Analysis of Wnt and Hedgehog Pathways Regulating Protein Kinases CK1 and CK2 in Acute Myeloid Leukemia Cells and Stem Cells: Correlation with the Expression of Wnt and Hedgehog Targets and Biological and Clinical Features

Background. Leukemia stem cells (LSC) constitute the reservoir of acute and chronic leukemias, from which the relapse of the disease takes origin. The Wnt/βcatenin and the Hedgehog (Hh) signaling pathways regulate the balance between self-renewal and commitment of both normal hematopoietic stem cells (HSC) and LSC. Serine-Threonine protein kinases CK1 and CK2 phosphorylate members of Wnt and Hh and are therefore potential regulators of HSC and LSC biology; however, their function in HSC and LSC is unknown. Purpose. In the present work we have investigated the mRNA and protein levels of CK1 and CK2 in acute myeloid leukemia (AML) blasts and LSC isolated from patients. We have also integrated the expression data of CK1 and CK2 with the expression data of Wnt and Hh signaling pathways gene targets and with biological and clinical parameters of a group of AML patients and cell lines. We also tested the effects on the growth of AML blasts of CK1 and CK2 small chemical inhibitors. The aim of this study was to gain insights into the function of these pivotal protein kinases and on their expression levels relative to biological, clinical and prognostic variables that dictate the outcome of AML patients. Methods. mRNA was extracted from HSC and FACS-sorted LSC (as defined as Lineage- CD34+ CD38- CD123+ CD90+); proteins were obtained from the bulk of blasts from fifteen AML patients. Quantitative RT-PCR was employed to assess mRNA levels of: CK1α, CK1γ, CK2α (catalytic) and CK2β (regulatory subunit); Lef1, FoxO1, FoxO3, CyclinD1 (Wnt targets); Ptch, Gli1, Gli2, Gli3, Bmi1 (Hh targets); western blot analysis was performed to determine CK1α, CK2α, CK2β, p53, phospho-Ser529 NF-κB and phospho-Ser13 Cdc37 (two direct CK2 target sites) protein levels. Normal CD34+ bone marrow cells were used as controls. CK2 chemical inhibitors (CX4945 and tTBB) were assayed on LSC and AML cells. Results. CK1α, CK2α and CK2β mRNA and proteins were found overexpressed in LSC and in AML blasts. CK1γ mRNA levels were barely detectable. Notably, some AML cases displayed low levels of CK2β but high levels of CK2α, indicating a possible CK2β-independent function of CK2α. FoxO1, FoxO3 mRNAs were found upregulated while Lef1 and CyclinD1 ones were found slightly and Axin2 one strongly diminished, respectively. Ptch, Smo, Gli1, Gli2 and Gli3 mRNAs were unchanged or reduced, while we observed a strong upregulation of that of Bmi1. A correlation between the overexpression of CK1α and CK2 and high-risk cytogenetic groups was observed. Also, in most of CK2α high-expressing cases, TP53 was found downregulated, while phospho-Ser529 p65 upregulated. Importantly, inhibition of CK2 with selective compounds caused AML cell and LSC growth arrest, a restoration of TP53 and a downregulation of phospho-Ser529-NF-kB p65 and phospho-Ser13 Cdc37 protein levels. Conclusions. The present study is the first to report on the expression of CK1 and CK2 kinases in normal HSC and LSC. CK1 (α) and CK2 (α and β) mRNA and protein levels were higher in LSC (especially from high risk groups) than in HSC. CK1γ mRNA levels were low. Wnt and Hh pathways genes were differentially upregulated pointing to a gene specific LSC-associated function. Moreover, our data with CK2 inhibitors suggest that CK2 could be a suitable therapeutic target to eradicate residual AML LSCs. Future research will assess more in depth the in vivo functional role of CK1 and CK2 in HSC and LSC.