Uncover the role of H3.3-G343R mutation in shaping the DNA damage response, anti-tumor immunity and mechanisms of resistance in glioma.

Pediatrichigh-grade gliomas (pHGGs) are one of the most aggressive forms of brain cancer, with a median
survival (MS) of ~18 months 1,2. The current standard of care for pHGG, consisting of tumor resection followed
by radiation and chemotherapy, elicits only a modest increase in MS 1-4. The main reasons for the limited
therapeutic outcomes are tumor recurrence, caused by the infiltrative nature of pHGG, and the development of
an immune-suppressive tumor microenvironment (TME). One of the main subtypes of hemispherical pHGG
encodes for G34R/V substitutions in the histone H3F3A 4, along with ATRX and TP53 inactivating mutations.
The investigation of the molecular pathways which play a role in the pathogenesis of pHGG requires accurate
mouse models which recapitulate the salient features of pHGG and develop within the brain's microenvironment
in an immune-competent host. Our lab created genetically engineered immune competent pHGG mouse models
employing the Sleeping Beauty (SB) transposase system 5,6. Tumors harbor genetic lesions encountered in a
subtype of pHGG, i.e., H3.3G34R co-expressed with ATRX and TP53 knock down. The host in this pHGG model
exhibits an intact immune system, thus enabling detailed mechanistic studies on all aspects of pHGG biology
in vivo, including interactions with the TME immune cells. Our preliminary RNA-Seq data on H3.3-G34R versus
H3.3-Wt pHGG revealed downregulation of gene ontologies (GO) related to DNA Damage Response (DDR)
in H3.3-G34R pHGG. We also observed upregulation of GOs related to the activation of the immune
response, such as “Regulation of Immune Response” and “Type I Interferon Production”, in the H3.3-G34R
pHGG model. Herein we propose to determine the cellular andmolecular mechanisms by which H3.3-G34R
regulates the response to radiotherapy and DDR inhibition in mouse and human H3.3G34R pHGG cells in vitro
and in vivo. We aim to elucidate the role played by H3.3-G34R in DNA repair processes, responseto DNA
damaging agents, and in TME immune cells' reprogramming. We propose to test the hypothesis that H3.3G34R
reshapes the epigenetic landscape, resulting in alterations in chromatin states and transcriptional changes.
These alterations cause DDR impairment and induce genomic instability, which in turn leads to cGAS-STING-
Pathway-mediated activation of the immune system within the H3.3G34R pHGG TME. We will assess
chromatin states by ATAC-seq, and establish whether reduced chromatin accessibility impairs DNA repair
in G34R pHGG. We will also evaluate whether genomic instability in G34R pHGG mediates the immune system
activation via cGAS-STING. We will also define at the molecular level the phenotypically diverse tumor and
infiltrating immune cell clusters within the H3.3-G34R pHGG microenvironment using scRNA-seq; this will allow
us to uncover mechanisms of therapeutic resistance. This information, will enable uncovering genetic makeup-
tailored therapeutic modalities for H3.3-G34R pHGG, such as cell cycle checkpoint or DDR inhibitors
combined with radiotherapy.

Grant Number: 5R01NS122165-05
NIH Institute/Center: NIH
Principal Investigator: Maria Castro