IL-7R-mediated signaling in T-cell acute lymphoblastic leukemia: an update

© 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/)Interleukin 7 (IL-7) and its receptor (IL-7R, a heterodimer of IL-7Rα and γc) are essential for normal lymphoid development. In their absence, severe combined immunodeficiency occurs. By contrast, excessive IL-7/IL-7R-mediated signaling can drive lymphoid leukemia development, disease acceleration and resistance to chemotherapy. IL-7 and IL-7R activate three main pathways: STAT5, PI3K/Akt/mTOR and MEK/Erk, ultimately leading to the promotion of leukemia cell viability, cell cycle progression and growth. However, the contribution of each of these pathways towards particular functional outcomes is still not completely known and appears to differ between normal and malignant states. For example, IL-7 upregulates Bcl-2 in a PI3K/Akt/mTOR-dependent and STAT5-independent manner in T-ALL cells. This is a 'symmetric image' of what apparently happens in normal lymphoid cells, where PI3K/Akt/mTOR does not impact on Bcl-2 and regulates proliferation rather than survival. In this review, we provide an updated summary of the knowledge on IL-7/IL-7R-mediated signaling in the context of cancer, focusing mainly on T-cell acute lymphoblastic leukemia, where this axis has been more extensively studied.Publication costs were supported by LISBOA-01-0145-FEDER-007391, project cofunded by FEDER, through POR Lisboa2020 Programa Operacional Regional de Lisboa, PORTUGAL 2020, and Fundação para a Ciência e a Tecnologia (FCT, Portugal). The research work in JTB's lab related to the present review was supported by the grants FAPESP/20015/2014 and PTDC/MEC-HEM/31588/2017, from FCT; and by the consolidator grant ERC CoG-648455 from the European Research Council, under the European Union's Horizon 2020 research and innovation programme. JTB is an FCT investigator (consolidator). MLO is a LisbonBioMed PhD student and received a fellowship from FCT. PA received a PhD fellowship from the EU Marie Sklodowska-Curie ITN Protein Conjugates.info:eu-repo/semantics/publishedVersio

Antibody based therapy for T-cell acute lymphoblastic leukemia : targeting IL-7 receptor

