CBFβ-SMMHC Impairs Erythroid Differentiation and Induces Expansion of Aberrant Megakaryocytic/Erythroid Progenitors Capable of Leukemia Initiation

Abstract

Approximately 12% of human acute myeloid leukemia (AMLs) harbor a recurrent chromosomal rearrangement inv(16)(p13q22). Inv(16) creates a fusion gene Cbfb-MYH11, encoding the fusion protein CBFß-SMMHC. Expressing CBFß-SMMHC in hematopoietic cells using a constitutive knock-in mouse model (Cbfb+/Cbfb-MYH11) or a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre; 129SvEv strain) causes defects in lymphoid and myeloid differentiation, and predisposes mice to AML. Previous studies with the constitutive knock-in mouse model showed impaired primitive erythropoiesis, however, Cbfb-MYH11 knocked-in cells were able to contribute to erythropoiesis in chimeric mice.To further delineate the effect of CBFß-SMMHC in adult erythropoiesis in the conditional knock-in mouse, we backcrossed Cbfb56M/+/Mx1-Cre into C57BL/6 and a Rosa26mT/mG Cre reporter strain. Induced expression of CBFß-SMMHC in adult mice leads to cell number dependent development of AML, consistent with previous studies in 129SvEv strain. Analysis of pre-leukemic bone marrow 2 weeks after induction revealed a 5.7-fold expansion of immunophenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E; Lin-cKit+Sca1-CD16-/loCD150+CD105-), and a 4.7 fold decrease of the erythroid progenitor (EP; Lin-cKit+Sca1-CD16-/loCD105hi) subset compared to similarly treated control mice. Both methylcellulose-based erythroid colony forming assay and in vitro erythroid differentiation culture showed that pre-leukemic Pre-Meg/Es expressing CBFß-SMMHC had an impaired differentiation potential for erythroid lineage. Using the Rosa26mT/mG Cre reporter allele, we tracked the proportions of CBFß-SMMHC- expressing cells (GFP+) in the Pre-Meg/E and EP subsets. We observed that the contribution of GFP+ cells sharply decreased in EPs but not in Pre-Meg/Es from primary pre-leukemic mice. Similar results were seen in transplant recipients engrafted with sorted GFP+ pre-leukemic Lin-cKit+Sca1+ cells. These results further confirmed that CBFß-SMMHC impairs cell-autonomous erythroid differentiation in vivo. Consistent with the impaired differentiation of Pre-Meg/Es, we observed altered expression pattern of erythroid regulatory genes, including Fog1, Gata2, and Gfi1b.The pre-leukemic Pre-Meg/Es exhibited increased colony forming and replating capacity in vitro and enhanced proliferation and survival in vivo. To determine whether these phenotypic Pre-Meg/E cells could be the cellular origin for leukemic transformation, we expressed a known cooperative onco-protein Mpl by retroviral transduction followed by transplantation. The majority of mice (83%) receiving 100,000 Pre-Meg/E cells developed leukemia with a medium onset of 92 days, suggesting that Pre-Meg/Es indeed are capable of leukemia initiation.In conclusion, the expression of CBFß-SMMHC impairs adult erythropoiesis at the transition of Pre-Meg/E to EPs, causing an expansion of Pre-Meg/E cells. These pre-leukemic Pre-Meg/Es could be the target cell of additional mutations contributing to leukemia transformation. DisclosuresNo relevant conflicts of interest to declare.