UMG1/CD3ε-bispecific T-cell engager redirects T-cell cytotoxicity against diffuse large B-cell lymphoma.

UMG1 is a unique epitope of CD43, not expressed by normal cells and tissues of haematopoietic and non-haematopoietic origin, except thymocytes and a minority (<5%) of peripheral blood T lymphocytes. By immunohistochemistry analysis of tissue microarray and pathology slides, we found high UMG1 expression in 20%-24% of diffuse large B-cell lymphomas (DLBCLs), including highly aggressive BCL2high and CD20low cases. UMG1 membrane expression was also found in DLBCL bone marrow-infiltrating cells and established cell lines. Targeting UMG1 with a novel asymmetric UMG1/CD3ε-bispecific T-cell engager (BTCE) induced redirected cytotoxicity against DLBCL cells and was synergistic with lenalidomide. We conclude that UMG1/CD3ε-BTCE is a promising therapeutic for DLBCLs.

Reactivation of telomerase reverse transcriptase expression in cancer: the role of TERT promoter mutations.

Telomerase activity and telomere elongation are essential conditions for the unlimited proliferation of neoplastic cells. Point mutations in the core promoter region of the telomerase reverse transcriptase (TERT) gene have been found to occur at high frequencies in several tumour types and considered a primary cause of telomerase reactivation in cancer cells. These mutations promote TERT gene expression by multiple mechanisms, including the generation of novel binding sites for nuclear transcription factors, displacement of negative regulators from DNA G-quadruplexes, recruitment of epigenetic activators and disruption of long-range interactions between TERT locus and telomeres. Furthermore, TERT promoter mutations cooperate with TPP1 promoter nucleotide changes to lengthen telomeres and with mutated BRAF and FGFR3 oncoproteins to enhance oncogenic signalling in cancer cells. TERT promoter mutations have been recognized as an early marker of tumour development or a major indicator of poor outcome and reduced patients survival in several cancer types. In this review, we summarize recent findings on the role of TERT promoter mutations, telomerase expression and telomeres elongation in cancer development, their clinical significance and therapeutic opportunities.

The Molecular Interplay between Human Oncoviruses and Telomerase in Cancer Development.

Human oncoviruses are able to subvert telomerase function in cancer cells through multiple strategies. The activity of the catalytic subunit of telomerase (TERT) is universally enhanced in virus-related cancers. Viral oncoproteins, such as high-risk human papillomavirus (HPV) E6, Epstein-Barr virus (EBV) LMP1, Kaposi's sarcoma-associated herpesvirus (HHV-8) LANA, hepatitis B virus (HBV) HBVx, hepatitis C virus (HCV) core protein and human T-cell leukemia virus-1 (HTLV-1) Tax protein, interact with regulatory elements in the infected cells and contribute to the transcriptional activation of TERT gene. Specifically, viral oncoproteins have been shown to bind TERT promoter, to induce post-transcriptional alterations of TERT mRNA and to cause epigenetic modifications, which have important effects on the regulation of telomeric and extra-telomeric functions of the telomerase. Other viruses, such as herpesviruses, operate by integrating their genomes within the telomeres or by inducing alternative lengthening of telomeres (ALT) in non-ALT cells. In this review, we recapitulate on recent findings on virus-telomerase/telomeres interplay and the importance of TERT-related oncogenic pathways activated by cancer-causing viruses.

A Novel Bispecific T-Cell Engager (CD1a x CD3ε) BTCE Is Effective against Cortical-Derived T Cell Acute Lymphoblastic Leukemia (T-ALL) Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy burdened by poor prognosis. While huge progress of immunotherapy has recently improved the outcome of B-cell malignancies, the lack of tumor-restricted T-cell antigens still hampers its progress in T-ALL. Therefore, innovative immunotherapeutic agents are eagerly awaited. To this end, we generated a novel asymmetric (2 + 1) bispecific T-cell engager (BTCE) targeting CD1a and CD3ε (CD1a x CD3ε) starting from the development of a novel mAb named UMG2. UMG2 mAb reacts against CD1a, a glycoprotein highly expressed by cortical T-ALL cells. Importantly, no UMG2 binding was found on normal T-cells. CD1a x CD3ε induced high T-cell mediated cytotoxicity against CD1a+ T-ALL cells in vitro, as demonstrated by the concentration-dependent increase of T-cell proliferation, degranulation, induction of cell surface activation markers, and secretion of pro-inflammatory cytokines. Most importantly, in a PBMC-reconstituted NGS mouse model bearing human T-ALL, CD1a x CD3ε significantly inhibited the growth of human T-ALL xenografts, translating into a significant survival advantage of treated animals. In conclusion, CD1a x CD3ε is a novel BTCE highly active against CD1a-expressing cortical-derived T-ALL cells suitable for clinical development as an effective therapeutic option for this rare and aggressive disease.

