crizotinib and Disease-Models--Animal

Cell based studies have suggested that the diabetes drug metformin may combine with the anaplastic lymphoma kinase receptor (ALK) inhibitor crizotinib to increase ALK positive lung cancer cell killing and overcome crizotinib resistance. We therefore tested metformin alone and in combination with crizotinib in vivo, by employing a xenograft mouse model of ALK positive lung cancer. We found that 14 days of daily oral metformin (100 mg/kg) alone had a moderate but statistically significant effect on tumour growth suppression, but in combination with crizotinib, produced no greater tumour suppression than crizotinib (25 mg/kg) alone. We also reassessed the effect of metformin on EML

Topics: A549 Cells; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Survival; Crizotinib; Disease Models, Animal; Dose-Response Relationship, Drug; Humans; Lung Neoplasms; Male; Metformin; Mice; Mice, Inbred BALB C; Mice, Nude; Oncogene Proteins, Fusion; Rodentia

Topics: Animals; Crizotinib; Disease Models, Animal; Hypertension, Pulmonary; Models, Animal; Pulmonary Arterial Hypertension; Pulmonary Artery

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer most commonly arises through EML4 (Echinoderm Microtuble Like 4)-ALK chromosomal fusion. We have previously demonstrated that combination of the ALK inhibitor crizotinib with the MEK inhibitor selumetinib was highly effective at reducing cell viability of ALK-positive non-small-cell lung cancer (H3122) cells. In this study, we further investigated the efficacy of crizotinib and selumetinib combination therapy in an in vivo xenograft model of ALK-positive lung cancer. Crizotinib decreased tumor volume by 52% compared with control, and the drug combination reduced tumor growth compared with crizotinib. In addition, MEK inhibition alone reduced tumor growth by 59% compared with control. Crizotinib and selumetinib alone and in combination were nontoxic at the dose of 25 mg/kg, with values for ALT (<80 U/l) and creatinine (<2 mg/dl) within the normal range. Our results support the combined use of crizotinib with selumetinib in ALK-positive lung cancer but raise the possibility that a sufficient dose of an MEK inhibitor alone may be as effective as adding an MEK inhibitor to an ALK inhibitor. SIGNIFICANCE STATEMENT: This study contains in vivo evidence supporting the use of combination MEK inhibitors in ALK+ lung cancer research, both singularly and in combination with ALK inhibitors. Contrary to previously published reports, our results suggest that it is possible to gain much of the benefit from combination treatment with an MEK inhibitor alone, at a tolerable dose.

Topics: Anaplastic Lymphoma Kinase; Animals; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Crizotinib; Disease Models, Animal; Drug Interactions; Humans; Lung Neoplasms; Mice; Mitogen-Activated Protein Kinase Kinases; Protein Kinase Inhibitors; Xenograft Model Antitumor Assays

Amid controversial reports that COVID-19 can be treated with a combination of the antimalarial drug hydroxychloroquine (HCQ) and the antibiotic azithromycin (AZI), a clinical trial (ONCOCOVID, NCT04341207) was launched at Gustave Roussy Cancer Campus to investigate the utility of this combination therapy in cancer patients. In this preclinical study, we investigated whether the combination of HCQ+AZI would be compatible with the therapeutic induction of anticancer immune responses. For this, we used doses of HCQ and AZI that affect whole-body physiology (as indicated by a partial blockade in cardiac and hepatic autophagic flux for HCQ and a reduction in body weight for AZI), showing that their combined administration did not interfere with tumor growth control induced by the immunogenic cell death inducer oxaliplatin. Moreover, the HCQ+AZI combination did not affect the capacity of a curative regimen (cisplatin + crizotinib + PD-1 blockade) to eradicate established orthotopic lung cancers in mice. In conclusion, it appears that HCQ+AZI does not interfere with the therapeutic induction of therapeutic anticancer immune responses.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Azithromycin; Cell Line, Tumor; Cisplatin; Clinical Trials, Phase II as Topic; COVID-19; COVID-19 Drug Treatment; Crizotinib; Disease Models, Animal; Drug Evaluation, Preclinical; Drug Interactions; Drug Therapy, Combination; Female; France; Humans; Hydroxychloroquine; Mice; Neoplasms; Oxaliplatin; Programmed Cell Death 1 Receptor; SARS-CoV-2

Anaplastic thyroid carcinomas (ATCs) have a high prevalence of BRAF and TP53 mutations. A trial of vemurafenib in nonmelanoma BRAFV600E-mutant cancers showed significant, although short-lived, responses in ATCs, indicating that these virulent tumors remain addicted to BRAF despite their high mutation burden. To explore the mechanisms mediating acquired resistance to BRAF blockade, we generated mice with thyroid-specific deletion of p53 and dox-dependent expression of BRAFV600E, 50% of which developed ATCs after dox treatment. Upon dox withdrawal there was complete regression in all mice, although recurrences were later detected in 85% of animals. The relapsed tumors had elevated MAPK transcriptional output, and retained responses to the MEK/RAF inhibitor CH5126766 in vivo and in vitro. Whole-exome sequencing identified recurrent focal amplifications of chromosome 6, with a minimal region of overlap that included Met. Met-amplified recurrences overexpressed the receptor as well as its ligand Hgf. Growth, signaling, and viability of Met-amplified tumor cells were suppressed in vitro and in vivo by the Met kinase inhibitors PF-04217903 and crizotinib, whereas primary ATCs and Met-diploid relapses were resistant. Hence, recurrences are the rule after BRAF suppression in murine ATCs, most commonly due to activation of HGF/MET signaling, which generates exquisite dependency to MET kinase inhibitors.

Topics: Amino Acid Substitution; Animals; Antineoplastic Agents; Cell Line, Tumor; Coumarins; Crizotinib; Disease Models, Animal; Drug Resistance, Neoplasm; Genes, p53; Humans; Indoles; MAP Kinase Signaling System; Mice; Mice, Transgenic; Mutation, Missense; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-met; Pyrazines; Sulfonamides; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Triazoles

MET inhibitors can be effective therapies in patients with. The patient-derived cell line and xenograft (PDX) DFCI358 were established from a crizotinib-resistant

Topics: Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Crizotinib; Disease Models, Animal; DNA Copy Number Variations; Drug Resistance, Neoplasm; Exons; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization, Fluorescence; Lung Neoplasms; Mice; Models, Biological; Mutation; Phosphatidylinositol 3-Kinases; Positron Emission Tomography Computed Tomography; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Proto-Oncogene Proteins p21(ras); Signal Transduction; Xenograft Model Antitumor Assays

The present study aimed to investigate the synergistic therapeutic effect of decreaseing cartilage angiogenesis via exposure to crizotinib encapsulated by chitosan microspheres and photo-crosslinked hydrogel, with the goal of evaluating crizotinib as a treatment for osteoarthritis. First, we developed and evaluated the characteristics of hydrogels and chitosan microspheres. Next, we measured the effect of crizotinib on the cartilage degeneration induced by interleukin-1β in chondrocytes. Crizotinib ameliorated the pathological changes induced by interleukin-1β via its anti-angiogenesis function. In addition, we surgically induced osteoarthritis in mice, which were then injected intra-articularly with crizotinib-loaded biomaterials. Cartilage matrix degradation, expression of vascular endothelial growth factor and extracellular signal-regulated kinases 1/2 were evaluated after surgery. Treatment with the combination of crizotinib-loaded biomaterials retarded the progression of surgically induced osteoarthritis. Crizotinib ameliorated cartilage matrix degradation by promoting anti-angiogenesis and impeding extracellular signal-regulated kinases 1/2 signaling pathway. Our results demonstrate that the combination of photo-crosslinked hydrogel and crizotinib-loaded chitosan microspheres might represent a promising strategy for osteoarthritis treatment.

Topics: Animals; Biomarkers; Cartilage; Chitosan; Chondrocytes; Crizotinib; Disease Models, Animal; Drug Carriers; Drug Liberation; Gene Expression; Humans; Hydrogel, Polyethylene Glycol Dimethacrylate; Interleukin-1beta; Mice; Microspheres; Neovascularization, Pathologic; Osteoarthritis; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Vascular Endothelial Growth Factor A

