Expression In Multiple Myeloma Cells Research Articles

Treg-derived TGF-β1 dampens cGAS-STING signaling to downregulate the expression of class I MHC complex in multiple myeloma

Multiple myeloma (MM) progression involves diminished tumor antigen presentation and an immunosuppressive microenvironment, characterized by diminished expression of major histocompatibility complexes (MHC) class I molecule and elevated programmed death ligand 1 (PDL1) in MM cells, along with an enriched population of regulatory T cells (Tregs). To investigate Treg's influence on MM cells, we established a co-culture system using Tregs from MM patients and the MM cell lines (MM.1S and SK-MM-1) in vitro and assessed the effects of intervening in the relevant pathways connecting Tregs and MM cells in vivo. In vitro, Tregs induced transforming growth factor beta-1 (TGF-β1) production, downregulated MHC I members, and increased PDL1 expression in MM cells. Treg-derived TGF-β1 suppressed the cGAS-STING pathway, contributing to the loss of MHC I molecule expression and PDL1 upregulation. Correspondingly, neutralizing TGF-β1 or activating the cGAS-STING pathway restored MHC I and PDL1 expression, effectively countering the pro-tumorigenic effect of Tregs on MM cells in vivo. These data elucidated how Tregs influence tumor antigen presentation and immunosuppressive signal in MM cells, potentially providing therapeutic strategies, such as neutralizing TGF-β1 or activating the cGAS-STING pathway, to address the immune escape and immunosuppressive dynamics in MM.

Abstract 7058: NSD2 overexpression drives multiple myeloma dependence on adenylate kinase 2 by diverting one-carbon metabolism to the epigenome

Abstract Chromosomal translocation (4;14), an adverse prognostic factor in multiple myeloma (MM), drives overexpression of the oncogenic histone methyltransferase NSD2. Genome-wide CRISPR screens in isogenic MM cells with high and low NSD2 expression identified adenylate kinase 2 (AK2), an enzyme critical for transport of ATP from the mitochondria, as an NSD2-driven vulnerability. AK2 suppression in t(4;14) MM cells decreased NADP(H) critical for conversion of ribonucleotides to deoxyribonucleosides by the enzyme ribonucleotide reductase (RNR). Consequently, metabolomic studies revealed depletion of deoxynucleotide triphosphates (dNTPs) in AK2-deficient t(4;14) MM cells, which resulted in replication stress, DNA damage and apoptosis. Exogenous supplementation with deoxynucleosides rescued AK2 depleted MM cells confirming the role of AK2 in dNTP homeostasis. In addition, NSD2 overexpression drives a large genome-wide increase in chromatin methylation, which depletes the methyl donor S-adenosylmethionine (SAM). Accordingly, we found that high NSD2 expression in MM cells disrupts creatine biosynthesis, a process dependent on SAM. The creatine/phosphocreatine system represents an alternative means of transporting high energy phosphate from the mitochondria. Hence, creatine supplementation restored NADP(H) levels and rescued AK2-deficient t(4;14) MM cells indicating that the hypersensitivity of these cells to AK2 depletion is driven by creatine deficiency due to NSD2 overexpression. Furthermore, AK2 inhibition impaired ATP-dependent protein folding increasing sensitivity of t(4;14) MM cells to proteasome inhibition. These findings delineate a novel mechanism in which aberrant transfer of carbon to the epigenome creates a metabolic vulnerability, with direct therapeutic implications for t(4;14) MM. Citation Format: Amin Sobh, Elena Encinas, Alisha Patel, Greeshma Surapaneni, Charlotte Kaestner, Janai Poullard, Monica Clerio, Karthik Vasan, Tzipporah Freeman, Dongwen Lv, Daphne Dupere-Richer, Alberto Riva, Benjamin Barwick, Daohong Zhou, Lawrence Boise, Constantine Mitsiades, Baek Kim, Richard Bennett, Navdeep Chandel, Jonathan Licht. NSD2 overexpression drives multiple myeloma dependence on adenylate kinase 2 by diverting one-carbon metabolism to the epigenome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7058.

