Mechanistic insights into the monotherapy and combination potential of FEN1 inhibition in cancer therapy.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease with critical functions in DNA replication and repair. FEN1 has been proposed as an anti-cancer drug target because of its synthetic lethal interaction with homologous recombination deficiency (HRD). However, the exploration of FEN1 in this context has been hampered by the lack of suitable small molecule tools. Here, we describe MSC778, a highly potent, specific and selective small molecule inhibitor of FEN1 nuclease activity. MSC778 directly engages FEN1 in cells, enhances its retention on chromatin, and selectively kills BRCA-deficient cells. Mechanistically, MSC778 suppresses DNA replication causing S-phase accumulation, DNA damage, and ultimately, cell death. Cancer cell panel screening identified Ewing sarcoma (EWS) cells as sensitive to MSC778, which is driven by the expression of SLFN11. In addition to HRD, CRISPR screening revealed a spectrum of synthetic lethal interactions between MSC778 and DNA damage response (DDR) factors, such as PARP1, USP1, PARG, and ATR. Furthermore, we demonstrate that combined inhibition of these factors with MSC778 induces synergistic killing of cancer cells. Together these data highlight FEN1 inhibition as an attractive precision oncology strategy either as monotherapy or as a combination therapy with a broad range of current and next generation DDR-targeting agents.