H3K27-mutant diffuse midline gliomas (DMGs) are defined as grade IV tumors by the World Health Organization. DMGs are inoperable and resistant to chemo/radio therapies. Median survival ranges from 8-11 months, with 2% of patients surviving beyond 5 years. H3K27M mutations lead to global epigenetic and transcriptional reprogramming driven by global loss of negative transcriptional regulator H3K27 trimethylation (H3K27me3). Loss of H3K27me3 is an initiating event in gliomagenesis. This disease lacks appropriate models to predict disease biology and response to treatment. Therefore, we developed a novel syngeneic H3K27M mouse model. An unbiased integrated systems biology approach identified that H3K27M but not isogenic controls relied on the amino acid methionine and the enzyme Methionine Adenosyltransferase 2A (MAT2A). MAT2A is a central regulator of one-carbon metabolism by converting methionine to S-adenosylmethionine (SAM), the universal methyl-donor for protein and nucleotide methylation reactions. In complementary genetic approaches, we applied these findings to patient-derived cell lines with the H3K27M mutation. We hypothesize that MAT2A abrogation, genetic/pharmacological, would alter DMG viability by disrupting the methylome. The current MAT2A sensitivity paradigm is based on Methylthioadenosine Phosphorylase (MTAP) deletion through a synthetic lethal mechanism. We provide a novel mechanism whereby H3K27M cells are sensitive to MAT2A loss, independent of MTAP and through Adenosylmethionine Decarboxylase 1 (AMD1) overexpression disrupting MAT2A regulation. This results in H3K27M cells having lower MAT2A protein levels, conferring a sensitivity by inhibiting residual MAT2A. Genetic/pharmacological aberrations to MAT2A resulted in reduced proliferation. Parallel H3K36me3 ChIP and RNA-sequencing identified loss of oncogenic and developmental transcriptional programs associated with MAT2A loss. In vivo syngeneic and patient-derived xenograft models with both inducible MAT2A knockdown or methionine restricted diets showed extended survival. These results suggest novel interactions between methionine metabolism and the epigenome of H3K27M gliomas and provide evidence that MAT2A, presents exploitable therapeutic vulnerabilities in histone mutant gliomas.
Primary central nervous system (CNS) tumors are now the most common cause of childhood cancer–related deaths. Pediatric high-grade gliomas (pHGGs) are among the most lethal brain tumors with a 5-year survival rate of only 20%. MYCN pHGGs represent one subgroup with an unmet need for therapeutics. MYCN belongs to the family of MYC transcription factors that regulate numerous cancer hallmarks such as proliferation, apoptosis, and metabolism. While no direct inhibitors of MYCN are in clinical trial, current strategies focus on targeting the MYCN mediated transcriptional machinery or cell cycle regulators. Lack of relevant pHGG models for pre-clinical testing contribute to limited therapeutic efficacy. To address these knowledge gaps, we developed a novel mouse model of MYCN pHGG using the FLEx-Cre switch system, whereby neural stem cells are selectively delivered with MYCN cDNA and shRNA targeting the tumor suppressor genes p53 and Pten and form tumors in vivo. We identified that this model harbors hyper-activation of the PI3K/AKT/mTOR signaling pathway. We demonstrate that dual PI3K-mTOR blood brain barrier penetrant inhibitors are effective in reducing pHGG growth and MYCN protein levels. Because treatment-resistance is a fundamental feature of pHGGs, we developed a novel drug-resistance model of MYCN pHGG as a mechanistic tool to identify relevant resistance mechanisms. Using transcriptome analysis, we identified the insulin growth factor signaling pathway as our top mechanism of resistance. We hypothesized that MYCN is a critical driver of pHGG and can be effectively targeted via dual inhibition of PI3K-mTOR and IGF/Insulin signaling pathways. We tested next generation inhibitors of IGF and PI3K/mTOR pathways and performed genetic and pharmacological assays in our MYCN pHGG and human MYCN pHGG cells. Inhibition of both pathways in our MYCN pHGG model and human MYCN cells were synergistic, leading to significant decreases in cell growth and MYCN signaling.
BACKGROUND High-grade gliomas (HGGs) are the most common fatal intrinsic brain tumors in pediatric patients. H3K27-altered diffuse midline gliomas (H3K27-DMGs), a subgroup of HGGs defined by a histone 3 position 27 alteration, are especially aggressive and result in the poorest patient outcomes. Despite in-depth genomic characterization, the 5-year survival rate has yet to improve beyond 2% following diagnosis. A common feature of H3K27-DMGs is infiltration of microglia, macrophages, other myeloid cells, collectively referred to as GAMs, and a small population of T-cells. The contribution of non-tumor cells in the tumor microenvironment (TME) can both promote and or inhibit tumor growth, thus representing an opportunity in the pursuit of novel therapeutics. Using bioinformatic analysis on a human H3K37-DMG single cell-RNA sequencing dataset, we reveal several cell-to-cell communication signaling networks, mediated by ligand and receptor pairs, between GAMs and tumor cells, respectively. HYPOTHESIS Microglial-derived growth factors activate oncogenic signaling pathways via paracrine signaling axes, thus promoting H3K27-DMG tumor cell proliferation and growth. METHODS I will validate these findings and test their therapeutic potential using co-culture studies, CRISPR and shRNA gene silencing, and phospho-proteomics technology. RELEVANCE This research provides further insights on the contribution of non-tumor cells in the TME towards H3K27-DMG cell proliferation and growth and could potentially inform future therapy paradigms.
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