1.1 Objectives
Based on the above-published findings, we hypothesized that H3.3 point mutations may trigger genome instability by altering the cellular response to DNA damage, thus leading to malignant transformation. Therefore, the overall research goal of this project was to characterize the function of H3.3 oncogenic mutations in the response to DSBs and in the recovery of damaged replication forks. By achieving this goal, we contributed to advance two main aspects of the current knowledge in the epigenetics of cancer: first, our findings helped to elucidate how histone variants and histone post-translational modifications (PTMs) contribute to the response to ongoing DNA damage, which is a feature of cancer cells. In addition, from a translational point of view, by dissecting how somatic H3.3 mutations contribute to the development of cancer-related features, such as genome instability, our work will uncover new players that specifically drive genome instability in H3.3-mutated cancers. In conclusion, our work will ultimately allow the identification of new targets that could be exploited for the sensitization of H3.3 mutated cancers to therapeutic treatments, such as chemotherapeutics.
Objective 1/ Work Package 1. Study the response of H3.3 mutated cell lines to DSBs or damaged replication forks in terms of: repair pathway activation, occurrence of genome instability and analysis of drug sensitivity.
In light of recent data showing that the H3.3 K36M mutation inhibits homologous recombination (HR) repair of DSBs and generates genome instability (9,10), we analyzed how H3.3 mutations other than K36M affect DSB-mediated genomic instability and DSB repair. H3.3 K36M expressing cells is used as a positive control in our assays. We induced DSBs with radiomimetic chemicals (bleomycin or neocarzinostatin) or with agents affecting DNA replication (hydroxyurea and camptothecin).
Our data indicate that some H3.3 mutant cells (K27M and G34R) show an aberrant activation of 53BP1-driven, classical end-joining repair pathway in S phase cells. This was confirmed by functional assays such as the HPRT assay to evaluate end-joining activity and the metaphase spreads to evaluate chromosomal aberrations, where K27M and G34R H3.3 mutants show increased end-joining activity and increased chromosomal aberrations.
Objective 2/Work Package 2. Validate the role of the newly identified players in genome stability and drug sensitivity of H3.3 mutated cancer.
Here and thanks to some interactome data generated in H3.3 wild-type and K27M, G34R mutant cells, we could identify DNA repair players that differentially interact with mutant H3.3 as compared to wild-type H3.3. More specifically, we focused our attention on three DNA repair enzymes that showed increased interaction with K27M and G34R as compared to wild-type H3.3. Thanks to the analysis of the function of the identifed DNA repair enzymes, we could dissecte that one of those three, contributes to the increased end-joining activity of H3.3 mutant cells. Future studies will be aimed at further characterizing the function of the identified enzyme in H3.3 mutant cells and to evaluate whether its inhibition can kill specifically H3.3 mutant cells.
In conclusion, in WP1 we identified a DNA repair defect in some H3.3 mutant cells and in WP2 we identified a DNA repair enzyme whose aberrant function may mediate the observed DNA repair defects. These results will be exploited by our group to try top target the abberrant enzyme’s activity in order to kill H3.3 mutant cancer cells. Moreover, we plan to disseminate the abovementioned findings through a publication and by participation to international scientific meetings.