Patient-derived xenograft (PDX) models are important tools in precision medicine and for the development of targeted therapies to treat cancer patients. This study aimed to evaluate our precision medicine strategy that integrates genomic profiling and preclinical drug-screening platforms, in order to personalize cancer treatments using PDX models.. We performed array-comparative genomic hybridization, microarray, and targeted next-generation sequencing analyses, in order to determine the oncogenic driver mutations. PDX cells were obtained from PDXs and subsequently screened in vitro with 17 targeted agents.. PDX tumors recapitulated the histopathologic and genetic features of the patient tumors. Among the samples from lung cancer patients that were molecularly-profiled, copy number analysis identified unique focal MET amplification in one sample, 033 T, without RTK/RAS/RAF oncogene mutations. Although HER2 amplification in 033 T was not detected in the cancer panel, the selection of HER2-amplified clones was found in PDXs and PDX cells. Additionally, MET and HER2 overexpression were found in patient tumors, PDXs, and PDX cells. Crizotinib or EGFR tyrosine kinase inhibitor treatments significantly inhibited cell growth and impaired tumor sphere formation in 033 T PDX cells.. We established PDX cell models using surgical samples from lung cancer patients, and investigated their preclinical and clinical implications for personalized targeted therapy. Additionally, we suggest that MET and EGFR inhibitor-based therapy can be used to treat MET and HER2-overexpressing lung cancers, without receptor tyrosine kinase /RAS/RAF pathway alterations.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Antineoplastic Agents; Cell Line; Crizotinib; Disease Models, Animal; ErbB Receptors; Female; Gene Amplification; Humans; Lung Neoplasms; Mice; Mice, SCID; Precision Medicine; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Receptor, ErbB-2; Xenograft Model Antitumor Assays

Colorectal cancer (CRC) is a leading cause of cancer-related deaths in the United States. We analyzed 26 MSI-High and 558 non-MSI-High CRC tumors. BRCA2 mutations were highly enriched (50%) in MSI-High CRC. Immunohistochemistry showed that BRCA2-mutated MSI-High CRC had high c-MET (64%) expression compared with BRCA-WT (17%). We hypothesized a mechanistic link between BRCA2-deficiency and c-MET overexpression and synergistic interaction between drugs that treat BRCA-deficient tumors (mitomycin C (MMC) or PARP inhibitors) and c-MET inhibitors (crizotinib). We tested CRC cell lines for sensitivity to MMC plus crizotinib or other drug combinations including PARP-inhibitors. Combined treatment of tumor cells with crizotinib and MMC led to increased apoptosis as compared with each drug alone. Additionally, combination treatment with increasing concentrations of both drugs demonstrated a synergistic anti-cancer effect (CI = 0.006-0.74). However, we found no evidence for c-MET upregulation upon effective BRCA2 knockdown in tumor cells -/+DNA damage. Although we found no mechanistic link between BRCA2 deficiency and c-MET overexpression, c-MET is frequently overexpressed in CRC and BRCA2 is mutated especially in MSI-H CRC. The combination of crizotinib with MMC appeared synergistic regardless of MSI or BRCA2 status. Using an in-vivo CRC xenograft model we found reduced tumor growth with combined crizotinib and MMC therapy (p = 0.0088). Our preclinical results support clinical testing of the combination of MMC and crizotinib in advanced CRC. Targeting cell survival mediated by c-MET in combination with targeting DNA repair may be a reasonable strategy for therapy development in CRC or other cancers.

Topics: Alleles; Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Crizotinib; Disease Models, Animal; Drug Synergism; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Silencing; Genes, BRCA2; Humans; Immunohistochemistry; Mice; Mitomycin; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Xenograft Model Antitumor Assays

Topics: Anaplastic Lymphoma Kinase; Animals; Blood Proteins; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Catechin; Cell Line, Tumor; Crizotinib; Disease Models, Animal; ErbB Receptors; Female; Gene Rearrangement; Heterografts; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; Oncogene Proteins, Fusion; Polyphenols; Protein S; Protein-Tyrosine Kinases; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Tea

Crizotinib, a first-generation anaplastic lymphoma kinase (ALK) tyrosine-kinase inhibitor, is known to be effective against echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive non-small cell lung cancers. Nonetheless, the tumors subsequently become resistant to crizotinib and recur in almost every case. The mechanism of the acquired resistance needs to be deciphered. In this study, we established crizotinib-resistant cells (A925LPE3-CR) via long-term administration of crizotinib to a mouse model of pleural carcinomatous effusions; this model involved implantation of the A925LPE3 cell line, which harbors the EML4-ALK gene rearrangement. The resistant cells did not have the secondary ALK mutations frequently occurring in crizotinib-resistant cells, and these cells were cross-resistant to alectinib and ceritinib as well. In cell clone #2, which is one of the clones of A925LPE3-CR, crizotinib sensitivity was restored via the inhibition of epidermal growth factor receptor (EGFR) by means of an EGFR tyrosine-kinase inhibitor (erlotinib) or an anti-EGFR antibody (cetuximab) in vitro and in the murine xenograft model. Cell clone #2 did not have an EGFR mutation, but the expression of amphiregulin (AREG), one of EGFR ligands, was significantly increased. A knockdown of AREG with small interfering RNAs restored the sensitivity to crizotinib. These data suggest that overexpression of EGFR ligands such as AREG can cause resistance to crizotinib, and that inhibition of EGFR signaling may be a promising strategy to overcome crizotinib resistance in EML4-ALK lung cancer.

