Mechanisms of asparaginase-associated hepatic steatosis and dysfunction
Creators: name: Zalma, Brian Andrew
Publisher: name: No Publisher Supplied
Year: 2025

description: Asparaginase is an enzyme that degrades asparagine and has been used as a chemotherapeutic agent to treat acute lymphoblastic leukemia (ALL) since the 1960s. As a chemotherapeutic, asparaginase has an unfavorable toxicity profile. Hepatic steatosis is a common adverse event in patients with overweight and obesity. The mechanistic understanding of how hepatic steatosis and dysfunction develops in response to asparaginase is unclear. Asparaginase activates the highly conserved integrated stress response (ISR). The ISR consists of four kinases which respond to different stress stimuli and phosphorylate the α subunit of the eukaryotic initiation factor 2 (eIF2α). The eIF2α kinases that respond to amino acid insufficiency and endoplasmic reticulum (ER) stress are general control nonderepressible 2 (GCN2) and protein kinase R-like ER-resident kinase (PERK), respectively. Phosphorylation of eIF2α suppresses global translation and concomitantly increases the synthesis of stress response transcription factors. These proteins bind DNA to regulate transcription of genes that are involved in cellular protection and recovery and include amino acid synthesis, lipid metabolism, and the cellular catabolic process, autophagy. The first aim of this dissertation sought to clarify the relative contributions of obesity and high-fat feeding to hepatic steatosis and ER stress during asparaginase exposure. To accomplish this, lean mice consumed a high-fat, obesogenic diet (OD) and obese mice consumed a low-fat, maintenance diet (MD) during exposure to asparaginase. Acute consumption of an OD to lean mice was sufficient to induce mild hepatic steatosis, but not ER stress. Consumption of a MD to obese mice did not prevent severe hepatic steatosis or ER stress in the liver. The severity of hepatic steatosis in both lean and obese mice corresponded with the magnitude of asparaginase-induced weight loss. Rates of hepatic triglyceride secretion were also measured and this revealed decreased triglyceride export from the liver in obese mice relative to lean mice and especially in combination with asparaginase. In addition, liver metabolomics showed an accumulation of sphingolipids in asparaginase-exposed obese mice, suggesting lipotoxicity and ER stress. Indeed, only obese mice showed activation of PERK during asparaginase exposure, and this activation correlated with a signature of lipid intermediates that included fatty acid metabolites, diacylglycerols, and sphingolipids. The contribution of lipolysis to steatosis was also tested by comparing mice exposed to asparaginase for 24 h to mice fasted the same amount of time, revealing that asparaginase increases hepatic steatosis similar to fasting. Taken together, the findings of this aim show that the greater magnitude of asparaginase-induced hepatic steatosis in obesity is driven by lipolysis associated with body weight loss in combination with reduced triglyceride secretion. ER stress in the liver likely develops as a consequence of increased adipose-derived fatty acid metabolites.The second aim of this dissertation was to define the role of autophagy in the hepatic stress response to asparaginase. Elevated sphingolipids are associated with dysfunctional autophagy, and the observed correlation between sphingolipids and PERK phosphorylation in Aim 1 suggested a possible role for autophagy in the hepatic stress response to asparaginase. To explore the role of autophagy, asparaginase was administered to mice with a global deletion of a core autophagy gene, autophagy related 7 (Atg7). Atg7 is necessary for expansion of the autophagosomal membrane and the systemic deletion of Atg7 in mice blocks autophagic flux. The deletion of Atg7 in adult mice induces hepatocellular injury, hepatic steatosis, and eventually leads to death. Surprisingly, Atg7-deficient mice (Atg7Δ/Δ) were protected from asparaginase-induced anorexia and weight loss. Interrogation of the ISR in the liver showed that the loss of Atg7 suppresses global translation and transcriptional execution of the ISR. To test the contribution of Atg7, specifically in the liver, to the modulation of food intake and body weight loss, asparaginase was administered to mice with a liver-specific deletion of Atg7 (ls-Atg7KO). Ls-Atg7KO mice were also protected from asparaginase-induced weight loss, but not anorexia. Transcriptional execution of the ISR in the livers of ls-Atg7KO mice was blunted in a similar manner to that of Atg7Δ/Δ mice. This dissertation provides evidence that obesity predisposes mice to asparaginase-induced hepatic steatosis by a combination of weight loss-induced lipolysis consequential to anorexia alongside reduced rates of hepatic TG secretion. Additionally, this dissertation shows that Atg7 is necessary for the development of hepatic steatosis during asparaginase exposure but does not play a critical role in the protection against asparaginase-induced liver dysfunction. Taken together, these insights provide a foundation for future research for both dietary and pharmaceutical interventions to mitigate this adverse event in patients with ALL.