Replication Stress Markers Research Articles

Embryonic genome instability upon DNA replication timing program emergence

Faithful DNA replication is essential for genome integrity1–4. Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis5–8. However, the regulation of DNA replication remains poorly understood in early mammalian embryos. Here we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and identified an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing program, and the entire genome replicated gradually and uniformly using extremely slow-moving replication forks. In 4-cell embryos, a somatic-cell-like replication timing program commenced abruptly. However, the fork speed was still slow, S phase was extended, and markers of replication stress, DNA damage and repair increased. This was followed by an increase in break-type chromosome segregation errors specifically during the 4-to-8-cell division with breakpoints enriched in late-replicating regions. These errors were rescued by nucleoside supplementation, which accelerated fork speed and reduced the replication stress. By the 8-cell stage, forks gained speed, S phase was no longer extended and chromosome aberrations decreased. Thus, a transient period of genomic instability exists during normal mouse development, preceded by an S phase lacking coordination between replisome-level regulation and megabase-scale replication timing regulation, implicating a link between their coordination and genome stability.

Transient Zn2+ deficiency induces replication stress and compromises daughter cell proliferation

Cells must replicate their genome quickly and accurately, and they require metabolites and cofactors to do so. Ionic zinc (Zn2+) is an essential micronutrient that is required for hundreds of cellular processes, including DNA synthesis and adequate proliferation. Deficiency in this micronutrient impairs DNA synthesis and inhibits proliferation, but the mechanism is unknown. Using fluorescent reporters to track single cells via long-term live-cell imaging, we find that Zn2+ is required at the G1/S transition and during S phase for timely completion of S phase. A short pulse of Zn2+ deficiency impairs DNA synthesis and increases markers of replication stress. These markers of replication stress are reversed upon resupply of Zn2+. Finally, we find that if Zn2+ is chelated during the mother cell's S phase, daughter cells enter a transient quiescent state, maintained by sustained expression of p21, which disappears upon reentry into the cell cycle. In summary, short pulses of mild Zn2+ deficiency in S phase specifically induce replication stress, which causes downstream proliferation impairments in daughter cells.

Abstract 4749: Acrivon predictive precision proteomics (AP3) uncovers mechanism of resistance to ACR-368, a clinical-stage CHK1/2 inhibitor, and identifies rational combination treatment

Abstract ACR-368 (prexasertib) is a clinically advanced CHK1/2 inhibitor which has demonstrated durable activity across a proportion of patients with advanced solid tumors. Genomic biomarkers have proven unsuccessful in predicting response to ACR-368, limiting its clinical success. Using AP3, we previously developed a response-predictive proteomics-based test for ACR-368 (ACR-368 OncoSignature) for the identification of patients sensitive to ACR-368 monotherapy treatment as demonstrated in blinded preclinical studies. A Phase 2 clinical trial is ongoing where patients are treated with ACR-368 monotherapy based on OncoSignature-predicted sensitivity (NCT05548296). Here, we demonstrate the utility of AP3 for the identification of a key druggable resistance mechanism to ACR-368 and how to overcome that with low dose gemcitabine (gem), providing OncoSignature negative patients with a new potential therapeutic option. Five ovarian cancer cell lines were rendered durably resistant to ACR-368 by culturing in the presence of clinically relevant concentrations of ACR-368. Matched parental and ACR-368 resistant cell line pairs were profiled using AP3 mass spectrometry. Comprehensive pathway reconstitution and kinase activity analyses were performed to identify drug resistance mechanisms in an unbiased manner. Downregulation of DNA damage repair pathway activity was causally linked to the ACR-368-resistant phenotype, suggesting agents that restore replication stress around the CHK1/2 signaling axis may re-sensitize to ACR-368. To test this, a panel of ovarian cancer cell lines with intrinsic or drug-induced resistance to ACR-368 were screened for cell growth inhibition by ACR-368 combined with gem. Gem synergized with ACR-368 in 12/13 cell lines at low doses (1-40 nM). Moreover, Western blot analysis demonstrated protein markers of replication stress were induced by low dose gem (3-30 nM), suggesting a correlation between gem-induced replication stress and synergy with ACR-368. Comet assays showed that DMSO, gem (3 nM), or ACR-368 (100 nM) had minimal impact (4.4%, 4.5%, and 11.4%, respectively) on % comet tail DNA in ACR-368 resistant cells, while the gem combination led to 35% comet tail DNA (p<0.001). Finally, in a human tumor xenograft mouse model, low dose gem demonstrated a dose-dependent increase in replication stress markers (Cyclin E, pCHK1 S345) from 0.3-3 mg/kg, which allometrically scales to 1-10 mg/m2 in humans. These data supported a dose escalation Phase 1b/2 clinical study of low dose gem with ACR-368 to evaluate the efficacy and safety of the combination in ACR-368 OncoSignature negative patients (NCT05548296). This shows the potential of AP3 for unbiased elucidation of actionable drug resistance mechanisms and rapid clinical implementation in our trials, which have recently confirmed clinical activity. Citation Format: Helén Nilsson, Lei Shi, Magnus E. Jakobsson, Joelle Baddour-Sousounis, Shahrzad Rafiei, Uthira Muralitharan, Zachary Best, Valentina Siino, Francisco J. Santana, Ignacio Arribas Diez, Kailash Singh, Portia Lombardo, William Dahlberg, Subodh Kumar, Ahmed Youssef, Reina Improgo, Corey Xu, Joon Jung, Jung-Min Lee, Ayesha Murshid, Michail Shipitsin, Jesper V. Olsen, Kristina Masson, David A. Proia, Caroline Wigerup, Peter Blume-Jensen. Acrivon predictive precision proteomics (AP3) uncovers mechanism of resistance to ACR-368, a clinical-stage CHK1/2 inhibitor, and identifies rational combination treatment [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 4749.

