Activating mutations in the FLT3 gene, including internal tandem duplications (ITDs) and missense point mutations of the tyrosine kinase domain (TKD), are frequently observed in AML patients and confer poor prognosis (1). Targeting FLT3 ITD mutations using the multi-kinase inhibitor Sorafenib (a type II kinase inhibitor, which binds to inactive conformation of a kinase ATP pocket)(2) showed impressive anti-leukemia effects in FLT3-ITD mutated AML in Phase I/II clinical trials (3) However, resistance/relapse develops regularly during prolonged Sorafenib therapy (4), in part through acquired point mutations of TKD domains. We postulated that the conformational change of FLT3 protein resulting from acquired point mutations limits the accessibility of sorafenib and leads to resistance (5, 6). Recently, Crenolanib, a novel PDGFRβ tyrosine kinase inhibitor, showed impressive anti-tumor effects by targeting the active conformation of a kinase ATP pocket of FLT3 protein (a type I kinase inhibitor). Therefore, we hypothesize that targeting different sites of FLT3 protein simultaneously using different types of kinase inhbitors may be effective in overcoming sorafenib resistance. We here report that Crenolanib has anti-leukemic activity in Sorafenib-resistant cells which harbor both ITD and acquired TKD point mutations i, and that its combination with Sorafenib in Sorafenib-resistant cells exerts synergistic pro-apoptotic effects.
The anti-leukemic activity of Crenolanib was assessed by measuring cell viability (trypan Blue exclusion) and apoptosis induction (annexin V/propidium iodide staining) in isogenic murine Ba/F3 AML cell lines with stable transfection of human FLT3-ITD mutations, in Sorafenib resistant Ba/F3-ITD-Res cells derived from long-term, low-dose exposure of Ba/F3-ITD to Sorafenib in vitro, which harbor N676D and Y842C mutations, and Sorafenib-resistant cell lines Ba/F3-ITD+676, Ba/F3-ITD+842 and Ba/F3-ITD+676/842 which carry ITD and TKD point mutations (N676D, Y842C and N676D/Y842C mutations, respectively). Effects of combinatorial regimen employing Crenolanib and Sorafenib were analyzed using CalcuSyn software (combination index (CI) : CI<1 = synergistic, CI>1 = antagonistic effects).
Results show that single agent Crenolanib induced cell growth arrest in leukemia cells Ba/F3-ITD, Ba/F3-ITD+676, Ba/F3-ITD+842 and Ba/f3-ITD+842/676, at IC50s of 0.012, 0.012 0.037 and 0.038uM, respectively, and induced apoptosis (EC50s) at 0.17, 0.23, 0.19, and 0.22uM, respectively, after 72 hours of treatment. Western Blot showed that Crenolanib profoundly suppressed phosphorylation levels of FLT3 protein and its downstream targets ERK and AKT and induced cleavage of caspase 3. Sorafenib-resistant cells Baf3-ITD+Res and Baf3-ITD+842/676 (EC50s for Sorafenib were 4.2 ± 1.50 and 6.6 ± 0.53 μM, respectively) were exposed to submicromolar concentrations of Crenolanib and Sorafenib concomitantly for 48 h, resulting in impressive synergistic pro-apoptotic effects (CIs were 0.56 ± 0.12 and 0.36 ± 0.04, respectively), implying high synergistic potency of Type I and Type II FLT3 kinase inhibitors, when given concomitantly. In vivo experiments are in progress.
Our findings provide therapeutic rationale for a combinatorial treatment strategy with Crenolanib and Sorafenib of FLT-ITD inhibitor-refractory AML.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.