Functional role of fatty acid synthase for signal transduction in Core binding factor-AML with activating c-Kit mutation

Functional role of fatty acid synthase for signal transduction in Core binding factor-AML with activating c-Kit mutation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Functional role of fatty acid synthase for signal transduction in Core binding factor-AML with activating c-Kit mutation Ruimeng Zhuang, Bente Siebels, Konstantin Hoffer, Anna Worthmann, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4648786/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background AML is a rare hematological malignancy still associated with poor prognosis. 5% of de novo AML and 30% of core binding factor (CBF) AML (translocation t(8;21)(q22;q22) or invasion (16)(p13;q22)), respectively, harbor activating c-Kit (CD117) mutations leading to an adverse clinical outcome. Posttranslational protein modifications, especially by myristolic and palmitic acid, are known to be important for diverse cell functions such as membrane organization, transduction signaling or regulation of apoptosis. However, most data come from solid tumor studies while its role in AML is still poorly understood. Fatty acid synthase (FASN) is one of the key palmitoyl-acyltransferases which controls subcellular localization, trafficking and degradation of various target proteins. H-Ras, N-Ras or FLT3-ITDmut receptors are known to be important target proteins for FASN in AML. Methods In this study, we investigated the role of FASN in two c-Kit-N822K mutated AML cell lines. Using FASN knockdown via shRNA and the FASN inhibitor TVB-3166. Functional implications including cell viability and proliferation were tracked in a combined approach integrating western blotting, mass spectrometry PamGene. Results In FASN-knockdown cells, we observed an increase in phosphorylation of c-Kit (p-c-Kit), Lyn kinase (pLyn) as well as of S6 kinase (pS6). Moreover, a downregulation of cathepsin Z (CTSZ), which belongs to endo-lysosomal proteases and is hence essential for degradation of cellular proteins within lysosomes was found. Conclusion Recent studies have suggested potential roles for palmitoylation in lysosomal function indirectly through its effects on proteins involved in lysosomal trafficking, membrane fusion, and signaling pathways. Therefore, our observation of the reduced expression of CTSZ due to the inhibition of FASN offers an explanation for the increased c-Kit, Lyn, and S6 kinase activity in CBF-AML with activating c-Kit mutation. fatty acid synthase palmitoylation TVB-3166 AML c-Kit mutation PI3K Akt mTOR S6 kinase Lyn Gli1 hedgehog signaling cathepsin Z Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background AML is a heterogeneous disease characterized by a block in differentiation and uncontrolled proliferation of myeloid blasts [ 1 , 2 ]. Currently, diverse recurrent chromosomal and molecular aberrations with prognostic and predictive impact are known and increasingly used in clinical practice [ 3 , 4 ]. 12–15% of AML have the chromosomal aberrations t(8;21)(q22;q22) and inv(16)(p13;q22) leading to abnormal function of the transcriptional CBF complex [ 5 ]. The long-term survival of patients with CBF-AML after intensive treatment is limited to 60% emphasizing the need for the development of further treatment options [ 5 ]. While only 5% patients with de novo AML have activating c-Kit mutations, their prevalence in the subgroup of CBF-AML is approximately 30% [ 6 ]. Mostly, the Exon 8 (extracellular domain) or the Exon 17 (internal tyrosine kinase domain) are affected by pathogenic mutations which are associated with adverse clinical outcome [ 7 ]. Trials investigating c-Kit inhibitors in addition to standard chemotherapy are ongoing [ 8 ]. CD117/c-Kit was initially called stem cell factor receptor because of its function in hematopoietic stem cell survival, self-renewal, and differentiation. It belongs to the receptor- tyrosine-kinases (RTKs) family type III consisting of an extracellular domain, a transmembrane domain, a juxtamembrane domain and an intracellular tyrosine kinase domain. Most human AML cells express wild-type c-Kit, which is constitutively autophosphorylated by binding of the ligand stem cell factor (SCF) [ 9 ]. In the case of activating mutations, a ligand-independent activation of the downstream c-Kit signaling takes place [ 10 , 11 ]. Various co-effector proteins such as Sos, GrB2, phosphatidylinositol-3 (PI3)-kinase, JAK2 or Src family kinases (SFK) bind to the juxtamembrane domain and C-terminal tail of c-Kit leading to an activation of the main downstream transduction elements Ras-Raf-MAP, JAK/STAT and PI3K/Akt pathway. Numerous authors reported several crosstalk mechanisms between these pathways [ 10 – 12 ]. Especially, the interaction between PI3K/AKT and Ras/Raf can synergistically mediate phosphorylation of the RPS6KB1(also called p70S6K) - RPS6 axis resulting in an up-regulation of ribosomal biosynthesis and cell growth [ 13 – 15 ]. Post-translational protein modification by covalent attachment of lipids (lipidation) plays an important role for membrane association and activity of various oncogenic drivers such as overexpressed or mutant epidermal growth factor receptor (EGFR), Ras or FLT3 receptors. Irreversible prenylation of Ras by farnesyl transferase (FTase) or geranylgeranyl transferase (GGTase) represents the most crucial mechanisms resulting in integration of the small GTPases into the plasma membrane [ 16 ]. However, the effectiveness of FTase and GGTase inhibitors is currently limited and combined treatments are too toxic for clinical use [ 17 ]. S-palmitoylation of cysteine sites, preferentially located to the C-termini of target proteins, represents another reversible mechanism. It is required for the localization of RTKs as well as of Non-RTKs, such as Src kinases, on the plasma and on the cytoplasmatic membranes as well as for the subsequent activation of cell signaling [ 18 ]. Several palmitoyl acyltransferases, for example, fatty acid synthase (FASN) or zinc finger DHHC-type palmitoyl acyltransferases (ZDHHCs) which are mostly localized on endoplasmatic reticulum (ER), can mediate palmitoylation. FASN activity can be regulated in response to cell metabolism and growth signals driven by SREBP-1, ZBTB7A and p53 downstream of the PI3K–Akt–mTOR as well as of the MAPK signaling [ 19 – 21 ]. In this study, we examined the role of FASN for c-Kit signaling in human and murine AML cell lines with activating c-Kit mutation. A strong increase of p-c-Kit, pLyn, a member of the Src kinase family, as well as of pS6 upon FASN inhibition either by the compound TVB-3166 or by using shRNA was observed. Moreover, we detected a down regulation of CTSZ in FASN-knockdown cells. CTSZ is essential for trafficking and endosomal degradation of various cell proteins, therefore, its downregulation due to the reduced FASN activity could explain the increase of p-cKit, pLyn and pS6 levels [ 22 ]. Methods Bacterial strain Xl1 blue from Pierce Thermo Fisher Scientific (Waltham, MA, USA) Kits DC-Protein Assay from Bio-Rad (Munich, Germany), NucleoBond Xtra Midi from Macherey-Nagel (Düren, Germany), Lipofectamine P3000 transfection reagents from Gibco/Thermo Fisher Scientific (Waltham, MA, USA), Super Signal West Dura chemiluminescence substrate from Thermo Fisher Scientific (Waltham, Massachusetts). Antibodies The monoclonal antibodies directed against panAkt (#4685S), pAkt S473 (#4060S), S6 (#2217S), pS6 (#2215S), MAPK (#4695S), pMAPK (#4377S), c-Kit (Ab81) (#3308) were purchased from Cell Signaling Technology (Beverly, MA, USA) and monoclonal antibodies directed against FASN (#48357) and Gli1 (#515751) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-mouse IgG HRP-linked antibody (#7076) and anti-rabbit IgG HRP-linked antibody (#7074) were from Cell Signaling Technology (Beverly, MA, USA). Vectors PLKO.1-puro vectors encoding FASN shRNA or non-target (scrambled, scr) shRNA were purchased from Sigma-Aldrich (Taufkirchen, Germany). The third generation lentiviral vector LeGO-iB2Zeo was used to express mutant KIT N822K in conjunction with a fusion of mTagBFP and Zeocin resistance (Sh ble) in Baf3 cells. Tyrosine kinase domain mutant N822K of human Kit was generated by in-vitro mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA) on wild type KIT cDNA (J. Cammenga, S. Horn, U. Bergholz, G. Sommer, P. Besmer, W. Fiedler, C. Stocking. Extracellular KIT receptor mutants, commonly found in core binding factor AML, are constitutively active and respond to imatinib mesylate. Blood, 106(12), 3958–3961 (2005). doi: 10.1182/blood-2005-02-0583. ) and N822K mutagenesis primers sthp289fw (5’-CTAGCCAGAGACATCAAGAATGATTCTAAGTATGT GGTTAAAGGAA-3’) and sthp290rv (5’-TTCCTTTAACCACATACTTAGAATCATTCTTGA TGTCTCTGGCTAG-3’). The sequence encoding KIT N822K was introduced into the LeGO vector through NotI cloning and verified by sequence analysis. Inhibitors TVB-3166 owned by Sagimet Biosciences Inc. (formerly 3-V-Biosciences) (San Mateo, California) and kindly provided for this work. Culturing of cells Thawing and freezing were performed according to the local directions. Cell density was maintained between 3 x 10^5 and 3 x 10^6 viable cells/mL. For evaluating cell concentrations, the standard trypan blue exclusion assay using a Neubauer chamber was performed. Kasumi1, a human AML cell line bearing translocation t(8;21) and Kit N822K gain-of-function mutation, and Baf3 with Kit N822K were grown in RPMI1640, supplemented with 20% FCS and 1% Penicillin/Streptomycin (P/S). For Baf3 wild type cells recombinant mouse interleukin-3 was added to a final concentration of 0.5ng/mL. HEK-293T cells were grown in DMEM supplemented with 10% FCS without P/S. Cells were protected from contamination by working under a safety cabinet class ll and cultivated in an incubator at 37°C with 5% CO2. Proliferation For inhibitor treatment, 10000 Kasumi1 cells were plated in 100µl per well in a 96-well plate (Greiner Bio-One, Frickenhausen, Germany). After 24 hours, 100µl of inhibitor solution was added. To compare proliferation of Kasumi1 cells with and without FASN knockdown, Kasumi1 SCR and FASN knockdown cells were plated accordingly in 200 µL medium containing 1.5µg/ml puromycin for different incubation times as annotated in the figures. Cell confluence was measured by the IncuCyte Zoom imaging system (Essen Bioscience, Ann Arbor, USA). Transformation