FIP1L1-PDGFRA p.T674I-D842L: a novel and ponatinib resistant compound mutation in  FIP1L1-PDGFRA positive eosinophilic leukemia 

Objectives
We report the molecular evolution of a FIP1L1-PDGFRA p.T674I positive leukemia in a patient who progressed under treatment with ponatinib. A novel ponatinib resistant FIP1L1-PDGFRA p.T674I-D842L double mutant was detected. Its pathogenicity and targetability were investigated.

Methods
The FIP1L1-PDGFRA fusion in the patient was cloned and constructs were made with the following mutations: p.T674I, p.D842L and p.T674I-D842L. The oncogenic potential of these constructs, as well as of the previously published p.D842V mutant was investigated in Ba/F3 cells. We tested the effect of sorafenib, ponatinib, quizartinib and crenolanib on the growth of transformed Ba/F3 cells and on the activity of FIP1L1-PDGFRA, STAT5 and ERK1/2.

Results
Expression of these fusions induced growth factor independent growth of Ba/F3 cells. Ba/F3 cells expressing the FIP1L1-PDGFRA p.T674I-D842L double mutant were sensitive to ponatinib in vitro at an IC50 exceeding the average steady-state plasma concentration. In contrast, crenolanib did inhibit the growth of the double mutant transformed Ba/F3 cells with a feasible IC50. The artificial FIP1L1-PDGFRA p.D842L mutant was sensitive to all tested inhibitors, whereas the p.D842V mutant did not respond to sorafenib as previously described. The effect of ponatinib and crenolanib on the phosphorylation of FIP1L1-PDGFRA mutants and downstream targets STAT5 and ERK1/2 was explored. Results were in line with the growth experiments.

Conclusion
Ponatinib is active in vitro against the FIP1L1-PDGFRA p.T674I and p.D842V mutants, but in vivo the emergence of a resistant FIP1L1-PDGFRA p.T674I-D842L compound mutation led to failure of ponatinib therapy. As such, we demonstrate yet another molecular mechanism of resistance development in FIP1L1-PDGFRA p.T674I positive leukemia. The availability of several compounds with in vitro activity against FIP1L1-PDGFRA p.T674I, has so far not substantially improved the grim prospects of patients with the FIP1L1-PDGFRA p.T674I mutation.