Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with median survival of less than one year and overall 5-year survival less than 5%. Gemcitabine has been the cornerstone in PDA treatment, yet its efficacy remains limited. Over the past decades, extensive investigations suggested a role of tumor microenvironment (TME) in rendering PDA resistance to gemcitabine, however, its underlying mechanism remained largely unknown. Cellular profiling has revealed the macrophages as a major immune cell component of PDA-TME. Our recent study showed that the expression of the homeobox protein VentX, a master regulator of macrophage plasticity, is significantly down-regulated in tumor associated macrophages (TAMs) of PDA. We demonstrated that restoration of VentX expression in PDA-TAMs polarizes the TAMs from a pro-tumor M2-like phenotype to an anti-tumor M1-like phenotype. We showed further that VentX-regulated-TAMs (VentX-TAMs) revert immune suppression of PDA-TME by inhibiting CD4 Treg differentiation and by promoting CD8 proliferation and activation. Using a newly developed tumor immune microenvironment-enabling model system (TIME-EMS), we showed that VentX-TAMs drastically promote efficacy of gemcitabine against PDA for about 4-fold but not cytotoxicity on normal pancreatic tissue. As such, our data suggested a function of VentX-TAMs to promote chemosensitivity of gemcitabine and to improve PDA prognosis. Citation Format: Joanna Le, Hong Gao, Yi Le, William Richards, Scott Radig, Ronald Bleday, Thomas Clancy, Zhenglun Zhu. VentX-modulated tumor associated macrophages revert immune suppression in tumor microenvironment and promote efficacy of gemcitabine against pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5111.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy that has no effective treatment. The tumor microenvironment (TME) of PDA employs a multitude of immune derangement strategies to protect PDA from immune elimination. Tumor-associated macrophages (TAMs) have been implicated in the pathogenesis of immune suppression of the PDA TME; however, its underlying mechanisms remained largely unknown. Using primary patient samples, our studies showed that, in comparison with macrophages isolated from normal pancreatic tissues, the phagocytosis activity of the PDA TAMs was significantly reduced. We found that the expression of homeobox protein VentX, a master regulator of macrophage plasticity, was significantly decreased in the PDA TAMs. We demonstrated that VentX was required for phagocytosis and that restoration of VentX expression in PDA TAMs promoted phagocytosis through the regulation of the signaling cascades involved in the process. Using an ex vivo culture model of primary human PDA, we showed that VentX-modulated TAMs transformed the PDA TME from a protumor milieu to an antitumor microenvironment by rectifying differentiation, proliferation, and activation of PDA-infiltrating immune cells. Using NSG-PDX models of primary human PDAs, we showed that VentX-modulated TAMs exerted strong inhibition on PDA tumorigenesis in vivo. Taken together, our data revealed a central mechanism underlying immune evasion of PDA and a potential novel venue to improve PDA prognosis.

Myelodysplastic syndromes (MDS) are clonal malignant hematopoietic disorders in the elderly characterized by ineffective hematopoiesis. This is accompanied by an altered bone microenvironment, which contributes to MDS progression and higher bone fragility. The underlying mechanisms remain largely unexplored. Here, we show that myelodysplastic NUP98‑HOXD13 (NHD13) transgenic mice display an abnormally high number of osteoblasts, yet a higher fraction of nonmineralized bone, indicating delayed bone mineralization. This was accompanied by high fibroblast growth factor-23 (FGF-23) serum levels, a phosphaturic hormone that inhibits bone mineralization and erythropoiesis. While Fgf23 mRNA expression was low in bone, brain, and kidney of NHD13 mice, its expression was increased in erythroid precursors. Coculturing these precursors with WT osteoblasts induced osteoblast marker gene expression, which was inhibited by blocking FGF-23. Finally, antibody-based neutralization of FGF-23 in myelodysplastic NHD13 mice improved bone mineralization and bone microarchitecture, and it ameliorated anemia. Importantly, higher serum levels of FGF‑23 and an elevated amount of nonmineralized bone in patients with MDS validated the findings. C‑terminal FGF‑23 correlated negatively with hemoglobin levels and positively with the amount of nonmineralized bone. Thus, our study identifies FGF-23 as a link between altered bone structure and ineffective erythropoiesis in MDS with the prospects of a targeted therapeutic intervention.