Topics: Amphiregulin; Anaplastic Lymphoma Kinase; Animals; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Line, Tumor; Crizotinib; Disease Models, Animal; Drug Resistance, Neoplasm; ErbB Receptors; Erlotinib Hydrochloride; Humans; Lung Neoplasms; Male; Mice; Microtubule-Associated Proteins; Mutation; Pleural Effusion; Pleural Neoplasms; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; RNA, Small Interfering; Serine Endopeptidases; Xenograft Model Antitumor Assays

To evaluate and compare the volumetric tumor burden changes during crizotinib therapy in mice and human cohorts with ALK-rearranged non-small-cell lung cancer (NSCLC).. Volumetric tumor burden was quantified on serial imaging studies in 8 bitransgenic mice with ALK-rearranged adenocarcinoma treated with crizotinib, and in 33 human subjects with ALK-rearranged NSCLC treated with crizotinib. The volumetric tumor burden changes and the time to maximal response were compared between mice and humans.. The median tumor volume decrease (%) at the maximal response was -40.4% (range: -79.5%-+11.7%) in mice, and -72.9% (range: -100%-+72%) in humans (Wilcoxon p=0.03). The median time from the initiation of therapy to maximal response was 6 weeks in mice, and 15.7 weeks in humans. Overall volumetric response rate was 50% in mice and 97% in humans. Spider plots of tumor volume changes during therapy demonstrated durable responses in the human cohort, with a median time on therapy of 13.1 months.. The present study described an initial attempt to evaluate quantitative tumor burden changes in co-clinical imaging studies of genomically-matched mice and human cohorts with ALK-rearranged NSCLC treated with crizotinib. Differences are noted in the degree of maximal volume response between the two cohorts in this well-established paradigm of targeted therapy, indicating a need for further studies to optimize co-clinical trial design and interpretation.

Topics: Adult; Aged; Aged, 80 and over; Anaplastic Lymphoma Kinase; Animals; Carcinoma, Non-Small-Cell Lung; Crizotinib; Disease Models, Animal; Female; Gene Rearrangement; Humans; Lung Neoplasms; Male; Mice; Middle Aged; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Tumor Burden

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

The aim of the present study was to evaluate the effect of crizotinib on visceral organs in an experimental rat model.. Eighteen Wistar albino rats were divided into three groups: experimental toxicity was induced with crizotinib (10 mg/kg) administered for 28 days (Group 1), 42 days (Group 2) orally by gavage. Control group received only distilled water. Rats in Group 1 and Group 2 were sacrificed after the collection of blood and tissue samples on the 28th and 42nd days, respectively.. Subjects in Group 1 and Group 2 had abnormal histology mainly in lung and liver. There were intraalveolar hemorrhage in lungs; mild portal inflammation, perivenular focal and confluent necrosis in liver; inflammatory reaction in renal pelvis and periureteral areas, and focal pancreatitis in pancreas.. This study is the first to evaluate the histopathological features of toxicity of crizotinib in a rat model.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Crizotinib; Disease Models, Animal; Dose-Response Relationship, Drug; Male; Organ Specificity; Pyrazoles; Pyridines; Rats; Rats, Wistar; Treatment Outcome; Viscera