SRRM2 may be a potential biomarker and immunotherapy target for multiple myeloma: a real-world study based on flow cytometry detection

Serine/arginine repetitive matrix 2 (SRRM2) has been implicated in tumorigenesis, cancer development, and drug resistance through aberrant splicing; however, its correlation with multiple myeloma (MM) has not been reported. We investigated the potential of SRRM2 as a biomarker and immunotherapeutic target in MM by examining its expression in MM cells using flow cytometry. Our study included 95 patients with plasma cell disease, including 80 MM cases, and we detected SRRM2 expression on plasma cells and normal blood cells to analyze its relationship with clinical profiles. We found widespread positive expression of SRRM2 on plasma cells with little expression on normal blood cells, and its expression on abnormal plasma cells was higher than that on normal plasma cells. Comparative analysis with clinical data suggests that SRRM2 expression on plasma cells correlates with MM treatment response. MM patients with high SRRM2 expression had higher levels of serum β2-mg and LDH, ISS staging, and plasma cell infiltration, as well as high-risk mSMART 3.0 stratification and cytogenetic abnormalities, particularly 1q21 amplification. In patients with previous MM, high SRRM2 expression on plasma cells was associated with higher plasma cell infiltration, high-risk mSMART 3.0 risk stratification, cytogenetic abnormalities, more relapses, and fewer autologous stem cell transplant treatments. In summary, SRRM2 may serve as a novel biomarker and immunotherapeutic target for MM. Its expression level on plasma cells can help in risk stratification of MM and monitoring of treatment response.

IGF2BP1 promotes multiple myeloma with chromosome 1q gain via increasing CDC5L expression in an m6A-dependent manner

Multiple myeloma (MM) patients with chromosome 1q gain (1q+) are clinically and biologically heterogeneous. The underlying molecular mechanisms are still under investigation, while the identification of targets for effective therapy of this subgroup of MM patients is urgently needed. We aimed to investigate the clinical significance and the regulatory mechanisms of insulin-like growth factor 2 messenger RNA (mRNA) binding protein 1 (IGF2BP1), a N6-methyladenosine (m6A) reader, in MM patients with 1q+. We found that MM patients with 1q+ exhibit a significantly higher level of IGF2BP1 mRNA than controls, while higher IGF2BP1 expression predicted a worse prognosis in MM patients with 1q+. IGF2BP1 overexpression promoted cell proliferation and G1-to-S phase transition of the cell cycle in NCI-H929 cells. Through comprehensive in silico analyses of existing public datasets and in-house generated high-throughput sequencing datasets, along with in vitro experiments, we identified CDC5L as a target of IGFBP1, which can bind to the m6A sites of CDC5L mRNA to up-regulate its protein abundance. Higher CDC5L expression also predicted a worse prognosis of MM patients with 1q+. Moreover, both knockdown and mutation of CDC5L attenuated the pro-proliferative effect of IGF2BP1. Furthermore, IGF2BP1 inhibitor BTYNB effectively inhibited CDC5L expression in MM cells with 1q+ and suppressed the proliferation of these cells in vitro and in vivo. Therefore, IGF2BP1 acts as a post-transcriptional enhancer of CDC5L in an m6A-dependent manner to promote the proliferation of MM cells with 1q+. Our work identified a novel IGF2BP1-CDC5L axis and provided new insight into developing targeted therapeutics for MM patients with 1q+.

