Cell fate decisions in malignant hematopoiesis: leukemia phenotype is determined by distinct functional domains of the MN1 oncogene

Abstract

Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia (T-ALL), share similar pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of leukemias. We dissected the functional aspects of different protein regions of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal region of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal region resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the N-terminal region. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active gene regions. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.

Figure 1. The N-terminal region of MN1 is required for its leukemogenic potential.

(A) MN1 mutation constructs for structure-function analysis. In strategy 1 distinct stretches of approximately 200 amino acids were deleted throughout wildtype MN1. In strategy 2, stretches of approximately 200 amino acids were cumulatively deleted starting from the MN1 N-terminus. In strategy 3, stretches of approximately 200 amino acids were cumulatively deleted starting from the MN1 C-terminus. (B–D) Percentage of transgene-positive white blood cells engrafting in peripheral blood of transplanted mice at 4-week intervals. P values are given for the comparison of the indicated construct with CTL-transduced cells. The average engraftment is shown. Number of analysed mice and standard error can be found in Table S1. (E-G) Survival of mice receiving transplants of cells transduced with (E) strategy 1, (F) strategy 2, and (G) strategy 3 MN1 deletions. P values are given for the comparison of the indicated construct with CTL-transduced cells. The number of analysed mice is detailed in Table S1. (H) Morphology of bone marrow cells at death of diseased mice. The cells were Wright-Giemsa stained. Images were visualised using a Nikon Eclipse 80i microscope (Nikon, Mississauga, ON, Canada) and a 20x/0.40 numerical aperture objective, or a 100x/1.25 numerical aperture objective and Nikon Immersion Oil (Nikon). A Nikon Coolpix 995 camera (Nikon) was used to capture images. § engraftment in peripheral blood at the indicated time point or at death in cases where a mouse died before that time point. † all mice were dead at this time point due to disease. * indicates P<0.05, ** indicates P<0.001.

Figure 2. The N-terminal region of MN1 is required to block megakaryocyte/erythroid differentiation.

(A–C) Percentage of transgene positive red blood cells engrafting in peripheral blood of transplanted mice at 4-week intervals. P values are calculated for the comparison of the indicated construct with CTL-transduced cells. The number of analysed mice and standard error is detailed in Table S3. (D–F) Proportion of red blood cells (RBC) compared to white blood cells (WBC) expressing (D) strategy 1, (E) strategy 2, or (F) strategy 3 MN1 deletion constructs after transplantation. P values are calculated for the comparison of the indicated construct with CTL-transduced cells. The number of analysed mice and standard error is detailed in Table S3. (G) Megakaryocyte colony-forming ability of mouse bone marrow cells transduced with MN1 deletion constructs (mean ± SD, n = 4). (H) Micrographs of representative CFC-Mk slides at the end of the first plating of bone marrow cells transduced with CTL vector, full-length MN1 or MN1Δ1. Images were visualised using a Nikon Eclipse 80i microscope (Nikon, Mississauga, ON, Canada) and a 20x/0.40 numerical aperture objective, or a 100x/1.25 numerical aperture objective and Nikon Immersion Oil (Nikon). A Nikon Coolpix 995 camera (Nikon) was used to capture images. * indicates P<0.05.

Figure 3. The C-terminal region of MN1 is required to block myeloid differentiation.

(A–F) In vitro sensitivity to ATRA of ND13-immortalized cells that were transduced with MN1 deletion constructs. Dose-response curves are shown in the left panels (A, C, E) and IC50 values are shown in the right panels (B, D, F) for each deletion strategy (mean ± SD, n≥6).

Figure 4. Hierarchical clustering of cells with N- and C-terminally deleted MN1.

(A) Heat map of differentially regulated pathways for enhanced proliferation and blocked differentiation. (B) Comparison of top 60 enriched gene ontology gene sets for the comparison of MN1Δ1 and MN1Δ7 with wildtype MN1.

Figure 5. A 606 amino-acid C-terminal portion of MN1 prevents T-lymphoid differentiation.

(A) Morphology of bone marrow cells from MN1Δ5-7 mice at death, showing a shift in leukemia from AML, as seen in MN1 leukemia, to an ALL leukemia upon loss of the C-terminal domains 5–7. (B) Representative immunophenotype of GFP⁺ MN1Δ5-7 bone marrow cells compared to wildtype MN1 bone marrow cells at death. (C) Representative immunophenotype of secondary transplants of GFP⁺ MN1Δ5-7 bone marrow cells at death. (D) Average cell surface marker expression for secondary transplants of GFP⁺ MN1Δ5-7 bone marrow cells at death (mean ± SEM, n = 3).

Figure 6. Functionally defined regions of MN1.