Specific Synthetic Lethal Killing of RAD54B-Deficient Human Colorectal Cancer Cells by FEN1 Silencing

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 to undergo meiosis. The resulting tetrads were dissected from the mitotic spreads (Ϯ SEM). Student’s t tests were performed between on YPD and later replica plated to additional selection media to identify the the mean chromosome number of the untransfected HCT and each of the genotypes indicated on the right. The combination of rad54::URA3 conditions. Conditions with statistically significant differences in means are rad27::KanMX within the same spore resulted in SL (indicated by boxes). identified by *, P Ͻ0.05 and ***, P Ͻ0.001.

hairpinRNAs (shRNA) targeting RAD54B. HCT116 colorectal small subset of cells were evident in the RAD54B depleted cells cells were specifically selected, because it is a near diploid cell that were not evident in the controls (HCT116, Non-silencing, or line that does not inherently exhibit CIN (i.e., is chromosomally eGFP) (Table 2). In agreement with the flow cytometry data, the stable). Before examining CS, RAD54B expression was assessed fraction of cells with elevated chromosome numbers (i.e., Ͼ46) at the protein level and determined to be absent or significantly correlated with decreased RAD54B expression. Furthermore, diminished in the knockout or knockdown cells, respectively the increases in the near polyploid populations observed by flow (Fig. 2A). Since most colorectal cancers exhibit increases in cytometry (Fig. 2B), were also apparent within the correspond- chromosome numbers (23, 24), we focused our attention to the ing chromosome spreads (Fig. 2D). Subsequent Student’s t tests proportion of cells with DNA contents in excess of the normal comparing the mean chromosome number for each treatment diploid G2/M peak (i.e., Ͼ4N). As RAD54B levels decreased, and the isogenic HCT116 control revealed statistically signifi- corresponding increases in the proportion of cells with DNA cant differences for the RAD54BϪ/Ϫ/Ϫ cells and the RAD54B-1- contents beyond the 4N peak were observed (Fig. 2B and Table and RAD54B-2-treated cells (Fig. 2E and Table S1). Not 1). In addition, small discrete peaks corresponding to increases surprisingly, these 3 conditions are those in which RAD54B in polyploidy were observed in both the RAD54B knockout and expression has decreased the most (see Fig. 2A). knockdown cells (Fig. 2B). To determine if increases in chromosome number could account for the increases in DNA Ectopic Expression of RAD54B Suppresses the CIN Phenotype. To content, mitotic chromosome spreads were generated and total more conclusively demonstrate that RAD54B expression is chromosome numbers were manually quantified (Fig. 2C and D causally linked to CIN, phenotypic rescue experiments were and Table 2). Although the modal number of chromosomes performed in RAD54BϪ/Ϫ/Ϫ cells. Briefly, V5 or EmGFP tagged remained at 45, increases in total chromosome numbers for a versions of RAD54B were ectopically expressed in

McManus et al. PNAS ͉ March 3, 2009 ͉ vol. 106 ͉ no. 9 ͉ 3277 Downloaded by guest on September 26, 2021 Table 1. DNA content analysis of RAD54B depleted colon cancer cells by ﬂow cytometry Percentage of total cells Fold increase Cell Line Ͻ2N 2N (G0/G1) S-phase 4N (G2/M) Ͼ4N over Untransfected (Ͼ4N)

Untransfected 1.0 31.4 39.9 21.8 5.9 NA Non-Silencing 1.2 33.3 36.8 21.2 7.5 1.3ϫ eGFP 1.2 30.1 37.9 24.3 6.5 1.1ϫ RAD54B-1 1.5 27.8 36.2 16.5 18.0 3.1ϫ RAD54B-2 1.5 27.5 33.4 22.3 15.3 2.6ϫ RAD54B-3 1.4 29.3 34.4 21.6 13.3 2.3ϫ P37 (RAD54Bϩ/ϩ/Ϫ) 0.9 30.5 36.4 22.0 10.2 1.8ϫ HPHB (RAD54BϪ/Ϫ/Ϫ) 1.0 28.8 31.6 21.5 17.1 2.9ϫ

Only a single set of values from a single representative dataset are shown; experiments were conducted in triplicate. NA, not applicable

