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

Stress granules form in response to diverse cellular perturbations to sequester translation components until the stress is resolved. Stress granules are composed of RNA-protein assemblies in membrane delimited structures and must be rapidly disassembled to release components to allow translation to resume. Disassembly of stress granules formed in response to heat stress is dependent on ubiquitiylation of stress granule components such as G3BP1. Ubiquitylation of stress granule proteins recruits the AAA-ATPase p97 (also known as VCP) to enable ubiquitin-dependent disassembly of these structures. Loss of p97 activity leads to the persistence of stress granules and is implicated in several age-related neurodegenerative diseases. Here we show that p97 recruitment to stress granules is dependent on its ubiquitin binding co-factor p47. p47 translocates to stress granules in response to a variety of cellular stressors and is required for the recruitment of p97 to stress granules. Loss of p47 leads to an inhibition in stress granule disassembly. We further show that p47 associates with G3BP1 in response to heat stress in a ubiquitin-dependent manner. Taken together our data adds to the growing list of p97 adaptors that are implicated in the recruitment of p97 for dissolution of stress granules.

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.

Replication stress (RS) is a major driver of genomic instability and a hallmark of cancer cells. Although dynamic heterochromatin remodeling has been implicated in RS response, the precise mechanisms remain unclear. The CHAMP1 complex, composed of CHAMP1, POGZ, HP1α, and the H3K9 methyltransferase SETDB1, is known to regulate heterochromatin assembly at multiple genomic sites. Interestingly, upon RS, the CHAMP1 complex is transiently recruited to stalled replication forks, where it facilitates H3K9me3 deposition and establishes a repressive chromatin environment. The complex is required for stabilization of replication forks, and it shields forks from MRE11-mediated degradation. The complex also reduces RS at specific chromosomal sites, such as the heterochromatin-rich telomeric sites in tumor cells which use the ALT pathway of telomere maintenance. Loss of the CHAMP1 complex results in increased micronuclei formation and heightened sensitivity to RS. Loss of the complex also leads to a compensatory increase in other pathways which reduce RS, such as the FA pathway and the ATR/CHK1 pathway. Notably, CHAMP1 deficiency induces synthetic lethality with FANCM inhibition in ALT-positive tumor cells, and the CHAMP1 complex is essential for the survival of CCNE1-amplified ovarian cancers. These findings uncover a heterochromatin-based mechanism of replication fork stabilization and suggest that CHAMP1 may represent a candidate therapeutic vulnerability in cancers with elevated RS.

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

AbstractStress granules form in response to diverse cellular perturbations to sequester translation components until the stress is resolved. Stress granules are composed of RNA–protein assemblies in membrane-delimited structures and must be rapidly disassembled to release components to allow translation to resume. Disassembly of stress granules formed in response to heat stress is dependent on ubiquitylation of stress granule components such as G3BP1. Ubiquitylation of stress granule proteins recruits the AAA-ATPase p97 (also known as VCP) to enable ubiquitin-dependent disassembly of these structures. Loss of p97 activity leads to the persistence of stress granules and is implicated in several age-related neurodegenerative diseases. Here, we show that p97 recruitment to stress granules is dependent on its ubiquitin-binding co-factor p47. p47 translocates to stress granules in response to a variety of cellular stressors and is required for the recruitment of p97 to stress granules. Loss of p47 leads to an inhibition in stress granule disassembly. We further show that p47 associates with G3BP1 in response to heat stress in a ubiquitin-dependent manner. Taken together, our data add to the growing list of p97 adaptors that are implicated in the recruitment of p97 for the dissolution of stress granules.

Ewing sarcoma (EwS) is a group of bone and soft-tissue cancers in children and young adults. Because EwS cells have pronounced sensitivity to radiation and chemotherapy-induced DNA damage, the oncoprotein EWS-FLI1 is likely to be involved in DNA repair. Here, we demonstrate that EWS-FLI1 causes a defect in microhomology-mediated end joining (MMEJ) repair. EWSR1 is a splicing factor that promotes the faithful splicing of the POLQ pre-mRNA, required for the expression of Polθ, a critical protein in the MMEJ pathway. Expression of EWS-FLI1 or depletion of EWSR1 causes increased POLQ exon 25 skipping, decreased Polθ expression, impaired MMEJ, and enhanced cellular sensitivity to inhibitors of the Fanconi anemia (FA), homologous recombination (HR), or non-homologous end joining (NHEJ) pathways, through the mechanism of synthetic lethality. Correction of POLQ exon 25 skipping restored Polθ expression and MMEJ activity in EwS. Inhibitors of the FA, HR, or NHEJ pathways may therefore provide a targeted therapy for EwS patients.

