DNA Damage Response Kinase Research Articles

Mitochondrial fatty acid oxidation drives senescence.

Cellular senescence is a stress-induced irreversible cell cycle arrest involved in tumor suppression and aging. Many stresses, such as telomere shortening and oncogene activation, induce senescence by damaging nuclear DNA. However, the mechanisms linking DNA damage to senescence remain unclear. Here, we show that DNA damage response (DDR) signaling to mitochondria triggers senescence. A genome-wide small interfering RNA screen implicated the outer mitochondrial transmembrane protein BNIP3 in senescence induction. We found that BNIP3 is phosphorylated by the DDR kinase ataxia telangiectasia mutated (ATM) and contributes to an increase in the number of mitochondrial cristae. Stable isotope labeling metabolomics indicated that the increase in cristae enhances fatty acid oxidation (FAO) to acetyl-coenzyme A (acetyl-CoA). This promotes histone acetylation and expression of the cyclin-dependent kinase inhibitor p16INK4a. Notably, pharmacological activation of FAO alone induced senescence both in vitro and in vivo. Thus, mitochondrial energy metabolism plays a critical role in senescence induction and is a potential intervention target to control senescence.

DNA double-strand break-capturing nuclear envelope tubules drive DNA repair.

Current models suggest that DNA double-strand breaks (DSBs) can move to the nuclear periphery for repair. It is unclear to what extent human DSBs display such repositioning. Here we show that the human nuclear envelope localizes to DSBs in a manner depending on DNA damage response (DDR) kinases and cytoplasmic microtubules acetylated by α-tubulin acetyltransferase-1 (ATAT1). These factors collaborate with the linker of nucleoskeleton and cytoskeleton complex (LINC), nuclear pore complex (NPC) protein NUP153, nuclear lamina and kinesins KIF5B and KIF13B to generate DSB-capturing nuclear envelope tubules (dsbNETs). dsbNETs are partly supported by nuclear actin filaments and the circadian factor PER1 and reversed by kinesin KIFC3. Although dsbNETs promote repair and survival, they are also co-opted during poly(ADP-ribose) polymerase (PARP) inhibition to restrain BRCA1-deficient breast cancer cells and are hyper-induced in cells expressing the aging-linked lamin A mutant progerin. In summary, our results advance understanding of nuclear structure-function relationships, uncover a nuclear-cytoplasmic DDR and identify dsbNETs as critical factors in genome organization and stability.

Physiological DNA damage promotes functional endoreplication of mammary gland alveolar cells during lactation

Lactation insufficiency affects many women worldwide. During lactation, a large portion of mammary gland alveolar cells become polyploid, but how these cells balance the hyperproliferation occurring during normal alveologenesis with terminal differentiation required for lactation is unknown. Here, we show that DNA damage accumulates due to replication stress during pregnancy, activating the DNA damage response. Modulation of DNA damage levels in vivo by intraductal injections of nucleosides or DNA damaging agents reveals that the degree of DNA damage accumulated during pregnancy governs endoreplication and milk production. We identify a mechanism involving early mitotic arrest through CDK1 inactivation, resulting in a heterogeneous alveolar population with regards to ploidy and nuclei number. The inactivation of CDK1 is mediated by the DNA damage response kinase WEE1 with homozygous loss of Wee1 resulting in decreased endoreplication, alveologenesis and milk production. Thus, we propose that the DNA damage response to replication stress couples proliferation and endoreplication during mammary gland alveologenesis. Our study sheds light on mechanisms governing lactogenesis and identifies non-hormonal means for increasing milk production.

A Novel Dual ATM/DNA-PK Inhibitor, XRD-0394, Potently Radiosensitizes and Potentiates PARP and Topoisomerase I Inhibitors.

