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Figure 2: Schematic of RCAS viral vector providing linked PDGFB and H3.3 delivery and expression.
Genomic-editing of H3.3 to revert the K27M mutation restores mitotic S31P (and K27Me3), and significantly decreases the rate of chromosome instability. Expression of H3.3 K27M or a non-phosphorylatable S31A mutant in normal, diploid cells and astrocytes results in chromosome missegregation and cell cycle checkpoint defects, and cells fail to undergo G1 cycle arrest in response to missegregation, leading to aneuploidy. This K27M effect is suppressed by co-expressing a phospho-mimetic S31E that restores S31P (K27M/S31E). Expression of K27M or S31A H3.3 inhibits p53 accumulation.
In our novel in vivo model of DIPG gliomas develop from neural stems cells following expression PDGFB linked to H3.3, PDGFB - H3.3K27M and H3.3S31A promoted the development of high-grade gliomas whereas H3.3WT controls did not. As H3.3S31A is WT for K27 methylation, our data demonstrates that loss of S31P is oncogenic. It is unclear if H3.3 mutations contribute to tumorigenesis via disruption of pericentromeric heterochromatin and chromosome missegregation via suppression of S31P or alterations in the epigenetic landscape via loss of lysine methylation or mostly likely via both.
Our research is conceptually innovative because it challenges the long-held notion that histone mutations drive pHGGs exclusively via changes in epigenetic regulation. In collaboration with Dr. Hinchcliffe we appear to be the only team in the world currently exploring mitotic phosphorylation/chromosome instability as additional driving events in pediatric gliomagenesis. Our work will aid in the development of new therapies and our team is well placed to pursue these in
in vivo model and clinical trials.
Figure 1: in vivo brains with metastatic melanoma induced with myrAKT1, BRAFCA and Cre in DCT-TVA/ Cdkn2aflox/flox in vivo. Immunohistochemistry for HA confirmed expression of myrAKT1 (54). Naturally occurring AKT mutations in melanoma (E17K) also promote brain metastasis in this model (55).
Brain tumor research
Gliomas are the most common malignant brain
tumors in children and represent the greatest
cause of cancer-related deaths under the age
of 19. Pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) are high-grade gliomas of children (pHGG). With a median overall survival of ~15 months, they are among the most lethal of any human cancers. To date, no clinical trial of a chemotherapeutic agent has shown any survival benefit for these tumors.
Mutations in histone variant H3.3 (H3F3A) occur as early events in DIPG (prior to p53 or ATRX mutations) and are sufficient to drive a global reduction of Lys 27 (H3 K27) bi- and tri- methylation (Me2, Me3) on all histone H3 variants: H3.1, 3.2, and 3.3. Histone H3 K27 methylation is responsible for epigenetic gene repression. The loss of K27 Me3 is associated with epigenetic dysregulation in H3.3K27M cells.
In addition to epigenetic reprogramming, in unpublished data (in final preparation
for publication), we find that the K27M mutations induce chromosome segregation and aneuploidy. Histone H3.3 has a unique Chk1 phosphorylation site, Ser31 (S31) that is critical for mitotic checkpoint regulation. Chromosome missegregation in non-transformed, diploid cells activates a p53-dependant G1 cell cycle arrest, which blocks the proliferation of normal cells that inadvertently become aneuploid. Masking of S31 phosphorylation (S31P) results in abrogation of cell cycle checkpoints that monitor the alignment and segregation of chromosomes. We find that that H3.3 K27M protein is defective for S31P by Chk1 kinase in vitro. DIPG cell lines also have decreased levels of mitotic S31P, and are chromosomally unstable and aneuploid.
Figure 3: A in vivo model of H3.3G34R pHGG. Survival and tumor grade and incidence
in Nestin-TVA in vivo infected with RCAS(A) H3.3G34R-P2A-PDGFβ, H3.3K27M or H3.3WT virus.
Publications:
• Grigore F, Yang H, Hanson ND, VanBrocklin MW, Sarver AL, Robinson JP. BRAF inhibition in melanoma is associated with the dysregulation of histone methylation and histone methyltransferases. Neoplasia. 2020 Sep;22(9):376-389. doi: 10.1016/j.neo.2020.06.006. Epub 2020 Jul 3. PMID: 32629178; PMCID: PMC7338995.
• Yang H, Kircher DA, Kim KH, Grossmann AH, VanBrocklin MW, Holmen SL, Robinson JP. Activated MEK cooperates with Cdkn2a and Pten loss to promote the development and maintenance of melanoma. Oncogene. 2017 Mar 6; PubMed PMID: 28263969.
• Shin CH, Grossmann AH, Holmen SL, Robinson JP. The BRAF kinase domain promotes the development of gliomas in vivo. Genes Cancer. 2015 Jan;6(1-2):9-18. PubMed PMID: 25821557; PubMed Central PMCID: PMC4362480.