Use of H3(K27M)-Driven Zebrafish Models to Identify Targetable Vulnerabilities in DIPG

Abstract The prognosis for children diagnosed with Diffuse Intrinsic Pontine Glioma has not changed in nearly 50 years—children have no chance for survival because there are no effective treatments for this cancer. DIPG is inoperable because it grows intertwined in the brainstem, and DIPG always becomes resistant to the current standard of care, radiation therapy. Although a key mutation in histone 3, H2(K27M) was recently identified in a majority of DIPG, this has not translated to new therapeutic options as originally hoped. Our long-term goal is to help develop new therapeutic approaches to treat DIPG using our expertise with zebrafish cancer modeling. The objective of this proposal is to use zebrafish that we have engineered to express H3(K27M) in glia to understand the effects of this mutation on DIPG progression and and to identify FDA-approved drugs that can be repurposed to block these phenotypes. The rationale for this project is that zebrafish models will fill critical gaps in our currently available tools to study DIPG. Unlike mouse models, fluorescently tagged tumor cells can be easily studied in the zebrafish brain from the earliest stages of tumor growth onwards to the development of radiation resistance. Cross species comparisons between human and zebrafish tumors can reveal critical proteins involved in tumor progression. Zebrafish are also an inexpensive animal model for rapid and large-scale drug screens; unlike cultured cells, DIPG in zebrafish grows in its native neural niche and under a blood-brain barrier, which can be more informative for drug testing. The objectives of this proposal will be met with the following specific aims: 1) Define the effects of H3(K27M) in DIPG onset and the acquisition of radio resistance using a zebrafish expressing H3(K27M) in glia, and 2) Perform large scale drug screens in zebrafish DIPG models to identify drugs that can revert DIPG phenotypes. Under the first aim, zebrafish with H3(K27M) mutation in their glia will be assessed for DIPG onset and progression both microscopically and histologically. Animals will also be irradiated to assess the effects of H3(K27M) in radioresistance mechanisms. Samples will be harvested for transcriptomics and to analyze the genome occupancy of H3(K27M) via chromatin immunoprecipitation sequencing. In the second aim, we will utilize both the H3(K27M) zebrafish model and patient derived xenografts in zebrafish for large scale screening of FDA-approved drugs to identify compounds that eliminate DIPG and/or revert the radioresistance phenotype. This research is innovative because it will develop and employ novel zebrafish models to define key events DIPG onset, which cannot be done in current mouse and cell culture models, and test the efficacy of large panels of drugs in targeting DIPG in its native neural niche. This proposed project is significant because zebrafish models can fill critical gaps in our understanding of DIPG biology. This knowledge ultimately has the potential to offer new strategies for the developments of the first successful treatment of DIPG.