Proliferation Of Multiple Myeloma Cells Research Articles

LncRNA MALAT1 silencing represses CXCL12-induced proliferation, invasion, and homing behavior in multiple myeloma by inhibiting CXCR4

ABSTRACT Background: Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been identified in multiple myeloma (MM); its influence on the homing behavior in MM cells is unclear. This study elucidates the impact and mechanism by which MALAT1 affects the homing behavior in MM cells. Methods: Bone marrow was obtained from patients with MM. MALAT1 and C-X-C motif chemokine receptor 4 (CXCR4) expression in cluster of differentiation 138-purified (CD138) plasma cells were detected by qRT-PCR and Western blotting. MALAT1, CXCR4, and C-X-C motif chemokine ligand 12 (CXCL12) influences on MM cell viability, proliferation, and invasion was monitored by CCK-8, 5-Ethynyl-2'-deoxyuridine, and Transwell assays. qRT-PCR, Western blotting, and ELISA were utilized to detect homing effect-related proteins in MM cells, including intercellular adhesion molecule 1 (ICAM-1) and very late antigen-4 (VLA-4). Results: MALAT1 and CXCR4 were overexpressed in CD138-purified plasma cells from bone marrow of MM patients, associating with bone damage. MALAT1 upregulated CXCR4 in MM cells. MALAT1 overexpression enhanced MM cell viability, proliferation, and invasion, whereas CXCR4 silencing reversed them. CXCR4 silencing attenuated MALAT1 induction on ICAM-1 and VLA-4 expression in MM cells. CXCL12 upregulation intensified MM cell proliferation and invasion and ICAM-1 and VLA-4 expression in MM cells. MALAT1 silencing counteracted the impact of CXCL12. Conclusion: MALAT1 silencing may repress CXCL12 induction on MM cell proliferation, invasion, and homing behavior by inhibiting CXCR4. MALAT1 may be a promising target for MM treatment.

ZNF655 involved in the progression of multiple myeloma via the activation of AKT.

Multiple myeloma (MM) is an incurable hematological malignancy, and the number of MM patients is increasing year by year. Zinc finger protein 655 (ZNF655) has been shown to regulate various biological processes and is implicated in the progression of many diseases. However, the roles of ZNF655 in MM progression remains unclear. In this study, we aimed to explore the effects of ZNF655 on progression by detecting the alteration of the phenotypes and tumorigenesis induced by ZNF655 knockdown in MM. The expression level of ZNF655 in MM was clarified by real-time quantitative polymerase chain reaction assays. Furthermore, loss-of-function assays in vitro and in vivo was investigated the biological functions of ZNF655 in MM. These findings revealed that ZNF655 depletion remarkably inhibited MM cell proliferation, arrested cell cycle, and induced cell apoptosis. Mechanistically, ZNF655 was found to regulate AKT in MM. In conclusion, this study indicated that ZNF655 regulated the progression of MM via AKT activation and downregulation of ZNF655 may be a promising antitumor strategy in MM.

Discovery of Novel SIRT3 Inhibitors for the Cancer Differentiation Therapy by Structural Modification.

Inhibition of SIRT3 triggered differentiation of multiple myeloma (MM) cells. In discovery of potent SIRT3 inhibitors for cancer differentiation therapy, structural modification was performed on the previously developed lead compound S27. A total of 49 compounds divided into two series were designed and synthesized. In the enzyme inhibitory assay, several molecules (A7, A13, B15, and B26) exhibited potent SIRT3 inhibitory activity and selectivity. Significantly, representative compounds, especially A7, promoted differentiation of MM cells from cancer phenotype to normal cells, accompanied by increased expression of antigen CD49e, human immunoglobulin light chain λ-IgLG and κ-IgLG. Additionally, molecule A7 reversed growth factor IL-6 induced MM cell proliferation, improved the antiproliferative activity of Ixazomib and increased the apoptotic rate of MM cells treated with Ixazomib. Collectively, potent SIRT3 inhibitors with MM cell differentiation potency were developed for the cancer therapy used alone or in combination.

