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Acute Myeloid Leukemia (AML) is a heterogenous hematopoietic cancer characterized by abnormal differentiation and uncontrolled proliferation of hematopoietic progenitor cells. Recent approaches to treatment, which aim to improve outcomes in refractory AML, include strategies to molecularly target specific receptor-mediated signaling pathways which regulate cell growth and proliferation. One target-specific approach is the inhibition of fms-like tyrosine kinase 3 (FLT3). FLT3 is expressed on the surface of hematopoietic progenitor cells and plays an important role in regulating proliferation, survival, and differentiation of multipotent stem cells. The presence of internal tandem duplications (ITDs) at the juxta membrane domain within the FLT3 receptor are common driver mutations in 30% of AML patients. In this study, we utilized 3 luciferase-expressing human AML models, MV4-11, MOLM-13, and, OCI-AML3 to determine the prognostic impact of FLT3 inhibitor Gilteritinib on the allelic ratio of ITD versus wildtype allele. OCI-AML3 expresses wild-type FLT3 whereas MOLM-13 is heterozygous for FLT3-ITD mutations, and MV4-11 possesses a homozygous in-frame tandem repeat insertion in the FLT3 allele. Here we demonstrate that FLT3-ITD mutational status impacts the efficacy of Gilteritinib in both in vitro and in vivo AML models. The in vitro assays showed that treatment with Gilteritinib resulted in significant dose dependent reduction in overall cell viability and bioluminescence in the MV4-11 cell line, while MOLM-13 was only moderately sensitive, and OCI-AML3 was least sensitive to Gilteritinib treatment. Additionally, we evaluated the downstream MAPK and PI3K signaling pathways as FLT3-ITD oncogenic mutations result in ligand-independent constitutive activation of signaling pathways mediating leukemic transformation. For in vivo validation, MV4-11, MOLM-13, and OCI-AML3 luciferase expressing cells were implanted systemically into NCG mice and once tumors were established, we evaluated the survival benefit in response to Gilteritinib and various standard of care agents. In accordance with our in vitro findings, the quantification of tumor progression by bioluminescent imaging showed the highest therapeutic activity of Gilteritinib in MV4-11 tumor bearing mice through selective targeting of FLT3-ITD mutations. Taken together, our in vitro and in vivo results demonstrate that differential FLT3-ITD mutational status in AML models impacts Gilteritinib treatment response and downstream signaling events with the highest activity in MV4-11 followed by MOLM-13, and lack of activity in OCI-AML3 harboring wild-type FLT3. Citation Format: Bincy John, Pasupathi Sundaramoorthy, Elizabeth Rainbolt, Andrew Wong, Zachary Ward, Justin Avery, Savannah Todd, Shasta Kidder, Diana Gietl, Chassidy Hall, David Harris. Differential flt3 mutational status in acute myeloid leukemia predicts sensitivity to flt3 inhibitor gilteritinib in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 8.