A majority of patients with cancer receive radiotherapy as part of their treatment regimens whether using external beam therapy or locally-delivered radioisotopes. While often effective, some tumors are inadequately controlled with radiation and radiotherapy has significant short-term and long-term toxicities for cancer survivors. Insights into molecular mechanisms involved in cellular responses to DNA breaks introduced by radiation or other cancer therapies have been gained in recent years and approaches to manipulate these responses to enhance tumor cell killing or reduce normal tissue toxicity are of great interest. Here, we report the identification and initial characterization of XRD-0394, a potent and specific dual inhibitor of two DNA damage response kinases, ATM and DNA-PKcs. This orally bioavailable molecule demonstrates significantly enhanced tumor cell kill in the setting of therapeutic ionizing irradiation in vitro and in vivo. XRD-0394 also potentiates the effectiveness of topoisomerase I inhibitors in vitro. In addition, in cells lacking BRCA1/2 XRD-0394 shows single-agent activity and synergy in combination with PARP inhibitors. A phase Ia clinical trial (NCT05002140) with XRD-0394 in combination with radiotherapy has completed. These results provide a rationale for future clinical trials with XRD-0394 in combination with radiotherapy, PARP inhibitors, and targeted delivery of topoisomerase I inhibitors.

DNA-PK and ATM drive phosphorylation signatures that antagonistically regulate cytokine responses to herpesvirus infection or DNA damage

The DNA-dependent protein kinase, DNA-PK, is an essential regulator of DNA damage repair. DNA-PK-driven phosphorylation events and the activated DNA damage response (DDR) pathways are also components of antiviral intrinsic and innate immune responses. Yet, it is not clear whether and how the DNA-PK response differs between these two forms of nucleic acid stress-DNA damage and DNA virus infection. Here, we define DNA-PK substrates and the signature cellular phosphoproteome response to DNA damage or infection with the nuclear-replicating DNA herpesvirus, HSV-1. We establish that DNA-PK negatively regulates the ataxia-telangiectasia-mutated (ATM) DDR kinase during viral infection. In turn, ATM blocks the binding of DNA-PK and the nuclear DNA sensor IFI16 to viral DNA, thereby inhibiting cytokine responses. However, following DNA damage, DNA-PK enhances ATM activity, which is required for IFN-β expression. These findings demonstrate that the DDR autoregulates cytokine expression through the opposing modulation of DDR kinases.

Abstract 5616: Targeting the DNA damage response sensor replication protein A for first in class cancer therapy

Abstract The DNA Damage Response (DDR) is an integrated network of DNA repair and cell signaling pathways that are critical towards maintaining genomic stability. The most successful DDR targeted therapies inhibit the DNA damage sensor, PARP, a protein that recognizes single strand DNA (ssDNA) breaks as well as other DNA secondary structures induced by single strand gaps that can initiate chromosome instability and cell death. Building on the initial clinical success of PARP inhibitors, development of DDR targeted therapeutics has become increasingly popular, however, the clinical outcomes with these therapeutics have not met expectations and thus a new approach to targeting the DDR pathway is needed. NERx Biosciences has developed a novel strategy of targeting the DDR by intervening upstream of the DDR kinases and targeting specific DDR sensors. The human ssDNA binding protein, replication protein A (RPA), is a critical sensor for the DDR, serving to sense the ssDNA at replication forks that arise from replication stress (RS) and is a novel target for cancer therapy. We have discovered, developed, and characterized a novel small molecule RPA inhibitor (RPAi) NERx-329 that blocks the RPA-DNA interaction and elicits a state of chemical RPA exhaustion that results in in vivo anticancer activity. Extensive chemical optimization enabled in vitro and in vivo analyses to elucidate mechanism of action, cellular engagement, and therapeutic activity of RPA-targeted agents. Biochemical reconstitution of the ATR signaling pathway shows that NERx-329 disrupts ATR kinase activity, further suggesting a novel mechanism of action driven by RPA inhibition induced replication catastrophe. In vivo analysis reveals activity in a series of lung and ovarian patient derived xenograft models of human cancer. Single agent activity results in a tumor growth delay, which can be enhanced in combination therapy with DDR inhibitors as well as traditional DNA damaging cancer therapeutics. Data demonstrate that tumor growth observed in vivo is a function of NERx329 bioavailability and not development of a specific resistance mechanism. In an effort to identify formulations effective in enhancing oral bioavailability, we have pursued the development of Spray-dried dispersions (SDD). PK analysis reveal a dose-dependent substantial increase in CMAX and AUC, allowing for optimal dosing regimens for efficacy and safety studies. In addition, specific genetic predictors of RPAi sensitivity have been identified and results demonstrate specific genetic alterations increase RPAi activity in vitro and in vivo. NERx-329 represents the first clinically viable agent to target the DDR pathway by disrupting the RPA-DNA interaction and holds the potential for significant impact in cancer treatment. Citation Format: Katherine Pawelczak, Pamela VanderVere-Carozza, Matthew Jordan, John Turchi. Targeting the DNA damage response sensor replication protein A for first in class cancer therapy [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 5616.