1Dept. of Nephrology and Institute Cardiovascular Research VU (ICaR-VU), VU University Medical Center, Amsterdam, The Netherlands, 2Dept. of Physiology, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands, 3Dept. of Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands, 4Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, United States. On behalf of the NIGRAM consortium

Squamous Lung Cancer (SC) which constitutes 30% of all non-small cell lung cancers (NSCLC) has few targeted therapy options for advanced disease. Surgery for early SC is the best treatment strategy; however, even patients who undergo surgery for stage IA or IB disease are still at a substantial risk for recurrence and death. Adjuvant therapy is not currently indicated for stage I SC smaller than 4 cm. Prior reports suggest gene expression-based signatures that may predict recurrence in patients with stage I SC, but none has been validated or is in clinical use. The SPECS2 Lung Cancer Consortium was assembled to compare and attempt to validate previously published prognostic signature(s) according to the guidelines proposed by Subramanian and Simon (J Natl Cancer Inst 2010; 7:327). The multi-institutional team assembled 249 frozen SC samples representing six participating institutions (cohort 1). These samples were fully annotated in a redcap database hosted by the independent statistical core. Cohort 2 was assembled utilizing 234 frozen SC samples from a prospective multi-institutional NCTN lung biobanking protocol (NCT00899782). RNA was extracted and profiled with U133A microarrays (Affymetrix) in independent core facilities. The data was transferred directly to the SPECS2 Lung statistical core in collaboration with the Alliance Statistical core and the performance of 6 most promising candidate signatures was evaluated relative to a base model that included only age, gender and AJCC stage (editions 6, 7, 8). Analysis of Cohort 1 demonstrated that only one signature (Raponi et al, Cancer Res 2006; 66:7466) significantly enhanced prognosis relative to the base model, independent of AJCC edition. This was also observed in Cohort 2, where Uno’s C index associated with AJCC 8th edition stage, sex and age (0.561; 0.468-0.654) was significantly (p <0.05) increased when the prognostic signature was added to the model (0.683; 0.611-0.755). The SPECS2 Lung Cancer Consortium was successful in validating a previously published prognostic molecular signature for early stage SC using rigorous experimental design. To our knowledge, this is the first unbiased validation of a lung cancer prognostic signature using multi-institutional prospective specimens. These results support a clinical trial designed to evaluate the potential role of adjuvant therapy in completely resected early stage SC.

Calcimimetics decrease parathyroid hormone (PTH) secretion in patients with secondary hyperparathyroidism. The decrease in PTH should cause a reduction in bone turnover; however, the direct effect of calcimimetics on bone cells, which express the calcium-sensing receptor (CaSR), has not been defined. In this study, we evaluated the direct bone effects of CaSR activation by a calcimimetic (AMG 641) in vitro and in vivo. To create a PTH "clamp," total parathyroidectomy was performed in rats with and without uremia induced by 5/6 nephrectomy, followed by a continuous subcutaneous infusion of PTH. Animals were then treated with either the calcimimetic or vehicle. Calcimimetic administration increased osteoblast number and osteoid volume in normal rats under a PTH clamp. In uremic rats, the elevated PTH concentration led to reduced bone volume and increased bone turnover, and calcimimetic administration decreased plasma PTH. In uremic rats exposed to PTH at 6-fold the usual replacement dose, calcimimetic administration increased osteoblast number, osteoid surface, and bone formation. A 9-fold higher dose of PTH caused an increase in bone turnover that was not altered by the administration of calcimimetic. In an osteosarcoma cell line, the calcimimetic induced Erk1/2 phosphorylation and the expression of osteoblast genes. The addition of a calcilytic resulted in the opposite effect. Moreover, the calcimimetic promoted the osteogenic differentiation and mineralization of human bone marrow mesenchymal stem cells in vitro. Thus, calcimimetic administration has a direct anabolic effect on bone that counteracts the decrease in PTH levels.

