P445: a systems-based approach to unveil drug resistance in flt3-itd aml

Background: Internal tandem duplications (ITDs) in FLT3 gene occurs in about 30% of patients with a de novo AML diagnosis and are generally associated with poor prognostic outcomes. The different FLT3-ITD insertion site impacts the sensitivity to tyrosine kinase inhibitors (TKIs) treatment, influencing patients’ clinical outcome. Specifically, the ITD insertion in the tyrosine kinase domain (TKD) is associated with chemoresistance, relapse and lower overall survival. Aims: Our work aims to elucidate the molecular mechanisms driving the different sensitivity of FLT3-ITD cells to tyrosine kinase inhibitors (TKIs). Additionally, we aim to investigate whether the different prognostic impact of ITD-mutations is due to a diverse cell responsiveness to one of the most commonly prescribed chemotherapeutic drugs, cytarabine. Our final goal is the identification of novel promising therapeutic targets that may improve the prognostic outcome of patients carrying the ITD in the TKD region Methods: Here we tested the hypothesis that the different sensitivity of FLT3-ITD cells to chemotherapeutic drugs may be caused by an extensive rewiring of cell signaling network. Thus, to obtain a comprehensive and unbiased picture of the FLT3-ITD signaling networks, we combined RNA sequencing and mass spectrometry (MS)-based (phospho)proteomics with a newly developed computational strategy, called “SignalingProfiler”. In vitro and in ex vivo assays were performed to in depth characterize the TKI and cytarabine-dependent signaling pathways in FLT3-ITD cells. Results: Our strategy revealed that the different ITD insertion site differently affects cell cycle progression, impairing the activation of crucial kinases. Specifically, we found that the WEE1-CDK1 axis plays a crucial role in TKI therapy failure in patients carrying the ITD in the TKD region. Remarkably, pharmacological inhibition of WEE1 completely rescued the ability of patient-derived primary blasts, carrying the ITD-TKD mutation, to undergo apoptosis in response to midostaurin treatment. Summary/Conclusion Finally, this study demonstrates the impact of ITD-location on cytarabine and TKI sensitivity, identifying clinically relevant effective drug combinations to increase the responsiveness of FLT3-ITD blasts. Additionally, we propose a novel strategy that can be applied to the study of drug resistance and mechanism of action, and can lead to the identification of novel therapeutic targets for combination therapy.Keywords: Proteomics, Signaling, Flt3-ITD, AML