A novel mechanism of action of HER2 targeted immunotherapy is explained by inhibition of NRF2 function in ovarian cancer cells

Nuclear erythroid related factor-2 (NRF2) is known to promote cancer therapeutic detoxification and crosstalk with growth promoting pathways. HER2 receptor tyrosine kinase is frequently overexpressed in cancers leading to uncontrolled receptor activation and signaling. A combination of HER2 targeting monoclonal antibodies shows greater anticancer efficacy than the single targeting antibodies, however, its mechanism of action is largely unclear. Here we report novel actions of anti-HER2 drugs, Trastuzumab and Pertuzumab, involving NRF2. HER2 targeting by antibodies inhibited growth in association with persistent generation of reactive oxygen species (ROS), glutathione (GSH) depletion, reduction in NRF2 levels and inhibition of NRF2 function in ovarian cancer cell lines. The combination of antibodies produced more potent effects than single alone; downregulated NRF2 substrates by repressing the Antioxidant Response (AR) pathway with concomitant transcriptional inhibition of NRF2. We showed the antibody combination produced increased methylation at the NRF2 promoter consistent with repression of NRF2 antioxidant function, as HDAC and methylation inhibitors reversed such produced transcriptional effects. These findings demonstrate a novel mechanism and role for NRF2 in mediating the response of cancer cells to the combination of Trastuzumab and Pertuzumab and reinforce the importance of NRF2 in drug resistance and as a key anticancer target.

