Targeting the antigen processing and presentation pathway to overcome resistance to immune checkpoint therapy

Despite the significant clinical advances with the use of immune checkpoint inhibitors (ICIs) in a wide range of cancer patients, response rates to the therapy are variable and do not always result in long-term tumor regression. The development of ICI-resistant disease is one of the pressing issue in clinical oncology, and the identification of new targets and combination therapies is a crucial point to improve response rates and duration. Antigen processing and presentation (APP) pathway is a key element for an efficient response to ICI therapy. Indeed, malignancies that do not express tumor antigens are typically poor infiltrated by T cells and unresponsive to ICIs. Therefore, improving tumor immunogenicity potentially increases the success rate of ICI therapy. In this review, we provide an overview of the key elements of the APP machinery that can be exploited to enhance tumor immunogenicity and increase the efficacy of ICI-based immunotherapy

Recent findings on the impact of ErbB receptors status on prognosis and therapy of head and neck squamous cell carcinoma

: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type, has often an aggressive course and is poorly responsive to current therapeutic approaches, so that 5-year survival rates for patients diagnosed with advanced disease is lower than 50%. The Epidermal Growth Factor Receptor (EGFR) has emerged as an established oncogene in HNSCC. Indeed, although HNSCCs are a heterogeneous group of cancers which differ for histological, molecular and clinical features, EGFR is overexpressed or mutated in a percentage of cases up to about 90%. Moreover, aberrant expression of the other members of the ErbB receptor family, ErbB2, ErbB3 and ErbB4, has also been reported in variable proportions of HNSCCs. Therefore, an increased expression/activity of one or multiple ErbB receptors is found in the vast majority of patients with HNSCC. While aberrant ErbB signaling has long been known to play a critical role in tumor growth, angiogenesis, invasion, metastatization and resistance to therapy, more recent evidence has revealed its impact on other features of cancer cells' biology, such as the ability to evade antitumor immunity. In this paper we will review recent findings on how ErbB receptors expression and activity, including that associated with non-canonical signaling mechanisms, impacts on prognosis and therapy of HNSCC

Proline-Rich Tyrosine Kinase 2 and Rac Activation by Chemokine and Integrin Receptors Controls NK Cell Transendothelial Migration

Abstract Protein tyrosine kinase activation is an important requisite for leukocyte migration. Herein we demonstrate that NK cell binding to endothelium activates proline-rich tyrosine kinase 2 (Pyk-2) and the small GTP binding protein Rac that are coupled to integrin and chemokine receptors. Chemokine-mediated, but not integrin-mediated, Pyk-2 and Rac activation was sensitive to pretreatment of NK cells with pertussis toxin, a pharmacological inhibitor of Gi protein-coupled receptors. Both Pyk-2 and Rac are functionally involved in chemokine-induced NK cell migration through endothelium or ICAM-1 or VCAM-1 adhesive proteins, as shown by the use of recombinant vaccinia viruses encoding dominant negative mutants of Pyk-2 and Rac. Moreover, we found that Pyk-2 is associated with the Rac guanine nucleotide exchange factor Vav, which undergoes tyrosine phosphorylation upon integrin triggering. Finally, we provide direct evidence for the involvement of Pyk-2 in the control of both chemokine- and integrin-mediated Rac activation. Collectively, our results indicate that Pyk-2 acts as a receptor-proximal link between integrin and chemokine receptor signaling, and the Pyk-2/Rac pathway plays a pivotal role in the control of NK cell transendothelial migration

DNAM-1-chimeric receptor-engineered NK cells, combined with Nutlin-3a, more effectively fight neuroblastoma cells in vitro: a proof-of-concept study

