Fatty Acid Synthase Inhibitor Research Articles

The role of ChREBP in carbohydrate sensing and NAFLD development.

Excessive sugar consumption and defective glucose sensing by hepatocytes contribute to the development of metabolic diseases including type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD). Hepatic metabolism of carbohydrates into lipids is largely dependent on the carbohydrate-responsive element binding protein (ChREBP), a transcription factor that senses intracellular carbohydrates and activates many different target genes, through the activation of de novo lipogenesis (DNL). This process is crucial for the storage of energy as triglycerides in hepatocytes. Furthermore, ChREBP and its downstream targets represent promising targets for the development of therapies for the treatment of NAFLD and T2DM. Although lipogenic inhibitors (for example, inhibitors of fatty acid synthase, acetyl-CoA carboxylase or ATP citrate lyase) are currently under investigation, targeting lipogenesis remains a topic of discussion for NAFLD treatment. In this Review, we discuss mechanisms that regulate ChREBP activity in a tissue-specific manner and their respective roles in controlling DNL and beyond. We also provide in-depth discussion of the roles of ChREBP in the onset and progression of NAFLD and consider emerging targets for NAFLD therapeutics.

Abstract 595: Targeting metabolic vulnerability in brain-metastatic HER2+ breast cancer

Abstract Introduction: Human epidermal growth factor receptor 2 positive (HER2+) breast cancer tumors have increased risk of brain metastases at an advanced stage. The brain’s microenvironment is relatively low in lipids; therefore, breast cancer cells that colonize the brain have an increased expression of fatty acid synthesis enzymes. This presents a potential vulnerability that could be targeted. There is an established feed-forward relationship between HER2 and fatty acid synthase (FASN) where HER2 signaling activates FASN signaling. FASN inhibition has been shown to decrease cell invasion in a preclinical breast cancer model. We hypothesize that targeting HER2+ breast cancer cells with a HER2 tyrosine kinase inhibitor (TKI) or HER2 antibody-drug conjugate (ADC) in combination with a FASN inhibitor will lead to synergistic cell death. We also hypothesize that FASN inhibitors alone impede outgrowth and invasion of HER2+ breast cancer cells. Methods: We performed combinatorial drug-screening using the Chou-Talalay method in vitro using HER2+ breast cancer cell spheroids and tested the impact of FASN inhibitors alone on the outgrowth of these spheroids embedded in Matrigel. The HER2+ cell lines we used include JIMT-1, JIMT-1-BR3, SUM190, and SUM-190-BR3. JIMT-1-BR3 and SUM190-BR3 are brain-seeking subclones of the primary cell lines generated by serial passaging through mice with collection of cells in the brain. In combinatorial drug-screening, we calculated combination index (CI) values for the combination of each of two FASN inhibitors (TVB-2640 and BI-99179) with each of three clinically approved HER2 TKIs (Tucatinib, Lapatinib, and Neratinib) and each of two ADCs (Trastuzumab deruxtecan and Trastuzumab emtansine). The experiments were performed in both media supplied with fetal bovine serum (FBS) and with lipid-stripped FBS, to mimic the brain microenvironment. Results: Drug screening revealed that FASN inhibitors are toxic only at high doses in cell viability assays. However, we calculated combination index (CI) values using the Chou-Talalay method indicative of synergy for the combinations of the FASN inhibitors with the HER2-targeting drugs. Moreover, we discovered that the FASN inhibitors TVB-2640 and BI-99179 alone significantly impede the outgrowth of the spheroids embedded in Matrigel. Conclusions: Based on these promising preliminary data, we will evaluate these results in vivo. Breast cancer patients harboring brain metastases are difficult to treat, and they suffer from severe symptoms impacting daily life and have poor survival rates. Our ultimate goal is to launch a clinical trial to test one or more of these drug combinations to provide an additional therapeutic option that may improve patients’ lives and decrease mortality. Citation Format: Habib Serhan, Liwei Bao, Xu Cheng, Sofia Merajver, Aki Morikawa, Nathan Merrill. Targeting metabolic vulnerability in brain-metastatic HER2+ breast 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 595.

Abstract 414: Targeting lipid metabolism to improve efficacy of braf-targeted therapy in colorectal cancer

