Formin-Like 1 (FMNL1) Associates with Rac1 and Negatively Regulates Neoplastic Growth and Migration in Leukemia Cell Lines.

Abstract 1030

FMNL1 belongs to a conserved family of formin-related proteins, has high restricted expression in malignant lymphoid derived cells and has been described as a tumor associated antigen in chronic lymphoid leukemia (CLL). Overexpression of FMNL1 in CLL has already been show to correlate with adverse clinical and biological parameters, particularly in young patients (E Calpe et al, ASH 2006, abstract submitted). Formins are known for their ability to act as effectors of Rho family GTPases, proteins that regulate multiple pathways involved in cell organization, adhesion, and proliferation. Deregulation of Rho GTPase family members has been associated with multiple human hematologic diseases such as leukemia. FMNL1 function and regulation have not yet been well characterized; its restricted expression suggests that FMNL1 represents an attractive target for novel immunotherapies in hematopoietic malignant disorders. Herein, we evaluated the association of FMNL1 with the three most studied Rho GTPase proteins (Rac1, RhoA and Cdc42) and the role of FMNL1, by gene silencing, in proliferation, migration and cell cycle in acute lymphoblastic leukemia cell lines. We also tested the cumulative effect of FMNL1 silencing and rapamycin treatment in proliferation assays. FMNL1 association with Rho GTPases proteins was analyzed by immunoprecipitation, followed by western blot. To inhibit FMNL1, specific shRNA-expressing lentiviral vector targeting the FMNL1 gene or no specific sequence (control) were used. Cell growth was measured using the MTT colorimetric reduction method in cells treated or not with different concentrations of rapamycin (10 or 100nM) in five replicates. Migration assays were performed in triplicate using 5-mm Transwells and the lower compartment was filled with 600 mL 0.5% BSA/RPMI containing 100ng/mL SDF-1. The experiments were performed in two cell lines: Jurkat and Namalwa. P value <0.05 was considered statistically significant. The levels of FMNL1 mRNA and protein in FMNL1 knockdown cells were reduced by approximately 70% for both cell lines tested. Inhibition of FMNL1 in Jurkat and Namalwa cells resulted in a significant decrease of proliferation by 40% for both cell lines when compared with control cells (P<0.01). Interesting, in both cell lines, the combination of FMNL1 inhibition/rapamycin treatment showed higher reduction in cell proliferation when compared with FMNL1 inhibited cells alone (P=0.008) or rapamycin treated cells (P=0.007). Inhibition of FMNL1 resulted in an accumulation of Namalwa cells in the S phase and a consequent decrease of cells in G2/M. FMNL1 silencing also resulted in a significant decrease by 62% of cell migration for both cell lines when compared to control cells (P=<0.01). Among the Rho GTPase proteins tested, FMNL1 was found to be associated with Rac1, but not with RhoA and Cdc42. The immunoprecipitation results suggest FMNL1 as an effector of Rac1. Indeed, similar effect obtained with FMNL1 silencing has already been shown with Rac1 knockdown, which also suppresses leukemia cell migration and growth. Our findings indicate that FMNL1 participates in the regulation of proliferation and migration of Jurkat and Namalwa cell lines, which suggests that FMNL1 represents an attractive therapeutic target. Based on the result of FMNL1 inhibition/rapamycin combination, we hypothesized that combinatorial inhibition of these pathways would be effective for the treatment of lymphoid malignancies. Supported by Fapesp and CNPq.