piRNA-30473 contributes to tumorigenesis and poor prognosis by regulating m6A RNA methylation in DLBCL.

The initiation and progression of diffuse large B-cell lymphoma (DLBCL) is governed by genetic and epigenetic aberrations. As the most abundant eukaryotic messenger RNA (mRNA) modification, N6-methyladenosine (m6A) is known to influence various fundamental bioprocesses by regulating the target gene; however, the function of m6A modifications in DLBCL is unclear. PIWI-interacting RNAs (piRNAs) have been indicated to be epigenetic effectors in cancer. Here, we show that high expression of piRNA-30473 supports the aggressive phenotype of DLBCL, and piRNA-30473 depletion decreases proliferation and induces cell cycle arrest in DLBCL cells. In xenograft DLBCL models, piRNA-30473 inhibition reduces tumor growth. Moreover, piRNA-30473 is significantly associated with overall survival in a univariate analysis and is statistically significant after adjusting for the National Comprehensive Cancer Network-International Prognostic Index in the multivariate analysis. Additional studies demonstrate that piRNA-30473 exerts its oncogenic role through a mechanism involving the upregulation of WTAP, an m6A mRNA methylase, and thus enhances the global m6A level. Integrating transcriptome and m6A-sequencing analyses reveals that WTAP increases the expression of its critical target gene, hexokinase 2 (HK2), by enhancing the HK2 m6A level, thereby promoting the progression of DLBCL. Together, the piRNA-30473/WTAP/HK2 axis contributes to tumorigenesis by regulating m6A RNA methylation in DLBCL. Furthermore, by comprehensively analyzing our clinical data and data sets, we discover that the m6A regulatory genes piRNA-30473 and WTAP improve survival prediction in DLBCL patients. Our study highlights the functional importance of the m6A modification in DLBCL and might assist in the development of a prognostic stratification and therapeutic approach for DLBCL.

Downregulation of ITGA6 confers to the invasion of multiple myeloma and promotes progression to plasma cell leukaemia.

BACKGROUND: Secondary plasma cell leukaemia (sPCL) is an aggressive form of multiple myeloma (MM), but the mechanism underlying MM progresses into PCL remains unknown. METHODS: Gene expression profiling of MM patients and PCL patients was analysed to identify the molecular differences between the two diseases. Cox survival regression and Kaplan-Meier analysis were performed to illustrate the impact of integrin subunit alpha 6 (ITGA6) on prognosis of MM. Invasion assays were performed to assess whether ITGA6 regulated the progression of MM to PCL. RESULTS: Gene expression profiling analyses showed that cell metastasis pathways were enriched in PCL and ITGA6 was differentially expressed between PCL and MM. ITGA6 expression was an independent prognostic factor for event-free survival (EFS) and overall survival (OS) of MM patients. Moreover, the stratification ability of the International Staging System (ISS) of MM was improved when including ITGA6 expression. Functional studies uncovered that increased ITGA6 reduced the myeloma cell invasion. Additionally, low expression of ITGA6 resulted from epigenetic downregulating of its anti-sense non-coding RNA, ITGA6-AS1. CONCLUSION: Our data reveal that ITGA6 gradually decreases during plasma cell dyscrasias progression and low expression of ITGA6 contributes to myeloma metastasis. Moreover, ITGA6 abundance might help develop MM prognostic stratification.

HOXC10 Regulates Osteogenesis of Mesenchymal Stromal Cells Through Interaction with Its Natural Antisense Transcript lncHOXC-AS3.

The characteristics of mesenchymal stromal cells (MSCs) which derived from multiple myeloma (MM) patients are typically impaired in osteogenic differentiation. However, the underlying molecular mechanisms need to be further investigated. lncRNAs are emerging as critical regulation molecules in oncogenic pathways. In this study, we identified that bioactive lncRNA HOXC-AS3, which is transcribed in opposite to HOXC10, was presented in MSCs derived from bone marrow (BM) of MM patients (MM-MSCs). HOXC-AS3 was able to interact with HOXC10 at the overlapping parts and this interaction increased HOXC10 stability, then promoted its expression, conferring osteogenesis repression to MM-MSCs. In mouse models, intravenously administered siHOXC-AS3 was proven to be effective in prevention of bone loss, sustained by both anticatabolic activities and bone-forming. These data showed that lncHOXC-AS3 was required for osteogenesis in BM-MSCs by enhancing HOXC10 expression. Our finding thus unveils a novel insight for the potential clinical significance of lncRNA HOXC-AS3 as a therapeutic target for bone disease in MM. Stem Cells 2019;37:247-256.