Enitociclib, a selective CDK9 inhibitor: in vitro and in vivo preclinical studies in multiple myeloma

Multiple myeloma (MM) is a cancer of the plasma cells that remains incurable despite advances in treatment options. In this study, a library of 216 clinically feasible small-molecule inhibitors was screened to identify agents that selectively inhibit MM cell proliferation. Enitociclib, a CDK9-specific small-molecule inhibitor, was found to be highly effective at decreasing cell viability and inducing apoptosis in four MM cell lines. Enitociclib inhibited the phosphorylation of the CTD of RNA Pol II at Ser2/Ser5 and repressed the protein expression of oncogenes c-Myc, Mcl-1, and PCNA in MM cells. Additionally, enitociclib demonstrated synergistic effects with several anti-MM agents, including bortezomib, lenalidomide, pomalidomide, and venetoclax. These results suggest that enitociclib may represent a promising therapeutic option for the treatment of MM, either as a single agent or in combination with other anti-MM agents.

Effect of JMJD3-IRF4 Signaling Pathway-Mediated Macrophage Polarization on the Malignant Biological Behavior of Multiple Myeloma Cells

To investigate the effect of macrophage polarization mediated by Jumonji domain containing-3 (JMJD3)-interferon regulatory factor 4 (IRF4) signaling pathway on the malignant biological behavior of multiple myeloma (MM) cells. THP-1 monocytes were induced to differentiate into macrophages by phorbol myristate acetate (PMA). THP-1 macrophages were divided into control group (normal culture), M2 induction group ［added recombinant human interleukin (IL) -4, IL-13 proteins］, M2+JMJD3 protein group (added recombinant human IL-4, IL-13 and JMJD3 proteins) and M2+JMJD3 inhibitor group (added recombinant human IL-4, IL-13 proteins and JMJD3 inhibitor), the proportion of CD206+ cells was detected by flow cytometry, the levels of IL-10 and transforming growth factor-β (TGF-β) in the culture supernatant were detected by ELISA assay, the expression levels of arginase-1 (Arg-1), JMJD3 and IRF4 mRNA were detected by real-time quantitative PCR (qRT-PCR), and the expression levels of Arg-1, JMJD3 and IRF4 proteins were detected by Western blot. Correspondingly, human MM cells U266 were cultured with THP-1 macrophage culture supernatant of each group, Methyl thiazolyl tetrazolium (MTT) method and plate colony formation assay were used to detect cell proliferation, cell apoptosis was detected by flow cytometry, Western blot was used to detect the expression levels of apoptosis-promoting protein Bcl-2-associated X protein (Bax) and cleaved caspase-3 in cells, and Transwell assay was used to detect cell migration and invasion. Compared with the control group, the proportion of CD206+ cells in THP-1 macrophages, the mRNA and protein expression levels of Arg-1, JMJD3 and IRF4, and the levels of IL-10 and TGF-β in the cell culture supernatant in M2 induction group were significantly increased (P <0.001), meanwhile, the proliferation activity and the number of clones of U266 cells were significantly increased (P <0.01), the apoptosis rate and the expression levels of apoptosis-promoting protein Bax and cleaved caspase-3 were significantly decreased (P <0.001), the numbers of migrated cells and invasive cells were increased (P <0.001). Compared with M2 induction group, the proportion of CD206+ cells in THP-1 macrophages, the mRNA and protein expression levels of Arg-1, JMJD3 and IRF4, and the levels of IL-10 and TGF-β in the cell culture supernatant in M2+JMJD3 protein group were further increased (P <0.01), meanwhile, the proliferation activity and the number of clones of U266 cells were further increased (P <0.05), the apoptosis rate and the expression levels of apoptosis-promoting protein Bax and cleaved caspase-3 were further decreased (P <0.01), the numbers of migrated cells and invasive cells were further increased (P <0.001); However, the change trends of the above indexes in M2+JMJD3 inhibitor group were opposite to those in M2+JMJD3 protein group. M2 polarization of macrophages mediated by JMJD3-IRF4 signaling pathway can promote the proliferation, migration and invasion of MM cells, and inhibit cell apoptosis.

DNp73 enhances tumor progression and immune evasion in multiple myeloma by targeting the MYC and MYCN pathways.

