High Levels Of Replication Stress Research Articles

Novel Amidine Derivative K1586 Sensitizes Colorectal Cancer Cells to Ionizing Radiation by Inducing Chk1 Instability.

Checkpoint kinase 1 (Chk1) is a key mediator of the DNA damage response that regulates cell cycle progression, DNA damage repair, and DNA replication. Small-molecule Chk1 inhibitors sensitize cancer cells to genotoxic agents and have shown preclinical activity as single agents in cancers characterized by high levels of replication stress. However, the underlying genetic determinants of Chk1-inhibitor sensitivity remain unclear. Although treatment options for advanced colorectal cancer are limited, radiotherapy is effective. Here, we report that exposure to a novel amidine derivative, K1586, leads to an initial reduction in the proliferative potential of colorectal cancer cells. Cell cycle analysis revealed that the length of the G2/M phase increased with K1586 exposure as a result of Chk1 instability. Exposure to K1586 enhanced the degradation of Chk1 in a time- and dose-dependent manner, increasing replication stress and sensitizing colorectal cancer cells to radiation. Taken together, the results suggest that a novel amidine derivative may have potential as a radiotherapy-sensitization agent that targets Chk1.

Abstract 2083: Discovery of ETX-19477, a novel and selective PARG inhibitor with high potency against tumors with underlying replication stress

Abstract DNA damage response (DDR) is an important cellular process that is regulated by poly ADP ribosylation (PARylation). PARP1 binds to DNA break sites and then catalyzes PARylation, which leads to recruitment of repair factors and initiation of base-excision and single-strand break (SSB) repair. Removal of PAR chains is performed by PARG and is also critical for successful completion of DNA repair. Suppression of PAR chain hydrolysis via PARG depletion or inhibition leads to defective single-strand break repair, reduced kinetics of repair, and hypersensitivity to certain DNA damaging agents. Cancer cells demonstrate high levels of replication stress, characterized as the slowing or stalling of replication forks during DNA replication. Replication stress generates DNA SSBs and exposes unligated Okazaki fragments which activate PARP1. PARP1 catalyzed PARylation slows replication fork progression as a protective mechanism, initiates damage repair, and promotes fork reversal. PAR chain removal, via PARG activity, enables completion of damage repair, replication fork restart, and recovery from replication stress. Under conditions of replication stress, PARP inhibition can result in unrestrained fork progression and the occurrence of DNA double strand breaks from fork runoff. In contrast, PARG inhibition causes prolonged slowing or stalling of replication fork progression, which can lead to replication catastrophe and cancer cell death. Because of these opposing roles, PARG inhibitors show distinct pharmacology as compared to PARP inhibitors in certain cancer cells including cancer cells that are BRCA wild type, homologous recombination proficient, and PARP inhibitor insensitive or resistant. ETX-19477 is a novel inhibitor of PARG that demonstrates low nM, on-target activity in cells as measured by the accumulation of PAR chains. ETX-19477 induces death and/or inhibits the proliferation of multiple cancer cell types including ER+HER2- breast, serous and mucinous ovarian, lung, and gastric cancers and induces pan-nuclear γH2AX expression, a hallmark of replication catastrophe. Preclinical studies demonstrate that ETX-19477 is orally bioavailable with favorable pharmacokinetics. Oral treatment of mice with ETX-19477 is well tolerated and shows robust dose-dependent in vivo anti-tumor activity in breast and ovarian cell-line derived and patient-derived xenograft models. Efficacy is observed with either once or twice daily dosing and with a discontinuous dosing regimen. In mouse xenograft models, induction of γH2AX expression in tumor tissue correlates with tumor growth inhibition. Preclinical data show ETX-19477 demonstrates potent and specific anti-proliferative effects by exposing underlying existing replication stress vulnerabilities in cancer cells across multiple tumor types. ETX-19477 will be advanced into clinical trials in 2024. Citation Format: John P. Holleran, Tamara S. Rodems, Shweta Sharma, Angelina M. Santini, Cindy Wuerz, Juping Liu, Lu Tian, Joseph Dosch, Joy Nader, Richard Rivera, Shyama Herath, Joseph Marakovits, Christopher McBride, Ryan Stansfield, Amy Salyards, James M. Veal, Jeffrey A. Stafford, Gretchen Bain. Discovery of ETX-19477, a novel and selective PARG inhibitor with high potency against tumors with underlying replication stress [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 2083.

