Accumulation Of Breaks Research Articles

Cell Senescence and the DNA Single-Strand Break Damage Repair Pathway

Cellular senescence is a response to endogenous and exogenous stresses, including telomere dysfunction, oncogene activation, and persistent DNA damage. In particular, radiation damage induces oxidative base damage and bond breaking in the DNA double-helix structure, which are treated by dedicated enzymatic repair pathways. In this review, we discuss the correlation between senescence and the accumulation of non-repaired single-strand breaks, as can occur during radiation therapy treatments. Recent in vitro cell irradiation experiments using high-energy photons have shown that single-strand breaks may be preferentially produced at the borders of the irradiated region, inducing senescence in competition with the apoptosis end-point typically induced by double-strand breaks. Such a particular response to radiation damage has been proposed as a possible cause of radiation-induced second primary cancer, as cells with an accumulation of non-repaired single-strand breaks might evade the senescent state at much later times. In addition, we highlight the peculiarities of strand-break repair pathways in relation to the base-excision pathway that repairs several different DNA oxidation defects.

Discovery of KPT-6566 as STAG1/2 Inhibitor sensitizing PARP and NHEJ Inhibitors to suppress tumor cells growth in vitro

Stromal antigen 1 and 2 (STAG1 and STAG2) are two mutually exclusive components of the cohesin complex that is crucial for centromeric and telomeric cohesion. Beyond its structural role, STAG2 also plays a pivotal role in homologous recombination (HR) repair and has emerged as a promising therapeutic target in cancer treatment. Here, we employed a fluorescence polarization (FP)-based high-throughput screening and identified KPT-6566 as a dual inhibitor of STAG1 and STAG2. Biochemical and biophysical analyses demonstrated that KPT-6566 directly binds to STAG1 and STAG2, disrupting their interactions with SCC1 and double-stranded DNA. A metaphase chromosome spread assay showed that KPT-6566 causes premature chromosome separation and induces chromosome damages in HeLa cells. Furthermore, KPT-6566 also impairs DNA damage repair, leading to the accumulation of double-strand breaks and cell apoptosis. Finally, KPT-6566 can sensitize HeLa and HepG2 cells to PARP inhibitor Olaparib and the NHEJ inhibitor UMI-77, exhibiting a synergistic effect in suppressing cell proliferation. Our findings highlight the potential of STAG1/2 as promising therapeutic targets in cancer treatment, particularly when they are targeted in combination with other DNA damage response inhibitors.

TORC2 inhibition triggers yeast chromosome fragmentation through misregulated Base Excision Repair of clustered oxidation events

Combinational therapies provoking cell death are of major interest in oncology. Combining TORC2 kinase inhibition with the radiomimetic drug Zeocin results in a rapid accumulation of double-strand breaks (DSB) in the budding yeast genome. This lethal Yeast Chromosome Shattering (YCS) requires conserved enzymes of base excision repair. YCS can be attenuated by eliminating three N-glycosylases or endonucleases Apn1/Apn2 and Rad1, which act to convert oxidized bases into abasic sites and single-strand nicks. Adjacent lesions must be repaired in a step-wise fashion to avoid generating DSBs. Artificially increasing nuclear actin by destabilizing cytoplasmic actin filaments or by expressing a nuclear export-deficient actin interferes with this step-wise repair and generates DSBs, while mutants that impair DNA polymerase processivity reduce them. Repair factors that bind actin include Apn1, RFA and the actin-dependent chromatin remodeler INO80C. During YCS, increased INO80C activity could enhance both DNA polymerase processivity and repair factor access to convert clustered lesions into DSBs.

RNA/DNA Binding Protein TDP43 Regulates DNA Mismatch Repair Genes with Implications for Genome Stability.