and plasmid preparation 100µl Xl1 blue bacteria were incubated with 100ng plasmid DNA following the manufacturer's instruction. The NucleoBond Xtra plasmid purification Kit (Sigma-Aldrich, Taufkirchen, Germany) was used to purify plasmid DNA (s. Supplemental). Lentiviral knockdown of FASN PLKO.1-puro vector encoding FASN-shRNA and PLKO.1 non-target (scrambled, scr) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Two FASN knockdown clones (kd1 and kd2) were established to check the reproducibility of the results. For virus production, HEK293T cells were plated in DMEM medium in one 10cm dish with 2 x 10^5 cells per dish. For the transfection of the viral vectors and helper plasmids, the lipofectamine kit was used following the manufacturer's instructions. In brief, 2.5µg DNA of each PLKO.1 vector were diluted in 20µl P3000 reagent, followed by addition of 8µg VSVG, gagPol and HIV1-Rev encoding plasmids. Target cells were seeded at a density of 3 x 10^5 cells per well in 2ml RPMI medium. The viral supernatants were harvested 24h and 48h after transfection and added immediately to the target cells. Selection of transduced target cells was carried out with 4µg/ml puromycin and considered as completed when all cells in the untransduced control wells were dead. All work with lentiviral particles was done in a S2 facility after approval according to German law. Immunoblotting Protein extracts were prepared with NP40 lysis buffer solution (BostonBioProducts). For determination of the protein concentration, the DC protein assay kit (Bio-Rad, Munich, Germany) was used. Protein lysates were separated according to their size by SDS-PAGE in 4–20% precasted gels (Thermo Fisher Scientific) using a voltage between 120V and 175V. After electrotransfer onto nitrocellulose membranes (Amersham / GE Healthcare, Amersham, England) at 65V for two hours, the membranes were stained in Ponceau staining solution until protein bands were visible, and then cut to useful sizes and pieces depending onthe proteins in focus. After addition of the respective primary antibody in a dilution of 1:1000, the membranes were incubated over night at 8°C. Secondary antibodies were added at a dilution of 1:5000 and incubated at room temperature for one hour. After washing, the membranes were developed using the LAS 4000 imager (GE Healthcare Bio-Sciences, Pittsburgh, Pennsylvania, USA) and Super Signal West Dura chemiluminescence substrate kit (Thermo Fisher Scientific). Lipidomic chromatography Fatty acid composition of cell extracts was determined by gas chromatography coupled with mass spectrometry. Cell pellets were resuspended in 50 µl of water and after adding 100 µl internal standard mix (tetradecanoate d27 and heptadecanoate d33, 200µg/ml each in Methanol/Toluol 4/1) as well as 1000 µl Methanol/Toluol 4/1 cells were vortexed. 100 µL acetyl chloride were added and samples were mixed vigorously. Subsequently, samples were heated for 1 h at 100°C to prepare fatty acid methyl esters. After cooling to room temperature, 3 mL of 6% sodium carbonate were added and samples were mixed vigorously. The mixture was centrifuged (1,800 g, 5 min) and the upper layer was transferred to auto sampler vials. Gas chromatography analyses were performed using a Trace 1310 gas chromatograph (Thermo fisher) equipped with following stationary phase: DB-225 30m x 0.25mm i.d., film thickness 0.25 µm (Agilent) coupled to a mass spectrometer (ISQ 7000 GC-MS, ThermoFisher Scientific, Dreieich, Germany). Peak identification and quantification were performed by comparing retention times and peak areas, respectively, to standard chromatograms and internal standards. Mass spectrometry-based differential quantitative proteome analysis Protein extraction and tryptic digestion The Kasumi1 cell line samples were dissolved in 100 mM triethyl ammonium bicarbonate and 1% w/v sodium deoxycholate buffer, boiled at 95°C for 5 min and sonicated with a probe sonicator. The protein concentration of denatured proteins was determined by the Pierce BCA Protein assay kit (Thermo Fisher). 20 µg of protein for each sample was diluted in 50 ell Buffer containing 0.1M TEAB and 1% (w/v) SDC in H 2 O. Disulfide bonds were reduced with 10mM DTT at 60°C for 30 minutes. Cysteine residues were alkylated with 20 mM iodoacetamide (IAA) for 30 minutes at 37°C in the dark. Tryptic digestion was performed for 16 hours at 37°C, using a trypsin / protein ratio of 1:100. After tryptic digestion the inhibition of trypsin activity as well as the precipitation of SDC was achieved by the addition of 1% formic acid (FA). Samples were centrifuged for 5 minutes at 16000 g. The supernatant was dried in a SpeedVac vacuum concentrator and stored at -20°C until further use. Prior to mass spectrometric analyses, peptides were resuspended in 0.1% FA to a final concentration of 1 µg/µl. 1 µg was used for LC-MS/MS acquisition. LC-MS/MS acquisition and data processing Chromatographic separation of peptides was achieved by nano UPLC (nanoAcquity system, Waters) with a two-buffer system (buffer A: 0.1% FA in water, buffer B: 0.1% FA in ACN). Attached to the UPLC was a peptide trap (100 µm × 20 mm, 100 Å pore size, 5 µm particle size, Acclaim PepMap, Thermo Scientific) for online desalting and purification followed by a 25-cm C18 reversed-phase column (75 µm × 200 mm, 130 Å pore size, 1.7 µm particle size, Peptide BEH C18, Waters). Peptides were separated using an 80-min method with a 60 min gradient elution from 2–30% buffer B. The eluting peptides were analyzed on a Quadrupole Orbitrap hybrid mass spectrometer (QExactive, Thermo Fisher Scientific). Here, the ions being responsible for the 15 highest signal intensities per precursor scan (1 × 10 6 ions, 70,000 Resolution, 240ms fill time) within a scan range from 400 to 1200 m/z were analyzed by MS/MS (HCD at 25 normalized collision energy, 1 × 10 5 ions, 17,500 Resolution, 50 ms fill time) starting at 120 m/z. A dynamic precursor exclusion of 20 s was used. LC-MS/MS data was searched with the Sequest algorithm integrated in the Proteome Discoverer software (Version 3.0.0.757), Thermo Fisher Scientific) against a reviewed human Swissprot database, obtained in December 2022. Carbamidomethylation was set as fixed modification for cysteine residues and the oxidation of methionine, and pyro-glutamate formation at glutamine residues at the peptide N-terminus, as well as acetylation of the protein N-terminus were allowed as variable modifications. A maximum number of 2 missing tryptic cleavages was set. Peptides between 6 and 144 amino acids where considered. A strict cutoff (FDR < 0.01) was set for peptide and protein identification. Quantification was performed using the Minora Algorithm, implemented in the Proteome Discoverer Software. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048252 [ 23 ]. Kinome analysis Functional kinome profiling of tyrosine as well as serin-threonin kinases has been described previously [ 24 ]. Here we used a PamStation®12 (located at the UCCH Kinomics Core Facility, Hamburg) and PTK-PamChip® arrays to profile tyrosine kinase according to the manufacturer’s instructions (PamGene International, ´s-Hertogenbosch, The Netherlands). In brief, whole cell lysates were made using 100 µl M-PER Mammalian Extraction Buffer containing Halt Phosphatase Inhibitor and ethylenediaminetetraacetic acid (EDTA)-free Halt Protease Inhibitor Cocktail (1:100 each; Pierce, Waltham, Massachusetts, USA) per 1x106 cells. The lysed sample were stored immediately in a -80°C freezer. Protein quantification was performed with the bicinchoninic acid assay according to the manufacturer´s instructions (BCA; Merck KGaA, Darmstadt, Germany). Per array 5 µg of protein and 400 µM ATP were applied. Sequence-specific peptide tyrosine and serin-threonin phosphorylation was detected by the fluorescein–labeled antibody PY20 (Exalpha, Maynard, Massachusetts, USA) and a CCD camera using the Evolve software (PamGene International, ´s-Hertogenbosch, The Netherlands). Data were analyzed using the BioNavigator software (PamGene International, 's-Hertogenbosch, The Netherlands). Statistical analysis For evaluating significance, unpaired t-Test was performed. Significance is presented in the graphs as * for p < 0.05, ** for p < 0.005, *** for p < 0.001. Standard deviation is presented as error bars. The ratio between p-protein and total protein is calculated in order to show the authentic changes of phosphorylation which is not due the differences in total protein that can be induced by protein expression. In order to better standardize the western blot quantification, the analysis of data is based on the total protein quantification using Ponceau red staining. Statistical analysis of proteomic data Normalized protein abundances were analyzed within the statistic software Perseus 1 . Abundances were log2 transformed and reduced to only valid values to perform linear principal component analysis. For statistical testing, data was reduced to proteins found in more than 2 replicates per phenotype (scr, kd1, kd2). Student's t-testing including permutation-based FDR correction was performed. As threshold for p- and adjusted p-values 0.05 was applied as well as a 2-fold change. Visualizations were performed in R software environment using an in-house script based on the ggplot 2 package. For analysis of FASN regulation, visualization was performed in GraphPad Prism (Version 8.0.2) as well as t-testing with significances of ** for p < 0.01 and *** for p < 0.001. Software: AIDA Image Analyzer: Version 3.44 Zotero: 6.0.20 Results Establishment of stable FASN knockdown in Kasumi1, detection of a reduced CTSZ expression by proteomic analysis The major goal of this project was the analysis of the functional role of FASN for signal transduction in CBF-AML cells. Therefore, stable knockdowns of FASN were established in the AML cell line Kasumi, an AML cell line with t(8;21) and c-Kit mutation at Asn822. As shown in Fig. 1 , expression of FASN was reduced by two independent lentiviral knockdown vectors by over 80% (see also Supplementary Fig. 1). The biological effect of FASN knockdown on the levels of fatty acids was analyzed in Kasumi1 cells by chromatography experiments. 