Induced Replication Stress Research Articles

P864: CTPS1 IS A NOVEL THERAPEUTIC TARGET IN MYELOMA - SELECTIVE SMALL MOLECULE INHIBITION DELIVERS SINGLE AGENT ACTIVITY AND SYNERGISES WITH ATR INHIBITION

Background: Recent years have seen major improvements in the treatment of myeloma; however, relapse occurs in the majority of patients. Novel treatment approaches are moving away from cytotoxic chemotherapy towards targeted therapies with defined mechanisms of action. Malignant cells have an increased demand for key cellular components and a reliance on de novo synthesis pathways. CTP synthetase 1 (CTPS1) plays a pivotal role in pyrimidine production, by catalysing the rate limiting step in the de novo synthesis of CTP, which is required for DNA, RNA and phospholipids. Human genetic studies have identified an essential and non-redundant role for CTPS1 in lymphoid cell proliferation which is complemented by the homologous CTPS2 isoform outside the haemopoietic system. Aims: To evaluate the role of CTPS1 as a novel target in myeloma, elucidate the molecular consequences of CTPS1 inhibition and test rationally designed combination therapy. Methods: STP938 is a potent small molecule CTPS1 inhibitor with >1,300-fold selectivity for CTPS1 over CTPS2. The effects of STP938 on cell proliferation (metabolic activity, tetrazolium indicator), apoptosis (annexin V) and cell cycle (propidium iodide) were assessed in vitro using 12 myeloma cell lines. The role of CTPS1 was further assessed in CRISPR knock-out (KO) experiments. Activation of proteins in the replication stress and DNA damage response pathways was analysed by western blotting. Additive/synergistic effects of combining STP938 with two different ATR inhibitors were assessed on cell line proliferation and enumerated by Bliss score (Synergyfinder 2.0). Results: STP938 showed single agent activity against 6 of 12 cell lines tested, with IC50 values for sensitive lines ranging from 19 to 128 nM (Figure A). STP938 showed cytotoxic activity against sensitive lines, evidenced by induction of apoptosis. KO experiments confirmed that MM cell lines depend on CTPS1 for proliferation. Exposure to STP938 was associated with accumulation of cells in S phase, induction of a replication stress response as evidenced by activation (phosphorylation) of CHEK1, and induction of the DNA damage response pathway as evidenced by activation of CHEK2 (albeit to a lesser extent than activation of CHEK1). Importantly, S phase accumulation, induction of replication stress and induction of DNA damage response was observed in cell lines where STP938 did not produce significant anti-proliferative activity (Figure B). Given the ability of STP938 to activate CHEK1, STP938 was tested in combination with an ATR inhibitor, as ATR is the main upstream activator of CHEK1. The ATR inhibitor ceralasertib demonstrated additive or synergistic activity when combined with STP938 (Figure C). Notably, synergy between STP938 and ATR inhibition, along with induction of apoptosis, was observed in cell lines resistant to single agent STP938. Results were confirmed with a second ATR inhibitor (VE-821, Figure C). Image:Summary/Conclusion: Inhibition of CTPS1 by single agent STP938 showed anti-proliferative activity against 6 of 12 myeloma cell lines, and induced replication stress in all cell lines. Combined STP938 and ATR inhibition showed anti-proliferative activity in all cell lines, including those resistant to single agent STP938. These data suggest a model whereby myeloma cells with high background replication stress are sensitive to killing by single agent STP938, whereas cells with lower background replication stress are sensitised by STP938 to cell death induced by ATR inhibition (Figure D). STP938 will shortly enter clinical development for patients with late stage lymphoid neoplasms.