Abstract A023: Preclinical development of PKMYT1 and WEE1 inhibitor combinations

Abstract Background: CCNE1-amplification or overexpression are prevalent alterations in multiple tumor types, including high-grade serous ovarian, endometrial, and gastro-esophageal cancers. CCNE1-amplification is associated with high levels of replication stress, genomic instability, and resistance to chemotherapy. Previously, CRISPR-enabled synthetic lethal screening identified that loss of the CDK1 regulator Membrane-associated tyrosine- and threonine-specific Cdc2-inhibitory kinase (PKMYT1) is synthetic lethal with CCNE1-overexpression, leading to the development of lunresertib - a first-in-class, potent, selective, orally bioavailable PKMYT1 inhibitor currently being investigated in phase I clinical trials (NCT04855656, NCT05147272, NCT05147350). In CCNE1 overexpressing cells, lunresertib treatment leads to reduction of inhibitory CDK1-T14 phosphorylation and activated CDK1 in S-phase, triggering premature mitosis, chromosome pulverization, and ultimately cell death. In CCNE1-amplified tumor models, lunresertib exhibits antitumor activity as monotherapy. We hypothesized that further activation of CDK1 by reducing the inhibitory phosphorylation site at CDK1-pY15 by WEE1 inhibition would increase the antitumor activity of lunresertib. Methods: In vitro cell viability studies were used to determine the level of synergy between lunresertib and the WEE1 inhibitor Debio-0123 in engineered as well as tumor-derived CCNE1-high models. The lunresertib/Debio-0123 combination mechanism of action was investigated using high content imaging and immunoblotting for DNA damage and cell cycle markers in cultured cells. Finally, the pre-clinical anti-tumor activity and tolerability of combination lunresertib/Debio-0123 was evaluated in vivo using CCNE1-amplified tumor xenografts in mice. Results: Combination of low doses of lunresertib with Debio-0123 selectively induced synergistic cytotoxicity and catastrophic DNA damage in CCNE1-overexpressing cells, without affecting wild type counterparts. Increased DNA damage resulted from robust activation of CDK1 in S-phase, causing rapid induction of premature mitosis. Combination of lunresertib and Debio-0123 abrogated phosphorylation of both Thr14 and Tyr15 inhibitory CDK1 sites at doses that led to only partial dephosphorylation in both monotherapy settings. Mechanistically, synergistic CDK1 dephosphorylation was partially mediated by activation of CDC25B phosphatase. In a mouse model, the combination of RP-6306 and Debio-0123 was well-tolerated and demonstrated tumor regressions in a CCNE1-overexpressing tumor xenograft model at sub-therapeutic monotherapy doses. Conclusions: Lunresertib and the WEE1 inhibitor Debio-0123 elicit strong synergistic activity in preclinical models of CCNE1-overexpressoin due to rapid and complete CDK1 activation. In a xenograft tumor model, intermittent dosing of lunresertib/WEE1i lead to tumor regressions at sub-therapeutic monotherapy doses. Together these findings provide mechanistic rationale for clinical development of PKMYT1 and WEE1 inhibitor combinations. Citation Format: David Gallo, Fenil Shah, Rino Stocco, Prasamit Baruah, Jimmy Fourtounis, Rosie Kryczka, Marc L Hyer, Anne Roulston, C Gary Marshall, Michal Zimmermann, Stephen Morris, Michael Zinda. Preclinical development of PKMYT1 and WEE1 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 A023.

