Synthetic lethal interaction between the tumour suppressor STAG2 and its paralog STAG1

Abstract

Cohesin is a multi-protein complex that tethers sister chromatids during mitosis and mediates DNA repair, genome compartmentalisation and regulation of gene expression. Cohesin subunits frequently acquire cancer loss-of-function alterations and act as tumour suppressors in several tumour types. This has led to increased interest in cohesin as potential target in anti-cancer therapy. Here we show that the loss-of-function of STAG2, a core component of cohesin and an emerging tumour suppressor, leads to synthetic dependency of mutated cancer cells on its paralog STAG1. STAG1 and STAG2 share high sequence identity, encode mutually exclusive cohesin subunits and retain partially overlapping functions. We inhibited STAG1 and STAG2 in several cancer cell lines where the two genes have variable mutation and copy number status. In all cases, we observed that the simultaneous blocking of STAG1 and STAG2 significantly reduces cell proliferation. We further confirmed the synthetic lethal interaction developing a vector-free CRISPR system to induce STAG1/STAG2 double gene knockout. We provide strong evidence that STAG1 is a promising therapeutic target in cancers with inactivating alterations of STAG2.

Keywords: cancer vulnerability; cohesin complex; paralog dependency; precision medicine; synthetic lethality.

Figure 1. Evidence of functional compensation between STAG1 and STAG2

Legend: (A) Sequence identity and domain architecture of STAG1 and STAG2 proteins as annotated in the SMART database [57]. (B) Composition and biological functions of cohesin SA1 and cohesin SA2. (C) Fraction of TCGA cancers with LoF alterations (homozygous gene deletions, truncating mutations and multiple hits) or damaging missense and splicing mutations in STAG1 or STAG2 divided by tumour type. The total number of sequenced samples in TCGA is reported in brackets. BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; COAD, colon adenocarcinoma; ESCA, oesophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukaemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumours; THCA, thyroid carcinoma; UCEC, uterine corpus endometrial carcinoma. (D) Expression profiles of STAG2 when it acquires damaging or LoF alterations as compared to when it is WT in cancer cell lines from the Cell Line Project (CLP, http://cancer.sanger.ac.uk/cell_lines) and in TCGA samples and. (E) Expression profiles of STAG1 when STAG2 acquires damaging or LoF alterations as compared to when it is WT in CLP cell lines and in TCGA samples. The numbers of mutated samples or cell lines are reported in brackets. Distributions were compared using the Wilcoxon test and corresponding p-values are shown; ns = not significant.

Figure 2. Effect of transient blocking of STAG1 and STAG2 on cell proliferation

Legend: (A) STAG1 and STAG2 gene (left) and protein (right) expression in CAL-51 cells 48 hours and 72 hours after siRNA transfection, respectively. (B) Proliferation curve of CAL-51 cells after transfection with negative, STAG1 and STAG2 siRNAs. Three biological replicates were done and the KD was repeated three times in each replicate. (C) Crystal violet staining of CAL-51 cells 120 hours after transfection with negative, STAG1 and STAG2 siRNAs. (D) STAG1 and STAG2 expression measured by quantitative RT-PCR in MCF-7 cells 72 hours after siRNA transfection. (E) Proliferation curve of MCF-7 cells after transfection with negative, STAG1 and STAG2 siRNAs. Two biological replicates were done and the KD was repeated three times in each replicate. (F) Sanger sequencing confirmation of STAG2 homozygous nonsense mutation in SK-ES-1 cells. (G) Immunoblots of STAG1 and STAG2 protein expression in untreated SK-ES-1 cells. (H) STAG1 mRNA expression in SK-ES-1 cells after siRNA transfection as compared to the control. (I) Number of SK-ES-1 cells 96 hours after transfection of STAG1 or STAG1 and STAG2 siRNAs as compared to the control. Each KD was repeated six times and cells were counted blindly and independently. Average number of cells and associated standard errors across replicates for each condition are shown. Means were compared using one-tailed Student's t-test. In all quantitative RT-PCR experiments, β-2-microglobulin was used for normalisation. Shown are mean and standard error of normalized expression values across replicates. In all proliferation curves, Relative Fluorescent Unit (RFU) values were normalised to the mean across replicates at 24 hours. Mean values at 96 hours were compared using the one-tailed Student's t-test; ns = not significant.