Abstract Background: Aberrant lipid metabolism is associated with poor prognosis in colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of lipogenesis, is overexpressed and a therapeutic target in CRC. Overexpression of CD36, a fatty acid translocase, plays a pro-tumorigenic role in CRC. BRAFV600E is the mutation occurring in about 10-15% of CRC cases. BRAF-targeted therapy is effective, but quickly developed resistance is an issue. Our preliminary data show that development of resistance to BRAF-targeted therapy is associated with an increase in expression of FASN, CD36, accumulation of triglycerides (TGs), and mitochondrial respiration. Therefore, our central hypothesis is that inhibition of lipid metabolism will sensitize CRC cells to BRAF inhibitors and overcome acquired resistance. Methods: We established HT29 cells and primary PT130 and PT2449pt cells resistant to PLX8394, a novel BRAF inhibitor. IC50 curves, PrestoBlue viability, CytoSelect™ 24-Well Cell Invasion, and Triglyceride Assays, Seahorse XF analysis, western blot, and confocal microscopy were used to evaluate differences between parental and resistant cells. RNA-seq and lipid analysis were used to evaluate changes in gene expression and lipids levels. Combination of PLX8394 and TVB3664 (FASN inhibitor) was tested on cell viability in parental and resistant cells. Results: PLX8394 resistant cells have a higher IC50, an increased proliferation, and invasion compared to parental cells. An increase in invasion of resistant cells is associated with a decrease in E-cadherin. RNA sequencing shows a significant increase in FASN expression. Western blot on resistant cells confirms upregulation of FASN, CD36, and other lipogenic markers. Consistently, resistant cells have an increase in levels of triglycerides as compared to parental cells. Seahorse XF Cell Mito Stress Test shows that resistant cells forgo the Warburg effect and instead rely more heavily on oxidative phosphorylation. To further support our hypothesis, FASN shRNA knockdown of FASN in parental HT29 cells is more susceptible to PLX8394 treatment compared to control. We also observed the synergetic effect of PLX8394 and TVB3664 treatment on cell viability in parental cells, but not in resistant cells. Conclusion: Our study demonstrates that resistance to BRAF inhibitors is associated with a significant increase in proliferation, metastasis, and upregulation of lipid metabolism. We show that combination of FASN and BRAF inhibitors has a combinational effect on inhibiting cell viability in parental but not resistant cells, suggesting that the addition of FASN inhibitor to the standard regimen for BRAFV600E mutation positive patients can improve efficacy of these therapies. Additional screening of lipid metabolism-targeted therapies in combination with standard BRAF regimens are needed to develop novel and more efficacious strategies for CRC patients with BRAF mutations. Citation Format: Mariah E. Geisen, Dang he, Chi Wang, Jill M. Kolesar, Yekaterina Zaytseva. Targeting lipid metabolism to improve efficacy of braf-targeted therapy in colorectal 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 414.

Abstract 2581: Fatty acid synthase regulates expression of Notum in colorectal cancer

Abstract Background: Upregulation of lipid synthesis has been associated with poor clinical outcomes in colorectal cancer (CRC). Fatty acid synthase (FASN) synthesizes 16-carbon fatty acid palmitate which can be utilized for post-translational modifications of various proteins. Notum, a palmitoleoyl-protein carboxylesterase, is involved in the negative regulation of the Wnt signaling via its role in de-palmitoylation of Wnt ligands and has been identified as a marker for poor prognosis in CRC. However, the crosstalk between FASN and Notum has not been reported. Our preliminary data suggest that FASN regulates β-catenin signaling and Notum expression. Therefore, the purpose of this study is to elucidate (I) the mechanisms of how FASN regulates expression of Notum and (II) the contribution of the FASN/Notum axis to CRC. Methods: Tumor and normal intestinal organoids were established from transgenic mice models, ApcMin and Apc/VillinCre-ERT2, with inducible hetero- and homozygous deletion of FASN. The effect of genetic deletion and pharmacological inhibition of FASN on organoid growth and viability was assessed by 4-hydroxytamoxifen (4-OHT) and TVB-3664 (a FASN inhibitor) treatments, respectively. LIVE/DEAD™ Viability/Cytotoxicity Cell Viability Kit and Cell Titer-Glo® 3D Cell Viability Assays were used for quantitative analysis of growth and viability. HCT116, NTC and FASN shRNA, and SW480, control and FASN overexpression, cells were used for analysis. Results: ERT2-mediated deletion of Apc leads to upregulation of FASN and Notum expression in mouse intestinal tissues and organoids. RNA-seq analysis of adenomas from Apc/VillinCre mice showed that hetero- and homozygous germline deletion of FASN is associated with a significant decrease in the number of adenomas, expression of Notum and CRC stem cell markers. Using qRT-PCR and Western blot, we confirmed that FASN downregulation is associated with a decrease in active β-catenin, Notum, and stem cell markers. Consistently with Apc/VillinCre model, downregulation of FASN results in a decrease in bud formation in Apc/VillinCre-ERT2 organoids, and viability and size in ApcMin organoids. Furthermore, overexpression of FASN increases the levels of active and total β-catenin, Notum, and stem cell markers expression in SW480 cells. In contrast, shRNA-mediated deletion of FASN decreases expression of Notum in HCT116 cells. Conclusion: Downregulation of FASN leads to a decrease in expression of Notum and is associated with morphological changes and a significant decrease in viability in organoid models. Conversely, FASN overexpression upregulates Notum expression suggesting a potential cross-talk between de novo lipid synthesis and Notum. Delineating the role of FASN regulation of stemness via altered β-catenin signaling and expression of Notum and other stem cell markers will provide the rationale for targeting the FASN/Notum axis as a preventative or early-stage therapeutic approach in CRC. Citation Format: Courtney Olivia Kelson, James Drury, Daheng He, Chi Wang, Yekaterina Zaytseva. Fatty acid synthase regulates expression of Notum in colorectal 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 2581.