Abstract 6206: Combined inhibition of AXL and ATR enhances replication stress, cell death and immune response in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung tumor. Despite high initial responses to frontline chemo-immunotherapy, therapeutic resistance develops rapidly. There are limited treatment options in the relapsed setting, where the prognosis remains dismal. SCLC tumors experience continuous and high levels of replication stress (RS) due to ubiquitous loss of key cell cycle checkpoints, RB1 and TP53. Frequent amplification and high expression of the transcription factor cMYC further contribute to increased RS. Thus, high levels of RS expose a potential SCLC vulnerability and provide a therapeutic opportunity. Our group and others have shown that AXL, a TAM family receptor tyrosine kinase that is highly expressed in mesenchymal tumors, mediates resistance to chemotherapy, radiation and targeted therapies in SCLC, non-small cell lung cancer and other cancers, through its role in driving epithelial to mesenchymal transition (EMT). More recently, a novel role for AXL in DNA damage repair and tolerance has emerged. Therefore, we hypothesize that AXL targeting may be a potential therapeutic approach in SCLC. We first investigated the transcriptomic expression profile of AXL in SCLC clinical cohorts. AXL-high tumors were seen in a subset of treatment-naïve SCLC tumors, frequently among, but not limited to, the inflamed SCLC subtype. AXL expression was also seen in many relapsed SCLC tumors. As expected, tumors with high AXL expression also expressed several mesenchymal genes and higher EMT scores. Interestingly, among the treatment-naïve SCLC tumors, AXL expression was inversely correlated with a RS signature (rho=-0.54, p<0.001). Next, we tested the effects of AXL inhibition in SCLC in vitro and in vivo models. In a panel of 30 SCLC cell lines, bemcentinib, a selective AXL inhibitor in clinical trials for various advanced solid tumors, exhibited a range of antiproliferative activity, with IC50 values ranging from 41 nM to 10 µM (median IC50 3.1 µM). Bemcentinib also significantly delayed tumor growth in in vivo SCLC models. Biomarkers associated with sensitivity to bemcentinib in SCLC cell lines included markers of RS (cMYC, replication stress score) and DNA damage response (phospho CHK1S345, phospho CHK2T68). Bemcentinib also induced RS, indicated by the activation of ATR/CHK1-mediated RS response pathway, and DNA damage, and the combination with an ATR inhibitor (ceralasertib) showed a greater than additive effect. In a syngeneic model of SCLC, the combination of bemcentinib, ceralasertib and an anti-PDL1 antibody induced significant tumor regression. Together, these promising findings demonstrate that AXL inhibition may be an effective strategy to target the RS vulnerability common in SCLC. Citation Format: Kavya Ramkumar, C. Allison Stewart, Azusa Tanimoto, Qi Wang, Yuanxin Xi, Benjamin B. Morris, Runsheng Wang, Li Shen, Robert J. Cardnell, Jing Wang, Carl M. Gay, Lauren A. Byers. Combined inhibition of AXL and ATR enhances replication stress, cell death and immune response in small cell lung cancer [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 6206.

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.

Correlative results from NCI protocol 10250: A phase II study of temozolomide and olaparib for the treatment of advanced uterine leiomyosarcoma.

11509 Background: uLMS is an aggressive sarcoma subtype of smooth muscle origin. Chemotherapy provides limited benefit for advanced disease. 18-25% of uLMS harbor deleterious alterations in homologous recombination (HR) DNA repair genes. uLMS exhibits high levels of replicative stress. These findings prompted a phase 2 study of O+T in pretreated uLMS where O+T demonstrated activity: ORR 27%, mPFS 6.9 mos (Ingham M. et. al. ASCO 2021: #11506) Methods: NCI protocol #10250 is a single-arm, multicenter, phase 2 trial evaluating O+T in advanced uLMS pts with progression on ≥1 prior line. Pre-treatment (Pre) and on-treatment (On) biopsies were collected from 22 pts. In prespecified analysis, we evaluated for a relationship between clinical outcomes and HR gene alterations by whole exome sequencing (WES), SLFN11/MGMT expression by RNAseq, and RAD51 foci formation (functional assay). HRD scores were calculated from WES using scarHRD. Gene expression was evaluated using a Spearman rank-order correlation analysis to identify genes associated with PFS (p < 0.01) and overexpressed in sensitive (S: PFS > 240d) or resistant (R: PFS < 240d) pts. Gene set enrichment analysis (GSEA) was performed (q = FDR-adjusted p value). Pts with available results: WES/RNAseq (16), Pre HRD score (13), Pre RAD51 foci (12). Results: 31% (5/16) pts had a mutation (Mut) or homozygous deletion (Hd) in the HR panel: ATRX Mut (2), ATR Mut, PALB2 Hd, RAD51B Hd. Pts with PALB2 and RAD51B Hd had longest PFS on study. Recurrent alterations also occurred in TP53 (56%) and RB1 (19%). Median HRD score in Pre samples was 51 (range 36-66) and 10/13 had HRD scores ≥ 42. Pre and On SLFN11 and MGMT RNA expression were not correlated with ORR/PFS. 6/13 Pre samples were HR-deficient by the RAD51 foci assay. Of pts with PFS ≥ 200d, 4/6 were HR-deficient. In Pre samples, 81 genes were overexpressed in S pts and 73 in R pts. In On samples, 146 genes were overexpressed in S pts and 127 in R pts. In On samples, GSEA identified the epithelial-mesenchymal transition enriched in S pts (q = 3.38e-7) and cell cycle pathways (E2F targets, G2M checkpoint) in R pts (q = 7.43e-4). Only 2 genes, CXCL10 and PCDH15, were differentially expressed between paired Pre and On samples (both increased in On). Gene expression signatures for replicative stress showed borderline association with worse PFS. Conclusions: Most uLMS tumors exhibit HR defects as measured by HRD scores. A subset of pts with greater benefit from O+T were identified by WES for HR genes and the RAD51 assay. There was no correlation between SLFN11 and MGMT expression and outcomes. GSEA identified pathways differentially expressed in S and R pts in On samples. O+T induced CXCL10 which has been associated with T-cell trafficking to tumors. A randomized phase 3 trial of O+T versus investigator’s choice is planned. These results provide insight into which pts may benefit most from this novel drug combination. Clinical trial information: NCT03880019.

