The metabolic pathways fueling tumor growth have been well characterized, but the specific impact of transforming events on network topology and enzyme essentiality remains poorly understood. Importantly, cancer cells must reprogram redox pathways to support increased biosynthesis and withstand environmental stresses. To this end, we performed combinatorial CRISPR-Cas9 screens on a set of 51 carbohydrate metabolism genes that represent glycolysis and the pentose phosphate pathway (PPP). This high-throughput methodology enabled systems-level interrogation of metabolic enzyme dispensability, interactions, and compensation within carbohydrate metabolism in two cell lines. The metabolic impact of specific combinatorial knockouts were validated using 13C and 2H isotope tracing. Quantitative analysis of fitness single knockout fitness in A549 and HeLa cells revealed a key role for the KEAP1-NRF2 signaling pathway in influencing the cellular response to knockout of oxidative pentose phosphate pathway (PPP) enzymes. Specifically, by augmenting NADPH regeneration and glutathione synthesis pathways, loss or mutation of KEAP1 facilitates growth by partially mitigating the deleterious effects of PPP gene knockout. Additionally, 2H tracing was used to understand how the PPP supports compartment-specific reprogramming of the redox network in response to routine hypoxic insults. These results together highlight the critical importance of the PPP in supporting redox homeostasis and demonstrate the need for further interrogation of redox metabolism at compartment- and network-level.

Interrogation of critical metabolic pathways important for compartment-specific redox homeostasis in cancer cells

255th National Meeting (2018)

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