Mll partial tandem duplication and Flt3 internal tandem duplication in a double knock-in mouse recapitulates features of counterpart human acute myeloid leukemias - PubMed (original) (raw)

. 2012 Aug 2;120(5):1130-6.

doi: 10.1182/blood-2012-03-415067. Epub 2012 Jun 6.

Kelsie M Bernot, Susan P Whitman, Ronald F Siebenaler, Elshafa H Ahmed, Gabriele G Marcucci, Daniel A Yanes, Kathleen K McConnell, Charlene Mao, Chidimma Kalu, Xiaoli Zhang, David Jarjoura, Adrienne M Dorrance, Nyla A Heerema, Benjamin H Lee, Gang Huang, Guido Marcucci, Michael A Caligiuri

Affiliations

Mll partial tandem duplication and Flt3 internal tandem duplication in a double knock-in mouse recapitulates features of counterpart human acute myeloid leukemias

Nicholas A Zorko et al. Blood. 2012.

Abstract

The MLL-partial tandem duplication (PTD) associates with high-risk cytogenetically normal acute myeloid leukemia (AML). Concurrent presence of FLT3-internal tandem duplication (ITD) is observed in 25% of patients with MLL-PTD AML. However, mice expressing either Mll-PTD or Flt3-ITD do not develop AML, suggesting that 2 mutations are necessary for the AML phenotype. Thus, we generated a mouse expressing both Mll-PTD and Flt3-ITD. Mll(PTD/WT):Flt3(ITD/WT) mice developed acute leukemia with 100% penetrance, at a median of 49 weeks. As in human MLL-PTD and/or the FLT3-ITD AML, mouse blasts exhibited normal cytogenetics, decreased Mll-WT-to-Mll-PTD ratio, loss of the Flt3-WT allele, and increased total Flt3. Highlighting the adverse impact of FLT3-ITD dosage on patient survival, mice with homozygous Flt3-ITD alleles, Mll(PTD/WT):Flt3(ITD/ITD), demonstrated a nearly 30-week reduction in latency to overt AML. Here we demonstrate, for the first time, that Mll-PTD contributes to leukemogenesis as a gain-of-function mutation and describe a novel murine model closely recapitulating human AML.

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Figures

Figure 1

Figure 1

MllPTD/WT:_Flt3_ITD/WT mice develop a predominance of AML. (A) Frequencies of acute leukemia immunophenotypes identified in _Mll_PTD/WT:_Flt3_ITD/WT mice (n = 33). (B) Representative flow cytometry plots from spleen and BM samples from age-matched controls and moribund _Mll_PTD/WT:_Flt3_ITD/WT mice killed with elevated WBC counts. AML with maturation (myeloperoxidase [MPO+]/CD3−/CD19−/B220−/CD11b+/Gr1+/CD117−; 15%), AML without maturation group A (MPO+/CD3−/B220lo/CD19−/CD11blo/−/Gr1−/CD117+/−; 34%), AML without maturation group B (MPO+/CD3−/B220+/CD19−/CD11b+/−/Gr1−/CD117+/−; 21%). Regions highlighted in the red rectangle emphasize key differences in populations for each of the AML immunophenotypes. MPO staining by IHC and flow cytometry results for B-cell and biphenotypic leukemias are not shown. (C) WBC counts of leukemic _Mll_PTD/WT:_Flt3_ITD/WT mice are significantly elevated at the time of death. Figure shows WBC counts from 3 age-matched cohorts of _Mll_PTD/WT:_Flt3_ITD/WT mice with AML and controls. (D) _Mll_PTD/WT:_Flt3_ITD/WT mice with leukemia exhibit significant splenomegaly at the time of death. Figure shows weights from 3 age-matched cohorts of _Mll_PTD/WT:_Flt3_ITD/WT mice with AML and controls. (E) Representative image of spleens from a _Mll_PTD/WT:_Flt3_ITD/WT mouse dying of AML with age-matched controls. (F) Representative blood smear stained with Wright-Giemsa and hematoxylin and eosin-stained organ preparations from a _Mll_PTD/WT:_Flt3_ITD/WT mouse dying of AML. Micrographs demonstrate blasts in peripheral blood (original magnification ×1000) and extensive infiltrations of myeloid blasts in liver (original magnification ×400), and adrenal gland (original magnification ×400). Micrograph images were recorded using a Zeiss Axioskop, Olympus Magnafire digital camera, using a 10× eyepiece, 40×/0.65 NA Acroplan objective lens or 100×/1.40 NA Plan-Apochromat and Zeiss Axiovision Version 4.7.2 software.

