Functional Genomics of Sarcoma

Division Of Basic Sciences - Nci

Investigators

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Abstract

Currently our efforts are focused primarily on pursuing leads arising from studies of osteosarcoma. While most tumors maintain telomeres using telomerase, a subset of tumors (notably sarcomas, certain brain tumors and some neuroblastomas) utilize a DNA-templated process termed Alternative Lengthening of Telomeres or ALT. ALT is associated with mutations in the ATRX/DAXX/H3.3 histone chaperone complex responsible for deposition of histone variant H3.3 at heterochromatic regions of the genome including telomeres. Most cancers expressing ALT have mutations in ATRX. DAXX mutations are less common but are relatively frequent in pancreatic neuroendocrine tumors. The G292 osteosarcoma cell line has a defect in the DAXX gene as the molecular basis of its ALT phenotype. We modified G292 cells with an inducible DAXX construct that has allowed us to investigate changes during the inducible suppression and re-expression of ALT. Using our published G292 iDAXX model (PMID: 30872698), we observed that gene expression changes are minimal, suggesting that post-transcriptional events are most important in transitions to and from the ALT state. Importantly, no assay has been able to text the functional impact of DAXX missense mutations prior to our development of the iDAXX system. While most cancer associated DAXX mutations result in truncations, missense mutations throughout DAXX have also been observed in tumors. Using iDAXX system, we tested the ability of disease-associated DAXX missense variants to suppress ALT. We found that specific mutations throughout the DAXX protein negatively impact the ability of DAXX to suppress ALT. Unexpectedly, we find that mutations in the DAXX histone binding domain lead to failure of ATRX localization. We conclude that a key function of DAXX in ALT suppression is the localization of ATRX to nuclear foci. These results shed new light on the essential role of correct ATRX localization in ALT suppression, as well as clarifying the significance of cancer-associated DAXX mutations. doi: https://doi.org/10.1101/2024.11.22.624895