data alluded to the benefits of combining survivin-inhibitors and Hsp90-inhibitors based on the expression of drug target genes. Hsp90 inhibitors indirectly target survivin by disrupting the interaction between survivin and Hsp90, resulting in a destabilized survivin protein that ultimately results in survivin degradation. In fact, we have earlier shown the effectiveness of Hsp90 inhibitors (17-AAG, AICAR, and CCT018159) as potent secDrugs against RRMM. Finally, high-dimensional mass cytometry or CyTOF analysis using heavy metal-conjugated proteins in patient bone marrow-derived primary myeloma cells ( ex vivo model system) showed that YM155 induced dose-dependent reduction of several protein markers implicated in cell survival and chemotherapeutic resistance in multiple myeloma, including myc, Ki-67, CD28, CD81, and CD274 (PD-L1). Previous studies have shown that CD28 and CD81 are high-risk immunophenotypes associated with significantly worse prognosis in patients with myeloma. PD-L1 (Programmed death-ligand 1) is a cell-surface glycoprotein expressed in a large number of malignancies that is involved in the termination of immune response and immune evasion. An earlier study has shown that YM155 enhances daratumumab-mediated cellular lysis of multiple myeloma cells. Our RNAseq and CyTOF results thus suggest a potential basis of synergy between YM155 with immunotherapies approved for myeloma, which we will test further. Our approach thus integrates in silico prediction with single-cell multi-ome analysis to identify molecular mechanisms potentially underlying subclonal response to novel combination therapy candidates (secDrugs) for the treatment of RRMM.

Overexpression of Integrin β7 in Newly Diagnosed Multiple Myeloma with High Risk Cytogenetic Abnormality and Poorer Prognosis

Introduction The second most prevalent malignant blood malignancy is multiple myeloma (MM), but the condition is still incurable. In the interaction between MM cells and the bone marrow microenvironment, adhesion molecules are known to mediate adhesion and promote the survival and proliferation of MM cells. Integrin β7 (ITGB7) is a member of the adhesion molecule family. However, there are few clinical studies on ITGB7 in MM and there is no prognostic analysis. By analyzing the relationship between ITGB7 expression and clinical signs and prognosis, this study thus provides a basis for evaluating the clinical and prognostic importance of ITGB7 expression in MM patients. Methods In 39 patients with newly diagnosed multiple myeloma from 2021.1 to 2022.9, we assessed the expression of ITGB7 in relation to clinical characteristics and prognosis. Flow cytometry was used to detect the median fluorescence of ITGB7, detecting that ITGB7 median fluorescence intensity (MFI) in abnormal plasma cell was significantly higher than that in mature B cells (18.76 vs 5.62, P<0.001). ROC curve defines that ITGB7 best indicator for predicting MM disease is the abnormal plasma cell to mature B cells median fluorescence intensity ratio (MFI P/B), with a cut-off value of 4.025. Results Compared to patients with low expression of ITGB7, patients with high expression of ITGB7 showed a significant increase in mSMART stratification risk (82.60% vs 50.00%, P=0.041), while those with IgH/FGFR3 showed a significant increase (38.9% vs 6.30%, P=0.043). Patients with high expression of ITGB7 had poorer prognostic trends in PFS and OS, (PFS: P=0.071; OS: P=0.070). Conclusion Overexpression of ITGB7 is prone to merging with high risk cytogenetic abnormality and poorer prognosis in newly diagnosed MM patients.

A SAGA Complex-Specific Dependency in Multiple Myeloma Orchestrates Oncogenic Transcriptional Programs

Multiple myeloma (MM) is a malignancy of antibody-producing plasma cells that reside within the bone marrow. Epigenetic factors contribute to the initiation, progression, relapse and clonal heterogeneity in MM, but our knowledge of epigenetic regulators involved in MM development is far from complete. Results from the Cancer Dependency Map (DepMap) Project revealed that several components of the SAGA complex are particularly important for MM cell growth and survival. The SAGA complex serves as a coactivator in transcription and also catalyzes histone acetylation and deubiquitination via its lysine acetyltransferase (KAT) and deubiquitinase (DUB) modules, respectively. Our analysis showed that each SAGA complex component is overexpressed in at least 50% of MM patients. To investigate SAGA-specific functions, we centered on ADA2B, a subunit in the KAT module that specifically functions in the SAGA complex. We observed reduced cell viability upon ADA2B knockdown and CRISPR/Cas9-mediated knockout in MM cell lines. To identify the underlying transcriptional pathways that ADA2B regulate, we performed transcriptome sequencing using MM cell lines with and without ADA2B depletion. Our results showed several oncogenic pathways, including MYC, E2F and MM-specific MAF oncogenic programs, are tightly regulated by ADA2B. We also discovered that the protein but not transcript levels of MAF and the KATs in SAGA, GCN5 and PCAF, as well as the global level of histone H3 lysine 9 acetylation (H3K9ac) are all prominently decreased upon ADA2B depletion. To investigate whether the global reduction in H3K9ac would alter chromatin accessibility, we performed assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and found decreased accessibility at many cancer marker and cancer-promoting genes. To decipher the protein domains that are essential for ADA2B function in MM, we generated and expressed mutated versions of ADA2B in MM cell lines. Excitingly, mutations of core hydrophobic amino acid residues in the SANT domain disrupt interactions with GCN5 and PCAF and negatively affect protein stability of ADA2B. Hence, drug inhibitors that target the SANT domain could achieve suppression of ADA2B and MM cell growth. Our findings uncover novel epigenetic and transcriptional mechanisms controlling MM development, and suggest potential for new epigenetic inhibitors targeting SAGA-specific ADA2B as future treatment modalities for MM.

