Pathways In Multiple Myeloma Cells Research Articles

A NOTCH3-CXCL12-driven myeloma-tumor niche signaling axis promotes chemoresistance in multiple myeloma.

Multiple myeloma (MM) remains incurable due to disease relapse and drug resistance. Notch signals from the tumor microenvironment (TME) confer chemoresistance, but the cellular and molecular mechanisms are not entirely understood. Using clinical and transcriptomic datasets, we found that NOTCH3 is upregulated in CD138+ cells from newly diagnosed MM (NDMM) patients compared to healthy individuals and increased in progression/relapsed MM (PRMM) patients. Further, NDMM patients with high NOTCH3 expression exhibited worse responses to Bortezomib (BOR)-based therapies. Cells of the TME, including osteocytes, upregulated NOTCH3 in MM cells and protected them from apoptosis induced by BOR. NOTCH3 activation (NOTCH3OE) in MM cells decreased BOR anti-MM efficacy and its ability to improve survival in in vivo myeloma models. Molecular analyses revealed that NDMM and PRMM patients with high NOTCH3 exhibit CXCL12 upregulation. TME cells upregulated CXCL12 and activated the CXCR4 pathway in MM cells in a NOTCH3-dependent manner. Moreover, genetic or pharmacologic inhibition of CXCL12 in NOTCH3OE MM cells restored sensitivity to BOR regimes in vitro and in human bones bearing NOTCH3OE MM tumors cultured ex vivo. Our clinical and preclinical data unravel a novel NOTCH3-CXCL12 pro-survival signaling axis in the TME and suggest that osteocytes transmit chemoresistance signals to MM cells.

Efficacy Evaluation of a Tetracyclic Triterpene Compound Targeting Multiple Apoptosis Proteins for the Development of a Multiple Myeloma Treatment

Introduction:Multiple myeloma (MM) is an incurable hematologic cancer that originates in plasma cells and occurs primarily in older patients over 60. This study screened for hit compounds for MM treatments targeting mitochondria and apoptosis proteins. Further, non-clinical testing, including molecular docking analysis, was performed. Methods: Based on our previous studies, candidate compounds targeting mitochondria were screened (www.medchemexpress.com). The killing effect of the compounds on MM cell lines (RPMI8226, IM9, and U266) was examined using a sulforhodamine B assay. Apoptosis mechanisms were investigated in vitro. Complex immunocompromised mice were established by injecting RPMI8226-RFP-FLuc cells through the tail vein. Efficacy of the KBB-N2 activity was assessed following intraperitoneal (15 or 30 mg/kg/day) and tail vein (7 mg/kg/day) infusions for three days a week at daily intervals. Toxicity, pharmacokinetics, and molecular docking analyses were performed according to the established protocols. Results: The hit compound, KBB-N2, a tetracyclic triterpene, was identified as an MM-targeted compound with cytotoxic effects. The primary mechanism of action of KBB-N2 in MM cells was inhibiting the activity of the mitochondrial electron transport (ETC) complex, resulting in the generation of excess intracellular reactive oxygen species (ROS), which led to tumor cell death by ROS-mediated apoptosis. In addition, exposure to KBB-N2 inhibited the activity of catalase, a powerful antioxidant enzyme in MM cells. Severe suppression of normal hematopoietic stem cell function was observed with lenalidomide administration at a concentration of 25 μM. However, this toxicity was not observed when KBB-N2 was administered at doses up to 60 μM. KBB-N2 activity involves PARP cleavage and caspase activation in the apoptotic pathway. Furthermore, KBB-N2 inhibited cell growth and exerted cytotoxicity. KBB-N2 induced apoptosis by activating the P53 apoptosis pathway in MM cells. In vivo, all untreated tumor-bearing mice showed rapid tumor growth and severe plasmacytomas, which led to death within seven weeks. Mice treated with KBB-N2 had significantly inhibited tumor growth and longer survival times. The molecular docking assay revealed the optimal docking conformation binding energy of KBB-N2 with docking scores (kcal/M) of -7.3 and -9.8 for ETC complexes I and III, respectively. Conclusions: The triterpene compound, KBB-N2, was identified as a hit compound and may act as a targeted agent for MM treatment. The antimyeloma activity of KBB-N2 involves interference with the ETC complex and leads to MM cell death.

Shutting off the fuel supply to target metabolic vulnerabilities in multiple myeloma.