Abstract C083: HDAC6 inhibition increases proteasome activity and modulates the myeloma immunopeptidome to promote cytotoxic T-cell activity

Abstract The proteasome functions as an essential component of immune surveillance by generating peptides from intracellular antigens that are then presented to cytotoxic T-cells. The vast majority of defined antitumor T-cell responses involves the proteasomal processing of intracellular proteins and their presentation in the context of major histocompatibility complex (MHC) class I molecules to antigenic peptide-specific CD8+ T-cells. Such antigenic peptides generated by the proteasome are generally 8-10 amino acids in length and mirror the linear sequence of the parental protein. However, immune evasion is a hallmark of cancer cells that disrupt the antigen processing and presentation machinery. Tumors develop strategies to evade host immunity and resist current immunotherapies, while others display a low tumor mutation burden limiting the presence of tumor antigenicity. Here, we postulated that pharmacologic enhancement of proteasome catalytic activity would increase MHC class I antigen presentation, modulate the myeloma immunopeptidome, and promote CD8+ T-cells anti-myeloma activity. A cell-based, high-throughput screen revealed that the histone deacetylase 6 (HDAC6) inhibitors tubastatin-A, ACY-738 and ACY-1215 increased proteasome activity in multiple myeloma (MM) cells. HDAC6 inhibitors increased pan-MHC class-I expression in MM cells as well as a panel of cancer cell lines. Treatment of MM patient tumor cells with HDAC6 inhibitors increased the cytotoxic effect of autologous patient-derived T-cells. Mechanism-based studies demonstrated that pharmacologic blockade or genetic ablation of the HDAC6 ubiquitin-binding (BUZ) domain disrupted HDAC6 association with HR23B, a substrate-shuttle factor that delivers ubiquitinylated cargo to proteasomes. HDAC6 inhibitors or HDAC6-BUZ domain deletion reduced the association of HR23B with HDAC6 and promoted binding of HR23B to proteasomes and a consequent increase in proteasome activity. The results validate the HDAC6-BUZ domain as an attractive drug discovery target and demonstrate that proteasome activators enhance antitumor immunity. Proteasome activators represent a paradigm-shifting approach to overcome mechanisms of immune escape in cancer cells. FDA-approved drugs that increase proteasome activity and boost antigen presentation and can be repositioned as cancer immunotherapeutics to overcome the existing bottlenecks in drug development. Taken together, our results highlight the breadth and magnitude of the proteasome in governing fundamental cellular processes and the immense potential of therapeutics that exploit proteasomes to treat human disease. Citation Format: Priyanka S. Rana, James J. Ignatz-Hoover, James J. Driscoll. HDAC6 inhibition increases proteasome activity and modulates the myeloma immunopeptidome to promote cytotoxic T-cell activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C083.

IGF2BP1 Promotes Multiple Myeloma with Chromosome 1q Gain Via Increasing CDC5L Expression in an m6A-Dependent Manner

Background: Multiple myeloma (MM) patients with chromosome 1q gain (1q+) are clinically and biologically heterogeneous. The underlying molecular mechanisms are still under investigation, while identification of targets for effective therapy of this subgroup of MM patients is urgently needed. We aimed to investigate the clinical significance and the regulatory mechanisms of insulin-like growth factor 2 messenger RNA (mRNA) binding proteins 1 (IGF2BP1), a N6-methyladenosine (m6A) reader, in MM patients with 1q+. Methods: Comprehensive in silico analyses of existed public datasets (MMRF CoMMpass study) and in-house generated high-throughput sequencing datasets (RNA-sequencing, methylated RNA immunoprecipitation sequencing, and crosslinking immunoprecipitation sequencing), along with routine molecular and cellular in vitro assays, as well as in vivo animal model studies, were conducted. Results: IGF2BP1 displays a high expression level in MM patients with 1q+ and its high expression predicts poor prognosis in these patients. IGF2BP1 overexpression promoted cell proliferation and G1-to-S phase transition of the cell cycle in H929 cells. Combinatory analyses of MeRIP-seq, RNA-seq, CLIP-seq and patients' survival data identified CDC5L as a target of IGF2BP1, which can bind the m6A sites of CDC5L mRNA to upregulate its protein abundance. Higher CDC5L expression also predicted worse prognosis of MM patients with 1q+. Moreover, both knockdown and mutation of CDC5L attenuated the pro-proliferative effect of IGF2BP1. Furthermore, IGF2BP1 inhibitor BTYNB effectively inhibited CDC5L expression in MM cells with 1q+, and suppressed the proliferation of these cells in vitro and in vivo. Conclusions: IGF2BP1 acts as a post-transcriptional enhancer of CDC5L in an m6A manner to promote the proliferation of MM cells with 1q+. Our work identified a novel IGF2BP1-CDC5L axis and provided new insight in developing targeted therapeutics for MM patients with 1q+.

