Intrastrand DNA Cross-links Research Articles

In Fanconi anemia, impaired accumulation of bone marrow neutrophils during emergency granulopoiesis induces hematopoietic stem cell stress

Fanconi Anemia (FA) is an inherited disorder of DNA-repair due to mutation in one of 20+ interrelated genes that repair intra-strand DNA crosslinks and rescue collapsed or stalled replication forks. The most common hematologic abnormality in FA is anemia, but progression to bone marrow failure (BMF), clonal hematopoiesis, or acute myeloid leukemia (AML) may also occur. In prior studies, we found that Fanconi DNA-repair is required for successful emergency granulopoiesis; the process for rapid neutrophil production during the innate immune response. Specifically, Fancc-/- mice did not develop neutrophilia in response to emergency granulopoiesis stimuli, but instead exhibited apoptosis of bone marrow hematopoietic stem cells (HSCs) and differentiating neutrophils. Repeated emergency granulopoiesis challenges induced BMF in most Fancc-/- mice, with AML in survivors. In contrast, we found equivalent neutrophilia during emergency granulopoiesis in Fancc-/-Tp53+/- mice and wild type (WT) mice, without BMF. Since termination of emergency granulopoiesis is triggered by accumulation of bone marrow neutrophils, we hypothesize neutrophilia protects Fancc-/-Tp53+/- bone marrow from the stress of a sustained inflammation that is experienced by Fancc-/- mice. In the current work, we found that blocking neutrophil accumulation during emergency granulopoiesis led to BMF in Fancc-/-Tp53+/- mice, consistent with this hypothesis. Blocking neutrophilia during emergency granulopoiesis in Fancc-/-Tp53+/- mice (but not WT) impaired cell cycle checkpoint activity, also found in Fancc-/- mice. Mechanisms for loss of cell cycle checkpoints during infections challenges may define molecular markers of FA progression, or suggest therapeutic targets for bone marrow protection in this disorder.

HEBP2 affects sensitivity to cisplatin and BCNU but not to paclitaxel in MDA-MB-231 breast cancer cells.

Breast cancer has the highest incidence of all cancer types in women. Triple-negative breast cancer (TNBC) accounts for 15% of all breast cancer cases and is the most aggressive type, with a poor prognosis and limited treatment. Treatment failure in patients is largely due to resistance to chemotherapy. In this study, we aimed to identify the novel factors contributing to chemoresistance in TNBC using cisplatin and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). We found that transactivation of the heme-binding protein 2 (HEBP2) gene was common in surviving colonies of cells after exposure to two types of chemotherapeutic agents, namely cisplatin and BCNU, from genome-scale transcriptional activation library screening in the TNBC cell line MDA-MB-231. Analysis of a public database (Proteogenomic Landscape of Breast Cancer, CPTAC) indicated that HEBP2 mRNA expression was elevated in TNBC tissues compared to that in non-TNBC tissues. HEBP2 facilitates necrotic cell death under oxidative stress; however, it is not yet known whether HEBP2 affects cancer cell survival following chemotherapy. Therefore, we investigated the effects of HEBP2 expression on the sensitivity to cisplatin and BCNU in MDA-MB-231 cells. Overexpression of HEBP2 significantly enhanced the viability of MDA-MB-231 cells in response to cisplatin and BCNU, but not methyl methanesulfonate (MMS) and paclitaxel. In contrast, CRISPR/Cas9-mediated HEBP2-knockout greatly reduced cell viability in response to cisplatin and BCNU, but not to MMS and paclitaxel, in MDA-MB-231 cells. Moreover, the exogenous introduction of HEBP2 restored the resistance of HEBP2-deficient cells to cisplatin and BCNU to wild-type levels. These findings suggest that HEBP2 may play a significant role in resistance to cisplatin and BCNU, which induce intrastrand and interstrand DNA crosslinks, but not to monoalkylating or microtubule-stabilizing agents in TNBC cells. The possibility exists that HEBP2 serves as a biomarker for predicting response or a therapeutic target for overcoming resistance to platinum-based and alkylating anticancer agents in TNBC.

