crizotinib and Melanoma

Current treatment modalities for disseminated cutaneous malignant melanoma (CMM) improve survival, however disease progression commonly ensues. In a previous study we identified afatinib and crizotinib in combination as a novel potential therapy for CMM independent of BRAF/NRAS mutation status. Herein, we elucidate the underlying mechanisms of the combination treatment effect to find biomarkers and novel targets for development of therapy that may provide clinical benefit by proteomic analysis of CMM cell lines and xenografts using mass spectrometry based analysis and reverse phase protein array. Identified candidates were validated using immunoblotting or immunofluorescence. Our analysis revealed that mTOR/Insulin signaling pathways were significantly decreased by the afatinib and crizotinib combination treatment. Both in vitro and in vivo analyses showed that the combination treatment downregulated pRPS6KB1 and pRPS6, downstream of mTOR signaling, and IRS-1 in the insulin signaling pathway, specifically ablating IRS-1 nuclear signal. Silencing of RPS6 and IRS-1 alone had a similar effect on cell death, which was further induced when IRS-1 and RPS6 were concomitantly silenced in the CMM cell lines. Silencing of IRS-1 and RPS6 resulted in reduced sensitivity towards combination treatment. Additionally, we found that IRS-1 and RPS6KB1 expression levels were increased in advanced stages of CMM clinical samples. We could demonstrate that induced resistance towards combination treatment was reversible by a drug holiday. CD171/L1CAM, mTOR and PI3K-p85 were induced in the combination resistant cells whereas AXL and EPHA2, previously identified mediators of resistance to MAPK inhibitor therapy in CMM were downregulated. We also found that CD171/L1CAM and mTOR were increased at progression in tumor biopsies from two matched cases of patients receiving targeted therapy with BRAFi. Overall, these findings provide insights into the molecular mechanisms behind the afatinib and crizotinib combination treatment effect and leverages a platform for discovering novel biomarkers and therapy regimes for CMM treatment.

Topics: Afatinib; Antineoplastic Agents; Crizotinib; Drug Resistance, Neoplasm; Humans; Melanoma; Melanoma, Cutaneous Malignant; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Signal Transduction; Skin Neoplasms; TOR Serine-Threonine Kinases

Current treatment modalities for disseminated cutaneous malignant melanoma (CMM) improve survival; however, relapses are common. A number of receptor tyrosine kinases (RTKs) including EGFR and MET have been reported to be involved in CMM metastasis and in the development of resistance to therapy, targeting the mitogen-activated protein kinase (MAPK pathway). IHC analysis showed that patients with higher MET protein expression had a significantly shorter overall survival. In addition, silencing of MET caused an upregulation of EGFR and p-AKT, which was abrogated by concomitant silencing of MET and EGFR in CMM cells resistant to MAPK-targeting drugs. We therefore explored novel treatment strategies using clinically approved drugs afatinib (ERBB family inhibitor) and crizotinib (MET inhibitor), to simultaneously block MET and ERBB family RTKs. The effects of the combination were assessed in cell culture and spheroid models using established CMM and patient-derived short-term cell lines, and an in vivo xenograft mouse model. The combination had a synergistic effect, promoting cell death, concomitant with a potent downregulation of migratory and invasive capacity independent of their BRAF/NRAS mutational status. Furthermore, the combination attenuated tumor growth rate, as ascertained by the significant reduction of Ki67 expression and induced DNA damage in vivo. Importantly, this combination therapy had minimal therapy-related toxicity in mice. Lastly, the cell cycle G2 checkpoint kinase WEE1 and the RTK IGF1R, non-canonical targets, were altered upon exposure to the combination. Knockdown of WEE1 abrogated the combination-mediated effects on cell migration and proliferation in BRAF mutant BRAF inhibitor-sensitive cells, whereas WEE1 silencing alone inhibited cell migration in NRAS mutant cells. In summary, our results show that afatinib and crizotinib in combination is a promising alternative targeted therapy option for CMM patients, irrespective of BRAF/NRAS mutational status, as well as for cases where resistance has developed towards BRAF inhibitors.

Topics: Afatinib; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Crizotinib; ErbB Receptors; Female; GTP Phosphohydrolases; Humans; Melanoma; Membrane Proteins; Mice; Mice, SCID; Mutation; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-met; Skin Neoplasms; 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

Topics: Antineoplastic Agents; Clinical Trials as Topic; Crizotinib; Diagnostic Test Approval; Drug Approval; Drug Labeling; Humans; Indoles; Melanoma; Molecular Targeted Therapy; Mutation; Neoplasms; Patient Selection; Precision Medicine; Pyrazoles; Pyridines; Sulfonamides; United States; United States Food and Drug Administration; Vemurafenib

Topics: Androstenes; Androstenols; Antibodies, Monoclonal; Antineoplastic Agents; Axitinib; Biomarkers, Tumor; Brentuximab Vedotin; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Crizotinib; Drug Approval; Female; Humans; Imidazoles; Immunoconjugates; Indazoles; Indoles; Ipilimumab; Lung Neoplasms; Male; Melanoma; Molecular Targeted Therapy; Neoplasms; Piperidines; Prostatic Neoplasms; Pyrazoles; Pyridines; Quinazolines; Rare Diseases; Sulfonamides; Survival Analysis; United States; United States Food and Drug Administration; Vemurafenib