CGAS-STING pathway expression correlates with genomic instability and immune cell infiltration in breast cancer

Genomic instability, as caused by oncogene-induced replication stress, can lead to the activation of inflammatory signaling, involving the cGAS-STING and JAK-STAT pathways. Inflammatory signaling has been associated with pro-tumorigenic features, but also with favorable response to treatment, including to immune checkpoint inhibition. In this study, we aim to explore relations between inflammatory signaling, markers of replication stress, and immune cell infiltration in breast cancer. Expression levels of cGAS-STING signaling components (STING, phospho-TBK1, and phospho-STAT1), replication stress markers (γH2AX and pRPA), replication stress-related proto-oncogenes (Cyclin E1 and c-Myc) and immune cell markers (CD20, CD4, and CD57) are determined immunohistochemically on primary breast cancer samples (n = 380). RNA-sequencing data from TCGA (n = 1082) and METABRIC (n = 1904) are used to calculate cGAS-STING scores. pTBK1, pSTAT1 expression and cGAS-STING pathway scores are all increased in triple-negative breast cancers compared to other subtypes. Expression of γH2AX, pRPA, Cyclin E1, c-Myc, and immune cell infiltration positively correlate with p-STAT1 expression (P < 0.001). Additionally, we observe significant positive associations between expression of pTBK1 and γH2AX, pRPA, c-Myc, and number of CD4+ cells and CD20+ cells. Also, cGAS-STING scores are correlated with genomic instability metrics, such as homologous recombination deficiency (P < 0.001) and tumor mutational burden (P < 0.01). Moreover, data from the I-SPY2 clinical trial (n = 71) confirms that higher cGAS-STING scores are observed in breast cancer patients who responded to immunotherapy combined with chemotherapy. In conclusion, the cGAS-STING pathway is highly expressed in TNBCs and is correlated with genomic instability and immune cell infiltration.

Reversion from basal histone H4 hypoacetylation at the replication fork increases DNA damage in FANCA deficient cells.

The FA/BRCA pathway safeguards DNA replication by repairing interstrand crosslinks (ICL) and maintaining replication fork stability. Chromatin structure, which is in part regulated by histones posttranslational modifications (PTMs), has a role in maintaining genomic integrity through stabilization of the DNA replication fork and promotion of DNA repair. An appropriate balance of PTMs, especially acetylation of histones H4 in nascent chromatin, is required to preserve a stable DNA replication fork. To evaluate the acetylation status of histone H4 at the replication fork of FANCA deficient cells, we compared histone acetylation status at the DNA replication fork of isogenic FANCA deficient and FANCA proficient cell lines by using accelerated native immunoprecipitation of nascent DNA (aniPOND) and in situ protein interactions in the replication fork (SIRF) assays. We found basal hypoacetylation of multiple residues of histone H4 in FA replication forks, together with increased levels of Histone Deacetylase 1 (HDAC1). Interestingly, high-dose short-term treatment with mitomycin C (MMC) had no effect over H4 acetylation abundance at the replication fork. However, chemical inhibition of histone deacetylases (HDAC) with Suberoylanilide hydroxamic acid (SAHA) induced acetylation of the FANCA deficient DNA replication forks to levels comparable to their isogenic control counterparts. This forced permanence of acetylation impacted FA cells homeostasis by inducing DNA damage and promoting G2 cell cycle arrest. Altogether, this caused reduced RAD51 foci formation and increased markers of replication stress, including phospho-RPA-S33. Hypoacetylation of the FANCA deficient replication fork, is part of the cellular phenotype, the perturbation of this feature by agents that prevent deacetylation, such as SAHA, have a deleterious effect over the delicate equilibrium they have reached to perdure despite a defective FA/BRCA pathway.

