Author Manuscript Published OnlineFirst on January 4, 2019; DOI: 10.1158/1078-0432.CCR-18-2363 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Exosome-Transmitted PSMA3 and PSMA3-AS1 Promotes Proteasome Inhibitors Resistance in Multiple Myeloma Hongxia Xu1,2, +, Huiying Han1, +, Sha Song1, Nengjun Yi3, Chen’ao Qian4, Yingchun Qiu1, Wenqi Zhou1, Yating Hong5, Wenyue Zhuang6, Zhengyi Li7, Bingzong Li5,*, Wenzhuo Zhuang1, * 1Department of Cell Biology, School of Biology & Basic Medical Sciences, Soochow University, Suzhou, China. 2Xiangyang No.1 People's Hospital, Hubei University of Medicine, Xiangyang, China. 3Department of Biostatistics, University of Alabama at Birmingham, Birmingham, AL 35294, USA. 4Department of Bioinformatics, School of Biology & Basic Medical Sciences, Soochow University, Suzhou, China. 5Department of Haematology, the Second Affiliated Hospital of Soochow University, Suzhou, China. 6Department of Molecular Biology, School of Laboratory Medicine of Beihua University, Jilin, China. 7Department of Clinical Examination Basis, Laboratory Acadamy of Jilin Medical College, Jilin, China. +: Contribute equally to this work *: Correspondence: Wenzhuo Zhuang, Email: [email protected], Tel.: 86−512−65880103, Address: School of Biology & Basic Medical Sciences, Soochow University, Ren Ai Road 199, Suzhou, 215123, China. Or Bingzong Li, Email: [email protected], Tel.: 86−512−67784069, Address: The Second Affiliated Hospital of Soochow University, San Xiang Road 1055, Suzhou, 215006, China. Running title: Exosomal PSMA3 and PSMA3-AS1 in PIs resistance Key words: Multiple myeloma, Proteasome inhibitors, Exosome, Long non-coding RNA, Drug resistance Financial support: This study was supported by Natural Science Foundation of Jiangsu Province China (BK20161218, BK20161223), the Science and Technology Development Project of Suzhou City (SS201856), National Natural Science Foundation of China (81670191, 81673448), The Applied Basic Research Programs of Suzhou City (SYS201546), Natural Science Foundation of Jilin Province (20160101234JC). Conflict of Interest statement: The authors declare no potential conflicts of interest. Text word count: 4830, Number of figures: 6, Number of tables: 0. 1 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 4, 2019; DOI: 10.1158/1078-0432.CCR-18-2363 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Translational Relevance PIs resistance is a major challenge for MM. The bone marrow microenvironment promotes myeloma cells survival and proliferation through the interaction between the MSCs and MM cells. The bone marrow microenvironment promotes the interactions between the MSCs and MM cells which permit MM to survive and proliferate. Here we discovered that exosome-mediated transfer of PSMA3 (encodes proteasome subunit α7) and lncPSMA3-AS1 from MSCs to MM cells contributed to PIs resistance. PSMA3-AS1, which arises from the antisense strand of PSMA3, was highly expressed in myeloma cells (r-MM) and MSCs (r-MSCs)-derived from bortezomib-resistant patients. As a pair of protein-coding/non-coding antisense transcripts, PSMA3 and PSMA3-AS1 were disordered concurrently and correlated positively in MM cells, driving PIs sensitivity in MM. These results provided in vitro and in vivo evidence that interference with exosome RNAs served as a promising approach to overcome PIs resistance in MM. Moreover, our data indicated that circulating exosomal PSMA3 and PSMA3-AS1 could develop the prognostic stratification of MM patients, in addition to the international staging system. 2 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 4, 2019; DOI: 10.1158/1078-0432.CCR-18-2363 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Purpose: How exosomal RNAs released within the bone marrow microenvironment affect proteasome inhibitors (PIs) sensitivity of multiple myeloma (MM) 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. Coculture experiments were performed to assess exosomal RNAs transferring from mesenchymal stem cells (MSCs) to MM cells. The role of PSMA3-AS1 in PIs sensitivity was further evaluated in vivo. To determine the prognostic significance of circulating exosomal PSMA3 and PSMA3-AS1, a cohort of newly diagnosed MM patients 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 promoted PIs 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 MM patients were significantly associated with PFS and OS. 3 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 4, 2019; DOI: 10.1158/1078-0432.CCR-18-2363 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Conclusion: This study suggested a unique role of exosomal PSMA3 and PSMA3-AS1 in transmitting PIs resistance from MSCs to MM cells, through a novel exosomal PSMA3-AS1/PSMA3 signaling pathway. Exosomal PSMA3 and PSMA3-AS1 might act as promising therapeutic targets for PIs resistance and prognostic predictors for clinical response. Introduction Multiple myeloma (MM) is a malignancy of the plasma cell characterized by proliferation of plasma cell clones (1). The proteasome inhibitor bortezomib (BTZ) has shown promise in the treatment of MM (2). However, its therapeutic activity was severely impeded by BTZ resistance (3). Multifactorial mechanisms underlying PIs resistance have been studied: genetic mutations, gene expression signatures, copy number abnormalities and bone marrow microenvironment (4). Nevertheless, MM is a multifaceted disease related with genetic, epigenetic and chromosomal alterations, and the mechanisms underlying PIs resistance in MM remains elusive. The proteasome is a 26S enzyme complex that consists of a 19S regulatory complex and a core 20S catalytic complex. The 20S complex is composed of two rings of seven α subunits and two rings of seven β subunits (5). PIs could slightly inhibit the peptidyl glutamyl–like activity at the β1 subunit (PSMB6) and mainly target the chymotrypsin-like activity of β5 subunit (PSMB5) (6, 7). The mutations in β5 subunit or the increased expression of β5 subunit was detected in BTZ-resistant hematologic tumor cell lines (8). However, the β5 mutations and overexpression are absent in clinical samples derived from BTZ resistant patients (9, 10). Therefore, the β5 subunit 4 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 4, 2019; DOI: 10.1158/1078-0432.CCR-18-2363 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. dysregulation may not be uniquely responsible for PIs resistance. It is conceivable that other mechanisms could be associated with PIs resistance. The bone marrow microenvironment activates many pathways leading to disease progression. MSCs support tumor cell growth, metastasis and evasion of the immune system (11). The interaction between MSCs and myeloma cells performs a critical role in MM pathogenesis, progress and chemotherapy resistance (12, 13). These findings indicate a need for developing novel drugs to counteract these cancer–stroma interactions. Exosomes act as key communicators between tumor microenvironment and cancer cells (14, 15). The interaction between MSCs and MM cells plays a crucial role in MM pathogenesis and drug resistance by exosomes (16, 17). Recently, lncRNAs have been reported to exist in exosomes (18, 19). LncRNAs are transcribed from thousands of loci in mammalian genomes and function in a wide range of biological processes (20, 21). In cancers, lncRNAs are emerging important regulators in oncogenic pathways (22, 23). However, the role of lncRNAs in the MM pathogenesis and progression has not been fully elucidated. In particular, how exosomal lncRNAs derived from the bone marrow microenvironment contribute to PIs resistance is unexplored in MM. Abundant bidirectional transcription of eukaryotic genes has been explored across genomes (24). Emerging evidence suggests that antisense lncRNAs play vital roles in regulating their sense transcripts partners, in particular, affecting the expression of their associated protein coding genes (24, 25). However, we know little about the 5 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript