Skwarska Anna, Augustin Ewa, Beffinger Michał, Wojtczyk Anita, Konicz Sonia, Laskowska Katarzyna, Polewska Joanna
Department of Pharmaceutical Technology and Biochemistry, Chemical Faculty, Gdańsk University of Technology, Gdańsk, Poland.
Biochem Pharmacol. 2015 Jun 15;95(4):238-52. doi: 10.1016/j.bcp.2015.04.006. Epub 2015 Apr 17.
Drugs targeting receptor tyrosine kinase FLT3 are of particular interest since activating FLT3-internal tandem duplication (ITD) mutations abundantly occur in fatal acute myeloid leukemias (AMLs). Imidazoacridinone C-1311, a DNA-reactive inhibitor of topoisomerase II, has been previously shown to be a potent and selective inhibitor of recombinant FLT3. Here, we expand those findings by studying its effect on leukemia cells with wild-type FLT3, FLT3-ITD mutant and no FLT3 receptor. While brief C-1311 exposure blocked wild-type and FLT3-ITD activity, profound and sustained inhibition was achieved only for FLT3-ITD mutants. C-1311 inhibited FLT3 downstream pathways (MAPK and AKT) independent of FLT3 status, yet translation to decreased viability was significant in FLT3-ITD cells. RNA interference against FLT3-ITD reduced cytotoxic effect and apoptosis induced by C-1311, indicating selective inhibition of FLT3-ITD crucial for high efficacy of drug against activated leukemia cells. Cellular responses in treated FLT3-ITD mutants included G1 and G2/M phase arrest, moderate inhibition of Bcl-2, caspase-3 activation, PARP cleavage, and depolarization of mitochondria. Consistent with selective decrease in FLT3-ITD activity, C-1311 remarkably reduced antiapoptotic survivin mRNA and protein expression, correlating well with enhanced apoptosis of FLT3-ITD cells. No survivin decrease and respectively lower level of apoptosis was found in wild-type and null-FLT3 cells. Combination of C-1311 with cytarabine or doxorubicin again showed distinct synergistic activity in FLT3-ITD-positive cells. The ability of C-1311 to selectively target constitutively active FLT3, suggests a favorable therapeutic index for AML carrying FLT3-ITD mutations. Thus further preclinical and clinical studies addressing its potency against FLT3-ITD kinase is well justified.
由于激活型FLT3内部串联重复（ITD）突变在致命性急性髓系白血病（AML）中大量出现，靶向受体酪氨酸激酶FLT3的药物备受关注。咪唑并吖啶酮C-1311是一种DNA反应性拓扑异构酶II抑制剂，先前已被证明是重组FLT3的强效选择性抑制剂。在此，我们通过研究其对具有野生型FLT3、FLT3-ITD突变体和无FLT3受体的白血病细胞的影响来扩展这些发现。虽然短暂暴露于C-1311可阻断野生型和FLT3-ITD的活性，但仅对FLT3-ITD突变体实现了深度且持续的抑制。C-1311抑制FLT3下游通路（MAPK和AKT），与FLT3状态无关，但在FLT3-ITD细胞中，其转化为细胞活力降低具有显著意义。针对FLT3-ITD的RNA干扰降低了C-1311诱导的细胞毒性作用和凋亡，表明对FLT3-ITD的选择性抑制对于药物对抗活化白血病细胞的高效性至关重要。经处理的FLT3-ITD突变体中的细胞反应包括G1期和G2/M期阻滞、对Bcl-2的适度抑制、caspase-3激活、PARP裂解以及线粒体去极化。与FLT3-ITD活性的选择性降低一致，C-1311显著降低了抗凋亡存活素mRNA和蛋白表达，这与FLT3-ITD细胞凋亡增强密切相关。在野生型和无FLT3细胞中未发现存活素降低以及相应较低水平的凋亡。C-1311与阿糖胞苷或多柔比星联合使用在FLT3-ITD阳性细胞中再次显示出明显的协同活性。C-1311选择性靶向组成型活性FLT3的能力表明，对于携带FLT3-ITD突变的AML具有良好的治疗指数。因此，进一步针对其对FLT3-ITD激酶效力的临床前和临床研究是完全合理的。