Abstract B045: Ataxia telangiectasia- and Rad3-related kinase inhibitor (ATRi) camonsertib in combination with low dose gemcitabine in patients with solid tumors with DNA damage response (DDR) aberrations: Preclinical and Phase 1b results

Abstract Background: Antitumor activity and good tolerability of camonsertib (cam) monotherapy in patients (pts) with recurrent solid tumors was previously reported. Preclinical data showed that cam-related tumor cell death is potentiated by the replication stress-inducing agent gemcitabine (gem) even at low doses, resulting in synergistic combination activity. We evaluated safety/tolerability and efficacy of cam+gem in this Ph1b trial (TRESR; NCT04497116). Methods: A tumor xenograft (Granta-519; ATM loss of function) was tested with a range of cam+gem doses. Pts &amp;gt;18yr, ECOG 0-1, with advanced solid tumors with deleterious DDR alterations were enrolled. Cam (80-120mg) + de-escalating gem doses (1000-100mg/m2) were given over 21- or 28-day (d) cycles (cam d1-2/3 and d8-9/10 or d15-16/17; gem d1 and d8 or d15). Dose decisions followed BOIN methodology. Endpoints were safety/tolerability, recommended Ph2 dose (RP2D), response rate (RECIST v1.1, CA-125 [GCIG], or PSA [PCWG3]), clinical benefit rate (CBR; response or treatment [Tx] duration ≥16 weeks [w] without progression), ctDNA molecular response rate (MRR; best reduction in mean variant allele frequency ≥50%), and cam PK. Replication stress markers were analyzed in surrogate normal tissue. Results: In a tumor xenograft model, low doses of cam (1/3 of maximum tolerated dose [MTD]) and gem (1/20 of MTD) resulted in tumor regression not observed in single agent arms, with good tolerability. As of May 2023, 54 pts were treated with cam+gem in TRESR (ovarian [n=19], pancreatic [n=8], breast [n=6], other [n=21] tumors); enrollment genes included ATM (n=12), BRCA1 (n=18), BRCA2 (n=16), other (n=8). No drug-drug interactions were noted. RECIST v1.1 or CA-125 responses were observed in 5 pts (40 evaluable): 3 cPR, 1 uPR in pts with ovarian (n=2; 1 ATM, 1 BRCA1), endometrial (gPALB2), and breast (gBRCA1) cancers; 1 CA-125 response in a pt (ovarian/gBRCA1) with SD (on Tx 21w+). Four additional pts (uterine carcinosarcoma, ovarian, liver, lung) with SD were on Tx ≥6 months. CBR was 42.4% (14/33) in evaluable pts and 66.7% (8/12) in pts with gynecological cancers. MRR was 58% (7/12) in evaluable pts. Myelotoxicity, the most frequent Tx-related toxicity, improved with decreasing gem doses. Responses were maintained in the 3 pts with cPR following reductions to ≤200mg/m2 gem; the pts with the uPR and CA-125 responses started Tx at 200 and 100mg/m2 gem, respectively. Preliminary RP2D is 80mg cam (50% of monotherapy RP2D) + 400mg/m2 gem, 28d cycle (1w on/1w off). Related Grade 3+ neutropenia, anemia, and thrombocytopenia occurred in 44%, 11%, and 0% pts, respectively (n=9). Importantly, neutrophil nadir occurred in the 1w off Tx with quick rebound, alleviating the need for dose modifications. Conclusion: The synergy of low doses of cam+gem, demonstrated in preclinical studies, was confirmed in the clinical setting. A safe and tolerable dose and schedule of the combination was identified. The antitumor efficacy supports further development in pts with gynecological cancers; evaluation at RP2D is ongoing. Citation Format: Ezra Rosen, Timothy A Yap, Elisa Fontana, Elizabeth K Lee, Devalingam Mahalingam, Martin Højgaard, Niharika B Mettu, Gregory M Cote, Ruth Plummer, Ian M Silverman, Joseph D Schonhoft, Emeline Bacque, Adrian J Fretland, Gabriela Gomez, Danielle Ulanet, Kezhen Fei, Julia Yang, Maria Koehler, Anne Roulston, Li Li, Michal Zimmermann, Benedito A Carneiro, Stephanie Lheureux. Ataxia telangiectasia- and Rad3-related kinase inhibitor (ATRi) camonsertib in combination with low dose gemcitabine in patients with solid tumors with DNA damage response (DDR) aberrations: Preclinical and Phase 1b results [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 B045.

Pharmacological Ascorbate Elicits Anti-Cancer Activities against Non-Small Cell Lung Cancer through Hydrogen-Peroxide-Induced-DNA-Damage.

Non-small cell lung cancer (NSCLC) poses a significant global health burden with unsatisfactory survival rates, despite advancements in diagnostic and therapeutic modalities. Novel therapeutic approaches are urgently required to improve patient outcomes. Pharmacological ascorbate (P-AscH-; ascorbate at millimolar concentration in plasma) emerged as a potential candidate for cancer therapy for recent decades. In this present study, we explore the anti-cancer effects of P-AscH- on NSCLC and elucidate its underlying mechanisms. P-AscH- treatment induces formation of cellular oxidative distress; disrupts cellular bioenergetics; and leads to induction of apoptotic cell death and ultimately reduction in clonogenic survival. Remarkably, DNA and DNA damage response machineries are identified as vulnerable targets for P-AscH- in NSCLC therapy. Treatments with P-AscH- increase the formation of DNA damage and replication stress markers while inducing mislocalization of DNA repair machineries. The cytotoxic and genotoxic effects of P-AscH- on NSCLC were reversed by co-treatment with catalase, highlighting the roles of extracellular hydrogen peroxide in anti-cancer activities of P-AscH-. The data from this current research advance our understanding of P-AscH- in cancer treatment and support its potential clinical use as a therapeutic option for NSCLC therapy.

