Synthetic lethality between the cohesin subunits STAG1 and STAG2 in diverse cancer contexts
- Vienna Biocenter, Austria
- Boehringer Ingelheim RCV GmbH & Co KG, Austria
- Spanish National Cancer Research Centre, Spain
- Centro de Investigación Biomédica en Red de Cáncer, Spain
- Children’s Cancer Research Institute, Austria
- Medical University of Vienna, Austria
- Universitat Pompeu Fabra, Spain
- Georgetown University School of Medicine, United States
Figures
Figure 1 with 5 supplements
Identification of STAG1 as a genetic vulnerability of STAG2 mutated cells.
(A) Engineering of an isogenic HCT 116 cell model by CRISPR/Cas9-mediated inactivation of STAG2. The position of the sgRNAs used to create deleterious insertion and deletion mutations in the STAG2 …
Figure 1—figure supplement 1
Characterization of CRISPR/Cas9-generated STAG2 mutated clones used in this study.
(A) Four different sgRNAs co-expressed with Cas9 from an all-in-one plasmid or a lentiviral vector were used to generate deleterious frameshift insertions or deletion mutations (indels) in STAG2 …
Figure 1—figure supplement 2
Rescue of the synthetic lethal interaction between STAG1 and STAG2 by expression of an siRNA-resistant FLAG-STAG1 transgene.
HCT 116 parental cells, a STAG2 wild-type clone (502wt), and two STAG2- clones (505c1 and 502c4) were transduced with a lentivirus encoding no transgene (empty vector) or an siRNA-resistant and …
Figure 1—figure supplement 3
Double depletion experiment confirms STAG1-STAG2 synthetic lethality.
(A) HCT 116 parental cells were co-transfected with NTC siRNA and siRNA duplexes targeting one of the following genes: NTC, PLK1, RAD21, CDCA5, SGOL1, STAG1 or STAG2. HCT 116 parental cells were …
Figure 1—figure supplement 4
Double depletion experiments indicate that the STAG1-STAG2 genetic interaction is independent of p53.
Parental HCT 116 cells and STAG2- 505c1 cells were transfected with NTC (-) or TP53 (+) siRNA duplexes. Protein extracts were prepared 4 days after transfection and analyzed by immunoblotting (left …
Figure 1—figure supplement 5
Depletion of STAG1 does not reduce viability in hTERT RPE-1 cells.
Human telomerase-immortalized retinal pigment epithelial cells (hTERT RPE-1) were transfected with the indicated siRNA duplexes. Protein extracts were prepared 4 days after transfection and analyzed …
Figure 2 with 2 supplements
Loss of STAG1 function causes severe mitotic defects, abrogates sister chromatid cohesion and triggers apoptosis in STAG2- but not parental HCT 116 cells.
(A) Parental and STAG2- 505c1 HCT 116 were transfected with NTC and STAG1 siRNA duplexes. Immunofluorescence analysis was performed 72 hr after transfection to determine the mitotic index by scoring …
Figure 2—figure supplement 1
The depletion of STAG1 prolongs mitosis in STAG2 mutated but not parental HCT 116 cells.
Parental and STAG2- 505c1 HCT 116 were transfected with NTC and STAG1 siRNA duplexes. Cells were tracked from 0.5 to 72 hr (imaging interval 30 min) after transfection by bright-field live-cell …
Figure 2—figure supplement 2
Aberrant nuclear morphology in STAG2-mutated but not parental HCT 116 cells upon depletion of STAG1.
Parental and STAG2- 505c1 HCT 116 were transfected with NTC and STAG1 siRNA duplexes, fixed and stained with Hoechst 72 hr after transfection. Nuclei were scored for aberrant size (>20 μm) or …
Figure 3 with 3 supplements
The synthetic lethal interaction between STAG1 and STAG2 is manifested in disease-relevant bladder cancer and Ewing sarcoma cell lines.
(A) The indicated bladder cancer cell lines were analyzed for STAG2 expression by immunoblotting. (B) The indicated bladder cancer cell lines were transfected with NTC, STAG1 and PLK1 siRNA …
Figure 3—figure supplement 1
Patient-derived STAG2 mutations cause STAG1 dependency in engineered isogenic HCT 116 cells.
HCT 116 cell lines engineered to harbor the indicated deleterious patient-derived STAG2 mutations were transfected with NTC, STAG1 and SGOL1 siRNA duplexes. (A) Protein extracts were prepared 48 hr …
Figure 3—figure supplement 2
Characterization of CRISPR/Cas9-generated STAG2 knockout in UM-UC-5 bladder cancer cell line.
(A) sgRNA 505 co-expressed with Cas9 from an all-in-one plasmid was used to generate deleterious frameshift insertions or deletion mutations (indels) in STAG2 coding exons. The cognate sgRNA target …
Figure 3—figure supplement 3
Restoration of STAG2 protein expression in UM-UC-3 bladder cancer cells.
STAG2-deficient UM-UC-3 cells were transduced with lentiviral particles encoding no transgene (empty vector) or a 3xFLAG-tagged STAG2 transgene (FLAG-STAG2). Stable selected cell pools were used for …
Figure 4 with 2 supplements
Model for the synthetic lethal interaction between STAG1 and STAG2.
In wild-type cells, both cohesin-STAG1 and cohesin-STAG2 complexes redundantly contribute to sister chromatid cohesion and successful cell division. Loss of STAG1 is tolerated in these cells as …
Figure 4—figure supplement 1
Analysis of STAG1 protein or mRNA expression in cancer cell lines and patient tumors.
(A), Immunoblot analysis of protein extracts prepared from the indicated cancer cell lines. (B), STAG1 mRNA expression levels (transcripts per million, TPM) in tumor samples obtained from The Cancer …
Figure 4—figure supplement 2
Expression levels of STAG1 and STAG2 mRNA in normal human tissues.
Expression data were obtained from The Genotype-Tissue Expression (GTEx) project (www.gtexportal.org). STAG1 and STAG2 mRNA expression levels (transcripts per million, TPM) in the indicated normal …
Additional files
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Supplementary file 1
Table of sgRNA sequences used in this study.
- https://doi.org/10.7554/eLife.26980.019
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Supplementary file 2
Table of cell lines used in this study.
Sources, STAG2 status and authentication information (STR fingerprinting) of cell lines are listed.
- https://doi.org/10.7554/eLife.26980.020