Multiple myeloma (MM) is an incurable hematological malignancy with high chromosome instability and heavy dependence on the immunosuppressive bone marrow microenvironment. P53 mutations are adverse prognostic factors in MM; however, clinically, some patients without P53 mutations also exhibit aggressive disease progression. DNp73, an inhibitor of TP53 tumor suppressor family members, drives drug resistance and cancer progression in several solid malignancies. Nevertheless, the biological functions of DNp73 and the molecular mechanisms in myelomagenesis remain unclear. The effects of DNp73 on proliferation and drug sensitivity were assessed using flow cytometry and xenograft models. To investigate the mechanisms of drug resistance, RNA-seq and ChIP-seq analyses were performed in MM cell lines, with validation by Western blot and RT-qPCR. Immunofluorescence and transwell assays were used to assess DNA damage and cell invasion in MM cells. Additionally, in vitro phagocytosis assays were conducted to confirm the role of DNp73 in immune evasion. Our study found that activation of NF-κB-p65 in multiple myeloma cells with different p53 mutation statuses upregulates DNp73 expression at the transcriptional level. Forced expression of DNp73 promoted aggressive proliferation and multidrug resistance in MM cells. Bulk RNA-seq analysis was conducted to assess the levels of MYCN, MYC, and CDK7. A ChIP-qPCR assay was used to reveal that DNp73 acts as a transcription factor regulating MYCN gene expression. Bulk RNA-seq analysis demonstrated increased levels of MYCN, MYC, and CDK7 with forced DNp73 expression in MM cells. A ChIP-qPCR assay revealed that DNp73 upregulates MYCN gene expression as a transcription factor. Additionally, DNp73 promoted immune evasion of MM cells by upregulating MYC target genes CD47 and PD-L1. Blockade of the CD47/SIRPα and PD-1/PD-L1 signaling pathways by the SIRPα-Fc fusion protein IMM01 and monoclonal antibody atezolizumab significantly restored the anti-MM activity of macrophages and T cells in the microenvironment, respectively. In summary, our study demonstrated for the first time that the p53 family member DNp73 remarkably induces proliferation, drug resistance, and immune escape of myeloma cells by directly targeting MYCN and regulating the MYC pathway. The oncogenic function of DNp73 is independent of p53 status in MM cells. These data contribute to a better understanding of the function of TP53 and its family members in tumorigenesis. Moreover, our study clarified that DNp73 overexpression not only promotes aggressive growth of tumor cells but, more importantly, promotes immune escape of MM cells through upregulation of immune checkpoints. DNp73 could serve as a biomarker for immunotherapy targeting PD-L1 and CD47 blockade in MM patients.

Molecular Signatures of CB-6644 Inhibition of the RUVBL1/2 Complex in Multiple Myeloma.

Multiple myeloma is the second most hematological cancer. RUVBL1 and RUVBL2 form a subcomplex of many chromatin remodeling complexes implicated in cancer progression. As an inhibitor specific to the RUVBL1/2 complex, CB-6644 exhibits remarkable anti-tumor activity in xenograft models of Burkitt's lymphoma and multiple myeloma (MM). In this work, we defined transcriptional signatures corresponding to CB-6644 treatment in MM cells and determined underlying epigenetic changes in terms of chromatin accessibility. CB-6644 upregulated biological processes related to interferon response and downregulated those linked to cell proliferation in MM cells. Transcriptional regulator inference identified E2Fs as regulators for downregulated genes and MED1 and MYC as regulators for upregulated genes. CB-6644-induced changes in chromatin accessibility occurred mostly in non-promoter regions. Footprinting analysis identified transcription factors implied in modulating chromatin accessibility in response to CB-6644 treatment, including ATF4/CEBP and IRF4. Lastly, integrative analysis of transcription responses to various chemical compounds of the molecular signature genes from public gene expression data identified CB-5083, a p97 inhibitor, as a synergistic candidate with CB-6644 in MM cells, but experimental validation refuted this hypothesis.