Abstract 5298: Activity and tolerability of combinations of trastuzumab deruxtecan (T-DXd) with inhibitors of the DNA damage response in preclinical models

Abstract Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate composed of an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a cytotoxic topoisomerase I inhibitor. T-DXd is approved for patients with HER2+ metastatic breast cancer who have received two or more anti-HER2 based regimens as well as HER2+ gastric cancer, and has demonstrated antitumor activity in both HER2+ and HER2-low cancers clinically. Trapping of Topo1cc by DXd in targeted cells, induces replication stress (RS) and double strand breaks (DSB), activating a DNA Damage Repair (DDR) response. Therefore we aimed to investigate if combination of T-DXd with DDR inhibitors enhances efficacy and durability of response. Methods: We evaluated the antiproliferative effect of the combination of T-DXd with AZD6738 (ATR inhibitor), AZD1775 (WEE1 inhibitor), AZD1390 (ATM inhibitor), in a panel of 27 breast cancer cell lines in an in vitro 7-day viability assay. Combinations were also evaluated in vivo in the HER2-high gastric NCI-N87 cell line xenograft. To evaluate the specificity of the combination activity in tumor cells (vs normal tissue), we further evaluated the combination in a 2D in vitro human bone marrow progenitor assay. Results: We found that the combination with all DDRi significantly enhanced in vitro cell killing activity over single agents in many of the models tested (12/27 for AZD6738, 9/27 for AZD1775, 6/27 for AZD1390). This was not limited to models with DNA damage repair mutations (e.g. BRCA1, BRCA2, or ATM). Mechanistically, T-DXd activated PARP and the ATR, Wee1, ATM pathways, inducing cell cycle arrest. Combination of T-DXd with AZD6738 and AZD1775 inhibited the RS response (pATR-T1989, pChk1-S345) and cell cycle arrest (pCdc2-Y15), while AZD1390 inhibited the DSB response pathway (pATM-S1981, pRad50-S685, pKap1-S824, pChk2-T68). All DDR inhibitors exacerbated DNA damage (pRPA-S4/8, gH2AX) and cell death (cCasp3). In the in vitro human bone marrow (BM) assay, T-DXd had no synergistic interaction with AZD6738 and AZD1775 inhibitors, but synergy with AZD1390. In vivo, combination therapy with all DDRi tested achieved superior Tumor Growth Inhibition (TGI) compared to monotherapy alone. At day 41 in NCI-N87, 3mg/kg of T-DXd provided TGI of 74% while monotherapy arms of 25mg/kg BID AZD6738, 60mg/kg (5-days on/2-days off) AZD1775 or 10mg/kg AZD1390 provided 68%, 47%, 31% TGI respectively. The combinations of T-DXd with AZD6738, AZD1775 and AZD1390 provided TGI of >100% (19% regression), 90%, and 99% respectively. Conclusions: These results suggest that AZD6738, AZD1775, and AZD1390 all potentiate T-DXd activity in vitro and in vivo, and the combination interactions with AZD6738 and AZD1775 were much stronger in some tumor models over bone marrow cells in vitro. These data suggest further investigation of these combinations in clinical studies may be warranted. Citation Format: Yann Wallez, Theresa Proia, Azadeh Cheraghchi-Bashi-Astaneh, Ankur Karmokar, Zena Wilson, Suzanne Randle, Mark Anderton, Stephen Durant, Elisabetta Leo, Alan Lau, Mark O'Connor, Jerome Mettetal. Activity and tolerability of combinations of trastuzumab deruxtecan (T-DXd) with inhibitors of the DNA damage response in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5298.

Abstract 2017: Therapeutic induction of replication stress in the context of salvage therapy in osteosarcoma

