Replication stress marker points to ovarian cancers that may yield to ALC1 targeting

Removing a single chromatin remodeling protein can make a wide spectrum of ovarian cancer cells dramatically more vulnerable to PARP inhibitor drugs, according to a new study published in EMBO Reports, and the key to predicting which tumors will respond lies in a measurable fingerprint of replication stress inside the cancer cell itself.

The protein, called ALC1 (Amplified in Liver Cancer 1), is a nucleosome sliding enzyme that helps repair chromatin-buried abasic sites, lesions that form during base excision repair or when a nucleobase is spontaneously lost from single-stranded DNA. A research team led by Lindsey N. Aubuchon and Priyanka Verma at Washington University in St. Louis showed that depleting ALC1 markedly sensitizes serous ovarian cancer cells, xenografts, and patient-derived models to PARP inhibitors across both homologous recombination-deficient and CCNE1-amplified contexts. Crucially, the same depletion had minimal to no impact on PARP inhibitor responses or genome stability in non-cancerous fallopian tube cells, including cells carrying a single pathogenic BRCA1 copy, revealing an unusually wide therapeutic window.

PARP inhibitors are already approved for homologous recombination-deficient high-grade serous ovarian cancer, but their durable benefit is limited by resistance and hematologic toxicity, and they are largely ineffective in homologous recombination-proficient tumors, particularly CCNE1-amplified cancers that remain therapeutically intractable. High-grade serous ovarian cancer carries the highest case-fatality rate among gynecologic malignancies. Whole-genome sequencing has shown that roughly half of these tumors show evidence of homologous recombination deficiency, while about 20 percent are homologous recombination-proficient and associated with amplification of the cyclin E1 gene. Cyclin E1-amplified tumors resist both platinum chemotherapy and PARP inhibitors, and there are currently no FDA-approved approaches to reduce effective PARP inhibitor dosage or to make the drugs work in that setting.

The mechanistic logic behind PARP inhibitor toxicity is well established. These drugs trap PARP1 and PARP2 enzymes on chromatin, and the trapped complexes impede replication fork progression, creating replication-coupled DNA gaps and breaks that increase the cell’s reliance on homologous recombination for survival. The trapping potency of individual PARP inhibitors correlates directly with cytotoxicity: talazoparib is the most potent trapper and the most cytotoxic, followed by niraparib, while olaparib and rucaparib share intermediate trapping potency and veliparib is a weak trapper with reduced potency. Stronger trapping also brings stronger side effects, with anemia and cytopenia more common with talazoparib and niraparib.

Building on a previous CRISPR screen that identified ALC1 as a top enhancer of olaparib sensitivity in the BRCA1-mutant UWB1.289 cell line, the team used a SaCas9-sgRNA strategy to deplete ALC1 and tested sensitivity to four PARP inhibitors. In BRCA1-mutant UWB1.289 and JHOS-4 cells, ALC1 loss significantly enhanced sensitivity to olaparib, rucaparib, and veliparib, with a relatively modest effect on niraparib. The enhanced olaparib and rucaparib sensitivity was recapitulated in patient-derived cells from tumors with a germline BRCA1 mutation and with BRCA1 promoter methylation. In BRCA2-mutant OVSAHO and Kuramochi cells, ALC1 loss also enhanced olaparib and rucaparib sensitivity, though to a lesser extent than in the BRCA1-mutant setting, and minimal enhancement of veliparib sensitivity was observed. Notably, ALC1 loss did not sensitize any of these cells to cisplatin, consistent with the remodeler being dispensable for transcription-coupled nucleotide excision repair and polymerase theta-mediated end joining.

The most striking result came from the homologous recombination-proficient, cyclin E1-high models. OVCAR3, OVCAR4, and OVCAR8 cells, which carry CCNE1 amplification or gain, retained the ability to load RAD51 at DNA breaks after olaparib treatment, confirming their HR proficiency. Yet ALC1 depletion significantly enhanced olaparib and rucaparib sensitivity in all three lines, and the same sensitization was reproduced in patient-derived cells with CCNE1 amplification and CCNE1 gain. In a xenograft experiment, OVCAR8 cells engineered to express sgRNAs targeting ALC1 or the safe-harbor AAVS1 locus were injected intraperitoneally into nude mice, and olaparib at 50 mg/kg was administered every second day for 35 days. Olaparib alone did not reduce tumor growth in the control group, but olaparib-treated ALC1-depleted cells showed significantly reduced tumor weight and nodule counts, with ten mice per group.

