Vaughan, Kevin T.

Abstract Diffuse gliomas are pediatric high-grade gliomas that frequently harbor heterozygous histone H3.3 K27M, G34R, or G34V mutations. These mutations alter histone methylations, although the precise mechanism(s) of gliomagenesis remains uncertain. H3.3 contains a unique Serine at position 31 that is phosphorylated in prophase and dephosphorylated in anaphase during normal cell division. Previously we have reported that chromosome missegregation triggers H3.3 S31 phosphorylation along chromosome arms in ana/telophase leading to p53-induced cell cycle arrest. Here we show, in vitro and in vivo, that H3.3 K27M, G34R, and G34V suppress S31 phosphorylation by its mitotic kinase, Chk1. In diploid cells, overexpression of H3.3 K27M, G34R, G34V, or non-phosphorylatable S31A significantly increases the frequency of chromosome missegregation, while H3.3 K36M did not. This missegregation phenotype could be rescued by overexpression of K27M/S31E or G34R/S31E double mutants. Next, we used CRISPR/Cas9 gene editing to revert K27M or G34V patient-derived DMG cells to WT or replace the mutant allele with S31A. Reversion to WT increased metaphase S31 phosphorylation and reduced chromosome missegregations, but inserting an S31A mutation caused more mitotic defects. Following a mitotic error, newly formed aneuploid cells overexpressing K27M, G34R, or G34V suppress p53 levels and continue to divide, while WT overexpressing cells always arrest. To test the effects of diminished S31 phosphorylation on gliomagenesis, we utilized a novel mouse model of glioma. Expression of PDGFß linked to H3.3K27M, H3.3G34R, and H3.3S31A in neural stem cells in vivo promoted the development of high-grade gliomas in under 100 days, whereas PDFß-H3.3WT did not. Notably, the S31A tumors had histologically normal levels of K27me3 and K36me3. This work shows that a single glioma-associated point mutation in H3.3 can generate chromosomal instability, and which is a major contributing factor to H3.3 K27M and G34-altered glioma formation. Citation Format: Charles A. Day, Florina Grigore, Alyssa Langfald, Faruck Hakkim, David Daniels, Kevin Vaughan, James Robinson, Edward Hinchcliffe. Histone H3.3 K27M and G34R/V glioma driver mutations inhibit mitotic phosphorylation of H3.3 S31, driving tumor formation by inducing chromosomal instability [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B033.

Abstract Diffuse midline gliomas (DMG) are high-grade pediatric gliomas that frequently harbor heterozygous histone H3.3 K27M, G34R, or G34V mutations. These mutations alter histone methylations, although the precise mechanism(s) of gliomagenesis remains uncertain. H3.3 contains a unique Serine at position 31 that is phosphorylated in prophase and dephosphorylated in anaphase during normal cell division. In an aberrant division, chromosome missegregation triggers S31 phosphorylation along chromosome arms in anaphase leading to p53-induced cell cycle arrest. Here we show in vitro and in vivo, that H3.3 K27M, G34R, and G34V significantly suppress S31 phosphorylation by Chk1 (the mitotic S31 kinase). In diploid cells, overexpression of H3.3 K27M, G34R, G34V, or non-phosphorylatable S31A significantly increases the frequency of chromosome missegregation, while H3.3 K36M did not. This missegregation phenotype could be rescued by overexpression of K27M/S31E or G34R/S31E double mutants. Next, we used genome editing to revert K27M or G34V patient-derived DMG cells to WT or replace the mutant allele with S31A. Reversion to WT increased metaphase S31 phosphorylation and reduced chromosome missegregations, but inserting an S31A mutation caused more mitotic defects. Following a mitotic error, newly formed aneuploid cells overexpressing K27M, G34R, or G34V suppress p53 levels and continue to divide, while WT overexpressing cells always arrest. To test the effects of diminished S31 phosphorylation on gliomagenesis, we utilized a novel mouse model of DMG. Expression of PDGFβ linked to H3.3K27M, H3.3G34R, and H3.3S31A in neural stem cells in vivo promoted the development of high-grade gliomas in under 100 days, whereas PDGFβ-H3.3WT did not. Notably, the S31A tumors had histologically normal levels of K27me3 and K36me3. This work shows that a single DMG-associated point mutation in H3.3 can generate chromosomal instability and suggests that chromosomal instability is a major contributing factor to DMG K27-altered and DMG G34-altered tumor formation.

Abstract Diffuse midline gliomas with the H3.3 K27M mutation are lethal brain tumors in children. H3 K27M causes global loss of Lys27 triple methylation (Lys27me3), inducing epigenetic reprograming. Here we show that H3.3 K27M also causes decreased H3.3 Ser31 phosphorylation on mitotic chromosomes. We show that H3.3 K27M DIPG cells have reduced pericentromeric phospho-Ser31 and increased rates of chromosome missegregation compared to normal, diploid human cells. CRISPR-editing K27M to M27K restored phospho-Ser31 to WT levels and dramatically decreased the rate of chromosome missegregation. We confirm that Chk1 is the H3.3 Ser31 kinase: K27M mutant H3.3 protein exhibits ~60% reduced Chk1 phosphorylation of Ser31 in vitro. Chk1 knockdown completely abolishes phospho-Ser31 in cells and these have increased rates of chromosome missegregation. In normal, diploid cells, expression of K27M or an S31A non-phosphorylatable mutant increased chromosome missegregation; this is suppressed by expressing a phosphomimetic double mutant (K27M/S31E) that restores phospho-Ser31. WT cells arrest following chromosome missegregation. However, cells expressing H3.3 K27M or S31A fail to arrest - despite having WT p53. Finally, we expressed H3F3AS31A and PDGFb in an RCAS/TVA mouse model of DIPG and ~80% developed diffuse high-grade brain tumors and show significantly decreased survival. Our results suggest that loss of phospho-Ser31 alone is oncogenic because H3.3 S31A-expressing cells are WT for K27me3. Our results demonstrate that H3.3 K27M inhibits Ser31 phosphorylation both in vitro and in vivo, leading to both chromosome missegregation and loss of subsequent G1 arrest – thus creating diffuse midline gliomas with both dynamic, complex karyotypes and epigenetic reprogramming.