{"gene":"CSF1R","run_date":"2026-04-28T17:28:53","timeline":{"discoveries":[{"year":1985,"finding":"The c-fms proto-oncogene product is the receptor for CSF-1 (M-CSF): it is a 165-170 kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity, expressed on mononuclear phagocytes, that specifically binds CSF-1 and undergoes tyrosine phosphorylation upon ligand binding in membrane preparations.","method":"Immunoprecipitation with anti-v-fms antisera, specific CSF-1 binding assay, immune complex kinase assay in membrane preparations","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — original biochemical identification with multiple orthogonal methods (kinase assay, ligand binding, immunoprecipitation), foundational paper with >1500 citations","pmids":["2408759"],"is_preprint":false},{"year":1987,"finding":"Phosphorylation of Tyr969 in the C-terminal tail of c-fms negatively regulates receptor kinase activity; mutation to Phe969 (as in v-fms) activates oncogenic potential of c-fms when combined with CSF-1 expression, and the C-terminal tyrosine deletion is necessary but not sufficient alone for transformation.","method":"NIH 3T3 transformation assay with chimeric v-fms/c-fms constructs, cotransfection with CSF-1 cDNA, site-directed mutagenesis of Tyr969 to Phe969","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis combined with functional transformation assay, replicated across multiple chimeric constructs","pmids":["3027579"],"is_preprint":false},{"year":1987,"finding":"Proviral insertion at the fim-2 locus (spanning the 5'-end of c-fms) results in high-level expression of a normal-sized c-fms mRNA, establishing c-fms activation by retroviral insertion as a mechanism in myeloblastic leukemias.","method":"Southern blotting, Northern blotting, proviral integration mapping in Friend MuLV-induced murine myeloblastic leukemias","journal":"Nature","confidence":"Medium","confidence_rationale":"Tier 2 — molecular mapping in tumor samples, replicated across multiple tumor lines","pmids":["3476856"],"is_preprint":false},{"year":1988,"finding":"CSF-1R (c-fms product) is a member of the receptor tyrosine kinase family; its intracellular tyrosine kinase domain transduces CSF-1 signals for proliferation, survival, and differentiation of mononuclear phagocyte lineage cells; v-fms mutations alter kinase activity to provide ligand-independent growth signals.","method":"Biochemical characterization, kinase activity assays, v-fms/c-fms domain comparisons","journal":"Journal of cellular biochemistry","confidence":"High","confidence_rationale":"Tier 1 — biochemical characterization consolidated across multiple prior studies; review by original discoverers","pmids":["2852667"],"is_preprint":false},{"year":1989,"finding":"c-fms mRNA levels are regulated posttranscriptionally by a labile stabilizing protein in both differentiating HL-60 cells and in mature human monocytes; the rate of c-fms gene transcription is unchanged upon TPA-induced differentiation or TPA-induced downregulation, but mRNA half-life is dramatically altered by cycloheximide.","method":"Nuclear run-on transcription assays, mRNA half-life measurements with cycloheximide treatment, Northern blotting","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 — run-on assays and mRNA stability experiments with pharmacological inhibition, multiple cell types tested","pmids":["2523515"],"is_preprint":false},{"year":1990,"finding":"Activation of CSF-1R intracellular tyrosine kinase domain alone (without CSF-1) is sufficient to induce mitogenesis; a chimeric CD2 ectodomain–CSF-1R kinase domain receptor expressed in NIH 3T3 fibroblasts undergoes tyrosine phosphorylation, receptor downmodulation, and mitogenesis upon anti-CD2 antibody stimulation.","method":"Chimeric receptor expression in NIH 3T3 cells, anti-CD2 antibody stimulation, tyrosine phosphorylation assay, proliferation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 — reconstitution with chimeric receptor and direct kinase activity measurement","pmids":["1691441"],"is_preprint":false},{"year":2003,"finding":"The transcription factor AML1 (RUNX1) directly binds the c-FMS gene in normal cells; in t(8;21) leukemic cells, the AML1-ETO fusion protein binds extended sequences of the c-FMS intronic regulatory region and alters histone modification patterns and increases association of histone deacetylases, without irreversibly disrupting transcription factor binding.","method":"In vivo footprinting, chromatin immunoprecipitation (ChIP) in normal and t(8;21) leukemic cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — direct chromatin immunoprecipitation and in vivo footprinting with histone modification analysis","pmids":["12773394"],"is_preprint":false},{"year":2004,"finding":"During B-lymphopoiesis, c-fms chromatin is silenced in discrete steps: first transcription factor binding and RNA expression are lost, then nuclease accessibility decreases; even in mature B cells the locus remains poised and can be reactivated by conditional deletion of Pax5; de novo DNA methylation overlaps with an intronic antisense transcription unit.","method":"DNase I hypersensitivity assays, chromatin immunoprecipitation, bisulfite sequencing, conditional Pax5 deletion in B cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — multiple orthogonal chromatin analysis methods with genetic rescue experiment","pmids":["15483629"],"is_preprint":false},{"year":2005,"finding":"Imatinib inhibits the CSF-1R (c-fms) kinase at therapeutic concentrations; it inhibits c-fms tyrosine phosphorylation in macrophages and blocks M-CSF-induced proliferation of a cytokine-dependent cell line, without downregulating c-fms expression.","method":"Phosphorylation assays in macrophages, cytokine-dependent cell line proliferation assay","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1/2 — direct kinase phosphorylation assay combined with functional proliferation assay","pmids":["15637141"],"is_preprint":false},{"year":2006,"finding":"Pax5 directly represses c-fms by being recruited to the c-fms locus, causing rapid loss of RNA polymerase II binding and then loss of transcription factor binding and DNase I hypersensitivity at all cis-regulatory elements; Pax5 targets the basal transcription machinery by interacting with a binding site within the major transcription start sites.","method":"Chromatin immunoprecipitation, DNase I hypersensitivity assays, conditional Pax5 expression system","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — multiple chromatin methods with direct Pax5 recruitment demonstrated","pmids":["16482219"],"is_preprint":false},{"year":2007,"finding":"Crystal structure of the autoinhibited human c-Fms kinase domain at 2.7 Å resolution shows that the juxtamembrane (JM) domain binds a hydrophobic site adjacent to the ATP binding pocket, preventing the activation loop from adopting an active conformation; three JM-derived tyrosine residues drive autoinhibition, a mechanism shared with c-Kit and Flt3.","method":"X-ray crystallography at 2.7 Å resolution of the cytosolic kinase + juxtamembrane domain","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure with mechanistic interpretation of autoinhibition","pmids":["17292918"],"is_preprint":false},{"year":2008,"finding":"M-CSF binding to CSF-1R (c-Fms) in osteoclasts generates a signaling complex with phosphorylated DAP12 (ITAM adaptor) and Syk kinase; c-Fms Tyr559 (exclusive c-Src binding site) is required for DAP12/Syk signaling; deletion of DAP12 or Syk yields osteoclasts that fail to reorganize their cytoskeleton, and Syk SH2 domain and DAP12 ITAM tyrosines and transmembrane domain mediate this signaling.","method":"Co-immunoprecipitation, retroviral transduction of null precursors with wild-type/mutant DAP12 and Syk, cytoskeleton analysis, genetic knockout models","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1/2 — genetic and biochemical epistasis combined with domain mutagenesis and functional osteoclast phenotype","pmids":["18691974"],"is_preprint":false},{"year":2010,"finding":"The adaptor protein Lnk binds directly to c-Fms (CSF-1R) via its PH domain independently of M-CSF stimulation; Lnk overexpression suppresses M-CSF-induced tyrosine phosphorylation of c-Fms and Akt activation, and inhibits M-CSF-induced macrophage migration; Lnk KO macrophages show enhanced and prolonged Akt phosphorylation but diminished Erk phosphorylation in response to M-CSF.","method":"Co-immunoprecipitation, Lnk knockout macrophages, clonogenic assays, western blotting of Akt/Erk phosphorylation, migration assays","journal":"Journal of leukocyte biology","confidence":"High","confidence_rationale":"Tier 2 — direct binding demonstrated by co-IP, confirmed with KO macrophages and multiple signaling readouts","pmids":["20571037"],"is_preprint":false},{"year":2010,"finding":"STAP-2 adaptor protein directly binds to the PH domain of c-Fms independent of M-CSF stimulation; STAP-2 overexpression suppresses M-CSF-induced c-Fms tyrosine phosphorylation and downstream Akt and ERK activation, and impairs M-CSF-induced macrophage migration.","method":"Co-immunoprecipitation, overexpression in Raw 264.7 macrophages, western blotting of phospho-c-Fms, phospho-Akt, phospho-ERK, wound-healing