The Role of circRNAs in Human Papillomavirus (HPV)-Associated Cancers.

Circular RNAs (circRNAs) are a new class of "non-coding RNAs" that originate from non-sequential back-splicing of exons and/or introns of precursor messenger RNAs (pre-mRNAs). These molecules are generally produced at low levels in a cell-type-specific manner in mammalian tissues, but due to their circular conformation they are unaffected by the cell mRNA decay machinery. circRNAs can sponge multiple microRNAs or RNA-binding proteins and play a crucial role in the regulation of gene expression and protein translation. Many circRNAs have been shown to be aberrantly expressed in several cancer types, and to sustain specific oncogenic processes. Particularly, in virus-associated malignancies such as human papillomavirus (HPV)-associated anogenital carcinoma and oropharyngeal and oral cancers, circRNAs have been shown to be involved in tumorigenesis and cancer progression, as well as in drug resistance, and some are useful diagnostic and prognostic markers. HPV-derived circRNAs, encompassing the HPV E7 oncogene, have been shown to be expressed and to serve as transcript for synthesis of the E7 oncoprotein, thus reinforcing the virus oncogenic activity in HPV-associated cancers. In this review, we summarize research advances in the biogenesis of cell and viral circRNAs, their features and functions in the pathophysiology of HPV-associated tumors, and their importance as diagnostic, prognostic, and therapeutic targets in anogenital and oropharyngeal and oral cancers.

Precision medicine in gastric cancer.

Gastric cancer (GC) is a complex disease linked to a series of environmental factors and unhealthy lifestyle habits, and especially to genetic alterations. GC represents the second leading cause of cancer-related deaths worldwide. Its onset is subtle, and the majority of patients are diagnosed once the cancer is already advanced. In recent years, there have been innovations in the management of advanced GC including the introduction of new classifications based on its molecular characteristics. Thanks to new technologies such as next-generation sequencing and microarray, the Cancer Genome Atlas and Asian Cancer Research Group classifications have also paved the way for precision medicine in GC, making it possible to integrate diagnostic and therapeutic methods. Among the objectives of the subdivision of GC into subtypes is to select patients in whom molecular targeted drugs can achieve the best results; many lines of research have been initiated to this end. After phase III clinical trials, trastuzumab, anti-Erb-B2 receptor tyrosine kinase 2 (commonly known as ERBB2) and ramucirumab, anti-vascular endothelial growth factor receptor 2 (commonly known as VEGFR2) monoclonal antibodies, were approved and introduced into first- and second-line therapies for patients with advanced/metastatic GC. However, the heterogeneity of this neoplasia makes the practical application of such approaches difficult. Unfortunately, scientific progress has not been matched by progress in clinical practice in terms of significant improvements in prognosis. Survival continues to be low in contrast to the reduction in deaths from many common cancers such as colorectal, lung, breast, and prostate cancers. Although several target molecules have been identified on which targeted drugs can act and novel products have been introduced into experimental therapeutic protocols, the overall approach to treating advanced stage GC has not substantially changed. Currently, surgical resection with adjuvant or neoadjuvant radiotherapy and chemotherapy are the most effective treatments for this disease. Future research should not underestimate the heterogeneity of GC when developing diagnostic and therapeutic strategies aimed toward improving patient survival.

Role of gut microbiota and oxidative stress in the progression of non-alcoholic fatty liver disease to hepatocarcinoma: Current and innovative therapeutic approaches.

Non-alcoholic fatty liver disease (NAFLD) represents the most common chronic liver disease in industrialized countries. NAFLD progresses through the inflammatory phase of non-alcoholic steatohepatitis (NASH) to fibrosis and cirrhosis, with some cases developing liver failure or hepatocellular carcinoma (HCC). Liver biopsy remains the gold standard approach to a definitive diagnosis of NAFLD and the distinction between simple steatosis and NASH. The pathogenesis of NASH is still not clear. Several theories have been proposed ranging from the "Two Hit Theory" to the "Multiple Hit Theory". However, the general consensus is that the gut microbiota, oxidative stress, and mitochondrial damage play key roles in the pathogenesis of NASH. The interaction between the gut epithelia and some commensal bacteria induces the rapid generation of reactive oxygen species (ROS). The main goal of any therapy addressing NASH is to reverse or prevent progression to liver fibrosis/cirrhosis. This problem represents the first "Achilles' heel" of the new molecules being evaluated in most ongoing clinical trials. The second is the inability of these molecules to reach the mitochondria, the primary sites of energy production and ROS generation. Recently, a variety of non-pharmacological and pharmacological treatment approaches for NASH have been evaluated including vitamin E, the thiazolidinediones, and novel molecules related to NASH pathogenesis (including obeticholic acid and elafibranor). Recently, a new isoform of human manganese superoxide dismutase (MnSOD) was isolated and obtained in a synthetic recombinant form designated rMnSOD. This protein has been shown to be a powerful antioxidant capable of mediating ROS dismutation, penetrating biological barriers via its uncleaved leader peptide, and reducing portal hypertension and fibrosis in rats affected by liver cirrhosis. Based on these distinctive characteristics, it can be hypothesized that this novel recombinant protein (rMnSOD) potentially represents a new and highly efficient adjuvant therapy to counteract the progression from NASH to HCC.

