Targeting glucose metabolism in cancer is an attractive therapeutic area, but efforts to develop successful glycolytic inhibitors have failed. A major barrier in the cancer metabolism field is a systems-­level understanding of the differential regulation of glycolysis that results from metabolic rewiring. This limitation prompted the investigation to determine whether metabolic control can be exploited for therapy. The hypothesis of the F99 phase is that rational targeting of pivotal enzymes that differentially regulate central carbon metabolism in cancer can result in anti-­tumor efficacy and characterization of resistance mechanisms to glycolysis inhibition can advance the development of combination therapy. To address this hypothesis, Aim 1 characterizes the role of two important enzymes in central carbon metabolism, 3-­ phosphoglycerate dehydrogenase (PHGDH) and glyceraldehyde-­3-­phosphate dehydrogenase (GAPDH) and how these enzymes can be targeted for cancer therapy. Sub-­aim 1.1 uses CRISPR/Cas9 mediated PHGDH knockout (KO) to validate the selectivity and activity of two novel allosteric PHGDH inhibitors. Sub-­aim 1.2 uses quantitative determinants of the Warburg Effect (WE) to reveal GAPDH as a rational therapeutic target, and comparative metabolomics to nominate KA as a potent GAPDH inhibitor in highly glycolytic cells, together uncovering metabolic predictors of drug response to GAPDH inhibition. These findings contribute to a shifting paradigm in the current understanding of metabolic cancer therapy and show the potential use of metabolic predictors, rather than genetic determinants, to predict response. Aim 2 will seek to characterize the resistance mechanisms to GAPDH inhibition in order to develop combination therapy and better understand the requirements of the WE. KA-­sensitive cells will be made resistant and a lentiviral CRISPR/Cas9 sgRNA library loss-­of-­function screen will be employed to determine the metabolic enzymes driving resistance. The outcome will result in characterization of resistance development to glucose metabolism inhibition and the mechanism by which the WE occurs. In addition, since tumor heterogeneity largely contributes to limitations in drug targeting, there is an urgent need to better characterize the molecular determinants of microenvironment formation. The interplay among environmental factors, genetics, and epigenetics have recently been appreciated. The hypothesis of the K00 phase is that a dynamic cross-­talk exists between oncogenic and epigenetic networks and is governed by tissue origin. Aim 3 will examine the role of oncogenic drivers and epigenetic regulators at different stages of cancer progression in different microenvironment settings to unravel the molecularcomponents contributing to tumor heterogeneity. The outcome will establish a better understanding of the relationship between tumor heterogeneity and therapeutic response.