FGFR inhibition blocks NF-ĸB-dependent glucose metabolism and confers metabolic vulnerabilities in cholangiocarcinoma.

Genomic alterations that activate Fibroblast Growth Factor Receptor 2 (FGFR2) are common in intrahepatic cholangiocarcinoma (ICC) and confer sensitivity to FGFR inhibition. However, the depth and duration of response is often limited. Here, we conduct integrative transcriptomics, metabolomics, and phosphoproteomics analysis of patient-derived models to define pathways downstream of oncogenic FGFR2 signaling that fuel ICC growth and to uncover compensatory mechanisms associated with pathway inhibition. We find that FGFR2-mediated activation of Nuclear factor-κB (NF-κB) maintains a highly glycolytic phenotype. Conversely, FGFR inhibition blocks glucose uptake and glycolysis while inciting adaptive changes, including switching fuel source utilization favoring fatty acid oxidation and increasing mitochondrial fusion and autophagy. Accordingly, FGFR inhibitor efficacy is potentiated by combined mitochondrial targeting, an effect enhanced in xenograft models by intermittent fasting. Thus, we show that oncogenic FGFR2 signaling drives NF-κB-dependent glycolysis in ICC and that metabolic reprogramming in response to FGFR inhibition confers new targetable vulnerabilities.

APOE2 gene therapy reduces amyloid deposition and improves markers of neuroinflammation and neurodegeneration in a mouse model of Alzheimer disease.

Epidemiological studies show that individuals who carry the relatively uncommon APOE ε2 allele rarely develop Alzheimer disease, and if they do, they have a later age of onset, milder clinical course, and less severe neuropathological findings than people without this allele. The contrast is especially stark when compared with the major genetic risk factor for Alzheimer disease, APOE ε4, which has an age of onset several decades earlier, a more aggressive clinical course and more severe neuropathological findings, especially in terms of the amount of amyloid deposition. Here, we demonstrate that brain exposure to APOE ε2 via a gene therapy approach, which bathes the entire cortical mantle in the gene product after transduction of the ependyma, reduces Aß plaque deposition, neurodegenerative synaptic loss, and, remarkably, reduces microglial activation in an APP/PS1 mouse model despite continued expression of human APOE ε4. This result suggests a promising protective effect of exogenous APOE ε2 and reveals a cell nonautonomous effect of the protein on microglial activation, which we show is similar to plaque-associated microglia in the brain of Alzheimer disease patients who inherit APOE ε2. These data increase the potential that an APOE ε2 therapeutic could be effective in Alzheimer disease, even in individuals born with the risky ε4 allele.

Genetic retargeting of E3 ligases to enhance CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapies are medical breakthroughs in cancer treatment. However, treatment failure is often caused by CAR T cell dysfunction. Additional approaches are needed to overcome inhibitory signals that limit anti-tumor potency. Here, we developed bifunctional fusion "degrader" proteins that bridge one or more target proteins and an E3 ligase complex to enforce target ubiquitination and degradation. Conditional degradation strategies were developed using inducible degrader transgene expression or small molecule-dependent E3 recruitment. We further engineered degraders to block SMAD-dependent TGFß signaling using a domain from the SARA protein to target both SMAD2 and SMAD3. SMAD degrader CAR T cells were less susceptible to suppression by TGFß and demonstrated enhanced anti-tumor potency in vivo. These results demonstrate a clinically suitable synthetic biology platform to reprogram E3 ligase target specificity for conditional, multi-specific endogenous protein degradation, with promising applications including enhancing the potency of CAR T cell therapy.

Wearable in-ear pulse oximetry validly measures oxygen saturation between 70% and 100%: A prospective agreement study.

