Activity of AMN107, a novel aminopyrimidine tyrosine kinase inhibitor, against human FIP1L1-PDGFR-alpha-expressing cells

Idiopathic hypereosinophilic syndrome (HES) is a myeloproliferative disorder characterized by tissue involvement and organ dysfunction due to abnormal eosinophil proliferation. In a subset of patients, this is caused by the FIP1L1-PDGFR-alpha fusion tyrosine kinase. Cumulative evidence indicates that the Bcr-Abl tyrosine kinase inhibitor imatinib mesylate (Gleevec) is active for the treatment of patients with HES, particularly those expressing the FIP1L1-PDGFR-alpha oncoprotein. The novel tyrosine kinase inhibitor AMN107 was initially developed as a potent Bcr-Abl inhibitor based on the molecular structure of imatinib. We tested the in vitro efficacy of imatinib and AMN107 in the EOL-1 cell line and in cells from a patient with HES harboring the FIP1L1-PDGFR-alpha fusion kinase. AMN107 was as potent as imatinib in inducing apoptosis and inhibiting proliferation of EOL-1 cells, with IC(50) values of 0.54 and 0.20 nM, respectively. In addition, both drugs inhibited the phosphorylation of PDGFR-alpha tyrosine kinase with equivalent efficacy. We conclude that AMN107 and imatinib are active and equipotent against cells expressing the FIP1L1-PDGFR-alpha fusion gene.     

Suppression of autophagy sensitizes multidrug resistant cells towards Src tyrosine kinase specific inhibitor PP2

Upregulated Src activity has been implicated in a variety of cancers. Thus, Src family tyrosine kinase (SFK) inhibitors are often effective cancer treatments. Here, we employed 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), a selective SFK inhibitor, to determine the possible involvement of tyrosine phosphorylation in the modulation of autophagy, for overcoming multidrug resistance. We found that multidrug-resistant v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr) were more susceptible to PP2 treatment than were their parental cells (Ras-NIH 3T3). The antiproliferative activity of PP2 appeared to be due to cell-cycle arrest at G1/S without induction of apoptosis. Interestingly, PP2 preferentially induced autophagy in Ras-NIH 3T3 cells but not in Ras-NIH 3T3/Mdr cells, which implies that a high level of autophagy may protect PP2-treated cells from undergoing cell death. PP2-induced autophagy in Ras-NIH 3T3 cells is accompanied by an inhibition of the mTOR signaling pathway. However, we found that in Ras-NIH 3T3/Mdr cells, PP2-induced mTOR inhibition was uncoupled from the induction of autophagy-likely due to the hyperactivation of AMPK by delayed Raf activation. We also found that PP2-induced dissociation of Beclin 1 from Bcl-2 leads to autophagy in Ras-NIH 3T3 cells. Taken together, these results suggest that functional autophagy in response to PP2 may lead to cell survival in Ras-NIH 3T3 cells, while defective autophagy may contribute to inhibition of growth in Ras-NIH 3T3/Mdr cells. Thus, modulators of autophagy may be used beneficially as adjunctive therapeutic agents during the treatment of cancers with SFK inhibitors.     

Simultaneous targeting of Src kinase and receptor tyrosine kinase results in synergistic inhibition of renal cell carcinoma proliferation and migration

