Synthetic lethal targeting of superoxide dismutase 1 selectively kills RAD54B-deficient colorectal cancer cells

Abstract

Synthetic lethality is a rational approach to identify candidate drug targets for selective killing of cancer cells harboring somatic mutations that cause chromosome instability (CIN). To identify a set of the most highly connected synthetic lethal partner genes in yeast for subsequent testing in mammalian cells, we used the entire set of 692 yeast CIN genes to query the genome-wide synthetic lethal datasets. Hierarchical clustering revealed a highly connected set of synthetic lethal partners of yeast genes whose human orthologs are somatically mutated in colorectal cancer. Testing of a small matrix of synthetic lethal gene pairs in mammalian cells suggested that members of a pathway that remove reactive oxygen species that cause DNA damage would be excellent candidates for further testing. We show that the synthetic lethal interaction between budding yeast rad54 and sod1 is conserved within a human colorectal cancer context. Specifically, we demonstrate RAD54B-deficient cells are selectively killed relative to controls via siRNA-based silencing and chemical inhibition and further demonstrate that this interaction is conserved in an unrelated cell type. We further show that the DNA double strand breaks, resulting from increased reactive oxygen species following SOD1 inhibition, persist within the RAD54B-deficient cells and result in apoptosis. Collectively, these data identify SOD1 as a novel candidate cancer drug target and suggest that SOD1 inhibition may have broad-spectrum applicability in a variety of tumor types exhibiting RAD54B deficiencies.

Keywords: RAD54B; colorectal cancer; reactive oxygen species; superoxide dismutase 1; synthetic lethality.

Figure 1

Analysis of yeast CIN gene SL interactions identify highly connected gene pairs. (A) Two-dimensional hierarchical gene clustering performed on 692 yeast CIN genes and their top 500 genetic interactors (left). A “data-rich” area of the clusterogram was used to identify a smaller (right) and highly focused network (bottom) of candidate interactions. Yeast sod1 is a SL interactor with genes whose human orthologs are mutated or deleted in CRC (red asterisks and Table S1). Yeast sod1 clusters with its copper chaperone, ccs1 (human CCS), and another ROS detoxifying enzyme tsa1 (human PRDX2). (B) Mini SL network comprising the human orthologs of yeast CIN genes (blue circles) that are somatically mutated in CRC and SL interactors evaluated in human cells (green circles). Putative conserved SL interactions are identified with a blue line, while those specific to yeast are shown in gray, and the single interaction identified only in HCT116 cells is shown in green. Human gene names are indicated with the corresponding yeast gene listed within parentheses. (C) Graphical depiction of a putative SL interaction observed between human RAD54B and PRDX2. Shown are the relative percentages of cells remaining ±SD, following siRNA treatments (x-axis). Note the enhanced killing (i.e., lower percentage of cells remaining) in the RAD54B-deficient cells treated with siPRDX2 relative to controls.

Figure 2

RAD54B and SOD1 are synthetic lethal interactors in human cells. (A) Western blot depicting diminished SOD1 expression following siRNA-mediated knockdown in HCT116 cells; α-tubulin is a loading control. Semiquantitative analysis was performed and the normalized SOD1 expression levels relative to siGAPDH (1.00) are shown. (B) Representative images depicting the qualitative decrease in cell numbers (i.e., DAPI-stained nuclei) following SOD1 silencing of RAD54B-deficient cells relative to controls. Arrowheads identify nuclei exhibiting hallmarks of cellular cytotoxicity. Bars, 25 μm. (C) Graphical depiction for the relative percentage of control and RAD54B-deficient cells (±SD) following treatment with various siRNAs (x-axis); statistical significance is indicated (***P-value <0.0001; *P-value <0.05; NS, not significant). Graphical depiction of the SL interaction observed following the simultaneous silencing of RAD54B and SOD1 in (D) RAD54B-proficient HCT116 and (E) hTERT cells. Depicted are the mean normalized percentages (±SD) for the individual silencing of either RAD54B (solid squares) or SOD1 (open triangles), and the expected value (shaded circles) calculated for the dual combined siRNAs calculated using a multiplicative model. Solid circles identify the actual observed values for the simultaneous dual silencing.

