crizotinib and Neuroblastoma

Neuroblastoma is an embryonic malignancy of early childhood originating from neural crest cells and showing heterogeneous biological, morphological, genetic and clinical characteristics. The correct stratification of neuroblastoma patients within risk groups (low, intermediate, high and ultra-high) is critical for the adequate treatment of the patients.High-throughput technologies in the Omics disciplines are leading to significant insights into the molecular pathogenesis of neuroblastoma. Nonetheless, further study of Omics data is necessary to better characterise neuroblastoma tumour biology. In the present review, we report an update of compounds that are used in preclinical tests and/or in Phase I-II trials for neuroblastoma. Furthermore, we recapitulate a number of compounds targeting proteins associated to neuroblastoma: MYCN (direct and indirect inhibitors) and downstream targets, Trk, ALK and its downstream signalling pathways. In particular, for the latter, given the frequency of ALK gene deregulation in neuroblastoma patients, we discuss on second-generation ALK inhibitors in preclinical or clinical phases developed for the treatment of neuroblastoma patients resistant to crizotinib.We summarise how Omics drive clinical trials for neuroblastoma treatment and how much the research of biological targets is useful for personalised medicine. Finally, we give an overview of the most recent druggable targets selected by Omics investigation and discuss how the Omics results can provide us additional advantages for overcoming tumour drug resistance.

Topics: Antineoplastic Agents; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Crizotinib; Drug Delivery Systems; Drug Resistance, Neoplasm; Genomics; Humans; Neoplasm Proteins; Neuroblastoma; Pyrazoles; Pyridines

Neuroblastoma is a childhood malignancy that has not yet benefitted from the rapid progress in the development of small-molecule therapeutics for cancer. An opportunity to take advantage of pharmaceutical innovation in this area arose when the identification of ALK fusion proteins in non-small cell lung cancer (NSCLC) occurred in parallel to the discovery of point mutations of ALK in neuroblastomas. ALK is now known to be a marker of poor outcome in neuroblastoma, and therefore, urgent development of specific ALK inhibitors to treat this devastating disease is a necessity. However, the translation of small molecules from adult directly into pediatric practice has thus far been challenging, due to mutation-specific structural variances in the ALK kinase domain. We discuss how the most recent structural and biological characterizations of ALK are directing preclinical and clinical studies of ALK inhibitors for both NSCLC and neuroblastoma.

Topics: Adult; Anaplastic Lymphoma Kinase; Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Child; Crizotinib; Drug Discovery; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; Neuroblastoma; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases

Treatment for neuroblastoma, the most common extracranial childhood tumor, spans a broad range of aggressiveness that mirrors the risk profiles of disease subtypes, with high-risk neuroblastoma still presenting a clinical challenge. Currently, most patients with relapsed neuro-blastoma die of disease and present a major challenge for treatment. New therapeutic options are urgently needed to improve patient survival. Activating mutations in the gene encoding the anaplastic lymphoma kinase (ALK) remain the most frequent druggable mutations identified in neuroblastomas to date. Preclinical data support an oncogene addiction of neuroblastoma cells to mutated ALK and demonstrate that ALK inhibitory therapy strongly combats tumor models. Most recently, pediatric phase I testing has been completed for the first approved ALK inhibitor, Crizotinib, showing very encouraging antitumoral results in neuroblastoma patients. Subsequently, an international phase I study with the second generation ALK inhibitor, LDK-378, will be launched that makes ALK inhibitory therapy also available to pediatric patients in Germany.

Topics: Anaplastic Lymphoma Kinase; Cell Transformation, Neoplastic; Child; Clinical Trials, Phase I as Topic; Crizotinib; DNA Mutational Analysis; Drug Approval; Drug Delivery Systems; Germany; Humans; Neoplasm Staging; Neuroblastoma; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Pyrimidines; Receptor Protein-Tyrosine Kinases; Sulfones

Neuroblastoma, the most common extracranial solid tumor in children, is derived from neural crest cells. Nearly half of patients present with metastatic disease and have a 5-year event-free survival of <50%. New approaches with targeted therapy may improve efficacy without increased toxicity. In this review we evaluate 3 promising targeted therapies: (i) (131)I-metaiodobenzylguanidine (MIBG), a radiopharmaceutical that is taken up by human norepinephrine transporter (hNET), which is expressed in 90% of neuroblastomas; (ii) immunotherapy with monoclonal antibodies targeting the GD2 ganglioside, which is expressed on 98% of neuroblastoma cells; and (iii) inhibitors of anaplastic lymphoma kinase (ALK), a tyrosine kinase that is mutated or amplified in ~10% of neuroblastomas and expressed on the surface of most neuroblastoma cells. Early-phase trials have confirmed the activity of (131)I-MIBG in relapsed neuroblastoma, with response rates of ~30%, but the technical aspects of administering large amounts of radioactivity in young children and limited access to this agent have hindered its incorporation into treatment of newly diagnosed patients. Anti-GD2 antibodies have also shown activity in relapsed disease, and a recent phase III randomized trial showed a significant improvement in event-free survival for patients receiving chimeric anti-GD2 (ch14.18) combined with cytokines and isotretinoin after myeloablative consolidation therapy. A recently approved small-molecule inhibitor of ALK has shown promising preclinical activity for neuroblastoma and is currently in phase I and II trials. This is the first agent directed to a specific mutation in neuroblastoma, and marks a new step toward personalized therapy for neuroblastoma. Further clinical development of targeted treatments offers new hope for children with neuroblastoma.

