Regulation of ABCB1 (p-glycoprotein) by the FOXO1 Transcription Factor in Acute Myeloid Leukemia.

Abstract 589

FOXO1 mediates a cellular stress response by inhibiting cell growth and up-regulating expression of molecules involved in apoptosis and antioxidant defences. The membrane pump p-glycoprotein (pgp, expressed by the ABCB1 gene) is responsible for the efflux of chemotherapeutic drugs and the redistribution of components of the ceramide signalling pathway. In AML, pgp over-expression is associated with failure to respond to remission-induction chemotherapy, but attempts to modulate pgp function have enjoyed little success and new approaches are needed. We studied transcriptional control of the ABCB1 gene in AML cells. The Pgp protein was measured using MRK-16; ABCB1 mRNA was measured using realtime PCR in patients entered into UK AML 14 and AML 15 clinical trials. 141 of these trial samples were also studied using the Affymetrix U133A (22,283 probesets) or the U133Plus2 (∼54,000 probesets) gene chips to establish correlations between pgp and factors which might influence expression. FOXO1 was one of the most closely correlated genes with pgp (P<0.001). FOXO3 and 4 did not correlate. To confirm these results FOXO1 mRNA levels were measured by real time PCR in a separate cohort of 178 trial samples: FOXO1 and ABCB1 were also strongly correlated in this cohort (P<0.001). Protein levels of FOXO1 were measured by flow cytometry, and corresponded to mRNA levels in patient samples. Using the TF1 cell line, which normally expresses high levels of FOXO1 and ABCB1 we studied the regulation of pgp by FOXO1 using siRNA. FOXO1 siRNA was found to decrease both FOXO1 and ABCB1 mRNA expression by 70% and 60% respectively. High pgp expression rarely occurs in a sample with a FLT3-ITD mutation. In this context we established that the presence of a FLT3-ITD in AML samples is associated with low FOXO1 expression (P<0.001, median FOXO1/B2M ratio = 0.51 in wildtype samples (n=268) and 0.18 in ITD samples (n=101)) and that siRNA to FLT3 in MV4.11 (FLT3-ITD) cells upregulates FOXO1 more than 2-fold. As the ABCB1 promoter is known to have a FOXO1 binding site, we conclude that FOXO1 is likely to be a major regulator of ABCB1 expression in AML and may therefore be a therapeutic target to modulate drug resistance. We further conclude that FOXO1 may be at least partially responsible for the distinction between two major poor risk phenotypes in AML–namely FLT3-ITD (causing low FOXO1, predictive for relapse) and pgp overexpression (induced by high FOXO1, predictive for chemoresistant disease).