Brincidofovir induces potent anti‐tumor activity in MYC‐driven Natural Killer/T‐cell Lymphoma with loss of transcriptional repressor TLE1

Introduction: Natural killer/T-cell lymphoma (NKTCL) is an aggressive Epstein-Barr virus (EBV)-associated malignancy which is prevalent in Asian countries. Brincidofovir (BCV), a lipid conjugated form of cidofovir, is a biologically-active acyclic nucleoside phosphonate recently shown to exhibit both antiviral and antitumor properties. Methods: We investigated BCV in NKTCL cell-lines (n = 11) and mouse xenograft models. Whole exome sequencing, bulk RNAseq and single cell RNAseq (scRNAseq) were employed to examine mechanisms of action, and validated using orthogonal assays. Results: BCV inhibited viability in all NKTCL cell-lines in a dose- and time-dependent manner. Highest sensitivity was demonstrated in four cell-lines KAI-3, NK-S1, NK-92 and KHYG-1 (IC50 36.0 to 303.6 ng/ml). Intraperitoneal BCV (40 mg/kg, 2X per week) inhibited tumor growth in NOD/SCID mice NK-S1 xenografts, compared with vehicle alone (p = 0.0005, two-tailed t-test). Whole exome sequencing of the cell-lines revealed widespread mutations in the JAK/STAT and DNA repair pathways, with no clear correlation observed with BCV sensitivity. RNAseq and Hallmark gene set enrichment analysis showed that MYC target pathways (FDR q < 0.001) were prominently upregulated in the four sensitive cell-lines compared to the rest. RNAseq of BCV-treated cell-lines (KAI-3 and NK-S1) revealed striking downregulation of MYC target pathways. scRNAseq revealed distinct temporal cell states evoked by BCV, including appearance of cell clusters enriched for p53 and apoptosis pathways, cell cycle and DNA repair pathways, as well as type I/II interferon and cytokine signaling. Western blot and quantitative PCR showed that markers of replication stress, DNA damage and STING signaling were potently upregulated, implicating an immunogenic form of cell death. Notably, TLE1, a known transcriptional repressor of the MYC oncogene, was the topmost downregulated gene (log2FoldChange -7.39, adjusted p = 3.51E-31) in the four BCV-sensitive cell-lines on RNAseq. This finding was verified on qPCR (p = 0.0061, Mann-Whitney test) and Western blot. In keeping with this result, RNAseq on NKTCL patient samples (n = 36) showed that TLE1-low tumors were enriched for MYC target pathways, which conferred worse progression-free survival (HR 3.44, 95% CI: 1.23–10.49, p = 0.0301). Conclusions: Taken together, these results suggest that BCV may play a novel role in the treatment of MYC-driven NKTCL. Loss of TLE1, a putative predictive biomarker, implies that BCV may favour patients in this poor prognostic subgroup. The research was funded by: Symbio Pharmaceuticals, Tanoto Foundation Professorship in Medical Oncology, New Century Foundation Limited, Ling Foundation, Singapore Ministry of Health’s National Medical Research Council Transition Award (TA21jun-0005), Research Training Fellowship Seed Fund (SEEDFD21jun-0002), Large Collaborative Grant (OFLCG18May-0028), and Collaborative Centre Grant (TETRAD II). Keywords: Diagnostic and Prognostic Biomarkers, Extranodal non-Hodgkin lymphoma, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. J. Y. Chan Consultant or advisory role: Symbio Pharmaceuticals Research funding: Symbio Pharmaceuticals M. Hazama Employment or leadership position: SymBio Pharmaceuticals Limited K. Fukushima Employment or leadership position: SymBio Pharmaceuticals Limited C. K. Ong Research funding: SymBio Pharmaceuticals Limited

Influence of 2-Nitroimidazoles in the Response of FaDu Cells to Ionizing Radiation and Hypoxia/Reoxygenation Stress

Cellular adaptations to hypoxia promote resistance to ionizing radiation (IR). This presents a challenge for treatment of head and neck cancer (HNC) that relies heavily on radiotherapy. Standard radiosensitizers often fail to reach diffusion-restricted hypoxic cells, whereas nitroimidazoles (NIs) [such as iodoazomycin arabinofuranoside (IAZA) and fluoroazomycin arabinofuranoside (FAZA)] can preferentially accumulate in hypoxic tumours. Here, we explored if the hypoxia-selective uptake of IAZA and FAZA could be harnessed to make HNC cells (FaDu) susceptible to radiation therapy. Cellular response to treatment was assessed through clonogenic survival assays and by monitoring DNA damage (immunofluorescence staining of DNA damage markers, γ-H2AX and p-53BP1, and by alkaline comet assay). The effects of reoxygenation were studied using the following assays: estimation of nucleoside incorporation to assess DNA synthesis rates, immunofluorescent imaging of chromatin-associated replication protein A as a marker of replication stress, and quantification of reactive oxygen species (ROS). Both IAZA and FAZA sensitized hypoxic HNC cells to IR, albeit the former is a better radiosensitizer. Radiosensitization by these compounds was restricted only to hypoxic cells, with no visible effects under normoxia. IAZA and FAZA impaired cellular adaptation to reoxygenation; high levels of ROS, reduced DNA synthesis capacity, and signs of replication stress were observed in reoxygenated cells. Overall, our data highlight the therapeutic potentials of IAZA and FAZA for targeting hypoxic HNC cells and provide rationale for future preclinical studies.

