Chromosome Breaks and Genome Stability
The aim of the Chromosome Integrity Group is to understand how genome stability is maintained in response to chromosomal breaks.
Exposure to ionizing radiation (IR) can cause chromosome breaks, in which both DNA strands are broken. In addition to causing cell death (the desired outcome during radiation therapy), such lesions can also cause chromosomal rearrangements and genome instability, a hallmark of cancer cells. We previously identified a role for H3K36me3 in homologous recombination repair of DNA double-strand breaks, and its loss sensitizes cells to ionizing radiation (1). Further, we found H3K36me3 loss to be synthetic lethal with WEE1 inactivation, identifying a role for SETD2 in facilitating DNA replication restart and defining WEE1 inhibition as causing CDK-induced replication stress (2). We have therefore focused on delineating the role of the H3K36me3 pathway in homologous recombination repair; determining how H3K36me3 maintains viability in response to replication stress; and translating our findings into the clinic, using both fission yeast and human cell systems, as summarised below.
DNA repair occurs within the context of chromatin, in which DNA is tightly wrapped around proteins called histones. We have studied a histone mark, SETD2-dependent histone H3 lysine 36 trimethylation (H3K36me3), which is associated with transcriptional elongation, and plays important roles in a range of cellular functions, including maintaining genome stability. Accordingly, SETD2 is a tumour suppressor and we find H3K36me3 to be frequently lost in some human cancers, including 60% of clear cell renal cell carcinomas (kidney cancers) and associated with poor prognosis. We and others previously identified a role for H3K36me3 in homologous recombination repair of DNA double-strand breaks, and its loss sensitizes cells to ionizing radiation (1). We found that loss of SETD2, and subsequently other HR repair factors, including RPA, BRCA1 or BRCA2, led to significantly elevated levels of DSB misrepair through use of the microhomology-mediated end joining (MMEJ) pathway. Our findings have identified a more general role for HR repair factors in maintaining genome stability by both promoting error-free HR repair and by suppressing mutagenic MMEJ repair of DSBs (3). We have further examined the role for the H3K36me3 axis in facilitating transcription-coupled homologous recombination repair and IR resistance. We find that IWS1, a transcription elongation factor previously found to control SETD2 mediated histone H3K36 methylation, is rapidly recruited to breaks, where we are examining its role in transcription-coupled HR repair and radiation resistance. Moreover, in collaboration with Fumiko Asashi’s lab, we find MRG15-mediated tethering of PALB2 to unperturbed chromatin via H3K36me3 protects active genes from genotoxic stress (4).
We have explored the role of Set2 in the replication stress response in both fission yeast and humans. In fission yeast, we find Set2 methyltransferase facilitates DNA replication and promotes genotoxic stress responses through MBF-dependent transcription (5). Further, we have identified an essential role for Set2-dependent dNTP homeostasis following CDK-induced replication stress induced by WEE1 inactivation (6). Analogous roles for H3K36me3 are being explored during normal cell cycle progression and in response to replication stress in mammalian cells. Separately, using fission yeast, we have identified a role for the homologous recombination machinery in preventing de novo telomere addition at DNA double-strand breaks (7) and are making important advances in our understanding of the relationship between DNA double-strand break metabolism and chromosome instability.
We have also advanced our translational studies in a number of important ways: We have exploited a powerful genetic approach (synthetic lethality) using yeast and human systems to identify drugs, which specifically kill histone H3K36me3-deficient cancers. We identified a conserved synthetic lethal interaction between loss of SETD2 and WEE1 inactivation. Surprisingly, we found that cell death in this context arises through replicative catastrophe, resulting from critically low deoxyribonucleotide (dNTP) levels, leading to DNA damage and apoptosis. Mechanistic analysis indicated that H3K36me3 loss resulted in reduced expression of the ribonucleotide reductase subunit, RRM2, and to reduced dNTP supply. Further, WEE1 inhibition, using the potent WEE1 inhibitor AZD1775, led to degradation of RRM2, thus depleting dNTPs, and to elevated DNA replication origin firing, thereby increasing dNTP demand. Importantly, we found that treatment of H3K36me3-deficient cancer xenografts with AZD1775 caused cancer regression without signs of toxicity (2). Notably, the use of the WEE1 inhibitor, AZD1775, has now been taken into clinical trials to treat cancer patients with SETD2-deficient solid tumours (Clinicaltrials.gov NCT03284385). Our research has also facilitated the inclusion of AZD1775 and new WEE1 inhibitors into other clinical trials. We have explored H3K36me3 loss more broadly and find SETD2-dependent histone H3K36 trimethylation is frequently lost or depleted in a number of common cancers, suggesting new indications that can be clinically targeted. Accordingly, we are developing biomarkers to detect H3K36me3 loss using both gene expression profiles and DNA methylation profiles. We are exploring the mechanisms by which SETD2 deficient cells can become resistant to WEE1i. Further, we have identified novel agents which profoundly sensitize SETD2-deficient cancer cells through synthetic lethality. We have also commented on the challenges of combining chemo-and radiotherapy with checkpoint kinase inhibitors (8).
1. Pfister, S. X. et al. SETD2-dependent histone H3K36 trimethylation is required for homologous recombination repair and genome stability. Cell Rep 7, 2006-2018, doi:10.1016/j.celrep.2014.05.026 (2014).
2. Pfister, S. X. et al. Inhibiting WEE1 Selectively Kills Histone H3K36me3-Deficient Cancers by dNTP Starvation. Cancer Cell 28, 557-568, doi:10.1016/j.ccell.2015.09.015 (2015).
3. Ahrabi, S. et al. A role for human homologous recombination factors in suppressing microhomology-mediated end joining. Nucleic Acids Res 44, 5743-5757, doi:10.1093/nar/gkw326 (2016).
4. Bleuyard, J. Y. et al. MRG15-mediated tethering of PALB2 to unperturbed chromatin protects active genes from genotoxic stress. Proc Natl Acad Sci U S A 114, 7671-7676, doi:10.1073/pnas.1620208114 (2017).
5. Pai, C. C. et al. Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription. Cell Rep 20, 2693-2705, doi:10.1016/j.celrep.2017.08.058 (2017).
6. Pai, C. C. et al. An essential role for dNTP homeostasis following CDK-induced replication stress. J Cell Sci 132, doi:10.1242/jcs.226969 (2019).
7. Dave, A. et al. Homologous recombination repair intermediates promote efficient de novo telomere addition at DNA double-strand breaks. Nucleic Acids Res 48, 1271-1284, doi:10.1093/nar/gkz1109 (2020).
8. van Bijsterveldt, L., Durley, S. C., Maughan, T. S. & Humphrey, T. C. The Challenge of Combining Chemo- and Radiotherapy with Checkpoint Kinase Inhibitors. Clin Cancer Res 27, 937-962, doi:10.1158/1078-0432.CCR-20-3358 (2021).