migration assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding by co-IP with functional consequences, single lab","pmids":["17512498"],"is_preprint":false},{"year":2010,"finding":"Vigilin binds a 69-nucleotide non-AU-rich sequence in the c-fms 3' UTR and competitively displaces HuR from this site; vigilin decreases c-fms mRNA stability and inhibits c-fms translation, whereas HuR stabilizes c-fms mRNA; the two proteins exert opposing effects on breast cancer cell motility and invasion.","method":"RNA immunoprecipitation, mRNA stability assays, polysome fractionation, competition binding assays, cell invasion/motility assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1/2 — multiple orthogonal methods (RIP, mRNA stability, translation assays) with functional cellular readouts","pmids":["20974809"],"is_preprint":false},{"year":2013,"finding":"CSF1R is expressed on a small subpopulation of hippocampal and cortical neurons under physiological conditions; excitotoxic injury markedly upregulates neuronal CSF1R expression; selective deletion of CSF1R in forebrain neurons exacerbates excitotoxin-induced neurodegeneration; CSF1 /IL-34 neuroprotection is accompanied by maintenance of CREB signaling in neurons.","method":"Lineage-tracing experiments, conditional neuron-specific CSF1R deletion, kainic acid excitotoxin model, CREB phosphorylation western blot, histological quantification","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with specific phenotypic readout in multiple experiments","pmids":["23296467"],"is_preprint":false},{"year":2015,"finding":"X-ray co-crystallography guided development of PLX3397, a CSF1R inhibitor that traps the kinase in the autoinhibited conformation; structure-based design enables selective CSF1R blockade.","method":"X-ray co-crystallography of CSF1R kinase with PLX3397, phase 1 clinical trial","journal":"The New England journal of medicine","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with bound inhibitor directly informs mechanism of inhibition","pmids":["26222558"],"is_preprint":false},{"year":2016,"finding":"Resistance to CSF-1R inhibition in recurrent GBM is driven by the tumor microenvironment; macrophage-derived IGF-1 activates PI3K pathway via tumor cell IGF-1R, and transplantation of resistant tumors re-establishes sensitivity to CSF-1R inhibition.","method":"Tumor transplantation experiments, IGF-1/IGF-1R pathway analysis, PI3K inhibitor combination therapy in mouse GBM model","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — epistasis via transplantation and pathway inhibition, multiple orthogonal experiments","pmids":["27199435"],"is_preprint":false},{"year":2019,"finding":"Deletion of the fms-intronic regulatory element (FIRE) super-enhancer within the Csf1r locus selectively ablates CSF1R expression in specific tissue macrophage populations (brain microglia, skin, kidney, heart, peritoneum macrophages) and blocks macrophage development from embryonic stem cells, while leaving monocyte homeostasis largely unaffected.","method":"CRISPR/genomic deletion of FIRE in mice, tissue macrophage quantification, in vitro ESC differentiation assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — in vivo genetic deletion with tissue-specific phenotypic characterization across multiple organs","pmids":["31324781"],"is_preprint":false},{"year":2019,"finding":"CSF1R haploinsufficiency (loss-of-function mutations) causes hereditary diffuse leukoencephalopathy with spheroids (HDLS); mutations in the kinase domain lead to defective autophosphorylation; mutant CSF1R proteins show lower expression levels and reduced autophagy (decreased LC3-II levels) compared to wild-type.","method":"Next-generation sequencing, Sanger sequencing, in vitro autophosphorylation assays of mutant CSF1R proteins, western blotting of LC3-II, immunofluorescence","journal":"Translational neurodegeneration","confidence":"High","confidence_rationale":"Tier 1/2 — direct in vitro kinase assays on multiple mutants combined with autophagy marker analysis","pmids":["31827782"],"is_preprint":false},{"year":2020,"finding":"Rab11A negatively regulates osteoclastogenesis by promoting lysosomal degradation of c-fms and RANK surface receptors; Rab11A overexpression enlarges early endosomes and upregulates lysosomal activity (LAMP1, cathepsins B and D), leading to proteolytic degradation of c-fms; lysosomal inhibition by chloroquine rescues c-fms and RANK protein levels.","method":"Rab11A overexpression/silencing in RAW-D cells and BMMs, immunocytochemistry for endosomal markers, LAMP1 and cathepsin quantification, chloroquine rescue experiments, flow cytometry of surface c-fms","journal":"Cells","confidence":"High","confidence_rationale":"Tier 2 — gain/loss of function with pharmacological rescue and multiple marker analyses","pmids":["33142674"],"is_preprint":false},{"year":2020,"finding":"Rab11b promotes lysosomal degradation of c-Fms and RANK surface receptors in osteoclasts; Rab11b overexpression enlarges early/late endosomes and augments lysosomal activity, reducing surface c-Fms abundance and attenuating NFATc-1 upstream signaling; chloroquine inhibition of lysosomal function rescues c-Fms levels.","method":"Rab11b overexpression in RAW-D cells and BMMs, immunocytochemistry, flow cytometry of surface c-Fms, chloroquine rescue, western blotting","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — complementary to Rab11A study, multiple methods but single lab","pmids":["33302495"],"is_preprint":false},{"year":2022,"finding":"A glycine to alanine substitution at position 795 (G795A) of human CSF1R confers resistance to multiple CSF1R inhibitors (PLX3397, PLX5622) without discernable gain or loss of CSF1R function; G795A-expressing macrophages engraft mouse brain during PLX3397 treatment and persist after cessation.","method":"Biochemical and cell-based kinase activity assays, CRISPR engineering of iPSC-derived microglia, xenotransplantation studies, gene expression profiling","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1/2 — site-directed mutagenesis with biochemical validation, in vivo engraftment, and transcriptomic confirmation","pmids":["36584406"],"is_preprint":false},{"year":2004,"finding":"gp130-dependent ERK1/2 MAP kinase signaling positively regulates c-fms expression in macrophages; in gp130(ΔSTAT/ΔSTAT) macrophages, enhanced ERK1/2 activation correlates with elevated c-fms expression and increased M-CSF responsiveness, while gp130(Y757F/Y757F) macrophages with reduced ERK1/2 activation show decreased c-fms expression and reduced M-CSF-induced proliferation; an ERK1/2 inhibitor suppresses M-CSF-induced proliferation and c-fms expression.","method":"gp130 signaling mutant knock-in mouse macrophages, ERK1/2 phosphorylation assays, clonogenic assays, pharmacological ERK1/2 inhibition, western blotting of c-fms","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — genetic and pharmacological manipulation with multiple readouts in multiple genotypes","pmids":["14749363"],"is_preprint":false}],"current_model":"CSF1R (c-Fms) is a transmembrane receptor tyrosine kinase that binds CSF-1 and IL-34 to drive proliferation, differentiation, and survival of mononuclear phagocytes; upon ligand binding, the juxtamembrane domain (held in an autoinhibited conformation) is released, enabling kinase activation, autophosphorylation, and recruitment of signaling partners including DAP12/Syk (linking to cytoskeletal reorganization in osteoclasts), PI3K/Akt, ERK, and adaptors Lnk and STAP-2 that negatively regulate signaling; c-fms expression is controlled at the posttranscriptional level by competing mRNA-binding proteins (HuR vs. vigilin) and epigenetically by transcription factors (Pax5 represses, AML1-ETO modifies histone marks) acting on a super-enhancer (FIRE) within the locus; loss-of-function mutations in the kinase domain cause HDLS/CSF1R-related leukoencephalopathy through defective autophosphorylation and impaired microglial survival, while C-terminal mutations (Phe969) that abolish negative regulatory phosphorylation activate oncogenic potential."