The Internal-Tandem-Duplication of Flt3 Mutation Generates Growth Inhibitory Signals in Mouse Hematopoietic Progenitor Cells by Down-Regulating Pbx1 Expression Via P21^cdkn1a.

Abstract 2340

Internal-Tandem-Duplication (ITD) Flt3 mutations are found in 30% of patients with AML; however, Flt3-inhibitors are not significantly efficacious in the therapy. We have reported that ITD-Flt3 down-regulates Pbx1 expression, a transcriptional factor that regulates the self-renewal of hematopoietic stem cells, in mouse bone marrow c-kit⁺-Sca-1⁺-Lin⁻ (KSL) cells, and that Pbx1 expression levels are reduced in patients with ITD-Flt3⁺AML compared with those lacking ITD-Flt3 (ASH 2011). However, the function of Pbx1 in ITD-Flt3 signaling remains unknown. In this report, we investigate the function of Pbx1 in hematopoietic progenitor cells transformed by ITD-Flt3.

ITD-Flt3 transduction in mouse marrow cells led to a decrease in Pbx1 mRNA expression levels in KSL cells (80±8% reduction compared with normal KSL cells, P<0.01, N=3), which coincided with the growth-factor-independent expansion of KSL cells. However, the transduction of shRNAs specific for Pbx1 (Pbx1-shRNA) in ITD-Flt3⁺ bone marrow cells led to a reduction in the number of CFU-GM colonies proliferating in the absence of growth factors compared with control shRNA-transduced ITD-Flt3⁺ cells (25 ± 9% reduction, P<0.05, N=3). Similarly, Pbx1-shRNA induced a decrease in the proliferation of ITD-Flt3⁺Ba/F3 cells upon cytokine deprivation (42±1% reduction, N=4, P<0.001). These results indicate that although Pbx1 is down-regulated by ITD-Flt3, the factor-independent proliferation of ITD-Flt3⁺cells is functionally supported by Pbx1. In contrast, p21^cdkn1a (p21)expression was up-regulated by ITD-Flt3 in both KSL cells (5.0±1.5-fold, P<0.05, N=3) and Ba/F3 cells (8.7±1.8-fold, P<0.05, N=3). However, the gene deletion of p21 resulted in an enhancement of ITD-Flt3⁺CFU-GM colony proliferation (2.0±0.3-fold increase, P<0.01, N=8), thereby indicating that an ITD-Flt3-mediated increase in p21 expression negatively modulates the aberrant proliferation of ITD-Flt3⁺ cells. Moreover, the loss of p21 upregulated Pbx1 mRNA expression in ITD-Flt3⁺KSL cells (2.1 ± 0.5-fold in p21^−/−compared with p21^+/+, P<0.05, N=3). However, the cell cycle fractionation of ITD-Flt3⁺KSL cells using Hoechst33314 and Pyronin-Y staining indicated that Pbx1 expression was reduced in G₁ phase cells compared with cells in the G₀ phase (0.2±0.1-fold, P<0.01, N=3), thereby suggesting that p21 negatively regulates Pbx1 in ITD-Flt3⁺cells, while the up-regulation of Pbx1 was not due to the cell cycle progression induced by p21 deletion. The transduction of Pbx1-shRNA in ITD-Flt3⁺p21^−/−bone marrow cells led to a decrease in the cytokine-independent hyperproliferation of CFU-GM colonies induced by p21 deletion compared with ITD-Flt3⁺p21^−/− cells containing the control shRNA (shRNA-1: 43±4% reduction; shRNA-2: 41±7% reduction, P<0.05, N=3). Similarly, anti-p21-shRNA treatment increased the growth-factor-independent proliferation of ITD-Flt3⁺Ba/F3 cells compared with the control-shRNA cells (5.3±0.3-fold increase, P<0.001, N=4), whereas the transduction of shRNA specific for Pbx1 in the ITD-Flt3⁺Ba/F3 cells harboring anti-p21-shRNA partially abrogated the enhanced proliferation that was induced by p21 knockdown (29±1% reduction compared with the control, P<0.001, N=4).

Our data indicates that expression of Pbx1, which supports the growth-factor-independent proliferation of ITD-Flt3⁺ cells, was down-regulated by p21, which was elevated in the presence of ITD-Flt3. The enhanced proliferation of ITD-Flt3⁺ cells induced by p21 deletion was partially abrogated by knocking down Pbx1, thereby indicating that the down-regulation of Pbx1 is secondary to the up-regulation of p21 that is induced by ITD-Flt3, which resulted in the growth inhibition of ITD-Flt3⁺cells. The data demonstrates that ITD-Flt3 can generate growth inhibitory signals by reducing the Pbx1 expression levels via p21.This implies that Flt3-inhibitors may destroy the Pbx1/p21 growth inhibitory signaling that is generated by ITD-Flt3, thereby contributing to subsequent recurrence of ITD-Flt3⁺AML cells.