1. Deoxyribonucleic Acid (DNA) Damage Response (DDR) Genes Are Epigenetically Regulated in Cancer, Affecting Chemosensitivity

Epigenetic modifications, such as gene promoter DNA hypermethylation, and subsequent changes in gene expression of DDR-associated genes, lead to a loss of DNA repair capacity and have been demonstrated in a variety of tumours and cell line models ^[1][2][1,2]. The loss of DNA repair activity in tumours may lead to chemosensitivity to DNA-damaging cytotoxic chemotherapy. One of the paradigms demonstrating the clinical relevance of epigenetic mechanisms involved in chemosensitivity is highlighted in glioblastoma. DNA methylation of MGMT, the DNA repair gene encoding O-6-methylguanine-DNA methyltransferase, leads to the loss of MGMT expression [1]. This leads to reduced DNA damage repair and subsequently increased sensitivity of cells to the alkylating agent, temozolomide [3]. Early clinical studies showed that glioma patients treated with temozolomide and radiotherapy and with a methylated MGMT gene promoter have a survival benefit compared to only radiotherapy. Patients with unmethylated MGMT promoters showed no statistically significant difference in survival [4]. Prospective randomised trials of glioblastoma patients for radiotherapy versus alkylating agent chemotherapy have demonstrated DNA methylation of MGMT is a clinically useful predictive biomarker to stratify patients, rather than just prognostic ^[3][5][6][3,5,6].

A second paradigm is the epigenetic alterations of homology recombination DNA repair (HR)-associated genes including breast cancer type 2 susceptibility proteins 1 and 2 (BRCA1/2) ^[7][8][7,8]. BRCA1 is frequently methylated in high-grade serous ovarian cancer (HGSOC) and can lead to HR-deficiency (HRD) which is associated with increased patient survival following platinum-based chemotherapy compared to patients with HR proficient tumours [8]. Sensitivity to platinum-based chemotherapy exploits HRD in HGSOC by introducing double-stranded breaks in DNA, leading to genomic instability and cell apoptosis [9]. However, non-homologous end joining (NHEJ) and base excision repair (BER) pathways, which require Poly (ADP-ribose) polymerase (PARP), can still be utilised to repair the damaged DNA. PARP inhibitors (PARPi) induce synthetic lethality in HR-deficient tumours by disrupting multiple DNA repair pathways simultaneously [10]. In breast and ovarian cancer, BRCA1/2 status is clinically useful to predict sensitivity to platinum-based chemotherapy and PARPi ^[11][12][11,12]. Furthermore, patients with BRCA1 methylated and HRD HGSOC have better prognosis than unmethylated HR proficient tumours [13].

In both the above paradigms, epigenetic regulation by DNA methylation during tumour development, prior to chemotherapy, leads to inactivation of DNA repair activity and drug sensitivity. However, epigenetic mechanisms have also been proposed as important drivers of acquired drug resistance adaptation during chemotherapy. This leads to increased epigenetic silencing in tumours at relapse compared to primary presentation [14]. For instance, loss of DNA mismatch repair (MMR) due to DNA methylation at the MutL Homolog 1 (MLH1) gene promoter has been associated with resistance to alkylating agents such as temozolomide and crosslinking agents such as cisplatin ^[15][16][17][15,16,17]. The presence of functional MMR has been proposed to lead to cell death due to futile repair cycles, generation of double-strand DNA breaks and engagement of apoptosis [18]. Thus, the absence of MMR leads to loss of engagement of cell death pathways by DDR pathways leading to drug resistance. In another example of epigenetic adaptation following chemotherapy, while sensitivity to PARP inhibitors of HGSOC is associated with DNA methylation at BRCA1, tumours recurring following chemotherapy restore BRCA1 expression associated with reduced DNA methylation. This supports a key role for DNA methylation changes during the acquisition of PARPi resistance [19]. FANCF, another DDR-associated gene closely linked to BRCA genes, is often methylated in several different cancer types including testicular [20], head and neck [21], lung [21], cervical [22] and ovarian ^[23][24][23,24]. Methylation of the FANCF promoter in ovarian cancer has been linked to platinum sensitivity, whereas demethylation of FANCF has been associated with platinum resistance and often occurs after platinum chemotherapy ^[24][25][24,25].

Whilst aberrant methylation of DDR genes has been shown in multiple cancers, other epigenetic mechanisms such as histone modifications at genomic regulatory regions, including enhancers and super enhancers, may also play an important role in response to chemotherapy. Studies show modest benefits of temozolmide treatment in patients with methylated MGMT in colorectal cancer and patient-derived xenograft (PDX) models of glioblastoma show high expression of MGMT linked to active enhancers, despite promoter methylation [26]. This suggests different epigenetic mechanisms are able to dynamically regulate gene expression. Furthermore, DDR-associated genes associated with drug response may themselves regulate the epigenetic landscape. BRCA1 mutations in breast cancer epithelial cells lead to the loss of H3K27ac at super enhancers and impair enhancer–promoter lopping [27], whereas BRCA2 depletion has been linked to chromatin remodelling [28]. MMR inactivation via MLH1 mutations has been shown to activate enhancers of genes associated with growth in colorectal cancer and may activate enhancers of genes associated with drug-resistance [29]. These observations highlight the potential interplay of different epigenetic mechanisms and DDR-associated genes in relation to chemosensitivity (Figure 1).

BRCA1/2 deficiency can impair the HR pathway leading to increased sensitivity to DNA-damaging agents such as platinum chemotherapy and PARPi. However, BRCA1/2 deficiency can also lead to chromatin remodelling and reduced H3K27ac at regulatory regions such as enhancers/super-enhancers, leading to the aberrant expression of genes including those associated with drug resistance/DDR-associated genes. Reactivation of BRCA1/2 and/or other DDR-associated genes can result in tumour cells which are drug resistant and have functional DNA damage repair.

