The year of 2005 was a watershed in the history of poly(ADP-ribose) polymerase (PARP) inhibitors due to the important findings of selective killing in BRCA-deficient cancers by PARP inhibition. The findings made PARP inhibition one of the most promising new therapeutic approaches to cancers, especially to those with specific defects. With AZD2281 and BSI-201 entering phase III clinical trials, the final application of PARP inhibitors in clinic would come true soon. This current paper will review the major advances in targeting PARP for cancer therapy and discuss the existing questions, the answers to which may influence the future of PARP inhibitors as cancer therapeutics.

Marine sponges have been recognized as a rich source of potential anti-proliferative metabolites. Currently, there are two sponge-derived anti-cancer agents (a macrolide and a nucleoside) isolated from the Porifera phylum, suggesting the great potential of this sponge as a rich source for anti-neoplastic agents. To search for more bioactive metabolites from this phylum, we examined the EtOAc extract of Theonella sp. sponge. We isolated seven compounds (1-7), including four 4-methylene sterols (1-4), two nucleosides (5 and 6), and one macrolide (7). Among them, theonellasterol L (1) was identified for the first time, while 5'-O-acetyl-2'-deoxyuridine (5) and 5'-O-acetylthymidine (6) were the first identified deoxyuridine and thymidine derivatives from the sponge Theonella sp. These structures were elucidated based on their spectroscopic data. The anti-proliferation activity of compounds 1-7 against the MCF-7, MDA-MB-231, T-47D, HCT-116, DLD-1, K562, and Molt 4 cancer cell lines was determined. The results indicated that the 14-/15-oxygenated moiety played an important role in the antiproliferative activity and the macrolide derivatives dominated the anti-proliferative effect of the sponge Theonella sp. The in silico analysis, using a chemical global positioning system for natural products (ChemGPS-NP), indicated an anti-proliferative mode of actions (MOA) suggesting the potential applications of the isolated active metabolites as anti-proliferative agents.

Conventional meta-analyses and randomized controlled trials have shown inconsistent results regarding the efficacy of immunosuppressants for pediatric steroid-resistant nephrotic syndrome (SRNS).

TRC102 inhibits base excision repair by binding abasic sites and preventing AP endonuclease processing; it potentiates the activity of alkylating agents, including temozolomide, in murine models. In published xenograft studies, TRC102 enhanced the antitumor effect of temozolomide regardless of cell line genetic characteristics, e.g., O6-methylguanine DNA methyltransferase (MGMT), mismatch repair (MMR), or p53 status.

Previous studies indicate that overexpression of N-methylpurine DNA glycosylase (MPG) dramatically sensitizes cells to alkylating agent-induced cytotoxicity. We recently demonstrated that this sensitivity is preceded by an increased production of AP sites and strand breaks, confirming that overexpression of MPG disrupts normal base excision repair and causes cell death through overproduction of toxic repair intermediates. Here we establish through site-directed mutagenesis that MPG-induced sensitivity to alkylation is dependent on enzyme glycosylase activity. However, in contrast to the sensitivity seen to heterogeneous alkylating agents, MPG overexpression generates no cellular sensitivity to MeOSO2(CH2)2-lexitropsin, an alkylator which exclusively induces 3-meA lesions. Indeed, MPG overexpression has been shown to increase the toxicity of alkylating agents that produce 7-meG adducts, and here we demonstrate that MPG-overexpressing cells have dramatically increased removal of 7-meG from their DNA. These data suggest that the mechanism of MPG-induced cytotoxicity involves the conversion of non-toxic 7-meG lesions into highly toxic repair intermediates. This study establishes a mechanism by which a benign DNA modification can be made toxic through the overexpression of an otherwise well-tolerated gene product, and the application of this principle could lead to improved chemotherapeutic strategies that reduce the peripheral toxicity of alkylating agents.

