CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials
- Cold Spring Harbor Laboratory, United States
- Stony Brook University, United States
Figures
Figure 1 with 4 supplements
Mutation of the MELK kinase domain does not affect cancer cell proliferation or anchorage-independent growth.
(A) Domain structure of MELK and locations of the sequences targeted by 7 MELK gRNAs. (B) Genomic DNA was purified from the indicated population of MDA-MB-231 cells and the targeted loci were …
Figure 1—figure supplement 1
Mutation of MELK using seven different guide RNAs in the A375 melanoma cell line.
Following sorting of GFP+ populations, genomic DNA was purified from each cell line and the targeted loci were amplified by PCR. The amplified fragments were sequenced using the forward and reverse …
Figure 1—figure supplement 2
Mutation of MELK using seven different guide RNAs in the Cal51 triple-negative breast cancer cell line.
Following sorting of GFP+ populations, genomic DNA was purified from each cell line and the targeted loci were amplified by PCR. The amplified fragments were sequenced using the forward and reverse …
Figure 1—figure supplement 3
Mutation of MELK using seven different guide RNAs in the MDA-MB-231 triple-negative breast cancer cell line.
Following sorting of GFP+ populations, genomic DNA was purified from each cell line and the targeted loci were amplified by PCR. The amplified fragments were sequenced using the forward and reverse …
Figure 1—figure supplement 4
Western blot analysis of MELK-disrupted cell populations.
Following sorting of GFP+ populations, whole-cell lysate was collected and analyzed for MELK expression using the Abcam ab108529 antibody. Alpha-tubulin was analyzed as a loading control.
Figure 2 with 2 supplements
Guide RNAs targeting MELK fail to drop out in triple-negative breast cancer cell line competition experiments.
(A) The fold change in the percentage of GFP+ cells, relative to the percentage of GFP+ cells at passage 1, is displayed for seven triple-negative breast cancer cell lines. (B) A table summarizing …
Figure 2—figure supplement 1
Guide RNAs targeting MELK fail to drop out in several cancer cell lines.
(A) The fold change in the percentage of GFP+ cells, relative to the percentage of GFP+ cells at passage 1, is displayed for six additional cancer cell lines. (B) A table summarizing the results …
Figure 2—figure supplement 2
Unbiased screens do not identify MELK as a cancer dependency.
Gene essentiality data were examined from a kinome-wide siRNA screen (Campbell et al., 2016), a genome-wide shRNA screen (Marcotte et al., 2012; Hart et al., 2014), and two genome-wide CRISPR …
Figure 3 with 2 supplements
Mutating MELK does not affect OTS167 sensitivity.
(A) Summary of GI50 values from OTS167 treatment of A375 cells harboring guide RNAs targeting Rosa26 or MELK. (B) 7 point dose-response curves of OTS167 in the indicated A375 cell lines. (C) Summary …
Figure 3—figure supplement 1
Receptor-positive breast cancer cell lines are sensitive to OTS167.
(A) Summary of GI50 values from OTS167 treatment of various breast cancer cell lines. (B and C) 7 point dose-response curves of OTS167 in the indicated cell lines. Note that the summary values in (A)…
Figure 3—figure supplement 2
OTS167 treatment, but not MELK mutation, causes the accumulation of multinucleate cells.
(A–C) Cells were either untreated or treated with the indicated drug for 24 hr. Subsequently, cells were stained with Hoechst dye and at least 200 cells for each condition were examined. (D) …
Figure 4 with 1 supplement
MELK-knockout cell lines proliferate at normal rates and remain sensitive to OTS167.
(A) Schematic of exons in the MDA-MB-231 MELK-KO g1/g6 knockout line. Half-arrows indicate positions of either cut-site or deletion-spanning primers used to screen these colonies. Primer sequences …
Figure 4—figure supplement 1
Generation and analysis of Cal51 MELK-KO cell lines.
(A) Sanger sequencing of 3 independent Cal51 MELK-KO clones transduced with single gRNAs targeting the MELK kinase domain. The highlighted regions indicate bases recognized by the gRNA. (B) Western …
Additional files
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Supplementary file 1
Guide RNA sequences.
The sequences of every guide RNA and the protein domain targeted by the guide RNAs are displayed.
- https://doi.org/10.7554/eLife.24179.016
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Supplementary file 2
PCR primers to amplify MELK gRNA cut sites and deletions.
The sequences of PCR primers used in this manuscript are displayed.
- https://doi.org/10.7554/eLife.24179.017
