Loss of the Wild-Type Allele Contributes to Myeloid Expansion and Disease Aggressiveness in FLT3/ITD Knock-in Mice

Abstract 866

Activating mutations of FLT3, either in the form of internal tandem duplication (ITD) mutations in the juxtamembrane domain or point mutations in the kinase domain, are one of the most frequent mutations in acute myeloid leukemia (AML). AML patients with FLT3/ITD mutations have poor prognosis. Loss of the wild-type FLT3 allele is associated with even worse prognosis when compared to those FLT3/ITD AML patients with the wild-type FLT3 allele still present. Also, FLT3/ITD patients with a high mutant-to-wild-type ratio have a significantly worse outcome than FLT3/ITD patients with a lower ratio. We have previously reported that heterozygous FLT3^wt/ITD “knock-in” mice develop a slowly fatal MPN. In order to study the roles wild-type FLT3 play in the development of leukemia associated with FLT3/ITD mutations, we crossed FLT3^wt/ITD mice with themselves or with FLT3 “knockout” (FLT3^−/−) mice to obtain hemizygous (FLT3^−/ITD) or homozygous (FLT3^ITD/ITD) FLT3/ITD mice. Investigating phenotypic differences among them reveals the impact of wild-type FLT3 on the development of MPN resulting from FLT3/ITD mutations, and by extension, the effect on acute leukemia.

FLT3^−/ITD mice, with the loss of the wild-type allele, displayed a more severe MPN, as evidenced by even larger spleen, higher white blood counts and shorter survival, compared to FLT3^wt/ITD mice. FLT3^ITD/ITD mice had an even severe MPN compared to the FLT3^−/ITD and FLT3^wt/ITD mice. Fully transformed leukemia developed in some of the FLT3^ITD/ITD (7%, 9/129), but not FLT3^wt/ITD or FLT3^−/ITD mice, with latency ranging from 139 to 304 days.

Compared to FLT3^wt/ITD mice, FLT3^−/ITD and FLT3^ITD/ITD mice displayed a further increase in the fraction of primitive hematopoietic cells, with notable increases in ST-HSCs and MPPs. Phosphorylation of STAT5, one of the key downstream targets for constitutively activated FLT3, was increased in FLT3^wt/ITD, FLT3^−/ITD and FLT3^ITD/ITD mice compared to the wild-type control. FLT3^wt/ITD, FLT3^−/ITD and FLT3^ITD/ITD BM also showed increased PU.1 expression and decreased GATA-1 expression, resulting in the subsequent expansion of granulocytic/monocytic/lymphocytic progenitors and a decrease in megakaryocytic/erythrocytic progenitors. It appears that the extent of myeloproliferation in FLT3/ITD mice correlates with loss of the wild-type allele (FLT3^wt/ITD vs. FLT3^−/ITD) and with the dose of mutant allele (FLT3^−/ITD vs. FLT3^ITD/ITD).

In order to further explore the potential moderating effect of wild-type FLT3 expression on FLT3/ITD-associated MPN, we transduced wild-type FLT3 (wtFLT3, with the lentiviral vector co-expressing GFP) into lineage-depleted FLT3^−/ITD CD45.2 BM cells and injected them into lethally irradiated CD45.1 recipients. When injected with sorted (GFP⁺) BM, vector alone-transduced GFP⁺FLT3^−/ITD BM recipients died of MPN, with a median survival of 62 days. 100% of the recipients in the other three groups, i.e., those injected with vector alone-transduced GFP⁺ wild-type BM, wtFLT3-transduced GFP⁺ wild-type BM or wtFLT3-transduced GFP⁺ FLT3^−/ITD BM, remained viable even after the point in time at which all of the recipients in the vector alone-transduced GFP⁺FLT3^−/ITD group died. Similarly, recipients transplanted with unselected (including GFP⁺ and GFP⁻ populations) vector alone-transduced FLT3^−/ITD BM also died early, with a median survival of 73 days and overt signs of MPN. The percentages of GFP⁺ and GFP⁻ cells in the BM of the dying recipients were comparable to those shortly after transplantation, indicative of the similar expansion ability of the GFP⁺ and GFP⁻ populations in the BM. In contrast to the wtFLT3-transduced GFP⁺FLT3^−/ITD BM recipients, which have a very prolonged survival, recipients injected with unselected wtFLT3-transduced FLT3^−/ITD BM died of MPN, with a median survival of 91 days. Interestingly, 99% of the BM cells in the BM of the dying recipients were GFP⁻, demonstrating a proliferative/survival advantage for the FLT3^−/ITD cells that had not been successfully transduced with wild-type FLT3. These results suggest that the presence of wild-type FLT3 delays and moderates the development of MPN caused by FLT3/ITD mutations.

These results suggest that loss of the wild-type allele contributes to the development of a more severe phenotype. Thus, the wild-type FLT3 allele seemingly functions as a “tumor suppressor” in leukemia harboring FLT3/ITD mutations.