Supplementary Figure S5. Chemical structures of USP1 inhibitors.

Supplementary Figure S4. USP1 inhibitor and a PARP inhibitor combination treatment is well tolerated in mice

Supplementary Figure S2. Olaparib sensitivity of BRCA1-mutated ovarian cancer patient-derived organoids

Clinical characteristics of tumor samples used to generate patient derived organoids

Supplementary Figure S3. USP1 inhibitor exhibits synergistic activity with a POLQ inhibitor in BRCA1-deficient preclinical models

Mass spectrometry of proteins captured using aniPOND method in samples treated with USP inhibitor versus untreated samples.

CRISPR screen results in UWB1.289 cells treated with USP1 inhibitor versus DMSO

Supplementary Materials and Methods

Supplementary Figure S1. USP1 inhibition causes accumulation of RAD18 at the replication fork in BRCA1-deficient cancer cells

Oncogene expression can cause replication stress (RS), leading to DNA double-strand breaks (DSBs) that require repair through pathways such as homologous recombination, nonhomologous end-joining, and microhomology-mediated end-joining (MMEJ). Cyclin D1 (encoded by CCND1) is a well-known oncoprotein overexpressed in cancer; however, its role in RS is unknown. Using mantle cell lymphoma (MCL) as a naturally occurring model of cyclin D1 overexpression, we examined the impact of cyclin D1 on RS and DSB repair mechanisms. Cyclin D1 overexpression elevated RS, increased DNA damage, especially during mitosis, and caused specific upregulation of MMEJ. Furthermore, cyclin D1 activated polymerase theta (POLQ) transcription by binding its promoter loci, driving POLΘ-mediated MMEJ that is essential to withstand cyclin D1-induced RS. Moreover, concurrent ATM deficiency further intensified RS, enhanced POLQ expression, and heightened reliance on MMEJ-mediated DNA damage repair. Consequently, inhibition of POLΘ in cyclin D1-overexpressed settings further exacerbated RS, causing single-strand DNA gap accumulations and chromosomal instability, ultimately leading to apoptosis, an effect amplified in ATM-deficient cells. Targeting MMEJ via POLΘ inhibition is therefore an effective strategy in the context of cyclin D1 overexpression and ATM deficiency and may provide a unique therapeutic approach for treating MCL and other malignancies characterized by similar alterations.

BRCA1-deficient tumors are unable to efficiently perform DNA repair by homologous recombination (HR) and are dependent on alternative DNA damage response pathways. PARP inhibitors have improved the progression-free survival of HR-deficient ovarian cancer patients in clinical settings. However, PARP inhibitor (PARPi) resistance often develops, and new therapies are required for these patients. USP1 is a deubiquitinating enzyme that controls DNA repair pathways through the deubiquitination of PCNA-Ub and the activation of Translesion DNA Synthesis (TLS). Inhibitors of USP1 result in a strong upregulation of PCNA-Ub levels and a dysregulation of TLS repair. Importantly, USP1 is synthetically lethal in BRCA1-deficient tumors (Lim et al., Mol Cell, 2018), and USP1 inhibitors (USP1is) are currently undergoing clinical development for the treatment of these cancers as monotherapy or in combination with a PARPi. USP1is kill BRCA1-mutated patient-derived ovarian cancer organoids (PDOs) and overcome PARPi resistance in BRCA1-WT PDOs (da Costa et al, Cancer Research, 2024). Sensitivity to USP1i strongly correlates with the accumulation of single strand DNA (ssDNA) gaps in both PARPi-sensitive and PARPi-resistant PDOs. Moreover, knockdown of RAD18, the E3 ubiquitin ligase required for PCNA ubiquitination, results in a reduction in PCNA-Ub levels and a corresponding reduction in ssDNA gaps, and in USP1i resistance. These findings suggest that ssDNA gaps and RAD18 levels in the tumors can predict the sensitivity to USP1 inhibitors. As BRCA1-mutated tumors express high levels of USP1 mRNA, we also investigated the relevance of this biomarker in predicting the sensitivity to USP1 inhibitors. Interestingly, the USP1i sensitivity correlated with increased USP1 mRNA expression, at baseline, in ovarian cancer PDO and patient-derived xenograft-derived organoid (PDXO) models. Similar results were obtained in BRCA1-deficient breast cancer cell line models with acquired PARPi resistance. USP1i sensitive ovarian cancer organoid models retained high USP1 mRNA levels upon PARPi treatment. The specific organoid models that were resistant to PARPi or USP1i monotherapy exhibited up to 40-fold increase in expression of USP1 mRNA and they were sensitive to a combination of USP1i plus PARPi. Taken together, in addition to the accumulation of ssDNA gaps and RAD18 levels, the relative expression of USP1 mRNA is a useful prognostic biomarker for patients who may respond to USP1 inhibitors, either as monotherapy or in combination with PARP inhibitors. These predictive biomarkers will be useful in monitoring the response of ovarian cancer patients enrolled in USP1i plus PARPi clinical trials. Ozge Somuncu, Nicholas Ashton, Alexandre A. B. A. da Costa, Sirisha Mukkavalli, Ramya Ravindranathan, Benjamin Lamarre, Huy Nguyen, Lisa Moreau, Joyce Liu, Geoffrey I. Shapiro, Kalindi Parmar, Alan D. D’Andrea. Identification of predictive biomarkers of USP1 inhibitor sensitivity using ovarian cancer organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4004.

