Abstract B68: Small GTPase RANBP16 as a novel target in acute lymphoblastic leukemia chemoresistance.
Authors: Silveira Vanessa S. , Suazo Veridiana K. , Borges Kleiton S. , Andrade Augusto F. , Cruzeiro Gustavo AV , Tone Luiz Gonzaga , Scrideli Carlos Alberto 
Published: August 2014

Background: Despite the improvements made on acute lymphoblastic leukemia (ALL) treatment around 20% of the patients still present treatment failure thus showing how the molecular mechanisms responsible for chemotherapy resistance still need to be clarified. The present study was undertaken to address the possible involvement of the small GTPase RANBP16 (a nuclear export receptor also known as Exportin 7) to acute lymphoblastic leukemia chemotherapy response.

Experimental procedures: global gene expression analysis was carried out among B-ALL patients classified as prednisone poor responders (PPR) versus prednisone good responders (PGR) using Agilent platform. Subsequent validation was performed by quantitative RT-PCR in childhood ALL patients under the Brazilian Group for Treatment of Childhood Leukemia - GBTLI 99. To address the role of RANBP16 in chemoresistance, the cell lines ReH (B-ALL) and Jurkat (T-ALL) were transduced with lentivirus containing either shRNA for the target gene or empty vector. Puromycin selection was performed to generate stable transformed lineages. Gene knockdown was confirmed by qRT-PCR and Western blot. Drug cytotoxic assay was performed in a 4-day XTT cell proliferation assay and IC50 value was calculated with Calcusyn software. Annexin V staining and Colony-forming assay (CFA) was tested in triplicate and t-test was used to analyze the data (alpha value was set at 5%).

Results: Initially, in a gene expression analysis by microarray we observed that RANBP16 was highly expressed in B-ALL samples from prednisone poor responders compared to good responder patients. Validation of this initial finding by quantitative RT-PCR in 87 B-ALL patients showed that RANBP16 up-regulation is indeed related to lower EFS (event free survival) rates, suggesting a role of this molecule in B-ALL poor outcome. RANBP16 knockdown induced a significantly reduction of IC50 values to prednisolone (0.5-150ug/mL) and daunorrubicin (10-150 nM) in both cell lines and a 7.0-fold increase in apoptosis rates in prednisolone resistant Jurkat cell line (as compared to the empty vector). Colony forming assays showed that RANBP16 knockdown significantly reduces the self-renewal capability after prednisolone treatment. Response to vincristine (1.0-20nM) was not modified by the knockdown condition.

Conclusions: This data suggests that the RANBP16 gene can play an important role in leukemic cells sensibility to the glucocorticoid prednisolone and could be considered as a target for further investigations on new therapeutic interventions for ALL treatment. Further experiments, are still need to clearly demonstrate the role of this specific RAN-GTPase in glucocorticoid-mediated response.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B68.

Citation Format: Vanessa S. Silveira, Veridiana K. Suazo, Kleiton S. Borges, Augusto F. Andrade, Gustavo AV Cruzeiro, Luiz Gonzaga Tone, Carlos Alberto Scrideli. Small GTPase RANBP16 as a novel target in acute lymphoblastic leukemia chemoresistance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B68.