Abstract 7113: Targeting the repair of DNA-protein crosslinks induced by bifunctional alkylating agents

Abstract Bifunctional alkylating agents, such as nitrosoureas, platinum-based therapeutics, and chloroethylhydrazines, have demonstrated clinical successes against cancer by inducing cytotoxic interstrand or intrastrand DNA crosslinks. However, the less well-characterized capacity of these agents to couple DNA to chromosomally-associated proteins may also contribute to these compounds’ observed cytotoxicities. Bulky DNA-protein crosslinks (DPCs) could obstruct the DNA metabolism critical to cancer proliferation, thus contribute to genomic instability and cell death. To endogenously repair DPCs, human cells often utilize a DPC-specific proteolytic mechanism dependent on the metalloprotease SPRTN. Complementary approaches for quantifying DPCs, generated ex vivo, to assess the capacity of several bifunctional alkylating agents to induce these lesions in cultured human leukemia cells are presented here. In the first approach, proteins were isolated from lysates of drug-treated cells and protein-bound DNA was quantified as a ratio to the total DNA present in the lysates. In the complementary approach, DNA was extracted from lysates of drug-treated cells using a modified ‘STAR’ assay in which genomic DNA is isolated and the DNA-bound proteins were quantified by fluorescein isothiocyanate labeling. Micromolar concentrations of the nitrogen mustard mechlorethamine and nitrosourea lomustine induced significant DPC formation while more modest DPC formation was observed using similar concentrations of the chloroethylating agents carmustine and laromustine, as well as cisplatin. Bifunctional alkylating agents dependent on epoxide functionality, including dianhydrogalacticol and diepoxybutane, required drug concentrations of at least one order of magnitude larger to produce similar DPC yields measurable by these techniques. A strategy to interfere with the DPC repair pathway via SPRTN knockdown and assess increased sensitivity of cancer cells to bifunctional alkylating agents is also presented. Chemotherapeutic sensitization by targeting SPRTN could suggest a therapeutic strategy to improve effectiveness of several DNA-targeted anticancer agents, especially if extended to other therapeutics that trap DPCs such as inhibitors of topoisomerase and poly(ADP-ribose) polymerase. Further, identifying structure-function relationships with respect to bifunctional alkylating agents and DPC formation could inform the future development and clinical utilization of these chemotherapies. Citation Format: Cole Turner, Hailey Cerrato, Victoria Melehov, Charlotte Healy, Kevin P. Rice. Targeting the repair of DNA-protein crosslinks induced by bifunctional alkylating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7113.

NCMP-26. PANCEREBELLITIS ASSOCIATED WITH CISPLATIN THERAPY IN A PATIENT WITH OROPHARYNGEAL/ NASOPHARYNGEAL SQUAMOUS CELL CARCINOMA

Abstract Cisplatin, a platinum-based drug, is one of the oldest and most widely used chemotherapeutic agents. It exerts its anti-tumor effect by forming DNA inter-strand and intra-strand cross-links, resulting in lesions that eventually activate apoptotic pathways. Cisplatin remains widely used for the treatment of several solid tumors, though this is often limited by its toxicity which can affect many organ systems, including nephrotoxicity, hepatotoxicity, ototoxicity, and neurotoxicity. A dose-dependent peripheral sensory neuropathy is the most reported cisplatin-induced neurotoxicity. Central nervous system (CNS) toxicity is less common, likely due to the large molecular size of cisplatin preventing its passage through the blood-brain barrier (BBB). There have been reports of cognitive impairment, posterior reversible encephalopathy syndrome, and optic neuritis associated with cisplatin. We present a case of a 38-year-old patient who was treated with concurrent cisplatin and radiation therapy for oropharyngeal/nasopharyngeal squamous cell carcinoma, and around a month after the end of therapy, was found to have imaging findings concerning for pancerebellitis. Extensive workup into infectious causes of cerebellitis was negative. Although there are no reports in the literature of cerebellitis due to cisplatin, it could be the culprit in this case. Animal studies have shown cerebellar dysfunction occurring in the setting of cisplatin exposure. Moreover, it is thought that the drug can reach the CNS if there is a disruption of the BBB, this is supported by the fact that CNS toxicity is more seen in patients who have primary CNS neoplasia, CNS metastatic disease, radiation exposure to the head, among other mechanisms that could potentially disrupt the BBB. Thus, it is important to consider CNS adverse effects, including cerebellitis, in patients receiving cisplatin, especially among those with a potentially compromised BBB.

FTO regulates the DNA damage response via effects on cell-cycle progression.

The fat mass and obesity-associated protein FTO is an "eraser" of N6-methyladenosine, the most abundant mRNA modification. FTO plays important roles in tumorigenesis. However, its activities have not been fully elucidated and its possible involvement in DNA damage - the early driving event in tumorigenesis - remains poorly characterized. Here, we have investigated the role of FTO in the DNA damage response (DDR) and its underlying mechanisms. We demonstrate that FTO responds to various DNA damage stimuli. FTO is overexpressed in mice following exposure to the promutagens aristolochic acid I and benzo[a]pyrene. Knockout of the FTO gene in TK6 cells, via CRISPR/Cas9, increased genotoxicity induced by DNA damage stimuli (micronucleus and TK mutation assays). Cisplatin- and diepoxybutane-induced micronucleus frequencies and methyl methanesulfonate- and azathioprine-induced TK mutant frequencies were also higher in FTO KO cells. We investigated the potential roles of FTO in DDR. RNA sequencing and enrichment analysis revealed that FTO deletion disrupted the p38 MAPK pathway and inhibited the activation of nucleotide excision repair and cell-cycle-related pathways following cisplatin (DNA intrastrand cross-links) treatment. These effects were confirmed by western blotting and qRT-PCR. FTO deletion impaired cell-cycle arrest at the G2/M phase following cisplatin and diepoxybutane treatment (flow cytometry analysis). Our findings demonstrated that FTO is involved in several aspects of DDR, acting, at least in part, by impairing cell cycle progression.

Can Cisplatin Therapy Be Improved? Pathways That Can Be Targeted.

Cisplatin (cis-diamminedichloroplatinum (II)) is the oldest known chemotherapeutic agent. Since the identification of its anti-tumour activity, it earned a remarkable place as a treatment of choice for several cancer types. It remains effective against testicular, bladder, lung, head and neck, ovarian, and other cancers. Cisplatin treatment triggers different cellular responses. However, it exerts its cytotoxic effects by generating inter-strand and intra-strand crosslinks in DNA. Tumour cells often develop tolerance mechanisms by effectively repairing cisplatin-induced DNA lesions or tolerate the damage by adopting translesion DNA synthesis. Cisplatin-associated nephrotoxicity is also a huge challenge for effective therapy. Several preclinical and clinical studies attempted to understand the major limitations associated with cisplatin therapy, and so far, there is no definitive solution. As such, a more comprehensive molecular and genetic profiling of patients is needed to identify those individuals that can benefit from platinum therapy. Additionally, the treatment regimen can be improved by combining cisplatin with certain molecular targeted therapies to achieve a balance between tumour toxicity and tolerance mechanisms. In this review, we discuss the importance of various biological processes that contribute to the resistance of cisplatin and its derivatives. We aim to highlight the processes that can be modulated to suppress cisplatin resistance and provide an insight into the role of uptake transporters in enhancing drug efficacy.

Overcoming Challenges in the Development of Anticancer ADCs

Overcoming Challenges in the Development of Anticancer ADCs

DNA- and DNA-Protein-Crosslink Repair in Plants.

DNA-crosslinks are one of the most severe types of DNA lesions. Crosslinks (CLs) can be subdivided into DNA-intrastrand CLs, DNA-interstrand CLs (ICLs) and DNA-protein crosslinks (DPCs), and arise by various exogenous and endogenous sources. If left unrepaired before the cell enters S-phase, ICLs and DPCs pose a major threat to genomic integrity by blocking replication. In order to prevent the collapse of replication forks and impairment of cell division, complex repair pathways have emerged. In mammals, ICLs are repaired by the so-called Fanconi anemia (FA) pathway, which includes 22 different FANC genes, while in plants only a few of these genes are conserved. In this context, two pathways of ICL repair have been defined, each requiring the interaction of a helicase (FANCJB/RTEL1) and a nuclease (FAN1/MUS81). Moreover, homologous recombination (HR) as well as postreplicative repair factors are also involved. Although DPCs possess a comparable toxic potential to cells, it has only recently been shown that at least three parallel pathways for DPC repair exist in plants, defined by the protease WSS1A, the endonuclease MUS81 and tyrosyl-DNA phosphodiesterase 1 (TDP1). The importance of crosslink repair processes are highlighted by the fact that deficiencies in the respective pathways are associated with diverse hereditary disorders.

