Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis

Abstract

Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. Missense alleles of the HXK2, REG1, GLC7 and SNF1 genes were shown to confer significant resistance to 2-deoxyglucose and all had the potential to alter the activity and or target selection of the Snf1 kinase signaling pathway. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Addition of 2DG promotes endocytosis of the glucose transporter Hxt3. All but one of the 2DG-resistant strains reduced the 2DG-mediated hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2deoxyglucose but induction of these genes is not associated with 2DG-resistance. RNAseq analysis of the transcriptional response to 2DG showed large scale, genome-wide changes in mRNA abundance that were greatly reduced in the 2DG resistant strains. These findings suggest the common adaptive response to 2DG is to limit the magnitude of the response. Genetic studies of 2DG resistance using the dominant SNF1-G53R allele in cells that are genetically compromised in both the endocytosis and DOG pathways suggest that at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.

Cellular toxicity of the metabolic inhibitor 2-deoxyglucose and associated resistance mechanisms

“…As would be expected from our current knowledge of 2-DG toxicity, in which 2-DG-6-P plays a pivotal role, the isolation of spontaneous 2-DG-resistant budding yeast strains consistently retrieved hexokinase mutants (Table 1) [196,32,19,64,71,186] and indeed, deletion of the main hexokinase gene HXK2 results in 2-DG resistance [35,64]. This is also the case in many other species, such as in the thermotolerant yeast Kluyveromyces marxianus [197], Kluyveromyces lactis [187], Schizosaccharomyces pombe [68] and others.…”

“…Previous work showed that the affinity of yeast hexokinase preparations towards 2-DG is in the same range as that for glucose [41,199,200] and recent work further showed that both hexokinases had a similar activity toward 2-DG in vitro [186]. This goes against the idea that 2-DG is not phosphorylated in hxk2∆ cells.…”

“…This goes against the idea that 2-DG is not phosphorylated in hxk2∆ cells. Accordingly, the deletion of phosphatases detoxicating 2-DG-6-P (discussed in the next paragraph) in a hxk2∆ mutant partially sensitizes this strain to 2-DG, suggesting that there is (at least) some phosphorylation of 2-DG in this strain and thus that (an)other sugar kinase(s) can perform this function [71,186]. Consequently, it remains unclear how the loss of HXK2, in the context of other enzymes competent for 2-DG phosphorylation, might lead to 2-DG resistance.…”

“…Consequently, it is unlikely that a screen would lead to mutations in all 7 genes known to import glucose. Additionally, more recent screens were performed in the context of using glucose as a carbon source [71,186], and given the structural similarities between glucose and 2-DG, mutation of glucose transporters that would affect 2-DG transport might equally impair glucose transport and thus prevent growth on glucose medium.…”

‘Sugarcoating’ 2-deoxyglucose: mechanisms that suppress its toxic effects

“…While Snf1 regulation of Mig1 (not show) alleviates repression of some genes in response to 2DG, other transcriptional responses to 2DG likely to occur via Snf1-independent signaling pathways Zimmermann and Scheel 1977). More recent studies in yeast have focused on defining mechanisms of 2DG-resistance in cells growing on glucose, which is more germane to what a cancerous cell would experience (Defenouillere et al 2019;McCartney et al 2014;O'Donnell et al 2015;Ralser et al 2008;Soncini et al 2020).…”