Cbfb/Runx1 repression-independent blockage of differentiation and accumulation of Csf2rb-expressing cells by Cbfb-MYH11

Abstract

It is known that CBFB-MYH11, the fusion gene generated by inversion of chromosome 16 in human acute myeloid leukemia, is causative for oncogenic transformation. However, the mechanism by which CBFB-MYH11 initiates leukemogenesis is not clear. Previously published reports showed that CBFB-MYH11 dominantly inhibits RUNX1 and CBFB, and such inhibition has been suggested as the mechanism for leukemogenesis. Here we show that Cbfb-MYH11 caused Cbfb/Runx1 repression-independent defects in both primitive and definitive hematopoiesis. During primitive hematopoiesis, Cbfb-MYH11 delayed differentiation characterized by sustained expression of Gata2, Il1rl1, and Csf2rb, a phenotype not found in Cbfb and Runx1 knockout mice. Expression of Cbfb-MYH11 in the bone marrow induced the accumulation of abnormal progenitor-like cells expressing Csf2rb in preleukemic mice. The expression of all 3 genes was detected in most human and murine CBFB-MYH11(+) leukemia samples. Interestingly, Cbfb-MYH11(+) preleukemic progenitors and leukemia-initiating cells did not express Csf2rb, although the majority of leukemia cells in our Cbfb-MYH11 knockin mice were Csf2rb(+). Therefore Csf2rb can be used as a negative selection marker to enrich preleukemic progenitor cells and leukemia-initiating cells from Cbfb-MYH11 mice. These results suggest that Cbfb/Runx1 repression-independent activities contribute to leukemogenesis by Cbfb-MYH11.

Figure 1

Cbfb-MYH11 causes Cbfb/Runx1 repression–independent defects during primitive hematopoiesis. (A) Representative FACS plots of Ter119 and c-Kit staining of the primitive blood from embryos of the indicated ages and genotypes. Percentage of cells in each gate is given. Line graphs of the percentage (%) of (B) BrdU-positive (+) and (C) annexin V⁺, 7AAD-low cells in the peripheral blood of littermate embryos of the indicated genotypes and ages. Bar graphs of the percentage of (D) BrdU⁺ and (E) annexin V⁺, 7AAD-low cells in the peripheral blood of Cbfb^+/+ and Cbfb^−/− littermate embryos of the indicated ages. Data from age-matched Cbfb^+/MYH1 embryos was included for comparison. *Statistically significant difference (P < .05) compared with Cbfb^+/+ embryos; **statistically significant difference (P < .05) compared with Cbfb^+/+ and Cbfb^−/− embryos. N ≥ 3 for all genotypes and ages. Error bars indicate SD within each genotype.

Figure 2

Csf2rb expression is related to Cbfb-MYH11–induced differentiation defects. (A) Representative FACS plots of Csf2rb and Ter119 staining in primitive blood cells from E10.5 embryos of the indicated genotypes. Percentage of cells in each gate is given. N ≥ 3 for all genotypes. (B) Representative FACS plots of Csf2rb and c-Kit staining in the primitive blood of E8.5 embryos of the indicated genotypes. N ≥ 3 for all genotypes.

Figure 3

Csf2rb, Il1rl1, and Gata2 are expressed in both mouse and human leukemic cells. (A) Representative FACS plots of Csf2rb and Il1rl1 staining in lineage-negative, c-Kit⁺ ScaI⁺ (LKS⁺) and c-Kit⁺, ScaI⁻ (LKS⁻) bone marrow and spleen from nonleukemic Cbfb^+/+ mice, and leukemic cells from the spleen of mice expressing a conditional allele of Cbfb-MYH11 (Cbfb^+/56M, Mx1-Cre). N ≥ 3 for Cbfb^+/56M, Mx1-Cre. Expression of Csf2rb (B) and Il1rl1 (C) in the peripheral blood of leukemic Cbfb^+/56, Mx1 Cre⁺ and control littermate mice. The control group contained Cbfb^+/+, Mx1-Cre⁻; Cbfb^+/56M, Mx1-Cre⁻; and Cbfb^+/+, Mx1-Cre⁺ mice. All mice were treated with pI:pC in the same way. (D) Western blot analysis of Gata2 expression in the peripheral blood of a nonleukemic Cbfb^+/+ adult mouse, 2 different leukemic adult mice expressing Cbfb-MYH11, and the human inv(16) AML-derived ME-1 cells. The bottom panel shows α-tubulin expression using the same blot. (E) Reverse transcription–PCR analysis using primers specific for human CSF2RB in ME-1 cells. An arrow indicates the band of the expected size. (F) FACS plot of ME-1 cells stained for C-KIT and IL1RL1. Percentage of cells in each gate is given.