ALC1 loss also restored PARP inhibitor sensitivity in clinically relevant models of acquired resistance. In two UWB1.289-derived lines, SYr12 and SYr14, that had acquired PARP inhibitor resistance through increased reliance on ATR-mediated RAD51 loading at DNA breaks and reversed forks, ALC1 depletion imparted sensitivity to olaparib and rucaparib. Similarly, in cells resistant through overexpression of the del11q BRCA1 hypomorph, ALC1 loss potentiated olaparib and rucaparib sensitivity. However, in cells overexpressing fully functional BRCA1 protein, ALC1 depletion did not enhance sensitivity to olaparib, rucaparib, niraparib, or cisplatin, though it did increase talazoparib sensitivity. The authors note that the impact of ALC1 on PARP inhibitor response was more profound in HR-deficient and cyclin E1-high settings than in acquired-resistance scenarios, suggesting that combining ALC1 targeting with PARP inhibitors upfront is likely to be more efficacious than waiting for chemoresistance to emerge.

The safety profile of the approach rests on experiments in normal fallopian tube epithelium, the tissue where the majority of high-grade serous ovarian cancers originate. In hTERT-immortalized FT190, FT194, and FT282 cells, ALC1 loss did not enhance sensitivity to platinum or any of the clinically approved PARP inhibitors. The team also generated two FT282 clones with a BRCA1 heterozygous genotype, confirmed by next-generation sequencing, to model the normal cells of individuals born with one pathogenic BRCA1 copy. ALC1 loss had minimal to no impact on PARP inhibitor or platinum responses in these cells. At the genome stability level, ALC1 loss did not elevate pS33-RPA2 or γH2AX in BRCA1-wild-type FT282 cells, and in BRCA1-heterozygous FT282 cells it produced only a 1.2-fold increase in pS33-RPA2 with no concomitant increase in γH2AX. By contrast, in BRCA1-mutant UWB1.289 cells, ALC1 loss drove 1.4-fold and 1.7-fold increases in pS33-RPA2 and γH2AX respectively. The authors propose that the modest replication stress increase in heterozygous cells is resolved by their intact homologous recombination capacity, whereas cancer cells with high intrinsic replication stress cannot cope without ALC1.

That intrinsic replication stress burden turned out to be the predictive variable. The team quantified endogenous pT21-RPA2, a phosphorylated form of the single-stranded DNA binding protein RPA2, co-staining with the S/G2 marker cyclin A to control for cell cycle distribution and normalizing foci counts to nuclear area. Any cell type with a median greater than 0.1 pT21-RPA2 foci per unit nuclear size proved sensitive to olaparib and rucaparib upon ALC1 depletion. The biological rationale is that replication stress generates single-stranded DNA, in which nucleobases are lost spontaneously at roughly four times the rate seen in double-stranded DNA, producing the abasic sites whose repair depends on ALC1-mediated chromatin remodeling. This explains why cyclin E1-high cells, which harbor elevated replication stress, respond to PARP inhibitors upon ALC1 loss despite their HR proficiency.

Patient-derived data lent cautious support to the biomarker concept. In a tissue microarray cohort of 48 PARP inhibitor-treated patients that included both HR-deficient and HR-proficient samples, no significant association was observed between ALC1 expression and overall survival in the full cohort. In the subgroup of 23 homologous recombination-proficient patients with high replication stress, those with ALC1-low tumors had longer overall survival than those with ALC1-high tumors, with an undefined median versus 92.1 months and a hazard ratio of 3.1, but the result did not reach statistical significance, likely because 87.5 percent of cases in the ALC1-low group were censored. Separately, proteomic data from nine HR-deficient patients on PARP inhibitors showed that low ALC1 protein levels trended with longer time on treatment, an inverse correlation not attributable to declining ALC1 levels over the course of therapy. The authors acknowledge that their studies used genetic ALC1 depletion rather than small-molecule inhibitors, which are not commercially available, but note that an ALC1 inhibitor has entered a Phase I clinical trial, and that the simplicity of an immunofluorescence assay for pT21-RPA2 makes it a practical candidate for further investigation as a functional biomarker of PARP inhibitor response.

Subject of Research: How ALC1 loss broadens PARP inhibitor efficacy in ovarian cancer models with high replication stress

Article Title: Endogenous replication stress creates a unique therapeutic window for ALC1 targeting

Article References: Aubuchon, L. N., Wong, D. H., Greenberg, S. F., Jackson, K. E., Reddy, R., Lomonosova, E., Compadre, A., Kemper, D., Mehta, V., Zoberi, K., Teklemariam, T. A., Khabele, D., Christie, E. L., Mullen, M. M., & Verma, P. (2026). Endogenous replication stress creates a unique therapeutic window for ALC1 targeting. EMBO Reports. https://doi.org/10.1038/s44319-026-00966-3

Image Credits: AI Generated

DOI: 10.1038/s44319-026-00966-3

Keywords: ALC1, PARP inhibitors, high-grade serous ovarian cancer, replication stress, pT21-RPA2, CCNE1 amplification, BRCA1, BRCA2, homologous recombination deficiency, drug resistance, fallopian tube cells, chromatin remodeling