Mounting evidence has shown that CETP has important physiological roles in adapting to chronic nutrient excess, specifically, to protect against diet-induced insulin resistance. However, the underlying mechanisms for the protective roles of CETP in metabolism are not yet clear. Mice naturally lack CETP expression. We used transgenic mice with a human CETP minigene (huCETP) controlled by its natural flanking region to further understand CETP-related physiology in response to obesity. Female huCETP mice and their wild-type littermates were fed a high-fat diet for 6 months. Blood lipid profile and liver lipid metabolism were studied. Insulin sensitivity was analyzed with euglycemic-hyperinsulinemic clamp studies combined with 3H-glucose tracer techniques. While high-fat diet feeding induced obesity for huCETP mice and their wild-type littermates lacking CETP expression, insulin sensitivity was higher for female huCETP mice than for their wild-type littermates. There was no difference in insulin sensitivity for male huCETP mice vs. littermates. The increased insulin sensitivity in females was largely caused by the better insulin-mediated suppression of hepatic glucose production. In huCETP females, CETP in the circulation decreased HDL-cholesterol content and increased liver cholesterol uptake and liver cholesterol and oxysterol contents, which was associated with the upregulation of LXR target genes in long-chain polyunsaturated fatty acid biosynthesis and PPARα target genes in fatty acid β-oxidation in the liver. The upregulated fatty acid β-oxidation may account for the improved fatty liver and liver insulin action in female huCETP mice. This study provides further evidence that CETP has beneficial physiological roles in the metabolic adaptation to nutrient excess by promoting liver fatty acid oxidation and hepatic insulin sensitivity, particularly for females.

We hypothesized that a healthy diet and exercise improve energy metabolism through different pathways in males and females. Understanding the mechanism for a how healthy diet vs. exercise improves metabolic outcomes is important for intervention through lifestyle change to target risk factors for obesity-associated diseases in specific populations. Mice were fed a high-fat diet (HFD) for 12 weeks, then divided into 4 groups, followed for 6 weeks: a. HFD; b. HFD + exercise; c. the diet was switched to chow; d. diet-switch + exercise. Instead of acute metabolic effects, we investigated sustained effects of exercise by examining tissue glucose uptake and metabolism 48 hours after the last exercise session. Diet-switch and exercise reduced obesity and fat contents in tissues in both sexes. Muscle glucose uptake was higher in males than females and not different between treatments 48 hours after exercise session. Liver glucose uptake was higher in males than females. Exercise, but not diet-switch, lowered liver glucose uptake, which effect was greater in males than females. Glucose uptake in subcutaneous, visceral, and brown adipose tissues was higher in females than males. Diet-switch, but not exercise, reduced glucose uptake in brown fat, which effect was greater in females than males. Furthermore, Diet-switch decreased the pathways for adipocyte differentiation (PPARγ) and lipogenesis (DGAT1/2) in subcutaneous adipose tissues in females. Muscle protein levels of CPT1β and pACC were increased by diet-switch in both sexes, and by exercise in males only. Our data indicate: 1) adipose tissue nutrient storage is the major energy reservoir that protects against obesity-related metabolic changes in females; 2) with regard to improving metabolic outcomes, diet-switch benefits females more and exercise benefits males more; for both sexes 3) exercise training is more effective in preventing fatty liver than diet-switch, and 4) both diet-switch and exercise promote fat oxidation in muscles. Disclosure J. An: None. L. Zhu: None. S. Yu: None. E. Edington: None. B. Litts: None. J. M. Stafford: None. Funding National Institutes of Health (R01HL144846)

Among the hallmarks of non-small cell lung cancer (NSCLC) is cancer immunoediting. Immunoediting results when cancer cells evade immune recognition via positive selection for cells that do not present immunogenic antigens recognized by CD8+ T cells. An epigenetic therapy-based approach has been developed in our lab to enhance expression of tumor antigens using combinatorial DNA methyltransferase inhibition (DNMTi) paired with histone deacetylase inhibition (HDACi). However, durable responses from the application of epigenetic therapy alone or in combination with other therapies have remained elusive in NSCLC. This may be because enhanced proliferation and infiltration of tumor-associated antigen specific CD8+ T cells are necessary to recognize epigenetically induced antigens to halt disease progression. In the current study, we identified epigenetic therapy inducible self-antigens that can potentially be used to activate and guide the differentiation of CD8+ T cells specific to NSCLC tumor antigens. The identification of these candidates was derived from analyses of paired RNAseq data from NSCLC patients treated with combination epigenetic therapy. From these analyses, differentially expressed transcripts selected from total transcriptome data were filtered to remove non-protein coding genes and genes with detectable expression in normal tissues as indicated by GTEx. HLA binding affinity (HLA-A*0201) of selected targets was defined by NetMHC 4.0 artificial neural network-based prediction to identify strong binding peptide candidates. The combined results of these data filtering facilitated the identification of 34 epigenetic therapy induced genes and associated peptides (9mers) with predicted high binding affinity for HLA-A*0201. The predicted peptides of target genes were selected for single peptide pulsing in allogenic dendritic cells to generate antigen specific CD8+ T cells for TCR sequencing (TCRseq). TCRseq libraries derived from peptide stimulation experiments revealed distinct V and J gene frequencies and emergence of antigen specific clonotypes, which suggest clonal expansion. To validate whether epigenetic therapy could induce target genes expression in an independent experimental model, in vitro human NSCLC cell lines were treated with DNMTi and HDACi to mimic the clinical drug paradigm and transcriptional augmentation was assessed by qRT-PCR. In this orthogonal system, we validated eight target genes that were induced by epigenetic therapy in vitro. Altogether, these data suggest that a conserved subset of genes, whose derived proteins are predicted to be presented in class I MHC and act as T cell epitopes, is induced by combination epigenetic treatment. Our studies provide a basis for the development of novel and personalized strategies to improve outcomes and quality of life for more patients with NSCLC. Citation Format: Julia An, Valsamo Anagnostou, Kristen A. Marrone, Victor E. Velculescu, Julie R. Brahmer, Stephen B. Baylin, Michael J. Topper. Identification of epigenetic therapy induced tumor antigens to promote proliferation and infiltration of antigen specific T cells in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3278.