Aberrant Activation of Epidermal Growth Factor Receptor in MPN May Respond to the Kinase Inhibitor Gefitinib

Abstract

Epidermal growth factor receptor (EGFR) expression is elevated in peripheral blood (PB) cells of polycythemia vera (PV) patients (Skov et al, Eur J Haematol 2011;87:54-60) and EGFR inhibitors (AEE788, erlotinib) inhibit erythroid burst-forming units (BFUE) from PV patients but not normal donors. The mechanism underlying the effect of EGFR inhibitors on MPN progenitor growth has not been established but could be due to an off-target effect on JAK2 activity (Li et al, J Biol Chem 2007;282:3428-32; Gaikwad et al, Exp Hematol 2007;35:1647-56). Therefore, we investigated the growth of BFUE in the presence and absence of the EGFR inhibitor gefitinib (10µM), which does not inhibit JAK2, and observed inhibition of growth of both erythropoietin (Epo)-dependent and -independent colonies from PB mononuclear cells (PBMNC) from PV patients but not from normal individuals. These results suggest a potential role for EGFR signalling in supporting growth and/or survival of PV progenitors. Therefore, to evaluate the possibility of somatic genetic abnormalities leading to EGFR hypersensitivity in MPN, we performed targeted exon capture and massively parallel sequencing, and sensitive mass array screening of 155 MPN patient samples. We identified a low-frequency, recurrent somatic variant of EGFR (p. C329R) in 3/155 MPN patients. The human EGFR C329 residue is homologous to the residue C359 of the C. elegans gene let-23, target of a known gain-of-function mutation (Katz et al, Mol Cell Biol 1996;16(2):529-37); it also aligns with the cysteine residue affected in the highly-transforming mutant ErbB2 C334S found in lung cancer (Greulich et al, PNAS 2012; 109:14476–14481); and it lies within the extracellular cysteine-rich region of EGFR that is the target of frequent somatic mutations in glioma. To confirm the hypothesis that the EGFR C329R mutant leads to altered cytokine response, we transduced Ba/F3 cells with empty vector, EGFR wild type (WT) or mutant constructs (BaF3/MIG, BaF3/EGFR and BaF3/EGFRC329R, respectively). Both WT and mutant receptors showed constitutive activation and transforming ability when expressed at high levels. However, BaF3/EGFRC329R cells display increased levels of STAT activation associated with a slight proliferation advantage when compared to BaF3/EGFR.Given that gefitinib inhibited the growth of both BaF3/EGFR and BaF3/EGFRC329R but did not affect BaF3/MIG cells grown in IL-3, we next compared the effect of gefitinib (10µM) on the growth of BFUE from PV patient samples with and without the EGFR C329R mutation. We observed significant inhibition of Epo-independent BFUE from all PV samples but not of Epo-dependent BFUE from normal controls (Figure A). Furthermore, genotyping of JAK2 and EGFR from the individual colonies obtained in BFUE assay (treated or not with gefitinib, 10µM) for a PV patient that is positive for EGFR C329R showed that drug treatment significantly reduced the proportion of JAK2 V617F heterozygous BFUE compared to the vehicle-treated control (chi-squared test = 0.0002, Figure B). This suggests that signalling from EGFR contributes to proliferation and/or survival in JAK2 V617F heterozygous BFUE from this patient.The results presented here are consistent with an EGFR signalling role in supporting growth of PV progenitors, particularly in the context of a heterozygous JAK2V617F mutation. STAT5 signalling is essential for PV (Walz et al, Blood 2012;119:3550-3560; Yan et al, Blood 2012;199:3539-3549) and JAK2-independent activation of STAT5 through EGFR (Quesnelle et al, J. Cell. Biochem. 2007; 102:311–319) via various mechanisms may contribute to the level of STAT5 activation required for the PV phenotype. A recent study demonstrating a role for EGFR in hematopoietic stem cells (Doan et al, Nat Med 2013;19:295-304) also highlights the potential of aberrant EGFR signalling to contribute to altered properties of MPN stem cells. Finally, given that gefitinib is currently in clinical use for treatment of solid tumors, these findings raise the possibility that gefitinib may have clinical utility in the context of MPN. [Display omitted] DisclosuresBranford:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding.