Knock-in of a FLT3 Internal Tandem Duplication Mutation Cooperates with a NUP98-HOXD13 Fusion to Generate Acute Myeloid Leukemia

Abstract 865

Acute myeloid leukemia (AML) with myelodysplastic related changes represents 24–35% of all cases of AML and has a poor response to chemotherapy and a dismal prognosis. Activating mutations of the FMS-like tyrosine kinase 3 (FLT3) receptor have been seen in 5% of myelodysplastic syndrome (MDS) cases, and an additional 10% of patients with MDS acquire FLT3 mutations during progression to AML. We have previously generated a knock-in mouse model in which an internal tandem duplication (ITD) mutation was inserted into the murine Flt3 gene, which induces a lethal myeloproliferative neoplasm, but not progression to overt leukemia. One mouse model of MDS involves the transgenic expression of a Nup98-HoxD13 (NHD13) fusion under the hematopoietic specific vav promoter. We bred the FLT3/ITD knock-in mice to the NHD13 transgenic mice to see if the two genetic alterations would cooperate. Strikingly, the FLT3/ITD-NHD13 mice on the FVB/N background developed acute leukemia with 100% penetrance and a mean survival of 95 ± 32 days (p<0.0001, n=20). This compares with a mean survival of 281 ± 94 days and 372 ± 84 days for the NHD13 alone and FLT3/ITD alone mice, respectively (both p<0.0001, n=20). FLT3/ITD-NHD13 mice generated on the C57Bl/6N background developed leukemia with a longer latency of 143 ± 37 days, but they still had a significantly shorter survival compared to the single mutants alone.

Competitive repopulation experiments showed that leukemic bone marrow was able to engraft in lethally irradiated recipients, with 1:200 cells in the bulk bone marrow having the potential to generate leukemia in recipient mice. To determine the identity of the leukemic initiating cell (LIC), the bulk bone marrow was further sorted into the multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte/macrophage progenitor (GMP), and megakaryocytic/erythroid progenitor (MEP) populations and limiting dilution transplantation was performed for each group. The highest engraftment potential was found in the MPP population (1:100 cells) with much rarer LICs from the CMP and GMP. The MEP population did not engraft in any recipient mice even at the highest cell dose used of 100,000 sorted cells.

RNA was extracted from the total bone marrow of mice 3 months prior to the development of disease and showed overexpression of HoxA7, HoxA9, HoxB4, HoxB6, HoxC4, and HoxC6 in the FLT3/ITD-NHD13 mice compared to the age-matched wildtype or FLT3/ITD mice. The overexpression of these genes appeared to be primarily driven by the NHD13 transgene, although there was also an increase in HoxA7 and HoxA9 expression observed in the FLT3/ITD mice. Since Hox genes are known to play an important role in stem cell self-renewal, we isolated RNA from 2-month-old littermates and stained for the cell surface markers characterizing the KSL, MPP, ST-HSC, and LT-HSC populations. FLT3/ITD-NHD13 and FLT3/ITD mice had a significant increase in the percentage of KSLs, long term HSCs, and short term HSCs. FLT3/ITD mice showed increased numbers of MPPs with a smaller but significant increase in the FLT3/ITD-NHD13 cohort.

A significant number of AML patients with FLT3/ITD mutations present with loss of the wildtype allele of FLT3 and additional patients acquire this loss at the time of relapse. Hemizygosity at the FLT3 locus in FLT3/ITD mutant AML patients is associated with an even more adverse prognosis compared to patients with an intact wild-type allele. We observed spontaneous loss of heterozygosity (LOH) occurring in 100% of the FLT3/ITD-NHD13 mice and resulting in loss of the wildtype Flt3 allele. The frequent LOH seen in the FLT3/ITD-NHD13 mice provides additional evidence that this transgenic mouse serves as an accurate model to further explore the molecular pathways that underlie the development of leukemias with activating FLT3 mutations. This mouse model is also one of the first models of MDS-related AML and should provide a means to explore the molecular pathways that underlie these devastating hematopoietic neoplasms.