The Role of the Constitutive Androstane Receptor in Cyclophosphamide-based Treatment of Lymphomas

Abstract

Cyclophosphamide (CPA) is an alkylating prodrug which has been utilized extensively in combination chemotherapies for the treatment of cancers and autoimmune disorders since its introduction to the market in the late 1950s. The metabolic conversion of CPA to its pharmacologically active metabolite 4-OH-CPA is catalyzed primarily by cytochrome P450 (CYP) 2B6. CPA is also subject to metabolism by CYP3A4 to an inactive metabolite, N-dechoroethyl-CPA (N-DCE-CPA), and a neurotoxic byproduct, chloroacetaldehyde. CPA is the backbone of the frontline chemotherapeutic regimen used for the treatment of non-Hodgkin lymphoma which combines the CHOP (CPA-doxorubicin-vincristine-prednisone) regimen with rituximab, a monoclonal CD20 antibody. The constitutive androstane receptor (CAR, NR1I3), an orphan nuclear receptor, is recognized as the key mediator of xenobiotic-induced expression of CYP2B6, Importantly, mounting evidence suggests that activation of hCAR leads to preferential induction of CYP2B6 over CYP3A4 which suggests that selective hCAR activation may enhance CPA bioactivation and enhance the efficacy:toxicity ratio of CHOP chemotherapy for NHL. CHOP chemotherapy has been associated with severe and cumulative cardiotoxicity arising from the doxorubicin component of the regimen and it is recommended that lymphoma patients with existing heart conditions avoid treatment with the full CHOP combination. Recently, it has been demonstrated that Nrf2 (nuclear factor (erythroid-derived 2))-like 2, NFE2L2) plays a key role in governing doxorubicin-induced cardiotoxicity. Nrf2 regulates the expression of important antioxidant genes and proteins which protect tissues from damage due to oxidative stress and inflammation. It has been shown both that insufficient Nrf2 expression results in hypersensitivity to doxorubicin cardiotoxicity and that stimulation of Nrf2 by small molecule activators can provide protection from doxorubicin-mediated toxicity. Our hypothesis was that hCAR activation will increase hepatic expression of CYP2B6 while having a negligible impact on other genes responsible for the disposition of CHOP drugs. Further, activation of Nrf2 in cardiac tissue may provide protection against cardiotoxicity induced by the doxorubicin component of CHOP. Together, these gene expression alterations will lead to augmented antineoplastic activity of CHOP in target lymphoma cells while alleviating the untoward cardiotoxicity associated with this regimen. This hypothesis was tested with a variety of methods including a novel hepatocyte-lymphoma-cardiomyocyte cell co-culture system as an in vitro model for studying the biotransformation of CPA and therapeutic effects of CHOP as well as the off-target toxicity in healthy tissues in an environment that closely resembles the in vivo condition. Using this system we successfully demonstrated that activation of hCAR with small molecule activators can significantly increase the anticancer activity of the CHOP regimen in lymphoma cells. Additionally, activation of Nrf2 in cardiomyocytes in co-culture significantly reduced the doxorubicin-induced cardiotoxicity of CHOP. Utilizing a hCAR-transgenic mouse model, we were able to show in vivo that the combination of a selective hCAR activator alongside CHOP significantly increases the anticancer activity of CHOP in a lymphoma tumor xenograft study. Taken together, these results implicate hCAR and Nrf2 as drug targets for facilitating CHOP-based treatment of lymphomas. We were able to identify several compounds from the NIH Chemical Genomics Center Pharmaceutical Collection which activate both hCAR and Nrf2 and have provided preliminary evidence for their utility in CHOP-based lymphoma treatment.