27 fatty acids were detected, their amounts (in µg/µl) were normalized to the total concentration of cellular fatty acids and quantified. In Fig. 2 , the relative amount of myristolic and of palmitic acid is shown in Kasumi1 cells after knockdown of FASN (kd1 and kd2) compared to Kasumi1 cells without knockdown (scr). A relative reduction of fatty acid levels from 100–64% and 86% for myristolic acid and from 100–85% and 85% for palmitic acid, respectively, was shown confirming successful knockdown of FASN in both cell clones. The other fatty acids were not down regulated by FASN knockdown (data not shown). We also reproduced FASN knockdowns by mass spectrometry (Fig. 3 ). In addition, CTSZ, cytochrome c oxidase (COX6C), the peptidase PEPD and the S100 calcium binding protein A4 (S100-A4) were found to be significantly downregulated, while the glucose transporter SLC2A1 (GLUT1) and the haemoglobin subunit delta (HBD) proteins, respectively, significantly upregulated in Kasumi1 FASN kd1 compared with Kasumi1 scr (Fig. 3 B). Further, we only focused on interpreting of the role of CTSZ due to its key function in endosomal protein recycling as well as to its overexpression and association with worst survival in pediatric acute leukaemia [ 22 , 25 ]. Up regulation of p- c-Kit, pS6 and of pLyn in Kasumi1 and in Baf3 c-Kit N822K cells after pharmacological and genetic FASN inhibition Using western blot, a significant upregulation of phosphor-S6 (pS6) was observed in both Kasumi1 FASN kd1 and kd2 cells. On the contrary, phospho-Akt (pAkt) and phospho-MAPK (pMAPK) showed a signal-increase under FASN knockdown, however, it was not significant (Fig. 4 A). We also observed a significant upregulation of pS6 was after treatment with the FASN inhibitor TVB-3166, however, the effect occurred 8h after treatment with TVB-3166 but not after 48h (Fig. 4 B). As mentioned above, we also investigated whether there is a link between FASN activity and the Hedgehog pathway, exemplified here by determining the expression of GLI1 in FASN knockdown cells. As shown in Fig. 4 C, we did not see any significant changes in GLI1 expression levels under the two stable FASN knockdowns in Kasumi1 cells (see also Supplementary Fig. 2). To verify our results, we examined the aberrant signaling of mutant Kit in two separate subcell lines of Baf3 cells (Fig. 5 ). Both cell lines ectopically expressed Kit-N822K, but were generated independently of each other. In the comparable design, the pharmacological inhibition of FASN by TVB-3166 treatment resulted in an upregulation of both, pAkt and pS6 (Fig. 5 ). Interestingly, upregulation of pAkt was detected 24 hours earlier than the upregulation of pS6, consistent with S6 being further downstream of Akt signaling. However, the agonistic effect of FASN inhibition occurred much later in the Baf3-N822K cells compared to the Kasumi1 cells (Fig. 4 ), suggesting that the time course of pharmacological FASN inhibition is different in the different cellular models and consequently the downstream signaling of the Kit mutant might also be affected, at least temporally differently. In further experiments, the tyrosine as well as serin-threonin kinases regulated by FASN in Kasumi1 cells were identified in detail by functional kinome profiling (Figs. 6 and 7 ). Expression of FASN was either reduced by knockdown or the activity of FASN was inhibited by TVB-3166. As shown in Fig. 6 A, 6 B, 7 A and 7 B, respectively, kinase activity was increased both after sustained knockdown as well as after treatment with 100nM FASN-inhibitor TVB-3166 for 5 days. However, it must be noted that only kd2 cells could be analyzed further for their regulatory effects on serin/threonin kinases, since basically no upregulation was detected in kd1 cells and only weak upregulation was detected in TVB-3166-treated cells (Fig. 7 B). Importantly, the kinome profile analysis clearly demonstrated higher activity of p-Kit, several members of SRC family kinases (SFK, including pLyn) and pS6 in both Kasumi1 FASN knockdowns as well as after treatment with TVB-3166 (Fig. 6 C and 7 C). No changes in proliferation activity of Kasumi1 cells after inhibition of FASN In the next experiments the effect of FASN on proliferation of Kasumi1 cells was analysed. Despite the repeatedly observed effect on the downstream signaling of KIT N822K as well as the activation status of other kinases, we did not observe significant changes in the proliferation of Kasumi1 cells after FASN knockdown or after treatment with 100nM TVB-3166 (Supplementary Fig. 3). Discussion Activating c-Kit mutations predominantly occur in CBF-AML causing an adverse clinical outcome [ 26 , 27 ]. To overcome these limitations in the treatment of CBF-AML patients with additional KIT mutations, in particular those in the kinase activation loop (exon 17), investigation of further treatment options is required. As mentioned above, active c-Kit mutants are usually localized on the Golgi network leading to an activation of the main signaling pathways STAT5, Akt and MAPK [ 11 ]. The Non-RTK Lyn is one of the members of the Src kinase family and an important co-effector of c-Kit. Lyn participates in c-Kit receptor-mediated downstream signaling by trafficking through the Golgi region and by associating with the juxtamembrane region of c-Kit. This mechanism is specified to the SH4 domain of Lyn [ 28 ]. Lyn was found to be highly expressed in a large series of AML patients playing an important role for leukemogenesis [ 29 ]. It has been reported to be distributed throughout the plasma membrane and the cytoplasm in AML cells where it was expressed in an active state [ 30 ]. Inhibition of Lyn efficiently reduced phosphorylation of mTOR and STAT5, however, it did not affect pAkt nor the ERK/MAPK signaling. Moreover, Lyn has been reported to play an important role in the FLT3-ITD mutated AML, in terms of a signaling component in the FLT3-ITD-STAT5 pathway [ 30 , 31 ]. FASN mediated palmitoylation of target proteins is considered to be an important mechanism for development of resistance, for example, in TKI-resistant, epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer. Combination of EGFR-TKIs and the FASN inhibitor Orlistat led to an interruption of the EGFR-TKI resistance in vivo [ 32 ]. In the meanwhile, diverse FASN inhibitors were developed for clinical studies [ 33 – 35 ]. Intriguingly, the interrelationship between S-palmitoylation and cell signaling transduction as well as its biological role are poorly understood in hematological malignancies. In mice with AML phenotype, it has been reported that S-palmitoylation of the GTPases H-Ras and N-Ras or of the mutated receptor FLT3-ITD is essential for their binding, localization and trafficking between the plasma membrane and different endosomes (Benjamin Cuiffo et al.) [ 36 , 37 ]. In 2019, it has been reported that in AML with an activating c-Kit N822K mutation, the mutant is active on the Golgi leading to an activation of Akt-, MAPK and STAT5 signaling while the inactive/dephosphorylated c-Kit form is localized in the ER [ 38 ]. However, the role of palmitoylation for the localization of c-Kit mutant on the Golgi lipid rafts and for its subsequent activity is currently under investigation. We were able to establish two stable FASN knockdowns in Kasumi1 cells by lentiviral transduction with FASN-shRNA vectors. Their statistical and biological significance was confirmed by immunoblotting, by mass spectrometry as well as by lipidomic analysis showing a strong level reduction of specifically myristolic and palmitic acid in both FASN knockdowns. Using two cell models of c-Kit-N822K mutated CBF-AML, we demonstrate that pharmacological and genetic FASN inhibition led to a rewiring of the c-Kit-mutant associated pathways, especially, to a significant upregulation of p-c-Kit, pLyn and pS6. Moreover, we found a down regulation of CTSZ in FASN-knockdown Kasumi1 cells by mass spectrometry. CTSZ is a critical component of the endocytic pathways in myelopoesis which is required for endosomal trafficking, degradation and recycling of numerous plasma membrane as well as of cytosolic proteins through the Golgi network [ 22 ]. Intriguingly, cathepsins are also known to be overexpressed and associated with poor clinical outcome in pediatric [ 25 ]. Recent studies have suggested potential role for S-palmitoylation in lysosomal functions indirectly through its effects on proteins involved in lysosomal trafficking, membrane fusion, signaling pathways and protein degradation [ 39 ]. Hence, inhibition of FASN can downregulate CTSZ, which then leads to a reduced degradation of c-Kit and p-c-Kit. In the next step, pLyn can upregulate pS6 which is a downstream target of mTOR. This mechanism can be suggested by the published data regarding the activation of mTOR by Lyn [ 30 , 31 ]. Via this mechanism, a subcellular reprogramming of c-Kit associated pathways could take place making AML cells stronger dependent on the pLyn-pS6 signalling. Remarkably, we were not able to detect any significant changes in proliferation of Kasumi1 cells upon treatment with the FASN inhibitor TVB-3166 nor in both FASN knockdowns which could be caused by persistently active pLyn. In view of the clinical approval of the Hedgehog signaling inhibitor Glasdegib for therapy of AML and in order to analyze a rationale for further treatment options, we also investigated whether the expression of the downstream Hedgehog effector Gli1 is dependent on the FASN activity. Our hypothesis is based on some reports in solid tumors which, for example, show that FASN knockdown via siRNA can reduce Gli1 levels in gastric cancer cells, suggesting that FASN may play a role in the tumorigenesis and metastasis [ 40 ]. However, we were not able to detect any significant changes in the expression of Gli1 by the inhibition of FASN. This observation could be explained in terms of a compensatory effect of the increased pS6 which would suggest the existence of SMO-independent regulation of Hedgehog-Gli signaling by key oncogenic drivers in various myeloid malignancies [ 41 ]. Conclusion To our knowledge, these findings offer new insights into a potential mechanism by which reduced expression of CTSZ due to the inhibition of FASN increases c-Kit, Lyn, and S6 kinase activity in CBF-AML with activating c-Kit mutation. In summary, our data suggest a rationale to further explore combined inhibition of FASN, c-Kit, Src/Lyn, and S6 kinase in the treatment of CBF-AML with activating c-Kit mutations. Abbreviations Acute Myeloid Leukemia (AML) FMS-like tyrosine kinase 3 (FLT3) wild-type FLT3 (FLT3-WT) Internal tandem duplication (FLT3-ITD) juxtamembrane domain (JM domain) tyrosine