The roles of H3.3 and ATRX abnormalities in driving alternative lengthening of telomeres and chromatin aberrations in cancers

Udugama M, Voon HP, Hii L and Wong LH

Biomedicine Discovery Institute, Monash University.

One striking finding in the area of cancer epigenetics has been 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 mutations always overlap with ATRX and p53 mutations, and these pGBMs are activated in the Alternative Lengthening of Telomeres (ALT) pathway, suggesting that H3.3G34R/ATRX/p53 mutations cooperate to drive ALT and GBM development. We have created cell models carrying H3.3G34R/ATRX/p53 mutations to recapitulate the initial driver epigenetic events that promote ALT. These mutants are compromised in heterochromatin formation at the telomeres, accompanied with telomere damage (and aberrant telomere protein binding) and formation of aberrant PML nuclear bodies (PML-NBs) that are irregular in size, shape and number. 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. As a result, it induces aberrant histone methylation pattern and affects telomere chromatin maintenance. IDH1 (which is also mutated in GBM) is also known to inhibit KDM4 function. We propose KDM4 chromatin network as a major driver that promotes ALT and the oncogenic process in GBMs. In the H3.3G34R/ATRX mutants, we also detect DNA copy loss at ATRX-bound ribosomal repeats, accompanied with severely reduced rRNA synthesis. ALT positive human sarcoma tumour samples 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 development of ALT cancers.