crizotinib and Cell-Transformation--Neoplastic

Anaplastic lymphoma kinase (ALK) has been found to fuse with other partners, such as echinoderm microtubule-associated protein-like 4 (EML4), leading to potent malignant transformation in lung cancer, specifically non-small-cell lung cancer (NSCLC). The frequency of the ALK rearrangement in patients with NSCLC is reported to be 4-7%, and the rearrangement is frequently observed in relatively younger patients, non- or light smokers and those with adenocarcinoma histology without other genetic disorders, such as mutations of the epidermal growth factor receptor gene. Crizotinib, which is a first-in-class ALK tyrosine kinase inhibitor (TKI), was shown to be effective and well tolerated in ALK-positive NSCLC patients by a single-arm phase I study. Furthermore, a phase III randomized study demonstrated the superiority of crizotinib to standard chemotherapy (pemetrexed or docetaxel) in the treatment of NSCLC patients harboring the ALK rearrangement who had received one prior platinum-based chemotherapy. However, the mechanisms of resistance to crizotinib are major concerns when administering crizotinib to ALK-positive NSCLC patients, and they include second mutations and a gain in the copy number of the ALK gene, activation of other oncogenes, etc. Treatment strategies to overcome these mechanisms of resistance have been developed, including the use of second-generation ALK inhibitors, such as alectinib and ceritinib, heat shock protein 90 inhibitors and so on. In this article, we review the pre-clinical and clinical data regarding the biologal and clinical significance of the ALK rearrangement in lung cancer.

Topics: Age Factors; Anaplastic Lymphoma Kinase; Carbazoles; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Transformation, Neoplastic; Crizotinib; ErbB Receptors; Gene Fusion; Gene Rearrangement; Humans; Lung Neoplasms; Microtubule-Associated Proteins; Molecular Targeted Therapy; Mutation; Oncogene Proteins, Fusion; Piperidines; Pyrazoles; Pyridines; Pyrimidines; Receptor Protein-Tyrosine Kinases; Serine Endopeptidases; Smoking; Sulfones

The burgeoning field of anaplastic lymphoma kinase (ALK) in cancer encompasses many cancer types, from very rare cancers to the more prevalent non-small-cell lung cancer (NSCLC). The common activation of ALK has led to the use of the ALK tyrosine kinase inhibitor (TKI) crizotinib in a range of patient populations and to the rapid development of second-generation drugs targeting ALK. In this Review, we discuss our current understanding of ALK function in human cancer and the implications for tumour treatment.

Topics: Anaplastic Lymphoma Kinase; Animals; Antineoplastic Agents; Caenorhabditis elegans Proteins; Cell Transformation, Neoplastic; Clinical Trials as Topic; Crizotinib; Drosophila Proteins; Drug Resistance, Neoplasm; Enzyme Induction; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Humans; Lymphoma, Large-Cell, Anaplastic; Mice; Models, Biological; Models, Molecular; Mutation; Neoplasm Proteins; Neoplasms; Oncogene Proteins, Fusion; Protein Conformation; Protein-Tyrosine Kinases; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Signal Transduction; Translocation, Genetic; Zebrafish Proteins

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

C-ros oncogene 1 receptor tyrosine kinase (ROS1) rearrangement has been detected in patients with advanced non-small cell lung cancer (NSCLC). Although ROS1 tyrosine kinase inhibitors (TKIs) provide a survival benefit for patients with ROS1-rearranged advanced NSCLC, subsequent therapy remains limited. Small cell transformation is an important mechanism of drug resistance in epidermal growth factor receptor-mutant NSCLC. However, its significance in mediating ROS1 resistance has not been determined yet. Here, we present the case of a 63-year-old man with ROS1-rearranged advanced NSCLC who had disease progression with small cell transformation of the mediastinal lymph node after 8 months of treatment with crizotinib. More importantly, fluorescence in situ hybridization of post-progression tumor biopsy demonstrated retention of ROS1 rearrangement. Tissue biopsy remains indispensable for patients who acquire resistance to ROS1 TKIs.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Transformation, Neoplastic; Crizotinib; Drug Resistance, Neoplasm; Gene Rearrangement; Humans; Lung Neoplasms; Male; Middle Aged; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Small Cell Lung Carcinoma