Transcriptional Co-Activator P300 Promotes Extra-Medullary Disease Formation and Myeloma Disease Progression

The transcription co-activator P300 regulates gene expression by histone H3 lysine 27 acetylation (H3K27ac) of chromatin and transcriptional regulators. P300 has been suggested to control a variety of biological functions, including differentiation, cell-cycle control, proliferation, apoptosis, and DNA damage response. The clinicopathologic/prognostic importance of P300 in multiple myeloma (MM) is unknown to date. P300 has been linked to the induction of epithelial-to-mesenchymal transition (EMT) and metastasis in a variety of solid tumor malignancies. Like EMT in solid tumors, extra-medullary disease (EMD) in MM relates to a poor prognosis and resistance to treatment. Similarly, the molecular changes concerning EMD in MM are poorly understood, and the role of P300 in MM EMD is yet to be investigated. We have employed chemical and functional genomics to unravel the role of P300 in MM disease progression and EMD formation. Six human MM cell lines-MM1S, KMS-18, H929, U266, OPM-2, and AMO-1-were used to construct lentiviral shRNA-mediated P300 knockdown (K/D) cells. In addition, to analyzing the effect of time-dependent P300 degradation, we have used a proteolysis-targeting chimera (PROTAC) compound termed “JQAD1” that selectively targets P300 for degradation. Treatment with JQAD1 effectively reduced the survival with an IC 50 of 50 nM in MM1S cells treated for 72 h. Furthermore, we wanted to understand the mechanism of P300 degradation-mediated inhibition of cell proliferation. Cell cycle analysis revealed a 40% increase in G0/G1 phase cell cycle arrest following treatment with 50 nM JQAD1 for 72 h. Blocking CD138 receptors is known to enhance MM sensitivity to proteasome inhibitors and significantly reduce tumor size. Immuno-flowcytometry analysis revealed that loss of P300 reduces CD138 expression in MM cells, suggesting their susceptibility to proteasome inhibitors. Next, we wanted to understand if P300 plays a role in developing EMD in MM. Boyden chamber-based migration and invasion assays exhibited reduced migration and invasion of P300 K/D cells with respect to control cells. Furthermore, to understand the distribution and localization of P300 silenced MM cells in vivo, we have used immune deficient NSG mice and studied MM progression after intravenous tumoral injection. In vivo studies revealed that P300 silencing led to reduced MM liver, kidney homing, and EMD formation, as observed through bioluminescence imaging, histology, and flow cytometry analysis, respectively. Further investigations are currently underway to find novel interaction partners and potential targets of P300 in MM and to delineate its mechanism of action. In conclusion, our research indicates that P300 is critical for the development of EMD and the progression of MM. This could potentially serve as a new therapeutic target for MM relapse and dissemination.

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

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

Identification of LPAR1/LPAR5 As Novel GPCR Partners of GPRC5D for the Efficient CAR-T Therapy of Multiple Myeloma

