Cancers have different nutritional requirements from their healthy counterparts. Herein, targeting the enzymes and thus the metabolic networks that constitute these different metabolic requirements is attracting numerous drug development efforts. While targeting enzymes in cancer-specific metabolic pathways has been successful, whether metabolism can be affected to similar extents by nutritional manipulation in specific and controlled manners is largely unknown. Methionine availability affects one-carbon cycle flux, DNA and histone methylation and thus epigenetic programming. Dietary methionine restriction (MR) also promotes metabolic health and extends insect and mammalian lifespan, two anti-cancer features. Thus, we hypothesize that dietary MR could inhibit tumor growth and enhance anti-cancer therapy. Our preliminary data show that dietary MR, a cost-effective approach, alters plasma methionine effectively in both healthy mice and humans. MR mediates promising tumor outcomes: in two RAS-driven colorectal cancer (CRC) patient-derived xenografts (PDX) models, it delays tumor growth and sensitizes tumor to 5-Fluorouracil, a frontline chemotherapy for CRC; and in an autochthonous KRASG12D+/-;TP53-/- soft tissue sarcoma model, it reaches a striking synergistic effect with radiation. In these models, cysteine and methionine metabolism is the most impacted metabolic pathway in the tumor and the plasma. However, the molecular mechanisms by which dietary MR interacts with metabolism and eventually mediates tumor outcome are unknown. To explore the mechanisms, I propose to focus on CRC PDX models through the following two aims. In Aim 1, I will first employ a state of the art metabolomics approach established in our laboratory to evaluate the metabolic alterations in tumor and non- tumor tissues by dietary MR or a combination of MR and 5-FU. Upon consolidation of the findings in vitro, I will conduct dietary interventions by supplementing cysteine or betaine to determine the contribution of methionine as a sulfur donor and a one-carbon donor to tumor growth in CRC PDX models, respectively. In Aim 2, I will first characterize the epigenetic and genetic adaptions to dietary MR in tumor and CRC cell linesusing bisulfite sequencing, ATAC- seq, ChIP-seq, and RNA-seq. With these data, I will construct a metabolic network and perform a functional screen of metabolic pathway genes using CRISPR/Cas9 technology. The outcome will reveal the metabolic, genetic, and epigenetic mechanisms underlying dietary MR-mediated tumor inhibition alone and in adjuvant with 5-FU in CRC. It is also anticipated to provide target metabolite and genes for future hypothesis generation and novel therapy targeting cancer metabolism.