There have been recent improvements in the treatment for metastatic renal cell carcinoma (RCC) with receptor tyrosine kinase (RTK) inhibitors being one of newer treatment options. We hypothesized that simultaneous targeting of Src kinase and the RTK may have synergistic effects to further improve therapies on metastatic RCC. The effects of Src kinase inhibitor saracatinib and multiple RTK inhibitor sunitinib on RCC cell line (ACHN) and Caki-1 were studied. Saracatinib alone or in combination with sunitinib inhibited the migration of ACHN and Caki-1 cells in vitro. Activation of migration related components FAK, P130Cas and Paxillin were blocked by saracatinib at 0.05- to 3-μM concentrations. Combined treatment resulted in improved growth inhibition, greater loss of the S phase cell population and decreased clonogenic colony formation compared to sunitinib alone in the metastatic Caki-1 line. Molecular studies in Caki-1 showed that saracatinib alone and in combination with sunitinib inhibited phosphorylation of the cell progression regulator c-Myc in a dose-dependent manner. Sunitinib alone or in combination suppressed cyclin-D1 expression with the combination showing greater dose-dependent effect. Sunitinib inhibited vascular endothelial growth factor (VEGF) secretion through the inhibition of STAT3 signaling and VEGF biosynthesis. HIF1-α expression in normoxic and hypoxic conditions in Caki-1 cells was inhibited by either saracatinib or sunitinib when administered alone, however, a greater reduction occurred when these compounds were given in combination. Targeting Src kinase and RTK simultaneously with saracatinib and sunitinib resulted in 70-80% blockade of RCC cell migration, synergistic inhibition of cell growth and reduction of acquired drug resistance in Caki-1 cells. The results show promise for combination targeted therapy of RCC.     

表皮生长因子受体酪氨酸激酶抑制剂的研究进展

表皮生长因子受体(epidermal growth factor receptor,EGFR)在细胞的信号转导、细胞增殖和分化中发挥着重要的作用,对多种肿瘤的发生和发展也具有重要影响,抑制该受体活性可以有效抑制肿瘤的生长。以此为靶点的抗肿瘤药物的开发,在多种肿瘤治疗中取得了令人鼓舞的疗效。     

The novel tyrosine kinase inhibitor AKN-028 has significant antileukemic activity in cell lines and primary cultures of acute myeloid leukemia

Aberrantly expressed tyrosine kinases have emerged as promising targets for drug development in acute myeloid leukemia (AML). We report that AKN-028, a novel tyrosine kinase inhibitor (TKI), is a potent FMS-like receptor tyrosine kinase 3 (FLT3) inhibitor (IC₅₀=6 nℳ), causing dose-dependent inhibition of FLT3 autophosphorylation. Inhibition of KIT autophosphorylation was shown in a human megakaryoblastic leukemia cell line overexpressing KIT. In a panel of 17 cell lines, AKN-028 showed cytotoxic activity in all five AML cell lines included. AKN-028 triggered apoptosis in MV4-11 by activation of caspase 3. In primary AML samples (n=15), AKN-028 induced a clear dose-dependent cytotoxic response (mean IC₅₀ 1 μℳ). However, no correlation between antileukemic activity and FLT3 mutation status, or to the quantitative expression of FLT3, was observed. Combination studies showed synergistic activity when cytarabine or daunorubicin was added simultaneously or 24 h before AKN-028. In mice, AKN-028 demonstrated high oral bioavailability and antileukemic effect in primary AML and MV4-11 cells, with no major toxicity observed in the experiment. In conclusion, AKN-028 is a novel TKI with significant preclinical antileukemic activity in AML. Possible sequence-dependent synergy with standard AML drugs and good oral bioavailability has made it a candidate drug for clinical trials (ongoing).     

表皮生长因子受体酪氨酸激酶抑制剂的研究进展

表皮生长因子受体（epidermal growth factor receptor，EGFR）在细胞的信号转导、细胞增殖和分化中发挥着重要的作用，对多种肿瘤的发生和发展也具有重要影响，抑制该受体活性可以有效抑制肿瘤的生长。以此为靶点的抗肿瘤药物的开发，在多种肿瘤治疗中取得了令人鼓舞的疗效。     

Lapatinib: a novel dual tyrosine kinase inhibitor

Lapatinib is an active and well-tolerated oral dual tyrosine kinase inhibitor that is able to inhibit epidermal growth factor receptor (EGFR) and HER2 with high specificity. It has shown promising activity in several cancers most notably metastatic breast cancer. Lapatinib is active in both trastuzumab-refractory and -naïve breast cancer and a number of phase III studies are ongoing to fully define its activity. Studies have also been conducted in a range of other cancers including renal cell carcinoma. It is essential that future studies incorporate predictive biomarker subgroup analysis. This will help to ensure that important subgroup activity is not missed and also allow the definition of groups most likely to benefit from lapatinib.     