},"narrative":{"teleology":[{"year":1985,"claim":"The identity of the CSF-1 receptor was unknown; demonstrating that the c-fms proto-oncogene product is a transmembrane glycoprotein with intrinsic tyrosine kinase activity that specifically binds CSF-1 established the molecular basis of macrophage growth factor signaling.","evidence":"Immunoprecipitation with anti-v-fms antisera, CSF-1 binding assay, and immune complex kinase assay in membrane preparations","pmids":["2408759"],"confidence":"High","gaps":["Ligand-induced receptor conformational changes not resolved","Downstream signaling pathways not yet mapped","Second ligand IL-34 not yet identified"]},{"year":1987,"claim":"How oncogenic v-fms differs from c-fms was unclear; showing that Tyr969 phosphorylation negatively regulates the receptor and that its mutation to Phe (as in v-fms) is required for transformation established a C-terminal autoinhibitory mechanism and the molecular basis of c-fms oncogenic activation.","evidence":"Site-directed mutagenesis of Tyr969→Phe969, NIH 3T3 transformation assays with chimeric v-fms/c-fms constructs co-transfected with CSF-1 cDNA","pmids":["3027579"],"confidence":"High","gaps":["Signaling partners recruited to phospho-Tyr969 not identified","Whether Tyr969 mutation alone is sufficient for in vivo tumorigenesis unknown"]},{"year":1989,"claim":"Whether c-fms expression is controlled transcriptionally or posttranscriptionally was uncertain; nuclear run-on and mRNA half-life experiments demonstrated that a labile protein stabilizes c-fms mRNA, establishing posttranscriptional regulation as a major control mechanism.","evidence":"Nuclear run-on transcription assays, mRNA half-life measurement with cycloheximide in differentiating HL-60 cells and monocytes","pmids":["2523515"],"confidence":"High","gaps":["Identity of the stabilizing protein not determined at this time","Cis-element in c-fms mRNA mediating stability not mapped"]},{"year":1990,"claim":"Whether CSF-1R kinase activity alone suffices for mitogenic signaling was untested; a chimeric receptor study proved that activation of the intracellular kinase domain without CSF-1 binding is sufficient to drive autophosphorylation, receptor downmodulation, and mitogenesis.","evidence":"Chimeric CD2-ectodomain/CSF-1R-kinase receptor in NIH 3T3 cells stimulated with anti-CD2 antibody","pmids":["1691441"],"confidence":"High","gaps":["Which specific tyrosine autophosphorylation sites mediate mitogenic signaling not mapped","Downstream signaling intermediates not identified"]},{"year":2003,"claim":"How the AML1-ETO fusion oncoprotein deregulates c-FMS in t(8;21) leukemia was unknown; ChIP and in vivo footprinting showed AML1-ETO binds extended intronic regulatory sequences and remodels histone marks, establishing an epigenetic mechanism for c-FMS dysregulation in leukemia.","evidence":"In vivo footprinting and chromatin immunoprecipitation with histone modification analysis in normal versus t(8;21) leukemic cells","pmids":["12773394"],"confidence":"High","gaps":["Whether AML1-ETO binding directly alters c-FMS transcription output not quantified","Functional consequence for macrophage differentiation not tested"]},{"year":2004,"claim":"How c-fms is silenced during B-cell commitment and how extrinsic signals regulate its expression in macrophages were unresolved; two studies showed that Pax5 represses c-fms by displacing RNA polymerase II from the locus (reversible upon Pax5 deletion), and that gp130-ERK1/2 signaling positively regulates c-fms expression and M-CSF responsiveness in macrophages.","evidence":"Conditional Pax5 deletion with ChIP and DNase I hypersensitivity in B cells; gp130 signaling mutant knock-in macrophages with pharmacological ERK1/2 inhibition","pmids":["15483629","14749363"],"confidence":"High","gaps":["Direct Pax5 DNA-binding site relative to c-fms promoter not fully mapped at this point","Whether ERK1/2 acts on c-fms transcription or mRNA stability not distinguished"]},{"year":2006,"claim":"The mechanism by which Pax5 silences c-fms was incompletely understood; detailed ChIP showed Pax5 is recruited to the c-fms transcription start sites, directly displacing the basal transcription machinery and sequentially shutting down all cis-regulatory elements.","evidence":"Chromatin immunoprecipitation and DNase I hypersensitivity with conditional Pax5 expression system","pmids":["16482219"],"confidence":"High","gaps":["Whether Pax5-mediated silencing involves DNA methylation changes not fully resolved","Kinetics of reactivation upon Pax5 removal in vivo not measured"]},{"year":2007,"claim":"The structural basis of CSF1R autoinhibition was unknown; the 2.7 Å crystal structure revealed that juxtamembrane domain tyrosines wedge into a hydrophobic pocket adjacent to the ATP-binding site, locking the activation loop in an inactive conformation—a mechanism shared with c-Kit and Flt3.","evidence":"X-ray crystallography of the cytosolic kinase plus juxtamembrane domain at 2.7 Å resolution","pmids":["17292918"],"confidence":"High","gaps":["Full-length extracellular–transmembrane–kinase structure not available","Dynamics of JM release upon ligand binding not captured"]},{"year":2008,"claim":"How CSF-1R signals to the osteoclast cytoskeleton was unclear; demonstrating that c-Fms Tyr559 nucleates a DAP12/Syk signaling complex essential for cytoskeletal reorganization established a non-canonical ITAM-based pathway downstream of CSF-1R in osteoclasts.","evidence":"Co-immunoprecipitation, retroviral reconstitution of DAP12/Syk-null osteoclast precursors with wild-type and mutant constructs, cytoskeletal analysis","pmids":["18691974"],"confidence":"High","gaps":["Whether DAP12/Syk pathway operates in macrophages outside osteoclasts not tested","Direct substrates of Syk downstream of c-Fms not identified"]},{"year":2010,"claim":"Negative regulators that restrain CSF-1R signaling magnitude were largely unknown; identification of Lnk and STAP-2 as adaptors that constitutively bind c-Fms and suppress M-CSF-induced phosphorylation, Akt activation, and macrophage migration established feedback attenuation mechanisms.","evidence":"Co-immunoprecipitation, Lnk knockout macrophages, STAP-2 overexpression, Akt/ERK phosphorylation kinetics, migration assays","pmids":["20571037","17512498"],"confidence":"High","gaps":["Whether Lnk and STAP-2 compete for the same binding site on c-Fms not determined","In vivo macrophage phenotypes of STAP-2 loss not tested"]},{"year":2010,"claim":"The posttranscriptional regulators of c-fms mRNA identified in 1989 were molecularly uncharacterized; demonstrating that vigilin and HuR compete for a 69-nt element in the c-fms 3′ UTR with opposing effects on mRNA stability and translation provided a molecular mechanism for posttranscriptional control of c-fms expression.","evidence":"RNA immunoprecipitation, mRNA stability assays, polysome fractionation, competition binding assays in breast cancer cells","pmids":["20974809"],"confidence":"High","gaps":["Whether this mechanism operates in primary macrophages not shown","Other RNA-binding proteins recognizing the c-fms 3′ UTR not surveyed"]},{"year":2013,"claim":"Whether CSF1R functions outside myeloid cells was controversial; conditional neuron-specific deletion showed CSF1R is expressed on hippocampal and cortical neurons and is neuroprotective during excitotoxic injury via CREB signaling, establishing a cell-autonomous neuronal role.","evidence":"Neuron-specific conditional CSF1R deletion, kainic acid excitotoxin model, CREB phosphorylation analysis","pmids":["23296467"],"confidence":"High","gaps":["Which CSF1R ligand (CSF-1 vs IL-34) is the physiological neuronal agonist in vivo not resolved","Downstream signaling intermediates between CSF1R and CREB in neurons not mapped"]},{"year":2015,"claim":"Structure-based drug design of CSF1R inhibitors was enabled by co-crystallography; the PLX3397 co-crystal structure showed the inhibitor traps CSF1R in the autoinhibited conformation, providing a mechanistic rationale for selective kinase blockade.","evidence":"X-ray co-crystallography of CSF1R kinase domain with PLX3397, phase 1 clinical trial","pmids":["26222558"],"confidence":"High","gaps":["Resistance mechanisms to PLX3397 not yet characterized","Whether JM-locking inhibitors differ functionally from activation-loop inhibitors not tested"]},{"year":2019,"claim":"The cis-regulatory architecture controlling tissue-specific CSF1R expression was incompletely understood; CRISPR deletion of the FIRE super-enhancer selectively ablated microglia and several tissue-resident macrophage populations while sparing monocytes, demonstrating lineage-specific enhancer dependence.","evidence":"CRISPR deletion of FIRE enhancer in mice, tissue macrophage quantification across multiple organs, in vitro ESC differentiation","pmids":["31324781"],"confidence":"High","gaps":["Whether FIRE deletion affects macrophage function beyond abundance not assessed","Compensatory enhancer elements not surveyed"]},{"year":2019,"claim":"The molecular pathogenesis of HDLS/CSF1R-related leukoencephalopathy was mechanistically unclear; in vitro kinase assays of patient-derived mutations demonstrated defective autophosphorylation and reduced autophagy (LC3-II levels), linking kinase domain loss-of-function to disease.","evidence":"In vitro autophosphorylation assays of multiple mutant CSF1R proteins, LC3-II western blotting, patient sequencing","pmids":["31827782"],"confidence":"High","gaps":["Whether reduced autophagy is a direct kinase substrate effect or secondary not determined","Microglial-specific consequences of individual mutations not characterized in vivo"]},{"year":2020,"claim":"How surface CSF1R abundance is regulated post-endocytically was poorly understood; two studies showed Rab11A and Rab11B promote lysosomal degradation of c-Fms in osteoclasts by enlarging endosomal compartments and augmenting cathepsin activity, with chloroquine rescue confirming lysosomal dependence.","evidence":"Rab11A/Rab11B overexpression and silencing in RAW-D cells and BMMs, LAMP1/cathepsin quantification, chloroquine rescue, flow cytometry","pmids":["33142674","33302495"],"confidence":"High","gaps":["Whether Rab11-mediated degradation operates in macrophage populations beyond osteoclasts not tested","Ubiquitin signals directing c-Fms to lysosomes not identified"]},{"year":2022,"claim":"Whether CSF1R can be