The common H232 STING allele shows impaired activities in DNA sensing, susceptibility to viral infection, and in monocyte cell function, while the HAQ variant possesses wild-type properties.

Different innate immune pathways converge to Stimulator of interferon genes (STING) and trigger type I interferon responses after recognition of abnormal nucleic acids in the cells. This non-redundant function renders STING a major player in immunosurveillance, and an emerging target for cancer and infectious diseases therapeutics. Beyond somatic mutations that often occur in cancer, the human gene encoding STING protein, TMEM173 (STING1), holds great genetic heterogeneity; R232, HAQ (R71H-G230A-R293Q) and H232 are the most common alleles. Although some of these alleles are likely to be hypomorphic, their function is still debated, due to the available functional assessments, which have been performed in biased biological systems. Here, by using genetic background-matched models, we report on the functional evaluation of R232, HAQ and H232 variants on STING function, and on how these genotypes affect the susceptibility to clinically relevant viruses, thus supporting a potential contributing cause to differences in inter-individual responses to infections. Our findings also demonstrate a novel toll-like receptor-independent role of STING in modulating monocytic cell function and differentiation into macrophages. We further supported the interplay of STING1 variants and human biology by demonstrating how monocytes bearing the H232 allele were impaired in M1/M2 differentiation, interferon response and antigen presentation. Finally, we assessed the response to PD-1 inhibitor in a small cohort of melanoma patients stratified according to STING genotype. Given the contribution of the STING protein in sensing DNA viruses, bacterial pathogens and misplaced cancer DNA, these data may support the development of novel therapeutic options for infectious diseases and cancer.

Carbonic anhydrase IX subcellular localization in normoxic and hypoxic SH-SY5Y neuroblastoma cells is assisted by its C-terminal protein interaction domain.

The human carbonic anhydrase IX (CA IX) is a hypoxia-induced transmembrane protein belonging to the α-CA enzyme family. It has a crucial role in pH regulation in hypoxic cells and acts by buffering intracellular acidosis induced by hypoxia. Indeed, it is frequently expressed in cancer cells, where it contributes to tumor progression. CA IX is also able to localize in the nucleus, where it contributes to 47S rRNA precursor genes transcription; however, the mechanisms assisting its nuclear translocation still remain unclear. The aim of our study was to deepen the understanding of the mechanisms involved in CA IX subcellular distribution. To this purpose, we implemented a site-directed mutagenesis approach targeting the C-terminal domain of CA IX and evaluated the subcellular distribution of the wild-type and mutant proteins in the SH-SY5Y cell line. The mutant proteins showed impaired binding ability and altered subcellular distribution in both normoxic and hypoxic conditions. Our data suggest that CA IX nuclear translocation depends on its transit through the secretory and the endocytic pathways.

Combined blockade of mTOR and p21-activated kinases pathways prevents tumour growth in KRAS-mutated colorectal cancer.

BACKGROUND: The identification of novel therapeutic strategies for metastatic colorectal cancer (mCRC) patients harbouring KRAS mutations represents an unmet clinical need. In this study, we aimed to clarify the role of p21-activated kinases (Paks) as therapeutic target for KRAS-mutated CRC. METHODS: Paks expression and activation levels were evaluated in a cohort of KRAS-WT or -mutated CRC patients by immunohistochemistry. The effects of Paks inhibition on tumour cell proliferation and signal transduction were assayed by RNAi and by the use of three pan-Paks inhibitors (PF-3758309, FRAX1036, GNE-2861), evaluating CRC cells, spheroids and tumour xenografts' growth. RESULTS: Paks activation positively correlated with KRAS mutational status in both patients and cell lines. Moreover, genetic modulation or pharmacological inhibition of Paks led to a robust impairment of KRAS-mut CRC cell proliferation. However, Paks prolonged blockade induced a rapid tumour adaptation through the hyper-activation of the mTOR/p70S6K pathway. The addition of everolimus (mTOR inhibitor) prevented the growth of KRAS-mut CRC tumours in vitro and in vivo, reverting the adaptive tumour resistance to Paks targeting. CONCLUSIONS: In conclusion, our results suggest the simultaneous blockade of mTOR and Pak pathways as a promising alternative therapeutic strategy for patients affected by KRAS-mut colorectal cancer.

A Novel Human Neutralizing mAb Recognizes Delta, Gamma and Omicron Variants of SARS-CoV-2 and Can Be Used in Combination with Sotrovimab.

