Specific synthetic lethal killing of RAD54B-deficient human colorectal cancer cells by FEN1 silencing Kirk J. McManus, Irene J. Barrett, Yasaman Nouhi, and Philip Hieter1 Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada, V6T 1Z4 Communicated by Thomas D. Petes, Duke University Medical Center, Durham, NC, January 5, 2009 (received for review October 29, 2008) Mutations that cause chromosome instability (CIN) in cancer cells hypothesized that the CIN phenotype associated with tumors, produce ‘‘sublethal’’ deficiencies in an essential process (chromo- but not normal cells, represents an excellent ‘‘Achilles’ heel’’ that some segregation) and, therefore, may represent a major un- would allow for the selective killing of cancer cells. Presumably, tapped resource that could be exploited for therapeutic benefit in if cross-species tests of candidate genes can be applied to identify the treatment of cancer. If second-site unlinked genes can be second site targets that exacerbate the sublethal defect associ- identified, that when knocked down, cause a synthetic lethal (SL) ated with a CIN-inducing mutation, a novel drug target will be phenotype in combination with a somatic mutation in a CIN gene, identified. Furthermore, by generating a SL interaction network novel candidate therapeutic targets will be identified. To test this for the set of yeast CIN genes whose human homologs are idea, we took a cross species SL candidate gene approach by somatically mutated in tumors (Fig. 1B), we can identify those recapitulating a SL interaction observed between rad54 and rad27 yeast genes that are positioned as SL ‘‘interaction nodes’’ and mutations in yeast, via knockdown of the highly sequence- and whose human homologs would then represent candidate thera- functionally-related proteins RAD54B and FEN1 in a cancer cell line. peutic targets for a broad spectrum of tumors (Fig. 1 C and D). We show that knockdown of RAD54B, a gene known to be In this study, we use both RAD54B knockout and RNAi- somatically mutated in cancer, causes CIN in mammalian cells. silenced cells, and isogenic controls, to demonstrate that de- Using high-content microscopy techniques, we demonstrate that creases in human RAD54B expression in colorectal cancer cells RAD54B-deficient human colorectal cancer cells are sensitive to SL correlates with increases in chromosome numbers. RAD54B was killing by reduced FEN1 expression, while isogenic RAD54B profi- chosen because homozygous mutations at highly conserved cient cells are not. This conserved SL interaction suggests that positions have been identified in human primary lymphoma and extrapolating SL interactions observed in model organisms for colon cancers (18), although the functional status of these homologous genes mutated in human cancers will aid in the mutant alleles has not been directly tested in a mammalian identification of novel therapeutic targets for specific killing of context (19). Furthermore, RAD54B exhibits a significant de- cancerous cells exhibiting CIN. gree of sequence and functional similarity with yeast Rdh54 and Rad54 which both exhibit strong CIN phenotypes in yeast (20). cancer therapeutics ͉ chromosome instability ͉ synthetic lethality Using a cross-species candidate gene approach and high-content digital imaging microscopy techniques, we show that the syn- enomic instability is now widely recognized as an important thetic lethality observed in yeast for rad54 rad27 double mutants Gfactor in the evolution of cancer and arises through either (see Fig. 1E and ref. 21) is conserved within a human colorectal of 2 mechanisms—increased mutation rate or chromosome cell line (by simultaneous down-regulation of the corresponding instability (CIN). CIN correlates with Ϸ85% of solid tumors and human gene products, RAD54B and FEN1). Decreases in cell is characterized by an increased error rate in the gain or loss of numbers with concomitant increases in cellular cytotoxicity were chromosomes during cell division (1). CIN is associated with observed in RAD54B deficient/FEN1 depleted cells that were numerous different tumor types including colon (2–5), ovarian not apparent in isogenic RAD54B proficient/FEN1 depleted (6, 7), and non-Hodgkin lymphoma (8–12), and it is believed to cells. These findings represent an example of a validated target be an early event in the etiology of tumorigenesis (13–15). for selective killing of mammalian cells as a prediction from a SL Conceptually, CIN promotes tumor heterogeneity by increasing interaction between a CIN gene mutation and an unlinked gene or decreasing chromosome numbers (16), and directly affects the mutation in yeast. We suggest that extrapolation of SL genetic expression levels of both oncogenes and tumor suppressor genes interaction