Utilizing lipidomics and fatty acid synthase inhibitors to explore lipid accumulation in two thraustochytrid species

Thraustochytrids are marine protists with an excellent ability to produce health-beneficial docosahexaenoic acid (DHA)-rich lipids. Since the demand for DHA sources for food and feed is increasing, a deeper understanding of the potential to enhance the DHA-production in thraustochytrids is important for a sustainable DHA supply. In most thraustochytrids, including the strains used in this work, DHA is synthesized by a polyketide synthase-like pathway (PKS), and not through the common fatty acid synthetase (FAS) pathway. Both pathways use the same precursors. The aim of this study was to determine if inhibiting the production of fatty acids (FA) through the FAS pathway under nitrogen starvation would result in higher net DHA production rates measured as DHA content, and if the channeling of the synthesized fatty acids to different lipids species is similar in two different thraustochytrid strains. We found that the DHA production rates in Aurantiochytrium limacinum SR21 and Aurantiochytrium sp. T66 were not affected when treated with the FAS-inhibitor cerulenin, despite a reduction of FAS-derived FAs. This indicates that precursor availability is less likely to be the limiting factor for DHA synthesis. Still, cerulenin-treatment in the lipid accumulation phase resulted in higher concentrations of the triacylglycerol (TAG) TG(22:6/22:6/22:6) than in the untreated cells after three hours, demonstrating that FAS inhibition is a possible strategy to enhance the content of DHA-rich lipids. Moreover, the relatively high abundance of the diacylglycerol DG(22:6/22:6) suggests the presence of an obstacle for using DG(22:6/22:6) as substrate to synthesize DHA-rich TAGs, a step that potentially can be improved by metabolic engineering.

Cassiae Semen improves non-alcoholic fatty liver disease through autophagy-related pathway

ObjectiveCassiae Semen (CS, Juemingzi in Chinese) has been used for thousands of years in ancient Chinese history for relieving constipation, improving liver function as well as preventing myopia. Here we aimed to elucidate the anti-steatosis effect and underlying mechanism of CS against non-alcoholic fatty liver disease (NAFLD). MethodsHigh-performance liquid chromatography (HPLC) was used to identify the major components of CS water extract. Mice were fed with a high-fat and sugar-water (HFSW) diet to induce hepatic steatosis and then treated with CS. The anti-NAFLD effect was determined by measuring serum biomarkers and histopathology staining. Additionally, the effects of CS on cell viability and lipid metabolism in oleic acid and palmitic acid (OAPA)-treated HepG2 cells were measured. The expression of essential genes and proteins involved in lipid metabolism and autophagy signalings were measured to uncover the underlying mechanism. ResultsFive compounds, including aurantio-obtusin, rubrofusarin gentiobioside, cassiaside C, emodin and rhein were simultaneously identified in CS extract. CS not only improved the diet-induced hepatic steatosis in vivo, as indicated by decreased number and size of lipid droplets, hepatic and serum triglycerides (TG) levels, but also markedly attenuated the OAPA-induced lipid accumulation in hepatocytes. These lipid-lowering effects induced by CS were largely dependent on the inhibition of fatty acid synthase (FASN) and the activation of autophagy-related signaling, including AMP-activated protein kinase (AMPK), light chain 3-II (LC3-II)/ LC3-1 and autophagy-related gene5 (ATG5). ConclusionOur study suggested that CS effectively protected liver steatosis via decreasing FASN-related fatty acid synthesis and activating AMPK-mediated autophagy, which might become a promising therapeutic strategy for relieving NAFLD.

Inhibition of fatty acid synthase protects obese mice from acute lung injury via ameliorating lung endothelial dysfunction

BackgroundObesity has been identified as a risk factor for acute lung injury/acute respiratory distress syndrome (ALI/ARDS). However, the underlying mechanisms remain elusive. This study aimed to investigate the role of fatty acid synthase (FASN) in lipopolysaccharide (LPS)-induced ALI under obesity.MethodsA high-fat diet-induced obese (DIO) mouse model was established and lean mice fed with regular chow diet were served as controls. LPS was intratracheally instilled to reproduce ALI in mice. In vitro, primary mouse lung endothelial cells (MLECs), treated by palmitic acid (PA) or co-cultured with 3T3-L1 adipocytes, were exposed to LPS. Chemical inhibitor C75 or shRNA targeting FASN was used for in vivo and in vitro loss-of-function studies for FASN.ResultsAfter LPS instillation, the protein levels of FASN in freshly isolated lung endothelial cells from DIO mice were significantly higher than those from lean mice. MLECs undergoing metabolic stress exhibited increased levels of FASN, decreased levels of VE-cadherin with increased p38 MAPK phosphorylation and NLRP3 expression, mitochondrial dysfunction, and impaired endothelial barrier compared with the control MLECs when exposed to LPS. However, these effects were attenuated by FASN inhibition with C75 or corresponding shRNA. In vivo, LPS-induced ALI, C75 pretreatment remarkably alleviated LPS-induced overproduction of lung inflammatory cytokines TNF-α, IL-6, and IL-1β, and lung vascular hyperpermeability in DIO mice as evidenced by increased VE-cadherin expression in lung endothelial cells and decreased lung vascular leakage.ConclusionsTaken together, FASN inhibition alleviated the exacerbation of LPS-induced lung injury under obesity via rescuing lung endothelial dysfunction. Therefore, targeting FASN may be a potential therapeutic target for ameliorating LPS-induced ALI in obese individuals.

UCP2-induced fatty acid synthase promotes NLRP3 inflammasome activation during sepsis.