Abstract PR010: Multiplex imaging reveals features of organotropism in pancreatic ductal adenocarcinoma

Abstract PDAC patients have a dismal prognosis with an 11% 5-year survival rate, driven in part by the fact that 90% of patients are diagnosed with metastatic disease. In a cohort of 300 PDAC patients, we observed that patients who had lung-only metastasis had longer overall survival than the majority of patients with metastasis to the liver. We used gene expression data to develop a signature based on gene expression in primary tumors of liver versus lung organotropism, which we called pORG. Our pORG signature predicts long term survival in PDAC patients and reveals unique features of the primary tumor, including increased interferon and immune activity in lung tropic tumors and increased proliferation and replication stress in liver-tropic tumors. To validate our findings, we generated cyclic immunofluorescence multiplex imaging data for a subset of 34 patients. Single cell image analysis revealed B and T cell infiltration and spatial clustering in low pORG/ lung tropic tumors, and markers of replication stress in high pORG patients. Our work suggests unique targetable tumor and microenvironment features linked to organotropism in PDAC. Citation Format: Jennifer Eng, Jason Link, Carl Pelz, Rosalie Sears. Multiplex imaging reveals features of organotropism in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR010.

Therapeutic Repurposing of Brincidofovir in Natural Killer/T-Cell Lymphoma Reveals Potent Induction of Replication Stress, Sting Pathway Activation and Immunogenic Cell Death

Introduction Natural killer/T-cell lymphoma (NKTCL) is an aggressive subtype of non-Hodgkin lymphoma associated with Epstein-Barr virus (EBV) infection. Brincidofovir (BCV), a lipid conjugated form of cidofovir, is a biologically active acyclic nucleoside phosphonate known for both antiviral and antiproliferative properties. Methods We investigated BCV in a panel of NKTCL cell lines (n=11) and mouse xenograft models. The mechanism of action was examined using bulk and single cell RNA sequencing (10X Chromium 3' Gene Expression Profiling), and validated in orthogonal assays. Results BCV evoked a dose- and time-dependent inhibition of cell viability in all NKTCL cell lines, independent of EBV presence. Exquisite sensitivity was observed in 4 cell lines KAI-3, NK-S1, NK-92 and KHYG-1 (IC50 range, 36.0 to 303.6 ng/ml), accompanied by increase in sub-G1 fraction and S-phase arrest on cell cycle analyses. Bulk RNA sequencing of BCV-treated cell lines (KAI-3 and NK-S1) revealed pervasive downregulation of MYC target pathways, along with upregulation of immune-related and DNA repair pathways. Single cell RNA sequencing revealed distinct temporal cell states evoked by BCV. In KAI-3 cells, a significant shift in transcriptomic cell states occurred upon BCV treatment for 48h, with appearance of cell clusters enriched for type I interferon and cytokine signaling pathways, as well as activation of p53 and apoptosis pathways. Cell cycle and DNA repair pathways were also evident. At 72h post-BCV treatment, type II interferon signaling and endoplasmic reticulum stress response pathways were prominent. Similarly in NK-S1 cells, cell clusters enriched for immune signals, including interleukin-18 and cytokine pathways emerged, as did cell cycle, DNA repair, and apoptosis pathways. Western blot demonstrated increase in replication stress markers (p-CHK1 S317 and S345, RRM2, p-RPA2 S33), p-H2AX (DNA double strand break) and p-TBK1 (STING pathway activation). In keeping with this result, BCV triggered micronuclei formation, DNA fragmentation, and cytoplasmic DNA release. Gene expression levels of type I (IFNA, IFNB1) and II (IFNY) interferons and cytokines (CCL5, CXCL10) were upregulated following BCV treatment, as did the proportion of calreticulin-expressing cells and amount of extracellular HMGB1 released, indicative of immunogenic cell death. Notably, nuclear and membranous protein expression of the immune checkpoint protein PD-L1 also markedly increased. Intraperitoneal administration of BCV (40 mg/kg, twice per week) inhibited tumor volume in NOD/SCID mouse xenografts of NKTCL, compared with vehicle alone (p=0.0005, two-tailed t-test). On immunohistochemistry, PD-L1 was significantly overexpressed in BCV-treated tumor xenografts compared with control, with a 1.9-fold higher median H-score (p=0.0007, Mann-Whitney test). Conclusions Taken together, these results demonstrate a novel repurposed role of BCV in the treatment of NKTCL. The activation of DNA damage-induced STING pathway and immune-related signals suggests a further potential for combination with checkpoint immunotherapy.