kinase domain (FLT3-TKD) plasma membrane (PM) RAS/extracellular signal–regulated kinase (ERK) cysteine at position 563 (C563) stem cell transplant (SCT) complete remission (CR) Core binding factor-AML (CBF-AML) mitogen-activated protein kinase (MAPK) Src family kinases (SFKs) Fatty Acid Synthase (FASN) Hedgehog (Hh) suppressor of fused homolog (SuFu) RPS6 (Ribosomal Protein S6) CD117 (cluster of differentiation 117) stem cell growth factor receptor (SCFR) core binding factor (CBF) hematopoietic stem cells (HSCs) European Leukemia Net (ELN) complete remission (CR) relapse-free survival (RFS) Small Nuclear Ribonucleoprotein Polypeptide G (SNRPG) Glutathione Peroxidase 7 (GPX7) Peptide Deformylase (PDF) Nebulin (NEB) reactive oxigen species (ROS) Non-alcoholic fatty liver disease (NAFLD) Declarations Disclosure of potential conflicts of interest All the authors of this article have declared ‘‘no conflict of interest”. Author contributions M.K. and M.J. planned and supervised the study. R.Z. planned and performed experiments and analyzed the data. M.Kr., K.H., H.V., B.S, S.H., A. W. performed experiments, wrote and reviewed the manuscript. N.vB., C.K., N.G., S.P-G. kindly provided financial support and reviewed the manuscript. W.F. and C.B. reviewed the manuscript. M. K. wrote the manuscript. All the authors have approved the manuscript. Corresponding authors Correspondence to Maxim Kebenko or Manfred Jücker Acknowledgments We thank Bettina Bettin for excellent technical assistance. The authors thank Sagimet Biosciences Inc. (formerly 3-V-Biosciences) ( San Mateo , California) for kindly providing of TVB-3166. This study was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (INST 337/15-1, INST 337/16-1, INST 152/837-1 and INST 152/947-1 FUGG). References Koenig KL, Sahasrabudhe KD, Sigmund AM, Bhatnagar B. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4648786","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":329331810,"identity":"948eb07e-fc8b-4819-adb8-355392837d2f","order_by":0,"name":"Ruimeng Zhuang","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Ruimeng","middleName":"","lastName":"Zhuang","suffix":""},{"id":329331811,"identity":"d0b9af5c-005d-44b5-bb6f-4e91e1eaa951","order_by":1,"name":"Bente Siebels","email":"","orcid":"","institution":"University Medical Center 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13:14:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4648786/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4648786/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61775908,"identity":"77f3c5ba-243c-4e78-8126-2262b91c8b2b","added_by":"auto","created_at":"2024-08-05 12:33:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":131484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eshRNA-mediated knockdown of endogenous FASN in Kasumi1 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFASN expression was detected in whole cell lysate by western blot. Kasumi1 FASN kd cell lines were transduced with FASN shRNA (either with vector1 which is kd1 or with vector2 which is kd2) or Kasumi1 scr cell line transduced with non-targeting vector as a control for the baseline expression of FASN in Kasumi1 cell line. HSC70 was used as loading control for the quantification of FASN expression. The western blot is shown on the left, the analysis on the right side with y axis of fold change normalized to the expression level of FASN in scr. Each sample was loaded 3 times and analyzed by t test. Significance is presented in the graphs as * for p\u0026lt;0.05. Ponceau staining of the same membrane was used to demonstrate equal loading of protein lysates (see also Supplementary Figure 1).\u003c/p\u003e","description":"","filename":"Figure01.png","url":"https://assets-eu.researchsquare.com/files/rs-4648786/v1/1b706b6fafeec45db18d6992.png"},{"id":61775909,"identity":"1edd4f32-3b1a-48fc-b74a-36f08ec34d41","added_by":"auto","created_at":"2024-08-05 12:33:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":208082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLipidomic analysis of Kasumi1 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePalmitic and myristolic acids after knockdown of FASN in Kasumi1 cells.\u003c/p\u003e","description":"","filename":"Figure02.png","url":"https://assets-eu.researchsquare.com/files/rs-4648786/v1/56556a7227e396e9b7559b79.png"},{"id":61776561,"identity":"acc94630-9a8a-4fe5-9d26-fd7c0eebeb60","added_by":"auto","created_at":"2024-08-05 12:41:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":301176,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteome analysis of Kasumi1 cells after FASN knockdown.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Scatter plot visualization of principle component analysis (PCA). Components 1 and 2 are shown and reveal phenotype-based clustering of scr, kd1 and kd2. Quantified proteins were reduced to only valid values (2155 proteins). B: FASN abundance in Log2 scale in scr, kd1 and kd2. \u0026nbsp;C: Volcano-plot visualization of Student’s t-testing results between kd1 and scr as well as D: \u0026nbsp;kd2 and scr based on 2314 proteins. Proteins were considered significantly differential abundant, if they exceeded the adj. p-value cutoff \u0026lt;0.05 as well as \u0026gt; 2-fold. Significance was tested based on an unpaired t-test (p-value \u0026lt; 0.01 =**, \u0026lt; 0.001=***).\u003c/p\u003e","description":"","filename":"Figure03.png","url":"https://assets-eu.researchsquare.com/files/rs-4648786/v1/2704c28e2a6802413d040672.png"},{"id":61775910,"identity":"b24dc40b-e588-4333-a960-15d495f5aee7","added_by":"auto","created_at":"2024-08-05 12:33:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":495392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of cKit signal transduction in Kasumi1 cells by western blotting.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Increased phosphorylation of S6 after stable knockdown of FASN in Kasumi1 cells, no significant regulation of Akt and MAPK detected in Kasumi1 cells with stable FASN-knockdown kd1 and kd2 and scr control cells. Samples were analysed in technical triplicates. One of each of the three triplicates are shown on the left and the statistic results are shown on the right. Densitometric quantification of phospho-protein/protein ratios after normalization to scr control cells (n=3, mean values with standard deviations). Significance is presented as * for p\u0026lt;0.05, ** for p\u0026lt;0.005, *** for p\u0026lt;0.001. B: Increased phosphorylation of S6 after treatment of Kasumi1 cells with 100 nM TVB-3166. Protein lysates were analysed for expression of pS6 and total S6 (S6) protein in technical replicates. Statistic bar charts used the optical density normalized to time 0h. Significance is presented in the graphs as **** = p\u0026lt;0.001. C: No significant regulation of Gli1 in Kasumi1 cells with stable FASN knockdown analyzed in technical triplicates. Densitometric quantification of Gli1 expression relative to scr control cells (n=3, mean values with standard deviation). Significance is shown as *** = p\u0026lt;0.001. Ponceau staining of the shown membranes was used to demonstrate equal loading of protein lysates (see also Supplementary Figure 2\u003c/p\u003e","description":"","filename":"Figure04.png","url":"https://assets-eu.researchsquare.com/files/rs-4648786/v1/30fe3100605fc94f6f690736.png"},{"id":61776920,"identity":"e2837586-624d-42bc-9e73-18e31262477f","added_by":"auto","created_at":"2024-08-05 12:49:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":543967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of cKit N822K signalling transduction in Baf3-cKit mutated Baf3 expressing the N822K mutant of Kit cells by western blotting.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein lysates were analysed for cKit overexpression in transduced Baf3 cells as well as for downstream activation of Akt and S6. Increased pAkt (S473) 24h and increased pS6 48h after treatment with 100 nM TVB-3166 were observed in Baf3 cells with c-Kit gain-of-function mutation in comparison to Baf3 wild type. Ponceau staining of the same membrane was used to demonstrate equal loading of protein lysates. Significant differences were not detected, therefore replicates were not carried out. Densitometric quantification of pAkt/Akt and pS6/S6 ratios normalized to time 0h (n=1).\u003c/p\u003e","description":"","filename":"Figure05.png","url":"https://assets-eu.researchsquare.com/files/rs-4648786/v1/374e61a4483440cc72d0ce51.png"},{"id":61776563,"identity":"9b60ba7c-c923-4b1b-b1ba-a6dafceeee70","added_by":"auto","created_at":"2024-08-05 12:41:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":84864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKinome analysis of tyrosine kinases in Kasumi1 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTyrosine kinases were analysed in Kasumi1 cells with FASN knockdowns (kd1 and kd2) or after TVB-3166 treatment using functional kinome profiling. (A) The heatmap of 110 analyzed peptides (the S100_log transformed values are depicted). (B) Boxplot quantifying the mean signal intensities. (C) Upstream kinase analysis of control vs. TVB-3166, FASN knockdown 1 or 2 respectively. (Normalized kinase statistic (log2) \u0026gt; 0: higher kinase activity in treated samples; specificity score (log2) \u0026gt;1.3 (white to red circles): statistically significant changes).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4648786/v1/f21f2739fd1006a91c96050a.png"},{"id":61776564,"identity":"9600410e-e5a0-47e3-84cb-30dc85d05501","added_by":"auto","created_at":"2024-08-05 12:41:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":325640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKinome analysis of serin/threonin kinases in Kasumi1 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerin/Threonin kinases were analysed in Kasumi1 cells with FASN knockdowns (kd1 and kd2) or after TVB-3166 treatment using functional kinome profiling. (A) The heatmap of 110 analyzed peptides (the S100_log transformed values are depicted). B) Volcano plot highlighting significantly altered peptides (scr control vs. treatment; x-axis: log fold change in peptide phosphorylation, dashed line = 0; y-axis: significance (plog) for each peptide, \u0026gt;1.3 (dashed/dotted line) significant changes). (C) Upstream kinase analysis of scr vs. kd2 (Normalized kinase statistic (log2) \u0026lt; 0: lower kinase activity in inhibitor treated sample; specificity score (log2) \u0026gt;1.3 (white to red circles): statistically significant changes).