PARP cells with intact homologous recombination (HR), DSBs

PARP Inhibitors

                PARP1 (poly(ADP-ribose) polymerase 1) is an abundant nuclear protein that catalyse the polymerization of ADP-ribose units resulting in the attachment of PAR polymers to itself or other target proteins. Its poly(ADP)ribosylation (PARylation) activity, which is one of the earliest steps of DNA damage recognition, is essential for initiating various forms of DNA repair and it becomes activated upon binding to DNA single-strand and double-strands breaks (SSBs and DSBs respectively), DNA crosslinks and stalled replication forks (Hui Ling Ko et al.). PARP1 is involved in the early recruitment of factors to facilitate DSB repair and its inhibition results in delayed activation of DDR proteins such as H2AX, p53  or SMC1(10.1038/nrm.2017.53). The critical role of PARP1 in DNA repair is reflected by its frequent upregulation in cancer, so blocking its activity with small molecules, can achieve synthetic lethality in combination with DNA damaging agents (Helgel et al.). In cells with intact homologous recombination (HR), DSBs that occur as a result of PARP inhibition can be resolved, but in tumour cells lacking homologous recombination, PARP inhibition leads to persistent double-strand breaks, inducing cell death (Farmer et al.). BRCA1 and BRCA2 are tumour suppressor genes encoding proteins that play important roles in the HR repair pathway. BRCA1- and BRCA2- deficient cells exhibit high sensitivity to PARP1 inhibitors drugs producing DNA lesions that would be normally repaired by HR and those high levels of DNA damage cause cell-cycle arrest and cell death. Partly, it is due to PARP inhibitor treatment was found to stimulate NHEJ in the HR-deficient cells, leading to genomic instability and cell death (10.1038/nature03445; 10.1155/2016/2346585). This synthetic lethality conferred by PARP inhibitors, especially in breast and ovarian cancers  has been tested in clinical trials since 2003. Currently, there are 3 FDA approved PARP inhibitors, olaparib, rucaparib and niraparib, and at least 8 PARP inhibitors under clinical trials, being proposed as a monotherapy or in combination with other drugs (see Table 1). The approved PARPi are indicated for use in adult patients with germline and/or somatic BRCA-mutated ovarian cancer and recently Olaparib has been approved for treatment of metastatic BRCA-mutated breast cancer. However there are PARPi in phase III trials for use in other carcinomas.  Those PARP inhibitors have a common mechanism of action by trapping PARP1 at the site of DNA damage, preventing autoPARylation and release of PARP1 (10.1126/science.aam7344), however they have different PARP trapping potency, for example, talazoparib being the most potent PARP1 trapping inhibitor and veliparib the least potent (10.1158/1535-7163.MCT-13-0803; 10.1126/scitranslmed.aaf9246). All of them bind within the nicotinamide-binding pocket in the ADP-ribosyl transferase catalytic site, making contact with the regulatory subdomains (10.1016/j.chembiol.2017.08.027; 10.1126/scitranslmed.aaf9246).

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                Different mechanisms of resistance to  PARPi have been identified, especially in ovarian cancer patients with BRCA-deficient tumors, many of them develop PARP inhibitor resistance within the first year (10.18632/oncotarget.14409). It has been shown that BRCA deficient tumours undergo PARPi resistance by deletion of a mutation in BRCA1 or BRCA2, restoring DNA repair by homologous recombination (10.1038/nature06548); another mechanism of resistance include inactivation of 53BP1, whose activity prevent end resection, crucial step for the initiation of HR (10.1126/science.aam7344), or the function of BRCA hypomorphs, where the residual activity of mutant proteins could make up for the absence of the wild-type protein (10.1016/j.chembiol.2017.08.027). On the other hand, overexpression of RAD51, key protein to DSB repair by HR, has been linked with therapy resistance to PARP inhibitors in triple-negative breast cancer cells, so by targeting RAD51, it may be possible to potentiate the sensitivity of cancer cells to PARPi (10.1158/1078-0432.CCR-15-1348).

