Inhibition of FLT3 kinase activity leads to an up-regulation of C/EBPα and PU.1 and an increase in the expression of differentiation markers in blasts of 2 of 3 FLT3/ITD-positive AML patients. (A) AML bone marrow blasts were collected before and after 4 weeks of treatment with CEP-701 from a FLT3/ITD-positive AML patient who was enrolled in the CEP-701 clinical trial and whose bone marrow blasts did not decrease with therapy. Total cellular RNA was prepared and 7 μg from each sample was subjected to sequential Northern blotting with a 0.7-kb EcoRI-HindIII fragment of human C/EBPα cDNA¹⁷  and actin cDNA probe. (B) Bone marrow blasts were obtained before and after 2 or 4 weeks of treatment with CEP-701 from 3 FLT3/ITD-positive AML patients whose bone marrow did not show a decrease of blasts in response to therapy. C/EBPα and PU.1 mRNA expression were analyzed using real-time RT-PCR analysis. Levels of C/EBPα and PU.1 were expressed relative to actin. Error bars indicate SD. (C-D) Bone marrow blasts were collected prior to and after 2 or 4 weeks of treatment with CEP-701 from 2 FLT3/ITD-positive AML patients. Antigen expression profiles of these blasts were determined by multiparameter flow cytometric analysis. AML blasts, characterized by low side-scattering value and expression of CD45, CD117, and CD33 (data not shown), were specifically gated (left). Expression of CD15 or CD11b by these blasts was then analyzed (right).