Tumor-infiltrating myeloid cells (TIMs) comprise monocytes, macrophages, dendritic cells, and neutrophils, and have emerged as key regulators of cancer growth. These cells can diversify into a spectrum of states, which might promote or limit tumor outgrowth but remain poorly understood. Here, we used single-cell RNA sequencing (scRNA-seq) to map TIMs in non-small-cell lung cancer patients. We uncovered 25 TIM states, most of which were reproducibly found across patients. To facilitate translational research of these populations, we also profiled TIMs in mice. In comparing TIMs across species, we identified a near-complete congruence of population structures among dendritic cells and monocytes; conserved neutrophil subsets; and species differences among macrophages. By contrast, myeloid cell population structures in patients' blood showed limited overlap with those of TIMs. This study determines the lung TIM landscape and sets the stage for future investigations into the potential of TIMs as immunotherapy targets.

Junho Choe, Shuibin Lin, Wencai Zhang, Qi Liu, Longfei Wang, Julia Ramirez7 Moya, Peng Du, Wantae Kim, Shaojun Tang, Piotr Sliz, Pilar Santisteban, Rani E. 8 George, William G. Richards, Kwok-Kin Wong, Nicolas Locker, Frank J. Slack, 9 and Richard I. Gregory 2, 10, 14, 15, 17 10 11 Stem Cell Program, Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA 02115, USA 12 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, 13 USA 14 Center for Translational Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 15 Guangdong 510080, China 16 Department of Pathology, Cancer Center, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA 17 Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad 18 Autónoma de Madrid (CSIC-UAM), Madrid, Spain 19 Harvard School of Dental Medicine, Boston MA 02115, USA 20 Innovation Center for Biomedical Informatics, Georgetown University Medical Center, Washington, DC20057, 21 USA 22 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 23 20057, USA 24 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA 25 Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA 26 Department of Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA 27 Division of Hematology and Medical Oncology, NYU School of Medicine, New York, NY 10016 28 School of Biosciences and Medicine, University of Surrey, Guildford, United Kingdom 29 Harvard Initiative for RNA Medicine, Boston MA 02115, USA 30 Harvard Stem Cell Institute, Cambridge, MA 02138, USA 31 Present address: Biomedical Translational Research Center, Korea Research Institute of Bioscience and 32 Biotechnology (KRIBB), Daejeon 34141, Republic of Korea. 33 34 35 Corresponding author: 36 Richard I. Gregory 37 Phone: (617) 919-2273 38 E-mail: rgregory@enders.tch.harvard.edu 39 40

Cardiovascular diseases account for ~50% of mortality in patients with chronic kidney disease (CKD). Fibroblast growth factor 23 (FGF23) is independently associated with endothelial dysfunction and cardiovascular mortality. We hypothesized that CKD impairs microvascular endothelial function and that this can be attributed to FGF23. Mice were subjected to partial nephrectomy (5/6Nx) or sham surgery. To evaluate the functional role of FGF23, non-CKD mice received FGF23 injections and CKD mice received FGF23-blocking antibodies after 5/6Nx surgery. To examine microvascular function, myocardial perfusion in vivo and vascular function of gracilis resistance arteries ex vivo were assessed in mice. 5/6Nx surgery blunted ex vivo vasodilator responses to acetylcholine, whereas responses to sodium nitroprusside or endothelin were normal. In vivo FGF23 injections in non-CKD mice mimicked this endothelial defect, and FGF23 antibodies in 5/6Nx mice prevented endothelial dysfunction. Stimulation of microvascular endothelial cells with FGF23 in vitro did not induce ERK phosphorylation. Increased plasma asymmetric dimethylarginine concentrations were increased by FGF23 and strongly correlated with endothelial dysfunction. Increased FGF23 concentration did not mimic impaired endothelial function in the myocardium of 5/6Nx mice. In conclusion, impaired peripheral endothelium-dependent vasodilatation in 5/6Nx mice is mediated by FGF23 and can be prevented by blocking FGF23. These data corroborate FGF23 as an important target to combat cardiovascular disease in CKD. NEW & NOTEWORTHY In the present study, we provide the first evidence that fibroblast growth factor 23 (FGF23) is a cause of peripheral endothelial dysfunction in a model of early chronic kidney disease (CKD) and that endothelial dysfunction in CKD can be prevented by blockade of FGF23. This pathological effect on endothelial cells was induced by long-term exposure of physiological levels of FGF23. Mechanistically, increased plasma asymmetric dimethylarginine concentrations were strongly associated with this endothelial dysfunction in CKD and were increased by FGF23.