Topics: Adenocarcinoma of Lung; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Crizotinib; Humans; Lung Neoplasms; Male; Middle Aged; Oncogene Proteins, Fusion; Prognosis

Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) treated with ALK tyrosine kinase inhibitor (TKI) eventually acquires resistance to the treatment. However, our current knowledge regarding the resistance mechanisms is based on non-synonymous mutation and amplification in ALK, with the reasons still unknown for nearly half of all such cases. Other than genomic alteration as a resistance mechanism, up to 10% of NSCLC with activating epithelial growth factor receptor (EGFR) mutation showed resistance to EGFR TKI through histologic transformation. Although limited in number, there are cases showing transformed samples retaining the initial genomic alteration, which support lineage transition as a novel resistance mechanism. In this report, we described the first case of squamous cell carcinoma (SCC) transformation from adenocarcinoma (ADC) in NSCLC with ALK rearrangement after treatment with ALK TKI.

Topics: Adenocarcinoma; Anaplastic Lymphoma Kinase; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Dedifferentiation; Cell Lineage; Cell Transformation, Neoplastic; Crizotinib; Drug Resistance, Neoplasm; Female; Gene Rearrangement; Humans; Lung Neoplasms; Middle Aged; Protein Kinase Inhibitors

Human tumors and liver cancer cell lines express the product of a fusion between the first 13 exons in the mannosidase α class 2A member 1 gene (MAN2A1) and the last 6 exons in the FER tyrosine kinase gene (FER), called MAN2A1-FER. We investigated whether MAN2A1-FER is expressed by human liver tumors and its role in liver carcinogenesis.. We performed reverse transcription polymerase chain reaction analyses of 102 non-small cell lung tumors, 61 ovarian tumors, 70 liver tumors, 156 glioblastoma multiform samples, 27 esophageal adenocarcinomas, and 269 prostate cancer samples, as well as 10 nontumor liver tissues and 20 nontumor prostate tissues, collected at the University of Pittsburgh. We also measured expression by 15 human cancer cell lines. We expressed a tagged form of MAN2A1-FER in NIH3T3 and HEP3B (liver cancer) cells; Golgi were isolated for analysis. MAN2A1-FER was also overexpressed in PC3 or DU145 (prostate cancer), NIH3T3 (fibroblast), H23 (lung cancer), and A-172 (glioblastoma multiforme) cell lines and knocked out in HUH7 (liver cancer) cells. Cells were analyzed for proliferation and in invasion assays, and/or injected into flanks of severe combined immunodeficient mice; xenograft tumor growth and metastasis were assessed. Mice with hepatic deletion of PTEN were given tail-vein injections of MAN2A1-FER.. We detected MAN2A1-FER messenger RNA and fusion protein (114 kD) in the hepatocellular carcinoma cell line HUH7, as well as in liver tumors, esophageal adenocarcinoma, glioblastoma multiforme, prostate tumors, non-small cell lung tumors, and ovarian tumors, but not nontumor prostate or liver tissues. MAN2A1-FER protein retained the signal peptide for Golgi localization from MAN2A1 and translocated from the cytoplasm to Golgi in cancer cell lines. MAN2A1-FER had tyrosine kinase activity almost 4-fold higher than that of wild-type FER, and phosphorylated the epidermal growth factor receptor at tyrosine 88 in its N-terminus. Expression of MAN2A1-FER in 4 cell lines led to epidermal growth factor receptor activation of BRAF, MEK, and AKT; HUH7 cells with MAN2A1-FER knockout had significant decreases in phosphorylation of these proteins. Cell lines that expressed MAN2A1-FER had increased proliferation, colony formation, and invasiveness and formed larger (>2-fold) xenograft tumors in mice, with more metastases, than cells not expressing the fusion protein. HUH7 cells with MAN2A1-FER knockout formed smaller xenograft tumors, with fewer metastases, than control HUH7 cells. HUH7, A-172, and PC3 cells that expressed MAN2A1-FER were about 2-fold more sensitive to the FER kinase inhibitor crizotinib and the epidermal growth factor receptor kinase inhibitor canertinib; these drugs slowed growth of xenograft tumors from MAN2A1-FER cells and prevented their metastasis in mice. Hydrodynamic tail-vein injection of MAN2A1-FER resulted in rapid development of liver cancer in mice with hepatic disruption of Pten.. Many human tumor types and cancer cell lines express the MAN2A1-FER fusion, which increases proliferation and invasiveness of cancer cell lines and has liver oncogenic activity in mice.