Approximately 25-40% of patients with lung cancer show bone metastasis. Bone modifying agents reduce skeletal-related events (SREs), but they do not significantly improve overall survival. Therefore, novel therapeutic approaches are urgently required. In this study, we investigated the anti-tumor effect of TAS-115, a VEGFRs and HGF receptor (MET)-targeted kinase inhibitor, in a tumor-induced bone disease model. A549-Luc-BM1 cells, an osteo-tropic clone of luciferase-transfected A549 human lung adenocarcinoma cells (A549-Luc), produced aggressive bone destruction associated with tumor progression after intra-tibial (IT) implantation into mice. TAS-115 significantly reduced IT tumor growth and bone destruction. Histopathological analysis showed a decrease in tumor vessels after TAS-115 treatment, which might be mediated through VEGFRs inhibition. Furthermore, the number of osteoclasts surrounding the tumor was decreased after TAS-115 treatment. In vitro studies demonstrated that TAS-115 inhibited HGF-, VEGF-, and macrophage-colony stimulating factor (M-CSF)-induced signaling pathways in osteoclasts. Moreover, TAS-115 inhibited Feline McDonough Sarcoma oncogene (FMS) kinase, as well as M-CSF and receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. Thus, VEGFRs/MET/FMS-triple inhibition in osteoclasts might contribute to the potent efficacy of TAS-115. The fact that concomitant dosing of sunitinib (VEGFRs/FMS inhibition) with crizotinib (MET inhibition) exerted comparable inhibitory efficacy for bone destruction to TAS-115 also supports this notion. In conclusion, TAS-115 inhibited tumor growth via VEGFR-kinase blockade, and also suppressed bone destruction possibly through VEGFRs/MET/FMS-kinase inhibition, which resulted in potent efficacy of TAS-115 in an A549-Luc-BM1 bone disease model. Thus, TAS-115 shows promise as a novel therapy for lung cancer patients with bone metastasis.

Topics: A549 Cells; Animals; Bone Neoplasms; Cell Differentiation; Cell Proliferation; Crizotinib; Disease Models, Animal; Humans; Indoles; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Osteoclasts; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Pyrroles; Quinolines; RANK Ligand; Receptor, Macrophage Colony-Stimulating Factor; Receptors, Vascular Endothelial Growth Factor; Signal Transduction; Sunitinib; Thiourea; Tibia; Transplantation, Heterologous; X-Ray Microtomography

Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1(G2032R) mutation and the ROS1(G2026M) gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. In vivo, PF-06463922 showed marked antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the CD74-ROS1(G2032R) mutation. Furthermore, PF-06463922 demonstrated antitumor activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.

Topics: Aminopyridines; Animals; Carcinogenesis; Cell Proliferation; Crizotinib; Crystallography, X-Ray; Disease Models, Animal; Drug Resistance, Neoplasm; Glioma; Humans; Lactams; Lactams, Macrocyclic; Mice; Models, Molecular; Mutation; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Pyrazoles; Pyridines; Signal Transduction

Papillary renal cell carcinoma (PRCC) is the second most common cancer of the kidney and carries a poor prognosis for patients with nonlocalized disease. The HGF receptor MET plays a central role in PRCC and aberrations, either through mutation, copy number gain, or trisomy of chromosome 7 occurring in the majority of cases. The development of effective therapies in PRCC has been hampered in part by a lack of available preclinical models. We determined the pharmacodynamic and antitumor response of the selective MET inhibitor AZD6094 in two PRCC patient-derived xenograft (PDX) models.. Two PRCC PDX models were identified and MET mutation status and copy number determined. Pharmacodynamic and antitumor activity of AZD6094 was tested using a dose response up to 25 mg/kg daily, representing clinically achievable exposures, and compared with the activity of the RCC standard-of-care sunitinib (in RCC43b) or the multikinase inhibitor crizotinib (in RCC47).. AZD6094 treatment resulted in tumor regressions, whereas sunitinib or crizotinib resulted in unsustained growth inhibition. Pharmacodynamic analysis of tumors revealed that AZD6094 could robustly suppress pMET and the duration of target inhibition was dose related. AZD6094 inhibited multiple signaling nodes, including MAPK, PI3K, and EGFR. Finally, at doses that induced tumor regression, AZD6094 resulted in a dose- and time-dependent induction of cleaved PARP, a marker of cell death.. Data presented provide the first report testing therapeutics in preclinical in vivo models of PRCC and support the clinical development of AZD6094 in this indication.