Adoptive transfer of engineered NK cells, one of clinical approaches to fight cancer, is gaining great interest in the last decade. However, the development of new strategies is needed to improve clinical efficacy and safety of NK cell-based immunotherapy. NK cell-mediated recognition and lysis of tumor cells are strictly dependent on the expression of ligands for NK cell-activating receptors NKG2D and DNAM-1 on tumor cells. Of note, the PVR/CD155 and Nectin-2/CD112 ligands for DNAM-1 are expressed primarily on solid tumor cells and poorly expressed in normal tissue cells. Here, we generated human NK cells expressing either the full length DNAM-1 receptor or three different DNAM-1-based chimeric receptor that provide the expression of DNAM-1 fused to a costimulatory molecule such as 2B4 and CD3ζ chain. Upon transfection into primary human NK cells isolated from healthy donors, we evaluated the surface expression of DNAM-1 and, as a functional readout, we assessed the extent of degranulation, cytotoxicity and the production of IFNγ and TNFα in response to human leukemic K562 cell line. In addition, we explored the effect of Nutlin-3a, a MDM2-targeting drug able of restoring p53 functions and known to have an immunomodulatory effect, on the degranulation of DNAM-1-engineered NK cells in response to human neuroblastoma (NB) LA-N-5 and SMS-KCNR cell lines. By comparing NK cells transfected with four different plasmid vectors and through blocking experiments, DNAM-1-CD3ζ-engineered NK cells showed the strongest response. Furthermore, both LA-N-5 and SMS-KCNR cells pretreated with Nutlin-3a were significantly more susceptible to DNAM-1-engineered NK cells than NK cells transfected with the empty vector. Our results provide a proof-of-concept suggesting that the combined use of DNAM-1-chimeric receptor-engineered NK cells and Nutlin-3a may represent a novel therapeutic approach for the treatment of solid tumors, such as NB, carrying dysfunctional p53

MYCN is an immunosuppressive oncogene dampening the expression of ligands for NK-cell-activating receptors in human high-risk neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor occurring in childhood. Amplification of the MYCN oncogene is associated with poor prognosis. Downregulation on NB cells of ligands recognized by Natural Killer (NK) cell-activating receptors, involved in tumor cell recognition and lysis, may contribute to tumor progression and relapse. Here, we demonstrate that in human NB cell lines MYCN expression inversely correlates with that of ligands recognized by NKG2D and DNAM1 activating receptors in human NB cell lines. In the MYCN-inducible Tet-21/N cell line, downregulation of MYCN resulted in enhanced expression of the activating ligands MICA, ULBPs and PVR, which rendered tumor cells more susceptible to recognition and lysis mediated by NK cells. Conversely, a MYCN non-amplified NB cell line transfected with MYCN showed an opposite behavior compared with control cells. Consistent with these findings, an inverse correlation was detected between the expression of MYCN and that of ligands for NK-cell-activating receptors in 12 NB patient specimens both at mRNA and protein levels. Taken together, these results provide the first demonstration that MYCN acts as an immunosuppressive oncogene in NB cells that negatively regulates the expression of ligands for NKG2D and DNAM-1 NK-cell-activating receptors. Our study provides a clue to exploit MYCN expression levels as a biomarker to predict the efficacy of NK-cell-based immunotherapy in NB patients. KEYWORDS: Immunosuppressive oncogene, MYCN oncogene, neuroblastoma, NK-cell-activating receptor ligands, tumor immune escap

DNAM-1 chimeric receptor-engineered NK cells: a new frontier for CAR-NK cell-based immunotherapy

DNAM-1 is a major NK cell activating receptor and, together with NKG2D and NCRs, by binding specific ligands, strongly contributes to mediating the killing of tumor or virus-infected cells. DNAM-1 specifically recognizes PVR and Nectin-2 ligands that are expressed on some virus-infected cells and on a broad spectrum of tumor cells of both hematological and solid malignancies. So far, while NK cells engineered for different antigen chimeric receptors (CARs) or chimeric NKG2D receptor have been extensively tested in preclinical and clinical studies, the use of DNAM-1 chimeric receptor-engineered NK cells has been proposed only in our recent proof-of-concept study and deserves further development. The aim of this perspective study is to describe the rationale for using this novel tool as a new anti-cancer immunotherapy

Combined treatment with inhibitors of ErbB Receptors and Hh signaling pathways is more effective than single treatment in reducing the growth of malignant mesothelioma both in vitro and in vivo

