DNA Interstrand Cross-links Research Articles

Potential Signaling Regulations of Stereisomeric DNA Interstrand Crosslinks Produced by Mitomycins in K562 Cells

Mitomycin C (MC) is a DNA alkylating agent broadly used in chemotherapy. MC and Decarbamoyl mitomycin C (DMC), a synthetic MC derivative which lacks the carbamoyl at the C10 position, can form interstrand crosslinks (ICLs) between the exocyclic amines of opposing deoxyguanosine moieties. These ICLs are responsible for the cytotoxicity of mitomycins towards cancer cells since they prevent replication of highly proliferating cells. MC and DMC can crosslink DNA in both 5’‐CpG and 5’‐GpC sequence contexts, and the crosslinking is diastereospecific: At 5’‐CpG, mitomycins produce trans‐ICLs whereas cis‐ICLs are found at 5’‐GpC steps. The major ICL formed by Mitomycin C in culture cells is the trans‐ICL whereas decarbamoylmitomycin C yields the stereoisomeric cis‐ICL preferentially. Our overarching goal is to synthesize oligonucleotides bearing the cisand trans isomeric crosslinks to investigate the relationship between the ICL structure and the cellular response. Our hypothesis is that the trans and cis‐ICLs trigger different cellular responses. There is evidence that the ICL produced by DMC triggers a p53 independent cell death and research uncovering mechanisms through which p53 independent cell death occurs is crucial to identify molecular targets to treat tumors with a mutant p53. These tumors represent at least 50% of all cancers. We present here the different synthetic routes to access cis and trans isomeric interstrand crosslinks formed by mitomycins in their reaction with DNA and preliminary label free proteomic studies to identify the stereoisomeric ICLs molecular targets. Proteomic studies were accomplished from K562 leukemia cells transfected with the purified ICLs for 24 hours and showed several interesting proteins differently regulated by the trans‐ and cis‐ICLs: (1) Histone‐lysine N‐methyltransferase EHMT2 which down‐regulates a cell cycle inhibitory gene expression network (decreased 46% upon treatment with the trans‐ICL, but no change was observed with the cis‐ICL); (2) Target of EGR1 protein 1 which inhibits cell growth rate and cell cycle (increased 69% upon treatment with the trans‐ICL, but decreased 30% with the cis‐ICL); (3) Poly(ADP‐ribose) glycohydrolase DNA repair, involved in DNA repair (decreased 19% upon treatment with the trans‐ICL, but increased 63% with the cis‐ICL); (4) Telomerases Cajal body protein 1, involved in cell cycle regulation (no change observed upon tratment with the trans‐ICL but decreased 75% with the cis‐ICL. In addition, Ingenuity Pathway Analysis was conducted to identify the major molecular pathways impacted by each ICL treatment. In conclusion, we have identified both ICL molecular targets and cellular responses involved in DNA repair and cell cycle regulation in K‐562cell lines. Further validations are needed to confirm the results.

The HMCES DNA-protein cross-link functions as an intermediate in DNA interstrand cross-link repair.

The 5-hydroxymethylcytosine binding, embryonic stem-cell-specific (HMCES) protein forms a covalent DNA-protein cross-link (DPC) with abasic (AP) sites in single-stranded DNA, and the resulting HMCES-DPC is thought to suppress double-strand break formation in S phase. However, the dynamics of HMCES cross-linking and whether any DNA repair pathways normally include an HMCES-DPC intermediate remain unknown. Here, we use Xenopus egg extracts to show that an HMCES-DPC forms on the AP site generated during replication-coupled DNA interstrand cross-link repair. We show that HMCES cross-links form on DNA after the replicative CDC45-MCM2-7-GINS (CMG) helicase has passed over the AP site, and that HMCES is subsequently removed by the SPRTN protease. The HMCES-DPC suppresses double-strand break formation, slows translesion synthesis past the AP site and introduces a bias for insertion of deoxyguanosine opposite the AP site. These data demonstrate that HMCES-DPCs form as intermediates in replication-coupled repair, and they suggest a general model of how HMCES protects AP sites during DNA replication.

