**7. Targeting DDC Is One of the Most E**ffi**cient Ways to Tackle Cancer Cells**

Cancer cells accumulate DNA damage in an increased fashion as compared to normal cells and rely on DDC to avoid that excessive damage is inherited after cell division, for this reason several inhibitors targeting this pathway have been developed or are in development (Table 1).

As previously mentioned, DNA damage determines cell cycle arrest before entering mitosis, if this arrest is prolonged enough, cells go through the apoptotic pathway. Sometimes, cancer cells can avoid mitotic arrest and inappropriately enter mitosis before completion of the S and G2 phase; this frequently determines an improper distribution of chromosomes leading to mitotic catastrophe, which is an onco-suppressive signaling cascade preceding cell death via apoptosis, senescence or necrosis, in the first or subsequent cell divisions [101]. Inhibition of proteins that control the S/G2 checkpoint promotes this pathway and in solid cancer this is the main cell death-leading pathway induced by IR treatment [102,103].

ATM mutations could induce synthetic lethality in association with other mutations or specific drugs; this could be exploited to develop more accurate therapies [22]. Since ATM inhibition determines uncontrolled cell entry into mitosis by removing several checkpoints control, the use of ATM inhibitors in cancer therapy has been reported to cause sensitization to radiotherapy. In addition, ATM inhibition enhances cell sensitivity to topoisomerase I and II inhibitors such as camptothecin, etoposide, and doxorubicin [104]. Several ATM inhibitors have been used in cancer therapy. AZD0156 is a strong radio-sensitizer that recently has been demonstrated to inhibit tumor growth after radiation treatment of lung xenograft [105]; in addition, a combinatory effect with PARP inhibitors (olaparib) is under study since sensitivity to these drugs is related to ATM deficiency [106]. Currently, AZD0156 is in phase 1 trial (NCT02588105). A phase I clinical trial with the inhibitor AZD1390 (NCT03423628) (Table 1), which has been proved to radio-sensitize brain tumor models in pre-clinical studies, has also been initiated [107]. Several other compounds have been produced such as KU-55933, KU-60019 and KU-59403 that are in pre-clinical studies, therefore targeting ATM is a promising strategy for cancer treatment [108].

DNA-PK inhibition promotes radio-sensitivity in gastric cancer [109]; accordingly, its overexpression is associated to radio-resistance in thyroid, cervix, and prostate cancer [110] (Table 1). DNA-PKs deficiency can sensitize cells to DNA-damaging agents such as topoisomerase I and II inhibitors. This has led to the development of several DNA-PK inhibitors such as M3814, which has been demonstrated to increase efficacy of topoisomerase II inhibition in ovarian cancer models [111]. A clinical trial is ongoing to study efficacy of this drug in combination with radiotherapy and capecitabine, a drug that interferes with DNA and RNA metabolism (NCT03770689). Similarly, the DNA-PK inhibitor VX-984 enhances radio-sensitivity of glioblastoma and a clinical trial has been completed to assess safety of this drug in combination with chemotherapy (NCT02644278) [112]. Other drugs, currently under study, are CC-115 and MSC2490484A (NCT02516813) (NCT02833883), whose efficacy has been suggested in ATM-deficient cells and in combination with radiotherapy [113] (Table 1).

Differently from the other kinases, ATR does not participate in the initial recognition phases of DNA damage, but its activity is essential to prevent replication fork collapse, thus ensuring faithful duplication by binding to ssDNA and to arrest cell cycle in G2/M phase by interacting with CHK1 [114]. Since cancer cells are often defective in G1 phase checkpoint due to mutation of p53 and Rb tumor suppressor genes, they arrive in S phase with replication stress that could stall replication fork; therefore, cells rely on S-G2 phase checkpoint to avoid cell death due to excessive DSBs. Thus, a deficiency of ATR activity determines sensitivity to DNA damage agents such as IR, DNA cross-linking agents, and topoisomerase poisons [115]; its inhibition combined with PARP inhibitors also reduces tumor growth in BRCAness models [116] (Table 1). Currently, the ATR inhibitors M6620, AZD6738, BAY1895344, and VE-821 have been shown to increase lethality of cancer cell lines ATM, defective such as gastric cancer, non-small cell lung cancer and chronic lymphocytic leukemia, strongly suggesting a synthetic lethality interaction between the ATM and ATR pathways [117,118] (Table 1). A synthetic lethality interaction has been observed in triple-negative breast cancer cell lines also by combining AZD6738 with WEE1 inhibition, and with CHK1 inhibitor AZD7762, since combination of ATR and CHK1 inhibition leads to ssDNA accumulation and replication stress [119,120] (Table 1). In addition, since ATR inhibition enhances replication stress, as anticipated in pre-clinical trials, several studies analyze cancer cell sensitization by these drugs to DNA damaging agents such as cisplatin, gemcitabine, and radiation. In particular, M6620 was studied in combination with radiation in patients with brain metastases (NCT02589522) and with cisplatin for head and neck squamous cell carcinoma (NCT02567422) (Table 1); AZD6738 in combination with radiotherapy in patients with solid tumors, VE-821 in combination with radiation and chemotherapeutics in pancreatic cancer patients [121,122]. The effect of BAY1895344 in combination with chemotherapeutics or radiotherapy on tumor cell growth and survival was studied in cancer xenograft models that carry DNA damage repair deficiencies [123].