Identification of USP2 as a novel target to induce degradation of KRAS in myeloma cells

Inducing the degradation of KRAS represents a novel strategy to combat cancers with KRAS mutation. In this study, we identify ubiquitin-specific protease 2 (USP2) as a novel deubiquitinating enzyme of KRAS in multiple myeloma (MM). Specifically, we demonstrate that gambogic acid (GA) forms a covalent bond with the Cysteine 284 residue of USP2 through an allosteric pocket, inhibiting its deubiquitinating activity. Inactivation or knockdown of USP2 leads to the degradation of KRAS, resulting in the suppression of MM cell proliferation in vitro and in vivo. Conversely, overexpressing USP2 stabilizes KRAS and partially abrogates GA-induced apoptosis in MM cells. Furthermore, elevated USP2 levels may be associated with poorer prognoses in MM patients. These findings highlight the potential of the USP2/KRAS axis as a therapeutic target in MM, suggesting that strategically inducing KRAS degradation via USP2 inhibition could be a promising approach for treating cancers with KRAS mutations.

Interference with PLA2G16 promotes cell cycle arrest and apoptosis and inhibits the reprogramming of glucose metabolism in multiple myeloma cells by modulating the Hippo/YAP signaling pathway.

Multiple myeloma, which is a clonal plasma cell tumor, derives from a postmitotic lymphoid B-cell lineage and remains untreatable. Group XVI phospholipase A2 (PLA2G16) can either be a tumor suppressor or an oncogene in different types of cancer. This study was intended to explore the role of PLA2G16 in multiple myeloma and to reveal the reaction mechanism. The mRNA and protein expressions of PLA2G16 in human bone marrow stromal cell line HS-5 and multiple myeloma cells were assessed using reverse transcription-quantitative PCR and western blot. The transfection efficacy of sh-PLA2G16 and oe-YAP was examined using reverse transcription-quantitative PCR and western blot. Through cell counting kit-8 assay and 5-ethynyl-2'- deoxyuridine staining, multiple myeloma cell viability and proliferation were detected. Flow cytometry was used to measure cell apoptosis and cell cycle distribution. Oxygen consumption rate, the activities of mitochondrial respiratory chain complexes I-V, and the activity of caspase-3 were estimated with Seahorse XF24 analyzer, oxidative phosphorylation activity assay kit, and caspase-3 assay kit, respectively. Lactate production and glucose consumption were evaluated usingcorresponding assay kits. Western blot was employed to meaure proteins associated with cell cycle, glycolysis, pentose phosphate pathway as well as Hippo/YAP signaling pathway. In this study, PLA2G16 expression was greatly increased in multiple myeloma cells and PLA2G16 silence inhibited cell proliferation, promoted cell apoptosis, facilitated cell cycle arrest, and suppressed the reprogramming of glucose metabolism in multiple myeloma. It was also identified that PLA2G16 depletion inhibited the Hippo/YAP signaling pathway. Further experiments revealed that the overexpression of YAP partially reversed the inhibitory effects of PLA2G16 silence on multiple myeloma cell malignant development and the reprogramming of glucose metabolism. Collectively, PLA2G16 silence impeded multiple myeloma progression and inhibited glucose metabolism reprogramming by blocking the Hippo/YAP signaling pathway.

LILRB4 regulates multiple myeloma development through STAT3-PFKFB1 pathway

Although multiple myeloma (MM) responds well to immunotherapeutic treatment, certain portions of MM are still unresponsive or relapse after immunotherapy. Other immune molecules are needed for the immunotherapy of MM. Here, we revealed that leukocyte immunoglobulin-like receptor B4 (LILRB4) was highly expressed in multiple myeloma cell lines and patient samples and that the expression of LILRB4 was adversely correlated with the overall survival of MM patients. Knockdown of LILRB4 efficiently delayed the growth of MM cells both in vitro and in vivo. Mechanistically, IKZF1 transactivated LILRB4 expression to trigger the downstream of STAT3-PFKFB1 pathways to support MM cell proliferation. Blockade of LILRB4 signaling by blocking antibodies can effectively inhibit MM progression. Our data show that targeting LILRB4 is potentially an additional therapeutic strategy for the immunotherapeutic treatment of MM.

