Cbfb-MYH11 Induces Expansion of a Lin-Kit+Sca1- Abnormal Progenitor Compartment that Predisposes Acute Myeloid Leukemia in Mice.

Abstract

Acute myeloid leukemia (AML) samples with chromosome 16 inversion express the CBFb-MYH11 fusion gene. One of three RUNX genes (RUNX1, RUNX2, and RUNX3) encode the α-subunit and CBFb encodes the β subunit of the heterodimeric transcription factor CBF. This transcription factor is a key regulator of multiple steps on hematopoietic differentiation. Studies in the mouse have determined that Cbfb-MYH11 expression impairs hematopoiesis, and that it induces AML in collaboration with other mutations. To further study the effects of Cbfb-MYH11 expression in hematopoiesis and leukemogenesis, we created a Cbfb-MYH11 conditional knock-in model (Cbfb56M), using the Cre-loxP recombination system. In this model, the floxed Cbfb allele expresses wildtype Cbfb, and the downstream Cbfb-MYH11 is induced upon Cre-mediated deletion. Floxed heterozygous and homozygous Cbfbfb56M mice are disease-free, indicating that Cbfb expressed from the floxed allele is functional. In addition, Cbfb-MYH11 was efficiently induced in over 80% of bone marrow cells from Cbfb56M/+/Mx1-Cre mice after pIpC injection. The preleukemic effects of Cbfb-MYH11 in hematopoiesis were analyzed using induced mice and non-competitive repopulation assays. First, circulating B-cells were reduced soon after Cbfb-MYH11 induction, and a significant differentiation block at the pre-pro B-cell stage was detected in the bone marrow. Second, thymic T-cell differentiation of induced mice showed impairment of DN2 to DN3 stage and reduction of thymic size in 3/10 induced mice analyzed. Interestingly, the number of circulating T cells was unaffected in repopulation assays. Third, platelets were reduced 50% in peripheral blood and megakaryocyte number was reduced in bone marrow. Fourth, we found an expanded abnormal progenitor compartment (Lin-kit+Sca1-) that accumulated in the bone marrow and spleen. In vitro differentiation assays showed a 2-to-3 fold increase of Cbfb-MYH11 colonies when compared to controls. The colony size was smaller, and showing partial differentiation deficiency. Interestingly, the colony numbers declined upon serial plating below controls. Taken together these results indicate that Cbfb-MYH11 induce accumulation of late progenitors (primarily myeloid progenitors) with limited self-renewal potential. Acute myeloid leukemia arised spontaneously 4 to 6 months after Cbfb-MYH11 induction, with expansion of blast- and monoblastic-like leukemic cells defined as Lin-kit+Sca1-. Leukemic mice showed infiltration in several tissues, including spleen, liver, brain, and lungs. To test whether a second “hit” is necessary in this model, we used bone marrow transduction assays to co-express Cbfb-MYH11 and the candidate cooperating gene Runx2. Recipient mice developed AML with similar phenotype 6 to 12 weeks post transplantation, while control mice remained healthy for 6 months. This study demonstrates that Cbfb-MYH11 expression (i) defines a preleukemic stage with hematopoietic differentiation block at stages associated with Runx function, (ii) the accumulation of an abnormal progenitor cell population (Lin-/kit+/Sca1-), (iii) induces additional mutations to efficiently develop AML, and (iv) synergizes with Runx2 in AML development.