Objectives: Postoperative monitoring outside intensive and post-anaesthesia care units is seldom, partly due to lack of suitable and approved systems. We therefore aim to validate the oxygen saturation (SpO2) and pulse rate measurement of the in-ear sensor c-med° alpha with a reference pulse oximeter. Methods: This prospective agreement study was conducted in 12 healthy (ASA 1) adult (18-50 years) volunteers according to the EN ISO 80601-2-61. The sitting volunteers were equipped with the finger pulse oximeter Rad-5 and two c-med° alpha sensors in each ear. The inspiratory oxygen content was reduced via a tight-fitting breathing mask to achieve five defined plateaus with stable SpO2 between 99% and 70%. The deviation of the SpO2 and pulse rate measurements of the c-med° alpha from those of the Rad-5 was calculated using the mean square error (Arms). Bias and limits of agreement between both devices were calculated using the Bland-Altman technique. The precision was compared based on the repeatability coefficients. Results: The c-med° alpha measured SpO2 had an Arms = 1.9% relative to the Rad-5, a non-significant bias (-0.1% (-0.2% to 0.0%)), levels of agreement from -4.0% to 3.8%, and the same repeatability coefficient (0.8% vs. 0.8%). The c-med° alpha measured pulse rate did not deviate from the one measured with the certified finger pulse oximeter (bias: 0.1 min-1 (0 to 0.1 min-1), level of agreement: -3.6 to 3.7 min-1, Arms: 1.8 min-1). Conclusions: The c-med° alpha fulfils the EN ISO 80601-2-61 standard and is sufficiently accurate for measuring SpO2 and pulse rate in healthy adults at rest. Trial registration: EUDAMED No. CIV-21-03-036033.

Regulation of RNA methylation by therapy treatment, promotes tumor survival.

Our data previously revealed that chemosurviving cancer cells translate specific genes. Here, we find that the m6A-RNA-methyltransferase, METTL3, increases transiently in chemotherapy-treated breast cancer and leukemic cells in vitro and in vivo. Consistently, m6A increases on RNA from chemo-treated cells, and is needed for chemosurvival. This is regulated by eIF2α phosphorylation and mTOR inhibition upon therapy treatment. METTL3 mRNA purification reveals that eIF3 promotes METTL3 translation that is reduced by mutating a 5'UTR m6A-motif or depleting METTL3. METTL3 increase is transient after therapy treatment, as metabolic enzymes that control methylation and thus m6A levels on METTL3 RNA, are altered over time after therapy. Increased METTL3 reduces proliferation and anti-viral immune response genes, and enhances invasion genes, which promote tumor survival. Consistently, overriding phospho-eIF2α prevents METTL3 elevation, and reduces chemosurvival and immune-cell migration. These data reveal that therapy-induced stress signals transiently upregulate METTL3 translation, to alter gene expression for tumor survival.

SULT1A1-dependent sulfonation of alkylators is a lineage-dependent vulnerability of liver cancers.

Adult liver malignancies, including intrahepatic cholangiocarcinoma and hepatocellular carcinoma, are the second leading cause of cancer-related deaths worldwide. Most individuals are treated with either combination chemotherapy or immunotherapy, respectively, without specific biomarkers for selection. Here using high-throughput screens, proteomics and in vitro resistance models, we identify the small molecule YC-1 as selectively active against a defined subset of cell lines derived from both liver cancer types. We demonstrate that selectivity is determined by expression of the liver-resident cytosolic sulfotransferase enzyme SULT1A1, which sulfonates YC-1. Sulfonation stimulates covalent binding of YC-1 to lysine residues in protein targets, enriching for RNA-binding factors. Computational analysis defined a wider group of structurally related SULT1A1-activated small molecules with distinct target profiles, which together constitute an untapped small-molecule class. These studies provide a foundation for preclinical development of these agents and point to the broader potential of exploiting SULT1A1 activity for selective targeting strategies.

Neutrophil profiles of pediatric COVID-19 and multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a delayed-onset, COVID-19-related hyperinflammatory illness characterized by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigenemia, cytokine storm, and immune dysregulation. In severe COVID-19, neutrophil activation is central to hyperinflammatory complications, yet the role of neutrophils in MIS-C is undefined. Here, we collect blood from 152 children: 31 cases of MIS-C, 43 cases of acute pediatric COVID-19, and 78 pediatric controls. We find that MIS-C neutrophils display a granulocytic myeloid-derived suppressor cell (G-MDSC) signature with highly altered metabolism that is distinct from the neutrophil interferon-stimulated gene (ISG) response we observe in pediatric COVID-19. Moreover, we observe extensive spontaneous neutrophil extracellular trap (NET) formation in MIS-C, and we identify neutrophil activation and degranulation signatures. Mechanistically, we determine that SARS-CoV-2 immune complexes are sufficient to trigger NETosis. Our findings suggest that hyperinflammatory presentation during MIS-C could be mechanistically linked to persistent SARS-CoV-2 antigenemia, driven by uncontrolled neutrophil activation and NET release in the vasculature.

