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

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 in contrast to D842H and F604+D842H did not increase the biochemical or cellular IC50 value to imatinib when compared to wild-type (wt). However, F604S and F604S+D842H transformed Ba/F3, NIH3T3 and mouse bone marrow more efficiently compared to wt and D842H, respectively. Also, F604S and F604S+D842H showed strong activation of Stat5, ERK and Akt compared to wt and D842H. Immunoprecipitation and immunoblotting indicated increased amounts of FIP1L1-PDGFRA protein in F604S versus wt cells. Moreover, SRC coimmunoprecipitated with FIP1L1-PDGFRA in wt, but not F604S cells. We hypothesized that F604S might interfere with FIP1L1-PDGFRA protein stability, and that SRC might be involved in this process. GST pull down experiments using SRC-SH2 domain showed lesser binding of FIP1L1-PDGFRA/F604S compared to wt. Similarly, using a GST-PDGFRA fragment, more SRC was precipitated with wt compared to F604S. Importantly both, the SRC inhibitor PD166326 and SRC siRNA mimicked the F604S phenotype and resulted in stabilization of the wt protein. Also, 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. Similar results were obtained with an exchange in near proximity to F604. Kinase-defective SRC had no effect on FIP1L1-PDGFRA stability, indicating that kinase activity of SRC is necessary for its effect on FIP1L1-PDGFRA stability. Moreover, kinase defective FIP1L1-PDGFRA (G610R) was not degraded indicating that kinase activity of FIP1l1-PDGFRA is necessary for its own degradation. Taken together, imatinib resistance screening in FIP1L1-PDGFRA identified a novel class of resistance mutations, that do not act by impeding drug binding to the target, but rather increase target protein levels by interfering with its SRC mediated degradation.