Balancing Proliferation, Differentiation, and Survival: Powerful Genetic and RNAi Technologies Reveal Essential microRNA Signaling for Leukemic Progenitor Cell Fitness

Acute myeloid leukemias (AML) are a heterogeneous group of malignancies with distinguishing gene and microRNA (miRNA or miR) expression profiles. In particular, expression of the miR-196 family of miRNA is significantly associated with a large fraction of AML expressing HOX gene signatures (e.g. NPM1c mutant, 11p15 and 11q23 cytogenetic abnormalities) and is prognostically instructive. However, the requirement for miR-196 in hematopoietic cell immortalization, malignant transformation, and leukemogenesis is not understood. We note that miR-196a-1 and miR-196b are both induced upon MLL-AF9 expression, and that miR-196b is a direct MLL-AF9 target gene. To genetically evaluate the necessity of miR-196 for MLL-AF9 tumorigenesis, we varied the number of miR196-encoding alleles and tested the capacity for marrow transformation by MLL-AF9. Specifically, we transduced bone marrow cells from wild-type (WT), miR-196b^+/-, and miR-196a-1^-/- b^-/- double-knockout (DKO) mice with retroviruses expressing MLL-AF9 to limit (miR-196b^+/-) or completely eliminate (DKO) miR-196 activity. All groups were immortalized in vitro, as evidenced by the formation of morphologically blast-like colonies, accompanied by serial replating in methylcellulose colony assays. Moreover, we found similar deregulation of HoxA9 and Meis1 expression. Since these are two essential MLL-AF9 target genes, we conclude that the MLL-oncoprotein complex must be functional without miR-196. However in vivo, despite similar levels of engraftment, only mice transplanted with WT or miR-196b^+/- MLL-AF9 cells formed leukemia (median latency 70 and 76.5 days, respectively; mice were followed for a total of 135 days). Flow cytometric analyses of leukemic granulocyte-monocyte progenitors (GMP) harvested from miR-196b^+/- MLL-AF9 moribund mice displayed a significant increase in CD11b expression as compared to WT MLL-AF9 controls. These miR-196 haploinsufficient and loss-of-function AML models genetically demonstrate that miR-196 activity is critical to fully transform and block differentiation of malignant progenitor cells. Next, we identified AML-relevant miR-196 targets by purifying miR-196b/RNA-target/RISC complexes in human 11q23-translocation AML cells, validating putative targets in luciferase reporter assays, then testing them in an in vivo leukemogenesisshRNA-enrichment screen. Knockdown of several miR-196b targets cooperates with MLL-AF9 to accelerate leukemogenesis, including Cdkn1b. Notably, Cdkn1b- knockdown cKit+ MLL-AF9 splenocytes from moribund mice displayed significantly decreased CD11b expression and increased colony forming potential in vitro. However, simply reducing Cdkn1b in MLL-AF9 leukemia cells did not alter the number of functional leukemia initiating cells (LIC) in an in vivo limiting-dilution analysis (suggesting that Cdkn1b- knockdown does not directly affect LIC biology). Instead, RNA-Seq analyses of Cdkn1b- knockdown MLL-AF9 leukemia cells from moribund animals showed increased expression of proliferation, cell cycle, and survival pathways with decreased expression of myeloid differentiation and apoptotic pathways. Taken together, these data suggest that during leukemogenesis miR-196 activity (through direct targets such as Cdkn1b) provides a leukemia cell fitness advantage, defined by the ability of a malignant cell to intrinsically balance the conflicting programs of proliferation/self-renewal and differentiation, resulting in survival. Given the critical role of miR-196 in MLL-AF9 transformation and leukemia maintenance, we asked whether the miR-196-Cdkn1b pathway might be a point of therapeutic intervention. Indeed, forced overexpression of Cdkn1b significantly diminished colony formation in vitro, and eliminated AML in vivo. Translating this into an RNAi therapeutic, we treated murine MLL-AF9 cells with locked nucleic acid (LNA) sequences designed to specifically block miR-196b binding to its target site in Cdkn1b mRNA. This resulted in not only significant de-repression of p27^Kip1 expression, but also reduced MLL-AF9 colony formation in vitro. In sum, we have established a critical genetic requirement for miR-196b in MLL-AF9 leukemogenesis through the balanced control of growth and differentiation, identified a relevant target, and demonstrated therapeutic potential of inhibiting miR-196 binding to this single target gene.