Malignant mesothelioma (MM) is a rare orphan aggressive neoplasia with low survival rates. Among the other signaling pathways, ErbB receptors and Hh signaling are deregulated in MM. Thus, molecules involved in these signaling pathways could be used for targeted therapy approaches. The aim of this study was to evaluate the effects of inhibitors of Hh- (GANT-61) and ErbB receptors (Afatinib)-mediated signaling pathways, when used alone or in combination, on growth, cell cycle, cell death and autophagy, modulation of molecules involved in transduction pathways, in three human MM cell lines of different histotypes. The efficacy of the combined treatment was also evaluated in a murine epithelioid MM cell line both in vitro and in vivo. This study demonstrated that combined treatment with two inhibitors counteracting the activation of two different signaling pathways involved in neoplastic transformation and progression, such as those activated by ErbB and Hh signaling, is more effective than the single treatments in reducing MM growth in vitro and in vivo. This study may have clinical implications for the development of targeted therapy approaches for MM

Antagomir-17-5p Abolishes the Growth of Therapy-Resistant Neuroblastoma through p21 and BIM

We identified a key oncogenic pathway underlying neuroblastoma progression: specifically, MYCN, expressed at elevated level, transactivates the miRNA 17-5p-92 cluster, which inhibits p21 and BIM translation by interaction with their mRNA 3′ UTRs. Overexpression of miRNA 17-5p-92 cluster in MYCN-not-amplified neuroblastoma cells strongly augments their in vitro and in vivo tumorigenesis. In vitro or in vivo treatment with antagomir-17-5p abolishes the growth of MYCN-amplified and therapy-resistant neuroblastoma through p21 and BIM upmodulation, leading to cell cycling blockade and activation of apoptosis, respectively. In primary neuroblastoma, the majority of cases show a rise of miR-17-5p level leading to p21 downmodulation, which is particularly severe in patients with MYCN amplification and poor prognosis. Altogether, our studies demonstrate for the first time that antagomir treatment can abolish tumor growth in vivo, specifically in therapy-resistant neuroblastoma

Targeted NGS Platforms for Genetic Screening and Gene Discovery in Primary Immunodeficiencies

Background: Primary Immunodeficiencies (PIDs) are a heterogeneous group of genetic immune disorders. While some PIDs can manifest with more than one phenotype, signs, and symptoms of various PIDs overlap considerably. Recently, novel defects in immune-related genes and additional variants in previously reported genes responsible for PIDs have been successfully identified by Next Generation Sequencing (NGS), allowing the recognition of a broad spectrum of disorders.Objective: To evaluate the strength and weakness of targeted NGS sequencing using custom-made Ion Torrent and Haloplex (Agilent) panels for diagnostics and research purposes.Methods: Five different panels including known and candidate genes were used to screen 105 patients with distinct PID features divided in three main PID categories: T cell defects, Humoral defects and Other PIDs. The Ion Torrent sequencing platform was used in 73 patients. Among these, 18 selected patients without a molecular diagnosis and 32 additional patients were analyzed by Haloplex enrichment technology.Results: The complementary use of the two custom-made targeted sequencing approaches allowed the identification of causative variants in 28.6% (n = 30) of patients. Twenty-two out of 73 (34.6%) patients were diagnosed by Ion Torrent. In this group 20 were included in the SCID/CID category. Eight out of 50 (16%) patients were diagnosed by Haloplex workflow. Ion Torrent method was highly successful for those cases with well-defined phenotypes for immunological and clinical presentation. The Haloplex approach was able to diagnose 4 SCID/CID patients and 4 additional patients with complex and extended phenotypes, embracing all three PID categories in which this approach was more efficient. Both technologies showed good gene coverage.Conclusions: NGS technology represents a powerful approach in the complex field of rare disorders but its different application should be weighted. A relatively small NGS target panel can be successfully applied for a robust diagnostic suspicion, while when the spectrum of clinical phenotypes overlaps more than one PID an in-depth NGS analysis is required, including also whole exome/genome sequencing to identify the causative gene