A phase I/II study of prexasertib in combination with irinotecan in patients with relapsed/refractory desmoplastic small round cell tumor and rhabdomyosarcoma.

11503 Background: Prexasertib (PRX) is an inhibitor of CHK1, prevents DNA repair leading to mitotic catastrophe, and can enhance the activity of DNA-damaging chemotherapy. Translocation driven sarcomas exhibit high levels of replication stress and have demonstrated susceptibility to CHK1 inhibition in preclinical models. Desmoplastic small round cell tumor (DSRCT) and rhabdomyosarcoma (RMS) are aggressive sarcomas of children, adolescents and young adults for which novel therapies are urgently required. Methods: We conducted a phase I/II trial of PRX with irinotecan (irino) in patients ≥ 12 months of age with relapsed or refractory DSRCT or RMS. Eligible patients could have any number of prior therapies, including irino. Dose level 1 was PRX 80 mg/m2 on day 1 + irino 20 mg/m2 for 10 days. Dose levels 2 and 2A were PRX 105 or 150 mg/m2 (>21 years or ≤ 21 years) on day 1 and irino 20 mg/m2 for 10 (level 2) or 5 (level 2A) days. All cycles were 21 days. The primary objectives were to determine the RP2D of PRX with irino, and to determine the best overall response rate (ORR) in 6 months at the RP2D (RECIST v1.1) in DSRCT, with 3 or more responses out of 16 considered promising. Results: 21 patients were enrolled (DSRCT: 19; 2 RMS:2). The RP2D was dose level 2A. Treatment was well tolerated with the most common adverse events being neutropenia (48%), nausea (48%), and fatigue (52%). Cytopenias were managed with the aid of growth factor support in all patients once the RP2D was established. The DSRCT expansion enrolled 13 of 16 planned patients due to discontinuation of PRX supply prior to study completion. Four patients remain on therapy at the time of this submission. Responses in DSRCT patients at all dose levels are shown in Table. Sixteen of 21 enrolled patients, and 5 of 6 patients achieving PR had previously received irino. The median (range) number of cycles was 7 (2-26). Both RMS patients treated at the RP2D experienced SD as best response. The estimated ORR at the RP2D was 23%, and lower boundary of the one-sided 90% confidence interval was 9%, exceeding the unpromising rate of 5%. The two-sided 90% confidence interval was 7 to 49%. In addition, 3 patients had a PR at doses lower than the RP2D, bringing the ORR for all dose levels (n = 19) to 32% (90%CI: 15 to 53%). Conclusions: The RP2D of PRX in combination with irino is PRX 105 or 150 mg/m2 (>21 years or ≤ 21 years) on day 1 and irino 20 mg/m2 for 5 days in 21 day cycles with myelosuppression successfully managed with growth factor support. The study met its primary objective to consider PRX + irino promising in DSRCT and should be further investigated. Clinical trial information: NCT04095221. [Table: see text]

An mRNA expression-based signature for oncogene-induced replication-stress.

Oncogene-induced replication stress characterizes many aggressive cancers. Several treatments are being developed that target replication stress, however, identification of tumors with high levels of replication stress remains challenging.We describe a gene expression signature of oncogene-induced replication stress. A panel of triple-negative breast cancer (TNBC) and non-transformed cell lines were engineered to overexpress CDC25A, CCNE1 or MYC, which resulted in slower replication kinetics. RNA sequencing analysis revealed a set of 52 commonly upregulated genes. In parallel, mRNA expression analysis of patient-derived tumor samples (TCGA, n=10,592) also revealed differential gene expression in tumors with amplification of oncogenes that trigger replication stress (CDC25A, CCNE1, MYC, CCND1, MYB, MOS, KRAS, ERBB2, and E2F1). Upon integration, we identified a six-gene signature of oncogene-induced replication stress (NAT10, DDX27, ZNF48, C8ORF33, MOCS3, and MPP6). Immunohistochemical analysis of NAT10 in breast cancer samples (n=330) showed strong correlation with expression of phospho-RPA (R=0.451, p=1.82x10-20) and γH2AX (R=0.304, p=2.95x10-9). Finally, we applied our oncogene-induced replication stress signature to patient samples from TCGA (n=8,862) and GEO (n=13,912) to define the levels of replication stress across 27 tumor subtypes, identifying diffuse large B cell lymphoma, ovarian cancer, TNBC and colorectal carcinoma as cancer subtypes with high levels of oncogene-induced replication stress.