Pathways that govern cellular bioenergetics are deregulated in tumor cells and represent a hallmark of cancer. Tumor cells have the capacity to reprogram pathways that control nutrient acquisition, anabolism and catabolism to enhance their growth and survival. Tumorigenesis requires the autonomous reprogramming of key metabolic pathways that obtain, generate and produce metabolites from a nutrient-deprived tumor microenvironment to meet the increased bioenergetic demands of cancer cells. Intra- and extracellular factors also have a profound effect on gene expression to drive metabolic pathway reprogramming in not only cancer cells but also surrounding cell types that contribute to anti-tumor immunity. Despite a vast amount of genetic and histologic heterogeneity within and between cancer types, a finite set of pathways are commonly deregulated to support anabolism, catabolism and redox balance. Multiple myeloma (MM) is the second most common hematologic malignancy in adults and remains incurable in the vast majority of patients. Genetic events and the hypoxic bone marrow milieu deregulate glycolysis, glutaminolysis and fatty acid synthesis in MM cells to promote their proliferation, survival, metastasis, drug resistance and evasion of immunosurveillance. Here, we discuss mechanisms that disrupt metabolic pathways in MM cells to support the development of therapeutic resistance and thwart the effects of anti-myeloma immunity. A better understanding of the events that reprogram metabolism in myeloma and immune cells may reveal unforeseen vulnerabilities and advance the rational design of drug cocktails that improve patient survival.

DNP73 PROMOTES DRUG RESISTANCE AND IMMUNE EVASION IN MULTIPLE MYELOMA VIA UPREGULATING MYC AND N‐MYC

Introduction: Multiple myeloma (MM) remains an incurable malignancy of plasma cells. The complex bone marrow microenvironment promotes resistance to both current anti-myeloma agents and emerging immunotherapies. Crosstalk between the NF-κB and p53 can play a pivotal role in various hematologic malignancies. DNp73, an inhibitor of the p53 tumor suppressor family, drives drug resistance and cancer progression in several solid malignancies. However, the biological functions and molecular mechanisms of DNp73 in MM remain unclear. In this study, we investigated the role of DNp73 in the drug resistance of MM, and disclosed the corresponding mechanism of how DNp73 promotes the immune escape of MM cells. Methods: We constructed DNp73 overexpression and sh-RNA interference plasmids to obtain stable cell lines. The effects of DNp73 on proliferation and drug sensitivity were determined by flow cytometry and xenotransplantation model. RNA-seq and CHIP-seq were performed to detect the mechanisms of drug resistance in MM cells. The DNA damage repair and invasion ability of MM cells were detected by immunofluorescence and transwell assay. To validate the role of DNp73 in immune escape, we performed phagocytosis assays. Results: Our previous study reported that miR-15a was downregulated in MM cells and correlated with the inferior outcome of MM patients. Further analysis demonstrated that DNp73 was a direct target of p65, loss of miR-15a led to the activation of NF-κB-p65 pathway in MM cells. In addition, the level of miR-15a was negatively correlated with the expression of DNp73 in MM cells of patients (r = −0.672, p < 0.05). After DNp73 was down-regulated, cell proliferation and drug resistance were effectively inhibited. Further in vivo study indicated the tumor volume in DNp73 knockdown group was reduced, and increased sensitivity to the carfilzomib, epirubicin and pomalidomide was observed (p < 0.01). Of note, RNA-seq analysis indicated that DNp73 expression was positively correlated with MYCN, MYC and CDK7 transcriptional programs in MM. GSEA analysis showed that MYC targets and DNA damage repair pathways were significantly enriched in DNp73-OE cells. Compared to control group, DNp73-OE cells were resistant to irradiation-induced cell death (p < 0.01). Meanwhile, DNp73 knockdown significantly reduced the migration and invasion of cells (p < 0.01). CHIP-seq data showed that DNp73 could bind to the promoter region of MYCN. DNp73 overexpression protects MM cells from phagocytosis, treated with anti-human CD47 antibody increased phagocytosis of macrophages (p < 0.01). Conclusions: We demonstrated that miR-15a downregulation activated NF-κB, which promoted DNp73 expression at transcription level. DNp73 promotes drug resistance and immune escape in multiple myeloma by modulating Myc and N-Myc signal pathway. The research was funded by: the CAMS Innovation Fund for Medical Sciences (2022-I2M-1-022), the Natural Science Foundation of China (82170194), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018RC320012), the Natural Science Foundation of China (81920108006), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018PT31006) Keywords: multiple myeloma, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

Combinations of ivermectin with proteasome inhibitors induce synergistic lethality in multiple myeloma

Multiple myeloma (MM) is an incurable malignancy of plasma cells. Ivermectin is a US Food and Drug Administration-approved drug for antiparasitic use. Here, we showed that ivermectin exerted anti-MM effects and significantly synergized with proteasome inhibitors in vitro and in vivo. Ivermectin alone exhibited mild anti-MM activity in vitro. Further investigation suggested that ivermectin inhibited proteasome activity in the nucleus by repressing the nuclear import of proteasome subunits, such as PSMB5-7 and PSMA3-4. Therefore, ivermectin treatment caused the accumulation of ubiquitylated proteins and the activation of the UPR pathway in MM cells. Furthermore, ivermectin treatment caused DNA damage and DNA damage response (DDR) signaling pathway activation in MM cells. Ivermectin and bortezomib exhibited synergized anti-MM activity in vitro. The dual-drug treatment resulted in synergistic inhibition of proteasome activity and increased DNA damage. An in vivo study using a human MM cell line xenograft mouse model showed that ivermectin and bortezomib efficiently repressed MM tumor growth in vivo, while the dual-drug treatment was well tolerated by experimental animals. Overall, our results demonstrated that ivermectin alone or cotreated with bortezomib might be promising in MM treatment.

