Activities Of Commonly Used Tyrosine Kinase Inhibitors Against Wild-Type C-KIT and Potential Impact On Normal Hematopoiesis

The receptor tyrosine kinase (RTK) c-KIT is important in early hematopoiesis, and impaired c-KIT activity is associated with abnormal erythroid and megakaryotic function in animal models. Several approved and investigational small molecule tyrosine kinase inhibitors have off-target activity against c-KIT, with resultant potential for hematopoietic toxicity. To investigate the association between c-KIT inhibition by TKIs and myelosuppression, we determined the inhibitory activity of a series of TKIs against c-KIT using immunoblot estimates of IC50 concentrations, and compared those results with the compounds’ effects on human hematopoiesis in progenitor cell assays. For this study, we used quizartinib, crenolanib, sorafenib, imatinib, pazopanib, and dasatinib. IC50s against FLT3 and c-Kit were established for each kinase with assays performed both in culture medium (RPMI/10%fetal bovine serum) and 100% human plasma. Colony formation assays with normal human bone marrow were performed in quadruplicate with clinically achievable levels of each drug, adjusting for plasma protein binding (and based on published pharmacokinetic data from plasma samples of human subjects). Out of the six TKIs studied, most showed limited to no inhibition of the formation of colony-forming unit-granulocyte macrophage (CFU-GM). Three TKIs, quizartinib, pazopanib, and dasatinib, demonstrated inhibition of burst-forming unit-erythroid (BFU-E) colonies. The highest doses of these drugs inhibited growth of BFU-E colonies by 30% versus untreated control. These same three drugs are also the most potent c-Kit inhibitors of this group, with IC50s of 35, 7.5, and 1.4 nM, respectively, in culture medium. The remaining TKIs, crenolanib, sorafenib, and imatinib, showed far less BFU-E inhibition, none inhibiting colony formation by more than 25%. These TKIs also had higher c-Kit IC50 values of 65, 395, and 122 nM, respectively. We conclude that c-KIT inhibition correlates with eythroid progenitor cell suppression in vitro, and that TKIs that achieve c-KIT inhibitory levels in vivo would be predicted to cause anemia. During clinical development of TKIs that have c-KIT inhibitory activity, attention should be paid to the therapeutic index between the targeted kinase (e.g., FLT3) and c-KIT, so as to avoid unnecessary myelosuppresive effects. Dose range determinations to spare c-Kit inhibition would lead to a decreased risk of disrupting normal hematopoiesis, making treatment far more manageable for these patients.