CBFB-Independent Activities of the Inv(16) Fusion Gene CBFB-MYH11.
Authors: Hyde R. Katherine , Kreycik Claire E. , Liu Pu Paul 
Published: October 2019

Abstract
Inv(16)(p13;q22), the chromosomal translocation commonly found in patients with AML M4Eo, results in the fusion gene CBFB-MYH11. We have shown previously that mice heterozygous for Cbfb-MYH11 (Cbfb+/MYH11) have severe central nervous system hemorrhage and a complete lack of definitive hematopoiesis. This phenotype is indistinguishable from that seen in mice homozygous for Cbfb null (Cbfb−/−), consistent with the idea that the Cbfb-MYH11 gene product prevents normal Cbfb activity. However, in the peripheral blood of Cbfb+/MYH11 embryos before embryonic day 12.5 (E12.5), we have observed an increase of morphologically immature primitive hematopoietic cells, which has not been described in the Cbfb−/− embryos. This finding raises the possibility that the Cbfb-MYH11 gene product has activities not related to the inhibition of normal Cbfb function. To address this hypothesis, we further characterized differentiation of primitive hematopoietic cells in Cbfb+/+, Cbfb+/MYH11, and Cbfb−/− embryos. By FACS, we found that over 90% of the primitive blood cells in Cbfb+/+ and Cbfb−/− E10.5 embryos showed high expression of the differentiation marker TER-119, and were negative for the progenitor marker, c-kit. In contrast, the primitive blood cells of Cbfb+/MYH11 embryos, had increased expression of c-kit, and decreased expression of TER-119 indicating an increase of immature, progenitor cells. We also saw this delay in differentiation in E11.5 and E12.5 Cbfb+/MYH11 embryos, although the severity decreased with age. In addition, we found increased BrdU incorporation and annexin V staining of primitive blood cells in E12.5 Cbfb+/MYH11 embryos as compared to blood from Cbfb+/+ embryos. The rates of BrdU incorporation and annexin V staining in the blood of Cbfb−/− embryos were indistinguishable from wildtype. Together, these results indicate that the Cbfb-MYH11 gene product causes defects in differentiation, proliferation, and apoptosis in primitive blood that is independent of its ability to inhibit normal Cbfb activity. In order to address potential targets of this Cbfb-independent activity, we performed microarray analysis with mRNA from the peripheral blood of Cbfb+/+ and Cbfb+/MYH11 E12.5 embryos. One gene that showed significant upregulation in the blood from Cbfb+/MYH11 embryos was Csfrβ, the common β dimerization partner of the IL-3, Il-5, and GM-CSF α receptors. By FACS, we confirmed that Csfrβ (also known as CD131) was also upregulated at the protein level. In contrast, expression of Csfrβ in the blood of Cbfb−/− embryos was indistinguishable from wildtype indicating that signaling through Csfrβ and its dimerization partners could play a role in the Cbfb-independent activities of the Cbfb-MYH11 gene product. This effect was not unique to embryonic blood as adult mice that conditionally express Cbfb-MYH11 developed a population of pre-monocytic, Csfrβ+ cells in their peripheral blood as early as one week after induction of the fusion protein. In addition, leukemic cells induced in the Cbfb-MYH11 knockin mice expressed high levels of Csfrβ. These results indicate that expression of Csfrβ is an almost immediate consequence of Cbfb-MYH11 expression and that Csfrβ may be a direct target of the fusion protein. In conclusion, we demonstrated that Cbfb-MYH11 is able to impair hematopoiesis in a Cbfb-independent manner, perhaps through the direct upregulation of Csfrβ and consequent activation of its downstream signaling pathways.