Microbial metabolism of tryptophan is associated with resistance to immune checkpoint (ICB) therapy in renal cell cancer (RCC).
Authors: Bari Shahla , Sarfraz Humaira , Darwin Alicia , Chahoud Jad , Muzaffar Jameel , Fishman Mayer N. , Jain Rohit K. , George Daniel J. , Armstrong Andrew J. , Chadha Juskaran , Powers John Joseph , Nguyen Tien , Wilches Milton , Jaglal Michael Vishal , Sur Melissa , Chrisopher Michael , Engelman Robert , Garrett Timothy , Conejo-Garcia Jose , Manley Brandon J. 
Published: June 2023

4564

Background: Metastatic RCC has a poor prognosis. Despite improvement in treatment outcomes with ICB and targeted therapy, many patients fail to respond to first line therapy and deep and durable responses remain elusive. The tryptophan metabolite kynurenine, an agonist of the immunosuppressive aryl hydrocarbon receptor (AhR), is associated with resistance to ICB. However, IDO (Indole 2,3 dioxygenase) blockers which inhibit tryptophan metabolism to kynurenine failed to show benefit in clinical trials, suggesting the presence of alternate AhR activation. We hypothesize that microbial metabolism of tryptophan to indole metabolites may be associated with AhR activation and ICB resistance. Methods: We prospectively collected stool and plasma of 79 treatment naïve metastatic RCC patients, treated with ICB +/- Tyrosine kinase inhibitors (TKI). Samples were collected at treatment initiation and at time of first response assessment (12+/-3 weeks). We evaluated stool metagenomics and untargeted stool and plasma metabolomics among responders (R) and non-responders (NR). We focused on kynurenine/tryptophan and indoles/tryptophan ratio to evaluate differential host and microbial metabolism of tryptophan. A responder was classified as progression free survival (PFS) greater than six months. We also performed global metabolomics on tumors of germ-free (GF) & specific pathogen free (SPF) mice to identify microbial & host metabolites. Results: Among 79 patients, 28 were treated with combination ICB, while 51 patients were treated with ICB + TKI. Median follow up for entire cohort was 9.5 months. 65% of patients had PFS > six months, while 27% of all patients had continued response at one year. Using unpaired t test comparing baseline relative abundance of metabolites between R vs NR - plasma Kynurenine/tryptophan ratio was significantly higher in NR vs R, at baseline (0.04 vs 0.02, p=0.017) and further increased in NR at 3 months (0.05 vs 0.02, p=0.02). Microbial metabolites of Tryptophan -Indole pyruvic acid (IPA), Indole carboxylic acid (IpA) and Indole acetaldehyde (IAA) were differentially associated with ICB resistance and was significantly higher in NR (IcA: 8.0 vs 4.96, p=0.03; IPA: 76.8 vs 64.9, p=0.02; IAA: 76.8 vs 64.6, p=0.026). We also noted differential enrichment of microbial KEGG enzymes associated with IPA, IAA and IcA production in the stool of NR patients compared to R. Microbial origin of IpyA, IAA and IcA was confirmed by its absence in tumor of GF mice. Conclusions: This is first study in metastatic RCC which shows the association of indoles (microbial metabolites of tryptophan) with resistance to ICB. These indoles are known to be potent AhR agonists and are associated with immunosuppression suggesting that microbial metabolism of tryptophan may represent a novel pathway for ICB resistance in RCC patients.