title: F604S exchange in FIP1L1-PDGFRA enhances FIP1L1-PDGFRA protein stability via SHP-2 and SRC: a novel mode of kinase inhibitor resistance.
abstract: FIP1L1-PDGFRA is a constitutively activated kinase described in chronic eosinophilic leukemia (CEL) and hypereosinophilic syndrome (HES). Imatinib is clinically active in FIP1L1-PDGFRA-positive diseases. Using in vitro screening to identify imatinib-resistant mutations, we frequently detected a Phe to Ser exchange at position 604 (F604S) of FIP1L1-PDGFRA alone or in combination with other exchanges. Surprisingly, FIP1L1-PDGFRA/F604S did not increase the biochemical or cellular IC50 value of imatinib when compared with unmutated FIP1L1-PDGFRA. However, FIP1L1-PDGFRA/F604S more efficiently induced growth factor independence in cell lines and primary mouse bone marrow cells. Pulse chase analysis revealed that the F604S exchange strongly stabilized FIP1L1-PDGFRA/F604S. The F604S mutation creates a binding site for the phosphatase domain of SHP-2, leading to lower autophosphorylation of FIP1L1-PDGFRA/F604S. This is associated with a reduced activation of SRC and CBL by FIP1L1-PDGFRA/F604S compared with the unmutated oncogene. As SRC inhibition and knockdown resulted in FIP1L1-PDGFRA stabilization, this explains the extended half-life of FIP1L1-PDGFRA/F604S. Interestingly, FIP1L1-PDGFRA/L629P, a recently identified mutation in an imatinib-resistant CEL patient, also showed protein stabilization similar to that observed with FIP1L1-PDGFRA/F604S. Therefore, resistance mutations in FIP1L1-PDGFRA that do not interfere with drug binding but rather increase target protein stability seem to be one of the drug-resistance mechanisms in FIP1L1-PDGFRA-positive disease.
authors.affiliation: 1] Department of Internal Medicine I, University Medical Center Freiburg, Freiburg, Germany [2] German Cancer Consortium (DKTK), Freiburg and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: S P
authors.initials: SP
authors.lastname: Gorantla
authors.affiliation: Department of Internal Medicine I, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Zirlik
authors.affiliation: Department of Hematology and Oncology, University Hospital Mannheim, Mannheim, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Reiter
authors.affiliation: Department of Internal Medicine I, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Yu
authors.affiliation: Department of Internal Medicine I, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: A L
authors.initials: AL
authors.lastname: Illert
authors.affiliation: Department of Internal Medicine I, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Von Bubnoff
authors.affiliation: 1] Department of Internal Medicine I, University Medical Center Freiburg, Freiburg, Germany [2] German Cancer Consortium (DKTK), Freiburg and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Duyster
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002478:Cells, Cultured; D057809:HEK293 Cells; D006801:Humans; D017681:Hypereosinophilic Syndrome; D051379:Mice; D009154:Mutation / Q000235:genetics*; D041681:NIH 3T3 Cells; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000737:chemistry* / Q000235:genetics*; D054448:Precursor Cells, B-Lymphoid; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D055550:Protein Stability / Q000187:drug effects*; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000737:chemistry* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D039221:mRNA Cleavage and Polyadenylation Factors / Q000737:chemistry* / Q000235:genetics*
chemical_list: D015514:Oncogene Proteins, Fusion; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D039221:mRNA Cleavage and Polyadenylation Factors; C479819:FIP1L1-PDGFRA fusion protein, human; D020796:Receptor, Platelet-Derived Growth Factor alpha; D015688:Oncogene Protein pp60(v-src); C516724:PTPN11 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11