MIR150 Is Involved in the Monocyte Subset Differentiation and Its Down-Regulation Leads to Classical Monocyte Accumulation in Chronic Myelomonocytic Leukemia

Background. Monocytes are a heterogeneous population of peripheral blood leukocytes. The expression of CD14 and CD16 distinguishes CD14⁺/CD16^- classical from CD14⁺/CD16⁺ intermediate and CD14^low/CD16⁺ non-classical monocytes. We have shown (Selimoglu-Buet D et al, Blood 2015) that monocytes that accumulate in the peripheral blood of patients with chronic myelomonocytic leukemia (CMML) are predominantly CD14⁺/CD16^- classical monocytes that typically represent more than 94% of total blood monocytes. Strikingly, this phenotypic signature efficiently distinguishes CMML from a reactive monocytosis. Importantly, the CMML-associated increase in classical monocyte fraction disappears in patients who respond to hypomethylating drugs. Whereas in the mouse, the transcription factor Nr4a1 is required for the development of the Ly6C^lowmonocytes, the molecular mechanisms that regulate the formation of the three human monocyte populations remain poorly understood. Analysis of the classical monocytes accumulation in CMML may provide insights into the regulation of monocyte subset differentiation.

Methods. A microarray screen of miRNA expression was performed in monocytes sorted from 33 CMML and 5 healthy donor blood samples. Validation was performed by qRT-PCR, in monocytes of a cohort of 160 CMML patients and 20 controls, and in CD34⁺ cells from 44 CMML patients and 19 controls. A mouse model of MIR150-knock-out (Mir150^-/-) was used to examine the consequences of the miRNA down-regulation. Multi-color flow cytometry assays were designed to explore mouse and human monocyte subsets.

Results. Microarray analyses and validation experiments identified a decreased expression of miR150 in monocytes and CD34⁺cells from CMML patients compared to controls.

Mir150^-/- mouse model does not generate monocytosis even in ageing animals. However, an increase in Ly6C^high inflammatory monocyte fraction at the expense of Ly6C^lowpatrolling monocytes was observed in the bone marrow and peripheral blood, leading to further explore the link between MIR150 and monocyte populations.

The abnormal repartition of monocyte populations in Mir150^-/- mice is a cell-autonomous phenotype as wild-type (WT) mice receiving bone marrow from Mir150^-/-mice demonstrated a reduced fraction of Ly6C^low monocytes. This phenotype was rescued by re-expression of MIR150 in LIN^- cells of Mir150^-/-mice before engraftment. The number of myeloid progenitors was normal in Mir150^-/-mice, and the remaining Ly6C^low monocytes did not demonstrate an increased sensitivity to apoptosis. Competitive reconstitution experiments combining WT and Mir150^-/-LIN^- cells did not identify any significant fitness advantage to Mir150^-/-cells, but Mir150^-/-donor cells developed reduced numbers of Ly6C^low monocytes than cells from WT donors. These data suggest that MIR150 is involved during late stages of monocyte development and has a key role in the generation of Ly6C^lowmonocytes.

Finally, TET2 is the main gene mutated in CMML, and Tet2^-/- animals develop a monocytosis. Mir150^-/- crossed with Tet2^-/-mice developed a CMML-like phenotype.

In human, the expression of MIR150 decreases along myeloid differentiation and is low in classical compared to intermediate and non-classical monocytes. Depletion or overexpression of MIR150 in human CD34⁺ cells alters the repartition of CD14⁺/CD16^- and CD14⁺/CD16⁺ cells generated in culture. In CMML patients who respond to hypomethylating agents, the normalization of monocyte subset repartition correlates with an increased expression of MIR150, suggesting an epigenetic regulation. MIR150 has several promoters. By combining ChIP-Seq and DNA methylation analyses, a differentially methylated region was detected in one of the MIR150 promoters in CMML patients compared to controls. Using monocyte differentiation conditions, RNA Sequencing performed in CD34⁺cells overexpressing MIR150, identified ID1 gene as a potential MIR150 target.

Conclusion: We demonstrate a role for MIR150 in the generation of intermediate and non-classical monocyte subsets, and its down-regulation in CMML accounts for the characteristic accumulation of classical monocytes.