Meiotic prophase length modulates Tel1-dependent DNA double-strand break interference.

During meiosis, genetic recombination is initiated by the formation of many DNA double-strand breaks (DSBs) catalysed by the evolutionarily conserved topoisomerase-like enzyme, Spo11, in preferred genomic sites known as hotspots. DSB formation activates the Tel1/ATM DNA damage responsive (DDR) kinase, locally inhibiting Spo11 activity in adjacent hotspots via a process known as DSB interference. Intriguingly, in S. cerevisiae, over short genomic distances (<15 kb), Spo11 activity displays characteristics of concerted activity or clustering, wherein the frequency of DSB formation in adjacent hotspots is greater than expected by chance. We have proposed that clustering is caused by a limited number of sub-chromosomal domains becoming primed for DSB formation. Here, we provide evidence that DSB clustering is abolished when meiotic prophase timing is extended via deletion of the NDT80 transcription factor. We propose that extension of meiotic prophase enables most cells, and therefore most chromosomal domains within them, to reach an equilibrium state of similar Spo11-DSB potential, reducing the impact that priming has on estimates of coincident DSB formation. Consistent with this view, when Tel1 is absent but Ndt80 is present and thus cells are able to rapidly exit meiotic prophase, genome-wide maps of Spo11-DSB formation are skewed towards pericentromeric regions and regions that load pro-DSB factors early-revealing regions of preferential priming-but this effect is abolished when NDT80 is deleted. Our work highlights how the stochastic nature of Spo11-DSB formation in individual cells within the limited temporal window of meiotic prophase can cause localised DSB clustering-a phenomenon that is exacerbated in tel1Δ cells due to the dual roles that Tel1 has in DSB interference and meiotic prophase checkpoint control.

Abstract IA022: Uracil DNA glycosylase activity limits deoxyuridine contamination in genomic DNA and is essential for the type-1 interferon response in cells treated with ATR kinase inhibitors

Abstract Most chemotherapies target DNA replication, and their efficacy depends on the DNA damage response that integrates DNA repair with the cell cycle. Widely used standard-of-care antimetabolites inhibit thymidylate synthase and increase the incorporation of deoxyuridine (dU) into DNA by polymerases. Contamination of dU in DNA is limited by the DNA damage response kinase ATR that induces cell cycle checkpoints and reduces the rate of DNA replication. ATR inhibitors (ATRi) induce origin firing across active replicons and cause ribonuclease reductase degradation in otherwise unperturbed cells. This increases both the amount of DNA replication and the amount of free dUTP in cells. Thus, ATRi increase the incorporation of dU into DNA by polymerases. Since ATRi also inhibit cell cycle checkpoints, ATRi induce more dU contamination than antimetabolites. ATRi-induced dU contamination in DNA is associated with an innate immune response. We showed this with the simple observation that ATRi-induced dU contamination and IFN-α/β expression is reversed by low doses of thymidine. Here we show that ATRi-induced IFN-α/β response is dependent on uracil DNA glycosylase (UNG) which removes dU from DNA. Our data are consistent with a model in which UNG-dependent base excision repair (BER) removes dU from DNA, ultimately generating cytoplasmic dsDNA that induces IFN-α/β. We propose that ATRi-induced dU contamination contributes to dose-limiting leukocytopenia and inflammation in the clinic and CD8+ T cell dependent anti-tumor responses in mouse models of cancer treated with radiation therapy. Citation Format: Pinakin Pandya, Frank P. Vendetti, Joseph A. Ghoubaira, Sudipta Pathak, Joshua J. Deppas, Reyna E. Jones, Yunqi Zhang, Daniel Ivanov, Raquel Buj, Katherine M. Aird, Jan H. Beumer, Robert W. Sobol, Christopher J. Bakkenist. Uracil DNA glycosylase activity limits deoxyuridine contamination in genomic DNA and is essential for the type-1 interferon response in cells treated with ATR kinase inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA022.

