Abstract
Internal tandem duplication (ITD) mutations in the Fms-related tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia (AML). Due to the development of drug resistance, few FLT3-ITD inhibitors are effective against FLT3-ITD+ AML. In this study, we show that FLT3-ITD activates a novel pathway involving p21Cdkn1a (p21) and pre-B cell leukemia transcription factor 1 (Pbx1) that attenuates FLT3-ITD cell proliferation and is involved in the development of drug resistance. FLT3-ITD up-regulated p21 expression in both mouse bone marrow c-kit+-Sca-1+-Lin- (KSL) cells and Ba/F3 cells. The loss of p21 expression enhanced growth factor-independent proliferation and sensitivity to cytarabine as a consequence of concomitantly enriching the S+G2/M phase population and significantly increasing the expression of Pbx1, but not Evi-1, in FLT3-ITD+ cells. This enhanced cell proliferation following the loss of p21 was partially abrogated when Pbx1 expression was silenced in FLT3-ITD+ primary bone marrow colony-forming cells and Ba/F3 cells. When FLT3-ITD was antagonized with AC220, a selective inhibitor of FLT3-ITD, p21 expression was decreased coincident with Pbx1 mRNA up-regulation and a rapid decline in the number of viable FLT3-ITD+ Ba/F3 cells; however, the cells eventually became refractory to AC220. Overexpressing p21 in FLT3-ITD+ Ba/F3 cells delayed the emergence of cells that were refractory to AC220, whereas p21 silencing accelerated their development. These data indicate that FLT3-ITD is capable of inhibiting FLT3-ITD+ cell proliferation through the p21/Pbx1 axis and that treatments that antagonize FLT3-ITD contribute to the subsequent development of cells that are refractory to a FLT3-ITD inhibitor by disrupting p21 expression.
Highlights
The cyclin-dependent kinase inhibitor p21Cdkn1a (p21) was originally identified as a mediator of p53-induced growth arrest [1,2] and regulates the proliferation of diverse cell types by modulating cell cycle, apoptosis and differentiation [3,4,5]
We previously showed that Fms-related tyrosine kinase 3 (FLT3)-Internal tandem duplication (ITD) mutations increase Survivin expression in mouse Ba/F3 cells [30] and that Survivin enhances the proliferation of primary mouse HPCs through p21-dependent and p21-independent mechanisms [9]
P21 negatively regulates cell proliferation in a variety of cell systems [6,7,8], these findings suggest that p21 may positively regulate FLT3-ITD signaling, similar to Survivin
Summary
The cyclin-dependent kinase inhibitor p21Cdkn1a (p21) was originally identified as a mediator of p53-induced growth arrest [1,2] and regulates the proliferation of diverse cell types by modulating cell cycle, apoptosis and differentiation [3,4,5]. P21 can facilitate the proliferation of normal hematopoietic progenitor cells (HPCs) ex vivo following stimulation with hematopoietic growth factors [9,10,11,12]. These findings suggest that p21 has a differentiation stage-specific function in the hematopoietic system. In addition to regulating the cell cycle [1,2,6,7,13], p21 can modulate the activity of a number of transcription factors and co-activators, such as Stat3 [13,14], estrogen receptorα[15], (Ccaat-enhancer-binding protein α (C/EBPα) [16,17] and c-Myc [18], suggesting that it may regulate cell fate by influencing gene expression [19]
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