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The Alternative Splicing Factor MBNL1 Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-Induced Stemness

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The Alternative Splicing Factor MBNL1 Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-Induced Stemness Author Manuscript Published OnlineFirst on September 14, 2020; DOI: 10.1158/0008-5472.CAN-20-1233 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. The Alternative Splicing Factor MBNL1 Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-induced Stemness Dillon M. Voss1*, Anthony Sloan1*, Raffaella Spina, PhD2,3,4, Heather M. Ames, MD PhD2,4, and Eli E. Bar, PhD2,3,4 1 Department of Neurological Surgery, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America. 2 Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America. 3 Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland, United States of America. 4 Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, United States of America. *Authors contributed equally to the study Running Title: MBNL1 Regulates GBM Initiation and Progression The authors declare no potential conflicts of interest. Correspondence to: Eli E. Bar, Ph.D. Departments of Pathology and Neurosurgery, Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine 655 W. Baltimore St., Bressler Research Bldg., Room 8-039, Baltimore, MD 21201; [email protected] (office) 410-706-4826, (lab) 410-706- 2299 Word Count: 7946 1 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 14, 2020; DOI: 10.1158/0008-5472.CAN-20-1233 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT Muscleblind-like-proteins (MBNL) belong to a family of tissue-specific regulators of RNA metabolism that control pre-messenger RNA-splicing (AS). Inactivation of MBNL causes an adult-to-fetal AS transition, resulting in the development of myotonic dystrophy. We have previously shown that the aggressive brain cancer glioblastoma (GBM) maintains stem-like features (GSC) through hypoxia- induced responses. Accordingly, we hypothesize here that hypoxia-induced responses in GBM might also include MBNL-based AS to promote tumor progression. When cultured in hypoxia, GSC rapidly exported MBNL1 out of the nucleus, resulting in significant inhibition of MBNL1 activity. Notably, hypoxia-regulated inhibition of MBNL1 also resulted in evidence of adult-to-fetal alternative splicing transitions. Forced expression of a constitutively active isoform of MBNL1 inhibited GSC self-renewal and tumor initiation in orthotopic transplantation models. Induced expression of MBNL1 in established orthotopic tumors dramatically inhibited tumor progression, resulting in significantly prolonged survival. This study reveals that MBNL1 plays an essential role in GBM stemness and tumor progression, where hypoxic responses within the tumor inhibit MBNL1 activity, promoting stem-like phenotypes and tumor growth. Reversing these effects on MBNL1 may therefore yield potent tumor suppressor activities, uncovering new therapeutic opportunities to counter this disease. Significance: This study describes an unexpected mechanism by which RNA-binding protein Muscleblind-Like-1(MBNL1) activity is inhibited in hypoxia by a simple isoform switch to regulate glioma stem cell self-renewal, tumorigenicity, and progression. 2 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 14, 2020; DOI: 10.1158/0008-5472.CAN-20-1233 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. INTRODUCTION Glioblastoma (GBM) is among the least curable cancers because of distinct sub-populations of proliferative and invasive cells that coordinately drive tumor growth, progression, and recurrence after therapy (1-3). The apparent genetic heterogeneity in GBM and other solid cancers, even within a single patient's cancer, demands the Identification and targeting of signaling nodes that are critical to multiple oncogenic pathways. Necrotic foci with surrounding hypoxic cellular pseudopalisades and microvascular hyperplasia are histological features found in GBM (4). Over the past several years, we have demonstrated that hypoxia promotes the expansion of aggressive subpopulations of cells, often referred to as glioma stem cells (GSCs) (5). However, the mechanisms by which hypoxia regulates GSC are not entirely clear. Oxygen levels play an essential role in regulating stem cells in multiple tissues, including the central nervous system (reviewed in (6)). Growth under low oxygen