Genome-wide CRISPR screens reveal synthetic lethality of RNASEH2 deficiency and ATR inhibition

Abstract

Ataxia telangiectasia mutated and RAD3 related (ATR) protein kinase plays critical roles in ensuring DNA replication, DNA repair, and cell cycle control in response to replication stress, making ATR inhibition a promising therapeutic strategy for cancer treatment. To identify genes whose loss makes tumor cells hypersensitive to ATR inhibition, we performed CRISPR/Cas9-based whole-genome screens in 3 independent cell lines treated with a highly selective ATR inhibitor, AZD6738. These screens uncovered a comprehensive genome-wide profile of ATR inhibitor sensitivity. From the candidate genes, we demonstrated that RNASEH2 deficiency is synthetic lethal with ATR inhibition both in vitro and in vivo. RNASEH2-deficient cells exhibited elevated levels of DNA damage and, when treated with AZD6738, underwent apoptosis (short-time treated) or senescence (long-time treated). Notably, RNASEH2 deficiency is frequently found in prostate adenocarcinoma; we found decreased RNASEH2B protein levels in prostate adenocarcinoma patient-derived xenograft (PDX) samples. Our findings suggest that ATR inhibition may be beneficial for cancer patients with reduced levels of RNASEH2 and that RNASEH2 merits further exploration as a potential biomarker for ATR inhibitor-based therapy.

Figure 1.. Pooled CRISPR/Cas9-based genome-wide screens were performed in 3 cell lines.

A. Schematic representation of the workflow for CRISPR screens performed in 293A, HCT116, and MCF10A cells. B. Ranking of ATRi co-essential genes based on drugZ analysis of the results of CRISPR/Cas9-based screening in 3 cell lines. The z-score was used to define a possible synthetic lethal interaction with ATR inhibition. All genes targeted by the Toronto Knock Out Library v3 were scored according to the fold change of levels of their sgRNAs (ATRi treatment vs. DMSO treatment). High-confidence candidate genes, those with a false discovery rate (FDR) of less than 0.05, are shaded in color. Genes whose loss of function led to ATRi sensitivity appear on the left side, and genes whose loss of function led to ATRi resistance appear on the right side. Some high-confidence genes are marked. C. The top10 significantly enriched Gene ontology (GO) terms (P< 0.01). High-confidence candidate genes from B in each cell lines are categorized based on the biological process. Different color represent different cell line. Blue: 293A, Orange: HCT116, Green: MCF10A. D. Candidate ATRi co-essential genes grouped according to their roles in specific pathways. The colored dots indicate the cell lines in which the candidate genes were identified. Blue: 293A, Orange: HCT116, Green: MCF10A. Abbreviations: TKOv3, Toronto Knock Out Library v3; MOI, multiplicity of infection; sgRNA, single guide RNA; DMSO, dimethyl sulfoxide; PCR, polymerase chain reaction; T0, time point 0 (baseline); ATRi, ATR inhibitor

Figure 2.. ATRi co-essential genes identified in CRISPR-based sgRNA screens.