Cellular lipid metabolism has been linked to immune responses; however, the precise mechanisms by which de novo fatty acid synthesis can regulate inflammatory responses remain unclear. The NLRP3 inflammasome serves as a platform for caspase-1–dependent maturation and secretion of proinflammatory cytokines. Here, we demonstrated that the mitochondrial uncoupling protein-2 (UCP2) regulates NLRP3-mediated caspase-1 activation through the stimulation of lipid synthesis in macrophages. UCP2-deficient mice displayed improved survival in a mouse model of polymicrobial sepsis. Moreover, UCP2 expression was increased in human sepsis. Consistently, UCP2-deficient mice displayed impaired lipid synthesis and decreased production of IL-1β and IL-18 in response to LPS challenge. In macrophages, UCP2 deficiency suppressed NLRP3-mediated caspase-1 activation and NLRP3 expression associated with inhibition of lipid synthesis. In UCP2-deficient macrophages, inhibition of lipid synthesis resulted from the downregulation of fatty acid synthase (FASN), a key regulator of fatty acid synthesis. FASN inhibition by shRNA and treatment with the chemical inhibitors C75 and cerulenin suppressed NLRP3-mediated caspase-1 activation and inhibited NLRP3 and pro–IL-1β gene expression in macrophages. In conclusion, our results suggest that UCP2 regulates the NLRP3 inflammasome by inducing the lipid synthesis pathway in macrophages. These results identify UCP2 as a potential therapeutic target in inflammatory diseases such as sepsis.

Abstract P3-11-07: Inhibition of FASN as a potential treatment of advanced endocrine therapy-resistant breast cancer

Abstract INTRODUCTION: The past decade has seen significant advancement in increasing survival in estrogen receptor alpha (ERα) positive breast cancer. The use of selective estrogen receptor down-regulators and modulators (SERMs) (e.g. fulvestrant and tamoxifen), mTOR inhibitors (e.g. everolimus), aromatase inhibitors (AIs), and cyclin-dependent kinase inhibitors (e.g. palbociclib, ribociclib, and abemaciclib) have helped to extend overall survival of breast cancer patients. Unfortunately, resistance to endocrine therapy is a common occurrence and all patients will eventually succumb to their disease. Fatty acid synthase (FASN) is a key enzyme in lipid biosynthesis and is overexpressed in more aggressive and therapy-resistant tumors, including breast cancers. FASN inhibitor, TVB-2640, has been evaluated in multiple tumor cell lines and in a phase 1 clinical study, and showed partial responses in 5 patients and multiple patients with prolonged stable disease (≥16 weeks). METHODS: We generated tamoxifen- and fulvestrant-resistant MCF7 cells by long term exposure to tamoxifen (MCF7/TamR cells) and fulvestrant (MCF7/FR cells), and palbociclib-resistant (MCF7/RB1Crispr and ZR75/RB1Crispr) cells were generated through CRISPR/Cas9 knockout of the retinoblastoma (RB) gene. We assessed the impact of TVB-3166 inhibitor (and analog of TVB-2640 with slightly lower molecular weight for in vitro use) on proliferation, viability, cell cycle, and apoptosis in these cells. We evaluated the impact of TVB on proliferation and ERα expression in patient derived explants, and tumor growth in xenografts. RNA sequencing of tamoxifen- and fulvestrant-resistant cells was performed to investigate alterations in gene expression. Subcellular localization of ERα was assessed using subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blotting after treatment with TVB with or without the addition of palmitate or BAPTA. RESULTS: TVB treatment leads to a marked inhibition of proliferation in tamoxifen-, fulvestrant- and palbociclib-resistant cells compared to the parental cells. RNA sequencing of explants of patients with ERα positive disease showed down regulation of ESR1 related genes and genes involved in invasiveness. RNA sequencing of fulvestrant-resistant cells showed that treatment with TVB results in down regulation of EMT and E2F target genes, cholesterol homeostasis genes and mTORC1 signaling. Additionally, TVB significantly inhibited tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB treatment is mediated through the induction of endoplasmic reticulum stress in tamoxifen-resistant cells. CONCLUSION: Our preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered. Citation Format: Henriette Balinda. Inhibition of FASN as a potential treatment of advanced endocrine therapy-resistant breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-07.

Fatty acid synthase as a new therapeutic target for HER2-positive gastric cancer

PurposeTrastuzumab is an HER2-specific agent approved as the gold-standard therapy for advanced HER2-positive (HER2+) gastric cancer (GC), but the high rate and rapid appearance of resistance limit its clinical efficacy, resulting in the need to identify new vulnerabilities. Defining the drivers influencing HER2+ cancer stem cell (CSC) maintenance/survival could represent a clinically useful strategy to counteract tumor growth and therapy resistance. Accumulating evidence show that targeting crucial metabolic hubs, as the fatty acid synthase (FASN), may be clinically relevant.MethodsFASN protein and transcript expression were examined by WB and FACS and by qRT-PCR and GEP analyses, respectively, in trastuzumab-sensitive and trastuzumab-resistant HER2+ GC cell lines cultured in adherent (2D) or gastrosphere promoting (3D) conditions. Molecular data were analyzed in silico in public HER2+ GC datasets. The effectiveness of the FASN inhibitor TVB3166 to overcome anti-HER2 therapy resistance was tested in vitro in gastrospheres forming efficiency bioassays and in vivo in mice bearing trastuzumab-resistant GC cells.ResultsWe compared the transcriptome profiles of HER2+ GC cells cultured in 2D versus 3D conditions finding a significant enrichment of FASN in 3D cultures. FASN upregulation significantly correlated with high stemness score and poor prognosis in HER2+ GC cases. TVB3166 treatment significantly decreased GCSCs in all cell targets. HER2 and FASN cotargeting significantly decreased the capability to form gastrospheres versus monotherapy and reduced the in vivo growth of trastuzumab-resistant GC cells.ConclusionOur findings indicate that cotargeting HER2 and FASN increase the benefit of anti-HER2 therapy representing a new opportunity for metabolically combating trastuzumab-resistant HER2+ GC.

