Over half of colorectal cancers (CRCs) harbor TP53 missense mutations (mutp53). We show that the most common mutp53 allele R248Q (p53) exerts gain of function (GOF) and creates tumor dependence in mouse CRC models. mutp53 protein binds Stat3 and enhances activating Stat3 phosphorylation by displacing the phosphatase SHP2. Ablation of the p53 allele suppressed Jak2/Stat3 signaling, growth, and invasiveness of established, mutp53-driven tumors. Treating tumor-bearing mice with an HSP90 inhibitor suppressed mutp53 levels and tumor growth. Importantly, human CRCs with stabilized mutp53 exhibit enhanced Jak2/Stat3 signaling and are associated with poorer patient survival. Cancers with TP53 are associated with a higher patient death risk than are those having nonR248 mutp53. These findings identify GOF mutp53 as a therapeutic target in CRC.

All current regimens for treating ovarian cancer center around carboplatin as standard first line. The HSP90 inhibitor ganetespib is currently being assessed in advanced clinical oncology trials. Thus, we tested the combined effects of ganetespib and carboplatin on a panel of 15 human ovarian cancer lines. Strikingly, the two drugs strongly synergized in cytotoxicity in tumor cells lacking wild-type p53. Mechanistically, ganetespib and carboplatin in combination, but not individually, induced persistent DNA damage causing massive global chromosome fragmentation. Live-cell microscopy revealed chromosome fragmentation occurring to a dramatic degree when cells condensed their chromatin in preparation for mitosis, followed by cell death in mitosis or upon aberrant exit from mitosis. HSP90 inhibition caused the rapid decay of key components of the Fanconi anemia pathway required for repair of carboplatin-induced interstrand crosslinks (ICLs), as well as of cell cycle checkpoint mediators. Overexpressing FancA rescued the DNA damage induced by the drug combination, indicating that FancA is indeed a key client of Hsp90 that enables cancer cell survival in the presence of ICLs. Conversely, depletion of nuclease DNA2 prevented chromosomal fragmentation, pointing to an imbalance of defective repair in the face of uncontrolled nuclease activity as mechanistic basis for the observed premitotic DNA fragmentation. Importantly, the drug combination induced robust antitumor activity in xenograft models, again accompanied with depletion of FancA. In sum, our findings indicate that ganetespib strongly potentiates the antitumor efficacy of carboplatin by causing combined inhibition of DNA repair and cell cycle control mechanisms, thus triggering global chromosome disruption, aberrant mitosis and cell death. 6 This provides a strong rationale for HSP90 inhibitors in cancer therapy 7 and raises the possibility that HSP90 inhibitors can be used in combination with DNAdamaging chemotherapeutics. 8 Ovarian cancer is among the most common gynecological tumor types and carries the worst prognosis. HSP90 is highly expressed in advanced ovarian carcinoma 9 and thus constitutes a potential therapeutic drug target. 10 In support, HSP90 inhibitors were found effective against ovarian cancer in preclinical models, 11-14 alone or in combination with conventional chemotherapy. 15,16 Carboplatin is the key component of all current first-line therapies for ovarian carcinoma. However, while initially often responding with regression, most tumors relapse and develop resistance within less than a year. 17,18 Like other platinum compounds, carboplatin acts by forming crosslinks within DNA. 17 Platinum compounds form intrastrand DNA lesions but also interstrand crosslinks (ICLs) by covalently linking opposite DNA strands. Such ICLs represent major obstacles to the DNA replication fork 19 and are therefore considered the principal toxic lesion of carboplatin's mode of action. The ICLs can only be removed by a sophisticated DNA repair systemthe Fanconi ...

Dipeptidyl peptidase 9 (DPP9) is a serine protease cleaving N-terminal dipeptides preferentially post-proline with (patho)physiological roles in the immune system and cancer. Only few DPP9 substrates are known. Here we identify an association of human DPP9 with the tumour suppressor BRCA2, a key player in repair of DNA double-strand breaks that promotes the formation of RAD51 filaments. This interaction is triggered by DNA-damage and requires access to the DPP9 active-site. We present crystallographic structures documenting the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in the DPP9 active-site. Mechanistically, DPP9 targets BRCA2 for degradation by the N-degron pathway, and promotes RAD51 foci formation. Both processes are phenocopied by BRCA2 N-terminal truncation mutants, indicating that DPP9 regulates both stability and the cellular stoichiometric interactome of BRCA2. Consistently, DPP9-deprived cells are hypersensitive to DNA-damage. Together, we identify DPP9 as a regulator of BRCA2, providing a possible explanation for DPP9 involvement in cancer development.

N‐terminal sequences are important sites for post‐translational modifications that alter protein localization, activity, and stability. Dipeptidyl peptidase 9 (DPP9) is a serine aminopeptidase with the rare ability to cleave off N‐terminal dipeptides with imino acid proline in the second position. Here, we identify the tumor‐suppressor BRCA2 as a DPP9 substrate and show this interaction to be induced by DNA damage. We present crystallographic structures documenting intracrystalline enzymatic activity of DPP9, with the N‐terminal Met1‐Pro2 of a BRCA21‐40 peptide captured in its active site. Intriguingly, DPP9‐depleted cells are hypersensitive to genotoxic agents and are impaired in the repair of DNA double‐strand breaks by homologous recombination. Mechanistically, DPP9 targets BRCA2 for degradation and promotes the formation of RAD51 foci, the downstream function of BRCA2. N‐terminal truncation mutants of BRCA2 that mimic a DPP9 product phenocopy reduced BRCA2 stability and rescue RAD51 foci formation in DPP9‐deficient cells. Taken together, we present DPP9 as a regulator of BRCA2 stability and propose that by fine‐tuning the cellular concentrations of BRCA2, DPP9 alters the BRCA2 interactome, providing a possible explanation for DPP9's role in cancer.

Since publication of this article, the authors reported that the online version is missing the links to most of the Supplementary data, specifically, Supplementary Figures S1-S9; Supplementary Table S1; all legends to Supplementary Material.

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