A. Venn diagram showing overlapping identified ATRi co-essential genes. The genes that overlapped in all 3 cell lines are listed below the diagram. B. Normalized sgRNA fold changes in DMSO treated group and AZD6738 treated group from the screen conducted in 293A cells. The counts of each sgRNA were divided by the counts of sgRNA in T0 (start point). Bar charts illustrating the log2 fold change of the indicated sgRNAs from DMSO-treated cells and ATRi-treated cells at Day21 (T21). Student T tests were performed to evaluate differences between the groups. ns, not significant; **P<0.01. Western blot were performed to determine the efficiency of sgRNAs. 293A cells were infected with lentiviruses expressing the indicated sgRNAs at low MOI and selected with puromycin. Cell lysates were blotted with the indicated antibodies. C. Validation of RNASEH2B and RNASEH2A knockout (KO) in HeLa cells by Western blotting using indicated antibodies. Note the marked decrease in the RNASEH2B protein level when RNASEH2A was knocked out. In contrast, RNASEH2A protein levels were only slightly decreased when RNASEH2B was knocked out. D. Loss of RNASEH2B/A sensitize cells to ATRi treatment in clonogenic assay. Images of colonies in colony formation assay were presented. HeLa wild-type (WT), RNASEH2B KO, and RNASEH2A KO cells were exposed to increasing concentrations of different ATR inhibitors and grew for 12 days. Results are representative of duplicate biological experiments. E. Dose-response survival curves of HeLa-WT, HeLa-RNASEH2BKO, HeLa-RNASEH2AKO cells exposed to increasing concentration of AZD6738 (upper) or VE822 (lower). Error bar represent s.d. (n=3), ANOVA p-value<0.01, results are representative of duplicate biological experiments. F. Loss of RNASEH2B sensitizes HeLa derived xenograft tumors to ATRi treatment. Two million of WT or RNASEH2BKO HeLa cells were subjected to xenograft assay and treated with AZD6738 (60mg/kg, 5 × weekly by oral gavage) or drug vehicle. Mice were treated for 3 weeks and then sacrificed. Xenograft tumors were shown. n=6. G. The weights of the tumors from F were quantified. n=6 mice, mean±s.d. Paired student t-test were used to analyze the result, *P<0.05, **P<0.01.

Figure 3.. ATRi treatment induced more DNA damage, apoptosis and senescence in RNASEH2 deficient cells.

A. Wild-type (WT) HeLa cells, RNASEH2B KO cells, or RNASEH2A KO cells were treated with DMSO or increasing concentrations of AZD6738 for 48 hrs. Representative immunofluorescence images of γ-H2A.X foci in each cell line were shown. Scale bar=10 μm. B. Quantification of γ-H2A.X foci in different groups of cells in A. n=100 cells, *P<0.05, **P<0.01, student t-test. C. ATRi treatment induced apoptosis in RNASEH2 deficient cells. HeLa WT cells, RNASEH2B KO cells, or RNASEH2A KO cells were treated with DMSO or increasing concentrations of AZD6738 for 48 hrs. Cell lysates were blotted with the indicated antibodies. Arrow marked the cleaved PARP1. D. Prolonged ATRi treatment induced senescence in RNASEH2 deficient cells. HeLa WT cells, RNASEH2B KO cells, or RNASEH2A KO cells were treated with DMSO or 0.8 μM AZD6738 for 6 Days. β-Galactosidase (β-Gal) staining was used to identify senescent cells. The boxed region is enlarged 3 times on the lower panel. Scale bar=10 μm. Results are representative of duplicate biological experiments. E. Quantification of β-Gal positive staining cells in D (mean±s.d.). ns=not significant, *P<0.05, **P<0.01. Student t-test.

Figure 4.. Frequent RNASEH2B depletion in prostate adenocarcinoma.

A. Genomic alternation of RNASEH2B and RNASEH2A in TCGA prostate cancer databases (914 samples from 5 independent studies). Blue: deep deletion; Red: amplification; Green: mutation. B. OncoPrint showing detailed genetic alterations from the prostate cancer samples in A. C. Low protein levels of RNASEH2B were frequently found in prostate adenocarcinoma cancer. Immunohistochemical staining of RNASEH2B was performed in normal prostate and prostate adenocarcinoma cancer samples. Gray staining indicates positive immunoreactivity. Representative cases of RNASEH2 staining were shown. The low RNASEH2B stained samples in prostate cancer tissues were summarized in the box. Statistical significance of low RNASEH2B protein levels and prostate adenocarcinoma was determined by Peason χ2 analysis. Scale bar=100 μm. D. Western blots showing decreased RNASEH2B protein levels in patient-derived prostate adenocarcinoma xenografts. E. Plots comparing RNASEH2A or RNASEH2B protein levels normalized to Actin in neuroendocrine prostate cancer and prostate adenocarcinoma samples. T tests were used to evaluate differences between the groups. n.s., not significant; *P < 0.05. *NEPC= neuroendocrine prostate cancer