西奥罗尼杀伤FLT3-ITD阳性急性髓系白血病细胞的作用及机制研究

目的 研究多靶点激酶抑制剂西奥罗尼对FLT3-ITD阳性急性髓系白血病（acute myeloid leukemia，AML）细胞的杀伤作用及其机制。方法 CCK8和集落形成试验检测不同浓度西奥罗尼对FLT3-ITD阳性AML细胞增殖和活力的影响；Annexin V/PI双染法检测西奥罗尼对FLT3-ITD阳性AML细胞株和原代细胞的凋亡诱导情况；Click-iT^® EdU试剂盒评估细胞周期的分布情况；裸鼠皮下瘤模型用于评估西奥罗尼对FLT3-ITD阳性AML细胞体内的杀伤作用；Western blotting探讨西奥罗尼杀伤FLT3-ITD阳性AML的潜在作用机制。结果 在FLT3-ITD阳性AML细胞株模型上，西奥罗尼具有降低细胞的活力、抑制细胞的集落形成、阻滞细胞周期和诱导凋亡的作用；通过8例原代FLT3-ITD阳性AML细胞研究发现，西奥罗尼对原代细胞同样具有诱导凋亡的潜能；裸鼠皮下瘤试验表明西奥罗尼降低FLT3-ITD阳性AML细胞体内增殖能力，延缓肿瘤的生长，而无明显不良反应；机制研究显示西奥罗尼降低VEGFR2的磷酸化水平，且下调其下游的MEK/Erk通路的磷酸化作用。结论 本研究明确了西奥罗尼在体外和体内具有杀伤FLT3-ITD阳性AML细胞的的潜能，抑制VEGFR2/MEK/Erk通路的活性可能是西奥罗尼发挥杀伤FLT3-ITD阳性AML细胞的潜在作用机制。     

FLT3基因突变在急性早幼粒细胞白血病中的临床意义

目的 分析FLT3基因突变在急性早幼粒细胞白血病(APL)中的临床意义.方法 抽取34例APL患者骨髓标本,采用PCR、RT-PCR和DNA酶切技术检测FLT3基因内部串连重复(FLT3-ITD)、FLT3基因15835突变(FLT3-D835),并分析它们与患者临床表现、症状、疗效的关系.结果 APL患者外周血高WBC数(wBC≥10×10~9/L)的阳性率,FLT3-ITD突变型明显高于FLT3-ITD野生型(P<0.05),而FLT3-D835突变与其野生型差异无显著性;骨髓原始细胞+早幼粒细胞之和高含量(≥80%)、B级出血症状、完全缓解(CR)阳性率,FLT3突变型与FLT3野生型患者差异均无统计学意义(P>0.05).结论 大部分FLT3-ITD突变型APL患者常表现高外周血WBC数,骨髓原始细胞与早幼粒细胞之和高含量,B级出血症状及CR率与野生型患者无显著差异.     

FLT3-ITD突变在急性髓细胞白血病中的临床意义

目的探讨FLT3-ITD基因突变在AML中的临床意义。方法回顾性分析我院2010年1月-2012年3月期间诊断为AML患者的临床资料。结果63例初治AML患者,FLT3-ITD基因突变突变率为17．5％;FLT3-ITD阳性组患者的外周血白细胞、幼稚细胞和骨髓原始细胞数显著高于FLT3-ITD阴性组,DFS、OS较阴性组明显缩短。结论FLT3-ITD阳性患者的主要临床特征为高原始细胞,低DFS、OS。     

急性髓系白血病CD123表达及其与FLT3-ITD突变的临床意义

目的:探讨急性髓系白血病(AML)患者中CD123表达及其与FLT3-ITD突变同步检测的临床意义。方法:采用流式细胞仪检测32例AML患者CD123表达,聚合酶链反应(PCR)检测FLT3-ITD突变。结果:32例AML中24例CD123表达阳性,阳性率75.0%,对照组表达阴性。CD123+患者诱导化疗完全缓解(CR)率37.5%,低于CD123-患者(75.0%)(P<0.05)。CD123+患者18个月生存率20.8%,低于CD123-患者(62.5%)(P<0.05)。32例AML中有6例FLT3-ITD突变阳性,阳性率18.8%,其中4例伴有CD123表达阳性,均未获完全缓解,生存期6～8个月。结论:CD123可能作为诊断急性髓系白血病独立参考依据之一及评估预后指标。CD123表达阳性及FLT3-ITD突变同时存在的AML患者生存期更短,预后更差。FLT3-ITD突变阳性患者CD123表达阳性率增高,FLT3-ITD突变在CD123+白血病干细胞最终形成中的意义值得深入探讨。     

