TARGETING MASH WITH NOVEL SMALL MOLECULE TXNIP INHIBITOR - Metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatitis (MASH) (aka NAFLD and NASH) affects 1 billion people, yet no approved therapy is available. Interestingly, our new antidiabetic small molecule thioredoxin-interacting protein (Txnip) inhibitor, TIX100, effectively protected obese and diabetic mice against steatosis and showed anti-inflammatory, anti-fibrotic effects in a non-diabetic, non-obese, model of MASH. Of note, Txnip is known to promote oxidative stress, apoptosis and inflammasome activation and to be elevated in livers of humans and mice with NAFLD/NASH. Thus, our overall hypothesis is that TIX100 protects against MASLD/MASH by improving glucose and lipid homeostasis and inhibiting inflammation and fibrosis. We propose 3 Specific Aims: #1: Determine the therapeutic effects of TIX100 in the context of diet-induced obesity and non-obese MASLD and MASH models. #2: Define the cellular TIX100 effects controlling MASH-associated inflammation and fibrosis and #3: Elucidate the molecular mechanisms underlying the beneficial effects of TIX100 in MASLD/MASH. To this end, we will assess liver histology, markers for lipogenesis, inflammation and fibrosis, hepatic and circulating lipids, and liver dysfunction, as well as any potential changes in whole body metabolism. This will be done in the context of MASLD, induced by high-fat diet obesity and of MASH and fibrosis, induced by a validated choline-deficient L-amino acid-defined high-fat diet. Involvement of carbohydrate response-element-binding protein, a key transcription factor of Txnip and lipogenic genes will also be determined by liver chromatin-immunoprecipitation studies. In addition, we will elucidate the specific TIX100 effects on hepatic stellate cells (HSC) and fibrogenesis, Kupffer cells and inflammation and cell autonomous effects using isolated primary hepatocytes. Using liver microRNA sequencing, we further discovered miR-34a as the top microRNA downregulated by TIX100. Intriguingly, miR-34a has been reported to promote lipogenesis and inflammation and to be upregulated in livers of humans and mice with MASLD/MASH. Now, our preliminary studies reveal that TIX100 treatment reduces the elevated miR-34a levels of MASLD/MASH and we will therefore continue to define the effects of TIX100 on miR-34a signaling in our liver, hepatocyte and HSC samples. To validate Txnip as a TIX100 target in the treatment of MASLD/MASH, we will take advantage of our whole body and liver cell-specific Txnip-deficient mouse models and expose them to DIO and CDAA-induced MASLD/MASH with and without TIX100 treatment. This will allow us to tease apart any Txnip-mediated (and potential Txnip- independent) effects and further define the mechanism(s) of TIX100 action in the treatment of MASLD/MASH. Thus, the results of these studies will provide mechanistic insight and critical preclinical data for the development of novel therapeutic approaches for MASLD/MASH at the intersection of metabolism, inflammation, and fibrosis. Since TIX100 has already undergone extensive preclinical safety toxicology and pharmacokinetic studies in preparation for first-in-man trials, the proposed studies also promise a high translational impact.
