Pooja Panwalkar

Lowered H3K27me3 and DNA hypomethylation define poorly prognostic pediatric posterior fossa ependymomas

A subset of childhood posterior fossa ependymomas with poor prognosis is epigenetically similar to H3K27M gliomas. Epigenetics helps find the bad tumors Ependymomas are brain tumors that can occur in people of all ages and in different parts of the central nervous system. The prognosis of these tumors does not necessarily correlate with clinical characteristics or even tumor grade, and there are no recurrent genetic mutations that can be used to classify these tumors. To address this problem, Bayliss et al. examined the epigenetics of ependymoma. By doing this, the authors identified some characteristic methylation patterns that correlate with prognosis, including one specific pattern that is also seen in childhood gliomas and associated with more invasive tumors. Childhood posterior fossa (PF) ependymomas cause substantial morbidity and mortality. These tumors lack recurrent genetic mutations, but a subset of these ependymomas exhibits CpG island (CpGi) hypermethylation [PF group A (PFA)], implicating epigenetic alterations in their pathogenesis. Further, histological grade does not reliably predict prognosis, highlighting the importance of developing more robust prognostic markers. We discovered global H3K27me3 reduction in a subset of these tumors (PF-ve ependymomas) analogous to H3K27M mutant gliomas. PF-ve tumors exhibited many clinical and biological similarities with PFA ependymomas. Genomic H3K27me3 distribution showed an inverse relationship with CpGi methylation, suggesting that CpGi hypermethylation drives low H3K27me3 in PF-ve ependymomas. Despite CpGi hypermethylation and global H3K27me3 reduction, these tumors showed DNA hypomethylation in the rest of the genome and exhibited increased H3K27me3 genomic enrichment at limited genomic loci similar to H3K27M mutant gliomas. Combined integrative analysis of PF-ve ependymomas with H3K27M gliomas uncovered common epigenetic deregulation of select factors that control radial glial biology, and PF radial glia in early human development exhibited reduced H3K27me3. Finally, H3K27me3 immunostaining served as a biomarker of poor prognosis and delineated radiologically invasive tumors, suggesting that reduced H3K27me3 may be a prognostic indicator in PF ependymomas.

MiR-206, a Cerebellum Enriched miRNA Is Downregulated in All Medulloblastoma Subgroups and Its Overexpression Is Necessary for Growth Inhibition of Medulloblastoma Cells

Medulloblastoma is the most common and a highly malignant pediatric brain tumor located in the cerebellar region of the brain. Medulloblastomas have recently been shown to consist of four distinct molecular subgroups, viz., WNT, SHH, group 3, and group 4. MiR-206, a miRNA first identified as a myomiR due to its enriched expression in skeletal muscle was found to be expressed specifically in the cerebellum, the site of medulloblastoma occurrence. MiR-206 expression was found to be downregulated in medulloblastomas belonging to all the four molecular subgroups as well as in established medulloblastoma cell lines. Further, the expression of murine homolog of miR-206 was also found to be downregulated in SHH subgroup medulloblastomas from the Smo+/+ transgenic mice and the Ptch1+/− knockout mice. MiR-206 downregulation in all the four medulloblastoma subgroups suggests tumor-suppressive role for miR-206 in medulloblastoma pathogenesis. The effect of miR-206 expression was analyzed in three established medulloblastoma cell lines, viz., Daoy, D425, and D283 belonging to distinct molecular subgroups. Restoration of miR-206 expression to the levels comparable to those in the normal cerebellum, however, was found to be insufficient to inhibit the growth of established medulloblastoma cell lines. OTX2, an oncogenic miR-206 target, overexpressed in all non-SHH medulloblastomas, is known to inhibit myogenic differentiation of medulloblastoma cells. Overexpression of miR-206 was necessary to downregulate OTX2 expression and inhibit growth of medulloblastoma cell lines.

Abstract 3863: A subset of poorly prognostic pediatric posterior fossa ependymomas exhibit lowered H3K27me3 and DNA hypomethylation and show epigenetic similarities with H3K27M mutant diffuse intrinsic pontine gliomas