Synthetic Lethal Screening Identifies CHK1 Inhibition As an Exploitable Vulnerability in EZH2 Deficient T-ALL

Loss of function mutations of Enhancer of Zeste (Drosophila) homolog 2 (EZH2), the enzymatic component of Polycomb Repressive Complex 2 (PRC2), are particularly frequent in the early T cell precursor (ETP) subtype of T cell Acute Lymphoblastic Leukaemia (T-ALL), and have been associated with chemotherapy-resistance and a poor prognosis. There is thus a need to identify novel treatment strategies for this subgroup of patients.We hypothesized that inactivation of EZH2 in T-ALL cells induces specific synthetic lethal vulnerabilities that can be exploited using drugs targeting other dependency pathways, potentially resulting in a clinically meaningful therapeutic strategy. Using isogenic Jurkat T-ALL cells, with and without EZH2 -inactivating mutations generated by double-nicking CRISPR Cas9, we performed cell-based drug screening using 220 well-characterized compounds, enriched for drugs targeting epigenetic, cell cycle and DNA repair machinery. Strikingly, we observed that EZH2 -knock out (KO) cells are hypersensitive to checkpoint kinase 1 (CHK1) inhibition by the clinical-grade small molecule MK8776, showing an IC50 reduction of over 2-fold. This effect was similarly observed when testing other CHK1 inhibitors (LY2603618 and CHIR-124), but was absent in the presence of inhibitors of other DNA damage-response kinases such as ATM, ATR and CHK2, demonstrating the specificity of this synthetic lethal interaction. Furthermore, pharmacological inhibition of EZH2 methyltransferase activity by GSK126 in human T-ALL cell lines resulted in sensitization to CHK1 inhibition. Notably, MK8776 resulted in apoptosis of EZH2-deficient cells as determined by Annexin V-PI staining.CHK1 inhibition has previously been shown to result in enhanced DNA damage and accumulation of cells with incompletely replicated DNA. In line with this, EZH2 -KO cells treated with MK8776 exhibited markedly elevated levels of the DNA damage marker yH2AX. Interestingly, the highest proportion of apoptotic cells were found at the G1 phase of the cell cycle when analyzed by TUNEL-PI, suggesting that MK8776 treated EZH2 -KO cells with accumulated DNA damage override the G2 checkpoint, exit mitosis and undergo mitotic catastrophe-related cell death during G1, a feature characteristic of mitotic slippage.To investigate the mechanism further, gene expression of two EZH2 -KO Jurkat clones was compared to their isogenic parental control by RNA-seq. EZH2 -KO cells showed a striking upregulation of genes associated with human ETP-ALL including LYL1, MYCN, KIT and HHEX, a finding that could be recapitulated with GSK126 treatment. Gene set enrichment analysis (GSEA) showed significant changes of expression in MYC target genes. Importantly, MYCN protein was markedly upregulated in both EZH2 -KO and GSK126 treated cells. We speculate that increased MYCN expression induces high levels of replication stress and increased dependency on CHK1 for cell survival. Our findings suggest that specific inhibition of the CHK1 pathway can preferentially target PRC2-deficient leukemic cells, providing a potentially less toxic and more effective treatment for this high-risk subgroup of patients. DisclosuresNo relevant conflicts of interest to declare.

RPA shields inherited DNA lesions for post-mitotic DNA synthesis

The paradigm that checkpoints halt cell cycle progression for genome repair has been challenged by the recent discovery of heritable DNA lesions escaping checkpoint control. How such inherited lesions affect genome function and integrity is not well understood. Here, we identify a new class of heritable DNA lesions, which is marked by replication protein A (RPA), a protein primarily known for shielding single-stranded DNA in S/G2. We demonstrate that post-mitotic RPA foci occur at low frequency during unperturbed cell cycle progression, originate from the previous cell cycle, and are exacerbated upon replication stress. RPA-marked inherited ssDNA lesions are found at telomeres, particularly of ALT-positive cancer cells. We reveal that RPA protects these replication remnants in G1 to allow for post-mitotic DNA synthesis (post-MiDAS). Given that ALT-positive cancer cells exhibit high levels of replication stress and telomere fragility, targeting post-MiDAS might be a new therapeutic opportunity.

MIF is a 3\u2019 flap nuclease that facilitates DNA replication and promotes tumor growth

How cancer cells cope with high levels of replication stress during rapid proliferation is currently unclear. Here, we show that macrophage migration inhibitory factor (MIF) is a 3’ flap nuclease that translocates to the nucleus in S phase. Poly(ADP-ribose) polymerase 1 co-localizes with MIF to the DNA replication fork, where MIF nuclease activity is required to resolve replication stress and facilitates tumor growth. MIF loss in cancer cells leads to mutation frequency increases, cell cycle delays and DNA synthesis and cell growth inhibition, which can be rescued by restoring MIF, but not nuclease-deficient MIF mutant. MIF is significantly upregulated in breast tumors and correlates with poor overall survival in patients. We propose that MIF is a unique 3’ nuclease, excises flaps at the immediate 3’ end during DNA synthesis and favors cancer cells evading replication stress-induced threat for their growth.