A Leucemia Linfoblástica Aguda de células T (LLA-T) é um cancro hematológico agressivo, para o qual a abordagem terapêutica atual consiste em regimes intensivos de quimioterapia multiagente, frequentemente levando a efeitos secundários a longo prazo que afetam a qualidade de vida dos doentes. Apesar do sucesso terapêutico em casos pediátricos, as recidivas ainda ocorrem em 10-20% dos casos, e o prognóstico é consideravelmente pior em adultos. Assim, existe uma necessidade de encontrar novos tratamentos mais seletivos e como tal, menos tóxicos, bem como de melhorar os resultados associados ao tratamento de LLA-T. A interleucina 7 (IL-7) e seu receptor, IL-7Rα, promovem o desenvolvimento de leucemia na maioria dos pacientes com LLA-T e à ativação mutacional de IL-7Rα, que ocorre em cerca de 10% dos casos, associa-se um elevado risco de recidiva. Utilizando bibliotecas de apresentação de fagos com combinações de scFv e reformatação de anticorpos, conseguimos produzir um anticorpo monoclonal IgG1, totalmente humano contra o IL-7Rα humano (denominado B12). B12 não apresenta reatividade cruzada contra o receptor de murganho e reconhece tanto a forma wild type como as formas mutantes de IL-7Rα, expressas naturalmente em linhas celulares de LLA-T e amostras de pacientes, bem como em células Ba/F3 transduzidas de forma estável com IL-7Rα humano. Simulações de dinâmica molecular sugerem que B12 forma um complexo estável com IL-7Rα num local diferente da IL-7. No entanto, B12 inibe a sinalização mediada por IL-7/IL-7R e induz morte celular per se em algumas linhas celulares de T-ALL, dependentes do eixo de sinalização IL-7/IL-7R (por exemplo, células TAIL7, dependentes de IL-7, e células DND4.1, que possuem IL7R mutante) e amostras de pacientes. Utilizando amostras de xenoenxertos derivados de doentes (PDX), células HPB-ALL e células D1 que sobrexpressam uma forma mutada para ganho de função de IL-7Rα, mostrámos, in vitro, que o anticorpo promove citotoxicidade mediada por células NK dependente deste e, in vivo, atrasos no desenvolvimento de leucemia de células T, reduzindo a carga tumoral e promovendo a sobrevivência do murganho. Além disso, B12 coopera com dexametasona na promoção da morte tanto de células HPB-ALL, resistentes a este fármaco, como de uma amostra de PDX, sensível ao mesmo. De notar, B12 é rapidamente internalizado através de poços revestidos de clatrina para o endossoma inicial, eventualmente traficando para o lisossoma - um efeito que é ligeiramente acelerado na presença de IL-7. Essas características tornam a B12 um veículo atraente para administração intracelular direcionada de uma carga altamente citotóxica. Como tal, desenvolvemos uma molécula de anticorpo conjugada com droga (ADC), B12-mono-metil-auristatina E (MMAE), na qual a conjugação sítio-específica de B12 foi realizada reduzindo-se as ligações dissulfeto inter-cadeias e reagindo o grupo tiol das cisteínas livres com um aceitador de Michael (derivado carbonil acrílico) ligado a um ligante clivável (valina-citrulina) e ao fármaco (MMAE). Testado contra diferentes linhagens celulares, células primárias de pacientes e amostras de PDX, o ADC B12-MMAE demonstra aumento da capacidade matar células leucémicas, in vitro, em comparação com o anticorpo não conjugado. No geral, este trabalho serve como ponto de partida para o desenvolvimento de novas estratégias terapêuticas direcionadas contra LLA-T e outras doenças, nas quais o IL-7Rα desempenha um papel patológico.T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer that is treated with intensive multi-agent chemotherapy, often leading to long-term side-effects impacting the quality of life of survivors. Despite the therapeutic success in children, relapses still occur in 10-20% of the cases, and adults face a considerably poorer prognosis. Novel, more selective treatments that contribute to reducing toxicities and improving outcome are thus in need. Interleukin 7 (IL-7) and its receptor IL-7Rα promote leukemia development in a majority of T-ALL patients and mutational activation of IL-7Rα, which occurs in around 10% of the cases, associates with very high risk in relapsed disease. Using combinatorial scFv phage display libraries and antibody reformatting we have now generated a fully human IgG1 monoclonal antibody (named B12) against human IL-7Rα. B12 does not display cross-reactivity against the mouse receptor and recognizes both wild type and mutant forms of IL-7Rα naturally expressed in T-ALL cell lines and patient samples, as well as in Ba/F3 cells stably transduced with human, but not mouse, IL-7Rα. Interestingly, molecular dynamics simulations suggest that B12 forms a stable complex with IL-7Rα at a different site from IL-7. Nonetheless, B12 inhibits IL-7/IL-7R-mediated signaling and induces cell death per se in at least some IL-7/IL-7Rreliant T-ALL cell lines (e.g. IL-7-dependent TAIL7 cells and mutant IL7R DND4.1 cells) and patient samples. Using patient-derived xenograft (PDX) samples, HPBALL cells and D1 cells overexpressing a mutated gain-of-function form of IL-7Rα, we show that the antibody also promotes antibody-dependent NK-mediated leukemia cytotoxicity in vitro and delays T-cell leukemia development In vivo, reducing tumor burden and promoting mouse survival. Moreover, B12 cooperates with dexamethasone in promoting the death of both dexamethasone-resistant HPBALL cells and a dexamethasone-sensitive PDX sample. Notably, B12 is rapidly internalized via clathrin-coated pits to the early endosome, eventually trafficking to the lysosome - an effect that is slightly accelerated in the presence of IL-7. These characteristics render B12 an attractive vehicle for targeted intracellular delivery of a highly cytotoxic warhead. As such, we engineered a B12- mono-methyl auristatin E (MMAE) antibody-drug conjugate (ADC) in which sitespecific conjugation of B12 was carried out by reducing inter-chain disulfide bonds and reacting the thiol group of the free cysteines with a Michael acceptor (carbonyl acrylic derivate) linked to a cleavable linker (valine-citrulline) and the drug (MMAE). Tested against different cell lines, primary patient cells and PDX samples, B12-MMAE ADC demonstrates increased leukemia cell killing ability in vitro as compared to the naked antibody. Altogether, our studies serve as a stepping stone towards the development of novel targeted therapeutic strategies in T-ALL and other diseases where IL-7Rα was shown to play a pathological role