Topics: 3-Iodobenzylguanidine; Anaplastic Lymphoma Kinase; Antibodies, Monoclonal; Child; Child, Preschool; Combined Modality Therapy; Crizotinib; Gangliosides; Humans; Molecular Targeted Therapy; Neural Crest; Neuroblastoma; Norepinephrine Plasma Membrane Transport Proteins; Precision Medicine; Pyrazoles; Pyridines; Radiopharmaceuticals; Receptor Protein-Tyrosine Kinases

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that affects a number of biological and biochemical functions through normal ligand-dependent signaling. It has oncogenic functions in a number of tumors including non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma, and neuroblastoma when altered by translocation or amplification or mutation. On August 2011, a small molecule inhibitor against ALK, crizotinib, was approved for therapy against NSCLC with ALK translocations. As we determine the molecular heterogeneity of tumors, the potential of ALK as a relevant therapeutic target in a number of malignancies has become apparent. This review will discuss some of the tumor types with oncogenic ALK alterations. The activity and unique toxicities of crizotinib are described, along with potential mechanisms of resistance and new therapies beyond crizotinib.

Topics: Anaplastic Lymphoma Kinase; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Crizotinib; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Lymphoma, Non-Hodgkin; Models, Genetic; Mutation; Neuroblastoma; Oncogene Proteins, Fusion; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Signal Transduction; Translocation, Genetic

Crizotinib is an inhibitor of multiple tyrosine kinases, including the anaplastic lymphoma kinase (ALK). Responses to crizotinib have also been reported in patients with ALK-positive anaplastic large-cell lymphoma (ALCL) and solid tumors with ALK-mutation, including neuroblastoma. Optimal treatment for patients with recurrent or refractory ALK-positive ALCL and neuroblastoma has not been established. There is a need to develop new drugs for these patients. The objectives of this trial are to evaluate the tolerability and safety of crizotinib in Japanese patients with recurrent/refractory ALK-positive ALCL or neuroblastoma (phase I) and its efficacy in recurrent/refractory ALK-positive ALCL (phase II).

Topics: Adolescent; Anaplastic Lymphoma Kinase; Antineoplastic Agents; Child; Child, Preschool; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Crizotinib; Humans; Infant; Lymphoma, Large-Cell, Anaplastic; Multicenter Studies as Topic; Neuroblastoma; Protein Kinase Inhibitors; Recurrence

Adult-onset neuroblastoma (AON) differs significantly in biology and clinical behavior from childhood-onset disease. AON poses therapeutic challenges since tolerance of intensive multimodality therapies that are standard of care for pediatric neuroblastoma (NB) is poor. AON is enriched for somatic mutations including anaplastic lymphoma kinase (. A single-center retrospective review of adults with NB receiving ALKi (2012-2022) was performed. Response was evaluated using International Neuroblastoma Response Criteria.. ALKis, particularly lorlatinib, are effective treatment options for AON. However, AEs necessitating dose reduction are common.

Topics: Adolescent; Adult; Aged; Anaplastic Lymphoma Kinase; Carcinoma, Non-Small-Cell Lung; Crizotinib; Humans; Lactams, Macrocyclic; Lung Neoplasms; Middle Aged; Neuroblastoma; Protein Kinase Inhibitors; Young Adult

Neuroblastoma (NB) is the second leading extracranial solid tumor of early childhood with about two-thirds of cases presenting before the age of 5, and accounts for roughly 15 percent of all pediatric cancer fatalities in the United States. Treatments against NB are lacking, resulting in a low survival rate in high-risk patients. A repurposing approach using already approved or clinical stage compounds can be used for diseases for which the patient population is small, and the commercial market limited. We have used Bayesian machine learning, in vitro cell assays, and combination analysis to identify molecules with potential use for NB. We demonstrated that pyronaridine (SH-SY5Y IC

Topics: Bayes Theorem; Cell Line, Tumor; Child; Child, Preschool; Crizotinib; Drug Repositioning; Etoposide; Fingolimod Hydrochloride; Humans; Neuroblastoma; Niclosamide

Topics: Alternative Splicing; Benzamides; Calmodulin; Cell Line, Tumor; Crizotinib; Doxorubicin; Humans; Indazoles; Neuroblastoma; Oxygen; Proto-Oncogene Proteins c-akt; Receptor, trkA; Ryanodine Receptor Calcium Release Channel

Topics: Anaplastic Lymphoma Kinase; Crizotinib; Humans; Lung Neoplasms; Mutation; Neuroblastoma; Protein Kinase Inhibitors

Anaplastic lymphoma kinase (ALK) aberrations are a promising target for patients with neuroblastoma. We assessed the activity of first-generation ALK inhibitor crizotinib in patients with no known curative treatments and whose tumors harbored an activating ALK alteration.. The objective response rate for patients with neuroblastoma was 15% [95% confidence interval (CI): 3.3%-34.3%]: two with partial responses and 1 with a complete response. All three patients had a somatic ALK Arg1275Gln mutation, the most common ALK hotspot mutation observed in neuroblastoma and the only mutation predicted to be sensitive to ALK inhibition with crizotinib. Two patients had prolonged stable disease (10 and 13 cycles, respectively); both harbored an ALK Arg1275Gln mutation. Three patients with ALK Phe1174Leu mutations progressed during cycle 1 of therapy, and one patient with an ALK Phe1174Val received three cycles before disease progression. The two patients with ALK amplification had no response. The most common adverse event was a decrease in neutrophil count.. Despite limited activity seen in this trial, we conclude that this is more likely due to an inability to reach the higher concentrations of crizotinib needed to overcome the competing ATP affinity.

Topics: Anaplastic Lymphoma Kinase; Child; Crizotinib; Humans; Lung Neoplasms; Neoplasm Recurrence, Local; Neuroblastoma; Protein Kinase Inhibitors

Topics: Adrenal Gland Neoplasms; Anaplastic Lymphoma Kinase; Antineoplastic Agents; Biopsy; Crizotinib; Fatal Outcome; Female; Gene Rearrangement; Humans; Lung Neoplasms; Neoplasm Staging; Neuroblastoma; Tomography, X-Ray Computed; Young Adult