Bispecific antibodies have become important formats for therapeutic discovery. They allow for potential synergy by simultaneously engaging two separate targets and enable new functions that are not possible to achieve by using a combination of two monospecific antibodies. Antagonistic antibodies dominate drug discovery today, but only a limited number of agonistic antibodies (i.e. those that activate receptor signaling) have been described. For receptors formed by two components, engaging both of these components simultaneously may be required for agonistic signaling. As such, bispecific antibodies may be particularly useful in activating multicomponent receptor complexes. Here, we describe a biparatopic (i.e. targeting two different epitopes on the same target) format that can activate the endocrine fibroblast growth factor (FGF) 21 receptor (FGFR) complex containing β-Klotho and FGFR1c. This format was constructed by grafting two different antigen-specific VH domains onto the VH and VL positions of an IgG, yielding a tetravalent binder with two potential geometries, a close and a distant, between the two paratopes. Our results revealed that the biparatopic molecule provides activities that are not observed with each paratope alone. Our approach could help address the challenges with heterogeneity inherent in other bispecific formats and could provide the means to adjust intramolecular distances of the antibody domains to drive optimal activity in a bispecific format. In conclusion, this format is versatile, is easy to construct and produce, and opens a new avenue for agonistic antibody discovery and development.

N6-methyladenosine (m6A) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered m6A homeostasis is linked to cancer1–5. m6A modification is catalysed by METTL3 and enriched in the 3′ untranslated region of a large subset of mRNAs at sites close to the stop codon5. METTL3 can promote translation but the mechanism and relevance of this process remain unknown1. Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon, supporting a mechanism of mRNA looping for ribosome recycling and translational control. Electron microscopy reveals the topology of individual polyribosomes with single METTL3 foci in close proximity to 5′ cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs—including bromodomain-containing protein 4—that is also m6A-modified in human primary lung tumours. The METTL3–eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mechanism of translation control that is based on mRNA looping and identify METTL3–eIF3h as a potential therapeutic target for patients with cancer. METTL3, the enzyme responsible for m6A modification, influences translation by interacting with eIF3h to mediate looping between the regions near the stop codon and 5′ cap of mRNA.

Endocrine fibroblast growth factors (FGFs) require Klotho transmembrane proteins as necessary co-receptors to activate FGF receptor (FGFR) signaling. In particular, FGF19 and FGF21 function through β-Klotho to regulate glucose and lipid metabolism. Recent research has focused on elucidating how these two FGFs interact with β-Klotho and FGFRs to activate downstream signaling. In this study, using hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS), we identified regions on the β-Klotho protein that likely participate in ligand interaction, and vice versa. Alanine and arginine mutagenesis were carried out to further probe the contributions of individual residues to receptor/ligand interactions. Using biochemical and cell-based signaling assays with full-length proteins, we show that both the KL1 and KL2 domains of β-Klotho participate in ligand interaction, and these binding sites on β-Klotho are shared by FGF19 and FGF21. In addition, we show that two highly conserved regions in the C-terminal tail of FGF19 and FGF21 are responsible for interaction with the co-receptor. Our results are consistent with recent publications on the crystal structures of the Klotho proteins and provide insight into how endocrine FGFs interact with co-receptors for signal transduction.

