Purpose: Hypoxia is a characteristic of solid tumors and a potentially important therapeutic target. Here, we characterize the mechanism of action and preclinical antitumor activity of a novel hypoxia-activated prodrug, the 3,5-dinitrobenzamide nitrogen mustard PR-104, which has recently entered clinical trials. Experimental Design: Cytotoxicity in vitro was evaluated using 10 human tumor cell lines. SiHa cells were used to characterize metabolism under hypoxia, by liquid chromatography-mass spectrometry, and DNA damage by comet assay and gH2AX formation. Antitumor activity was evaluated in multiple xenograft models (PR-104 F radiation or chemotherapy) by clonogenic assay 18 h after treatment or by tumor growth delay. Results: The phosphate ester ''pre-prodrug'' PR-104 was well tolerated in mice and converted rapidly to the corresponding prodrug PR-104A. The cytotoxicity of PR-104A was increased 10-to 100-fold by hypoxia in vitro. Reduction to the major intracellular metabolite, hydroxylamine PR-104H, resulted in DNA cross-linking selectively under hypoxia. Reaction of PR-104H with chloride ion gave lipophilic cytotoxic metabolites potentially able to provide bystander effects. In tumor excision assays, PR-104 provided greater killing of hypoxic (radioresistant) and aerobic cells in xenografts (HT29, SiHa, and H460) than tirapazamine or conventional mustards at equivalent host toxicity. PR-104 showed single-agent activity in six of eight xenograft models and greater than additive antitumor activity in combination with drugs likely to spare hypoxic cells (gemcitabine with Panc-01pancreatic tumors and docetaxel with 22RV1prostate tumors). Conclusions: PR-104 is a novel hypoxia-activated DNA cross-linking agent with marked activity against human tumor xenografts, both as monotherapy and combined with radiotherapy and chemotherapy.Hypoxia is a uniquely attractive target in oncology for two reasons. The first is that hypoxic cells are obstacles to curative cancer therapy with all major treatment modalities. Hypoxia can compromise outcomes of surgery by increasing tumor metastasis (1 -3). It is also a major cause of radioresistance because oxygen is a radiosensitizer, and multiple clinical studies have documented the importance of hypoxia determining local tumor control in radiotherapy (4 -6). Hypoxia also contributes to chemoresistance through multiple mechanisms (7), including limitations on delivery of blood-borne drugs to hypoxic regions of tumors (8,9). The second reason for targeting hypoxia is that it is a common feature of a wide variety of human tumors and is typically more severe in tumors than in normal tissues, thus providing a basis for tumor selectivity (10,11).Several strategies for exploiting tumor hypoxia are now in preclinical or clinical development (7), with the main focus on prodrugs that are activated by metabolic reduction under hypoxic conditions to form cytotoxins. Early efforts focused on quinone bioreductive drugs, such as porfiromycin (12), and 2-nitroimidazole -linked alkylating a...

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.

PDLIM2 integrates cytoskeletal signaling with gene expression to enable reversible differentiation of epithelial cancer cells. PDLIM2 associates with the COP9 signalosome and controls its nuclear translocation and the stability of key transcription factors necessary for either a mesenchymal or an epithelial phenotype.

Crohn's disease is an inflammatory disorder of the bowel, believed to arise from the dysregulation of intestinal mucosal immunity. The interleukin-10-deficient (IL10-/-) mouse, which develops intestinal inflammation in the presence of gut microflora, serves as a mouse model of Crohn's disease. Nontargeted urinary metabolite profiling was carried out to identify systemic metabolic changes associated with the development of intestinal inflammation caused by IL10-deficiency. Spot urine samples, collected from IL10-/- and wildtype mice at ages 5.5, 7, 8.5, and 10.5 weeks old were analyzed by gas chromatography-mass spectrometry (GCMS). The data were analyzed using XCMS software, multiple t tests, and ANOVA. Among the key metabolic differences detected were elevated urinary levels of xanthurenic acid and fucose in IL10-/- mice relative to wildtype, indicating upregulation of tryptophan catabolism and perturbed fucosylation in IL10-/- mice. Three short-chain dicarboxylic acid metabolites were decreased in urine of IL10-/- mice relative to wildtype, suggesting the downregulation of fatty acid oxidation in IL10-/- mice. These metabolic differences were reproducible in an independent set of mice. This study demonstrates that nontargeted GCMS metabolite profiling of IL10-/- mice can provide insights into the metabolic effects of IL10-deficiency and identify potential markers of intestinal inflammation.