Topics: Animals; Antineoplastic Agents; Carcinoma, Papillary; Carcinoma, Renal Cell; Cell Line, Tumor; Crizotinib; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Humans; Indoles; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Pyrazines; Pyrazoles; Pyridines; Pyrroles; Sunitinib; Triazines; Tumor Burden; Xenograft Model Antitumor Assays

Topics: Adenocarcinoma; Anaplastic Lymphoma Kinase; Animals; Carcinoma, Non-Small-Cell Lung; Chromosome Inversion; CRISPR-Cas Systems; Crizotinib; Disease Models, Animal; Gene Transfer Techniques; Genes, ras; Genomic Structural Variation; Humans; Lung Neoplasms; Mice; NIH 3T3 Cells; Oncogene Proteins, Fusion; Phosphorylation; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; RNA Editing; Translocation, Genetic

To extend the results of a phase III trial in patients with non-small cell lung cancer with adenocarcinomas harboring EML4-ALK fusion.. We conducted a co-clinical trial in a mouse model comparing the ALK inhibitor crizotinib to the standard-of-care cytotoxic agents docetaxel or pemetrexed.. Concordant with the clinical outcome in humans, crizotinib produced a substantially higher response rate compared with chemotherapy, associated with significantly longer progression-free survival. Overall survival was also prolonged in crizotinib- compared with chemotherapy-treated mice. Pemetrexed produced superior overall survival compared with docetaxel, suggesting that this agent may be the preferred chemotherapy in the ALK population. In addition, in the EML4-ALK-driven mouse lung adenocarcinoma model, HSP90 inhibition can overcome both primary and acquired crizotinib resistance. Furthermore, HSP90 inhibition, as well as the second-generation ALK inhibitor TAE684, demonstrated activity in newly developed lung adenocarcinoma models driven by crizotinib-insensitive EML4-ALK L1196M or F1174L.. Our findings suggest that crizotinib is superior to standard chemotherapy in ALK inhibitor-naïve disease and support further clinical investigation of HSP90 inhibitors and second-generation ALK inhibitors in tumors with primary or acquired crizotinib resistance.

Topics: Anaplastic Lymphoma Kinase; Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Crizotinib; Disease Models, Animal; Drug Resistance, Neoplasm; HSP90 Heat-Shock Proteins; Humans; Lung Neoplasms; Magnetic Resonance Imaging; Mice; Oncogene Proteins, Fusion; Positron-Emission Tomography; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Treatment Outcome

Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.

Topics: Aminoquinolines; Animals; Antineoplastic Agents; Colonic Neoplasms; Crizotinib; Crystallization; Disease Models, Animal; DNA Breaks, Single-Stranded; DNA Repair; DNA Repair Enzymes; Female; Homeostasis; Humans; Mice; Mice, SCID; Models, Molecular; Nucleotides; Phosphoric Monoester Hydrolases; Protein Conformation; Protein Kinase Inhibitors; Proteomics; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Pyrazoles; Pyridines; ras Proteins; Substrate Specificity; Xenograft Model Antitumor Assays

Positron emission tomography (PET) is used to monitor the uptake of the labeled glucose analogue fluorodeoxyglucose (¹⁸F-FDG) by solid tumor cells, a process generally believed to reflect viable tumor cell mass. The use of ¹⁸F-FDG exploits the high demand for glucose in tumor cells, and serves to document over time the response of a solid tumor to an inducer of apoptosis. The apoptosis inducer crizotinib is a small-molecule inhibitor of c-Met, a receptor tyrosine kinase that is often dysregulated in human tumors. In this protocol, we describe how to monitor the response of a solid tumor to crizotinib. Human gastric tumor cells (GTL-16 cells) are injected into recipient mice and, on tumor formation, the mice are treated with crizotinib. The tracer ¹⁸F-FDG is then injected into the mice at several time points, and its uptake is monitored using PET. Because ¹⁸F-FDG uptake varies widely among different tumor models, preliminary experiments should be performed with each new model to determine its basal level of ¹⁸F-FDG uptake. Verifying that the basal level of uptake is sufficiently above background levels will assure accurate quantitation. Because ¹⁸F-FDG uptake is not a direct measure of apoptosis, it is advisable to carry out an additional direct method to show the presence of apoptotic cells.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Crizotinib; Disease Models, Animal; Fluorodeoxyglucose F18; Humans; Mice; Positron-Emission Tomography; Pyrazoles; Pyridines; Stomach Neoplasms; Xenograft Model Antitumor Assays

Chromosomal rearrangements have a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions. A recently discovered example is a fusion between the genes echinoderm microtubule-associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK), generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4-ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC) and is clinically relevant because it confers sensitivity to ALK inhibitors. Despite their importance, modelling such genetic events in mice has proven challenging and requires complex manipulation of the germ line. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4-Alk-driven lung cancer. The resulting tumours invariably harbour the Eml4-Alk inversion, express the Eml4-Alk fusion gene, display histopathological and molecular features typical of ALK(+) human NSCLCs, and respond to treatment with ALK inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentially in other organisms.