Abstract B020: Targeting the DNA damage response sensor Replication Protein A for first in class cancer therapy

Abstract The DNA Damage Response (DDR) is a DNA repair and cell signaling pathway crucial in maintaining genomic stability. The most successful DDR targeted therapies inhibit the DNA damage sensor, PARP, a protein that recognizes single strand DNA (ssDNA) breaks as well as other DNA secondary structures induced by single strand gaps that can initiate chromosome instability and cell death. Building on the initial clinical success of PARP inhibitors, development of DDR targeted therapeutics has become increasingly popular with the majority of new agents targeting protein kinases downstream of the DNA damage sensors. The clinical outcomes with these therapeutics have not met expectations and thus a new approach to targeting the DDR pathway is needed. NERx Biosciences has developed a novel strategy of targeting the DDR by intervening upstream of the DDR kinases and targeting specific DDR sensors. The human ssDNA binding protein, replication protein A (RPA), is a critical sensor for the DDR, serving to sense ssDNA at replication forks that arise from replication stress (RS) and is a novel target for cancer therapy. We have discovered, developed, and characterized a novel small molecule RPA inhibitor (RPAi) NERx-329 that blocks the RPA-DNA interaction and elicits a state of chemical RPA exhaustion that results in in vivo anticancer activity. Chemical optimization enabled in vitro and in vivo analyses to elucidate mechanism of action, cellular engagement, and therapeutic activity of RPA-targeted agents. Biochemical reconstitution of the ATR signaling pathway shows that NERx-329 disrupts ATR kinase activity, further suggesting a novel mechanism of action driven by RPA inhibition induced replication catastrophe. NERx-329 elicits single agent in vitro anticancer activity across a broad spectrum of cancers and the cellular response suggests existence of a threshold before chemical RPA exhaustion induces cell death. In vivo analysis reveals activity in a series of lung and ovarian PDX models of human cancer. Single agent activity results in a tumor growth delay, which can be enhanced in combination therapy with DDR inhibitors as well as traditional DNA damaging cancer therapeutics. Data demonstrate that tumor growth observed in vivo is a function of NERx329 bioavailability and not development of a specific resistance mechanism. In addition, specific genetic predictors of RPAi sensitivity have been identified and results demonstrate specific genetic alterations increase RPAi activity in vitro and in vivo. NERx-329 represents the first clinically viable agent to target the DDR pathway by disrupting the RPA-DNA interaction and holds potential for significant impact in cancer treatment. This is supported by data from a retrospective clinical assessment of RPA expression to assess treatment outcomes in lung cancer patients, revealing that high RPA expression is a negative prognostic biomarker correlating with worse overall patient survival. NERx 329 offers a novel therapeutic approach for these cancers that are not effectively treated with existing therapeutics. Citation Format: Katherine Pawelczak, Pamela VanderVere-Carozza, Matthew Jordon, Navnath Gavande, John Turchi. Targeting the DNA damage response sensor Replication Protein A for first in class cancer therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B020.

Kaposi's sarcoma herpesvirus exploits the DNA damage response to circularize its genome.

To establish lifelong, latent infection, herpesviruses circularize their linear, double-stranded, DNA genomes through an unknown mechanism. Kaposi's sarcoma (KS) herpesvirus (KSHV), a gamma herpesvirus, is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV persists in latently infected cells as a multi-copy, extrachromosomal episome. Here, we show the KSHV genome rapidly circularizes following infection, and viral protein expression is unnecessary for this process. The DNA damage response (DDR) kinases, ATM and DNA-PKcs, each exert roles, and absence of both severely compromises circularization and latency. These deficiencies were rescued by expression of ATM and DNA-PKcs, but not catalytically inactive mutants. In contrast, γH2AX did not function in KSHV circularization. The linear viral genomic ends resemble a DNA double strand break, and non-homologous DNA end joining (NHEJ) and homologous recombination (HR) reporters indicate both NHEJ and HR contribute to KSHV circularization. Last, we show, similar to KSHV, ATM and DNA-PKcs have roles in circularization of the alpha herpesvirus, herpes simplex virus-1 (HSV-1), while γH2AX does not. Therefore, the DDR mediates KSHV and HSV-1 circularization. This strategy may serve as a general herpesvirus mechanism to initiate latency, and its disruption may provide new opportunities for prevention of herpesvirus disease.