Ribosome changes reprogram translation for chemosurvival in G0 leukemic cells.

Quiescent leukemic cells survive chemotherapy, with translation changes. Our data reveal that FXR1, a protein amplified in several aggressive cancers, is elevated in quiescent and chemo-treated leukemic cells and promotes chemosurvival. This suggests undiscovered roles for this RNA- and ribosome-associated protein in chemosurvival. We find that FXR1 depletion reduces translation, with altered rRNAs, snoRNAs, and ribosomal proteins (RPs). FXR1 regulates factors that promote transcription and processing of ribosomal genes and snoRNAs. Ribosome changes in FXR1-overexpressing cells, including RPLP0/uL10 levels, activate eIF2α kinases. Accordingly, phospho-eIF2α increases, enabling selective translation of survival and immune regulators in FXR1-overexpressing cells. Overriding these genes or phospho-eIF2α with inhibitors reduces chemosurvival. Thus, elevated FXR1 in quiescent or chemo-treated leukemic cells alters ribosomes that trigger stress signals to redirect translation for chemosurvival.

CD4+ T helper 2 cells suppress breast cancer by inducing terminal differentiation.

Cancer immunology research is largely focused on the role of cytotoxic immune responses against advanced cancers. Herein, we demonstrate that CD4+ T helper (Th2) cells directly block spontaneous breast carcinogenesis by inducing the terminal differentiation of the cancer cells. Th2 cell immunity, stimulated by thymic stromal lymphopoietin, caused the epigenetic reprogramming of the tumor cells, activating mammary gland differentiation and suppressing epithelial-mesenchymal transition. Th2 polarization was required for this tumor antigen-specific immunity, which persisted in the absence of CD8+ T and B cells. Th2 cells directly blocked breast carcinogenesis by secreting IL-3, IL-5, and GM-CSF, which signaled to their common receptor expressed on breast tumor cells. Importantly, Th2 cell immunity permanently reverted high-grade breast tumors into low-grade, fibrocystic-like structures. Our findings reveal a critical role for CD4+ Th2 cells in immunity against breast cancer, which is mediated by terminal differentiation as a distinct effector mechanism for cancer immunoprevention and therapy.

Global characterization of macrophage polarization mechanisms and identification of M2-type polarization inhibitors.

Macrophages undergoing M1- versus M2-type polarization differ significantly in their cell metabolism and cellular functions. Here, global quantitative time-course proteomics and phosphoproteomics paired with transcriptomics provide a comprehensive characterization of temporal changes in cell metabolism, cellular functions, and signaling pathways that occur during the induction phase of M1- versus M2-type polarization. Significant differences in, especially, metabolic pathways are observed, including changes in glucose metabolism, glycosaminoglycan metabolism, and retinoic acid signaling. Kinase-enrichment analysis shows activation patterns of specific kinases that are distinct in M1- versus M2-type polarization. M2-type polarization inhibitor drug screens identify drugs that selectively block M2- but not M1-type polarization, including mitogen-activated protein kinase kinase (MEK) and histone deacetylase (HDAC) inhibitors. These datasets provide a comprehensive resource to identify specific signaling and metabolic pathways that are critical for macrophage polarization. In a proof-of-principle approach, we use these datasets to show that MEK signaling is required for M2-type polarization by promoting peroxisome proliferator-activated receptor-γ (PPARγ)-induced retinoic acid signaling.

Synaptic proteins associated with cognitive performance and neuropathology in older humans revealed by multiplexed fractionated proteomics.

