Targeted inhibition of histone demethylase activity in brainstem glioma

Pediatric brainstem gliomas often harbor oncogenic K27M mutation of histone H3.3. The K27M mutation causes substantial reduction of wild-type histone H3K27 methylation in cellular chromatin, and I have recently shown that inhibition of the H3K27 demethylase JMJD3 acts to restore K27 methylation in brainstem glioma cells, while demonstrating potent anti-tumor activity, both in vitro and in vivo (Hashizume et al., Nature Medicine. 2014). These results, as well as results published by others (Ntziachristos et al., Nature. 2014), support JMJD3 as having pro-tumorigenic activity in at least certain types of cancer: specifically T-cell acute lymphoblastic leukemia (T-cell ALL), and diffuse intrinsic pontine glioma (DIPG) with histone H3.3 K27M mutation. However, it is likely that K27 methylation status in brainstem tumors is influenced by other histone-modifying enzyme activities, and whether these activities act in concert with JMJD3, to promote brainstem tumor development, or, alternatively, oppose tumor development, is largely unknown. This application will examine how different H3K27 methyltransferase and demethylase activities influence brainstem tumor growth, and in so doing will determine whether JMJD3’s role in tumor maintenance is dependent upon other proteins that regulate K27 methylation (aim1). This project, in addition, will examine the effects of JMJD3 inhibition when used in combination with radiation (aim 2), as one of the cellular properties affected by JMJD3 inhibition is DNA repair. JMJD3 inhibition, therefore, may enhance the cytotoxic effects of radiation. Aim 1. Investigate the tumor biologic activities of K27 methyltransferase expression alterations in K27M and K27 wild-type glioma cells. In this aim we will use siRNAs and shRNAs to suppress endogenous methylatransferase and demethylase activities in K27M and K27 wild-type glioma cells, and we will transfer corresponding cDNAs into these cells to increase demethylase and methyltransferase activities, in order to examine the effects of enzyme expression alterations on K27M and K27 wild-type glioma cell proliferation, apoptosis, migration, and tumorigenicity, as well as on K27 methylation. Aim 2. Investigate the anti-tumor activity, and associated molecular biology, of combined GSKJ4 + radiation therapy (RT) of K27M gliomas. We will examine whether GSKJ4 inhibits DNA repair process and enhance the radiation response of K27M gliomas. We will test different therapeutic administration sequences in human orthotopic DIPG xenografts, that we have found to be an important consideration in studying other chemo-RT combinations. Intracranial (brainstem) tumor growth and response to therapy will be quantitatively measured by bioluminescence imaging (BLI), and efficacy will be assessed by survival analysis.