Crenolanib is a selective type I pan-FLT3 inhibitor

Abstract

Tyrosine kinase inhibitors (TKIs) represent transformative therapies for several malignancies. Two critical features necessary for maximizing TKI tolerability and response duration are kinase selectivity and invulnerability to resistance-conferring kinase domain (KD) mutations in the intended target. No prior TKI has demonstrated both of these properties. Aiming to maximize selectivity, medicinal chemists have largely sought to create TKIs that bind to an inactive (type II) kinase conformation. Here we demonstrate that the investigational type I TKI crenolanib is a potent inhibitor of Fms tyrosine kinase-3 (FLT3) internal tandem duplication, a validated therapeutic target in human acute myeloid leukemia (AML), as well as all secondary KD mutants previously shown to confer resistance to the first highly active FLT3 TKI quizartinib. Moreover, crenolanib is highly selective for FLT3 relative to the closely related protein tyrosine kinase KIT, demonstrating that simultaneous FLT3/KIT inhibition, a prominent feature of other clinically active FLT3 TKIs, is not required for AML cell cytotoxicity in vitro and may contribute to undesirable toxicity in patients. A saturation mutagenesis screen of FLT3-internal tandem duplication failed to recover any resistant colonies in the presence of a crenolanib concentration well below what has been safely achieved in humans, suggesting that crenolanib has the potential to suppress KD mutation-mediated clinical resistance. Crenolanib represents the first TKI to exhibit both kinase selectivity and invulnerability to resistance-conferring KD mutations, which is unexpected of a type I inhibitor. Crenolanib has significant promise for achieving deep and durable responses in FLT3-mutant AML, and may have a profound impact upon future medicinal chemistry efforts in oncology.

Keywords: D835 mutations; activation-loop mutations; sorafenib.

Fig. 1.

Activity of crenolanib in cells expressing FLT3 D835 mutations. (A) Western blot analysis using 4G10 and anti-FLT3 antibody after immunoprecipitation with anti-FLT3 antibody and Western blot analysis of phospho-ERK (pERK) and ERK performed on whole cell lysates from HB119 and Molm14 cells. Cells were exposed to 100 nM crenolanib for 60 min. (B) Average normalized percentage of live cells as assessed by caspase-3 activation at 72 h and 96 h following treatment with 100 nM crenolanib for HB119, HL60, K562, and Molm14 cells (error bars represent SD of triplicate experiments). (C) Western blot analysis using 4G10 and anti-FLT3 antibody after immunoprecipitation with anti-FLT3 antibody on lysates prepared from MV4;11 cells or primary patient blasts exposed for 60 min to DMSO or 50 nM crenolanib as indicated: 2830, patient FLT3–ITD+/D835Y+, refractory quizartinib; 1727, newly diagnosed patient FLT3–ITD+. (D) Western blot analysis as described in A, including phospho-STAT5 (pSTAT5), phospho-S6 (pS6), STAT5, and S6 on whole cell lysates from parental Molm14 cells and Molm14 cells expressing the D835Y mutation. Cells were exposed to crenolanib in human plasma for 120 min.

Fig. 2.

Crenolanib Is 100-fold more selective for FLT3 compared with KIT. (A) Normalized cell viability of Molm14, MV4;11, HMC 1.1, HMC 1.2, and K562 cells after 48 h in various concentrations of crenolanib (error bars represent SD of triplicates from the same experiment). (B) Western blot analysis using 4G10 and anti-FLT3 antibody after immunoprecipitation with anti-FLT3 antibody and Western blot analysis of pSTAT5, pERK, anti-STAT5, and anti-ERK antibody performed on whole cell lysates from MV4;11 and Molm14 cells. Cells were exposed to crenolanib for 60 min. (C) Western blot analysis of pKIT (Y703), pERK, pS6, KIT, ERK, and S6 antibody performed on whole cell lysates from HMC 1.2 cells. Cells were exposed to crenolanib for 60 min.

Fig. 3.

Activity of crenolanib against quizartinib resistance-causing FLT3–ITD KD mutations. (A) Normalized cell viability of Ba/F3 populations stably expressing FLT3–ITD mutant isoforms after 48 h in various concentrations of crenolanib (error bars represent SD of triplicates from the same experiment). (B) Western blot analysis using pFLT3, pSTAT5, pERK, pS6, FLT3, STAT5, ERK, and S6 performed on lysates from IL-3–independent Ba/F3 populations expressing the FLT3–ITD mutant isoforms indicated. Cells were exposed to crenolanib for 90 min.

Fig. 4.

Activity of crenolanib against FLT3–ITD KD mutations identified in an in vitro mutagenesis screen. (A) Normalized cell viability of Ba/F3 populations stably expressing FLT3–ITD mutant isoforms after 48 h in various concentrations of crenolanib (error bars represent SD of triplicates from the same experiment). (B) Western blot analysis of pFLT3, pSTAT5, pERK, pS6, FLT3, STAT5, ERK, and S6 performed on lysates from IL-3–independent Ba/F3 populations expressing the FLT3–ITD mutant isoforms indicated. Cells were exposed to crenolanib for 90 min.

Fig. 5.

Modeling of FLT3–crenolanib interactions. (A) Cartoon presentation of modeled FLT3 KD in an active conformation. Two orthogonal views are shown. The AL, Helix-C, and P-loop are colored in yellow, pink, and green, respectively. Blue-colored crenolanib is presented in stick and surface mode. The figures were made by PyMOL. (B) Two orthogonal views of coordination of crenolanib in the top scored docking pose. Crenolanib is in blue. The FLT3 KD residues that coordinate crenolanib binding are colored in yellow and white.