NAT10 contributes to the progression of multiple myeloma through ac4C modification of GPR37

Novel therapies for multiple myeloma.

Stroma-Derived Exosomes Mediate Oncogenesis in Multiple Myeloma

Background. Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) support multiple myeloma (MM) cell growth, but little is known about the putative mechanisms that may regulate the interaction between clonal MM plasma cells and the surrounding BM milieu. We characterized the role BM-MSC-derived exosomes as key regulators of MM pathogenesis. Purpose. 1) To determine the ability of BM-MSCs to release and transfer exosomes to MM cells. 2) To determine the role of BM-MSC-derived exosomes in inducing MM tumor growth and MM cell dissemination Methods. MSCs were collected from BM of healthy subjects and MM patients, showing a multipotent MSC phenotype (CD34-; CD14-; CD45-; CD19-; CD138-; CD73+; CD90+; CD105+; CD106+). Exosomes were collected from conditioned-medium of normal-BM-MSCs and MM-BM-MSCs, or HS-5 cells; and studied using electron microscopy, immunogold-labeling, and western-blot for CD63 and CD81 detection. Transfer of PKH67-fluorescently-labeled-exosomes to MM cells was evaluated by time-lapse confocal microscopy. Transfer of murine-derived miRNA-containing exosomes into human MM cells was evaluated by qRT-PCR (exosomes were collected from BM-MSCs of C57BL/6 miRNA-15a/16-1-/- or C57BL/6 mice). miRNA expression profiling was obtained from normal (n=4) and MM (n=9) BM-MSCs-derived exosomes (TaqMan-human-miRNA-profiling). Normal and MM BM-MSCs-derived exosomes were loaded into tissue-engineered bones (TEB) with MM.1S-GFP+/Luc+ cells: MM cell homing and MM tumor growth have been tested in vivo by using confocal-microscopy and bioluminescence-imaging (BLI), respectively. Loss- and gain-of-function studies were performed using normal-BM-MSCs, MM-BM-MSCs and HS-5 cells transfected with either pre- or anti-miRNA-15a. Results. Normal-BM-MSCs and MM-BM-MSCs released CD63+/CD81+ exosomes, as confirmed by electron microscopy, immunogold labeling, and western blot. BM-MSCs exosomes are transferred into MM cells, as shown by confocal microscopy; and further validated by qRT-PCR in human MM cell lines incubated with murine (C57BL/6 miRNA-15a/16-1-/- and wild-type) BM-MSCs-derived exosomes. The impact of normal-BM-MSC- and MM-BM-MSC-derived exosomes on MM cell behavior in vivo was next evaluated. MM cells co-cultured with MM BM-MSC-derived exosomes induced rapid tumor growth at the site of the TEB scaffold, as well as rapid dissemination to distant BM niches, as compared to MM cells co-cultured with exosomes derived from normal BM-MSCs. We next performed miRNA expression profiling on exosomes isolated from MSCs, and found increased expression of 24 miRNAs and reduced expression of 3 miRNAs in MM-BM-MSCs-derived exosomes versus normal (1.5 fold-change; P<0.05). Specifically, miRNA15a was significantly lower in MM-BM-MSC-derived exosomes, similarly to primary MM cells that present with reduced miRNA-15a expression. We therefore sought to examine whether lack of transfer of the tumor suppressor miRNA15a can lead to significant change in tumor growth and dissemination in MM, and found that by over-expressing miRNA-15a in normal BM-MSCs and HS-5 cells inhibited MM cell proliferation and adhesion to fibronectin. Next MM cells were cultured in presence of BM-MSCs isolated from either C57BL/6 mice or C57BL/6 miRNA15a/16-1-/-: miRNA15a-deficient BM-MSCs significantly induced MM cell proliferation (P<0.05). Moreover, exosomes isolated from HS-5 pre-miRNA15a-transfected cells both inhibited MM cell proliferation and reduced their adhesion properties. Conclusion. These findings demonstrate the existence of exosome-driven interactions between the BM milieu and MM cells, suggesting that exosomes might constitute a novel mechanism for intercellular transfer of miRNAs to MM cells. Citation Format: Aldo M. Roccaro, Antonio Sacco, Patricia Maiso, Michele Moschetta, Salomon Manier, Yuji Mishima, Michaela Reagan, Yosra Aljawai, Irene M. Ghobrial. Stroma-derived exosomes mediate progression in multiple meyloma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B62. doi:10.1158/1538-7445.CHTME14-B62