TAR DNA-binding protein 43 (TDP43) is increasingly recognized for its involvement in neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP43 proteinopathy, characterized by dysregulated nuclear export and cytoplasmic aggregation, is present in most ALS/FTD cases and is associated with a loss of nuclear function and genomic instability in neurons. Building on prior evidence linking TDP43 pathology to DNA double-strand breaks (DSBs), this study identifies a novel regulatory role for TDP43 in the DNA mismatch repair (MMR) pathway. We demonstrate that depletion or overexpression of TDP43 affects the expression of key MMR genes, including MLH1, MSH6, MSH2, MSH3, and PMS2. Specifically, TDP43 modulates the expression of MLH1 and MSH6 proteins through alternative splicing and transcript stability. These findings are validated in ALS mice models, patient-derived neural progenitor cells and autopsied brain tissues from ALS patients. Furthermore, MMR depletion showed a partial rescue of TDP43-induced DNA damage in neuronal cells. Bioinformatics analysis of TCGA cancer database reveals significant correlations between TDP43 and MMR gene expressions and mutational burden across various cancer subtypes. These results collectively establish TDP43 as a critical regulator of the MMR pathway, with broad implications for understanding the genomic instability underlying neurodegenerative and neoplastic diseases.

Investigation of an MCM8 gene variant in women with premature ovarian insufficiency.

The MCM8 gene is involved in the homologous recombination and repair of double-stranded DNA breaks. It maintains the meiotic process continuously. If the MCM8-9 helicase does not function, the accumulation of DNA breaks can result in cell death. Studies have reported MCM8 gene suppression with primary ovarian insufficiency. In the present study, a variant of the MCM8 gene was investigated in women with primary ovarian insufficiency to elucidate the role of MCM8 in this pathology. To evaluate the frequency of the NG_042869.1:g.40270G>A variant of the MCM8 gene in the study population. The MCM8 gene variant was analyzed via real-time polymerase chain reaction using a hydrolysis probe in DNA samples from women diagnosed with primary ovarian insufficiency, with a normal karyotype and without FMR1 gene permutation, and from a Control Group, who had menopause after 50 years of age. Frequencies were compared using Fisher's exact test. A total of 100 samples from the Case Group and 100 samples from the Control Group were selected. The variant was detected in heterozygosity in a Case Group sample but was not identified in the Control Group. This variant was first described in a consanguineous Arab family. This variant is classified as pathogenic and has a prevalence of 1% in women with primary ovarian insufficiency. This study is a pioneering investigation of this variant in Brazilian women. These findings suggest that the rs138761187 variant of the MCM8 gene is rare in Brazilian women.

DNA methylation drives hematopoietic stem cell aging phenotypes after proliferative stress.

Aging of hematopoietic stem cells (HSCs) is implicated in various aging phenotypes, including immune dysfunction, anemia, and malignancies. The role of HSC proliferation in driving these aging phenotypes, particularly under stress conditions, remains unclear. Therefore, we induced forced replications of HSCs in vivo by a cyclical treatment with low-dose fluorouracil (5FU) and examined the impact on HSC aging. Our findings show that proliferative stress induces several aging phenotypes, including altered leukocyte counts, decreased lymphoid progenitors, accumulation of HSCs with high expression of Slamf1, and reduced reconstitution potential, without affecting stem cell self-renewal capacity. The divisional history of HSCs was imprinted in the DNA methylome, consistent with functional decline. Specifically, DNA methylation changes included global hypermethylation in non-coding regions and similar frequencies of hypo- and hyper-methylation at promoter regions, particularly affecting genes targeted by the PRC2 complex. Importantly, initial forced replication promoted DNA damage repair accumulated with age, but continuous proliferative stress led to the accumulation of double-strand breaks, independent of functional decline. Overall, our results suggest that HSC proliferation can drive some aging phenotypes primarily through epigenetic mechanisms, including DNA methylation changes.

