Page 37 - Hormel Report 2021

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Pediatric Brain tumors: Pediatric glioblastomas are high-grade childhood brain tumors that are infiltrative, resistant to conventional therapies, and carry a dismal prognosis, with 5-year survival rates well below 10%. Pediatric glioblastomas, particularly diffuse intrinsic pontine glioma (DIPG), are clinically and biologically distinct from the adult disease and defined in part by highly specific heterozygous mutations K27M (or G34R) in the genes that encode histone H3, primarily in the variant H3.3. Methylation of a key lysine residue (Lys27) on H3 acts as an epigenetic regulator. Loss of Lys27 triple methylation (Me3) – caused by the dominant negative K27M mutation – reprograms the epigenome and is proposed as a driving event in pediatric gliomas.
Our recent work suggests that K27M (or G34R) also contribute to tumorigenesis via inhibition of Ser31 phosphorylation and the induction of chromosome instability. In histone H3.3, Ser31 is one of five amino acid substitutions that differentiate it from canonical H3.1. H3.3 Ser31 is phosphorylated during mitosis specifically at pericentromeric heterochromatin,
where it plays a role in chromosome segregation. We find that K27M or G34R mutations inhibit H3.3 Ser31 phosphorylation in vitro. Pediatric DIPG cell lines with the K27M mutation exhibit a significant decrease in Ser31 phosphorylation in vivo, and that these cells exhibit chromosome instability. CRISPR-Cas9 editing of the K27M mutation (H3.3M27K) restores WT levels of Ser31 phosphorylation during mitosis, and revertants show significant decreases in chromosomal instability. These revertants also regain WT levels of Lys27 triple methylation. In diploid cells, expression of K27M,
G34R, or S31A non-phosphorylatable mutant triggers chromosome instability.
Our long-term goal is to understand how mutations in an H3.3 gene contribute to the generation of pediatric gliomas. Our central hypothesis is reduction in total available phosphorylatable H3.3 – caused by the het- erozygous mutations in residues flanking Ser31 in one of the two H3.3 genes – partially abrogates cell cycle checkpoint function.
Figure 2: Localization of cGAS to a micronucleus (red)
Cell cycle response to CIN – “Aneuploidy Fail-Safe” and cGAS-STING: In normal, diploid cells chromosome missegregation is extremely rare because of robust cell cycle checkpoint mechanisms that monitor the alignment of sister chromatids to the metaphase plate. This checkpoint feeds back upon the signals that drive sister chromatid segregation at anaphase (33). However, chromosome missegregation can occur in normal cells. These inadvertent/rare missegregation events are monitored by “Fail-Safe” devices active in G1: the “Aneuploidy Fail-Safe” and the cGAS-STING innate immune response. The “Aneuploidy Fail-Safe” detects single chromosome missegregation, and activates a p53-dependant cell cycle arrest. The result is a lack of proliferation of aneuploid daughter cells.
The cGAS-STING pathway is part of the innate immune response that detects double stranded DNA in the cytoplasm. dsDNA can be the result of genomic DNA leaking from the nucleus. In the case of chromosome missegregation, a small micronucleus – adjacent to the main nucleus, forms around the lagging chromosome(s). These micronuclei are defective, and “leak” – exposing the chromatin to the cytoplasm. The exposed dsDNA recruit cyclic GMP-AMP Synthase (cGAS) and activates Stimulator of Interferon Genes (the STING protein) turning on the IRF3 transcription factor and expression of pro-inflammatory genes, such as type I interferons like INF. The cGAS- STING pathway induces both an immune signal- ing response and triggers senescence of neigh- boring cells. Thus, leaky micronuclei block the proliferation of chromosomally unstable tumor cells, while also initiating regional tumor immune editing.
Publication:
• Day, CA, A Langfald, and EH. Hinchcliffe (2020) Manipulating cultured mammalian cells for mitosis research. Methods Cell Biol. 158:43-61. PMID: 32423650