MAF Translocations Are Enriched in NDMM Patients with Elevated Levels of Circulating Tumor Cells Suggesting a Genetic Basis for Aggressive Disease Course

Introduction A subset of Multiple Myeloma (MM) patients responds poorly to treatment, highlighting the need to understand the pathobiology of high-risk MM. The correlation between aggressive disease and elevated levels of circulating tumor cells (CTCs) has been recognized. We recently identified a subset of high-risk newly diagnosed MM (NDMM) patients based on a transcriptomic classifier derived from patients with high levels of CTC, indicating that CTC levels are a relevant reflection of tumor cell biology. Here, we hypothesized that comparing bone marrow plasma cells (BM PCs) of MM patients with high and low CTC levels will provide an opportunity to identify mechanistic drivers of aggressive disease. Results We first set out to exclude the presence of a circulating aggressive subclone of MM cells in patients with high levels of CTCs. Paired single cell transcriptomes from BM and peripheral blood MM cells of 7 NDMM patients were generated. Analysis of 55,230 CTCs and BM PCs revealed patient specific clustering, but with large transcriptional overlap between individual paired CTCs and BM PCs. Since only 11 genes were significantly upregulated in the BM PCs (P < 0.05) compared to paired CTCs, we excluded the presence of circulating aggressive subclones in patients with high CTC levels. Next, we aimed to identify differences in MM cell pathobiology between patients with high and low levels of CTCs, we generated single cell transcriptomes from 89,092 BM MM cells from 8 NDMM patients with high (≥0.5%) and 14 patients with low (0.004-0.006%) percentages of CTCs. Single cell data integration across all patients led to the identification of 8 distinct PC clusters. Gene set enrichment analysis between both patient groups revealed upregulation of cell cycle pathways in MM cells of patients with high CTC levels. Based on cell cycle analyses an increase in cycling PCs (53% vs 32%, p=0.007) was associated with high CTC levels (Figure 1). Ki67 labeling of BM biopsies validated this finding. Interestingly, proliferation was increased across all transcriptomically-defined plasma cell clusters, suggesting a common cell-intrinsic driver of increased proliferation. To analyze whether the prevalence of genetic lesions previously associated with proliferation was increased in patients with high CTC levels, WGS was performed on the same 22 patients. This revealed an elevated mutation burden and a predilection for MAF translocations, as 50% (4/8) of the patients with high CTC levels carried MAF translocations versus none in the patients with low CTC levels (0/14). We also observed increased transcription of cyclin D2 (CCND2), CCR1 and IntegrinB7 (ITGB7) in patients with high CTC levels. These proliferation or tumor survival genes have been shown to be increased in patients with t(14;16), a translocation also resulting in overexpression of MAF. To validate these findings in a larger patient cohort, clinical, genomic and CTC data were retrieved from the MMRF-CoMMpass database (n=725). Strikingly, 21% of patients with CTC levels above 0.3% showed MAF translocations vs only 3% in patients with CTC levels below 0.3% (p<0.001), solidifying the correlation between MAF translocations and high levels of CTCs in NDMM patients with a high-risk disease profile. Conclusion Single cell RNA sequencing and WGS of MM cells from patients with high and low CTC levels identified a correlation between CTC levels and overrepresentation of MAF translocations in NDMM patients. This finding suggests that MAF target genes and downstream proliferation could be drivers of aggressive disease and might be used for future patient stratification. Figure 1. Based on cell cycle analyses in single cell transcriptomics an increase in cycling PCs was associated with high CTC levels. a) UMAP of cell cycle analysis of 89,902 BM MM cells from 22 patients with high (n=8) and low (n=14) CTC levels. Colors represent cell cycle phase. b) Bar graph representing percentage of cycling cells. Data points are individual patients (high CTC levels, n=8; low CTC levels, n=14). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Brucein D imparts a growth inhibitory effect in multiple myeloma cells by abrogating the Akt-driven signaling pathway.

