Characterizing and Overriding the Structural Mechanism of the Quizartinib-Resistant FLT3 “Gatekeeper” F691L Mutation with PLX3397

Abstract

Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKIs) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase “gatekeeper” residues, which control access to an allosteric pocket adjacent to the ATP-binding site, have been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first co-crystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved DFG motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position.

Identification of a germline F692L drug resistance variant in cis with Flt3-internal tandem duplication in knock-in mice

“…15 Moreover, our findings emphasize the importance of careful characterization of genetically modified mice before drawing conclusions about the pathologies they model, particularly when targeted therapeutic interventions are being investigated. Although the genetic variant influencing drug response was not present in wild-type mice in our study, in other cases it may represent a consistent human -mouse difference, as was recently described for lenalidomide and a mousespecific variant in Cereblon (Crbn) that makes murine cells insusceptible to the drug.…”

“…As the murine F692L variant is equivalent to the human F691L gatekeeper mutation, which confers AML resistance to multiple FLT3 tyrosine kinase inhibitors, 3,15 we proceeded to test whether this was also true for the murine variant. To do this we cultured AML cells from two independent Npm1 cA/+ ; Flt3…”

Selective Inhibition of the Myeloid Src-Family Kinase Fgr Potently Suppresses AML Cell Growth in Vitro and in Vivo

“…One important example is quizartinib (also known as AC220), a very potent and selective Flt3 inhibitor that was among the first to show efficacy as a single agent against Flt3-ITD + AML 5,31 . Quizartinib often induces rapid remission in Flt3-ITD + AML, but acquired drug resistance, commonly in the form of Flt3 kinase domain mutations, has limited its effectiveness 4,5,32–34 . Unlike TL02–59, we found that quizartinib does not inhibit Fgr or other AML-associated kinases, including Syk, Fes, or Hck in vitro; the same is true for other Flt3 inhibitors tested in the clinic, including sorafenib, sunitinib, and tandutinib (data not shown).…”

The role of targeted therapy in the management of patients with AML

“…Although the findings validated that the mechanism of response to quizartinib was due to FLT3 inhibition, they also pointed to a need to develop drugs that target both FLT3-ITD and common TKD resistance mutations as a method to circumvent resistance and maximize the efficacy of FLT3 inhibition. 13,14 Two such agents quickly followed, each showing in vitro activity against both FLT3-ITD and FLT3-D835, the most common TKD mutation conferring resistance to quizartinib. First to enter trials was crenolanib, which was quickly followed by gilteritinib (ASP2215).…”