MYBL2 and ATM suppress replication stress in pluripotent stem cells

Replication stress, a major cause of genome instability in cycling cells, is mainly prevented by the ATR‐dependent replication stress response pathway in somatic cells. However, the replication stress response pathway in embryonic stem cells (ESCs) may be different due to alterations in cell cycle phase length. The transcription factor MYBL2, which is implicated in cell cycle regulation, is expressed a hundred to a thousand‐fold more in ESCs compared with somatic cells. Here we show that MYBL2 activates ATM and suppresses replication stress in ESCs. Consequently, loss of MYBL2 or inhibition of ATM or Mre11 in ESCs results in replication fork slowing, increased fork stalling and elevated origin firing. Additionally, we demonstrate that inhibition of CDC7 activity rescues replication stress induced by MYBL2 loss and ATM inhibition, suggesting that uncontrolled new origin firing may underlie the replication stress phenotype resulting from loss/inhibition of MYBL2 and ATM. Overall, our study proposes that in addition to ATR, a MYBL2‐MRN‐ATM replication stress response pathway functions in ESCs to control DNA replication initiation and prevent genome instability.

Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors

Oncogene-induced replication stress, for instance as a result of Cyclin E1 overexpression, causes genomic instability and has been linked to tumorigenesis. To survive high levels of replication stress, tumors depend on pathways to deal with these DNA lesions, which represent a therapeutically actionable vulnerability. We aimed to uncover the consequences of Cyclin E1 or Cdc25A overexpression on replication kinetics, mitotic progression, and the sensitivity to inhibitors of the WEE1 and ATR replication checkpoint kinases. We modeled oncogene-induced replication stress using inducible expression of Cyclin E1 or Cdc25A in non-transformed RPE-1 cells, either in a TP53 wild-type or TP53-mutant background. DNA fiber analysis showed Cyclin E1 or Cdc25A overexpression to slow replication speed. The resulting replication-derived DNA lesions were transmitted into mitosis causing chromosome segregation defects. Single cell sequencing revealed that replication stress and mitotic defects upon Cyclin E1 or Cdc25A overexpression resulted in genomic instability. ATR or WEE1 inhibition exacerbated the mitotic aberrancies induced by Cyclin E1 or Cdc25A overexpression, and caused cytotoxicity. Both these phenotypes were exacerbated upon p53 inactivation. Conversely, downregulation of Cyclin E1 rescued both replication kinetics, as well as sensitivity to ATR and WEE1 inhibitors. Taken together, Cyclin E1 or Cdc25A-induced replication stress leads to mitotic segregation defects and genomic instability. These mitotic defects are exacerbated by inhibition of ATR or WEE1 and therefore point to mitotic catastrophe as an underlying mechanism. Importantly, our data suggest that Cyclin E1 overexpression can be used to select patients for treatment with replication checkpoint inhibitors.

Cyclin E expression is associated with high levels of replication stress in triple-negative breast cancer

Replication stress entails the improper progression of DNA replication. In cancer cells, including breast cancer cells, an important cause of replication stress is oncogene activation. Importantly, tumors with high levels of replication stress may have different clinical behavior, and high levels of replication stress appear to be a vulnerability of cancer cells, which may be therapeutically targeted by novel molecularly targeted agents. Unfortunately, data on replication stress is largely based on experimental models. Further investigation of replication stress in clinical samples is required to optimally implement novel therapeutics. To uncover the relation between oncogene expression, replication stress, and clinical features of breast cancer subgroups, we immunohistochemically analyzed the expression of a panel of oncogenes (Cyclin E, c-Myc, and Cdc25A,) and markers of replication stress (phospho-Ser33-RPA32 and γ-H2AX) in breast tumor tissues prior to treatment (n = 384). Triple-negative breast cancers (TNBCs) exhibited the highest levels of phospho-Ser33-RPA32 (P < 0.001 for all tests) and γ-H2AX (P < 0.05 for all tests). Moreover, expression levels of Cyclin E (P < 0.001 for all tests) and c-Myc (P < 0.001 for all tests) were highest in TNBCs. Expression of Cyclin E positively correlated with phospho-RPA32 (Spearman correlation r = 0.37, P < 0.001) and γ-H2AX (Spearman correlation r = 0.63, P < 0.001). Combined, these data indicate that, among breast cancers, replication stress is predominantly observed in TNBCs, and is associated with expression levels of Cyclin E. These results indicate that Cyclin E overexpression may be used as a biomarker for patient selection in the clinical evaluation of drugs that target the DNA replication stress response.

