The loss of p53-AS isoforms does not alter cellular stress responses or survival to spontaneous tumors.

(A) mRNAs for p53-α and p53-AS isoforms from thymocytes of irradiated mice were quantified by RT-qPCR, and p53-α levels in p53+/+ mice were assigned a value of 1. Means ± SEM (n=3). (B) Protein extracts from thymocytes of irradiated mice were immunoblotted with p53 or actin antibodies. After normalisation to actin, full-length (FL) p53-α levels in p53+/+ thymocytes were assigned a value of 1. (C) mRNA levels of p53 target genes in thymocytes, before or after ψ-irradiation. Means ± SEM (n=3). (D) Thymocyte apoptotic response to ψ-irradiation. Means ± SEM (n=6). (E) Cell cycle control in mouse embryonic fibroblasts (MEFs) after ψ-irradiation. Asynchronous MEFs were exposed to 0-10 Gy ψ-irradiation, and after 24 hr, cells were labelled with BrdU for 1 hr and analyzed by FACS. Means ± SEM from >3 independent experiments with at least 2 independent MEF clones per genotype. (F) mRNA levels of p53 target genes in MEFs untreated or treated with 0.5 μg/ml of clastogenic Doxorubicin (Doxo) or 10 μM of the Mdm2 antagonist Nutlin. Means ± SEM from > 3 experiments with ≥ 2 independent MEF clones. (G) MEFs proliferation under hyperoxic conditions. Cells were grown according to a 3T3 protocol. Each point is the mean from 4 independent MEF clones, the value for each clone resulting from triplicates. (H) Growth of tumor xenografts. E1A+Ras-expressing MEFs were injected into the flanks of nude mice and tumor volumes were determined after 1-25 days. Means ± SD (n=4 per timepoint and genotype). (I) Tumor-free survival of p53+/+ and p53ΔAS/ΔAS mice (n=cohort size). (J) Incidence of the indicated tumor types, determined at death after macroscopic examination and histological analysis. In A, D, E, ns=non-significant in Student’s t-test.

Male-specific acceleration of Myc-induced B-cell lymphomagenesis in mice lacking p53-AS isoforms.

(A) Tumor-free survival of p53+/+ Eμ-Myc and p53ΔAS/ΔAS Eμ-Myc mice, classified according to sex. (n=cohort size). (B) Tumor volumes upon dissection of p53+/+ Eμ-Myc and p53ΔAS/ΔAS Eμ-Myc mice, classified according to sex. Means ± SEM from 100 lymph nodes from p53+/+ Eμ-Myc males, 96 from p53+/+ Eμ-Myc females, 148 from p53ΔAS/ΔAS Eμ-Myc males and 124 from p53ΔAS/ΔAS Eμ-Myc females. (C) Myc mRNA and protein levels in lymph node tumors. (D) Levels of p53-α and p53-AS transcripts and p53 protein levels in lymph node tumors. (E) Transcript levels of the indicated p53 target genes. Means ± SEM (n=6 per genotype). (F-G) Apoptosis (F) and cell proliferation (G) in tumor lymph nodes from Eμ-Myc males were determined by immunohistochemistry with antibodies against cleaved caspase-3 and ki67, respectively. Positive cells were counted and normalized to the analyzed areas. Means ± SEM (n=6 mice per assay and genotype). In F, G scale bars=50 μm. Statistical analyses with Mantel-Cox (A) and Student’s t (B-G) tests. ***P<0.001, *P<0.05, ns: non-significant.

The loss of p53-AS isoforms affects Ackr4 expression in Eμ-Myc male mice.

(A) B-cell subpopulations in spleens of 6 weeks-old p53+/+ Eμ-Myc and p53ΔAS/ΔAS Eμ-Myc mice. Means ± SEM (n=6 per genotype). (B-C) RNAseq analysis of spleens from p53+/+ Eμ-Myc (n=3) and p53ΔAS/ΔAS Eμ-Myc (n=4) 4-6 weeks-old male mice. Volcano plot (B), with differentially expressed genes (DEGs) in red. Unsupervised clustering heat-map plot (C), with DEGs ranked according to mean fold changes, and protein-coding genes in bold. (D) RT-qPCR analysis of candidate DEGs from spleens of p53+/+ Eμ-Myc males and p53ΔAS/ΔAS Eμ-Myc males. Means ± SEM (n=3-4 per genotype). (E) RT-qPCR analysis of indicated DEGs from spleens of 4-6 weeks-old p53+/+ Eμ-Myc males, p53ΔAS/ΔAS Eμ-Myc males, p53+/+ Eμ-Myc females and p53ΔAS/ΔAS Eμ-Myc females. Means ± SEM (n=3-4 per sex and genotype). (F) Ackr4 is transactivated by p53 in response to stress. Ackr4 mRNAs in untreated or doxorubicin-treated WT and p53-/- MEFs. Data from 2-3 MEFs per genotype (Younger et al., 2015). (G) A putative p53 response element in Ackr4 intron 1. Top: Map of the Ackr4 gene. (boxes: exons (brown box: translated region), black line: intron 1); middle: p53 ChIP in doxorubicin-treated MEFs according to ChIP-Atlas (SRX270554) (Oki et al., 2018); bottom: p53 Response Element (p53RE) consensus sequence (R=G or A, W=A or T, Y=C or T), the putative p53RE and its mutated counterpart. (H) Luciferase assays of the candidate p53RE. A 1.5 kb fragment containing the WT or mutant p53RE was cloned upstream a luciferase reporter, then transfected into p53-/- MEFs together with an expression plasmid for full length p53 (FL), p53-AS or the DNA-binding mutant p53R270H (RH). Means ± SEM (n=4-6). (I) In MEFs, p53 activation leads to an increased Ackr4 expression attenuated by estradiol. Ackr4 and Cdkn1a mRNAs were quantified by RT-qPCR from p53+/+ and p53ΔAS/ΔAS MEFs, untreated or treated with 10 μM Nutlin and/or 5 μg/ml 17-β estradiol (E2). Means ± SEM from 4 independent experiments. In A, D, E, F, H, I ***P<0.001, **P<0.01, *P<0.05, °P≤0.057, ns: non-significant in Student’s t or Mann-Whitney tests.

