Histone H3K27 methylation modulates the dynamics of FANCD2 on chromatin to facilitate NHEJ and genome stability

Abstract

Dysregulation of the homeostatic balance of histone H3 di- and tri-methyl lysine 27 (H3K27me2/3) levels caused by the mis-sense mutation of histone H3 (H3K27M) is reported to be associated with various types of cancers. In this study, we found that reduction in H3K27me2/3 caused by H3.1K27M, a mutation of H3 variants found in patients with diffuse intrinsic pontine glioma (DIPG), dramatically attenuated the presence of 53BP1 (also known as TP53BP1) foci and the capability of non-homologous end joining (NHEJ) in human dermal fibroblasts. H3.1K27M mutant cells showed increased rates of genomic insertions/deletions and copy number variations, as well as an increase in p53-dependent apoptosis. We further showed that both hypo-H3K27me2/3 and H3.1K27M interacted with FANCD2, a central player in the choice of DNA repair pathway. H3.1K27M triggered the accumulation of FANCD2 on chromatin, suggesting an interaction between H3.1K27M and FANCD2. Interestingly, knockdown of FANCD2 in H3.1K27M cells recovered the number of 53BP1-positive foci, NHEJ efficiency and apoptosis rate. Although these findings in HDF cells may differ from the endogenous regulation of the H3.1K27M mutant in the specific tumor context of DIPG, our results suggest a new model by which H3K27me2/3 facilitates NHEJ and the maintenance of genome stability.This article has an associated First Person interview with the first author of the paper.

Combined Targeting of Mutant p53 and Jumonji Family Histone Demethylase Augments Therapeutic Efficacy of Radiation in H3K27M DIPG

“…Gamma-H2AX, p53 (DO-1) and GAPDH-specific antibodies were obtained from Cell Signaling Technology (CST) (Danvers, MA, USA). Proliferation and clonogenic survival assays were performed as previously described [22] with modifications. For proliferation assays, DIPG cells harboring the histone H3.3 K27M mutation were pre-treated for 5 days with indicated concentrations of GSK-J4 (0-3 µM) or with APR-246 (0-25 µM), or EZH2 inhibitor EP005687 (0-3 µM), or mock-pretreated with DMSO, were then seeded in 24-well plates, at concentration 10,000 cells/well, then irradiated with 0 or 4 Gy dose of XRT, and harvested at 96 h after treatments.…”

“…Furthermore, growth inhibitory effects of GSK-J4 appeared to be independent of TP53 status ( Figure S4). Since radiation is the standard treatment modality for pediatric DIPG, and given the recently described important role of H3K27 trimethylation in non-homologous end joining (NHEJ) type of DNA damage repair [22], we To determine whether the effects of GSK-J4 inhibitor are specific for H3K27M mutation, we evaluated the effects of this drug on H3K27 wild type cells ( Figure S4). Intriguingly, we found that GSK-J4 displayed a significant growth inhibitory effect in H3K27 wild type cell lines, raising the possibility of H3K27M-independent effects of this drug.…”

“…Furthermore, growth inhibitory effects of GSK-J4 appeared to be independent of TP53 status ( Figure S4). Since radiation is the standard treatment modality for pediatric DIPG, and given the recently described important role of H3K27 tri-methylation in non-homologous end joining (NHEJ) type of DNA damage repair [22], we investigated the effects of GSK-J4 inhibitor combined with radiation in clonogenic survival assays ( Figure 4). investigated the effects of GSK-J4 inhibitor combined with radiation in clonogenic survival assays (Figure 4).…”

“…Clinical evaluation of APR-246 in p53 mutant myelodysplastic syndromes (MDS) revealed a dramatic 82% rate of complete response, leading to a fast track designation and an orphan drug designation of APR-246 by Food and Drug Administration (FDA) in April 2019 [19,20]. Given the important role of p53 tumor suppressor activities and H3K27 methylation in DNA damage response [21,22], we investigated the efficacy of mutant p53 targeting, oxidative stress induction, and Jumonji family histone demethylase JMJD3 inhibition combined with therapeutic radiation in human DIPG cells. Our hypothesis was that dual targeting of the proposed epigenetic mechanisms of disease pathogenesis mediated by H3K27M and TP53 mutations would sensitize DIPG cells to therapeutic radiation.…”

Combined Targeting of Mutant p53 and Jumonji Family Histone Demethylase Augments Therapeutic Efficacy of Radiation in H3K27M DIPG

“…Clinical evaluation of APR-246 in p53 mutant myelodysplastic syndromes (MDS) revealed a dramatic 82% rate of complete response, leading to a fast track designation and an orphan drug designation of APR-246 by FDA in April 2019 [18,19]. Given the important role of p53 tumor suppressor activities and H3K27 methylation in DNA damage response [20,21], we investigated the efficacy of mutant p53 targeting, oxidative stress induction, and Jumonji family histone demethylase JMJD3 inhibition combined with therapeutic radiation in human DIPG cells. Our hypothesis was that dual targeting of the proposed epigenetic mechanisms of disease pathogenesis mediated by H3K27M and TP53 mutations would sensitize DIPG cells to therapeutic radiation.…”