Primary acute myelogenous leukaemia (AML) samples are heterogeneous in clonogenicity, both among patients and within the leukaemic cell population of a single patient. To explain this heterogeneity the leukaemia stem cell model postulates that leukaemic hematopoiesis is organized in a hierarchy, sustained by leukaemia stem cells that may either self-renew or differentiate aberrantly to give rise to blasts that can no longer proliferate. This process is akin to the irreversible growth arrest entered by terminally differentiating normal blood cells.
We wished to identify genes associated with clonogenicity in AML. To obtain pure populations of cells of defined growth abilities, we analyzed low passage cultures of the cell line OCI-AML4. This cell line resembles primary AML cells in several important respects; it is growth factor-dependent, contains a low proportion of clonogenic cells, and has a relatively simple karyotype. 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 EAR-2 to be expressed four-fold lower in leukemia single cells that spontaneously lose proliferative ability, compared to single cells with greater proliferative capacity.
EAR-2 is a member of the COUP transcription factor family, which play roles in various developmental processes through interactions with nuclear receptors and other transcription factors. We assessed expression of EAR-2 in monoblastic leukaemia U937 cells induced to differentiate with a variety of induction agents. Treatment with dimethylsulfoxide, phorbol ester, vitamin D3, and all trans retinoic acid (ATRA) all induced significant decreases in EAR-2 expression. This phenomenon was also seen in a mouse model of acute promyelocytic leukaemia (APL). When primary bone marrow cultures of hCG-NuMA-RAR transgenic mice were induced to differentiate with ATRA, an average decrement in EAR-2 expression of 5.58 fold was observed (p<0.005). Since aberrant differentiation is an invariant feature of AML, we hypothesized that the overall expression of EAR-2 would be greater in AML patients relative to healthy controls. Analysis by quantitative RT-PCR of 15 AML, 10 CMML, 12 MDS and 16 normal bone marrow samples showed that EAR-2 is overexpressed in all three disease categories (p<0.0009 AML, 0.03 CMML, 0.0003 MDS).
To characterize the effect of forced expression of EAR-2 on clonogenicity we transduced U937 cells with a retrovirus encoding either EAR-2 (U937-EAR2) or EGFP (U937-GFP). Analysis of FACS-purified U937-EAR2 and U937-GFP cultures showed that forced expression of EAR-2 reduces the doubling time of these populations (U937-EAR2 = 24h; U937-GFP = 34h; p<< 0.001), while no significant difference was observed in cell cycle profile. The decrease in doubling time of U937-EAR2 cells may reflect a decrease in the rate of cell loss in the population, consistent with the hypothesis that EAR-2 functions as a repressor of terminal differentiation.
We have observed that expression of the orphan nuclear receptor EAR-2 is positively associated with maintenance of proliferative capacity and negatively associated with differentiation. These observations establish the importance of EAR-2 in the regulation of clonogenicity and terminal differention.