Breast cancer cell line proliferation blocked by the Src-related Rak tyrosine kinase

Rak is a 54 kDa protein tyrosine kinase originally isolated from breast cancer cells and expressed in epithelial cells. It resembles the protooncogene Src structurally but lacks an amino-terminal myristylation site and localizes to the nuclear and perinuclear regions of the cell. We report here that expression of Rak in 2 different breast cancer cell lines inhibits growth and causes G(1) arrest of the cell cycle. This growth inhibition is kinase-dependent but does not require the Rak SH2 or SH3 domain. Rak also binds to the pRb tumor-suppressor protein but inhibits growth even in cells that lack pRb. These results suggest that Rak regulates cell growth by phosphorylating perinuclear proteins and has a function that is distinct from the Src-related kinase family.     

MGMT modulates glioblastoma angiogenesis and response to the tyrosine kinase inhibitor sunitinib

Angiogenesis inhibitors, such as sunitinib, represent a promising strategy to improve glioblastoma (GBM) tumor response. In this study, we used the O⁶-methylguanine methyltransferase (MGMT)-negative GBM cell line U87MG stably transfected with MGMT (U87/MGMT) to assess whether MGMT expression affects the response to sunitinib. We showed that the addition of sunitinib to standard therapy (temozolomide [TMZ] and radiation therapy [RT]) significantly improved the response of MGMT-positive but not of MGMT-negative cells. Gene expression profiling revealed alterations in the angiogenic profile, as well as differential expression of several receptor tyrosine kinases targeted by sunitinib. MGMT-positive cells displayed higher levels of vascular endothelial growth factor receptor 1 (VEGFR-1) compared with U87/EV cells, whereas they displayed decreased levels of VEGFR-2. Depleting MGMT using O⁶-benzylguanine suggested that the expression of these receptors was directly related to the MGMT status. Also, we showed that MGMT expression was associated with a dramatic increase in the soluble VEGFR-1/VEGFA ratio, thereby suggesting a decrease in bioactive VEGFA and a shift towards an antiangiogenic profile. The reduced angiogenic potential of MGMT-positive cells is supported by: (i) the decreased ability of their secreted factors to induce endothelial tube formation in vitro and (ii) their low tumorigenicity in vivo compared with the MGMT-negative cells. Our study is the first to show a direct link between MGMT expression and decreased angiogenicity and tumorigenicity of GBM cells and suggests the combination of sunitinib and standard therapy as an alternative strategy for GBM patients with MGMT-positive tumors.     

Bruton酪氨酸激酶抑制剂在套细胞淋巴瘤中的研究进展

套细胞淋巴瘤（MCL）是一种高度异质性的疾病,其临床表现多种多样,对常规化疗药物多不敏感,预后相对较差。以依鲁替尼（ibrutinib）为代表的新型Bruton酪氨酸激酶（BTK）靶向药物的出现为MCL的治疗提供了新希望。文章就BTK抑制剂在MCL治疗中的最新研究进展进行综述。     

ZD1839 (Iressa), a novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, potently inhibits the growth of EGFR-positive cancer cell lines with or without erbB2 overexpression.

Overexpression of the growth factor receptors EGFR and erbB2 occurs frequently in several human cancers and is associated with aggressive tumour behaviour and poor patient prognosis. We have investigated the effects of ZD1839 (Iressa), a novel EGFR tyrosine kinase inhibitor, on the growth, in vitro and in vivo, of human cancer cell lines expressing various levels of EGFR and erbB2. Proliferation of EGFR-overexpressing A431 and MDA-MB-231 cells in vitro was potently inhibited (50%-70%) by ZD1839 with half-maximally effective doses in the low nanomolar range. In parallel, ZD1839 blocked autophosphorylation of EGFR and prevented activation of PLC-gamma 1, ERK MAP kinases and PKB/Akt by EGF. It also inhibited proliferation in EGFR(+) cancer cell lines overexpressing erbB2 (SKBr3, SKOV3, BT474) by between 20% and 80%, effects which correlated with inhibition of EGF-dependent erbB2 phosphorylation and activation of ERK MAP kinase and PKB/Akt in SKOV3 cells. Oral administration of ZD1839 inhibited the growth of MDA-MB-231 and SKOV3 tumours, established as xenografts in athymic mice, by 71% and 32%, respectively. Growth inhibition coincided with reduced proliferation but no change in apoptotic index. Collectively, these results show that ZD1839, at the doses studied, is a potent inhibitor of proliferation not only in cells overexpressing EGFR but also in EGFR(+) cells that overexpress erbB2.     

