PROJECT SUMMARY / ABSTRACT Background and long-term objectives: Pediatric high-grade glioma (pHGG) is among the most lethal pediatric cancers, and new targeted therapies are desperately needed. Approved therapies for pHGG remain non- targeted and 2-year survival rates are less than 20%. Loss of function mutations in the chromatin remodeling protein ATRX are found in 30% of pHGG and DIPG, usually with concurrent mutation in the histone variant H3F3A (H3.3). We previously developed a mouse model of ATRX-deficient GBM and showed that loss of ATRX results in increased sensitivity to radiation treatment. We recently discovered that HGG cells with isogenic ATRX loss demonstrate inappropriate release of G1/S and G2/M checkpoint after irradiation and radio-sensitization with inhibitors of the master cell cycle regulator ATM. However, the mechanism driving this phenotype has not been established, and no models utilizing a background of pHGG mutations (e.g. H3.3) have been employed to study ATRX loss. Thus, there is a critical need to determine how ATRX loss deregulates cell cycle checkpoints, and to clarify the impact of concurrent H3F3A mutation on cell cycle regulation and radiation sensitizing therapy. In the absence of such knowledge, the ability to translate therapies targeted to the cell cycle checkpoint deficit in ATRX-deficient pHGG will remain unlikely. Our overall objective in this proposal is to determine the epigenetic mechanism of cell cycle dysfunction in ATRX mutated-pHGG and the impact/targetability of concurrent H3F3A mutation. Our central hypothesis is that ATRX mutation in pHGG results in reduced H3.3-promotor binding and expression of the cell cycle checkpoint regulator Checkpoint Kinase 1 (CHK1), leading to permissive cell cycle checkpoints after DNA damage. We propose that co-occurrence of H3K27M mutation will enhance this deficit and increase radio-sensitization with ATM inhibition. This is based on our preliminary data demonstrating (i) ATRX/H3.3 deposition at CHEK1 promoter sites, (ii) reduction in Chk1 expression and checkpoint maintenance after irradiation in ATRX deficient models, and (iii) increased cell cycle release with ATM inhibition in H3K27M cells compared to controls. Specific Aim 1: Determine the mechanism of cell-cycle phase dysfunction in ATRX-deficient pHGG. We will accomplish this by integrating complementary experimental approaches of multiple human and mouse pre- clinical models of ATRX loss in pHGG, including epigenetic, cell cycle and DNA-damage repair experiments. Specific Aim 2: Determine the impact of co-occurring H3F3A mutation on the targetability of ATRX- deficient pHGG. We will accomplish this Aim by integrating multiple human and mouse pre-clinical models of ATRX loss in pHGG, including a novel genetically engineered mouse model with isogenic control of H3F3A and ATRX, to isolate contribution of each driver on cell-cycle deficit and targetability. Our integrative experimental approach will establish the...