Gain-of-Function Mutations of Receptor Tyrosine Kinases in Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in human gastrointestinal tract. We first found that most GISTs expressed KIT, a receptor tyrosine kinase encoded by protooncogene c-kit and that approximately 90% of the sporadic GISTs had somatic gain-of-function mutations of the c-kit gene. Since both GISTs and interstitial cells of Cajal (ICCs) were double-positive for KIT and CD34, GISTs were considered to originate from ICCs or their precursor cells. We also found that germline gain-of-function mutations of the c-kit gene resulted in familial and multiple GISTs with diffuse hyperplasia of ICCs as the preexisting lesion. Moreover, we found that about half of the sporadic GISTs without c-kit gene mutations had gain-of-function mutations of platelet-derived growth factor receptor alpha (PDGFRA) gene that encodes another receptor tyrosine kinase. Imatinib which is known to inhibit constitutively activated BCR-ABL tyrosine kinase in chronic myelogenous leukemia also inhibits constitutive activation of mutated KIT and PDGFRA, and is now being used for metastatic or unresectable GISTs as a molecular target drug. Mutational analyses of c-kit and PDGFRA genes are considered to be significant for prediction of effectiveness of imatinib and newly developed/developing other agents on GISTs. Some mouse models of familial and multiple GISTs have been genetically created, and may be useful for further investigation of GIST biology.     

ALK Kinase Domain Mutations in Primary Anaplastic Large Cell Lymphoma: Consequences on NPM-ALK Activity and Sensitivity to Tyrosine Kinase Inhibitors

ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.     

Antitumor Effect of the Tyrosine Kinase Inhibitor Nilotinib on Gastrointestinal Stromal Tumor (GIST) and Imatinib-Resistant GIST Cells

Despite the benefits of imatinib for treating gastrointestinal stromal tumors (GIST), the prognosis for high risk GIST and imatinib-resistant (IR) GIST remains poor. The mechanisms of imatinib resistance have not yet been fully clarified. The aim of the study was to establish imatinib-resistant cell lines and investigate nilotinib, a second generation tyrosine kinase inhibitor (TKI), in preclinical models of GIST and imatinib-resistant GIST. For a model of imatinib-resistant GIST, we generated resistant cells from GK1C and GK3C cell lines by exposing them to imatinib for 6 months. The parent cell lines GK1C and GK3C showed imatinib sensitivity with IC₅₀ of 4.59±0.97 µM and 11.15±1.48 µM, respectively. The imatinib-resistant cell lines GK1C-IR and GK3C-IR showed imatinib resistance with IC₅₀ values of 11.74±0.17 µM (P<0.001) and 41.37±1.07 µM (P<0.001), respectively. The phosphorylation status of key cell signaling pathways, receptor tyrosine kinase KIT (CD117), platelet-derived growth factor receptor alpha (PDGFRA) and downstream signaling kinases: serine-threonine kinase Akt (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) or the non-receptor tyrosine kinase: proto-oncogene tyrosine-protein kinase Src (SRC), was analyzed in established cell lines and ERK1/2 phosphorylation was found to be increased compared to the parental cells. Nilotinib demonstrated significant antitumor efficacy against GIST xenograft lines and imatinib-resistant GIST cell lines. Thus, nilotinib may have clinical potential for patients with GIST or imatinib-resistant GIST.     

肺癌EGFR突变与酪氨酸激酶抑制剂临床敏感性的关系

表皮生长因子受体(EGFR)酪氨酸激酶抑制剂(TKI)是近年来在临床中使用的一类新的小分子靶向药物,主要用于晚期非小细胞肺癌(NSCLC)的治疗,然而并非所有的NSCLC患者对TKI敏感。近期研究发现,在NSCLC治疗过程中,EGFR突变与TKI临床敏感性密切相关,通过检测肺癌EGFR突变状况可以预测TKI治疗的效果。     