The Akt signaling pathway is an oncogenic cascade activated in the bone marrow microenvironment of multiple myeloma (MM) cells and contributes to their uncontrolled proliferation. Abrogation of Akt signaling has been presentedas one of the prime therapeutic targets in the treatment of MM. In the present report, we have investigated the effect of Brucein D (BD) on Akt-driven signaling events in MM cells. BD (300 nM) substantially inhibited cell viability and imparted growth-inhibitory effects in U266 cells as evidenced by cell viability assays and flow cytometric analysis. Effect of BD on cell viability was evaluated by MTT assay. Apoptotic cells and cell cycle arrest by BD were analyzed by flow cytometer. The results of the TUNEL assay and western blotting showed that BD induces apoptosis of MM cells by activating caspase-8 and 9 with subsequent reduction in the expression of antiapoptotic proteins (Bcl-2, Bcl-xl, survivin, cyclin D1, COX-2, VEGF, MMP-9). Analysis of activated kinases by Phospho-Kinase Array Kit revealed that Akt, p70S6K, HSP60, p53, and WNK1 were strongly expressed in untreated cells and BD treatment reversed this effect. Using transfection experiments, AKT depletion led to a decrease in phosphorylation of Akt, mTOR, p70S6K, and WNK. However, Akt overexpression led to increase in phosphorylation of these proteins. Depletion of Akt potentiated the apoptosis-inducing effect of BD whereas overexpression displayed resistance to BD-induced apoptosis suggesting the role of Akt in chemoresistance. Taken together, BD mitigates Akt-dependent signaling pathways in MM cells to impart its anticancer activity.

The Influence of Cannabinoids on Multiple Myeloma Cells: A Scoping Review

Multiple myeloma (MM) is one of the most common hematological malignancies. There is a clear need for research into new treatment options that can improve the life expectancy and quality of life for MM patients; this is particularly salient for those with relapsed/refractory disease. Cannabinoids (CB) have shown potential in treatment regimens for a number of cancers, but little is currently known about their effectiveness against MM. Hence, we conducted a scoping review regarding the usage of CB against MM cells. For our review, searches were conducted in PubMed, Web of Science, and OVID Medline. After screening, six articles were eligible for inclusion, all of which were laboratory studies. It was demonstrated that CB decrease MM cell viability, and this was consistently shown to occur alongside the activation of apoptotic pathways in MM cells. These effects were shown to continue to occur in dexamethasone-resistant MM cells. The effects of CB on MM cells were enhanced when used in combination with standard treatments for MM. Critically, these marked decreases in MM cell viability induced by CB did not occur in non-MM cells. Overall, these findings indicate a clear need for future clinical trials of the integration of CB into MM treatment regimens.

The effect of proteasome inhibitor Carfilzomib on pyroptosis cell death pathway in multiple myeloma cells

Amaç: Multipl miyelom (MM), monoklonal antikor salgılayan anormal plazma hücrelerinin kemik iliğinde aşırı birikimi ile karakterize bir B hücre malignitesidir. Klinik uygulamalarda ikinci nesil proteozom inhibitörü Carfilzomib (CFZ), relaps veya tedaviye dirençli hastaların tedavi rejimlerinde kullanılmaktadır. Ancak, MM hücrelerinde CFZ'in tetiklediği hücre ölümü mekanizmaları tam olarak aydınlatılamamıştır. Bu çalışmanın amacı, MM hücrelerinde CFZ'in apoptotik olmayan düzenli hücre ölüm yolaklarından biri olan piroptozis üzerine olan etkisinin araştırılmasıdır. Yöntem: İnsan RPMI 8226, U266 ve NCI H929 MM hücre hatları, CFZ’in IC50 dozları ile 48 saat süre boyunca muamele edildi. Muamele edilen hücrelerde piroptozisin önemli substratları olan GSDMD ve GSDME ile BAX ve BCL-2 genlerinin mRNA düzeylerindeki farklılıklar kantitatif eş zamanlı PCR (qPCR) yöntemiyle belirlendi. Bulgular: NCI H929 ve RPMI 8226 hücrelerinde CFZ uygulamasının hem GSDMD hem de GSDME mRNA düzeylerinde anlamlı artışa neden olduğu belirlenirken, U266 hücrelerinde ise sadece GSDME mRNA seviyesinde anlamlı bir artış tespit edildi (p<0.05). MM hücrelerinde CFZ uygulaması BAX mRNA ifadesinde genel bir artışa neden olurken sadece RPMI 8226 hücrelerinde bu artış istatistiksel olarak anlamlı bulundu. CFZ ile muamele edilen MM hücrelerinde BCL-2 mRNA seviyesinde ise bir değişiklik saptanmadı. Sonuç: Bu çalışmada, ilk kez CFZ’in MM hücrelerinde piroptozis ölüm yolağını da tetikleyerek anti-miyelom özellik gösterebileceği belirlenmiştir.