Figure 2

Figure 2

BM samples from _Mll_PTD/WT:_Flt3_ITD/WT mice dying of AML are cytogenetically normal as are the age-matched, WT, and single-mutant controls. G-banded karyotype analysis of WT, single-mutant, and leukemic _Mll_PTD/WT:_Flt3_ITD/WT mice was conducted on whole BM samples.

Figure 3

Figure 3

MllPTD/WT:_Flt3_ITD/WT mice develop a fatal AML that is transplantable. (A) Survival of WT and single knock-in (MllPTD/WT:_Flt3_WT/WT or _Mll_WT/WT:_Flt3_ITD/WT) control mice and mice with the heterozygous alleles of _Mll_PTD/WT:_Flt3_ITD/WT. (B) _Mll_PTD/WT:_Flt3_ITD/WT cells from spleen containing AML are serially transplantable and lethal. Serially transplanted cells develop into progressively more aggressive AML with each successive passage.

Figure 4

Figure 4

AML from _Mll_PTD/WT:_Flt3_ITD/WT mice have molecular features similar to those reported for human MLL-PTD+ and/or FLT3-ITD+ AML. (A) Quantitative real-time RT-PCR was performed using CD117+ BM cells sorted to > 95% purity from age-matched mice ≥ 50 weeks of age to measure the copy numbers of the mouse _Mll_-PTD and the _Mll-_WT transcripts. Results demonstrate reduced _Mll_-WT copy number only in leukemic _Mll_PTD/WT:_Flt3_ITD/WT mice. Figure is representative of 2 age-matched cohorts of mice. (B) Relative real-time RT-PCR measuring HoxA9 expression in whole BM cells from age-matched mice > 50 weeks of age. (C) Total Flt3 mRNA was measured by relative real-time RT-PCR using BM cells of age-matched mice ≥ 50 weeks of age. (D) Genomic PCR reactions using 150 ng DNA and genotyping primers to amplify both the _Flt3_-ITD and _Flt3_-WT loci were performed to demonstrate a reduction or loss of _Flt3_-WT in primary _Mll_PTD/WT:_Flt3_ITD/WT AML samples taken from moribund mice. Multiple samples from each animal are defined as follows: lane 1, tail DNA from genotyping at 4 weeks; lane 2, whole BM DNA from the time of death when moribund with AML (50-80 weeks); and lane 3, sorted leukemic Ly5.2 _Mll_PTD/WT:_Flt3_ITD/WT AML blasts from secondary transplant of _Mll_PTD/WT:_Flt3_ITD/WT AML blasts into Ly5.1 recipients. The Ly5.2 → Ly5.1 adoptive transfer was used to obtain a pure population of _Mll_PTD/WT:_Flt3_ITD/WT AML blasts for analysis. Five representative samples are shown. Reactions that did not produce any bands for _Flt3_-WT and _Flt3_-ITD were removed as noted by the black bar.

Figure 5

Figure 5

Acute leukemia in _Mll_PTD/WT:_Flt3_ITD/ITD mice. (A) Only AML and B-cell leukemias developed in _Mll_PTD/WT:_Flt3_ITD/ITD mice. Nineteen mice were analyzed. (B) _Mll_PTD/WT:_Flt3_ITD/ITD mice with leukemia exhibited significant splenomegaly at time of death. (C) WBC counts (median ± SEM) for leukemic _Mll_PTD/WT:_Flt3_ITD/ITD and age-matched control genotypes at the time of death. (D) _Mll_PTD/WT:_Flt3_ITD/ITD mice died of AML with a significantly shorter latency than _Mll_PTD/WT:_Flt3_ITD/WT mice. (E) Copy number ratio of _Mll_-WT:_Mll_-PTD was calculated for age-matched nonleukemic 15- to 25-week-old _Mll_PTD/WT:_Flt3_ITD/WT and leukemic _Mll_PTD/WT:_Flt3_ITD/ITD mice. Results are representative of 2 age-matched cohorts of mice.

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