FAM193A is a positive regulator of p53 activity

Abstract

Inactivation of the p53 tumor suppressor, either by mutations or through hyperactivation of repressors such as MDM2 and MDM4, is a hallmark of cancer. Although many inhibitors of the p53-MDM2/4 interaction have been developed, such as Nutlin, their therapeutic value is limited by highly heterogeneous cellular responses. We report here a multi-omics investigation of the cellular response to MDM2/4 inhibitors, leading to identification of FAM193A as a widespread regulator of p53 function. CRISPR screening identified FAM193A as necessary for the response to Nutlin. FAM193A expression correlates with Nutlin sensitivity across hundreds of cell lines. Furthermore, genetic codependency data highlight FAM193A as a component of the p53 pathway across diverse tumor types. Mechanistically, FAM193A interacts with MDM4, and FAM193A depletion stabilizes MDM4 and inhibits the p53 transcriptional program. Last, FAM193A expression is associated with better prognosis in multiple malignancies. Altogether, these results identify FAM193A as a positive regulator of p53.

Keywords: CP: Cancer; CP: Molecular biology; CRISPR; MDM2; MDM4; MDMX; Nutlin; apoptosis; cell-cycle arrest; p53.

Figure 1.. Sensitivity to MDM2 inhibitors is associated with high basal activity in the p53 network

(A) Distribution of Nutlin IC₅₀ values (micromolar, natural logarithm) of TP53 WT and TP53 mutant cell lines. Drug sensitivity data were obtained from the GDSC database. The number of biological replicates for the TP53 WT and MUT cell lines was 179 and 313, respectively; IC₅₀ values were calculated based on data obtained from 2 technical replicates as described previously. The notches indicate medians, and whiskers extend to the largest/smallest value but no further than the 1.5× IQR (interquartile range) from the hinge. Statistical significance was calculated with unpaired two-sample Wilcoxon test. (B) Ranking of TP53 WT cell lines based on their sensitivity to Nutlin, with demarcation of cells carrying MDM2 gene amplification. Also annotated on the rank plot are cell lines used in this study. The number of biological replicates for cell lines with amplified and without amplified MDM2 was 11 and 168, respectively; IC₅₀ values were calculated based on data obtained from 2 technical replicates as described previously. (C) Distribution of p values (negative log₁₀) and Spearman correlation coefficients (rho) calculated between Nutlin IC₅₀ and gene expression (basal RNA levels, obtained from the GDSC database) in TP53 WT cell lines. Nutlin IC₅₀ data were obtained based on biological and technical replicates for TP53 WT cell lines as described in (A). Gene expression profiling was performed for each cell line in 1 replicate. The top 10 genes with highest negative correlations and smallest p values are highlighted in red. (D) Scatter plots and linear regression curves between Nutlin IC₅₀ (micromolar, natural logarithm) values and RNA levels for the 4 most negatively correlated genes highlighted in (C). Statistical analysis was performed as indicated in (C), and p values were adjusted with the Benjamini-Hochberg procedure (q values). Biological and technical replicates for IC₅₀ values and expression data are as described in (C). (E) Upstream transcriptional regulators predicted by the Upstream Regulator tool of the Ingenuity Pathway Analysis (IPA) software of the top 100, 250, 500, and 1,000 genes with the highest negative correlations shown in (C). (F) Gene set enrichment analysis (GSEA) comparison between correlations shown in (C) and (l)core genes induced by Nutlin or (2) a gene signature predictive of sensitivity to the MDM2 inhibitor NVP-CGM09 or (3) a gene signature predictive of sensitivity to the MDM2 inhibitor DS-3032b. See also Figure S1.