\u003c/p\u003e","description":"","filename":"Figure07.png","url":"https://assets-eu.researchsquare.com/files/rs-4648786/v1/5d60cac8cc780ee112ab01cd.png"},{"id":61958102,"identity":"7e661ae2-0b23-434d-bb5c-1b53a1ce55c6","added_by":"auto","created_at":"2024-08-07 14:05:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2813108,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4648786/v1/f89aea30-9df5-451a-b8ce-930879fe77a6.pdf"},{"id":61775912,"identity":"ec3476ad-0c18-420d-a629-0fec417266b1","added_by":"auto","created_at":"2024-08-05 12:33:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":348581,"visible":true,"origin":"","legend":"","description":"","filename":"Supplemental.docx","url":"https://assets-eu.researchsquare.com/files/rs-4648786/v1/21b7601b56c2d9390a623ddb.docx"},{"id":61775915,"identity":"cb44368f-2446-40e0-9306-0589d24a1ed8","added_by":"auto","created_at":"2024-08-05 12:33:22","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4184831,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaluncroppedwesternblotts.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4648786/v1/80728efa86c10f89f8d80f9c.pptx"},{"id":61775914,"identity":"6df53df5-fcba-4e85-8087-6a9a067ab38f","added_by":"auto","created_at":"2024-08-05 12:33:22","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":397638,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTablesMassspec.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4648786/v1/4f8f3f0a1b95e206dff56d39.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functional role of fatty acid synthase for signal transduction in Core binding factor-AML with activating c-Kit mutation","fulltext":[{"header":"Background","content":"\u003cp\u003eAML is a heterogeneous disease characterized by a block in differentiation and uncontrolled proliferation of myeloid blasts [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Currently, diverse recurrent chromosomal and molecular aberrations with prognostic and predictive impact are known and increasingly used in clinical practice [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e12\u0026ndash;15% of AML have the chromosomal aberrations t(8;21)(q22;q22) and inv(16)(p13;q22) leading to abnormal function of the transcriptional CBF complex [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The long-term survival of patients with CBF-AML after intensive treatment is limited to 60% emphasizing the need for the development of further treatment options [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. While only 5% patients with de novo AML have activating c-Kit mutations, their prevalence in the subgroup of CBF-AML is approximately 30% [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Mostly, the Exon 8 (extracellular domain) or the Exon 17 (internal tyrosine kinase domain) are affected by pathogenic mutations which are associated with adverse clinical outcome [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Trials investigating c-Kit inhibitors in addition to standard chemotherapy are ongoing [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCD117/c-Kit was initially called stem cell factor receptor because of its function in hematopoietic stem cell survival, self-renewal, and differentiation. It belongs to the receptor- tyrosine-kinases (RTKs) family type III consisting of an extracellular domain, a transmembrane domain, a juxtamembrane domain and an intracellular tyrosine kinase domain. Most human AML cells express wild-type c-Kit, which is constitutively autophosphorylated by binding of the ligand stem cell factor (SCF) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the case of activating mutations, a ligand-independent activation of the downstream c-Kit signaling takes place [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Various co-effector proteins such as Sos, GrB2, phosphatidylinositol-3 (PI3)-kinase, JAK2 or Src family kinases (SFK) bind to the juxtamembrane domain and C-terminal tail of c-Kit leading to an activation of the main downstream transduction elements Ras-Raf-MAP, JAK/STAT and PI3K/Akt pathway. Numerous authors reported several crosstalk mechanisms between these pathways [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Especially, the interaction between PI3K/AKT and Ras/Raf can synergistically mediate phosphorylation of the RPS6KB1(also called p70S6K) - RPS6 axis resulting in an up-regulation of ribosomal biosynthesis and cell growth [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePost-translational protein modification by covalent attachment of lipids (lipidation) plays an important role for membrane association and activity of various oncogenic drivers such as overexpressed or mutant epidermal growth factor receptor (EGFR), Ras or FLT3 receptors. Irreversible prenylation of Ras by farnesyl transferase (FTase) or geranylgeranyl transferase (GGTase) represents the most crucial mechanisms resulting in integration of the small GTPases into the plasma membrane [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the effectiveness of FTase and GGTase inhibitors is currently limited and combined treatments are too toxic for clinical use [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. S-palmitoylation of cysteine sites, preferentially located to the C-termini of target proteins, represents another reversible mechanism. It is required for the localization of RTKs as well as of Non-RTKs, such as Src kinases, on the plasma and on the cytoplasmatic membranes as well as for the subsequent activation of cell signaling [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Several palmitoyl acyltransferases, for example, fatty acid synthase (FASN) or zinc finger DHHC-type palmitoyl acyltransferases (ZDHHCs) which are mostly localized on endoplasmatic reticulum (ER), can mediate palmitoylation. FASN activity can be regulated in response to cell metabolism and growth signals driven by SREBP-1, ZBTB7A and p53 downstream of the PI3K\u0026ndash;Akt\u0026ndash;mTOR as well as of the MAPK signaling [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we examined the role of FASN for c-Kit signaling in human and murine AML cell lines with activating c-Kit mutation. A strong increase of p-c-Kit, pLyn, a member of the Src kinase family, as well as of pS6 upon FASN inhibition either by the compound TVB-3166 or by using shRNA was observed. Moreover, we detected a down regulation of CTSZ in FASN-knockdown cells. CTSZ is essential for trafficking and endosomal degradation of various cell proteins, therefore, its downregulation due to the reduced FASN activity could explain the increase of p-cKit, pLyn and pS6 levels [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strain\u003c/h2\u003e \u003cp\u003eXl1 blue from Pierce Thermo Fisher Scientific (Waltham, MA, USA)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eKits\u003c/h2\u003e \u003cp\u003eDC-Protein Assay from Bio-Rad (Munich, Germany), NucleoBond Xtra Midi from Macherey-Nagel (D\u0026uuml;ren, Germany), Lipofectamine P3000 transfection reagents from Gibco/Thermo Fisher Scientific (Waltham, MA, USA), Super Signal West Dura\u003c/p\u003e \u003cp\u003echemiluminescence substrate from Thermo Fisher Scientific (Waltham, Massachusetts).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003eThe monoclonal antibodies directed against panAkt (#4685S), pAkt S473 (#4060S), S6 (#2217S), pS6 (#2215S), MAPK (#4695S), pMAPK (#4377S), c-Kit (Ab81) (#3308) were purchased from Cell Signaling Technology (Beverly, MA, USA) and monoclonal antibodies directed against FASN (#48357) and Gli1 (#515751) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-mouse IgG HRP-linked antibody (#7076) and anti-rabbit IgG HRP-linked antibody (#7074) were from Cell Signaling Technology (Beverly, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eVectors\u003c/h2\u003e \u003cp\u003ePLKO.1-puro vectors encoding FASN shRNA or non-target (scrambled, scr) shRNA were purchased from Sigma-Aldrich (Taufkirchen, Germany). The third generation lentiviral vector LeGO-iB2Zeo was used to express mutant KIT N822K in conjunction with a fusion of mTagBFP and Zeocin resistance (Sh ble) in Baf3 cells. Tyrosine kinase domain mutant N822K of human Kit was generated by in-vitro mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA) on wild type \u003cem\u003eKIT\u003c/em\u003e cDNA (J. Cammenga, S. Horn, U. Bergholz, G. Sommer, P. Besmer, W. Fiedler, C. Stocking. Extracellular KIT receptor mutants, commonly found in core binding factor AML, are constitutively active and respond to imatinib mesylate. Blood, 106(12), 3958\u0026ndash;3961 (2005). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1182/blood-2005-02-0583.\u003c/span\u003e\u003cspan address=\"10.1182/blood-2005-02-0583.\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and N822K mutagenesis primers sthp289fw (5\u0026rsquo;-CTAGCCAGAGACATCAAGAATGATTCTAAGTATGT GGTTAAAGGAA-3\u0026rsquo;) and sthp290rv (5\u0026rsquo;-TTCCTTTAACCACATACTTAGAATCATTCTTGA TGTCTCTGGCTAG-3\u0026rsquo;). The sequence encoding KIT N822K was introduced into the LeGO vector through NotI cloning and verified by sequence analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eInhibitors\u003c/h2\u003e \u003cp\u003eTVB-3166 owned by Sagimet Biosciences Inc. (formerly 3-V-Biosciences) (San Mateo, California) and kindly provided for this work.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCulturing of cells\u003c/h2\u003e \u003cp\u003eThawing and freezing were performed according to the local directions. Cell density was maintained between 3 x 10^5 and 3 x 10^6 viable cells/mL. For evaluating cell concentrations, the standard trypan blue exclusion assay using a Neubauer chamber was performed. Kasumi1, a human AML cell line bearing translocation t(8;21) and Kit N822K gain-of-function mutation, and Baf3 with Kit N822K were grown in RPMI1640, supplemented with 20% FCS and 1% Penicillin/Streptomycin (P/S). For Baf3 wild type cells recombinant mouse interleukin-3 was added to a final concentration of 0.5ng/mL. HEK-293T cells were grown in DMEM supplemented with 10% FCS without P/S. Cells were protected from contamination by working under a safety cabinet class ll and cultivated in an incubator at 37\u0026deg;C with 5% CO2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eProliferation\u003c/h2\u003e \u003cp\u003eFor inhibitor treatment, 10000 Kasumi1 cells were plated in 100\u0026micro;l per well in a 96-well plate (Greiner Bio-One, Frickenhausen, Germany). After 24 hours, 100\u0026micro;l of inhibitor solution was added. To compare proliferation of Kasumi1 cells with and without FASN knockdown, Kasumi1 SCR and FASN knockdown cells were plated accordingly in 200 \u0026micro;L medium containing 1.5\u0026micro;g/ml puromycin for different incubation times as annotated in the figures. Cell confluence was measured by the IncuCyte Zoom imaging system (Essen Bioscience, Ann Arbor, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTransformation and plasmid preparation\u003c/h2\u003e \u003cp\u003e100\u0026micro;l Xl1 blue bacteria were incubated with 100ng plasmid DNA following the manufacturer's instruction. The NucleoBond Xtra plasmid purification Kit (Sigma-Aldrich, Taufkirchen, Germany) was used to purify plasmid DNA (s. Supplemental).