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Histone H3.3 oncogenic mutations: a role in genome instability through altered DNA repair and replication fork stability?

Periodic Reporting for period 1 - HiMIN (Histone H3.3 oncogenic mutations: a role in genome instability through altered DNA repair and replication fork stability?)

Reporting period: 2018-07-01 to 2020-06-30

Background
Besides chromatin modifiers, also histone proteins themselves are involved in chromatin dynamics and participate in the preservation of genome stability(1–3). Interestingly, point mutations were initially identified in human genes encoding histone H3 variants in specific cancer types including gliomas and bone tumors(4) and more recently in a broader range of tumors, even if at lower frequency(5). Particularly frequent in H3.3 these mutations (K27M, G34R/V/W and K36M), are dominant events in cancer. Although not sufficient for neoplastic transformation, as they frequently associate with p53 loss-of-function, H3.3 mutations do promote tumorigenesis at least in part through alterations in histone modifications impacting gene expression(4). However, further investigations are needed to fully elucidate how these histone mutations drive tumor progression. Indeed, tumors with H3.3 mutations display chromosomal abnormalities including copy number alterations(6) but the molecular basis of this genomic instability is not known.
When we started the project, it was still unclear whether cancer-associated H3.3 mutations drive tumor progression only via alterations in H3 modifications impacting gene expression. An alternative/non-mutually exclusive hypothesis would be that these mutations also directly affect genome stability independently of their function in gene expression. Indeed, recent data indicate that H3.3 is deposited de novo at sites of DNA damage(7,8) and the H3.3 K36M mutation inhibits DSB repair by homologous recombination(9,10). Furthermore, H3.3 and one of its specific chaperones are implicated in replication fork progression and stability in chicken and human cells(11,12) and the G34R mutation was recently shown to impair replication fork stability in yeast cells(13). These findings called for a more systematic characterization of the impact of H3.3 point mutations on genome instability, that is what our project aims at. In addition, there is currently no therapy in place for H3.3 mutated tumors. Based on this premises, our work added to the current research efforts by systematically characterizing the contribution of H3.3 oncomutations in genome stability maintenace and in DNA damage repair. By doing so, it also contributed at opening up therapeutic angles for H3.3 mutant tumors(14–16).
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.
Methodology and main findings
The stable expression of mutant H3.3 transgenes is sufficient to recapitulate some of the molecular defects observed in patients with H3.3-mutated tumors22. Thus, this represents a reliable and easily tractable system to dissect the mechanisms whereby H3.3 mutations may enhance genome instability. For our experiments, we so far used U2OS and glioma cell lines established in the laboratory that stably express wild-type or mutant H3.3 (with K27M, G34R/V/W or K36M point mutation, see section II for more details). These histones are also fused to a SNAP tag to allow tracking of their de novo deposition into chromatin20. Upon the generation of human cell lines that stably express the most frequently found H3.3 mutations, we analyzed their response to a panel of DNA damaging agents inducing DSBs or disrupting fork stability. Interestingly, we observed that the repair of DSBs in S phase was altered upon expression of several H3.3 mutants, and it correlated with a differential sensitivity to DNA damaging agents (see section II for more details). We are now dissecting the mechanisms whereby H3.3 mutations impact DSB repair, and we are investigating whether and how altered DSB repair drives genome instability in H3.3-mutated cells.
model for alteration of DNA repair in H3.3 mutated cells