The dramatic experience with SARS-CoV-2 has alerted the scientific community to be ready to face new epidemics/pandemics caused by new variants. Among the therapies against the pandemic SARS-CoV-2 virus, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein have represented good drugs to interfere in the Spike/ Angiotensin Converting Enzyme-2 (ACE-2) interaction, preventing virus cell entry and subsequent infection, especially in patients with a defective immune system. We obtained, by an innovative phage display selection strategy, specific binders recognizing different epitopes of Spike. The novel human antibodies specifically bind to Spike-Receptor Binding Domain (RBD) in a nanomolar range and interfere in the interaction of Spike with the ACE-2 receptor. We report here that one of these mAbs, named D3, shows neutralizing activity for virus infection in cell cultures by different SARS-CoV-2 variants and retains the ability to recognize the Omicron-derived recombinant RBD differently from the antibodies Casirivimab or Imdevimab. Since anti-Spike mAbs, used individually, might be unable to block the virus cell entry especially in the case of resistant variants, we investigated the possibility to combine D3 with the antibody in clinical use Sotrovimab, and we found that they recognize distinct epitopes and show additive inhibitory effects on the interaction of Omicron-RBD with ACE-2 receptor. Thus, we propose to exploit these mAbs in combinatorial treatments to enhance their potential for both diagnostic and therapeutic applications in the current and future pandemic waves of coronavirus.

High-Throughput Monoclonal Antibody Discovery from Phage Libraries: Challenging the Current Preclinical Pipeline to Keep the Pace with the Increasing mAb Demand.

Monoclonal antibodies are among the most powerful therapeutics in modern medicine. Since the approval of the first therapeutic antibody in 1986, monoclonal antibodies keep holding great expectations for application in a range of clinical indications, highlighting the need to provide timely and sustainable access to powerful screening options. However, their application in the past has been limited by time-consuming and expensive steps of discovery and production. The screening of antibody repertoires is a laborious step; however, the implementation of next-generation sequencing-guided screening of single-chain antibody fragments has now largely overcome this issue. This review provides a detailed overview of the current strategies for the identification of monoclonal antibodies from phage display-based libraries. We also discuss the challenges and the possible solutions to improve the limiting selection and screening steps, in order to keep pace with the increasing demand for monoclonal antibodies.

Functional annotation and investigation of the 10q24.33 melanoma risk locus identifies a common variant that influences transcriptional regulation of OBFC1.

The 10q24.33 locus is known to be associated with susceptibility to cutaneous malignant melanoma (CMM), but the mechanisms underlying this association have been not extensively investigated. We carried out an integrative genomic analysis of 10q24.33 using epigenomic annotations and in vitro reporter gene assays to identify regulatory variants. We found two putative functional single nucleotide polymorphisms (SNPs) in an enhancer and in the promoter of OBFC1, respectively, in neural crest and CMM cells, one, rs2995264, altering enhancer activity. The minor allele G of rs2995264 correlated with lower OBFC1 expression in 470 CMM tumors and was confirmed to increase the CMM risk in a cohort of 484 CMM cases and 1801 controls of Italian origin. Hi-C and chromosome conformation capture (3C) experiments showed the interaction between the enhancer-SNP region and the promoter of OBFC1 and an isogenic model characterized by CRISPR-Cas9 deletion of the enhancer-SNP region confirmed the potential regulatory effect of rs2995264 on OBFC1 transcription. Moreover, the presence of G-rs2995264 risk allele reduced the binding affinity of the transcription factor MEOX2. Biologic investigations showed significant cell viability upon depletion of OBFC1, specifically in CMM cells that were homozygous for the protective allele. Clinically, high levels of OBFC1 expression associated with histologically favorable CMM tumors. Finally, preliminary results suggested the potential effect of decreased OBFC1 expression on telomerase activity in tumorigenic conditions. Our results support the hypothesis that reduced expression of OBFC1 gene through functional heritable DNA variation can contribute to malignant transformation of normal melanocytes.

Generation of a Retargeted Oncolytic Herpes Virus Encoding Adenosine Deaminase for Tumor Adenosine Clearance.