ATM and ATR Inhibitors

                As PARP1 is involved in the recruitment of transducer proteins in DDR, its substrates like ATM contain PAR-binding domains and its interaction with PAR stimulate its activity (10.1074/jbc.M608406200). ATM (Ataxia-telangiectasia mutated) and ATR (Ataxia and Rad3-related)  belong to the PIKK family (phosphatidylinositol 3-kinase-related kinase) of serine/threonine protein kinases, which also comprises DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Both ATM and ATR are activated after DNA damage and they are the master transducers of DNA signals, which propagate upon DNA damage signalling cascades through phosphorylation of numerous targets, such as H2AX, p53 and the checkpoint kinases Chk1 and Chk2; although ATM and ATR downstream effects and functions are distinct and not redundant (10.1016/B978-0-12-380888-2.00003-0). Whereas ATM is primarily activated by DSBs, ATR responds to a wide spectrum of DNA damage, including SSBs. Activation of these kinases is essential  for the proper coordination of cell cycle checkpoints and DNA repair processes, nevertheless they can modulate other biological outcomes such as apoptosis or senescence. It has been shown that inhibition of ATM and ATR functions, sensitises cancer cell  to the lethal effects of genotoxic agents, so they became therapeutically interesting as possible chemotherapeutic targets. The high level of sequence similarity between both kinases, has constituted a difficult challenge for the development of specific ATM and ATR inhibitors. ATM inhibition represents an exciting clinical opportunity as a target to hyper-sensitive tumours to chemo/radiotherapy and most of the ATM inhibitors which show specificity for ATM share the same mechanism of action by binding to the ATP binding pocket of ATM, blocking its kinase function and ATM-mediated signaling (see Table 1). One of the most ATM inhibitors used in research laboratories is KU-55933, which was the first potent and selective ATM inhibitor. KU-55933 is a cell-permeable, potent, selective and ATP-competitive inhibitor of ATM and exposure of cells to this inhibitor induce a significant sensitization to the cytotoxic effects of ionizing radiation and to the DNA DSBs-inducing chemotherapeutic agents (10.1158/0008-5472.CAN-04-2727). However, lipophilicity issues complicate its clinical potential, so it is not enrolled in clinical trials. In 2009, an improved analogue of KU-55933, called KU-60019, was designed with more efficiency at blocking radiation-induced phosphorylation of key ATM downstream targets, in particular in human glioma cells (10.3390/biom5043204) and like KU-55933, is an ATP-competitive ATM inhibitor. It has also been demonstrated that besides inhibiting DDR, it also reduces prosurvival signaling, migration and invasion of tumour cells and radiosensitizes human glioma cells (10.1158/1535-7163.MCT-09-0519). Moreover, it was demonstrated that KU-60019 suppress the proliferation of MCF-7 cells and increase chemosensitization in combination with doxorubicin, therefore, it could be a promising strategy for non-invasive breast cancer (10.3892/ol.2014.2444). Even though KU-60019 showed better solubility than KU-55933, bio-availability is still poor (10.1016/j.pharmthera.2014.12.001). In 2016, Degorce et al. published a new developed ATM inhibitor, a novel 3-quinole carboxamides, known as AZD-0156 (10.1021/acs.jmedchem.6b00519), a first in class orally available ATM inhibitor, which is the only ATM inhibitor enrolled in clinical trials, specifically in phase I trial in combination with Olaparib in patients with locally advanced/metastatic cancer (see Table 1). AZD-0156 is a permeable, highly soluble compound showing a robust efficacy in mouse xenograft models after oral administration with certain DSB inducing agents (http://dx.doi.org/10.1016/S0959-8049(16)61417-X) and it has been proved that in mice models represent potential therapeutic strategies for the treatment of acute myeloid leukemia (10.1126/scisignal.aad8243).

