Identifying molecular vulnerabilities of ALT-dependent cancers

PROJECT SUMMARY Telomeres are specialized protein-DNA structures that protect the ends of linear chromosomes and maintain genomic stability. Maintenance of adequate telomere length is thus essential for cellular immortalization and tumorigenesis. While most human cancers achieve this through telomerase activation, ~ 5% - 10% of them adopt a recombination-based mechanism termed alternative lengthening of telomeres (ALT) to elongate their telomeres. Although the ALT-driven cancers are generally aggressive with poor prognosis, there are currently no targeted therapies. In human cancers, ALT is strongly associated with genetic alterations that affect histone H3.3 chaperone ATRX-DAXX complex. We and others previously demonstrated that the ATRX-DAXX complex is essential for normal telomere maintenance. We showed that ATRX or DAXX loss, while promoting tumorigenesis by potentiating the ALT-driven immortalization, also creates a persistent telomere replication dysfunction. We therefore hypothesize that ALT-immortalized cancers must have adopted special mechanism(s) to offset their innate telomere DNA replication defects, and thus would be selectively vulnerable to the inhibition of those compensatory pathways. By combining our unique isogenic ALT-immortalization model system and customized domain-focused CRISPR screen platform, we have uncovered a list of selective molecular vulnerabilities including histone lysine demethylase KDM2A for ALT-dependent cells. We demonstrate that KDM2A-mediated H3K36me2 demethylation is required for ALT-directed telomere maintenance. Inactivation of KDM2A impairs ALT-specific multitelomere cluster dissolution, leading to chromosome missegregation and mitotic cell death. The objectives of this proposal are to delineate the molecular mechanism underlying ALT-directed telomere maintenance and to identify mechanism-based therapeutic targets against ALT-driven human cancers. To meet those goals, we will pursue the following three specific aims. In Aim 1, we will investigate the molecular functions of KDM2A in ALT-directed telomere maintenance. In Aim 2, we will define the potential adverse effects and off-tumor toxicities of future KDM2A-targeted therapies. In Aim 3, we will establish an in vivo platform to explore the synthetic- lethal interactions of ALT-driven ATRX-mutant malignant gliomas. Completion of the proposed studies will uncover the molecular mechanisms and critical dependencies underlying ALT-directed telomere maintenance and thus drive the development of novel mechanism-based therapeutics against ALT- dependent cancers including the ATRX mutant malignant gliomas.