Suppressed Multiple Myeloma Cell Research Articles

Phenylalanine deprivation inhibits multiple myeloma progression by perturbing endoplasmic reticulum homeostasis

Amino acid metabolic remodeling is a hallmark of cancer, driving an increased nutritional demand for amino acids. Amino acids are pivotal for energetic regulation, biosynthetic support, and homeostatic maintenance to stimulate cancer progression. However, the role of phenylalanine in multiple myeloma (MM) remains unknown. Here, we demonstrate that phenylalanine levels in MM patients are decreased in plasma but elevated in bone marrow (BM) cells. After the treatment, phenylalanine levels increase in plasma and decrease in BM. This suggests that changes in phenylalanine have diagnostic value and that phenylalanine in the BM microenvironment is an essential source of nutrients for MM progression. The requirement for phenylalanine by MM cells exhibits a similar pattern. Inhibiting phenylalanine utilization suppresses MM cell growth and provides a synergistic effect with Bortezomib (BTZ) treatment in vitro and murine models. Mechanistically, phenylalanine deprivation induces excessive endoplasmic reticulum stress and leads to MM cell apoptosis through the ATF3–CHOP–DR5 pathway. Interference with ATF3 significantly affects phenylalanine deprivation therapy. In conclusion, we have identified phenylalanine metabolism as a characteristic feature of MM metabolic remodeling. Phenylalanine is necessary for MM proliferation, and its aberrant demand highlights the importance of low-phenylalanine diets as an adjuvant treatment for MM.

Metformin and chidamide synergistically suppress multiple myeloma progression and enhance lenalidomide/bortezomib sensitivity.

Multiple myeloma (MM) is a common hematological malignancy, and patients with MM are recommended to take immunomodulatory drugs such as lenalidomide along with proteasome inhibitors such as bortezomib to extend survival. However, drug resistance influences the efficacy of treatment for MM. In our study, we found that metformin and chidamide both suppressed MM cell growth in a concentration- and time-dependent way (p < .001). Moreover, combined therapy with metformin and chidamide exhibited enhanced inhibition of the growth of MM cells compared with monotherapy (p < .05). Additionally, the triple-drug combination of metformin and chidamide with lenalidomide or bortezomib was used to stimulate the MM cells, and the results revealed that metformin and chidamide treatment sensitized MM cells to lenalidomide and bortezomib. As a result, the apoptosis (p < .001) together with cell cycle arrest at G0/G1 phase (p < .05) was stimulated by lenalidomide and bortezomib, and showed significant elevation in the triple-drug combination group compared with the lenalidomide or bortezomib treatment alone group (p < .05). Furthermore, the impacts of different drugs on glycolysis in MM cells were examined. We found that metformin and chidamide combined treatment significantly promoted glucose uptake and reduced energy production in MM cells treated with lenalidomide and bortezomib (p < .001), suggesting that metformin and chidamide affected glycolysis in MM cells and enhanced the sensitivity of lenalidomide and bortezomib in MM by regulating glucose metabolism. In conclusion, metformin and chidamide synergistically hindered MM cell growth and sensitized cells to lenalidomide/bortezomib. The findings of this study might provide novel clues to improve MM therapy.

MiR-125b targeted regulation of MKNK2 inhibits multiple myeloma proliferation and invasion.

An increasing number of studies demonstrate that abnormal miRNA expression contributes to the advancement of many tumors. Nonetheless, the potential role of miR-125b in multiple myeloma (MM) remains unknown. To explore the potential effects and mechanism of miR-125b in MM. Real-time quantitative PCR was used to measure the expression levels of miR-125b and MKNK2 in a variety of MM samples. Colony formation and cell counting Kit-8 (CCK-8) assays were used to assess cell proliferation, the transwell assay was used to evaluate the cell invasion capability, and dual luciferase reporter gene assay and Western blot were used to examine the interaction between miR-125b and MKNK2. The levels of miR-125b were higher in MM tissue samples, alongside increased expression of MKNK2. There was a negative correlation between MKNK2 and miR-125b expression in MM tissues. MKNK2 was identified as a direct target gene of miR-125b in MM cells. Overexpression of miR-125b suppressed MM cell growth, colony formation, and invasion. In addition, MKNK2 was found to mediate the effects of miR-125b on cell proliferation, colony formation, and invasion in MM. miR-125b acts as a suppressive factor in multiple myeloma and can affect the malignant behavior of MM by regulating the expression of MKNK2.