Soft tissue sarcoma: biology and therapeutic correlates

Soft tissue sarcomas (STS) comprise a heterogenenous group of greater than 50 malignancies of putative mesenchymal cell origin and as such they may arise in diverse tissue types in various anatomical locations throughout the whole body. Collectively they account for approximately 1% of all human malignancies yet have a spectrum of aggressive behaviours amongst their subtypes. They thus pose a particular challenge to manage and remain an under investigated group of cancers with no generally applicable new therapies in the past 40 years and an overall 5-year survival rate that remains stagnant at around 50%. From September 2000 to July 2006 I undertook a full time post-doctoral level research fellowship at the MD Anderson Cancer Center, Houston, Texas, USA in the department of Surgical Oncology to investigate the biology of soft tissue sarcoma and test novel anti- sarcoma adenovirus-based therapy in the preclinical nude rat model of isolated limb perfusion against human sarcoma xenografts. This work, in collaboration with colleagues as indicated herein, led to a number of publications in the scientific literature furthering our understanding of the malignant phenotype of sarcoma and reported preclinical studies with wild-type p53, in a replication deficient adenovirus vector, and oncolytic adenoviruses administered by isolated limb perfusion. Additional collaborative and pioneering preclinical studies reported the molecular imaging of sarcoma response to systemically delivered therapeutic phage RGD-4c AAVP. Doxorubicin chemotherapy is the single most active broadly applicable anti-sarcoma chemotherapeutic yet only has an approximate 30% overall response rate with additional breakthrough tumour progression and recurrence after initial chemo-responsiveness further problematic features in STS management. Doxorubicin is a substrate for the multi- drug resistance (mdr) gene product p-glycoprotein drug efflux pump and exerts its main mode of action by induction of DNA double-strand breaks during the S-phase of the cell cycle. Two papers in my thesis characterise different aspects of chemoresistance in sarcoma. The first shows that wild-type p53 suppresses Protein Kinase Calpha (PKCα) phosphorylation (and activation) of p-glycoprotein by transcriptional repression of PKCα through a Sp-1 transcription factor binding site in its -244/-234 promoter region. The second paper demonstrates that Rad51 (a central mediator of homologous recombination repair of double strand breaks) has elevated levels in sarcoma and particularly in the S- G2 phase of the cell cycle. Suppression of Rad51 with small interfering RNA in sarcoma cell culture led to doxorubicin chemosensitisation. Reintroduction of wild-type p53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression via transcriptional repression of the Rad51 promoter through increased AP-2 binding. In light of poor response rates to chemotherapy, escape from local control portends a poor prognosis for patients with sarcoma. Two papers in my thesis characterise aspects of sarcoma angiogenesis, invasion and metastasis. Human sarcoma samples were found to have high levels of matrix metalloproteinase-9 (MMP-9) with expression levels that correlated with p53 mutational status. MMP-9 is known to degrade extracellular collagen, contribute to the control of the angiogenic switch necessary in primary tumour progression and facilitate invasion and metastasis. Reconstitution of wild-type p53 function led to decreased levels of MMP-9 protein and mRNA as well as zymography-assessed MMP-9 proteolytic activity and decreased tumour cell invasiveness. Reintroduction of wild-type p53 into human sarcoma xenografts in-vivo decreased tumour growth and MMP-9 protein expression. Wild-type p53 was found to suppress mmp-9 transcription via decreased binding of NF-κB to its -607/-595 mmp-9 promoter element. Studies on the role of the VEGF165 in sarcoma found that sarcoma cells stably transfected with VEGF165 formed more aggressive xenografted tumours with increased vascularity, growth rate, metastasis, and resistance to chemotherapy. Use of the anti-VEGFR2 antibody DC101 enhanced doxorubicin sensitivity at sub-conventional dosing, inhibited tumour growth, decreased development of metastases, and reduced tumour micro-vessel density while increasing the vessel maturation index. These effects were explained primarily through effects on endothelial cells (e.c.s), rather than the tumour cells per se, where DC101 induced e.c. sensitivity to doxorubicin and suppressed e.c. production of MMPs. The p53 tumour suppressor pathway is the most frequently mutated pathway in sarcoma. Recapitulation of wild-type p53 function in sarcoma exerts a number of anti-cancer outcomes such as growth arrest, resensitisation to chemotherapy, suppression of invasion, and attenuation of angiogenesis. Using a modified nude rat-human sarcoma xenograft model for isolated limb perfusion (ILP) delivery of wild-type p53 in a replication deficient adenovirus vector I showed that functionally competent wild-type p53 could be delivered to and detected in human leiomyosarcoma xenografts confirming preclinical feasibility - although not efficacious due to low transgene expression. Viral fibre modification to express the RGD tripeptide motif led to greater viral uptake by sarcoma cells in vitro (transductional targeting) and changing the transgene promoter to a response element active in cells with active telomerase expression restricted the transgene expression to the tumour intracellular environment (transcriptional targeting). Delivery of the fibre-modified, selectively replication proficient oncolytic adenovirus Ad.hTC.GFP/ E1a.RGD by ILP demonstrated a more robust, and tumour-restricted, transgene expression with evidence of anti-sarcoma effect confirmed microscopically. Collaborative studies using the fibre modified phage RGD-4C AAVP confirmed that systemic delivery specifically, efficiently, and repeatedly targets human sarcoma xenografts, binds to αv integrins in tumours, and demonstrates a durable, though heterogeneous, transgene expression of 1-4 weeks. Incorporation of the Herpes Simplex Virus thymidine kinase (HSVtk) transgene into RGD-4C AAVP permitted CT-PET spatial and temporal molecular imaging in vivo of transgene expression and allowed quantification of tumour metabolic activity both before and after interval administration of a systemic cytotoxic with predictable and measurable response to treatment before becoming apparent clinically. These papers further the medical and scientific community’s understanding of the biology of soft tissue sarcoma and report preclinical studies with novel and promising anti- sarcoma therapeutics.

Genetic control of development in the parasitic nematode Haemonchus contortus by microRNAs

The parasitic nematode Haemonchus contortus has a major impact on the welfare and economic sustainability of small ruminant farming throughout the world. Increasing drug resistance requires the development of novel therapeutic agents. To further this process, we examined the fundamental biology of development in H. contortus, specifically, the potential role of microRNAs (miRNAs). miRNAs are short, non-coding RNA molecules that negatively regulate gene expression. In the free-living nematode Caenorhabditis elegans, miRNAs regulate a variety of genes including those involved in development. This thesis describes the expression patterns, potential targets and possible functions of miRNAs in H. contortus throughout development.

Proteomics in the assessment of the therapeutic response of antineoplastic drugs: strategies and practical applications

Uncovering mechanisms of unknown pathological mechanisms and body response to applied medication are the drive forces toward personalized medicine. In this post-genomic era, all eyes are tuned to proteomic field, searching for the answers and explanations by investigating the final physiological functional units – proteins and their proteoforms. Development of cutting-edge mass spectrometric technologies and powerful bioinformatics tools, allowed life-science community mining of disease-specific proteins as biomarkers, which are often hidden by high complexity of the samples and/or small abundance. Nowadays, there are several proteomics-based approaches to study the proteome. This chapter focuses on gold standard proteomics strategies and related issues towards candidate biomarker discovery, which may have diagnostic/prognostic as well as mechanistic utility.