Fructose-2,6-bisphosphate restores DNA repair activity of PNKP and ameliorates neurodegenerative symptoms in Huntington’s disease

Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3) are the two most prevalent polyglutamine (polyQ) neurodegenerative diseases, caused by CAG (encoding glutamine) repeat expansion in the coding region of the huntingtin (HTT) and ataxin-3 (ATXN3) proteins, respectively. We have earlier reported that the activity, but not the protein level, of an essential DNA repair enzyme, polynucleotide kinase 3'-phosphatase (PNKP), is severely abrogated in both HD and SCA3 resulting in accumulation of double-strand breaks in patients' brain genome. While investigating the mechanistic basis for the loss of PNKP activity and accumulation of DNA double-strand breaks leading to neuronal death, we observed that PNKP interacts with the nuclear isoform of 6-phosphofructo-2-kinase fructose-2,6-bisphosphatase 3 (PFKFB3). Depletion of PFKFB3 markedly abrogates PNKP activity without changing its protein level. Notably, the levels of both PFKFB3 and its product fructose-2,6 bisphosphate (F2,6BP), an allosteric modulator of glycolysis, are significantly lower in the nuclear extracts of postmortem brain tissues of HD and SCA3 patients. Supplementation of F2,6BP restored PNKP activity in the nuclear extracts of patients' brain. Moreover, intracellular delivery of F2,6BP restored both the activity of PNKP and the integrity of transcribed genome in neuronal cells derived from the striatum of the HD mouse. Importantly, supplementing F2,6BP rescued the HD phenotype in Drosophila, suggesting F2,6BP to serve in vivo as a cofactor for the proper functionality of PNKP and thereby, of brain health. Our results thus provide a compelling rationale for exploring the therapeutic use of F2,6BP and structurally related compounds for treating polyQ diseases.

CDK12 controls transcription at damaged genes and prevents MYC-induced transcription-replication conflicts

The identification of genes involved in replicative stress is key to understanding cancer evolution and to identify therapeutic targets. Here, we show that CDK12 prevents transcription-replication conflicts (TRCs) and the activation of cytotoxic replicative stress upon deregulation of the MYC oncogene. CDK12 was recruited at damaged genes by PARP-dependent DDR-signaling and elongation-competent RNAPII, to repress transcription. Either loss or chemical inhibition of CDK12 led to DDR-resistant transcription of damaged genes. Loss of CDK12 exacerbated TRCs in MYC-overexpressing cells and led to the accumulation of double-strand DNA breaks, occurring between co-directional early-replicating regions and transcribed genes. Overall, our data demonstrate that CDK12 protects genome integrity by repressing transcription of damaged genes, which is required for proper resolution of DSBs at oncogene-induced TRCs. This provides a rationale that explains both how CDK12 deficiency can promote tandem duplications of early-replicated regions during tumor evolution, and how CDK12 targeting can exacerbate replicative-stress in tumors.

Genotype-Directed Synthetic Cytotoxicity of ATR Inhibition with Radiotherapy.

The importance of the DNA damage response in mediating effects of radiotherapy (RT) has galvanized efforts to target this pathway with radiosensitizers. Yet early clinical trials of this approach have failed to yield a benefit in unselected populations. We hypothesized that ataxia-telangiectasia mutated (Atm)-null tumors would demonstrate genotype-specific synergy between RT and an inhibitor of the DNA damage response protein ataxia-telangiectasia and Rad3-related (ATR) kinase. We investigated the synergistic potential of the ATR inhibitor (ATRi) RP-3500 and RT in two Atm-null and isogenic murine models, both in vitro and in vivo. Staining of γ-H2AX foci, characterization of the immune response via flow cytometry, and tumor rechallenge experiments were performed to elucidate the mechanism of interaction. To examine genotype specificity, we tested the interaction of ATRi and RT in a Brca1-null model. Finally, patients with advanced cancer with ATM alterations were enrolled in a phase I/II clinical trial to validate preclinical findings. Synergy between RP-3500 and RT was confirmed in Atm-null lines in vitro, characterized by an accumulation of DNA double-strand breaks. In vivo, Atm-null tumor models had higher rates of durable control with RT and ATRi than controls. In contrast, there was no synergy in tumors lacking Brca1. Analysis of the immunologic response indicated that efficacy is largely mediated by cell-intrinsic mechanisms. Lastly, early results from our clinical trial showed complete responses in patients. Genotype-directed radiosensitization with ATRi and RT can unleash significant therapeutic benefit and could represent a novel approach to develop more effective combinatorial synthetic cytotoxic RT-based treatments.