Clotrimazole inhibits growth of multiple myeloma cells in vitro via G0/G1 arrest and mitochondrial apoptosis

Patients with multiple myeloma (MM) experience relapse and drug resistance; therefore, novel treatments are essential. Clotrimazole (CTZ) is a wide-spectrum antifungal drug with antitumor activity. However, CTZ’s effects on MM are unclear. We investigated CTZ’s effect on MM cell proliferation and apoptosis induction mechanisms. CTZ’s effects on MM.1S, NCI- H929, KMS-11, and U266 cell growth were investigated using Cell Counting Kit-8 (CCK-8) assay. The apoptotic cell percentage was quantified with annexin V-fluorescein isothiocyanate/7-amino actinomycin D staining. Mitochondrial membrane potential (MMP) and cell cycle progression were evaluated. Reactive oxygen species (ROS) levels were measured via fluorescence microscopy. Expression of apoptosis-related and nuclear factor (NF)-κB signaling proteins was analyzed using western blotting. The CCK-8 assay indicated that CTZ inhibited cell proliferation based on both dose and exposure time. Flow cytometry revealed that CTZ decreased apoptosis and MMP and induced G0/G1 arrest. Immunofluorescence demonstrated that CTZ dose-dependently elevated in both total and mitochondrial ROS production. Western blotting showed that CTZ enhanced Bax and cleaved poly ADP-ribose polymerase and caspase-3 while decreasing Bcl-2, p-p65, and p-IκBα. Therefore, CTZ inhibits MM cell proliferation by promoting ROS-mediated mitochondrial apoptosis, inducing G0/G1 arrest, inhibiting the NF-κB pathway, and has the potential for treating MM.

PRAME promotes proliferation of multiple myeloma cells through CTMP/Akt/p21/CCND3 axis by ubiquitinating CTMP and p21

Multiple myeloma (MM) is a Ubiquitin Proteasome System (UPS)-dysfunction disease. We previously reported that high PRAME transcript levels associated with unfavorable progression free survival (PFS) in patients with no bortezomib therapy, and bortezomib-containing regimen significantly improved PFS in patients with high PRAME transcript levels, which indicated that PRAME expression was prognostic for MM patients, and was related to proteasome inhibitor treatment. However, molecular mechanisms underlying the above clinical performance remain unclear. In the present study, MM cell models with PRAME knockdown and overexpression were established, and PRAME was identified to play the role of promoting proliferation in MM cells. P-Akt signaling was found to be activated as PRAME overexpressed. As a substrate recognizing subunit (SRS) of the E3 ubiquitin ligase, PRAME targets substrate proteins and mediates their degradation. CTMP and p21 were found to be the novel targets of PRAME in the Cul2-dependent substrate recognition process. PRAME interacted with and mediated ubiquitination and degradation of CTMP and p21, which led to accumulation of p-Akt and CCND3 proteins, and thus promoted cell proliferation and increased bortezomib sensitivity in MM cells.

Ginsenoside Rg1 inhibits multiple myeloma and overcomes bortezomib resistance through AMPK-mTOR pathway

BackgroundThe resistance of multiple myeloma (MM) to bortezomib (BTZ) has brought multiple challenges to its clinical use. Numerous ginsenosides have potential anti-tumor effects, however, the research on the role of Rg1 in MM has not been reported. ObjectiveTo examine the inhibitory impact of Rg1 on the growth of MM and reduce the drug resistance of MM to BTZ through in vivo and in vitro experiments, and to explore their potential mechanism. MethodsBTZ drug-resistant cell line RPMI8226R was constructed. Mouse tumor-bearing model was developed by abdominal subcutaneous injection of MM cells. MM cells were treated with AMPK inhibitor Compound C or autophagy inhibitor Chloroquine together with Rg1. RPMI8226R cells were treated with BTZ and Rg1. Cell multiplication was detected using Methylthiazolyldiphenyl-tetrazolium bromide assay. Apoptosis was assessed using flow cytometry. Immunofluorescence assay was employed to assess the autophagy markers LC3. Western blot was utilized to assess the protein expression. Immunohistochemistry was used to detect cell proliferation and apoptosis in tumor tissues. ResultsIn vitro experiments demonstrated that Rg1 could hinder the proliferation of MM cells, promote apoptosis and enhance autophagy. Rg1 could also increase the sensitivity of RPMI8226R to BTZ. In vivo experiments illustrated that Rg1 could hinder the development of MM cells in mice, weaken the proliferation of tumor cells and enhance their apoptosis. Further study found that the anti-MM impact of Rg1 was linked to AMPK-mTOR pathway, the autophagy degree of RPMI8226R was higher than that of RPMI8226, and that Rg1 could inhibit MM and overcome drug resistance through autophagy induced by AMPK-mTOR pathway. ConclusionRg1 has significant anti-MM effect and can overcome BTZ resistance, and its potential mechanism is related to the regulation of autophagy induced by AMPK-mTOR pathway. Rg1 is a promising adjuvant drug for the treatment of MM.