Comparative transcription profiling of mRNA and lncRNA in pulmonary arterial hypertension after C75 treatment

ObjectivesTo investigate mRNA and long non-coding RNA (lncRNA) expression profiles in monocrotaline (MCT)- mice.Materials and methodsLung tissues (Control-Vehicle, MCT-Vehicle, and MCT-C75) were examined by high-throughput sequencing (HTS). Aberrantly expressed mRNAs and lncRNAs were analyzed by bioinformatics. Cell proliferation and cell cycle analysis were performed to detect the potential protective effects of C75, an inhibitor of fatty acid synthase. The signaling pathways associated with inflammatory responses were verified by real time-PCR.ResultsRNA sequencing data reveals 285 differentially expressed genes (DEGs) and 147 lncRNAs in the MCT-Vehicle group compared to the control. After five-week of C75 treatment, 514 DEGs and 84 lncRNAs are aberrant compared to the MCT-Vehicle group. Analysis of DEGs and lncRNA target genes reveals that they were enriched in pathways related to cell cycle, cell division, and vascular smooth muscle contraction that contributes to the PAH pathological process. Subsequently, the expression of eight DEGs and three lncRNAs is verified using RT-PCR. Differentially expressed lncRNAs (ENSMUSG00000110393.2, Gm38850, ENSMUSG00000100465.1, ENSMUSG00000110399.1) may associate in PAH pathogenesis as suggested by co-expression network analysis. C75 can protect against MCT-induced PAH through its anti-inflammatory and anti-proliferation.ConclusionsThese DEGs and lncRNAs can be considered as novel candidate regulators of PAH pathogenesis. We propose that C75 treatment can partially reverse PAH pathogenesis through modulating cell cycle, cell proliferation, and anti-inflammatory.

P019 Host-Enterobacteriaceae interactions and mitochondrial dysfunction in Crohn’s colitis

Abstract Background Crohn’s disease (CD) is characterized by chronic gastrointestinal inflammation and unfavourable changes in the composition of intestinal microbiota (‘dysbiosis’). Members of Enterobacteriaceae such as K. pneumoniae and E. coli are often over-represented in colonic mucosa of CD patients. These bacteria contribute to vicious circle of dysbiosis and inflammation by expressing multiple virulence factors. Further, mitochondrial dysfunction in Paneth cells has recently been reported in ileal CD. In agreement, high-fat diet and repeated use of antibiotics increases susceptibility to colitis by damaging mitochondria. However, it remains unknown whether impaired mitochondrial bioenergetics could enhance mucosal colonization by Enterobacteriaceae and thus, contribute to the pathophysiology of colonic CD. Furthermore, the molecular mechanism leading to mitochondrial damage in colonic CD are incompletely understood. Methods Right colonic biopsies were collected from 74 adult CD patients and 28 healthy participants at St. Paul’s Hospital (Vancouver, BC, Canada). Microbes in colonic biopsies were characterized by 16S rDNA sequencing using Illumina MiSeq platform, followed by analysis of differentially abundant families via ANCOM-BC methodology. Further, high-throughput data-independent acquisition (DIA) proteomics was performed in colonic biopsies (Bruker Daltonics TimsTof Pro2 LC-MS/MS), and later analyzed via DIA-neural network software. Spearman's rank correlation approach was used for host proteomics- Enterobacteriaceae correlational analysis. Microbiome and proteomic data were corrected for false discovery rate (FDR; < 0.5 for statistical significance). For validation studies, human colonic TC7 epithelial cells and patient-derived organoid cultures were infected with CD-patient derived E. coli ± C75, a chemical inhibitor of fatty acid synthase (FASN; de novo fatty acid synthesis enzyme). Results We found an increased abundance of Enterobacteriaceae members in colonic mucosa of CD patients compared to healthy controls (Fig A). Next, our proteomics data revealed a significant downregulation in a group of proteins which were functionally related to mitochondrial health and activity, in CD patients compared to controls (Fig B). Further, Spearman’s analysis showed a positive correlation between Enterobacteriaceae and host FASN enzyme (Fig C), a protein directly related to mitochondrial metabolism. Interestingly, we found that blocking FASN with chemical inhibitor (C75) significantly enhanced colonization by E. coli in TC7 cells (Fig D). Conclusion Mechanistically, our study reports that aberrant activity of host FASN enzyme may cause mitochondrial dysfunction and thus, facilitates intestinal colonization by Enterobacteriaceae in CD patients.

