OBJECTIVES/GOALS: MGMT methylation status is used to predict the response to TMZ. However, a subpopulation of patients lacking MGMT methylation still respond to TMZ. We applied omics approaches and functional studies to a cohort of GBM patients to identify novel markers that may more accurately predict TMZ response. METHODS/STUDY POPULATION: We applied a combination of omics approaches and functional studies to a cohort of GBM patients to search for novel markers that would predict the response to TMZ treatment more accurately than traditional markers. Using a set of 47 primary and secondary GBM tumor samples, we employed comparative transcriptomics, whole exome sequencing, data independent acquisition (DIA) proteomics, and phosphoproteomics to look for DNA mutations and changes in gene expression and/or protein expression that correlated to response or non-response to TMZ. Subsequently, we performed functional studies and analyzed patient treatment data to validate our results. RESULTS/ANTICIPATED RESULTS: This study is in early stage, but we anticipate that our combination of methods may allow us to identify and validate at least one novel biomarker for TMZ response in patient GBM. For example, comparative transcriptomics or phosphoproteomics may identify a previously unrecognized gene or protein over/under-expression in a subset of patients. In this case we will validate findings using western blotting, IHC staining, and through siRNA of target gene/protein on patient derived GBM cells to examine if removal of this marker leads to TMZ sensitivity. We will further confirm prediction of marker correlation through comparison with matched patient treatment data. DISCUSSION/SIGNIFICANCE: The identification of improved biomarkers to predict response to TMZ treatment is a discovery that could rapidly become standard of care for GBM patients. It would ensure that all responders receive TMZ and avoid exposing nonresponders unnecessarily to TMZ and its potential side effects.