Inhibition of glutaminolysis alone and in combination with HDAC inhibitor has anti-myeloma therapeutic effects.

Aim: This study aimed to investigate drug candidates and their efficacy in treating refractory multiple myeloma (MM) despite significant therapeutic advances and the introduction of novel agents. Our study focused on how myeloma cells mediate the metabolic pathways essential for survival. Therefore, we examined the role of glutaminolysis in this process. Methods: We investigated the role of glutaminolysis in myeloma cell growth. In addition, we analyzed the ability of CB-839 (telaglenastat), a glutaminase (GLS) inhibitor, to suppress myeloma cell proliferation and enhance the sensitivity to histone deacetylase (HDAC) inhibitors. Results: Glutamate deprivation significantly reduced MM cell proliferation. We observed an upregulation of GLS1 expression in MM cell lines compared to that in normal controls. CB-839 inhibits MM cell proliferation in a dose-dependent manner, resulting in enhanced cytotoxicity. Additionally, intracellular α-ketoglutarate and nicotinamide adenine dinucleotide phosphate levels decreased after CB-839 administration. Combining panobinostat with CB-839 resulted in enhanced cytotoxicity and increased caspase 3/7 activity. Cells transfected with GLS shRNA exhibited reduced cell viability and elevated sub-G1 phase according to cell cycle analysis results. Compared to control cells, these cells also showed increased sensitivity to panobinostat. Conclusion: Glutaminolysis contributes to the viability of MM cells, and the GLS inhibitor CB-839 has been proven to be an effective treatment for enhancing the cytotoxic effect of HDAC inhibition. These results are clinically relevant and suggest that CB-839 is a potential therapeutic candidate for patients with MM.

Irisin prevents trabecular bone damage and tumor invasion in a mouse model of multiple myeloma

Abstract Bone disease associated with multiple myeloma (MM) is characterized by osteolytic lesions and pathological fractures, which remain a therapeutic priority despite new drugs improving MM patient survival. Antiresorptive molecules represent the main option for the treatment of MM-associated bone disease (MMBD), whereas osteoanabolic molecules are under investigation. Among these latter, we here focused on the myokine irisin, which is able to enhance bone mass in healthy mice, prevent bone loss in osteoporotic mouse models, and accelerate fracture healing in mice. Therefore, we investigated irisin effect on MMBD in a mouse model of MM induced by intratibial injection of myeloma cells followed by weekly administration of 100 μg/kg of recombinant irisin for 5 wk. By micro-Ct analysis, we demonstrated that irisin improves MM-induced trabecular bone damage by partially preventing the reduction of femur Trabecular Bone Volume/Total Volume (P = .0028), Trabecular Number (P = .0076), Trabecular Fractal Dimension (P = .0044), and increasing Trabecular Separation (P = .0003) in MM mice. In cortical bone, irisin downregulates the expression of Sclerostin, a bone formation inhibitor, and RankL, a pro-osteoclastogenic molecule, while in BM it upregulates Opg, an anti-osteoclastogenic cytokine. We found that in the BM tibia of irisin-treated MM mice, the percentage of MM cells displays a reduction trend, while in the femur it decreases significantly. This is in line with the in vitro reduction of myeloma cell viability after 48 h of irisin stimulation at both 200 and 500 ng/mL and, after 72 h already at 100 ng/mL rec-irisin. These results could be due to irisin ability to downregulate the expression of Notch 3, which is important for cell-to-cell communication in the tumor niche, and Cyclin D1, supporting an inhibitory effect of irisin on MM cell proliferation. Overall, our findings suggest that irisin could be a new promising strategy to counteract MMBD and tumor burden in one shot.