ACKR4 is a male-specific prognostic factor in Burkitt lymphoma.

(A) In human cells, p53 activation leads to an increased ACKR4 expression abrogated by estradiol. ACKR4 and CDKN1A mRNAs were quantified by RT-qPCR from p53-proficient (MRC5) and p53-deficient (MRC5-SV40) human fibroblasts, untreated or treated with Nutlin and/or estradiol (E2). Means ± SEM from 4 independent experiments. (B-C) Analysis of lymphoma dataset #GSE4475. ACKR4 gene expression was plotted for all lymphoma patients with clinical follow-up (91 men [M], 68 women [W]), classified according to sex (B, left). Gene expression (B, right) or survival curves (C) were plotted for the 30% patients (27 men, 20 women) with the highest or lowest ACKR4 expression, classified according to sex. (D-E) Analysis of Burkitt lymphoma-specific dataset #phs00235. ACKR4 gene expression was plotted for all patients with a Burkitt lymphoma diagnosed at age 0-17 (48 males, 29 females), classified according to sex (D, left). Gene expression (D, right) or survival curves (E) were plotted for the 30% patients (15 men, 9 women) with the highest or lowest ACKR4 expression, classified according to sex. Statistical analyses by Student’s t or Mann-Whitney tests (A, B, D) and Mantel-Cox (C, E) test. ***P<0.001, **P<0.01, * P<0.01, °P=0.054, ns: non-significant.

Description of the p53ΔAS mouse model and analysis of p53ΔAS/ΔAS thymocytes and fibroblasts.

(A) Relative expression of p53 isoforms with an α or AS C-terminus in tissues of wild-type mice. RNAs were prepared from the indicated tissues of wild-type mice, then p53-AS and p53-α mRNAs were quantified by RT-qPCR. Results are expressed as mean AS/a ratios ± SD, from 3 independent experiments. (B) Comparative maps of the wild-type (WT) and ΔAS Trp53 alleles. Left, top: the 3’ end of the WT Trp53 gene is shown – black line: intron 10; boxes are for exons, with greytones for translated regions from exon 10 (light gray), exon 11 (dark gray), and exon AS (black). Left, below: the WT Trp53 allele encodes proteins with two different C-termini. (4D: tetramerization domain, CTD: C-terminal domain). Right, top: the 3’ end of the ΔAS Trp53 allele is shown; the AS exon was deleted and replaced by a SpeI restriction site. Right, below: the ΔAS Trp53 allele only enables the synthesis of proteins with the ‘canonical’ α C-terminus. (C) Quantifications of p53 isoforms with an AS or an α C-terminus, in mouse embryonic fibroblasts (MEFs) of the indicated genotypes, that were either untreated, or treated for 24hr with 0.5 μg/ml doxorubicin (Doxo) or 10 μM Nutlin. Means ± SEM from >3 experiments with ≥ 2 independent MEF clones of each genotype are shown. ***P<0.001, *P<0.05, °P=0.07 by Student’s t-test. (D) Apoptotic responses of p53+/+ and p53ΔAS/ΔAS thymocytes. p53+/+ and p53ΔAS/ΔAS mice were irradiated and their thymocytes were recovered and analyzed by FACS after annexin V-FITC staining. A typical experiment for cells of each genotype and condition is shown. Numbers indicate % cells, apopt.: apoptotic cells. (E) Cell cycle control of p53-/-, p53+/+ and p53ΔAS/ΔAS fibroblasts. Asynchronous MEFs were exposed to 0-10 Gy ψ-irradiation, then after 24 hr cells were labelled with BrdU for 1 hr and analyzed by FACS. A typical experiment for cells of each genotype and condition is shown, with % of cells in G1, S and G2/M mentioned in each panel. (F) mRNA levels of the indicated genes were quantified in MEFs of the indicated genotypes left untreated or treated for 24 hr with 0.5 μg/ml Doxorubicin (Doxo), 10 μM Nutlin-3 (Nutlin). Means ± SEM from at least 3 experiments with 2 independent MEF clones of p53+/+ and p53ΔAS/ΔAS genotypes are shown. (G) mRNA levels of the indicated genes were quantified in MEFs of the indicated genotypes left untreated or treated for 24 hr with 15 μM etoposide (Eto). Means ± SEM from at least 3 experiments with 2 independent MEF clones per genotype.

