FIP1L1-PDGFRα D842V, a Novel Panresistant Mutant, Emerges after Treatment of FIP1L1-PDGFRα T674I Eosinophilic Leukemia with Single Agent Sorafenib

Abstract

Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm characterized by the FIP1L1-PDGFRA fusion gene, variant PDGFRA fusions, or other genetic lesions. Most FIP1L1-PDGFRA positive patients enjoy durable complete molecular responses to low-dose imatinib, but resistance mediated by a T674I mutation in the ATP-binding pocket of PDGFRA has been reported in advanced disease. Sorafenib, a potent inhibitor of RAF-1, B-RAF, VEGFR and PDGFR, has been shown to be active against this mutant in vitro. We explored a case of FIP1L1-PDGFRα T674I CEL in blast crisis that was treated with sorafenib as single agent. A partial hematological response was induced, but three months later, progression to blast crisis again occurred. At this time point, sequencing of FIP1L1-PDGFRA revealed the presence of a novel FIP1L1-PDGFRα D842V mutant, while FIP1L1-PDGFRα T674I was no longer detected. The sensitivity of this mutant to different inhibitors was further explored using FIP1L1-PDGFRα D842V transformed Ba/F3 cells. The growth of FIP1L1-PDGFRα D842V transformed Ba/F3 cells was highly resistant to sorafenib and PKC412, in addition to imatinib and dasatinib (IC50 ≥ 1000 nM for imatinib, sorafenib and dasatinib; IC50 of PKC412 not reached due to toxicity above 500 nM). Consistent with these dose response curves, FIP1L1-PDGFRα D842V cells did not undergo apoptosis when cultured in 500 nM sorafenib, imatinib or dasatinib, while 30% of FIP1L1-PDGFRα cells did under the same conditions. Analysis of FIP1L1-PDGFRα autophosphorylation and phosphorylation of the downstream signaling proteins ERK1 and ERK2 confirmed that the D842V mutant protein was not inhibited by sorafenib, imatinib or dasatinib at concentrations up to 1000 nM, in contrast to FIP1L1-PDGFRα itself or the T674I mutant. Intriguingly, FIP1L1-PDGFRα D842V cells are significantly less sensitive to dasatinib than PDGFRα D842V expressing cells. Finally, an ENU-mutagenesis screen indeed identified this mutant as a major sorafenib resistant mutant. In summary, this case represents the fifth reported case of acquired resistance to imatinib in FIP1L1-PDGFRA positive CEL. Our data illustrate the efficacy of sorafenib against FIP1L1-PDGFRα T674I as a single agent in vivo. Yet, selection of secondary sorafenib resistant clones is likely to occur, as has also been observed in imatinib resistant CML treated with second line tyrosine kinase inhibitors. While FIP1L1-PDGFRα D842V is a novel panresistant mutation in CEL, the PDGFRα D842V mutation is a known activating mutation of PDGFRα and causes primary imatinib resistance in a small percentage of gastro-intestinal stromal tumors. The homologous KIT D816V mutation in systemic mastocytosis is also associated with imatinib resistance. Of note, the latter two mutations respond better to dasatinib than FIP1L1-PDGFRα D842V. Our observation highlights the difficult challenge of treating resistant mutations and provides a basis for further proactive development of inhibitors with activity against sorafenib resistance mutants.