Topics: alpha-Mannosidase; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Crizotinib; Dose-Response Relationship, Drug; Enzyme Activation; ErbB Receptors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Fusion; Golgi Apparatus; Humans; Liver Neoplasms; Mice; Mice, Knockout; Mice, SCID; Morpholines; Neoplasm Invasiveness; Neoplasm Transplantation; NIH 3T3 Cells; Oncogene Proteins, Fusion; Oncogenes; Phosphorylation; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; PTEN Phosphohydrolase; Pyrazoles; Pyridines; RNA Interference; Time Factors; Transfection; Tumor Burden

Development of the acquired ALK G1202R solvent front mutation and small cell lung cancer (SCLC) transformation have both been independently reported as resistance mechanisms to ALK inhibitors in ALK-rearranged (ALK+) non-small cell lung cancer (NSCLC) patients but have not been reported in the same patient. Here we report an ALK+ NSCLC patient who had disease progression after ceritinib and then alectinib where an ALK G1202R mutation was detected on circulating tumor (ct) DNA prior to enrollment onto a trial of another next generation ALK inhibitor, lorlatinib. The patient's central nervous system (CNS) metastases responded to lorlatinib together with clearance of ALK G1202R mutation by repeat ctDNA assay. However, the patient developed a new large pericardial effusion. Resected pericardium from the pericardial window revealed SCLC transformation with positive immunostaining for synaptophysin, chromogranin, and ALK (D5F3 antibody). Comprehensive genomic profiling (CGP) of the tumor infiltrating pericardium revealed the retainment of an ALK rearrangement with emergence of an inactivating Rb1 mutation (C706Y) and loss of exons 1-11 in p53 that was not detected in the original tumor tissue at diagnosis. The patient was subsequently treated with carboplatin/etoposide and alectinib, but had rapid clinical deterioration and died. The patient never received crizotinib. This case illustrates that multiple/compound resistance mechanisms to ALK inhibitors can occur and provide supporting information that loss of p53 and Rb1 are important in SCLC transformation. If clinically feasible, tissue-based re-biopsy allowing histological examination and CGP remains the gold standard to assess resistance mechanism(s) and to direct subsequent rational clinical care.

Topics: Adult; Aged; Aminopyridines; Anaplastic Lymphoma Kinase; Carcinoma, Non-Small-Cell Lung; Cell Transformation, Neoplastic; Crizotinib; Disease Progression; Drug Resistance, Neoplasm; Fatal Outcome; Female; Humans; Lactams; Lactams, Macrocyclic; Liquid Biopsy; Lung Neoplasms; Male; Middle Aged; Mutation; Neoplastic Cells, Circulating; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Retinoblastoma Binding Proteins; Small Cell Lung Carcinoma; Ubiquitin-Protein Ligases

A 56-year-old woman, a never-smoker, had postoperative recurrence of anaplastic lymphoma kinase rearranged lung cancer. She achieved a partial response to treatment with an anaplastic lymphoma kinase tyrosine kinase inhibitor, crizotinib. After the tumor regrowth, crizotinib was switched to alectinib; once again a partial response was observed. At the second recurrence, transbronchial needle aspiration of the right paratracheal node was performed, which revealed cytological findings of small-cell carcinoma. While treatment with cisplatin-irinotecan chemotherapy made reduction of some tumor shadows, including the biopsied mediastinal lymph nodes, new, small, nodular shadows, highly suggestive of pulmonary metastases, were detected in both lung fields. This case may show proof of the transformation to small-cell lung cancer as a mechanism of resistance to anaplastic lymphoma kinase tyrosine kinase inhibitors in anaplastic lymphoma kinase rearranged tumor. However, this transformation may also be only one part of the resistance mechanism of the heterogeneous tumor.

Topics: Anaplastic Lymphoma Kinase; Antineoplastic Agents; Carbazoles; Carcinoma, Non-Small-Cell Lung; Cell Transformation, Neoplastic; Crizotinib; Drug Resistance, Neoplasm; Female; Humans; Lung Neoplasms; Lymph Nodes; Middle Aged; Neoplasm Recurrence, Local; Piperidines; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Small Cell Lung Carcinoma

Histologic changes can be involved in resistance to anticancer drugs. Transformation to small cell lung carcinoma (SCLC) following treatment with EGFR tyrosine kinase inhibitors has been reported in patients with EGFR-mutant lung adenocarcinoma. Herein, we report a case of ALK-rearranged lung adenocarcinoma with SCLC-like histology in a metastatic abdominal nodule that was resistant to crizotinib therapy.