Introduction: The emergence of chimeric antigen receptor T-cell (CAR T) therapy has brought hope to the treatment of refractory or relapsed multiple myeloma (RRMM). GPRC5D is an orphan G protein-coupled receptor (GPCR), which has a seven-transmembrane structure, and really high expression in multiple myeloma cells. So that CAR-T cells that target GPRC5D and this has been validated in a phase I/II clinical trials with encouraging clinical efficacy and manageable safety profiles of anti-GPRC5D CAR T-cell therapy for RRMM. It is of great clinical significance to find ways to further improve the efficacy of anti-GPRC5D CAR T-cell therapy. Methods: Our previous study based on a single-cell transcriptome analysis revealed a strong co-expression correlation of GPRC5D and lysophosphatidic acid receptor 1/5 (LPAR1/5). To further confirm the interaction with GPRC5D, LPAR1 and LPAR5 were tested by co-immunoprecipitation (co-IP) screening, bimolecular fluorescence complementation (BiFC), multiple pharmacological ELISA assays, and flow cytometry (FACS) experiments. The MM cell lines (RPMI 8226, L363 and KMS11) were treated with activator or antagonists of LPAR1 or LPAR5, and then co-cultured with anti-GPRC5D CAR T-cells. The cytotoxicity and cytokine analysis were determined by luciferase-base killing assay and FACS. Results: The results showed the obvious interactions between LPAR1/LPAR5 and GPRC5D from co-IP and BiFC assays, strongly supported the prediction from the bioinformatic analysis. The heterodimers of GPRC5D and LPAR1/LPAR5 influenced membrane expression of GPRC5D to different degrees based on ELISA assay. The HEK293T surface expression of GPRC5D was reduced in the presence of LPA receptors activator (1-Oleoyl lysophosphatidic acid, 1-Oleoyl-LPA), which indicated that both LPAR1 and LPAR5 had trafficking effect on GPRC5D. To further confirm these effects, the MM cell lines, RPMI 8226, L363 and KMS11, were cultured with 1-Oleoyl-LPA or combining antagonists of LPAR1 (AM966) or LPAR5 (TC LPA5 4). The cell-surface change of GPRC5D expression had no statistical difference when MM cells were only treated with 1-Oleoyl-LPA. However, the surface expression of GPRC5D of MM cell lines was increased with the combination of LPAR1/LPAR5 activator and antagonists by FACS detection To further investigate the above effects could promote the efficacy of anti-GPRC5D CAR T-cell therapy, MM cell lines and anti-GPRC5D CAR T-cells were co-cultured, with or without the treatment of activator and antagonists. The results showed that the CAR T-cell killing capacity to MM cell lines was reduced after the treatment of 1-Oleoyl-LPA, while its killing capacity was significantly recovered after performing a combination of 1-Oleoyl-LPA and AM966/TC LPA5 4. Notably, the levels of IL-2 and TNF-α in 1-Oleoyl-LPA groups were lower than the mock groups, and increased after the combination of AM966/TC LPA5 4. But, IFN-γ did not display significant difference. These data indicated that the regulation of LPAR1 and LPAR5 function might have enhance the efficacity in anti-GPRC5D CAR T-cell therapy. Conclusions: Our study provides hint evidence that LPAR1 and LPAR5 increase the MM cell-surface expression of GPRC5D and improve the efficacy of anti-GPRC5D CAR T-cell therapy, which might be as novel GPCR partners of GPRC5D for the efficient CAR-T therapy of multiple myeloma.

