The FLT3-Inhibitors Midostaurin, Sunitinib, Sorafenib, and TKI258 Differentially Affect NK Cell-Mediated Immunesurveillance of Acute Myeloid Leukemia.
Authors: Salih Julia , Kanz Lothar , Salih Helmut R , Krusch Matthias 
Published: October 2019

Abstract
Abstract 3785
Poster Board III-721
FLT3 is a receptor tyrosine kinase with an important role in hematopoietic progenitor cell survival and proliferation. The discovery of internal tandem duplication mutations (ITD) in FLT3 was a major breakthrough in understanding the role of abnormally activated FLT3 in myeloid transformation. Between 15% and 34% of AML patients show FLT3-ITD mutations, and thus the inhibition of FLT3 in combination with chemotherapeutic agents may be a promising stragety in the treatment of Acute Myeloid Leukemia (AML). Several protein kinase inhibitors (PKI) targeting FLT3 like e.g. Midostaurin, Sunitinib, Sorafenib, and TKI258 are currently under preclinical and/or clinical evaluation (http://clinicaltrials.gov/ct2/results?term=AML+and+FLT3). Since those PKI, besides targeting their eponymous enzyme FLT3, also inhibit signaling via other molecules they may impair the effector function of various components of anti-tumor immunity. NK cells as part of the innate immune system play an important role in the immune surveillance of tumors due to their ability to directly kill target cells and to shape adaptive immune responses by secreting cytokines like IFN-γ. Clinical evidence for the particularly important role of NK cells in leukemia has recently been provided by studies of haploidentical stem cell transplantation (Ruggeri et al., Science 2002). We report here that CD107a expression as a surrogate marker for degranulation of NK cells within PBMC is inhibited by pharmacological concentrations of Sorafenib (10μg/ml) and Midostaurin (2μg/ml), but not by Sunitinib (200ng/ml) and TKI258 (125ng/ml). In line, pharmacological concentrations of Sunitinib and TKI258 did not affect NK cell cytotoxicity and IFN-γ production in cocultures with leukemia cells. Sorafenib and Midostaurin caused a clear concentration-dependent inhibition of NK cell cytokine production in response to target cells both in resting and in IL-2 activated state (92% and 66%, respectively at plasma peak levels). Furthermore, pharmacological concentrations of Sorafenib and Midostaurin also reduced lysis of leukemia cells by NK cells (54% and 58%, respectively, E:T ratio 10:1) and thus generally compromised NK cell reactivity. Analysis of NK cell signaling revealed that Sorafenib, but not Midostaurin decreased phosphorylation of PI3K and ERK which are important regulators of NK cell reactivity. Thus, Midostaurin inhibits yet undefined signaling events which are crucial for NK effector functions, but are independent of the “classical” PI3K – Rac – PAK – MEK – ERK pathway and are presently under study. Moreover, in light of the important role of NK cells in the immune surveillance of leukemia and the differential influence of clinically used FLT3-inhibitors on NK cell functions our data indicate that the choice and dosing of the most suitable compound in the treatment of AML requires further characterization and careful consideration.

Disclosures:
No relevant conflicts of interest to declare.