Group spike-and-slab lasso generalized linear models for disease prediction and associated genes detection by incorporating pathway information.

Motivation: Large-scale molecular data have been increasingly used as an important resource for prognostic prediction of diseases and detection of associated genes. However, standard approaches for omics data analysis ignore the group structure among genes encoded in functional relationships or pathway information. Results: We propose new Bayesian hierarchical generalized linear models, called group spike-and-slab lasso GLMs, for predicting disease outcomes and detecting associated genes by incorporating large-scale molecular data and group structures. The proposed model employs a mixture double-exponential prior for coefficients that induces self-adaptive shrinkage amount on different coefficients. The group information is incorporated into the model by setting group-specific parameters. We have developed a fast and stable deterministic algorithm to fit the proposed hierarchal GLMs, which can perform variable selection within groups. We assess the performance of the proposed method on several simulated scenarios, by varying the overlap among groups, group size, number of non-null groups, and the correlation within group. Compared with existing methods, the proposed method provides not only more accurate estimates of the parameters but also better prediction. We further demonstrate the application of the proposed procedure on three cancer datasets by utilizing pathway structures of genes. Our results show that the proposed method generates powerful models for predicting disease outcomes and detecting associated genes. Availability and implementation: The methods have been implemented in a freely available R package BhGLM (http://www.ssg.uab.edu/bhglm/). Contact: nyi@uab.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Potent Activity of the Bromodomain Inhibitor OTX015 in Multiple Myeloma.

Several studies demonstrate that the bromodomain inhibitor OTX015 has an antitumor activity in cancers. However, translation of these data to molecules suitable for clinical development has yet to be accomplished in multiple myeloma (MM). Here, we identified genes and biologic processes that substantiated the antimyeloma activity of OTX015 with global transcriptomics. OTX015 exerted a strong antiproliferative effect and induced cell cycle arrest in vitro. Gene expression profiling uncovered that OTX015 targeted NF-κB, EGFR, cell cycle regulation, and the cancer proliferation signaling pathway. Gene expression signatures displaying various levels of sensitivity to OTX015 were also identified. The data also showed that oral administration of OTX015 displayed significant antitumor activity in the mice model of disseminated human myeloma. In addition, our study provided the first evidence and rationale that OTX015 could promote osteoblast differentiation of mesenchymal stem cells (MSCs) and inhibited osteoclast formation and resorption in vivo experiments. Herein our results expanded the understanding of the mechanism for BET inhibitors OTX015 in MM. Our study provided an impressive basis for the clinical application of the novel antimyeloma agent OTX015 and uncovered signaling pathways that may play key roles in myeloma cell proliferation.

The BET Bromodomain Inhibitor OTX015 Synergizes with Targeted Agents in Multiple Myeloma.

Treatment failure remains a main challenge in the management of high-risk multiple myeloma (MM) even with the expanding repertoire of new drugs. Combinatorial therapy is considered an encouraging strategy that can overcome the compensatory mechanisms and undesirable off-target effects that limit the benefits of many prospective agents. Preliminary results of a current phase I trial have indicated that the new BET bromodomain inhibitor OTX015 has favorable activity and tolerability. However, OTX015 is not efficacious enough as a monotherapy. Here, we provide evidence that synergistic drug combinations with OTX015 were generally more specific to particular cellular contexts than single agent activities. In addition, pairing OTX015 with three classes of drugs dramatically enhanced the antitumor activity in mouse models of disseminated human myeloma. Our studies further underscored that the BET inhibitor OTX015 sensitized MM cells by interrupting several pathways and genes critical for MM cell proliferation and drug response, which provided the rationale for multiple myeloma therapy with OTX015 combined with conventional chemotherapeutic drugs. Thus, the context specificity of synergistic combinations not only provide profound insights into therapeutically relevant selectivity but also improve control of complex biological systems.

IDH2 contributes to tumorigenesis and poor prognosis by regulating m6A RNA methylation in multiple myeloma.