FASN multi-omic characterization reveals metabolic heterogeneity in pancreatic and prostate adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) and prostate cancer (PCa) are among the most prevalent malignant tumors worldwide. There is now a comprehensive understanding of metabolic reprogramming as a hallmark of cancer. Fatty acid synthase (FASN) is a key regulator of the lipid metabolic network, providing energy to favor tumor proliferation and development. Whereas the biological role of FASN is known, its response and sensitivity to inhibition have not yet been fully established in these two cancer settings.MethodsTo evaluate the association between FASN expression, methylation, prognosis, and mutational profile in PDAC and PCa, we interrogated public databases and surveyed online platforms using TCGA data. The STRING database was used to investigate FASN interactors, and the Gene Set Enrichment Analysis platform Reactome database was used to perform an enrichment analysis using data from RNA sequencing public databases of PDAC and PCa. In vitro models using PDAC and PCa cell lines were used to corroborate the expression of FASN, as shown by Western blot, and the effects of FASN inhibition on cell proliferation/cell cycle progression and mitochondrial respiration were investigated with MTT, colony formation assay, cell cycle analysis and MitoStress Test.ResultsThe expression of FASN was not modulated in PDAC compared to normal pancreatic tissues, while it was overexpressed in PCa, which also displayed a different level of promoter methylation. Based on tumor grade, FASN expression decreased in advanced stages of PDAC, but increased in PCa. A low incidence of FASN mutations was found for both tumors. FASN was overexpressed in PCa, despite not reaching statistical significance, and was associated with a worse prognosis than in PDAC. The biological role of FASN interactors correlated with lipid metabolism, and GSEA indicated that lipid-mediated mitochondrial respiration was enriched in PCa. Following validation of FASN overexpression in PCa compared to PDAC in vitro, we tested TVB-2640 as a FASN inhibitor. PCa proliferation arrest was modulated by FASN inhibition in a dose- and time-dependent manner, whereas PDAC proliferation was not altered. In line with this finding, mitochondrial respiration was found to be more affected in PCa than in PDAC. FASN inhibition interfered with metabolic signaling causing lipid accumulation and affecting cell viability with an impact on the replicative processes.ConclusionsFASN exhibited differential expression patterns in PDAC and PCa, suggesting a different evolution during cancer progression. This was corroborated by the fact that both tumors responded differently to FASN inhibition in terms of proliferative potential and mitochondrial respiration, indicating that its use should reflect context specificity.

Enhanced De Novo Lipid Synthesis Mediated by FASN Induces Chemoresistance in Colorectal Cancer.

Oxaliplatin is one of the most widely used chemotherapy drugs for colorectal cancer (CRC). Resistance to oxaliplatin threatens the prognosis of CRC. Since previous studies have aroused interest in fatty acid metabolism in cancer, in this study, we determined whether fatty acid biosynthesis and the related regulating mechanism contribute to oxaliplatin resistance in CRC. The effect of the fatty acid synthase (FASN) and its inhibitor Orlistat was characterized in Gene Expression Omnibus (GEO) databases, oxaliplatin-resistant cell lines, and xenografts. MRNA-seq and analysis identified related pathway changes after the application of Orlistat, which was verified by Western blotting. By leveraging the GEO databases, FASN and closely related gene signatures were identified as being correlated with the response to oxaliplatin-based chemotherapy and poor prognosis. Additionally, FASN-upregulated expression promoted oxaliplatin resistance in CRC cell lines. We then applied Orlistat, a typical FASN inhibitor, in cell culture and xenograft models of oxaliplatin-resistant CRC, which attenuated the resistance to oxaliplatin. Additionally, the combination of the FASN inhibitor and oxaliplatin significantly increased cell cycle arrest and facilitated apoptosis, partly due to the diminished phosphorylation of the MAPK/ERK and PI3K/AKT pathways. In vivo studies showed that inhibiting fatty acid biosynthesis with Orlistat restrained the growth of xenograft tumors and increased the responsiveness to oxaliplatin. Our study revealed that FASN enhanced resistance to oxaliplatin in CRC. The inhibition of FASN could rescue the response to oxaliplatin by regulating MAPK/ERK and PI3K/AKT pathways.

Abstract IA020: Improving treatment of primary and metastatic brain tumors: Emerging insights and novel strategies

Abstract Approximately 200,000 patients develop metastasis each year in US alone. This incidence is approximately 20 times higher than glioblastoma – a uniformly fatal disease where all Phase III immune-checkpoint trials have failed to date. Patients diagnosed with metastatic breast cancer have a median survival of two years and account for approximately 40,000 deaths annually in the US alone. About 1/3rd of women with advanced breast cancer are at risk of developing brain metastases (BMs). A notable example is human epidermal growth factor receptor 2 (HER2)-positive breast cancer, where BMs from this disease are refractory to drugs that otherwise control disease in extracranial sites, for two reasons: i) the blood-brain/blood-tumor barrier (BBB/BTB) hinders the delivery of drugs to BMs, and ii) the brain tumor microenvironment (TME) reduces the efficacy of these therapies even when they accrue in BMs (Arvanitis et al, Nature Reviews Cancer 2020). By employing clinically relevant murine models, we demonstrated that theanti-HER2 antibody trastuzumab can upregulate VEGF in the microenvironment of BM (Izumi et al, Nature 2002), and thus, adding anti-VEGF/R agents can improve the efficacy of anti-HER2 therapies (Kodack et al, PNAS 2012). We further showed that HER3 is overexpressed in BMs and confers de novo resistance to anti-HER2 therapies. Blocking HER3 enhanced the efficacy of FDA-approved targeted therapies including trastuzumab and neratinib - a finding supported by independent clinical trials (Kodack et al, Science Translational Medicine 2017). In our attempt to improve survival further, we discovered that breast cancer cells implanted in the brain exhibit increased de novo lipid synthesis compared to breast cancer cells implanted in the breast or liver. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reducesHER2+ breast tumor growth in the brain – unlike those grown in the breast or liver, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies (Ferraro et al, Nature Cancer 2021). In parallel, we have employed focused ultrasound to improve delivery of various therapeutics to BMs by transiently disrupting BBB/BTB (Arvanitis et al, PNAS 2018). Finally, by recognizing the similarities between the TME of BM and glioblastoma, we have developed 3 new strategies to reprogram the immunosuppressive microenvironment of glioblastoma to improve the efficacy of immune-check point blockers against these uniformly fatal tumors (Amoozgar et al, Nature Communications 2021, Amoozgar et al, bioarxiv 2022, Datta et al. bioarxiv 2022). These strategies are likely to also benefit patients with brain metastases that have now become a leading cause of morbidity and mortality with improved detection and extracranial treatments. Citation Format: Rakesh K. Jain. Improving treatment of primary and metastatic brain tumors: Emerging insights and novel strategies [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr IA020.

