A noncanonical FLT3 gatekeeper mutation disrupts gilteritinib binding and confers resistance

Abstract

Acute myeloid leukemia (AML) is a genetically heterogenous disease with approximately 20 000 new cases per year in the United States. 1 Patients with AML have a 5-year survival of <25%, and intense efforts are underway to develop new treatments to improve survival. 2 Mutations in the FMS-like tyrosine kinase-3 (FLT3) gene are among the most common genomic aberrations in AML. Internal tandem duplication (ITD) in the juxtamembrane domain of FLT3 are present in approximately 20% of patients with AML. These mutations cause constitutive kinase activity, and lead to an increased risk of relapse and reduced survival. Another set of mutations in the tyrosine kinase domain (TKD) of FLT3 occur in 5-10% of AML patients and also result in activation of FLT3. 3 Multiple FLT3 tyrosine kinase inhibitors have been developed and can be separated into two classes. Type I inhibitors are canonical ATP competitors that bind the ATP binding site of FLT3 in the active conformation and are effective against both ITD and TKD mutations. By contrast, type II inhibitors bind the hydrophobic region adjacent to the ATP binding domain in the inactive conformation. Type II inhibitors are effective against FLT3-ITD, but do not inhibit FLT3-TKD mutations. Quizartinib, a type II inhibitor, has potent activity against FLT3, KIT, and RET. Despite high response rates as a monotherapy in

The FLT3N701K mutation causes clinical AML resistance to gilteritinib and triggers TKI sensitivity switch to quizartinib

“…22 Although FLT3 N701K mutations act as a gatekeeper for gilteritinib, the mutation retains a surprising sensitivity to quizartinib and other type II inhibitors. 10 Although we initially found this mutation in gilteritinibresistant cell lines, we now report a patient with the FLT3 ITD+N701K mutation with ex vivo sensitivity that mirrors our cell lines. In silico modeling suggests that the N701K mutation is predicted to sterically disrupt the binding of gilteritinib, and trigger a conformational switch that favors adoption of the inactive kinase conformation.…”

“…2 In addition to NRAS mutations, we also identified a novel FLT3 N701K mutation in three resistant cultures that promoted gilteritinib resistance. 10 We demonstrated that FLT3 N701K effectively acts as a gatekeeper mutation, blocking gilteritinib from binding to FLT3. Notably, FLT3 N701K did not block quizartinib binding, indicating that FLT3 N701K mutations are more specific for type I FLT3 inhibitors and, thereby serve as noncanonical gatekeepers unlike canonical gatekeepers, such as FLT3 F691L , which block binding of type I and II inhibitors.…”

Targeting Gatekeeper Mutations for Kinase Drug Discovery

“…The first-generation multitarget kinase inhibitor midostaurin and two second-generation FLT3 inhibitors, gilteritinib and quizartinib (Figure ), have been approved for the treatment of AML patients harboring FLT3 mutations. Despite their high response rates as monotherapy, the clinical effectiveness of gilteritinib and quizartinib is limited due to acquired resistance associated with TKD mutations, such as the activation loop (D835Y/V/F) and/or the gatekeeper Phe691-to-Leu691 (F691L) mutations. − To date, there is still no approved drug to overcome clinical resistance mediated by the gatekeeper FLT3 F691L mutation.…”

Therapeutic Targeting of FLT3 in Acute Myeloid Leukemia: Current Status and Novel Approaches

“… 134 A novel gatekeeper mutation, N701K was detected in gilteritinib-resistant FLT3-ITD cell lines by sterically interfering with the binding of gilteritinib. 144 …”