Mutational Activation of the NRF2 Pathway Upregulates Kynureninase Resulting in Tumor Immunosuppression and Poor Outcome in Lung Adenocarcinoma

SIMPLE SUMMARY: Activation of the Nuclear factor-erythroid factor 2-related factor 2 (NRF2) pathway through gain-of-function mutations or loss-of-function of its suppressor Kelch-like ECH-associated protein 1 (KEAP1) is frequent in lung cancer. NRF2 activation has also been reported to alter the tumor microenvironment. Proteomic profiles of 47 lung adenocarcinoma (LUAD) cell lines (11 KEAP1 mutant and 36 KEAP1 wild-type) revealed the tryptophan-kynurenine enzyme kynureninase (KYNU) as a top overexpressed protein associated with activated NRF2. Mechanistic studies demonstrated that NRF2 is a regulator of enzymatically functional KYNU in LUAD. Analysis of multiple independent gene expression datasets of human lung cancer and a LUAD tumor microarray demonstrated that elevated tumor KYNU expression was associated with immunosuppression, including potent induction of T-regulatory cells, increased levels of PD1 and PD-L1, and poorer overall survival. Our findings indicate a novel mechanism of NRF2 tumoral immunosuppression through upregulation of KYNU. ABSTRACT: Activation of the NRF2 pathway through gain-of-function mutations or loss-of-function of its suppressor KEAP1 is a frequent finding in lung cancer. NRF2 activation has been reported to alter the tumor microenvironment. Here, we demonstrated that NRF2 alters tryptophan metabolism through the kynurenine pathway that is associated with a tumor-promoting, immune suppressed microenvironment. Specifically, proteomic profiles of 47 lung adenocarcinoma (LUAD) cell lines (11 KEAP1 mutant and 36 KEAP1 wild-type) revealed the tryptophan-kynurenine enzyme kynureninase (KYNU) as a top overexpressed protein associated with activated NRF2. The siRNA-mediated knockdown of NFE2L2, the gene encoding for NRF2, or activation of the NRF2 pathway through siRNA-mediated knockdown of KEAP1 or via chemical induction with the NRF2-activator CDDO-Me confirmed that NRF2 is a regulator of KYNU expression in LUAD. Metabolomic analyses confirmed KYNU to be enzymatically functional. Analysis of multiple independent gene expression datasets of LUAD, as well as a LUAD tumor microarray demonstrated that elevated KYNU was associated with immunosuppression, including potent induction of T-regulatory cells, increased levels of PD1 and PD-L1, and resulted in poorer survival. Our findings indicate a novel mechanism of NRF2 tumoral immunosuppression through upregulation of KYNU.