Abstract 546: PARG and WEE1 inhibition in GI cancers: A novel synergistic, targeted therapeutic approach

Abstract Colorectal carcinomas (CRC) and pancreatic ductal adenocarcinomas (PDAC) are the 2nd and 3rd leading causes of cancer-related deaths in the U.S. respectively. Recent work from our lab highlighted Poly ADP-Ribose Glycohydrolase (PARG) as a target in PDAC, and PARG inhibition (PARGi) sensitized PDAC cells to the DNA damaging agents, 5-FU and oxaliplatin. Other labs have shown that PARG inhibition causes replication stress in cancer cells. CRC and PDAC cells, while cellularly different, share a high frequency of KRAS and P53 inactivating mutations. High levels of replication stress (RS) has also been well documented in these tumor types. Targeting DNA Damage Response Kinases (DDRKs) in combination with chemotherapy (mostly DNA damage inducers like 5-FU, oxaliplatin, etc.) has gained recent interest in solid tumors but overlapping toxicities and tolerability have been issues clinically. Accordingly, we hypothesized that combined inhibition of PARG and DDRKs could yield synergistic effects in tumors with high replication stress while alleviating adverse events seen in other combination strategies. We determined in drug sensitivity assays that PARGi did not synergize with either the CHK1 inhibitor prexasertib or the ATR inhibitor VE-821 in our cell lines but did synergize with Wee1i (AZD1775) in both PDAC and CRC cell lines (Comb. Analysis, p&amp;lt;.05). Additionally, we assessed long-term cell survival using colony formation assays, which yielded a dramatic reduction in cell survival for Panc1, MiaPaCa-2, CFPAC1 (PDAC cells), HCT116 (CRC cells) and SW480 (CRC cells) cell lines with the combination of PARGi and Wee1i as compared to single-agent controls (p&amp;lt;.001). To validate the targeted synergy between AZD1775 and PARGi, we utilized genetic models of PARG and Wee1 silencing, including CRISPR PARG knockout cell lines, siRNA, and shRNA knockdown models. Genetic knockdown of Wee1 and PARG in PDAC and CRC cell lines yielded strikingly similar results in colony formation assays, with 2 and 12-fold changes respectively compared to Wee1i alone (P-value &amp;lt; .0001). HCT116 CRISPR PARG knockouts yielded a 5-fold difference in Wee1i sensitivity in colony formation assays. Mechanistically, western blot analysis demonstrated PARGi and Wee1i led to &amp;gt;2-fold increases in PARylation, yH2AX, and cleaved-caspase 3 as compared to single-agent controls. In conclusion, combined PARG - Wee1 inhibition is a novel targeting strategy in PDAC and CRC tumors that results in decreased cell viability. We are currently testing the efficacy of AZD1775 in PARG CRISPR-knockout tumors in in vivo xenograft models. Ongoing studies are focused on assessing the effect of Wee1i + PARGi on chromosomal aberrations, disruption in replication fork dynamics via DNA fiber labeling, and cell cycle dependencies. Citation Format: Lebaron C. Agostini, Aditi Jain, Michael Pishvaian, Charles Yeo, Jonathan Brody. PARG and WEE1 inhibition in GI cancers: A novel synergistic, targeted therapeutic approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 546.

Nucleosides Rescue Replication-Mediated Genome Instability of Human Pluripotent Stem Cells.

SummaryHuman pluripotent stem cells (PSCs) are subject to the appearance of recurrent genetic variants on prolonged culture. We have now found that, compared with isogenic differentiated cells, PSCs exhibit evidence of considerably more DNA damage during the S phase of the cell cycle, apparently as a consequence of DNA replication stress marked by slower progression of DNA replication, activation of latent origins of replication, and collapse of replication forks. As in many cancers, which, like PSCs, exhibit a shortened G1 phase and DNA replication stress, the resulting DNA damage may underlie the higher incidence of abnormal and abortive mitoses in PSCs, resulting in chromosomal non-dysjunction or cell death. However, we have found that the extent of DNA replication stress, DNA damage, and consequent aberrant mitoses can be substantially reduced by culturing PSCs in the presence of exogenous nucleosides, resulting in improved survival, clonogenicity, and population growth.

CHK1 Inhibition Is Synthetically Lethal with Loss of B-Family DNA Polymerase Function in Human Lung and Colorectal Cancer Cells.

Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (POLA1, POLE, and POLE2) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies.

Supraphysiological protection from replication stress does not extend mammalian lifespan.

Replication Stress (RS) is a type of DNA damage generated at the replication fork, characterized by single-stranded DNA (ssDNA) accumulation, and which can be caused by a variety of factors. Previous studies have reported elevated RS levels in aged cells. In addition, mouse models with a deficient RS response show accelerated aging. However, the relevance of endogenous or physiological RS, compared to other sources of genomic instability, for the normal onset of aging is unknown. We have performed long term survival studies of transgenic mice with extra copies of the Chk1 and/or Rrm2 genes, which we previously showed extend the lifespan of a progeroid ATR-hypomorphic model suffering from high levels of RS. In contrast to their effect in the context of progeria, the lifespan of Chk1, Rrm2 and Chk1/Rrm2 transgenic mice was similar to WT littermates in physiological settings. Most mice studied died due to tumors -mainly lymphomas- irrespective of their genetic background. Interestingly, a higher but not statistically significant percentage of transgenic mice developed tumors compared to WT mice. Our results indicate that supraphysiological protection from RS does not extend lifespan, indicating that RS may not be a relevant source of genomic instability on the onset of normal aging.

Abstract B103: Torin2 and related analogs exploit replicative and checkpoint vulnerabilities to induce death of triple-negative breast cancer cells

Abstract Frequent mutation of genes in the PI3K/AKT/mTOR signaling pathway in human cancers has stimulated large investments in over 40 small molecule therapeutic drugs but most have low efficacy in patients. As a result, cancers with high PI3K pathway activity such as triple-negative breast cancer (TNBC) are still treated primarily with conventional chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors with varying degrees of polyselectivity, we find that the preclinical compound Torin2 and related analogs are unusually effective because they inhibit both mTORC1/2 and structurally related PI3K-like kinases (PIKKs). The activity of Torin2 in TNBC cells is most closely mimicked by combining selective inhibitors of mTORC1/2 and Chk1, a kinase that acts downstream of the PIKKs ATR and DNA-PK in the signaling response to replication stress. Torin2 is more cytotoxic in TNBC/basal-like cell lines (N=19) than clinical grade mTORC1/2 inhibitors such as sapanisertib, but produces no cytotoxicity in non-transformed cells with basal-like gene expression (N=2). The high activity of Torin2 in TNBC is associated with greater activation of caspase-dependent apoptosis and stronger inhibition of cell proliferation than conventional PI3K/AKT/mTOR inhibitors, which do not inhibit PIKKs. Pulse-labeling studies with thymidine analogs and live-cell imaging of cells expressing fluorescent cell cycle reporters show that unlike other PI3K pathway drugs, Torin2 blocks progression of S phase, thereby causing accumulation of single-stranded DNA, DNA damage and death by replication catastrophe or mitotic failure. These phenotypes appear to result from co-targeting distinct pathways required during S phase in TNBC cells: inhibiting mTOR perturbs de novo biosynthesis and salvage of pyrimidines and purines, and inhibiting PIKKs causes high levels of replication stress and DNA damage. Moreover, computational models developed to better understand the complex cell cycle mechanism of action of Torin2 demonstrate the importance of co-targeting S-phase vulnerabilities for cytotoxicity. Thus, Torin2 and related analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC and possibly to other cancer cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors. Citation Format: Sameer S. Chopra, Annie Jenney, Adam Palmer, Mario Niepel, Mirra Chung, Caitlin Mills, Sindhu Carmen Sivakumaren, Qingsong Liu, Jia-Yun Chen, Clarence Yapp, Nathanael S. Gray, Peter K. Sorger. Torin2 and related analogs exploit replicative and checkpoint vulnerabilities to induce death of triple-negative breast cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B103. doi:10.1158/1535-7163.TARG-19-B103

Triggering mitosis.

Entry into mitosis is triggered by the activation of cyclin-dependent kinase 1 (Cdk1). This simple reaction rapidly and irreversibly sets the cell up for division. Even though the core step in triggering mitosis is so simple, the regulation of this cellular switch is highly complex, involving a large number of interconnected signalling cascades. We do have a detailed knowledge of most of the components of this network, but only a poor understanding of how they work together to create a precise and robust system that ensures that mitosis is triggered at the right time and in an orderly fashion. In this review, we will give an overview of the literature that describes the Cdk1 activation network and then address questions relating to the systems biology of this switch. How is the timing of the trigger controlled? How is mitosis insulated from interphase? What determines the sequence of events, following the initial trigger of Cdk1 activation? Which elements ensure robustness in the timing and execution of the switch? How has this system been adapted to the high levels of replication stress in cancer cells?