EVI1 and GATA2 misexpression induced by inv(3)(q21q26) contribute to megakaryocyte-lineage skewing and leukemogenesis

Abstract

Chromosomal rearrangements between 3q21 and 3q26 elicit high-risk acute myeloid leukemia (AML), which is often associated with elevated platelet and megakaryocyte (Mk) numbers. The 3q rearrangements reposition a GATA2 enhancer near the EVI1 (or MECOM) locus, which results in both EVI1 overexpression and GATA2 haploinsufficiency. However, the mechanisms explaining how the misexpression of these 2 genes individually contribute to leukemogenesis are unknown. To clarify the characteristics of differentiation defects caused by EVI1 and GATA2 misexpression and to identify the cellular origin of leukemic cells, we generated a system to monitor both inv(3) allele-driven EVI1 and Gata2 expression in 3q-rearranged AML model mice. A cell population in which both EVI1 and Gata2 were highly induced appeared in the bone marrows before the onset of frank leukemia. This population had acquired serial colony-forming potential. Because hematopoietic stem/progenitor cells (HSPCs) and Mks were enriched in this peculiar population, we analyzed the independent EVI1 and GATA2 contributions to HSPC and Mk. We found that inv(3)-driven EVI1 promotes accumulation of Mk-biased and myeloid-biased progenitors, Mks, and platelets, and that Gata2 heterozygous deletion enhanced Mk-lineage skewing of EVI1-expressing progenitors. Notably, inv(3)-directed EVI1 expression and Gata2 haploinsufficient expression cooperatively provoke a leukemia characterized by abundant Mks and platelets. These hematological features of the mouse model phenocopy those observed in human 3q AML. On the basis of these results, we conclude that inv(3)-driven EVI1 expression in HSPCs and Mks collaborates with Gata2 haploinsufficiency to provoke Mk-lineage skewing and leukemogenesis with excessive platelets, thus mimicking an important feature of human AML.

Graphical abstract

Figure 1.

An EVI1 plus Gata2 High population has replicative potential. (A) Structures of the inv(3) allele, the 3q21q26-EVI1 BAC, and the 3q21q26-tdTomato BAC. In the 3q21q26-tdTomato BAC, a tdTomato reporter gene was introduced at the translation start site of the EVI1 gene in the parental 3q21q26-EVI1 BAC. (B) Dual-reporter mouse to monitor the effects of inv(3)-driven EVI1 and endogenous Gata2 expression by tdTomato and green fluorescent protein (GFP) fluorescence, respectively. (C) Representative flow cytometric profiles of bone marrow cells recovered from Gata2^+/gfp::Tom or 3q21q26-EVI1::Gata2^+/gfp::Tom mice. (D) Relative intensities of tdTomato (left) and GFP (right) fluorescence in High, Low, and Negative sorted populations (C). The average values for the High population in Gata2^+/gfp::Tom mice was set as 1. (E) Relative mRNA levels of transgenic human plus endogenous mouse EVI1 (left) and mouse endogenous Gata2 mRNA (right). mRNA abundance was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA. The average values for the High population in Gata2^+/gfp::Tom mice was set as 1. (F) Absolute number of High, Low, and Negative cells in Gata2 haploinsufficient mice either containing or lacking the 3q21q26-EVI1 BAC. (G) Percentages of “High” population cells in the bone marrows of Gata2^+/gfp::Tom and 3q21q26-EVI1::Gata2^+/gfp::Tom mice. (H) Colony-forming potential of High, Low, and Negative populations of Gata2^+/gfp::Tom and 3q21q26-EVI1::Gata2^+/gfp::Tom mice. Note that the EVI1-/Gata2-High population has high colony-forming potential, and that EVI1 overexpression alone confers serial replating potential on the High cell population. (I) Percentages of High population cells in primary and subsequent passage colonies derived from High populations of Gata2^+/gfp::Tom and 3q21q26-EVI1::Gata2^+/gfp::Tom mice. Values represent the means ± standard deviations (SDs). *P < .05; **P < .01  (unpaired Student t test). n.s., not significant.

Figure 2.