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

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

The Spop/BRD4/c-Myc Axis Modulates IMiDs Sensitivity in Multiple Myeloma

The development of novel agents including immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) has led to improved patient outcomes in multiple myeloma (MM); however, MM patients inevitably experience relapse and drug resistance. The expression level of CRBN, CRBN-binding proteins, and CRL4 CRBN ubiquitin ligase is considered to be associated with IMiDs resistance. Clinical studies have shown that MM patients can exhibit IMiDs resistance even without CRBN abnormalities, suggesting the molecular mechanisms of intrinsic resistance to IMiDs are not fully understood. To delineate the molecular mechanisms underlying IMiDs resistance, we performed genome-wide knockout (KO) screening in IMiDs-sensitive MM cells. For in vitro work, we used the MM cell lines MM1S, and H929. We used sgRNA to KO SPOP and BRD4 in MM cells. After performing a CRISPR KO screen in MM cells, we discovered that in addition to CRBN and its associated genes, sgRNAs targeting SPOP (speckle-type POZ protein, a speckled zinc finger protein) were highly enriched after treatment with Poma. Importantly, SPOP KO MM cell lines acquired significant resistance to Pom and Len treatment. To examine whether SPOP KO-induced IMiDs resistance was CRBN-pathway dependent, we assessed CRBN and its downstream interacting protein levels. SPOP KO showed no effect on CRBN expression; moreover, IMiDs can still trigger IKZF1 and IKZF3 degradation, associated with the downregulation of IRF4, suggesting that SPOP mediates sensitivity to IMiDs in a mechanism independent of CRBN-IKZF1/3 axis.Compared to plasma cells from normal donors, we found that the expression levels of SPOP were significantly decreased in MGUS patients, smoldering myeloma patients, newly diagnosed MM patients, and refractory relapsed MM patients (p-values: 2.4e-2, 2.0e-4, 9.9e-3, 3.17e-6, respectively). SPOP is the adaptor protein of the Cullin3-RING ubiquitin ligase complex. Therefore, the function of SPOP is determined by the characteristics of the substrates it recruits. We performed immunoprecipitation with overexpressed SPOP in MM cells, followed by mass spectrometry analysis, and identified BRD4 as a candidate protein binding to SPOP in MM, which was confirmed by Co-IP. KO of SPOP led to an upregulation of BRD4 protein levels in MM cells. As a downstream regulator of BRD4, the oncogene c-Myc showed a significant increase in protein expression in IMiDs-resistant MM patients. Furthermore, the KO of SPOP notably enhanced c-Myc expression in MM cells, while the KO of BRD4 in SPOP KO MM cells decreaed c-Myc expression and restored MM cell sensitivity to IMiD treatment. These findings suggest that SPOP modulates c-Myc expression through BRD4 in a CRBN-IKZF1/3 independent manner, thereby mediating MM cell sensitivity to IMiDs. BRD4 plays a significant role in the development and progression of various tumors. The BRD4 inhibitor JQ1 has been shown to exhibit anti-tumor effects in multiple types of cancer. We found that combination treatment with JQ-1 and IMiDs reversed IMiDs resistance of SPOP KO MM cell lines and primary MM cells from relapsed patients. Our data show that SPOP is a CRBN-independent modulator of IMiDs sensitivity by regulating the BRD4/c-Myc axis and provides the preclinical rationale for combining IMiDs with BRD4 inhibitors to overcome IMiDs resistance and improve patient outcomes.

Unrevealed roles of extracellular enolase‑1 (ENO1) in promoting glycolysis and pro‑cancer activities in multiple myeloma via hypoxia‑inducible factor 1α.

The involvement of enolase‑1 (ENO1), intracellularly or extracellularly, has been implicated in cancer development. Moreover, anticancer activities of an ENO1‑targeting antibody has demonstrated the pathological roles of extracellular ENO1 (surface or secreted forms). However, although ENO1 was first identified as a glycolytic enzyme in the cytosol, to the best of our knowledge, extracellular ENO1 has not been implicated in glycolysis thus far. In the present study, the effects of extracellular ENO1 on glycolysis and other related pro‑cancer activities were investigated in multiple myeloma (MM) cells invitro and invivo. Knockdown of ENO1 expression reduced lactate production, cell viability, cell migration and surface ENO1 expression in MM cells. Notably, addition of extracellular ENO1 protein in cancer cell culture enhanced glycolytic activity, hypoxia‑inducible factor 1‑α (HIF‑1α) expression, glycolysis‑related gene (GRG) expression and pro‑cancer activities, such as cell migration, cell viability and tumor‑promoting cytokine secretion. Consistently, these extracellular ENO1‑induced cellular effects were inhibited by an ENO1‑specific monoclonal antibody (mAb). In addition, extracellular ENO1‑mediated glycolysis, GRG expression and pro‑cancer activities were also reduced by HIF‑1α silencing. Lastly, administration of an ENO1 mAb reduced tumor growth and serum lactate levels in an MM xenograft model. These results suggested that extracellular ENO1 (surface or secreted forms) enhanced a HIF‑1α‑mediated glycolytic pathway, in addition to its already identified roles. Therefore, the results of the present study highlighted the therapeutic potential of ENO1‑specific antibodies in treating MM, possibly via glycolysis inhibition, and warrant further studies in other types of cancer.

