Metabolic flexibility and rewiring is a distinctive feature of the cellular stress response and oncogenesis. The staggering breadth of adaptive pathways simultaneously promises a wealth of therapeutic targets while also acting as obstacles to effective therapies. Therefore, interest centers on linchpin metabolic actors as accessible handles for throttling cellular growth and survival. This idea is exemplified by a study appearing in the October 2016 edition of Cell Reports by Steven Zhao, fourth-year graduate student in the Cancer Biology program in the laboratory of Dr. Kathryn Wellen.
The Wellen laboratory studies the pathways of production and utilization of the metabolite acetyl-CoA, which is an essential unit for carbon flux, macromolecular biosynthesis, and epigenetic protein modification. The lab uses cell line and animal models through genetic, metabolomic, and proteomic analyses to interrogate the cycling of acetyl-CoA as an essential process that can be a chokepoint and a lever for controlling cancer cell growth. Here Zhao, et al. trace the compensation mechanisms for glucose-derived acetyl-CoA production. The study argues that extracellular acetate can somewhat compensate as an alternative source for acetyl-CoA and is sufficient for restoring some acetyl-CoA dependent processes and viability to transformed cells.
Nuclear and cytosolic acetyl-CoA primarily derives from glucose through the TCA cycle and the activity of ATP-citrate lyase (ACLY). Actively proliferating cells are known to be dependent on ACLY for de novo lipid synthesis and other processes to the point that ACLY inhibition can arrest the growth of immortalized cells. Zhao, et al. observe that immortalized MEF and glioblastoma cells drastically slow down cycling upon ACLY ablation. Unfortunately, cells escaping this ablation have a selective advantage that causes them to repopulate the niche with wild-type ACLY cells.
The backbone of the paper consists of the surprising phenotypes the immortalized cells exhibit upon ACLY deletion. At once, it was apparent that the stable knockout clones were extraordinarily debilitated. That there was any cell survival was itself surprising, as previous whole-organism deletions showed that ACLY loss was embryonic lethal. “Frankly, I wasn't even sure the cells were going to be viable long term without it,” says Zhao. For his persevering cultivation of the mutants Zhao was rewarded with the clearest picture yet of cell dependence on ACLY and the ability of other acetyl-CoA pathways to compensate for ACLY loss as a model for ACLY-oriented therapy.
Zhao thoroughly demonstrates that total ACLY loss causes a major increase in acetyl-CoA production by acetyl-CoA synthetase (ACSS2) from nonexistent to predominant in both the MEF and glioblastoma models. He confirms that the ACLY knockouts cease slowly dying and begin to proliferate again at partial speed only when the cells are exposed to physiological concentrations of extracellular acetate, the substrate for acetyl-CoA synthesis by ACSS2. Stable isotope tracing experiments corroborate the near cessation of acetyl-CoA production, fatty acid synthesis, and ketone body synthesis from glucose-derived carbon. The tracing experiments present an almost complete switch to acetate-derived carbon to restore nucleocytosolic acetyl-CoA levels back to wild-type levels. Together, these findings indicate a major reprogramming event when the key ACLY enzyme is lost, shifting the burden of acetyl-CoA production and fatty acid synthesis onto ACSS2. Interestingly, the combined deletion of ACLY and ACSS2 produces a pronounced toxic effect. This promising phenotype is ripe for further investigation and therapeutic leveraging.
Another known consequence of ACLY inhibition is a profound decrease in global histone acetylation. Surprisingly, compensation by ACSS2 could not rescue this observed decrease either globally or at specific histone tail residues. Although the levels of available nucleocytosolic acetyl-CoA were restored, acetylation at H4K5, H3K14, H3K18, H3K23 and H3K27 remained low until extracellular acetate was brought to ten-fold excess over physiological concentrations. This result is both frustrating and stimulating for Zhao, remaining unanswered and yet presenting an enticing avenue for further study. Zhao elaborates that “[the acetylation result] tells us there's obviously much more that ACLY does for the cell besides strictly acetyl-CoA production that we're still unclear about,” and ranks elucidating the underlying mechanism of action as one of his top priorities as the work continues. Further priorities for Zhao include understanding the extent and profile of the compensation for ACLY ablation in vivo. As seen in the cell models, ACSS2 levels increased because of ACLY deletion in epididymal and inguinal white adipocyte deposits. Moreover, the same enrichment was seen for acetate-derived acetyl-CoA and fatty acids. Nonetheless, de novo lipid synthesis decreased and remained decreased in some of the tested adipocyte populations, indicating a complex set of tissue-specific responses wherein glucose and acetate utilization may have differing roles. The elucidation of how these different tissues respond to ACLY loss, through ACSS2 or other unknown mechanisms, would go far to identifying the most promising pathways for ACLY therapies.
ATP-citrate lyase (ACLY) is the predominant means of acetyl-CoA production, which is important for lipogenesis, maintenance of histone acetylation marks, and cell proliferation. ACLY deficiency causes upregulation of acetyl-CoA synthetase (ACSS2). Under ACLY deficiency, ACSS2 requires exogenous acetate to restore acetyl-CoA levels. This leads to a partial rescue of lipid synthesis and cell viability but not histone acetylation.
Steven Zhao continues to study acetyl-CoA regulation and metabolism and is currently focusing on examining the in vivo contexts for these assembled findings.
Zhao, S., Torres, A., Henry, R.A., Trefely, S., Wallace, M., Lee, J.V., Carrer, A., Sengupta, A., Campbell, S.L., Kuo, Y.-M., et al. (2016). ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch. Cell Rep. 17, 1037–1052.