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

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

Characterization of Multiple Myeloma Subpopulations Associated with Treatment Resistance By Single-Cell RNA Sequencing

Multiple myeloma (MM) has extensive genomic and transcriptomic heterogeneity with identical variable-diversity-joining (VDJ) region recombination of immunoglobulin loci (called repertoire). Even though new treatment approaches have been developed, most cases of MM remain incurable. Recent evidence suggests that epigenetic cell plasticity and changes in cell state underlie the therapy resistance, but the mechanisms remain largely unknown. We attempted to understand the comprehensive architecture of the entire tumor cell populations using repertoire clonality as a fingerprint. First, we subdivided bone marrow samples of primary MM at different stages (n=8) into 11 fractions and performed bulk RNA sequencing (bulk RNA-seq). Analysis of the repertoire revealed that tumor cells were present at very low frequencies in some minor fractions other than the main fraction (Lineage marker - (Lin -)/CD19 -/CD38 +/CD138 +) as the disease stage progressed. Importantly, MM cells were consistently present in CD138 negative fraction (Lin -/CD19 -/CD38 +/CD138 -) regardless of disease stage. Flow cytometry (FCM) analysis confirmed that CD138negative MM cells were present at a rate of less than 10% across the disease stages. Compared with CD138 positive cells, they were slowly cycling and showed unique morphology with large cytoplasm (n=59). To achieve a selective and comprehensive evaluation of MM population, we performed single-cell RNA sequencing (scRNA-seq) coupled with VDJ targeted sequencing (scVDJ-seq) on Lin -/CD19 -/CD38 +(without CD138 gating) samples from 11 MM patients, including those at diagnosis and in remission with minimal residual disease (MRD). This method allowed us to discriminate MM cells (20,084 cells) clearly from others by clonal repertoire sequence even in the post-treatment samples with extremely small populations of tumor cells. Because mRNA levels of CD138 did not correspond to the differential intensity in FCM, we defined gene signatures of CD138 positive and negative cells respectively by bulk RNA-seq (n=6). As a result of integration of these gene signatures using Gene Set Enrichment Analysis (GSEA), the heterogeneity on the CD138 axis was also unraveled in scRNA-seq data. Interestingly, gene ontology (GO) analysis of bulk RNA-seq data revealed that several terms related to H3K4 methylation were positively enriched in CD138 negative fraction, which included KMT2A, KMT2B, KMT2C and KMT2D. Western blotting confirmed significant reductions in H3K4me1 and H3K4me3 levels in CD138 negative cells. Furthermore, primary MM cell culture indicated the plasticity between CD138 positive and negative cells, which was also suggested by trajectory/velocity analysis of scRNA-seq data. Collectively, it was suggested that epigenetic changes, including those in histone modifications, contribute to the heterogeneity along the CD138 axis. We next compared the size of each cluster before and after treatment in scRNA-seq data to identify the MM populations associated with treatment resistance. Remarkably, we identified unique clusters which tended to remain at high proportions against treatment in the CD138 negative population. They were characterized by low expression of MHC class-I genes and TNFRSF17/BCMA, as well as 198 significantly upregulated genes including MCL1, MALAT1 and NEAT1, which have been previously implicated in treatment resistance of MM. To systematically interrogate their contribution in MM cell survival, we performed CRISPR/Cas9 knockout screening of selected 100 genes, which were differentially up-regulated in the resistant clusters and associated with poor prognosis in a MMRF CoMMpass Study database. GO analysis of indispensable genes reproducibly identified among three MM cell lines revealed enrichment of several pathways related to mRNA splicing. These results suggested that the RNA splicing pathway is a promising therapeutic target to eliminate resistant populations. Indeed, we confirmed that inhibition of a splicing factor by CRISPR/Cas9-mediated knockout and pharmacological protein degradation suppresses MM cell proliferation effectively. Taken together, we achieved a complete view of MM diversity by using repertoire clonality and confirmed heterogeneity along the CD138 axis, which may be characterized by epigenetic plasticity. Our results highlight a novel subpopulation as a candidate therapeutic target for cure.

Neosetophomone B induces apoptosis in multiple myeloma cells via targeting of AKT/SKP2 signaling pathway.