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLentiviral knockdown of FASN\u003c/h2\u003e \u003cp\u003ePLKO.1-puro vector encoding FASN-shRNA and PLKO.1 non-target (scrambled, scr) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Two FASN knockdown clones (kd1 and kd2) were established to check the reproducibility of the results. For virus production, HEK293T cells were plated in DMEM medium in one 10cm dish with 2 x 10^5 cells per dish. For the transfection of the viral vectors and helper plasmids, the lipofectamine kit was used following the manufacturer's instructions. In brief, 2.5\u0026micro;g DNA of each PLKO.1 vector were\u003c/p\u003e \u003cp\u003ediluted in 20\u0026micro;l P3000 reagent, followed by addition of 8\u0026micro;g VSVG, gagPol and HIV1-Rev encoding plasmids. Target cells were seeded at a density of 3 x 10^5 cells per well in 2ml RPMI medium. The viral supernatants were harvested 24h and 48h after transfection and added immediately to the target cells. Selection of transduced target cells was carried out with 4\u0026micro;g/ml puromycin and considered as completed when all cells in the untransduced control wells were dead. All work with lentiviral particles was done in a S2 facility after approval according to German law.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eProtein extracts were prepared with NP40 lysis buffer solution (BostonBioProducts). For determination of the protein concentration, the DC protein assay kit (Bio-Rad, Munich, Germany) was used. Protein lysates were separated according to their size by SDS-PAGE in 4\u0026ndash;20% precasted gels (Thermo Fisher Scientific) using a voltage between 120V and 175V. After electrotransfer onto nitrocellulose membranes (Amersham / GE Healthcare, Amersham, England) at 65V for two hours, the membranes were stained in Ponceau staining solution until protein bands were visible, and then cut to useful sizes and pieces depending onthe proteins in focus. After addition of the respective primary antibody in a dilution of 1:1000, the membranes were incubated over night at 8\u0026deg;C. Secondary antibodies were added at a dilution of 1:5000 and incubated at room temperature for one hour. After washing, the membranes were developed using the LAS 4000 imager (GE Healthcare Bio-Sciences, Pittsburgh, Pennsylvania, USA) and Super Signal West Dura chemiluminescence substrate kit (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLipidomic chromatography\u003c/h2\u003e \u003cp\u003eFatty acid composition of cell extracts was determined by gas chromatography coupled with mass spectrometry. Cell pellets were resuspended in 50 \u0026micro;l of water and after adding 100 \u0026micro;l internal standard mix (tetradecanoate d27 and heptadecanoate d33, 200\u0026micro;g/ml each in Methanol/Toluol 4/1) as well as 1000 \u0026micro;l Methanol/Toluol 4/1 cells were vortexed. 100 \u0026micro;L acetyl chloride were added and samples were mixed vigorously. Subsequently, samples were heated for 1 h at 100\u0026deg;C to prepare fatty acid methyl esters. After cooling to room temperature, 3 mL of 6% sodium carbonate were added and samples were mixed vigorously. The mixture was centrifuged (1,800 g, 5 min) and the upper layer was transferred to auto sampler vials. Gas chromatography analyses were performed using a Trace 1310 gas chromatograph (Thermo fisher) equipped with following stationary phase: DB-225 30m x 0.25mm i.d., film thickness 0.25 \u0026micro;m (Agilent) coupled to a mass spectrometer (ISQ 7000 GC-MS, ThermoFisher Scientific, Dreieich, Germany). Peak identification and quantification were performed by comparing retention times and peak areas, respectively, to standard chromatograms and internal standards.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometry-based differential quantitative proteome analysis\u003c/h2\u003e \u003cp\u003eProtein extraction and tryptic digestion\u003c/p\u003e \u003cp\u003eThe Kasumi1 cell line samples were dissolved in 100 mM triethyl ammonium bicarbonate and 1% w/v sodium deoxycholate buffer, boiled at 95\u0026deg;C for 5 min and sonicated with a probe sonicator. The protein concentration of denatured proteins was determined by the Pierce BCA Protein assay kit (Thermo Fisher). 20 \u0026micro;g of protein for each sample was diluted in 50 ell Buffer containing 0.1M TEAB and 1% (w/v) SDC in H\u003csub\u003e2\u003c/sub\u003eO. Disulfide bonds were reduced with 10mM DTT at 60\u0026deg;C for 30 minutes. Cysteine residues were alkylated with 20 mM iodoacetamide (IAA) for 30 minutes at 37\u0026deg;C in the dark. Tryptic digestion was performed for 16 hours at 37\u0026deg;C, using a trypsin / protein ratio of 1:100. After tryptic digestion the inhibition of trypsin activity as well as the precipitation of SDC was achieved by the addition of 1% formic acid (FA). Samples were centrifuged for 5 minutes at 16000 g. The supernatant was dried in a SpeedVac vacuum concentrator and stored at -20\u0026deg;C until further use. Prior to mass spectrometric analyses, peptides were resuspended in 0.1% FA to a final concentration of 1 \u0026micro;g/\u0026micro;l. 1 \u0026micro;g was used for LC-MS/MS acquisition.\u003c/p\u003e \u003cp\u003eLC-MS/MS acquisition and data processing\u003c/p\u003e \u003cp\u003eChromatographic separation of peptides was achieved by nano UPLC (nanoAcquity system, Waters) with a two-buffer system (buffer A: 0.1% FA in water, buffer B: 0.1% FA in ACN). Attached to the UPLC was a peptide trap (100 \u0026micro;m \u0026times; 20 mm, 100 \u0026Aring; pore size, 5 \u0026micro;m particle size, Acclaim PepMap, Thermo Scientific) for online desalting and purification followed by a 25-cm C18 reversed-phase column (75 \u0026micro;m \u0026times; 200 mm, 130 \u0026Aring; pore size, 1.7 \u0026micro;m particle size, Peptide BEH C18, Waters). Peptides were separated using an 80-min method with a 60 min gradient elution from 2\u0026ndash;30% buffer B. The eluting peptides were analyzed on a Quadrupole Orbitrap hybrid mass spectrometer (QExactive, Thermo Fisher Scientific). Here, the ions being responsible for the 15 highest signal intensities per precursor scan (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e ions, 70,000 Resolution, 240ms fill time) within a scan range from 400 to 1200 m/z were analyzed by MS/MS (HCD at 25 normalized collision energy, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e ions, 17,500 Resolution, 50 ms fill time) starting at 120 m/z. A dynamic precursor exclusion of 20 s was used.\u003c/p\u003e \u003cp\u003eLC-MS/MS data was searched with the Sequest algorithm integrated in the Proteome Discoverer software (Version 3.0.0.757), Thermo Fisher Scientific) against a reviewed human Swissprot database, obtained in December 2022. Carbamidomethylation was set as fixed modification for cysteine residues and the oxidation of methionine, and pyro-glutamate formation at glutamine residues at the peptide N-terminus, as well as acetylation of the protein N-terminus were allowed as variable modifications. A maximum number of 2 missing tryptic cleavages was set. Peptides between 6 and 144 amino acids where considered. A strict cutoff (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.01) was set for peptide and protein identification. Quantification was performed using the Minora Algorithm, implemented in the Proteome Discoverer Software.\u003c/p\u003e \u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048252 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eKinome analysis\u003c/h2\u003e \u003cp\u003eFunctional kinome profiling of tyrosine as well as serin-threonin kinases has been described previously [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Here we used a PamStation\u0026reg;12 (located at the UCCH Kinomics Core Facility, Hamburg) and PTK-PamChip\u0026reg; arrays to profile tyrosine kinase according to the manufacturer\u0026rsquo;s instructions (PamGene International, \u0026acute;s-Hertogenbosch, The Netherlands). In brief, whole cell lysates were made using 100 \u0026micro;l M-PER Mammalian Extraction Buffer containing Halt Phosphatase Inhibitor and ethylenediaminetetraacetic acid (EDTA)-free Halt Protease Inhibitor Cocktail (1:100 each; Pierce, Waltham, Massachusetts, USA) per 1x106 cells. The lysed sample were stored immediately in a -80\u0026deg;C freezer. Protein quantification was performed with the bicinchoninic acid assay according to the manufacturer\u0026acute;s instructions (BCA; Merck KGaA, Darmstadt, Germany). Per array 5 \u0026micro;g of protein and 400 \u0026micro;M ATP were applied. Sequence-specific peptide tyrosine and serin-threonin phosphorylation was detected by the fluorescein\u0026ndash;labeled antibody PY20 (Exalpha, Maynard, Massachusetts, USA) and a CCD camera using the Evolve software (PamGene International, \u0026acute;s-Hertogenbosch, The Netherlands). Data were analyzed using the BioNavigator software (PamGene International, 's-Hertogenbosch, The Netherlands).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor evaluating significance, unpaired t-Test was performed. Significance is presented in the graphs as * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.005, *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Standard deviation is presented as error bars. The ratio between p-protein and total protein is calculated in order to show the authentic changes of phosphorylation which is not due the differences in total protein that can be induced by protein expression. In order to better standardize the western blot quantification, the analysis of data is based on the total protein quantification using Ponceau red staining.