Mechanisms of cytotoxicity in six classes of ionic liquids: Evaluating cell cycle impact and genotoxic and apoptotic effects

Ionic liquids (ILs), earlier praised for their eco-friendliness, have emerged as key chemicals in advancing green chemistry, catalysis, solvent development, and more. However, the discovery of their notable toxicity has led to a controversial reputation of ILs and has shifted the research landscape towards understanding their biological impacts. The present study examines the mechanism of cytotoxicity of 32 ILs across six classes, highlighting their effects on the cell cycle of the Jurkat cell line. Focusing on five ILs with pronounced cytotoxicity, we uncover their genotoxic effects and their role in inducing apoptosis. Our findings suggest intricate interplay between the extrinsic and intrinsic apoptotic pathways at different time points after exposure to ILs. Moreover, the ILs studied displayed marked genotoxicity, likely stemming from the accumulation of double-strand DNA breaks in the Jurkat cells.This investigation offers a comprehensive view on interactions of ILs with eukaryotic cells, thereby providing new guidelines for developing safer pharmaceutical and industrial applications of these chemicals. The results not only broaden and enhance the previous perceptions but also open new avenues in research, emphasizing the dual potential of ILs in innovation and safety, and marking a significant step towards integrating chemical innovations with biological safety.

Progerin Can Induce DNA Damage in the Absence of Global Changes in Replication or Cell Proliferation.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging, and individuals with HGPS seldom live beyond their mid-teens. The syndrome is commonly caused by a point mutation in the LMNA gene which codes for lamin A and its splice variant lamin C, components of the nuclear lamina. The mutation causing HGPS leads to production of a truncated, farnesylated form of lamin A referred to as "progerin." Progerin is also expressed at low levels in healthy individuals and appears to play a role in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs) and alterations in the nature of DSB repair. The source of DSBs in HGPS is often attributed to stalling and subsequent collapse of replication forks in conjunction with faulty recruitment of repair factors to damage sites. In this work, we used a model system involving immortalized human cell lines to investigate progerin-induced genomic damage. Using an immunofluorescence approach to visualize phosphorylated histone H2AX foci which mark sites of genomic damage, we report that cells engineered to express progerin displayed a significant elevation of endogenous damage in the absence of any change in the cell cycle profile or doubling time of cells. Genomic damage was enhanced and persistent in progerin-expressing cells treated with hydroxyurea. Overexpression of wild-type lamin A did not elicit the outcomes associated with progerin expression. Our results show that DNA damage caused by progerin can occur independently from global changes in replication or cell proliferation.

Sustained inactivation of the Polycomb PRC1 complex induces DNA repair defects and genomic instability in epigenetic tumors

Cancer initiation and progression are typically associated with the accumulation of driver mutations and genomic instability. However, recent studies demonstrated that cancer can also be driven purely by epigenetic alterations, without driver mutations. Specifically, a 24-h transient downregulation of polyhomeotic (ph-KD), a core component of the Polycomb complex PRC1, is sufficient to induce epigenetically initiated cancers (EICs) in Drosophila, which are proficient in DNA repair and characterized by a stable genome. Whether genomic instability eventually occurs when PRC1 downregulation is performed for extended periods of time remains unclear. Here, we show that prolonged depletion of PH, which mimics cancer initiating events, results in broad dysregulation of DNA replication and repair genes, along with the accumulation of DNA breaks, defective repair, and widespread genomic instability in the cancer tissue. A broad misregulation of H2AK118 ubiquitylation and to a lesser extent of H3K27 trimethylation also occurs and might contribute to these phenotypes. Together, this study supports a model where DNA repair and replication defects accumulate during the tumorigenic transformation epigenetically induced by PRC1 loss, resulting in genomic instability and cancer progression.

FANCD2 counteracts O6-methylguanine-induced mismatch repair-dependent apoptosis.