Multiple myeloma (MM) is a hematologic malignancy associated with malignant plasma cell proliferation in the bone marrow. Despite the available treatments, drug resistance and adverse side effects pose significant challenges, underscoring the need for alternative therapeutic strategies. Natural products, like the fungal metabolite neosetophomone B (NSP-B), have emerged as potential therapeutic agents due to their bioactive properties. Our study investigated NSP-B's antitumor effects on MM cell lines (U266 and RPMI8226) and the involved molecular mechanisms. NSP-B demonstrated significant growth inhibition and apoptotic induction, triggered by reduced AKT activation and downregulation of the inhibitors of apoptotic proteins and S-phase kinase protein. This was accompanied by an upregulation of p21Kip1 and p27Cip1 and an elevated Bax/BCL2 ratio, culminating in caspase-dependent apoptosis. Interestingly, NSP-B also enhanced the cytotoxicity of bortezomib (BTZ), an existing MM treatment. Overall, our findings demonstrated that NSP-B induces caspase-dependent apoptosis, increases cell damage, and suppresses MM cell proliferation while improving the cytotoxic impact of BTZ. These findings suggest that NSP-B can be used alone or in combination with other medicines to treat MM, highlighting its importance as a promising phytoconstituent in cancer therapy.

Inhibition of VCP modulates NF-κB signaling pathway to suppress multiple myeloma cell proliferation and osteoclast differentiation.

Multiple myeloma (MM) is the second most common hematological malignancy, in which the dysfunction of the ubiquitin-proteasome pathway is associated with the pathogenesis. The valosin containing protein (VCP)/p97, a member of the AAA+ ATPase family, possesses multiple functions to regulate the protein quality control including ubiquitin-proteasome system and molecular chaperone. VCP is involved in the occurrence and development of various tumors while still elusive in MM. VCP inhibitors have gradually shown great potential for cancer treatment. This study aims to identify if VCP is a therapeutic target in MM and confirm the effect of a novel inhibitor of VCP (VCP20) on MM. We found that VCP was elevated in MM patients and correlated with shorter survival in clinical TT2 cohort. Silencing VCP using siRNA resulted in decreased MM cell proliferation via NF-κB signaling pathway. VCP20 evidently inhibited MM cell proliferation and osteoclast differentiation. Moreover, exosomes containing VCP derived from MM cells partially alleviated the inhibitory effect of VCP20 on cell proliferation and osteoclast differentiation. Mechanism study revealed that VCP20 inactivated the NF-κB signaling pathway by inhibiting ubiquitination degradation of IκBα. Furthermore, VCP20 suppressed MM cell proliferation, prolonged the survival of MM model mice and improved bone destruction in vivo. Collectively, our findings suggest that VCP is a novel target in MM progression. Targeting VCP with VCP20 suppresses malignancy progression of MM via inhibition of NF-κB signaling pathway.

MALAT1 regulates network of microRNA-15a/16-VEGFA to promote tumorigenesis and angiogenesis in multiple myeloma.

MALAT1 is one of the most hopeful members implicated in angiogenesis in a variety of non-malignant diseases. In multiple myeloma (MM), MALAT1 is recognized as the most highly expressed long non-coding RNA. However, the functional roles of MALAT1 in angiogenesis and the responsible mechanisms have not yet been explored. Herein, we discovered a novel regulatory network dependent on MALAT1 in relation to MM tumorigenesis and angiogenesis. We observed that MALAT1 was upregulated in MM and significantly associated with poor overall survival. MALAT1 knockdown suppressed MM cell proliferation and promoted apoptosis, while restricting endothelial cells angiogenesis. Moreover, MALAT1 directly targeted microRNA-15a/16, and microRNA-15a/16 suppression partly reverted the effects of MALAT1 deletion on MM cells in vitro as well as tumor growth and angiogenesis in vivo. In addition, further study indicated that MALAT1 functioned as a competing endogenous RNA for microRNA-15a/16 to regulate vascular endothelial growth factor A (VEGFA) expression. Our results suggest that MALAT1 plays an important role in the regulatory axis of microRNA-15a/16-VEGFA to promote tumorigenicity and angiogenesis in MM. Consequently, MALAT1 could serve as a novel promising biomarker and a potential antiangiogenic target against MM.