Targeting DDC pathway is an attractive strategy in cancer therapy. In fact, it has proven to be particularly effective in combination with radiotherapy determining cell death by induction of mitotic catastrophe pathway. Moreover, as DDC is upstream to several DNA repair pathways, different synthetic lethality combinations, currently under study, could be exploited taking advantage of this network.


For each target it is reported the name of the drugs currently in analysis; combination of target inhibition and classic anti-cancer therapy under study; possible synthetic lethality interaction; ongoing clinical trials with the drugs; references.

### **8. Combination of DSB Inducers and DSB-Repair Proteins Inhibition Is Greatly E**ff**ective**

A large number of DNA damaging agents exert their activity by inducing DSBs; therefore, combining these agents with compounds inhibiting DSB repair-involved players produced promising results. Particularly, inhibition of HR-involved proteins combined with DNA damage induction forces cells to use more error-prone DNA repair pathways, thus determining accumulation of DNA aberrations. Nevertheless, upregulation of mutagenic repair pathways could promote therapy resistance ultimately leading to disease progression [125]; for this reason, compounds inhibiting NHEJ or MMEJ players are in development to help in overcoming this problem and improve clinical outcomes of classic therapies.

Regarding HR, several inhibitors have been developed, targeting MRN complex and among them, mirin is the most studied (Table 2).

Mirin inhibits MRE11-associated exonuclease activity preventing MRN-dependent ATM activation. This inhibitor potentially sensitizes different cancers to genotoxic agents, such as malignant glioma, prostate cancer, and neuroblastoma [24,126,127]. In addition, several studies suggest that MRN defects could synergize with PARP inhibition, indicating a possible synthetic lethality interaction that could be exploited in specific cancer therapy [128].

As overexpression of *RAD51* stimulates cancer aggressiveness, it has been shown that lowering its expression or activity (through inhibition) can sensitize cancer cells to chemotherapeutics [113]. To date, different classes of RAD51 inhibitors which take advantage of different inhibition mechanisms have

been developed (Table 2): some inhibitors interfere with RAD51 ssDNA binding ability and could lead to the formation of toxic RAD51 complexes; other inhibitors interfere with RAD51 ability to interact with other DNA repair factors such as BRCA2 and finally drugs that inhibit the RAD51 capacity to migrate through the DNA strand and form foci after DNA damage. B02 is a RAD51 inhibitor which interferes with ssDNA binding ability and has proved to sensitize cancer cells to several DNA damage agents in preclinical studies also in combination with PARP inhibitors [129–131]. The RAD51 inhibitor RI-1 destabilizes RAD51 oligomerization/filament formation on DNA strand leading to the formation of cytotoxic complexes. This compound specifically works on those cancer cells where *RAD51* is overexpressed, sensitizing them to chemo/radiotherapy and PARP inhibitors [132,133]. IBR120 is an inhibitor that disrupts RAD51 interaction with BRCA2 sensitizing cancer cells to IR [134,135]. Finally, CYT-0851 is a compound that reduced RAD51 migration to damaged DNA ends [125]. A clinical trial to test this inhibitor has already started, (NCT03997968) (Table 2). Anyhow, even if the other inhibitors are not in clinical trials, they proved to have great potential in pre-clinical studies.

Since BRCA1 and BRCA2 are large proteins that interact with several partners with their functional domains and act as hubs in DNA damage signaling/repair, it is difficult to design specific compounds to inhibit their activities [136]. Some BRCA1/BRCA2 inhibitors acting on the BRCT domain, which is essential for the interaction with DNA repair partners, such as RAD51, determine an increase of cell sensitivity to drugs such as PARP inhibitors [137,138]. Moreover, small molecules acting on the RING domain of BRCA1 have been developed and this could provide novel ways to inhibit HR [24].


**Table 2.** HR repair proteins targeted in cancer therapy.

For each target it is reported: name of the drug currently in analysis; combination of target inhibition and classic anti-cancer therapy under study; possible synthetic lethality interaction; ongoing clinical trials with the drug; references.