\u003c/p\u003e \u003cp\u003eStatistical analysis of proteomic data\u003c/p\u003e \u003cp\u003eNormalized protein abundances were analyzed within the statistic software Perseus\u003csup\u003e1\u003c/sup\u003e. Abundances were log2 transformed and reduced to only valid values to perform linear principal component analysis. For statistical testing, data was reduced to proteins found in more than 2 replicates per phenotype (scr, kd1, kd2). Student's t-testing including permutation-based FDR correction was performed. As threshold for p- and adjusted p-values 0.05 was applied as well as a 2-fold change. Visualizations were performed in R software environment using an in-house script based on the ggplot\u003csup\u003e2\u003c/sup\u003e package. For analysis of FASN regulation, visualization was performed in GraphPad Prism (Version 8.0.2) as well as t-testing with significances of ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSoftware:\u003c/h2\u003e \u003cp\u003eAIDA Image Analyzer: Version 3.44\u003c/p\u003e \u003cp\u003eZotero: 6.0.20\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEstablishment of stable FASN knockdown in Kasumi1, detection of a reduced CTSZ expression by proteomic analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe major goal of this project was the analysis of the functional role of FASN for signal transduction in CBF-AML cells. Therefore, stable knockdowns of FASN were established in the AML cell line Kasumi, an AML cell line with t(8;21) and c-Kit mutation at Asn822. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, expression of FASN was reduced by two independent lentiviral knockdown vectors by over 80% (see also Supplementary Fig.\u0026nbsp;1). The biological effect of FASN knockdown on the levels of fatty acids was analyzed in Kasumi1 cells by chromatography experiments. 27 fatty acids were detected, their amounts (in \u0026micro;g/\u0026micro;l) were normalized to the total concentration of cellular fatty acids and quantified. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the relative amount of myristolic and of palmitic acid is shown in Kasumi1 cells after knockdown of FASN (kd1 and kd2) compared to Kasumi1 cells without knockdown (scr). A relative reduction of fatty acid levels from 100\u0026ndash;64% and 86% for myristolic acid and from 100\u0026ndash;85% and 85% for palmitic acid, respectively, was shown confirming successful knockdown of FASN in both cell clones. The other fatty acids were not down regulated by FASN knockdown (data not shown). We also reproduced FASN knockdowns by mass spectrometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In addition, CTSZ, cytochrome c oxidase (COX6C), the peptidase PEPD and the S100 calcium binding protein A4 (S100-A4) were found to be significantly downregulated, while the glucose transporter SLC2A1 (GLUT1) and the haemoglobin subunit delta (HBD) proteins, respectively, significantly upregulated in Kasumi1 FASN kd1 compared with Kasumi1 scr (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Further, we only focused on interpreting of the role of CTSZ due to its key function in endosomal protein recycling as well as to its overexpression and association with worst survival in pediatric acute leukaemia [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eUp regulation of p- c-Kit, pS6 and of pLyn in Kasumi1 and in Baf3 c-Kit N822K cells after pharmacological and genetic FASN inhibition\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUsing western blot, a significant upregulation of phosphor-S6 (pS6) was observed in both Kasumi1 FASN kd1 and kd2 cells. On the contrary, phospho-Akt (pAkt) and phospho-MAPK (pMAPK) showed a signal-increase under FASN knockdown, however, it was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We also observed a significant upregulation of pS6 was after treatment with the FASN inhibitor TVB-3166, however, the effect occurred 8h after treatment with TVB-3166 but not after 48h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). As mentioned above, we also investigated whether there is a link between FASN activity and the Hedgehog pathway, exemplified here by determining the expression of GLI1 in FASN knockdown cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, we did not see any significant changes in GLI1 expression levels under the two stable FASN knockdowns in Kasumi1 cells (see also Supplementary Fig.\u0026nbsp;2). To verify our results, we examined the aberrant signaling of mutant Kit in two separate subcell lines of Baf3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Both cell lines ectopically expressed Kit-N822K, but were generated independently of each other. In the comparable design, the pharmacological inhibition of FASN by TVB-3166 treatment resulted in an upregulation of both, pAkt and pS6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Interestingly, upregulation of pAkt was detected 24 hours earlier than the upregulation of pS6, consistent with S6 being further downstream of Akt signaling. However, the agonistic effect of FASN inhibition occurred much later in the Baf3-N822K cells compared to the Kasumi1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), suggesting that the time course of pharmacological FASN inhibition is different in the different cellular models and consequently the downstream signaling of the Kit mutant might also be affected, at least temporally differently.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn further experiments, the tyrosine as well as serin-threonin kinases regulated by FASN in Kasumi1 cells were identified in detail by functional kinome profiling (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Expression of FASN was either reduced by knockdown or the activity of FASN was inhibited by TVB-3166. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, respectively, kinase activity was increased both after sustained knockdown as well as after treatment with 100nM FASN-inhibitor TVB-3166 for 5 days. However, it must be noted that only kd2 cells could be analyzed further for their regulatory effects on serin/threonin kinases, since basically no upregulation was detected in kd1 cells and only weak upregulation was detected in TVB-3166-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Importantly, the kinome profile analysis clearly demonstrated higher activity of p-Kit, several members of SRC family kinases (SFK, including pLyn) and pS6 in both Kasumi1 FASN knockdowns as well as after treatment with TVB-3166 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eNo changes in proliferation activity of Kasumi1 cells after inhibition of FASN\u003c/h2\u003e \u003cp\u003eIn the next experiments the effect of FASN on proliferation of Kasumi1 cells was analysed.\u003c/p\u003e \u003cp\u003eDespite the repeatedly observed effect on the downstream signaling of KIT N822K as well as the activation status of other kinases, we did not observe significant changes in the proliferation of Kasumi1 cells after FASN knockdown or after treatment with 100nM TVB-3166 (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eActivating c-Kit mutations predominantly occur in CBF-AML causing an adverse clinical outcome [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To overcome these limitations in the treatment of CBF-AML patients with additional KIT mutations, in particular those in the kinase activation loop (exon 17), investigation of further treatment options is required.\u003c/p\u003e \u003cp\u003eAs mentioned above, active c-Kit mutants are usually localized on the Golgi network leading to an activation of the main signaling pathways STAT5, Akt and MAPK [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The Non-RTK Lyn is one of the members of the Src kinase family and an important co-effector of c-Kit. Lyn participates in c-Kit receptor-mediated downstream signaling by trafficking through the Golgi region and by associating with the juxtamembrane region of c-Kit. This mechanism is specified to the SH4 domain of Lyn [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Lyn was found to be highly expressed in a large series of AML patients playing an important role for leukemogenesis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. It has been reported to be distributed throughout the plasma membrane and the cytoplasm in AML cells where it was expressed in an active state [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Inhibition of Lyn efficiently reduced phosphorylation of mTOR and STAT5, however, it did not affect pAkt nor the ERK/MAPK signaling. Moreover, Lyn has been reported to play an important role in the FLT3-ITD mutated AML, in terms of a signaling component in the FLT3-ITD-STAT5 pathway [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFASN mediated palmitoylation of target proteins is considered to be an important mechanism for development of resistance, for example, in TKI-resistant, epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer. Combination of EGFR-TKIs and the FASN inhibitor Orlistat led to an interruption of the EGFR-TKI resistance in vivo [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In the meanwhile, diverse FASN inhibitors were developed for clinical studies [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Intriguingly, the interrelationship between S-palmitoylation and cell signaling transduction as well as its biological role are poorly understood in hematological malignancies. In mice with AML phenotype, it has been reported that S-palmitoylation of the GTPases H-Ras and N-Ras or of the mutated receptor FLT3-ITD is essential for their binding, localization and trafficking between the plasma membrane and different endosomes (Benjamin Cuiffo et al.) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In 2019, it has been reported that in AML with an activating c-Kit N822K mutation, the mutant is active on the Golgi leading to an activation of Akt-, MAPK and STAT5 signaling while the inactive/dephosphorylated c-Kit form is localized in the ER [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, the role of palmitoylation for the localization of c-Kit mutant on the Golgi lipid rafts and for its subsequent activity is currently under investigation.