Figure 3

RAD54B-deficient cells are selectively killed following ATTM and 2ME2 treatment. (A) Dose response curves for cells treated with varying concentrations of ATTM (left) and 2ME2 (right). Data are normalized to the respective DMSO-treated controls. (B) Real-time growth curves for control (left) and RAD54B-deficient (right) cells treated with DMSO, ATTM, or 2ME2. Chemical compounds were added 20 hr postseeding (arrow). (C) Graph depicting an increase in the mean proliferation defects (±SD) for RAD54B-deficient cells treated with either ATTM or 2ME2 (x-axis), relative to controls. Student’s t-tests identified highly statistically significant increases in mean proliferation defects (***P-value <0.0001) for the RAD54B-deficient treated cells. (D) Graphs depicting the relative number (±SD) of colonies formed in soft agar for cells treated with DMSO, ATTM, or 2ME2 for 28 days (***P-value <0.0001).

Figure 4

ATTM and 2ME2 treatments induce ROS production. (A) Representative images depicting the abundance of ROS as reflected by qualitative changes in the global abundance of fluorescein total signal intensity (green) in RAD54B-proficient and RAD54B-deficient cells treated with DMSO, ATTM, or 2ME2. All images were acquired using the identical exposure times and thus changes in fluorescence intensities reflect changes in the abundance of ROS in these cells. Images were acquired following a 6-hr incubation with the chemicals. Note that both RAD54B-proficient and RAD54B-deficient cells show an increase in ROS after treatment with ATTM or 2ME2. Nuclei (blue) are counterstained with Hoechst. Bar, 10 μm. (B) Graphical depiction of the changes in normalized ROS total signal intensity following DMSO, ATTM, and 2ME2 treatments (***P-value <0.0001).

Figure 5

DNA double strand breaks persist in RAD54B-deficient cells following ATTM and 2ME2 treatment. (A) Graphs depicting the changes in normalized γ-H2AX total signal intensities in cells treated with DMSO, ATTM, or 2ME2. Semiquantitative imaging microscopy was performed on cells prior to treatment (t = 0 hr), following a 6-hr treatment (t = 6 hr), and following a 36-hr recovery, following washout (t = 42 hr) of drug (***P-value <0.0001). (B) Graphs depicting the semiquantitative changes in normalized 53BP1 total signal intensities following DMSO, ATTM, and 2ME2 treatments. Graphs are labeled as indicated in A.

Figure 6

ATTM and 2ME2 selectively induce apoptosis in RAD54B-deficient cells. (A) Representative images depicting the abundance of apoptosis as reflected by qualitative increases in cleaved Caspase 3 in cells treated with DMSO, ATTM, or 2ME2. Staurosporine induces apoptosis and is included as a positive control. All images were acquired with identical exposure times and thus changes in fluorescence intensities identify changes in the global abundance of the cleaved Caspase 3 (apoptosis). Images were acquired immediately prior to treatment (t = 0 hr) and following a 12-hr treatment (t = 12 hr). The nuclei and cleaved Caspase 3 are pseudocolored red and green, respectively, within the merged images. Note the striking increase in cleaved Caspase 3 signal intensities within RAD54B-deficient cells treated with ATTM or 2ME2 relative to controls. Bar, 30 μm. (B) Graphs depicting the semiquantitative changes in the normalized cleaved Caspase 3 total signal intensities following DMSO, ATTM, and 2ME2 treatments (NS, not significant; ***P-value <0.0001). Quantitative imaging microscopy was performed on cells prior to treatment (t = 0 hr) and following a 12-hr treatment (t = 12 hr).