Crizotinib is an oral tyrosine kinase inhibitor, approved by the FDA in 2011, for use in anaplastic lymphoma kinase positive, metastatic, non-small cell lung cancer. Crizotinib inhibits oncogenic protein expression and impairs cellular proliferation in tumors with an overexpressed anaplastic lymphoma kinase gene. Currently used most frequently in the adult patient population, pediatric use is becoming more prominent, specifically in disease states exhibiting anaplastic lymphoma kinase-positive, metastatic disease, such as neuroblastoma. Approximately 8% of neuroblastomas have activating anaplastic lymphoma kinase-mutations, making this a promising target for a difficult-to-treat disease. Studies in the pediatric population are limited. However, targeted anaplastic lymphoma kinase-inhibitor therapies have shown improved outcomes at both one-year and two-year marks in both overall survival and progression free survival in anaplastic lymphoma kinase-positive adult patients with non-small cell lung cancer. One Children's Oncology Group phase I trial examined toxicities associated with anaplastic lymphoma kinase inhibitor therapy in pediatric patients. Results revealed varying grades in severity of neutropenia, dizziness, and liver function test elevation. In the adult population, severe toxicities reported by the manufacturer include effects on liver, cardiac and lung function. Additionally, several cases of severe, erosive, pill-esophagitis due to crizotinib therapy have been documented in the adult population. Erosive esophagitis is common in the pediatric population due to a variety of factors. Ingestion of medications or other corrosive agents accounts for approximately 3-5% (5000-10,000 cases per year) of esophagitis presentation in the pediatric population. Common causative medications include non-steroidal anti-inflammatory drugs, antibiotics such as doxycycline and tetracycline, and ferrous sulfate. Presented here is the first reported case of crizotinib-induced pill esophagitis in a pediatric patient.

Topics: Anaplastic Lymphoma Kinase; Antineoplastic Agents; Child; Crizotinib; Esophagitis; Humans; Male; Neuroblastoma; Protein Kinase Inhibitors; Receptor Protein-Tyrosine Kinases

The prognosis of advanced stage neuroblastoma patients remains poor and, despite intensive therapy, the 5-year survival rate remains less than 50%. We previously identified histone deacetylase (HDAC) 8 as an indicator of poor clinical outcome and a selective drug target for differentiation therapy in vitro and in vivo. Here, we performed kinome-wide RNAi screening to identify genes that are synthetically lethal with HDAC8 inhibitors. These experiments identified the neuroblastoma predisposition gene ALK as a candidate gene. Accordingly, the combination of the ALK/MET inhibitor crizotinib and selective HDAC8 inhibitors (3-6 µM PCI-34051 or 10 µM 20a) efficiently killed neuroblastoma cell lines carrying wildtype ALK (SK-N-BE(2)-C, IMR5/75), amplified ALK (NB-1), and those carrying the activating ALK F1174L mutation (Kelly), and, in cells carrying the activating R1275Q mutation (LAN-5), combination treatment decreased viable cell count. The effective dose of crizotinib in neuroblastoma cell lines ranged from 0.05 µM (ALK-amplified) to 0.8 µM (wildtype ALK). The combinatorial inhibition of ALK and HDAC8 also decreased tumor growth in an in vivo zebrafish xenograft model. Bioinformatic analyses revealed that the mRNA expression level of HDAC8 was significantly correlated with that of ALK in two independent patient cohorts, the Academic Medical Center cohort (n = 88) and the German Neuroblastoma Trial cohort (n = 649), and co-expression of both target genes identified patients with very poor outcome. Mechanistically, HDAC8 and ALK converge at the level of receptor tyrosine kinase (RTK) signaling and their downstream survival pathways, such as ERK signaling. Combination treatment of HDAC8 inhibitor with crizotinib efficiently blocked the activation of growth receptor survival signaling and shifted the cell cycle arrest and differentiation phenotype toward effective cell death of neuroblastoma cell lines, including sensitization of resistant models, but not of normal cells. These findings reveal combined targeting of ALK and HDAC8 as a novel strategy for the treatment of neuroblastoma.

Topics: Anaplastic Lymphoma Kinase; Animals; Antineoplastic Agents; Cell Cycle Checkpoints; Cell Differentiation; Cell Proliferation; Crizotinib; Drug Screening Assays, Antitumor; Histone Deacetylases; Humans; Hydroxamic Acids; Indoles; Neuroblastoma; Protein Kinase Inhibitors; Repressor Proteins; RNA Interference; Tumor Cells, Cultured; Zebrafish

Aberrant activation of anaplastic lymphoma kinase (ALK) can cause sporadic and familial neuroblastoma. Using a proteomics approach, we identified Bruton's tyrosine kinase (BTK) as a novel ALK interaction partner, and the physical interaction was confirmed by co-immunoprecipitation. BTK is expressed in neuroblastoma cell lines and tumor tissues. Its high expression correlates with poor relapse-free survival probability of neuroblastoma patients. Mechanistically, we demonstrated that BTK potentiates ALK-mediated signaling in neuroblastoma, and increases ALK stability by reducing ALK ubiquitination. Both ALK

Topics: Adenine; Agammaglobulinaemia Tyrosine Kinase; Anaplastic Lymphoma Kinase; Animals; Antineoplastic Agents; Crizotinib; Humans; Mice; Mice, Nude; Neuroblastoma; Piperidines; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Signal Transduction; Xenograft Model Antitumor Assays

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is a clinical target of major interest in cancer. Mutations and rearrangements in

Topics: Anaplastic Lymphoma Kinase; Biomarkers, Tumor; Cell Line, Tumor; Crizotinib; Dual-Specificity Phosphatases; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mass Spectrometry; Mitogen-Activated Protein Kinase Phosphatases; Neuroblastoma; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Interaction Mapping; Protein Kinase Inhibitors; Proteome; Proteomics; Receptor, Insulin; RNA, Small Interfering; Sequence Analysis, RNA; Signal Transduction

Targeted inhibition of anaplastic lymphoma kinase (ALK) is a successful approach for the treatment of many ALK-aberrant malignancies; however, the presence of resistant mutations necessitates both the development of more potent compounds and pharmacodynamic methods with which to determine their efficacy. We describe immunoassays designed to quantitate phosphorylation of ALK, and their use in preclinical models of neuroblastoma, a pediatric malignancy in which gain-of-function ALK mutations predict a poor overall outcome to conventional treatment. Validation of the immunoassays is presented using a panel of neuroblastoma cell lines and evidence of on-target ALK inhibition provided by treatment of a genetically engineered murine model of neuroblastoma with two clinical ALK inhibitors, crizotinib and ceritinib, highlighting the superior efficacy of ceritinib.