RAD54BϪ/Ϫ/Ϫ cells. After a brief selection process, cells were RAD54B Deficient Cells Exhibit Proliferation Defects when FEN1 Ex- divided into 2 groups; 1 group was used for protein quantifica- pression Is Reduced. Yeast rdh54 and rad54 have each previously tion and the second group was harvested for DNA content been shown to exhibit SL/SGD interactions with rad27 (21, analysis by flow cytometry as above. Total RAD54B expression 25–27) (Fig. S1A). To determine if a similar genetic interaction was assayed by either standard western blot analysis (V5- is conserved in human cells, RAD54B proficient and deficient RAD54B expression level) or quantitative imaging microscopy cells were transiently transfected with siRNA duplexes specifi- (QIM; EmGFP-RAD54B expression levels) which quantify ex- cally targeting FEN1, the homolog of yeast rad27. All FEN1 pression levels in cell populations, or single cell levels, respec- duplexes (including the pools) specifically target unique non- tively (Fig. 3A). In both cases, ectopic RAD54B expression levels overlapping regions within the FEN1 coding region and cause were determined to be slightly elevated over wild-type RAD54B reduced FEN1 expression 24 h to at least 7 days post-transfection levels, but still remained predominantly within the endogenous (Fig. S1B). The 2 most effective independent siRNA duplexes expression range (Fig. 3B). In fact, QIM demonstrated that the (FEN1–2 and FEN1–3) were used to demonstrate the specificity predominant proportion (i.e., Ͼ80%) of cells ectopically ex- of the phenotype, while a FEN1-pool was used to decrease the pressing EmGFP-RAD54B had expression levels within the number of off-target effects that are potentially observed when normal distribution range of isogenic HCT116 cells expressing using a single duplex. The mitotic kinase, PLK1, was included as a positive control as it is an essential mitotic kinase known to endogenous RAD54B levels. Next, we wished to determine if decrease cellular proliferation through increased cytotoxicity ectopic RAD54B expression could rescue the CIN phenotype. that is independent of any known SL interaction (28, 29). High- RAD54B expressing cells and controls were subjected to FACS content digital imaging microscopy (HC-DIM) was performed analysis as detailed above, and DNA content profiles are pre- on fixed cells and the total numbers of Hoechst positive cells sented in Fig. 3C. Interestingly, the DNA content profiles for the imaged were calculated (Table S3). The percentages of cells RAD54B rescued cells more closely resembled the profiles of the relative to GAPD were determined for each of the conditions, wild-type (RAD54B proficient) HCT116 cells than the parental and are presented in Fig. 4A. As anticipated, PLK1 silencing Ϫ/Ϫ/Ϫ RAD54B into which RAD54B was reintroduced (Table diminished the relative total number of cells significantly, irre- S2). More specifically, the near polyploid populations previously spective of RAD54B status. However, visually striking decreases Ϫ/Ϫ/Ϫ present within the RAD54B parental line are visually in relative cell numbers were also apparent in RAD54B-deficient diminished (Fig. 3C). Since it is highly unlikely that ectopic cells in which FEN1 expression had been diminished, that were RAD54B expression reverts karyotypically abnormal cells to not apparent in RAD54B-proficient cells. Moreover, the large karyotypically normal cells, these observations are most likely difference in relative cell numbers observed in the RAD54B- attributable to either natural apoptotic mechanisms affecting the deficient cells for the various FEN1 conditions appears to reflect near polyploid populations or through diminished cell cycle the efficiency of FEN1 knock-down in general (Fig. S1B). For progression and/or proliferation which would effectively dilute example, the FEN1-pool was visually the most effective down- those near polyploid cells within the actively growing normal regulator and exhibited the greatest decrease in relative cell diploid cells. numbers (23.5% Ϯ 10.7% of GAPD-silenced total cell num-

Table 2. Increased Chromosome Numbers Following Diminished RAD54B Expression Percentage of Mitotic Spreads

