Analysis and Targeting of the Transcriptional Regulator BRD4 in Diffuse Intrinsic Pontine Glioma (DIPG)

Grants and Contracts Details

Description

The prognosis for children diagnosed with Diffuse Intrinsic Pontine Glioma (DIPG) has not changed in nearly 50 years—children with DIPG have no chance at survival because there are no effective treatments for this cancer. DIPG is inoperable because it grows intertwined in the brainstem, and all DIPG will become resistant radiation, the current therapy. Recently, researchers discovered that nearly 80% of DIPG harbor a mutation in Histone 3, H3K27M, which prevents methylation of the histone and leads to expression of genes that are normally silenced. Although only one allele of H3 is mutated in DIPG, and H3K27M makes up only 20% of the histone produced by the cells, H3K27M is found disproportionally and non-randomly localized to sites of active transcription. H3K27M histones also co-localize with the BRD4, a transcriptional and epigenetic regulator with well-established oncogenic roles in a variety of cancers. However, while these findings have provided insight into drivers of DIPG formation, they have not translated into useful therapeutic advances. The mechanisms through which BRD4/H3K27M leads to DIPG pathology are still unknown. While transcription is globally altered, critical gene targets responsible for DIPG onset, progression, and radiation resistance have not been identified. Additionally, the BRD4 inhibitor JQ1 has strong anti-proliferative and pro-differentiation effects in DIPG cells in culture, but this compound been relegated to research tool status because of undesirable off-target effects in animals. Less toxic BRD4 inhibitors are needed before any pre-clinical trials can begin. Our long-term goal is to help develop new therapeutic approaches to treat DIPG using the combined expertise of the Blackburn and Glazer labs in zebrafish cancer modelling and medicinal inorganic chemistry, respectively. In our preliminary data, the Blackburn lab has developed transgenic zebrafish expressing h3k27m-GFP in glial cells. These fish develop GFP+ tumors in the brain that resemble human DIPG. Benefits of zebrafish are that, unlike mouse models, DIPG onset can be visualized and tracked in living animals from the earliest stages of tumor onset, which can provide new information on the role of oncogenes like BRD4 and H3K27M in DIPG development. Zebrafish also have benefits compared to DIPG cell culture, as zebrafish DIPG can be studied in the context of its tumor microenvironment, and zebrafish models provide a blood-brain barrier for more informative drug testing. Additionally, zebrafish brd4 is 93% identical to human BRD4 at the bromodomains, and research from other groups indicate that it functions similarly to human BRD4 and can be targeted by inhibitors like JQ1. The Glazer lab has used a JQ1 based Proteolysis Targeting Chimera, in which is JQ1 is coupled to a ligand for E3 ubiquitin ligase to target BRD4 for proteolytic degradation, to create a novel PHOTAC. In this compound, the BRD4 PROTAC is coupled to heavy metals ruthenium or platinum, which block binding of the E3 ligand to the E3 ligase. The metal is released upon light stimulation, and BRD4 is degraded. This system has the potential to significantly enhance the efficacy and reduce the off-target toxicity of BRD4 inhibition compared to JQ1. Ruthenium and platinum are also radiosensitizers, so they could be an ideal targeted therapy to combine with radiation, the current DIPG standard of care. Building on research from both of our labs, the objectives in this application are to define the key effects of BRD4/H3K27M in DIPG and to optimize the BRD4 PHOTAC in BRD4 inhibition and as a DIPG therapeutic. The rationale for this project is that a better understanding of how BRD4 collaborates with H3K27M to drive DIPG development will provide new insights into DIPG biology and can identify novel drug targets. Additionally, the BRD4 PHOTAC could be a useful therapeutic in its own right, and the photocaging methods that will be developed by the Glazer lab can be applied to other PROTACs once additional drug targets are identified. We plan to meet our objectives with the following two specific aims. 1) Define the transcriptional effects BRD4 in H3K27M-driven DIPG in early DIPG onset and in the acquisition of radiation resistance, and 2) Evaluate the therapeutic efficacy of light and radiation activated BRD4 PHOTACs. Under the first aim, we will use ChIP to assess the genome occupancy of BRD4 in 5 day old zebrafish whose glial cells express H3K27M-GFP or wild-type H3-GFP. These data will be coupled with RNAseq to identify BRD4/H3K27M gene targets in DIPG onset. Animals will also be irradiated for similar experiments to determine how BRD4/H3K27M gene occupancy changes after immediately after irradiation to promote DIPG survival. Hit will be validated using a nuclease dead Cas9 system to block BRD4-mediated transcription and identify key genes in DIPG onset and radiation resistance. For the second aim, panels of light and gamma- irradiation activated BRD4 PHOTACS will be produced by varying the amine groups of the previously developed BRD4 PHOTAC. Stability, photochemistry, ligand release will be assessed. Human DIPG cell cultures will be treated with compounds, and BRD4 degradation, expression of known BRD4 transcriptional targets, and cell survival will be assessed with and without light/irradiation activation. Finally, the compounds will be assessed for toxicity and efficacy will be quantified in zebrafish. In total, the proposed research will significantly enhance our understanding of BRD4/H3K27M in DIPG and develop novel methods to safely target BRD4 in cancer that could ultimately be applied to other DIPG drug targets. These efforts are important steps in the path towards new therapies and better survival rates for children with DIPG.
StatusActive
Effective start/end date7/1/226/30/24

Funding

  • KY Cabinet for Health and Family Services: $479,497.00

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