Topics: Anaplastic Lymphoma Kinase; Crizotinib; Drug Screening Assays, Antitumor; HeLa Cells; Humans; Immunoassay; Neoplasm Proteins; Neuroblastoma; Phosphorylation; Pyrazoles; Pyridines; Pyrimidines; Receptor Protein-Tyrosine Kinases; Sulfones

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 β-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 β-catenin expression, since siRNA knockdown of β-catenin restored the crizotinib-ALK binding and lowered the crizotinib resistance to the level of RU cells. Enforced expression of β-catenin in RU cells resulted in the opposite effects. To conclude, high expression of β-catenin in the stem-like NB cells contributes to their crizotinib resistance. Combining β-catenin inhibitors and ALK inhibitors may be useful in treating NB patients.

Topics: AC133 Antigen; Anaplastic Lymphoma Kinase; beta Catenin; Cell Line, Tumor; Cell Survival; Crizotinib; Drug Resistance, Neoplasm; Gene Expression; Humans; Neuroblastoma; Phenotype; Protein Binding; Protein Kinase Inhibitors; RNA Interference; RNA, Small Interfering

The presence of an ALK aberration correlates with inferior survival for patients with high-risk neuroblastoma. The emergence of ALK inhibitors such as crizotinib has provided novel treatment opportunities. However, certain ALK mutations result in de novo crizotinib resistance, and a phase I trial of crizotinib showed a lack of response in patients harboring those ALK mutations. Thus, understanding mechanisms of resistance and defining circumvention strategies for the clinic is critical.. The sensitivity of human neuroblastoma-derived cell lines, cell line-derived, and patient-derived xenograft (PDX) models with varying ALK statuses to crizotinib combined with topotecan and cyclophosphamide (topo/cyclo) was examined. Cultured cells and xenografts were evaluated for effects of these drugs on proliferation, signaling, and cell death, and assessment of synergy.. In neuroblastoma murine xenografts harboring the most common ALK mutations, including those mutations associated with resistance to crizotinib (but not in those with wild-type ALK), crizotinib combined with topo/cyclo enhanced tumor responses and mouse event-free survival. Crizotinib + topo/cyclo showed synergistic cytotoxicity and higher caspase-dependent apoptosis than crizotinib or topo/cyclo alone in neuroblastoma cell lines with ALK aberrations (mutation or amplification).. Combining crizotinib with chemotherapeutic agents commonly used in treating newly diagnosed patients with high-risk neuroblastoma restores sensitivity in preclinical models harboring both sensitive ALK aberrations and de novo-resistant ALK mutations. These data support clinical testing of crizotinib and conventional chemotherapy with the goal of integrating ALK inhibition into multiagent therapy for ALK-aberrant neuroblastoma patients.

Topics: Anaplastic Lymphoma Kinase; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Survival; Crizotinib; Cyclophosphamide; Dose-Response Relationship, Drug; Drug Synergism; Female; Humans; Inhibitory Concentration 50; Mice, SCID; Mutation; Neuroblastoma; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Topotecan; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Neuroblastomas harboring activating point mutations in anaplastic lymphoma kinase (ALK) are differentially sensitive to the ALK inhibitor crizotinib, with certain mutations conferring intrinsic crizotinib resistance. To overcome this clinical obstacle, our goal was to identify inhibitors with improved potency that can target intractable ALK variants such as F1174L. We find that PF-06463922 has high potency across ALK variants and inhibits ALK more effectively than crizotinib in vitro. Most importantly, PF-06463922 induces complete tumor regression in both crizotinib-resistant and crizotinib-sensitive xenograft mouse models of neuroblastoma, as well as in patient-derived xenografts harboring the crizotinib-resistant F1174L or F1245C mutations. These studies demonstrate that PF-06463922 has the potential to overcome crizotinib resistance and exerts unprecedented activity as a single targeted agent against F1174L and F1245C ALK-mutated xenograft tumors, while also inducing responses in an R1275Q xenograft model. Taken together, these results provide the rationale to move PF-06463922 into clinical trials for treatment of patients with ALK-mutated neuroblastoma.. The next-generation ALK/ROS1 inhibitor PF-06463922 exerts unparalleled activity in ALK-driven neuroblastoma models with primary crizotinib resistance. Our biochemical and in vivo data provide the preclinical rationale for fast-tracking the development of this agent in children with relapsed/refractory ALK-mutant neuroblastoma.

Topics: Aminopyridines; Anaplastic Lymphoma Kinase; Animals; Cell Line, Tumor; Crizotinib; Drug Resistance, Neoplasm; Humans; Lactams; Lactams, Macrocyclic; Mice; Mutation; Neuroblastoma; Phosphorylation; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Treatment Outcome; Xenograft Model Antitumor Assays

The crizotinib-resistant ALK(F1174L) mutation arises de novo in neuroblastoma (NB) and is acquired in ALK translocation-driven cancers, lending impetus to the development of novel anaplastic lymphoma kinase (ALK) inhibitors with different modes of action. The diaminopyrimidine TAE684 and its derivative ceritinib (LDK378), which are structurally distinct from crizotinib, are active against NB cells expressing ALK(F1174L). Here we demonstrate acquired resistance to TAE684 and LDK378 in ALK(F1174L)-driven human NB cells that is linked to overexpression and activation of the AXL tyrosine kinase and epithelial-to-mesenchymal transition (EMT). AXL phosphorylation conferred TAE684 resistance to NB cells through upregulated extracellular signal-regulated kinase (ERK) signaling. Inhibition of AXL partly rescued TAE684 resistance, resensitizing these cells to this compound. AXL activation in resistant cells was mediated through increased expression of the active form of its ligand, GAS6, that also served to stabilize the AXL protein. Although ectopic expression of AXL and TWIST2 individually in TAE684-sensitive parental cells led to the elevated expression of mesenchymal markers and invasive capacity, only AXL overexpression induced resistance to TAE684 as well. TAE684-resistant cells showed greater sensitivity to HSP90 inhibition than did their parental counterparts, with downregulation of AXL and AXL-mediated ERK signaling. Our studies indicate that aberrant AXL signaling and development of an EMT phenotype underlie resistance of ALK(F1174L)-driven NB cells to TAE684 and its derivatives. We suggest that the combination of ALK and AXL or HSP90 inhibitors be considered to delay the emergence of such resistance.

