Werner syndrome helicase is a selective vulnerability of microsatellite instability-high tumor cells
- Boehringer Ingelheim RCV GmbH & Co KG, Austria
- Research Institute of Molecular Pathology, Austria
- University of Michigan, United States
- Baylor University Medical Center, United States
Figures
Figure 1 with 1 supplement
WRN is a selective dependency in MSI-H cancer cell models.
(A) WRN shRNA activity by RSA score in pooled shRNA depletion screens from Project DRIVE (McDonald et al., 2017). Cell lines were binned according to tumor type. (B) MSS/MSI-H status and WRN RSA of …
Figure 1—figure supplement 1
WRN dependency correlates with MMR gene mutation status and MLH1 expression.
(A) Receiver operating characteristic curve and variable importance plot for Random Forest model. Note: <gene >_st denotes the mutational status of respective gene whereas <gene > denotes the …
Figure 2 with 3 supplements
Loss of WRN selectively impairs viability of MSI-H CRC and endometrial cancer cell models.
(A) MSS and MSI-H CRC cell lines were transfected with the indicated siRNAs. Cell viability was determined 7 days after transfection and is shown relative to non-targeting control (NTC) siRNA. WRN …
Figure 2—figure supplement 1
Non-transformed cells do not display WRN dependency.
(A) WRN siRNA knock-down efficacy in endometrial carcinoma cell models was analyzed by qRT-PCR. RNA lysates were prepared 72 hr after transfection. WRN mRNA expression is normalized to 18S rRNA …
Figure 2—figure supplement 2
MLH1/MSH3 reconstitution in HCT 116 CRC cells does not alleviate WRN dependency.
(A) HCT 116 cell lines were transfected with the indicated siRNAs. Cell viability was determined 7 days after transfection and is shown relative to non-targeting control (NTC) siRNA. (B) WRN siRNA …
Figure 2—figure supplement 3
WRN inactivation does not elicit dependency on MLH1 or MSH3 in MSS SW480 CRC cells.
(A) SW480 monoclonal cell lines were transfected with the indicated siRNAs. Cell viability was determined 7 days after transfection and is shown relative to non-targeting control (NTC) siRNA. …
Figure 3 with 1 supplement
CRISPR-Cas9-mediated knock-out of WRN confirms the selective dependency of MSI-H CRC models on WRN.
(A) Schematic representation of CRISPR-Cas9 depletion assays. Cas9 expressing cells were transduced with a lentivirus encoding GFP and sgRNAs. The percentage of GFP-positive cells was determined …
Figure 3—figure supplement 1
CRISPR-Cas9-mediated knock-out of WRN confirms the selective dependency of MSI-H CRC models on WRN.
Cas9-GFP expressing MSS or MSI-H CRC cells were transduced with a lentivirus encoding GFP and sgRNAs targeting multiple domains in WRN as indicated. The percentage of GFP-positive cells was …
Figure 4 with 1 supplement
WRN dependency in MSI-H cancer cell lines is linked to its helicase function.
(A) Schematic representation of WRN domain structure. Location of nuclease- and ATPase-inactivating mutations in siRNA-resistant WRN (WRNr) expression constructs is indicated. (B) MSI-H CRC HCT 116 …
Figure 4—figure supplement 1
WRN dependency in MSI-H cancer cell lines is linked to its helicase function.
(A) MSI-H CRC RKO cells were stably transduced with FLAG-tagged wild-type or mutant forms of WRNr. Anti-FLAG immunofluorescence analysis was performed to monitor homogenous expression of WRNr. …
Figure 5
WRN loss-of-function in MSI-H CRC results in nuclear morphology and integrity defects.
(A) MSS and MSI-H CRC cell lines were transfected with the indicated siRNAs. Immunofluorescence analysis was performed 96 hr after transfection to determine the fraction of cells with chromosomal …
Figure 6
Time-lapse analysis of mitosis in WRN-depleted MSS and MSI-H CRC models.
Mitotic live cell imaging in WRN-depleted MSS and MSI-H CRC cell lines. Cells were transfected with WRN siRNA #1. Cells were stained with SiR-Hoechst dye and were analyzed 24 hr post siRNA …
Figure 7 with 1 supplement
Loss of WRN function in MSI-H CRC causes severe chromosomal defects.
(A) MSS and MSI-H CRC cell lines were transfected with the indicated siRNAs. Mitotic chromosome spreads were prepared 72 hr after transfection and visualized by microscopy. Non-homologous radial …
Figure 7—figure supplement 1
Elevated levels of the DNA damage marker γ-H2AX upon loss of WRN function in MSI-H CRC cells.
(A) MSS and MSI-H CRC cell lines were transfected with the indicated siRNAs or treated with 5 µM etoposide. Immunofluorescence analysis was performed 72 hr after transfection or 24 hr post etoposide …
Author response image 1
SW480 (upper panel) and CaCo-2 (lower panel) parental and MLH1-knock-out monoclonal CRC cell lines were transfected with the indicated siRNAs.
Cell viability was determined 7 days after transfection and is shown relative to non-targeting control (NTC) siRNA (n=3 biological replicates; error bars denote standard deviation). Knock-out of …
Author response image 2
Left panel: List of MSI target genes and positive/negative control genes included in the siRNA knock-down analysis.
