Significant Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPα Mutations.

Transcription factor CCAAT enhancer binding protein α (C/EBPα) is crucial for the differentiation of granulocytes. Experimental data from animal models as well as patient samples suggest that loss of function or expression of C/EBPα provides a platform on which acute myeloid leukemia (AML) develops. C/EBPα is mutated in around 9% of acute myeloid leukemia. The mutations reported in C/EBPα are frame shift mutations at N-terminal domain and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. Peptidyl-prolyl cis/trans isomerase, Pin1 binds to and isomerizes the peptidyl-prolyl bond in specific phosphorylated Ser/Thr-Pro motifs. A growing number of studies show that Pin1 is overexpressed in many cancers and has significant role in tumorigenesis. In the present study we investigated the role of Pin1 in acute myeloid leukemia with C/EBPα mutation. Here we report C/EBPα-p30 could induce Pin1 transcription as assessed by quantitative Real-Time RT-PCR analysis. Affymetrix mRNA expression analysis show that Pin1 is upregulated in patients with acute myeloid leukemia. Silencing of Pin1 could overcome the dominant negative action of the C/EBPα-p30 over the C/EBPα-p42 transactivation capacity as analyzed by promoter assay. By silencing Pin1 with inhibitor against Pin1 (PiB), we could show myeloid differentiation in Kasumi-6 cells by FACS analysis with granulocytic specific markers. Western blot analysis shows that Pin1 inhibition by PiB could upregulate wild type C/EBPα protein level. Luciferase promoter assay for the Pin1 promoter shows that C/EBPα-p30 induces Pin1 promoter activity in association with E2F1. Mean while, wild type C/EBPα interferes with transactivation of the Pin1 promoter and downregulates Pin1 mRNA expression. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We have previously shown that c-Jun expression is high in AML patients with C/EBPα mutation and c-Jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that overexpression of Pin1 induces c-Jun mRNA expression, while inhibition of Pin1 by the inhibitor PiB downregulates c-Jun mRNA expression. We show that c-Jun blocks the DNA binding and transactivation of wild type C/EBPα protein as assessed by gel shift assay and promoter assay respectively. In conclusion, inhibition of Pin1 leads to granulocytic differentiation of human myeloid cells. Our findings suggest inhibition of Pin1 as a novel strategy in treating AML patients with C/EBPα mutation.