Membrane Active Vancomycin Analogues: A Strategy to Combat Bacterial Resistance

The alarming growth of antibiotic resistant superbugs such as vancomycin-resistant Enterococci and Staphylococci has become a major global health hazard. To address this issue, we report the development of lipophilic cationic vancomycin analogues possessing excellent antibacterial activity against several drug-resistant strains. Compared to vancomycin, efficacy greater than 1000-fold was demonstrated against vancomycin-resistant Enterococci (VRE). Significantly, unlike vancomycin, these compounds were shown to be bactericidal at low concentrations and did not induce bacterial resistance. An optimized compound in the series, compared to vancomycin, showed higher activity in methicillin-resistant Staphylococcus aureus (MRSA) infected mouse model and exhibited superior antibacterial activity in whole blood with no observed toxicity. The remarkable activity of these compounds is attributed to the incorporation of a new membrane disruption mechanism into vancomycin and opens up a great opportunity for the development of novel antibiotics

A Biodegradable Polycationic Paint that Kills Bacteria <i>in Vitro</i> and <i>in Vivo</i>

Bacterial colonization and subsequent formation of biofilms onto surfaces of medical devices and implants is a major source of nosocomial infections. Most antibacterial coatings to combat infections are either metal-based or nondegradable-polymer-based and hence limited by their nondegradability and unpredictable toxicity. Moreover, to combat infections effectively, the coatings are required to display simultaneous antibacterial and antibiofilm activity. Herein we report biocompatible and biodegradable coatings based on organo-soluble quaternary chitin polymers which were immobilized noncovalently onto surfaces as bactericidal paint. The polycationic paint was found to be active against both drug-sensitive and -resistant bacteria such as methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), vancomycin-resistant <i>Enterococcus faecium</i> (VRE), and β-lactam-resistant <i>Klebsiella pneumoniae</i>. The cationic polymers were shown to interact with the negatively charged bacterial cell membrane and disrupt the membrane integrity, thereby causing leakage of intracellular constituents and cell death upon contact. Importantly, surfaces coated with the polymers inhibited formation of biofilms against both Gram-positive <i>S. aureus</i> and Gram-negative <i>E. coli</i>, two of the most clinically important bacteria that form biofilms. Surfaces coated with the polymers displayed negligible toxicity against human erythrocytes and embryo kidney cells. Notably, the polymers were shown to be susceptible toward lysozyme. Furthermore, subcutaneous implantation of polymer discs in rats led to 15–20% degradation in 4 weeks thereby displaying their biodegradability. In a murine model of subcutaneous infection, polymer-coated medical-grade catheter reduced MRSA burden by 3.7 log compared to that of noncoated catheter. Furthermore, no biofilm development was observed on the coated catheters under <i>in vivo</i> conditions. The polycationic materials thus developed herein represent a novel class of safe and effective coating agents for the prevention of device-associated infections

Cleavable Cationic Antibacterial Amphiphiles: Synthesis, Mechanism of Action, and Cytotoxicities

The development of novel antimicrobial agents having high selectivity toward bacterial cells over mammalian cells is urgently required to curb the widespread emergence of infectious diseases caused by pathogenic bacteria. Toward this end, we have developed a set of cationic dimeric amphiphiles (bearing cleavable amide linkages between the headgroup and the hydrocarbon tail with different methylene spacers) that showed high antibacterial activity against human pathogenic bacteria (<i>Escherichia coli</i> and <i>Staphylococcus aureus</i>) and low cytotoxicity. The Minimum Inhibitory Concentrations (MIC) were found to be very low for the dimeric amphiphiles and were lower or comparable to the monomeric counterpart. In the case of dimeric amphiphiles, MIC was found to decrease with the increase in the spacer chain length (<i>n</i> = 2 to 6) and again to increase at higher spacer length (<i>n</i> > 6). It was found that the compound with six methylene spacers was the most active among all of the amphiphiles (MICs = 10–13 μM). By fluorescence spectroscopy, fluorescence microscopy, and field-emission scanning electron microscopy (FESEM), it was revealed that these cationic amphiphiles interact with the negatively charged bacterial cell membrane and disrupt the membrane integrity, thus killing the bacteria. All of the cationic amphiphiles showed low hemolytic activity (HC₅₀) and high selectivity against both gram-positive and gram-negative bacteria. The most active amphiphile (<i>n</i> = 6) had a 10–13-fold higher HC₅₀ than did the MIC. Also, this amphiphile did not show any cytotoxicity against mammalian cells (HeLa cells) even at a concentration above the MIC (20 μM). The critical micellar concentration (CMC) values of gemini surfactants were found to be very low (CMC = 0.30–0.11 mM) and were 10–27 times smaller than the corresponding monomeric analogue (CMC = 2.9 mM). Chemical hydrolysis and thermogravimetric analysis (TGA) proved that these amphiphiles are quite stable under both acidic and thermal conditions. Collectively, these properties make the newly synthesized amphiphiles potentially superior disinfectants and antiseptics for various biomedical and biotechnological applications

