Abstract 6606: HK2 is conditionally essential for growth in human cancer cells

Abstract

Abstract The genetic drivers of cell growth can depend on an interplay of intrinsic factors and environmental context. However, there is often little consideration into how nutrient availability affects the genetic dependencies of proliferating human cells. Moreover, efforts such as DepMap have catalogued cell fitness genes using in vitro CRISPR screens across hundreds of cancer cell lines cultured in traditional media, which poorly replicate nutrient conditions found in the body. We recently tested the hypothesis that the nutrient environment can affect gene essentiality in human cells. By performing CRISPR screens with blood cancer cell lines, we identified sets of genes differentially required for cells cultured in traditional (e.g., RPMI) versus Human Plasma-Like Medium (HPLM). Among the strongest HPLM-essential hits was HK2, which encodes for the glycolytic enzyme hexokinase 2. Notably, most cancer cell lines co-express two hexokinases (HK1; HK2) that can each catalyze the phosphorylation of glucose to glucose-6-phosphate. Interestingly, while these two HKs share high degrees of structural and biochemical similarity, only HK2 deletion had differentially stronger effects in HPLM. Further, both HK1 and HK2 contain an N-terminal region that permits association with the outer mitochondrial membrane (OMM). To investigate the conditional phenotype for loss of HK2, we generated HK2-knockout K562 leukemia cells, which displayed HK1 expression levels comparable to those measured in control cells. We also confirmed that our HK2-knockout cells showed a stronger relative growth defect in HPLM versus RPMI. Importantly, the expression of HK2 cDNA normalized this defect. By contrast, the expression of kinase-dead HK2 did not provide any rescue effects, suggesting that HK activity is required for the conditionally essential role of HK2. We also engineered a cytosol-restricted HK2 by truncating its N-terminal OMM binding region. When we expressed this cDNA in our HK2-knockout cells, we observed complete rescue of the relative growth defect, suggesting that subcellular HK2 activity contributes to conditional HK2 essentiality. We are now purifying mitochondria to ask how the subcellular distributions of HK1 and HK2 vary with nutrient conditions. In addition, we are also comparing the tracing patterns of 13C glucose in HK2-knockout and control cells to ask how HK2 deletion affects glucose utilization. Finally, given that we normalized glucose levels across our screening conditions, we are applying a systematic approach to determine the gene-nutrient interaction that underlies the conditional phenotype for HK2 deletion. Collectively, our data suggest that HK2 serves a non-redundant and conditionally essential role. We expect to generate novel insights into how HK2 supports human cell growth and why this role can further depend on nutrient availability. Citation Format: Kimberly S. Huggler, Kyle M. Flickinger, Jason R. Cantor. HK2 is conditionally essential for growth in human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6606.