PROJECT SUMMARY Increased uptake and synthesis of macromolecules is essential to sustain the demands of hyperproliferative cancer cells. Therefore, developing approaches to limit biomass production specifically in tumors could have profound implications for cancer treatment. While seminal studies have shown that the universal oncogene MYC promotes protein synthesis in cancer cells, there is still a gap in our understanding of how MYC molecularly controls translation during oncogenic transformation. We found that the transcription factor aryl hydrocarbon receptor (AHR) and its ligand kynurenine, a byproduct of the amino acid tryptophan, are induced by MYC in colon cancer cells. Our preliminary results indicate that AHR is necessary for the expression of genes involved in ribosomal biogenesis and protein synthesis in proliferating cells. Our hypothesis is that AHR senses and responds to tryptophan-derived kynurenine by translocating into the nucleus and inducing the transcription of genes that mediate ribosome biogenesis and translation in colon cancer cells. Aim 1 will directly test the role of AHR in protein synthesis in colon cancer cells, using genetic silencing in isogenic lines of human colonic epithelial cells and of mouse organoids progressed to colon cancer. Aim 2 will establish the contribution of kynurenine to AHR-regulated protein synthesis and cell proliferation by examining the requirement for kynurenine for the expression of AHR target genes, protein synthesis, and growth. We will utilize AHR knockout cells and animals and a competitive inhibitor that prevents the binding of kynurenine to AHR to define AHR- specific functions regulated by kynurenine. Aim 3 will directly test the importance of tryptophan-metabolizing enzymes in generating kynurenine and in regulating proliferation of colonic cells. We will determine the effects of knocking down or knocking out enzymes in the kynurenine pathway to define their requirement for AHR activity, protein synthesis, and proliferation of colon cancer cells and organoids. This study has the potential to define a direct physiological role for the kynurenine-AHR pathway in driving increased biomass production and cell proliferation in colon cancer. Moreover, this study will broaden the understanding of the role of kynurenine as an oncometabolite. Our findings could become the basis for the development of novel approaches to limit kynurenine production and AHR activity as a means to treat MYC-dependent tumors.