Mirna-21 Targets Sprouty2 to Activate SYK Mediated BCR and MAPK-Erk Signaling in Chronic Lymphocytic Leukemia

INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) represents the most common adult leukemia in the western world. Several reports have demonstrated that the constitutively activated MAPK-Erk signaling pathways promote CLL cell proliferation and survival. However, the precise molecular mechanisms that lead to deregulated MAPK signaling in CLL initiation and progression are not fully understood. We have previously reported that Sprouty2 (Spry2) is a negative regulator of BCR and MAPK-Erk signaling in poor prognosis CLL (American Society of Hematology, 56^th Annual Meeting, San Francisco, CA, 2014). Here in follow up studies, we set to further determine molecular basis deregulation of Spry2 in patients with poor prognosis CLL. Spry2 is either epigenetically silenced or targeted by Microrna-21 (miR21) in several malignancies. MiR-21 plays a role of oncomir in CLL, which is significantly upregulated in CLL patients with poor prognosis specifically with Del 17p, High 38 and Zap70 expression. Notably, we observed a decrease in Spry2 expression in CLL cells expressing high levels of miR-21. Also, it is already reported that miR21 is regulated by STAT3 transcription factor in CLL patients with poor prognosis in refractory phase. However the precise molecular mechanism by which the miR21 leads to leukemic progression in poor prognosis CLL is not known. Here we demonstrate the molecular mechanism of miR21 to activate Syk mediated BCR and MAPK-Erk signaling by targeting Spry2 in CLL cells from poor prognosis patients.

METHODOLOGY: We isolated CLL cells from peripheral blood of newly diagnosed high CD38 poor prognosis patients. For molecular analysis, in addition to primary CLL cells we use Mec-1 CLL cell line. To measure the levels of miR21 we used Taqman assay qPCR from Invitrogen. To decrease miR21 levels we used miR21 inhibitor (Invitrogen) and STAT3 inhibitor (SantaCruz). To overexpress miR21 we used pcDNAmir-21 construct from Addgene. Activation of BCR and MAPK-Erk signaling pathway was measured by levels of p-Erk and p-Syk. Expression of Spry2, Erk and Syk was measured by western blot analysis.

RESULTS: To study if miR21 targets Spry2 in CLL cells, we first overexpressed miR21 in Mec-1 cells. We observed a significant decrease in Spry2 expression in Mec-1 cells expressing high levels of miR-21 compared to empty vector control. In our previous studies we observed Spry2 acts as a negative regulator of BCR and MAPK-Erk signaling. Therefore, we next studied the activity of Spry2 targeting signaling pathways in miR21-overexpressing CLL cells. We observed elevated levels of p-Erk and p-Syk in CLL cells overexpressing miR21. For control we used pcDNA-empty vector. Whereas upon miR21 knockdown in CLL cells resulted in stabilization of Spry2 expression and decrease in BCR and MAPK-Erk signaling. Also, targeting miR21 by STAT3 inhibitor leads to induction of spontaneous apoptosis in CLL cells. We next tested the effect of STAT3 inhibitor on primary CLL cells from 13 different patients. We measured the viability of CLL cells using MTT assay, we observed higher efficacy of STAT3 inhibitor over Syk and BRAF inhibitors on CLL cells.

CONCLUSION: We conclude that Spry2 is a target of miR21 in CLL cells from poor prognosis patients. MiR21 targets Spry2 to activate Syk mediated BCR and MAPK signaling in CLL patients. Also, STAT3 can be used as therapeutic target for poor prognosis CLL patients with high miR21 expression.