Combined CDK6 and FLT3 kinase inhibition reveals synergistic effects. (A) Cells were sensitized to palbociclib administration by a single dose of FLT3 inhibitor TCS-359 for 3 days. Cell viability and proliferation were assessed by using the CTG assay. Analysis was carried out in triplicate. Error bars indicate ± SEM. A one-way analysis of variance was used for statistical comparison. (B-C) Combined effects of palbociclib with different FLT3 inhibitors tested (TCS-359, quizartinib, and tandutinib) exceeds Bliss prediction indicating synergy. Dose-response surfaces are centered on the half maximal effective concentration (EC₅₀) of each compound in the MOLM-14 cells (B, upper panel). Analysis was carried out in triplicate. Values depicted represent absolute deviations. Observed values were divided through standard deviations (SDs) plus 15th percentile. Needle graphs indicated deviation from Bliss predicted additivity in AML cells carrying mutant FLT3 kinase (MOLM-14) (B, lower panel and C). (D) Potential synergistic drug combination was evaluated in MOLM-14 cells by isobologram analysis using CompuSyn software. The obtained combination index values (<1) indicated synergy. Analysis was performed in triplicate. (E) Dose-response curve with FLT3 inhibitor TCS-359 alone or in the presence of 30 nM palbociclib (based on the isobologram analysis) in the MOLM-14 cell line. Three independent experiments were carried out. Error bars indicate ± SEM. ****P < .0001.