Microrna (miR)-17-92 Contributes to Therapy Resistance in Burkitt Lymphoma Cells

Background

Burkitt lymphoma (BL) is curable, but patients with relapse or refractory disease have very poor outcomes. The underlying biological differences leading to relapse/refractory disease are unknown. We previously identified a potential association between microRNA (miR)-17-92 and relapse in patient samples. MiRs encoded by this gene have been implicated in therapy resistance, so we are seeking relationships between miRNA expression and therapy resistance in BL cells.

Methods

The miR-17-92 locus was targeted for deletion in Raji BL cells using custom CRISPR-Cas9 lentiviral vectors. Single cell-derived clones were established and locus deletion was determined by genomic DNA PCR. Inducible miR-17-92 expression in Raji cells was established by transduction of a cumate switch inducible lentivector system containing the miR-17-92 gene along with a CymR repressor expression vector (Raji/CymR/miR-17-92). MiR expression was assessed by TaqMan assay, and protein expression and caspase-3 cleavage were assessed by western blotting. Cells were treated with 4HC, the active metabolite of cyclophosphamide, or doxorubicin, and viability was assessed by AlamarBlue assay.

Results

Genomic DNA PCR verified hemizygous deletion of the miR-17-92 gene in several Raji BL clones; no homozygous deleted clones were identified. In two hemizygous clones R5 and R24, miR-17 expression was decreased to 0.24-fold and 0.37-fold, respectively, relative to the parental Raji line. The miR-17-92 target proteins PTEN and Bim were increased in the R5 and R24 clones by western blotting. Compared to Raji, R5 and R24 showed increased basal caspase-3 cleavage and much greater induction of caspase-3 cleavage with cytotoxic chemotherapy (10 µM 4HC). In addition, 0.5 and 1 µM doxorubicin showed a dose dependent induction of cleaved caspase in R5 and R24 cells that was much greater than in parental Raji cells. The IC50 for doxorubicin in R5 and R24 miR-17-92 hemizygous cells was markedly decreased (0.00025 and 0.00032 µM, respectively) compared to parental Raji cells (0.064 µM). Finally, miR-17 expression was inducible by treating cumate repressor vector transduced Raji/CymR/miR-17-92 cells with 30 µg/mL cumate (+2.8-fold at 3 days, +7.5-fold at 5 days).

Conclusion

We established miR-17-92 hemizygous-deleted cell lines that show increased PTEN and pro-apoptotic Bim, increased basal caspase-3 cleavage, and increased chemosensitivity. We have established Raji BL cell lines with inducible miR-17-92 expression to extend these experiments by determining the effect of increased miR-17-92 on chemosensitivity. Our findings show that high miR-17-92 expression in BL cells can contribute to therapy resistance, which may be important for understanding refractory BL in patients and could lead to more effective targeted therapies.