High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma

Multiple myeloma (MM) growth is supported by an immune-tolerant bone marrow microenvironment. Here, we find that loss of Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) in tumor microenvironmental cells is associated with MM growth suppression. The absence of NEK2 leads to both fewer tumor-associated macrophages (TAMs) and inhibitory Tcells. NEK2 expression in myeloid progenitor cells promotes the generation of functional TAMs when stimulated with MM conditional medium. Clinically, high NEK2 expression in MM cells is associated with increased CD8+ T effector memory cells, while low NEK2 is associated with an IFN-γ gene signature and activated Tcell response. Inhibition of NEK2 upregulates PD-L1 expression in MM cells and myeloid cells. In a mouse model, the combination of NEK2 inhibitor INH154 with PD-L1 blockade effectively eliminates MM cells and prolongs survival. Our results provide strong evidence that NEK2 inhibition may overcome tumor immune escape and support its further clinical development.

Inhibitory Effect of Resveratrol on the Proliferation of Multiple Myeloma Cells and the Underlying Mechanism

To investigate the effect of resveratrol (RSV) on the proliferation of multiple myeloma (MM) cells and its molecular mechanism. MM cells (MM1.S, RPMI-8226 and U266) were treated with different concentrations of RSV for 24-72 h. The effect of RSV on the proliferation of MM cells was detected by CCK-8 (cell counting kit-8) assay. RPMI-8226 cells were divided into RSV, miR-21 mimic, RSV+miR-21 mimic, miR-21 inhibitor and RSV+miR-21 inhibitor groups, and transfected with corresponding plasmids. The cell cycle distribution of each group was detected by flow cytometry with propidium iodide (PI) single staining. The cell apoptosis of each group was detected by AnnexinV-FITC/PE-PI double staining. The expression of miR-21 in MM cells treated with RSV and the expression of KLF5 mRNA in each group were detected by qRT-PCR. The expression of KLF5 protein in each group was detected by Western blot. RSV inhibited the proliferation and induced apoptosis of MM cells in a time- and dose-dependent manner. After the MM cells were treated with RSV, the number of cells in sub-G1 phase was increased, and that in G2/M phase was decreased. Moreover, RSV significantly downregulated the expression of miR-21 in MM cells, and the inhibitory effect of miR-21 mimic on KLF5 expression in MM cells was counteracted by RSV. RSV may inhibit the proliferation and induce apoptosis of MM cells by inhibiting miR-21 and up-regulating KLF5 expression.

Ginsenoside Rh4 inhibits the malignant progression of multiple myeloma and induces ferroptosis by regulating SIRT2.

Multiple myeloma (MM) has a high mortality rate, and the exploration of therapeutic drugs for MM with low side effects is a hot topic at the moment. Ginsenoside Rh4 has been shown to inhibit tumour growth in many cancers. However, the role of ginsenoside Rh4 in MM and its reaction mechanism have not been reported so far. After the treatment with different concentrations of ginsenoside Rh4, the proliferation of NCI-H929 cells was detected by Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine staining. The cell apoptosis and cycle arrest were detected by flow cytometry and western blot. The thiobarbituric acid-reactive substances (TBARS) production was assessed with TBARS assay, whereas Fe2+ fluorescence assay was used for the measurement of Fe2+ level. The production of reactive oxygen species was evaluated with dichloro-dihydro-fluorescein diacetate staining, and western blot was applied for the estimation of ferroptosis-related proteins. The potential targets of ginsenoside Rh4 were predicted by molecular docking technology and verified by western blot. The transfection efficacy of overexpression-SIRT2 was examined with quantitative reverse transcription polymerase chain reaction and western blot. To figure out the detailed reaction mechanism between ginsenoside Rh4 and SIRT2 in MM, rescue experiments were conducted. We found that ginsenoside Rh4 inhibited cell proliferation, induced cell apoptosis, promoted cycle arrest and facilitated ferroptosis in MM. Moreover, ginsenoside Rh4 inhibited SIRT2 expression in MM cells. The overexpression of SIRT2 reversed the effects of ginsenoside Rh4 on cell proliferation, cell apoptosis, cycle arrest and ferroptosis in MM. Overall, ginsenoside Rh4 inhibited the malignant progression of MM and induced ferroptosis by regulating SIRT2.

