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The deubiquitylase (DUB) USP37 is a component of the ubiquitin system and controls cell proliferation by regulating the stability of the cyclin-dependent kinase inhibitor 1B, (CDKN1B/p27Kip1). The expression of USP37 is downregulated in human medulloblastoma tumor specimens. In the current study, we show that USP37 prevents medulloblastoma growth in mouse orthotopic models, suggesting that it has tumor-suppressive properties in this neural cancer. Here, we also report on the mechanism underlying USP37 loss in medulloblastoma. Previously, we observed that the expression of USP37 is transcriptionally repressed by the RE1 silencing transcription factor (REST), which requires chromatin remodeling factors for its activity. Genetic and pharmacologic approaches were employed to identify a specific role for G9a, a histone methyltransferase (HMT), in promoting methylation of histone H3 lysine-9 (H3K9) mono- and dimethylation, and surprisingly trimethylation, at the USP37 promoter to repress its gene expression. G9a inhibition also blocked the tumorigenic potential of medulloblastoma cells in vivo. Using isogenic low- and high-REST medulloblastoma cells, we further showed a REST-dependent elevation in G9a activity, which further increased mono- and trimethylation of histone H3K9, accompanied by downregulation of USP37 expression. Together, these findings reveal a role for REST-associated G9a and histone H3K9 methylation in the repression of USP37 expression in medulloblastoma. Implications: Reactivation of USP37 by G9a inhibition has the potential for therapeutic applications in REST-expressing medulloblastomas. Mol Cancer Res; 15(8); 1073–84. ©2017 AACR.

A major goal of our work is to study medulloblastoma biology to facilitate the development of targeted therapy. Here, we have dissected the contribution of epigenetics to tumor development. Epigenetic perturbations are pharmacologically reversible and may have therapeutic potential. The RE1-Silencing Transcription Factor (REST) is an epigenetic modulator, and a repressor of neuronal differentiation genes. Its expression is significantly elevated in human medulloblastomas, and is associated with poor prognosis. REST is a driver of medulloblastoma in mouse orthotopic models. To understand REST's contribution to tumor development, we created a novel genetically engineered mouse model in which REST transgene is conditionally elevated in the cerebellar granule progenitors (GNPs), the cells of origin of some medulloblastoma. Transgene induction caused GNP hyperproliferation, accompanied by a failure to express the cell cycle regulator-p27. Using genetic and biochemical approaches, we attributed the lack of p27 expression to direct REST-mediated repression of a novel deubiquitylase, USP37. We also identified a role for USP37 in p27 protein stabilization, and terminal cell cycle exit. USP37 and p27 levels were significantly correlated in patient samples, and their loss was associated with poor overall patient survival. Importantly, constitutive USP37 expression blocked the tumorigenic potential of high-REST human medulloblastomas, suggesting a tumor suppressive function for USP37. REST binding to the USP37 promoter caused increased histone H3 lysine (K)-9 tri-methylation, an epigenetic mark associated with gene silencing. The histone methyl-transferase G9a, which promotes this modification, is a known REST co-repressor. Importantly, genetic ablation of G9a or pharmacological inhibition of its activity upregulated USP37 expression in a REST-dependent manner, and blocked tumor growth in mice. Our work has provided the first link between epigenetic deregulation of the proteasome and medulloblastoma development, and supports the manipulation of the REST-epigenome for future therapeutic application.

