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.

Using a combination of mass-spectrometry and aptamer array-based proteomics, we characterized the protein features of circulating extracellular vesicles (EVs) in the context of lung (LUAD) and pancreatic ductal (PDAC) adenocarcinomas. We profiled EVs isolated from conditioned media of LUAD and PDAC cell lines to identify EV-associated protein cargoes released by these cancer cell types. Analysis of the resulting data identified LUAD and PDAC specific and pan-adenocarcinoma EV protein signatures. Bioinformatic analyses confirmed enrichment of proteins annotated to vesicle-associated processes and intracellular compartments, as well as representation of cancer hallmark functions and processes. Analysis of upstream regulator networks indicated significant enrichment of TP53, MYC, TGFB1 and KRAS-driven network effectors (p = 1.69 × 10⁻⁷⁷–2.93 × 10⁻⁴⁹) manifest in the adenocarcinoma sEV protein cargoes. We extended these findings by profiling the proteome of EVs isolated from lung (N = 15) and pancreatic ductal (N = 6) adenocarcinoma patient plasmas obtained at time of diagnosis, along with EVs derived from matched healthy controls (N = 21). Exploration of these proteomic data revealed abundant protein features in the plasma EVs with capacity to distinguish LUAD and PDAC cases from controls, including features yielding higher performance in the plasma EV isolates relative to unfractionated plasmas.

Small-cell-lung cancer (SCLC) is associated with overexpression of oncogenes including Myc family genes and YAP1 and inactivation of tumor suppressor genes. We performed in-depth proteomic profiling of plasmas collected from 15 individuals with newly diagnosed early stage SCLC and from 15 individuals before the diagnosis of SCLC and compared findings with plasma proteomic profiles of 30 matched controls to determine the occurrence of signatures that reflect disease pathogenesis. A total of 272 proteins were elevated (area under the receiver operating characteristic curve (AUC) ≥ 0.60) among newly diagnosed cases compared to matched controls of which 31 proteins were also elevated (AUC ≥ 0.60) in case plasmas collected within one year prior to diagnosis. Ingenuity Pathway analyses of SCLC-associated proteins revealed enrichment of signatures of oncogenic MYC and YAP1. Intersection of proteins elevated in case plasmas with proteomic profiles of conditioned medium from 17 SCLC cell lines yielded 52 overlapping proteins characterized by YAP1-associated signatures of cytoskeletal re-arrangement and epithelial-to-mesenchymal transition. Among samples collected more than one year prior to diagnosis there was a predominance of inflammatory markers. Our integrated analyses identified novel circulating protein features in early stage SCLC associated with oncogenic drivers.