Applying Mathematical Models to Study the Role of the Immune System in Chronic Myelogenous Leukemia

Although tyrosine kinase inhibitors (TKIs) such as imatinib have transformed chronic myelogenous leukemia (CML) into a chronic condition, these therapies are not curative in the majority of cases. Most patients must continue TKI therapy indefinitely, a requirement that is both expensive and that compromises a patient's quality of life. While TKIs are known to reduce leukemic cells' proliferative capacity and to induce apoptosis, their effects on leukemic stem cells, the immune system, and the microenvironment are not fully understood. A more complete understanding of their global therapeutic effects would help us to identify any limitations of TKI monotherapy and to address these issues through novel combination therapies. Mathematical models are a complementary tool to experimental and clinical data that can provide valuable insights into the underlying mechanisms of TKI therapy. Previous modeling efforts have focused on CML patients who show biphasic and triphasic exponential declines in BCR-ABL ratio during therapy. However, our patient data indicates that many patients treated with TKIs show fluctuations in BCR-ABL ratio yet are able to achieve durable remissions. To investigate these fluctuations, we construct a mathematical model that integrates CML with a patient's autologous immune response to the disease. In our model, we define an immune window, which is an intermediate range of leukemic concentrations that lead to an effective immune response against CML. While small leukemic concentrations provide insufficient stimulus, large leukemic concentrations actively suppress a patient's immune system, thus limiting it's ability to respond. Our patient data and modeling results suggest that at diagnosis, a patient's high leukemic concentration is able to suppress their immune system. TKI therapy drives the leukemic population into the immune window, allowing the patient's immune cells to expand and eventually mount an efficient response against the residual CML. This response drives the leukemic population below the immune window, causing the immune population to contract and allowing the leukemia to partially recover. The leukemia eventually reenters the immune window, thus stimulating a sequence of weaker immune responses as the two populations approach equilibrium. We hypothesize that a patient's autologous immune response to CML may explain the fluctuations in BCR-ABL ratio that are regularly seen during TKI therapy. These fluctuations may serve as a signature of a patient's individual immune response to CML. By applying our modeling framework to patient data, we are able to construct an immune profile that can then be used to propose patient-specific combination therapies aimed at further reducing a patient's leukemic burden. Our characterization of a patient's anti-leukemia immune response may be especially valuable in the study of drug resistance, treatment cessation, and combination therapy.

Intercepting Cyclic Dinucleotide Signaling with Small Molecules

Bacterial infections, especially the ones that are caused by multidrug-resistant strains, are becoming increasingly difficult to treat and put enormous stress on healthcare systems. Recently President Obama announced a new initiative to combat the growing problem of antibiotic resistance. New types of antibiotic drugs are always in need to catch up with the rapid speed of bacterial drug-resistance acquisition. Bacterial second messengers, cyclic dinucleotides, play important roles in signal transduction and therefore are currently generating great buzz in the microbiology community because it is believed that small molecules that inhibit cyclic dinucleotide signaling could become next-generation antibacterial agents. The first identified cyclic dinucleotide, c-di-GMP, has now been shown to regulate a large number of processes, such as virulence, biofilm formation, cell cycle, quorum sensing, etc. Recently, another cyclic dinucleotide, c-di-AMP, has emerged as a regulator of key processes in Gram-positive and mycobacteria. C-di-AMP is now known to regulate DNA damage sensing, fatty acid synthesis, potassium ion transport, cell wall homeostasis and host type I interferon response induction. Due to the central roles that cyclic dinucleotides play in bacteria, we are interested in small molecules that intercept cyclic dinucleotide signaling with the hope that these molecules would help us learn more details about cyclic dinucleotide signaling or could be used to inhibit bacterial viability or virulence. This dissertation documents the development of several small molecule inhibitors of a cyclic dinucleotide synthase (DisA from B. subtilis) and phosphodiesterases (RocR from P. aeruginosa and CdnP from M. tuberculosis). We also demonstrate that an inhibitor of RocR PDE can inhibit bacterial swarming motility, which is a virulence factor.

