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. 2004 Apr 6;101(14):4924-9.
doi: 10.1073/pnas.0400930101. Epub 2004 Mar 24.

Identification of genes that synergize with Cbfb-MYH11 in the pathogenesis of acute myeloid leukemia

L H Castilla  1 , P Perrat, N J Martinez, S F Landrette, R Keys, S Oikemus, J Flanegan, S Heilman, L Garrett, A Dutra, S Anderson, G A Pihan, L Wolff, P P Liu
Affiliations

Identification of genes that synergize with Cbfb-MYH11 in the pathogenesis of acute myeloid leukemia

L H Castilla et al. Proc Natl Acad Sci U S A. .

Abstract

Acute myeloid leukemia subtype M4 with eosinophilia is associated with a chromosome 16 inversion that creates a fusion gene CBFB-MYH11. We have previously shown that CBFB-MYH11 is necessary but not sufficient for leukemogenesis. Here, we report the identification of genes that specifically cooperate with CBFB-MYH11 in leukemogenesis. Neonatal injection of Cbfb-MYH11 knock-in chimeric mice with retrovirus 4070A led to the development of acute myeloid leukemia in 2-5 months. Each leukemia sample contained one or a few viral insertions, suggesting that alteration of one gene could be sufficient to synergize with Cbfb-MYH11. The chromosomal position of 67 independent retroviral insertion sites (RISs) was determined, and 90% of the RISs mapped within 10 kb of a flanking gene. In total, 54 candidate genes were identified; six of them were common insertion sites (CISs). CIS genes included members of a zinc finger transcription factors family, Plag1 and Plagl2, with eight and two independent insertions, respectively. CIS genes also included Runx2, Myb, H2T24, and D6Mm5e. Comparison of the remaining 48 genes with single insertion sites with known leukemia-associated RISs indicated that 18 coincide with known RISs. To our knowledge, this retroviral genetic screen is the first to identify genes that cooperate with a fusion gene important for human myeloid leukemia.

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Figures

Fig. 4.
Fig. 4.
Ideogram representation of chromosomal location of genes flanking RISs. CISs are red and SISs are black. Uncharacterized genes are identified by their Cri number.
Fig. 1.
Fig. 1.
Induction of AML in Cbfb-MYH11 knock-in chimeras by RIM. Kaplan-Mayer survival plot of 40 wild-type mice (dashed line) and 43 Cbfb+/MYH11 chimeras (solid line) after neonatal injection with MuLV-4070A retrovirus. Test and control mice were followed up to 1 year after treatment.
Fig. 2.
Fig. 2.
Histological features of leukemic cells. (A) Wright-Giemsa staining of peripheral blood (×40 magnification) from an affected mouse showing a high count of white blood cells (dark blue) and a decreased number of red blood cells. (B) Wright-Giemsa staining of leukemic cells (×100 magnification) showing the presence of predominantly myeloblasts and monoblasts in peripheral blood. Hematoxylin/eosin stained histology sections at ×60 magnification of enlarged spleen (C) and infiltrated liver (D; at ×40 magnification) show focal infiltration of leukemic cells.
Fig. 3.
Fig. 3.
Southern blot analysis of retroviral insertions in spleen DNA of Cbfb+/MYH11 chimeras with AML. (A) A representative EcoRI-digested DNA panel of 11 AML samples (lanes 2-12) and two wild-type controls (lanes 1 and 13) was hybridized with the 4070A envelop probe. (B)An EcoRI-digested DNA panel of primary leukemia samples (lanes 1 and 4) and of secondary leukemia samples after transplantation (lanes 2, 3, 5, and 6) hybridized with the 4070A envelope probe. Asterisks indicate bands present in both the primary (donor) and transplanted leukemic samples. (C) Cri10 insertions map back to the expected DNA fragment. DNA from leukemia sample VC2 digested with BamHI (lanes 1 and 3) or EcoRI (lanes 2 and 4). Viral probe envelope (env, lanes 1 and 2) and Cri10 clone Vc2.5 genomic probe (vc2.5, lanes 3 and 4) were used to sequentially hybridize to the same blot. Bands shown include wild-type allele (wa), inserted allele (ins), and an internal viral fragment (asterisks). Note that the band representing the inserted allele is expected to be fainter than the wild-type allele, because the spleen sample used includes a large number of wild-type cells, in addition to the leukemic cells carrying one inserted allele and one wild-type allele.
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