Topics: Anaplastic Lymphoma Kinase; Axl Receptor Tyrosine Kinase; Blotting, Western; Cell Line, Tumor; Cell Survival; Crizotinib; Drug Resistance, Neoplasm; Enzyme Activation; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Mutation; Neuroblastoma; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Pyrazoles; Pyridines; Pyrimidines; Receptor Protein-Tyrosine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sulfones

ALK receptor tyrosine kinase has been shown to be a therapeutic target in neuroblastoma. Germline ALK activating mutations are responsible for the majority of hereditary neuroblastoma and somatic ALK activating mutations are also frequently observed in sporadic cases of advanced NB. Crizotinib, a first-line therapy in the treatment of advanced non-small cell lung cancer (NSCLC) harboring ALK rearrangements, demonstrates striking efficacy against ALK-rearranged NB. However, crizotinib fails to effectively inhibit the activity of ALK when activating mutations are present within its kinase domain, as with the F1174L mutation. Here we show that a new ALK inhibitor AZD3463 effectively suppressed the proliferation of NB cell lines with wild type ALK (WT) as well as ALK activating mutations (F1174L and D1091N) by blocking the ALK-mediated PI3K/AKT/mTOR pathway and ultimately induced apoptosis and autophagy. In addition, AZD3463 enhanced the cytotoxic effects of doxorubicin on NB cells. AZD3463 also exhibited significant therapeutic efficacy on the growth of the NB tumors with WT and F1174L activating mutation ALK in orthotopic xenograft mouse models. These results indicate that AZD3463 is a promising therapeutic agent in the treatment of NB.

Topics: Anaplastic Lymphoma Kinase; Animals; Apoptosis; Autophagy; Cell Line, Tumor; Cell Proliferation; Cell Survival; Crizotinib; Disease Models, Animal; Drug Resistance, Neoplasm; Female; Humans; Mice; Mutation; Neuroblastoma; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Signal Transduction; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK(R1275Q), an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK(R1275Q) knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK(R1275Q) globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK(R1275Q)/MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK(R1275Q)-expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK(R1275Q)/MYCN mice. These results collectively indicate that ALK(R1275Q) confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK(R1275Q)/MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK(R1275Q)-positive tumors.

Topics: Anaplastic Lymphoma Kinase; Animals; Crizotinib; Extracellular Matrix; Mice; Mice, Inbred C57BL; Mutation; N-Myc Proto-Oncogene Protein; Neoplasm Invasiveness; Neuroblastoma; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases

The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALK(F1174L)/MYCN Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients.

Topics: Aminopyridines; Anaplastic Lymphoma Kinase; Animals; Cell Line, Tumor; Cell Proliferation; Clinical Trials as Topic; Crizotinib; Lactams; Lactams, Macrocyclic; Mice, Inbred BALB C; Mice, Nude; Mutation; N-Myc Proto-Oncogene Protein; Neuroblastoma; PC12 Cells; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Rats; Receptor Protein-Tyrosine Kinases; Xenograft Model Antitumor Assays

The treatment of patients with advanced non-small-cell lung cancer harboring chromosomal rearrangements of anaplastic lymphoma kinase (ALK) has been revolutionized by the development of crizotinib, a small-molecule inhibitor of ALK, ROS1, and MET. However, resistance to crizotinib inevitably develops through a variety of mechanisms, leading to relapse both systemically and in the central nervous system (CNS). This has motivated the development of "second-generation" ALK inhibitors, including alectinib and ceritinib, that overcome some of the mutations leading to resistance. However, most of the reported ALK inhibitors do not show inhibition of the G1202R mutant, which is one of the most common mutations. Herein, we report the development of a structural analogue of alectinib (JH-VIII-157-02) that is potent against the G1202R mutant as well as a variety of other frequently observed mutants. In addition, JH-VIII-157-02 is capable of penetrating the CNS of mice following oral dosing.

Topics: Anaplastic Lymphoma Kinase; Animals; Carbazoles; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Lung; Lung Neoplasms; Mice; Molecular Docking Simulation; Neuroblastoma; NIH 3T3 Cells; Piperidines; Point Mutation; Protein Kinase Inhibitors; Receptor Protein-Tyrosine Kinases

Current methods for treatment of high-risk neuroblastoma patients include surgical intervention, in addition to systemic chemotherapy. However, only limited therapeutic tools are available to pediatric surgeons involved in neuroblastoma care, so the development of intraoperative treatment modalities is highly desirable. This study presents a silk film library generated for focal therapy of neuroblastoma; these films were loaded with either the chemotherapeutic agent doxorubicin or the targeted drug crizotinib. Drug release kinetics from the silk films were fine-tuned by changing the amount and physical crosslinking of silk; doxorubicin loaded films were further refined by applying a gold nanocoating. Doxorubicin-loaded, physically crosslinked silk films showed the best in vitro activity and superior in vivo activity in orthotopic neuroblastoma studies when compared to the doxorubicin-equivalent dose administered intravenously. Silk films were also suitable for delivery of the targeted drug crizotinib, as crizotinib-loaded silk films showed an extended release profile and an improved response both in vitro and in vivo when compared to freely diffusible crizotinib. These findings, when combined with prior in vivo data on silk, support a viable future for silk-based anticancer drug delivery systems.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Crizotinib; Doxorubicin; Drug Delivery Systems; Female; Humans; Mice, Nude; Neuroblastoma; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Silk