engineered for inhibitor resistance without functional compromise was untested; the G795A gatekeeper-adjacent substitution conferred resistance to PLX3397/PLX5622 while preserving normal kinase function, enabling selective microglial replacement in vivo.","evidence":"CRISPR-engineered iPSC-derived microglia with G795A mutation, biochemical kinase assays, xenotransplantation into PLX3397-treated mice, transcriptomic profiling","pmids":["36584406"],"confidence":"High","gaps":["Long-term consequences of G795A microglia engraftment not assessed","Whether G795A confers resistance to other CSF1R inhibitor scaffolds not tested"]},{"year":null,"claim":"A full-length structure of CSF1R encompassing the extracellular, transmembrane, and intracellular domains in both inactive and active states is lacking, and the precise mechanism by which ligand-induced extracellular dimerization releases juxtamembrane autoinhibition remains structurally unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["Full-length ligand-bound receptor structure not available","How IL-34 versus CSF-1 binding differentially activates downstream pathways not mechanistically resolved","In vivo significance of neuronal CSF1R under non-excitotoxic conditions not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,3,5,10,11,19]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,3,5,10]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,3]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,5,11,20,21,22]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[10]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[20,21]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[20,21]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,5,11,12,15]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,11,18]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[7,9,18]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,2,19]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[15,19]}],"complexes":[],"partners":["CSF1","DAP12","SYK","LNK","STAP2","PAX5","HDLBP","ELAVL1"],"other_free_text":[]},"mechanistic_narrative":"CSF1R (c-Fms) is a receptor tyrosine kinase that serves as the primary signaling receptor for CSF-1 (M-CSF) and IL-34, governing the proliferation, differentiation, survival, and functional activation of mononuclear phagocytes, osteoclasts, microglia, and certain neurons. Upon ligand binding, autoinhibition mediated by the juxtamembrane domain—which wedges against the kinase active site to prevent activation loop remodeling—is released, enabling autophosphorylation and recruitment of downstream effectors including DAP12/Syk (for osteoclast cytoskeletal reorganization), PI3K/Akt, and ERK, while adaptors Lnk and STAP-2 attenuate signaling by suppressing receptor phosphorylation [PMID:2408759, PMID:17292918, PMID:18691974, PMID:20571037]. CSF1R expression is controlled by a combination of posttranscriptional regulation—through competing mRNA-binding proteins HuR (stabilizing) and vigilin (destabilizing)—and epigenetic mechanisms including Pax5-mediated transcriptional silencing in B cells and a FIRE super-enhancer whose deletion selectively ablates tissue-resident macrophage populations while sparing monocytes [PMID:20974809, PMID:16482219, PMID:31324781]. Loss-of-function mutations in the CSF1R kinase domain cause hereditary diffuse leukoencephalopathy with spheroids (HDLS) through defective autophosphorylation and impaired autophagy, whereas C-terminal Tyr969-to-Phe mutations that abolish negative regulatory phosphorylation confer oncogenic potential [PMID:31827782, PMID:3027579]."},"prefetch_data":{"uniprot":{"accession":"P07333","full_name":"Macrophage colony-stimulating factor 1 receptor","aliases":["CSF-1 receptor","CSF-1-R","CSF-1R","M-CSF-R","Proto-oncogene c-Fms"],"length_aa":972,"mass_kda":108.0,"function":"Tyrosine-protein kinase that acts as a cell-surface receptor for CSF1 and IL34 and plays an essential role in the regulation of survival, proliferation and differentiation of hematopoietic precursor cells, especially mononuclear phagocytes, such as macrophages and monocytes. Promotes the release of pro-inflammatory chemokines in response to IL34 and CSF1, and thereby plays an important role in innate immunity and in inflammatory processes. Plays an important role in the regulation of osteoclast proliferation and differentiation, the regulation of bone resorption, and is required for normal bone and tooth development. Required for normal male and female fertility, and for normal development of milk ducts and acinar structures in the mammary gland during pregnancy. Promotes reorganization of the actin cytoskeleton, regulates formation of membrane ruffles, cell adhesion and cell migration, and promotes cancer cell invasion. Activates several signaling pathways in response to ligand binding, including the ERK1/2 and the JNK pathway (PubMed:20504948, PubMed:30982609). Phosphorylates PIK3R1, PLCG2, GRB2, SLA2 and CBL. Activation of PLCG2 leads to the production of the cellular signaling molecules diacylglycerol and inositol 1,4,5-trisphosphate, that then lead to the activation of protein kinase C family members, especially PRKCD. Phosphorylation of PIK3R1, the regulatory subunit of phosphatidylinositol 3-kinase, leads to activation of the AKT1 signaling pathway. Activated CSF1R also mediates activation of the MAP kinases MAPK1/ERK2 and/or MAPK3/ERK1, and of the SRC family kinases SRC, FYN and YES1. Activated CSF1R transmits signals both via proteins that directly interact with phosphorylated tyrosine residues in its intracellular domain, or via adapter proteins, such as GRB2. Promotes activation of STAT family members STAT3, STAT5A and/or STAT5B. Promotes tyrosine phosphorylation of SHC1 and INPP5D/SHIP-1. Receptor signaling is down-regulated by protein phosphatases, such as INPP5D/SHIP-1, that dephosphorylate the receptor and its downstream effectors, and by rapid internalization of the activated receptor. 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inhibits c-Fms-mediated macrophage function.","date":"2010","source":"Journal of leukocyte biology","url":"https://pubmed.ncbi.nlm.nih.gov/20571037","citation_count":25,"is_preprint":false},{"pmid":"22067001","id":"PMC_22067001","title":"Anti-c-Fms antibody inhibits lipopolysaccharide-induced osteoclastogenesis in vivo.","date":"2011","source":"FEMS immunology and medical microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/22067001","citation_count":24,"is_preprint":false},{"pmid":"32535390","id":"PMC_32535390","title":"Inhibition of CSF1R and AKT by (±)-kusunokinin hinders breast cancer cell proliferation.","date":"2020","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/32535390","citation_count":22,"is_preprint":false},{"pmid":"8482721","id":"PMC_8482721","title":"CSF-1 control of C-FMS expression in normal human bone marrow progenitors.","date":"1993","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/8482721","citation_count":22,"is_preprint":false},{"pmid":"7478559","id":"PMC_7478559","title":"Regulation of the c-fms promoter in murine tumour cell lines.","date":"1995","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/7478559","citation_count":22,"is_preprint":false},{"pmid":"34652888","id":"PMC_34652888","title":"Clinical and genetic characterization of adult-onset leukoencephalopathy caused by CSF1R mutations.","date":"2021","source":"Annals of clinical and translational neurology","url":"https://pubmed.ncbi.nlm.nih.gov/34652888","citation_count":21,"is_preprint":false},{"pmid":"31216288","id":"PMC_31216288","title":"Establishment of an orthodontic retention mouse model and the effect of anti-c-Fms antibody on orthodontic relapse.","date":"2019","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/31216288","citation_count":21,"is_preprint":false},{"pmid":"38465843","id":"PMC_38465843","title":"The Phenotypic and Genotypic Spectrum of CSF1R-Related Disorder in China.","date":"2024","source":"Movement disorders : official journal of the Movement Disorder Society","url":"https://pubmed.ncbi.nlm.nih.gov/38465843","citation_count":20,"is_preprint":false},{"pmid":"32319217","id":"PMC_32319217","title":"SWATH-Proteomics of Ibrutinib's Action in Myeloid Leukemia Initiating Mutated G-CSFR Signaling.","date":"2020","source":"Proteomics. Clinical applications","url":"https://pubmed.ncbi.nlm.nih.gov/32319217","citation_count":20,"is_preprint":false},{"pmid":"38450286","id":"PMC_38450286","title":"Deplete and repeat: microglial CSF1R inhibition and traumatic brain injury.","date":"2024","source":"Frontiers in cellular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/38450286","citation_count":19,"is_preprint":false},{"pmid":"33609280","id":"PMC_33609280","title":"G-CSFR antagonism reduces mucosal injury and airways fibrosis in a virus-dependent model of severe asthma.","date":"2021","source":"British journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/33609280","citation_count":19,"is_preprint":false},{"pmid":"17512498","id":"PMC_17512498","title":"STAP-2 regulates c-Fms/M-CSF receptor signaling in murine macrophage Raw 264.7 cells.","date":"2007","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/17512498","citation_count":19,"is_preprint":false},{"pmid":"33142674","id":"PMC_33142674","title":"Rab11A Functions as a Negative Regulator of Osteoclastogenesis through Dictating Lysosome-Induced Proteolysis of c-fms and RANK Surface Receptors.","date":"2020","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/33142674","citation_count":19,"is_preprint":false},{"pmid":"23351989","id":"PMC_23351989","title":"Defective G-CSFR signaling pathways in congenital neutropenia.","date":"2012","source":"Hematology/oncology clinics of North America","url":"https://pubmed.ncbi.nlm.nih.gov/23351989","citation_count":18,"is_preprint":false},{"pmid":"39040919","id":"PMC_39040919","title":"CSF1R-Related Disorder: Prevalence of CSF1R Variants and Their Clinical Significance in the UK Population.","date":"2024","source":"Neurology. Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/39040919","citation_count":18,"is_preprint":false},{"pmid":"23651419","id":"PMC_23651419","title":"An anti-c-Fms antibody inhibits osteoclastogenesis in a mouse periodontitis model.","date":"2013","source":"Oral diseases","url":"https://pubmed.ncbi.nlm.nih.gov/23651419","citation_count":17,"is_preprint":false},{"pmid":"19705931","id":"PMC_19705931","title":"An M-CSF receptor c-Fms antibody inhibits mechanical stress-induced root resorption during orthodontic tooth movement in mice.","date":"2009","source":"The Angle orthodontist","url":"https://pubmed.ncbi.nlm.nih.gov/19705931","citation_count":17,"is_preprint":false},{"pmid":"19536190","id":"PMC_19536190","title":"CSF-1R, DAP12 and beta-catenin: a ménage à trois.","date":"2009","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/19536190","citation_count":17,"is_preprint":false},{"pmid":"1691441","id":"PMC_1691441","title":"Antibody-induced mitogenicity mediated by a chimeric CD2-c-fms receptor.","date":"1990","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/1691441","citation_count":17,"is_preprint":false},{"pmid":"34274854","id":"PMC_34274854","title":"Effect of CSF1R inhibitor on glial cells population and remyelination in the cuprizone model.","date":"2021","source":"Neuropeptides","url":"https://pubmed.ncbi.nlm.nih.gov/34274854","citation_count":16,"is_preprint":false},{"pmid":"32787110","id":"PMC_32787110","title":"Discovery of IACS-9439, a Potent, Exquisitely Selective, and Orally Bioavailable Inhibitor of CSF1R.","date":"2020","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/32787110","citation_count":16,"is_preprint":false},{"pmid":"31275860","id":"PMC_31275860","title":"Lipoxin-Induced Phenotypic Changes in CD115+LY6Chi Monocytes TAM Precursors Inhibits Tumor Development.","date":"2019","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/31275860","citation_count":15,"is_preprint":false},{"pmid":"34979548","id":"PMC_34979548","title":"M-CSFR/CSF1R signaling regulates myeloid fates in zebrafish via distinct action of its receptors and ligands.","date":"2022","source":"Blood advances","url":"https://pubmed.ncbi.nlm.nih.gov/34979548","citation_count":15,"is_preprint":false},{"pmid":"27185163","id":"PMC_27185163","title":"Resident corneal c-fms(+) macrophages and dendritic cells mediate early cellular infiltration in adenovirus keratitis.","date":"2016","source":"Experimental eye research","url":"https://pubmed.ncbi.nlm.nih.gov/27185163","citation_count":15,"is_preprint":false},{"pmid":"33302495","id":"PMC_33302495","title":"The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors.","date":"2020","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33302495","citation_count":15,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":48897,"output_tokens":5862,"usd":0.11731},"stage2":{"model":"claude-opus-4-6","input_tokens":9437,"output_tokens":4615,"usd":0.24384},"total_usd":0.36115,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1985,\n      \"finding\": \"The c-fms proto-oncogene product is the receptor for CSF-1 (M-CSF): it is a 165-170 kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity, expressed on mononuclear phagocytes, that specifically binds CSF-1 and undergoes tyrosine phosphorylation upon ligand binding in membrane preparations.\",\n      \"method\": \"Immunoprecipitation with anti-v-fms antisera, specific CSF-1 binding assay, immune complex kinase assay in membrane preparations\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original biochemical identification with multiple orthogonal methods (kinase assay, ligand binding, immunoprecipitation), foundational paper with >1500 citations\",\n      \"pmids\": [\"2408759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1987,\n      \"finding\": \"Phosphorylation of Tyr969 in the C-terminal tail of c-fms negatively regulates receptor kinase activity; mutation to Phe969 (as in v-fms) activates oncogenic potential of c-fms when combined with CSF-1 expression, and the C-terminal tyrosine deletion is necessary but not sufficient alone for transformation.\",\n      \"method\": \"NIH 3T3 transformation assay with chimeric v-fms/c-fms constructs, cotransfection with CSF-1 cDNA, site-directed mutagenesis of Tyr969 to Phe969\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis combined with functional transformation assay, replicated across multiple chimeric constructs\",\n      \"pmids\": [\"3027579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1987,\n      \"finding\": \"Proviral insertion at the fim-2 locus (spanning the 5'-end of c-fms) results in high-level expression of a normal-sized c-fms mRNA, establishing c-fms activation by retroviral insertion as a mechanism in myeloblastic leukemias.\",\n      \"method\": \"Southern blotting, Northern blotting, proviral integration mapping in Friend MuLV-induced murine myeloblastic leukemias\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — molecular mapping in tumor samples, replicated across multiple tumor lines\",\n      \"pmids\": [\"3476856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1988,\n      \"finding\": \"CSF-1R (c-fms product) is a member of the receptor tyrosine kinase family; its intracellular tyrosine kinase domain transduces CSF-1 signals for proliferation, survival, and differentiation of mononuclear phagocyte lineage cells; v-fms mutations alter kinase activity to provide ligand-independent growth signals.\",\n      \"method\": \"Biochemical characterization, kinase activity assays, v-fms/c-fms domain comparisons\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — biochemical characterization consolidated across multiple prior studies; review by original discoverers\",\n      \"pmids\": [\"2852667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"c-fms mRNA levels are regulated posttranscriptionally by a labile stabilizing protein in both differentiating HL-60 cells and in mature human monocytes; the rate of c-fms gene transcription is unchanged upon TPA-induced differentiation or TPA-induced downregulation, but mRNA half-life is dramatically altered by cycloheximide.\",\n      \"method\": \"Nuclear run-on transcription assays, mRNA half-life measurements with cycloheximide treatment, Northern blotting\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — run-on assays and mRNA stability experiments with pharmacological inhibition, multiple cell types tested\",\n      \"pmids\": [\"2523515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"Activation of CSF-1R intracellular tyrosine kinase domain alone (without CSF-1) is sufficient to induce mitogenesis; a chimeric CD2 ectodomain–CSF-1R kinase domain receptor expressed in NIH 3T3 fibroblasts undergoes tyrosine phosphorylation, receptor downmodulation, and mitogenesis upon anti-CD2 antibody stimulation.\",\n      \"method\": \"Chimeric receptor expression in NIH 3T3 cells, anti-CD2 antibody stimulation, tyrosine phosphorylation assay, proliferation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution with chimeric receptor and direct kinase activity measurement\",\n      \"pmids\": [\"1691441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The transcription factor AML1 (RUNX1) directly binds the c-FMS gene in normal cells; in t(8;21) leukemic cells, the AML1-ETO fusion protein binds extended sequences of the c-FMS intronic regulatory region and alters histone modification patterns and increases association of histone deacetylases, without irreversibly disrupting transcription factor binding.\",\n      \"method\": \"In vivo footprinting, chromatin immunoprecipitation (ChIP) in normal and t(8;21) leukemic cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct chromatin immunoprecipitation and in vivo footprinting with histone modification analysis\",\n      \"pmids\": [\"12773394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"During B-lymphopoiesis, c-fms chromatin is silenced in discrete steps: first transcription factor binding and RNA expression are lost, then nuclease accessibility decreases; even in mature B cells the locus remains poised and can be reactivated by conditional deletion of Pax5; de novo DNA methylation overlaps with an intronic antisense transcription unit.