Acetaldehyde forms covalent GG intrastrand crosslinks in DNA

Carcinogens often generate mutable DNA lesions that contribute to cancer and aging. However, the chemical structure of tumorigenic DNA lesions formed by acetaldehyde remains unknown, although it has long been considered an environmental mutagen in alcohol, tobacco, and food. Here, we identify an aldehyde-induced DNA lesion, forming an intrastrand crosslink between adjacent guanine bases, but not in single guanine bases or in other combinations of nucleotides. The GG intrastrand crosslink exists in equilibrium in the presence of aldehyde, and therefore it has not been detected or analyzed in the previous investigations. The newly identified GG intrastrand crosslinks might explain the toxicity and mutagenicity of acetaldehyde in DNA metabolism.

Detection of UV-Induced Thymine Dimers.

Ultraviolet rays induce interstrand and intrastrand DNA cross-links, usually thymine-thymine cyclobutane dimer (T-T) and thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct (T (6-4) T). These DNA cross-links, if left unrepaired, increase the risk of these mutation being incorporated in the genetic material (i.e., DNA). Numerous studies have reported the mutagenic potential of above mentioned DNA adducts in prokaryotes, yeast and mammalian cells. Different techniques have been developed to identify such DNA adducts such as immuno-Southern blotting. This is a routinely used quantitative method to determine especially the amount of thymine dimers formed, following irradiation. In this chapter, the detailed methodology to identify thymine dimers formation is provided, using specific antibody against these adducts.

Theoretical studies on the purine radical induced purine-purine type intrastrand cross-links.

At the density functional theory (DFT) level, addition reactions between the guanine-8-yl radical and its 3'/5' neighboring purine deoxynucleosides forming the purine-purine type intrastrand cross-links were studied. It is found that addition of the guanine-8-yl radical to the C8 site of its 5' neighboring deoxyguanosine or deoxyadenosine is a two-step reaction consisting of a structurally relatively unfavourable conformational transformation step, while the corresponding 3' C8 addition is straightforward and kinetically more efficient. The 3' C8 preference of the guanine-8-yl radical additions indicates the existence of an obvious sequence effect, which is completely opposite to that observed in the formation of pyrimidine radicals induced DNA intrastrand cross-links. The detrimental effects from steric hindrance and stabilizing weak interactions make these addition reactions markedly suppressed in double stranded DNA. This work broadens our knowledge about the possible types of DNA intrastrand cross-links.

Abstract A01: Distinct mechanism of action of DNA-damaging agent dianhydrogalactitol (VAL-083) suggests combination therapy with PARP inhibitors

