Abstract

Acute myeloid leukemia (AML) is a genetically heterogeneous disease where multiple mutations coincide in hematopoietic stem and progenitor cells leading to malignant transformation. One important class of mutations alters the function of signaling intermediates such as Fms-like tyrosine kinase 3 (FLT3), thereby helping AML cells to overcome the physiological communication with their microenvironment. Activating mutations in FLT3 are found in approximately 30% of adult AML cases. Particularly common are internal tandem duplications (ITD) in the juxtamembrane domain of FLT3, which are associated with poor clinical outcome. Recently, a phase II study of the second-generation FLT3 inhibitor AC220 (quizartinib) showed a complete remission rate of 44% to 54% in relapsed/chemotherapy refractory AML. However, secondary point mutations in the FLT3 tyrosine kinase domain have been reported as common causes of acquired clinical resistance to the FLT3 inhibitor AC220.

We used quantitative mass-spectrometry-based phosphoproteomics to elucidate and compare the signaling out-put of FLT3-ITD and its AC220-resistant mutants harbouring either the F691L 'gatekeeper' substitution or the D835V activation loop mutant in AML cell models. Our comprehensive signaling analyses profiled thousands of phosphorylation events in a site-specific manner and revealed marked differences in the signaling profiles of the FLT3 mutant variants. In general, we found differential activation of signal transducer and activator of transcription 5 (STAT5) and mitogen-activated protein (MAP) kinase signaling when comparing FLT3-ITD and the AC220-resistant mutants. Interestingly, some cytosolic tyrosine kinases showed differential activation patterns. For instance, spleen tyrosine kinase (SYK) signaling was significantly enhanced downstream of FLT3-ITD-F691L and cells harbouring this mutant showed increased responsiveness to compounds targeting SYK. Our resource study shows, how point mutations conferring resistance to AC220 impact the signaling output of FLT3-ITD and uncovers pathways whose inhibition might be useful to disrupt oncogenic signaling elicited by FLT3-ITD-F691L and -D835V mutants.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.