Targeting fatty acid synthase modulates sensitivity of hepatocellular carcinoma to sorafenib via ferroptosis

BackgroundSorafenib resistance is a key impediment to successful treatment of patients with advanced hepatocellular carcinoma (HCC) and recent studies have reported reversal of drug resistance by targeting ferroptosis. The present study aimed to explore the association of fatty acid synthase (FASN) with sorafenib resistance via regulation of ferroptosis and provide a novel treatment strategy to overcome the sorafenib resistance of HCC patients.MethodsIntracellular levels of lipid peroxides, glutathione, malondialdehyde, and Fe2+ were measured as indicators of ferroptosis status. Biological information analyses, immunofluorescence assays, western blot assays, and co-immunoprecipitation analyses were conducted to elucidate the functions of FASN in HCC. Both in vitro and in vivo studies were conducted to examine the antitumor effects of the combination of orlistat and sorafenib and CalcuSyn software was used to calculate the combination index.ResultsSolute carrier family 7 member 11 (SLC7A11) was found to play an important role in mediating sorafenib resistance. The up-regulation of FASN antagonize of SLC7A11-mediated ferroptosis and thereby promoted sorafenib resistance. Mechanistically, FASN enhanced sorafenib-induced ferroptosis resistance by binding to hypoxia-inducible factor 1-alpha (HIF1α), promoting HIF1α nuclear translocation, inhibiting ubiquitination and proteasomal degradation of HIF1α, and subsequently enhancing transcription of SLC7A11. Orlistat, an inhibitor of FASN, with sorafenib had significant synergistic antitumor effects and reversed sorafenib resistance both in vitro and in vivo.ConclusionTargeting the FASN/HIF1α/SLC7A11 pathway resensitized HCC cells to sorafenib. The combination of orlistat and sorafenib had superior synergistic antitumor effects in sorafenib-resistant HCC cells.

RF13 | PMON48 A novel missense variant in the gene encoding Fatty Acid Synthase (FASN) associated with a unique multi-system disorder including hypopituitarism and hypoparathyroidism

Abstract A male patient presented with a unique complex phenotype including panhypopituitarism, short stature with failure to respond to GH [IGF-1 generation test: no response to 2.8 mg/m2/day GH], sensorineural deafness, hypoparathyroidism, retinal dystrophy, and intellectual disability. He was 127cm tall at the age of 21 and was completely prepubertal (LH 0.8 U/L, FSH 2.6 U/L, Testosterone <0.69 nmol/L) with a bone age of 12 years. A trial of recombinant IGF-1 failed to improve growth. He died at the age of 26 years following a collapse of unknown etiology. Exome sequencing and subsequent variant calling on this patient revealed a novel heterozygous de novo missense variant (c.6395C>T, p.A2132V) at a highly conserved residue in FASN encoding Fatty Acid Synthase. The variant was not present in any control database, including the GnomAD browser. Human embryonic brain expression analysis using in situ hybridisation, revealed FASN mRNA transcript staining in the diencephalon and hypothalamus at Carnegie stages (CS) 16, 19, 20 and 23, and in Rathke's pouch (primordium of the anterior pituitary) at CS16 only. Fatty acid synthase (FASN), a crucial multienzyme with seven different catalytic activities, converts acetyl-CoA and malonyl-CoA into long-chain saturated fatty acids such as palmitate, in the presence of NADPH. Murine null mutant Fasn-/- embryos die before implantation and Fasn+/- embryos at various stages in the early postnatal period. Fasn is expressed in multiple tissues including the brain, parathyroids, liver and adrenal. Biochemical investigation of the patient revealed high triglyceride concentrations (3.81-6.49 mmol/L; NR 0.38-2) at all time points during an 18 hour fast. He mobilised free fatty acids but did not generate an increase in 3-hydroxybutyrate, suggesting a defect in fatty acid oxidation or ketone body synthesis. He maintained normoglycaemia with normal hormone responses to fasting, except for an undetectable IGF-1 (<25 ng/ml) and IGFBP-3 (1.17 mg/L). We measured de novo fatty acid synthesis in cultured patient and control fibroblast cells with 13C-Glucose, with and without the C75 fatty acid synthase inhibitor. Cell pellets were collected at three time-points, and 13C-fatty acids derived from 13C-glucose were analysed by mass spectrometry. Preliminary data showed that more C13-glucose was incorporated into de novo synthesised palmitic acid in control cells, suggesting reduced fatty acid synthesis in the patient. Our data suggest that FASN p.A2132V is pathogenic and contributed significantly to the complex patient phenotype, consistent with recent studies suggesting that neural stem/progenitor cells (NSPCs) in the rodent brain are governed by Fasn-dependent de novo lipogenesis for proliferation. Presentation: Sunday, June 12, 2022 12:36 p.m. - 12:41 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Cerulenin suppresses ErbB2-overexpressing breast cancer by targeting ErbB2/PKM2 pathway.

