PDTM-38. HISTONE H3.3 SER31 PHOSPHORYLATION REGULATES CHROMOSOME SEGREGATION AND CELL CYCLE CHECKPOINT CONTROL AND IS IMPAIRED IN PEDIATRIC GLIOMAS. (11th November 2019)

• Record Type: Journal Article
• Authors: Day, Charles; Langfald, Alyssa; Fadness, Sela; Zykova, Tatyana; Sepaniac, Leslie; Stumpff, Jason; Dong, Zigang; Vaughan, Kevin; Robinson, James; Hinchcliffe, Edward
• Is Part Of: Neuro-oncology. Volume 21(2019)Supplement 6
• Page Start: vi195
• Page End: vi196
• Publication Date: 2019-11-11
• Subjects: Brain Neoplasms -- Periodicals; Brain -- Tumors -- Periodicals; Brain -- Cancer -- Periodicals; Nervous system -- Cancer -- Periodicals; 616.99481
• DOI: 10.1093/neuonc/noz175.814
• Languages: English
• ISSNs: 1522-8517

Abstract

Abstract: Diffuse intrinsic pontine glioma (DIPG) carries a signature mutation in histone H3.3 (K27M) that induces epigenetic reprogramming via suppressed Lys27 triple methylation (K27me3). H3.3 has a Ser at position 31, which is adjacent to the K27M DIPG mutation. H3.3 Ser31 is phosphorylated beginning in prophase. This phosphorylation is restricted to pericentromeric heterochromatin and becomes dephosphorylated in anaphase. Surprisingly, chromosome missegregation triggers Ser31 hyperphosphorylation on the lagging chromosome, and masking phosphoS31 prevents p53 accumulation; part of the G1 checkpoint response to aneuploidy. Whether the K27M mutation influences Ser31 phosphorylation, chromosome segregation or G1 checkpoint control is unknown. We show that K27M DIPG cells have reduced pericentromeric phosphoS31 and increased chromosome instability (CIN) compared to normal, diploid human cells. CRISPR-editing of K27M to M27K restored phosphoS31 to WT levels and dramatically decreased the rate of CIN. We also demonstrate that Chk1 is the H3.3 Ser31 kinase. S31A abolishes Ser31 phosphorylation by Chk1, while K27M reduces it by ~60% in vitro. Chk1 knockdown abolishes phosphoS31 in normal, diploid mitotic cells, and induces CIN. In normal, diploid cells, expression of S31A or K27M mutations increased chromosome missegregation, whereas cells expressing a phosphomimetic double mutant (K27M/S31E) divide normally. WT cells arrest following chromosome missegregation. Yet, normal cells expressing H3.3 K27M or S31A do not arrest - despite having wild-type p53. Finally, using RCAS/TVA, we expressed H3F3A S31A in the brains of BRAF V600E mice, and ~50% developed tumors, suggesting that loss of phosphoS31 alone is oncogenic. Importantly, H3.3 S31A-expressing cells are WT for K27me3. Our results demonstrate that expressing H3.3 K27M reduces the population of H3.3 that is phosphorylatable at Ser31, leading to chromosome missegregation and preventing G1 arrest – thus creating proliferating CIN cells. We propose that a single amino acid substitution drives oncogenesis of DIPG, at least impart, by triggering the evolution of complex karyotypes.