Saccharomyces cerevisiae Genetics Predicts Candidate Therapeutic Genetic Interactions at the Mammalian Replication Fork

The concept of synthetic lethality has gained popularity as a rational guide for predicting chemotherapeutic targets based on negative genetic interactions between tumor-specific somatic mutations and a second-site target gene. One hallmark of most cancers that can be exploited by chemotherapies is chromosome instability (CIN). Because chromosome replication, maintenance, and segregation represent conserved and cell-essential processes, they can be modeled effectively in simpler eukaryotes such as Saccharomyces cerevisiae. Here we analyze and extend genetic networks of CIN cancer gene orthologs in yeast, focusing on essential genes. This identifies hub genes and processes that are candidate targets for synthetic lethal killing of cancer cells with defined somatic mutations. One hub process in these networks is DNA replication. A nonessential, fork-associated scaffold, CTF4, is among the most highly connected genes. As Ctf4 lacks enzymatic activity, potentially limiting its development as a therapeutic target, we exploited its function as a physical interaction hub to rationally predict synthetic lethal interactions between essential Ctf4-binding proteins and CIN cancer gene orthologs. We then validated a subset of predicted genetic interactions in a human colorectal cancer cell line, showing that siRNA-mediated knockdown of MRE11A sensitizes cells to depletion of various replication fork-associated proteins. Overall, this work describes methods to identify, predict, and validate in cancer cells candidate therapeutic targets for tumors with known somatic mutations in CIN genes using data from yeast. We affirm not only replication stress but also the targeting of DNA replication fork proteins themselves as potential targets for anticancer therapeutic development.


Functional analysis of Ctf4 mutant alleles
Two proteins of disparate functions that bind Ctf4 are the GINS complex member and DNA replication protein Sld5, and Mms22, a subunit of an E3--ubiquitin ligase required for double strand break repair (Ben--Aroya et al. 2010;Duro et al. 2010;O'Donnell et al. 2010). We performed a yeast two--hybrid assay assessing the ability of the nine Ctf4 alleles to bind Sld5 and Mms22. As in published reports, the interaction between Ctf4 and Sld5 is sensitive to perturbation by mutation at all points tested except the extreme N--terminus (Gambus et al. 2009); on the other hand, the interaction between Ctf4 and Mms22 appears to be most sensitive to mutation or deletion in the middle third of the Ctf4 primary sequence ( Figure S2).
Knowing that there were differences in protein interactions between the Ctf4 alleles, we wanted to correlate phenotypic severity with the differing physical interactions. We first compared the sensitivity of the allele series to bleomycin, a radiomimetic that induces DNA double strand breaks, and HU ( Figure S2). Alleles that retained interactions with both Sld5 and Mms22 were not sensitive to genotoxic stress (i.e. ctf4--107 and ctf4--41). Conversely, alleles with disrupted Sld5 binding had a range of sensitivities from very subtle phenotypes in ctf4--154, to intermediate phenotypes in ctf4--46, --43 and --66 to null phenotypes in ctf4--25, --50 and --65 ( Figure S2C). Binding to Mms22 did not predict a consistent trend in drug sensitivity. This experiment is complicated by the fact that the status of the many other Ctf4 physical interactions is unknown and the expression levels and protein stability of the various alleles are unknown. We quantitated the genome stability defects in each CTF4 allele first using a quantitative Chromosome Transmission Fidelity (CTF) assay ). While all alleles tested had a strong increase in chromosome loss, there were no significant differences between the alleles in this assay ( Table S6). The CTF method represents a sensitized assay in which the chromosome fragment is prone to loss and therefore may have prevented our identification of subtle differences between the alleles. We also performed a sister chromatid cohesion assay (Michaelis et al. 1997). Again, all but one of the alleles had an elevated frequency of cells with separated chromatids similar to or greater than ctf4Δ ( Table S6). The presence of cohesion defects greater than ctf4Δ hints that some of the alleles may have dominant negative phenotypic effects. ctf4--41 exhibited lower rates of cohesion loss than the other mutants (Table S6).
Interestingly, ctf4--41 was the only mutant protein that was able to physically interact with both Sld5 and Mms22 ( Figure S2).
Taken together, these data suggest that the ability to bind efficiently to Sld5 is critical to the cohesion establishment and genotoxin resistance functions Ctf4. Furthermore, the ability of a Ctf4 mutant protein to interact with Mms22 is predictive of neither its cohesion establishing ability, nor of its ability to function in the response to DNA damage.

Expanding the therapeutic target range of Ctf4/WDHD1 inhibitors
CTF4 is a genetic interaction hub connected to the yeast orthologs of CIN cancer genes (Yuen et al. 2007). Thus, Ctf4/WDHD1 represents a potential broad--spectrum target for anticancer therapeutic development. As genetic interaction data is not yet available for a large proportion of the essential yeast genome, we sought to expand the range of genotypes targetable with potential inhibitors of Ctf4/WDHD1 by carrying out an SGA screen of Ctf4 against collections of essential gene mutants (Breslow et al. 2008;Li et al. 2011). In addition to yielding candidate gene--drug target interactions, this approach also defines the potential off--target effects of Ctf4 inhibition by revealing the complement of cellular pathways sensitized to Ctf4 perturbation. Figure S3 shows the network of essential genes sensitized to CTF4 deletion. A handful of interactions were validated by tetrad analysis and spot--dilution assays (Table S5). GO Figure S2 Ctf4 is physically and functionally linked to several replication protein complexes. (A) Schematic of CTF4 alleles used. Numbers represent the amino acid number and asterisks indicate the relative position of point mutations. (B) CTF4 alleles confer differential ability to interact with Sld5 and Mms22 by yeast--two--hybrid. Cells carrying the indicated plasmids were grown to log phase, subjected to ten--fold serial dilution, plated on the indicated medium, and imaged after five days' growth. (C) CTF4 alleles confer differential sensitivity to DNA damaging drugs. Experiment was conducted as in (B) on plates containing the indicated drug and concentration.  Figure S3 Expanding the therapeutic value of CTF4. (A) Genetic interactions between CTF4 and essential genes determined by SGA analysis. Edge thickness represents relative strength of interaction. Red edges, interaction validated by tetrad or spot dilution analysis (Table S5). Green edge, interaction did not validate by tetrad or spot dilution analysis. Red node, human ortholog appears in the cancer gene census. Tables S1--S8 Available for download as Excel files at http://www.g3journal.org/lookup/suppl/doi:10.1534/g3.112.004754/--/DC1. Table S1 Yeast strains used in this study. Table S2 Chemical--genetic interaction screening raw data. Table S3 Compiled chemical sensitivity data from SGD and this study. Table S4 SGA raw scores for essential miniarray screens and taf1--1 whole genome screen. Table S5 Summary of tetrad analysis for all SGA screens. Table S6 Quantitation of colony sectoring and chromatid separation assay for CTF4 mutants (on next page). Table S7 Compiled CTF4 genetic interactors and their mutational status in cancer. Table S8 SGA raw scores ctf4Δ essential miniarray screen.