ALBERT

Description: The EPHB4 receptor is implicated in the development of several epithelial tumors and is a promising therapeutic target, including in prostate tumors in which EPHB4 is overexpressed and promotes tumorigenicity. Here, we show that high expression of EPHB4 correlated with poor survival in prostate cancer patients and EPHB4 inhibition induced cell death in both hormone sensitive and castration-resistant prostate cancer cells. EPHB4 inhibition reduced expression of the glucose transporter, GLUT3, impaired glucose uptake, and reduced cellular ATP levels. This was associated with the activation of endoplasmic reticulum stress and tumor cell death with features of immunogenic cell death (ICD), including phosphorylation of eIF2α, increased cell surface calreticulin levels, and release of HMGB1 and ATP. The changes in tumor cell metabolism after EPHB4 inhibition were associated with MYC downregulation, likely mediated by the SRC/p38 MAPK/4EBP1 signaling cascade, known to impair cap-dependent translation. Together, our study indicates a role for EPHB4 inhibition in the induction of immunogenic cell death with implication for prostate cancer therapy.

Description: The initiation and progression of diffuse large B-cell lymphoma (DLBCL) is governed by genetic and epigenetic aberrations. As the most abundant eukaryotic message RNA modification, N6-methyladenosine (m6A) is known to influence various fundamental bioprocesses by regulating 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 (OS) in a univariate analysis, and is statistically significant after adjusting for the National Comprehensive Cancer Network-International Prognostic Index (NCCN-IPI) 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, thus enhances the global m6A level. Integrating transcriptome and m6A-seq analyses reveal that WTAP increases the expression of its critical target gene 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 datasets, 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.

Description: PIs resistance is a major challenge for multiple myeloma (MM). The bone marrow microenvironment facilitates crucial interactions between the myeloma cells and mesenchymal stem cells (MSCs) that permit MM to survive and proliferate progression. Exosomes are involved in intercellular communication, and in this study we investigated how the transfer of exosomic PMSA3 (encodes proteasome subunit α7) and lncPSMA3-AS1 from MSCs to MM cells affected proteasome inhibitors resistance (Figure 1). We firstly underscored that exosomes derived from r-MSCs (MSCs derived from bortezomib-resistant patients), but not from s-MSCs (MSCs derived from bortezomib-resistant patients) reduced the proteasome inhibitors sensitivity in MM cells (Figure 2). To further elucidate mechanisms of Proteasome inhibitors (PIs) resistance, we retrieved a database containing gene expression profile of 169 myeloma cases with clinical response and disease prognosis (GSE9782). The analysis of this dataset showed that the mRNA levels of PSMA3 and PSMA3-AS1 in CD138+ cells are upregulated in bortezomib-resistant patients (Figure 3A-3D). Moreover, Kaplan-Meier analysis showed that high PSMA3 levels in CD138+ MM cells were correlated with reduced progression-free survival (PFS) (p = 0.0307) and overall survival (OS) (p = 0.0328) (Figure 3E). Cox proportional hazards regression analysis further demonstrated that high PSMA3 was an independent prognostic factor for MM patients with bortezomib therapy in a multivariate analysis (p = 0.0013, HR = 1.3104, 95%CI = 1.1113-1.545). Further analysis of Oncomine data showed that the PSMA3 levels appeared a progressive increase in MGUS, SM, MM and PCL (Figure 3F-3H). Similarly, our PIs resistant models (U266BR, U266CR, U266IR, MM.1SBR, MM.1SCR, MM.1SIR) consistently displayed up-regulation of PSMA3 and PSMA3-AS1 expression (Figure 3J). Consistent with this previously published study, our clinical data showed that the mRNA levels of PSMA3 and PSMA3-AS1 are upregulated in CD138+ MM cells derived from bortezomib resistant patients relative to those from bortezomib sensitive patients (Figure 3I). In addition, r-MSCs had increased expression of PSMA3 and PSMA3-AS1 compared to s-MSCs (Figure 3K). Moreover, the expression of PSMA3 and PSMA3-AS1 in MSCs were positively correlated with that in CD138+ myeloma cells (Figure 3L). These data suggested that high levels of PSMA3 and PSMA3-AS1 were correlated with proteasome inhibitors resistance in MM. We further identified that PSMA3 and PSMA3-AS1 in MSCs could be incorporated into exosomes and transmitted to myeloma cells, thus promoting PIs resistance (Figure not shown). PSMA3-AS1 was capable of forming an RNA duplex with PSMA3 pre-mRNA at overlapping regions and this duplex transcriptionally promoted PSMA3 expression by increasing its stability, conferring bortezomib resistance to myeloma cells (Figure not shown). To evaluate the therapeutic potential of PSMA3-AS1 in MM in vivo, bioluminescent MM models (U266-luc), which recapitulates the clinical sequelae, anatomic distribution of MM lesions, and hallmark bone pathophysiology observed in MM patients were established. Intravenously administered siPSMA3-AS1 was found to be effective in increasing bortezomib sensitive (Figure 4). Moreover, circulating exosomal PSMA3 and PSMA3-AS1 derived from the plasma of MM patients were significantly associated with both progression-free survival (PFS) and overall survival (OS) in the univariate analysis, and were still statistically significant after adjusting for the international staging system (ISS) and several other clinical variables in the multivariate analysis (Figure not shown). In summary, our results indicated a unique role of exosomic lncPSMA3-AS1 in transferring proteasome inhibitors resistance from MSCs to MM cells, through a novel exosomic lncPSMA3-AS1/PSMA3 signaling pathway. Exosomic PSMA3 and PSMA3-AS1 may serve as a potential therapeutic target for proteasome inhibitors resistance and a prognostic predictor for clinical response. Disclosures No relevant conflicts of interest to declare.