Abstract Osteosarcoma (OS) is an aggressive pediatric cancer with ~35% of patients developing metastasis over time. The survival rate for metastatic and relapsed OS patients is <30% and there is currently no standardized salvage therapy. Lack of efficacy is attributed to extensive genetic complexity present in OS that is partly due to moderate levels of replication stress (RS). While high levels of RS can induce cell death, moderate RS levels may cause genomic instability that contributes to progression of OS. Therefore, induction of RS to high levels, especially in genetically complex cancers like OS, could be a promising therapeutic strategy. Bromodomain and extra-terminal domain (BET) proteins (BRD2,3,4) are a family of epigenetic readers that not only regulate gene expression networks, but also regulate DNA replication and RS. BRD4 directly regulates major factors involved in DNA replication and checkpoint signaling. Thus, disruption of BRD4 function should exacerbate RS to levels that cause cell death. The objective of this study is to test the hypothesis that BET inhibition potentiates the efficacy of current salvage therapy through RS induction in aggressive OS. The effects of BET inhibitors (BETi), AZD5153 and OTX-015, as single agents and in combination with drugs used in salvage therapy such as topotecan were evaluated for effects on OS cell growth, PARP cleavage, and the DNA damage repair network. BET knockdown experiments were performed to evaluate target selectivity and dependency. In vivo efficacy and safety studies focused on patient-derived xenografts (PDXs) of relapsed OS. TT2-77 xenoline, Saos2, G292, and U2OS cell lines were selected for in vitro experiments. Combination index and Bliss independence analyses demonstrated additive to synergistic cell growth inhibition upon treatment with clinically relevant concentrations of BETi+topotecan. Significant increase in PARP cleavage was observed in the combination compared to single agent, indicating enhancement of apoptosis. Moreover, Western analyses demonstrated that BETi induces its effect, at least partly, via decreased CHK1 activation and increased DNA damage. Selective siRNA treatments illustrated that transient knockdown of individual BET proteins was not sufficient for potentiation of topotecan-induced cell death in OS cells, indicating that simultaneous knockdown of BETs may be required. Dose-finding studies of AZD5153 in relapsed OS PDXs that harbor replication stress signatures (TT2-77 and PDX96) indicated that daily doses of 1.25 or 2.5 mg/kg AZD5153 were well tolerated and effective in partially suppressing tumor growth compared to vehicle (p<0.05, Two-way ANOVA; Holm-Sidak). In vivo combination treatments of BETi+topotecan are in progress. These data collectively suggest that BET inhibition alongside salvage therapy holds promise as a novel treatment strategy for inducing RS-mediated cell death in aggressive OS. Citation Format: Niknam Riyahi, Pankita H. Pandya, M. Reza Saadatzadeh, Khadijeh Bijangi-Vishehsaraei, Barbara J. Bailey, Erika A. Dobrota, Courtney Young, Melissa A. Trowbridge, Kathy Coy, Henry Mang, Reagan K. Wohlford, Anthony L. Sinn, Emily S. Sims, Matt J. Repass, Nuri Damayanti, Farinaz Barghi, Harlan E. Shannon, Michael J. Ferguson, Jamie L. Renbarger, Karen E. Pollok. Therapeutic induction of replication stress in the context of salvage therapy in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2017.

Abstract 315: B7H3 is a potential therapeutic target in advanced metastatic prostate cancer

Abstract B7H3 is an immunomodulatory protein of the B7 family which inhibits T cell function. B7H3 mRNA is ubiquitously expressed in normal human tissues, but the protein is expressed at low levels, suggesting a tight post-transcriptional regulation. By contrast, B7H3 protein is overexpressed in several solid tumors and often correlates with poor survival, higher tumor grade, and metastasis independently of immune regulation. A few studies also have reported an association between B7H3 overexpression and therapy resistance. Since B7H3 protein is preferentially expressed on cancer cells and tumor vasculature, it is an attractive target for developing novel cancer therapies. Here we analyzed the expression of B7H3 in 26 mCRPC organoid models and examined whether B7H3 can serve as a novel therapeutic target for advanced mPCa patients that fail current treatment regimens. The B7H3 protein expression in PDXOs of various lineages including adenocarcinoma (n=19), double-negative (DNPC, n= 2), and neuroendocrine (NE, n =5) lineages, was analyzed using simple western™ assay, immunofluorescence, and flow cytometry. We tested the ex-vivo drug response of PDXOs and performed PDX preclinical trials using humanized B7H3-ADC armed with a pyrrolobenzodiazepine (PBD) warhead. PBD dimers are DNA interstrand cross-linking agents and are a poor substrate of P-gp drug transporters. Our results showed that B7-H3 was overexpressed in several PDX derived organoids of mPCa. Organoids with adenocarcinoma phenotype (n=19) demonstrated a range of B7H3 protein expression whereas RB1/TP53 mutated small cell neuroendocrine or double negative prostate cancer showed a relatively more uniform and low expression. Nonetheless, PDXOs of neuroendocrine, double negative or adenocarcinoma origin responded with maximum efficacy to B7H3-ADC ex vivo. Adenocarcinoma organoid models (n=19) displayed a wide range of responses, which was only partially correlated with B7H3 protein levels. Genetically-engineered loss of B7-H3 converted a responder to a non-responder phenotype. Our investigation for potential biomarkers of B7H3-PBD sensitivity suggested that RB1-deficiency induced replication stress and/or expression of SLFN11, an interferon pathway transcriptional target, to be the strong predictors of B7H3-PBD response, independent of tumor lineage. We further performed preclinical trials using four PDX models based on their RB1 and SLFN11 status, including a model of advanced adenocarcinoma lineage with low B7H3 expression. B7H3-PBD eradicated large established subcutaneous tumors and metastases and improved long-term overall survival in either RB1 deficient and/or SLFN11HIGH models, but showed no response in RB1+/SLFN11- tumors. In conclusion, B7H3-PBD treatment has potential to produce complete and durable response in appropriately-selected, heavily-pretreated metastatic CRPC and NEPC. Citation Format: Supreet Agarwal, Lei Fang, Juan Juan Yin, Elaine Hurt, Kathleen Kelly. B7H3 is a potential therapeutic target in advanced metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 315.