Condition n Յ43 44 45 46 47 48 49 Ն50 FIϾ46

Untransfected 322 4.0 16.1 63.8 12.7 2.2 0.0 0.0 1.2 NA Nonsilencing 221 6.3 16.7 58.8 12.7 1.8 0.0 0.5 3.2 1.59ϫ eGFP 201 5.5 18.9 56.2 13.9 3.5 1.0 0.0 1.0 1.61ϫ RAD54B-1 213 7.0 11.7 52.6 12.2 7.0 2.8 1.4 5.2 4.83ϫ RAD54B-2 215 9.3 13.4 50.5 12.5 5.1 3.2 0.9 5.1 4.21ϫ RAD54B-3 207 9.7 14.5 53.1 11.6 4.8 1.9 0.5 3.9 3.26ϫ RAD54Bϩ/ϩ/Ϫ 203 5.9 16.3 55.7 12.8 3.9 1.5 1.5 2.5 2.75ϫ RAD54BϪ/Ϫ/Ϫ 606 7.3 15.7 56.3 10.4 3.8 0.7 0.3 5.6 3.05ϫ

n ϭ number of mitotic spreads quantified. FIϾ46 identifies the fold increase in frequency of cells harboring Ͼ 46 chromosomes (sum of the percentages for those mitotic spreads with 47, 48, 49 and Ն 50 chromosomes) relative to the Untransfected control HCT116 (first line). NA, not applicable