Analysis of tumors from p53+/+ Eμ-myc and p53ΔAS/ΔAS Eμ-Myc mice.

(A) Histological analyses of Eμ-Myc-induced tumor lymph nodes. Tumor lymph nodes were analyzed by Hematoxilin-Eosin staining (H & E), or antibodies against B220 (a B-cell specific marker) or CD3 (a T-cell specific marker). p53+/+ Eμ-Myc and p53ΔAS/ΔAS Eμ-Myc mice developed similar B-cell lymphomas, characterized by massive tissue homogenization of the lymph node by B-cells. Scale bars=50 μM. (B) Tumor-free survival of p53+/+ Eμ-myc and p53ΔAS/ΔAS Eμ-Myc mice are similar when sexes are not considered (n=cohort size). (C) Increased tumor-free survival of p53+/+ Eμ-myc males compared to p53ΔAS/ΔAS Eμ-Myc males, p53+/+ Eμ-Myc females and p53ΔAS/ΔAS Eμ-Myc females (n=cohort size). Statistical analysis with Mantel-Cox test. (D) Trp53 DNA sequencing of p53+/+ Eμ-myc and p53ΔAS/ΔAS Eμ-Myc tumors. The portion of the Trp53 gene encoding the DNA Binding domain (DBD), most frequently mutated in cancers, was sequenced for 14 tumors from p53+/+ (WT) Eμ-myc males and 18 tumors from p53ΔAS/ΔAS (ΔAS) Eμ-myc males, and Trp53 mutations were found in 3/14 and 1/18 tumors, respectively. (E) Details on the 4 p53 mutations identified in (D).

Analysis of pre-tumoral spleens.

(A) Cell sorting of B-cell subpopulations by FACS. Splenocytes were incubated with DAPI and the following antibodies: APC rat anti-mouse CD45R/B220, FITC rat anti-mouse CD43, PE rat anti-mouse IgM and BV605 rat anti-mouse IgD. First, the B220+CD43-cells were selected from DAPI negative living cells, subsequently yielding 4 different B subpopulations based on IgM and IgD labeling: IgM-/IgD-pre-B cells (box 1), IgM low/IgD-immature B cells (box 2), IgM high/IgD-transitional B cells (box 3) and IgM+/IgD+ mature B cells (box 4). Typical results with a p53+/+ Eμ-Myc male mouse and a p53ΔAS/ΔAS Eμ-Myc male mouse are shown. (B) The loss of p53-AS isoforms does not impair B-cell differentiation in 6 weeks-old non-transgenic male mice. Means ± SEM from 4 mice per genotype. ns: non-significant by Student’s t-test. (C) Control for expression of p53-FL, p53-AS and p53R270H in luciferase assays. As a control to luciferase experiments reported in Fig. 3H, protein extracts from p53-/- MEFs transfected with expression plasmids for p53-FL, p53-AS or p53R270H were immunoblotted with antibodies against p53, p21 and actin. Quantifications are relative to actin. p53-FL and p53-AS were expressed at similar levels and both transactivated p21, whereas the DNA-binding mutant p53R270H was expressed at higher amounts but failed to transactivate p21, as expected. nd: not determined. (D) Evidence for Myc binding at the Mt2 promoter in B-cells. ChIP-Atlas reports Myc binding to Mt2 promoter sequences in primary B-cells from the lymph nodes of Eμ-Myc mice (SRX353785, SRX353783) and in Eμ-Myc-induced lymphoma cells (SRX522383). Chr: chromosome.

Further analyses of human B-cell lymphoma datasets.

(A) MT2A gene expression is not a prognostic marker in human B-cell lymphomas. Survival curves for the 30% patients with the highest MT2A mRNA levels and the 30% patients with the lowest MT2A mRNA levels according to sex (n=cohort sizes), for patients from dataset #GSE4475. Statistical analyses by Mantel-Cox tests. (B) ACKR4 is a prognostic factor in Burkitt lymphomas but not in diffuse large B-cell lymphomas. Survival curves of patients from dataset #GSE181063, for the 30% patients with the highest ACKR4 mRNA levels and the 30% patients with the lowest ACKR4 mRNA levels, classified according to sex (n=cohort sizes), and diagnosed with either a diffuse large B-cell (top) or a Burkitt (bottom) lymphoma. Statistical analyses by Mantel-Cox tests.

Expression of Ackr4, Cdkn1a and Mdm2 in p53+/+ Eμ-Myc and p53ΔAS/ΔAS Eμ-Myc male splenocytes.

Read numbers for the indicated genes, obtained by Bulk RNA-seq from the spleens of three p53+/+ Eμ-Myc (WT_Myc) and four p53ΔAS/ΔAS Eμ-Myc (ΔAS_Myc) male mice.

Primer Sequences.