Impacts of arsenic on Rad18 and translesion synthesis

Arsenite interferes with DNA repair protein function resulting in the retention of UV-induced DNA damage. Accumulated DNA damage promotes replication stress which is bypassed by DNA damage tolerance pathways such as translesion synthesis (TLS). Rad18 is an essential factor in initiating TLS through PCNA monoubiquitination and contains two functionally and structurally distinct zinc fingers that are potential targets for arsenite binding. Arsenite treatment displaced zinc from endogenous Rad18 protein and mass spectrometry analysis revealed arsenite binding to both the Rad18 RING finger and UBZ domains. Consequently, arsenite inhibited Rad18 RING finger dependent PCNA monoubiquitination and polymerase eta recruitment to DNA damage in UV exposed keratinocytes, both of which enhance the bypass of cyclobutane pyrimidine dimers during replication. Further analysis demonstrated multiple effects of arsenite, including the reduction in nuclear localization and UV-induced chromatin recruitment of Rad18 and its binding partner Rad6, which may also negatively impact TLS initiation. Arsenite and Rad18 knockdown in UV exposed keratinocytes significantly increased markers of replication stress and DNA strand breaks to a similar degree, suggesting arsenite mediates its effects through Rad18. Comet assay analysis confirmed an increase in both UV-induced single-stranded DNA and DNA double-strand breaks in arsenite treated keratinocytes compared to UV alone. Altogether, this study supports a mechanism by which arsenite inhibits TLS through the altered activity and regulation of Rad18. Arsenite elevated the levels of UV-induced replication stress and consequently, single-stranded DNA gaps and DNA double-strand breaks. These potentially mutagenic outcomes support a role for TLS in the cocarcinogenicity of arsenite.

Identification of a Molecularly-Defined Subset of Breast and Ovarian Cancer Models that Respond to WEE1 or ATR Inhibition, Overcoming PARP Inhibitor Resistance.

PARP inhibitors (PARPi) induce synthetic lethality in homologous recombination repair (HRR)-deficient tumors and are used to treat breast, ovarian, pancreatic, and prostate cancers. Multiple PARPi resistance mechanisms exist, most resulting in restoration of HRR and protection of stalled replication forks. ATR inhibition was highlighted as a unique approach to reverse both aspects of resistance. Recently, however, a PARPi/WEE1 inhibitor (WEE1i) combination demonstrated enhanced antitumor activity associated with the induction of replication stress, suggesting another approach to tackling PARPi resistance. We analyzed breast and ovarian patient-derived xenoimplant models resistant to PARPi to quantify WEE1i and ATR inhibitor (ATRi) responses as single agents and in combination with PARPi. Biomarker analysis was conducted at the genetic and protein level. Metabolite analysis by mass spectrometry and nucleoside rescue experiments ex vivo were also conducted in patient-derived models. Although WEE1i response was linked to markers of replication stress, including STK11/RB1 and phospho-RPA, ATRi response associated with ATM mutation. When combined with olaparib, WEE1i could be differentiated from the ATRi/olaparib combination, providing distinct therapeutic strategies to overcome PARPi resistance by targeting the replication stress response. Mechanistically, WEE1i sensitivity was associated with shortage of the dNTP pool and a concomitant increase in replication stress. Targeting the replication stress response is a valid therapeutic option to overcome PARPi resistance including tumors without an underlying HRR deficiency. These preclinical insights are now being tested in several clinical trials where the PARPi is administered with either the WEE1i or the ATRi.

DNA methylating drug temozolomide sensitizes ARID1A-mutated tumors to PARP inhibitors (010)

<b>Objectives:</b> Somatic mutations of ARID1A are present in greater than 50% of ovarian clear cell carcinoma (OCCC). ARID1A and BRG1 are key subunits that form the SWI/SNF complex to regulate chromatin configuration and other key cellular functions. Somatic mutation of TP53 or BRG1 is rare in OCCC and when either mutation occurs, it is often mutually exclusive with ARID1A mutations, suggesting an overlapping function of these tumor suppressor genes. ARID1A-SWI/SNF complex was involved in DNA damage repair. Compromised DNA repairs are identified in ARID1A-deficient tumors, rendering ARID1A-deficient tumors hypersensitive to PARP inhibitors when primed with low-dose ionizing radiation (IR). The methylating chemotherapeutic drug, Temozolomide (TMZ), induces DNA lesions like IR. Thus, we propose to re-purpose this drug for gynecological cancers. <b>Methods:</b> As a proof-of-principle study, we employed four pairs of isogenic human cell lines, hEM3, MCF10a, HCT116, and OVCA429, engineered with/without ARID1A knockout. HCT116 and OVCA429 have DNA mismatch repair (MMR) deficiency while hEM3 and MCF10a do not. Different concentrations of the PARP inhibitor, Olaparib (Olap), and TMZ were co-applied <i>in vitro</i> to determine cytotoxic synergism. OCCC xenografts were established in athymic <i>nu/ nu</i> mice and treated with TMZ, Olap, TMZ+Olap, or vehicle control. Efficacy was measured by changes in tumor volume. Biomarker and pathological analyses were performed on tumor samples.Fig. 1 <b>Results:</b> MMR-proficient ARID1A-/- cells were more sensitive to TMZ+Olap treatment compared to the isogenic MMR-proficient ARID1A+/+ cells. Additionally, ARID1A-/- cells could not effectively repair DNA lesions induced by the DNA alkylating drug, TMZ, because of a compromised base excision repair. DNA single-strand breaks in ARID1A-deficienct cells were associated with replication fork instability, manifested by fork collapse and replication stress. Moreover, the TMZ+Olap combination treatment delayed the <i>in vivo</i> growth of OCCC xenograft tumors with ARID1A-inactivating mutations. The treatment induced DNA breaks, replication stress, and apoptosis on the xenografted tumors. The markers of replication stress including gH2Ax and pRPA and a marker of apoptosis, cleaved caspase 3, were significantly elevated. Proteomic analysis of ARID1A knockout cells using an anti-BRG1 antibody to isolate the SWI/SNF complex indicated an upregulation of DNA damage repair proteins and RNA helicases, suggesting dysregulation of these processes in ARID1A-deficient cells. <b>Conclusions:</b> Our findings suggest a synergistic relationship between TMZ and Olap in treating ARID1A-deficient tumors. The data demonstrated accumulated DNA lesions and breaks induced by TMZ that cannot be effectively repaired by ARID1A-deficient tumor cells because of hampered base excision repair-related machinery. Thus, ARID1A-deficient tumor cells are highly reliant on PARP-dependent repair. Our data provide pre-clinical supports for a DNA break-primed and PARP inhibitor-based treatment strategy for OCCC.