The clinically relevant examples above demonstrate how promoter DNA methylation may confer tumour chemosensitivity or chemoresistance. However, there is a wide spectrum of DDR genes whose epigenetic regulation can influence chemosensitivity [2]. Epigenetic therapies such as DNA-demethylating agents and inhibitors of maintenance of histone post-translational modification (for instance, histone deacetylase inhibitors) are now registered for clinical use, particularly in haematological malignancies. Furthermore, they remain the focus of many clinical trials in epithelial cancers [30]. However, careful patient selection will be key to demonstrating clinical efficacy of epigenetic therapies, especially when used in combination with other therapies.

This is exemplified in early clinical trials of DNA-demethylating agents in HGSOC. In cell line models, the loss of MMR due to MLH1 methylation results in failure to engage apoptotic responses and resistance to platinum coordination complexes and alkylating agents which can be reversed by DNA-demethylating agents such as 5-azacytidine and its derivatives [31]. DNA methylation has been used as a pharmacodynamic marker in surrogate tissues, such as blood, to demonstrate biological activity and guide the scheduling of combination studies with other therapies [32]. There have been two randomised phase II studies of DNA-demethylating agents and carboplatin in HGSOC with contrasting outcomes ^[33][34][33,34]. One study was closed early due to unacceptable toxicity and lack of efficacy of the combination compared to single-agent carboplatin [33]. The other trial showed an improvement in the 6-month progression-free survival of patients treated with the combination. However, this trial did not show statistically significant superiority for the primary endpoint of progression-free survival, potentially due to being statistically underpowered for the latter endpoint [34]. Both studies explored the combination of carboplatin with a DNA-demethylating agent during second-line chemotherapy. Glasspool et al., recruited partially platinum-sensitive patients recurring 6–12 months following the initial response to platinum-based chemotherapy while Oza et al., recruited women with recurrence within 6 months of the last platinum-containing regimen. It is possible that partially platinum sensitive patients may have a different proportion of women with tumours who are sensitive due to the methylation of HR genes, such as BRCA1, and for whom a demethylating agent may have an adverse effect. Neither study selected patient recruitment based on the methylation status of the patients’ tumours.

As previously mentioned, BRCA1 is frequently methylated in high-grade serous ovarian cancer and associated with increased patient survival following platinum-based and PARP inhibitor chemotherapy compared to patients with HR-proficient tumours. BRCA1/2 deficiency remains the strongest predictor of PARPi sensitivity [35] although abrogation of other key HR genes including FA Complementation Group A (FANCA) [36], DNA Repair Protein RAD51 homolog 1 (RAD51) [37], X-ray Repair Cross Complementing 2 (XRCC2) and X-ray Repair Cross Complementing 3 (XRCC3) [38] and DNA Polymerase Delta 4 (POLD4) [39] have been linked to platinum and/or PARPi responses. Furthermore, not all BRCA mutant tumours are HR deficient and many HR-proficient tumours can initially respond well to PARPi ^{[40][41][42][43][44]}[40,41,42,43,44] which has been attributed to the involvement of PARP in other non-DDR associated mechanisms including chromatin remodelling [45]. Unfortunately, as with platinum-based chemotherapy, primary and acquired resistance to PARPi is common ^{[35][40][46][47]}[35,40,46,47]. Reversion mutations can restore the function of HR-associated genes frequently mutated in HGSOC, including BRCA1/2 ^{[48][49][50][51]}[48,49,50,51] and RAD51C/D [52]. Epigenetic mechanisms including histone modifications may also contribute to PARPi resistance although the exact mechanisms remain poorly understood ^{[12][52][53][54][55][56][57]}[12,52,53,54,55,56,57].

Histone methylation has been linked to PARP inhibitor sensitivity in multiple cancers [58]. Enhancer of Zeste Homolog 2 (EZH2) and Euchromatic Histone Lysine Methyltransferase 2 (EHMT2) both maintain repressive H3K27 and H3K9 methylation histone marks, respectively, and are frequently overexpressed in cancer [59]. The inhibition of EZH2 alone has previously been linked to reducing the expression of multiple genes associated with DDR pathways in multiple cancers including prostate [60] and ovarian [61]. Furthermore, EZH2 inhibition has been shown to sensitise breast cancer cells to PARPi ^[58][61][58,61] and PARPi can regulate EZH2 expression [62] via PARPylation. EHMT2 has been linked with directly recruiting HR-associated factors, including BRCA1 to promote DNA damage repair [63]. The inhibition of EHMT2 promotes increased DNA damage and altered cell cycle regulation [64] and PARPi resistant cells treated with an EHMT1/2 inhibitor show significantly altered gene expression changes enriched in pro-survival pathways including, phosphatidylinositol 3 kinase(PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR) [64]. In BRCA1-depleted SUM149 breast cancer cells and PDX models, treatment with an EZH2i and PARPi reduced tumour growth more than single PARPi treatment; however, this effect was not seen in a BRCA2-depleted mouse model of breast cancer [65]. EZH2 inhibition alone may not be sufficient to modulate chromatin conformation [66] therefore dual EZH2/EHMT2 inhibitors in combination with PARPi would perhaps be of future interest. A clear theme regarding epigenetic therapies in combination with chemotherapy and their ability to modulate epigenetically driven drug resistance is that any future combination therapies must have clear stratification markers. The global epigenetic and mutational profile of DDR-associated genes must also be considered in order to maximise the positive outcomes for patients. A summary of key DDR genes associated with drug resistance and/or regulated by epigenetic mechanisms are shown in Table 1.