Synergy of molecular targeted approaches and immunotherapy in melanoma: preclinical basis and clinical perspectives.

INTRODUCTION: Targeted therapy and immunotherapies are the novel pharmacologic treatment strategies for metastatic melanoma. BRAF and MEK inhibitors effectively block the hyperactivation of the MAPK pathway in BRAF mutant melanomas and also have several other effects on melanoma cells and on the immune response. The aim of this work is to discuss the rationale, evidence and perspectives of approaches combining target and immunotherapy against melanoma. AREAS COVERED: We first review the effects of BRAF and MEK inhibitors on melanoma cells and on the different components of the immune system. Afterwards, we summarize the results of the preclinical and clinical studies that have combined targeted therapy and immunotherapy for the treatment of melanoma. EXPERT OPINION: Clinical applications of immunotherapy strategies have recently changed the therapeutic mainstay for metastatic melanoma. Biologic and initial preclinical data support their integration with innovative molecular targeted therapies, opening enormous perspectives for researchers in the effort of finding a definitive cure. Main open challenges are the definition of reliable research models, assessment of effective schedules, safety issues and designing of personalized approaches.

The Combination of Sorafenib and Everolimus Abrogates mTORC1 and mTORC2 upregulation in osteosarcoma preclinical models.

PURPOSE: The multikinase inhibitor sorafenib displays antitumor activity in preclinical models of osteosarcoma. However, in sorafenib-treated patients with metastatic-relapsed osteosarcoma, disease stabilization and tumor shrinkage were short-lived and drug resistance occurred. We explored the sorafenib treatment escape mechanisms to overcome their drawbacks. EXPERIMENTAL DESIGN: Immunoprecipitation, Western blotting, and immunohistochemistry were used to analyze the mTOR pathway [mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2)]. Cell viability, colony growth, and cell migration were evaluated in different osteosarcoma cell lines (MNNG-HOS, HOS, KHOS/NP, MG63, U-2OS, SJSA-1, and SAOS-2) after scalar dose treatment with sorafenib (10-0.625 µmol/L), rapamycin-analog everolimus (100-6.25 nmol/L), and combinations of the two. Cell cycle, reactive oxygen species (ROS) production, and apoptosis were assessed by flow cytometry. Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice injected with MNNG-HOS cells were used to determine antitumor and antimetastatic effects. Angiogenesis and vascularization were evaluated in vitro by exploiting endothelial branching morphogenesis assays and in vivo in xenografted mice and chorioallantoic membranes. RESULTS: After sorafenib treatment, mTORC1 signaling was reduced (downstream target P-S6), whereas mTORC2 was increased (phospho-mTOR Ser2481) in MNNG-HOS xenografts compared with vehicle-treated mice. Combining sorafenib with everolimus resulted in complete abrogation of both mTORC1 [through ROS-mediated AMP-activated kinase (AMPK) activation] and mTORC2 (through complex disassembly). The sorafenib/everolimus combination yielded: (i) enhanced antiproliferative and proapoptotic effects, (ii) impaired tumor growth, (iii) potentiated antiangiogenesis, and (iv) reduced migratory and metastatic potential. CONCLUSION: mTORC2 activation is an escape mechanism from sorafenib treatment. When sorafenib is combined with everolimus, its antitumor activity is increased by complete inhibition of the mTOR pathway in the preclinical setting.

Sorafenib blocks tumour growth, angiogenesis and metastatic potential in preclinical models of osteosarcoma through a mechanism potentially involving the inhibition of ERK1/2, MCL-1 and ezrin pathways.

BACKGROUND: Osteosarcoma (OS) is the most common primary bone tumour in children and young adults. Despite improved prognosis, metastatic or relapsed OS remains largely incurable and no significant improvement has been observed in the last 20 years. Therefore, the search for alternative agents in OS is mandatory. RESULTS: We investigated phospho-ERK 1/2, MCL-1, and phospho-Ezrin/Radixin/Moesin (P-ERM) as potential therapeutic targets in OS. Activation of these pathways was shown by immunohistochemistry in about 70% of cases and in all OS cell lines analyzed. Mutational analysis revealed no activating mutations in KRAS whereas BRAF gene was found to be mutated in 4/30 OS samples from patients. Based on these results we tested the multi-kinase inhibitor sorafenib (BAY 43-9006) in preclinical models of OS. Sorafenib inhibited OS cell line proliferation, induced apoptosis and downregulated P-ERK1/2, MCL-1, and P-ERM in a dose-dependent manner. The dephosphorylation of ERM was not due to ERK inhibition. The downregulation of MCL-1 led to an increase in apoptosis in OS cell lines. In chick embryo chorioallantoic membranes, OS supernatants induced angiogenesis, which was blocked by sorafenib and it was also shown that sorafenib reduced VEGF and MMP2 production. In addition, sorafenib treatment dramatically reduced tumour volume of OS xenografts and lung metastasis in SCID mice. CONCLUSION: In conclusion, ERK1/2, MCL-1 and ERM pathways are shown to be active in OS. Sorafenib is able to inhibit their signal transduction, both in vitro and in vivo, displaying anti-tumoural activity, anti-angiogenic effects, and reducing metastatic colony formation in lungs. These data support the testing of sorafenib as a potential therapeutic option in metastatic or relapsed OS patients unresponsive to standard treatments.