Abstract DNA damage response (DDR) is a network of intracellular pathways designed to minimize the impact of DNA damage. Multiple DNA repair pathways are known, including mismatch repair (MMR), O6-methylguanine DNA methyltransferase (MGMT), non-homologous end joining (NHEJ), and homologous recombination (HR), which act either by repairing the damage, arresting cell growth or, if necessary, promoting cell death. DDR defects are a hallmark of cancer development, rendering the cancer cells highly sensitive to targeted DNA-damaging agents. Ovarian cancer is normally treated with platinum (Pt)-based chemotherapy, which forms predominantly intrastrand DNA crosslinks; however, most advanced ovarian cancer patients develop episodes of recurrent disease with progressively shorter disease-free intervals. Dysfunctional p53 diminishes the therapeutic cytotoxicity of many DNA-targeting drugs, including Pt-based agents, comprising a therapeutic challenge in high-grade serous ovarian cancer (HGSOC), where p53 is almost universally mutated (~96%). Attempts to overcome Pt-resistance in HGSOC include PARP inhibitors (PARPi), which target tumors with deficiencies in the HR pathway (50% of HGSOC), responsible for the repair of DNA double-strand breaks (DSBs). Unfortunately, PARPi resistance frequently arises, leading to a 5-year survival rate of only 40% in this cancer. VAL-083 is a first-in-class DNA-damaging agent that has demonstrated clinical activity against a range of tumor types, including ovarian cancer, in historical NCI-sponsored clinical studies. VAL-083 rapidly induces interstrand crosslinks at guanine-N7, causing DNA DSBs that, if left unrepaired, are lethal to the cancer cell. We have shown that VAL-083 treatment leads to DNA damage that activates HR and is independent of common DNA repair pathways, including MGMT, MMR, and NHEJ. Notably, VAL-083 induces persistent S/G2 phase cell cycle arrest through two parallel pathways--one p53-independent and one p53-dependent. Taken together, these data propose the potential for targeting HR-impaired tumors and for combination treatment with S-phase specific DNA-targeting agents, including PARPi. We have previously seen VAL-083 cytotoxicity against a panel of ovarian cancer cells with differing p53 status and superadditivity between VAL-083 and PARPi olaparib, in vitro. The purpose of this study was to expand on these studies by examining the cytotoxicity of VAL-083 against HR-impaired ovarian cancer cells, the impact of p53 status, and the potential for VAL-083 combination with PARPi as part of a treatment strategy for HR-impaired, p53 dysfunctional HGSOC. VAL-083 cytotoxicity was investigated using the 5-day MTT assay in A2780 ovarian cancer cells. HR-impaired A2780 were examined using BRCA1 siRNA oligos prior to VAL-083 exposure. Combinations with PARPi veliparib (catalytic) and talazoparib (trapping) were examined in HR-proficient and HR–impaired A2780. The impact of p53 status was investigated by CRISPR/cas 9 knockout of p53 in the wildtype (wt) A2780 cells and in p53-mutated OVCAR3 cancer cells. We report increased VAL-083 cytotoxicity against HR-impaired A2780 cancer cells, further supporting HR as the main repair pathway for VAL-083-induced DNA damage, and suggesting HR-impaired HGSOC as a target for VAL-083 treatment. We further report superadditivity between VAL-083 and PARPi veliparib and talazoparib. Significantly, VAL-083 cytotoxicity against wt and knockout p53 A2780 cells and against p53-mutated OVCAR3 cells differed only by a factor of ~2, supporting a p53-independent mechanism of action for VAL-083. In conclusion, our results demonstrate a distinct DNA-damaging mechanism for VAL-083, resulting in the ability to target HR-impaired tumors and overcome MGMT- , MMR- , and NHEJ-related chemoresistance. VAL-083 activity was independent of p53 status, and superadditivity with PARPi was identified in HR-proficient A2780. Studies in HR-impaired A2780 are ongoing and will also be presented. Citation Format: Jeffrey A. Bacha, Guangan He, Xiaolei Xie, Anne Steino, Dennis M. Brown, Zahid H. Siddik. Distinct mechanism of action of DNA-damaging agent dianhydrogalactitol (VAL-083) suggests combination therapy with PARP inhibitors. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A01.

Force-induced rupture of double-stranded DNA in the absence and presence of covalently bonded anti-tumor drugs: Insights from molecular dynamics simulations.

DNA intra-strand cross-link (ICL) agents are widely used in the treatment of cancer. ICLs are thought to form a link between the same strand (intra-strand) or complimentary strand (inter-strand) and thereby increase the stability of DNA, which forbids the processes like replication and transcription. As a result, cell death occurs. In this work, we have studied the enhanced stability of a double stranded DNA in the presence of ICLs and compared our findings with the results obtained in the absence of these links. Using atomistic simulations with explicit solvent, a force is applied along and perpendicular to the direction of the helix and we measured the rupture force and the unzipping force of DNA-ICL complexes. Our results show that the rupture and the unzipping forces increase significantly in the presence of these links. The ICLs bind to the minor groove of DNA, which enhance the DNA stabilisation. Such information may be used to design alternative drugs that can stall replication and transcription that are critical to a growing number of anticancer drug discovery efforts.

5-(Halomethyl)uridine derivatives as potential antitumor radiosensitizers: A DFT study

Considering the fact that the efficiency of the uridine-5-methyl radical in producing cytotoxic DNA intrastrand cross-link lesions is greatly higher than that of the uridine-5-yl radical, the radiosensitizing action of 5-(halomethyl)uridines (5-XCH2U, X = F, Cl, or Br) is studied in the present work. It is found that 5-XCH2U has sufficient electron affinity to capture a pre-hydrated or a hydrated electron, and electron attachment leads to significantly facile X− elimination forming the uridine-5-methyl radical. All these three halogenated uridine derivatives are shown to be potential radiosensitizers, with their radiosensitizing abilities increased in an order 5-FCH2U < 5-ClCH2U ≈ 5-BrCH2U.