急性髓细胞白血病FLT3-ITD基因的表达及临床意义

目的 通过对初诊急性髓细胞白血病(acute myeloid leukemia,AML)患者进行FLT3 -ITD基因突变检测,探讨FLT3-ITD基因突变与AML临床特征的相关性,为AML的临床分层治疗、分子靶向治疗及预后判断提供理论依据.方法 采用实时定量聚合酶链反应(RT-PCB)检测60例初治AML患者和10例对照组患者骨髓FLT3-ITD基因突变.结果 60例初治AML患者突变阳性率为25.0％( 15/60),10例对照组患者骨髓均未检测到该突变;依据法美英协作组(FAB)分型标准备亚型FLT3 -ITD突变率不同,其中以M3、M5型突变率较高;60例初治AML中,28例老年AML组与32例非老年AML组FLT3-ITD基因表达水平比较,差异有统计学意义(P＜0.05);FLT3-ITD阳性组患者具有外周血白细胞数、骨髓白血病细胞比例高的临床特点,并且FLT3-ITD阳性组化疗后完全缓解率(CR)低于FLT3-ITD阴性组(P＜0.05).结论 FLT3-ITD基因突变在AML患者中存在一定的突变率,且多见于M3、M5型患者,FLT3-ITD阳性患者具有外周血白细胞数、骨髓白血病细胞比例高,CR低的临床特点.     

老年急性髓细胞白血病患者FLT3表达的意义

目的研究老年急性髓细胞白血病（AML）患者Fms样的酪氨酸激酶3（FLT3）的表达水平,探讨其在老年AML中的作用机制及临床意义。方法采用半定量RT-PCR方法检测36例老年AML和11例良性血液病为对照组FLT3表达水平。结果36例老年AML组表达水平明显高于对照组,差异有统计学意义（P〈0．001）;复发难治组最高,初治组居中,完全缓解组最低,三者表达水平具有显著性差异（P〈0．05）。结论FLT3在老年AML中存在过度表达,提示FLT3在老年AML的发病机制中有一定作用。FLT3表达水平与疾病进展及预后相关。在检测微小残留病,估计残留白血病细胞负荷量,也可能有一定价值。     

治疗急性髓系白血病新药:FLT3抑制剂 gilteritinib

FMS样酪氨酸激酶3 (FLT3)的基因在造血干细胞增殖、分化及存活方面发挥重要作用。FLT3内部串联重复突变(FLT3-ITD)在急性髓系白血病(AML)中常见,且与迅速复发及生存率低密切相关。gilteritinib (Gil)为新型FLT3抑制剂,由Astellas制药公司生产,于2018年11月经美国食品和药物管理局批准用于FLT3突变型复发难治性AML的治疗。临床试验表明对其他FLT3抑制剂耐药的AML患者,采用Gil治疗仍可取得较好疗效,其最常见的不良反应包括肌肉痛、关节痛、转氨酶升高、疲劳、发热等。     

阿可拉定下调STAT5基因表达对急性髓性白血病发挥抗肿瘤作用

目的研究阿可拉定在体内外对急性髓性白血病(AML)的抗肿瘤作用及其对AML的重要靶点的影响。方法用MTT法测定阿可拉定在体外对MV-4-11细胞生长抑制作用,流式细胞分析不同浓度阿可拉定对MV-4-11细胞的凋亡和周期的影响,RT-PCR检测阿可拉定对FLT3和STAT5基因表达的影响,采用MV-4-11细胞NOD/SCID小鼠皮下移植瘤模型评价阿可拉定在体内抑制肿瘤生长的作用。结果MTT结果表明,阿可拉定能够明显抑制MV-4-11细胞生长,IC50为7.48μmol·L-1;在体外不同浓度的阿可拉定能够明显诱导MV-4-11细胞凋亡,使G0/G1期细胞比例升高且呈现浓度依赖性;体内实验表明,阿可拉定明显抑制荷瘤小鼠皮下瘤块的生长,且用药时间越早对控制肿瘤进展越有利。结论阿可拉定明显下调STAT5基因的表达,在体外能够明显抑制含FLT3-ITD突变体的MV-4-11细胞生长,在体内也表现出良好的抑制肿瘤生长的作用。     

Fms样酪氨酸激酶3表达对评估急性髓系白血病预后的意义

目的 探讨Fms样酪氨酸激酶3(FLT3)表达对评估急性髓系白血病(AML)预后的意义.方法 选取AML患者50例,其中核型正常的AML患者20例,核型异常的AML患者30例.分别于化疗前抽取骨髓3 ml,用PCR方法检测白血病细胞FLT3的表达情况.结果 核型正常的AMLFLT3的表达率为5.0％,核型异常的AMLFLT3的表达率为26.7％.难治复发的AMLFLT3的表达率是33.3％,持续完全缓解的AMLFLT3的表达率是4.5％.FLT3的表达情况与骨髓中高白血病细胞所占比例和高外周血白细胞计数有关,与FAB亚型无关.有FLT3表达的AML患者无病生存(DFS)和总体生存(OS)时间短,无FLT3表达的AML患者DFS和OS时间长,二者差异有统计学意义(x2=4.17,P＜0.05).结论 FLT3是AML患者预后不好的因素,可以指导临床个体化治疗AML.     

