Combination of antibodies directed against different ErbB3 surface epitopes prevents the establishment of resistance to BRAF/MEK inhibitors in melanoma

Patients with metastatic melanoma bearing V600 mutations in BRAF oncogene clinically benefit from the treatment with BRAF inhibitors alone or in combination with MEK inhibitors. However, a limitation to such treatment is the occurrence of resistance. Tackling the adaptive changes helping cells survive from drug treatment may offer new therapeutic opportunities. Very recently the ErbB3 receptor has been shown to act as a central node promoting survival of BRAF mutated melanoma. In this paper we first demonstrate that ErbB3/AKT hyperphosphorylation occurs in BRAF mutated melanoma cell lines following exposure to BRAF and/or MEK inhibitors. This strongly correlates with increased transcriptional activation of its ligand neuregulin. Anti-ErbB3 antibodies impair the establishment of de novo cell resistance to BRAF inhibition in vitro. In order to more potently ablate ErbB3 activity we used a combination of two anti-ErbB3 antibodies directed against distinct epitopes of its extracellular domain. These two antibodies in combo with BRAF/MEK inhibitors potently inhibit in vitro cell growth and tumor regrowth after drug withdrawal in an in vivo xenograft model. Importantly, residual tumor masses from mice treated by the antibodies and BRAF/ERK inhibitors combo are characterized almost exclusively by large necrotic areas with limited residual areas of tumor growth. Taken together, our findings support the concept that triple therapy directed against BRAF/MEK/ErbB3 may be able to provide durable control of BRAF mutated metastatic melanoma

Activation of an early feedback survival loop involving phospho-ErbB3 is a general response of melanoma cells to RAF/MEK inhibition and is abrogated by anti-ErbB3 antibodies

BACKGROUND: Treatment of advanced melanoma has been improved with the advent of the BRAF inhibitors. However, a limitation to such treatment is the occurrence of resistance. Several mechanisms have been identified to be responsible for the development of resistance, either MEK-dependent or MEK-independent. In order to overcome resistance due to reactivation of MEK signaling, MEK inhibitors are being clinically developed with promising results. However, also in this case resistance inevitably occurs. It has been recently reported that ErbB3, a member of the EGFR receptor family, may be involved in the establishment of drug resistance. METHODS: Three melanoma cell lines were tested: LOX IMVI (BRAF V600E), MST-L (BRAF V600R) and WM266 (BRAF V600D). Phosphorylation of Receptor Tyrosine Kinases (RTKs) was assessed by an RTK array. Western blot analysis was performed on total protein extracts using anti-ErbB3, anti-AKT and anti-ERK 1/2 antibodies. The expression of neuregulin after vemurafenib treatment was assessed by Real Time PCR and Western blotting. The growth inhibitory effects of vemurafenib, GSK1120212b and/or anti-ErbB3 mAbs were evaluated by in vitro colony formation assays. RESULTS: In the present study we demonstrate that ErbB3 is the main RTK undergoing rapidly hyperphosphorylation upon either treatment with a BRAF inhibitor or with a MEK inhibitor in a panel of melanoma cell lines harboring a variety of V600BRAF mutations and that this results in a strong activation of phospho-AKT. Importantly, ErbB3 activation is fully abrogated by the simultaneous use of anti-ErbB3 monoclonal antibodies, which are also shown to potently synergize with BRAF inhibitors in the inactivation of both AKT and ERK pathways and in the inhibition of melanoma cell growth. We show that upregulation of phospho-ErbB3 is due to an autocrine loop involving increased transcription and production of neuregulin by melanoma cells. CONCLUSIONS: On the basis of these results, we propose that initial co-treatment with BRAF and/or MEK inhibitors and anti-ErbB3 antibodies should be pursued as a strategy to reduce the ErbB3-dependent feedback survival mechanism and enhance duration of clinical response

Human lung adenocarcinoma cell cultures derived from malignant pleural effusions as model system to predict patients chemosensitivity