Figure 3. Synthetic lethality between STAG1 and STAG2 in stably edited STAG2 cells

Legend: (A) Schematic diagram to derive STAG2 KO clones via vector-free (vf) CRISPR editing. CAL-51 cells are first infected with a Cas9 containing lentiviral vector to induce Cas9 expression and then transfected with a universal trans-activating RNA (tracrRNA) and gene-specific CRISPR targeting RNAs (crRNAs). Finally, edited clones are isolated via single cell cloning. (B) Evidence of Cas9 mRNA expression in CAL-51 Cas9 cells. (C) T7 endonuclease 1 assay (T7E1) assay on the edited regions of STAG2 after transfection with three STAG2-crRNAs. STAG2b (red box) was selected because of its higher editing efficiency. (D) High Resolution Melting Assay on isolated clones after single cell cloning from a heterogeneous population of STAG2 edited cells. The assay was used to identify clones with homozygous STAG2 editing. (E) Sanger sequencing confirmation of the eight-base-pair-long homozygous deletion in CAL-51 L161fs-STAG2 cells. (F) Expression of STAG2 via quantitative RT-PCR in CAL-51 L161fs-STAG2 cells and in CAL-51 Cas9 cells. β-2-microglobulin was used for normalisation. Shown are mean and standard error of normalized expression values across replicates. (G) Western blots of STAG2 protein expression in CAL-51 L161fs-STAG2 cells and CAL-51 Cas9 cells. (H) Proliferation curve of CAL-51 L161fs-STAG2 cells after transfection with negative or STAG1 siRNAs. Three biological replicates were done and the KD was repeated three times in each replicate. Relative Fluorescent Unit (RFU) values were normalised to the mean across replicates at 24 hours. Mean values at 96 hours were compared using the one-tailed Student's t-test.

Figure 4. Synthetic lethality between STAG1 and STAG2 in stably edited STAG1 cells

Legend: (A) Schematic representation of vector-mediated STAG1 editing. Cells were infected (CAL-51) or transfected (U2OS, MEF-319, RT-112) with a STAG1-Cas9 vector. Resulting STAG1 edited cells were subsequently isolated (see Methods). (B) T7E1 assay on STAG1 edited region in crVector-STAG1 CAL-51 cells. (C) STAG1 expression in CAL-51 Cas9 and crVector-STAG1 cells. β-2-microglobulin was used for normalisation. Shown are mean and standard error of normalized expression values across replicates. (D) Western blots of STAG1 in CAL-51 Cas9 cells and crVector-STAG1 cells. (E) Proliferation curve of CAL-51-Cas9 and crVector-STAG1 cells after transfection with negative or STAG2 siRNAs. (F) T7E1 assay on STAG1 edited region in crVector-STAG1 U2OS, MFE-319, and RT-112 cells, respectively. (G) Proliferation curve of U2OS, MFE-319, and RT-112-Cas9 and corresponding crVector-STAG1 cells after transfection with negative or STAG2 siRNAs. All proliferation assays were done in triplicates, except for MFE-319 where two replicates were performed, and the KD was repeated three times in each replicate. Relative Fluorescent Unit (RFU) values or Optical Density at 570 nm (OD-570) values were normalised to the mean across replicates at 24 hours and log2 transformed. Mean values at 96 hours were compared using the one-tailed Student's t-test; ns = not significant.

Figure 5. Synthetic lethality between STAG1 and STAG2 via double gene editing

Legend: (A) Schematic representation of STAG1 and STAG2 double gene editing using a lentiviral vector. CAL-51 L161fs-STAG2 and CAL-51 Cas9 cellswere infected with the STAG1-Cas9 lentiviral vector and subjected to puromycin selection to produce STAG1-STAG2 and STAG1 edited cells, respectively. (B) Crystal violet staining of CAL-51 STAG1 and STAG1-STAG2 edited cells ten days after puromycin selection. Less than 150 cells were counted in CAL-51 STAG1-STAG2 edited cells as compared to around 200,000 CAL-51 STAG1 edited cells. (C) Schematic representation of the STAG1 and STAG2 double gene editing using the vf-CRISPR system. STAG1 or EMX1 were edited using the vf-CRISPR system on CAL-51 L161fs-STAG2 and CAL-51 Cas9 cells to generate STAG1-STAG2 or EMX1-STAG2 or STAG1 or EMX1 edited cells, respectively. (D) Representative T7E1 assay on EMX1 and STAG1 edited regions in CAL-51 Cas9 and CAL-51 L161fs-STAG2 cells. (E) Quantification of EMX1 and STAG1 gene editing in CAL-51 Cas9 and CAL-51 L161fs-STAG2 cells. Each gene editing was repeated three times and each time the percentage of editing was quantified using ImageJ. Barplots show the mean percentage of gene editing and associated standard errors across replicates. One-tailed Student's t-test was used to assess statistical significance and corresponding p-values are shown; ns = not significant.