Pharmacologic targeting of the p62 ZZ domain enhances both anti-tumor and bone-anabolic effects of bortezomib in multiple myeloma.

Multiple myeloma (MM) is a malignancy of plasma cells whose antibody secretion creates proteotoxic stress relieved by the N-end rule pathway, a proteolytic system that degrades N-arginylated proteins in the proteasome. When the proteasome is inhibited, protein cargo is alternatively targeted for autophagic degradation by binding to the ZZ-domain of p62/ sequestosome-1. Here, we demonstrate that XRK3F2, a selective ligand for the ZZ-domain, dramatically improved two major responses to the proteasome inhibitor bortezomib (Btz) by increasing: i) killing of human MM cells by stimulating both Btz-mediated apoptosis and necroptosis, a process regulated by p62; and ii) preservation of bone mass by stimulating osteoblast differentiation and inhibiting osteoclastic bone destruction. Co-administration of Btz and XRK3F2 inhibited both branches of the bimodal N-end rule pathway exhibited synergistic anti-MM effects on MM cell lines and CD138+ cells from MM patients, and prevented stromal-mediated MM cell survival. In mice with established human MM, co-administration of Btz and XRK3F2 decreased tumor burden and prevented the progression of MM-induced osteolytic disease by inducing new bone formation more effectively than either single agent alone. The results suggest that p62-ZZ ligands enhance the anti- MM efficacy of proteasome inhibitors and can reduce MM morbidity and mortality by improving bone health.

Non-Toxicological Role of Aryl Hydrocarbon Receptor in Obesity-Associated Multiple Myeloma Cell Growth and Survival.

Obesity is not only a risk factor for multiple myeloma (MM) incidence, but it is also associated with an increased risk of progression from myeloma precursors-monoclonal gammopathy of undetermined significance-and smoldering myeloma. Adipocytes in the bone marrow (BMAs) microenvironment have been shown to facilitate MM cell growth via secreted factors, but the nature of these secreted factors and their mechanism of action have not been fully elucidated. The elevated expression of aryl hydrocarbon receptor (AhR) is associated with a variety of different cancers, including MM; however, the role of AhR activity in obesity-associated MM cell growth and survival has not been explored. Indeed, this is of particular interest as it has been recently shown that bone marrow adipocytes are a source of endogenous AhR ligands. Using multiple in vitro models of tumor-adipocyte crosstalk to mimic the bone microenvironment, we identified a novel, non-toxicological role of the adipocyte-secreted factors in the suppression of AhR activity in MM cells. A panel of six MM cell lines were cultured in the presence of bone marrow adipocytes in (1) a direct co-culture, (2) a transwell co-culture, or (3) an adipocyte-conditioned media to interrogate the effects of the secreted factors on MM cell AhR activity. Nuclear localization and the transcriptional activity of the AhR, as measured by CYP1A1 and CYP1B1 gene induction, were suppressed by exposure to BMA-derived factors. Additionally, decreased AhR target gene expression was associated with worse clinical outcomes. The knockdown of AhR resulted in reduced CYP1B1 expression and increased cellular growth. This tumor-suppressing role of CYP1A1 and CYP1B1 was supported by patient data which demonstrated an association between reduced target gene expression and worse overall survival. These data demonstrated a novel mechanism by which bone marrow adipocytes promote MM progression.

Ribosomal protein S3 mediates drug resistance of proteasome inhibitor: potential therapeutic application in multiple myeloma.

Multiple myeloma (MM) remains incurable due to drug resistance. Ribosomal protein S3 (RPS3) has been identified as a non-Rel subunit of NF-κB. However, the detailed biological roles of RPS3 remain unclear. Here, we report for the first time that RPS3 is necessary for MM survival and drug resistance. RPS3 was highly expressed in MM, and knockout of RPS3 in MM inhibited cell growth and induced cell apoptosis both in vitro and in vivo. Overexpression of RPS3 mediated the proteasome inhibitor resistance of MM and shortened the survival of MM tumor-bearing animals. Moreover, our present study found an interaction between RPS3 and the thyroid hormone receptor interactor 13 (TRIP13), an oncogene related to MM tumorigenesis and drug resistance. We demonstrated that the phosphorylation of RPS3 was mediated by TRIP13 via PKCδ, which played an important role in activating the canonical NF-κB signaling and inducing cell survival and drug resistance in MM. Notably, the inhibition of NF-κB signaling by the small-molecule inhibitor targeting TRIP13, DCZ0415, was capable of triggering synergistic cytotoxicity when combined with bortezomib in drug-resistant MM. This study identifies RPS3 as a novel biomarker and therapeutic target in MM.