Aberrant R-loop-induced replication stress in MED12-mutant uterine fibroids

Uterine fibroid (UF) driver mutations in Mediator complex subunit 12 (MED12) trigger genomic instability and tumor development through unknown mechanisms. Herein, we show that MED12 mutations trigger aberrant R-loop-induced replication stress, suggesting a possible route to genomic instability and a novel therapeutic vulnerability in this dominant UF subclass. Immunohistochemical analyses of patient-matched tissue samples revealed that MED12 mutation-positive UFs, compared to MED12 mutation-negative UFs and myometrium, exhibited significantly higher levels of R-loops and activated markers of Ataxia Telangiectasia and Rad3-related (ATR) kinase-dependent replication stress signaling in situ. Single molecule DNA fiber analysis revealed that primary cells from MED12 mutation-positive UFs, compared to those from patient-matched MED12 mutation-negative UFs and myometrium, exhibited defects in replication fork dynamics, including reduced fork speeds, increased and decreased numbers of stalled and restarted forks, respectively, and increased asymmetrical bidirectional forks. Notably, these phenotypes were recapitulated and functionally linked in cultured uterine smooth muscle cells following chemical inhibition of Mediator-associated CDK8/19 kinase activity that is known to be disrupted by UF driver mutations in MED12. Thus, Mediator kinase inhibition triggered enhanced R-loop formation and replication stress leading to an S-phase cell cycle delay, phenotypes that were rescued by overexpression of the R-loop resolving enzyme RNaseH. Altogether, these findings reveal MED12-mutant UFs to be uniquely characterized by aberrant R-loop induced replication stress, suggesting a possible basis for genomic instability and new avenues for therapeutic intervention that involve the replication stress phenotype in this dominant UF subtype.

A clinically relevant heterozygous ATR mutation sensitizes colorectal cancer cells to replication stress

Colorectal cancer (CRC) ranks third among the most frequent malignancies and represents the second most common cause of cancer-related deaths worldwide. By interfering with the DNA replication process of cancer cells, several chemotherapeutic molecules used in CRC therapy induce replication stress (RS). At the cellular level, this stress is managed by the ATR-CHK1 pathway, which activates the replication checkpoint. In recent years, the therapeutic value of targeting this pathway has been demonstrated. Moreover, MSI + (microsatellite instability) tumors frequently harbor a nonsense, heterozygous mutation in the ATR gene. Using isogenic HCT116 clones, we showed that this mutation of ATR sensitizes the cells to several drugs, including SN-38 (topoisomerase I inhibitor) and VE-822 (ATR inhibitor) and exacerbates their synergistic effects. We showed that this mutation bottlenecks the replication checkpoint leading to extensive DNA damage. The combination of VE-822 and SN-38 induces an exhaustion of RPA and a subsequent replication catastrophe. Surviving cells complete replication and accumulate in G2 in a DNA-PK-dependent manner, protecting them from cell death. Together, our results suggest that RPA and DNA-PK represent promising therapeutic targets to optimize the inhibition of the ATR-CHK1 pathway in oncology. Ultimately, ATR frameshift mutations found in patients may also represent important prognostic factors.