A ferrocene-containing nucleoside analogue targets DNA replication in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is a disease that remains refractory to existing treatments including the nucleoside analogue gemcitabine. In the current study we demonstrate that an organometallic nucleoside analogue, the ferronucleoside 1-(S,Rp), is cytotoxic in a panel of PDAC cell lines including gemcitabine-resistant MIAPaCa2, with IC50 values comparable to cisplatin. Biochemical studies show that the mechanism of action is inhibition of DNA replication, S-phase cell cycle arrest and stalling of DNA-replication forks, which were directly observed at single molecule resolution by DNA-fibre fluorography. In agreement with this, transcriptional changes following treatment with 1-(S,Rp) include activation of three of the four genes (HUS1, RAD1, RAD17) of the 9-1-1 check point complex clamp and two of the three genes (MRE11, NBN) that form the MRN complex as well as activation of multiple downstream targets. Furthermore, there was evidence of phosphorylation of checkpoint kinases 1 and 2 as well as RPA1 and gamma H2AX, all of which are considered biochemical markers of replication stress. Studies in p53-deficient cell lines showed activation of CDKN1A (p21) and GADD45A by 1-(S,Rp) was at least partially independent of p53. In conclusion, because of its potency and activity in gemcitabine-resistant cells, 1-(S,Rp) is a promising candidate molecule for development of new treatments for PDAC.

A Comparative Assessment of Replication Stress Markers in the Context of Telomerase.

Simple SummaryGenetic alterations such as oncogenic- or aneuploidy-inducing mutations can induce replication stress as a tumor protection mechansim. Previous data indicated that telomerase may ameliorate the cellular responses that induce replication stress. However, the mechanisms how this may occur are still unclear. In order to address this question, the accurate evaluation of replication stress in the presence and absence of telomerase is crucial. Therefore, we used telomerase negative normal human fibroblasts, as well as their telomerase positive counterparts to compare the suitability of three protein markers (pRPA2, γ-H2AX and 53BP1), which were previously reported to accumulate in response to harmful conditions leading to replication stress in cells. In summary, we find that pRPA2 is the most consistent and reliable marker for the detection of replication stress. Further, we demonstrated that the inhibition of the DNA-damage activated ATM and ATR kinases by specific small compounds impaired the accumulation of pRPA2 foci in the absence of telomerase. These data suggest that telomerase rescues the cells from replication stress upon supression of DNA damage induction by modulating the ATM and ATR signaling pathways, and may therefore support tumor formation of genetically unstable cells.Aberrant replication stress (RS) is a source of genome instability and has serious implications for cell survival and tumourigenesis. Therefore, the detection of RS and the identification of the underlying molecular mechanisms are crucial for the understanding of tumourigenesis. Currently, three protein markers—p33-phosphorylated replication protein A2 (pRPA2), γ-phosphorylated H2AX (γ-H2AX), and Tumor Protein P53 Binding Protein 1 (53BP1)—are frequently used to detect RS. However, to our knowledge, there is no report that compares their suitability for the detection of different sources of RS. Therefore, in this study, we evaluate the suitability of pRPA2, γ-H2AX, and 53BP1 for the detection of RS caused by different sources of RS. In addition, we examine their suitability as markers of the telomerase-mediated alleviation of RS. For these purposes, we use here telomerase-negative human fibroblasts (BJ) and their telomerase-immortalized counterparts (BJ-hTERT). Replication stress was induced by the ectopic expression of the oncogenic RAS mutant RASG12V (OI-RS), by the knockdown of ploidy-control genes ORP3 or MAD2 (AI-RS), and by treatment with hydrogen peroxide (ROS-induced RS). The level of RS was determined by immunofluorescence staining for pRPA2, γ-H2AX, and 53BP1. Evaluation of the staining results revealed that pRPA2- and γ-H2AX provide a significant and reliable assessment of OI-RS and AI-RS compared to 53BP1. On the other hand, 53BP1 and pRPA2 proved to be superior to γ-H2AX for the evaluation of ROS-induced RS. Moreover, the data showed that among the tested markers, pRPA2 is best suited to evaluate the telomerase-mediated suppression of all three types of RS. In summary, the data indicate that the choice of marker is important for the evaluation of RS activated through different conditions.

