ARHGAP21 Is Overexpressed in Bone Marrow of Acute Leukemia Patients and Interacts with Focal Adhesion Kinase (FAK) in Hematopoietic Cells.

ARHGAP21 is a new protein recently described by us and characterized as a Rho-GTPase activating protein (Rho-GAPs), a negative regulator of RhoGTPase signaling pathways. RhoGTPases mediate many aspects of cell biology, including proliferation, apoptosis, survival, adhesion and actin cytoskeleton dynamics. Recently, ARHGAP21 was identified as a new component of cell-cell junctions that controls α-catenin recruitment and is able to interact with α-catenin, ARF1 and ARF6, important proteins in the cytoskeleton assembly and adherent junctions. Cell-to-cell adhesion is one of the major factors that restrict cellular invasion and metastasis in malignant diseases. In the attempt to verify the role of ARHGAP21 in leukemogenesis, we aimed to verify the expression level of ARHGAP21 mRNA and protein in bone marrow samples from adults with diagnosis of acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL). In addition, we attempted to verify a possible interaction of ARHGAP21 with proteins involved in cell adhesion, as Focal adhesion kinase (FAK). FAK is a non-receptor tyrosine kinase, and coordinates signals from integrins, cytokines, growth factor receptors, and oncogenes. In AML cells, FAK has been found to be overexpressed and associated with enhanced blast migration, increased cellularity, and poor prognosis. The National Ethical Committee Board approved the study and informed-written consent was obtained from all subjects. Quantitative real-time PCR analysis was performed using specific primers to ARHGAP21 transcript, normalized by β-actin control. ARHGAP21 mRNA expression was found to be significantly higher in AML (n = 37) and ALL (n = 9) samples when compared with normal hematopoietic cells (n= 5) (medians; AML: 3.01 vs 0.25, P < 0.0001; ALL: 4.47 vs 0.25, P = 0.0010; Mann-Whitney test). ARHGAP21 mRNA expression showed a positive correlation with bone marrow blast cell counts (Spearman test, P = 0.0362). Western blotting analysis showed higher ARHGAP21 expression in acute leukemia cell lines: Jurkat, Molt-4, K562 and HL60 in comparison to normal peripheral blood mononuclear cells (PBMC). Immunoprecipitation and Western blotting assays using anti-ARHGAP21 and anti-FAK antibodies detected the interaction between ARHGAP21 and FAK in protein extracts of Jurkat cells and normal PBMC. Pull down assays using three different FAK-GST fusion proteins: C-terminal (residues 687–1054), FERM domain (residues 60–349) and catalytic domain (residues 390–696) in protein cell lysates of HL-60 and normal PBMC showed that ARHGAP21 is associated with the C-terminal region of FAK. In conclusion, our data show that ARHGAP21 is overexpressed in AML and ALL cells and is associated with FAK in leukemia cell lines and in normal PBMC. These findings give rise to the hypothesis that ARHGAP21 may be involved in leukemogenesis, aiming this gene as a candidate for anti-tumor therapy.