The vast majority of human tumors with p53 mutations undergo loss of the remaining wildtype p53 allele (loss-of-heterozygosity, p53LOH). p53LOH has watershed significance in promoting tumor progression. However, driving forces for p53LOH are poorly understood. Here we identify the repressive WTp53–HSF1 axis as one driver of p53LOH. We find that the WTp53 allele in AOM/DSS chemically-induced colorectal tumors (CRC) of p53^R248Q/+ mice retains partial activity and represses heat-shock factor 1 (HSF1), the master regulator of the proteotoxic stress response (HSR) that is ubiquitously activated in cancer. HSR is critical for stabilizing oncogenic proteins including mutp53. WTp53-retaining CRC tumors, tumor-derived organoids and human CRC cells all suppress the tumor-promoting HSF1 program. Mechanistically, retained WTp53 activates CDKN1A/p21, causing cell cycle inhibition and suppression of E2F target MLK3. MLK3 links cell cycle with the MAPK stress pathway to activate the HSR response. In p53^R248Q/+ tumors WTp53 activation by constitutive stress represses MLK3, thereby weakening the MAPK-HSF1 response necessary for tumor survival. This creates selection pressure for p53LOH which eliminates the repressive WTp53-MAPK-HSF1 axis and unleashes tumor-promoting HSF1 functions, inducing mutp53 stabilization enabling invasion.

Flow cytometry of T cells in tdLNs and the TME

Lymphatic PD-L1 regulates proliferating CD44+ CD62L+ PD-1+ CD8+ T cells within YUMMER1.7-draining LNs. A, Study design overview. PD-L1LECKO mice were treated with tamoxifen for five consecutive days before being inoculated intradermally with YUMMER1.7 melanoma cells (indicated by the arrow). On day 7, 8, and 9, mice received FTY720 intraperitoneally (IP) before being sacrificed on day 10. B and C, Count per LN or mg of tumor for CD3+ T cells, CD4+ T cells, CD8+ T cells, and Ki67+ CD62L+ CD44+ PD-1+ CD8+ T cells in the tdLNs (B) and primary tumors (C) of the YUMMER1.7 melanoma model under the indicated treatment. N = 3–5 mice/group. *, P < 0.05; **, P < 0.01; one-way ANOVA with a Šídák post test. D, Schematic representation of the proposed mode of action. In Cre-mice (top), PD-L1 expressed by LECs in tdLNs inhibits activation, proliferation, or LN egress of early activated CD8+ T cells, limiting their migration and accumulation within the TME. In Cre+ mice lacking lymphatic PD-L1 expression, early activated CD8+ T cell output of tdLNs is enhanced, resulting in increased infiltration of the TME. BV, blood vessel; LV, lymphatic vessel.

Lymphatic PD-L1 deletion reduces PD-1+ Th1 cells in the YUMMER1.7 primary tumor specifically. A, UMAP with unsupervised clustering of CD4+ T cells in the tdLNs and individual UMAP for each melanoma model and condition. B, Heatmap depicting mean marker expression across all clusters. C, Heatmap of the cluster frequencies of CD4+ T cells in the tdLNs under the indicated treatment. D, UMAP with unsupervised clustering of CD4+ T cells in the TME and individual UMAP for each melanoma model and condition. E, Heatmap depicting mean marker expression across all clusters. F, Heatmap of the cluster frequencies of CD4+ T cells in the TME under the indicated treatment. *, P < 0.05; Student t test. G, Frequency of PD-1+ CD44+ Foxp3− CD4+ T cells (cluster 7) in the TME under the indicated conditions determined by classic flow cytometry gating. N = 4–7 mice/group. *, P < 0.05; Student t test. APO, apotracker.

Lymphatic PD-L1 does not affect melanoma tumor growth or metastasis in tdLNs. A, Study design overview. PD-L1LECKO mice were treated with tamoxifen for five consecutive days before being inoculated intradermally (ID) with syngeneic melanoma cells (indicated by the arrow). Mice were sacrificed when the tumor volume reached 1 cm3. B, Frequency of LECs (CD45− podoplanin+ CD31+) and PD-L1+ LECs in the YUMMER1.7-, YUMM1.7- and YUMM5.2-draining LNs of PD-L1LECKO mice treated with tamoxifen. ****, P < 0.0001; two-way ANOVA with a Šídák post test. C, Tumor growth curve of YUMMER1.7, YUMM1.7, and YUMM5.2 in PD-L1LECKO mice treated with tamoxifen. Tumor growth was monitored by caliper measurements. N = 4–7 mice/group. D, Representative microscopy images of immunofluorescence staining for BRAFV600E (red), CD45 (green), and nuclei (blue) of LNs draining the indicated melanoma mouse models. Only the Cre- group is shown. Arrows indicate metastatic nodules. E, Quantification of metastatic areas in naïve and melanoma-draining LNs. N = 4–7 LNs/group. Dashed line represents background staining obtained using naïve LNs. Student t test.