EVI1 plus Gata2 High fraction contains HSPC and Mks. (A-E) Representative flow cytometric profiles of High and Low/Negative populations in Gata2^+/gfp::Tom and 3q21q26-EVI1::Gata2^+/gfp::Tom mouse bone marrows. Red circles and boxes in each panel represent populations of Mk cells (A), monocytes and neutrophils (B), B cells (C), erythrocytes (D), and HSPC (E). (F) Percentages of High population cells falling into cell type-specific gated fractions from Gata2^+/gfp::Tom (green circle) and 3q21q26-EVI1::Gata2^+/gfp::Tom (blue circle) mouse bone marrows; monocytes (Mono), neutrophils (Neu), B cells (B), erythroid cells (Ery), and Mks. (G) Percentages of High populations in LK and LSK fractions from Gata2^+/gfp::Tom (green circle) and 3q21q26-EVI1::Gata2^+/gfp::Tom (blue circle) mouse bone marrows. (H) Cell populations contained in High population (yellow background ellipse). Note that HSPC and Mk-lineage cells are both enriched in the High population. Values represent the means ± SD. **P < .01 (unpaired Student t test).

Figure 3.

Inv(3)-driven EVI1 expression promotes expansion of multipotent progenitors, but not LT-HSC. (A) Mouse models used for evaluation of the individual or combinatorial contributions of EVI1 overexpression and Gata2 haploinsufficiency to leukemogenesis. Twelve-week-old mice were used for this study. (B) Representative flow cytometric profiles of lineage-negative fractions from 12-week-old WT, Gata2^+/gfp, 3q21q26-EVI1, and 3q21q26-EVI1::Gata2^+/gfp bone marrows. Further analysis of the LSK fraction (top) is shown in the middle panels, whereas analyses of the Flt3-low fraction in the middle panels are shown in the bottom panels. (C) Absolute numbers of LSK cells in the bone marrows from 12-week-old WT (blue circle), Gata2^+/gfp (red square), 3q21q26-EVI1 (green triangle), and 3q21q26-EVI1::Gata2^+/gfp (purple reverse triangle) mice. (D) Percentages of LT-HSC, ST-HSC, MPP2, MPP3, and MPP4 in LSK fractions from 12-week-old WT (blue circle), Gata2^+/gfp (red square), 3q21q26-EVI1 (green triangle), and 3q21q26-EVI1::Gata2^+/gfp (purple reverse triangle) mice. (E) Absolute numbers of LT-HSC, ST-HSC, MPP2, MPP3, and MPP4 in the bone marrows from 12-week-old WT (blue circle), Gata2^+/gfp (red square), 3q21q26-EVI1 (green triangle), and 3q21q26-EVI1::Gata2^+/gfp (purple reverse triangle) mice. Values represent the means ± SD. *P < .05; **P < .01 (1-way analysis of variance [ANOVA]).

Figure 4.

Inv(3)-driven EVI1 expression promotes Mk-lineage skewing. (A-B) Absolute numbers of MEP (A) and Mks (B) in the bone marrows of 12-week-old WT (blue circle), Gata2^+/gfp (red square), 3q21q26-EVI1 (green triangle), and 3q21q26-EVI1::Gata2^+/gfp (purple reverse triangle) mice. (C) Percentages of c-Kit⁺ cells in (CD41⁺CD61⁺) Mk of WT (blue circle), Gata2^+/gfp (red square), 3q21q26-EVI1 (green triangle), and 3q21q26-EVI1::Gata2^+/gfp (purple reverse triangle) mice. (D) Platelet (PLT) counts in the peripheral blood of WT (blue circle), Gata2^+/gfp (red square), 3q21q26-EVI1 (green triangle), and 3q21q26-EVI1::Gata2^+/gfp (purple reverse triangle) mice. Note that inv(3)-driven EVI1 expression promotes Mk and platelet numbers through increased MEP abundance. (E) Absolute numbers of erythroid cells in the bone marrows. (F) Red blood cell (RBC) counts in the peripheral blood of WT (blue circle), Gata2^+/gfp (red square), 3q21q26-EVI1 (green triangle), and 3q21q26-EVI1::Gata2^+/gfp (purple reverse triangle) mice. (G-J) Absolute numbers of neutrophils (G), monocytes (H), B cells (I), and pDCs (J) in the bone marrows of WT (blue circle), Gata2^+/gfp (red square), 3q21q26-EVI1 (green triangle), and 3q21q26-EVI1::Gata2^+/gfp (purple reverse triangle) mice. (K) Absolute numbers of thymocytes from WT (blue circle), Gata2^+/gfp (red square), 3q21q26-EVI1 (green triangle), and 3q21q26-EVI1::Gata2^+/gfp (purple reverse triangle) mice. (L) A model for lineage skewing by misexpression of EVI1 (red arrows) and Gata2 (green circle). Values represent the means ± SD. *P < .05; **P < .01 (1-way ANOVA).