The association of genetic variations in DNA repair pathways with severe toxicities in NSCLC patients undergoing platinum-based chemotherapy.

Genetic variations in genes involved in repairing platinum-induced DNA lesions may contribute to the toxicity of platinum-based chemotherapy. The role of single-nucleotide polymorphisms (SNPs) within DNA repair pathways in the occurrence of severe toxicity is not yet understood. Current studies prefer to do original works rather than analyze previously published data. Our study aimed to replicate associations between previously investigated SNPs and toxicities and to identify new genetic makers. We systematically examined the relevance of 97 SNPs in 54 candidate genes responsible for repairing DNA interstrand and intrastrand cross-links to severe toxicity in a discovery cohort of 437 NSCLC patients receiving platinum-based chemotherapy. Statistically significant SNPs were then assessed for replication in an independent validation cohort of 781 NSCLC patients. We found that 7 SNPs were significant at p < 0.01 (RRM1 rs12806698, XPC rs2228000, XPF rs1799801, hMLH1 rs1800734, PMS2 rs1062372, REV3L rs462779 and FANCC rs4647554) in the discovery cohort. Among them, two SNPs (RRM1 rs12806698 and hMLH1 rs1800734) remained significant after Bonferroni correction. XPC rs2228000 showed a significant relationship with severe gastrointestinal toxicity in the validation cohort. When the two cohorts were combined, XPC rs2228000 presented better tolerance of severe hematologic toxicity, gastrointestinal toxicity and leukopenia (OR = 0.677, 95% CI: 0.510-0.899, p = 0.007; OR = 0.565, 95% CI: 0.368-0.869, p = 0.009; OR = 0.628, 95% CI: 0.439-0.899, p = 0.011, respectively). Our findings can offer comprehensive pharmacogenetic information for platinum-induced toxicities.

Abstract NTOC-108: ACTIVITY OF DIANHYDROGALACTITOL (VAL–083) IN OVARIAN TUMOR MODELS, SENSITIVE OR RESISTANT TO CISPLATIN

Abstract PURPOSE: Platinum drug resistance is a major clinical impediment that is normally ascribed to several mechanisms, with dysfunctional p53 playing a critical role, particularly in the highly lethal high-grade ovarian serous carcinoma, where p53 mutations can be &amp;gt;80%. Since restoration of p53 function could restore drug-dependent cytotoxicity, the goal of this study was to examine dianhydrogalactitol (VAL-083) for its potential in an ovarian tumor panel representing different p53 statuses and cisplatin-resistance. VAL-083 has demonstrated clinical activity against a range of tumor types, including ovarian cancer, in historical NCI-sponsored clinical studies, but its activity in a cisplatin-resistance setting is not known. VAL-083 is a bi-functional alkylating agent with a distinct mechanism of action, forming DNA interstrand cross links at the N7 position of guanine, whereas cisplatin and carboplatin predominantly form intrastrand DNA cross-links. METHODS: The tumor panel was composed of cisplatin-sensitive wild type (wt) p53 ovarian cancer cell line A2780, cisplatin-resistant A2780-derived heterozygous p53-V172F mutant 2780CP/Cl-16 cells, and three other cisplatin-resistant models harboring mutant (OVCAR-10) or wt p53 (Hey and OVCA-433). Cells were exposed to cisplatin or VAL-083 and the IC50 cytotoxic parameter was determined by fitting the 5-day MTT cell survival data to a 4-parameter sigmoidal curve. MTT assay was used in conjunction with Bliss Independence Model and combination index approaches to assess VAL-083/cisplatin combinations. Immunoblot analysis assessed p53 response. RESULTS: The IC50 of cisplatin in the cisplatin-sensitive A2780 model was 0.2-0.3 µM. In contrast, the IC50 in the four cisplatin-resistant models (2780CP/Cl-16, OVCAR-10, Hey and OVCA-433) was 3-8 µM; a 10- to 27-fold increase. The corresponding IC50 for VAL-083 was about 0.5 µM in A2780 cells and 2-4 µM in the four cisplatin-resistant models; a 4- to 7-fold increase, suggesting a distinct mode of action of VAL-083 and the ability to circumvent cisplatin resistance. In addition, these results further indicate that VAL-083 activity is less dependent on p53 status. However, immunoblots of 2780CP/Cl-16 cells suggest partial dependency on p53 by demonstrating that VAL-083, unlike cisplatin, can activate mutant p53-V172F through Ser20 phosphorylation, thereby preventing binding to p53 of inhibitory MDM4. VAL-083 thus restores p53 function, which is consistent with its greater potency in p53 mutated cisplatin-resistant ovarian cancer. These results suggest that VAL-083 have two mechanisms: one dependent on p53 and another on a different pathway. The apparent differences in the mechanism between cisplatin and VAL-083 prompted a combination study with the two agents, and consistent synergy was demonstrated. CONCLUSIONS: VAL-083 can circumvent cisplatin-resistance in ovarian tumor models, and its activity is less dependent on p53 status. Together with overt synergy between VAL-083 and cisplatin, our results demonstrate the effectiveness of VAL-083 against cisplatin-resistant ovarian cancer as a single agent or in combination with cisplatin. Citation Format: Anne Steino, Michelle Martinez-Rivera, Guanghan He, Xiaolei Xie, Jeffrey A. Bacha, Dennis M. Brown and Zahid H. Siddik. ACTIVITY OF DIANHYDROGALACTITOL (VAL–083) IN OVARIAN TUMOR MODELS, SENSITIVE OR RESISTANT TO CISPLATIN [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-108.