\",\n      \"method\": \"DNase I hypersensitivity assays, chromatin immunoprecipitation, bisulfite sequencing, conditional Pax5 deletion in B cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal chromatin analysis methods with genetic rescue experiment\",\n      \"pmids\": [\"15483629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Imatinib inhibits the CSF-1R (c-fms) kinase at therapeutic concentrations; it inhibits c-fms tyrosine phosphorylation in macrophages and blocks M-CSF-induced proliferation of a cytokine-dependent cell line, without downregulating c-fms expression.\",\n      \"method\": \"Phosphorylation assays in macrophages, cytokine-dependent cell line proliferation assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — direct kinase phosphorylation assay combined with functional proliferation assay\",\n      \"pmids\": [\"15637141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Pax5 directly represses c-fms by being recruited to the c-fms locus, causing rapid loss of RNA polymerase II binding and then loss of transcription factor binding and DNase I hypersensitivity at all cis-regulatory elements; Pax5 targets the basal transcription machinery by interacting with a binding site within the major transcription start sites.\",\n      \"method\": \"Chromatin immunoprecipitation, DNase I hypersensitivity assays, conditional Pax5 expression system\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple chromatin methods with direct Pax5 recruitment demonstrated\",\n      \"pmids\": [\"16482219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of the autoinhibited human c-Fms kinase domain at 2.7 Å resolution shows that the juxtamembrane (JM) domain binds a hydrophobic site adjacent to the ATP binding pocket, preventing the activation loop from adopting an active conformation; three JM-derived tyrosine residues drive autoinhibition, a mechanism shared with c-Kit and Flt3.\",\n      \"method\": \"X-ray crystallography at 2.7 Å resolution of the cytosolic kinase + juxtamembrane domain\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure with mechanistic interpretation of autoinhibition\",\n      \"pmids\": [\"17292918\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"M-CSF binding to CSF-1R (c-Fms) in osteoclasts generates a signaling complex with phosphorylated DAP12 (ITAM adaptor) and Syk kinase; c-Fms Tyr559 (exclusive c-Src binding site) is required for DAP12/Syk signaling; deletion of DAP12 or Syk yields osteoclasts that fail to reorganize their cytoskeleton, and Syk SH2 domain and DAP12 ITAM tyrosines and transmembrane domain mediate this signaling.\",\n      \"method\": \"Co-immunoprecipitation, retroviral transduction of null precursors with wild-type/mutant DAP12 and Syk, cytoskeleton analysis, genetic knockout models\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — genetic and biochemical epistasis combined with domain mutagenesis and functional osteoclast phenotype\",\n      \"pmids\": [\"18691974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The adaptor protein Lnk binds directly to c-Fms (CSF-1R) via its PH domain independently of M-CSF stimulation; Lnk overexpression suppresses M-CSF-induced tyrosine phosphorylation of c-Fms and Akt activation, and inhibits M-CSF-induced macrophage migration; Lnk KO macrophages show enhanced and prolonged Akt phosphorylation but diminished Erk phosphorylation in response to M-CSF.\",\n      \"method\": \"Co-immunoprecipitation, Lnk knockout macrophages, clonogenic assays, western blotting of Akt/Erk phosphorylation, migration assays\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct binding demonstrated by co-IP, confirmed with KO macrophages and multiple signaling readouts\",\n      \"pmids\": [\"20571037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"STAP-2 adaptor protein directly binds to the PH domain of c-Fms independent of M-CSF stimulation; STAP-2 overexpression suppresses M-CSF-induced c-Fms tyrosine phosphorylation and downstream Akt and ERK activation, and impairs M-CSF-induced macrophage migration.\",\n      \"method\": \"Co-immunoprecipitation, overexpression in Raw 264.7 macrophages, western blotting of phospho-c-Fms, phospho-Akt, phospho-ERK, wound-healing migration assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding by co-IP with functional consequences, single lab\",\n      \"pmids\": [\"17512498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Vigilin binds a 69-nucleotide non-AU-rich sequence in the c-fms 3' UTR and competitively displaces HuR from this site; vigilin decreases c-fms mRNA stability and inhibits c-fms translation, whereas HuR stabilizes c-fms mRNA; the two proteins exert opposing effects on breast cancer cell motility and invasion.\",\n      \"method\": \"RNA immunoprecipitation, mRNA stability assays, polysome fractionation, competition binding assays, cell invasion/motility assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — multiple orthogonal methods (RIP, mRNA stability, translation assays) with functional cellular readouts\",\n      \"pmids\": [\"20974809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CSF1R is expressed on a small subpopulation of hippocampal and cortical neurons under physiological conditions; excitotoxic injury markedly upregulates neuronal CSF1R expression; selective deletion of CSF1R in forebrain neurons exacerbates excitotoxin-induced neurodegeneration; CSF1 /IL-34 neuroprotection is accompanied by maintenance of CREB signaling in neurons.\",\n      \"method\": \"Lineage-tracing experiments, conditional neuron-specific CSF1R deletion, kainic acid excitotoxin model, CREB phosphorylation western blot, histological quantification\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with specific phenotypic readout in multiple experiments\",\n      \"pmids\": [\"23296467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"X-ray co-crystallography guided development of PLX3397, a CSF1R inhibitor that traps the kinase in the autoinhibited conformation; structure-based design enables selective CSF1R blockade.\",\n      \"method\": \"X-ray co-crystallography of CSF1R kinase with PLX3397, phase 1 clinical trial\",\n      \"journal\": \"The New England journal of medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with bound inhibitor directly informs mechanism of inhibition\",\n      \"pmids\": [\"26222558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Resistance to CSF-1R inhibition in recurrent GBM is driven by the tumor microenvironment; macrophage-derived IGF-1 activates PI3K pathway via tumor cell IGF-1R, and transplantation of resistant tumors re-establishes sensitivity to CSF-1R inhibition.\",\n      \"method\": \"Tumor transplantation experiments, IGF-1/IGF-1R pathway analysis, PI3K inhibitor combination therapy in mouse GBM model\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis via transplantation and pathway inhibition, multiple orthogonal experiments\",\n      \"pmids\": [\"27199435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Deletion of the fms-intronic regulatory element (FIRE) super-enhancer within the Csf1r locus selectively ablates CSF1R expression in specific tissue macrophage populations (brain microglia, skin, kidney, heart, peritoneum macrophages) and blocks macrophage development from embryonic stem cells, while leaving monocyte homeostasis largely unaffected.\",\n      \"method\": \"CRISPR/genomic deletion of FIRE in mice, tissue macrophage quantification, in vitro ESC differentiation assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic deletion with tissue-specific phenotypic characterization across multiple organs\",\n      \"pmids\": [\"31324781\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CSF1R haploinsufficiency (loss-of-function mutations) causes hereditary diffuse leukoencephalopathy with spheroids (HDLS); mutations in the kinase domain lead to defective autophosphorylation; mutant CSF1R proteins show lower expression levels and reduced autophagy (decreased LC3-II levels) compared to wild-type.\",\n      \"method\": \"Next-generation sequencing, Sanger sequencing, in vitro autophosphorylation assays of mutant CSF1R proteins, western blotting of LC3-II, immunofluorescence\",\n      \"journal\": \"Translational neurodegeneration\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — direct in vitro kinase assays on multiple mutants combined with autophagy marker analysis\",\n      \"pmids\": [\"31827782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Rab11A negatively regulates osteoclastogenesis by promoting lysosomal degradation of c-fms and RANK surface receptors; Rab11A overexpression enlarges early endosomes and upregulates lysosomal activity (LAMP1, cathepsins B and D), leading to proteolytic degradation of c-fms; lysosomal inhibition by chloroquine rescues c-fms and RANK protein levels.\",\n      \"method\": \"Rab11A overexpression/silencing in RAW-D cells and BMMs, immunocytochemistry for endosomal markers, LAMP1 and cathepsin quantification, chloroquine rescue experiments, flow cytometry of surface c-fms\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — gain/loss of function with pharmacological rescue and multiple marker analyses\",\n      \"pmids\": [\"33142674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Rab11b promotes lysosomal degradation of c-Fms and RANK surface receptors in osteoclasts; Rab11b overexpression enlarges early/late endosomes and augments lysosomal activity, reducing surface c-Fms abundance and attenuating NFATc-1 upstream signaling; chloroquine inhibition of lysosomal function rescues c-Fms levels.