RAD52 is another therapeutic target in breast and ovarian cancer because its depletion is synthetically lethal to cells BRCA2 or BRCA1/PALB2 deficient [139,140]. Different groups have developed several RAD52 inhibitors (F79, 6-OH-dopa, A5MP, AICAR/ZMP, D-I03, NP-004255, F779-0434) that act either by inhibiting oligomerization or blocking ssDNA binding activities of the protein, similar to some RAD51 inhibitors [141] (Table 1). Recently, to potentiate the effect of RAD52 drugs and reduce resistance, a new strategy named "dual synthetic lethality" has been proposed with the idea to combine them with PARP inhibitors, in BRCA1-deficient cancer cells [142].

Regarding NHEJ, several chemical compounds have been developed for cancer therapy, but they are all in preclinical studies. These studies are analyzing how their inhibition could influence cancer development and drugs resistance (Table 3). It has been reported that Ku70/80 inhibition sensitizes cells to radiation treatment, XLF inhibition contributes to overcome chemoresistance in colorectal cancer cells, and LIG IV silencing results in increased cellular sensitivity to chemotherapeutic alkylating agents [143–145].

Some studies are developing small ligands able to inhibit TP53BP1 by binding to its interacting domain and competing with regular substrates, making it unable to coordinate downstream effectors [146,147], moreover a synthetic lethal interaction between TP53BP1 loss and treatment with ATR inhibitors and cisplatin has been demonstrated [148] (Table 3).

MMEJ pathway is still far from being fully characterized and several groups are working to identify drugs able to inhibit Pol-θ activity and to exploit this pathway in cancer therapy [149]. Recently, it has been discovered that Pol-θ inhibition sensitizes cells to replication stress caused by agents such as topoisomerases poisons or ATR inhibitors, suggesting novel cancer treatments [150] (Table 3). In addition, thanks to a CRISPR genetic screen, 140 genes whose deletion is synthetically lethal with *POLQ* mutations have been discovered. Among them, several HR and NHEJ-involved genes and components of the TP53BP1 pathway have been identified. Moreover, in the same study, it has been discovered that 30% of breast cancer cases deposited in TCGA (the Cancer Genome Atlas) have alteration in at least one of these 140 genes; the proteins encoded by these genes could be potential targets to develop new strategies for cancer patients who have Pol-θ alteration [151]. Moreover, knockdown of Pol-θ in ovarian cancer cell lines deficient for FA factor FANCD2 enhances cell death [152]. Finally, the antibiotic novobicin is now under study as specific Pol-θ inhibitor, and preliminary results showed that this drug is able to kill HR-deficient tumor cells due to accumulation of toxic RAD51 foci [153] (Table 3).


For each target it is reported: name of the drugs currently in analysis; combination of target inhibition and classic anti-cancer therapy under study; possible synthetic lethality interaction; ongoing clinical trials with the drug; references.

#### **9. Targeting DNA Helicases Increases Sensitivity to Several Treatments**

BLM is one of the helicases that have an essential role in HR pathway, promoting DNA end resection, RAD51 filament and D-loop formation [154]. The inhibitor ML216 targets this helicase (Table 4), by competing for ATP binding and for BLM binding to DNA; anyhow, this compound showed poor aqueous solubility and cell permeability, therefore it will require further optimization [155].

DNA2 helicase allows cancer cells to resist DNA replication stresses induced by chemotherapy or radiotherapy, by promoting flap removal during DNA replication, DNA resection during repair, and stabilization and restart of stalled replication forks [156]. C5, a potent DNA2 inhibitor, has been

identified by a high throughput screening; this inhibitor sensitizes cells to camptothecin and PARP inhibitors, making it interesting for cancer therapy [157] (Table 4).

*WRN* downregulation leads to mitotic catastrophe and cancer cell death [158]. Interestingly, evidences show that treatment with a WRN inhibitor (NSC 19630) induces apoptosis in leukemia cell lines [159]. NSC617145 is an improved derivative of the previous drug, which likely traps WRN on the DNA substrate, leading to sensitization of cancer cells to DNA-damaging agents. This drug sensitizes cells deficient of the FA and NHEJ pathways to mitomycin C, suggesting the possibility of a synthetic lethality approach [160] (Table 4). Recently, new small molecule WRN inhibitors, identified by a high throughput screen, have been shown to reduce proliferation of cancer cells [161].

These data suggest that DNA helicases are potential targets for cancer therapy to be exploited as novel synthetic lethal interactions. Several studies are now ongoing to identify new drugs that could provide alternative and useful strategies to sensitize several cancers to DNA damage agents [155,162] (Table 4).


**Table 4.** Helicases and nucleases targeted in cancer therapy.

For each target it is reported: name of the drugs currently in analysis; combination of target inhibition and classic anti-cancer therapy under study; possible synthetic lethality interaction; references.
