Small molecule inhibitors uncover synthetic genetic interactions of human flap endonuclease 1 (FEN1) with DNA damage response genes

Abstract

Flap endonuclease 1 (FEN1) is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI) as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T) microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.

The Importance of Poly(ADP-Ribose) Polymerase as a Sensor of Unligated Okazaki Fragments during DNA Replication

“…We suggest that unligated Okazaki fragments that are trapped by the PARP inhibitor require HR-mediated repair for their removal, either directly as single-strand gaps or following their conversion into DSBs by nucleases or DNA replication fork collapse, as has been demonstrated in E. coli ( Kouzminova and Kuzminov, 2012 , Kuzminov, 2001 ). That unligated Okazaki fragments can also trigger HR-mediated repair in human cells is consistent with the observation that LIG1 -mutated 46BR cells exhibit elevated levels of baseline sister chromatid exchange ( Henderson et al., 1985 ) and with the observation that the FEN1 inhibitor induces RAD51 focus formation or cell death in BRCA2 -proficient and -deficient cells, respectively ( Ward et al., 2017 ).…”

Cross-Species Complementation of Nonessential Yeast Genes Establishes Platforms for Testing Inhibitors of Human Proteins

“…While this inhibitor did display some mild off-target effects, it appeared to be a potent hFEN1 inhibitor. In support of our results, other HU-based derivatives have been shown to increase human cell line sensitivity to MMS treatment (Tumey et al 2005;Exell et al 2016) and selectively impair the proliferation of HR-defective cancer cell lines (Exell et al 2016;He et al 2016;Ward et al 2017). Further, in vitro assays have demonstrated that some HU-based compounds are able to enzymatically inhibit the related endonucleases hXPG (Tumey et al 2005) and hEXO1 (Exell et al 2016), but showed species specificity upon testing with bacteriophage T5 FEN and Kluyveromyces lactis XRN1 (Exell et al 2016).…”

<p>miR-193b Increases the Chemosensitivity of Osteosarcoma Cells by Promoting FEN1-Mediated Autophagy</p>

“…42 More importantly, FEN1 was shown to participate in the repairment of DNA damage agent induced Top2-SSB complexes then maintain DNA stability, 43 and disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. 8 Autophagy is reportedly activated in response to DNA damage, which may mediate survival at low levels of DNA damage; however, excessive lesions that cannot be repaired may lead to persistent, unrestrained autophagy, that in turn induces a form of cell death known as "autophagic cell death" (ACD). [44][45][46] Consequently, we suspect that FEN1 knockdown can sensitize OS cells to epirubicin through the disruption of DNA damage repair processes.…”

“…Flap structure-specific endonuclease 1 (FEN1) participates in numerous DNA processing pathways, including Okazaki fragment maturation, stalled replication fork rescue, telomere maintenance, long-patch base excision repair, and apoptotic DNA fragmentation, 6,7 and disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. 8 Moreover, FEN1 is considered a marker for metastasis and poor prognosis in multiple cancers; [9][10][11] additionally, recent studies have revealed that FEN1 has a role in regulating sensitivity to chemotherapy. 12,13 Autophagy is a stress-relieving/homeostatic cellular recycling process involved in the maintenance of cellular homeostasis through the lysosomal degradation of various proteins and damaged organelles.…”