Aquaporin 3 inhibition suppresses the mitochondrial respiration rate and viability of multiple myeloma cells

Aquaporin 3 (AQP3) is a member of the aquaporin water channel family expressed by numerous cell types, including some cancer cells. Accumulating evidence suggests that AQP3 inhibition may impede cancer progression, but drugs targeting AQP3 are still in the early pre-clinical stage of development. Here, we examined the effect of AQP3 inhibition on multiple myeloma (MM), an incurable plasma cell malignancy. Four MM cell lines were cultured in the presence of an anti-AQP3 monoclonal antibody (mAb), the AQP3 inhibitor DFP00173, or corresponding controls, and the effects on cell viability, proliferation, apoptosis, and mitochondrial respiration capacity were compared. Both anti-AQP3 mAb and DFP00173 reduced cell growth, mitochondrial respiration rate, and electron transport chain complex I activity. Both agents also potentiated the antiproliferative efficacy of the anticancer drug venetoclax. Administration of the anti-AQP3 mAb to immunodeficient mice inoculated with RPMI8226 or KMS-11 MM cells significantly suppressed tumor growth. These data provide evidence that AQP3 blockade can suppress MM cell growth in vitro and tumor growth in mice. Thus, AQP3 inhibition may be an effective therapeutic strategy for MM.

ISG20L2 suppresses bortezomib antimyeloma activity by attenuating bortezomib binding to PSMB5.

The proteasome inhibitors (PIs) bortezomib and carfilzomib, which target proteasome 20S subunit beta 5 (PSMB5) in cells, are widely used in multiple myeloma (MM) treatment. In this study, we demonstrated the role of interferon-stimulated 20 kDa exonuclease-like 2 (ISG20L2) in MM PI resistance. Gain- and loss-of-function studies showed that ISG20L2 suppressed MM cell sensitivity to PIs in vitro and in vivo. Patients with ISG20L2lo MM had a better response to PIs and a longer overall survival than patients with ISG20L2hi MM. Biotinylated bortezomib pull-down assays showed that ISG20L2 competed with PSMB5 in binding to bortezomib. The surface plasmon resonance assay confirmed the direct binding of bortezomib to ISG20L2. In ISG20L2hi MM cells, ISG20L2 attenuated the binding of bortezomib to PSMB5, resulting in lower inhibition of proteasome activity and therefore less bortezomib-induced cell death. Overall, we identified a potentially novel mechanism by which ISG20L2 conferred bortezomib resistance on MM. The expression of ISG20L2 correlated with MM PI responses and patient treatment outcomes.

NAT10 acetylates BCL-XL mRNA to promote the proliferation of multiple myeloma cells through PI3K-AKT pathway.

Multiple myeloma (MM) is a clinically distinctive plasma cell malignancy in the bone marrow (BM), in which epigenetic abnormalities are featured prominently. Epigenetic modifications including acetylation have been deemed to contribute to tumorigenesis. N-acetyltransferase 10 (NAT10) is an important regulator of mRNA acetylation in many cancers, however its function in MM is poorly studied. We first analyzed MM clinical databases and found that elevated NAT10 expression conferred a poor prognosis in MM patients. Furthermore, overexpression of NAT10 promoted MM cell proliferation. The correlation analysis of acRIP-seq screened BCL-XL (BCL2L1) as a significant downstream target of NAT10. Further RNA decay assay showed that increased NAT10 improved the stability of BCL-XL mRNA and promoted protein translation to suppress cell apoptosis. NAT10 activated PI3K-AKT pathway and upregulated CDK4/CDK6 to accelerate cellular proliferation. Importantly, inhibition of NAT10 by Remodelin suppressed MM cell growth and induced cell apoptosis. Our findings show the important role of NAT10/BCL-XL axis in promoting MM cell proliferation. Further explorations are needed to fully define the potential of targeting NAT10 therapy in MM treatment.

Blocking glycine utilization inhibits multiple myeloma progression by disrupting glutathione balance

Metabolites in the tumor microenvironment are a critical factor for tumor progression. However, the lack of knowledge about the metabolic profile in the bone marrow (BM) microenvironment of multiple myeloma (MM) limits our understanding of MM progression. Here, we show that the glycine concentration in the BM microenvironment is elevated due to bone collagen degradation mediated by MM cell-secreted matrix metallopeptidase 13 (MMP13), while the elevated glycine level is linked to MM progression. MM cells utilize the channel protein solute carrier family 6 member 9 (SLC6A9) to absorb extrinsic glycine subsequently involved in the synthesis of glutathione (GSH) and purines. Inhibiting glycine utilization via SLC6A9 knockdown or the treatment with betaine suppresses MM cell proliferation and enhances the effects of bortezomib on MM cells. Together, we identify glycine as a key metabolic regulator of MM, unveil molecular mechanisms governing MM progression, and provide a promising therapeutic strategy for MM treatment.

