Aberrant Megakaryocytic/Erythroid Progenitors Contributes To Transformation Of Cbfb-SMMHC Induced Acute Myeloid Leukemia

Inv(16)(p13q22) is a recurrent chromosomal rearrangement found in approximately 12% of human acute myeloid leukemia (AML) cases and creates a fusion gene between CBFb and MYH11. The fusion gene encodes a fusion protein CBFß-SMMHC which causes defects in lymphoid and myeloid differentiation. Previous studies also showed that primitive erythropoiesis is impaired by CBFß-SMMHC, however, CBFß-SMMHC knocked-in cells was able to contribute to adult erythropoiesis in chimeric mice. Expressing CBFß-SMMHC in the hematopoietic cells using a conditional knock-in mouse model (Cbfb^56M/+/Mx1-Cre; 129SvEv strain) recapitulates inv(16)-associated AML. Previous studies in this model showed that CBFß-SMMHC expression leads to pre-leukemic hematopoietic alterations, and together with additional cooperative mutations, result in spontaneous myeloid leukemia in mice with a 3-6 month latency. We hypothesized that an expanded cell population at the pre-leukemic stage could be the target of additional mutations, and hence the cell of origin of leukemia initiating cells.

To further delineate the pre-leukemic progenitors and leukemia initiating cells, we backcrossed Cbfb^56M/+/Mx1-Cre into C57BL/6 for more than 10 generations. Similar to previous studies in the129SvEv strain, expressing CBFß-SMMHC in adult C57BL/6 mice leads to cell number dependent development of AML. Analysis of pre-leukemic bone marrow as early as 2 weeks after induction revealed a 5.7-fold expansion of Pre-Meg/E cells (Pre-Megakaryocyte/Erythrocyte: Lin^-cKit⁺Sca1^-CD16^-/loCD150⁺CD105^-) compared to similarly treated control mice. While there was a significant increase in Pre-Meg/E population, we did not find significant increase in their proliferation but observed a 4.7 fold decrease of the erythroid progenitor (EP; Lin^-cKit⁺Sca1^-CD16^-/loCD105^hi) subset. Methylcellulose-based colony forming assay showed that pre-leukemic Pre-Meg/E had an impaired differentiation potential for erythroid lineage. In vitro erythroid differentiation assay also showed a partial block of differentiation from pre-leukemic Pre-Meg/E progenitors. These Pre-Meg/E like progenitors were able to induce leukemia in the presence of a known cooperative oncoprotein MPL when transplanted in lethally irradiated congenic recipient mice. In summary, our Results suggest that expression of CBFß-SMMHC impairs adult erythropoiesis at the transition of Pre-Meg/E to EPs, causing an expansion of Pre-Meg/E cells, which can be the target cell of additional mutations contributing to leukemia transformation.