ALK is a receptor tyrosine kinase with an oncogenic role in various types of human malignancies. Despite constitutive activation of the kinase through gene alterations, such as chromosomal translocation, gene amplification or mutation, treatments with kinase inhibitors invariably lead to the development of resistance. Aiming to develop new tools for ALK targeting, we took advantage of our previous demonstration identifying ALK as a dependence receptor, implying that in the absence of ligand the kinase-inactive ALK triggers or enhances apoptosis. Here, we synthesized peptides mimicking the proapoptotic domain of ALK and investigated their biological effects on tumor cells. We found that an ALK-derived peptide of 36 amino acids (P36) was cytotoxic for ALK-positive anaplastic large-cell lymphoma and neuroblastoma cell lines. In contrast, ALK-negative tumor cells and normal peripheral blood mononuclear cells were insensitive to P36. The cytotoxic effect was due to caspase-dependent apoptosis and required N-myristoylation of the peptide. Two P36-derived shorter peptides as well as a cyclic peptide also induced apoptosis. Surface plasmon resonance and mass spectrometry analysis of P36-interacting proteins from two responsive cell lines, Cost lymphoma and SH-SY5Y neuroblastoma, uncovered partners that could involve p53-dependent signaling and pre-mRNA splicing. Furthermore, siRNA-mediated knockdown of p53 rescued these cells from P36-induced apoptosis. Finally, we observed that a treatment combining P36 with the ALK-specific inhibitor crizotinib resulted in additive cytotoxicity. Therefore, ALK-derived peptides could represent a novel targeted therapy for ALK-positive tumors.

Topics: Anaplastic Lymphoma Kinase; Apoptosis; Biomimetic Materials; Cell Line, Tumor; Crizotinib; HeLa Cells; Humans; Jurkat Cells; Neoplasms; Neuroblastoma; Peptide Fragments; Phosphorylation; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Signal Transduction

Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.

Topics: Anaplastic Lymphoma Kinase; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Crizotinib; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Gene Amplification; Humans; Mechanistic Target of Rapamycin Complex 1; Mice, Inbred NOD; Mice, SCID; Molecular Targeted Therapy; Multiprotein Complexes; Mutation; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Ribosomal Protein S6; RNA Interference; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Transfection; Xenograft Model Antitumor Assays

Neuroblastoma (NB) patients harboring mutated ALK can be expected to potentially benefit from targeted therapy based on ALK tyrosine kinase inhibitor (TKI), such as crizotinib and ceritinib. However, the effect of the treatment varies with different individuals, although with the same genic changes. Axl receptor tyrosine kinase is expressed in a variety of human cancers, but little data are reported in NB, particularly in which carrying mutated ALK. In this study, we focus on the roles of Axl in ALK-mutated NB for investigating rational therapeutic strategy. We found that Axl is expressed in ALK-positive NB tissues and cell lines, and could be effectively activated by its ligand GAS6. Ligand-dependent Axl activation obviously rescued crizotinib-mediated suppression of cell proliferation in ALK-mutated NB cells. Genetic inhibition of Axl with specific small interfering RNA markedly increased the sensitivity of cells to ALK-TKIs. Furthermore, a small-molecule inhibitor of Axl significantly enhanced ALK-targeted therapy, as an increased frequency of apoptosis was observed in NB cells co-expressing ALK and Axl. Taken together, our results demonstrated that activation of Axl could lead to insensitivity to ALK inhibitors, and dual inhibition of ALK and Axl might be a potential therapeutic strategy against ALK-mutated NB.

Topics: Anaplastic Lymphoma Kinase; Animals; Axl Receptor Tyrosine Kinase; Benzocycloheptenes; Crizotinib; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Experimental; Neuroblastoma; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Structure-Activity Relationship; Triazoles; Tumor Cells, Cultured

Genetic studies have established anaplastic lymphoma kinase (ALK), a cell surface receptor tyrosine kinase, as a tractable molecular target in neuroblastoma. We describe comprehensive genomic, biochemical, and computational analyses of ALK mutations across 1,596 diagnostic neuroblastoma samples. ALK tyrosine kinase domain mutations occurred in 8% of samples--at three hot spots and 13 minor sites--and correlated significantly with poorer survival in high- and intermediate-risk neuroblastoma. Biochemical and computational studies distinguished oncogenic (constitutively activating) from nononcogenic mutations and allowed robust computational prediction of their effects. The mutated variants also showed differential in vitro crizotinib sensitivities. Our studies identify ALK genomic status as a clinically important therapeutic stratification tool in neuroblastoma and will allow tailoring of ALK-targeted therapy to specific mutations.

Topics: Anaplastic Lymphoma Kinase; Antineoplastic Agents; Crizotinib; Disease-Free Survival; Drug Resistance, Neoplasm; Humans; Hydrogen Bonding; Infant; Kaplan-Meier Estimate; Kinetics; Models, Molecular; Molecular Targeted Therapy; Mutation, Missense; Neuroblastoma; Oncogenes; Protein Binding; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases

Crizotinib, a dual anaplastic lymphoma kinase (ALK) and mesenchymal-epithelial transition (MET) tyrosine kinase inhibitor, is currently being evaluated for the treatment of neuroblastoma. Its effects are thought to be mediated mainly via its activity against ALK. Although MET genomic/protein expression status might conceivably affect crizotinib efficacy, this issue has hitherto not received attention in neuroblastomas.. MET genomic and protein expression status was characterised by silver in situ hybridisation and immunohistochemistry (IHC) respectively, in a cohort of 54 neuroblastoma samples. MET splice isoforms were characterised in 15 of these samples by quantitative PCR.. One case (1/54; prevalence 1.85%) displayed MET genomic amplification, while another case (1/54; prevalence 1.85%) displayed strong membranous MET protein expression (IHC score 3+). Alternative exon 10-deleted and exon 14-deleted MET splice isoforms were identified.. MET amplification and protein expression, although low in prevalence, are present in neuroblastomas. This has implications when crizotinib is employed as a therapeutic agent in neuroblastomas. Additionally, the existence of alternatively spliced MET isoforms may have clinical and biological implications in neuroblastomas.

