The use of cellular thermal shift assay (CETSA) to study Crizotinib resistance in ALK-expressing human cancers.
ABSTRACT: Various forms of oncogenic ALK proteins have been identified in various types of human cancers. While Crizotinib, an ALK inhibitor, has been found to be therapeutically useful against a subset of ALK(+) tumours, clinical resistance to this drug has been well recognized and the mechanism of this phenomenon is incompletely understood. Using the cellular thermal shift assay (CETSA), we measured the Crizotinib-ALK binding in a panel of ALK(+) cell lines, and correlated the findings with the ALK structure and its interactions with specific binding proteins. The Crizotinib IC50 significantly correlated with Crizotinib-ALK binding. The suboptimal Crizotinib-ALK binding in Crizotinib-resistant cells is not due to the cell-specific environment, since transfection of NPM-ALK into these cells revealed substantial Crizotinib-NPM-ALK binding. Interestingly, we found that the resistant cells expressed higher protein level of ?-catenin and siRNA knockdown restored Crizotinib-ALK binding (correlated with a significant lowering of IC50). Computational analysis of the crystal structures suggests that ?-catenin exerts steric hindrance to the Crizotinib-ALK binding. In conclusion, the Crizotinib-ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity, and Crizotinib-ALK binding is in turn dictated by the structure of ALK and some of its binding partners.
Project description:Anaplastic large cell lymphomas (ALCL) are mainly characterized by harboring the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). The ALK inhibitor, crizotinib specifically induced apoptosis in Ba/F3 cells expressing NPM-ALK by inhibiting the activation of NPM-ALK and its downstream molecule, signal transducer and activator of transcription factor 3 (STAT3). We found that ?-tocopherol, a major component of vitamin E, attenuated the effects of crizotinib independently of its anti-oxidant properties. Although ?-tocopherol suppressed the inhibitory effects of crizotinib on the signaling axis including NPM-ALK and STAT3, it had no influence on the intake of crizotinib into cells. Crizotinib also directly inhibited the kinase activity of NPM-ALK; however, this inhibitory effect was not altered by the co-treatment with ?-tocopherol. Whereas the nuclear localization of NPM-ALK was disappeared by the treatment with crizotinib, the co-treatment with ?-tocopherol swept the effect of crizotinib and caused the localization of NPM-ALK in nucleus. The administration of ?-tocopherol attenuated the anti-tumor activity of crizotinib against NPM-ALK-provoked tumorigenesis in vivo. Furthermore, the ?-tocopherol-induced inhibition of crizotinib-caused apoptosis was also observed in NPM-ALK-positive cells derived from ALCL patients, namely, SUDHL-1 and Ki-JK. Collectively, these results not only revealed the novel mechanism underlying crizotinib-induced apoptosis in NPM-ALK-positive cells, but also suggest that the anti-tumor effects of crizotinib are attenuated when it is taken in combination with vitamin E.
Project description:The regulatory microRNA miR-150 is involved in the development of hemopathies and is downregulated in T-lymphomas, such as anaplastic large-cell lymphoma (ALCL) tumors. ALCL is defined by the presence or absence of translocations that activate the anaplastic lymphoma kinase (ALK), with nucleophosmin-ALK (NPM-ALK) fusions being the most common. Here, we compared samples of primary NPM-ALK(+) and NPM-ALK(-) ALCL to investigate the role of miR-150 downstream of NPM-ALK. Methylation of the MIR150 gene was substantially elevated in NPM-ALK(+) biopsies and correlated with reduced miR-150 expression. In NPM-ALK(+) cell lines, DNA hypermethylation-mediated miR-150 repression required ALK-dependent pathways, as ALK inhibition restored miR-150 expression. Moreover, epigenetic silencing of miR-150 was due to the activation of STAT3, a major downstream substrate of NPM-ALK, in cooperation with DNA methyltransferase 1 (DNMT1). Accordingly, miR-150 repression was turned off following treatment with the DNMT inhibitor, decitabine. In murine NPM-ALK(+) xenograft models, miR-150 upregulation induced antineoplastic activity. Treatment of crizotinib-resistant NPM-ALK(+) KARPAS-299-CR06 cells with decitabine or ectopic miR-150 expression reduced viability and growth. Altogether, our results suggest that hypomethylating drugs, alone or in combination with other agents, may benefit ALK(+) patients harboring tumors resistant to crizotinib and other anti-ALK tyrosine kinase inhibitors (TKIs). Moreover, these results support further work on miR-150 in these and other ALK(+) malignancies.
