Although clonal neo-antigen burden is associated with improved response to immune therapy, the functional basis for this remains unclear.,Here we study this question in a novel controlled mouse melanoma model that enables us to explore the effects of intra-tumor heterogeneity (ITH) on tumor aggressiveness and immunity independent of tumor mutational burden.,Induction of UVB-derived mutations yields highly aggressive tumors with decreased anti-tumor activity.,However, single-cell-derived tumors with reduced ITH are swiftly rejected.,Their rejection is accompanied by increased T cell reactivity and a less suppressive microenvironment.,Using phylogenetic analyses and mixing experiments of single-cell clones, we dissect two characteristics of ITH: the number of clones forming the tumor and their clonal diversity.,Our analysis of melanoma patient tumor data recapitulates our results in terms of overall survival and response to immune checkpoint therapy.,These findings highlight the importance of clonal mutations in robust immune surveillance and the need to quantify patient ITH to determine the response to checkpoint blockade.,•Novel mouse system to uncouple tumor mutational load and tumor heterogeneity•Lower tumor heterogeneity leads to decreased tumor growth because of immune rejection•Both clone numbers and their genetic diversity mediate tumor growth and rejection•Tumor heterogeneity is linked to patient survival and checkpoint blockade response,Novel mouse system to uncouple tumor mutational load and tumor heterogeneity,Lower tumor heterogeneity leads to decreased tumor growth because of immune rejection,Both clone numbers and their genetic diversity mediate tumor growth and rejection,Tumor heterogeneity is linked to patient survival and checkpoint blockade response,Syngeneic mouse models of melanoma and human patient data are used to explore the effect of intra-tumor heterogeneity on immune response and demonstrate that heterogeneity predicts immunotherapy outcomes better than mutational burden.
Metastatic melanoma represents a major clinical problem.,Its incidence continues to rise in western countries and there are currently no curative treatments.,While mutation of the P53 tumour suppressor gene is a common feature of many types of cancer, mutational inactivation of P53 in melanoma is uncommon; however, its function often appears abnormal.,In this study whole genome bead arrays were used to examine the transcript expression of P53 target genes in extracts from 82 melanoma metastases and 6 melanoma cell lines, to provide a global assessment of aberrant P53 function.,The expression of these genes was also examined in extracts derived from diploid human melanocytes and fibroblasts.,The results indicated that P53 target transcripts involved in apoptosis were under-expressed in melanoma metastases and melanoma cell lines, while those involved in the cell cycle were over-expressed in melanoma cell lines.,There was little difference in the transcript expression of P53 target genes between cell lines with null/mutant P53 compared to those with wild-type P53, suggesting that altered expression in melanoma was not related to P53 status.,Similarly, down-regulation of P53 by short-hairpin RNA (shRNA) had limited effect on P53 target gene expression in melanoma cells, whereas there were a large number of P53 target genes whose mRNA expression was significantly altered by P53 inhibition in melanocytes.,Analysis of whole genome gene expression profiles indicated that the ability of P53 to regulate genes involved in the cell cycle was significantly reduced in melanoma cells.,Moreover, inhibition of P53 in melanocytes induced changes in gene expression profiles that were characteristic of melanoma cells and resulted in increased proliferation.,Conversely, knockdown of P53 in melanoma cells resulted in decreased proliferation.,These results indicate that P53 target genes involved in apoptosis and cell cycle regulation are aberrantly expressed in melanoma and that this aberrant functional activity of P53 may contribute to the proliferation of melanoma.