SCIO-469, a Potent and Selective Inhibitor of p38a MAPK, Normalizes the Bone Marrow Microenvironment and Inhibits Multiple Myeloma Cell Proliferation in In Vitro and In Vivo Models.

Anti-Estrogens Induce Apoptosis of Multiple Myeloma Cells

Anti-Estrogens Induce Apoptosis of Multiple Myeloma Cells

We have shown that thalidomide (Thal) and its immunomodulatory derivatives (IMiDs), proteasome inhibitor PS-341, and As(2)O(3) act directly on multiple myeloma (MM) cells and in the bone marrow (BM) milieu to overcome drug resistance. Although Thal/IMiDs, PS-341, and As(2)O(3) inhibit nuclear factor (NF)-kappaB activation, they also have multiple and varied other actions. In this study, we therefore specifically address the role of NF-kappaB blockade in mediating anti-MM activity. To characterize the effect of specific NF-kappaB blockade on MM cell growth and survival in vitro, we used an IkappaB kinase (IKK) inhibitor (PS-1145). Our studies demonstrate that PS-1145 and PS-341 block TNFalpha-induced NF-kappaB activation in a dose- and time-dependent fashion in MM cells through inhibition of IkappaBalpha phosphorylation and degradation of IkappaBalpha, respectively. Dexamethasone (Dex), which up-regulates IkappaBalpha protein, enhances blockade of NF-kappaB activation by PS-1145. Moreover, PS-1145 blocks the protective effect of IL-6 against Dex-induced apotosis. TNFalpha-induced intracellular adhesion molecule (ICAM)-1 expression on both RPMI8226 and MM.1S cells is also inhibited by PS-1145. Moreover, PS-1145 inhibits both IL-6 secretion from BMSCs triggered by MM cell adhesion and proliferation of MM cells adherent to BMSCs. However, in contrast to PS-341, PS-1145 only partially (20-50%) inhibits MM cell proliferation, suggesting that NF-kappaB blockade cannot account for all of the anti-MM activity of PS-341. Importantly, however, TNFalpha induces MM cell toxicity in the presence of PS-1145. These studies demonstrate that specific targeting of NF-kappaB can overcome the growth and survival advantage conferred both by tumor cell binding to BMSCs and cytokine secretion in the BM milieu. Furthermore, they provide the framework for clinical evaluation of novel MM therapies based upon targeting NF-kappaB.