Identification of Potential Cellular Responses Triggered by Stereoisomeric DNA Interstrand Crosslinks Produced by Mitomycins in MCF‐7 Cells

Mitomycin C (MC) is a DNA alkylating agent broadly used in chemotherapy. MC and Decarbamoylmitomycin C (DMC), a synthetic MC derivative which lacks the carbamoyl at the C10 position, can form interstrand crosslinks (ICLs) between the exocyclic amines of opposing deoxyguanosine moieties. These ICLs are responsible for the cytotoxicity of mitomycins towards cancer cells since they prevent replication of highly proliferating cells. MC and DMC can crosslink DNA in both 5’‐CpG and 5’‐GpC sequence contexts, and the crosslinking is diastereospecific: At 5’‐CpG, mitomycins produce trans‐ICLs whereas cis‐ICLs are found at 5’‐GpC steps. The major ICL formed by Mitomycin C in culture cells is the trans‐ICL whereas decarbamoylmitomycin C yields the stereoisomeric cis‐ICL preferentially. Our overarching goal is to synthesize oligonucleotides bearing the cisand trans isomeric crosslinks to investigate the relationship between ICL structure and cellular response. Our hypothesis is that the trans and cis‐ICLs trigger different cellular responses. There is evidence that the ICL produced by DMC triggers a p53 independent cell death and research uncovering mechanisms through which p53 independent cell death occurs is crucial to identify molecular targets to treat tumors with a mutant p53. These tumors represent at least 50% of all cancers. We present here the different synthetic routes to access cis and trans isomeric interstrand crosslinks formed by mitomycins in their reaction with DNA and preliminary label free proteomic studies to identify the stereoisomeric ICLs molecular targets. Proteomic studies were accomplished from MCF‐7 cells transfected with the purified ICLs for 24 hours. They showed several interesting proteins were differently regulated by the trans‐ and cis‐ICLs: (1) TBC1 domain family member 9 which regulates cell proliferation (increased 41% upon treatment with the trans‐ICL treatment, but decreased 50% with the cis‐ICL); (2) Core histone macro‐H2A.2, involved in DNA repair (decreased 75% upon treatment with the trans‐ICL, but increased 89% with the cis‐ICL); (3) Aurora kinase B which inhibits p21 expression and regulates cell cycle (increased 50% upon treatment with the trans‐ICL, but decreased 73% by the cis‐ICL); and (4) Membrane‐associated tyrosine‐ and threonine‐specific cdc2‐inhibitory kinase which acts a negative regulator of G2 to M transition (decreased 36% upon treatment with the trans‐ICL, but increased 5% with the cis‐ICL). In addition, Ingenuity Pathway Analysis was conducted to identify the major molecular pathways impacted by each ICL treatment. In conclusion, we now have identified both ICL molecular targets and cellular responses involved in DNA repair and cell cycle regulation in MCF7 cell lines. Further validations are needed to confirm the results.

Rapid DNA interstrand cross-linking of Pt(IV) compound

Pt(IV) anticancer compounds have been developed for several decades to overcome the drawbacks of their Pt(II) congeners, and the reduction of Pt(IV) to Pt(II) has been commonly regarded as a necessary step in the activation of Pt(IV) compounds prior to targeting DNA. However, blockage of glutathione (GSH) biosynthesis resulted in a slight effect on the cytotoxicity of oxoplatin in yeast Saccharomyces cerevisiae strains, urging us to reconsider the mechanism of actions for the “inert” Pt(IV) complexes. Using X-ray absorption near-edge spectroscopy (XANES), our data demonstrated that Pt(IV) complex oxoplatin could bind to DNA in a tetravalent state. Both alkaline denaturing agarose electrophoresis and thermal denaturation-renaturation assay revealed that oxoplatin could rapidly produce stable interstrand crosslinks (ICLs), which can further translate into a fast cell-killing process in cancer cells. Using quantitative real-time PCR and immunofluorescence analysis, we also proved that Pt(IV) complex oxoplatin could induce a quick intracellular response of the FA/BRCA pathway in cancer cells that involves the DNA interstrand crosslinking repair system, and this quick response to ICLs was independent with the intracellular GSH levels. Cell cycle analysis showed that short incubation with oxoplatin can induce a strong S phase arrest in HeLa cells, indicating that the rapid interstrand crosslinks produced by oxoplatin might stall the replication fork, result in the double-strand breaks, and eventually induce cell death. Our results implied that, besides the reduction mechanism to release the Pt(II) congeners, direct and rapid interstrand cross-linking with DNA by Pt(IV) compounds might be a unique mechanism for Pt(IV) compounds, which may provide new insight for the development of next-generation platinum-based drugs.