Overexpression of the Axl tyrosine kinase receptor in cutaneous SCC-derived cell lines and tumours

The molecular mechanisms that underlie the development of squamous cell skin cancers (SSC) are poorly understood. We have used oligonucleotide microarrays to compare the differences in cellular gene expression between a series of keratinocyte cell that mimic disease progression with the aim of identifying genes that may potentially contribute towards squamous cell carcinoma (SCC) progression in vivo, and in particular to identify markers that may serve as potential therapeutic targets for SCC treatment. Gene expression differences were corroborated by polymerase chain reaction and Western blotting. We identified Axl, a receptor tyrosine kinase with transforming potential that has also been shown to have a role in cell survival, adhesion and chemotaxis, was upregulated in vitro in SCC-derived cells compared to premalignant cells. Extending the investigation to tumour biopsies showed that the Axl protein was overexpressed in vivo in a series of SCCs.     

Induction of cell cycle arrest and apoptosis in human nasopharyngeal carcinoma cells by ZD6474, an inhibitor of VEGFR tyrosine kinase with additional activity against EGFR tyrosine kinase

ZD6474 is a vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. The present study was undertaken to investigate the direct antiproliferative effect of ZD6474 on human nasopharyngeal carcinoma (NPC) in vitro and the antitumor activity on NPC xenografts in vivo. Results indicated that ZD6474 treatment inhibited EGFR phosphorylation and led to a dose- and time-dependent decrease in NPC cell (CNE-1, CNE-2 and C666-1) proliferation. Further investigation demonstrated G0/G1 cell cycle arrest in all 3 cell lines, which was associated with an upregulation of p21 and/or p27, and downregulation of CDK4, CDK6 and CDK2. ZD6474 treatment also induced apoptosis in CNE-1 and CNE-2 cells. The apoptosis mechanisms involved reduction of Bcl-2 and/or Bcl-XL, induction of Bak and/or Bax, and activation of caspases-3, -9 and/or -8. The in vivo antitumor activity was evaluated in CNE-2 and C666-1 xenografted nude mice. Administration of ZD6474 (25-100 mg/kg/day, once-daily, p.o.) produced a dose-dependent inhibition of tumor growth and prolonged survival in both models. This study suggests that ZD6474 exerts direct antiproliferative effects on NPC cell lines in vitro by inducing G0/G1 arrest and apoptosis, and potent antitumor effects on NPC xenografts in vivo. It indicates that ZD6474 may offer a new and effective treatment for human NPC.     

Hepatic resection for metastatic gastrointestinal stromal tumors in the tyrosine kinase inhibitor era

BACKGROUND:

Before the advent of tyrosine kinase inhibitors (TKIs), surgical resection was the primary treatment for hepatic gastrointestinal stromal tumor (GIST) metastases. Although TKIs have improved survival in the metastatic setting, outcomes after multimodal therapy comprised of hepatectomy and TKIs for GIST are unknown. The objective of this study was to determine whether combination therapy for hepatic GIST metastases is associated with improved overall survival compared with reported outcomes from surgery or TKI therapy alone.

METHODS:

Demographics, clinicopathologic tumor characteristics, treatments, and outcomes of patients who underwent hepatic resection at 3 high‐volume centers from 1995 to 2010 were reviewed.