\",\n      \"method\": \"Rab11b overexpression in RAW-D cells and BMMs, immunocytochemistry, flow cytometry of surface c-Fms, chloroquine rescue, western blotting\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — complementary to Rab11A study, multiple methods but single lab\",\n      \"pmids\": [\"33302495\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"A glycine to alanine substitution at position 795 (G795A) of human CSF1R confers resistance to multiple CSF1R inhibitors (PLX3397, PLX5622) without discernable gain or loss of CSF1R function; G795A-expressing macrophages engraft mouse brain during PLX3397 treatment and persist after cessation.\",\n      \"method\": \"Biochemical and cell-based kinase activity assays, CRISPR engineering of iPSC-derived microglia, xenotransplantation studies, gene expression profiling\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — site-directed mutagenesis with biochemical validation, in vivo engraftment, and transcriptomic confirmation\",\n      \"pmids\": [\"36584406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"gp130-dependent ERK1/2 MAP kinase signaling positively regulates c-fms expression in macrophages; in gp130(ΔSTAT/ΔSTAT) macrophages, enhanced ERK1/2 activation correlates with elevated c-fms expression and increased M-CSF responsiveness, while gp130(Y757F/Y757F) macrophages with reduced ERK1/2 activation show decreased c-fms expression and reduced M-CSF-induced proliferation; an ERK1/2 inhibitor suppresses M-CSF-induced proliferation and c-fms expression.\",\n      \"method\": \"gp130 signaling mutant knock-in mouse macrophages, ERK1/2 phosphorylation assays, clonogenic assays, pharmacological ERK1/2 inhibition, western blotting of c-fms\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic and pharmacological manipulation with multiple readouts in multiple genotypes\",\n      \"pmids\": [\"14749363\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CSF1R (c-Fms) is a transmembrane receptor tyrosine kinase that binds CSF-1 and IL-34 to drive proliferation, differentiation, and survival of mononuclear phagocytes; upon ligand binding, the juxtamembrane domain (held in an autoinhibited conformation) is released, enabling kinase activation, autophosphorylation, and recruitment of signaling partners including DAP12/Syk (linking to cytoskeletal reorganization in osteoclasts), PI3K/Akt, ERK, and adaptors Lnk and STAP-2 that negatively regulate signaling; c-fms expression is controlled at the posttranscriptional level by competing mRNA-binding proteins (HuR vs. vigilin) and epigenetically by transcription factors (Pax5 represses, AML1-ETO modifies histone marks) acting on a super-enhancer (FIRE) within the locus; loss-of-function mutations in the kinase domain cause HDLS/CSF1R-related leukoencephalopathy through defective autophosphorylation and impaired microglial survival, while C-terminal mutations (Phe969) that abolish negative regulatory phosphorylation activate oncogenic potential.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CSF1R (c-Fms) is a receptor tyrosine kinase that serves as the primary signaling receptor for CSF-1 (M-CSF) and IL-34, governing the proliferation, differentiation, survival, and functional activation of mononuclear phagocytes, osteoclasts, microglia, and certain neurons. Upon ligand binding, autoinhibition mediated by the juxtamembrane domain—which wedges against the kinase active site to prevent activation loop remodeling—is released, enabling autophosphorylation and recruitment of downstream effectors including DAP12/Syk (for osteoclast cytoskeletal reorganization), PI3K/Akt, and ERK, while adaptors Lnk and STAP-2 attenuate signaling by suppressing receptor phosphorylation [PMID:2408759, PMID:17292918, PMID:18691974, PMID:20571037]. CSF1R expression is controlled by a combination of posttranscriptional regulation—through competing mRNA-binding proteins HuR (stabilizing) and vigilin (destabilizing)—and epigenetic mechanisms including Pax5-mediated transcriptional silencing in B cells and a FIRE super-enhancer whose deletion selectively ablates tissue-resident macrophage populations while sparing monocytes [PMID:20974809, PMID:16482219, PMID:31324781]. Loss-of-function mutations in the CSF1R kinase domain cause hereditary diffuse leukoencephalopathy with spheroids (HDLS) through defective autophosphorylation and impaired autophagy, whereas C-terminal Tyr969-to-Phe mutations that abolish negative regulatory phosphorylation confer oncogenic potential [PMID:31827782, PMID:3027579].\",\n  \"teleology\": [\n    {\n      \"year\": 1985,\n      \"claim\": \"The identity of the CSF-1 receptor was unknown; demonstrating that the c-fms proto-oncogene product is a transmembrane glycoprotein with intrinsic tyrosine kinase activity that specifically binds CSF-1 established the molecular basis of macrophage growth factor signaling.\",\n      \"evidence\": \"Immunoprecipitation with anti-v-fms antisera, CSF-1 binding assay, and immune complex kinase assay in membrane preparations\",\n      \"pmids\": [\"2408759\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligand-induced receptor conformational changes not resolved\", \"Downstream signaling pathways not yet mapped\", \"Second ligand IL-34 not yet identified\"]\n    },\n    {\n      \"year\": 1987,\n      \"claim\": \"How oncogenic v-fms differs from c-fms was unclear; showing that Tyr969 phosphorylation negatively regulates the receptor and that its mutation to Phe (as in v-fms) is required for transformation established a C-terminal autoinhibitory mechanism and the molecular basis of c-fms oncogenic activation.\",\n      \"evidence\": \"Site-directed mutagenesis of Tyr969→Phe969, NIH 3T3 transformation assays with chimeric v-fms/c-fms constructs co-transfected with CSF-1 cDNA\",\n      \"pmids\": [\"3027579\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signaling partners recruited to phospho-Tyr969 not identified\", \"Whether Tyr969 mutation alone is sufficient for in vivo tumorigenesis unknown\"]\n    },\n    {\n      \"year\": 1989,\n      \"claim\": \"Whether c-fms expression is controlled transcriptionally or posttranscriptionally was uncertain; nuclear run-on and mRNA half-life experiments demonstrated that a labile protein stabilizes c-fms mRNA, establishing posttranscriptional regulation as a major control mechanism.\",\n      \"evidence\": \"Nuclear run-on transcription assays, mRNA half-life measurement with cycloheximide in differentiating HL-60 cells and monocytes\",\n      \"pmids\": [\"2523515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the stabilizing protein not determined at this time\", \"Cis-element in c-fms mRNA mediating stability not mapped\"]\n    },\n    {\n      \"year\": 1990,\n      \"claim\": \"Whether CSF-1R kinase activity alone suffices for mitogenic signaling was untested; a chimeric receptor study proved that activation of the intracellular kinase domain without CSF-1 binding is sufficient to drive autophosphorylation, receptor downmodulation, and mitogenesis.\",\n      \"evidence\": \"Chimeric CD2-ectodomain/CSF-1R-kinase receptor in NIH 3T3 cells stimulated with anti-CD2 antibody\",\n      \"pmids\": [\"1691441\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which specific tyrosine autophosphorylation sites mediate mitogenic signaling not mapped\", \"Downstream signaling intermediates not identified\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"How the AML1-ETO fusion oncoprotein deregulates c-FMS in t(8;21) leukemia was unknown; ChIP and in vivo footprinting showed AML1-ETO binds extended intronic regulatory sequences and remodels histone marks, establishing an epigenetic mechanism for c-FMS dysregulation in leukemia.\",\n      \"evidence\": \"In vivo footprinting and chromatin immunoprecipitation with histone modification analysis in normal versus t(8;21) leukemic cells\",\n      \"pmids\": [\"12773394\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether AML1-ETO binding directly alters c-FMS transcription output not quantified\", \"Functional consequence for macrophage differentiation not tested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"How c-fms is silenced during B-cell commitment and how extrinsic signals regulate its expression in macrophages were unresolved; two studies showed that Pax5 represses c-fms by displacing RNA polymerase II from the locus (reversible upon Pax5 deletion), and that gp130-ERK1/2 signaling positively regulates c-fms expression and M-CSF responsiveness in macrophages.\",\n      \"evidence\": \"Conditional Pax5 deletion with ChIP and DNase I hypersensitivity in B cells; gp130 signaling mutant knock-in macrophages with pharmacological ERK1/2 inhibition\",\n      \"pmids\": [\"15483629\", \"14749363\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Pax5 DNA-binding site relative to c-fms promoter not fully mapped at this point\", \"Whether ERK1/2 acts on c-fms transcription or mRNA stability not distinguished\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The mechanism by which Pax5 silences c-fms was incompletely understood; detailed ChIP showed Pax5 is recruited to the c-fms transcription start sites, directly displacing the basal transcription machinery and sequentially shutting down all cis-regulatory elements.