GENOMIC INTEGRITY IN THE CONTEXT OF H3.3K27M DIFFUSE MIDLINE GLIOMAS

INTRODUCTION AND OBJECTIVE: Gliomas are the most frequent primary brain malignancies in children. Regarding pediatric high grade gliomas (pHGG), the identification of unique mutations in histone H3 genes, which account for ~50% of pHGG, led to the recognition of ‘diffuse midline glioma, H3 K27-altered’ as a defined pathological identity by the WHO (2021). H3K27-altered gliomas arise either from a point mutation in one allele of H3F3A, causing the substitution of lysine 27 (K27) to methionine (M), or from protein expression changes (i.e. overexpression of EZHIP). In both cases, genome-wide epigenetic rearrangements (i.e. H3K27me3 loss/H3K27Ac gain) lead to misregulation of gene expression, potentially triggering tumorigenesis. However, recent studies demonstrated that transcriptional changes are limited (Krug et al. 2019, Harutyunyan et al. 2019), raising the possibility that processes unrelated to gene regulation contribute to cancer development. An often overlooked aspect of H3.3K27M tumors is their high degree of genomic and chromosomal instability (GIN/CIN) compared to H3-wildtype tumors. In addition, it is known that mutations in DNA damage response (DDR) pathways are common in H3.3K27M tumors (Mackay et al. 2017). Considering the physiological importance of normal H3.3 in the maintenance of genome integrity, we hypothesized that H3.3K27M promotes GIN/CIN, leading to tumorigenesis.

MATERIALS AND METHODS: Levels of GIN/CIN in H3.3K27M pHGG patient-derived biopsies were measured by shallow single-cell whole genome sequencing (sWGS). To investigate causes of GIN, an inducible FLAG-H3.3K27M overexpression model was developed in RPE1-hTERT cells. Protein-H3.3K27M interactions during mitosis were assessed with FLAG-Immunoprecipitation coupled with Mass spectrometry (FLAG-IP/MS). Mitotic abnormalities were quantified with live-cell imaging. DNA ultrafine bridges (UFB) and 53BP1 nuclear bodies (NB) formation were measured with immunofluorescence. Aphidicolin was used as replication stress (RS) inducing agent. CRISPR-KO of H3.3K27M was performed in patient-derived pHGG cells to verify consequences of mutation loss. All patient-derived material used was approved by local ethics committees.

RESULTS AND CONCLUSION: sWGS demonstrated that H3.3K27M tumors are highly aneuploid with recurrent chromosomal abnormalities, and provided evidence for intra-tumoral clonal heterogeneity. Moreover, mitotic FLAG-IP/MS showed enhanced interaction of DNA replication factors with H3.3K27M. Furthermore, following RS induction, H3.3K27M cells exhibited more mitotic abnormalities and increased formation of DNA UFBs. Reciprocally, reduced formation of UFBs was noted in primary pHGG cells, upon H3.3K27M CRISPR-KO. Additionally, following RS, lower than expected 53BP1-NBs were formed in both H3.3K27M RPE1 cells and primary pHGG, indicating that the DDR is affected by the mutation. Our findings uncover previously undescribed roles for H3.3K27M in pHGG development.