A new hexapeptide from the leader peptide of rMnSOD enters cells through the oestrogen receptor to deliver therapeutic molecules.

A 24-amino acid leader peptide of a new human recombinant manganese superoxide dismutase can enter cells and carry molecules. Here, we demonstrated that six of the 24 amino acids penetrate cells through a particular gate represented by a specific amino acid sequence of the oestrogen receptor (ER). We analysed the internalization of the synthetic hexapeptide and the cytotoxic activity of the hexapeptide conjugated to cisplatin on a cell line panel. In most cell lines, the hexapeptide delivered an amount of cisplatin that was 2 to 8 times greater than that released by cisplatin when the drug was used alone. This increased delivery increases the therapeutic index of cisplatin and reduces side effects caused by a high dosage or long-term treatment times. We may consider this hexapeptide a new molecular carrier to deliver molecules with therapeutic activity into ER(+) cells for diagnostic purposes and clinical or immune therapy.

Impairment of T cell development and acute inflammatory response in HIV-1 Tat transgenic mice.

Immune activation and chronic inflammation are hallmark features of HIV infection causing T-cell depletion and cellular immune dysfunction in AIDS. Here, we addressed the issue whether HIV-1 Tat could affect T cell development and acute inflammatory response by generating a transgenic mouse expressing Tat in lymphoid tissue. Tat-Tg mice showed thymus atrophy and the maturation block from DN4 to DP thymic subpopulations, resulting in CD4(+) and CD8(+) T cells depletion in peripheral blood. In Tat-positive thymus, we observed the increased p65/NF-κB activity and deregulated expression of cytokines/chemokines and microRNA-181a-1, which are involved in T-lymphopoiesis. Upon LPS intraperitoneal injection, Tat-Tg mice developed an abnormal acute inflammatory response, which was characterized by enhanced lethality and production of inflammatory cytokines. Based on these findings, Tat-Tg mouse could represent an animal model for testing adjunctive therapies of HIV-1-associated inflammation and immune deregulation.

Aberrant glycosylation as biomarker for cancer: focus on CD43.

Glycosylation is a posttranslational modification of proteins playing a major role in cell signalling, immune recognition, and cell-cell interaction because of their glycan branches conferring structure variability and binding specificity to lectin ligands. Aberrant expression of glycan structures as well as occurrence of truncated structures, precursors, or novel structures of glycan may affect ligand-receptor interactions and thus interfere with regulation of cell adhesion, migration, and proliferation. Indeed, aberrant glycosylation represents a hallmark of cancer, reflecting cancer-specific changes in glycan biosynthesis pathways such as the altered expression of glycosyltransferases and glycosidases. Most studies have been carried out to identify changes in serum glycan structures. In most cancers, fucosylation and sialylation are significantly modified. Thus, aberrations in glycan structures can be used as targets to improve existing serum cancer biomarkers. The ability to distinguish differences in the glycosylation of proteins between cancer and control patients emphasizes glycobiology as a promising field for potential biomarker identification. In this review, we discuss the aberrant protein glycosylation associated with human cancer and the identification of protein glycoforms as cancer biomarkers. In particular, we will focus on the aberrant CD43 glycosylation as cancer biomarker and the potential to exploit the UN1 monoclonal antibody (UN1 mAb) to identify aberrant CD43 glycoforms.

CDK/CCN and CDKI alterations for cancer prognosis and therapeutic predictivity.