A Hyaluronan and Proteoglycan Link Protein 1 Matrikine: Role of Matrix Metalloproteinase 2 in Multiple Myeloma NF-κB Activation and Drug Resistance.

HAPLN1 and MMP2 produced by BMSCs obtained from patients with multiple myeloma promote NF-κB activity and resistance to bortezomib toxicity in multiple myeloma cells, uncovering their potential as biomarkers or therapeutic targets to address bortezomib resistance in patients with multiple myeloma.

Radotinib inhibits multiple myeloma cell proliferation via suppression of STAT3 signaling.

Multiple myeloma (MM) is a hematological cancer causing from accumulated abnormal plasma cells. STAT3 overexpression in MM appears to be mediated by a variety of factors, and it may be associated with an adverse prognosis and play a role in microenvironment-dependent treatment resistance. Unfortunately, MM remains an incurable disease, as relapse is very common. Therefore, there is urgent need to develop new treatment options for MM. Radotinib is a novel anti-cancer drug, currently approved in South Korea for the treatment of chronic myeloid leukemia patients. It is an oral, multitargeted inhibitor of receptor tyrosine kinases, including BCR-ABL, c-KIT, PDGFR, and Src family kinases. However, little is known about the effects of radotinib on multiple myeloma cells. However, little is known about the effects of radotinib on multiple myeloma cells. But even tinip almost not known about the impact of multiple myeloma cells. Moreover, nothing is known about how it affects STAT3 and JAK2. In this study, we analyzed the effect of radotinib on multiple myeloma cells. Herein, Moreover, nothing is known about how it. Moreover, not all is known about how the affects STAT3 and JAK2. We investigated the effect of radotinib on the STAT3 signaling pathway in MM cells, including several MM cell lines and mouse models. So we investigated the effect of radotinib on MM cells, including several MM cell lines and mouse models. Interestingly, radotinib induced apoptosis, and inhibited cell proliferation in MM cells including RPMI-8226, MM.1S, U266B1, and IM-9 cells. Moreover, radotinib treatment significantly increased the number Annexin V-positive cells and G0/G1-phase cells. In addition, radotinib treatment in various MM cells strongly suppressed the activity and expression of STAT3 and JAK2 proteins. We also observed that diverse proteins related to the STAT3 signaling pathway, including c-Myc, Bcl-xL, Mcl-1, cyclin D1 and cyclin D3, were powerfully inhibited by radotinib treatment in MM cells. Furthermore, radotinib significantly suppressed MM cell growth in a xenograft animal model using IM-9 cells. In conclusion, radotinib may play an important role as a candidate agent for MM treatment.

Myeloma cells regulate miRNA transfer from fibroblast-derived exosomes by expression of lncRNAs.

Multiple myeloma (MM) progression and drug resistance depend on the crosstalk between MM cells and bone marrow (BM) fibroblasts (FBs). During monoclonal gammopathy of undetermined significance (MGUS) to MM transition, MM cell-derived exosomes (EXOs) reprogram the miRNA (miR) profile of FBs, inducing the overexpression miR-23b-3p, miR-27b-3p, miR-125b-5p, miR-214-3p, and miR-5100. Here, we demonstrate that the miR content of MM FB-derived EXOs (FB-EXOs) overlaps the miR profile of parental FBs by overexpressing comparable levels of miR-23b-3p, miR-27b-3p, miR-125b-5p, miR-214-3p, and miR-5100. Recipient MM cells co-cultured with MM FB-EXOs selectively overexpress only miR-214-3p and miR-5100 but not miR-23b-3p, miR-27b-3p, and miR-125b-5p, suggesting a putative selective transfer. MM cells express HOTAIR, TOB1-AS1, and MALAT1 lncRNAs. Transient transfection of MM cells with lnc·siRNAs demonstrates that HOTAIR, TOB1-AS1, and MALAT1 lncRNAs are sponges for miR-23b-3p, miR-27b-3p, and miR-125b-5p. Indeed, lncRNA knockdown significantly increased miR levels in U266 MM cells co-cultured with MM FB-EXOs. Selective miR-214-3p and miR-5100 overexpression modulates MAPK, PI3K/AKT/mTOR, and p53 pathways in MM cells. Interrogation using the DIANA tools algorithm and transient overexpression using miR mimic probes confirmed the involvement of miR-214-3p and miR-5100 and their target genes, PTEN and DUSP16, respectively, in the modulation of these intracellular pathways. Finally, the uptake of EXOs as well as miR-214-3p and miR-5100 overexpression increase MM cell proliferation and resistance to bortezomib-induced apoptosis by switching the balance between pro-/anti-apoptotic proteins. Overall, these data show that MM cells are not simply a container into which EXOs empty their cargo. On the contrary, tumour cells finely neutralize exosomal miRs via lncRNA expression to ensure their survival. © 2021 The Pathological Society of Great Britain and Ireland.

