Cyclin Dependent Kinase Inhibitor P21 Negatively Regulates PBX-1 Via Internal Tandem Duplication of Flt3 Dependent Manner, a Novel Mechanism That Does Not Exist in Normal Hematopoietic Progenitor Cells

Abstract 1310

The cyclin dependent kinase inhibitor p21 CDKN1 (p21) regulates proliferation of diverse cell types by modulating cell cycle and apoptosis in variety of ways, however, its molecular mechanism underlying cell fate decisions remains to be explored. We previously reported that p21 expression is up-regulated in mouse Ba/F3 cells by Internal Tandem Duplication mutations on the Flt3 gene (ITD-Flt3), which induce constitutive activation of Flt3 signaling and associate with poor prognosis of patients with acute myeloid leukemia (AML). However, despite up-regulation of p21 by ITD-Flt3, deletion of p21 gene accentuated growth factor independent proliferation of primary mouse hematopoietic progenitor cells (HPC) transformed by ITD-Flt3 (Abe et al. ASH 2010). The data suggests that the paradoxical increase of p21 induced by ITD-Flt3 negatively regulates oncogenic proliferation of ITD-Flt3⁺ HPC. Although p21 is generally considered to reduce cell proliferation by inhibiting G₁/S transition, p21 can enhance proliferation of normal HPC stimulated by hematopoietic growth factors (Mantel et al. 1995, Fukuda et al. 2004). These data suggest that differential mechanism is involved in regulating p21 function between normal versus transformed HPC. Herein in order to determine the mechanism responsible for growth factor independent proliferation of ITD-Flt3⁺HPC regulated by p21, we identified selective molecules regulated by p21 exclusively in ITD-Flt3⁺ HPC that are distinct from normal HPC.

Deletion of p21 gene modulated expression of 247 genes in c-kit⁺, Sca-1⁺, lineage⁻ (KSL) cells over-expressing ITD-Flt3. Out of 247 genes modulated by p21 deletion in ITD-Flt3⁺ KSL cells, 112 genes were either not modulated or inversely regulated by p21 in normal mouse marrow KSL cells in vivo. The results implicate that alteration of 112 mRNA expression by p21 may be specific for transformed KSL cells by ITD-Flt3, which is distinct from normal KSL cells. Out of 112 genes, 13 genes are listed in the known genes deregulated in human AML stem cells (LSC) (Majeti et al. 2009). Among the 13 molecules that are also deregulated in AML LSC, pbx-1, a transcriptional factor, which is profoundly associated with hematopoietic stem cell function but down-regulated in AML LSC (Ficara et al. 2008), was up-regulated by 1.6±0.2 fold in ITD-Flt3⁺ KSL cells lacking p21 (P<0.05, N=3) compared to p21^+/+ counterparts. Whereas, loss of p21 did not affect pbx-1 expression in normal KSL cells (1.1±0.1 fold compared to p21^+/+ KSL cells, NS). Pbx-1 mRNA expression was significantly lower in ITD-Flt3 transduced KSL cells compared to freshly isolated KSL cells (P<0.05, N=3), regardless of p21 status, a finding consistent with down-regulation of pbx-1 in AML LSC compared to normal CD34⁺CD38⁻ cells and presence of ITD-Flt3 in AML LSC (Levis et al. 2005). In contrast, p21 mRNA expression was coincidently elevated in ITD-Flt3⁺KSL cells (P<0.05) compared to normal KSL cells, indicating that p21 and pbx-1 are concomitantly but inversely modulated by ITD-Flt3 in ITD-Flt3⁺ KSL cells. Consistently, p21 expression was negatively correlated with pbx-1 expression in 293 AML subjects obtained from a public database Gene Expression Omnibus (GEO: GSE1159). However, the association between p21 and pbx-1 was not observed in the gene expression profile of normal human CD34⁺CD38⁻ cells. In addition, pbx-1 expression was significantly lower in ITD-Flt3⁺ AML cells (N=78) compared to the ITD-Flt3 negative AML cells (N=215, P<0.05) listed in GSE1159, providing additional evidence for the negative regulation of pbx-1 by ITD-Flt3 in AML cells.

These results suggest that p21 and ITD-Flt3 down-regulate pbx-1 expression, but the effect of p21 on pbx-1 is ITD-Flt3 dependent. As opposed to p21/pbx1 axis identified in transformed HPC and AML, pbx-1 and p21 is not associated in normal KSL cells and CD34⁺CD38⁻ cells. The data suggests that p21/pbx-1 axis selectively exist in AML cells, a mechanism that is not present in normal HPC. Enhancement of growth factor independent proliferation of ITD-Flt3⁺ KSL cells resulting from p21 deletion may be partly due to up-regulation of pbx-1induced by p21 deletion, rather than merely a consequence of modulation of G₁/S transition by p21. In this regard, manipulation of selective p21/pbx-1 axis that is dependent on ITD-Flt3 may represent a novel therapeutic strategy for ITD-Flt3⁺AML that are refractory to ITD-Flt3 inhibitors.