One striking finding in the cancer epigenetics was the identification of mutated histone genes (oncohistones) in paediatric glioblastomas (pGBMs). Two H3.3 mutations are found. The first mutation replaces lysine 27 with a methionine. The second one replaces glycine 34 by an arginine (G34R). H3.3G34R overlaps with ATRX and p53 mutations, and these pGBMs are activated in the Alternative Lengthening of Telomeres (ALT) telomere maintenance pathway, suggesting that H3.3G34R/ATRX/p53 mutations cooperate to drive ALT and pGBM development.
We have created cell models carrying H3.3G34R/ATRX/p53 mutations to recapitulate the initial driver epigenetic events that promote ALT. KDM4 proteins are demethylases or epigenetic erasers that remove the methyl group from trimethylated H3K9 and H3K36. We find that the H3.3G34R inhibits KDM4 catalytic function of and drives its aberrant distribution. Thus, it induces aberrant histone methylation pattern and affects telomere chromatin maintenance. Our success in inducing ALT in H3.3G34R/ATRX/TP53/TERT cell model verifies the roles of H3.3 and ATRX in ALT activation and ALT is a multifactorial process. We propose KDM4 chromatin network as a major driver that promotes ALT and the oncogenic process.
In the H3.3G34R/ATRX mutants, we detect DNA copy loss at ATRX-bound ribosomal DNA (rDNA) repeats, accompanied with severely reduced rRNA synthesis. ALT positive human sarcoma tumours are substantially reduced in rDNA copy. Moreover, ALT cancer cells show increased sensitivity to RNA Polymerase I transcription inhibitor, suggesting the therapeutic potential of targeting Pol I transcription in ALT cancers. Our study provides insights into chromatin defects associated with ATRX/H3.3 mutations and ALT.