#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.

Abstract 6046: CFT8634, a BRD9 BiDAC™ degrader, is active in a subset of multiple myeloma cell line models and synergistic when combined with pomalidomide or dexamethasone

Abstract Recent literature has shown that loss-of-function of BRD9, a component of the noncanonical SWI/SNF (ncSWI/SNF) complex, mediated by RNA interference or by BRD9 degrader compounds can inhibit multiple myeloma (MM) cell lines and primary MM cell proliferation in vitro, as well as inhibit mouse MM xenograft tumor growth in vivo. Additionally, in vitro synergy was observed when a BRD9 degrader was combined with either dexamethasone or pomalidomide. CFT8634 is a potent and selective oral BiDAC™ degrader of BRD9 that entered a clinical trial for the treatment of SMARCB1-perturbed cancers, including synovial sarcoma and SMARCB1-null tumors. Pharmacokinetic and pharmacodynamic data from this trial demonstrated dose-proportional human plasma exposure and robust BRD9 degradation in patients. Here we show that CFT8634 has anti-proliferative activity in a subset of MM cell lines that translates into tumor growth inhibition in MM xenograft models at clinically relevant CFT8634 exposures. We observe that cell lines less sensitive to pomalidomide tend to be significantly more sensitive to CFT8634 treatment. As pomalidomide is a SoC treatment in MM, we interrogated the ability to combine CFT8634 and pomalidomide. CFT8634 demonstrates synergy with pomalidomide in MM cell lines where CFT8634 is anti-proliferative as a single agent. We further show that clinically relevant exposures of CFT8634 demonstrate synergy with pomalidomide in vivo in the NCI-H929 mouse xenograft model. This model is relatively unresponsive to pomalidomide alone, suggesting that CFT8634 can render otherwise recalcitrant MM tumors sensitive to the effects of pomalidomide in combination. Pharmacokinetic and pharmacodynamic analyses show that in combination, CFT8634 and pomalidomide do not interfere with the ability to degrade each other’s targets (BRD9 and IKZF1/3, respectively) despite both utilizing the E3 ligase cereblon. As the corticosteroid dexamethasone is another SoC treatment given in combination with pomalidomide, we explored the ability to combine dexamethasone with CFT8634. CFT8634 demonstrates synergy with dexamethasone in the RPMI-8226 in vivo xenograft model. This combination leads to tumor regressions at clinically relevant doses and outperforms the current combination of pomalidomide with dexamethasone. In conclusion, we demonstrate in vitro and in vivo activity in an expanded set of MM models using the clinical oral BRD9 degrader CFT8634 as a single agent and show in vivo synergy between CFT8634 and SoC agents pomalidomide and dexamethasone at clinically relevant doses. Citation Format: Laura L. Poling, David Cocozziello, Minsng he, Eunju Hurh, Riadh Lobbardi, Katrina L. Jackson, Stewart L. Fisher, Roy M. Pollock. CFT8634, a BRD9 BiDAC™ degrader, is active in a subset of multiple myeloma cell line models and synergistic when combined with pomalidomide or dexamethasone [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 6046.

Abstract 3345: PP1 phosphatase complex at the hub of the integrated stress response: A potential therapeutic target in multiple myeloma