Topics: Alternative Splicing; Anaplastic Lymphoma Kinase; Cell Line, Tumor; Child; Child, Preschool; Crizotinib; Epithelial-Mesenchymal Transition; Exons; Female; Genomics; Humans; In Situ Hybridization; Infant; Isoenzymes; Male; Mutation; Neuroblastoma; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Real-Time Polymerase Chain Reaction; Receptor Protein-Tyrosine Kinases; Tissue Array Analysis; Treatment Outcome

Neuroblastoma is a childhood extracranial solid tumour that is associated with a number of genetic changes. Included in these genetic alterations are mutations in the kinase domain of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), which have been found in both somatic and familial neuroblastoma. In order to treat patients accordingly requires characterisation of these mutations in terms of their response to ALK tyrosine kinase inhibitors (TKIs). Here, we report the identification and characterisation of two novel neuroblastoma ALK mutations (A1099T and R1464STOP), which we have investigated together with several previously reported but uncharacterised ALK mutations (T1087I, D1091N, T1151M, M1166R, F1174I and A1234T). In order to understand the potential role of these ALK mutations in neuroblastoma progression, we have employed cell culture-based systems together with the model organism Drosophila as a readout for ligand-independent activity. Mutation of ALK at position 1174 (F1174I) generates a gain-of-function receptor capable of activating intracellular targets such as ERK (extracellular signal regulated kinase) and STAT3 (signal transducer and activator of transcription 3) in a ligand-independent manner. Analysis of these previously uncharacterised ALK mutants and comparison with ALK(F1174) mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These classes are: (i) gain-of-function ligand-independent mutations such as ALK(F1174l), (ii) kinase-dead ALK mutants, e.g. ALK(I1250T) (Schönherr et al., 2011a) and (iii) ALK mutations that are ligand-dependent in nature. Irrespective of the nature of the observed ALK mutants, in every case the activity of the mutant ALK receptors could be abrogated by the ALK inhibitor crizotinib (Xalkori/PF-02341066), albeit with differing levels of sensitivity.

Topics: Anaplastic Lymphoma Kinase; Animals; Cell Culture Techniques; Cell Proliferation; Cell Transformation, Neoplastic; Crizotinib; Disease Models, Animal; Drosophila melanogaster; Humans; Inhibitory Concentration 50; Mutant Proteins; Mutation; Neurites; Neuroblastoma; PC12 Cells; Phenotype; Phosphorylation; Protein Structure, Tertiary; Pyrazoles; Pyridines; Rats; Receptor Protein-Tyrosine Kinases

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in neuroblastoma, a devastating pediatric cancer of the sympathetic nervous system. Germline and somatically acquired ALK aberrations induce increased autophosphorylation, constitutive ALK activation and increased downstream signaling. Thus, ALK is a tractable therapeutic target in neuroblastoma, likely to be susceptible to both small-molecule tyrosine kinase inhibitors and therapeutic antibodies-as has been shown for other receptor tyrosine kinases in malignancies such as breast and lung cancer. Small-molecule inhibitors of ALK are currently being studied in the clinic, but common ALK mutations in neuroblastoma appear to show de novo insensitivity, arguing that complementary therapeutic approaches must be developed. We therefore hypothesized that antibody targeting of ALK may be a relevant strategy for the majority of neuroblastoma patients likely to have ALK-positive tumors. We show here that an antagonistic ALK antibody inhibits cell growth and induces in vitro antibody-dependent cellular cytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity was induced in cell lines harboring either wild type or mutated forms of ALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitor crizotinib sensitized cells to antibody-induced growth inhibition by promoting cell surface accumulation of ALK and thus increasing the accessibility of antigen for antibody binding. These data support the concept of ALK-targeted immunotherapy as a highly promising therapeutic strategy for neuroblastomas with mutated or wild-type ALK.

Topics: Anaplastic Lymphoma Kinase; Antibodies, Monoclonal; Antigens, Neoplasm; Cell Death; Cell Line, Tumor; Cell Proliferation; Crizotinib; Humans; Mutation; Neuroblastoma; Phosphorylation; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Signal Transduction

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that, when genetically altered by mutation, amplification, chromosomal translocation or inversion, has been shown to play an oncogenic role in certain cancers. Small molecule inhibitors targeting the kinase activity of ALK have proven to be effective therapies in certain ALK-driven malignancies and one such inhibitor, crizotinib, is now approved for the treatment of EML4-ALK-driven, non-small cell lung cancer. In neuroblastoma, activating point mutations in the ALK kinase domain can drive disease progression, with the two most common mutations being F1174L and R1275Q. We report here crystal structures of the ALK kinase domain containing the F1174L and R1275Q mutations. Also included are crystal structures of ALK in complex with novel small molecule ALK inhibitors, including a classic type II inhibitor, that stabilize previously unobserved conformations of the ALK activation loop. Collectively, these structures illustrate a different series of activation loop conformations than has been observed in previous ALK crystal structures and provide insight into the activating nature of the R1275Q mutation. The novel active site topologies presented here may also aid the structure-based drug design of a new generation of ALK inhibitors.

Topics: Adenosine Triphosphate; Amino Acid Motifs; Anaplastic Lymphoma Kinase; Animals; Benzoxazoles; Binding, Competitive; Catalytic Domain; Cell Line; Crizotinib; Crystallography, X-Ray; Humans; Hydrogen Bonding; Models, Molecular; Mutation, Missense; Neuroblastoma; Protein Binding; Proteolysis; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Spodoptera; Structural Homology, Protein

Mutations in the kinase domain of ALK (anaplastic lymphoma kinase) have recently been shown to be important for the progression of the childhood tumour neuroblastoma. In the present study we investigate six of the putative reported constitutively active ALK mutations, in positions G1128A, I1171N, F1174L, R1192P, F1245C and R1275Q. Our analyses were performed in cell-culture-based systems with both mouse and human ALK mutant variants and subsequently in a Drosophila melanogaster model system. Our investigation addressed the transforming potential of the putative gain-of-function ALK mutations as well as their signalling potential and the ability of two ATP-competitive inhibitors, Crizotinib (PF-02341066) and NVP-TAE684, to abrogate the activity of ALK. The results of the present study indicate that all mutations tested are of an activating nature and thus are implicated in tumour initiation or progression of neuroblastoma. Importantly for neuroblastoma patients, all ALK mutations used in the present study can be blocked by the inhibitors, although some mutants exhibited higher levels of drug sensitivity than others.

