INTRODUCTION AND OBJECTIVES: Acute lymphoblastic leukemia (ALL) is the most diagnosed pediatric cancer. Within ALL, those originating from B precursor cells are the most frequent (B-ALL). The occurrence of specific gene rearrangements is associated with prognostic risk stratification. Among several gene products presenting aberrant expression in ALL, osteopontin (OPN) has been reported. OPN is a matricellular glycophosphoprotein involved in several steps of tumor progression. In B-ALL, our previous data demonstrated that OPNc is the most upregulated OPN splice variant (OPN-SV) in patients harbouring KMT2A-AFF1 fusion, which is a poor prognostic risk biomarker associated with increased relapse risk and central nervous system (CNS) involvement. Moreover, OPNc expression levels were found to be associated with poor prognostic risk features, such as high white blood cell counting and CNS infiltration. In this scenario, the current study aimed to investigate the functional roles of OPN-c in a B-ALL cell line presenting KMT2A-AFF1. MATERIAL AND METHODS: Using the strategy to specifically knock-down OPNc using an antisense DNA oligomer (ASO) anti-OPNc targeting this splice variant, the RS4;11 cell line was transfected with this ASO or with the scrambled DNA control sequence (ASO scramble) using the 4D Nucleofector system. OPNc- silenced cells were then evaluated by RT-qPCR, immunofluorescence, cell adhesion, viability, transmigration and invasion assays.RESULTS AND CONCLUSIONS:. The RS4;11 cells transfected with the ASO anti-OPNc displayed a decrease in 85% of transcriptional expression levels of OPNc in relation to control cells. OPNc-silenced cells also presented an increase of 76% in proliferation rates and a decrease of 41% in cell viability rates at 24h time point. Moreover, these cells displayed a decrease of 55% and 17% in adhesion to matrigel and osteoblasts cell matrix, respectively. Corroborating these results, we found that RS4;11 OPNc-silenced cell also presented a decrease in 54% and 51% of transmigration and invasion capacity through matrigel, respectively. In conclusion, we found that OPNc expression levels in B-ALL cells presenting KMT2A-AFF1 can modulate B-ALL cell proliferation, adhesion, migration and invasion properties. We then hypothesize that upregulated OPNc expression in this leukemia model may control the ability of B-ALL cells to adhere in the bone marrow matrix, which could be a factor related to leukemia relapse, while acquiring invasive properties, possibly associated CNS and other extramedullary sites infiltration, contributing to more aggressive B-ALL phenotypes. Further work should be conducted to better understand OPN-SI mechanism in the CNS involvement.