Dual targeting of Saccharomyces cerevisiae Pso2 to mitochondria and the nucleus, and its functional relevance in the repair of DNA interstrand crosslinks.

Repair of DNA interstrand crosslinks involves a functional interplay among different DNA surveillance and repair pathways. Previous work has shown that interstrand crosslink-inducing agents cause damage to Saccharomyces cerevisiae nuclear and mitochondrial DNA, and its pso2/snm1 mutants exhibit a petite phenotype followed by loss of mitochondrial DNA integrity and copy number. Complex as it is, the cause and underlying molecular mechanisms remains elusive. Here, by combining a wide range of approaches with in vitro and in vivo analyses, we interrogated the subcellular localization and function of Pso2. We found evidence that the nuclear-encoded Pso2 contains 1 mitochondrial targeting sequence and 2 nuclear localization signals (NLS1 and NLS2), although NLS1 resides within the mitochondrial targeting sequence. Further analysis revealed that Pso2 is a dual-localized interstrand crosslink repair protein; it can be imported into both nucleus and mitochondria and that genotoxic agents enhance its abundance in the latter. While mitochondrial targeting sequence is essential for mitochondrial Pso2 import, either NLS1 or NLS2 is sufficient for its nuclear import; this implies that the 2 nuclear localization signal motifs are functionally redundant. Ablation of mitochondrial targeting sequence abrogated mitochondrial Pso2 import, and concomitantly, raised its levels in the nucleus. Strikingly, mutational disruption of both nuclear localization signal motifs blocked the nuclear Pso2 import; at the same time, they enhanced its translocation into the mitochondria, consistent with the notion that the relationship between mitochondrial targeting sequence and nuclear localization signal motifs is competitive. However, the nuclease activity of import-deficient species of Pso2 was not impaired. The potential relevance of dual targeting of Pso2 into 2 DNA-bearing organelles is discussed.

Head and Neck Cancer Susceptibility and Metabolism in Fanconi Anemia.

Simple SummaryGermline loss-of-function mutations in any of over 20 known Fanconi anemia (FA) genes cause the disorder FA. The FA pathway plays a key role in the repair of DNA interstrand crosslinks (ICLs) and stabilization of stalled replication fork. Epidemiological studies have shown that persons with FA have a uniquely increased susceptibility to squamous cell carcinomas (SCCs) and increased human papilloma virus (HPV) prevalence. Genomic and transcriptomic studies are informative, but there is a paucity of data regarding metabolic deregulation in FA deficient systems. Metabolism is an essential component of cancer development and has significant impact on therapeutic outcomes. Therefore, a deeper understanding of the metabolic architecture of FA may aid in our understanding of SCC development, and allow for the discovery of metabolism-based diagnostic biomarkers and new therapeutic targets. In this review, we discuss known metabolic consequences of FA pathway loss on cancer pathogenesis, and in particular in normal and transformed keratinocytes—the cells of origin for SCC.Fanconi anemia (FA) is a rare inherited, generally autosomal recessive syndrome, but it displays X-linked or dominant negative inheritance for certain genes. FA is characterized by a deficiency in DNA damage repair that results in bone marrow failure, and in an increased risk for various epithelial tumors, most commonly squamous cell carcinomas of the head and neck (HNSCC) and of the esophagus, anogenital tract and skin. Individuals with FA exhibit increased human papilloma virus (HPV) prevalence. Furthermore, a subset of anogenital squamous cell carcinomas (SCCs) in FA harbor HPV sequences and FA-deficient laboratory models reveal molecular crosstalk between HPV and FA proteins. However, a definitive role for HPV in HNSCC development in the FA patient population is unproven. Cellular metabolism plays an integral role in tissue homeostasis, and metabolic deregulation is a known hallmark of cancer progression that supports uncontrolled proliferation, tumor development and metastatic dissemination. The metabolic consequences of FA deficiency in keratinocytes and associated impact on the development of SCC in the FA population is poorly understood. Herein, we review the current literature on the metabolic consequences of FA deficiency and potential effects of resulting metabolic reprogramming on FA cancer phenotypes.