Abstract Recent studies suggest that PARP inhibitors and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulation of single-stranded DNA gaps (ssDNA gaps) at replication forks. Loss of USP1, a deubiquitinating enzyme, is also synthetic lethal with BRCA1 deficiency, and USP1 inhibitors are now undergoing clinical development for these cancers. Here, we show that USP1 inhibitors also promote the accumulation of ssDNA gaps during replication in BRCA1-deficient cells. USP1 inhibition increased monoubiquitinated PCNA at replication forks and resulted in accumulation of ssDNA gaps in BRCA1-deficient cancer cells. Knockdown of RAD18, the E3 ubiquitin ligase known to ubiquitinate PCNA, caused USP1 inhibitor resistance and suppression of ssDNA gaps. ssDNA gap accumulation induced by USP1 inhibition correlates with drug sensitivity of BRCA1-mutated cancer cells and overcomes PARP inhibitor resistance in cell line and xenograft models. Furthermore, USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. A set of patient-derived ovarian cancer organoids (PDOs) were used to confirm the sensitivity of BRCA1-deficient cells to USP1 inhibition. Five PDOs were derived from patients with high grade-serous ovarian cancer (HGSOC). Among the five PDOs, two of them were from tumors harboring germline BRCA1 pathogenic mutations. BRCA1-mutant PDOs, but not BRCA1-WT PDOs, were sensitive to a USP1 inhibitor and to PARP inhibitor monotherapies. The accumulation of ssDNA gaps after treatment with a USP1 inhibitor or a PARP inhibitor correlated with the sensitivity to these drugs in all the models tested. Moreover, the combination of a PARP inhibitor and a USP1 inhibitor showed synergy in a BRCA1-WT model, which was resistant to both monotherapies. Interestingly, ssDNA gaps accumulated with the combination treatment and not with monotherapy in this model. Ovarian cancer PDOs therefore provide a powerful tool for rapid in vitro sensitivity testing. The detection of ssDNA gap accumulation may be a useful predictive biomarker for response to USP1 inhibition as monotherapy or in combination in ongoing clinical trials. Citation Format: Alexandre Andre B. A. Da Costa, Ozge Somuncu, Ramya Ravindranathan, Sirisha Mukkavalli, David Martigneti, Huy Nguyen, Yuqing Jiao, Benjamin Lamarre, Golbahar Sadatrezai, Lisa Moreau, Joyce Liu, Divya Iyer, Jean-Bernard Lazaro, Geoffrey Shapiro, Kalindi Parmar, Alan D. D'Andrea. Single strand DNA GAP accumulation as a functional biomarker for USP1 inhibitor sensitivity in ovarian cancer patient-derived organoid models [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 4644.

Abstract Recent studies suggest that PARP and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulation of single-stranded DNA (ssDNA) gaps at replication forks. Loss of USP1, a deubiquitinating enzyme, is also synthetically lethal with BRCA1 deficiency, and USP1 inhibitors are now undergoing clinical development for these cancers. Herein, we show that USP1 inhibitors also promote the accumulation of ssDNA gaps during replication in BRCA1-deficient cells, and this phenotype correlates with drug sensitivity. USP1 inhibition increased monoubiquitinated proliferating cell nuclear antigen at replication forks, mediated by the ubiquitin ligase RAD18, and knockdown of RAD18 caused USP1 inhibitor resistance and suppression of ssDNA gaps. USP1 inhibition overcame PARP inhibitor resistance in a BRCA1-mutated xenograft model and induced ssDNA gaps. Furthermore, USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. Finally, in patient-derived ovarian tumor organoids, sensitivity to USP1 inhibition alone or in combination correlated with the accumulation of ssDNA gaps. Assessment of ssDNA gaps in ovarian tumor organoids represents a rapid approach for predicting response to USP1 inhibition in ongoing clinical trials. Significance: USP1 inhibitors kill BRCA1-deficient cells and cause ssDNA gap accumulation, supporting the potential of using ssDNA gap detection as a functional biomarker for clinical trials on USP1 inhibitors.