Surgical resection is offered to some patients with malignant pleural mesothelioma (MPM), depending on fitness for surgery and apparent technical resectability of the tumor. Due to risk of surgery-related morbidity and mortality in patients with a fatal malignancy, life expectancy is also a primary consideration in the surgical decision. Prognosis is related to the stage and histological subtype of the disease, neither of which can be known with certainty prior to definitive pathological review of the tumor specimen. Therefore, radiology and pleural biopsy, respectively, are used to predict these prognostic factors and to determine potential benefit from surgical intervention. In this review, we discuss the current limitations of histologic subtyping using pleural biopsy as a preoperative predictor. In addition, we explore available and emerging non-histologic analyses of biopsy specimens to evaluate molecular biomarkers, predicted neoantigens and the immune microenvironment. In the context of novel biological and immune-based treatment strategies currently under investigation for mesothelioma, accurate prognostic and predictive information available from biopsy material will likely influence treatment strategy including the surgical decision.

With the recent clinical success of immunotherapy in non-small cell lung carcinoma, the character of the inflammatory infiltrate associated with these tumors is now the subject of increasing interest. Molecular studies have suggested that tumors can be stratified by the character of their inflammatory infiltrate. We now describe the detailed histological appearances of a multi-institutional series of early stage squamous carcinomas and correlate them with mutation burden, PDL1 expression patterns and clinical outcome. Histologic sections of from 250 tumors were evaluated by two pathologists independently for squamous subtype (WHO classification), percentage and character of intratumoral inflammatory cells, percentage and character of para-tumoral infiltrate and presence or absence of scalloping at tumor cell/stromal interface by inflammatory cells along the edges of tumor cell nests, a feature possibly related to existing immune reaction. The ratios of infiltrating inflammatory cells to tumor cells were estimated in 10% increments by microscopic inspection. Quantity and character of infiltrates was assessed by Kaplan-Meir testing for effect on survival and by Pearson bivariate testing for relationships among variables. The character and extent of inflammatory infiltrates were highly heterogeneous. The infiltrates could be divided into intratumoral and paratumoral patterns according to their location in relation to microscopic tumor cell nests. Intratumoral infiltrates could be further subdivided into two patterns: one consisted exclusively lymphocytes, usually few in number; a second polymorphous pattern contained many inflammatory cell types including polymorphonuclear leukocytes (PMNs). In paratumoral tissue, three patterns could be discerned: lymphocytic, plasmacytic and polymorphous. Inflammatory cell infiltrate quantity or character did not correlate with survival for either intratumoral or paratumoral infiltrates and there was no evident relationship to mutational burden or to PDL1 expression by IHC. Scalloping at the tumor cell stromal interface was also not prognostically significant. The inflammatory infiltrates in early stage squamous lung carcinoma are highly heterogenous and are not associated with outcome. However, the complexity of tumor infiltrating inflammatory cells is worthy of further evaluation in future immunotherapeutic trials.

Tumor volumetrics is currently being explored as a means of enhancing the accuracy of clinical staging of malignant pleural mesothelioma (MPM). In a recent multicenter study (JTO 2016, 11(8):1335-1344) evaluating feasibility of volume estimation using CT (VolCT), clinical staging by CT was poorly correlated between readers and underestimated pathological stage, but correlation of VolCT estimates between two experienced readers was high (rho = 0.82). MR has been found superior to CT in evaluating certain staging parameters, and may enhance volumetrics based on improved contrast. Here we evaluate the relative performance and reproducibility of MR for volumetrics (VolMR) and clinical staging of MPM in a single institution cohort.