Evaluation of conformational transitions of h-BRCA2 functional domain and unclassified variant Arg2502Cys using multimodal approach

Breast cancer type 2 susceptibility (BRCA2) protein plays an essential role in the repair mechanism of DNA double-strand breaks and interstrand cross-links by Homologous recombination. Germline mutations identified in the BRCA2 gene confer an increased risk of hereditary breast and ovarian cancer. Missense mutations are identified all over the gene, including the DNA binding region of BRCA2 that interacts with FANCD2. However, the majority of these missense mutations are classified as ‘Variants of Uncertain Significance’ due to a lack of structural, functional and clinical correlations. Therefore, multi-disciplinary in-silico, in-vitro and biophysical approaches have been explored to characterize an unclassified missense mutation, BRCA2 Arg2502Cys, identified from a case-control study. Circular-dichroism and Fluorescence spectroscopy show that the Arg2502Cys mutation in hBRCA2 (residues 2350–2545) decreases the α-helical/β-sheet propensity of the wild-type protein and perturb the tertiary structure conformation. Molecular dynamics simulations revealed alteration in the intramolecular H-bonds, overall compactness and stability of the hydrophobic core were observed in the mutant protein. Principle component analysis indicated that Arg2502Cys mutant exhibited comparatively large conformational transitions and periodic fluctuation. Therefore, to our conclusion, BRCA2 Arg2502Cys mutant perturbed the structural integrity and conformational dynamics of BRCA2.

Selective Antitumor Activity and Photocytotoxicity of Glutathione-Activated Abasic Site Trapping Agents.

Abasic (AP) sites are one of the most common DNA lesions in cells. Aldehyde-reactive alkoxyamines capture AP sites and block the activity of APE1, the enzyme responsible for initiating their repair. Blocking the APE1 repair of AP sites leads to cell death, and it is an actively investigated approach for treating cancer. However, unselective AP site capture in different cells produces side effects and limits the application of alkoxyamines in chemotherapy. Herein we take advantage of the higher glutathione (GSH) concentration in cancer cells over normal cells to develop GSH-inducible agents that selectively kill cancer cells. 2,4-Dinitrobenzenesulfonamide caged coumarin-based alkoxyamines 1 and 2 are selectively revealed by GSH to release SO2 and fluorescent coumarin-based alkoxyamines 3 and 4 that trap AP sites in cells. GSH-directed AP site trapping and SO2 release result in selective cytotoxicity (defined as IC50WI38/IC50H1299) against H1299 lung cancer cells over normal WI38 lung cells, ranging from 1.8 to 2.8 for 1 and 2. The alkylating agent methylmethanesulfonate (MMS) promotes the formation of AP sites in cells and enhances the cytotoxicity of agent 1 in a dose-dependent way. Moreover, the comet assay and γH2AX assay suggest that AP adducts form a highly toxic DNA interstrand cross-link (ICL) upon photolysis, leading to further cell death. DNA flow cytometric analysis showed that 1 promoted cell apoptosis in the early stage and induced G2/M phase cell-cycle arrest. The 2,4-dinitrobenzenesulfonamide-caged alkoxyamines exhibited selective antitumor activity and photocytotoxicity in cancer cells, illuminating their potential as GSH-directed chemotherapeutic agents.