"Mutações no gene OCT4 em células eritroleucêmicas humanas: implicações sobre o fenótipo de resistência a múltiplas drogas (MDR)”

O fator de transcrição OCT4 é um importante marcador de células tronco e tem sido relacionado com o conceito de células tronco tumorais (CTTs). Recentemente, ele tem sido também relacionado ao fenótipo de resistência a múltiplas drogas (MDR). O objetivo deste trabalho foi testar se o OCT4 está ligado ao fenótipo MDR em células eritroleucêmicas derivadas da linhagem LMC-K562. Para isso, foi realizada a análise de expressão de genes associados à superfamília de transportadores ABC (ATP Binding Cassette) e, também, de fatores de transcrição relacionados a células tronco. Os primeiros resultados apontaram uma relação direta entre OCT4 e transportadores ABC na linhagem MDR derivada de K562 (Lucena). O sequenciamento de promotores ABC não revelou qualquer mutação que pudesse explicar a expressão diferenciada do OCT4 na linhagem MDR. Posteriormente, o sequenciamento da região contendo o domínio homeobox do gene OCT4 de ambas as linhagens celulares evidenciou, pela primeira vez, que este fator de transcrição é alvo de mutações que podem estar relacionados com o fenótipo MDR. As mutações encontradas implicam substituições de vários aminoácidos em ambas as linhagens celulares. K562 teve sete substituições (três exclusivas), enquanto Lucena teve 13 (nove exclusivas). Além disso, um busca in silico por motivos de fosforilação dentro da sequência de aminoácidos comparada, revelou que o OCT4 humano normal possui sete motivos potenciais de fosforilação. Entretanto, K562 perdeu um motivo e Lucena dois deles. Moléculas com diferentes padrões de fosforilação podem ter sua função modificada. Estes resultados indicam o OCT4 com uma alvo potencial para o tratamento do câncer, especialmente aqueles resistentes à quimioterapia.

Avaliação da atividade antitumoral de amidas graxas análogas de canabinóides

A atividade antiproliferativa in vitro de uma série de amidas graxas sintéticas, em sete linhagens de células tumorais foi investigada. Baseado em GI50, TGI e LC50, os ensaios preliminares mostraram que a maior parte dos compostos mostrou atividade antiproliferativa moderada a boa contra as linhagem de células tumorais testadas, principalmente em células de glioma humano (U251) e câncer de ovário humano com fenótipo de resistentencia a múltiplos fármacos (NCI-ADR/RES). A amida (R,S)-3d, derivada do ácido ricinoleico, mostrou uma elevada seletividade com potência de inibição do crescimento e morte celular para a linhagem de células de glioma. Além disso, as amidas (S)-3c e (S)-3e, derivadas dos ácidos oleico e linoleico respectivamente, foram especificas para glioma e ovário com fenótipo de resistência a múltiplos fármacos com inibição potente do crescimento celular. Estes resultados aliados a um perfil de segurança relativo quando analisado o efeito sobre as linhagens celulares não–tumorais, apontam para que estes compostos sirvam como modelos para o desenvolvimento de candidatos a fármacos para o tratamento de câncer, incluindo cânceres com fenótipo de resistência a múltiplos fármacos.

Predicted residual activity of rilpivirine in HIV-1 infected patients failing therapy including NNRTIs efavirenz or nevirapine

This is an open access article under the CC BY-NC-ND license - http://creativecommons.org/licenses/by-nc-nd/4.0/

The contribution of Angola to the early spread of the HIV-1 epidemic

Poster presented at the 22nd International HIV Dynamics and Evolution. Budapest, Hungary, 13-16 May 2015

Anti-vibrio potentials of acetone and aqueous leaf extracts of Ocimum gratissimum (Linn)

Purpose: To evaluate the anti-vibrio potentials of acetone and aqueous leaf extracts of Ocimum gratissimum and determine its relevance in the treatment of vibrios infection. Methods: The agar-well diffusion method was used for screening the extracts for their anti-vibrio activity. Broth micro-dilution assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts. Time-kill assay was used to assess bactericidal and/or bacteriostatic activity. Results: The acetone extract showed activity against 47.5 % (19/40) of the test bacteria, while the aqueous extract had activity against 30 % (12/40). MIC and MBC values range for the acetone extract were 0.625 – 5.0 mg/mL and 2.5 – 10 mg/mL respectively. The range of MIC exhibited by the antibiotic (gentamicin) against the vibrios is 0.002 mg/mL and >0.256 mg/mL. Significant reduction in the bacterial density was at 2 × MIC after a 4 h interaction period, while bacterial density after 6 and 8 h interactions with extract was highly bactericidal. Growth inhibition and efficacy of the crude acetone extract were observed to be both concentration- and time-dependent. Conclusion: The bacteriostatic and bactericidal activities observed for Ocimum gratissimum leaf suggest that the plant is a potential source of bioactive components that may be effective in the treatment of vibrios infections.