RESULTS:

In total, 39 patients underwent hepatectomy for metastatic GISTs, and 27 patients received postoperative TKI therapy. At a median follow‐up of 39.7 months, 23 patients (59%) experienced recurrence at a median of 18 months. The 1‐year, 2‐year, and 3‐year overall survival rates were 96.7%, 76.8%, and 67.9%, respectively. Median survival was not reached at 5 years. The rates of severe complication and mortality were 10.2% (4 patients) and 2.5% (1 patient), respectively. When controlling for confounders, postoperative TKI therapy was associated with improved survival (hazard ratio, 0.04; 95% confidence interval, 0.01‐0.50; P = .006), and extrahepatic disease was associated with worse survival (hazard ratio, 9.51; 95% confidence interval, 1.63‐55.7; P = .012).

CONCLUSIONS:

Overall survival after combination therapy exceeded previous reports for the treatment of metastatic GIST with hepatic resection or TKI therapy alone and was significantly enhanced by postoperative TKI therapy. The results from this study support findings that combination therapy for GIST liver metastases comprised of surgical resection and TKI therapy is more effective than surgery or TKI therapy alone. Cancer 2012;3571–3578. © 2011 American Cancer Society.     

Population pharmacokinetics of a HER2 tyrosine kinase inhibitor CP-724,714 in patients with advanced malignant HER2 positive solid tumors

Purpose To develop a population pharmacokinetic (popPK) model for CP-724,714, a novel HER2 tyrosine kinase inhibitor is under development for the treatment of advanced HER2 positive cancers. Methods Concentration-time data (n = 484) from 30 cancer patients receiving daily oral CP-724,714 at doses of 250 mg QD, 250 mg BID, 250 mg TID, and 400 mg BID in 21-day cycles in an open label First-in-Human dose-escalation study were evaluated. Serum concentrations of CP-724,714 were obtained after single dose and at steady state. Nonlinear mixed effect analysis in NONMEM using first order conditional estimation with interaction (FOCEI) was performed. The effect of covariates was assessed. Diagnostic plots, decrease of objective function value (>7.8), bootstrapping, and predictive check were used as model selection criteria. Results A 2-compartment first-order absorption pharmacokinetic (PK) model with inter-subject variability (ISV) on the apparent oral elimination clearance (CL/F), apparent central volume of distribution (V1/F), apparent peripheral volume of distribution (V2/F), and first-order oral absorption rate constant (ka), interoccasion variability (IOV) on CL/F, and body weight (WT) as covariate on CL/F was developed. There was no evidence of dose-dependent and/or time-dependent PK. CL/F increased with increasing WT. The ISV of CL/F was reduced by approximately 20% with WT as a covariate. Age, race, and liver metastasis did not influence CP-724,714 disposition. Conclusions A popPK model was developed that adequately described the pharmacokinetics of CP-724,714. WT was identified as a covariate on CL/F that reduced ISV and improved model performance. Future studies will further assess the importance of WT as a pharmacokinetic covariate. The proposed popPK model can be used to simulate CP-724,714 Phase 2/3 trials.     

c-Met小分子酪氨酸激酶抑制剂ARQ197对肺癌H1299细胞放射增敏作用的实验研究

目的 观察酪氨酸激酶抑制ARQ197对肺癌H1299细胞的放射增敏作用及机制。方法 首先用不同浓度的重组人肝细胞生长因子(HGF)和ARQ197分别处理H1299 细胞,应用克隆形成实验法检测细胞增殖,筛选出用于放射敏感性研究的HGF和ARQ197的合适浓度。随后将细胞分为对照组、HGF处理组、ARQ197处理组、HGF+ARQ197处理组,观察不同组别之间差异。最后用蛋白印记检测HGF、单纯放射或联合应用ARQ197对c-Met及下游Akt和Erk1/2蛋白表达和活化的影响。结果 H1299细胞的克隆形成率随着HGF浓度增加呈进行性升高,而ARQ197则进行性下降。LQ模型细胞存活曲线示HGF、HGF+ARQ197处理及ARQ197对H1299细胞的放射增益比分别为0.85、1.20、1.27(存活分数为0.1时的剂量比)。H1299细胞在HGF刺激后p-cMet、p-Akt、p-Erk1/2高表达,HGF+ARQ197中随着ARQ197浓度增加p-cMet、p-Akt、p-Erk1/2蛋白表达进行性下降。细胞接受2 Gy照射后p-cMet、p-Akt、p-Erk1/2高表达,但照射+ARQ197后p-cMet、p-Akt、p-Erk1/2蛋白表达显著下降,但总的cMet、Akt、Erk1/2蛋白表达无明显变化。结论 ARQ197通过抑制HGF/c-Met及其下游传导通路的活化对离体肺癌H1299细胞有显著放射增敏作用。     

