Human KIT-D816Y Stable Cell Line - Ba/F3

Activating D816 mutations in the class III receptor tyrosine kinase KIT are associated with the majority of patients with systemic mastocytosis (SM) but are also associated with core binding factor (CBF) AML, making KIT mutations an attractive therapeutic target for the treatment of these cancers. Crenolanib is a potent and selective inhibitor of wild-type and mutant isoforms of the class III receptor tyrosine kinases FLT3 and PDGFRα/β. Notably, crenolanib inhibits a constitutively active mutant FLT3 isoform generated by an aspartate amino acid substitution at codon 835, which is homologous to codon 816 in the KIT gene—indicating sensitivity to the mutant KIT D816 isoform as well. Here, researchers demonstrate that crenolanib targets KIT D816 in SM and CBF AML models: crenolanib inhibits cell proliferation and initiates apoptosis in mastocytosis cell lines expressing these mutations. Targeting specificity was confirmed using syngeneic cell models. The KIT D816 mutation can be targeted with clinically achievable doses of crenolanib. In addition, a rationale is provided for combining crenolanib with cladribine (2-CDA), a standard of care in SM.

The potency of crenolanib in inhibiting cell proliferation and inducing apoptosis in engineered Ba/F3 cell lines (carrying KIT D816V, KIT D816Y, or FLT3 ITD) showed strong correlation with the corresponding tumor cell lines (HMC1.2, p815, or MOLM14, respectively). In contrast, parental IL-3-dependent Ba/F3 cells did not show any significant sensitivity to crenolanib, exhibiting off-target toxicity at 1000 nM (Figures 1A-1F). Notably, comparative analysis of crenolanib versus dasatinib in inducing apoptosis did reveal a higher sensitivity of crenolanib to the mutant KIT D816V target (Figure 1G). This finding suggests that crenolanib could be used to treat mutant KIT malignancies.

Figure 1. Cellular effects of crenolanib are tyrosine kinase-mediated. (Kampa-Schittenhelm K M, et al., 2017)