High-throughput gene expression profiling led to proposal of multiple expression-based prognostic signatures for squamous cell lung carcinoma (SCC), but none has been validated. A multi-institutional squamous lung cancer consortium of investigators is developing prognostic signatures through the US NCI Lung SPECS (Strategic Partnership for Evaluation of Cancer Signatures) program. Six institutions contributed tumor specimens and published/unpublished expression-based prognostic signatures for validation using standardized sample cohorts (a primary validation cohort comprising institutional cases, and additional validation cohorts from two prospective cooperative group studies) and quality controlled assessment in independent laboratory and statistical cores. Here, we report the results of the primary validation. Cases of primary SCC (by central pathology review) meeting clinical (Stage I-II; surgical treatment only; 3-year followup) and specimen quality criteria (Tumor cellularity ≥ 50%; necrosis ≤ 20%) were submitted. Clinical, pathological and outcome data were uploaded to a central database. Frozen tumor samples underwent centralized mRNA extraction (Qiagen Symphony), quality control (RIN ≥ 6.0) and microarray profiling (Affymetrix U133) in core labs. An independent statistical core assessed validation of 7 pre-existing mRNA signatures and generated new models using MCP clustering. Among 250 cases meeting entry criteria, median age was 70 (43-92), 161 (65%) were male, and most were former (70%) or current (28%) smokers. Surgery was pneumonectomy: 5%; bilobectomy: 2%; lobectomy: 74%; sublobar: 18%. Pathologic staging was T1: 49%; T2: 50%; T3: 1%; N0: 88%; N1: 12%, and grade was G1: 4%; G2: 50%; G3: 44%. At followup, 148 (59%) were deceased. Three mRNA signatures demonstrated significant univariable association with OS and added independent prognostic value (see Figure) to a multivariable model accounting for age, sex and stage (c-index = 0.641). The validated signatures, along with two novel signatures generated from the current dataset, are currently undergoing further validation studies using two prospective co-operative group cohorts.

In renal failure, hyperphosphatemia occurs despite a marked elevation in serum fibroblast growth factor (FGF)-23. Abnormal regulation of the FGFR1-Klotho receptor complex may cause a resistance to the phosphaturic action of FGF23. The purpose of the present study was to investigate the regulation of renal Klotho and FGF receptor (FEFR)-1 in healthy and uremic rats induced by 5/6 nephrectomy. In normal rats, the infusion of rat recombinant FGF23 enhanced phosphaturia and increased renal FGFR1 expression; however, Klotho expression was reduced. Uremic rats on a high-phosphate (HP) diet presented hyperphosphatemia with marked elevation of FGF23 and an increased fractional excretion of phosphate (P) that was associated with a marked reduction of Klotho expression and an increase in FGFR1. After neutralization of FGF23 by anti-FGF23 administration, phosphaturia was still abundant, Klotho expression remained low, and the FGFR1 level was reduced. These results suggest that the expression of renal Klotho is modulated by phosphaturia, whereas the FGFR1 expression is regulated by FGF23. Calcitriol (CTR) administration prevented a decrease in renal Klotho expression. In HEK293 cells HP produced nuclear translocation of β-catenin, together with a reduction in Klotho. Wnt/β-catenin inhibition with Dkk-1 prevented the P-induced down-regulation of Klotho. The addition of CTR to HP medium was able to recover Klotho expression. In summary, high FGF23 levels increase FGFR1, whereas phosphaturia decreases Klotho expression through the activation of Wnt/β-catenin pathway.-Muñoz-Castañeda, J. R., Herencia, C., Pendón-Ruiz de Mier, M. V., Rodriguez-Ortiz, M. E., Diaz-Tocados, J. M., Vergara, N., Martínez-Moreno, J. M., Salmerón, M. D., Richards, W. G., Felsenfeld, A., Kuro-O, M., Almadén, Y., Rodríguez, M. Differential regulation of renal Klotho and FGFR1 in normal and uremic rats.

Pharmacological doses of fibroblast growth factor (FGF) 21 effectively normalize glucose, lipid and energy homeostasis in multiple animal models with many benefits translating to obese humans with type 2 diabetes. However, a role for FGF21 in the regulation of bile acid metabolism has not been reported. Herein, we demonstrate AAV-mediated FGF21 overexpression in mice increases liver expression of the key bile acid producing enzyme, Cyp7a1, resulting in an increased bile acid pool. Furthermore, in cholecystectomized mice, FGF21-mediated bile acid pool increase led to increased transit of bile acids into colon. We elucidate that the mechanism of FGF21 induced bile acid changes is mainly through antagonizing FGF15/19 function on liver βKlotho/FGFR4 receptor complex; thus inhibiting FGF15/19-mediated suppression of Cyp7a1 expression. In conclusion, these data reveal a previously unidentified role for FGF21 on bile acid metabolism and may be relevant to understand the effects of FGF21 analogs in clinical studies.