Abstract Targeting BRCA-deficient tumors with PARP inhibitors has improved the progression-free survival of ovarian and breast cancer patients in the clinic. However, the majority of patients develop acquired resistance. Recent studies suggest that PARP and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulating single-stranded DNA (ssDNA) gaps at replication forks. We have previously shown that USP1 deficiency is synthetic lethal with BRCA1 deficiency, suggesting USP1 is a potential therapeutic target for overcoming PARPi resistance (Lim et al., Mol Cell, 2018). Accordingly, USP1 inhibitors (USPi) are now undergoing clinical development. Here, we show that USP1i also promote ssDNA gap accumulation during replication in BRCA1-deficient cells. USP1 inhibition results in the accumulation of monoubiquitinated PCNA at the replication fork and increased levels of non-cleaved USP1 and RAD18 at the replication fork. Accumulation of ssDNA gaps is dependent on RAD18 activity and monoubiquitinated PCNA levels. Knockdown of RAD18, the E3 ubiquitin ligase known to ubiquitinate PCNA, resulted in reduced PCNA-Ub levels, resistance to the USP1i, and suppression of ssDNA gaps. Moreover, the accumulation of ssDNA gaps is associated with increased S-phase PAR levels and is independent of PRIMPOL, suggesting defects on the lagging strand of the replication fork. Interestingly, the induction of ssDNA gaps was detected in USP1i-sensitive but not USP1i-resistant BRCA1-deficient cells, suggesting this is the mechanism of cytotoxicity by USP1 inhibition. Indeed, USP1i also sensitized BRCA1-deficient cancer cells to other ssDNA gap-inducing agents such as POLQ and PARP inhibitors. Notably, USP1i exhibited monotherapy activity in ovarian and breast cancer cell lines with acquired PARPi resistance and re-sensitized them to PARP inhibition. PARPi-resistant BRCA1-mutated patient-derived ovarian cancer organoids were also sensitized by USP1 inhibition. Similar to the cell lines, the ssDNA gap accumulation induced by USP1i in organoids strongly correlated with the drug sensitivity of both PARPi-sensitive and PARPi-resistant models. Finally, USPi overcame PARPi resistance in a BRCA1-mutant PARPi-resistant PDX model. In conclusion, USPi exhibits monotherapy activity in both PARPi-sensitive. Citation Format: Alexandre A. B. A. da Costa, Ozge Somuncu, Ramya Ravindranathan, Sirisha Mukkavalli, David B. Martignetti, Huy Nguyen, Yuqing Jiao, Benjamin Lamarre, Lisa Moreau, Joyce Liu, Divya Iyer, Jean-Bernard Lazaro, Geoffrey I. Shapiro, Kalindi Parmar, Alan D. D’Andrea. USP1 inhibition induces single strand DNA gap accumulation and overcomes PARP inhibitor resistance in BRCA1 deficient cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B032.

The twenty-three Fanconi anemia (FA) proteins cooperate in the FA/BRCA pathway to repair DNA interstrand cross-links (ICLs). The cell division cycle and apoptosis regulator 1 (CCAR1) protein is also a regulator of ICL repair, though its possible function in the FA/BRCA pathway remains unknown. Here, we demonstrate that CCAR1 plays a unique upstream role in the FA/BRCA pathway and is required for FANCA protein expression in human cells. Interestingly, CCAR1 co-immunoprecipitates with FANCA pre-mRNA and is required for FANCA mRNA processing. Loss of CCAR1 results in retention of a poison exon in the FANCA transcript, thereby leading to reduced FANCA protein expression. A unique domain of CCAR1, the EF hand domain, is required for interaction with the U2AF heterodimer of the spliceosome and for excision of the poison exon. Taken together, CCAR1 is a splicing modulator required for normal splicing of the FANCA mRNA and other mRNAs involved in various cellular pathways.

Supplementary Table 1 showing reagents and antibodies used in the study.

Supplementary Table 2 showing the positive and negative GSEA results for each cell line.

Supplementary Figure 2. Functional analysis of the BRCA1 (p.Arg1699Trp) mutation.