Phenyl Selenide-Based Precursors as Hydrogen Peroxide Inducible DNA Interstrand Cross-Linkers.

DNA interstrand crosslinks (ICLs) are highly toxic DNA lesions, and induce cell death by blocking DNA strand separation. Most ICL agents aiming to kill cancer cells, also generate adverse side effects to normal cells. H2 O2 -inducible DNA ICL agents are highly selective for targeting cancer cells, as the concentration of H2 O2 is higher in cancer cells than normal cells. Previous studies have focused on arylboronate-based precursors, reacting with H2 O2 to generate reactive quinone methides (QMs) crosslinking DNA. Here we explore phenyl selenide-based precursors 1-3 as H2 O2 -inducible DNA ICL agents. The precursors 1-3 can be activated by H2 O2 to generate the good benzylic leaving group and promote production of reactive QMs to crosslink DNA. Moreover, the DNA cross-linking ability is enhanced by the introduction of substituents in the para-position of the phenolic hydroxyl group. From the substituents explored (H, OMe, F), the introduction of electron donating group (OMe) shows a pronounced elevating effect. Further mechanistic studies at the molecular and DNA levels confirm alkylation sites located mainly at dAs, dCs and dGs in DNA. Additionally, cellular experiments reveal that agents 1-3 exhibit higher cytotoxicity toward H1299 human lung cancer cells compared to clinically used drugs, by inducing cellular DNA damage, apoptosis and G0/G1 cell cycle arrest. This study provides a strategy to develop H2 O2 -inducible DNA interstrand cross-linkers.

Formation of bifunctional cross-linked products due to reaction of NAMI-A with DNA bases – a DFT study

It is reported that NAMI-A and other Ru-anticancer complexes preferably bind with the N7 site of guanine and can also form DNA inter-strand cross-links. Therefore, in order to understand the DNA cross-link formation capability of NAMI-A, we have investigated here the structure and energetics of the reactions of the GN7-NAMI-A (a monofunctional adduct of NAMI-A with the N7 site of guanine) with the N3, N7, and O6 sites of guanine; the N1, N3, and N7 sites of adenine; the O2 and N3 sites of cytosine; and the O2 and O4 sites of thymine, using the M06-2X functional of density functional theory. It is found that the GN7-NAMI-A can form stable cross-linked products at all the sites studied here except at the N3 site of cytosine and O2 site of thymine. The calculated reaction free energies and reaction enthalpies indicate that the N3 site of adenine (AN3) and N7 site of guanine (GN7) are most exothermic among all the studied reactions. This study shows that NAMI-A would favorably form the cross-linked products involving the N7 site of guanine at one side and the N7 site of guanine or the N3 site of adenine at the other side.

SCAI promotes error‐free repair of DNA interstrand crosslinks via the Fanconi anemia pathway

DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI‐FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase ζ (Polζ)‐mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error‐free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error‐free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Polζ and localizes to ICLs during DNA replication. SCAI‐deficient cells are exquisitely sensitive to ICL‐inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR‐mediated ICL repair is defective, and breaks are instead re‐ligated by polymerase θ‐dependent microhomology‐mediated end‐joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error‐free ICL repair.

The rate of formation and stability of abasic site interstrand crosslinks in the DNA duplex

DNA interstrand crosslinks (ICLs) strands pose an impenetrable barrier for DNA replication. Different ICLs are known to recruit distinct DNA repair pathways. NEIL3 glycosylase has been known to remove an abasic (Ap) site derived DNA crosslink (Ap-ICL). An Ap-ICL forms spontaneously from the Ap site with an adjacent adenine in the opposite strand. Lack of genetic models and a poor understanding of the fate of these lesions leads to many questions about the occurrence and the toxicity of Ap-ICL in cells.Here, we investigate the circumstances of Ap-ICL formation. With an array of different oligos, we have investigated the rates of formation, the yields, and the stability of Ap-ICL. Our findings point out how different bases in the vicinity of the Ap site change crosslink formation in vitro. We reveal that AT-rich rather than GC-rich regions in the surrounding Ap site lead to higher rates of Ap-ICL formation. Overall, our data reveal that Ap-ICL can be formed in virtually any DNA sequence context surrounding a hot spot of a 5′-Ap-dT pair, albeit with significantly different rates and yields. Based on Ap-ICL formation in vitro, we attempt to predict the number of Ap-ICLs in the cell.

