Identification of leukemia-initiating cells by transcriptome profiling in a mouse model

Transcriptome profiling and identification of leukemia-initiating cells in a mouse model expressing the fusion gene CBFB-MYH11, which results from chromosome 16 inversion, a common chromosome abnormality in human acute myeloid leukemia.
Published in Cancer

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Acute myeloid leukemia (AML) is a devastating blood cancer with a high morbidity and mortality; around 20,000 new cases are reported annually and just over 30% of patients survive after five years*. Different subtypes of AML stratify patients into varying prognostic categories. While patients with an inversion of chromosome 16 (inv(16); the FAB M4 eos subtype) are considered to have a favorable prognosis, more than half of the patients who achieve clinical remission will relapse and not survive. The inversion of chromosome 16 leads to an in-frame fusion of the Core-Binding Factor Subunit Beta (CBFβ) gene (CBFB) with Smooth Muscle Myosin Heavy Chain (SMMHC) gene, MYH11. The resultant fusion protein (CBFb-SMMHC) is a driver of inv(16) AML but is not sufficient to induce leukemia by itself. Other genetic mutations or alterations occur that ultimately give rise to overt disease. Due to the clonal nature of leukemia, techniques that track changes in individual cells have become critical for tracking disease evolution and remission status, as well as defining relapsed disease.

In this paper, we used flow cytometry to isolate the hematopoietic stem and progenitor cells  from bone marrow in the CBFB-MYH11 conditional knock-in mice during pre-leukemic and leukemic phases and performed single-cell RNA-sequencing (scRNA-Seq). The data was then compared with the data from HSPCs isolated from wild-type mice. Overall, we identified cell populations that precede the previously-defined Abnormal Myeloid Progenitor (AMP) population in this mouse model, which may be the cells that truly initiate and drive disease progression.


We obtained scRNA-Seq data for >14,000 cells, which included >4,000 Kit+ bone marrow cells per condition (pre-leukemic and leukemic from the CBFB-MYH11 mice, and the wildtype control). We determined that the cells from mice with overt leukemia mostly resemble the AMP cells, and that the pre-leukemic cells housed previously undescribed populations of cells. Analysis of  single cell data in pseudotime 1 highlighted a population of cells that are largely pre-leukemic that lie directly upstream of overt leukemia, marking the original leukemia-initiating population of cells.

 The inclusion of the pre-leukemic state, made possible by the inducible expression of the CBFB-MYH11 fusion protein, allowed us to see the initial gene expression changes in HPSCs driven by the expression of the fusion protein and the quantification of the first cell type shifts that arise during leukemogenesis in this model.

 In previous reports of this mouse model, the AMPs were described as having an immunophenotype of myeloid-erythroid progenitors (MEPs) 2,3. With flow cytometry, the AMPs fall in the same gate as MEPs, forming an expanded population that grows as the leukemia progresses. In this paper, we showed how wild-type MEPs represent a separate population from any pre-leukemic or leukemic cell population. We used flow cytometry to validate Cd59a as one of the earliest markers of leukemia progression. Although Cd59a is expressed in a subset of wild-type cells, its expression in pre-leukemic and leukemic cells is far higher than in any wild-type progenitors. Overall, this study identified disease-specific markers and verified their lack of expression in wild-type MEPs for the first time, which we hope will provide insight into earlier detection and targeted therapies of inv(16) leukemia.

 *NCI SEER Program;

 1          Chen, H. et al. Single-cell trajectories reconstruction, exploration and mapping of omics data with STREAM. Nat Commun 10, 1903 (2019).

2          Castilla, L. H. et al. The fusion gene Cbfb-MYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia. Nat Genet 23, 144-146 (1999).

3          Zhen, T. et al. RUNX1 and CBFbeta-SMMHC transactivate target genes together in abnormal myeloid progenitors for leukemia development. Blood 136, 2373-2385 (2020).

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