Cyclophilin was included as a negative control gene; PLK1, PSMA1 and SGO1 are used as positive control genes. siRNA targeting WRN was also include in the analysis. NTC, non-targeting control siRNA. …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | Werner Syndrome RecQ Like Helicase (WRN) | N/A | Entrez Gene: 7486 | |
| Genetic reagent (Homo sapiens) | NTC siRNA | Dharmacon | D-001810–10 | non-targeting siRNA pool |
| Genetic reagent (Homo sapiens) | WRN siRNA pool | Dharmacon | L-010378–00 | WRN-targeting siRNA pool |
| Genetic reagent (Homo sapiens) | WRN siRNA | Dharmacon | J-010378–05 | WRN-targeting siRNA |
| Cell line (Homo sapiens) | HCT 116 | ATCC | RRID:CVCL_0291 | MSI-H CRC cell line |
| Cell line (Homo sapiens) | RKO | ATCC | RRID:CVCL_0504 | MSI-H CRC cell line |
| Cell line (Homo sapiens) | SW480 | ATCC | RRID:CVCL_0546 | MSS CRC cell line |
| Cell line (Homo sapiens) | SK-CO-1 | ATCC | RRID:CVCL_0626 | MSS CRC cell line |
| Cell line (Homo sapiens) | hTERT RPE-1 | ATCC | RRID:CVCL_4388 | Non-transformed telomerase immortalized cell line |
| Antibody | mouse anti-WRN | Cell Signaling | RRID:AB_10692114 | 1/1000 dilution for immunoblot |
| Antibody | mouse anti-GAPDH | Abcam | RRID:AB_2107448 | 1/30000 dilution for immunoblot |
| Antibody | mouse anti-FLAG | SIGMA | RRID:AB_262044 | 1/1000 dilution for immunoblot |
| Antibody | mouse anti-LAP2ß | BD Transduction Laboratories | RRID:AB_398313 | 1/100 for immunofluorescence analysis |
| Recombinant DNA reagent | pLVX-WRN-3x FLAG-IRES-Puro | This study | Lentiviral vector for stable expression of WRN wild-type | |
| Recombinant DNA reagent | pLVX-WRN-3x FLAG-IRES-Puro E84A | This study | Lentiviral vector for stable expression of WRN E84A mutant | |
| Recombinant DNA reagent | pLVX-WRN-3x FLAG-IRES-Puro K577M | This study | Lentiviral vector for stable expression of WRN K577M mutant | |
| Recombinant DNA reagent | pLVX-WRN-3x FLAG-IRES-Puro E84A _K577M | This study | Lentiviral vector for stable expression of WRN E84A/K577M double mutant | |
| Other | Drive database | PMID 28753431 | Functional genomics database on cancer cell line dependencies |
Author response table 1
Cell lines were plated at initial densities of 1000 or 2000 cells per well of a 96-well plate.
The following day, cells were treated with the indicated inhibitors at concentrations ranging from 10 to 0.04 µM (0.5 to 0.002 µM for panobinostat). Cell viability was determined 7 days after …
| Tumor Type | MSS/MSI status | Cell line | NSC 617145 IC50 [µM] | NSC 19630 IC50 [µM] | ML-216 IC50 [µM] | Panobinostat IC50 [µM] |
| CRC | MSS | SK-CO-1 | 7.09 | 3.11 | >10 | 0.011 |
| MSS | SW480 | 2.14 | 3.32 | >10 | 0.028 | |
| MSS | SW480_wt_clone | 1.83 | 4.09 | >10 | 0.018 | |
| MSS | SW480_WRN_KO_clone#1 | 1.73 | 2.64 | >10 | 0.016 | |
| MSS | SW480_WRN_KO_clone#2 | 1.38 | 3.73 | >10 | 0.022 | |
| MSI | HCT 116 | 8.87 | 4.99 | 4.40 | 0.012 | |
| MSI | RKO | >10 | 6.39 | >10 | 0.044 | |
| Endometrial carcinoma | MSS | MFE-280 | 4.54 | 3.38 | 5.71 | 0.009 |
| MSI | HEC-265 | 7.74 | 6.80 | >10 | 0.021 | |
| MSI | ISHIKAWA | >10 | 5.55 | >10 | 0.016 | |
| Non-transformed | MSS | hTERT-RPE-1 | 4.34 | 5.54 | >10 | 0.035 |
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[Editors' note: further revisions were requested prior to acceptance, as described below.]
Additional files
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Supplementary file 1
MSS/MSI-H status analysis of CRC, endometrial and gastric carcinoma cell lines.
MSS/MSI-H status was analyzed using fluorescent PCR-based analysis of the mononucleotide microsatellite markers NR-21, BAT-26, BAT-25, NR-24 and MONO-27. Main peak sizes for the mononucleotide microsatellite markers are shown for the MSS control cell line K562 and CRC, endometrial and gastric carcinoma cell lines. Cell models were classified as MSS (blue) or MSI-H (red) according to the indicated size range classification of MSS alleles.
- https://doi.org/10.7554/eLife.43333.016
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Supplementary file 2
Overview of cell lines used in this study.
Cell lines used in this study are listed with tumor type of origin, MSS/MSI-H status, vendor source, and STR confirmation status. Variable STR profiles are reported for ISHIKAWA cells, consistent with MSI-H status (Korch et al., 2012).
- https://doi.org/10.7554/eLife.43333.017
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Supplementary file 3
Sequences of sgRNAs used for CRISPR depletion studies.
Sequences of sgRNAs used for targeting WRN are listed in N- to C-terminal order according to the representation in Figure 3 and Expanded View Figure 3. Domains are annotated according to PFAM entry Q14191. RQC, RecQ helicase family DNA-binding domain; HRDC, Helicase and RNase D C-terminal, HTH, helix-turn-helix motif. Negative and positive control sgRNA sequences are also listed.
- https://doi.org/10.7554/eLife.43333.018
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Transparent reporting form
- https://doi.org/10.7554/eLife.43333.019
