Effect Of Epidermal Growth Factor Receptor Inhibitor On Proliferation Of JAK2V617F-positive Cells In Vitro

Objective:1. To study the effect of erlotinib, a kind of epidermal growth factor receptor(EGFR) tyrosine kinase inhibitor, on the proliferation of hematopoietic progenitor cells with JAK2V617F mutation in polycythemia vera patients.2. To detect proliferation activity of the HEL cell line under the action of erlotinib and gefinitib in vitro.3. To discuss epidermal growth factor receptor inhibitor for the potential targeted therapy of polycythemia vera.Methods:1. Case screening:Select patients diagnosed with polycythemia vera by WHO2008revised diagnostic criteria from the Shandong Provincial Hospital, and use allele specific PCR screening cases harboring JAK2V617F mutation and JAK2wild type simultaneously for the study. The total of cases is12.2. Colony formation assays in vitro:Separate bone marrow mononuclear cells through lymphocyte separation medium, and IMDM complete medium are used for culture.4hours later, vaccinate in methylcellulose semi-solid medium, and then culture in sterile incubator for14d (including the control group and the intervention group which is intervene with the erlotinib diluted solution, the final concentration of erlotinib in the system is5μM/L), identification of CFU is completed under the microscope. Use AS-PCR identifying colonies containing JAK2V617F mutation, and count the total number of two kinds of colonies respectively.3MTT assays:Detect HEL cell line proliferation activity under the effect of different concentrations of erlotinib and gefitinib respectively, and detect K562cells proliferation activity.under the effect of different concentrations of erlotinib. HEL cell line has been proven to contain natural JAK2homozygous mutation, while K562cell line comes from chronic myeloid leukemia (JAK2V617F-negative). Gefitinib is an EGFR inhibitor, and its mechanism and clinical applications are similar to erlotinib.Results:1. To the intervention group and the control group, the CFU property distribution had a significant difference. The JAK2V617F-positive colonies proportion of the intervention group declined comparing to the control group. It shows that erlotinib in5μM/L concentration may inhibit proliferation of hematopoietic progenitor cells from polycythemia vera patients with JAK2V617F mutation, while it plays a small role with normal hematopoietic progenitor cells from the patients.2Erlotinib inhibit HEL cell proliferation activity in a dose-dependent manner, and the IC50was4.1μM/L. Gefitinib inhibit HEL cell proliferation activity in a dose-dependent manner, too, and the IC50was6.2μM/L. It reveals that gefitinib’s inhibition activity of JAK2V617F-positive cells is weaker than erlotinib. Erlotinib didn’t inhibit K562cells proliferation significantly in these concentration levels, confirming that it has no inhibition ability on JAK2V617F-negative leukemia cell lines.Conclusions:Erlotinib, epidermal growth factor receptor(EGFR) inhibitor, could inhibit proliferation of JAK2V617F-positive cells in vitro, and it may be a potential JAK2V617F inhibitor.