Profiling ubiquitin signalling with UBIMAX reveals DNA damage- and SCFβ-Trcp1-dependent ubiquitylation of the actin-organizing protein Dbn1

Ubiquitin widely modifies proteins, thereby regulating most cellular functions. The complexity of ubiquitin signalling necessitates unbiased methods enabling global detection of dynamic protein ubiquitylation. Here, we describe UBIMAX (UBiquitin target Identification by Mass spectrometry in Xenopus egg extracts), which enriches ubiquitin-conjugated proteins and quantifies regulation of protein ubiquitylation under precise and adaptable conditions. We benchmark UBIMAX by investigating DNA double-strand break-responsive ubiquitylation events, identifying previously known targets and revealing the actin-organizing protein Dbn1 as a major target of DNA damage-induced ubiquitylation. We find that Dbn1 is targeted for proteasomal degradation by the SCFβ-Trcp1 ubiquitin ligase, in a conserved mechanism driven by ATM-mediated phosphorylation of a previously uncharacterized β-Trcp1 degron containing an SQ motif. We further show that this degron is sufficient to induce DNA damage-dependent protein degradation of a model substrate. Collectively, we demonstrate UBIMAX’s ability to identify targets of stimulus-regulated ubiquitylation and reveal an SCFβ-Trcp1-mediated ubiquitylation mechanism controlled directly by the apical DNA damage response kinases.

Mapping combinatorial drug effects to DNA damage response kinase inhibitors

One fundamental principle that underlies various cancer treatments, such as traditional chemotherapy and radiotherapy, involves the induction of catastrophic DNA damage, leading to the apoptosis of cancer cells. In our study, we conduct a comprehensive dose-response combination screening focused on inhibitors that target key kinases involved in the DNA damage response (DDR): ATR, ATM, and DNA-PK. This screening involves 87 anti-cancer agents, including six DDR inhibitors, and encompasses 62 different cell lines spanning 12 types of tumors, resulting in a total of 17,912 combination treatment experiments. Within these combinations, we analyze the most effective and synergistic drug pairs across all tested cell lines, considering the variations among cancers originating from different tissues. Our analysis reveals inhibitors of five DDR-related pathways (DNA topoisomerase, PLK1 kinase, p53-inducible ribonucleotide reductase, PARP, and cell cycle checkpoint proteins) that exhibit strong combinatorial efficacy and synergy when used alongside ATM/ATR/DNA-PK inhibitors.

Abstract B057: Preclinical development of PKMYT1 and ATR inhibitor combinations

Abstract Background: Inactivation of the PKMYT1 kinase is synthetically lethal in tumor cells with genomic alterations leading to high replication stress, such as CCNE1 amplification frequently found in high-grade serous ovarian, uterine, and gastro-esophageal cancers. On this basis, we developed lunresertib (RP-6306), a first-in-class, potent and selective oral PKMYT1 inhibitor currently in Phase I clinical trials (NCT04855656). We have shown previously that PKMYT1 inhibition abrogates an inhibitory phosphorylation of the cyclin-dependent kinase CDK1 at threonine 14 (CDK1 pT14), leading to premature mitotic entry and catastrophic chromosome shattering specifically in cyclin E-high tumor cells, but not in cells with normal cyclin E levels. This leads to robust tumor growth inhibition by lunresertib in cyclin E-high pre-clinical models. We hypothesized that inhibition of the DNA damage response kinase ATR may further potentiate the tumor cytotoxicity of lunresertib, as ATR is known to regulate CDK1 pT14 levels in a parallel fashion to PKMYT1 through inhibition of the CDC25 family of phosphatases. Here we present the pre-clinical validation of this hypothesis. Methods: We used in vitro cell viability studies to determine the level of synergy between lunresertib and the ATR inhibitor camonsertib (RP-3500) in various engineered as well as tumor-derived models. We investigated the mechanism of action of the lunresertib/camonsertib combination using high content imaging and immunoblotting for DNA damage and cell cycle markers in cultured cells. Finally, we have evaluated the pre-clinical efficacy and tolerability of lunresertib/camonsertib combinations in vivo using mouse xenograft models. Results: In CCNE1-overexpressing cells, combining lunresertib with camonsertib led to a synergistic increase in cytotoxicity and catastrophic DNA damage at doses that did not affect wild type counterparts. Increased DNA damage resulted primarily from enhanced CDK1 dephosphorylation when lunresertib was combined with camonsertib, leading to robust and rapid induction of premature mitosis. Altogether these data suggest that camonsertib synergistically potentiates lunresertib’s mechanism of action. Consistent with that, we observed synergy between camonsertib and lunresertib in models carrying genomic biomarkers predictive of lunresertib sensitivity (CCNE1 amplification, FBXW7 loss-of-function), and not unrelated DNA repair defects (ATM or BRCA1 loss-of-function). In mouse xenograft models of tumors carrying predictive molecular alterations, the combination of lunresertib/camonsertib was well tolerated, enhanced CDK1 dephosphorylation and DNA damage, and was highly efficacious, including generating tumor regressions at doses sub-therapeutic as monotherapy. Conclusions: Lunresertib and camonsertib elicit synergistic activity in pre-clinical models of CCNE1-amplified and cyclin E-overexpressing tumors due to a complementary mechanism of action. These data provide a rationale for clinical development of PKMYT1 and ATR inhibitor combinations. Citation Format: David Gallo, Marc L. Hyer, Giovanni Martino, Adam Pertrone, Elia Aguado-Fraile, Rosie Kryczka, Jimmy Fourtounis, Rino Stocco, Stephen J. Morris, C. Gary Marshall, Anne Roulston, Michael Zinda, Michal Zimmermann. Preclinical development of PKMYT1 and ATR inhibitor combinations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B057.

Metabolomic Profiling Confirms the Key Role of Oxidative Stress in the Pathophysiology of Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA), a rare congenital bone marrow failure syndrome, characterized by severe anemia, erythroblastopenia, and congenital malformations is mostly associated with pathogenic variants in ribosomal protein (RP) genes, which cause their haploinsufficiency. The marked heterogeneity of the disease, related to different severity of hematological and non-hematological symptoms and diverse response to treatment, even in patients harboring the same RP mutation is not fully understood. Similarly, the mechanisms responsible for the predisposition of DBA patients to cancer await elucidation. We previously showed that the formation of reactive oxygen species (ROS) exceeds the anti-oxidant capacity of DBA erythrocytes leading to their higher susceptibility to destruction. Using DBA patients' samples and CRISPR/Cas9-created Rps19- and Rpl5-haploinsufficient MEL cellular models we also showed, that excessive oxidative stress, DNA damage, and activation of DNA damage response of erythroblasts is associated with an increased production of pro-/inflammatory cytokines and induction of apoptosis and senescence. In recent years, the use of metabolomic analysis appears as a promising tool that could contribute to a better understanding of the pathophysiology of the disease and discovery of novel biochemical markers with their potential use in diagnostics and prognostication. We therefore performed targeted metabolomic analysis of serum and erythrocyte samples from DBA patients (n=26) and healthy controls (n=10) using hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) where 129 metabolites were detected. Significant changes in the metabolome of DBA patients were observed, with the key deregulated metabolites involved in the antioxidant defense and energy metabolism; the reduction of amine compounds (creatinine, guanidinoacetate) and hippurate in DBA patients compared to controls was also noted. In two DBA families analyzed, the altered erythrocyte metabolism and markers of oxidative stress distinguished the patients from their asymptomatic family members with the same RP mutation. The incubation of erythrocytes from symptomatic DBA patients with antioxidant N-acetylcysteine in vitro led to normalization of ROS levels in erythrocytes. Similarly, the treatment of Rpl5- and Rps19-haploinsufficienct MEL cells with a pro-inflammatory cytokine inhibitor pomalidomide, resulted in the amelioration of oxidative DNA damage (assessed by the presence of 8-oxoguanine, 8-oxoG) and reduction of p53 activation. In addition, targeted metabolomic analysis of cellular extracts showed clear separation of Rps19- and Rpl5-haploinsufficient MEL cells from control cells with significant changes affecting the level of many carbohydrates and acylcarnitines. Elevated levels of glutathione and pentose phosphate cycle intermediates in Rps19- and Rpl5-haploinsufficient MEL cells correspond with their need to cope with increased oxidative stress. Significantly increased levels of deoxynucleotides, mainly in Rpl5-haploinsufficient MEL cells, could be related to DNA damage response (DDR) process to promote repair of ROS-mediated damaged DNA. Nucleotide formation is an energy intensive process and therefore the observed decrease in acylcarnitines could be related to increased beta oxidation and increased formation of reduced coenzymes. This result from model MEL cells is consistent with 8-oxoG foci and activation of ATM (a DDR kinase) observed in erythroblasts of DBA patients expanded in vitro. Importantly, immunoreactivity for 8-oxoG and phospho-ATM S1981 was detected also in non-erythroid bone marrow cells of two DBA patients, who recently developed myelodysplastic neoplasia (MDS). Collectively, our data support the involvement of oxidative stress and pro-/inflammatory cytokines in the pathophysiology of DBA and suggest that the treatment aiming to reduce oxidative stress and/or suppress inflammation could alleviate the severity of the disease, though additional studies are needed. The potential link between oxidative damage, inflammatory cytokines, and cancer risk in DBA also deserves further research. Grant support: The Ministry of Education, Youth and Sports of the Czech Republic (8F20005), European Union - Next Generation EU, Program EXCELES (LX22NPO5102); Internal grant of Palacky University (IGA_LF_2023_002).

