The MN1 Oncogene Blocks Differentiation of Multiple Hematopoietic Lineages.
Authors: Heuser Michael , Argiropoulos Bob , Kuchenbauer Florian , Humphries R. Keith 
Published: November 2019

Abstract
Overexpression of wild-type MN1 (meningioma 1) is a negative prognostic factor in normal karyotype acute myeloid leukemia (AML). Recently we demonstrated that MN1 overexpression is sufficient to induce a rapidly lethal AML in mice by promoting proliferation of transduced bone marrow (BM) cells and by blocking myeloid differentiation. In fact, MN1 increased resistance to all-trans retinoic acid (ATRA)-induced cell-cycle arrest and differentiation by more than 3,000-fold in an in vitro differentiation model. We have shown that the block in differentiation can be overcome by fusion of a transcriptional activator (VP16) to MN1. MN1VP16-transduced BM cells gave rise to immortalized cell lines, however, these cells differentiated to mature granulocytes and underwent cell cycle arrest upon treatment with ATRA. The functional characteristics of MN1VP16GFP-expressing cells were further investigated in the present study using retroviral gene transfer and the murine BM transplantation model. Mean engraftment in peripheral blood (PB) of transduced (GFP+) cells was 27 and 37% at 11 and 21 weeks (n=9) respectively, and 76% at time of death (n=5). In two mice engraftment peaked at 21 weeks and then decreased, and 2 mice are alive at 260 days post transplantation with 0.2 and 2% GFP+ cells in PB. MN1VP16 mice had a median survival of 143 days (n=16) compared to 35 days of MN1 mice (n=18) (p〈.001). At time of death sick mice had normal or increased WBC counts (mean WBC counts 37.5 × 10e6/mL), severe anemia (mean RBC counts 1.3 × 10e9/mL), thrombocytopenia (mean platelet count 30 × 10e6/mL), and splenomegaly (mean weight 0.43 g). Analysis of the immunophenotype of GFP+ cells from PB, BM, and spleen revealed a lympho-myeloid reconstitution with 24 and 64 percent Gr-1 and Mac-1 expressing cells in BM, respectively, while 53% of cells in BM expressed c-kit. B220 and CD4 or CD8 expressing cells were predominantly found in spleen (22 and 18 percent, respectively), suggesting that MN1VP16-transduced cells were at least partly responsive to homing signals and to their microenvironment. Interestingly, morphologic analysis of BM revealed in most cases a predominance of mature granulocytes but immmature forms of less than 20 percent; absence of maturing erythroid precursors, and absence of megakaryocytes, consistent with a diagnosis of myeloproliferative-like disease according to Bethesda criteria. The key difference between MN1- and MN1VP16-induced disease is that myeloid cells are blocked at an early stage in MN1-expressing cells as evidenced by large increases in myeloid blasts in MN1 diseased mice, but can terminally differentiate in MN1VP16-expressing cells. The common feature of these two models is the severe anemia and thrombocytopenia. In BM cell lines overexpressing MN1 or MN1VP16 gene expression of Gata-1, Jak2, and c-mpl is decreased compared to total BM whereas expression of Gata-2 is increased. These findings suggest that MN1 not only blocks myeloid differentiation but blocks megakaryocytic/erythroid differentiation as well. By selectively enabling myeloid differentiation through fusion of a transcriptional activator to MN1 we were able to functionally separate the myeloid- from the megakaryocytic/erythroid-differentiation blocking function. It is therefore likely that MN1 blocks myeloid and megakaryocytic/erythroid differentiation through distinct mechanisms. Our findings suggest that anemia and thrombocytopenia seen in AML patients may not be secondary to myeloproliferation and space constraints in the BM but may in fact be transcriptionally encoded.