3278 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813414106 McManus et al. Downloaded by guest on September 26, 2021 Fig. 3. Ectopic RAD54B expression rescues CIN. (A) Western blot analysis of RAD54B expression in the RAD54BϪ/Ϫ/Ϫ cells ectopically expressing V5- RAD54B was determined to be near wild-type levels (HCT116) at the popula- ␣ tions level. An -tubulin loading control is included. (B) GFP-RAD54B expres- Fig. 4. FEN1 down-regulation underlies synthetic lethality in RAD54B- sion levels at single cell resolution as determined by QIM. Note that the entire deficient human cells. (A) Graphical representation of the percentages of cells range of the normalized RAD54B signal intensities are shown for both the relative to GAPD knockdown (Ϯ SEM) are shown for the isogenic RAD54Bϩ/ϩ/ϩ Ϫ/Ϫ/Ϫ wild-type HCT116 cells and the isogenic RAD54B cells ectopically express- and RAD54BϪ/Ϫ/Ϫ cells treated with the various siRNAs indicated (x-axis). A ing EmGFP-RAD54B. Although the distribution range is larger in the trans- single representative data series collected in sextuplet and compiled from 1 of th fected cells, the regions indicated in the boxes [25 percentile (bottom line), 3 experiments is shown (see Table S3). Highly statistically significant differ- th mean (middle line), and 75 percentile (top line)] overlap to a large degree, ences (P Ͻ0.0001) in the mean percentage of cells relative to GAPD knock- indicating that protein expression levels are similar, albeit slightly elevated. down as determined by Student’s t- test (see Table S4) are identified (***). (B) (C) Asynchronous and sub-confluent cells were PI-labeled and subjected to Live cell imaging coupled with PI incorporation into dead/dying cells reveals flow cytometry. The DNA content profiles were determined for wild-type an increase in death in RAD54B deficient (black) cells treated with FEN1 siRNAs Ϫ/Ϫ/Ϫ HCT116 cells (red) and RAD54B cells (green) ectopically expressing V5- versus RAD54B-proficient (gray) cells treated similarly. Five non-overlapping RAD54B (blue), EmGFP-RAD54B (brown), or empty EmGFP vector alone (pur- images from each well were collected every 2 h for 48 h and the total number ple). The arrows highlight the near polyploid populations that exist within the of PI-positive nuclei were scored. All data were normalized to the first time- RAD54B-deficient cells. point (t ϭ 0) to permit easy comparisons between the respective siRNA treatments indicated at the top. Each graph depicts a single representative experiment performed in triplicate and repeated at least once. Note that the bers). FEN1–3 however, was visually less efficient at silencing relative death index (y-axis) scale is different for the PLK1 positive control. and exhibited a less profound, but still significant, effect on relative cell numbers (47.6% Ϯ 5.5%), while the FEN1–2 knockdown efficiency was intermediate, as was its effect on pression status (Fig. S3 and Table S7). These results strongly relative cell numbers (40.5% Ϯ 6.4%). Student’s t tests (Table suggest that diminished FEN1 expression in a RAD54B deficient S4) comparing mean relative cell numbers identified highly background decreases cellular proliferation and/or increases GENETICS statistically significant differences (P Ͻ0.0001) for PLK1 in both cellular cytotoxicity in a manner analogous to the yeast SL RAD54B-proficient and deficient cells, while highly significant interactions described for the homologous yeast gene mutations, differences were only observed after FEN1 silencing (i.e., rdh54/rad54 and rad27. FEN1–2, FEN1–3, and FEN1-pool) in RAD54B deficient cells. Because HCT116 cells are MLH1-deficient, there is a possi- Increased Cellular Cytotoxicity Underlies the RAD54B/FEN1 Genetic bility that a second-site gene mutation (unrelated to RAD54B Interaction. To determine if an increase in cellular cytotoxicity knockout) was clonally fixed in the background of the RAD54 could account for the decreased cell numbers identified above, knockout cell line that is actually responsible for the SL inter- HC-DIM was performed on live RAD54B-proficient and defi- action with FEN1 knockdown. Accordingly, we performed dual cient cells treated with FEN1 or control siRNAs. Cells were RNAi against RAD54B and FEN1 in the parental HCT116 cell transfected as above, however, medium was supplemented with line. In an analogous fashion to that described above, diminished propidium iodide (PI). Since PI is normally membrane- RAD54B and FEN1 expression in concert resulted in a highly impermeable, only nuclei with compromised biological mem- statistically significant decrease in total cell numbers relative to branes (e.g., necrotic or late stage apoptotic cells) will become a GAPD-silenced control (Fig. S2 and Table S5) that is not fluorescently labeled (30). Therefore, an increase in the number observed for either of the independent knockdowns (RAD54B- of PI-stained nuclei over time was used as a metric for cellular pool or FEN1-pool). The extent of the decrease in total cell death (30–32). HC-DIM was streamlined by only including numbers for the dual RNAi system was not as great as with the FEN1–2 and FEN1-pool as they produce the greatest degree of RAD54B knockout cell line, and is likely due to the residual FEN1 silencing (Fig. S1) and cellular proliferation defects (Fig. RAD54B and FEN1 expression levels. 4A). Live cell images were acquired every 2 h for a total of 48 h To further assess the specificity of the RAD54B/FEN1 syn- and the total number of PI-positive nuclei were determined and thetic interaction, 12 randomly selected human gene targets (see normalized to 1 at t ϭ 0 h for each treatment. PLK1 was selected Table S6) were subjected to silencing in the RAD54B-deficient as a positive cytotoxicity control (see above) and under these background. None of the 12 silenced targets produced a statis- conditions it consistently exhibited a 12- to 19-fold increase in tically significant decrease in overall cell numbers relative to the PI-staining nuclei over the course of the experiment (Fig. 4B). GAPD-silenced control that was dependent on RAD54B ex- In agreement with the fixed HC-DIM presented above, increases