                In 2009 Nishida et al. reported the first ATR-selective small-molecule inhibitor, Schisandrin B which was able to abrogate UV-induced intra-S-phase and G2/M cell cycle checkpoints and increase the cytotoxicity of UV radiation in human lung cancer cells but high drug concentration of this drug was needed for its inhibitory potency (10.1093/nar/gkp593). A more potent ATR inhibitor, VE-822, was developed in 2011  by Fokas et al. that  in vitro and in vivo radiosensitized pancreatic cancer cells (10.1038/cddis.2012.181). Other study reported that in vivo VE-822 blocked ATR activity in tumours and dramatically enhance the efficacy of cisplatin across a panel of patients derived primary lung xenograft (10.18632/oncotarget.2158). VE-822 was the first ATR inhibitor to enter clinical development and currently is included in phase I/II trial in combination with Topotecan treating small cell lung cancer and in combination with other chemotherapeutics in patients with advanced refractory solid tumours (see Table 1).  AZD-6739 is another ATM inhibitor in clinical trials, in particular phase I/II trial in combination with Acalabrutinib in subjects with relapse or refractory high-risk chronic lymphocytic leukemia. As a analogue of A720, AZD-6738 possesses significantly improved solubility, bioavailability and pharmacokinetics properties (10.1158/1538-7445.AM2013-2348). A third and recently inhibitor undergoing clinical trials is BAY-1895344,  a potent, orally available and selective ATR inhibitor, that in vitro exhibits strong anti-tumour efficacy in monotherapy in a variety of xenograft models of different indications that are characterized by DDR deficiencies, inducing stable disease in ovarian and colorectal cancer or even complete tumor remission in cell lymphoma models (10.1158/1538-7445.AM2017-836). Currently, BAY-1895344 is in phase I trial as a monotherapy treatment in advanced solid tumours and lymphomas.

DNA Helicase inhibitors

                DNA helicases have a key role in genomic stability, helping cells to deal with endogenous or exogenous stress to  prevent chromosomal instability and to maintain cellular homeostasis. Many DNA helicases have their gene expression upregulated in several tumour tissues which is required for cancer cell proliferation or enhance resistance to  DNA damage chemotherapeutic agents. On the other hand, chromosomal instability caused by downregulation of DNA helicases promotes carcinogenesis. The RecQ DNA helicases, a family conserved from bacteria to humans is made up of five helicases  RecQL1, BLM, WRN and RecQL4 and RecQL5 and all of them are required for genomic stability (10.1146/annurev-genet-102209-163602). It has been described genetic diseases that predispose to cancer in three of these RecQ DNA helicases: BLM in Bloom syndrome (PubMed: 7585968), WRN in Werner syndrome (PubMed: 8602509) and RecQL4 in Rothmund-Thomson syndrome (10.1038/8788). Although there is not genetic disorder related to RecQL1 or RecQL5 mutations, these RecQ DNA helicases have important roles in cancer, since RecQL1 is highly expressed in various cancers and its depletion reduces tumour cell proliferation (https://doi.org/10.3892/ijmm_00000375; 10.1111/j.1349-7006.2007.00647.x; 10.1186/1476-4598-10-83) and RecQL5 is also necessary for cancer cell proliferation (10.1093/nar/gkr844) and RecQL5 knockout mice display increased cancer rates (10.1101/gad.1609107). RecQL1 is a DNA repair protein whose activity is regulated by PARP1, and play a key role in the recovery of DNA replication forks, which are slowed down and reversed into chicken-foot structures as result of replication stress induce by topoisomerase I inhibitors. This aspect is crucial to avoid redundant DNA repair mechanisms, by combining different essential inhibitors.

                WRN (Werner syndrome protein) is an example of RecQ DNA helicase and nuclease whose inhibition by small molecules has been targeted, as WRN plays a prominent role in replication fork progression after DNA damage or fork arrest that might allow rapidly dividing tumour cells to deal with replicative lesions; nevertheless to date, there is not any WRN inhibitor in clinical trials. Chemical inhibition of WRN helicase activity in human cells defective in the Fanconi anemia (FA) DNA repair pathway are 

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