concentrations is known to maintain pluripotency and inhibit the differentiation of embryonic stem cells (7). In the rodent brain, hypoxia regulates self-renewal, survival, and differentiation of stem and progenitor cells (8, 9). In humans, hypoxia promotes the expansion of progenitor cells (10). We have recently shown that low oxygen levels commonly found in human tumors, promote the expansion of the cancer stem cell pool, as well as those grown in vitro for more extended periods (5). Along these lines, we and others have demonstrated the pivotal roles of hypoxia-inducible factors (HIFs) -1alpha and -2alpha in orchestrating the transcriptional and metabolic adaptations to hypoxia, and in inducing stem cell phenotypes in the hypoxic microenvironment ((5, 11-16), also reviewed in (17)). Thus, while the contributions of HIFs and hypoxia to promote self-renewal and expansion of glioma stem cells (GSCs) are established, the identity of additional regulators of GSC biology remains to be elucidated. Precursor messenger RNA (pre-mRNA) splicing is a fundamental process in the regulation of eukaryotic gene expression. The mammalian nervous system makes extensive use of splicing regulation to generate specialized protein isoforms that affect all aspects of neuronal development and function (18- 3 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 14, 2020; DOI: 10.1158/0008-5472.CAN-20-1233 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 21). While splicing defects are increasingly implicated in neurological diseases and several types of cancer such as myelodysplastic syndrome and AML, a clear role for specific alternative splicing events in GBM and their regulators remain to be established (22). Alternative splicing patterns are regulated by specialized RNA binding proteins that alter spliceosome assembly at specific splice sites (21, 23, 24). The Muscle-Blind-Like (MBNL) family of RNA binding proteins have been studied extensively in the context of the neuromuscular disorder myotonic dystrophy, where inactivation of MBNL proteins result in a shift in splicing from an adult- to fetal-like patterns (25, 26). Alternative mRNA splicing can substantially alter the functions of the proteins encoded by the mRNA (27). The Muscleblind-like (MBNL) family of sequence-specific pre-mRNA splicing factors bind RNA through pairs of highly conserved zinc finger binding domains that recognize YGCY (where Y = C or U) and similar motifs (28-32). MBNL proteins are predominantly expressed in skeletal muscle, neuronal tissues, thymus, liver, and kidney and are essential for terminal differentiation of myocytes and neurons (33). In the brain, MBNL1 levels are significantly higher in astrocytes as compared with neural stem cells (34). MBNL1 is also involved in pluripotent stem cell differentiation, thereby linking isoform expression and the pluripotent and differentiation states (35). Importantly, Mbnl1 transcripts themselves undergo extensive alternative splicing, generating numerous protein isoforms. The inclusion of the highly conserved exon 5 is essential for nuclear localization and splicing activity of the MBNL1 protein (36, 37). The knockdown of Mbnl1 in cultured murine fetal liver progenitors blocks erythroid differentiation (38). Inactivation of the MBNL1 protein is critical in the etiology of myotonic dystrophy, resulting in cataract formation, abnormal muscle relaxation, heart and nerve dysfunction, and other pathologies (25, 39). Importantly, MBNL1 inactivation results in an adult-to-fetal alternative splicing shift in numerous MBNL1 target genes. The function and expression of MBNL1 in gliomas and specifically in GSCs are currently unknown. So too is the role of the hypoxic GBM microenvironment in regulating MBNL1 activity. Accordingly, this study explores the impact of hypoxia on MBNL1 activity in GSCs. In doing so, we 4 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 14, 2020; DOI: 10.1158/0008-5472.CAN-20-1233 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. found that while MBNL1 is expressed in all gliomas, MBNL1 activity is inhibited in hypoxia, mimicking myotonic dystrophy-like adult-to-fetal alternative splicing switching in multiple MBNL1 target genes. These included the fetal isoform of the GSC marker integrin alpha 6 (ITGA6) and CD47, a novel macrophage immune checkpoint protein that plays a broad role in cancer immune evasion across multiple cancer types, suggesting
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