H3K36 Trimethylation Mediated By SETD2 Regulates Cell Proliferation and Cell Cycle By Modulating BCMA-JNK and c-Myc Pathways in Multiple Myeloma

In addition to genetic aberrations, accumulating evidence indicates that deregulation of histone methyltransferases, such as MMSET, EZH2 and KDM6A, plays crucial roles in the oncogenic transformation and development of multiple myeloma (MM). For example, overexpression of MMSET leading to a global increase in H3K36me2, is believed to be the driving force in the pathogenesis of t (4;14) MM. However, as the histone methyltransferase is responsible for H3K36me3, the role of SETD2 is not been known in myeloma.To explore the possible clinical value of SETD2 in MM, we first examined the gene expression profile from GEO database, which indicated that the SETD2 expression was significantly decreased in MM patients when compared with monoclonal gammopathy of undetermined significance and smoldering multiple myeloma patients (GSE6477). Moreover, the expression of SETD2 decreased with the advanced international staging system stage of MM patients (GSE19784). The Kaplan-Meier analysis showed that low expression of SETD2 was significantly associated with a poor overall survival in MM patients (GSE2658, P<0.05; GSE9782, P<0.001). Thus, our analysis suggests that SETD2 might participate in cancer progression and could become a biomarker for the prognosis of patients with MM.We then investigated the biological role and the underlying mechanism of SETD2 in MM. Firstly, we used lentiviral-mediated RNA interference to knockdown SETD2 (SETD2 KD) in MM cell lines (RPMI8226 and MM.1S). CCK8 and colony-forming assays showed that reduced expression of SETD2 significantly promoted MM cell proliferation and colony growth. BrdU incorporation assay revealed increased DNA synthesis in SETD2 KD MM cells. Then, treatment with JIB-04, a small molecule inhibitor targeting H3K36me3 loss in SETD2 KD MM cells showed that H3K36me3 recovery was capable of reversing the tumor-promoting effect due to SETD2 down-regulation of MM cells in vitro. Moreover, the xenograft growth assay revealed that SETD2 down-regulation facilitated tumor growth and JIB-04 treatment exhibited anti-myeloma activity in vivo. Therefore, we conclude that SETD2 plays an important role in MM maintenance, and inhibition of H3K36me3 shows therapeutic efficacy for MM.To further explore the underlying mechanisms, we performed the RNA-Seq analysis and discovered that low H3K36me3 level was associated with reduced expression of CDKN1A and increased expression of TNFRSF17 (BCMA) and c-Myc in MM cells. The Gene Set Enrichment Analysis (GSEA) revealed that MAPK signaling pathway was enriched in SETD2 knockdown and JIB-04 treated MM cells. Subsequent Western blotting analysis further confirmed that SETD2 KD cells had increased JNK activation, while JIB-04 treated group showed decreased level of p-JNK. To investigate whether BCMA was the directly transcriptional target for H3K36me3, we carried out a dual-luciferase reporter assay in both SETD2 KD and JIB-04 treated MM cells, and confirmed that H3K36me3 inhibited the expression of BCMA through physically interacting with motifs in its promoter. Furthermore, down-regulation of BCMA in SETD2 KD MM cells could lead to a reduction of p-JNK and an up-regulation of CDKN1A, and resulting inhibited cell proliferation and cell cycle progression. Furthermore, blockage of the JNK pathway by its inhibitor SP600125 resulted in significant inhibition of MM cell proliferation induced by SETD2 knockdown. Additionally, suppression of c‐Myc using 10058‐F4 inhibited proliferation, and induced cell cycle arrest as well as CDKN1A expression in SETD2 KD MM cells. These results indicate that BCMA/JNK and c-Myc pathways were involved in STED2 and H3K36me3 mediated cell proliferation in MM.Together, our data delineate that SETD2-dependent H3K36me3 modification plays a critical role in regulation cell proliferation and cell cycle by BCMA-JNK and c-Myc pathways in MM cells. Targeting the SETD2-H3K36me3 pathway represents a promising therapy for MM. DisclosuresNo relevant conflicts of interest to declare.

