A dominant-negative effect drives selection of TP53 missense mutations in myeloid malignancies

Abstract

TP53, which encodes the tumor suppressor p53, is the most frequently mutated gene in human cancer. The selective pressures shaping its mutational spectrum, dominated by missense mutations, are enigmatic, and neomorphic gain-of-function (GOF) activities have been implicated. We used CRISPR-Cas9 to generate isogenic human leukemia cell lines of the most common TP53 missense mutations. Functional, DNA-binding, and transcriptional analyses revealed loss of function but no GOF effects. Comprehensive mutational scanning of p53 single-amino acid variants demonstrated that missense variants in the DNA-binding domain exert a dominant-negative effect (DNE). In mice, the DNE of p53 missense variants confers a selective advantage to hematopoietic cells on DNA damage. Analysis of clinical outcomes in patients with acute myeloid leukemia showed no evidence of GOF for TP53 missense mutations. Thus, a DNE is the primary unit of selection for TP53 missense mutations in myeloid malignancies.

Fig. 1.. TP53 hotspot missense and null mutations in isogenic AML cell lines show similar oncogenic phenotypes.

(A) Schematic of the experimental workflow for generating K562-TP53 and MOLM13-TP53 isogenic AML cell lines. CRISPR-HDR, CRISPR-Cas9-mediated homology-directed repair; CRISPR-KO, CRISPR-Cas9-mediated gene knock-out (B) MOLM13-TP53 isogenic AML cell lines were treated with 100nM daunorubicin for up to 72 hours. At the indicated time points, cells were stained with Annexin V and analyzed by flow cytometry to assess total apoptotic cells (replicates n=3, symbols represent averages of experimental replicates, error bars indicate s.e.m., *** p<0.001, **** p<0.0001, two-tailed Student’s t-test). (C) MOLM13-TP53 isogenic AML cell lines were treated with DMSO (−) or 100nM daunorubicin (+) for 6 hours, after which whole cell protein lysates were collected, run on a polyacrylamide gel, and immunoblotted for p53, p21 and actin (replicates n=3, representative images are shown). (D) Growth kinetics of MOLM13-TP53 isogenic AML cell lines (replicates n=3, symbols represent averages of experimental replicates, error bars indicate s.e.m.). (E) MOLM13-TP53 isogenic AML cell lines were treated with DMSO or indicated drugs at increasing concentrations for 72 hours, after which cell viability was assessed using CellTiter-Glo® luminescent assay, (replicates n=3, symbols represent averages of experimental replicates, error bars indicate s.e.m.).

Fig. 2.. Transcriptional consequences of TP53 hotspot missense mutations in isogenic AML cell lines.

(A) Genome-wide relative enrichment of wildtype and missense mutant p53 variants (ChIP for wild-type or missense mutant p53 over ChIP in p53^−/− cells) over transcriptional start site (TSS)-proximal regions (−10kb – first intron) in K562-TP53 isogenic cell lines upon treatment with DMSO or 100nM daunorubicin for 24 or 6 hours, respectively. (B) Heatmap depicting normalized expression of genes associated with WT-specific (left), shared (middle), and p53 mutant-specific (right) ChIP-seq peaks in K562-TP53 isogenic cell lines treated with 100nM daunorubicin for 24 hours (experimental replicates n=3). (C) Heatmap of the pooled top 30 (left) and pooled bottom 30 (right) genes relative to wild-type p53 in K562-TP53 isogenic cell lines treated with 100nM daunorubicin for 24 hours (RNA-seq experimental replicates n=3).

Fig. 3.. TP53 missense mutations within the DNA-binding domain confer dominant-negative effects.

(A) MOLM13-TP53 isogenic AML cell lines with p53^+/+, p53^+/−, p53^−/− as well as p53^R248Q/+ and p53^R248Q/− were treated with DMSO (−) or 100nM daunorubicin (+) for 6 hours, after which whole cell protein lysates were collected, run on a polyacrylamide gel, and immunoblotted for p53, p21 and actin (replicates n=3, representative images are shown). (B) MOLM13-TP53 isogenic AML cell lines were treated 100nM daunorubicin for up to 72 hours. At the indicated time points, cells were stained with Annexin V and analyzed by flow cytometry to assess total apoptotic cells (replicates n=3, symbols represent averages of experimental replicates, error bars indicate s.e.m., * p,0.05, **p,0.01, ***p<0.001, **** p<0.0001, two-tailed Student’s t-test). (C) MOLM13-TP53 isogenic AML cell lines were treated with DMSO, nutlin-3a or daunorubicin at increasing concentrations for 72 hours, after which cell viability was assessed using CellTiter-Glo® luminescent assay. (replicates n=3, symbols represent averages of experimental replicates, error bars indicate s.e.m.). (D) Heatmap depicting the TP53 saturation mutagenesis screen results after nutlin-3a treatment shown as log10 of the ratio of normalized read counts in GFP^lo over GFP^hi cells per TP53 variant (top panel) overlaid on a lollipop plot demonstrating TP53 mutational data from 1,040 patients with myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and AML (bottom panel). Missense mutations (green circles) and truncating mutations (black circles) comprising frame-shift, nonsense, and splice mutations are shown. TAD, transactivation domain; PRD, proline-rich domain; OD, oligomerization domain; CTD, C-terminal domain.

Fig. 4.. Heterozygous Trp53 missense mutations confer a competitive advantage over Trp53^+/− HSPCs upon sublethal gamma-irradiation.

(A) Schematic of the experimental workflow for hematopoietic competition assays in mixed chimeric mice to assess the relative competitive fitness of Trp53 genotypes upon sublethal gamma-irradiation. Trp53^+/+-CD45.1/2 recipient mice were engrafted with a 1:1 mixture of Trp53 genotypes from either CD45.1 or CD45.2 mice. Following hematopoietic reconstitution, mixed chimeric mice were sublethally gamma-irradiated (single dose of 2.5 Gy), and thereafter, peripheral blood (PB) chimerism was assessed by flow-cytometry every 4 weeks. (B-D) PB chimerism in mixed chimeric mice of the indicated genotypes in non-irradiated control mice (black squares) and mice treated with a single dose of 2.5 Gy gamma-irradiation (red squares). (experimental replicates n=2-3 per group, n=14-20 mice per group, symbols represent averages of individual mice across experimental replicates, error bars indicate s.e.m., * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, Mann-Whitney test) (E) Schematic summary of the results obtained from hematopoietic competition assays depicting the relative competitive fitness of the indicated Trp53 genotypes towards sublethal DNA damage. (F) Kaplan-Meier analysis for event-free and (G) overall survival in AML patients according to TP53 mutational status (missense mutations, blue line or truncating mutations comprising frame-shift, nonsense, and splice mutation, red line, log-rank (Mantel-Cox) test).