Project description:Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor involved in both solid and hematological tumors. About 80% of ALK-positive anaplastic large-cell lymphoma (ALCL) cases are characterized by the t(2;5)(p23;q35) translocation, encoding for the aberrant fusion protein nucleophosmin (NPM)-ALK, whereas 5% of non-small-cell lung cancer (NSCLC) patients carry the inv(2)(p21;p23) rearrangement, encoding for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion. The ALK/c-MET/ROS inhibitor crizotinib successfully improved the treatment of ALK-driven diseases. However, several cases of resistance appeared in NSCLC patients, and ALK amino acid substitutions were identified as a leading cause of resistance to crizotinib. Second-generation ALK inhibitors have been developed in order to overcome crizotinib resistance. In this work, we profiled in vitro the activity of crizotinib, AP26113, ASP3026, alectinib, and ceritinib against six mutated forms of ALK associated with clinical resistance to crizotinib (C1156Y, L1196M, L1152R, G1202R, G1269A, and S1206Y) and provide a classification of mutants according to their level of sensitivity/resistance to the drugs. Since the biological activity of ALK mutations extends beyond the specific type of fusion, both NPM-ALK- and EML4-ALK-positive cellular models were used. Our data revealed that most mutants may be targeted by using different inhibitors. One relevant exception is represented by the G1202R substitution, which was highly resistant to all drugs (>10-fold increased IC50 compared to wild type) and may represent the most challenging mutation to overcome. These results provide a prediction of cross-resistance of known crizotinib-resistant mutations against all second-generation tyrosine kinase inhibitors (TKIs) clinically available, and therefore could be a useful tool to help clinicians in the management of crizotinib-resistance cases.
Project description:ALK has been identified as a novel therapeutic target in neuroblastoma (NB), but resistance to ALK inhibitors (such as crizotinib) is well recognized. We recently published that the crizotinib sensitivity in NB cells strongly correlates with the crizotinib-ALK binding, and ?-catenin effectively hinders this interaction and confers crizotinib resistance. Here, we asked if these observations hold true for the stem-like cells in NB cells, which were purified based on their responsiveness to a Sox2 reporter. Compared to bulk, reporter unresponsive (RU) cells, reporter responsive (RR) cells had significantly higher neurosphere formation ability, expression of CD133/nestin and chemo-resistance. Using the cellular thermal shift assay, we found that RR cells exhibited significantly weaker crizotinib-ALK binding and higher crizotinib resistance than RU cells. The suboptimal crizotinib-ALK binding in RR cells can be attributed to their high ?-catenin expression, since siRNA knockdown of ?-catenin restored the crizotinib-ALK binding and lowered the crizotinib resistance to the level of RU cells. Enforced expression of ?-catenin in RU cells resulted in the opposite effects. To conclude, high expression of ?-catenin in the stem-like NB cells contributes to their crizotinib resistance. Combining ?-catenin inhibitors and ALK inhibitors may be useful in treating NB patients.
Project description:Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that has been recognized as a therapeutic target for EML4-ALK fusion-positive nonsmall cell lung cancer (NSCLC) treatment using type I kinase inhibitors such as crizotinib to take over the ATP binding site. According to Shaw's measurements, ALK carrying G1202R mutation shows reduced response to crizotinib (IC50?=?382?nM vs. IC50?=?20?nM for wild-type), whereas L1198F mutant is more responsive (IC50?=?0.4?nM). Interestingly, the double mutant L1198F/G1202R maintains a similar response (IC50?=?31?nM) to the wild-type. Herein we conducted molecular modeling simulations to elucidate the varied crizotinib sensitivities in three mutants carrying L1198F and/or G1202R. Both L1198 and G1202 are near the ATP pocket. Mutation G1202R causes steric hindrance that blocks crizotinib accessibility, which greatly reduces efficacy, whereas mutation L1198F enlarges the binding pocket entrance and hydrophobically interacts with crizotinib to enhance sensitivity. With respect to the double mutant L1198F/G1202R, F1198 indirectly pulls R1202 away from the binding entrance and consequently alleviates the steric obstacle introduced by R1202. These results demonstrated how the mutated residues tune the crizotinib response and may assist kinase inhibitor development especially for ALK G1202R, analogous to the ROS1 G2302R and MET G1163R mutations that are also resistant to crizotinib treatment in NSCLC.