The Repressor Element-1 Silencing Transcription Factor (REST) negatively regulates neurogenesis. It epigenetically silences the expression of neuronal differentiation genes through modulation of histone acetylation and histone methylation. REST levels are elevated in over 80% of human medulloblastomas, and spans all four molecular sub-groups. Interestingly, a subset of patients with sonic hedgehog (Shh)-driven medulloblastomas with high REST expression (+3/ + 4 on a scale of 0-4) had a significantly elevated incidence of metastatic disease compared to patients with low-REST or no-REST expressing Shh-driven tumors. To understand if REST is a driver of metastatic disease, we generated a novel REST knock-in (RESTKI) mouse model in which transgene expression is conditionally elevated in cerebellar granule neuron progenitors (GNPs), the cells of origin of Shh-driven medulloblastomas. RESTKI mice were then crossed with mice exhibiting constitutive activation of Shh signaling (Ptc +/−), and REST transgene expression was induced in the progeny (RESTKI/Ptc +/−) on postnatal day 2. While RESTKI mice exhibited pre-neoplastic lesions, both Ptc +/− and RESTKI/Ptc +/− mice formed cerebellar tumors. However, a significant increase in tumor penetrance (100%) was seen in RESTKI/Ptc +/− mice compared to 20% in Ptc +/− mice. A substantial decrease in tumor latency (10-40 days) was also observed in RESTKI/Ptc +/− in contrast to 3-7 months in Ptc +/− mice. Histopathological analyses of mice brains revealed leptomeningeal and infiltrative medulloblastoma with significant changes in the tumor microenvironment in RESTKI/Ptc +/− mice. In contrast, cerebella of Ptc +/− mice exhibited localized disease. Importantly, RESTKI/ Ptc +/− tumors expressed very high levels of neural stem cell (NSC) markers such as Nestin, GFAP and Vimentin when compared with Ptc +/− tumors, suggesting that REST elevation in GNPs may have resulted in their de-differentiation into a more primitive state, and driven medulloblastoma genesis in the context of Shh hyperactivation. Our studies implicate REST in the modulation of the oncogenic potential of Shh signaling and medulloblastoma development.

Medulloblastoma is a poorly differentiated and hyperproliferative malignant pediatric brain tumor. It frequently arises in the cerebellum. Current therapies while efficacious against primary tumors not only cause severe neurologic deficits in children with brain tumors, but are also ineffective against recurrences. The overall goal of our work is to gain a better understanding of tumor biology so that therapies that target tumor specific events can be developed. This approach would also spare the normal brain from therapy-related toxicity. The RE1 Silencing Transcription Factor (REST) is an epigenetic modulator of gene expression and a repressor of neuronal differentiation. While normal cerebellar neurons did not express REST, its levels were significantly elevated in human medulloblastoma samples, and was associated with poor prognosis for patients. To examine the feasibility of developing REST as a therapeutic target, we performed genetic and pharmacological experiments to explore its role in driving medulloblastoma formation. We observed that REST loss in human tumor cells abrogates their tumorigenic potential in mouse intracranial models. Conversely, REST elevation in neural progenitors promoted tumor formation in the context of Myc overexpression. REST-expressing tumor cells failed to undergo differentiation and had sustained proliferation. REST is a known repressor of genes required for terminal neurogenesis. Interestingly, we made a novel observation that REST also controlled tumor cell proliferation through direct transcriptional repression of a gene encoding a novel deubiquitylase called USP37. Using purified USP37 protein, we demonstrated that it deubiquitylated the cell cycle regulator p27, a critical mediator of normal cell cycle exit during neurogenesis. In patient samples, we noted a significant correlation between loss of USP37, decreased p27 protein levels and poor prognosis. Importantly, constitutive expression of wildtype USP37, but not mutant USP37, promoted a decrease in tumor cell proliferation in vitro and a loss of tumorigenic potential in mouse orthotopic models. Since REST-mediated transcriptional silencing is effected by its associated histone deacetylase (HDAC) and histone methyltransferase (HMT)-G9a chromatin remodeling complexes, we performed genetic and pharmacological assays to identify the specific epigenetic activity promoting USP37 down-regulation in medulloblastoma cells. These studies revealed that G9a loss or inhibition of its activity upregulated USP37 expression and blocked tumor cell proliferation in vitro and tumor growth in vivo. Together, these findings suggest that USP37 is a novel tumor suppressor gene in medulloblastoma and that its epigenetic modulation in REST-high tumors may have potential therapeutic application. Citation Format: Rashieda Jonine Hatcher, Vrushali Datar, Chandra M. Das, Jason Fangusaro, Stewart Goldman, Rishi Lulla, Veena Rajaram, Vidya Gopalakrishnan. Epigenetic deregulation of a novel deubiquitylase promotes medulloblastoma genesis. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B2.