Figure 4

Cbfb-MYH11 expression results in an abnormal Csf2rb⁺ population with reduced progenitor activity. Representative FACS plots of c-Kit and ScaI (A), and Csf2rb (B) staining in lineage-depleted (lin⁻) bone marrow from mice of Cbfb^+/56/M, Mx1-Cre⁺ and Cbfb^+/+ mice at the indicated number of days after treatment with pI:pC. Percentage of cells in each gate is given. N ≥ 3 for all genotypes. (C) Representative FACS plots of c-Kit and ScaI staining of the Csf2rb⁻ and Csf2rb⁺ populations from lin⁻ bone marrow from Cbfb^+/56M, Mx1-Cre⁺ mice 10 days after Cbfb-MYH11 induction. (D) Bar graphs of the relative total numbers of colonies seen from lin⁻ bone marrow cells from Cbfb^+/56M, Mx1-Cre⁺ mice 10 days after induction of Cbfb-MYH11 expression, sorted for Csf2rb expression and grown in culture for 12 days. N ≥ 3. *Statistically significant difference (P < .01). (E) FACS staining for Csf2rb of colonies derived from the Csf2rb⁻ cells described in panel C. Percentage of Csf2rb⁺ cells is indicated. Error bars indicate SD within the indicated cell population.

Figure 5

A transient population of Csf2rb⁺ cells is seen in the peripheral blood after Cbfb-MYH11 induction. (A) Line graphs showing percentage (%) of Csf2rb⁺ cells in the peripheral blood at the indicated number of weeks after pI:pC treatment in 3 representative mice. The line marked with circles represents a wild-type mouse after pI:pC treatment. The line marked with triangles represents a conditional Cbfb-MYH11 knockin mouse after pI:pC treatment that did not develop leukemia within the observed time period (7 weeks). The line marked with diamonds represents a conditional Cbfb-MYH11 knockin mouse after pI:pC treatment that spontaneously developed AML (without ENU). (B) Percentage of Csf2rb⁺ cells in a larger cohort of mice at the indicated number of weeks after pI:pC treatments. *Statistically significant difference between Cbfb^+/56M, Mx1-Cre and control mice (P < .02). Wright-Giemsa staining of sorted Csf2rb⁺ peripheral blood cells from (C) a preleukemic Cbfb^+/56M, Mx1-Cre mouse 2 weeks after Cbfb-MYH11 induction and (D) a leukemic Cbfb^+/56M, Mx1-Cre mouse. Magnification, 1000×.

Figure 6

Csf2rb⁻/lin⁻ BM cells in mice expressing Cbfb-MYH11 are enriched for preleukemic progenitors and leukemia-initiating cells. (A) FACS staining of lineage-depleted bone marrow cells from ENU-treated, preleukemic Cbfb^+/56M, Mx1-Cre mice 14 days after induction of Cbfb-MYH11. Cells were sorted for Csf2rb expression as indicated by the boxes, and transplanted into sublethally irradiated mice via retro-orbital injection. (B) Kaplan-Meier survival curves of mice that received a transplant of preleukemic Csf2rb⁻ or Csf2rb⁺ cells. (C) FACS staining for the indicated differentiation markers in the Csf2rb⁻ and Csf2rb⁺ leukemic spleen cells from a representative mouse that developed AML after transplantation of preleukemic Csf2rb⁻ cells. (D) Western blot of CBFβ-SMMHC expression in the Csf2rb⁻ and Csf2rb⁺ leukemic cells of 2 different recipient animals. (E) Kaplan-Meier survival curves of secondary transplant recipient mice that underwent transplantation as described in panel A with leukemic Csf2rb⁻ and Csf2rb⁺ cells.

Figure 7

Model of Cbfb-MYH11–induced aberrant differentiation and leukemia initiation. We propose that expression of Cbfb-MYH11 in HSCs or early progenitors causes an abnormal and incomplete differentiation that culminates in a Csf2rb⁺ cell population. Our results indicate that by the time the Cbfb-MYH11–expressing cells have reached the Csf2rb⁺ stage, they are no longer capable of initiating leukemia.