CSIG-30. HISTONE H3.3 K27M AND G34R/V GLIOMA DRIVER MUTATIONS BLOCK H3.3 PHOSPHORYLATION AND TRAP CHK1 – LEADING TO ANEUPLOIDY

Abstract Diffuse midline and diffuse hemispheric glioma contain the driver mutations H3.3 K27M and G34R/V, respectively. These mutations alter histone methylations, suggesting that dysregulation of gene expression may contribute to tumor formation. We recently reported that these mutations also inhibit the phosphorylation of H3.3 at Serine 31. Importantly, H3.3 S31A mice developed high-grade gliomas – similar to those from G34R and K27M mice – indicating that loss of phosphoS31 promotes gliomagenesis. Chk1, a key kinase in DNA damage response and mitotic fidelity, phosphorylates H3.3 S31. H3.3 S31 is phosphorylated at the pericentromere in metaphase and is typically absent during interphase. Reduction of S31 phosphorylation by H3.3 K27M, G34R, G34V, or S31A was sufficient to induce mitotic errors leading to aneuploidy. Using CRISPR/Cas9, we generated novel H3.3 WT/WT and WT/S31A lines from glioma patient-derived SF-8628 (H3.3 WT/K27M) and KNS-42 (H3.3 WT/G34V) cells. Immunofluorescence of metaphase spreads revealed H3.3 phosphoS31 at the pericentromeres as expected. Interestingly, total Chk1 and active Chk1 (pS296) were significantly enriched at the pericentromeres of H3.3 K27M and G34V cells, as compared to H3.3 WT cells. While total ATR and active ATR (pT1989) were present at the pericentromere, their levels were unaltered by the H3.3 mutations. Surface plasmon resonance of purified proteins revealed that Chk1 has two binding affinities for H3.3 - likely corresponding to the previously identified Chk1 phosphorylation sites (T11 and S31) on H3.3. Chk1 binding at one site was unaffected by H3.3 K27M, while the second site showed an altered dissociation from H3.3 K27M. In summary, our data suggest that the low pericentromeric pS31 levels associated with H3.3 glioma mutations result from Chk1 recognizing the mutant histone, but without completing a phosphorylation cycle, becoming trapped at the pericentromere. This result suggests that H3.3 mutations, by trapping Chk1, may impede Chk1 activities beyond just reducing H3.3 phosphorylation.