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 γ-irradiation. Means ± SEM (n=3). (D) Thymocyte apoptotic response to γ-irradiation. Means ± SEM (n=6). (E) Cell cycle control in mouse embryonic fibroblasts (MEFs) after γ-irradiation. Asynchronous MEFs were exposed to 0-10 Gy γ-irradiation, and after 24h, cells were labelled with BrdU for 1h 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. (J) 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. (K) Gene set enrichment analysis (GSEA). GSEA, performed in p53+/+ Eμ-Myc and p53ΔAS/ΔAS Eμ-Myc male splenic cells, indicated an enrichment of hallmark Myc targets in p53ΔAS/ΔAS Eμ-Myc cells. 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. (F) The knockout of ACKR4 in Burkitt lymphoma Raji cells increases their CCL21-guided migration. Chemotaxis was assayed by using Boyden chambers with bare polycarbonate membranes as previously described (Calpe et al., 2011). Equal number of cells were deposited on the membrane of a transwell insert, then migration was determined by counting cells in the lower compartment, after 15h of culture with or without CCL21 added to the lower chamber. Statistical analyses by Student’s t or Mann-Whitney tests (A, B, D, F) and Mantel-Cox (C, E) test. ***P<0.001, **P<0.01, * P<0.01, °P=0.054, ns: non-significant.