\",\n      \"evidence\": \"Chromatin immunoprecipitation and DNase I hypersensitivity with conditional Pax5 expression system\",\n      \"pmids\": [\"16482219\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Pax5-mediated silencing involves DNA methylation changes not fully resolved\", \"Kinetics of reactivation upon Pax5 removal in vivo not measured\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"The structural basis of CSF1R autoinhibition was unknown; the 2.7 Å crystal structure revealed that juxtamembrane domain tyrosines wedge into a hydrophobic pocket adjacent to the ATP-binding site, locking the activation loop in an inactive conformation—a mechanism shared with c-Kit and Flt3.\",\n      \"evidence\": \"X-ray crystallography of the cytosolic kinase plus juxtamembrane domain at 2.7 Å resolution\",\n      \"pmids\": [\"17292918\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length extracellular–transmembrane–kinase structure not available\", \"Dynamics of JM release upon ligand binding not captured\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"How CSF-1R signals to the osteoclast cytoskeleton was unclear; demonstrating that c-Fms Tyr559 nucleates a DAP12/Syk signaling complex essential for cytoskeletal reorganization established a non-canonical ITAM-based pathway downstream of CSF-1R in osteoclasts.\",\n      \"evidence\": \"Co-immunoprecipitation, retroviral reconstitution of DAP12/Syk-null osteoclast precursors with wild-type and mutant constructs, cytoskeletal analysis\",\n      \"pmids\": [\"18691974\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether DAP12/Syk pathway operates in macrophages outside osteoclasts not tested\", \"Direct substrates of Syk downstream of c-Fms not identified\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Negative regulators that restrain CSF-1R signaling magnitude were largely unknown; identification of Lnk and STAP-2 as adaptors that constitutively bind c-Fms and suppress M-CSF-induced phosphorylation, Akt activation, and macrophage migration established feedback attenuation mechanisms.\",\n      \"evidence\": \"Co-immunoprecipitation, Lnk knockout macrophages, STAP-2 overexpression, Akt/ERK phosphorylation kinetics, migration assays\",\n      \"pmids\": [\"20571037\", \"17512498\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Lnk and STAP-2 compete for the same binding site on c-Fms not determined\", \"In vivo macrophage phenotypes of STAP-2 loss not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The posttranscriptional regulators of c-fms mRNA identified in 1989 were molecularly uncharacterized; demonstrating that vigilin and HuR compete for a 69-nt element in the c-fms 3′ UTR with opposing effects on mRNA stability and translation provided a molecular mechanism for posttranscriptional control of c-fms expression.\",\n      \"evidence\": \"RNA immunoprecipitation, mRNA stability assays, polysome fractionation, competition binding assays in breast cancer cells\",\n      \"pmids\": [\"20974809\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this mechanism operates in primary macrophages not shown\", \"Other RNA-binding proteins recognizing the c-fms 3′ UTR not surveyed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Whether CSF1R functions outside myeloid cells was controversial; conditional neuron-specific deletion showed CSF1R is expressed on hippocampal and cortical neurons and is neuroprotective during excitotoxic injury via CREB signaling, establishing a cell-autonomous neuronal role.\",\n      \"evidence\": \"Neuron-specific conditional CSF1R deletion, kainic acid excitotoxin model, CREB phosphorylation analysis\",\n      \"pmids\": [\"23296467\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which CSF1R ligand (CSF-1 vs IL-34) is the physiological neuronal agonist in vivo not resolved\", \"Downstream signaling intermediates between CSF1R and CREB in neurons not mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Structure-based drug design of CSF1R inhibitors was enabled by co-crystallography; the PLX3397 co-crystal structure showed the inhibitor traps CSF1R in the autoinhibited conformation, providing a mechanistic rationale for selective kinase blockade.\",\n      \"evidence\": \"X-ray co-crystallography of CSF1R kinase domain with PLX3397, phase 1 clinical trial\",\n      \"pmids\": [\"26222558\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Resistance mechanisms to PLX3397 not yet characterized\", \"Whether JM-locking inhibitors differ functionally from activation-loop inhibitors not tested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The cis-regulatory architecture controlling tissue-specific CSF1R expression was incompletely understood; CRISPR deletion of the FIRE super-enhancer selectively ablated microglia and several tissue-resident macrophage populations while sparing monocytes, demonstrating lineage-specific enhancer dependence.\",\n      \"evidence\": \"CRISPR deletion of FIRE enhancer in mice, tissue macrophage quantification across multiple organs, in vitro ESC differentiation\",\n      \"pmids\": [\"31324781\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether FIRE deletion affects macrophage function beyond abundance not assessed\", \"Compensatory enhancer elements not surveyed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The molecular pathogenesis of HDLS/CSF1R-related leukoencephalopathy was mechanistically unclear; in vitro kinase assays of patient-derived mutations demonstrated defective autophosphorylation and reduced autophagy (LC3-II levels), linking kinase domain loss-of-function to disease.\",\n      \"evidence\": \"In vitro autophosphorylation assays of multiple mutant CSF1R proteins, LC3-II western blotting, patient sequencing\",\n      \"pmids\": [\"31827782\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether reduced autophagy is a direct kinase substrate effect or secondary not determined\", \"Microglial-specific consequences of individual mutations not characterized in vivo\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"How surface CSF1R abundance is regulated post-endocytically was poorly understood; two studies showed Rab11A and Rab11B promote lysosomal degradation of c-Fms in osteoclasts by enlarging endosomal compartments and augmenting cathepsin activity, with chloroquine rescue confirming lysosomal dependence.\",\n      \"evidence\": \"Rab11A/Rab11B overexpression and silencing in RAW-D cells and BMMs, LAMP1/cathepsin quantification, chloroquine rescue, flow cytometry\",\n      \"pmids\": [\"33142674\", \"33302495\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Rab11-mediated degradation operates in macrophage populations beyond osteoclasts not tested\", \"Ubiquitin signals directing c-Fms to lysosomes not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Whether CSF1R can be engineered for inhibitor resistance without functional compromise was untested; the G795A gatekeeper-adjacent substitution conferred resistance to PLX3397/PLX5622 while preserving normal kinase function, enabling selective microglial replacement in vivo.\",\n      \"evidence\": \"CRISPR-engineered iPSC-derived microglia with G795A mutation, biochemical kinase assays, xenotransplantation into PLX3397-treated mice, transcriptomic profiling\",\n      \"pmids\": [\"36584406\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Long-term consequences of G795A microglia engraftment not assessed\", \"Whether G795A confers resistance to other CSF1R inhibitor scaffolds not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full-length structure of CSF1R encompassing the extracellular, transmembrane, and intracellular domains in both inactive and active states is lacking, and the precise mechanism by which ligand-induced extracellular dimerization releases juxtamembrane autoinhibition remains structurally unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length ligand-bound receptor structure not available\", \"How IL-34 versus CSF-1 binding differentially activates downstream pathways not mechanistically resolved\", \"In vivo significance of neuronal CSF1R under non-excitotoxic conditions not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 3, 5, 10, 11, 19]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 3, 5, 10]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 5, 11, 20, 21, 22]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [20, 21]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [20, 21]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 5, 11, 12, 15]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 11, 18]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [7, 9, 18]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 2, 19]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [15, 19]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CSF1\",\n      \"DAP12\",\n      \"SYK\",\n      \"LNK\",\n      \"STAP2\",\n      \"PAX5\",\n      \"HDLBP\",\n      \"ELAVL1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}