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.2010 Feb 11;115(6):1194-203.
doi: 10.1182/blood-2009-04-216184. Epub 2009 Dec 9.

Retroviral insertional mutagenesis identifies Zeb2 activation as a novel leukemogenic collaborating event in CALM-AF10 transgenic mice

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Retroviral insertional mutagenesis identifies Zeb2 activation as a novel leukemogenic collaborating event in CALM-AF10 transgenic mice

David Caudell et al. Blood..

Abstract

The t(10;11) translocation results in a CALM-AF10 fusion gene in a subset of leukemia patients. Expression of a CALM-AF10 transgene results in leukemia, with prolonged latency and incomplete penetrance, suggesting that additional events are necessary for leukemic transformation. CALM-AF10 mice infected with the MOL4070LTR retrovirus developed acute leukemia, and ligation-mediated polymerase chain reaction was used to identify retroviral insertions at 19 common insertion sites, including Zeb2, Nf1, Mn1, Evi1, Ift57, Mpl, Plag1, Kras, Erg, Vav1, and Gata1. A total of 26% (11 of 42) of the mice had retroviral integrations near Zeb2, a transcriptional corepressor leading to overexpression of the Zeb2-transcript. A total of 91% (10 of 11) of mice with Zeb2 insertions developed B-lineage acute lymphoblastic leukemia, suggesting that Zeb2 activation promotes the transformation of CALM-AF10 hematopoietic precursors toward B-lineage leukemias. More than half of the mice with Zeb2 integrations also had Nf1 integrations, suggesting cooperativity among CALM-AF10, Zeb2, and Ras pathway mutations. We searched for Nras, Kras, and Ptpn11 point mutations in the CALM-AF10 leukemic mice. Three mutations were identified, all of which occurred in mice with Zeb2 integrations, consistent with the hypothesis that Zeb2 and Ras pathway activation promotes B-lineage leukemic transformation in concert with CALM-AF10.

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Figures

Figure 1
Figure 1
MOL4070LTR infection accelerates leukemic transformation inCALM-AF10 mice. (A) Survival of MOL4070LTR-infectedCALM-AF10 mice (■, N = 45), MOL4070LTR-infected WT (○, n = 31), and noninfectedCALM-AF10 transgenic mice (●, N = 20). *P < .001, comparing infectedCALM-AF10 with noninfectedCALM-AF10, or infectedCALM-AF10 with WT mice using a log-rank test. (B) Distribution of leukemia by immunophenotype in WT andCALM-AF10 mice infected with MOL4070LTR retrovirus. Note the predominance of B-cell and myeloid (AML) tumors, as well as the lack of pre-T LBL, in theCALM-AF10 mice compared with the WT control mice. (C) Retroviral integration analysis. Southern blot ofEcoRI-digested DNA extracted from leukemic spleens hybridized to a viralenv probe. Mouse identification numbers are indicated; C indicates germline control tissue; size standards are given in kilobases. The proviral genome is 8.7 kb and does not contain anEcoRI site.
Figure 2
Figure 2
Nf1 analysis. (A) Southern blot ofBamHI-digested DNA from leukemic spleens hybridized to anNf1 probe; 2 germline bands are seen as the probe contains aBamH1 site. Mouse identification numbers are indicated; C indicates germline control tissue; size standards are given in kilobases. *Germline bands. Note loss of the germline allele in mice 17 and 23. (B) Protein lysates from leukemic spleens with a knownNf1 CIS were compared with spleens from clinically healthyCALM-AF10 and WT mice. The Nf1 protein is indicated withformula image; sizes are in kDa. The blot was reprobed with anti–α-tubulin as a loading control. Intensity of the Nf1 and α-tubulin signals were quantified with ImageJ software, and the ratio compared with that of the WT mouse.
Figure 3
Figure 3
Zeb2 integration analysis. (A) Mouse chromosome 2. Insertions identified by LM-PCR are indicated by black arrows. Insertion events reported in the RTCGD are indicated by red arrows. The vertical red arrow represents an insertion event for which the integration orientation was not reported in the RTCGD. (●) Insertion events identified by Southern blot. Probes A and B are indicated by black bars. Numbers above black arrows and circles represent mice withZeb2 intregrations. (B) Southern blot ofStuI-digested genomic DNA from infiltrated spleen hybridized to aZeb2 probe. Mouse identifications are indicated; C indicates germline control tissue; size standards are given in kilobases. *Germline bands. Note the equal intensity of germline and rearranged bands indicating that the leukemias were predominately clonal, except for mouse 98. (C) Acute lymphoid leukemia (B-ALL) inCALM-AF10 mouse 17 infected with MOL4070LTR. Hematoxylin and eosin–stained liver (Ci inset), B220 stained liver (Cii inset), and myeloperoxidase-stained liver (Ciii inset). (D) mRNA levels determined by quantitative PCR of leukemic spleens withZeb2 retrovirus insertions (N = 9).Zeb2 expression was normalized to the 18S ribosomal control and compared with WT spleen (N = 5 independent samples), WT bone marrow (N = 5 independent samples), or B-lineage ALL samples withoutZeb2 integrations (N = 4, mice 8, 14, 38, and 39). *P < .03. **P < .01. ***P < .001. (E) RelativeZEB2 expression in pediatric B-lineage leukemias determined by expression array from supplemental data reported by Yeoh et al (http://www.stjuderesearch.org/data/ALL1). Asterisks denote comparison of each group to theMLL group; for all comparisons,P < .001.
Figure 4
Figure 4
Increased expression of CIS genes. (A) Expression levels ofMn1 were evaluated in mice withMn1 insertions (N = 4), mice withoutMn1 orEvi1 insertions (N = 5), mice withEvi1 insertions but withoutMn1 insertions (N = 5), WT spleen (N = 5), and WT BM (N = 5). *P < .03. **P < .01. ns indicates not significant. (B) Expression levels ofEvi1 in mice withEvi1 insertions (N = 5), mice withoutEvi1 insertions (N = 4), WT spleen (N = 5), and WT BM (N = 5). (C) Evaluation of 8 additional CIS (Plag1,Mpl,Kras,Erg,Ift57,Cd47,Vav1, andGata1) compared with either WT bone marrow (N = 5) or WT spleen (N = 5). Expression of each gene was determined by quantitative RT-PCR and normalized to the 18S ribosomal RNA.
Figure 5
Figure 5
Inhibition ofZEB2 leads to decreased proliferation of MV4-11 cells. (A) Expression ofZEB2 mRNA in MV4-11 and B-ALL1 cell lines. (B) Inhibition ofZEB2 mRNA expression in MV4-11 cell line by siRNA leads to decreased proliferation. (Inset)ZEB2 quantitative RT-PCR assay at 24 hours. (C) Treatment of B-ALL1 cell line withZEB2 siRNA does not lead to decreased proliferation. Quantitative RT-PCR values are plotted as minimum/maximum values from triplicate assays. *P < .03. ***P < .001. Error bars for 120-hour Zeb2 siRNA time point are very small and not visible.
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References

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