The regulation of cell growth and division occurs in an accurate sequential manner. It is dictated by the accumulation of cyclins (CCNs) and cyclin-dependent kinases (CDKs) complexes and degradation of CCNs. In human tumors, instead, the cell cycle is deregulated, causing absence of differentiation and aberrant cell growth. Oncogenic alterations of CCNs, CDKs, and CDKIs have been reported in more than 90% of human cancers, and the most frequent are those related to the G1 phase. Several molecular mechanisms, including gene overexpression, chromosomal translocations, point mutations, insertions and deletions, missense and frame shift mutation, splicing, or methylation, may be responsible for these alterations. The cell cycle regulators are involved in tumor progression given their association with cancers characterized by higher incidence of relapses and chemotherapy resistance. In the last decade anticancer drug researches focused on new compounds, able to target molecules related to changes in genes associated with tumor status. Recently, the studies have focused on the restoration of cell cycle control modulating molecular targets involved in cancer-cell alterations. This paper aims to correlate alterations of cell cycle regulators with human cancers and therapeutic responsivity.

The functional role of MnSOD as a biomarker of human diseases and therapeutic potential of a new isoform of a human recombinant MnSOD.

Reactive oxygen species (ROS) are generated as a consequence of metabolic reactions in the mitochondria of eukaryotic cells. This work describes the role of the manganese superoxide dismutase (MnSOD) as a biomarker of different human diseases and proposes a new therapeutic application for the prevention of cancer and its treatment. The paper also describes how a new form of human MnSOD was discovered, its initial application, and its clinical potentials. The MnSOD isolated from a human liposarcoma cell line (LSA) was able to kill cancer cells expressing estrogen receptors, but it did not have cytotoxic effects on normal cells. Together with its oncotoxic activity, the recombinant MnSOD (rMnSOD) exerts a radioprotective effect on normal cells irradiated with X-rays. The rMnSOD is characterized by the presence of a leader peptide, which allows the protein to enter cells: this unique property can be used in the radiodiagnosis of cancer or chemotherapy, conjugating radioactive substances or chemotherapic drugs to the leader peptide of the MnSOD. Compared to traditional chemotherapic agents, the drugs conjugated with the leader peptide of MnSOD can selectively reach and enter cancer cells, thus reducing the side effects of traditional treatments.

Cancer-associated CD43 glycoforms as target of immunotherapy.

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer-mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy.

Mass spectrometry-based identification of the tumor antigen UN1 as the transmembrane CD43 sialoglycoprotein.

The UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated protein with the apparent molecular weight of 100-120 kDa; this antigen was peculiarly expressed in fetal tissues and several cancer tissues, including leukemic T cells, breast, and colon carcinomas. However, the lack of primary structure information has limited further investigation on the role of the UN1 antigen in neoplastic transformation. In this study, we have identified the UN1 antigen as CD43, a transmembrane sialoglycoprotein involved in cell adhesion, differentiation, and apoptosis. Indeed, mass spectrometry detected two tryptic peptides of the membrane-purified UN1 antigen that matched the amino acidic sequence of the CD43 intracellular domain. Immunological cross-reactivity, migration pattern in mono- and bi-dimensional electrophoresis, and CD43 gene-dependent expression proved the CD43 identity of the UN1 antigen. Moreover, the monosaccharide GalNAc-O-linked to the CD43 peptide core was identified as an essential component of the UN1 epitope by glycosidase digestion of specific glycan branches. UN1-type CD43 glycoforms were detected in colon, sigmoid colon, and breast carcinomas, whereas undetected in normal tissues from the same patients, confirming the cancer-association of the UN1 epitope. Our results highlight UN1 monoclonal antibody as a suitable tool for cancer immunophenotyping and analysis of CD43 glycosylation in tumorigenesis.

In vivo targeting and growth inhibition of the A20 murine B-cell lymphoma by an idiotype-specific peptide binder.

B-cell lymphoma is a clonal expansion of neoplastic cells that may result in fatal outcomes. Here, we report the in vivo targeting and growth inhibition of aggressive A20 murine B-cell lymphoma by idiotype-specific peptide pA20-36. pA20-36 was selected from random peptide libraries and bound specifically to the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, as shown by histology and positron emission tomographic analysis. BCR cross-linking of A20 cells with pA20-36 resulted in massive apoptosis of targeted tumor cells and in an increased survival of the diseased animals without any detectable evidence of toxicity. The pA20-36 treatment reverted the immune suppression of the tumor microenvironment as shown by reduced expression of vascular endothelial growth factor, interleukin-10, and transforming growth factor-beta cytokines together with a lower number of CD11b+Gr-1+ inhibitor myeloid-derived suppressor cells and Foxp3+CD4+ Treg cells. Furthermore, pA20-36 treatment was associated with an increased number of tumor-infiltrating, activated CD8+ T cells that exerted a tumor-specific cytolytic activity. These findings show that a short peptide that binds specifically to the complementarity-determining regions of the A20 BCR allows in vivo detection of neoplastic cells together with significant inhibition of tumor growth in vivo.