\u003c/p\u003e \u003cp\u003eWe were able to establish two stable FASN knockdowns in Kasumi1 cells by lentiviral transduction with FASN-shRNA vectors. Their statistical and biological significance was confirmed by immunoblotting, by mass spectrometry as well as by lipidomic analysis showing a strong level reduction of specifically myristolic and palmitic acid in both FASN knockdowns. Using two cell models of c-Kit-N822K mutated CBF-AML, we demonstrate that pharmacological and genetic FASN inhibition led to a rewiring of the c-Kit-mutant associated pathways, especially, to a significant upregulation of p-c-Kit, pLyn and pS6. Moreover, we found a down regulation of CTSZ in FASN-knockdown Kasumi1 cells by mass spectrometry.\u003c/p\u003e \u003cp\u003eCTSZ is a critical component of the endocytic pathways in myelopoesis which is required for endosomal trafficking, degradation and recycling of numerous plasma membrane as well as of cytosolic proteins through the Golgi network [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Intriguingly, cathepsins are also known to be overexpressed and associated with poor clinical outcome in pediatric [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Recent studies have suggested potential role for S-palmitoylation in lysosomal functions indirectly through its effects on proteins involved in lysosomal trafficking, membrane fusion, signaling pathways and protein degradation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Hence, inhibition of FASN can downregulate CTSZ, which then leads to a reduced degradation of c-Kit and p-c-Kit. In the next step, pLyn can upregulate pS6 which is a downstream target of mTOR. This mechanism can be suggested by the published data regarding the activation of mTOR by Lyn [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Via this mechanism, a subcellular reprogramming of c-Kit associated pathways could take place making AML cells stronger dependent on the pLyn-pS6 signalling. Remarkably, we were not able to detect any significant changes in proliferation of Kasumi1 cells upon treatment with the FASN inhibitor TVB-3166 nor in both FASN knockdowns which could be caused by persistently active pLyn.\u003c/p\u003e \u003cp\u003eIn view of the clinical approval of the Hedgehog signaling inhibitor Glasdegib for therapy of AML and in order to analyze a rationale for further treatment options, we also investigated whether the expression of the downstream Hedgehog effector Gli1 is dependent on the FASN activity. Our hypothesis is based on some reports in solid tumors which, for example, show that FASN knockdown via siRNA can reduce Gli1 levels in gastric cancer cells, suggesting that FASN may play a role in the tumorigenesis and metastasis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, we were not able to detect any significant changes in the expression of Gli1 by the inhibition of FASN. This observation could be explained in terms of a compensatory effect of the increased pS6 which would suggest the existence of SMO-independent regulation of Hedgehog-Gli signaling by key oncogenic drivers in various myeloid malignancies [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo our knowledge, these findings offer new insights into a potential mechanism by which reduced expression of CTSZ due to the inhibition of FASN increases c-Kit, Lyn, and S6 kinase activity in CBF-AML with activating c-Kit mutation. In summary, our data suggest a rationale to further explore combined inhibition of FASN, c-Kit, Src/Lyn, and S6 kinase in the treatment of CBF-AML with activating c-Kit mutations.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAcute Myeloid Leukemia (AML)\u003c/p\u003e\n\u003cp\u003eFMS-like tyrosine kinase 3 (FLT3)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ewild-type FLT3 (FLT3-WT)\u003c/p\u003e\n\u003cp\u003eInternal tandem duplication (FLT3-ITD)\u003c/p\u003e\n\u003cp\u003ejuxtamembrane domain (JM domain)\u003c/p\u003e\n\u003cp\u003etyrosine kinase domain (FLT3-TKD)\u003c/p\u003e\n\u003cp\u003eplasma membrane (PM)\u003c/p\u003e\n\u003cp\u003eRAS/extracellular signal\u0026ndash;regulated kinase (ERK)\u003c/p\u003e\n\u003cp\u003ecysteine at position 563 (C563)\u003c/p\u003e\n\u003cp\u003estem cell transplant (SCT)\u003c/p\u003e\n\u003cp\u003ecomplete remission (CR)\u003c/p\u003e\n\u003cp\u003eCore binding factor-AML (CBF-AML)\u003c/p\u003e\n\u003cp\u003emitogen-activated protein kinase (MAPK)\u003c/p\u003e\n\u003cp\u003eSrc family kinases (SFKs)\u003c/p\u003e\n\u003cp\u003eFatty Acid Synthase (FASN)\u003c/p\u003e\n\u003cp\u003eHedgehog (Hh)\u003c/p\u003e\n\u003cp\u003esuppressor of fused homolog (SuFu)\u003c/p\u003e\n\u003cp\u003eRPS6 (Ribosomal Protein S6)\u003c/p\u003e\n\u003cp\u003eCD117 (cluster of differentiation 117)\u003c/p\u003e\n\u003cp\u003estem cell growth factor receptor (SCFR)\u003c/p\u003e\n\u003cp\u003ecore binding factor (CBF)\u003c/p\u003e\n\u003cp\u003ehematopoietic stem cells (HSCs)\u003c/p\u003e\n\u003cp\u003eEuropean Leukemia Net (ELN)\u003c/p\u003e\n\u003cp\u003ecomplete remission (CR)\u003c/p\u003e\n\u003cp\u003erelapse-free survival (RFS)\u003c/p\u003e\n\u003cp\u003eSmall Nuclear Ribonucleoprotein Polypeptide G (SNRPG)\u003c/p\u003e\n\u003cp\u003eGlutathione Peroxidase 7 (GPX7)\u003c/p\u003e\n\u003cp\u003ePeptide Deformylase (PDF)\u003c/p\u003e\n\u003cp\u003eNebulin (NEB)\u003c/p\u003e\n\u003cp\u003ereactive oxigen species (ROS)\u003c/p\u003e\n\u003cp\u003eNon-alcoholic fatty liver disease (NAFLD)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure of potential conflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors of this article have declared \u0026lsquo;\u0026lsquo;no conflict of interest\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.K. and M.J. planned and supervised the study. R.Z. planned and performed experiments and analyzed the data. M.Kr., K.H., H.V., B.S, S.H., A. W. performed experiments, wrote and reviewed the manuscript. N.vB., C.K., N.G., S.P-G. kindly provided financial support and reviewed the manuscript. W.F. and C.B. reviewed the manuscript. M. K. wrote the manuscript. All the authors have approved the manuscript.\u0026nbsp;\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Maxim Kebenko or Manfred J\u0026uuml;cker\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Bettina Bettin for excellent technical assistance.\u0026nbsp;The authors thank\u0026nbsp;Sagimet Biosciences Inc. (formerly 3-V-Biosciences)\u0026nbsp;(\u003cem\u003eSan Mateo\u003c/em\u003e, California) for kindly providing of TVB-3166.\u0026nbsp;This study was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (INST 337/15-1, INST 337/16-1, INST 152/837-1 and INST 152/947-1 FUGG).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKoenig KL, Sahasrabudhe KD, Sigmund AM, Bhatnagar B. AML with Myelodysplasia-Related Changes: Development, Challenges, and Treatment Advances. Genes (Basel). 2020;11(8). doi: 10.3390/genes11080845.\u003c/li\u003e\n\u003cli\u003eSwaminathan M, Wang ES. Novel therapies for AML: a round-up for clinicians. Expert Rev Clin Pharmacol. 2020;13(12):1389-400. doi: 10.1080/17512433.2020.1850255.\u003c/li\u003e\n\u003cli\u003eDohner H, Wei AH, Appelbaum FR, Craddock C, DiNardo CD, Dombret H, et al. Diagnosis and management of AML in adults: 2022 recommendations from an international expert panel on behalf of the ELN. 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Cancers (Basel). 2021;13(19). doi: 10.3390/cancers13194888.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"fatty acid synthase, palmitoylation, TVB-3166, AML, c-Kit mutation, PI3K, Akt, mTOR, S6 kinase, Lyn, Gli1, hedgehog signaling, cathepsin Z","lastPublishedDoi":"10.21203/rs.3.rs-4648786/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4648786/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAML is a rare hematological malignancy still associated with poor prognosis. 5% of de novo AML and 30% of core binding factor (CBF) AML (translocation t(8;21)(q22;q22) or invasion (16)(p13;q22)), respectively, harbor activating c-Kit (CD117) mutations leading to an adverse clinical outcome. Posttranslational protein modifications, especially by myristolic and palmitic acid, are known to be important for diverse cell functions such as membrane organization, transduction signaling or regulation of apoptosis. However, most data come from solid tumor studies while its role in AML is still poorly understood. Fatty acid synthase (FASN) is one of the key palmitoyl-acyltransferases which controls subcellular localization, trafficking and degradation of various target proteins. H-Ras, N-Ras or FLT3-ITDmut receptors are known to be important target proteins for FASN in AML.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study, we investigated the role of FASN in two c-Kit-N822K mutated AML cell lines. Using FASN knockdown via shRNA and the FASN inhibitor TVB-3166. Functional implications including cell viability and proliferation were tracked in a combined approach integrating western blotting, mass spectrometry PamGene.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn FASN-knockdown cells, we observed an increase in phosphorylation of c-Kit (p-c-Kit), Lyn kinase (pLyn) as well as of S6 kinase (pS6). Moreover, a downregulation of cathepsin Z (CTSZ), which belongs to endo-lysosomal proteases and is hence essential for degradation of cellular proteins within lysosomes was found.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eRecent studies have suggested potential roles for palmitoylation in lysosomal function indirectly through its effects on proteins involved in lysosomal trafficking, membrane fusion, and signaling pathways. Therefore, our observation of the reduced expression of CTSZ due to the inhibition of FASN offers an explanation for the increased c-Kit, Lyn, and S6 kinase activity in CBF-AML with activating c-Kit mutation.\u003c/p\u003e","manuscriptTitle":"Functional role of fatty acid synthase for signal transduction in Core binding factor-AML with activating c-Kit mutation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-05 12:33:17","doi":"10.21203/rs.3.rs-4648786/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b22aa4e3-3acc-44ce-a063-16574526a1ba","owner":[],"postedDate":"August 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-07T13:57:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-05 12:33:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4648786","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4648786","identity":"rs-4648786","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}