Anti-biofilm and antimicrobial activity of Mentha pulegium L essential oil against multidrug-resistant Acinetobacter baumannii

Purpose: To investigate the antimicrobial and anti-biofilm activities of essential oil from Mentha pulegium L. (EOMP) on multi-drug resistant (MDR) isolates of A. baumannii , as well as its phytochemical composition, antioxidant properties and cytotoxic activity. Methods: The phytochemical composition of EOMP was analyzed by gas chromatography, while its antimicrobial activities were determined by disc diffusion and broth micro-dilution methods. Minimal biofilm inhibition concentration (MBIC) and minimal biofilm eradication concentration (MBEC) tests were used for assessment of its anti-biofilm properties. Viability in the biofilm was studied using 2,3-bis (2- methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, while colorimetric assay was used to assess its cytotoxicity on L929 cells. Results: D-isomenthone, pulegone, isopulegone, menthol and piperitenone were the major components of the plant extract. EOMP produced > 22 mm inhibition zone for the isolates, with minimum inhibitory concentration (MIC) and MBIC of 0.6 - 2.5 and 0.6 - 1.25 μL/mL, respectively, while MBEC was ≥ 10 μL/msL. EOMP damaged biofilm structures formed by A. baumannii strains at MIC by 26 – 91 %. Conclusion: These results suggest that EOMP contains agents that may be useful in the development of new drugs against A. baumannii infections.

Influence de l’hypoxie dans la régulation du pH et les conséquences dans la progression tumorale et la résistance à la chimiothérapie

Résumé : La formation de métastases s’inscrit comme la finalité d’un processus darwinien dans lequel les cellules tumorales subissent des altérations génétiques et épigénétiques dans l’unique but de préserver un avantage prolifératif. L’environnement hypoxique, caractéristique des tumeurs solides, se révèle comme une pression de sélection et un facteur déterminant dans la progression tumorale. Face à l’hypoxie, une des adaptations majeures des cellules tumorales est le déséquilibre du pH cellulaire qui mène à la formation de métastases et à la résistance à la chimiothérapie. Cette thèse met en lumière de nouveaux liens moléculaires entre l’hypoxie et la régulation du pH dans des contextes d’invasion cellulaire et de chimiorésistance. Les échangeurs d’ions NHE1 et NHE6 sont au cœur de ces études où de nouveaux rôles dans la progression du cancer leur ont été attribués. Premièrement, nous avons observé l’influence de l’hypoxie sur la régulation de NHE1 par p90RSK et les conséquences fonctionnelles de cette interaction dans l’invasion cellulaire par les invadopodes. En conditions hypoxiques, NHE1 est activé par p90RSK résultant en une acidification extracellulaire. En modifiant le pH, NHE1 stimule la formation des invadopodes et la dégradation de la matrice extracellulaire. Ainsi, la phosphorylation de NHE1 par p90RSK en hypoxie apparaît comme un biomarqueur potentiel des cancers métastatiques. Peu étudié, le pH endosomal peut intervenir dans la chimiorésistance mais les mécanismes sont inconnus. Nous avons développé une méthode pour mesurer précisément le pH endosomal par microscopie. Ceci a permis d’illuminer un nouveau mécanisme de résistance induit par l’hypoxie et mettant en vedette l’échangeur NHE6. L’hypoxie favorise l’interaction de NHE6 avec RACK1 à la membrane plasmique empêchant la localisation endosomale de l’échangeur. Cette interaction mène à la séquestration de la doxorubicine dans des endosomes sur-acidifiés. Ces travaux mettent en évidence pour la première fois le rôle du pH endosomal et l’échangeur NHE6 comme des éléments centraux de la chimiorésistance induite par l’hypoxie. Cette thèse renforce donc l’idée voulant que les interactions entre les cellules tumorales et le microenvironnement hypoxique sont le « talon d’Achille » du cancer et la régulation du pH cellulaire est primordiale dans l’adaptation des cellules à l’hypoxie et l’instauration du phénotype malin du cancer. La découverte de nouveaux rôles pro-tumoraux pour NHE1 et NHE6 les placent à l’avant-plan pour le développement de stratégies thérapeutiques orientées contre la formation de métastases et la chimiorésistance.

