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. 2020 Sep 29;9:e57761. doi: 10.7554/eLife.57761

Figure 3. YAP1 and WWTR1 of the Hippo pathway are selectively essential in a distinct subset of OSCC cell lines.

(A) Essentiality profile (depicted with CRISPR scores heatmap) of YAP1, WWTR1, PIK3CA, TP63 and SOX2 across 21 OSCC cell lines derived from the CRISPR/Cas9 screen. Dependency on these genes were depicted as grey box in the bottom panel, according to the MAGeCK definition of significant depletion at FDR ≤ 0.05. No cell lines were dependent on TP63 or SOX2. The degree of essentiality differs across the lines. Some subsets of the cell lines are only dependent on either YAP1 or WWTR1, while neither gene appears to be essential in another subset of cell lines. PIK3CA, TP63 and SOX2 are genes implicated in HNSCC carcinogenesis that are often co-amplified with WWTR1, located on chromosome 3q25-28. All WWTR1-dependent cell lines had copy number amplification on these genes while all PIK3CA mutated cell lines are not dependent on either YAP1 or WWTR1. (B) Western blot images showing the protein level of YAP1 and WWTR1 on day 4 upon transducing the Cas-9 expressing cell lines with lentivirus carrying gene-specific sgRNA. Two sgRNAs were used per target gene. (C) Co-competition assay was used to validate the essentiality of YAP1 and WWTR1 on the selected cell lines. The growth of the BFP-positive transduced population was compared to the non-transduced population throughout the 18 days assay. The percentage of BFP-positive cells obtained at different time points were normalized to the day 4 readings for respective sgRNA (except ORL-204 which had time points normalized to the day 6 readings for respective sgRNA). PLK1 is a core essential gene included as a positive control. Negative controls include CHAT which is a non-essential gene across the panel of cell lines, and NT serves as a non-targeting control. Data are shown as mean ± SD (n = 2 biological repeats). (D) qPCR results show suppression of downstream targets of YAP1 and WWTR1 only when the respective fitness gene is being knocked-out. Down-regulation of CTGF and CYR61 gene expression was observed when YAP1 is knocked-out in the YAP1-dependent cell lines (ORL-48 and ORL-204). In the WWTR1-dependent cell lines (ORL-214, PE/CA-PJ15), CTGF and CYR61 expression is only suppressed when WWTR1 is knocked-out. Data are shown as mean ± SD (n = 2 independent experiments with technical triplicates).

Figure 3—source data 1. All raw data related to Figure 3 and its figure supplements on analysis result of YAP1 and WWTR1 as fitness genes for OSCC.

Figure 3.

Figure 3—figure supplement 1. Fitness genes and copy number amplification.

Figure 3—figure supplement 1.

(A) Barcharts showing proportion of screened genes identified by MAGeCK as fitness genes, before and after CRISPRcleanR correction, for those that mapped onto chromosomal region with frequent copy number amplification. (B) Distribution of MAGeCK fitness genes across all chromosomes. As expected, Y chromosome have the least percentage of genes being essential. For the rest of the chromosomes, proportion of MAGeCK fitness genes among screened genes ranged from 12 to 19%. Chromosome with frequent copy number amplification did not show significantly higher number of fitness genes.
Figure 3—figure supplement 2. YAP1 and WWTR1 from the Hippo signaling pathway are fitness genes for OSCC.

Figure 3—figure supplement 2.

(A) KEGG pathway analysis of essential genes found on copy number amplified chromosome shows enrichment of pro-tumorigenic pathway including the Hippo signaling pathway. (B) The Hippo signaling pathway and annotation of genes that are OSCC fitness genes. (C) Oncoprint downloaded from cBioportal showing the copy number alterations of YAP1, WWTR1, PIK3CA, TP63, and SOX2 in the HNSCC samples (n = 295) (TCGA1). Co-amplification of WWTR1 with PIK3CA can be seen in majority of the cases, whereby 40/59 (68%) tumors with PIK3CA amplification also have WWTR1 amplification. (D) Boxplots of CRISPR scores for the five genes, classified into two groups with (n = 11) or without (n = 10) copy number amplification of genes on chromosome 3q25-28. OSCC with copy number amplification showed significantly more negative CRISPR score for WWTR1, i.e. more dependent on WWTR1. This correlation was not found for PIK3CA. TP63 and SOX2 were not essential in any of the 21 OSCC cell lines. Whiskers of boxplot show minimum and maximum values, lines representing median. Unpaired two-tailed t-test was used for calculating statistical difference between two groups. (E) Baseline mRNA expressions of YAP1 and WWTR1 in selected OSCC cell lines were quantified by qPCR. YAP1-dependent ORL-48 and ORL-204 showed upregulation of YAP1 expression. While in other OSCC cell lines, the extend of WWTR1 overexpression is higher than YAP1. OKF6/htert1, an immortalized normal oral epithelial cell line is used as reference control for calculation of relative fold difference of gene expression. Data are shown as mean ± SD (n = 3 technical repeats). (F) Baseline protein expression of YAP1 and WWTR1 protein in selected OSCC cell lines. Actin was probed as a loading control.
Figure 3—figure supplement 3. Correlation of gene essentiality and gene expression for YAP1/WWTR1.

Figure 3—figure supplement 3.

(A) Plot of differential essentiality between YAP1 and WWTR1 reveal correlation of essentiality with gene expression. Gene expression and CRISPR score of YAP1 and WWTR1 from Project Score on 273 cancer cell lines were extracted. Those lines that are more dependent on YAP1 over WWTR1 (negative differential CRISPR score) are mostly with high YAP1 (blue) and low WWTR1 (red) expression; On the other hand, cell lines that are more dependent on WWTR1 are among those with overexpression of WWTR1 (red). (B) Significant negative correlation between WWTR1 gene expression and gene essentiality was evident in the 273 cancer cell lines screened in Project Score. Those cell lines that are dependent on WWTR1 (negative CRISPR score) tend to have higher WWTR1 expression. (C) WWTR1 gene expression and gene essentiality also showed non-significant negative correlation among the 21 OSCC lines screened. (D) Other cancer types showing significant negative correlation between WWTR1 gene expression and gene essentiality include non-small cell lung carcinoma, squamous cell lung carcinoma, glioblastoma, breast carcinoma, and lung adenocarcinoma (data from Project Score).
Figure 3—figure supplement 4. Clonogenic assay of representative lines and validation of YAP1 dependency in SAS.

Figure 3—figure supplement 4.

(A) Clonogenic assay showing inhibitory effect upon knocking out respective fitness genes in YAP1- and WWTR1-dependent lines. In BICR10 and HSC-2 knocking out of either YAP1 or WWTR1 with sgRNAs did not have any impact on clonogenicity. Experiment is performed in technical triplicate and repeated twice. Representative image of one technical repeat is shown. (B) sgRNA level log fold change from triplicate CRISPR screens of SAS. Depletion were seen only in sgRNAs targeting exon 1–4 of YAP1, which were present in YAP1-MAML2 fusion protein, but not seen in sgRNA 6–6 which targets exon 6. Data are shown as mean ± SD (n = 3 biological repeats). (C) Western blot to check efficacy of target protein knockout using CRISPR/Cas9 on SAS. Y1K is one of the sgRNA from the KY library v1, targeting exon 4, while Y2B is an independent sgRNA targeting exon 1 of YAP1. (D) Co-competition assay results for SAS, validating its dependency on YAP1. Previous work by Picco et al., 20192 revealed oncogenic fusion of YAP1 with MAML2 in SAS, rendering the cells to be dependent on the fusion protein. Both our genome-wide screening data and validation data have also confirmed the preferential dependency of SAS on YAP1, over its paralog, WWTR1. (E) Clonogenicity assay confirmed preferential dependency of SAS on YAP1. (F) qPCR of downstream target genes showed relatively stronger suppression when the fitness gene, YAP1 is depleted with sgRNA Y1K or Y2B. Data are shown as mean ± SD (n = 2 independent experiments with technical triplicates).