Topics: Anaplastic Lymphoma Kinase; Animals; Antineoplastic Agents; Cells, Cultured; Chromosome Inversion; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Associated Proteins; CRISPR-Cas Systems; Crizotinib; Disease Models, Animal; Genetic Engineering; Lung Neoplasms; Mice; NIH 3T3 Cells; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; Translocation, Genetic

Genetic rearrangement of the ROS1 receptor tyrosine kinase was recently identified as a distinct molecular signature for human non-small cell lung cancer (NSCLC). However, direct evidence of lung carcinogenesis induced by ROS1 fusion genes remains to be verified. The present study shows that EZR-ROS1 plays an essential role in the oncogenesis of NSCLC harboring the fusion gene. EZR-ROS1 was identified in four female patients of lung adenocarcinoma. Three of them were never smokers. Interstitial deletion of 6q22-q25 resulted in gene fusion. Expression of the fusion kinase in NIH3T3 cells induced anchorage-independent growth in vitro, and subcutaneous tumors in nude mice. This transforming ability was attributable to its kinase activity. The ALK/MET/ROS1 kinase inhibitor, crizotinib, suppressed fusion-induced anchorage-independent growth of NIH3T3 cells. Most importantly, established transgenic mouse lines specifically expressing EZR-ROS1 in lung alveolar epithelial cells developed multiple adenocarcinoma nodules in both lungs at an early age. These data suggest that the EZR-ROS1 is a pivotal oncogene in human NSCLC, and that this animal model could be valuable for exploring therapeutic agents against ROS1-rearranged lung cancer.

Topics: Adenocarcinoma; Animals; Carcinoma, Non-Small-Cell Lung; Crizotinib; Disease Models, Animal; Lung Neoplasms; Mice; Mice, Nude; Mice, Transgenic; Oncogene Fusion; Oncogene Proteins, Fusion; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases

The synthesis of bis-ortho-alkoxy-para-piperazinesubstituted-2,4-dianilinopyrimidines is described and their structure-activity-relationship to anaplastic lymphoma kinase (ALK) is presented. KRCA-0008 is selective and potent to ALK and Ack1, and displays drug-like properties without hERG liability. KRCA-0008 demonstrates in vivo efficacy comparable to Crizotinib in xenograft mice model.

Topics: Anaplastic Lymphoma Kinase; Animals; Cell Line, Tumor; Crizotinib; Disease Models, Animal; Lung Neoplasms; Mice; Piperazines; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Pyrimidines; Receptor Protein-Tyrosine Kinases; Structure-Activity Relationship; Xenograft Model Antitumor Assays

Uveal melanoma is the most common primary intraocular malignant tumor in adults and half of the primary tumors will develop fatal metastatic disease to the liver and the lung. Crizotinib, an inhibitor of c-Met, anaplastic lymphoma kinase (ALK), and ROS1, inhibited the phosphorylation of the c-Met receptor but not of ALK or ROS1 in uveal melanoma cells and tumor tissue. Consequently, migration of uveal melanoma cells was suppressed in vitro at a concentration associated with the specific inhibition of c-Met phosphorylation. This effect on cell migration could be recapitulated with siRNA specific to c-Met but not to ALK or ROS1. Therefore, we developed a uveal melanoma metastatic mouse model with EGFP-luciferase-labeled uveal melanoma cells transplanted by retro-orbital injections to test the effect of crizotinib on metastasis. In this model, there was development of melanoma within the eye and also metastases to the liver and lung at 7 weeks after the initial transplantation. When mice were treated with crizotinib starting 1 week after the transplantation, we observed a significant reduction in the development of metastases as compared with untreated control sets. These results indicate that the inhibition of c-Met activity alone may be sufficient to strongly inhibit metastasis of uveal melanoma from forming, suggesting crizotinib as a potential adjuvant therapy for patients with primary uveal melanoma who are at high risk for the development of metastatic disease.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Crizotinib; Disease Models, Animal; Gene Expression; Gene Knockdown Techniques; Hepatocyte Growth Factor; Humans; Male; Melanoma; Mice; Neoplasm Metastasis; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; RNA, Small Interfering; Tumor Burden; Uveal Neoplasms; 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