Alzheimer's disease (AD) is defined by the presence of abundant amyloid-ß (Aß) and tau neuropathology. While this neuropathology is necessary for AD diagnosis, it is not sufficient for causing cognitive impairment. Up to one third of community dwelling older adults harbor intermediate to high levels of AD neuropathology at death yet demonstrate no significant cognitive impairment. Conversely, there are individuals who exhibit dementia with no gross explanatory neuropathology. In prior studies, synapse loss correlated with cognitive impairment. To understand how synaptic composition changes in relation to neuropathology and cognition, multiplexed liquid chromatography mass-spectrometry was used to quantify enriched synaptic proteins from the parietal association cortex of 100 subjects with contrasting levels of AD pathology and cognitive performance. 123 unique proteins were significantly associated with diagnostic category. Functional analysis showed enrichment of serotonin release and oxidative phosphorylation categories in normal (cognitively unimpaired, low neuropathology) and "resilient" (unimpaired despite AD pathology) individuals. In contrast, frail individuals, (low pathology, impaired cognition) showed a metabolic shift towards glycolysis and increased presence of proteasome subunits.

Multisystem inflammatory syndrome in children is driven by zonulin-dependent loss of gut mucosal barrier.

BACKGROUNDWeeks after SARS-CoV-2 infection or exposure, some children develop a severe, life-threatening illness called multisystem inflammatory syndrome in children (MIS-C). Gastrointestinal (GI) symptoms are common in patients with MIS-C, and a severe hyperinflammatory response ensues with potential for cardiac complications. The cause of MIS-C has not been identified to date.METHODSHere, we analyzed biospecimens from 100 children: 19 with MIS-C, 26 with acute COVID-19, and 55 controls. Stools were assessed for SARS-CoV-2 by reverse transcription PCR (RT-PCR), and plasma was examined for markers of breakdown of mucosal barrier integrity, including zonulin. Ultrasensitive antigen detection was used to probe for SARS-CoV-2 antigenemia in plasma, and immune responses were characterized. As a proof of concept, we treated a patient with MIS-C with larazotide, a zonulin antagonist, and monitored the effect on antigenemia and the patient's clinical response.RESULTSWe showed that in children with MIS-C, a prolonged presence of SARS-CoV-2 in the GI tract led to the release of zonulin, a biomarker of intestinal permeability, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation. The patient with MIS-C treated with larazotide had a coinciding decrease in plasma SARS-CoV-2 spike antigen levels and inflammatory markers and a resultant clinical improvement above that achieved with currently available treatments.CONCLUSIONThese mechanistic data on MIS-C pathogenesis provide insight into targets for diagnosing, treating, and preventing MIS-C, which are urgently needed for this increasingly common severe COVID-19-related disease in children.

Targeted degradation of the enhancer lysine acetyltransferases CBP and p300.

The enhancer factors CREB-binding protein (CBP) and p300 (also known as KAT3A and KAT3B) maintain gene expression programs through lysine acetylation of chromatin and transcriptional regulators and by scaffolding functions mediated by several protein-protein interaction domains. Small molecule inhibitors that target some of these domains have been developed; however, they cannot completely ablate p300/CBP function in cells. Here we describe a chemical degrader of p300/CBP, dCBP-1. Leveraging structures of ligand-bound p300/CBP domains, we use in silico modeling of ternary complex formation with the E3 ubiquitin ligase cereblon to enable degrader design. dCBP-1 is exceptionally potent at killing multiple myeloma cells and can abolish the enhancer that drives MYC oncogene expression. As an efficient degrader of this unique class of acetyltransferases, dCBP-1 is a useful tool alongside domain inhibitors for dissecting the mechanism by which these factors coordinate enhancer activity in normal and diseased cells.

Neutrophil Profiles of Pediatric COVID-19 and Multisystem Inflammatory Syndrome in Children.