ATR activation by Cr-DNA damage is a major survival response establishing late S and G2 checkpoints after Cr(VI) exposure

Inhalation exposure to hexavalent chromium is known to cause lung cancer and other pulmonary toxicity. Cellular metabolism of chromium(VI) entering cells as chromate anion produces different amounts of reactive Cr(V) intermediates and finally yields Cr(III). Direct reduction of Cr(VI) by ascorbate (Asc), the dominant metabolic reaction in vivo but not in standard cell cultures, skips production of Cr(V) but still permits extensive formation of Cr-DNA damage. To understand the importance of different forms of biological injury in Cr(VI) toxicity, we examined activation of several protein- and DNA damage-sensitive stress responses in human lung cells under Asc-restored conditions. We found that Asc-restored cells suppressed upregulation of oxidant-sensitive stress systems by Cr(VI) but showed a strong activation of the apical DNA damage-responsive kinase ATR. ATR signaling was triggered in late S phase and persisted upon entry of cells into G2 phase. Inhibition of ATR prevented the establishment of late-S and G2 cell cycle checkpoints and did not lead to a compensatory activation of a related kinase ATM. Inactivation of ATR also strongly impaired viability of Cr(VI)-treated lung cells including stem-like cells and revealed a significant formation of toxic Cr-DNA damage at low Cr(VI) doses. Our findings identified a major Cr(VI) resistance mechanism involving sensing of Cr-DNA damage by ATR in late S phase and a subsequent establishment of protective cell cycle checkpoints.

Targeting the DNA damage response kinase CHK1 in TP53-mutated thyroid cancer: in vitro studies

Targeting the DNA damage response kinase CHK1 in TP53-mutated thyroid cancer: <i>in vitro</i> studies

226-LB: Repurposing a Cancer Drug, AZD6738 (ATRi), for the Prevention of Type 1 Diabetes

Type 1 diabetes (T1D) is a chronic, incurable autoimmune disorder caused by the destruction of pancreatic β cells by diabetogenic T cells, leading to insulin deficiency and hyperglycemia. It affects 3 million Americans and 80 new people are diagnosed daily. Despite treatment with insulin, patients have a significantly reduced life expectancy. This study proposes a new approach to prevent T1D by specifically targeting diabetogenic T cells undergoing the Warburg effect, a metabolic process similar to cancer cells, with a cancer drug approved by the FDA. The use of FDA-approved drugs accelerates the development of new therapies as these agents have already undergone initial efficacy and toxicity tests in humans. One promising drug for this purpose is AZD6738 (ATRi), a cancer drug that is currently in clinical trials. ATRi is a small molecule inhibitor that targets ATR, a central DNA damage response kinase, whose inhibition blocks DNA replication. We demonstrate that ATRi treatment significantly delays T1D onset in an adoptive transfer mouse model by reducing the survival of diabetogenic CD4+ T cells by inhibiting their proliferation. Furthermore, an FDA-approved glycolysis inhibitor PFK158 potentiates inhibition of diabetogenic CD4+ T cells by ATRi. These agents target different properties of the Warburg effect. While ATRi inhibits rapid proliferation, PFK158 blocks glycolysis. Combination therapy allows for increased efficacy while reducing potential toxicity by allowing the use of lower doses of each agent. These data show promise in the rapid establishment of novel T1D prevention methods by repurposing FDA-approved cancer drugs. Disclosure N. Sugitani: None. H. R. Mason: None. S. Roy: None. J. D. Piganelli: None. Funding National Institutes of Health (R01DK132583); JDRF (2-SRA2020-910-S-B)

