Therapeutic effect of microRNA-15/16 cluster in a mouse model of chronic lymphocytic leukemia

Background: Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in adults in the Western world. Previously, we generated transgenic mice over-expressing the TCL1 oncogene in B cells. These mice developed a disease very similar to human CLL. However, TCL1 is over-expressed in only 25-30% of all human CLL cases. These findings support the idea that other genes might be involved in CLL. Recent studies showed that CLL harbors alterations in the microRNAs genes miR-15a and miR-16-1 that are frequently deleted and/or down-regulated in CLL patients. Both microRNAs negatively regulate Bcl2, an anti-apoptotic protein over-expressed in human as well as mouse CLL B cells. Repression of Bcl2 by microRNAs is enough to induce apoptosis. Therefore, miR-15 and -16 are natural antisense Bcl2 interactors that could be used for therapy of Bcl2-overexpressing malignancies. Since mouse CLL lymphocytes, like their human counterpart, are mostly resting CD5+ B cells, in a previous study we did transplantation experiments of mouse malignant lymphocytes into syngeneic mice in order to expand CLLs in vivo. Purpose: We exploited the in vivo transplant system to verify the potential ability of miRs 15/16 to delay or stop the development of mouse CLL. Experimental Design: Ex vivo treatments of CLL cells with miRs 15/16 were performed as follows. White cells with expanded CD5+/IgM+ populations (70 to 95%) were isolated from the enlarged spleen of two syngeneic mice previously transplanted with CLL # 450 and 504 and incubated in vitro in 10% FBS in RPMI medium 1640 in three different conditions: a) regular medium, b) transfected with pSR-GFP-Neo expression lentiviral vector containing both miRNAs (TWEEN-miR15/16), and c) transfected with control empty lentiviral vector. After two days incubation, CLL cells were collected, washed twice, and resuspended in PBS. Ten million cells were then injected intraperitoneally into new syngeneic mice to monitor the evolution of the disease over time. Results: Eight mice were used as controls (injected with untreated or empty virus-treated cells) and eight more animals were injected with miR-treated cells. After 4 months from CLL cells injection, seven control mice were dead (87%) while only one miR-treated mouse died (13%; Fisher’s exact test, p = 0.01). These findings show a significant delay in the onset of the disease due to miR-treatment. Conclusions: This is the first study showing an inhibitory effect of specific microRNAs on CLL phenotype with a therapeutic potential. These promising, though still preliminary, results from the ex vivo experiments encourage us to try, in the next step, a direct in vivo approach to demonstrate the tumor suppressor effect of miR 15/16 in the TCL1 mouse model of CLL.