Discovery of Prolyl-tRNA Synthetase As a Novel Target in Multiple Myeloma

Discovery of Prolyl-tRNA Synthetase As a Novel Target in Multiple Myeloma

Aberrant CDK7 Activity Drives the Cell Cycle and Transcriptional Dysregulation to Support Multiple Myeloma Growth: An Attractive Molecular Vulnerability

Multiple myeloma (MM) cells are characterized by cell cycle dysregulation, epigenetic heterogeneity, and perturbation of the transcriptional landscape. We have previously shown that chemical and genetic perturbation of transcriptional regulator CDK7 significantly and selectively impacted MM cell growth and viability, supporting it as a pharmacologically relevant target for MM. Indeed, selective CDK7 inhibitor YKL-5-124 was active against a large panel of MM cell lines and primary MM cells, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs). The efficacy of YKL-5-124 was confirmed in vivo in several murine models of MM, including disseminated models.Gene expression analysis after CDK7 inhibition in several MM cell lines revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells, supporting CDK7 as a central hub in the oncogenic CDK-pRb-E2F pathway in MM cells, with its expression and activity positively correlated with E2F transcriptional output in patient MM cells. Importantly, dual inhibition with low doses of YKL-5-124 and BRD4 inhibitor JQ1, displayed superior activity against a panel of MM cell lines and primary MM cells compared to single perturbation alone by both converging on a subset of key SE-associated dependencies as well as impacting distinct oncogenic expression programs.To identify synthetically lethal targets and mechanisms of resistance to CDK7 inhibition, we performed a genome-wide CRISPR-Cas9 knockout screen in the MM1S cells treated with YKL-5-124 or DMSO. We found that BCL6, NFKBIA and B, TRAF2, TSC1 and CSNK2A1, a subunit of CK2, were top synthetically lethal hits; whereas deletion of RB1, SF3B3 and the DNA-binding transcriptional activator TADA2A that regulates RNA-pol II transcription, led to resistance to YKL-5-124. Molecular mechanisms of intrinsic and acquired resistance to CDK7 inhibition are now under investigation and will be presented.In conclusion, our study demonstrates CDK7 as an attractive molecular vulnerability in MM that can be exploited therapeutically alone or in combination. DisclosuresShirasaki: FIMECS: Consultancy. Chesi: Novartis: Consultancy, Patents & Royalties: human CRBN transgenic mouse; Pfizer: Consultancy; Pi Therapeutics: Patents & Royalties: Genetically engineered mouse model of myeloma; Abcuro: Patents & Royalties: Genetically engineered mouse model of myeloma; Palleon Pharmaceuticals: Patents & Royalties: Genetically engineered mouse model of myeloma. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Mitsiades: Arch Oncology: Research Funding; Karyopharm: Research Funding; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Honoraria; Abbvie: Research Funding; FIMECS: Consultancy, Honoraria; Nurix: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; BMS: Research Funding; TEVA: Research Funding. Munshi: Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy.

Apoptotic Extracellular Vesicles Ameliorate Multiple Myeloma by Restoring Fas-Mediated Apoptosis.

Apoptosis is critical for maintaining bodily homeostasis and produces a large number of apoptotic extracellular vesicles (apoEVs). Several types of cancer cells display reduced expression of Fas on the cell surface and are thus capable of escaping Fas ligand-induced apoptosis. However, it is unknown whether normal cell-derived apoEVs can regulate tumor growth. In this study, we show that apoEVs can induce multiple myeloma (MM) cell apoptosis and inhibit MM cell growth. Systemic infusion of mesenchymal stem cell (MSC)-derived apoEVs significantly prolongs the lifespan of MM mice. Mechanistically, apoEVs directly contact MM cells to facilitate Fas trafficking from the cytoplasm to the cell membrane by evoking Ca2+ influx and elevation of cytosolic Ca2+. Subsequently, apoEVs use their Fas ligand to activate the Fas pathway in MM cells, leading to the initiation of apoptosis. This study identifies the role of apoEVs in inducing MM apoptosis and suggests a potential for apoEVs to treat MM.

Blockage of the JAK/STAT3 signaling pathway in multiple myeloma by leelamine

BackgroundLeelamine (LEE) is a lipophilic diterpene amine phytochemical, which can be naturally extracted from pine's bark trees. It has been extensively studied recently for its promising chemopreventive and anti-cancer effects against various cancers such as that of prostate and breast. HypothesisWe examined the potential impact of LEE in affecting the activation of signal transducer and activator of transcription 3 (STAT3) and promoting apoptosis in human multiple myeloma (MM) cells. MethodsWe evaluated the effect of LEE on STAT3 signaling pathway in MM cells by using Western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Thereafter, apoptosis was evaluated using cell cycle analysis and Annexin V assay. ResultsWe noted that LEE could attenuate the phosphorylation of STAT3 and other up-stream signaling molecules such as JAK1, JAK2, and Src activation in U266 and MM.1S cells. It also diminished STAT3 translocation into the nucleus and enhanced the expression of protein-tyrosine phosphatase epsilon (PTPε). Additionally, LEE caused cell cycle arrest and synergistically augmented the apoptotic actions of bortezomib against MM cells. ConclusionsOur data indicates that LEE could block STAT3 signaling cascade linked to tumorigenesis and can be used in combination with approved anti-cancer agents in attenuating MM growth and survival.