Type-I Interferon Signaling in Fanconi Anemia.

Fanconi Anemia (FA) is a genome instability syndrome caused by mutations in one of the 23 repair genes of the Fanconi pathway. This heterogenous disease is usually characterized by congenital abnormalities, premature ageing and bone marrow failure. FA patients also show a high predisposition to hematological and solid cancers. The Fanconi pathway ensures the repair of interstrand crosslinks (ICLs) DNA damage. Defect in one of its proteins prevents functional DNA repair, leading to the accumulation of DNA breaks and genome instability. Accumulating evidence has documented a close relationship between genome instability and inflammation, including the production of type-I Interferon. In this context, type-I Interferon is produced upon activation of pattern recognition receptors by nucleic acids including by the cyclic GMP-AMP synthase (cGAS) that detects DNA. In mouse models of diseases displaying genome instability, type-I Interferon response is responsible for an important part of the pathological symptoms, including premature aging, short stature, and neurodegeneration. This is illustrated in mouse models of Ataxia-telangiectasia and Aicardi-Goutières Syndrome in which genetic depletion of either Interferon Receptor IFNAR, cGAS or STING relieves pathological symptoms. FA is also a genetic instability syndrome with symptoms such as premature aging and predisposition to cancer. In this review we will focus on the different molecular mechanisms potentially leading to type-I Interferon activation. A better understanding of the molecular mechanisms engaging type-I Interferon signaling in FA may ultimately lead to the discovery of new therapeutic targets to rescue the pathological inflammation and premature aging associated with Fanconi Anemia.

Facile preparation of model DNA interstrand cross-link repair intermediates using ribonucleotide-containing DNA

DNA interstrand cross-links (ICLs) are lesions with a covalent bond formed between DNA strands. ICLs are extremely toxic to cells because they prevent the separation of the two strands, which are necessary for the genetic interpretation of DNA. ICLs are repaired via Fanconi anemia and replication-independent pathways. The formation of so-called unhooked repair intermediates via a dual strand incision flanking the ICL site on one strand is an essential step in nearly all ICL repair pathways. Recently, ICLs derived from endogenous sources, such as those from ubiquitous DNA lesions, abasic (AP) sites, have emerged as an important class of ICLs. Despite the earlier efforts in preparing AP-ICLs in high yield using nucleotide analogs, little information is available for preparing AP-ICL unhooked intermediates with varying lengths of overhangs. In this study, we devise a simple approach to prepare model ICL unhooked intermediates derived from AP sites. We exploited the alkaline lability of ribonucleotides (rNMPs) and the high cross-linking efficiency between an AP lesion and a nucleotide analog, 2-aminopurine, via reductive amination. We designed chimeric DNA/RNA substrates with rNMPs flanking the cross-linking residue (2-aminopurine) to facilitate subsequent strand cleavage under our optimized conditions. Mass spectrometric analysis and primer extension assays confirmed the structures of ICL substrates. The method is straightforward, requires no synthetic chemistry expertise, and should be broadly accessible to all researchers in the DNA repair community. For step-by-step descriptions of the method, please refer to the companion manuscript in MethodsX.

Preclinical Development of ADCT-601, a Novel Pyrrolobenzodiazepine Dimer-based Antibody-drug Conjugate Targeting AXL-expressing Cancers.

