Lawal Bashir, Kuo Yu-Cheng, Khedkar Harshita, Mokgautsi Ntlotlang, Sumitra Maryam Rachmawati, Wu Alexander Th, Huang Hsu-Shan
Department of Pathology, University of Pittsburgh Pittsburgh, PA 15232, U.S.A.
Graduate Institute for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University Taipei 11031, Taiwan.
Am J Cancer Res. 2022 Nov 15;12(11):5140-5159. eCollection 2022.
Acute myeloid leukemia (AML) is a type of leukemia with an aggressive phenotype, that commonly occurs in adults and with disappointing treatment outcomes. Genetic alterations were implicated in the etiology of cancers and form the basis for defining patient prognoses and guiding targeted therapies. In the present study, we leveraged bulk and single-cell RNA sequencing datasets from AML patients to determine the clinical significance of Fms-related receptor tyrosine kinase 3 (FLT3) alterations on the T-cell phenotype and immune response of AML patients. Subsequently, we evaluated the therapeutic potential of Lwk-n019, a novel small-molecule derivative of thiochromeno[2,3-c]quinolin-12-one. Our results suggested that FLT3 plays an important role in the progression, aggressive phenotype, and worse immune response of patients. An FLT3 mutation was associated with dysfunctional T-cell phenotypes, and high risk and shorter survival of AML patients. Our findings further suggested that the aggressiveness of AML and the prognostic role of FLT3 are associated with the co-occurrence of NPM1 and DNMT3A mutations. Lwk-n019 demonstrated dose-dependent anticancer activities against various leukemia cancer cell lines. Lwk-n019 demonstrated highly selective kinase inhibitory activities against the wild-type FLT3 (D835V) and mutant FLT3 (internal tandem duplication (ITD), D835V) with >95% and 99% inhibitory levels, respectively. Moreover, the compound demonstrated the best binding constant (Kd value) of 0.77 µM against FLT3 (ITD, 835V). In addition, Lwk-n019 significantly inhibited the activities of both the topoisomerase I (TOPI) and TOPII enzymes, with higher TOPI inhibitory activity than camptothecin, a clinical inhibitor. While the jejunum, duodenum, cecum, and colon were prime sites of absorption, Lwk-n019 achieved maximum concentration (Cmax), Vd, blood/plasma ratio, time to maximum concentration (Tmax), area under the receiver operating concentration curve (AUC), and AUC values of 0.665 µg/mL, 5.21 Vc, L/kg, 1.5 h, 6634.7, and 6909.2, respectively. In conclusion, Lwk-n019 demonstrated anticancer activities via multi-target inhibition of TOPs and kinases with high inhibition preference for mutant ITD-FLT3. The present pioneer study provides a basis for advanced optimization of drug potency, selectivity, specificity, and other properties desired of anticancer drug leads. Studies are ongoing to determine the full therapeutic properties of Lwk-n019 and the detailed mechanisms of FLT3 in TOP inhibition.
急性髓系白血病（AML）是一种具有侵袭性表型的白血病，常见于成年人，治疗效果令人失望。基因改变与癌症病因有关，是定义患者预后和指导靶向治疗的基础。在本研究中，我们利用AML患者的批量和单细胞RNA测序数据集，以确定Fms相关受体酪氨酸激酶3（FLT3）改变对AML患者T细胞表型和免疫反应的临床意义。随后，我们评估了硫代色烯并[2,3-c]喹啉-12-酮的新型小分子衍生物Lwk-n019的治疗潜力。我们的结果表明，FLT3在患者的病情进展、侵袭性表型和较差的免疫反应中起重要作用。FLT3突变与T细胞表型功能失调、AML患者的高风险和较短生存期相关。我们的研究结果进一步表明，AML的侵袭性和FLT3的预后作用与NPM1和DNMT3A突变的同时出现有关。Lwk-n019对各种白血病癌细胞系表现出剂量依赖性的抗癌活性。Lwk-n019对野生型FLT3（D835V）和突变型FLT3（内部串联重复（ITD），D835V）表现出高度选择性的激酶抑制活性，抑制水平分别>95%和99%。此外，该化合物对FLT3（ITD，835V）的最佳结合常数（Kd值）为0.77µM。此外，Lwk-n019显著抑制拓扑异构酶I（TOPI）和TOPII酶的活性，其TOPI抑制活性高于临床抑制剂喜树碱。虽然空肠、十二指肠、盲肠和结肠是主要吸收部位，但Lwk-n019的最大浓度（Cmax）、分布容积（Vd）、血/浆比、达峰时间（Tmax）、接受者操作浓度曲线下面积（AUC）和AUC值分别为0.665µg/mL、5.21 Vc、L/kg、1.5 h、6634.7和6909.2。总之，Lwk-n019通过对TOPs和激酶的多靶点抑制表现出抗癌活性，对突变型ITD-FLT3具有高度抑制偏好。本开创性研究为抗癌药物先导物所需的药物效力、选择性、特异性和其他特性的进一步优化提供了基础。正在进行研究以确定Lwk-n019的完整治疗特性以及FLT3在TOP抑制中的详细机制。