Cellular mechanism of action of 2-nitroimidazoles as hypoxia-selective therapeutic agents

Solid tumours are often poorly oxygenated, which confers resistance to standard treatment modalities. Targeting hypoxic tumours requires compounds, such as nitroimidazoles (NIs), equipped with the ability to reach and become activated within diffusion limited tumour niches. NIs become selectively entrapped in hypoxic cells through bioreductive activation, and have shown promise as hypoxia directed therapeutics. However, little is known about their mechanism of action, hindering the broader clinical usage of NIs. Iodoazomycin arabinofuranoside (IAZA) and fluoroazomycin arabinofuranoside (FAZA) are clinically validated 2-NI hypoxic radiotracers with excellent tumour uptake properties. Hypoxic cancer cells have also shown preferential susceptibility to IAZA and FAZA treatment, making them ideal candidates for an in-depth study in a therapeutic setting. Using a head and neck cancer model, we show that hypoxic cells display higher sensitivity to IAZA and FAZA, where the drugs alter cell morphology, compromise DNA replication, slow down cell cycle progression and induce replication stress, ultimately leading to cytostasis. Effects of IAZA and FAZA on target cellular macromolecules (DNA, proteins and glutathione) were characterized to uncover potential mechanism(s) of action. Covalent binding of these NIs was only observed to cellular proteins, but not to DNA, under hypoxia. While protein levels remained unaffected, catalytic activities of NI target proteins, such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the detoxification enzyme glutathione S-transferase (GST) were significantly curtailed in response to drug treatment under hypoxia. Intraperitoneal administration of IAZA was well-tolerated in mice and produced early (but transient) growth inhibition of subcutaneous mouse tumours.

Combined Inactivation of CTPS1 and ATR Is Synthetically Lethal to MYC-Overexpressing Cancer Cells.

Inhibition of CTPS in MYC-overexpressing cells blocks pyrimidine synthesis while maintaining ribosome synthesis activity to create an anabolic imbalance that induces replication stress, providing a new approach to selectively target MYC-driven cancer. See related commentary by Chabanon and Postel-Vinay, p. 969.

Telomeric replication stress: the beginning and the end for alternative lengthening of telomeres cancers.

Telomeres are nucleoprotein structures that cap the ends of linear chromosomes. Telomeric DNA comprises terminal tracts of G-rich tandem repeats, which are inherently difficult for the replication machinery to navigate. Structural aberrations that promote activation of the alternative lengthening of telomeres (ALT) pathway of telomere maintenance exacerbate replication stress at ALT telomeres, driving fork stalling and fork collapse. This form of telomeric DNA damage perpetuates recombination-mediated repair pathways and break-induced telomere synthesis. The relationship between replication stress and DNA repair is tightly coordinated for the purpose of regulating telomere length in ALT cells, but has been shown to be experimentally manipulatable. This raises the intriguing possibility that induction of replication stress can be used as a means to cause toxic levels of DNA damage at ALT telomeres, thereby selectively disrupting the viability of ALT cancers.

Human cytomegalovirus hijacks host stress response fueling replication stress and genome instability.