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.

YTHDF2 promotes multiple myeloma cell proliferation via STAT5A/MAP2K2/p-ERK axis.

Multiple myeloma (MM) is still incurable partially due to lacking effective therapeutic targets. Aberrant N6-methyladenosine (m6A) RNA modification plays a vital role in many cancers, however few researches are executed in MM. We first screened the m6A-related genes in MM patient cohorts and correlated these genes with patient outcomes. We found that YTHDF2, a well-recognized m6A reader, was increased in MM patients and associated with poor outcomes. Decreased YTHDF2 expression hampered MM cell proliferation in vitro and in vivo, while enforced YTHDF2 expression reversed those effects. The analyses of m6A-RIP-seq and RIP-PCR indicated that STAT5A was the downstream target of YTHDF2, which was binding to the m6A modification site of STAT5A to promote its mRNA degradation. ChIP-seq and PCR assays revealed that STAT5A suppressed MM cell proliferation by occupying the transcription site of MAP2K2 to decrease ERK phosphorylation. In addition, we confirmed that YTHDF2 mediated the unphosphorylated form of STAT5A to inhibit the expression of MAP2K2/p-ERK. In conclusion, our study highlights that YTHDF2/STAT5A/MAP2K2/p-ERK axis plays a key role in MM proliferation and targeting YTHDF2 may be a promising therapeutic strategy.

Aplidin (Plitidepsin) Regulates Viability and Function of Myeloma Cells and Bone Cells in Combination with Other Anti-MM Drugs

In multiple myeloma (MM) the clonal expansion of malignant plasma cells within the bone marrow results in the development of lytic bone lesions due to exacerbated bone resorption. Despite recent progress in its treatment, MM remains incurable and the associated bone disease persists even after complete remission. Thus, identification of new therapeutic approaches that suppress MM cell growth and protect the bone is an unmet need. Aplidin, a novel anti-cancer compound isolated from the marine tunicate Aplidium albicans, exhibits anti-MM activity even in MM cells resistant to conventional chemotherapy and is currently in phase III clinical trials in Europe. However, the effects of Aplidin on bone cells and MM-induced bone disease are unknown. The goal of the current study was to examine the effect of Aplidin, alone or in combination with other anti-MM drugs, on myeloma cells and bone cells (osteocytes, osteoblasts, and osteoclasts) using in vitro approaches.Aplidin decreased the number of human JJN3 and murine 5TGM1 MM cells with an EC50~10nM, determined by MTT assay, due to decreased cell viability, assessed by Trypan blue exclusion, reaching a 20% increase in MM cell death after 48h. Treatment with glucocorticoids (GC) (10-5M) or Bortezomib (3nM) also decreased MM viability and enhanced the anti-MM activity of Aplidin by 1.6-fold and 2.5-fold, respectively. In addition, Aplidin at >10nM induced MLO-A5 osteocytic dead up to 15% and in combination with GC up to 50%; whereas, in contrast, Bortezomib did not affect osteocyte viability and partially prevented the increase in MLO-A5 cell death induced by Aplidin. Further, 1nM Aplidin increased OB-6 osteoblastic cell death up to 20% and decreased their matrix mineralization capacity, quantified by Alizarin red. These effects were enhanced when Aplidin was combined with GC, but partially prevented by combination of Aplidin with Bortezomib. Cells of the osteoclastic lineage exhibited higher sensitivity to Aplidin in comparison to MM cells or osteoblastic cells, and thus it was tested at lower concentrations. Specifically, 50 pM Aplidin decreased the number of hematopoietic precursors by 15-20%, as quantified by enumerating GM-CSF colonies in cultures established from murine bone marrow. Moreover, Aplidin, at concentrations as low as 0.01 pM, reduced osteoclast precursor commitment and differentiation by 10%, as quantified by enumerating the number of osteoclast precursors maintained in the presence of M-CSF for 3 days with or without Aplidin. In addition, Aplidin decreased by 40% the number of TRAP positive osteoclasts generated after 4 days in the presence of M-CSF and sRANKL; and blunted the rapid (5-15min) increased in ERK phosphorylation induced by Rankl, but did not affect RANKL-induced p38 phosphorylation, suggesting that Aplidin inhibited RANKL-induced differentiation of osteoclast precursors by preventing ERK activation. Lastly, to test the effects of Aplidin on osteoclasts function, mature osteoclasts generated in the presence of M-CSF/sRANKL were detached by trypsinization, replated on bovine dentin slides and exposed to Aplidin for 3 and 7 days. 10pM Aplidin reduced the number of mature osteoclasts by 60% and markedly decreased the number of resorption pits.In summary, Aplidin (1nM and up) decreases MM cell viability and GC or Borteozmib enhances its anti-MM activity. In addition, Aplidin (10nM and up) decreases osteocytic cell viability and this effect is exacerbated by GC but partially prevented by Bortezomib. Moreover, Aplidin (1nM and up) induces osteoblastic cell death and decreases their mineral production, and these effects are increased by GC, but partially prevented by Bortezomib. Furthermore, Aplidin (0.01pM and up) reduces osteoclast precursor commitment and differentiation, and inhibits mature osteoclast resorption activity. Taken together these findings suggest that combining Aplidin with proteosomal inhibitors may be a novel therapy for MM to enhance anti-tumor responses, decrease bone destruction by inhibiting bone resorption, and prevent potential side effects of Aplidin on cells of the osteoblastic lineage. DisclosuresDelgado-Calle:PharmaMar: Research Funding. Galmarini:PharmaMar: Employment. Roodman:PharmaMar: Research Funding. Bellido:PharmaMar: Research Funding.