Topics: Anaplastic Lymphoma Kinase; Animals; Animals, Genetically Modified; Antineoplastic Agents; Cell Proliferation; Cell Transformation, Neoplastic; Compound Eye, Arthropod; Crizotinib; Drosophila melanogaster; Extracellular Signal-Regulated MAP Kinases; Humans; Mice; Mutation, Missense; Neurites; Neuroblastoma; PC12 Cells; Phenotype; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyridines; Pyrimidines; Rats; Receptor Protein-Tyrosine Kinases; STAT3 Transcription Factor

EML4-ALK fusions define a subset of lung cancers that can be effectively treated with anaplastic lymphoma kinase (ALK) inhibitors. Unfortunately, the duration of response is heterogeneous and acquired resistance limits their ultimate efficacy. Thus, a better understanding of resistance mechanisms will help to enhance tumor control in EML4-ALK-positive tumors.. By applying orthogonal functional mutagenesis screening approaches, we screened for mutations inducing resistance to the aminopyridine PF02341066 (crizotinib) and/or the diaminopyrimidine TAE684.. Here, we show that the resistance mutation, L1196M, as well as other crizotinib resistance mutations (F1174L and G1269S), are highly sensitive to the structurally unrelated ALK inhibitor TAE684. In addition, we identified two novel EML4-ALK resistance mutations (L1198P and D1203N), which unlike previously reported mutations, induced resistance to both ALK inhibitors. An independent resistance screen in ALK-mutant neuroblastoma cells yielded the same L1198P resistance mutation but defined two additional mutations conferring resistance to TAE684 but not to PF02341066.. Our results show that different ALK resistance mutations as well as different ALK inhibitors impact the therapeutic efficacy in the setting of EML4-ALK fusions and ALK mutations.

Topics: Anaplastic Lymphoma Kinase; Antineoplastic Agents; Cell Cycle Proteins; Cell Line, Tumor; Crizotinib; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; Microtubule-Associated Proteins; Mutation; Neuroblastoma; Oncogene Proteins, Fusion; Polymorphism, Single Nucleotide; Pyrazoles; Pyridines; Pyrimidines; Receptor Protein-Tyrosine Kinases; Serine Endopeptidases

Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neuroblastoma, a cancer of the developing autonomic nervous system that is the most commonly diagnosed malignancy in the first year of life. The most frequent ALK mutations in neuroblastoma cause amino acid substitutions (F1174L and R1275Q) in the intracellular tyrosine kinase domain of the intact ALK receptor. Identification of ALK as an oncogenic driver in neuroblastoma suggests that crizotinib (PF-02341066), a dual-specific inhibitor of the ALK and Met tyrosine kinases, will be useful in treating this malignancy. Here, we assessed the ability of crizotinib to inhibit proliferation of neuroblastoma cell lines and xenografts expressing mutated or wild-type ALK. Crizotinib inhibited proliferation of cell lines expressing either R1275Q-mutated ALK or amplified wild-type ALK. In contrast, cell lines harboring F1174L-mutated ALK were relatively resistant to crizotinib. Biochemical analyses revealed that this reduced susceptibility of F1174L-mutated ALK to crizotinib inhibition resulted from an increased adenosine triphosphate-binding affinity (as also seen in acquired resistance to epidermal growth factor receptor inhibitors). Thus, this effect should be surmountable with higher doses of crizotinib and/or with higher-affinity inhibitors.

Topics: Adenosine Triphosphate; Anaplastic Lymphoma Kinase; Cell Line, Tumor; Crizotinib; Drug Resistance, Neoplasm; Genome, Human; Humans; Kinetics; Models, Molecular; Mutant Proteins; Mutation; Neuroblastoma; Phosphorylation; Protein Kinase Inhibitors; Protein Structure, Tertiary; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases

Topics: Anaplastic Lymphoma Kinase; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Crizotinib; Drug Resistance, Neoplasm; ErbB Receptors; Humans; Lung Neoplasms; Mutation; Neuroblastoma; Phosphatidylinositol 3-Kinases; Piperidines; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Pyrazoles; Pyridines; ras Proteins; Receptor Protein-Tyrosine Kinases; Translocation, Genetic

The ALK kinase inhibitor crizotinib (PF-02341066) is clinically effective in patients with ALK-translocated cancers, but its efficacy will ultimately be limited by acquired drug resistance. Here we report the identification of a secondary mutation in ALK, F1174L, as one cause of crizotinib resistance in a patient with an inflammatory myofibroblastic tumor (IMT) harboring a RANBP2-ALK translocation who progressed while on crizotinib therapy. When present in cis with an ALK translocation, this mutation (also detected in neuroblastomas) causes an increase in ALK phosphorylation, cell growth, and downstream signaling. Furthermore, the F1174L mutation inhibits crizotinib-mediated downregulation of ALK signaling and blocks apoptosis in RANBP2-ALK Ba/F3 cells. A chemically distinct ALK inhibitor, TAE684, and the HSP90 inhibitor 17-AAG are both effective in models harboring the F1174L ALK mutation. Our findings highlight the importance of studying drug resistance mechanisms in order to develop effective clinical treatments for patients with ALK-translocated cancers.

Topics: Anaplastic Lymphoma Kinase; Cell Line; Crizotinib; Drug Resistance, Neoplasm; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Mutation; Neoplasms, Muscle Tissue; Neuroblastoma; Oncogene Proteins, Fusion; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Reverse Transcriptase Polymerase Chain Reaction; Translocation, Genetic