Identification of a Novel Mode of Kinase Inhibitor Resistance: An F604S Exchange in FIP1L1-PDGFRA Modulates FIP1L1-PDGFRA Protein Stability in a SHP-2 and SRC-Dependent Manner

Abstract 1739

FIP1L1-PDGFR alpha is a constitutively activated protein kinase which was reported in chronic eosinophilic leukemia (CEL) and in cases of hypereosinophilic syndrome and mastocytosis with eosinophilia. Imatinib is clinically active against FIP1L1-PDGFRA positive disease. However, clinical resistance to imatinib has been observed in FIP1L1-PDGFRA positive leukemia and was shown to occur due to a secondary mutation (T674I) in the PDGFR alpha kinase domain. Using a screening strategy to identify imatinib resistant mutations, we generated numerous imatinib resistant cell clones. Analysis of the PDGFRA kinase domain in these cell clones revealed a broad spectrum of resistance mutations including the clinically reported exchange T674I. Interestingly, one of the abundant mutations was a Phe to Ser exchange at position 604 (F604S), which occurred alone or in combination with other exchanges. Surprisingly, FIP1L1-PDGFRA/F604S did not increase the biochemical or cellular IC50 value to imatinib when compared to wild-type (WT FP). However, F604S and F604S+D842H transformed Ba/F3 and mouse bone marrow more efficiently compared to WT and D842H, respectively. Immunoprecipitation and immunoblotting indicated increased amounts of FIP1L1-PDGFRA protein in F604S versus WT cells. Pulse chase analysis revealed that FIP1L1-PDGFRA/F604S is strongly stabilized compared to WT. SRC coimmunoprecipitated with FIP1L1-PDGFRA in WT, but not F604S cells. Co-expression of SRC in 293T cells augmented degradation of WT, but not F604S FIP1L1-PDGFRA, indicating that SRC is a negative regulator of FIP1L1-PDGFRA protein stability. Importantly both, the SRC inhibitor PD166326 and SRC siRNA mimicked the F604S phenotype and resulted in stabilization of the WT protein. Importantly, phosphatase inhibitor treatment of FIP1L1-PDGFRA/F604S led to destabilization and SRC recruitment indicating that phosphatases might be responsible for the enhanced stability of FIP1L1-PDGFRA/F604S. In fact, coimmunuprecipitaion experiments identified the phosphatase SHP2 as a specific binding partner of F604S and mapping experiments revealed that the phosphatase domain of SHP-2 directly interacted with FIP1L1-PDGFRA/F604S but not with wt- FIP1L1-PDGFRA. Together, these results suggest that stabilization of FIP1L1-PDGFRA/F604S is due to dephosphorylation by SHP-2 leading to lesser activation of the SRC and Cbl mediated ubiquitination machinery. Finally a novel exchange (L629P) identified in imatinib resistance CEL patient also leads to the stabilization of FIP1L1-PDGFRA protein similar to F604S. This indicates that stabilization of FIP1L1-PDGFRA is a common mode of drug resistance in FIP1L1-PDGFRA positive HES or CEL. In summary, imatinib resistance screening identified a novel class of resistance mutations in FIP1L1-PDGFRA, that do not act by impeding drug binding to the target, but increased target protein stability and abundance by interfering with SRC- mediated degradation.