Enzyme Catalyzed Decomposition of 4-Hydroxycyclophosphamide

According to general doctrine [1] canceroselectivity of Cyclophosphamide is based on different activities of the 4-hydroxycyclophosphamide (OHCP) detoxifying cellular enzyme aldehyde dehydrogenase in tumor and normal cells. Aldehyde dehydrogenase converts the OHCP tautomere aldophosphamide (ALDO) to the non-cytotoxic carboxyphosphamide. Due to different activities of the detoxifying enzyme more cytotoxic phosporamide mustard (PAM) is spontaneously released from OHCP/ALDO in tumor cells. PAM unfolds its cytotoxic activity by forming intrastrand and interstrand DNA crosslinks. This hypothesis is supported by in vitro experiments which show inverse correlations of aldehyde dehydrogenase activity and sensitivity of tumor cells against activated congeners of cyclophosphamide like mafosfamide which hydrolyses within a few minutes to OHCP. In protein free rat serum ultrafiltrate however free OHCP and its coexisting tautomer ALDO are stable compounds. Its half-life in protein free rat serum ultrafiltrate (pH7, 37oC) is more than 20 h. Contrary to protein free ultrafiltrate in whole serum ALDO is enzymatically decomposed to PAM and 3-hydroxypropionaldehyde (HPA) within minutes. The decomposing enzyme was identified as 3´-5´ phosphodiesterase, the Michaelis constant was determined to be 10-3M in human serum.The experiments presented clearly demonstrate that ALDO is not only cleaved base catalyzed yielding acrolein and PAM [2, 3] but also cleaved enzymatically by serum phosphodiesterases yielding HPA and PAM. It is discussed that the reason of the high canceroselectivity of cyclophosphamide is not only due to enrichment of OHCP/ALDO in tumor cells due to less detoxification of ALDO in tumor cells than in normal cells. It is discussed that there is a good reason for an additional mechanism namely the amplification of apoptosis of PAM damaged cells by HPA.A two step mechanism for the mechanism of action of OHCP/ALDO is discussed. During the first step, the DNA is damaged by alkylation by PAM. During the second step the cell containing damaged DNA is eliminated by apoptosis, supported by HPA.

DNA intrastrand cross-links induced by the purine-type deoxyguanosine-8-yl radical: a DFT study.

Currently, all known DNA intrastrand cross-links are found to be induced by pyrimidine-type radicals; however, whether or not purine-type radicals are able to cause DNA intrastrand cross-links remains unclear. In the present study, probable additions of the highly reactive deoxyguanosine-8-yl radical to its 3'/5' neighboring pyrimidine nucleotides in four model compounds, 5'-G˙T-3', 5'-TG˙-3', 5'-G˙C-3', and 5'-CG˙-3', were studied using density functional theory (DFT) methods. In single-stranded DNA, the deoxyguanosine-8-yl radical is preferred to efficiently attack the C5 site of its 3' neighboring deoxythymidine or deoxycytidine, forming the G[8-5]T or G[8-5]C intrastrand cross-link rather than the C6 site forming the G[8-6]T or G[8-6]C intrastrand cross-link. The four corresponding sequence isomers, namely T[5-8]G, T[6-8]G, C[5-8]G, and C[6-8]G, formed by additions of deoxyguanosine-8-yl radical to its 5' neighboring pyrimidine nucleotides are predicted to be formed inefficiently. In double-stranded DNA, considering the detrimental effects of stabilizing weak interactions on related structural adjustments required in each addition reaction path, relatively lower reaction yields are suggested for the G[8-5]T and G[8-5]C intrastrand cross-links, while the formation of the other six intrastrand cross-links becomes quite difficult. All calculations definitely demonstrate that, in addition to pyrimidine-type radicals, the purine-type deoxyguanosine-8-yl radical is able to attack its 3'/5' neighboring pyrimidine nucleotides forming several DNA intrastrand cross-links.

