Background & Aims Nonalcoholic fatty liver disease (NAFLD) is a common consequence of human and rodent obesity. Disruptions in lipid metabolism lead to accumulation of triglycerides and fatty acids, which can promote inflammation and fibrosis and lead to nonalcoholic steatohepatitis (NASH). Circulating levels of fibroblast growth factor (FGF)21 increase in patients with NAFLD or NASH, so we assessed the role of FGF21 in the progression of murine fatty liver disease, independent of obesity, caused by methionine and choline deficiency. Methods C57BL/6 wild-type and FGF21-knockout (FGF21-KO) mice were placed on methionine- and choline-deficient (MCD), high-fat, or control diets for 8–16 weeks. Mice were weighed; serum and liver tissues were collected and analyzed for histology, levels of malondialdehyde and liver enzymes, gene expression, and lipid content. Results The MCD diet increased hepatic levels of FGF21 mRNA more than 50-fold and serum levels 16-fold, compared with the control diet. FGF21-KO mice had more severe steatosis, fibrosis, inflammation, and peroxidative damage than wild-type C57BL/6 mice. FGF21-KO mice had reduced hepatic fatty acid activation and β oxidation, resulting in increased levels of free fatty acid. FGF21-KO mice given continuous subcutaneous infusions of FGF21 for 4 weeks while on MCD diets had reduced steatosis and peroxidative damage, compared with mice not receiving FGF21. The expression of genes that regulate inflammation and fibrosis were reduced in FGF21-KO mice given FGF21, similar to those of wild-type mice. Conclusions FGF21 regulates fatty acid activation and oxidation in livers of mice. In the absence of FGF21, accumulation of inactivated fatty acids results in lipotoxic damage and increased steatosis.

ObjectiveIncreased fructose consumption is a contributor to the burgeoning epidemic of non-alcoholic fatty liver disease (NAFLD). Recent evidence indicates that the metabolic hormone FGF21 is regulated by fructose consumption in humans and rodents and may play a functional role in this nutritional context. Here, we sought to define the mechanism by which fructose ingestion regulates FGF21 and determine whether FGF21 contributes to an adaptive metabolic response to fructose consumption.MethodsWe tested the role of the transcription factor carbohydrate responsive-element binding protein (ChREBP) in fructose-mediated regulation of FGF21 using ChREBP knockout mice. Using FGF21 knockout mice, we investigated whether FGF21 has a metabolic function in the context of fructose consumption. Additionally, we tested whether a ChREBP-FGF21 interaction is likely conserved in human subjects.ResultsHepatic expression of ChREBP-β and Fgf21 acutely increased 2-fold and 3-fold, respectively, following fructose gavage, and this was accompanied by increased circulating FGF21. The acute increase in circulating FGF21 following fructose gavage was absent in ChREBP knockout mice. Induction of ChREBP-β and its glycolytic, fructolytic, and lipogenic gene targets were attenuated in FGF21 knockout mice fed high-fructose diets, and this was accompanied by a 50% reduction in de novo lipogenesis a, 30% reduction VLDL secretion, and a 25% reduction in liver fat compared to fructose-fed controls. In human subjects, serum FGF21 correlates with de novo lipogenic rates measured by stable isotopic tracers (R = 0.55, P = 0.04) consistent with conservation of a ChREBP-FGF21 interaction. After 8 weeks of high-fructose diet, livers from FGF21 knockout mice demonstrate atrophy and fibrosis accompanied by molecular markers of inflammation and stellate cell activation; whereas, this did not occur in controls.ConclusionsIn summary, ChREBP and FGF21 constitute a signaling axis likely conserved in humans that mediates an essential adaptive response to fructose ingestion that may participate in the pathogenesis of NAFLD and liver fibrosis.

Tazemetostat (EZM6438) is a potent, orally bioavailable small molecule inhibitor of EZH2, the enzymatic subunit of the polycomb repressive complex 2, which has been approved for treatment of epithelioid sarcoma and relapsed/refractory follicular lymphoma. EZH2 has been shown to play a key role in B-cell maturation and multiple B-cell malignancies are dependent on EZH2 for survival. Mantle cell lymphoma (MCL) is a rare subtype of mature B cell non-Hodgkin lymphoma characterized by the t(11;14)(q13;q32) translocation leading to overexpression of cyclin D1, which plays a significant role in tumor cell proliferation via cell cycle dysregulation, chromosomal instability, and epigenetic regulation. This disease most often presents at an advanced stage and while initial responses occur, most cases relapse to frontline therapy, including Bruton's Tyrosine Kinase (BTK) inhibitors. The rate of intrinsic and acquired resistance to these treatments results in a high unmet medical need. We previously reported that treatment with EZH2 inhibitors, as monotherapy or in combination with BTK inhibitors, elicited in vitro anti-proliferative activity and in vivo tumor growth inhibition in MCL models, demonstrating that EZH2 may be a promising therapeutic target in this indication. To extend our previous in vitro combination studies, we demonstrated that the recently approved BTK inhibitor, zanubrutinib synergized with tazemetostat and demonstrated that tazemetostat resensitized a subset of cell lines intrinsically resistant to BTK inhibitors. In vivo studies confirmed these findings, showing a significant tumor growth delay when tazemetostat was combined with zanubrutinib in the MCL MINO cell line-derived murine xenograft model compared to the single agent treatments. To understand if EZH2 inhibition offered potential therapeutic benefit to the MCL BTK inhibitor-resistant population, we generated in vitro MCL models of acquired resistance to the BTK inhibitors ibrutinib and zanubrutinib. These cell lines retained the in vitro sensitivity to tazemetostat observed in the parental cell line, suggesting a potential therapeutic option for tazemetostat in the setting of acquired resistance to BTK inhibitors. Additionally, we showed that tazemetostat induced anti-proliferative effects ex vivo in samples derived from MCL patients that were relapsed or refractory to one or more current standard of care agents, including the BTK inhibitor ibrutinib. Subsequent studies aim to identify the mechanisms of growth inhibition driven by tazemetostat in MCL models sensitive and resistant to BTK inhibitors. In summary, these data suggest that tazemetostat treatment (alone or in combination with a BTK inhibitor) could be a potential therapeutic option in the treatment of the MCL patient population that is relapsed/refractory to BTK inhibitor therapy. Citation Format: Jeffrey A. Keats, Arleide Lee, Jeremy C. Cunniff, Weiqing Chen, Revonda Mehovic, Vania Estanek, Crag Markwood, Cuyue Tang, Daniel T. Dransfield, Veronica Gibaja, Alejandra Raimondi. EZH2 inhibitor tazemetostat demonstrates activity in preclinical models of Bruton's tyrosine kinase inhibitor-resistant relapsed/refractory mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1161.