Abstract Protein phosphatase-1 (PP1) is a class of eukaryotic enzyme that catalyzes more than half of the phosphoserine/threonine dephosphorylation reactions, and plays essential roles in cell division, glycogen metabolism and protein synthesis. The substrate specificity of PP1 holoenzyme complex is mediated by PP1-interacting proteins and its catalytic subunits. Of note, PPP1R15B/CReP and PPP1R15A/GADD34 are regulatory subunits of PP1 complex that regulates global protein synthesis and mRNA translation through eIF2α dephosphorylation. Notably, PPP1R15B is constitutively expressed, whereas the expression of PPP1R15A is stress-inducible. Previous studies showed that multiple myeloma (MM), a hematological malignancy arising from immunoglobulin-secreting plasma cells, is characterized by continual ER stress and high dependency on the unfolded protein response (UPR). The integrated stress response (ISR) activated by eIF2α phosphorylation is the fastest reaction pathway of the UPR; however, its functional role and therapeutic potential in MM remains largely unknown. In this study, we performed gene expression analysis and super-enhancer (SE) profiling in MM cell lines, primary patient samples, normal CD138+ plasma cells and memory B cells. we found that PPP1R15B is highly expressed in MM cells as compared to healthy controls, but did not observe significant upregulation of PPP1R15A in MM cells. In addition, higher expression of PPP1R15B predicted poor overall survival of MM patients, suggesting its clinical relevance in MM pathogenesis. Depletion of PPP1R15B significantly reduced cell viability, proliferation and induced G2/M phase cell cycle arrest. Gene Ontology analysis of downregulated genes in response to PPP1R15B knockdown indicates significant association with the UPR pathway. Immunoblot analysis showed activation of ER stress pro-apoptotic executors PUMA, NOXA, and cleavage of caspase-12 in PPP1R15B knockdown cells. Furthermore, we examined the therapeutic effects of eIF2α dephosphorylation inhibitors, Salubrinal and Raphin1, on MM cells. Raphin1 is a selective inhibitor of PPP1R15B that binds strongly to the PPP1R15B-PP1c complex, whereas Salubrinal inhibits both PPP1R15A-PP1c and PPP1R15B-PP1c complex. Our data showed that both Salubrinal and Raphin1 inhibited MM cell proliferation in a dose-dependent manner, while sparing normal plasma cells. MM cell lines with elevated PPP1R15B expression level were more sensitive to Salubrinal or Raphin1 than those with lower PPP1R15B level. Similar changes in the expression patterns of ISR-related genes were observed in response to eIF2α dephosphorylation inhibitors. Taken together, our study suggests that targeting PPP1R15B-PP1c complex or maintaining eIF2α hyperphosphorylation may serve as a new therapeutic approach in MM, which warrants further investigation. Citation Format: Sinan Xiong, Jianbiao Zhou, Tze King Tan, Sabrina Hui-Min Toh, Tae-Hoon Chung, Takaomi Sanda, Wee Joo Chng. PP1 phosphatase complex at the hub of the integrated stress response: A potential therapeutic target in multiple myeloma [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 3345.

LncRNA NEAT1 contributes to multiple myeloma progression via upregulating ARPC5 by sponging miR-133a

IntroductionLong non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) is confirmed to be involved in regulation of the multiple myeloma (MM) process. However, its underlying molecular mechanism deserves further investigation.Material and methodsQuantitative real-time PCR was used to examine NEAT1, microRNA (miR)-133a and actin-related protein 2/3 complex subunit 5 (ARPC5) expression. Cell proliferation and apoptosis were evaluated by cell counting kit 8 assay, soft-agar colony formation assay and flow cytometry. Dual-luciferase reporter assay was used to confirm the interaction between miR-133a and NEAT1 or ARPC5. The localization of NEAT1 and miR-133a in MM cells was determined by fluorescent in situ hybridization assay. In addition, animal experiments were conducted to explore the effect of NEAT1 knockdown on MM tumor growth in vivo.ResultsNEAT1 had increased expression in MM patients and cells. NEAT1 knockdown could repress MM cell proliferation and enhance apoptosis. NEAT1 could sponge miR-133a, and miR-133a could target ARPC5. MiR-133a was lowly expressed and ARPC5 was highly expressed in MM patients. MiR-133a inhibitor could abolish the suppressive effect of NEAT1 knockdown on MM cell growth, and these effects also could be reversed by ARPC5 silencing.ConclusionsAnimal experiments showed that NEAT1 downregulation reduced MM tumor growth by regulating miR-133a/ARPC5 axis. NEAT1 facilitated MM progression through the regulation of miR-133a/ARPC5, which provided new evidence that NEAT1 was a potential therapeutic target for MM.