BACKGROUND: Lung cancer is the leading cause of cancer related deaths and Malignant Pleural Effusion (MPE) is a frequent complication. Current therapies suffer from lack of efficacy in a great percentage of cases, especially when cancer is diagnosed at a late stage. Moreover patients' responses vary and the outcome is unpredictable. Therefore, the identification of patients who will benefit most of chemotherapy treatment is important for accurate prognostication and better outcome. In this study, using malignant pleural effusions (MPE) from non-small cell lung cancer (NSCLC) patients, we established a collection of patient-derived Adenocarcinoma cultures which were characterized for their sensitivity to chemotherapeutic drugs used in the clinical practice. METHODS: Tumor cells present in MPEs of patients with NSCLC were isolated by density gradient centrifugation, placed in culture and genotyped by next generation sequencing. In a subset of cases patient derived xenografts (PDX) were obtained upon tumor cell inoculation in rag2/IL2 knock-out mice. Isolated primary cultures were characterized and tested for drug sensitivity by in vitro proliferation assays. Additivity, antagonism or synergy for combinatorial treatments were determined by analysis with the Calcusyn software. RESULTS: We have optimized isolation procedures and culture conditions to expand in vitro primary cultures from Malignant Pleural Effusions (MPEs) of patients affected by lung adenocarcinomas, the most frequent form of non small cell lung cancer. Using this approach we have been able to establish 16 primary cultures from MPEs. Cells were banked at low passages and were characterized for their mutational pattern by next generation sequencing for most common driver mutations in lung cancer. Moreover, amplified cultures were shown to engraft with high efficiency when injected in immunocompromised mice. Cancer cell sensitivity to drugs used in standard chemotherapy regimens was assessed either individually or in combination. Differential chemosensitivity and different mutation profiles were observed which suggests that this isolation method could provide a platform for predicting the efficacy of chemotherapy in the clinical setting. Most importantly for six patients it was possible to establish a correlation between drug response in vitro and response to therapy in the clinic. CONCLUSIONS: Results obtained using primary cultured cells from MPEs underscore the heterogeneity of NSCLC in advanced stage as indicated by drug response and mutation profile. Comparison of data obtained from in vitro assays with patients' responses to therapy leads to the conclusion that this strategy may provide a potentially useful approach for evaluating individual chemosensitivity profile and tailor the therapy accordingly. Furthermore, combining MPE-derived primary cultures with their genomic testing allows to identify patients eligible to trials with novel targeted agents

Spheres Derived from Lung Adenocarcinoma Pleural Effusions: Molecular Characterization and Tumor Engraftment

Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells

SYNTHESIS, SELF-ASSEMBLY AND OPTOELECTRONIC PROPERTIES OF HETEROAROMATIC CHROMOPHORES

Conjugated molecules constitute the elemental bricks of molecular materials that have given rise to considerable research efforts in basic and applied material science to develop new materials for information, communication and lighting technologies. The ambitions of the present work consisted to have a detailed understanding on the relationships between properties and the structure of π-conjugated molecules. To do this end, different classes of π-conjugated organic compounds were investigated delving into their synthetic processes and chimico-physical features. The first part of the present doctoral thesis describes the synthesis and unprecedented photophysical properties of novel linear π-conjugated organic molecules to design wavelength shifters for an important medical imaging technique, the Positron Emission Tomography. Specifically, to achieve the wavelength conversion two approaches were considered, the push-pull approach and the heteroatom effect. The second part of this work addressed instead a different category of π-conjugates, namely the extended π-conjugated porphyrin macrocycle. Porphyrins were here exploited to study their interactions with metallic surfaces and their ability to form patterned surface based on unique two-dimensional supramolecular assemblies. Specifically, the self-assembly processes were formed through non-covalent interactions between novel pyridyl-substituted porphyrins and investigated by means of the Scanning Tunneling Microscopy (STM) technique. The third and last part focused on the preparation and physical characterization of diverse porphyrin-derived Double-Walled Carbon Nanotubes (DWCNTs) conjugates with the aim of get insights on the structural and electronic properties of these materials. In particular, COOH-derived DWCNTs, produced by means of a three-step oxidative protocol, were covalently linked to a porphyrin molecule. The synthesized DWCNTs-conjugates were also implemented in multilayered-type devices, fabricated with the aim of investigating the interaction of the porphyrin-derived CNTs with polymeric poly(3-hexylthiophene)-pyrene containing small amounts of [60]PCBM.Le molecole coniugate costituiscono gli elementi fondamentali dei cosiddetti materiali molecolari avendo dato luogo a un notevole sforzo della ricerca scientifica di base e applicata per sviluppare nuovi materiali per l’informazione, per la comunicazione e per l’illuminazione. L’interesse dietro le molecole coniugate è aumentato anche grazie allo sviluppo di metodologie sintetiche avanzate, come le reazioni di cross-coupling catalizzate da metalli di transizione, e di nuove tecniche di analisi che permettono un’accurata caratterizzazione di ciascuna molecola. Le ambizioni di questo lavoro sono state quelle di fornire un’analisi dettagliata del rapporto che esiste fra le proprietà e la struttura di molecole coniugate. Per questo obiettivo, sono state prese in considerazione diverse classi di composti organici π-coniugati, approfondendo i loro aspetti sintetici e le loro caratteristiche chimico-fisiche. Nelle prime parti di questo lavoro sono presentate molecole organiche π-coniugate per ideare convertitori di lunghezza d’onda, per un’importante tecnica medica d’imaging, la Tomografia ad Emissione di Positroni (acronimo: PET). In particolare, sono stati considerati due diversi approcci per produrre i suddetti convertitori, l’approccio donatore-accettore (o push-pull) e quello che sfrutta l’effetto dell’insersione di un eteroatomo. Alcuni di questi sistemi si sono rivelati degli ottimi convertitori di lunghezza d'onda. Successivamente è stat affrontata una diversa classe di composti organici π-coniugati, le porfirine. Queste sono state studiate nell’ambito delle loro interazioni con superfici metalliche approfondendo le loro abilità di auto-assemblaggio bisimensionale (2D) per formare superfici con molecole organizzate che interagiscono attraverso interazioni supramolecolari. Queste superfici sono state poi analizzate attraverso la tecnica Scanning Tunneling Microscopy (STM). In particolare, per formare superifici di porfirine ordinate sfruttando un’interazione di tipo metallo-ligando, sono stati sintetizzati e studiati tre derivati caratterizzate da gruppi tipo fenil-etinil-piridina sostituiti nelle posizioni meso del macrociclo. Le ultime sezioni di questo lavoro hanno riguardato la preparazione e la caratterizzazione fotofisica di diversi coniugati porfirina-Nanotubi di Carbonio di tipo double-wall (DWCNTs) con l’obiettivo di approfondire le relazioni struttura-proprietà di questi materiali. In particolare, DWCNTs ossidati, prodotti attraverso tre passaggi sintetici, sono stati covalentemente uniti ad una molecola di porfirina producendo tre differenti derivati che sono stati caratterizzati in maniera estensiva dal punto di vista strutturale e fotofisico. I materiali prodotti sono stati poi introdotti in un prototipo elettronico di tipo multistrato, fabricato allo scopo di studiare le interazioni fra i coniugati porfirina-nanotubi e la matrice polimerica inserita a sua volta nel dispositivo.(DOCSC02) -- FUNDP, 201

Iron porphyrins-catalysed oxidation of alpha-alkyl substituted mono and dimethoxylated benzyl alcohols

The mechanisms of oxidation of a series of a-alkyl substituted mono and dimethoxylated benzyl alcohols catalysed by mesotetrakis(4-N-methylpyridynium)porphyrin iron (III) chloride (FeTMPyPCl) and meso-tetrakis(4-sulfonatophenyl)porphyrin iron (III) chloride (FeTSPPCl) in aqueous solution with KHSO5 as oxygen atom donor and by meso-tetrakis(pentafluorophenyl)-porphyrin iron (III) chloride (FeTPFPPCl) in dichloromethane employing iodosylbenzene as oxidant have been investigated. In the highly polar aqueous medium an electron transfer mechanism is operating. With FeTMPyPCl, which is a much more efficient catalyst than FeTSPPCl due to the presence of stronger electron withdrawing substituents, formation of side-chain oxidation products accompanies generation of nuclear oxidation products. In the low polar solvent dichloromethane, two competing mechanism have been suggested: hydrogen atom transfer and formation of a complex between the active species iron-oxo porphyrin radical cation and the substrate

Thermal and photochemical racemization of chiral aromatic sulfoxides via the intermediacy of sulfoxide radical cations

Efficient racemization of enantiomerically pure methyl aryl sulfoxides was obtained by N-methylquinolinium tetrafluoborate (NMQ+) sensitized photolysis and by one-electron oxidation catalyzed by tris(2,2′-bipyridyl)ruthenium(III) hexafluorophosphate. © 2007 American Chemical Society