Viral infections enhance cancer risk and threaten host genome integrity. Although human cytomegalovirus (HCMV) proteins have been detected in a wide spectrum of human malignancies and HCMV infections have been implicated in tumorigenesis, the underlying mechanisms remain poorly understood. Here, we employed a range of experimental approaches, including single-molecule DNA fiber analysis, and showed that infection by any of the four commonly used HCMV strains: AD169, Towne, TB40E or VR1814 induced replication stress (RS), as documented by host-cell replication fork asymmetry and formation of 53BP1 foci. The HCMV-evoked RS triggered an ensuing host DNA damage response (DDR) and chromosomal instability in both permissive and non-permissive human cells, the latter being particularly relevant in the context of tumorigenesis, as such cells can survive and proliferate after HCMV infection. The viral major immediate early enhancer and promoter (MIEP) that controls expression of the viral genes IE72 (IE-1) and IE86 (IE-2), contains transcription-factor binding sites shared by promoters of cellular stress-response genes. We found that DNA damaging insults, including those relevant for cancer therapy, enhanced IE72/86 expression. Thus, MIEP has been evolutionary shaped to exploit host DDR. Ectopically expressed IE72 and IE86 also induced RS and increased genomic instability. Of clinical relevance, we show that undergoing standard-of-care genotoxic radio-chemotherapy in patients with HCMV-positive glioblastomas correlated with elevated HCMV protein markers after tumor recurrence. Collectively, these results are consistent with our proposed concept of HCMV hijacking transcription-factor binding sites shared with host stress-response genes. We present a model to explain the potential oncomodulatory effects of HCMV infections through enhanced replication stress, subverted DNA damage response and induced genomic instability.

Targeting the DNA Damage Response for Cancer Therapy by Inhibiting the Kinase Wee1.

Cancer cells typically heavily rely on the G2/M checkpoint to survive endogenous and exogenous DNA damage, such as genotoxic stress due to genome instability or radiation and chemotherapy. The key regulator of the G2/M checkpoint, the cyclin-dependent kinase 1 (CDK1), is tightly controlled, including by its phosphorylation state. This posttranslational modification, which is determined by the opposing activities of the phosphatase cdc25 and the kinase Wee1, allows for a more rapid response to cellular stress than via the synthesis or degradation of modulatory interacting proteins, such as p21 or cyclin B. Reducing Wee1 activity results in ectopic activation of CDK1 activity and drives premature entry into mitosis with unrepaired or under-replicated DNA and causing mitotic catastrophe. Here, we review efforts to use small molecule inhibitors of Wee1 for therapeutic purposes, including strategies to combine Wee1 inhibition with genotoxic agents, such as radiation therapy or drugs inducing replication stress, or inhibitors of pathways that show synthetic lethality with Wee1. Furthermore, it become increasingly clear that Wee1 inhibition can also modulate therapeutic immune responses. We will discuss the mechanisms underlying combination treatments identifying both cell intrinsic and systemic anti-tumor activities.

LINE-1 expression in cancer correlates with p53 mutation, copy number alteration, and S phase checkpoint

Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expression. Induction of LINE-1 expression in an invitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.

Rev1 deficiency induces replication stress to cause metabolic dysfunction differently in males and females.

DNA damage responses compete for cellular resources with metabolic pathways, but little is known about the metabolic consequences of impaired DNA replication, a process called replication stress. Here we characterized the metabolic consequences of DNA replication stress at endogenous DNA lesions by using mice with a disruption of Rev1, a translesion DNA polymerase specialized in the mutagenic replication of damaged DNA. Male and female Rev1 knockout (KO) mice were compared with wild-type (WT) mice and followed over time to study the natural course of body weight gain and glucose tolerance. Follow-up measurements were performed in female mice for in-depth metabolic characterization. Body weight and fat mass were only increased in female KO mice versus WT mice, whereas glucose intolerance and a reduction in lean mass were observed in both sexes. Female KO mice showed reduced locomotor activity while male KO mice showed increased activity as compared with their WT littermates. Further characterization of female mice revealed that lipid handling was unaffected by Rev1 deletion. An increased respiratory exchange ratio, combined with elevated plasma lactate levels and increased hepatic gluconeogenesis indicated problems with aerobic oxidation and increased reliance on anaerobic glycolysis. Supplementation with the NAD+ precursor nicotinamide riboside to stimulate aerobic respiration failed to restore the metabolic phenotype. In conclusion, replication stress at endogenous DNA lesions induces a complex metabolic phenotype, most likely initiated by muscular metabolic dysfunction and increased dependence on anaerobic glycolysis. Nicotinamide riboside supplementation after the onset of the metabolic impairment did not rescue this phenotype.NEW & NOTEWORTHY An increasing number of DNA lesions interferes with cellular replication leading to metabolic inflexibility. We utilized Rev1 knockout mice as a model for replication stress, and show a sex-dependent metabolic phenotype, with a pronounced reduction of lean mass and glucose tolerance. These data indicate that in obesity, we may end up in an infinite loop where metabolic disturbance promotes the formation of DNA lesions, which in turn interferes with cellular replication causing further metabolic disturbances.

