Lisa Moreau

Supplementary Figure S5. Chemical structures of USP1 inhibitors.

Supplementary Materials and Methods

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

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

Clinical characteristics of tumor samples used to generate patient derived organoids

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

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

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

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

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.