Identification of P450 Oxidoreductase as a Major Determinant of Sensitivity to Hypoxia-Activated Prodrugs

Abstract

Hypoxia is a prevalent feature of many tumors contributing to disease progression and treatment resistance, and therefore constitutes an attractive therapeutic target. Several hypoxiaactivated 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); 4211-23. Ó2015 AACR.

The hypoxia-activated prodrug evofosfamide in combination with multiple regimens of radiotherapy

“…Interestingly, A549 cells were also more sensitive than UT-SCC-14 to increasing concentrations of evofosfamide (Supplementary Figure 2). The cytochrome P450 oxidoreductase (POR) has previously been identified as major determinant for the sensitivity of hypoxia-activated prodrugs [18, 19]. Therefore, the expression level of POR was determined on the cellular and tumor level by western blotting and immunohistochemistry, respectively.…”

“…Several studies on the activation of HAPs previously demonstrated that cytochrome P450 oxidoreductase is indispensable for several HAPs [18, 19, 25]. However, its relevance for the nitroimidazole mustard evofosfamide is less clear.…”

“…Su et al knocked-out POR in multiple tumor cell lines that resulted in resistance to several one-electron reductase substrates but not to evofosfamide, suggesting the existence of structure-dependent oxidoreductase redundancies [26]. On the other hand, Hunter et al recently demonstrated reduced cytotoxicty of evofosfamide in head&neck carcinoma cells with shRNA-downregulated POR-expression [18]. More important, a heterogeneous POR expression status was retrospectively determined in head&neck squamous cell carcinoma patient samples, and suggests that POR might be a major predictive determinant for HAPs including evofosfamide [18].…”

“…On the other hand, Hunter et al recently demonstrated reduced cytotoxicty of evofosfamide in head&neck carcinoma cells with shRNA-downregulated POR-expression [18]. More important, a heterogeneous POR expression status was retrospectively determined in head&neck squamous cell carcinoma patient samples, and suggests that POR might be a major predictive determinant for HAPs including evofosfamide [18]. Our own studies, which were performed on the cellular level and to our best knowledge for the first time also in vivo , strongly suggest that POR co-determines the potency of evofosfamide.…”

“…Our in vitro data also corroborate that evofosfamide in combination with IR is more potent in BRCA2-deficient tumor cells than in their BRCA2-wildtype counterpart cells. Previous studies were only performed in genetically-defined CHO-cells and in combination with cisplatinum against tumor cells [18, 19]. …”