The c-Abl inhibitor, radotinib induces apoptosis in multiple myeloma cells via mitochondrial-dependent pathway

Multiple myeloma (MM) is a hematological cancer resulting from accumulated abnormal plasma cells. 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. Its mechanism of action involves inhibition of the tyrosine kinase Bcr-Abl and the platelet-derived growth factor receptor. Generally, the mechanism of inhibition of non-receptor tyrosine kinase c-Abl has played an essential role in the inhibition of cancer progression. However, little is known regarding the effects of the c-Abl inhibitor, radotinib on MM cells. In this study, we analyzed the effect of radotinib on multiple myeloma cells. Interestingly, radotinib caused apoptosis in MM cells including RPMI-8226, MM.1S, and IM-9 cells, even in the absence of c-kit expression in 2 of these lines. Radotinib treatment significantly increased the number Annexin V-positive cells and decreased the mitochondrial membrane potential in MM cells. Additionally, we observed that cytochrome C was localized in the cytosol of radotinib-treated MM cells. Moreover, radotinib decreased the expression of Bcl-2 and Bcl-xL, and increased the expression of Bax and Bak in MM cells. Furthermore, radotinib promoted caspase pathway activation by inducing the expression and activity of caspase-3, -7, and -9. Expression of cleaved PARP-1 was also increased by radotinib treatment in various MM cells. In addition, radotinib significantly suppressed MM cell growth in a xenograft animal model using RPMI-8226 cells, and killed ex vivo myeloma cells from patients. In conclusion, radotinib may play an important role as a candidate agent or chemosensitizer for the treatment of MM.

KDM2A Targets PFKFB3 for Ubiquitylation to Inhibit the Proliferation and Angiogenesis of Multiple Myeloma Cells.

The lysine demethylase KDM2A (also known as JHDM1A or FBXL11) demethylates histone H3 at lysine K36 which lead to epigenetic regulation of cell proliferation and tumorigenesis. However, many biological processes are mediated by KDM2A independently by its histone demethylation activity. In the present study, we aimed to characterize the functional significance of KDM2A in multiple myeloma (MM) disease progression. Specifically, we defined that one of the key enzymes of glycolysis PFKFB3 (6-phosphofructo-2-kinase) is ubiquitylated by KDM2A which suppresses MM cell proliferation. Previous study showed that KDM2A and PFKFB3 promoted angiogenesis in various tumor cells. We further reveal that KDM2A targets PFKFB3 for ubiquitination and degradation to inhibit angiogenesis. Several angiogenic cytokines are also downregulated in MM. Clinically, MM patients with low KDM2A and high PFKFB3 levels have shown worse prognosis. These results reveal a novel function of KDM2A through ubiquitin ligase activity by targeting PFKFB3 to induce proliferation, glycolysis and angiogenesis in MM cells. The data provides a new potential mechanism and strategy for MM treatment.

LncRNA transcription factor 7 is related to deteriorating clinical features and poor prognosis in multiple myeloma, and its knockdown suppresses disease progression by regulating the miR-203-mediated Jagged1-Notch1 signaling pathway.