Cerulenin is a fungal metabolite and a specific inhibitor of fatty acid synthase (FASN), which has shown a potential anticancer activity. 20-25% of breast cancer patients with ErbB2-overexpressing develop resistance to treatment. Therefore, it is urgent to find an effective new target for the treatment of ErbB2-overexpressing breast cancer. Our previous study found that cerulenin inhibits the glycolysis and migration of SK-BR-3 cells, but the effect of cerulenin on other malignant phenotypes of breast cancer is still unknown. Furthermore, the mechanism by which cerulenin displays its inhibitory effects is not fully understood. In this study, we systematically investigate the inhibitory effects of cerulenin on proliferation, migration, invasion and glycolysis of ErbB2-overexpressing breast cancer cells and its molecular mechanism. We found that cerulenin obviously suppresses the proliferation, migration, invasion as well as glycolysis. Through bioinformatic analyses, we found that PKM2 might be a target of cerulenin. In addition, ErbB2 and its signaling pathway upregulated PKM2 protein levels. Furthermore, we demonstrated that cerulenin downregulated the protein levels of ErbB2, PKM2 and EMT markers (MMP9, MMP2 and Snail2) in a dose- and time-dependent manner. Finally, the inhibitory of cerulenin on colony formation, migration, invasion and glycolysis, as well as protein levels of EMT markers were rescued by replenishing with PKM2. These findings illustrated that cerulenin inhibits proliferation, migration, invasion and glycolysis by targeting ErbB2/PKM2 pathway in ErbB2-overexpressing breast cancer cells.

AZ12756122, a novel fatty acid synthase inhibitor, decreases resistance features in EGFR-TKI resistant EGFR-mutated NSCLC cell models

Different EGFR tyrosine kinase inhibitors (TKIs) have been developed for the treatment of non-small cell lung cancer (NSCLC) patients harboring sensitizing mutations in the EGFR gene. Apart from acquired secondary mutations, multiple resistance mechanisms have been reported, such as the overexpression of fatty acid synthase (FASN), a multi-functional enzyme essential for the de novo lipogenesis, or the increase of cancer stem cells, a small subpopulation within the tumor responsible for relapse, metastasis, and resistance to therapies. Hence, the purpose of this work is to evaluate the novel FASN inhibitor AZ12756122, both alone and in combination with gefitinib and osimertinib, in EGFR-mutated (EGFRm) lung adenocarcinoma cell models sensitive and resistant to EGFR-TKIs. The molecular effect of AZ12756122 (alone and in combination with EGFR-TKI) on FASN, EGFR/STAT3, Akt/mTOR, and MAPK signaling pathways was analyzed using RT-qPCR and Western blot. FASN expression was also evaluated in samples from patients with EGFRm NSCLC through immunohistochemistry. Our findings revealed that AZ12756122 caused cytotoxic effects inducing apoptosis, downregulated FASN expression and activity, decreased the activation of EGFR and Akt/mTOR pathway, and reduced cancer stem-like cells. Furthermore, the combination of AZ12756122 and osimertinib sensitized cells to EGFR-TKI, showing a synergistic effect that resulted in a reduction in the activation of EGFR, Akt/mTOR, and MAPK signaling pathways. Our study also showed that FASN+ EGFRm NSCLC patients exhibited a longer mPFS in patients who responded to EGFR-TKI treatment. In conclusion, FASN inhibition should be further studied for the treatment, alone or in combination with EGFR-TKIs, for EGFRm NSCLC patients.

Fatty Acid Biosynthesis Pathways Are Downregulated during Stigma Development and Are Critical during Self-Incompatible Responses in Ornamental Kale.

In Brassicaceae, the papillary cells of the stigma are the primary site of the self-incompatibility (SI) responses. SI preserves the genetic diversity by selectively rejecting irrelevant or incompatible pollen, thus promoting cross fertilization and species fitness. Mechanisms that regulate SI responses in Brassica have been studied mainly on the mature stigma that often undermines how stigma papillary cells attain the state of SI during development. To understand this, we integrated PacBio SMRT-seq with Illumina RNA-seq to construct a de novo full-length transcriptomic database for different stages of stigma development in ornamental kale. A total of 48,800 non-redundant transcripts, 31,269 novel transcripts, 24,015 genes, 13,390 alternative splicing, 22,389 simple sequence repeats, 21,816 complete ORF sequences, and 4591 lncRNAs were identified and analyzed using PacBio SMRT-seq. The Illumina RNA-seq revealed 15,712 differentially expressed genes (DEGs) and 8619 transcription factors. The KEGG enrichment analysis of 4038 DEGs in the "incompatibility" group revealed that the flavonoid and fatty acid biosynthesis pathways were significantly enriched. The cluster and qRT-PCR analysis indicated that 11 and 14 candidate genes for the flavonoid and fatty acid biosynthesis pathways have the lowest expression levels at stigma maturation, respectively. To understand the physiological relevance of the downregulation of fatty acid biosynthesis pathways, we performed inhibitor feeding assays on the mature stigma. The compatible pollination response was drastically reduced when mature stigmas were pre-treated with a fatty acid synthase inhibitor. This finding suggested that fatty acid accumulation in the stigmas may be essential for compatible pollination and its downregulation during maturity must have evolved as a support module to discourage the mounting of self-incompatible pollen.