Impact of CAR and Nrf2 dual activation on cyclophosphamide bioactivation and doxorubicin detoxification

Project Summary Cyclophosphamide (CPA) and doxorubicin (DOX)-containing regimens are extensively utilized in treating various malignancies, including triple-negative breast cancer (TNBC), a particularly aggressive form of cancer that is not responsive to hormonal and targeted therapies. Although the administration of the CPA/DOX combination has resulted in improved therapeutic outcomes, a significant number of patients succumb to their disease due to drug resistance and/or intolerable side toxicities, highlighting the need for further optimization. The aim of this project is to enhance the therapeutic efficacy of CPA and DOX-based treatment while minimizing side toxicity. This will be achieved by improving the metabolic conversion of CPA, an alkylating prodrug, to its pharmacologically active metabolite via cytochrome P450 (CYP) 2B6, while mitigating DOX-induced cardiotoxicity through the activation of Nrf2 antioxidant signaling. We have shown that activation of the human constitutive androstane receptor (hCAR) preferentially induces the expression of hepatic CYP2B6 over other CYP enzymes, resulting in heightened metabolic activation and improved therapeutic efficacy of CPA. Concurrently, activation of Nrf2-antioxidant signaling represents a key mechanism counteracting DOX-induced cardiotoxicity. Recently, we identified novel hCAR and Nrf2 dual activators that can simultaneously induce CYP2B6 in hepatocytes and activate Nrf2 in cardiomyocytes. In this proposal, we hypothesize that dual activation of hCAR and Nrf2 enhances the bioactivation of CPA while concurrently reducing DOX-mediated cardiotoxicity, thereby improving the therapeutic efficacy of CPA/DOX-based treatment. This central hypothesis will be tested by the following specific aims: Aim #1. Determine the effects of chemical optimization of novel hCAR/Nrf2 dual activators on CPA metabolism and DOX detoxification; Aim #2. Evaluate the antiproliferative activity and cardiotoxicity of the CPA/DOX combination with and without hCAR/Nrf2 dual activators in a multicellular co- culture model; and Aim #3. Examine the role of hCAR/Nrf2 dual activation in CPA/DOX-based TNBC treatment in a hCAR-transgenic mouse model. The proposed studies aim to provide novel mechanistic insights into the beneficial effects of hCAR/Nrf2 on CPA/DOX-based treatment. The outcomes from this project are expected to establish hCAR and Nrf2 as promising combined therapeutic targets, ultimately enhancing the clinical efficacy of CPA/DOX-containing regimens.