Project description:Anaplastic large cell lymphoma (ALCL) is a rare aggresive non-Hodgkin lymphoma, of which over 50% of cases have an aberrant nucleophosmin (NPM)?anaplastic lymphoma kinase (ALK) fusion protein. Both mechanistic target of rapamycin (mTOR) inhibitor everolimus and ALK inhibitor crizotinib have shown promising antitumor activity in ALK-positive cancer cell lines. However, their combined effect has not yet been investigated.We evaluated the anti-proliferative effects of everolimus and/or crizotinib in ALK-positive ALCL cell lines, Karpas 299 and SU-DHL-1, and lung adenocarcinoma cell line, NCI-H2228.We found that individually, both everolimus and crizotinib potently inhibited cell growth in a dose-dependent manner in both Karpas 299 and SU-DHL-1 cells. A combination of these agents synergistically inhibited proliferation in the two cell lines. Crizotinib down-regulated aberrant AKT and ERK phosphorylation induced by everolimus. Combination treatment also significantly increased G0/G1 cell-cycle arrest, DNA damage, and apoptosis compared with everolimus or crizotinib alone in ALK-positive ALCL cells. In the Karpas 299 xenograft model, the combination treatment exerted a stronger antitumor effect than monotherapies, without significant change in body weight. The synergistic effect of everolimus and crizotinib was also reproduced in the ALK-positive lung adenocarcinoma cell line NCI-H2228. The combination treatment abrogated phosphoinositide 3-kinase/AKT and mTOR signaling pathways with little effect on the Ras/ERK pathway in NCI-H2228 cells.Crizotinib combinedwith everolimus synergistically inhibits proliferation of ALK-positive ALCL cells. Our results suggest that this novel combination is worthy of further clinical development in patients with ALK-positive ALCL.
Project description:Purpose Fusions involving the ALK gene are the predominant genetic lesion underlying pediatric anaplastic large cell lymphomas (ALCL) and inflammatory myofibroblastic tumors (IMTs). We assessed the activity of the ALK inhibitor crizotinib in patients who had no known curative treatment options at diagnosis or with relapsed/recurrent disease. Methods In this study, 26 patients with relapsed/refractory ALK-positive ALCL and 14 patients with metastatic or inoperable ALK-positive IMT received crizotinib orally twice daily. Study objectives were measurement of efficacy and safety. Correlative studies evaluated the serial detection of NPM-ALK fusion transcripts in patients with ALCL. Results The overall response rates for patients with ALCL treated at doses of 165 (ALCL165) and 280 (ALCL280) mg/m2 were 83% and 90%, respectively. The overall response rate for patients with IMT (treated at 100, 165, and 280 mg/m2/dose) was 86%. A complete response was observed in 83% (five of six) of ALCL165, 80% (16 of 20) of ALCL280, and 36% (five of 14) of patients with IMT. Partial response rates were 0% (none of six), 10% (two of 20), and 50% (seven of 14), respectively. The median duration of therapy was 2.79, 0.4, and 1.63 years, respectively, with 12 patients ceasing protocol therapy to proceed to transplantation. The most common drug-related adverse event was decrease in neutrophil count in 33% and 70% of the ALCL165 and ALCL280 groups, respectively, and in 43% of patients with IMT. Levels of NPM-ALK decreased during therapy in most patients with ALCL. Conclusion The robust and sustained clinical responses to crizotinib therapy in patients with relapsed ALCL and metastatic or unresectable IMT highlight the importance of the ALK pathway in these diseases.
Project description:NPM-ALK? T-cell anaplastic large-cell lymphoma (ALCL) is an aggressive type of cancer. Standard treatment of NPM-ALK? ALCL is CHOP polychemotherapy. Although patients initially respond favorably to CHOP, resistance, relapse, and death frequently occur. Recently, selective targeting of ALK has emerged as an alternative therapeutic strategy. ASP3026 is a second-generation ALK inhibitor that can overcome crizotinib resistance in non-small cell lung cancer, and is currently being evaluated in clinical trials of patients with ALK? solid tumors. However, NPM-ALK? ALCL patients are not included in these trials. We studied the effects of ASP3026 on NPM-ALK? ALCL cell lines in vitro and on systemic lymphoma growth in vivo. ASP3026 decreased the viability, proliferation, and colony formation, as well as induced apoptotic cell death of NPM-ALK? ALCL cells. In addition, ASP3026 significantly reduced the proliferation of 293T cells transfected with NPM-ALK mutants that are resistant to crizotinib and downregulated tyrosine phosphorylation of these mutants. Moreover, ASP3026 abrogated systemic NPM-ALK? ALCL growth in mice. Importantly, the survival of ASP3026-treated mice was superior to that of control and CHOP-treated mice. Our data suggest that ASP3026 is an effective treatment for NPM-ALK? ALCL, and support the enrollment of patients with this lymphoma in the ongoing clinical trials.
Project description:In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor. When her tumor relapsed, sequencing of the resistant tumor revealed an ALK L1198F mutation in addition to the C1156Y mutation. The L1198F substitution confers resistance to lorlatinib through steric interference with drug binding. However, L1198F paradoxically enhances binding to crizotinib, negating the effect of C1156Y and resensitizing resistant cancers to crizotinib. The patient received crizotinib again, and her cancer-related symptoms and liver failure resolved. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT01970865.).
Project description: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.