Epigenetic alterations have been previously shown to contribute to multiple myeloma (MM) pathogenesis via DNA methylations and histone modifications. RNA methylation, a novel epigenetic modification, is required for cancer cell survival, and targeting this pathway has been proposed as a new therapeutic strategy. The extent to the N6-methyladenosine (m6A)-regulatory pathway functions in MM remains unknown. Here, we show that an imbalance of RNA methylation may underlies the tumorigenesis of MM. Mechanistically, isocitrate dehydrogenase 2 (IDH2) is highly expressed in CD138+ cells from MM and its levels appear a progressive increase in the progression of plasma cell dyscrasias. Downregulation of IDH2 increases global m6A RNA levels and reduces myeloma cell growth in vitro, decreases the burden of disease and prolongs overall survival in vivo. IDH2 regulates RNA methylation by activating the RNA demethylase FTO, which is an α-KG-dependent dioxygenase. Furthermore, IDH2-mediated FTO activation decreases the m6A level on WNT7B transcripts, then increases WNT7B expression and thus activated Wnt signaling pathway. Moreover, survival analysis indicates that the elevated expression of IDH2 predicts a poor prognosis. Higher expression of FTO is related to higher International Staging System (ISS) stage and higher Revised-ISS (R-ISS) stage of MM. Collectively, our studies reveal that IDH2 regulates global m6A RNA modification in MM via targeting RNA demethylases FTO. The imbalance of m6A methylation activates the Wnt signaling pathway by enhancing the WNT7B expression, and thus promoting tumorigenesis and progression of MM. IDH2 might be used as a therapeutic target and a possible prognostic factor for MM.

Exosome-Transmitted PSMA3 and PSMA3-AS1 Promote Proteasome Inhibitor Resistance in Multiple Myeloma.

PURPOSE: How exosomal RNAs released within the bone marrow microenvironment affect proteasome inhibitors' (PI) sensitivity of multiple myeloma is currently unknown. This study aims to evaluate which exosomal RNAs are involved and by which molecular mechanisms they exert this function.Experimental Design: Exosomes were characterized by dynamic light scattering, transmission electron microscopy, and Western blot analysis. Coculture experiments were performed to assess exosomal RNAs transferring from mesenchymal stem cells (MSC) to multiple myeloma cells. The role of PSMA3-AS1 in PI sensitivity was further evaluated in vivo. To determine the prognostic significance of circulating exosomal PSMA3 and PSMA3-AS1, a cohort of patients with newly diagnosed multiple myeloma was enrolled to study. Cox regression models and Kaplan-Meier curves were used to analyze progression-free survival (PFS) and overall survival (OS). RESULTS: We identified that PSMA3 and PSMA3-AS1 in MSCs could be packaged into exosomes and transferred to myeloma cells, thus promoting PI resistance. PSMA3-AS1 could form an RNA duplex with pre-PSMA3, which transcriptionally promoted PSMA3 expression by increasing its stability. In xenograft models, intravenously administered siPSMA3-AS1 was found to be effective in increasing carfilzomib sensitivity. Moreover, plasma circulating exosomal PSMA3 and PSMA3-AS1 derived from patients with multiple myeloma were significantly associated with PFS and OS. CONCLUSIONS: This study suggested a unique role of exosomal PSMA3 and PSMA3-AS1 in transmitting PI resistance from MSCs to multiple myeloma cells, through a novel exosomal PSMA3-AS1/PSMA3 signaling pathway. Exosomal PSMA3 and PSMA3-AS1 might act as promising therapeutic targets for PI resistance and prognostic predictors for clinical response.

Exosome-mediated transfer of lncRUNX2-AS1 from multiple myeloma cells to MSCs contributes to osteogenesis.

Multiple myeloma (MM) is characterized by the decreased osteogenic potential of mesenchymal stem cells (MSCs). Communication between cancer cells and cancer stromal cells is a driving factor in tumor progression. Understanding the myeloma-stroma interactions is critical to the development of effective strategies that can reverse bone diseases. Here we identified that bioactive lncRNA RUNX2-AS1 in myeloma cells could be packed into exosomes and transmitted to MSCs, thus repressing the osteogenesis of MSCs. RUNX2-AS1, which arises from the antisense strand of RUNX2, was enriched in MSCs derived from MM patients (MM-MSCs). RUNX2-AS1 was capable of forming an RNA duplex with RUNX2 pre-mRNA at overlapping regions and this duplex transcriptionally repressed RUNX2 expression by reducing the splicing efficiency, resulting in decreased osteogenic potential of MSCs. In vivo mouse models, administered an inhibitor of exosome secretion, GW4869, was found to be effective in preventing bone loss, sustained by both bone formation and anticatabolic activities. Therefore, exosomal lncRNA RUNX2-AS1 may serve as a potential therapeutic target for bone lesions in MM. In summary, our results indicated a key role of exosomal lncRUNX2-AS1 in transferring from MM cells to MSCs in osteogenic differentiation, through a unique exosomal lncRUNX2-AS1/RUNX2 pathway.