Topics: Abdominal Neoplasms; Adenocarcinoma; Aged; Anaplastic Lymphoma Kinase; Cell Transformation, Neoplastic; Crizotinib; Drug Resistance, Neoplasm; Gene Rearrangement; Humans; Lung Neoplasms; Male; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Small Cell Lung Carcinoma

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Adult; Anaplastic Lymphoma Kinase; Cell Transformation, Neoplastic; Crizotinib; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Rearrangement; Humans; Lung Neoplasms; Male; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Sarcoma

Pancreatic ductal adenocarcinoma (PDAC) remains a major unsolved health problem. Most drugs that pass preclinical tests fail in these patients, emphasizing the need of improved preclinical models to test novel anticancer strategies. Here, we developed four orthotopic mouse models using primary human PDAC cells genetically engineered to express firefly- and Gaussia luciferase, simplifying the ability to monitor tumor growth and metastasis longitudinally in individual animals with MRI and high-frequency ultrasound. In these models, we conducted detailed histopathologic and immunohistochemical analyses on paraffin-embedded pancreatic tissues and metastatic lesions in liver, lungs, and lymph nodes. Genetic characteristics were compared with the originator tumor and primary tumor cells using array-based comparative genomic hybridization, using frozen specimens obtained by laser microdissection. Notably, the orthotopic human xenografts in these models recapitulated the phenotype of human PDACs, including hypovascular and hypoxic areas. Pursuing genomic and immunohistochemical evidence revealed an increased copy number and overexpression of c-Met in one of the models; we examined the preclinical efficacy of c-Met inhibitors in vitro and in vivo. In particular, we found that crizotinib decreased tumor dimension, prolonged survival, and increased blood and tissue concentrations of gemcitabine, synergizing with a cytidine deaminase-mediated mechanism of action. Together, these more readily imaged orthotopic PDAC models displayed genetic, histopathologic, and metastatic features similar to their human tumors of origin. Moreover, their use pointed to c-Met as a candidate therapeutic target in PDAC and highlighted crizotinib and gemcitabine as a synergistic combination of drugs warranting clinical evaluation for PDAC treatment.

Topics: Animals; Antineoplastic Agents; Carcinoma, Pancreatic Ductal; Cell Transformation, Neoplastic; Crizotinib; Deoxycytidine; Female; Gemcitabine; Humans; Inactivation, Metabolic; Mice; Mice, Nude; Pancreatic Neoplasms; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The proto-oncogene MET is aberrantly activated via overexpression or mutation in numerous cancers, making it a prime anticancer molecular target. However, the clinical success of MET-directed tyrosine kinase inhibitors (TKI) has been limited due, in part, to mutations in the MET kinase domain that confer therapeutic resistance. Circumventing this problem remains a key challenge to improving durable responses in patients receiving MET-targeted therapy. MET is an HSP90-dependent kinase, and in this report we show that HSP90 preferentially interacts with and stabilizes activated MET, regardless of whether the activation is ligand-dependent or is a consequence of kinase domain mutation. In contrast, many MET-TKI show a preference for the inactive form of the kinase, and activating mutations in MET can confer resistance. Combining the HSP90 inhibitor ganetespib with the MET-TKI crizotinib achieves synergistic inhibition of MET, its downstream signaling pathways, and tumor growth in both TKI-sensitive and -resistant MET-driven tumor models. These data suggest that inclusion of an HSP90 inhibitor can partially restore TKI sensitivity to previously resistant MET mutants, and they provide the foundation for clinical evaluation of this therapeutic combination in patients with MET-driven cancers.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Transformation, Neoplastic; Cells, Cultured; Crizotinib; Drug Resistance, Neoplasm; Drug Synergism; Female; HEK293 Cells; HSP90 Heat-Shock Proteins; Humans; Mice; Mice, Nude; Neoplasms; NIH 3T3 Cells; Proto-Oncogene Mas; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Triazoles; Xenograft Model Antitumor Assays

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

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