Comparison of Elisa-Based Tyrosine Kinase Assays for Screening EGFR Inhibitors

Abstract

Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer, and therefore PTK inhibitors are currently under intense investigation as potential drug candidates. PTK inhibitor screening data are, however, poorly comparable because of the different assay technologies used. Here we report a comparison of ELISA-based assays for screening epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitory compound libraries to study interassay variations. All assays were based on the same protocol, except for the source of EGFR-TK enzymes. In the first protocol, the enzyme was isolated from A431 cells without affinity purification. In the second protocol, commercial EGFR-TK (Sigma) isolated from A431 cells by affinity-purification was employed. In the third protocol, an enzyme preparation obtained from a recombinant (Baculovirus transfected Sf9 cells) expression system was used. All assays employed the synthetic peptide substrate poly-(Glu, Tyr)_1:4 and an ELISA-based system to detect phosphorylated tyrosine residues by a monoclonal antibody. We observed significant differences in both the activity of the enzymes and in the EGFR-TK inhibitory effect of our reference compound PD153035. The differences were significant in case of A431 cell lysate compared to affinity purified EGFR-TKs derived from either A431cells or Baculovirus transfected Sf9 cells, whereas the latter two showed comparable results. Our data suggest that differences in terms of interassay variation are not related to the source of the enzyme but to its purity; changes in the mode of detection can markedly influence the reproducibility of results. In conclusion, normalization of the EGFR activity used for inhibitor screening and standardization of detection methods enable safe comparison of data.     

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.     

小分子酪氨酸激酶抑制剂耐药机制

表皮生长因子受体（epidermal growth factor receptor,EGFR）通路异常在肿瘤发生、发展过程中起到非常重要的作用,特异性抑制该通路的小分子受体酪氨酸激酶抑制剂在肿瘤治疗上取得了显著的效果,但是该药在临床上已经出现耐药现象,现将有关EGFR基因突变、EGFR旁路信号通路的激活、下游信号分子的结构性活化3个方面对EGFR抑制剂耐药机制的研究进展进行综述。     

Reduced Immunosuppressive Properties of Axitinib in Comparison with Other Tyrosine Kinase Inhibitors

The multikinase inhibitors sunitinib, sorafenib, and axitinib have an impact not only on tumor growth and angiogenesis, but also on the activity and function of immune effector cells. In this study, a comparative analysis of the growth inhibitory properties and apoptosis induction potentials of tyrosine kinase inhibitors on T cells was performed. Tyrosine kinase inhibitor treatment resulted in a dramatic decrease in T cell proliferation along with distinct impacts on the cell cycle progression. This was at least partially associated with an enhanced induction of apoptosis although triggered by distinct apoptotic mechanisms. In contrast to sunitinib and sorafenib, axitinib did not affect the mitochondrial membrane potential (Δψ_m) but resulted in an induction or stabilization of the induced myeloid leukemia cell differentiation protein (Mcl-1), leading to an irreversible arrest in the G₂/M cell cycle phase and delayed apoptosis. Furthermore, the sorafenib-mediated suppression of immune effector cells, in particular the reduction of the CD8⁺ T cell subset along with the down-regulation of key immune cell markers such as chemokine CC motif receptor 7 (CCR7), CD26, CD69, CD25, and CXCR3, was not observed in axitinib-treated immune effector cells. Therefore, axitinib rather than sorafenib seems to be suitable for implementation in complex treatment regimens of cancer patients including immunotherapy.     

小分子 Fms 样酪氨酸激酶 3 抑制剂的研究进展

Fms 样酪氨酸激酶 3（FLT3）是一种重要的Ⅲ型受体酪氨酸激酶,对造血细胞和淋巴细胞的增殖起关键作用,其突变以及过度表 达是造成多种恶性肿瘤的关键因素。通过外源性抑制剂阻断细胞增殖信号的传导来促使肿瘤细胞凋亡是当前治疗肿瘤的重要手段。FLT3 小 分子抑制剂作为一类重要的外源性受体酪氨酸激酶抑制剂已应用于多种恶性肿瘤的治疗并引起广泛关注。综述近 5 年来 FLT3 小分子抑制剂 的研究进展。     

Therapeutic Efficacy Assessment of CK6, a Monoclonal KIT Antibody,in a Panel of Gastrointestinal Stromal Tumor Xenograft Models