Formation of pyrimidine-pyrimidine type DNA intrastrand cross-links: a theoretical verification.

Pyrimidine-type radicals have been demonstrated to be able to attack their 3' or 5' neighboring purine nucleotides forming diverse DNA intrastrand cross-links, but whether or not these radicals can attack their surrounding pyrimidine nucleotides forming pyrimidine-pyrimidine type DNA intrastrand cross-links remains unclear. To resolve this question, probable additions of the uracil-5-methyl (˙UCH2) radical to the C5[double bond, length as m-dash]C6 double bond of its 3'/5' neighboring pyrimidine nucleotides in the four models, 5'-T(˙UCH2)-3', 5'-C(˙UCH2)-3', 5'-(˙UCH2)T-3', and 5'-(˙UCH2)C-3', are explored in the present work employing density functional theory (DFT) methods. The C6 site of its 5' neighboring thymidine is the preferred target for ˙UCH2 radical addition, while additions of the ˙UCH2 radical to the C6 and C5 sites of its 5' neighboring deoxycytidine are found to be competitive reactions. The ˙UCH2 radical can react with both the C6 and C5 sites of its 3' neighboring pyrimidine nucleotides, but the efficiencies of these reactions are predicted to be much lower than those of the corresponding addition reactions to its 5' neighboring pyrimidine nucleotides, indicating the existence of an obvious sequence effect. All the addition products could be finally transformed into closed-shell intrastrand cross-links, the molecular masses of which are found to be exactly the same as certain MS values determined in a recent study of an X-irradiated deoxygenated aqueous solution of calf thymus DNA. The present study thus not only definitely corroborates the fact that the reactive ˙UCH2 radical can attack its 3'/5' neighboring pyrimidine nucleotides forming several pyrimidine-pyrimidine type DNA intrastrand cross-links, but also provides a plausible explanation for the identities of these structurally unknown intrastrand cross-links.

Co-inhibition of pol θ and HR genes efficiently synergize with cisplatin to suppress cisplatin-resistant lung cancer cells survival.

Cisplatin exert its anticancer effect by creating intrastrand and interstrand DNA cross-links which block DNA replication and is a major drug used to treat lung cancer. However, the main obstacle of the efficacy of treatment is drug resistance. Here, we show that expression of translesion synthesis (TLS) polymerase Q (POLQ) was significantly elevated by exposure of lung cancer cells A549/DR (a cisplatin-resistant A549 cell line) to cisplatin. POLQ expression correlated inversely with homologous recombination (HR) activity. Co-depletion of BRCA2 and POLQ by siRNA markedly increased sensitivity of A549/DR cells to cisplatin, which was accompanied with impairment of double strand breaks (DSBs) repair reflected by prominent cell cycle checkpoint response, increased chromosomal aberrations and persistent colocalization of p-ATM and 53BP1 foci induced by cisplatin. Thus, co-knockdown of POLQ and HR can efficiently synergize with cisplatin to inhibit A549/DR cell survival by inhibiting DNA DSBs repair. Similar results were observed in A549/DR cells co-depleted of BRCA2 and POLQ following BMN673 (a PARP inhibitor) treatment. Importantly, the sensitization effects to cisplatin and BMN673 in A549/DR cells by co-depleting BRCA2 and POLQ was stronger than those by co-depleting BRCA2 and other TLS factors including POLH, REV3, or REV1. Our results indicate that there is a synthetic lethal relationship between pol θ-mediated DNA repair and HR pathways. Pol θ may be considered as a novel target for lung cancer therapy.