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The de novo design of small molecule-binding proteins has seen exciting recent progress; however, high-affinity binding and tunable specificity typically require laborious screening and optimization after computational design. We developed a computational procedure to design a protein that recognizes a common pharmacophore in a series of poly(ADP-ribose) polymerase-1 inhibitors. One of three designed proteins bound different inhibitors with affinities ranging from

Mutations in the BRCA2 gene are associated with sporadic and familial cancer, cause genomic instability and sensitize cancer cells to inhibition by the poly(ADP-ribose) polymerase (PARP). Here we show that human pluripotent stem cells (hPSCs) with one copy of BRCA2 deleted can be used to annotate variants of this gene and to test their sensitivities to PARP inhibition. By using Cas9 to edit the functional BRCA2 allele in the locally haploid hPSCs and in fibroblasts differentiated from them, we characterized essential regions in the gene to identify permissive and loss-of-function mutations. We also used Cas9 to directly test the function of individual amino acids, including amino acids encoded by clinical BRCA2 variants of uncertain significance, and identified alleles that are sensitive to PARP inhibitors used as a standard of care in BRCA2-deficient cancers. Locally haploid human pluripotent stem cells can facilitate detailed structure-function analyses of genes and the rapid functional evaluation of clinically observed mutations.

Approximately 50% of patients diagnosed with ovarian cancer harbor tumors with mutations in BRCA1, BRCA2, or other genes involved in homologous recombination repair (HR). The presence of homologous recombination deficiency (HRD) is an approved biomarker for poly-ADP-ribose polymerase inhibitors (PARPis) as a maintenance treatment following a positive response to initial platinum-based chemotherapy. Despite this treatment option, the development of resistance to PARPis is common among recurrent disease patients, leading to a poor prognosis. In this study, we conducted a comprehensive analysis using publicly available datasets to elucidate the molecular mechanisms driving PARPi resistance in BRCA1-deficient ovarian cancer. Our findings reveal a central role for the interferon (IFN) pathway in mediating resistance in the context of BRCA1 deficiency. Through integrative bioinformatics approaches, we identified LY6E, an interferon-stimulated gene, as a key mediator of PARPi resistance, with its expression linked to an immunosuppressive tumor microenvironment (TME) encouraging tumor progression and invasion. LY6E amplification correlates with poor prognosis and increased expression of immune-related gene signatures, which is predictive of immunotherapy response. Interestingly, LY6E expression upon PARPi treatment resistance was found to be dependent on BRCA1 status. Gene expression analysis in the Orien/cBioPortal database revealed an association between LY6E and genes involved in DNA repair, such as Rad21 and PUF60, emphasizing the interplay between DNA repair pathways and immune modulation. Moreover, PUF60, Rad21, and LY6E are located on chromosome 8q24, a locus often amplified and associated with the progression of ovarian cancer. Overall, our study provides novel insights into the molecular determinants of PARPi resistance and highlights LY6E as a promising prognostic biomarker in the management of HRD ovarian cancer. Future studies are needed to fully elucidate the molecular mechanisms underlying the role of LY6E in PARPi resistance. [ABSTRACT FROM AUTHOR]