BackgroundThe therapeutic benefits of mesenchymal stromal cells (MSCs) are largely dependent on paracrine factors, but the supernatants of the different MSCs may have different effects on multiple myeloma (MM) cells. Therefore, this study compared supernatants of bone marrow-derived mesenchymal stromal cells (BM-MSCs) with umbilical cord wharton’s jelly’s mesenchymal stem cells (UC-WJ MSCs) in different states (non-senescent and replicative senescence) on the MM cells.MethodsWe extracted human BM-MSCs and UC-WJ MSCs in vitro and used H2O2 to induce replicative senescence. Concentrated supernatants from MSCs and senescent MSCs (SMSCs) were added to MM cells. Cell proliferation, the cell cycle, apoptosis, cell migration, tumor stemness factor expression, and cytokine expression levels were analyzed. Transcription regulation of signaling pathways was discussed.ResultsWe successfully isolated and identified BM-MSCs, UC-WJ MSCs, and SMSCs. When concentrated supernatants from BM-MSCs, UC-WJ MSCs, senescent BM-MSCs (SBM-MSCs), senescent UCWJ MSCs (SUC-WJ MSCs) were used to treat MM cells, BMMSCs and SBM-MSCs supernatants promoted the proliferation of MM cells, with a more pronounced effect by SBM-MSCs. UC-WJ MSCs and SUC-WJ MSCs supernatants inhibited the viability and proliferation of MM cells. BM-MSCs and SBM-MSCs supernatants increased the proportion of MM cells in the S-phase, with the effect of SBM-MSCs being more evident. UC-WJ MSCs and SUC-WJ MSCs supernatants arrested MM cells in the G0/G1 phase. BM-MSCs and SBM-MSCs supernatants enhanced the migration and tumor stemness of MM cells, with SBMMSCs having a more dramatic effect. UC-WJ MSCs and SUC-WJ MSCs supernatants inhibited the migration and tumor stemness of MM cells, with UC-WJ MSCs having a more inhibitory effect. IL-6 and VEGFA expression correlated negatively with the survival of patients with MM according to online database analysis, in addition, we found that the expression of IL-6 and VEGFA was higher in MM patients through GEO database analysis. BM-MSCs and SBM-MSCs supernatants treatment increased the expression of IL-6 and VEGFA on MM cells, while UC-WJ MSCs and SUC-WJ MSCs supernatants inhibited their expression. Signal pathway validation showed that the biological function of MSCs in MM is closely related to the PI3K/AKT/NF-κB pathway.ConclusionThe supernatants of BM-MSCs promote the proliferation of MM cells, On the contrary, the supernatants of UC-WJ MSCs inhibit MM cell proliferation. We observed that MSCs from different sources and different states have contrasting biological functions in MM cells. Furthermore, this research was provided to the optimal cancer gene therapy vector for MM was UC-WJ MSCs, even UC-WJ MSCs was in the state of senescence.

Targeting IKK Inhibits Multiple Myeloma (MM) Cell Growth in the Bone Marrow Microenvironment.

Targeting MM at the Nexus between Cell Cycle and Transcriptional Regulation Via CDK7 Inhibition

Blockage of TRAF6 by Dominant Negative Peptides to Inhibit Multiple Myeloma (MM) Cell Proliferation and Osteoclast Formation through NF-κB, JNK and AKT Signal Transduction Pathways

C/EBPb Is a Critical Mediator of Resistance to IMiD® Immunomodulatory Compounds and Affected by IMiD Compounds Via Control of Protein Translation

To investigate the effect of different isoforms of RBBP6 on the proliferation of multiple myeloma (MM) cells after alternative splicing mediated by splicing factor SRSF1 . RT-PCR was used to detect the expression levels of RBBP6 mRNA splicing isoforms regulated by SRSF1 . The GEO database was used to analyze the changes of RBBP6 isoform 1 in the progression of plasma cell disease, and survival analysis was used to evaluate the value of this gene in the prognosis of MM patients. in vitro loss-of-function and gain-of-function experiments were conducted by transfecting control siRNA, RBBP6 isoform 1 siRNA, empty vector (EV), OE-RBBP6 isoform 3 into MM.1S cells, then the expression levels of RBBP6 isoform 1 and RBBP6 isoform 3 were detected by real-time PCR and Western blot. CCK-8 assay was performed to detect the cell proliferation ability. Knockdown of SRSF1 increased the expression of RBBP6 isoform 3 and decreased the expression of RBBP6 isoform 1. RBBP6 isoform 1 was closely related to the progression of plasma cell disease, and the high expression of RBBP6 isoform 1 was associated with poor prognosis in patients with MM. Downregulation of RBBP6 isoform 1 expression and overexpression of RBBP6 isoform 3 both reduced the proliferation ability of MM cells. And after downregulating the expression of RBBP6 isoform 1, the level of p53 protein in MM cells was significantly increased (P < 0.05). In MM, splicing factor SRSF1 can cause alternative splicing abnormalities in RBBP6 . The RBBP6 isoform 1 promotes MM cell proliferation, while the RBBP6 isoform 3 inhibits MM cell proliferation, and the mechanism may be related to regulating the p53 pathway.

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

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.