We evaluated the efficacy of CK6, a KIT monoclonal antibody, in a panel of human gastrointestinal stromal tumor (GIST) xenograft models. Nude mice were bilaterally transplanted with human GIST xenografts (four patient derived and two cell line derived), treated for 3 weeks, and grouped as follows: control (untreated); CK6 (40 mg/kg, 3 × weekly); imatinib (50 mg/kg, twice daily); sunitinib (40 mg/kg, once daily); imatinib + CK6; sunitinib + CK6 (same doses and schedules as in the single-agent treatments). Tumor volume assessment, Western blot analysis, and histopathology were used for evaluation of efficacy. Statistical analysis was performed using Mann-Whitney U (MWU) and Wilcoxon matched-pairs tests. CK6 as a single agent only reduced tumor growth rate in the UZLX-GIST3 model (P = .053, MWU compared to control), while in none of the other GIST models an effect on tumor growth rate was observed. CK6 did not result in significant anti-proliferative or pro-apoptotic effects in any of the GIST models, and moreover, CK6 did not induce a remarkable inhibition of KIT activation. Furthermore, no synergistic effect of combining CK6 with tyrosine kinase inhibitors (TKIs) was observed. Conversely, in certain GIST xenografts, anti-tumor effects seemed to be inferior under combination treatment compared to single-agent TKI treatment. In the GIST xenografts tested, the anti-tumor efficacy of CK6 was limited. No synergy was observed on combination of CK6 with TKIs in these GIST models. Our findings highlight the importance of using relevant in vivo human tumor xenograft models in the preclinical assessment of drug combination strategies.     

The Tyrosine Kinase Csk Dimerizes through Its SH3 Domain

The Src family kinases possess two sites of tyrosine phosphorylation that are critical to the regulation of kinase activity. Autophosphorylation on an activation loop tyrosine residue (Tyr 416 in commonly used chicken c-Src numbering) increases catalytic activity, while phosphorylation of a C-terminal tyrosine (Tyr 527 in c-Src) inhibits activity. The latter modification is achieved by the tyrosine kinase Csk (C-terminal Src Kinase), but the complete inactivation of the Src family kinases also requires the dephosphorylation of the activation loop tyrosine. The SH3 domain of Csk recruits the tyrosine phosphatase PEP, allowing for the coordinated inhibition of Src family kinase activity. We have discovered that Csk forms homodimers through interactions mediated by the SH3 domain in a manner that buries the recognition surface for SH3 ligands. The formation of this dimer would therefore block the recruitment of tyrosine phosphatases and may have important implications for the regulation of Src kinase activity.     

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.     

Gastrointestinal Stromal Tumors,Somatic Mutations and Candidate Genetic Risk Variants

Gastrointestinal stromal tumors (GISTs) are rare but treatable soft tissue sarcomas. Nearly all GISTs have somatic mutations in either the KIT or PDGFRA gene, but there are no known inherited genetic risk factors. We assessed the relationship between KIT/PDGFRA mutations and select deletions or single nucleotide polymorphisms (SNPs) in 279 participants from a clinical trial of adjuvant imatinib mesylate. Given previous evidence that certain susceptibility loci and carcinogens are associated with characteristic mutations, or “signatures” in other cancers, we hypothesized that the characteristic somatic mutations in the KIT and PDGFRA genes in GIST tumors may similarly be mutational signatures that are causally linked to specific mutagens or susceptibility loci. As previous epidemiologic studies suggest environmental risk factors such as dioxin and radiation exposure may be linked to sarcomas, we chose 208 variants in 39 candidate genes related to DNA repair and dioxin metabolism or response. We calculated adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for the association between each variant and 7 categories of tumor mutation using logistic regression. We also evaluated gene-level effects using the sequence kernel association test (SKAT). Although none of the association p-values were statistically significant after adjustment for multiple comparisons, SNPs in CYP1B1 were strongly associated with KIT exon 11 codon 557-8 deletions (OR = 1.9, 95% CI: 1.3-2.9 for rs2855658 and OR = 1.8, 95% CI: 1.2-2.7 for rs1056836) and wild type GISTs (OR = 2.7, 95% CI: 1.5-4.8 for rs1800440 and OR = 0.5, 95% CI: 0.3-0.9 for rs1056836). CYP1B1 was also associated with these mutations categories in the SKAT analysis (p = 0.002 and p = 0.003, respectively). Other potential risk variants included GSTM1, RAD23B and ERCC2. This preliminary analysis of inherited genetic risk factors for GIST offers some clues about the disease''s genetic origins and provides a starting point for future candidate gene or gene-environment research.     

Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT

Background

The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT.