Suppression of steroid 5\u03b1-reductase type I promotes cellular apoptosis and autophagy via PI3K/Akt/mTOR pathway in multiple myeloma

Steroid 5α-reductase type I (SRD5A1) is a validated oncogene in many sex hormone-related cancers, but its role in multiple myeloma (MM) remains unknown. Based on gene expression profiling (GEP) of sequential MM samples during the disease course, we found that the aberrant expression of SRD5A1 was correlated with progression and poor prognosis in MM patients. In this study, the oncogenic roles of SRD5A1 were validated in human MM cell lines (ARP1 and H929) and the xenograft MM model as well as the 5TMM mouse model. MTT and flow cytometry were used to assess MM cell proliferation, cell cycle, and apoptosis post inducible knockdown SRD5A1 by lentivirus-mediated short-hairpin RNA (shRNA). Transcriptomic sequencing, immunofluorescence, and western blot were used to investigate the effects of SRD5A1 suppression on cell apoptosis and autophagy. Mechanistically, SRD5A1 downregulation simultaneously regulated both the Bcl-2 family protein-mediated apoptosis and the autophagic process via PI3K/Akt/mTOR signaling pathway in MM cells. Meanwhile, the autophagy inhibitor (3-methyladenine) and SRD5A1 inhibitor (Dutasteride) were utilized to evaluate their anti-myeloma effect. Thus, our results demonstrated that SRD5A1 downregulation simultaneously regulated both the apoptosis and the autophagic process in MM cells. The dual autophagy–apoptosis regulatory SRD5A1 may serve as a biomarker and potential target for MM progression and prognosis.

Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling.

Aberrant expression of lymphoid enhancer-binding factor-1 (LEF1) has been identified in various hematological malignancies including multiple myeloma (MM). However, the exact role of LEF1 in MM remains largely unknown. Here, we showed that knockdown of LEF1 could apparently impair the proliferation, induce apoptosis and promote the ROS production in MM cell lines, suggesting that LEF1 might be involved in maintaining MM cell growth and survival. Moreover, we observed that the mRNA level of the deubiquitinase cylindromatosis (CYLD), a well-recognized tumor suppressor in MM, was significantly increased following LEF1 depletion in myeloma cells. Further study showed that LEF1 could directly associate with the promoter of CYLD gene and thus repress its transcription in MM cells. Intriguingly, LEF1 depletion-mediated CYLD upregulation was sufficient to negatively modulate NF-κB signaling pathway in MM cells. Moreover, the decrease in NF-κB activity following LEF1 knockdown could be largely rescued when CYLD was silenced in MM cells. Taken together, our study provided the compelling evidence to show that LEF1 may augment the proliferation and survival of MM cells through direct repression of CYLD transcription and subsequent activation of NF-κB signaling pathway, corroborating that LEF1 may become a potential therapeutic target against MM.

FTY720 induces ferroptosis and autophagy via PP2A/AMPK pathway in multiple myeloma cells

AimsMultiple myeloma (MM) is the second hematological plasma cell malignany and sensitive to fingolimod (FTY720), a novel immunosuppressant. Previous study shows FTY720-induced apoptosis and autophagy can cause cell death in MM cells, however, the high death rate cannot fully be explained. The study aims to investigate further mechanism of how FTY720 kills MM cells. Materials and methodsExperiments are performed on 25 human primary cell samples and two MM cell lines by flow cytometry, fluorescence microscopy, and transmission electron microscopy. Expressions of relative factors are tested by qRT-PCR or western blot. Key findingsFerroptosis-specific inhibitors, deferoxamine mesylate (DFOM) and ferropstatin-1 (Fer-1), reverse FTY720-induced cell death in MM cells. Glutathione peroxidase 4 (GPX4) and soluble carrier family 7 member 11 (SLC7A11), key regulators of ferroptosis, are highly expressed in primary MM cells and can be decreased by FTY720 at the mRNA and protein level in MM cells. In addition, FTY720 induces other characteristic changes of ferroptosis. Furthermore, FTY720 can dephosphorylate AMP-activated protein kinase subunit ɑ (AMPKɑ) at the Thr172 site by activating protein phosphatase 2A (PP2A) and reduce the expression of phosphorylated eukaryotic elongation factor 2 (eEF2), finally cause MM cell death. Using LB-100, a PP2A inhibitor, AICAR, an agonist of AMPK, and bafilomycin A1 (Baf-A1), an autophagy inhibitor, we discover that FTY720 induces ferroptosis and autophagy through the PP2A/AMPK pathway, and ferroptosis and autophagy can reinforce each other. SignificanceThese results provide a new perspective on the treatment of MM.