Characterization of a Hierarchy of Proliferative Ability in AML: Identification of a Role for NR2F6 in the Maintenance of the Undifferentiated State.

AML cells are heterogeneous in their capacity to proliferate and form colonies in vitro. Determination of the genes whose expression is necessary for clonogenicity has been hindered by the inability to isolate pure populations of AML cells with defined proliferative abilities. By analyzing the growth of clonal siblings we established that the hierarchical organization of AML can be resolved into distinct strata, permitting the use of clonal siblings as biological reporters of the transcriptional signature of single cells with defined proliferative abilities. We then analyzed low passage cultures of the cell line OCI-AML4. Clones consisting of four cells were micromanipulated so that a single cell was sampled for global RT-PCR while its three clonal siblings served as reporters of clonogenicity. By microarray analysis we found the orphan nuclear receptor NR2F6 to be expressed four-fold lower in leukaemia single cells that spontaneously lose proliferative ability, compared to single cells with greater proliferative capacity. NR2F6 is a poorly characterized member of the COUP transcription factor family. We observed that NR2F6 is overexpressed in AML, CMML and MDS relative to normal bone marrow (BM). We then assessed expression of NR2F6 in U937 leukaemia cells induced to differentiate with a variety of induction agents. All differentiation agents induced significant decreases in NR2F6 expression. To characterize the effect of forced expression of NR2F6 we transduced U937 cells with a retrovirus encoding either NR2F6 or EGFP. Forced expression of NR2F6 reduced the doubling time of these populations, and inhibited retinoic acid induction of U937 cell differentiation. NR2F6 overexpressing cells did not acquire expression of the maturation marker CD11b nor were capable of reducing nitroblue tetrazolium, indicating functional maturity. Assessment of DNA content and analysis of growth kinetics revealed that NR2F6 overexpressing cells did not undergo the proliferative arrest associated with terminally differentiating U937 cells. We then investigated the role of NR2F6 in normal haematopoiesis in the C57/BL6 and Balb/c strains of mice. In both strains we observed that overexpression of NR2F6 resulted in a significant reduction in BFU-E and CFU-GM colony numbers, and colony size. These data are consistent with the notion that NR2F6 inhibits maturation of normal BM. More specifically, analysis of the immunophenotype of BM cultured in suspension for 10 days showed that NR2F6 overexpression resulted in a significant reduction in monocytes, and an increase in mast cells. An excess of mast cells was also observed in cytospin preparations of CFU-GM colonies. Additionally, we have demonstrated that overexpression of NR2F6 augments the in vitro self-renewal ability of BM, using serial replating of semi-solid cultures. Competitive BM transplantation experiments were employed to confirm the role of NR2F6 on stem cell self-renewal and differentiation in vivo. While these investigations are in progress, progenitor assays and analysis of serial-replating in all animals examined have recapitulate our ex vivo observations. Overall, these data establish the importance of NR2F6 in the regulation of normal and leukaemic haematopoietic cell self-renewal and differentiation. Furthermore, our results establish that analysis of clonal siblings allows the elucidation of differences in gene expression within the AML hierarchy.