AXL, a tyrosine kinase receptor that is overexpressed in many solid and hematologic malignancies, facilitates cancer progression and is associated with poor clinical outcomes. Importantly, drug-induced expression of AXL results in resistance to conventional chemotherapy and targeted therapies. Together with its presence on multiple cell types in the tumor immune microenvironment, these features make it an attractive therapeutic target for AXL-expressing malignancies. ADCT-601 (mipasetamab uzoptirine) is an AXL-targeted antibody-drug conjugate (ADC) comprising a humanized anti-AXL antibody site specifically conjugated using GlycoConnect technology to PL1601, which contains HydraSpace, a Val-Ala cleavable linker and the potent pyrrolobenzodiazepine (PBD) dimer cytotoxin SG3199. This study aimed to validate the ADCT-601 mode of action and evaluate its efficacy in vitro and in vivo, as well as its tolerability and pharmacokinetics. ADCT-601 bound to both soluble and membranous AXL, and was rapidly internalized by AXL-expressing tumor cells, allowing release of PBD dimer, DNA interstrand cross-linking, and subsequent cell killing. In vivo, ADCT-601 had potent and durable antitumor activity in a wide variety of human cancer xenograft mouse models, including patient-derived xenograft models with heterogeneous AXL expression where ADCT-601 antitumor activity was markedly superior to an auristatin-based comparator ADC. Notably, ADCT-601 had antitumor activity in a monomethyl auristatin E-resistant lung-cancer model and synergized with the PARP inhibitor olaparib in a BRCA1-mutated ovarian cancer model. ADCT-601 was well tolerated at doses of up to 6 mg/kg and showed excellent stability in vivo. These preclinical results warrant further evaluation of ADCT-601 in the clinic.

Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites

DNA interstrand cross-links (ICLs) are extremely deleterious DNA lesions, which can block different DNA transactions. A major step in ICL repair involves strand cleavage activities flanking the cross-linking site, also known as unhooking. The cleavage generates a single-stranded DNA remnant attached to the unbroken strand, often referred to as the unhooked ICL repair intermediates. The unhooked ICLs are substrates for specialized DNA polymerases, leading to the eventual restoration of the duplex DNA structure. Although these repair events have been outlined, the understanding of molecular details of the repair pathways has been hindered by the difficulty of preparing structurally defined ICL repair intermediates. Here, we present a straightforward method to prepare model ICL repair intermediates derived from a ubiquitous type of endogenous DNA modification, abasic (AP) sites. AP-derived ICLs have emerged as an important type of endogenous ICLs. We developed the method based on commercially available materials without the requirement of synthetic chemistry expertise. The method is expected to be accessible to any interested labs in the DNA repair community.• The method exploits the alkaline lability of ribonucleotides and uses designer oligonucleotides to create ICL repair intermediates with varying lengths of the unhooked strand.• Strand cleavage at ribonucleotides is achieved using NaOH, which avoids the potential for incomplete digestion during enzymatic workup due to specific substrate structures.• The method is grounded on the high cross-linking yield between an AP lesion and a nucleotide analog, 2-aminopurine, via reductive amination, developed by Gates and colleagues.

DNA damage and repair in the hematopoietic system.

Although hematopoietic stem cells (HSCs) in the bone marrow are in a state of quiescence, they harbor the self-renewal capacity and the pluripotency to differentiate into mature blood cells when needed, which is key to maintain hematopoietic homeostasis. Importantly, HSCs are characterized by their long lifespan ( e. g., up to 60 months for mice), display characteristics of aging, and are vulnerable to various endogenous and exogenous genotoxic stresses. Generally, DNA damage in HSCs is endogenous, which is typically induced by reactive oxygen species (ROS), aldehydes, and replication stress. Mammalian cells have evolved a complex and efficient DNA repair system to cope with various DNA lesions to maintain genomic stability. The repair machinery for DNA damage in HSCs has its own characteristics. For instance, the Fanconi anemia (FA)/BRCA pathway is particularly important for the hematopoietic system, as it can limit the damage caused by DNA inter-strand crosslinks, oxidative stress, and replication stress to HSCs to prevent FA occurrence. In addition, HSCs prefer to utilize the classical non-homologous end-joining pathway, which is essential for the V(D)J rearrangement in developing lymphocytes and is involved in double-strand break repair to maintain genomic stability in the long-term quiescent state. In contrast, the base excision repair pathway is less involved in the hematopoietic system. In this review, we summarize the impact of various types of DNA damage on HSC function and review our knowledge of the corresponding repair mechanisms and related human genetic diseases.