Alkylating drugs are among the most often used chemotherapeutics. While cancer cells frequently develop resistance to alkylation treatments, detailed understanding of mechanisms that lead to the resistance is limited. Here, by using genome-wide CRISPR-Cas9 based screen, we identify transcriptional Mediator complex subunit 13 (MED13) as a novel modulator of alkylation response. The alkylation exposure causes significant MED13 downregulation, while complete loss of MED13 results in reduced apoptosis and resistance to alkylating agents. Transcriptome analysis identified cyclin D1 (CCND1) as one of the highly overexpressed genes in MED13 knock-out (KO) cells, characterized by shorter G1 phase. MED13 is able to bind to CCND1 regulatory elements thus influencing the expression. The resistance of MED13 KO cells is directly dependent on the cyclin D1 overexpression, and its down-regulation is sufficient to re-sensitize the cells to alkylating agents. We further demonstrate the therapeutic potential of MED13-mediated response, by applying combinatory treatment with CDK8/19 inhibitor Senexin A. Importantly, the treatment with Senexin A stabilizes MED13, and in combination with alkylating agents significantly reduces viability of cancer cells. In summary, our findings identify novel alkylation stress response mechanism dependent on MED13 and cyclin D1 that can serve as basis for development of innovative therapeutic strategies.

Considering that HTB140 melanoma cells have shown a poor response to either protons or alkylating agents, the effects of a combined use of these agents have been analysed.

Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada) response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i) the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii) the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB), characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB), PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems against alkylating agents of exo- and endogenous origin.

Treatment of glioblastoma (GB), the most common malignant primary brain tumor in adults, can include alkylating chemo-therapeutic agents. Two molecular biomarkers of treatment response are MGMT (O6-methylguanine-DNA methyltransferase) promoter methylation and IDH (isocitrate dehydrogenase) mutations, which prevent repair of tumor cell DNA damage caused by alkylating chemotherapy. The status of MGMT promoter methylation and IDH mutation are associated with longer survival and a better response to chemotherapy.

O-6-methylguanine-DNA methyltransferase (MGMT) is an abundantly expressed nuclear protein dealkylating O6-methylguanine (O6-MG) DNA residue, thus correcting the mismatches of O6-MG with a thymine residue during DNA replication. The dealkylating effect of MGMT is relevant not only in repairing DNA mismatches produced by environmental alkylating agents promoting tumor pathogenesis, but also when alkylating molecules are applied in the chemotherapy of different cancers, including glioma, the most common primary tumor of the central nervous system. Elevated MGMT gene expression is known to confer resistance to the treatment with the alkylating drug temozolomide in patients affected by gliomas and, on the contrary, methylation of MGMT gene promoter, which causes reduction of MGMT protein expression, is known to predict a favourable response to temozolomide. Thus, detecting expression levels of MGMT gene is crucial to indicate the option of alkylating agents or to select patients directly for a second line targeted therapy. Further study is required to gain insights into MGMT expression regulation, that has attracted growing interest recently in MGMT promoter methylation, histone acetylation and microRNAs expression. The review will focus on the epigenetic regulation of MGMT gene, with translational applications to the identification of biomarkers predicting response to therapy and prognosis.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA damage sensor and contributes to both DNA repair and cell death processes. However, how PARP-1 signaling is regulated to switch its function from DNA repair to cell death remains largely unknown. Here, we found that PARP-1 plays a central role in alkylating agent-induced PARthanatic cancer cell death. Lysine demethylase 6B (KDM6B) was identified as a key regulator of PARthanatos. Loss of KDM6B protein or its demethylase activity conferred cancer cell resistance to PARthanatic cell death in response to alkylating agents. Mechanistically, KDM6B knockout suppressed methylation at the promoter of O6-methylguanine-DNA methyltransferase (MGMT) to enhance MGMT expression and its direct DNA repair function, thereby inhibiting DNA damage-evoked PARP-1 hyperactivation and subsequent cell death. Moreover, KDM6B knockout triggered sustained Chk1 phosphorylation and activated a second XRCC1-dependent repair machinery to fix DNA damage evading from MGMT repair. Inhibition of MGMT or checkpoint response re-sensitized KDM6B deficient cells to PARthanatos induced by alkylating agents. These findings provide new molecular insights into epigenetic regulation of PARP-1 signaling mediating DNA repair or cell death and identify KDM6B as a biomarker for prediction of cancer cell vulnerability to alkylating agent treatment.

Ifosfamide (IFO) is widely used DNA-alkylating agents in cancer chemotherapy for management of solid tumors and hematological malignancies. However, hemorrhagic cystitis limits the use of IFO.

Breast cancer chemotherapy with high dose alkylating agents is severely limited by their collateral toxicity to crucial normal tissues such as immune and gut cells. Taking advantage of the selective dependence of cancer cells on high glucose and combining glucose deprivation with these agents could produce therapeutic synergy.