Pediatric posterior fossa ependymomas are poorly understood childhood brain tumors and have no effective treatments. The biology of these tumors is obscure as recent sequencing efforts suggest that they lack recurrent genetic alterations. A subset of these tumors termed PF-A ependymomas exhibits CpG-island hypermethylation implicating epigenetic alterations in their pathogenesis. Through comprehensive analyses of histone modification, we discovered global H3K27me3 reduction in a subset of these tumors. Tumors with lowered H3K27me3 showed many clinical and biologic similarities with PFA-ependymomas. Global reduction in H3K27me3 is likewise observed in pediatric gliomas that bear histone H3K27M mutations termed diffuse intrinsic pontine gliomas (DIPG) that also arise in the posterior fossa of young children. Analyses of ependymomas with reduced H3K27me3 and H3K27M mutant DIPGs showed many similarities in DNA methylation and enrichment of H3K27me3 in many genomic loci important for neuroglial specification. Combined integrative analysis of both tumor types uncovered common epigenetic deregulation of select factors that control radial glial biology and radial glia in the developing posterior fossa showed reduced H3K27me3. Finally, PF ependymomas with lowered H3K27me3 were more invasive radiologically and exhibited poor prognosis in three independent cohorts (P 300). These data have clinical implications for biomarker development and to inform epigenetic approaches to treat PF ependymomas. Citation Format: Sriram Venneti, Jill Bayliss, Piali Mukherjee, Chao Lu, Siddhant Jain, Chan Chung, Daniel Martinez, Benjamin Sabari, Ashley Margol, Pooja Panwalkar, Abhijit Paroloia, Melike Pekmezci, Richard Mc Eachin, Marcin Cieslik, Benita Tamrazi, Benjamin Garcia, Gaspare La Rocca, Mariarita Santi, Peter Lewis, Cynthia Hawkins, Ari Melnick, C David Allis, Craig B. Thompson, Arul Chinnaiyan, Alexander R. Judkins. A subset of poorly prognostic pediatric posterior fossa ependymomas exhibit lowered H3K27me3 and DNA hypomethylation and show epigenetic similarities with H3K27M mutant diffuse intrinsic pontine gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3863. doi:10.1158/1538-7445.AM2017-3863

Abstract B41: miR-148a functions as tumor-suppressor microRNA in medulloblastoma cell lines by targeting Neuropilin1

Purpose: Medulloblastoma, a most common pediatric malignant brain tumor consists of four molecular subgroups viz. WNT, SHH, Group 3 and Group 4. MiR-148a is over-expressed in the WNT subgroup tumors, which have the lowest incidence of metastasis and excellent survival among all medulloblastomas. Therefore, the role of miR-148a in medulloblastoma biology was investigated. Experimental Design: MiR-148a was expressed either in a transient manner using synthetic mimic or in a stable doxycycline inducible manner using a lentiviral vector. Effect of miR-148a expression on the growth and malignant behavior of medulloblastoma cell lines was investigated. CpG Methylation status of region upstream to pri-miR-148a was determined by performing bisulphite sequencing of this region in medulloblastoma cell lines and tissues. Results: Expression of miR-148a to the levels comparable to that in the WNT subgroup tumors was found to inhibit proliferation, clonogenic potential and in vitro invasion potential of medulloblastoma cells. In vivo tumorigenicity of medulloblastoma cells Daoy, D425 and D283, as seen by either generating subcutaneous xenografts or orthotopic xenografts in immunodeficient mouse, was found to be reduced significantly upon miR-148a expression. NRP1, ROCK1 and DNMT1 were identified as direct targets of miR-148a with NRP1 being novel target. Restoration of NRP1 expression in medulloblastoma cells was found to rescue the reduction in the invasion potential and tumorigenicity brought about by miR-148a expression. NRP1expression in medulloblastomas was found to be associated with poor survival, with little or no expression in majority of the WNT tumors. This observation is consistent with high miR-148a expression and low incidence of metastasis and excellent survival of the WNT subgroup tumors. miR-148a is known to be down-regulated as a result of promoter hypermethylation in colon, lung, breast, head and neck carcinomas and melanoma. Bisulphite sequencing of the region upstream to pri-miR-148a revealed presence of CpG methylation in four medulloblastoma cell lines Daoy, D283, D341 and D425 and non WNT medulloblastoma tissues, while it was found to be un-methylated in WNT medulloblastomas. Conclusions: The tumor suppressive effect of miR-148a expression in medulloblastoma cells accompanied by the down-regulation of NRP1, ROCK1 and DNMT1 makes miR-148a an attractive therapeutic agent for the treatment of medulloblastomas. Preliminary findings provided by bisulphite sequencing suggest that under-expression of miR-148a may be as a result of CpG methylation of upstream region to pri-miR-148a sequence. Citation Format: Kedar Narsinha Yogi, Epari Sridhar, Naina Goel, Rakesh Jalali, Atul Goel, Aliasgar Moiyadi, Rahul Thorat, Pooja Panwalkar, Atul Khire, Archya Dasgupta, Prakash Shetty, Neelam Shirsat. miR-148a functions as tumor-suppressor microRNA in medulloblastoma cell lines by targeting Neuropilin1. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B41.