Targeting Cellular Iron Homeostasis with Ironomycin in Diffuse Large B-cell Lymphoma.

Iron homeostasis represents a potential therapeutic target for high-risk patients with DLBCL that can be targeted with ironomycin to induce cell death and to sensitize tumor cells to conventional treatments.

Increased replication stress and R-loop accumulation in EGFRvIII-expressing glioblastoma present new therapeutic opportunities.

BackgroundThe oncogene epidermal growth factor receptor variant III (EGFRvIII) is expressed in approximately one-third of all glioblastomas (GBMs). So far it is not clear if EGFRvIII expression induces replication stress in GBM cells, which might serve as a therapeutical target.MethodsIsogenetic EGFRvIII− and EGFRvIII+ cell lines with endogenous EGFRvIII expression were used. Markers of oncogenic and replication stress such as γH2AX, RPA, 53BP1, ATR, and CHK1 were analyzed using western blot, immunofluorescence, and flow cytometry. The DNA fiber assay was performed to analyze replication, transcription was measured by incorporation of EU, and genomic instability was investigated by micronuclei and CGH-Array analysis. Immunohistochemistry staining was used to detect replication stress markers and R-loops in human GBM samples.ResultsEGFRvIII+ cells exhibit an activated replication stress response, increased spontaneous DNA damage, elevated levels of single-stranded DNA, and reduced DNA replication velocity, which are all indicative characteristics of replication stress. Furthermore, we show here that EGFRvIII expression is linked to increased genomic instability. EGFRvIII-expressing cells display elevated RNA synthesis and R-loop formation, which could also be confirmed in EGFRvIII-positive GBM patient samples. Targeting replication stress by irinotecan resulted in increased sensitivity of EGFRvIII+ cells.ConclusionThis study demonstrates that EGFRvIII expression is associated with increased replication stress, R-loop accumulation, and genomic instability. This might contribute to intratumoral heterogeneity but may also be exploited for individualized therapy approaches.

MMB-FOXM1-driven premature mitosis is required for CHK1 inhibitor sensitivity

SUMMARYTo identify genes whose loss confers resistance to CHK1 inhibitors, we perform genome-wide CRISPR-Cas9 screens in non-small-cell lung cancer (NSCLC) cell lines treated with the CHK1 inhibitor prexasertib (CHK1i). Five of the top six hits of the screens, MYBL2 (B-MYB), LIN54, FOXM1, cyclin A2 (CCNA2), and CDC25B, are cell-cycle-regulated genes that contribute to entry into mitosis. Knockout of MMB-FOXM1 complex components LIN54 and FOXM1 reduce CHK1i-induced DNA replication stress markers and premature mitosis during Late S phase. Activation of a feedback loop between the MMB-FOXM1 complex and CDK1 is required for CHK1i-induced premature mitosis in Late S phase and subsequent replication catastrophe, indicating that dysregulation of the S to M transition is necessary for CHK1 inhibitor sensitivity. These findings provide mechanistic insights into small molecule inhibitors currently studied in clinical trials and provide rationale for combination therapies.

Targeting the PI3K/mTOR Pathway Augments CHK1 Inhibitor-Induced Replication Stress and Antitumor Activity in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecologic malignancy in industrialized countries and has limited treatment options. Targeting ataxia-telangiectasia and Rad3-related/cell-cycle checkpoint kinase 1 (CHK1)-mediated S-phase and G2-M-phase cell-cycle checkpoints has been a promising therapeutic strategy in HGSOC. To improve the efficacy of CHK1 inhibitor (CHK1i), we conducted a high-throughput drug combination screening in HGSOC cells. PI3K/mTOR pathway inhibitors (PI3K/mTORi) showed supra-additive cytotoxicity with CHK1i. Combined treatment with CHK1i and PI3K/mTORi significantly attenuated cell viability and increased DNA damage, chromosomal breaks, and mitotic catastrophe compared with monotherapy. PI3K/mTORi decelerated fork speed by promoting new origin firing via increased CDC45, thus potentiating CHK1i-induced replication stress. PI3K/mTORi also augmented CHK1i-induced DNA damage by attenuating DNA homologous recombination repair activity and RAD51 foci formation. High expression of replication stress markers was associated with poor prognosis in patients with HGSOC. Our findings indicate that combined PI3K/mTORi and CHK1i induces greater cell death in HGSOC cells and in vivo models by causing lethal replication stress and DNA damage. This insight can be translated therapeutically by further developing combinations of PI3K and cell-cycle pathway inhibitors in HGSOC. SIGNIFICANCE: Dual inhibition of CHK1 and PI3K/mTOR pathways yields potent synthetic lethality by causing lethal replication stress and DNA damage in HGSOC, warranting further clinical development.