Pharmacological targeting of MTHFD2 suppresses acute myeloid leukemia by inducing thymidine depletion and replication stress

The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.

Metnase and EEPD1: DNA Repair Functions and Potential Targets in Cancer Therapy.

Cells respond to DNA damage by activating signaling and DNA repair systems, described as the DNA damage response (DDR). Clarifying DDR pathways and their dysregulation in cancer are important for understanding cancer etiology, how cancer cells exploit the DDR to survive endogenous and treatment-related stress, and to identify DDR targets as therapeutic targets. Cancer is often treated with genotoxic chemicals and/or ionizing radiation. These agents are cytotoxic because they induce DNA double-strand breaks (DSBs) directly, or indirectly by inducing replication stress which causes replication fork collapse to DSBs. EEPD1 and Metnase are structure-specific nucleases, and Metnase is also a protein methyl transferase that methylates histone H3 and itself. EEPD1 and Metnase promote repair of frank, two-ended DSBs, and both promote the timely and accurate restart of replication forks that have collapsed to single-ended DSBs. In addition to its roles in HR, Metnase also promotes DSB repair by classical non-homologous recombination, and chromosome decatenation mediated by TopoIIα. Although mutations in Metnase and EEPD1 are not common in cancer, both proteins are frequently overexpressed, which may help tumor cells manage oncogenic stress or confer resistance to therapeutics. Here we focus on Metnase and EEPD1 DNA repair pathways, and discuss opportunities for targeting these pathways to enhance cancer therapy.

Proline-specific aminopeptidase P prevents replication-associated genome instability.

Genotoxic stress during DNA replication constitutes a serious threat to genome integrity and causes human diseases. Defects at different steps of DNA metabolism are known to induce replication stress, but the contribution of other aspects of cellular metabolism is less understood. We show that aminopeptidase P (APP1), a metalloprotease involved in the catabolism of peptides containing proline residues near their N-terminus, prevents replication-associated genome instability. Functional analysis of C. elegans mutants lacking APP-1 demonstrates that germ cells display replication defects including reduced proliferation, cell cycle arrest, and accumulation of mitotic DSBs. Despite these defects, app-1 mutants are competent in repairing DSBs induced by gamma irradiation, as well as SPO-11-dependent DSBs that initiate meiotic recombination. Moreover, in the absence of SPO-11, spontaneous DSBs arising in app-1 mutants are repaired as inter-homologue crossover events during meiosis, confirming that APP-1 is not required for homologous recombination. Thus, APP-1 prevents replication stress without having an apparent role in DSB repair. Depletion of APP1 (XPNPEP1) also causes DSB accumulation in mitotically-proliferating human cells, suggesting that APP1's role in genome stability is evolutionarily conserved. Our findings uncover an unexpected role for APP1 in genome stability, suggesting functional connections between aminopeptidase-mediated protein catabolism and DNA replication.