McManus et al. PNAS ͉ March 3, 2009 ͉ vol. 106 ͉ no. 9 ͉ 3279 Downloaded by guest on September 26, 2021 in PI-labeled cells over time were readily apparent for RAD54B- cancers, because it would only adversely affect tumor cells deficient cells (3- to 5-fold) treated with FEN1 siRNAs, but not harboring the primary sensitizing somatic mutations, and leave for similarly treated RAD54B-proficient cells. In fact, the FEN1- the normal tissue unaffected. silenced RAD54B-proficient cells exhibited similar kinetics to SL/SGD interactions have been studied extensively in yeast those of the negative GAPD controls (Ͻ2-fold). Of particular (26, 27, 36, 37) and much data are currently available for yeast note is the observation that the greatest increases in cytotoxicity genes whose (putative) human orthologs are known to be generally occur within the first 24 h of imaging (Ϸ24–48 h somatically mutated in human CIN tumors. Although several post-transfection), with only slight increases, or a plateau effect, groups have used rad54 as a gene query (21, 25–27), rdh54 has observed from t ϭ 24 to 48 h. never been used as a query, but rather has only been identified as a hit (26). Fig. S1 graphically depicts all of the known SL/SGD Discussion interactions for both yeast rad54 and rdh54. Of note, only 3 of The concept of using model organism genetics (in particular, those characterized SL/SGD interactions are shared in common, synthetic lethality screens) to predict evolutionarily conserved namely rad27, pop2, and ccr4. Of those, rad27 is of particular proteins that could be targeted to selectively kill cancer cells was interest because it has a known functional human ortholog, first articulated by Hartwell and Friend in 1997 (17). However, FEN1, which exhibits a high degree of sequence identity (58%) over the past decade, very little experimental evidence in favor and similarity (73%) and has a BLAST value of eϪ104. FEN1 is or against this concept has appeared. In this regard, enhancing an essential protein that is required for both DNA synthesis and the phenotype of a somatic mutation causing genetic instability repair. In addition to its flap endonuclease and nick exonuclease to a lethal phenotype through the specific down-regulation of a activities, it also exhibits gap endonuclease activity. During second unlinked gene product predicted from a yeast SL genetic S-phase, FEN1 processes the 5Ј ends of Okazaki fragments in interaction has not previously been reported in human cells. In lagging strand synthesis and following DNA damage, it is fact, very few examples of SL in humans have been described involved in both base excision and homologous recombination (33–35) and were primarily based on characterized biology repair where it removes the 5Ј overhanging flaps [reviewed in involving DNA repair pathways rather than cross-species can- (38, 39)]. Most recently, FEN1 was shown to be involved in didate approaches. Here, we report that human RAD54B (18) telomere stability through its contribution to lagging strand exhibits a role in maintaining CS in human colorectal cancer DNA replication at the telomeres which has direct implications cells. We demonstrate that diminished RAD54B expression on CIN (40). Of particular interest, RAD54B also exhibits roles correlates with increasing DNA content and chromosome num- in DNA repair (22, 41) and CS (this manuscript). Since SL/SGD bers. We also show that reexpression of an epitope-tagged interactions frequently identify genes whose products impinge version of RAD54B in RAD54B-deficient cells is sufficient to on the same essential biological process, we reasoned that restore DNA content profiles back to those of wild-type diminished FEN1 expression/activity in a RAD54B-deficient RAD54B-proficient cells. Most importantly, we provide mam- background would represent an excellent test candidate. malian data demonstrating a conserved SL/SGD interaction To determine if SL/SGD interactions are conserved between initially observed in yeast. Specifically, using HC-DIM we dem- species and potentially identify a new therapeutic target for onstrate that a RAD54B-deficient background genetically sen- cancer therapy, we specifically targeted FEN1 for RNAi- medi- sitizes cells to selective killing in combination with diminished ated silencing in isogenic RAD54B-proficient and deficient cells. FEN1 expression. To eliminate the characterization of artifacts arising through In this work, we describe an experimental paradigm using off-target effects, 3 independent FEN1 silencing conditions were the HCT116 colon cancer cell line for assessing synthetic lethal used—2 independent siRNA duplexes (FEN1–2 and FEN1–3) interactions between candidate CIN genes mutated in tumors and a FEN1-pool comprised of 4 independent siRNA duplexes and candidate synthetic lethal partner genes predicted from (effectively quartering the concentration of each duplex), that yeast genetic network analysis. Because the HCT116 cell line has been shown to greatly diminish off-target effects (42–46). is MLH1-deficient, we cannot exclude the formal possibility Using fixed and live cell HC-DIM, we showed that diminished that the synthetic lethality observed when FEN1 and RAD54B FEN1 expression adversely affects overall cell numbers, which are simultaneously knocked down in HCT116 could be de- presumably occurs through the corresponding increases in cel- pendent on the defective MLH1 allele in the HCT116 back- lular cytotoxicity. The underlying reason for the apparent di- ground (formally a 3-way synthetic lethal interaction between minishment in relative death from t ϭ 24 to 48 h is currently FEN1, RAD54B, and MLH1). However, because no known unknown and under investigation. However, it may simply reflect synthetic growth defects have been characterized between a decrease in the efficiency of silencing produced by the siRNA yeast mlh1 and rad54, rdh54, or rad27, we believe that the duplexes, perhaps by degradation and/or dilution effects. Alter- increased cellular cytotoxicity observed after FEN1 down- natively, it may signal the presence of a subpopulation of cells in regulation in RAD54B-silenced HCT116 cells (Fig. S2) and in which initial doses of siRNA duplexes were not sufficient to the RAD54B-deficient cells (Fig. 4), results from a 2-way SL diminish protein expression past a specific required threshold interaction (i.e., RAD54B/FEN1) rather than a 3-way inter- value, or, the existence of a subpopulation of cells that are action (i.e., MLH1/RAD54B/FEN1). refractory to siRNA treatment. In any case, FEN1 depletion was Somatic CIN mutations underlie aberrant chromosome seg- demonstrated to significantly enhance cellular cytotoxicity in a regation and therefore represent ‘sublethal’ hits on an essential synthetic genetic manner analogous to that of rad54/rdh54 and process. Conceivably, if this genetic sensitization could be sub- rad27. sequently exploited by enhancing the ‘sublethal’ phenotype to a The results presented here suggest that in the context of a ‘lethal’ phenotype, then selective killing could be invoked. RAD54B-deficient cancer cell, diminished FEN1 activity Following this logic, any cells (i.e., normal cells) not harboring through either siRNA-mediated silencing or a small molecule the genetically sensitizing CIN mutations would be left unaf- inhibitor could adversely affect the proliferation of cancer cells, fected or relatively unaffected. Therefore, uncovering the ge- while leaving the normal surrounding cells relatively unaffected. netic vulnerabilities, or the known CIN mutational spectrum, for Importantly, these data support the conservation in mammalian a given tumor type could conceivably lead to the identification cells, at least in part, of SL/SGD networks identified in model of potential therapeutic targets through the identification of organisms such as S. cerevisiae. Combining these data with an unlinked gene product (SL/SGD) interaction networks. Concep- increased understanding of the mutational spectrum for any CIN tually, SL/SGD is of particular interest in the treatment of tumor type and the continually expanding SL/SGD data emerg-

