PLX3397 Is An Investigational Selective FLT3 Inhibitor That Retains Activity Against the Clinically-Relevant FLT3-ITD/F691L “Gatekeeper” Mutation in Vitro
Authors: Smith Catherine C. , Perl Alexander E , Lasater Elisabeth , Zhang Chao , Jeschke Grace R , Damon Lauren E. , Carroll Martin , Shah Neil P. 
Published: October 2019

Abstract
Abstract 764

Background:
Activating mutations in FLT3 are detected in approximately 30% of adult acute myeloid leukemia (AML) cases, most commonly involving internal tandem duplication (ITD) events. The clinically-active FLT3 inhibitor AC220 (quizartinib) was recently reported to be associated with a composite complete remission (CR) rate of 43% in relapsed/refractory FLT3-ITD+ AML patients in an interim analysis of a phase II study (Cortes, et al, EHA 2011, abstract #1019). AC220 is vulnerable in vitro to a limited number of resistance-conferring mutations in the FLT3 kinase domain (Smith et al, AACR 2011, abstract #4737). Mutations at two of these residues, F691 and D835, have been detected in 9/9 patients with AML with loss of response to AC220 analyzed to date (Smith et al, ASH 2011, submitted). Mutations at “gatekeeper” residues such as F691 have repeatedly surfaced as important mediators of clinical resistance to inhibitors of BCR-ABL, EGFR, ALK and KIT kinases. Identifying tyrosine kinase inhibitors that retain activity against these substitutions has proven challenging. PLX3397 is a potent and selective inhibitor of FMS, KIT and oncogenic FLT3 with an elimination half-life of 20 hours in humans. Steady-state plasma concentrations of 10–20 uM have been achieved in solid tumor patients enrolled in a phase I study, where minimal myelosuppression has been observed (Anthony et al, J Clin Oncol 29: 2011, suppl abstr 3093).

Results:
PLX3397 selectively inhibited the proliferation of the human FLT3-ITD+ AML cell lines MV4-11 and MOLM-14 with a 50% inhibitory concentration (IC50) in the submicromolar range (0.09–0.2 uM). PLX3397 inhibited phosphorylation of FLT3-ITD in cells with a dose response similar to the growth inhibition range. We next evaluated the ability of PLX3397 to inhibit the proliferation of Ba/F3 cells transformed with FLT3-ITD and AC220-resistant FLT3-ITD mutant isoforms F691L, D835V/Y, and Y842C/H. PLX3397 inhibited the proliferation of Ba/F3/FLT3-ITD cells (IC50 0.07 uM) at a concentration comparable to that needed to inhibit the growth of MV4-11. Encouragingly, PLX3397 retained activity against Ba/F3 cells expressing the clinically-relevant F691L gatekeeper mutation at a similar concentration (IC50 0.37 uM). Other AC220-resistant mutations evaluated conferred substantial cross-resistance to PLX3397 (IC50 >200x IC50 of FLT3-ITD alone for all mutations; range 14.3–130,000 uM). Western blot analysis of FLT3 autophosphorylation demonstrated dose responsiveness in the same concentration range as observed in cellular proliferation results for each FLT3-ITD mutant isoform. Based upon molecular docking studies, PLX3397 forms non-specific hydrophobic interactions with the gatekeeper side-chain, and thus is less sensitive to the F691L mutation than AC220, for which the combined effects of steric hindrance and polarity mismatch impede its binding to the FLT3-ITD/F691L. To further assess if PLX3397 warrants clinical evaluation as a FLT3 inhibitor in AML, we performed a modified plasma inhibitory assay by incubating MOLM-14 cells in either normal donor or AML patient plasma spiked with increasing concentrations of PLX3397 as well as unmanipulated, steady-state plasma samples from the solid tumor PLX3397 phase I trial. Using phospho-specific flow cytometry to evaluate FLT3 signaling through the downstream protein ribosomal S6, we observed near-maximal reductions in phospho-S6 in both normal and AML patient plasma containing >10 uM PLX3397 as well as plasma samples obtained from the solid tumor trial.

Conclusions:
PLX3397 harbors substantial promise for the treatment of FLT3-ITD+ AML. Our data demonstrate that potent FLT3 inhibitory concentrations are achieved in humans. Moreover, while mutations in the FLT3-ITD kinase domain represent an important cause of loss of response to clinically-active FLT3 inhibitors, PLX3397 retains activity against the FLT3-ITD/F691L “gatekeeper” mutation, which we have found to be a common cause of preclinical and acquired clinical resistance to AC220. A multi-site phase I/II study of PLX3397 in FLT3-ITD+ AML has been initiated.

Disclosures:
Off Label Use: Investigational agent PLX3397 will be discussed. Zhang:Plexxikon Inc.: Employment. Carroll:Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding. Shah:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Research Funding.