CDK4/6 inhibitors induce replication stress to cause long‐term cell cycle withdrawal

CDK4/6 inhibitors arrest the cell cycle in G1‐phase. They are approved to treat breast cancer and are also undergoing clinical trials against a range of other tumour types. To facilitate these efforts, it is important to understand why a cytostatic arrest in G1 causes long‐lasting effects on tumour growth. Here, we demonstrate that a prolonged G1 arrest following CDK4/6 inhibition downregulates replisome components and impairs origin licencing. Upon release from that arrest, many cells fail to complete DNA replication and exit the cell cycle in a p53‐dependent manner. If cells fail to withdraw from the cell cycle following DNA replication problems, they enter mitosis and missegregate chromosomes causing excessive DNA damage, which further limits their proliferative potential. These effects are observed in a range of tumour types, including breast cancer, implying that genotoxic stress is a common outcome of CDK4/6 inhibition. This unanticipated ability of CDK4/6 inhibitors to induce DNA damage now provides a rationale to better predict responsive tumour types and effective combination therapies, as demonstrated by the fact that CDK4/6 inhibition induces sensitivity to chemotherapeutics that also cause replication stress.

Molecular Insights into How the Dimetal Center in Dihydropyrimidinase Can Bind the Thymine Antagonist 5-Aminouracil: A Different Binding Mode from the Anticancer Drug 5-Fluorouracil.

Dihydropyrimidinase (DHPase) is a key enzyme for pyrimidine degradation. DHPase contains a binuclear metal center in which two Zn ions are bridged by a posttranslationally carbamylated lysine. DHPase catalyzes the hydrolysis of dihydrouracil to N-carbamoyl-β-alanine. Whether 5-aminouracil (5-AU), a thymine antagonist and an anticancer drug that can block DNA synthesis and induce replication stress, can interact with DHPase remains to be investigated. In this study, we determined the crystal structure of Pseudomonas aeruginosa DHPase (PaDHPase) complexed with 5-AU at 2.1 Å resolution (PDB entry 7E3U). This complexed structure revealed that 5-AU interacts with Znα (3.2 Å), Znβ (3.0 Å), the main chains of residues Ser289 (2.8 Å) and Asn337 (3.3 Å), and the side chain of residue Tyr155 (2.8 Å). These residues are also known as the substrate-binding sites of DHPase. Dynamic loop I (amino acid residues Pro65-Val70) in PaDHPase is not involved in the binding of 5-AU. The fluorescence quenching analysis and site-directed mutagenesis were used to confirm the binding mode revealed by the complexed crystal structure. The 5-AU binding mode of PaDHPase is, however, different from that of 5-fluorouracil, the best-known fluoropyrimidine used for anticancer therapy. These results provide molecular insights that may facilitate the development of new inhibitors targeting DHPase and constitute the 5-AU interactome.

Oncogenic C-Myc Induces Replication Stress by Increasing Cohesins Chromatin Occupancy at Ctcf Sites

Oncogenic C-Myc Induces Replication Stress by Increasing Cohesins Chromatin Occupancy at Ctcf Sites

Cockayne syndrome group B protein regulates fork restart, fork progressionand MRE11-dependent fork degradation in BRCA1/2-deficient cells.

Cockayne syndrome group B (CSB) protein has been implicated in the repair of a variety of DNA lesions that induce replication stress. However, little is known about its role at stalled replication forks. Here, we report that CSB is recruited to stalled forks in a manner dependent upon its T1031 phosphorylation by CDK. While dispensable for MRE11 association with stalled forks in wild-type cells, CSB is required for further accumulation of MRE11 at stalled forks in BRCA1/2-deficient cells. CSB promotes MRE11-mediated fork degradation in BRCA1/2-deficient cells. CSB possesses an intrinsic ATP-dependent fork reversal activity in vitro, which is activated upon removal of its N-terminal region that is known to autoinhibit CSB’s ATPase domain. CSB functions similarly to fork reversal factors SMARCAL1, ZRANB3 and HLTF to regulate slowdown in fork progression upon exposure to replication stress, indicative of a role of CSB in fork reversal in vivo. Furthermore, CSB not only acts epistatically with MRE11 to facilitate fork restart but also promotes RAD52-mediated break-induced replication repair of double-strand breaks arising from cleavage of stalled forks by MUS81 in BRCA1/2-deficient cells. Loss of CSB exacerbates chemosensitivity in BRCA1/2-deficient cells, underscoring an important role of CSB in the treatment of cancer lacking functional BRCA1/2.