3280 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813414106 McManus et al. Downloaded by guest on September 26, 2021 ing from yeast screens (26, 27) could provide critical insights into content imaging (HCI) was performed with a Cellomics ArrayScan V HCS the identification of therapeutic targets in which human CIN Reader equipped with a 20ϫ dry lens. Three days post-transfection all nuclei tumors are efficiently and exclusively eliminated through ther- were stained with Hoechst, and 10 images per well were collected. Total apeutic intervention. Integration of knowledge among emerging nuclear counts per well were summed and normalized to a GAPD-silenced control. Live cell HCI was performed on a Cellomics KineticScan equipped with high-throughput datasets in model organisms such as S. cerevi- a live cell chamber and a 10ϫ dry lens. To visualize dead and/or dying cells, siae and Caenorhabditis elegans, will stimulate new research complete growth media was supplemented with PI. All HCI experiments were directions and solutions to current challenges in combating conducted in sextuplet and repeated at least once. Further details can be human cancer. found in SI Materials and Methods.

Materials and Methods ACKNOWLEDGMENTS. We thank Dr. Miyagawa (Hiroshima University, Japan) Retroviral shRNAs or siRNA pools and independent duplexes were purchased for generously providing the RAD54B reagents (cells, cDNA clone, and anti- from Open Biosystems or Dharmacon, respectively, and transfected with body) and Abcam for providing the tubulin and FEN1 antibodies. We thank Drs. Vogelstein, Koshland, and Aparicio for helpful suggestions, Drs. Roberge, RNAiMax (Invitrogen). Western blots were conducted on proteins extracted Underhill, and Sampaio and Ms. Aruna Balgi for technical assistance with the from asynchronous and sub-conﬂuent cells 5 days post-transfection, essen- HC-DIM, and Mr. Jan Stoepel and Ms. Payal Sipahimalani for the tetrad analysis tially as described elsewhere (47). Flow cytometry, mitotic chromosome work. KJM is a Lymphoma Foundation Canada Fellow and was previously spreads, and microscopy were performed as described in ref. 48. Fixed high- funded by CIHR and MSFHR. Operational funds were provided by CIHR to PAH.

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