Methodology/Principal Findings

In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC₅₀) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC₅₀ of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant.

Conclusions

Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity.     

Comparison of Endoscopic and Open Resection for Small Gastric Gastrointestinal Stromal Tumor

The National Comprehensive Cancer Network recommends conservative follow-up for gastric gastrointestinal stromal tumors (GISTs) less than 2 cm. We have previously reported that the mitotic index of 22.22% of small gastric GISTs exceeded 5 per 50 high-power fields and recommended that all small gastric GISTs should be resected once diagnosed. The aim of the present study is to compare the safety and outcomes of endoscopic and open resection of small gastric GISTs. From May 2010 to March 2014, a total of 90 small gastric GIST patients were enrolled in the present study, including 40 patients who underwent surgical resection and 50 patients who underwent endoscopic resection. The clinicopathological characteristics, resection-related factors, and clinical outcomes were recorded and analyzed. The clinicopathological characteristics were comparable between the two groups except for tumor location and DOG-1 expression. Compared with the surgical resection group, the operation time was shorter (P = .000), blood loss was less (P = .000), pain intensity was lower (P < .05), duration of first flatus and defecation was shorter (P < .05), and medical cost of hospitalization was lower (P = .027) in the endoscopic resection group. The complications and postoperative hospital stay were comparable between the two groups. No in situ recurrence or liver metastasis was observed during follow-up. Endoscopic resection of small gastric GISTs is safe and feasible compared with surgical resection, although perforation could not be totally avoided during and after resection. The clinical outcome of endoscopic resection is also favorable.     

Imatinib Mesylate for Patients with Recurrent or Metastatic Gastrointestinal Stromal Tumors Expressing KIT: A Decade Experience from Taiwan

PURPOSE: Our preliminary report of imatinib mesylate (IM) in gastrointestinal stromal tumor (GIST) patients detailed a high response rate; however, the long-term result is still unknown. We conducted an analysis of Taiwan advanced inoperable/metastatic GIST patients treated on IM regarding survival, pattern of failure, potential prognostic factors, and mutational status. PATIENTS AND METHODS: From 2001 to 2010, patients with pathologically proven advanced inoperable/metastatic GIST receiving IM were enrolled onto this study. Data on KIT mutational status, measurable tumor size, and other potential prognostic factors were prospectively collected. Patients were followed up for a median of 33.6 months. RESULTS: There were 171 patients (106 men and 65 women) with response rate, and their clinical benefit for IM was 57.3% and 87.1%, respectively. Median progression-free survival (PFS) and overall survival (OS) for these 171 patients are 37.6 and 71.0 months, respectively. Of 171 patients, 120 (70.2%) remained on long-term IM use. Poor performance status, tumor larger than 11.5 cm, primary resistance, and the presence of an exon 9 mutation were independently associated with unfavorable PFS. Regarding OS, poor performance status, primary resistance, and tumor larger than 11.5 cm were three independently unfavorable predictors. CONCLUSIONS: The median PFS and OS of 171 GIST patients are 37.6 and 71.0 months, respectively. Poor performance status, tumor size larger than 11.5 cm, primary resistance, and an exon 9 mutation were independently associated with unfavorable PFS. Regarding OS, poor performance status, primary resistance, and tumor size larger than 11.5 cm were three independent unfavorable predictors.     

Depression of Neuronal Protein Synthesis Initiation by Protein Tyrosine Kinase Inhibitors

Abstract— Growth factors stimulate cellular protein synthesis, but the intracellular signaling mechanisms that regulate initiation of mRNA translation in neurons have not been clarified. A rate-limiting step in the initiation of protein synthesis is the formation of the ternary complex among GTP, eukaryotic initiation factor 2 (elF-2), and the initiator tRNA. Here we report that genistein, a specific tyrosine kinase inhibitor, decreases tyrosine kinase activity and the content of phosphotyrosine proteins in cultured primary cortical neurons. Genistein inhibits protein synthesis by >80% in a dose-dependent manner (10–80 μg/ml) and concurrently decreases ternary complex formation by 60%. At the doses investigated, genistein depresses tyrosine kinase activity and concomitantly stimulates PKC activity. We propose that a protein tyrosine kinase participates in the initiation of protein synthesis in neurons, by affecting the activity of elF-2 directly or through a protein kinase cascade.