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 p53R248Q/+ 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 p53R248Q/+ 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.

HIGHLIGHTS 14 15• heterozygous p53 R248Q/+ tumors retain p53 transcriptional activity in a mouse model of 16 colorectal cancer (CRC) 17• wildtype p53 actively represses the tumor-promoting HSF1-regulated chaperone system 18 and proteotoxic stress response 19• the repressive WTp53 -HSF1 axis creates a selective pressure for WTp53 loss-of-20 heterozygosity in CRC tumors 21• p53 loss-of-heterozygosity enables stabilization of the gain-of-function p53 R248Q mutant 22 protein which in turn enables CRC invasion 23 24 25 26 27 28 29 30 2 31 Abstract 32A prerequisite for gain-of-function (GOF) p53 missense mutants (mutp53) is protein 33 stabilization. Moreover, a prerequisite for mutp53 stabilization is loss of the remaining wildtype 34 (WT) p53 allele (loss-of-heterozygosity, p53LOH) in mutp53/+ tumors. Thus, GOF, mutp53 35 stabilization and p53LOH are strictly linked. However, the driving force for p53LOH is unknown. 36Typically, heterozygous tumors are an instable transition state. Here we identify the repressive 37WTp53-HSF1 axis as the driver of p53LOH. 38We find that the WTp53 allele in AOM/DSS-induced colorectal tumors (CRC) of p53 R248Q/+ mice 39 retains its haploid transcriptional activity. Notably, WTp53 represses heat-shock factor 1 (HSF1) 40 activity, the master transcription factor of the proteotoxic stress defense response (HSR) that is 41 ubiquitously and constitutively activated in cancer tissues. HSR is critical for stabilizing 42 oncogenic proteins including mutp53. WTp53-retaining murine CRC tumors and tumor-derived 43 organoids and human CRC cells all suppress the tumor-promoting HSF1 transcriptional 44 program. 45Mechanistically, the retained WTp53 allele activates CDKN1A/p21, leading to cell cycle 46 inhibition and suppression of the E2F target gene MLK3. MLK3 links cell cycle to the MAPK 47 stress pathway to activate the HSR response. We show that in p53 R248Q/+ tumors WTp53 48 activation by constitutive stress (emanating from proliferative/metabolic stresses and genomic 49 instability) represses MLK3, consequently inactivating the MAPK-HSF1 response necessary to 50 ensure tumor survival. This creates strong selection pressure for p53LOH which eliminates the 51 repressive WTp53-HSF1 axis and unleashes the tumor-promoting HSF1 functions, inducing 52 mutp53 stabilization and enabling invasion. 53 54 Keywords 55 mutp53, HSF1, Hsp90, Hsp70, CDK4, MLK3, MAPK, AOM/DSS, colorectal cancer, organoids, 56 Idasanutlin 57 58 59 60 RESULTS 101 p53LOH is a prerequisite for mutp53 stabilization and invasion in colorectal cancer 102Stabilization of missense mutant p53 (mutp53) proteins specifically in tumor but not normal cells 103 is a key feature and prerequisite of GOF 16, 42 . Since p53LOH is a critical prerequisite for mutp53 104 stabilization in sarcomas and breast cancer 41 , we examined mutp53 stabilization before and 105 after p53LOH in the colorectal AOM/DSS model 9 . Briefly, we combined the humanized GOF 106

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