Chromosome 16 inversion, inv(16)(p13q22), is one of the most frequent chromosome abnormalities in human acute myeloid leukemia (AML), comprising almost 100% of subtype M4Eo and up to 15% of all AML. This inv(16) leads to an in-frame fusion of CBFB and MYH11 genes. CBFB-MYH11 encodes a fusion protein between CBF&#946;, which is an obligate partner of RUNX1 or AML1, and smooth muscle myosin heavy chain (SMMHC). Using knock-in mouse models we have previously demonstrated that Cbfb-MYH11 dominantly blocks Runx1/Cbfb function in hematopoiesis and predisposes mice to AML (requiring a second hit from ENU or retroviral mutagenesis). However, the molecular mechanism underlying these findings remains unclear. Current hypotheses, which are based on previous in vitro studies, focus on the ability of CBF&#946;-SMMHC to dominantly inhibit RUNX1/CBF&#946; and include (1) CBF&#946;-SMMHC binding RUNX1 with higher affinity than CBF&#946;; (2) CBF&#946;-SMMHC sequestration of RUNX1 in the cytoplasm; (3) CBF&#946;-SMMHC stabilization of RUNX1 by decreasing RUNX1 ubiquitination; and (4) CBF&#946;-SMMHC repression of RUNX1 transactivation, which is dependent on SMMHC multimerization and repressor recruitment. To test these hypotheses in vivo, we generated knock in chimeric and F1 heterozygous mice expressing CBF&#946;-SMMHC with C-terminal and internal deletions. One of the deletions removes only the domain responsible for high affinity binding of RUNX1 (HABD). The CBF&#946;-SMMHC protein with HABD deletion did not bind RUNX1 with higher affinity than CBF&#946; and was hypothesized to be unable to dominantly repress RUNX1. Consistent with this hypothesis, the HABD-deleted protein was less efficient in sequestering RUNX1 and caused less severe hematopoietic defects in F1 embryos than full length CBF&#946;-SMMHC. In contrast to mice expressing full length CBF&#946;-SMMHC, which develop AML only after ENU or retroviral mutagenesis, most HABD-deleted chimeric and all HABD-deleted F1 mice developed AML spontaneously shortly after birth. A larger deletion removed both the HABD and the RUNX1 stabilization domain (RSD). CBF&#946;-SMMHC with the HABD-RSD double deletion did not bind RUNX1 with high affinity and could not sequester RUNX1 in the cytoplasm. Mice expressing HABD-RSD double deleted CBF&#946;-SMMHC had normal hematopoiesis, did not develop leukemia spontaneously and developed T cell (not myeloid) malignancies after ENU treatment. These data suggest that HABD and RSD are important for CBF&#946;-SMMHC to block RUNX1 function and to dominantly impair hematopoiesis. The accelerated leukemogenesis associated with HABD deletion and induction of T cell malignancies in mice expressing HABD-RSD double deleted CBF&#946;-SMMHC strongly support the hypothesis that CBF&#946;-SMMHC can induce leukemia through RUNX1-inhibition-independent pathways.[unreadable] [unreadable] Using the Cbfb-MYH11 knockin mouse model we have further characterized hematopoietic defects induced by Cbfb-MYH11 and discovered novel mechanisms for such defects. In the peripheral blood of Cbfb+/MYH11 embryos before embryonic day 12.5 (E12.5), we have observed the 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 of 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, in the blood of Cbfb+/MYH11 embryos, we found 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 the blood of 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 in the peripheral blood of 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 Csfr&#946;, the common &#946; dimerization partner of the IL-3, Il-5, and GM-CSF&#945; receptors. By FACS, we confirmed that Csfr&#946; (also known as CD131) is also upregulated at the protein level. In contrast, expression of Csfr&#946; in the blood of Cbfb-/- embryos is indistinguishable from wildtype indicating that signaling through Csfr&#946; and its dimerization partners could play a role in the Cbfb-independent activities of the Cbfb-MYH11 gene product. This effect is not unique to embryonic blood as adult mice that conditionally express Cbfb-MYH11 develop a population of pre-monocytic, Csfr&#946;+ 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 express high levels of Csfr&#946;. These results indicate that expression of Csfr&#946; is an almost immediate consequence of Cbfb-MYH11 expression and that Csfr&#946; may be a direct target of the fusion protein. In conclusion we demonstrated that Cbfb-MYH11 is able to impair hematopoiesis through Cbfb-independent pathways and can affect cell proliferation and survival in addition to blockage of differentiation. We also showed evidence that Cbfb-MYH11 may directly affect signaling pathways through upregulation of Csfr&#946;.