Sn1-type alkylating agents methylate the oxygen atom on guanine bases thereby producing O6-methylguanine. This modified base could pair with thymine and cytosine, resulting in the formation of O6-methylguanine/thymine mismatch during DNA replication, recognized by the mismatch repair (MMR) complex, which then initiates the DNA damage response and subsequent apoptotic processes. In our investigation of the molecular mechanisms underlying MMR-dependent apoptosis, we observed FANCD2 modification upon the activity of alkylating agent N-methyl-N-nitrosourea (MNU). This observation led us to hypothesize a relevant role for FANCD2 in the apoptosis induction process. We generated FANCD2 knockout cells using the CRISPR/Cas9 method in the human cervical cancer cell line HeLa MR. FANCD2-deficient cells exhibited MNU hypersensitivity. Upon MNU exposure, FANCD2 colocalized with the MMR complex. MNU-treated FANCD2 knockout cells displayed severe S phase delay followed by increased G2/M arrest and MMR-dependent apoptotic cell death. Moreover, FANCD2 knockout cells exhibited impaired CtIP and RAD51 recruitment to the damaged chromatin and DNA double-strand break accumulation, indicated by simultaneously observed increased γH2AX signal and 53BP1 foci. Our data suggest that FANCD2 is crucial for recruiting homologous recombination factors to the sites of the MMR-dependent replication stress to resolve the arrested replication fork and counteract O6-methylguanine-triggered MMR-dependent apoptosis.

Bloom syndrome DNA helicase mitigates mismatch repair-dependent apoptosis

Generation of O6-methylguanine (O6-meG) by DNA-alkylating agents such as N-methyl N-nitrosourea (MNU) activates the multiprotein mismatch repair (MMR) complex and the checkpoint response involving ATR/CHK1 and ATM/CHK2 kinases, which may in turn trigger cell cycle arrest and apoptosis. The Bloom syndrome DNA helicase BLM interacts with the MMR complex, suggesting functional relevance to repair and checkpoint responses. We observed a strong interaction of BLM with MMR proteins in HeLa cells upon treatment with MNU as evidenced by co-immunoprecipitation as well as colocalization in the nucleus as revealed by dual immunofluorescence staining. Knockout of BLM sensitized HeLa MR cells to MNU-induced cell cycle disruption and enhanced expression of the apoptosis markers cleaved caspase-9 and PARP1. MNU-treated BLM-deficient cells also exhibited a greater number of 53BP1 foci and greater phosphorylation levels of H2AX at S139 and RPA32 at S8, indicating the accumulation of DNA double-strand breaks. These findings suggest that BLM prevents double-strand DNA breaks during the MMR-dependent DNA damage response and mitigates O6-meG-induced apoptosis.

Nature-inspired substituted 3-(imidazol-2-yl) morpholines targeting human topoisomerase IIα: Dynophore-derived discovery

The molecular nanomachine, human DNA topoisomerase IIα, plays a crucial role in replication, transcription, and recombination by catalyzing topological changes in the DNA, rendering it an optimal target for cancer chemotherapy. Current clinical topoisomerase II poisons often cause secondary tumors as side effects due to the accumulation of double-strand breaks in the DNA, spurring the development of catalytic inhibitors. Here, we used a dynamic pharmacophore approach to develop catalytic inhibitors targeting the ATP binding site of human DNA topoisomerase IIα. Our screening of a library of nature-inspired compounds led to the discovery of a class of 3-(imidazol-2-yl) morpholines as potent catalytic inhibitors that bind to the ATPase domain. Further experimental and computational studies identified hit compound 17, which exhibited selectivity against the human DNA topoisomerase IIα versus human protein kinases, cytotoxicity against several human cancer cells, and did not induce DNA double-strand breaks, making it distinct from clinical topoisomerase II poisons. This study integrates an innovative natural product-inspired chemistry and successful implementation of a molecular design strategy that incorporates a dynamic component of ligand-target molecular recognition, with comprehensive experimental characterization leading to hit compounds with potential impact on the development of more efficient chemotherapies.

VRK1 Regulates Sensitivity to Oxidative Stress by Altering Histone Epigenetic Modifications and the Nuclear Phosphoproteome in Tumor Cells.

