In Vitro and In Vivo downregulation of Leukemogenesis by FMNL1 Inhibition In Namalwa Cell Line

Abstract 1485

Formins represent a family of proteins indispensable for fundamental actin-dependent processes; it has been suggested that they represent a group of attractive drug targets for the treatment of inflammation and malignant diseases. Formins are known for their ability to act as effectors of Rho family GTPases, proteins involved in cell organization, adhesion, and proliferation. Deregulation of Rho GTPase family members has been associated with multiple human hematological diseases such as leukemia. FMNL1 is a formin-related protein that is restrictedly expressed in hematopoietic-lineage-derived cells and overexpressed in malignant lymphoid cells such as chronic and acute lymphoid leukemia. This restricted expression supports the idea that FMNL1 may be an attractive target for novel therapies in such diseases. FMNL1 has recently been shown to interact with srGAP2, a RhoGAP family member, and this complex is temporally regulated by Rac1. Herein, we evaluated the role of FMNL1, by gene silencing, in proliferation, cell cycle, clonal growth, migration and transendothelial migration (TEM), and apoptosis in Namalwa cells, an acute lymphoblastic leukemia cell line. We also accessed the role of FMNL1 in tumor growth using a NOD/SCID xenograft model. Moreover, we confirmed the endogenous association of FMNL1 with Rac1, and studied Rac1 activity in leukemic cells lacking FMNL1 expression for the first time. To inhibit FMNL1 in the Namalwa cell line, specific shRNA-expressing lentiviral vectors targeting the FMNL1 gene (shFMNL1) or no specific sequence (shControl) were used. Cell growth was measured using the MTT colorimetric reduction in five replicates. Colony formation was carried out in semisolid methylcellulose medium and was detected after 8 days of culture by adding 1mg/mL of MTT reagent and scored by Image J quantification software. Both assays were carried out in lentiviral transduced cells treated, or not, with different concentrations of rapamycin (10 or 100nM). Migration assays were performed in triplicate using 8-mm transwells, and for TEM, HUVECs were seeded onto fibronectin-coated 5-mm transwell filters. The lower compartment for both assays was filled with SDF-1. Annexin-PI was used to access apoptosis. NOD/SCID mice were implanted with lentiviral transduced Namalwa cells in the dorsal subcutaneous site of the animal. FMNL1 association with Rac1 protein was analyzed by immunoprecipitation, and Rac1 activity was measured by a PAK-pull down assay, both followed by Western blotting. P value <0.05 was considered statistically significant. Inhibition of FMNL1 in Namalwa cells resulted in a significant decrease in proliferation of 40%, when compared with control cells (P <0.01). Interestingly, the combination of FMNL1 inhibition/rapamycin treatment showed a significantly higher reduction in cell proliferation (50%) when compared with FMNL1 inhibited cells alone (P =0.008) or rapamycin treated cells (P =0.007). Inhibition of FMNL1 induced cell cycle delay in the S phase in Namalwa cells and a consequent decrease in the number of cells in G2/M. A significant decrease of 30% in the number of colonies was observed in shFMNL1, as compared to shControl cells (P <0.0001). Again, rapamycin treatment resulted in the synergic inhibition of colony formation by 50% (P =0.007). FMNL1 silencing also resulted in a significant decrease (62%, P <0.01) in transwell and a decrease (20%) in the TEM assay. Apoptosis was unaffected by FMNL1 knockdown. In vivo experiments, the ShFMNL1 cell injected group showed a significant decrease in tumor growth during the experimental period (15 days after injection), when compared to the shControl cell injected group (P =0.002). FMNL1 was found to be associated with Rac1 in Namalwa cells, by immunoprecipitation, performed also in a reverse assay. Interestingly, Rac1 presented a four-fold increase in its activity in shFMNL1 cells in three independent PAK-pull down experiments. Aberrant Rac1 activity is known in tumorigenesis; however, in this leukemia cell model, inhibition of FMNL1 bypassed Rac1 activity. Our findings indicate that FMNL1 participates in the regulation of leukemogenesis, through different cell activities, including proliferation, colony formation, migration and tranwell migration of Namalwa cells, suggesting that FMNL1 represents a possible therapeutic target together with rapamycin, based on their synergic effects.