Description: N6-methyladenosine (m6A), the most abundant modification on eukaryote messenger RNA (mRNA), functions in various fundamental bioprocesses. However, the role of m6A in diffuse large B-cell lymphoma (DLBCL) remains poorly understood. Here, we reported that piRNA-30473, which expression supported the aggressive phenotype of DLBCL and was correlated with poor prognosis, was a potent regulator of m6a modification through targeting m6a methyltransferase WTAP (Figure 1). Piwi-interacting RNAs (piRNAs), a novel class of small non-coding RNAs, have been documented to be involved in the epigenetic regulation of cancer. A cohort of 42 patients with newly diagnosed DLBCL, uniformly treated and followed, was studied. We show that piRNA-30473 was significantly upregulated in high-risk DLBCL patients compared to low-risk DLBCL patients by microarray assay on 6 samples, further confirmed by qPCR analysis on 42 samples (Figure 2). Moreover, silencing of piRNA-30473 expression reduced proliferation and induced cell cycle arrest, but not apoptosis in SU-DHL-8 and Toledo lymphoma cells. In "human-in-mouse" xenograft DLBCL models, injection of antagomir-30473 into mice led to a significant reduction in tumor volume compared with control(Figure 3). Our data further indicated that the combination of piRNA-30473 signature with the National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI) and cytogenetics status had a better prediction for PFS and OS than those without the piRNA-30473 signature in the univariate and multivariate analyses by an AUC analysis with cross-validation. Silencing of piRNA-30473 diminished global m6A level in SU-DHL-8 and Toledo cells and decreased m6a methyltransferase WTAP in mRNA and protein levels. We further identified that piRNA-30473 enhanced WTAP expression through direct binding to its 3' UTR. Consistently, WTAP knockdown decreased the global m6A level and induced cell cycle arrest and growth inhibition in SU-DHL-8 and Toledo cells. Moreover, Silencing of piRNA-30473 displayed decreased cell proliferation, which could be abrogated by WTAP overexpression. Kaplan-Meier analysis showed that high WTAP levels in DLBCL patients were correlated with OS by analyzing the gene expression profiling of DLBCL patients from GEO database (GSE10846) (Figure 4). Next, we mapped the m6A methylomes of siCtrl and siWTAP SU-DHL-8 cells by m6A sequencing (m6A-seq) with independent biological replicates. The RGACH motif (R = G/A; H = A/C/U) were identified to be highly enriched within m6A sites in the SU-DHL-8 cells. M6A peaks in siCtrl and siWTAP SU-DHL-8 cells were abundant in coding sequences (CDSs), 3′ untranslated regions (UTRs), and near stop codons. Remarkably, 85% m6A-Hyper transcripts identified from siCtrl SU-DHL-8 cells turned into m6A-Hypo in siWTAP SU-DHL-8 cells, with approximately 66% of the Hypo-down and 65% of Hypo-up transcripts became Hyperup and Hyper-down, respectively, which might be genuine targets of WTAP. Eleven genes listed in Table 2 showed a significant change between siCtrl and siWTAP SU-DHL-8 cells in m6A peak levels, and abundance of the corresponding mRNA transcript, and were also significantly positively or negatively correlated with WTAP in expression in four datasets of large cohorts of DLBCL. Collectively, our data demonstrate that an enzyme hexokinase II (HK2), which was reported to be a key metabolic driver of the DLBCL phenotype, were functionally important targets of WTAP. WTAP-mediated regulation of HK2 depended on its m6A demethylase activity and the m6A modifications in the target mRNA transcripts (Figure 5). In summary, our finding demonstrate that piRNA-30473 were significantly associated with both PFS and OS in the univariate analysis, and were still statistically significant after adjusting for NCCN-IPI, and adverse cytogenetics in the multivariate analysis. Moreover, we provide compelling evidence demonstrating that WTAP, which was downregulated by piRNA-30473, played a critical oncogenic role in DLBCL, through enhancing m6A levels in mRNA transcripts of its critical target gene HK2 and thereby triggering corresponding signaling cascades. Our study highlights the functional importance of the m6A modification machinery in DLBCL, and provides profound insights into the molecular mechanisms underlying tumorigenesis by revealing a previously unrecognized mechanism of gene regulation in DLBCL. Disclosures No relevant conflicts of interest to declare.