A sterol‐PI(4)P exchanger modulates the Tel1/ATM axis of the DNA damage response

Upon DNA damage, cells activate the DNA damage response (DDR) to coordinate proliferation and DNA repair. Dietary, metabolic, and environmental inputs are emerging as modulators of how DNA surveillance and repair take place. Lipids hold potential to convey these cues, although little is known about how. We observed that lipid droplet (LD) number specifically increased in response to DNA breaks. Using Saccharomyces cerevisiae and cultured human cells, we show that the selective storage of sterols into these LD concomitantly stabilizes phosphatidylinositol‐4‐phosphate (PI(4)P) at the Golgi, where it binds the DDR kinase ATM. In turn, this titration attenuates the initial nuclear ATM‐driven response to DNA breaks, thus allowing processive repair. Furthermore, manipulating this loop impacts the kinetics of DNA damage signaling and repair in a predictable manner. Thus, our findings have major implications for tackling genetic instability pathologies through dietary and pharmacological interventions.

P53-dependent DNA repair during the DNA damage response requires actin nucleation by JMY

The tumour suppressor p53 is a nuclear transcription factor with key roles during DNA damage to enable a variety of cellular responses including cell cycle arrest, apoptosis and DNA repair. JMY is an actin nucleator and DNA damage-responsive protein whose sub-cellular localisation is responsive to stress and during DNA damage JMY undergoes nuclear accumulation. To gain an understanding of the wider role for nuclear JMY in transcriptional regulation, we performed transcriptomics to identify JMY-mediated changes in gene expression during the DNA damage response. We show that JMY is required for effective regulation of key p53 target genes involved in DNA repair, including XPC, XRCC5 (Ku80) and TP53I3 (PIG3). Moreover, JMY depletion or knockout leads to increased DNA damage and nuclear JMY requires its Arp2/3-dependent actin nucleation function to promote the clearance of DNA lesions. In human patient samples a lack of JMY is associated with increased tumour mutation count and in cells results in reduced cell survival and increased sensitivity to DNA damage response kinase inhibition. Collectively, we demonstrate that JMY enables p53-dependent DNA repair under genotoxic stress and suggest a role for actin in JMY nuclear activity during the DNA damage response.

Abstract 6189: Targeting DNA damage responsive pathways in cancer therapy

Abstract DNA double strand breaks (DSBs) result in activation of several key DNA damage response (DDR) kinases including ATM, ATR, and DNA-PK. These protein kinases not only promote DNA damage-induced checkpoint control, but also facilitate DSB repair in humans. Thus, these DDR kinases have become promising drug targets for cancer therapy. However, the benefits of targeting DDR kinases remain to be realized, in part due to the lack of predictive biomarkers. By undertaking CRISPR screens with inhibitors targeting key DDR kinases, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that an ATM inhibitor plus a PARP inhibitor induced dramatic levels of cell death, probably through promoting apoptosis. Our results provide a better understanding of DDR pathways, which may facilitate the use of these DDR-targeting agents in cancer therapy. Moreover, To provide comprehensive and unbiased perspective of DDR signaling pathways, we performed 30 fluorescence-activated cell sorting-based genome-wide CRISPR screens with antibodies recognizing distinct endogenous DNA damage-signaling proteins to identify new regulators involved in DNA damage response (DDR). We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as new modulators that regulate ATM protein level. Moreover, we discovered that GNB1L is a master regulator of DDR signaling via its role as a co-chaperone for PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes. Citation Format: Junjie Chen. Targeting DNA damage responsive pathways in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6189.