Identification of cofactor-dependent enzymes driving cell growth and differentiation

Abstract

Biological phenomena are orchestrated through the actions of active enzymes, which interact with ~40,000 metabolites. By contrast to protein abundance, enzyme activity is dynamically regulated by small molecule cofactors (e.g., iron, NAD), at their catalytic active sites, capable of directly mediating metabolic reactions. Cofactors often rely on co-substrates from microenvironments for their functionality (e.g., O2 and 2oxoglutarate), thus, cofactor-dependent enzymes are able to ‘sense’ their microenvironment. Within the landscape of oncology, cancer cells exploit unique microenvironments that regulate a number of activities of cofactor-dependent enzymes for development and growth. For example, in glioblastoma (GBM), cells isolated from hypoxic regions display slow growth, while cells from the normoxic edge display invasive growth leading to relapse. Additionally, mutant isocitrate dehydrogenases (IDH) often found in cancer cells produces 2-D-hyroxygluturate (2-HG) in place of 2-oxoglutarate (2-OG), an essential co-substrate required for diverse oxygen-dependent enzymes. In GBM, mutant IDH leads to slow growth and better prognosis while in acute myeloid leukemia (AML) it leads to aberrant cell differentiation and worse prognosis. Despite the significance of these findings, the fundamental question of “how these unique environments regulate cancer in cofactor-dependent fashion?” has been poorly addressed due to a lack of technologies capable of comprehensively evaluating cofactor-dependent enzyme activities. To this goal, we have developed and pioneered Reverse- Polarity Activity-Based Protein Profiling (RP-ABPP) which, for the first time, enables us to map enzyme activities regulated by ‘environment-sensitive’ cofactors on a proteome-wide scale. In our proposed study, we aim to address the following questions by combining RP-ABPP with in vitro cancer models that faithfully recapitulates tumor microenvironments: Aim 1:“Which mutant-IDH sensitive enzyme(s) contribute to divergent outcome between AML vs. GBM? ”, Aim 2: “How do environmental cofactors (oxygen) impact the growth of AML and GBM?”. We will evaluate enzyme activities by RP-ABPP in the different conditions above (wildtype vs. IDH mutation and normoxia vs. hypoxia) to map cofactor-dependent enzymes driving cell growth and differentiation. This study will be the first landscape of enzyme activity governed by cellular microenvironment in aberrant cell growth and development. Due to the pervasive influences of the local cellular environments across cancer types numerous homeostatic/developmental settings, we anticipate our studies will have a broad impact across many domains of biomedical sciences.

Amount Recommended: $300,000

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