Figure 2.. A genetic screen for positive regulators of p53 function

(A) Western blot analysis of components of the p53 network and apoptosis markers in A549, MCF7, HCT116, SJSA, and CHP212cellstreated for48 h with 10 μM Nutlin. (B) Apoptosis induction measured by Annexin V binding in cells treated with Nutlin as in (A). The graph represents the average of 3 biological replicates ± SEM. Statistical analysis was performed with ANOVA with post hoc Tukey test. (C) Scatterplot of mean counts per million (cpm) and fold changes (FCs) of sgRNAs detected by next-generation sequencing following 2 rounds of Nutlin or DMSO treatment (2 biological replicates each). A dashed line indicates cutoff values of the screen analysis pipeline. Red, blue, green, and cyan dots indicate sgRNAs targeting genes that passed the final criterium of a minimum of 2 sgRNAs enrichment per gene. (D) Volcano plot showing p values (log₁₀) and Spearman’s correlation coefficients calculated for Nutlin IC₅₀ values and gene expression levels (as presented and with statistics calculated as in Figure 1C). Genes targeted by sgRNAs enriched in the CRISPR screen are marked in red. (E) Heatmap of correlation coefficients of the top-10 screen hits listed on the plot shown in (D). (F) Volcano plot showing p values (log₁₀) and Spearman’s correlation coefficients for TP53 codependencies calculated with data obtained from the DepMap database (261 TP53 WT cell lines with 4 technical replicates each were used to calculate gene effect scores). Top positive and negative TP53 codependent genes are labeled. Additionally, CRISPR screen hits are indicated in red, and the top 10 TP53 codependent CRISPR hits are highlighted. (G) Heatmap of TP53 codependency rho values and gene ranks of top-10 screen hits listed on the plot shown in (F). (H) Scatterplot for correlation coefficients of gene expression levels and Nutlin IC₅₀ values (on the x axis) vs. TP53 gene codependencies (on the y axis). CRISPR screen hit genes are highlighted in red. (I) Volcano plot of FAM193A gene codependencies (rho vs. p values). TP53 and major p53 regulators and effectors found as top positive and negative FAM193A codependent genes are labeled on the plot (biological and technical replicates as described in F). (J) Scatterplots with linear regression lines of FAM193A gene effects plotted against TP53, TP53BP1, PPM1D, and MDM2 gene effects as calculated in (I) with p values adjusted with the Benjamini-Hochberg procedure (q values, biological and technical replicates as described in F). See also Figure S2.

Figure 3.. FAM193 is a positive regulator of p53 activity

(A) Boxplot of the percentages of cells in S phase of the cell cycle in control non-targeting (WT), FAM193A KO, and CDKN1A KO cells treated with the indicated doses of Nutlin for 24 h. The percentage of cells in S phase was measured through immunofluorescence detection of incorporated bromodeoxyuridine (BrdU). The plot represents a summary of data from independent single cell clones (biological replicates represented by individual single cell clones are 4, 5, and 4 per group for the WT, FAM193A KO, and CDKN1A KO, respectively; each sample was obtained from 2 technical replicates). The horizontal line represents the median, and whiskers extend to the largest/smallest value but no further than the 1.5× IQR from the median. Statistical analysis was performed with ANOVA with post hoc Tukey test. (B) Plot of the percentage of cells in G1 phase in samples as described in (A). Quantification of cells in G1 was based on detection of DNA content (stained with propidium iodide [PI]) and immunofluorescence detection of BrdU. (A and B) *p < 0.05, **p < 0.01, ***p < 0.001. (C) Western blot analysis of p53, p53 target genes, and MDM4 in nontargeting control (WT) and FAM193A KO CHP212 cells treated with vehicle or0.625,1.25, or 2.5 μM Nutlin for 24 h. (D) Volcano plots of gene expression changes (FCs [log₂FC; Nutlin/DMSO] vs. adjusted p value [q]) induced upon 12 h of 10 μM Nutlin treatment. Genes that did not pass gating for subsequent analysis (with |log₂FC| < 1.5 and/or q > 0.05) are marked in gray. (E) Rank plot of mRNA FCs of DEGs in FAM193A WT cells (black dots). Corresponding FCs in FAM193A KO cells are marked in red (for upregulated genes) and blue (for downregulated genes). (F) Scatterplot of expression FCs in WT and FAM193A KO cells of core p53 target genes. Regions of the plane above and below the gray dashed lines include data points with differences in FCs between WT and FAM193A KO that are greater than 1. (G) Heatmap of relative expression levels of top 20 Nutlin-induced p53 core target genes. (D–G) RNA-seq was performed on 2 biological replicates per condition. See also Figure S3.

