Nuclear NFATc1 Causes Development of Fms-like Receptor Tyrosine Kinase 3 Receptor (FLT3) Internal Tandem Duplication Mutation-Positive (FLT3-ITD) Acute Myeloid Leukemia (AML) and Mediates Drug Resistance

Background: Although an internal tandem duplication (ITD) of the Fms-like receptor tyrosine kinase 3 receptor (FLT3) confers a very poor prognosis in acute myeloid leukemia (AML), it is insufficient to cause AML, suggesting important cooperating genetic events. NFATc1 is a member of the nuclear factor of activated T-cells (NFAT) transcription factor family, whose activation is dependent on nuclear translocation. NFATc1 is well known for its central role in regulating T-cell development and function, but was also shown to contribute to neoplastic transformation in diverse types of cancers. We have recently demonstrated that NFATc1 is overexpressed in primary FLT3-ITD⁺ AML causing resistance to the FLT3-kinase inhibitor sorafenib in vitro. Here we asked, whether NFATc1 induces AML transformation and drug resistance when expressed in the context of FLT3-ITD⁺ in vivo.

Methods: Using the stem cell like (SCL)-promoter, Cre-recombinase inducible expression of a constitutively nuclear NFATc1 protein (cnNFATc1) was targeted to the early hematopoietic stem cell compartment. The SCL-Cre/cnNFATc1 mice were subsequently crossed with transgenic FLT3-ITD mice to yield Scl-Cre^+/-/cnNFATc1^+/+/FLT3-ITD^+/+ mice (FCN^+/+), which co-express cnNFAT and FLT3-ITD in the stem cell compartment.

Results: Whereas FLT3-ITD mice developed a non-lethal, mild myeloproliferative disease, co-expression of FLT3-ITD with cnNFATc1 led to rapidly fatal AML. The median survival of FCN mice was only four months (range, 2 to 8 ). In median, FCN mice had 15-fold (range, 10 to 20) increased white blood cell count, a high proportion of circulating peripheral blasts, severe anemia and thrombocytopenia. There was also a significant B-cell developmental block. As opposed to F^+/+ animals, FCN^+/+ mice revealed a severe spleno- and hepatomegaly secondary to a gross leukemic infiltration, which disrupted the normal anatomical architecture of the involved organs. Fluorescence activating cell sorting and colony forming unit (CFU) assays demonstrated that cnNFATc1, when expressed in the context of FLT3-ITD, leads to a significant expansion of the lin^-Sca⁺c-kit⁺ (LSK) stem cell-, progenitor cell (LK)-, the Gr-1^-/CD11b⁺ monocytic and Gr-1^low/CD11b⁺ immature myeloid compartments in bone marrow and spleen. FCN^+/+-AML was polyclonal, and re-transplantable in secondary wild-type recipient mice. In vivo, cnNFATc1 caused not only potent FLT3-ITD inhibitor resistance to sorafenib and to the more FLT3-ITD-selective compound quizartinib, but also to chemotherapy. Comparative mRNA-sequencing of sorted LK and LSK compartments in wild-type, F^+/+, C^+/- N^+/+, and FCN^+/+ mice will reveal the genetic basis of the biological cooperativity between NFATc1 and FLT3-ITD in stem and progenitor cell compartments. Consistent with the murine data, a data set of AML patients (n=163) revealed that NFATc1 overexpression alone or when combined with FLT3-overexpression was associated with a significantly decreased overall survival (hazard ratio [HR] 1.91, CI 1.29-2.18, p=0.001 versus HR 2.57, CI 1.62-4.08, p<0.0001).

Conclusions: These results provide for the first time evidence for an important role of nuclear NFATc1 overexpression in the molecular pathogenesis of FLT3-ITD positive AML and the induction of drug resistance. These data also underscore that the mutational landscape in AML insufficiently describes AML biology.