Multisystem Inflammatory Syndrome in Children (MIS-C) is a delayed-onset, COVID-19-related hyperinflammatory systemic illness characterized by SARS-CoV-2 antigenemia, cytokine storm and immune dysregulation; however, the role of the neutrophil has yet to be defined. In adults with severe COVID-19, neutrophil activation has been shown to be central to overactive inflammatory responses and complications. Thus, we sought to define neutrophil activation in children with MIS-C and acute COVID-19. We collected samples from 141 children: 31 cases of MIS-C, 43 cases of acute pediatric COVID-19, and 67 pediatric controls. We found that MIS-C neutrophils display a granulocytic myeloid-derived suppressor cell (G-MDSC) signature with highly altered metabolism, which is markedly different than the neutrophil interferon-stimulated gene (ISG) response observed in pediatric patients during acute SARS-CoV-2 infection. Moreover, we identified signatures of neutrophil activation and degranulation with high levels of spontaneous neutrophil extracellular trap (NET) formation in neutrophils isolated from fresh whole blood of MIS-C patients. Mechanistically, we determined that SARS-CoV-2 immune complexes are sufficient to trigger NETosis. Overall, our findings suggest that the hyperinflammatory presentation of MIS-C could be mechanistically linked to persistent SARS-CoV-2 antigenemia through uncontrolled neutrophil activation and NET release in the vasculature. ONE SENTENCE SUMMARY: Circulating SARS-CoV-2 antigen:antibody immune complexes in Multisystem Inflammatory Syndrome in Children (MIS-C) drive hyperinflammatory and coagulopathic neutrophil extracellular trap (NET) formation and neutrophil activation pathways, providing insight into disease pathology and establishing a divergence from neutrophil signaling seen in acute pediatric COVID-19.

Deregulation of ribosomal protein expression and translation promotes breast cancer metastasis.

Circulating tumor cells (CTCs) are shed into the bloodstream from primary tumors, but only a small subset of these cells generates metastases. We conducted an in vivo genome-wide CRISPR activation screen in CTCs from breast cancer patients to identify genes that promote distant metastasis in mice. Genes coding for ribosomal proteins and regulators of translation were enriched in this screen. Overexpression of RPL15, which encodes a component of the large ribosomal subunit, increased metastatic growth in multiple organs and selectively enhanced translation of other ribosomal proteins and cell cycle regulators. RNA sequencing of freshly isolated CTCs from breast cancer patients revealed a subset with strong ribosome and protein synthesis signatures; these CTCs expressed proliferation and epithelial markers and correlated with poor clinical outcome. Therapies targeting this aggressive subset of CTCs may merit exploration as potential suppressors of metastatic progression.

Multiplexed quantitative phosphoproteomics of cell line and tissue samples.

Post-translational modifications (PTMs) of proteins increase a biological system's repertoire of regulatory tools to control cellular mechanisms. Protein phosphorylation is the most studied PTM and known to be dysregulated in many diseases, including cancer, and protein kinases are among the most important drug targets. Many proteins across the eukaryotic proteome are phosphorylated, and more than 50,000 unique protein phosphorylation sites have been identified in a single human cell line. Understanding the vast biological networks directed by protein phosphorylation requires deep quantitative mapping of the phosphoproteome across many samples. Multiplexed proteomics using isobaric labeling reagents to barcode proteome samples for simultaneous quantification has greatly increased the throughput of mass spectrometry-based proteomics and enabled the number of analyses required to understand complex biological systems. We are presenting a detailed protocol to use multiplexed proteomics for mapping phosphoproteomes in samples from cell culture experiments and in tissue samples. The protocol includes phosphopeptide enrichment with TiO2 and phosphotyrosine antibody technology. We are using tandem mass tag (TMT) reagents for barcoding the samples allowing parallel quantification of up to 11 samples. The mass spectrometry method is based on the MultiNotch MS3 method to generate quantitative data of high accuracy and reproducibility. Tandem mass spectrometry (MS2) based on regular collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) is used to maximize the number of quantified phosphopeptides. The protocol typically enables the quantification of more than 20,000 unique phosphoforms (unique patterns of peptide phosphorylations) from proteome samples of human origin requiring less than 8h of mass spectrometry time per sample.

Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

Mitochondrial Permeability Uncouples Elevated Autophagy and Lifespan Extension.

