**4. Mechanism of action for NQO1-bioactivatable therapies**

### **4.1 NQO1 vs. catalase ratio and specificity**

NQO1 is a Phase II detoxification enzyme that reduces ROS levels in cancer cells. NQO1 converts quinones into stable intermediate hydroquinones that are exported out of the cell by conjugation [10]. Most solid cancers, including non-small cell lung and pancreatic cancers (>85%), prostate, colon, and breast cancers (60%) and head and neck cancers (40%) overexpress NQO1 5- to 200- fold above normal tissue. Corresponding levels of catalase in these cancers were strikingly reduced, impacting the ability of cancer cells to eliminate ROS [14]. Overexpression of NQO1 appears to stabilize HIF-1alpha and promotes metastasis [15].

Though NQO1 detoxifies most quinones through two-electron oxidoreduction, a few quinones undergo a rapid futile redox cycle response, generating an unstable intermediate hydroquinone that spontaneously reverts back to its original form using two oxygenation steps and creating two superoxides. Deoxynyboquiones (DNQ ), KP372 agents, and β-lapachone are three classes of NQO1-bioactivatable drugs currently known [16]. Recently, Napabucasin, an orphan drug in clinical trials for pancreatic and cervical cancer, has also been reported to be bioactivated by NQO1 [17]. Though mitomycin C and streptonigrin are metabolized by NQO1,

these agents can also be activated by other drug metabolizing enzymes [18]. Human cancer cells overexpressing NQO1 have been shown to be sensitive to NQO1 bioactivatable drugs alone and in combination with PARP inhibitors, cisplatin, radiation, and NAMPT inhibitors both in cell culture and xenograft models [14, 19].