Figure 5.

Gata2 heterozygous loss increases the incidence of EVI1-driven leukemia with high platelets and Mks. (A) PLT counts in the peripheral blood of leukemic 3q21q26-EVI1 (red circle) and 3q21q26-EVI1::Gata2^+/gfp (blue square) mice. An orange dotted line represents PLT levels of age-matched WT mice. (B) Percentages of Mks in the bone marrows of leukemic 3q21q26-EVI1 (red circle) and 3q21q26-EVI1::Gata2^+/gfp (blue square) mice. (C) Kaplan-Meier survival curves of 3q21q26-EVI1 (High-Mk type leukemia [orange dotted line], Low-Mk type leukemia [red line]), and 3q21q26-EVI1::Gata2^+/gfp mice (High-Mk type leukemia [turquoise line] or Low-Mk type leukemia [blue dashed line]). *P < .05 (log-rank test).

Figure 6.

Leukemia in the 3q21q26-EVI1::Gata2^+/gfpmice with high platelets has characteristics similar to those of human patients with AML harboring 3q rearrangements. (A) Representative peripheral blood smears (Wright-Giemsa staining) from leukemic 3q21q26-EVI1::Gata2^+/gfp mice (High-Mk type leukemia). Note that Blast cells (white arrowheads), myeloid cells (yellow arrowhead), micromegakaryocytes (black arrowheads), and giant platelets (arrow) are observed. (B) Representative flow cytometric profiles of bone marrow cells from leukemic 3q21q26-EVI1::Gata2^+/gfp mice (High-Mk type leukemia). CD41^– and CD41⁺CD61⁺ fractions were further subdivided to assess granulocytic and blast cell characteristics. (C) Representative c-Kit expression profile in each fraction from leukemic 3q21q26-EVI1::Gata2^+/gfp mice (High-Mk type leukemia). (D) Wright-Giemsa staining of leukemic cells from 3q21q26-EVI1::Gata2^+/gfp bone marrows. (E) Relative mRNA levels of EVI1 and mouse endogenous Gata2 mRNA in each population. B220⁺Gr1^–CD41^– (red), B220^–Gr1⁺CD41^– (orange), B220^–Gr1^–CD41^– (green), and B220^–Gr1^–CD41⁺CD61⁺ (blue) cells are shown. The abundance of the mRNA was normalized to glyceraldehyde-3-phosphate dehydrogenase. Average values of B220⁺Gr1^–CD41^– population were set to 1. Values represent the means ± SD. *P < .05; **P < .01 (1-way ANOVA). (F) Schema for the transplantation analysis; 5 × 10⁴ CD41⁺B220^–Gr1^–, CD41^–B220⁺Gr1^–, and CD41^–B220^–Gr1⁺ cells from leukemic 3q21q26-EVI1::Gata2^+/gfp (High-Mk type leukemia, CD45.1/CD45.2 heterozygotes or CD45.1 homozygous) mice were independently transplanted into sublethally irradiated CD45.2 homozygous WT mice. (G) Kaplan-Meier survival curves of WT mice receiving CD41⁺B220^–Gr1^–, CD41^–B220⁺Gr1^–, and CD41^–B220^–Gr1⁺ cells from leukemic 3q21q26-EVI1::Gata2^+/gfp mice (High-Mk type leukemia). (H) Representative flow cytometric patterns of the bone marrows of recipient mice receiving CD41^–B220⁺Gr1^– cells.

Figure 7.

A model for lineage skewing and leukemogenesis induced by inv(3)-driven EVI1 expression and GATA2 haploinsufficiency. Inv(3)-driven EVI1 expression promotes expansion of MPP2 and MPP3, and also induces subsequent megakaryopoiesis (all cells encompassed by the red ellipse circle). Gata2 haploinsufficiency has a mild enhancement effect on MPP2 and ST-HSC (green circle). Based on the results that the 3q21q26-EVI1::Gata2^+/gfp-induced leukemias nicely recapitulate major hematological features of patients with 3q AML with elevated Mk and platelet numbers, it is implied that the High Mk/platelet leukemia arises from a cell that could be ST-HSC, MPP2, or a progenitor somewhere between these 2 (purple arrow).