Ocratossina A, metalli, insetticidi e fitoestratti: valutazione degli effetti e degli aspetti residuali nel pollame e in alimenti destinati all'uomo.

La presente tesi di Dottorato di Ricerca descrive le attività di ricerca riguardanti diversi ambiti della sicurezza alimentare, della farmacologia e tossicologia veterinaria. Sono presentati i dati relativi alla ricerca di ocratossina A (OTA) in campioni di bile, rene e fegato di polli, salami artigianali e diversi tipi di formaggio; le concentrazioni di diversi metalli pesanti in uova di galline rurali e 5 tipologie di uova industriali; i dati preliminari ottenuti dalle analisi dei primi campioni di uova e penne provenienti da allevamenti amatoriali di pollame, analizzati per la ricerca del fipronil e metaboliti. Sempre per fipronil, e la molecola foxim, sono presentati dati inerenti a studi di potenziale farmaco-resistenza di popolazioni naturali di Dermanyssus gallinae, e dell’attività insetticida di alcuni fitocomposti a base di tannini. Riguardo all’uso di fitoestratti, sono presentati i dati preliminari ottenuti nei primi mesi di una prova sperimentale in galline di razza Livornese allevata con metodo free-range, finalizzata allo studio della attività anti-infiammatoria e immuno-stimolante, nonché alla valutazione della sicurezza della integrazione nella dieta di Boswellia serrata e Salix alba. Sono stati condotti anche studi traslazionali con altri settori scientifici, relativamente la prevalenza sierologica e microbiologica per Salmonella enterica var. enteritidis e var. thiphymurium in allevamenti ornamentali e da auto-consumo di pollame, e di prevalenza e caratterizzazione di potenziali varianti virali e batteriche (incluse zoonosiche) di alcune malattie infettive del pollame ornamentale. In specie avicole ornamentali è riportato anche uno studio di campo sul trattamento off-label con Fenbendazolo e Flubendazolo in infestazioni massive da Cyathostoma bronchialis in oche esotiche ornamentali. A seguito di una collaborazione con l’Università di Firenze, sono presentati anche i risultati ottenuti da uno studio su modelli murini, dell’azione e funzionamento dei recettori Beta3, espressi nelle neoplasie più frequenti che si verificano nelle donne in gravidanza.

Preclinical evaluation of a MYCN-specific inhibitor for the treatment of small cell lung cancer

Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, characterized by rapid growth, early metastasis and acquired drug resistance. SCLC is usually sensitive to initial treatment, however, most patients relapse within few months; thus more effective therapies are urgently needed. Key genetic alterations very frequently observed in SCLC include loss of TP53 and RB1 and mutations in the MYC family genes (MYC, MYCL or MYCN). One of them is amplified and overexpressed in a mutually exclusive manner and represents the most prominent activating oncogene alteration in this malignancy. In particular, MYCN amplification is associated with tumor progression, treatment failure and poor prognosis. Given the role of MYCN in SCLC and its restricted expression profile, MYCN represents a promising therapeutic target; although it is considered undruggable by traditional approaches. An innovative approach to target the oncogene concerns specific MYCN expression inhibition, acting directly at the level of DNA, through an antigene peptide nucleic acid (agPNA) oligonucleotide, called BGA002. This thesis focused on the study of BGA002, as a possible targeted therapeutic strategy for the treatment of MYCN-related SCLC. In this context, BGA002 proved to be a specific and highly effective inhibitor. Furthermore, MYCN silencing induced alterations in many downstream pathways and led to apoptosis, in concomitance with autophagy reactivation. Moreover, systemic administration of BGA002 was effective in vivo as well, significantly increasing survival in MNA mouse models, even in the scenario of multidrug-resistance. In addition, BGA002 treatment successfully reduced N-Myc protein expression and, more importantly, caused a massive diminishment in tumor vascularization in the multidrug-resistant model. Overall, these results proved that MYCN inhibition by BGA002 may represent a new promising precision medicine approach, to treat MYCN-related SCLC.