A fully human anti-IL-7Rα antibody promotes antitumor activity against T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer for which treatment options often result in incomplete therapeutic efficacy and long-term side-effects. Interleukin 7 (IL-7) and its receptor IL-7Rα promote T-ALL development and mutational activation of IL-7Rα associates with very high risk in relapsed disease. Using combinatorial phage-display libraries and antibody reformatting, we generated a fully human IgG1 monoclonal antibody (named B12) against both wild-type and mutant human IL-7Rα, predicted to form a stable complex with IL-7Rα at a different site from IL-7. B12 impairs IL-7/IL-7R-mediated signaling, sensitizes T-ALL cells to treatment with dexamethasone and can induce cell death per se. The antibody also promotes antibody-dependent natural killer-mediated leukemia cytotoxicity in vitro and delays T-cell leukemia development in vivo, reducing tumor burden and promoting mouse survival. B12 is rapidly internalized and traffics to the lysosome, rendering it an attractive vehicle for targeted intracellular delivery of cytotoxic cargo. Consequently, we engineered a B12–MMAE antibody–drug conjugate and provide proof-of-concept evidence that it has increased leukemia cell killing abilities as compared with the naked antibody. Our studies serve as a stepping stone for the development of novel targeted therapies in T-ALL and other diseases where IL-7Rα has a pathological role

Small Molecular Antibacterial Peptoid Mimics: The Simpler the Better!

The emergence of multidrug resistant bacteria compounded by the depleting arsenal of antibiotics has accelerated efforts toward development of antibiotics with novel mechanisms of action. In this report, we present a series of small molecular antibacterial peptoid mimics which exhibit high in vitro potency against a variety of Gram-positive and Gram-negative bacteria, including drug-resistant species such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. The highlight of these compounds is their superior activity against the major nosocomial pathogen Pseudomonas aeruginosa. Nontoxic toward mammalian cells, these rapidly bactericidal compounds primarily act by permeabilization and depolarization of bacterial membrane. Synthetically simple and selectively antibacterial, these compounds can be developed into a newer class of therapeutic agents against multidrug resistant bacterial species

Broad Spectrum Antibacterial and Antifungal Polymeric Paint Materials: Synthesis, Structure–Activity Relationship, and Membrane-Active Mode of Action

Microbial attachment and subsequent colonization onto surfaces lead to the spread of deadly community-acquired and hospital-acquired (nosocomial) infections. Noncovalent immobilization of water insoluble and organo-soluble cationic polymers onto a surface is a facile approach to prevent microbial contamination. In the present study, we described the synthesis of water insoluble and organo-soluble polymeric materials and demonstrated their structure–activity relationship against various human pathogenic bacteria including drug-resistant strains such as methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), vancomycin-resistant enterococci (VRE), and beta lactam-resistant <i>Klebsiella pneumoniae</i> as well as pathogenic fungi such as <i>Candida</i> spp. and <i>Cryptococcus</i> spp. The polymer coated surfaces completely inactivated both bacteria and fungi upon contact (5 log reduction with respect to control). Linear polymers were more active and found to have a higher killing rate than the branched polymers. The polymer coated surfaces also exhibited significant activity in various complex mammalian fluids such as serum, plasma, and blood and showed negligible hemolysis at an amount much higher than minimum inhibitory amounts (MIAs). These polymers were found to have excellent compatibility with other medically relevant polymers (polylactic acid, PLA) and commercial paint. The cationic hydrophobic polymer coatings disrupted the lipid membrane of both bacteria and fungi and thus showed a membrane-active mode of action. Further, bacteria did not develop resistance against these membrane-active polymers in sharp contrast to conventional antibiotics and lipopeptides, thus the polymers hold great promise to be used as coating materials for developing permanent antimicrobial paint