Supplementary Materials and Methods

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

Ataxia telangiectasia and Rad3-related (ATR) inhibition is under evaluation for the treatment of high-grade serous ovarian cancer (HGSOC) to reverse acquired resistance to poly (ADP-ribose) polymerase (PARP) inhibition and to exacerbate chemotherapy-induced replicative stress. Here, we define PTEN deficiency as a predictive biomarker for the response to ATR inhibition, as monotherapy and in combination with PARP inhibition or gemcitabine. In response to ATR inhibition and compared with PTEN-proficient cells, PTEN-deficient cells are prone to (a) uncoupling of DNA polymerase and helicase activities, leading to excessive ssDNA and replication stress; (b) cytoplasmic sequestration of checkpoint kinase 1 (CHK1), compromising cell-cycle checkpoint control with reduced compensatory effects by ataxia-telangiectasia mutated (ATM) and DNA-dependent proteinase K (DNA-PK), leading to mitotic catastrophe; and (c) reduced DNA repair protein RAD51 homolog 1 (RAD51) recruitment, exacerbating replication fork instability, also leading to lethality. Retrospective analyses revealed that patients with HGSOC who expressed low PTEN levels experienced greater clinical benefit on ATR inhibitor-based trials than did those with high PTEN levels. These results justify prospective trials evaluating ATR inhibition as a therapeutic strategy for PTEN-deficient tumors.

Integration of the high-risk human papillomavirus 16 (HPV16) genome into the host chromosome, frequently driven by microhomology-mediated end joining (MMEJ), is a critical step in the carcinogenesis of HPV-associated tumors. However, the mechanisms by which viral oncoproteins manipulate the error-prone MMEJ pathway remain poorly defined. Here, we demonstrate that the HPV16 E6 oncoprotein upregulates MMEJ to facilitate viral genome integration. This heightened MMEJ activity is driven by a marked increase in the protein levels of DNA polymerase theta (PolΘ), a central enzyme of the MMEJ pathway. Mechanistically, we show that the elevation of PolΘ levels in response to HPV16 E6 expression is dependent on the host E3 ubiquitin ligase UBE3A/E6AP but is independent of p53 degradation. E6 redirects UBE3A to enhance the ubiquitination and degradation of RAD23A, a shuttle protein required for delivering polyubiquitinated PolΘ to the proteasome. Consequently, the loss of functional RAD23A phenocopies the effect of HPV16 E6, leading to PolΘ protein stabilization and increased MMEJ activity. By elucidating the E6-UBE3A-RAD23A-PolΘ axis, our findings reveal a mechanism through which HPV manipulates the host DNA repair machinery to promote its integration and oncogenic potential.

ATR is a critical kinase in coordinating genomic stability with cellular division that can be targeted in tumors with high replication stress. ATM can compensate for loss of ATR signaling, raising the possibility that ATM deficiency could serve as a potential biomarker for ATR inhibition. However, ATR inhibitor monotherapy has produced only modest response rates in ATM-deficient tumors in clinical trials. The failure of ATM deficiency as a robust ATR inhibitor biomarker suggests that additional compensation pathways for ATR inhibition may be active in these tumors. DNA-PK has also been reported to compensate for ATR inhibition but the dynamic between ATM and DNA-PK in compensating for the loss of ATR signaling is not well understood. Here, we have examined ATM and DNA-PK activities in ATM-proficient and -deficient gastric cancer cells treated with ATR inhibitors to investigate their interplay in ATR pathway compensation. Following ATR inhibition, both ATM and DNA-PK are activated in a cell cycle-dependent manner at the S/G2 boundary by increased CDK1/2 activity that triggers premature activation of the mitotic cellular program. However, ATM and DNA-PK have distinct compensatory roles during ATR inhibition. DNA-PK detects DNA damage in early S phase and phosphorylates CHK1 to antagonize the S to G2 transition. In contrast, ATM detects DNA damage in late S phase and G2/M, where it activates the MRN complex to promote end resection and phosphorylates CHK1 and CHK2 to promote mitotic arrest. In ATM-deficient cells and patient tumors, DNA-PK is activated following ATR inhibition. DNA damage in ATM-deficient cells treated with an ATR inhibitor is detected by DNA-PK in early S phase where it facilitates DNA replication and reenforces the S/G2 checkpoint. Consequently, DNA-PK inhibition synergizes with ATR inhibition in ATM-deficient cells and significantly enhances ATR inhibitor efficacy in multiple ATM-deficient gastric and non-small cell lung cancer PDX models. Together, these data offer a cell cycle-dependent framework for understanding how ATM and DNA-PK compensate for the loss of ATR signaling and provide a compelling rationale for evaluating combined ATR and DNA-PK inhibitor therapy in ATM-deficient cancers in the clinic. Timothy B. Branigan, Roshen Alharthi, Bose S. Kochpurakkal, Jie Hao, Steffie Revia, Mu-Yan Cai, Yuqing Jiao, David B. Martignetti, Sirisha Mukkavalli, Ian A. Roundtree, Gregory M. Cote, Prafulla C. Gokhale, Kalindi Parmar, Alan D. D'Andrea, Astrid Zimmermann, Geoffrey I. Shapiro. DNA-PK reenforces the S/G2 checkpoint in ATM-deficient cells to limit ATR inhibitor efficacy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A031.

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.