Constitutive activation of FLT3 in acute myeloid leukaemia and its consequences for growth of 32D cells

The receptor tyrosine kinase Flt3 is expressed on leukaemic blasts of most cases with acute myeloid leukaemia (AML). In order to evaluate the presence and significance of constitutive activation of Flt3 for leukaemogenesis, w e (1) analysed the expression and activation status of the receptor in AML blasts; and (2) evaluated the functional consequences of constitutively act ive Flt3 in a myeloid progenitor cell line. Immunoprecipitation studies rev ealed Flt3 expression in a high proportion of AML cases (27/32) with ligand -dependent Flt3 autophosphorylation in 18, constitutive autophosphorylation in three and no autophosphorylation in six cases. Only one out of three sa mples with constitutively active Flt3 but 3/18 samples with ligand-dependen t autophosphorylated Flt3 contained the recently described internal tandem repeat (ITR) mutations. To test the significance of Flt3 activation in myel oid cell function, we also characterized the biochemical and biological eff ects of the activating mutation D838V of Flt3 (FLt3(D838V)) on the factor-d ependent myeloid progenitor cell line 32Dcl3: cells transfected with wild-t ype Flt3 (32D/Flt3) grew FLt3 ligand (FL) dependent, and the receptor was l igand dependently autophosphorylated. In contrast, the receptor was constit utively autophosphorylated in 32D/Flt3(D838V) cells, which grew independent ly of FL. We conclude that, in some AML samples, Flt3 is constitutively act ivated and that; this does not correlate with ITR mutations in the juxtamem brane domain. Furthermore, constitutively active Flt3 confers factor indepe ndence to the myeloid progenitor cell line 32D, It remains to be determined whether activation of Flt3 is leukaemogenic in vivo and whether strategies aimed at inhibition of Flt3 activation could inhibit leukaemogenesis.