Figure 4.. FAM193A interacts with the RING domain of MDM4

(A) Western blot analysis post Immunoprecipitation of 3×FLAG-FAM193A In CHP212 cells showing co-immunoprecipitation of MDM4 along with FAM193A. (B) Western blot analysis of samples immunoprecipitated with an MDM4 antibody showing co-immunoprecipitation of 3×FLAG-FAM193A along with MDM4 in CHP212 cells. (C) Western blot analysis of Halo-FAM193A pulldown in transfected HCT116 cells in Nutlin-treated and control cells reveals formation of a ternary complex between FAM193A, MDM4, and MDM2. (D and E) Western blot analysis of MDM4 immunoprecipitation or (E) p53 and MDM2 immunoprecipitation in non-targeting control (WT) and FAM193A KO (KO) CHP212 cells treated with Nutlin. (F) Schematics of the MDM4c3 construct. The MDM4 open reading frame was modified to include insertions of 3 PreScission (PreS) protease cleavage sites. Additionally, 3 of the4 fragments were tagged with FLAG, myc, or HA tags. Digestion of MDM4c3 with PreS leads to generation of4protein fragments containing (1)the p53-binding domain recognized bythe MDM4–82 monoclonal antibody, (2) the acidic and zinc-finger(ZF) RanBP2-type domains labeled with a FLAG tag, (3) the domain containing the SQ motif labeled with a myc tag, and (4) the RING domain labeled with an HA tag. (G) Western blot analysis following pull-down of Halo-tagged FAM193A in transfected HEK293FT cells along with full-length MDM4c3 or with PreS-cleaved MDM4c3 fragments reveals interaction between FAM193A and the MDM4 RING domain. (H) Secondary structures of FAM193A protein predicted by MobiDB with demarcated FAM193A fragments assayed for MDM4 and MDM2 interaction. (I) Co-pulldown of Halo-tagged FAM193A fragments (as indicated in H) with MDM4 (ectopically expressed) or MDM2 (expression induced with 10 μM Nutlin for16 h) in HCT116 cells. (J) Proposed model of FAM193A function in supporting the p53 transcriptional program. See also Figure S4.

Figure 5.. High FAM193A expression is associated with better prognosis in multiple cancer types

(A) FAM193A normalized gene expression levels(FPKM) in tumor samples and corresponding normal tissues (from the GTEx and TCGA databases). Cancer types marked in orange or teal are tumor-normal tissue pairs with significant differences in FAM193A expression. The horizontal lines represent the median, and whiskers extend to the largest/smallest value but no farther than the 1.5× IQR from the median. Statistical analysis was performed with Wilcoxon rank-sum test with Benjamini-Hochberg correction. The number of normal and tumor tissues profiled are as follows (cancer type: normal/tumor): BLCA: 28/362, BRCA: 199/982, CESC: 13/259, CHOL: 9/31,COAD: 235/285, ESCA: 670/183, HNSC: 97/460, KICH: 25/60, KIRC: 104/475, KIRP: 29/236, LIHC: 163/295, LUAD: 372/503, LUSC: 154/426, READ: 153/87, STAD: 225/380, THCA: 371/441, UCEC: 105/141. (B) Heatmap of FCs of mean FAM193A mRNA expression between normal and cancer tissues depicted in (A). Statistical significance is denoted by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001). (C) Distribution of FAM193A alterations (mutations, deletions, and/or amplifications) by cancer type in TP53 WT tumors (data source: TCGA Pan-Cancer Atlas Studies). (D) Plot of overall survival (OS) ratios of patients with high FAM193A-expressing tumors vs. low FAM193A-expressing tumors (all tumors are TP53 WT) and log rank test p values adjusted with the Benjamini-Hochberg procedure (q values). (E) Kaplan-Meier plots of OS of patients with TP53 WT tumors with high or low expression of FAM193A in cancer types that surpassed the q value cutoff marked in (D). The numbers of FAM193A high- and low-expressing tumors are as follows(cancer type: normal/tumor): BLCA: 129/70, HNSC: 35/118, UCEC: 273/44, LUSC: 49/30, PAAD: 7/61,THCA: 306/176. See also Figure S5.