Males exhibit higher incidence and worse prognosis for the majority of cancers, including glioblastoma (GBM). Disparate survival may be related to sex-biased responses to treatment, including radiation. Using a mouse model of GBM, we show that female cells are more sensitive to radiation, and that senescence represents a major component of the radiation therapeutic response in both sexes. Correlation analyses revealed that the CDK inhibitor p21 and irradiation induced senescence were differentially regulated between male and female cells. Indeed, female cellular senescence was more sensitive to changes in p21 levels, a finding that was observed in wildtype and transformed murine astrocytes, as well as patient-derived GBM cell lines. Using a novel Four Core Genotypes model of GBM, we further show that sex differences in p21-induced senescence are patterned during early development by gonadal sex. These data provide a rationale for the further study of sex differences in radiation response and how senescence might be enhanced for radiation sensitization. The determination that p21 and gonadal sex are required for sex differences in radiation response will serve as a foundation for these future mechanistic studies.

Background Neurodevelopmental disorders increase brain tumor risk, suggesting that normal brain development may have protective properties. Mutations in epigenetic regulators are common in pediatric brain tumors, highlighting a potentially central role for disrupted epigenetic regulation of normal brain development in tumorigenesis. For example, lysine 27 to methionine mutation (H3K27M) in the H3F3A gene occurs frequently in Diffuse Intrinsic Pontine Gliomas (DIPGs), the most aggressive pediatric glioma. As H3K27M mutation is necessary but insufficient to cause DIPGs, it is accompanied by additional mutations in tumors. However, how H3K27M alone increases vulnerability to DIPG tumorigenesis remains unclear. Results Here, we used human embryonic stem cell models with this mutation, in the absence of other DIPG contributory mutations, to investigate how H3K27M alters cellular proliferation and differentiation. We found that H3K27M increased stem cell proliferation and stem cell properties. It interfered with differentiation, promoting anomalous mesodermal and ectodermal gene expression during both multi-lineage and germ layer-specific cell specification, and blocking normal differentiation into neuroectoderm. H3K27M mutant clones exhibited transcriptomic diversity relative to the more homogeneous wildtype population, suggesting reduced fidelity of gene regulation, with aberrant expression of genes involved in stem cell regulation, differentiation, and tumorigenesis. These phenomena were associated with global loss of H3K27me3 and concordant loss of DNA methylation at specific genes in H3K27M-expressing cells. Conclusions Together, these data suggest that H3K27M mutation disrupts normal differentiation, maintaining a partially differentiated state with elevated clonogenicity during early development. This disrupted response to early developmental cues could promote tissue properties that enable acquisition of additional mutations that cooperate with H3K27M mutation in genesis of DMG/DIPG. Therefore, this work demonstrates for the first time that H3K27M mutation confers vulnerability to gliomagenesis through persistent clonogenicity and aberrant differentiation and defines associated alterations of histone and DNA methylation.

Sex can be an important determinant of cancer phenotype and exploring sex-specific tumor biology holds promise for identifying novel therapeutic targets and new approaches to cancer treatment. In an established model of glioblastoma, we discovered transcriptome-wide sexual dimorphism in gene-expression that was concordant with sex differences in H3K27ac marks, large Brd4-bound enhancer usage, and Brd4 co-localization with Myc and p53. The sex-specific enhancer usage drove sex differences in stem cell function and tumorigenicity. Moreover, male and female GBM cells exhibited opposing responses to pharmacological or genetic inhibition of Brd4. Brd4 knockdown or inhibition decreased male GBM cell clonogenicity and in vivo tumorigenesis, while increasing both in female GBM cells. These results were validated in male and female patient-derived GBM cell lines. Thus, for the first time, Brd4 activity is revealed to drive a sexually dimorphic stem cell and tumorigenic phenotype, resulting in diametrically opposite responses to BET inhibition in male and female glioblastoma cells. This has critical implications for the clinical evaluation and use of BET inhibitors. Citation Format: Najla Kfoury-Beaumont, Zongtai Qi, Michael Wilkinson, Lauren Broestl, Kristopher Berrett, Arnav Moudgil, Sumithra Sankararaman, Xuhua Chen, Jay Gertz, Robi Mitra, Joshua B. Rubin. Brd4-bound enhancers drive critical sex differences in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3658.