The Mir-17/92 Cluster Is Involved in the Molecular Etiology of the SCLL Syndrome Driven By the BCR-FGFR1 Chimeric Kinase

MicroRNAs have pathogenic roles in the development of various types of leukemia. Here we identify miRNAs that have important roles in the development of stem cell leukemia/lymphoma syndrome (SCLL) resulting from the expression of the chimeric BCR-FGFR1 kinase. Comparison of BBC2 cells, a murine pre-B-cell lymphoma models of SCLL, with FACS sorted normal murine splenic CD19+ B cells, led to identification of 191 upregulated and 59 downregulated in BBC2 cells. We next investigated which of these miRNAs directly respond to FGFR1 through inhibition of FGFR1 signaling with BGJ398 in BBC2 cells, and finally defined 46 core miRNAs, with 33 activated in BBC2 downregulated by BGJ398, and 13 suppressed upregulated by BGJ398.

Further analysis of these 46 miRNAs showed that the miR-17/92 cluster was particularly implicated and forced expression resulted in increased cell proliferation, while inhibiting its function using microRNA sponges reduced cell growth and induced apoptosis. In addition, overexpression of miR-17/92 partially rescued the FGFR1 signaling blockage by BGJ398. Cell treatment with the potent BGJ389 FGFR1 inhibitor led to miR-17/92 downregulation, suggesting direct regulation by FGFR1. To further validate this direct regulation, both transient luciferase reporter assays and qRT-PCR detection of endogenous miR-17/92 expression in transduced stable cell lines were used. The reporter assay showed that cotransfection of the microRNA promoter with BCR-FGFR1 led to increased luciferase expression proportional to BCR-FGFR1 levels, confirming this microRNA cluster was directly activated by the fusion kinase. NIH3T3 and BaF3 cells stably expressing BCR-FGFR1 showed upregulation of endogenous miR-19b-3p, miR-20a-5p, demonstrating that BCR-FGFR1 can directly regulate miR-17/92 expression. In addition, this positive association of miR-17/92 with BCR-FGFR1 was also confirmed in primary mouse SCLL tissues and primary human CLL samples.

Our data further revealed that miR-17/92 promoted cell proliferation and survival by targeting CDKN1A and PTEN in B-lymphoma cell lines and primary tumors from BCR-FGFR1 mouse model, with decreased mRNA and protein expression in miR-17/92 overexpressed cells and tumor tissues, while increased mRNA and protein expression in miR-17/92 inhibited cells. An inverse correlation in expression levels was seen between miR-17/92 and both CDKN1A and PTEN in two cohorts of CLL patients. Finally, in vivo engraftment studies demonstrated that manipulation of miR-17/92 was sufficient to affect disease progression of BCR-FGFR1 driven leukemogenesis, in which overexpression of miR-17/92 increased the proportion of GFP+ BBC2 cells in peripheral blood, enlarged the spleen size and shorten the survival time of recipient mice, while inhibition of miR-17/92 showed reverse effects. Overall, our results define miR-17/92 as a direct downstream effector of FGFR1 in BCR-FGFR1 driven B cell lymphoblastic leukemia.