DNA Interstrand Crosslink Repair by Xpf-Ercc1 Homologue Confers Ultraviolet Resistance in Neurospora Crassa

DNA Interstrand Crosslink Repair by Xpf-Ercc1 Homologue Confers Ultraviolet Resistance in Neurospora Crassa

Abstract LBA041: Identification and characterization of potent RAD51 inhibitors targeting RAD51-BRCA2 interaction

Abstract The DNA damage response (DDR) is a complex cellular network that acts as a cell defense mechanism and exerts signal for repair of DNA lesions. It repairs different types of DNA damage such as single-strand (ssDNA) breaks, DNA inter-strand crosslinks and double strand breaks (DSB) caused by endogenous and exogenous factors. These damages are mainly repaired by homologous recombination (HR) repair, and non-homologous end joining (NHEJ). DNA double-strand breaks (DSBs) are the most severe DNA damage and predominantly repaired by HR. As the DDR pathway is critical for tumorigenesis, a high degree of interest has been generated in the recent past to target this network. RAD51 is one of the pivotal enzymes for DNA double-strand break repair by the HR pathway. While PARP inhibitors are extensively used for BRCA2 mutated cancers, their responses in BRCA2 WT is limited. Targeting RAD51 will open a new avenue to treat BRCA2 WT cancer patients. BRCA2 interaction with RAD51 is a prerequisite for RAD51 nuclear translocation to exert its function by binding to DNA. The disruption of this interaction leads to inhibition of DNA repair and in turn inhibits proliferation and tumor growth. As this leads to BRCAness in an otherwise BRCA2 WT background, it is expected to have synergy with PARP1 inhibitors. We report the identification of novel and potent RAD51 inhibitors which disrupt the RAD51:BRCA2 interaction. Our hit compounds show anticancer activity in relevant cancer cell lines across various indications. Further, our compounds inhibit RAD51 nuclear translocation upon induction of exogenous DNA damage. Multiple series have been identified, with a good SAR trend and correlating well with cell based and PD activity. Further profiling of these compounds is ongoing. Citation Format: Sanjita Sasmal, Sukanya Patra, Venkatesham Boorgu, Mahesh Yanamandra, Shankar Chithaluri, Hema Sankar Pathange, Deepika Hiremath, Sandeep M Girme, Amit Kumar Pawar. Identification and characterization of potent RAD51 inhibitors targeting RAD51-BRCA2 interaction [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA041.

RAD52: Paradigm of Synthetic Lethality and New Developments.

DNA double-strand breaks and inter-strand cross-links are the most harmful types of DNA damage that cause genomic instability that lead to cancer development. The highest fidelity pathway for repairing damaged double-stranded DNA is termed Homologous recombination (HR). Rad52 is one of the key HR proteins in eukaryotes. Although it is critical for most DNA repair and recombination events in yeast, knockouts of mammalian RAD52 lack any discernable phenotypes. As a consequence, mammalian RAD52 has been long overlooked. That is changing now, as recent work has shown RAD52 to be critical for backup DNA repair pathways in HR-deficient cancer cells. Novel findings have shed light on RAD52’s biochemical activities. RAD52 promotes DNA pairing (D-loop formation), single-strand DNA and DNA:RNA annealing, and inverse strand exchange. These activities contribute to its multiple roles in DNA damage repair including HR, single-strand annealing, break-induced replication, and RNA-mediated repair of DNA. The contributions of RAD52 that are essential to the viability of HR-deficient cancer cells are currently under investigation. These new findings make RAD52 an attractive target for the development of anti-cancer therapies against BRCA-deficient cancers.