The chromatin organization and its dynamic remodeling determine its accessibility and sensitivity to DNA damage oxidative stress, the main source of endogenous DNA damage. We studied the role of the VRK1 chromatin kinase in the response to oxidative stress. which alters the nuclear pattern of histone epigenetic modifications and phosphoproteome pathways. The early effect of oxidative stress on chromatin was studied by determining the levels of 8-oxoG lesions and the alteration of the epigenetic modification of histones. Oxidative stress caused an accumulation of 8-oxoG DNA lesions that were increased by VRK1 depletion, causing a significant accumulation of DNA strand breaks detected by labeling free 3'-DNA ends. In addition, oxidative stress altered the pattern of chromatin epigenetic marks and the nuclear phosphoproteome pathways that were impaired by VRK1 depletion. Oxidative stress induced the acetylation of H4K16ac and H3K9 and the loss of H3K4me3. The depletion of VRK1 altered all these modifications induced by oxidative stress and resulted in losses of H4K16ac and H3K9ac and increases in the H3K9me3 and H3K4me3 levels. All these changes were induced by the oxidative stress in the epigenetic pattern of histones and impaired by VRK1 depletion, indicating that VRK1 plays a major role in the functional reorganization of chromatin in the response to oxidative stress. The analysis of the nuclear phosphoproteome in response to oxidative stress detected an enrichment of the phosphorylated proteins associated with the chromosome organization and chromatin remodeling pathways, which were significantly decreased by VRK1 depletion. VRK1 depletion alters the histone epigenetic pattern and nuclear phosphoproteome pathways in response to oxidative stress. The enzymes performing post-translational epigenetic modifications are potential targets in synthetic lethality strategies for cancer therapies.

TRIM33 loss in multiple myeloma is associated with genomic instability and sensitivity to PARP inhibitors

Deletions of chromosome 1p (del(1p)) are a recurrent genomic aberration associated with poor outcome in Multiple myeloma (MM.) TRIM33, an E3 ligase and transcriptional co-repressor, is located within a commonly deleted region at 1p13.2. TRIM33 is reported to play a role in the regulation of mitosis and PARP-dependent DNA damage response (DDR), both of which are important for maintenance of genome stability. Here, we demonstrate that MM patients with loss of TRIM33 exhibit increased chromosomal instability and poor outcome. Through knockdown studies, we show that TRIM33 loss induces a DDR defect, leading to accumulation of DNA double strand breaks (DSBs) and slower DNA repair kinetics, along with reduced efficiency of non-homologous end joining (NHEJ). Furthermore, TRIM33 loss results in dysregulated ubiquitination of ALC1, an important regulator of response to PARP inhibition. We show that TRIM33 knockdown sensitizes MM cells to the PARP inhibitor Olaparib, and this is synergistic with the standard of care therapy bortezomib, even in co-culture with bone marrow stromal cells (BMSCs). These findings suggest that TRIM33 loss contributes to the pathogenesis of high-risk MM and that this may be therapeutically exploited through the use of PARP inhibitors.

