Pediatric high-grade gliomas still have no effective treatments (1, 2). Our overall goal is to be a game changer in this area by advancing knowledge and catalyzing innovative therapies. Mutant histone H3.3 is a promising new therapeutic target in these fatal childhood tumors (3-10) as it appears specific to pediatric cancers and its coding gene H3F3A is one of the most commonly mutated genes there. There are two types of codon- changing, point mutations prevailing in H3F3A: H3.3 K27M and H3.3 G34R/V. The cellular and molecular mechanisms by which these mutations contribute to brain cancer remain largely open questions and together represent a key gap in the field. Our specific objectives in the proposed study are to determine novel oncogenic mechanisms of the H3.3 mutants, define specific downstream effector pathways that contribute to glioma, and determine effects of two specific classes of drugs (NOTCH and BET inhibitors) as novel potential therapies. To attain these objectives, we will use innovative approaches including a human brain cancer in a dish model together with CRISPR gene editing. Our initial studies enhance the feasibility and premise of our proposal via abundant, rigorous preliminary data. We have adapted the brain organoid system to produce human mini-brains from human induced pluripotent stem cells (IPSC) that also contain a range of ?spiked in? pediatric glioma cells, which led to growth of discrete tumors inside the organoids, and other tumor- like phenotypes. This is an ideal, innovative, and highly tractable system for studying our panel of gene edited cells and childhood brain cancer more generally. Our hypothesis is that the mutant H3.3 pathway alters specific epigenomic outcomes including aberrant neurogenesis and NOTCH pathway activation, and that we can effectively target this overall mutant H3.3 pathway. We will test this hypothesis using a rigorous study design combining human cellular and mouse xenograft studies. This proposal has a high degree of innovation both conceptually and technically for the following reasons: modeling and targeting brain cancer in a dish via human brain organoids made from IPSC seeded with pediatric glioma cells is an exciting new technology; the model system of CRISPR-modified (gain and loss of specific H3.3 mutations) human primary cells is novel; we have identified new mutant H3.3 targets; and brain organoid-based drug studies are unique. These studies by our strong team of investigators will have a transformative impact by advancing knowledge of core childhood glioma oncogenic mechanisms and via definition of predicted positive effects of targeted drugs. The new technologies, tools, and knowledge produced will also boost impact.
The proposed research is relevant to human health because it will advance our understanding of common mechanisms in fatal childhood gliomas associated with mutant histone H3.3. It will have substantial impact on human health by catalyzing innovative treatments for these largely incurable cancers and provide key insights into other tumors as well.