networks identified in yeast to a human context will encoded on the mis-segregated chromosomes. Most importantly provide a productive strategy to identify a broad range of novel to the work presented here, CIN gene mutations genetically cancer therapeutic targets. distinguish tumor cells from normal cells and may therefore represent a genetic susceptibility that could be exploited for Results selective killing (see below). Consequently, identifying the gene Diminished Rad54B Expression Causes Chromosome Instability in products that regulate chromosome stability (CS) will not only Human Tissue Culture. Having previously demonstrated in yeast provide insights into the molecular mechanisms of chromosome that both RAD54 and RDH54 play important roles in main- segregation and tumorigenesis, but it will also provide a list of taining CS (20), we wished to determine if RAD54B exhibits a similar role in humans. Accordingly, we used genomic knockouts candidate cancer CIN genes that may be exploited to identify Ϫ Ϫ Ϫ novel therapeutic targets for the treatment of cancer. [RAD54B / / (see SI Materials and Methods for description)] In 1997, Hartwell and colleagues (17) posited that cancer cells generously provided by Dr. Miyagawa (22) and short- harboring somatic mutations or deletions represent genetically sensitized cells, relative to normal surrounding cells, that may be Author contributions: K.J.M. and P.H. designed research; K.J.M., I.J.B., and Y.N. performed susceptible to drug therapies selectively targeting a second research; K.J.M. contributed new reagents/analytic tools; K.J.M., I.J.B., and Y.N. analyzed unlinked gene product. They suggested that synthetic lethality data; and K.J.M. and P.H. wrote the paper. (SL), which refers to the lethal combination of 2 independently The authors declare no conflict of interest. viable mutations or deletions in 2 unlinked genes (Fig. 1A), could 1To whom correspondence should be addressed at: Michael Smith Laboratories, 2185 East be used in model organisms such as yeast to identify candidate Mall, Vancouver, BC, Canada V6T 1Z4. Email: [email protected]. SL interactions that may be conserved in humans. Because This article contains supporting information online at www.pnas.org/cgi/content/full/ chromosome segregation is an essential cellular process, we 0813414106/DCSupplemental. 3276–3281 ͉ PNAS ͉ March 3, 2009 ͉ vol. 106 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813414106 Downloaded by guest on September 26, 2021 Fig. 1. Synthetic lethality in model organisms and human cancer. (A)ASL Fig. 2. RAD54B depletion underlies CIN. (A) Western blots depicting RAD54B interaction occurs when 2 independently viable gene mutations/deletions expression levels in knockout (RAD54Bϩ/ϩ/Ϫ and RAD54BϪ/Ϫ/Ϫ), knockdown [i.e., yfg1 (e.g., rad54) and yfg2 (e.g., rad27)] are combined to produce a lethal (RAD54B-1, RAD54B-2, and RAD54B-3), and control (Untransfected, Non- phenotype. If slow growth is observed, a synthetic growth defect (SGD) is silencing, and eGFP) cells. An ␣-tubulin loading control has been included. (B) defined. (B) A representative example of a genetic interaction network gen- DNA content analysis of RAD54B knockout and knockdown cells. Asynchro- erated from yeast data available in Biogrid (49), where circles identify genes nous cells were PI-labeled and subjected to flow cytometry. The diploid 2N and lines represent SL/SGD interactions. Note that rad27 intersects with both (G0/G1), 4N (G2/M) and Ͼ4N (aneuploid/polyploid) populations have been rad54 and rdh54. (C) Schematic representation of SL/SGD in a human cancer identified. The various cell lines/conditions are indicated in the legend. (C) context. A mutation or deletion of yfg1 (e.g., RAD54B CIN mutation) genet- Representative images of DAPI counterstained chromosomes found in mitotic ically sensitizes a cancer cell to SL attack through down-regulation of a second spreads generated from untransfected HCT116 (top left), RAD54B-1 trans- unlinked gene product [yfg2 (e.g., FEN1)], while leaving the normal adjacent fected (top right and bottom left) and RAD54BϪ/Ϫ/Ϫ (bottom right) cells. The cell(s) unaffected. (D) The yeast network presented in (B) has been humanized total chromosome numbers are indicated. (D) Scatter plots depicting the total by identifying the top hit human homolog for the respective yeast genes and chromosome distribution for cells RAD54B knockout and knockdown cells and is presented. Note that the lines only identify candidate interactions assuming controls. (E) Graphical representation of the mean chromosomes numbers evolutionary conservation. (E) Haploid rad54::URA3 and rad27::KanMX were determined for each of the conditions indicated on the x axis as quantified GENETICS mated and induced