Glioblastomas (GBMs) are highly aggressive, invasive brain tumors with bad prognosis and unmet medical need. These tumors are heterogeneous being constituted by a variety of cells in different states of differentiation. Among these, cells endowed with stem properties, tumor initiating/propagating properties and particularly resistant to chemo- and radiotherapies are designed as the real culprits for tumor maintenance and relapse after treatment. These cells, termed cancer stem-like cells, have been designed as prominent targets for new and more efficient cancer therapies. G-protein coupled receptors (GPCRs), a family of membrane receptors, play a prominent role in cell signaling, cell communication and crosstalk with the microenvironment. Their role in cancer has been highlighted but remains largely unexplored. Here, we report a descriptive study of the differential expression of the endo-GPCR repertoire in human glioblastoma cancer stem-like cells (GSCs), U-87 MG cells, human astrocytes and fetal neural stem cells (f-NSCs). The endo-GPCR transcriptome has been studied using Taqman Low Density Arrays. Of the 356 GPCRs investigated, 138 were retained for comparative studies between the different cell types. At the transcriptomic level, eight GPCRs were specifically expressed/overexpressed in GSCs. Seventeen GPCRs appeared specifically expressed in cells with stem properties (GSCs and f-NSCs). Results of GPCR expression at the protein level using mass spectrometry and proteomic analysis are also presented. The comparative GPCR expression study presented here gives clues for new pathways specifically used by GSCs and unveils novel potential therapeutic targets.

Breast cancer (BC) is a heterogeneous disease for which the commonly used chemotherapeutic agents primarily include the anthracyclines (doxorubicin, epirubicin), microtubule inhibitors (paclitaxel, docetaxel, eribulin), and alkylating agents (cyclophosphamide). While these drugs can be highly effective, metastatic tumours are frequently refractory to treatment or become resistant upon tumour relapse.

DNA alkylguanine DNA alkyltransferases (AGTs) are evolutionary conserved proteins that repair alkylation damage in DNA, counteracting the effects of agents inducing such lesions. Over the last years AGTs have raised considerable interest for both the peculiarity of their molecular mechanism and their relevance in cancer biology. AGT knock out mice show increased tumour incidence in response to alkylating agents, and over-expression of the human AGT protein in cancer cells is frequently associated with resistance to alkylating chemotherapy. While all data available point to a function of AGT proteins in the cell response to alkylation lesions, we report for the first time that one of the two AGT paralogs of the model organism C. elegans, called AGT-2, also plays unexpected roles in meiosis and early development under physiological conditions. Our data suggest a role for AGT-2 in conversion of homologous recombination intermediates into post-strand exchange products in meiosis, and show that agt-2 gene down-regulation, or treatment of animals with an AGT inhibitor results in increased number of germ cells that are incompatible with producing viable offspring and are eliminated by apoptosis. These results suggest possible functions for AGTs in cell processes distinct from repair of alkylating damage.

Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, and this class of drugs has been studied as protective agents against DNA damages. Alkylating agents (AAs) are able to induce alkylation in macromolecules, causing DNA damage, as DNA methylation. Our objective was to evaluate atorvastatin (AVA) antimutagenic, cytoprotective, and antigenotoxic potentials against DNA lesions caused by AA. AVA chemopreventive ability was evaluated using antimutagenicity assays (Salmonella/microsome assay), cytotoxicity, cell cycle, and genotoxicity assays in HepG2 cells. The cells were cotreated with AVA and the AA methyl methanesulfonate (MMS) or cyclophosphamide (CPA). Our datum showed that AVA reduces the alkylation-mediated DNA damage in different in vitro experimental models. Cytoprotection of AVA at low doses (0.1-1.0 μM) was observed after 24 h of cotreatment with MMS or CPA at their LC50, causing an increase in HepG2 survival rates. After all, AVA at 10 μM and 25 μM had decreased effect in micronucleus formation in HepG2 cells and restored cell cycle alterations induced by MMS and CPA. This study supports the hypothesis that statins can be chemopreventive agents, acting as antimutagenic, antigenotoxic, and cytoprotective components, specifically against alkylating agents of DNA.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) causes resistance of tumor cells to alkylating agents. It is a predictive biomarker in high-grade gliomas treated with temozolomide, however, there is no consensus on which test method, methylation sites, and cutoff values to use.

Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O(6)-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-N'-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gammaH2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks.

O6-methylguanine-DNA methyltransferase (MGMT) plays a crucial role in repairing damaged DNA caused by alkylating agents. A number of cancer susceptibility loci have been recognized as enhancer variants. This study aimed to explore the significance of enhancer variants of MGMT in glioma susceptibility.