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M. Spagnuolo et al.
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Figure 6. Effect of FRZB expression on leukemogenesis and β-catenin activity of Philadelphia-positive (Ph+) BV173 cells. (A) Survival of mice injected with 2x106 BV173-ShMYB 7TFP pUltra-Empty Vector (EV) or BV173-ShMYB 7TFP pUltra-hot-FRZB cells (FRZB). (B) Luciferase reporter assay for β-catenin activity in GFP+ cells isolated from the bone marrow (bm) or spleen (sp) of a NOD scid gamma (NSG) mouse injected with (EV)- or FRZB-BV173 cells and sacrificed when terminally ill.
left panel). To assess whether FRZB is a direct target of miR-17-92, a human FRZB 3’UTR fragment containing wild-type or mutated miR-17 or miR-19a seed sequences (Figure 4F, middle panel) was cloned downstream of the firefly luciferase reporter gene and co-transfected with miR-17 or miR-19a mimics in 293T cells. The relative luciferase activity of the reporter with wild-type 3’UTR was decreased by 27% upon expression of the miR-17 mimic and by 29% upon expression of the miR-19a mimic; in contrast, there was no decrease in luciferase activity of the mutant reporter (Figure 4F, right panel), sug- gesting that FRZB is a direct target of miR-17 as well as of miR-19a.
To investigate whether FRZB has a role as a miR-17-92 target gene in the “MYB addiction” of BCR-ABL-trans- formed cells, we assessed the relative expression of FRZB, the miR-17-92 cluster and MYB in blast cells from 2 Ph+ leukemia patients (n=1: p210BCR/ABL CML-myeloid blast crisis; n=1: p190BCR/ABL ALL). High expression of the miR-17-92 cluster correlated with that of MYB and was more abundant than in CD34+ or peripheral blood mononuclear cells from healthy donors (Figure 5A and B, left panel). In contrast, levels of FRZB were much higher in cells from healthy donors than in blast cells from the Ph+ leukemia patients (Figure 5B, right panel). In agreement with these findings, we found that, in a microarray dataset of 122 Ph+ ALL samples, MYB mRNA levels were more abundant in Ph+ ALL cells compared to normal B cells, while the opposite was found for FRZB expression (Figure 5C).
To investigate directly whether expression of FRZB has a negative effect for leukemia development, NOD scid gamma (NSG) mice were injected with EV-transduced or FRZB-expressing BV173 cells carrying the β-catenin-Luc reporter plasmid and assessed for overall survival. Survival of the two groups was identical (Figure 6A); however, β-catenin activity was markedly reduced in BV173 cells isolated from the bone marrow or spleen of a mouse injected with FRZB-expressing compared to EV-trans- duced cells (Figure 6B). These data suggest that leukemia induced by Ph+ BV173 cells is β-catenin-independent but do not exclude the possibility that FRZB-dependent regu- lation of β-catenin activity is important for leukemia induced by primary Ph+ ALL cells.
Discussion
The expression of MYB is critical for the proliferation and survival of many leukemic cells, including BCR-ABL1- transformed myeloid and lymphoid cells;6,12 however, the mechanisms responsible for the “MYB addiction” of these cells are only partially understood.
In this study, we assessed the miRNA expression profile of MYB-silenced BV173 and K562 CML-blast crisis cells with the goal of identifying miRNAs whose modulation might explain the impaired proliferation and survival asso- ciated with MYB knockdown in BCR-ABL1-transformed lymphoid or myeloid precursors.6,12 Interestingly, MYB appears to have broad effects, directly or indirectly, on the levels of miRNAs since approximately 24% and 13% of those expressed in BV173 and K562 cells, respectively, were modulated by MYB silencing. Although many miRNAs regulated by MYB exhibited changes in both cell lines, a high number of the modulated miRNAs exhibited cell-type specificity.
We speculated that those modulated by MYB in a cell- type specific manner may regulate pathways required for more specialized cell functions, while those regulated in both cell lines may be involved in more general biological processes, such as cell proliferation and survival. Within the miRNAs regulated by MYB in both cell lines, we focused on the miR-17-92 cluster because of its oncogenic role in many tumors,28,43,44 its involvement in BCR-ABL1- transformed cells,45 and its regulation by MYC,44 a known MYB target.46 We found that MYB bound directly to the miR-17-92 promoter, suggesting that its effects on the expression of several members of the miR-17-92 cluster are direct, although an indirect effect through other tran- scription factors (eg. c-Myc) and/or co-activators cannot be excluded.47 On the other hand, silencing MYB alone does not abolish expression of the miR-17-92 cluster, sug- gesting that other transcription factors also regulate the expression of the miR-17-92 cluster in BCR-ABL1-trans- formed cells.16 Compared to control cells, MYB-silenced BV173 cells exhibit a marked inhibition of cell growth which is due to cell-cycle arrest and induction of apopto- sis.48 Thus, we asked whether restoring expression of the miR-17-92 cluster would rescue the impaired growth of
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haematologica | 2019; 104(1)