The ability of PET to image functional changes in tumors is increasingly being used to evaluate response and predict clinical benefit to conventional and novel cancer therapies. Although the use of (18)F-FDG PET is well established, 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET has potential advantages as a more specific marker of cellular proliferation. c-MET signaling is frequently dysregulated in cancer and is therefore an attractive therapeutic target. Crizotinib (PF-2341066) is a novel adenosine triphosphate-competitive c-MET kinase inhibitor with antitumor activity in a range of tumor models. The aim of this study was to investigate the utility of PET of glucose metabolism and cell proliferation to monitor tumor response to crizotinib in 2 cell lines with aberrant c-MET signaling.. Mice bearing GTL-16 or U87MG xenografts were evaluated for changes in tumor volume and (18)F-FDG and (18)F-FLT uptake after daily oral treatment with up to 50 mg/kg crizotinib. GTL-16 and U87MG cells were treated with crizotinib in vitro and analyzed for (3)H-2-deoxyglucose uptake and expression of activated MET, AKT, and ERK by immunoblotting.. Treatment of c-MET-amplified GTL-16 xenografts with 50 mg/kg crizotinib caused tumor regression that was associated with a slow reduction in (18)F-FDG uptake (P < 0.05, day 13) and reduced expression of the glucose transporter 1, GLUT-1. Although baseline (18)F-FDG uptake into U87MG tumors was substantially higher than in GTL-16 tumors, (18)F-FDG uptake into U87MG tumors remained unchanged on treatment at 50 mg/kg crizotinib, despite tumor growth inhibition of 93% on day 8 of treatment. These findings were confirmed in vitro, where treatment of U87MG cells with 1 μM crizotinib had no demonstrable effect on glucose uptake. Furthermore, these cells demonstrated constitutive, crizotinib-independent phosphoinositide 3-kinase pathway signaling as demonstrated by phosphorylated AKT and ribosomal protein S6. Both U87MG and GTL-16 tumors showed high baseline uptake of (18)F-FLT, which was reduced by 50% and 53% on days 4 and 8 of treatment, respectively.. While the results provide a strong rationale to investigate the use of (18)F-FLT PET as a clinical biomarker for monitoring tumor response to c-MET inhibition, (18)F-FDG PET may be a less robust marker.

Topics: Animals; Biomarkers; Cell Line, Tumor; Cell Proliferation; Crizotinib; Dideoxynucleosides; Disease Models, Animal; Fluorodeoxyglucose F18; Glucose; Glucose Transporter Type 1; Humans; Mice; Mice, Inbred BALB C; Mice, SCID; Neoplasm Transplantation; Piperidines; Positron-Emission Tomography; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Signal Transduction

Aberrant expression of HGF/SF and its receptor, c-Met, often correlates with advanced prostate cancer. Our previous study showed that expression of c-Met in prostate cancer cells was increased after attenuation of androgen receptor (AR) signalling. This suggested that current androgen ablation therapy for prostate cancer activates c-Met expression and may contribute to development of more aggressive, castration resistant prostate cancer (CRPC). Therefore, we directly assessed the efficacy of c-Met inhibition during androgen ablation on the growth and progression of prostate cancer.. We tested two c-Met small molecule inhibitors, PHA-665752 and PF-2341066, for anti-proliferative activity by MTS assay and cell proliferation assay on human prostate cancer cell lines with different levels of androgen sensitivity. We also used renal subcapsular and castrated orthotopic xenograft mouse models to assess the effect of the inhibitors on prostate tumor formation and progression.. We demonstrated a dose-dependent inhibitory effect of PHA-665752 and PF-2341066 on the proliferation of human prostate cancer cells and the phosphorylation of c-Met. The effect on cell proliferation was stronger in androgen insensitive cells. The c-Met inhibitor, PF-2341066, significantly reduced growth of prostate tumor cells in the renal subcapsular mouse model and the castrated orthotopic mouse model. The effect on cell proliferation was greater following castration.. The c-Met inhibitors demonstrated anti-proliferative efficacy when combined with androgen ablation therapy for advanced prostate cancer.

Topics: Animals; Cell Line, Tumor; Crizotinib; Disease Models, Animal; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Humans; Indoles; Male; Mice; Neoplasm Transplantation; Phosphorylation; Piperidines; Prostatic Neoplasms; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Signal Transduction; Sulfones