Bosutinib: a third generation tyrosine kinase inhibitor for the treatment of chronic myeloid leukemia

Bosutinib is an oral tyrosine kinase inhibitor (TKI) with very potent dual inhibitory activity against SRC and abelson gene. Bosutinib was approved in 2012 for the treatment of resistant Philadelphia chromosome positive chronic myeloid leukemia (CML). Bosutinib is a very effective TKI against all phases of intolerant or resistant CML regardless of the presence or absence of an abelson gene domain mutation, except for cases with detectable T315I or V299L. Bosutinib is overall well tolerated and associated with a unique, but manageable toxicity profile. Factors that influence the prescribing pattern of this drug are complex and include physicians’, and increasingly patients and families’ preference, patients’ comorbid conditions, schedule of administration, as well as financial factors. This paper provides an overview of CML, the TKI market, pharmacokinetics, pharmacodynamics, clinical efficacy, safety and tolerability of bosutinib.     

酪氨酸激酶抑制剂在费城染色体阳性急性淋巴细胞白血病诱导治疗中的作用

目的 探讨低剂量化疗联合酪氨酸激酶抑制剂(TKI)作为初诊费城染色体阳性急性淋巴细胞白血病(Ph+ ALL)一线诱导治疗方案的可行性.方法 回顾性分析61例初诊Ph+ ALL患者接受不同诱导治疗方案的疗效与不良反应.结果 全部患者初次诱导治疗的完全缓解(CR)率为73.8％(45/61),再次诱导CR率为86.7％(13/15),两程诱导治疗总的CR率为95.1％(58/61),治疗相关死亡1例(1.6％).无论是否联用TKI,常规剂量组与低剂量组有效率差异无统计学意义[未联用组:65.5 ％(19/29)比60.0％(3/5),P=0.812;联用组:90.5％(19/21)比100.0％(6/6),P=0.432];低剂量化疗联合TKI的有效率与单用常规剂量化疗比较,差异亦无统计学意义(P=0.089).无论化疗强度如何,联合TKI均能提高初次诱导治疗有效率(常规剂量组:P=0.041;低剂量组:P=0.087);联用TKI方案总有效率显著高于未联用TKI方案[92.6％(25/27)比64.7％(22/34),P=0.010].对于未获CR患者,初次诱导时未联用TKI者再次诱导治疗联用TKI的有效率明显高于初次诱导曾联用TKI者[100.0％(8/8)比33.3％(1/3),P=0.011].不同遗传学亚组间初次诱导治疗的有效率差异无统计学意义(均P＞0.05),联用TKI可在一定程度上提高有效率,但差异均无统计学意义(均P＞0.05).全部患者初次诱导治疗的感染率为50.8％(31/61),出血发生率为4.9％(3/61).常规剂量组总感染率[56.0％(28/50)]高于低剂量组[27.3％(3/11)],但差异无统计学意义(P=0.084),两组总出血发生率差异亦无统计学意义[6.0％(3/50)比0(0/11),P=0.405].低剂量化疗联合TKI组的感染率低于常规剂量化疗联合TKI组[0(0/6)比71.4％(15/21),P=0.002],也低于单用常规剂量化疗组[0(0/6)比44.8％(13/29),P=0.039],组间出血发生率差异均无统计学意义(均P＞ 0.05).结论 低剂量化疗联合TKI作为Ph+ ALL一线诱导治疗方案值得进一步探索.