VSVG/HIV-1_(NL4-3)Luc假病毒筛选抗HIV-1药物的条件优化及应用

目的建立并优化VSVG/HIV-1_(NL4-3)Luc假病毒筛选抗HIV药物药效模型。方法参照Promega公司的荧光素酶活性分析系统,比较VSVG/HIV-1_(NL4-3)Luc对4种不同细胞的感染力。通过采用3种不同实验条件对不同类型HIV-1上市药物进行活性评价,进一步验证了该模型的可行性与有效性。最后,应用该系统对特定靶点化合物进行筛选,并与HIV-1_(ⅢB)病毒筛选体系所得结果进行比较。结果 VSVG/HIV-1_(NL4-3)Luc对CRFK细胞的感染力最强,报告基因的表达水平与加入的病毒量呈剂量依赖关系。比较3种实验条件下不同阳性药物抑制VSVG/HIV-1_(NL4-3)Luc的半数有效剂量浓度EC50,发现第3种条件最优。而用该假病毒对合成化合物进行筛选,发现其EC50与病毒HIV-1_(ⅢB)所得EC_(50)基本一致。结论成功建立并优化了基于VSVG/HIV-1_(NL4-3)Luc假病毒的抗HIV药物筛选系统。     

Wu-5, a novel USP10 inhibitor,enhances crenolanib-induced FLT3-ITD-positive AML cell death via inhibiting FLT3 and AMPK pathways

The kinase FLT3 internal tandem duplication(FLT3-ITD)is related to poor clinical outcomes of acute myeloid leukemia(AML).FLT3 inhibitors have provided novel strategies for the treatment of FLT3-ITD-positive AML.But they are limited by rapid development of acquired resistance and refractory in monotherapy.Recent evidence shows that inducing the degradation of FLT3-mutated protein is an attractive strategy for the treatment of FLT3-ITD-positive AML,especially those with FLT3 inhibitor resistance.In this study we identified Wu-5 as a novel USP10 inhibitor inducing the degradation of FLT3-mutated protein.We showed that Wu-5 selectively inhibited the viability of FLT3 inhibitor-sensitive(MV4-11,Molm13)and-resistant(MV4-11R)FLT3-ITD-positive AML cells with IC50 of 3.794,5.056,and 8.386μM,respectively.Wu-5(1?10μM)dose-dependently induced apoptosis of MV4-11,Molm13,and MV4-11R cells through the proteasome-mediated degradation of FLT3-ITD.We further demonstrated that Wu-5 directly interacted with and inactivated USP10,the deubiquitinase for FLT3-ITD in vitro(IC50 value=8.3μM)and in FLT3-ITD-positive AML cells.Overexpression of USP10 abrogated Wu-5-induced FLT3-ITD degradation and cell death.Also,the combined treatment of Wu-5 and crenolanib produced synergistic cell death in FLT3-ITD-positive cells via the reduction of both FLT3 and AMPKαproteins.In support of this,AMPKαinhibitor compound C synergistically enhanced the anti-leukemia effect of crenolanib,while AMPKαactivator metformin inhibited the anti-leukemia effect of crenolanib.In summary,we demonstrate that Wu-5,a novel USP10 inhibitor,can overcome FLT3 inhibitor resistance and synergistically enhance the anti-AML effect of crenolanib through targeting FLT3 and AMPKαpathway.     

Targeting of FLT3-ITD kinase contributes to high selectivity of imidazoacridinone C-1311 against FLT3-activated leukemia cells

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.     

Discovery of 4-(4-aminophenyl)-6-phenylisoxazolo[3,4-b]pyridine-3-amine derivatives as novel FLT3 covalent inhibitors for the intervention of acute myeloid leukemia

Small molecule covalent drugs have proved to be desirable therapies especially on drug resistance related to point mutations. Secondary mutations of FLT3 have become the main mechanism of FLT3 inhibitors resistance which further causes the failure of treatment. Herein, a series of 4-(4-aminophenyl)-6-phenylisoxazolo[3,4-b]pyridine-3-amine covalent derivatives were synthesized and optimized to overcome the common secondary resistance mutations of FLT3. Among these derivatives, compound F15 displayed potent inhibition activities against FLT3 (IC₅₀ = 123 nM) and FLT3-internal tandem duplication (ITD) by 80% and 26.06%, respectively, at the concentration of 1 μM. Besides, F15 exhibited potent activity against FLT3-dependent human acute myeloid leukemia (AML) cell lines MOLM-13 (IC₅₀ = 253 nM) and MV4-11 (IC₅₀ = 91 nM), as well as BaF3 cells with variety of secondary mutations. Furthermore, cellular mechanism assays indicated that F15 inhibited phosphorylation of FLT3 and its downstream signaling factors. Notably, F15 could be considered for further development as potential drug candidate to treat AML.     