Multiple myeloma (MM) remains a challenge to treat, and its precise pathogenic mechanisms have not been fully clarified. The present study aimed to evaluate the relation between long non-coding RNA transcription factor 7 (lnc-TCF7) and clinical features, as well as the prognosis of patients with MM, and to determine the effects of lnc-TCF7-knockdown on the regulation (and regulatory mechanisms) of MM progression. lnc-TCF7 expression was detected in the bone marrow plasma cells of 86 patients with MM and 30 healthy controls. In patients with MM, the clinical data were collected, and event-free survival (EFS) and overall survival (OS) analyses were conducted. In vitro, lnc-TCF7 expression was detected in MM cell lines and normal bone marrow plasma cells. Using Roswell Park Memorial Institute 8226 cells, functional experiments were conducted following lnc-TCF7 short hairpin (sh)RNA transfection, and compensation experiments were performed after lnc-TCF7 shRNA transfection alone and in combination with a microRNA (miR)-203 inhibitor. lnc-TCF7 expression was increased in patients with MM compared with the healthy controls and was positively related to β-2-microglobulin expression and International Staging System stage, while negatively associated with complete response, EFS and OS. In vitro, lnc-TCF7 was upregulated in MM cells compared with normal bone marrow plasma cells, and its knockdown suppressed MM cell proliferation while promoting apoptosis. Compensation experiments showed that miR-203 inhibition promoted MM progression by regulating the Jagged1-Notch1 signaling pathway in lnc-TCF7-knockdown cells. In conclusion, increased lnc-TCF7 expression was related to deteriorating clinical features and prognosis, and lnc-TCF7-knockdown inhibited disease progression by regulating the miR-203-mediated Jagged1-Notch1 signaling pathway activation in MM.

Dual EZH2 and G9a inhibition suppresses multiple myeloma cell proliferation by regulating the interferon signal and IRF4-MYC axis

Epigenetic mechanisms such as histone modification play key roles in the pathogenesis of multiple myeloma (MM). We previously showed that EZH2, a histone H3 lysine 27 (H3K27) methyltransferase, and G9, a H3K9 methyltransferase, are potential therapeutic targets in MM. Moreover, recent studies suggest EZH2 and G9a cooperate to regulate gene expression. We therefore evaluated the antitumor effect of dual EZH2 and G9a inhibition in MM. A combination of an EZH2 inhibitor and a G9a inhibitor strongly suppressed MM cell proliferation in vitro by inducing cell cycle arrest and apoptosis. Dual EZH2/G9a inhibition also suppressed xenograft formation by MM cells in vivo. In datasets from the Gene Expression Omnibus, higher EZH2 and EHMT2 (encoding G9a) expression was significantly associated with poorer prognoses in MM patients. Microarray analysis revealed that EZH2/G9a inhibition significantly upregulated interferon (IFN)-stimulated genes and suppressed IRF4-MYC axis genes in MM cells. Notably, dual EZH2/G9a inhibition reduced H3K27/H3K9 methylation levels in MM cells and increased expression of endogenous retrovirus (ERV) genes, which suggests that activation of ERV genes may induce the IFN response. These results suggest that dual targeting of EZH2 and G9a may be an effective therapeutic strategy for MM.

CAMKIIγ is a targetable driver of multiple myeloma through CaMKIIγ/ Stat3 axis.

Aberrant activation of CAMKIIγ has been linked to leukemia and T-cell lymphoma, but not multiple myeloma (MM). The purpose of this study was to explore the role of CaMKIIγ in the pathogenesis and therapy of MM. In this study, we found that CaMKIIγ was aberrantly activated in human MM and its expression level was positively correlated with malignant progression and poor prognosis. Ectopic expression of CaMKIIγ promoted cell growth, colony formation, cell cycle progress and inhibited apoptosis of MM cell lines, whereas, knockdown of CAMKIIγ expression suppressed MM cell growth in vitro and in vivo. Mechanically, we observed that CaMKIIγ overexpression upregulated p-ERK and p-Stat3 levels and suppression of CaMKIIγ had opposite effects. CaMKIIγ is frequently dysregulated in MM and plays a critical role in maintaining MM cell growth through upregulating STAT3 signaling pathway. Furthermore, our preclinical studies suggest that CaMKIIγ is a potential therapeutic target in MM, and could be intervened pharmacologically by small-molecule berbamine analogues.