A comparative genomic approach for identifying synthetic lethal interactions in human cancer

Abstract

Synthetic lethal interactions enable a novel approach for discovering specific genetic vulnerabilities in cancer cells that can be exploited for the development of therapeutics. Despite successes in model organisms such as yeast, discovering synthetic lethal interactions on a large scale in human cells remains a significant challenge. We describe a comparative genomic strategy for identifying cancer-relevant synthetic lethal interactions whereby candidate interactions are prioritized on the basis of genetic interaction data available in yeast, followed by targeted testing of candidate interactions in human cell lines. As a proof of principle, we describe two novel synthetic lethal interactions in human cells discovered by this approach, one between the tumor suppressor gene SMARCB1 and PSMA4, and another between alveolar soft-part sarcoma-associated ASPSCR1 and PSMC2. These results suggest therapeutic targets for cancers harboring mutations in SMARCB1 or ASPSCR1 and highlight the potential of a targeted, cross-species strategy for identifying synthetic lethal interactions relevant to human cancer.

Figure 1

Comparative genomic approach for discovering cancer related synthetic sick/lethal interactions in human. (A) Flowchart describing steps to use the wealth of synthetic sick/lethal interactions available in yeast and knowledge of genes commonly mutated in cancer (Sanger Institute Cancer Gene Census) for discovery of novel cancer drug targets in human. (B) Summary of yeast synthetic sick/lethal interaction network statistics and mapping of interactions between human orthologs. The “Complete set” contains all significant synthetic sick or lethal interaction pairs at an intermediate confidence cutoff as described in (11) (ε < −0.08; p-value < 0.05), and human totals include any genes with human orthologs. The “Filtered set” contains only high confidence interactions (ε < −0.2; p-value < 0.05) or interactions replicated in two independent experiments, and human totals include only gene pairs with one-to-one orthologs (see Methods – Processing yeast genetic interaction data).

Figure 2

Interaction testing of 21 selected candidate synthetic sick/lethal interactions in human fibroblast cell lines. (A) The interaction scores for all human interactions tested. The interaction score is the difference between observed and expected growth rate based on a multiplicative model. The significant negative genetic interactions are colored red and strength of the significance is denoted by the number of asterisks, according to the legend shown. (B) Results for each significant interaction tested. The number of days the fibroblast cells were grown in the presence of shRNAs is indicated in each plot. The error bars for both (A) and (B) represent twice the width of the standard error in the interaction scores and growth rates.

Figure 3

Yeast data for the candidate synthetic sick/lethal interaction between SNF5-PRE9 (human SMARCB1- PSMA4) and UBX4-RPT1 (human ASPSCR1-PSMC2). (A) Fitnesses of the single and double mutants relative to wild-type for the SNF5-PRE9 interaction. The interaction score (ε) was estimated by comparing the observed double mutant fitness with the fitness expected based on the single mutant fitnesses. (B) Confirmation of the synthetic sick/lethal interaction using tetrad dissection analysis for the SNF5-PRE9 double mutant. Each tetrad is oriented horizontally and represents four meiotic progeny of a heterozygous double mutant between pre9Δ::natMX4/PRE9 and snf5Δ::kanMX4/SNF5. Four representative tetrads are shown. The genes knocked-out are identified by the presence of the natMX and kanMX markers, respectively. The identified double knock-out spore colonies are enclosed in circles while single gene knock-out strains are enclosed in squares or diamonds, and wild type strains are not enclosed. (C) and (D) present similar data for query mutant rpt1-1, a temperature-sensitive conditional mutant of RPT1, and yeast ubx4Δ

Figure 4

Validation of two candidate synthetic sick/lethal interactions in human fibroblast cell lines. (A) A Western blot of IMR90 cells transduced with PSMA4 and SMARCB1 shRNA virus showing down-regulation of respective protein expression. (B and C) Cell viability analyses of PSMA4 and SMARCB1 interaction using two different clones which showed decreased cell survival in cells depleted of both PSMA4 and SMARCB1 compared to cells expressing shRNAs of individual genes and compared to the expected effect from depletion of both genes. (D) Immunoblotting showing knockdown of PSMC2 and ASPSCR1 expression in IMR90 cells treated with viral particles encoding PSMC2 or ASPSCR1 shRNA. (E,F) Synthetic lethal interaction effect of PSMC2 and ASPSCR1 in IMR90 cells as shown by significantly decreased survival in cells expressing shRNAs of both genes compared to the expected effect from depletion of both genes.

Figure 5

Validation of the PSMA4-SMARCB1 synthetic lethal interaction in cancer cell lines harboring SMARCB1 loss-of-function mutations. (A) Cell viability analyses of cell lines with (A-204) or without (293) endogenous SMARCB1 mutation, grown with or without PSMA4 shRNA knock-down (shPSMA4-1), demonstrate the therapeutic potential for this cancer associated synthetic lethal interaction. (B) The experiment in (A) is repeated with a different PSMA shRNA construct (shPSMA4-2). (C,D) The experiment in A,B is repeated with a different SMARCB1 deficient cell line, G-401. (E) A Western blot showing the complete absence of SMARCB1 protein in cell lines, A-204 and G-401, which have endogenous null SMARCB1 mutations, and normal expression of SMARCB1 in the control 293 cell line. The endogenous PSMA4 and β-actin protein levels detected serve as loading controls.