Autophagy is required in diverse paradigms of lifespan extension, leading to the prevailing notion that autophagy is beneficial for longevity. However, why autophagy is harmful in certain contexts remains unexplained. Here, we show that mitochondrial permeability defines the impact of autophagy on aging. Elevated autophagy unexpectedly shortens lifespan in C. elegans lacking serum/glucocorticoid regulated kinase-1 (sgk-1) because of increased mitochondrial permeability. In sgk-1 mutants, reducing levels of autophagy or mitochondrial permeability transition pore (mPTP) opening restores normal lifespan. Remarkably, low mitochondrial permeability is required across all paradigms examined of autophagy-dependent lifespan extension. Genetically induced mPTP opening blocks autophagy-dependent lifespan extension resulting from caloric restriction or loss of germline stem cells. Mitochondrial permeability similarly transforms autophagy into a destructive force in mammals, as liver-specific Sgk knockout mice demonstrate marked enhancement of hepatocyte autophagy, mPTP opening, and death with ischemia/reperfusion injury. Targeting mitochondrial permeability may maximize benefits of autophagy in aging.

Identification of galectin-3 as an autoantigen in patients with IgG4-related disease.

BACKGROUND: The antigenic trigger that drives expansion of circulating plasmablasts and CD4+ cytotoxic T cells in patients with IgG4-related disease (IgG4-RD) is presently unknown. OBJECTIVE: We sought to sequence immunoglobulin genes from single-cell clones of dominantly expanded plasmablasts and generate recombinant human mAbs to identify relevant antigens in patients with IgG4-RD by using mass spectrometry. METHODS: Paired heavy and light chain cDNAs from dominant plasmablast clones were expressed as mAbs and used to purify antigens by using immunoaffinity chromatography. Affinity-purified antigens were identified by using mass spectrometry and validated by means of ELISA. Plasma levels of the antigen of interest were also determined by using ELISA. RESULTS: mAbs expressed from the 2 dominant plasmablast clones of a patient with multiorgan IgG4-RD stained human pancreatic tissue sections. Galectin-3 was identified as the antigen specifically recognized by both mAbs. Anti-galectin-3 autoantibody responses were predominantly of the IgG4 isotype (28% of the IgG4-RD cohort, P = .0001) and IgE isotype (11% of the IgG4-RD cohort, P = .009). No significant responses were seen from the IgG1, IgG2, or IgG3 isotypes. IgG4 anti-galectin-3 autoantibodies correlated with increased plasma galectin-3 levels (P = .001), lymphadenopathy (P = .04), total IgG level increase (P = .05), and IgG4 level increase (P = .03). CONCLUSION: Affinity chromatography using patient-derived mAbs identifies relevant autoantigens in patients with IgG4-RD. IgG4 galectin-3 autoantibodies are present in a subset of patients with IgG4-RD and correlate with galectin-3 plasma levels. The marked increases in levels of circulating IgG4 and IgE observed clinically are, at least in part, caused by the development of IgG4- and IgE-specific autoantibody responses.

Auto-fatty acylation of transcription factor RFX3 regulates ciliogenesis.

Defects in cilia have been associated with an expanding human disease spectrum known as ciliopathies. Regulatory Factor X 3 (RFX3) is one of the major transcription factors required for ciliogenesis and cilia functions. In addition, RFX3 regulates pancreatic islet cell differentiation and mature ß-cell functions. However, how RFX3 protein is regulated at the posttranslational level remains poorly understood. Using chemical reporters of protein fatty acylation and mass spectrometry analysis, here we show that RFX3 transcriptional activity is regulated by S-fatty acylation at a highly conserved cysteine residue in the dimerization domain. Surprisingly, RFX3 undergoes enzyme-independent, "self-catalyzed" auto-fatty acylation and displays preferences for 18-carbon stearic acid and oleic acid. The fatty acylation-deficient mutant of RFX3 shows decreased homodimerization; fails to promote ciliary gene expression, ciliogenesis, and elongation; and impairs Hedgehog signaling. Our findings reveal a regulation of RFX3 transcription factor and link fatty acid metabolism and protein lipidation to the regulation of ciliogenesis.