Abstract 2505: PYCR1 inhibition in multiple myeloma-associated stroma limits tumor growth

Abstract Introduction Multiple myeloma (MM) is a hematological cancer, characterized by the accumulation of monoclonal plasma cells in the bone marrow. It remains an incurable cancer due to drug resistance, wherein the bone marrow microenvironment plays a crucial role. We have previously shown that glutamine-to-proline conversion is upregulated in MM cells upon hypoxic culture. Moreover, inhibition of proline production by blocking its converting enzyme PYCR1 successfully reduced proliferation and viability in vitro. Importantly, PYCR1 inhibition combined with standard-of-care agent bortezomib decreased tumor load in vivo. As PYCR1 is also highly expressed in stromal cells and proline an important component of extracellular matrix proteins, we investigated whether PYCR1 targeting in stromal cells affects MM viability and its structural microenvironment. Material and methods PYCR1 expression was investigated by the use of microarray data from the Heidelberg/Montpellier cohort. For in vitro experiments, the human MM cell line OPM-2 and human stromal cell line HS-5 were used. CD138+ and CD138- fractions from primary patient samples were separated by MACS. To obtain primary stromal cells, the CD138- fraction was plated out in fresh medium. After 48h, the medium was refreshed and all adherent cells were further cultured and used as primary stromal cells. PYCR1 expression in HS-5 cells was reduced through siRNA. Hypoxic culture (<1% O2) was established through chambers. RNA expression was measured by RT-qPCR, while protein expression was measured by western blot. Viability was assessed by CellTiterGlo assay. Results Gene expression analysis shows high RNA expression of PYCR1 in healthy bone marrow plasma cells (BMPCs), plasma cells from monoclonal gammopathy of undetermined significance (MGUS) and MM patients, but also in bone marrow stromal cells (BMSCs). PYCR1 expression was low or absent in other cell types, including T cells, osteoclasts, monocytic and granulocytic cells. On protein level, we also confirmed PYCR1 expression in MM cells (CD138+), CD138- fraction (including stromal cells) and cultured primary stromal cells. Moreover, PYCR1 expression increased upon hypoxic culture in primary stromal cells and stromal cell line HS-5. PYCR1 knockdown in stromal cell line HS-5 did not affect viability of the stromal cells, but its conditioned medium did reduce its protective effects when OPM-2 MM cells were treated with standard-of-care agent bortezomib. Further investigation revealed that knockdown of PYCR1 in HS-5 reduces RNA expression of structural proteins col1a1, col1a2, col3a1, ctgf and acta2 in HS-5. Conclusion PYCR1 is highly expressed in myeloma cells and stromal cells. PYCR1 inhibition in stromal cells reduces its proliferative effect on myeloma cells when combined with bortezomib. Preliminary data indicates a link between PYCR expression in stromal cells and rearrangement of extracellular matrix proteins. Citation Format: Inge Oudaert, Catharina Muylaert, Hatice Satilmis, Sylvia Faict, Kim De Veirman, Elke De Bruyne, Karin Vanderkerken, Eline Menu. PYCR1 inhibition in multiple myeloma-associated stroma limits tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2505.

All-trans retinoic acid improves NSD2-mediated RARα phase separation and efficacy of anti-CD38 CAR T-cell therapy in multiple myeloma

BackgroundImmunotherapies targeting CD38 have demonstrated salient efficacy in relapsed/refractory multiple myeloma (MM). However, loss of CD38 antigen and outgrowth of CD38 negative plasma cells have emerged as a major obstacle...

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

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

Suppression of Multiple Myeloma Via Inhibition of EFNB2 Reverse Signaling

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

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

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