Hypoxia is a prevalent feature of many tumors contributing to disease progression and treatment resistance, and therefore constitutes an attractive therapeutic target. Several hypoxia-activated prodrugs (HAP) have been developed, including the phase III candidate TH-302 (evofosfamide) and the preclinical agent SN30000, which is an optimized analogue of the well-studied HAP tirapazamine. Experience with this therapeutic class highlights an urgent need to identify biomarkers of HAP sensitivity, including enzymes responsible for prodrug activation during hypoxia. Using genome-scale shRNA screens and a high-representation library enriched for oxidoreductases, we identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of sensitivity to SN30000 in three different genetic backgrounds. No other genes consistently modified SN30000 sensitivity, even within a POR-negative background. Knockdown or genetic knockout of POR reduced SN30000 reductive metabolism and clonogenic cell death and similarly reduced sensitivity to TH-302 under hypoxia. A retrospective evaluation of head and neck squamous cell carcinomas showed heterogeneous POR expression and suggested a possible relationship between human papillomavirus status and HAP sensitivity. Taken together, our study identifies POR as a potential predictive biomarker of HAP sensitivity that should be explored during the clinical development of SN30000, TH-302, and other hypoxia-directed agents. Cancer Res; 75(19); 1–13. ©2015 AACR.

Hypoxia is a prevalent feature of many tumors contributing to disease progression and treatment resistance, and therefore constitutes an attractive therapeutic target. Several hypoxia-activated prodrugs (HAPs) have been developed including the Phase III candidate TH-302 (evofosfamide) and the preclinical agent SN30000, which is an optimized analogue of the well-studied HAP tirapazamine. Experience with this therapeutic class highlights an urgent need to identify biomarkers of HAP sensitivity, including enzymes responsible for prodrug activation during hypoxia. Using genome-scale shRNA screens and a high-representation library enriched for oxidoreductases, we identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of sensitivity to SN30000 in three different genetic backgrounds. No other genes consistently modified SN30000 sensitivity, even within a POR-negative background. Knockdown or genetic knockout of POR reduced SN30000 reductive metabolism and clonogenic cell death and similarly sensitized cells to TH-302 under hypoxia. A retrospective evaluation of head and neck squamous cell carcinomas showed heterogeneous POR expression and suggested a possible relationship between human papillomavirus status and HAP sensitivity. Taken together, our study identifies POR as a potential predictive biomarker of HAP sensitivity that should be explored during the clinical development of SN30000, TH-302, and other hypoxia directed agents.

The tumor suppressor p53 prevents cancer development via initiating cell cycle arrest, cell death, repair, or anti-angiogenesis processes. Over 50% of human cancers harbor cancer-causing mutant p53. p53 mutations not only abrogate its tumor suppressor function, but also endow mutant p53 with a gain-of-function (GOF), creating a proto-oncogene that contributes to tumorigenesis, tumor progression and chemo- or radiotherapy resistance. Thus, targeting mutant p53 to restore a wild-type p53 signaling pathway provides an attractive strategy for cancer therapy. We demonstrate that small molecule NSC59984 not only restores wild-type p53 signaling, but also depletes mutant p53 GOF. NSC59984 induces mutant p53 protein degradation via MDM2 and the ubiquitin-proteasome pathway. NSC59984 restores wild-type p53 signaling via p73 activation specifically in mutant p53-expressing colorectal cancer cells. At therapeutic doses, NSC59984 induces p73-dependent cell death in cancer cells with minimal genotoxicity and without evident toxicity towards normal cells. NSC59984 synergizes with CPT11 to induce cell death in mutant p53-expressing colorectal cancer cells and inhibits mutant p53-associated colon tumor xenograft growth in a p73-¬dependent manner in vivo. We hypothesize that specific targeting of mutant p53 may be essential for anti-cancer strategies that involve the stimulation of p73 in order to efficiently restore tumor suppression. Taken together, our data identify NSC59984 as a promising lead compound for anti-cancer therapy that acts by targeting GOF mutant p53 and stimulates p73 to restore the p53 pathway signaling.

Diffuse malignant mesothelioma (DMM) is a tumor of serosal membranes with propensity for progressive local disease. Because current treatment options are largely ineffective, novel therapeutic strategies based on molecular mechanisms and the disease characteristics are needed to improve the outcomes of patients with this disease. Akt kinase interacting protein 1 (Aki1; Freud-1/CC2D1A) is a scaffold protein for the PI3K–PDK1–Akt signaling module that helps determine receptor signal selectivity for EGFR. Aki1 has been suggested as a therapeutic target, but its potential has yet to be evaluated in a tumor setting. Here, we report evidence supporting its definition as a therapeutic target in DMM. In cell-based assays, Aki1 silencing decreased cell viability and caused cell-cycle arrest of multiple DMM cell lines via effects on the PKA–CREB1 signaling pathway. Blocking CREB activity phenocopied Aki1 silencing. Clinically, Aki1 was expressed in most human DMM specimens where its expression correlated with phosphorylated CREB1. Notably, Aki1 siRNA potently blocked tumor growth in an orthotopic implantation model of DMM when administered directly into the pleural cavity of tumor-bearing mice. Our findings suggest an important role for the Aki1–CREB axis in DMM pathogenesis and provide a preclinical rationale to target Aki1 by intrathoracic therapy in locally advanced tumors. Cancer Res; 75(19); 1–10. ©2015 AACR.

Diffuse malignant mesothelioma (DMM) is a tumor of serosal membranes with propensity for progressive local disease. Since current treatment options are largely ineffective, novel therapeutic strategies based on molecular mechanisms and the disease characteristics are needed to improve the outcomes of patients with this disease. Akt kinase-interacting protein Aki1 (Freud-1/CC2D1A) is a scaffold protein for the PI3K/PDK1/Akt signaling module that helps determines receptor signal selectivity for EGFR. Aki1 has been suggested as a therapeutic target but its potential has yet to be evaluated in a tumor setting. Here we report evidence supporting its definition as a therapeutic target in DMM. In cell based assays, Aki1 silencing decreased cell viability and caused cell cycle arrest of multiple DMM cell lines via effects on the PKA/CREB1 signaling pathway. Blocking CREB activity phenocopied Aki1 silencing. Clinically, Aki1 was expressed in most human DMM specimens where its expression correlated with phosphorylated CREB1. Notably, Aki1 siRNA potently blocked tumor growth in an orthotopic implantation model of DMM when administered directly into the pleural cavity of tumor-bearing mice. Our findings suggest an important role for the Aki1/CREB axis in DMM pathogenesis and provide a preclinical rationale to target Aki1 by intrathoracic therapy in locally advanced tumors.