An Evolutionarily Conserved Synthetic Lethal Interaction Network Identifies FEN1 as a Broad-Spectrum Target for Anticancer Therapeutic Development

<div><p>Harnessing genetic differences between cancerous and noncancerous cells offers a strategy for the development of new therapies. Extrapolating from yeast genetic interaction data, we used cultured human cells and siRNA to construct and evaluate a synthetic lethal interaction network comprised of chromosome instability (CIN) genes that are frequently mutated in colorectal cancer. A small number of genes in this network were found to have synthetic lethal interactions with a large number of cancer CIN genes; these genes are thus attractive targets for anticancer therapeutic development. The protein product of one highly connected gene, the flap endonuclease <em>FEN1</em>, was used as a target for small-molecule inhibitor screening using a newly developed fluorescence-based assay for enzyme activity. Thirteen initial hits identified through <em>in vitro</em> biochemical screening were tested in cells, and it was found that two compounds could selectively inhibit the proliferation of cultured cancer cells carrying inactivating mutations in <em>CDC4</em>, a gene frequently mutated in a variety of cancers. Inhibition of flap endonuclease activity was also found to recapitulate a genetic interaction between <em>FEN1</em> and <em>MRE11A</em>, another gene frequently mutated in colorectal cancers, and to lead to increased endogenous DNA damage. These chemical-genetic interactions in mammalian cells validate evolutionarily conserved synthetic lethal interactions and demonstrate that a cross-species candidate gene approach is successful in identifying small-molecule inhibitors that prove effective in a cell-based cancer model.</p> </div

RF00974 recapitulates the interaction between <i>FEN1</i> and <i>MRE11A</i>, and leads to increased endogenous DNA damage.

<p>(A) siRNA-mediated knockdown of MRE11A sensitizes HCT116 cells to treatment with RF00974. siRNA transfection experiments were carried out as described in Materials and Methods. Cells were fixed and imaged four days following siRNA transfection, after cells had been incubated in compound for 48 hours. Data were analyzed by one-way ANOVA followed by a Tukey test. Shown is mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. (B) Chemical inhibition of MRE11A sensitizes cells to RF00974. Cells were incubated in the indicated compounds at the indicated concentrations for three days prior to fixation and imaging as described in Materials and Methods. Data were analyzed by one-way ANOVA followed by a Tukey test. Shown is mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. (C) Inhibition of flap endonuclease activity with RF00974 mimics siRNA-mediated knockdown of FEN1 by increasing endogenous DNA damage. Data were analyzed by Student's <i>t</i> test. Shown is mean ± SEM.</p

Yeast and human gene orthologs.^A

A<p>Names indicated are the names used in this work. Names in parentheses indicate common alternative gene names. Members of the cohesin complex (and <i>SCC2/NIPBL</i>, a cohesin loader) are indicated in boldface type.</p>B<p>Identified with DELTA BLAST algorithm.</p

Evolutionary conservation of synthetic lethal interactions in HCT116 cells.

<p>(A) A yeast cancer-ortholog synthetic lethal network. Lines indicate synthetic lethal genetic interactions. Yeast genes and human orthologs are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003254#pgen-1003254-t001" target="_blank">Table 1</a>. Red circles represent <i>S. cerevisiae</i> orthologs of genes mutated in cancer; blue circles indicate common interacting partners, which are referred to as “central” genes. (B–D) Representative data depicting mean percentage of remaining HCT116 cells (± SEM) treated with pooled siRNAs targeting central genes (top) and cancer genes (along <i>x</i>-axis) relative to GAPDH-silenced controls. Blue circles, siRNA targeting central gene alone. Red circles, siRNA targeting cancer gene alone. Yellow triangles, predicted viability of double siRNA treatment. Green circles, observed viability of double siRNA treatment. For raw data, please refer to . GAPDH siRNA does not significantly reduce viability relative to a non-silencing siRNA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003254#pgen.1003254.s002" target="_blank">Figure S2A</a>). (E) Representative data depicting mean percentage viability of hTERT cells (± SEM) treated with pooled siRNAs targeting FEN1 and cancer genes (along <i>x</i>-axis) relative to GAPDH-silenced controls. Symbols are as in B. (F) Mammalian genetic interaction network. Solid grey line, interaction observed in both <i>S. cerevisiae</i> and HCT116 cells; green dashed line, interaction observed only in <i>S. cerevisiae</i>; orange dotted line, interaction observed only in HCT116 cells.</p

IC₅₀ curves of flap endonuclease inhibitors.

<p>FEN1 assays were carried out as described in Materials and Methods. Compound names are indicated above each graph, and structures are given to the right of each graph. Tumey 16 (top-left panel) was included as a positive control for flap endonuclease inhibition.</p

Cell-based assays for flap endonuclease inhibitor activity reveal two compounds that selectively inhibit the proliferation of cells deficient in <i>CDC4</i>.

<p>(A) siRNA-mediated knockdown of FEN1 selectively inhibits the proliferation of <i>CDC4</i>-knockout HCT116 cells. siRNA transfections were carried out as described in Materials and Methods. Cells were fixed and imaged four days following siRNA transfection. Data were analyzed by one-way ANOVA followed by a Tukey test. Shown is mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. (B) Some flap endonuclease inhibitors recapitulate the genetic interaction between <i>FEN1</i> and <i>CDC4</i> in HCT116 cells. Cells were incubated with compound at the indicated concentration for 72 hours in optically clear 96-well plates prior to fixation and imaging as described in Materials and Methods. Data were analyzed by one-way ANOVA followed by a Tukey test. Shown is mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. (C) RF00974 and NSC645851 recapitulate the genetic interaction between <i>FEN1</i> and <i>CDC4</i> in DLD-1 cells. Experiments were carried out as in (B). Data were analyzed by one-way ANOVA followed by a Tukey test. Shown is mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.</p

Screening for FEN1 inhibitors <i>in vitro</i>.

<p>Schematic representation of the fluorescence-based assay employed to identify FEN1 inhibitors. In the absence of inhibitor, FEN1 cleaves the 5′ flap to which the 6-FAM fluorophore is attached, allowing it to diffuse away from the BHQ-1 quencher and fluoresce. Activity is read as increasing fluorescence over time.</p

NSC645851 and RF00974 selectively inhibit the proliferation of HCT116 and DLD-1 cells with both homozygous and heterozygous inactivating mutations of <i>CDC4</i>.

<p>Experiments were carried out as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003254#pgen-1003254-g004" target="_blank">Figure 4B</a>. Data were analyzed by one-way ANOVA followed by a Tukey test. Shown is mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.</p