Abstract 1467: Contribution of estrogen metabolites to never-smoking lung cancer

Abstract There has been a consistent rise in the incidence of non-small cell lung cancer (NSCLC) among individuals who have never smoked. Remarkably, over 50% of diagnosed female cases and 15-20% of male cases are never-smokers. Notably, NSCLC in never-smokers exhibits distinctive clinical and pathological features, notably a higher incidence of tumors with EGFR mutations, setting it apart from its counterpart in smokers. Despite the improved disease-free survival observed with EGFR-tyrosine kinase inhibitors, eventual relapse and limited responsiveness to immunotherapy are common, underscoring the urgency to comprehend the molecular mechanisms driving the escalating prevalence of NSCLC in never-smokers and develop targeted preventive and interceptive therapeutic strategies. The preponderance of females among NSCLC patients who never smoked has prompted an exploration into the potential contribution of estrogen to lung tumorigenesis. In contrast to prevailing research focused on estrogen receptor-mediated signaling, our investigations have yielded compelling evidence supporting the involvement of estrogen metabolism in lung carcinogenesis among never-smokers. This group is the first to report that estrogen is metabolized extensively in both mouse and human lungs, resulting in the generation of various derivatives, including the potential carcinogen 4-hydroxyestrogen (4-OHE). Notably, recent findings from our laboratory indicate that prolonged exposure of nonneoplastic human bronchial epithelial cells to 17β estradiol or 4-OHE, at concentrations approximating physiological levels, induces the accumulation of double-strand DNA breaks and cellular transformation. A comprehensive analysis was performed to further elucidate the mechanisms underlying the transformation process. Isolated transformed clones obtained after 9, 18, and 22 wks of exposure to 4-OHE were subjected to karyotyping, F.I.S.H., and RNA and exome sequencing. A longitudinal progression of aberrant chromosomal rearrangements and copy number alterations was observed, that increased with time of 4-OHE exposure. In addition, RNA-Seq analyses revealed the epithelial-mesenchymal transition pathway was highly enriched at the early timepoint (9 wks), while replicative stress-related signaling pathways were induced at all timepoints. To determine if the differentially expressed genes were also dysregulated in clinical lung cancer cases, TCGA lung adenocarcinoma (LUAD) data were examined. Interestingly, many of the genes upregulated after estrogen exposure were similarly overexpressed in the LUAD samples and often associated with a worse prognosis. In conclusion, our data support the role of estrogen metabolites in promoting lung cancer among never-smokers. (Supported by CA217161 and generous contributions from the Hellendall Family Foundation, Gregory G. Lawton and the Timothy and Aurora Hughes Cancer Research Fund.) Citation Format: Mitchell Cheung, Lisa A. Vanderveer, Daniel Krzizike, J. Nicholas Bodor, Joseph Treat, Joseph R. Testa, Margie L. Clapper. Contribution of estrogen metabolites to never-smoking lung cancer [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 1467.

A synergistic two-drug therapy specifically targets a DNA repair dysregulation that occurs in p53-deficient colorectal and pancreatic cancers

The tumor-suppressor p53 is commonly inactivated in colorectal cancer and pancreatic ductal adenocarcinoma, but existing treatment options for p53-mutant (p53Mut) cancer are largely ineffective. Here, we report a therapeutic strategy for p53Mut tumors based on abnormalities in the DNA repair response. Investigation of DNA repair upon challenge with thymidine analogs reveals a dysregulation in DNA repair response in p53Mut cells that leads to accumulation of DNA breaks. Thymidine analogs do not interrupt DNA synthesis but induce DNA repair that involves a p53-dependent checkpoint. Inhibitors of poly(ADP-ribose) polymerase (PARPis) markedly enhance DNA double-strand breaks and cell death induced by thymidine analogs in p53Mut cells, whereas p53 wild-type cells respond with p53-dependent inhibition of the cell cycle. Combinations of trifluorothymidine and PARPi agents demonstrate superior anti-neoplastic activity in p53Mut cancer models. These findings support a two-drug combination strategy to improve outcomes for patients with p53Mut cancer.

DNA damage-induced allosteric activation of protein phosphatase PP1:NIPP1 through Src kinase-induced circularization of NIPP1.

Protein phosphatase-1 (PP1) complexed to nuclear inhibitor of PP1 (NIPP1) limits DNA repair through dephosphorylation of NIPP1-recruited substrates. However, the PP1:NIPP1 holoenzyme is completely inactive under basal conditions, hinting at a DNA damage-regulated activation mechanism. Here, we report that DNA damage caused the activation of PP1:NIPP1 after a time delay of several hours through phosphorylation of NIPP1 at the C-terminal tyrosine 335 (Y335) by a Src-family kinase. PP1:NIPP1 activation partially resulted from the dissociation of the C terminus of NIPP1 from the active site of PP1. In addition, the released Y335-phosphorylated C terminus interacted with the N terminus of NIPP1 to enhance substrate recruitment by the flanking forkhead-associated (FHA) domain. Constitutive activation of PP1:NIPP1 by knock-in of a phospho-mimicking (Y335E) NIPP1 mutant led to the hypo-phosphorylation of FHA ligands and an accumulation of DNA double-strand breaks. Our data indicate that PP1:NIPP1 activation through circularization of NIPP1 is a late response to DNA damage that contributes to the timely recovery from damage repair.