VX-322: a novel dual receptor tyrosine kinase inhibitor for the treatment of acute myelogenous leukemia

In acute myelogenous leukemia (AML), the FLT3 receptor tyrosine kinase (RTK) is highly expressed with 30% of patients expressing a mutated, constitutively active form of this protein. To inhibit this receptor, VX-322 was developed and found to be very potent against both the FLT3 and c-KIT RTKs with enzyme K(i) values of <1 nM and a cellular IC(50) between 1 and 5 nM. It was efficacious in a FLT3-ITD dependent myeloproliferative mouse model, doubling survival compared to other FLT3 inhibitors, with 25% of the mice cured. Upon treatment of primary AML patient blast cells, the dual inhibition of FLT3 and c-KIT was superior to inhibitors targeting a single RTK. Thus, this compound may represent an improved pharmacologic and selectivity profile that could be effective in the treatment of AML.     

The antitumor compound triazoloacridinone C-1305 inhibits FLT3 kinase activity and potentiates apoptosis in mutant FLT3-ITD leukemia cells

Aim:

FMS-like receptor tyrosine kinase (FLT3) is expressed in some normal hematopoietic cell types and plays an important role in the pathogenesis of acute myeloid leukemia (AML). In this study, we examined the effects of triazoloacridinone C-1305, an antitumor compound, on AML cells with different FLT3 status in vitro.

Methods:

A panel of human leukemic cell lines with different FLT3 status was used, including FLT3 internal tandem duplication mutations (FLT3-ITD, MV-4-11), wild-type FLT3 (RS-4-11) and null-FLT3 (U937) cells. Cell proliferation was estimated using MTT assays, and apoptosis was studied with flow cytometry and fluorescence microscopy. FLT3 kinase activity (phosphorylation of FLT3 at Tyr591) was determined with ELISA and Western blotting. FLT3 downstream signaling proteins involving AKT, MAPK and STAT5 were examined by Western blotting. RNA silencing was used to decrease the endogenous FLT3.

Results:

The mutant FLT3-ITD cells were more sensitive to C-1305 than the wild-type FLT3 and null-FLT3 cells (the IC₅₀ values measured at 24 h were 1.2±0.17, 2.0±09, 7.6±1.6 μmol/L, respectively). C-1305 (1–10 μmol/L) dose-dependently inhibited the kinase activity of FLT3, which was more pronounced in the mutant FLT3-ITD cells than in the wild-type FLT3 cells. Furthermore, C-1305 dose-dependently decreased the phosphorylation of STAT5 and MAPK and the inhibitory phosphorylation of Bad, and induced time- and dose-dependent apoptosis in the 3 cell lines with the null-FLT3 cells being the least susceptible to C-1305-induced apoptosis. Knockdown of FLT3 with siRNA significantly decreased C-1305-induced cytotoxicity in the mutant FLT3-ITD cells.

Conclusion:

C-1305 induces apoptosis in FLT3-ITD-expressing human leukemia cells in vitro, suggesting that mutated FLT3 kinase can be a new target for C-1305, and C-1305 may be a drug candidate for the therapeutic intervention in FLT3-associated AML.     

Novel bis(1H-indol-2-yl)methanones as potent inhibitors of FLT3 and platelet-derived growth factor receptor tyrosine kinase

FLT3 receptor tyrosine kinase is aberrantly active in many cases of acute myeloid leukemia (AML). Recently, bis(1H-indol-2-yl)methanones were found to inhibit FLT3 and PDGFR kinases. To optimize FLT3 activity and selectivity, 35 novel derivatives were synthesized and tested for inhibition of FLT3 and PDGFR autophosphorylation. The most potent FLT3 inhibitors 98 and 102 show IC50 values of 0.06 and 0.04 microM, respectively, and 1 order of magnitude lower PDGFR inhibiting activity. The derivatives 76 and 82 are 20- to 40-fold PDGFR selective. Docking at the recent FLT3 structure suggests a bidentate binding mode with the backbone of Cys-694. Activity and selectivity can be related to interactions of one indole moiety with a hydrophobic pocket including Phe-691, the only different binding site residue (PDGFR Thr-681). Compound 102 inhibited the proliferation of 32D cells expressing wildtype FLT3 or FLT3-ITD similarly as FLT3 autophosphorylation, and induced apoptosis in primary AML patient blasts.