Fig 1.
Chemical structure of N-hydroxyurea series inhibitors of FEN1 used in this study.
Fig 2.
Model for the repair of DNA double-strand breaks.
DSBs are recognised by the MRN complex (i.) which binds to the blunt DNA ends, holding them in close proximity. The exonuclease activity of MRE11A (or EXO1 in concert with BLM) is able to resect blunt ends, creating a 3’ ssDNA overhang, which becomes coated by the single-strand binding protein RPA (ii.), signalling for cell-cycle checkpoint arrest via ATR. RPA is displaced by RAD51 (iii.) to allow for HR. Alternatively, the MRN complex can be replaced by KU78/KU80 complex (iv.), protecting DNA ends from resection and promoting NHEJ through the binding of the DNA-PK catalytic subunit (DNA-PKcs). DSBs formed as a consequence of replication fork collapse require HR for their repair. Fork stalling, following replication stress for example, activates the FA pathway in an attempt to stabilise and protect the fork (v.). The FA core complex recognises the stalled fork and ubiquitinates the FANCD2-FANCI heterodimer. Stalled forks can be further processed by structure selective endonucleases to restore the replication fork (vi.) or cleave the fork to produce a DSB (vii.). Alternatively, the post-replication machinery can bypass damaged bases at stalled forks. One such pathway leads to template switching in a HR-mediated pathway.
Table 1.
Summary of a high-throughput study to identify cell-lines sensitive to FEN1 inhibitors.
Fig 3.
Cell-lines with MSI are specifically sensitive to chemical inhibition of FEN1 by compounds 1 and 3.
A. Box and whisker diagram to show the variation in GI50 values for cell-lines subject to treatment with 1 and 3. B. Correlation between the sensitivity of cell-lines to 1 and 3. C-H. The influence of MSI on sensitivity to DNA repair inhibitors. ns = not significant * p < 0.05. ** p < 0.005.
Table 2.
Tissue-specific sensitivity to compound 1.
a Resistance was defined as GI50 greater than the maximum dose used in this study (30 μM).
Table 3.
Tissue-specific sensitivity to compound 2.
a Resistance was defined as GI50 greater than the maximum dose used in this study (30 μM).
Table 4.
Tissue-specific sensitivity to compound 3.
a Resistance was defined as GI50 greater than the maximum dose used in this study (30 μM).
Table 5.
MRE11A poly(T)11 mutations associated with MSI.
Fig 4.
Sensitivity of MSI cell-lines to FEN1 inhibition is through MRE11A deficiency.
A. Cells down-regulated for MRE11A by shRNA are sensitive to FEN1 inhibitor 1. B. Confirmation that the MSI cell-line HCT-116 is disrupted for the MRN complex. Re-introduction of chromosome 3 (HCT-116 chr3) restores MLH1 without affecting MRE11A protein level. C-F. Down-regulation of FEN1 by siRNA is toxic in MSI cells devoid of MRN complex by clonogenic survival (C) or by measuring proliferation (D-F). Each data point is the mean of at least 3 individual repeats and the error bars represent the standard error. Significance was determined by student t-test. ns = not significant * p < 0.05. ** p < 0.005 G. HCT-116 cells fail to signal for the DNA damage response upon treatment with 1.
Fig 5.
Cells disrupted for pathways required to maintain replication fork stability are sensitive to inhibition of FEN1.
A. Clonogenic survival of cells stably expressing shRNA against MRE11A, ATM or a non-targeting control when treated with 1. B. Clonogenic survival of FaDu cells or FaDu cells with all three ATM alleles knocked-out when treated with 1. C. Clonogenic survival of FaDu cells or FaDu cells with all three ATM alleles knocked-out when treated with siRNA against FEN1. D. DNA damage response induced in cells expressing shRNA against FEN1, ATM BRCA2, FANCD2 and a non-target control. E-F. Clonogenic survival of cells disrupted for FANCD2 by shRNA (E) or genes required for PRR (UBC13, HLTF) by siRNA (F) compared to a non-target control. (G) Accumulation of Rad51 foci in cells treated with 1. Data is collected from at least 500 cells per treatment. H. Clonogenic survival of cells disrupted for genes required for HR (BRCA2, BLM) and NHEJ (DNA-PKcs) by shRNA compared to a non-target control. In each clonogenic assay, data points represent the mean of at least 3 individual repeats and the error bars represent the standard error. Significance was determined by Student t-test. ns = not significant * p < 0.05. ** p < 0.005 D. DNA damage response induced in cells expressing shRNA against FEN1, ATM and a non-target control.
Fig 6.
FEN1 is epistatic with FANCD2 for the repair of olaparib- and cisplatin-induced DNA damage.
A. Olaparib sensitivity of cells disrupted for FEN1, FANCD2 or BRCA2 by shRNA compared to a non-target control. B. Sensitisation of cells to olaparib following treatment with 10 μM 1. C. Epistasis analysis of FEN1 inhibition and FANCD2 depletion following exposure to olaparib. D. Cisplatin sensitivity of cells disrupted for FEN1, XPF and FANCD2 by shRNA compared to a non-target control. E. Sensitisation of cells to cisplatin following treatment with 5 μM 1. F. Epistasis analysis of FEN1 inhibition and FANCD2 depletion following exposure to cisplatin. G-I. Sensitivity of Saccharomyces cerevisiae strains deleted for rad27, pso2, msh2 singularly and in combination either in asynchronous culture (G) or synchronised in S-phase (H-I). In all cases, each data point is the mean of at least 3 individual repeats and the error bars represent the standard error. Significance was determined by student t-test. ns = not significant * p < 0.05. ** p < 0.005.
Fig 7.
Current model for the formation and repair of DNA damage following FEN1 inhibition.
The inhibition of FEN1 leads to the accumulation of immature Okazaki fragments bound by RPA, accumulating aberrant replication structures that destabilise the replication fork. The PRR machinery is thought to allow for the tolerance of such structures by switching template strand, however failure to do so in a timely manner could lead to the stalling and, ultimately, collapse of the fork. The broken fork would require processing by endonucleases to create a 3’ overhang able to invade into back into the dsDNA and re-start replication. Persistent inhibition of FEN1 would ultimately lead to an overwhelming level of DNA damage and, ultimately, cell death.