Minimal Myc functional threshold for tumorigenesis

Division Of Basic Sciences - Nci

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Abstract

We designed a genetic test of the hypothesis that a minimum level of Myc function is required to "amplify" the transcriptional programs necessary for promoting and sustaining tumor formation. We employed the mouse p53 null mutant (p53 KO) as a robust tumor model to test for Myc dependency by introducing modest changes in the endogenous cMyc level (cMycHet). Tumor free survival times were compared in genetically similar p53 KO sibling mice that were either cMycWT (Myc WT; p53 KO), or cMycHet (Myc Het; p53 KO). Median tumor free survival times doubled in the MycHet; p53 KO relative to MycWT (significant at P 0.0001). This difference was independent of the tumor type, with hemangiosarcoma and thymic lymphoma being the most common (83%) tumor types in both groups. Analyses of cMyc genomic alterations and expression levels in p53 KO tumors, using Spectral Karyotyping (SKY), FISH, and quantitative RNA ISH and immunohistochemistry, revealed that compensation for the initially reduced endogenous cMyc dosage had occurred in tumors arising in MycHet;p53KO mice. Notably, in hemangiosarcomas, genome amplification achieved by several rounds of genome tetraploidization was consistently higher in the MycHet;p53 KO than in the MycWT;p53 KO tumors. Although thymic lymphomas of either genotype showed no genomic amplification, expression of Myc RNA and Myc protein were nevertheless comparably elevated in both the MycWT and MycHet tumors, suggesting that compensation for reduced Myc gene dosage had occurred at the transcriptional level. These results indicate that reduced endogenous cMyc dosage substantially delays tumor development in mice that are genetically predisposed to neoplasia and that, in order for MycHet;p53 KO mice to develop tumors, a compensatory increase in expression of Myc, which can occur by multiple mechanisms, is required. We have also used allografts using of thymic lymphomas from p53 KO mice carrying conditional cMyc floxed and tamoxifen dependent Cre alleles and a dual fluorescent reporter to monitor recombination efficacy, in order to test whether reducing cMyc will adversely impact established tumors (i.e. growth, progression) and found that after recovery from recombination induced apoptosis, tumor growth rate is substantially slowed when endogenous Myc dosage is acutely reduced. Our results strongly suggest that a modest reduction in Myc can curtail cancer growth and has important implications, particularly for extending tumor free survival in patients with Li Fraumeni syndrome (germline mutations in p53), as well as sporadic cancers. This study provides a framework and model for future analyses of cMyc role in tumorigenesis and tumor progression and can be readily extended to other tumor types using analogous strategies, and other tumor promoters, such as oncogenic Ras. We are currently testing whether p53 loss driven tumorigenesis can occur at all in the context of severely reduced or absent endogenous cMyc expression levels using a conditional knockout approach to model hemangiosarcomas by removing both p53 and cMyc genes together, selectively from endothelial tissues (Cdh5CreER) in young (2 week age) mice. Surprisingly, we have found that hemagiosarcomas still occur in p53KO mice with cmyc loss in endothelial cells; however, these develop at a much slower pace than in cmycWT mice, with a latency period of well over a year, suggesting two likely possible explanations: 1) incomplete recombination with removal of p53, but not both copies of the cmyc allele removed in rare endothelial cells, 2) compensatory activation of another endogenous myc gene (eg. Nmyc) in a small fraction of p53KO endothelial cells that compensates for cMyc loss. We are currently analyzing tumors that develop in these mice to determine the basis for tumor formation in the absence of cMyc. So far, we have found that all of the tumors developing in mice that are homozygous null for cMyc in endothelial cells express NMyc at an elevated level and do not appear to express cMyc. This indicates that the tumor occurrence is not due to incomplete recombination in the cMyc conditional knockout, but occurs because of compensation by the activation of another Myc gene (NMyc), supporting the model that tumorigenesis absolutely requires a minimum Myc threshold to occur. In the absence of functional cMyc, there is selective pressure favoring cells that activate NMyc to survive and proliferate following loss of the Trp53 tumor suppressor gene. We are now preparing these results to submit for publication and have deposited a draft of the manuscript at BioRXiv preprint server (https://www.biorxiv.org/cgi/content/short/2025.07.28.667174v1).