BACKGROUND: Oncolytic viruses are immunotherapeutic agents that can be engineered to encode payloads of interest within the tumor microenvironment to enhance therapeutic efficacy. Their therapeutic potential could be limited by many avenues for immune evasion exerted by the tumor. One such is mediated by adenosine, which induces pleiotropic immunosuppression by inhibiting antitumor immune populations as well as activating tolerogenic stimuli. Adenosine is produced starting from the highly immunostimulatory ATP, which is progressively hydrolyzed to ADP and adenosine by CD39 and CD73. Cancer cells express high levels of CD39 and CD73 ectoenzymes, thus converting immunostimulatory purinergic signal of ATP into an immunosuppressive signal. For this reason, CD39, CD73 and adenosine receptors are currently investigated in clinical trials as targets for metabolic cancer immunotherapy. This is of particular relevance in the context of oncovirotherapy, as immunogenic cell death induced by oncolytic viruses causes the secretion of a high amount of ATP which is available to be quickly converted into adenosine. METHODS: Here, we took advantage of adenosine deaminase enzyme that naturally converts adenosine into the corresponding inosine derivative, devoid of immunoregulatory function. We encoded ADA into an oncolytic targeted herpes virus redirected to human HER2. An engineered ADA with an ectopic signal peptide was also generated to improve enzyme secretion (ADA-SP). RESULTS: Insertion of the expression cassette was not detrimental for viral yield and cancer cell cytotoxicity. The THV_ADA and THV_ADA-SP successfully mediated the secretion of functional ADA enzyme. In in vitro model of human monocytes THP1, this ability of THV_ADA and THV_ADA-SP resulted in the retrieval of eADO-exposed monocytes replication rate, suggesting the proficiency of the viruses in rescuing the immune function. CONCLUSIONS: Encoding ADA into oncolytic viruses revealed promising properties for preclinical exploitation.

Immunomodulatory mAbs as Tools to Investigate on Cis-Interaction of PD-1/PD-L1 on Tumor Cells and to Set Up Methods for Early Screening of Safe and Potent Combinatorial Treatments.

Antibodies targeting Immune Checkpoints (IC) on tumor infiltrating lymphocytes improve immune responses against cancer. Recently, the expression of some ICs has also been reported on cancer cells. We used the clinically validated Ipilimumab and Nivolumab and other novel human antibodies targeting Cytotoxic T- lymphocyte-antigen 4 (CTLA-4), Programmed Death receptor-1 (PD-1) and Programmed Death Ligand 1 (PD-L1) to shed light on the functions of these ICs in cancer cells. We show here for the first time that all these antagonistic mAbs are able to reduce Erk phosphorylation and, unexpectedly, to induce a significant increase of ICs expression on tumor cells, involving a hyperphosphorylation of NF-kB. On the contrary, agonistic PD-L1 and PD-1 recombinant proteins showed opposite effects by leading to a significant reduction of PD-1 and PD-L1, thus also suggesting the existence of a crosstalk in tumor cells between multiple ICs. Since the immunomodulatory mAbs show their higher anti-tumor efficacy by activating lymphocytes against cancer cells, we also investigated whether it was possible to identify the most efficient combinations of immunomodulatory mAbs for achieving potent anti-tumor efficacy associated with the lowest adverse side effects by setting up novel simple and predictive in vitro models based on co-cultures of tumor cells or human fetal cardiomyocytes with lymphocytes. We demonstrate here that novel combinations of immunomodulatory mAbs with more potent anti-cancer activity than Ipilimumab and Nivolumab combination can be identified with no or lower cardiotoxic side effects. Thus, we propose these co-cultures-based assays as useful tools to test also other combinatorial treatments of emerging immunomodulatory mAbs against different ICs for the early screening of most potent and safe combinatorial therapeutic regimens.

Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2.

Among the therapies against the pandemic SARS-CoV-2 virus, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein represent good candidates to interfere in the Spike/ACE2 interaction, preventing virus cell entry. Since anti-spike mAbs, used individually, might be unable to block the virus entry in the case of resistant mutations, we designed an innovative strategy for the isolation of multiple novel human scFvs specific for the binding domain (RBD) of Spike. By panning a large phage display antibody library on immobilized RBD, we obtained specific binders by eluting with ACE2 in order to identify those scFvs recognizing the epitope of Spike interacting with its receptor. We converted the novel scFvs into full size IgG4, differently from the previously isolated IgG1 mAbs, to avoid unwanted potential side effects of IgG1 potent effector functions on immune system. The novel antibodies specifically bind to RBD in a nanomolar range and interfere in the interaction of Spike with ACE2 receptor, either used as purified protein or when expressed on cells in its native conformation. Furthermore, some of them have neutralizing activity for virus infection in cell cultures by using two different SARS-CoV-2 isolates including the highly contagious VOC 202012/01 variant and could become useful therapeutic tools to fight against the SARS-CoV-2 virus.

Generation of a Novel Mesothelin-Targeted Oncolytic Herpes Virus and Implemented Strategies for Manufacturing.

BACKGROUND: HER2-based retargeted viruses are in advanced phases of preclinical development of breast cancer models. Mesothelin (MSLN) is a cell-surface tumor antigen expressed in different subtypes of breast and non-breast cancer. Its recent identification as a marker of some triple-negative breast tumors renders it an attractive target, presently investigated in clinical trials employing antibody drug conjugates and CAR-T cells. The availability of MSLN-retargeted oncolytic viruses may complement the current immunotherapeutic panel of biological drugs against HER2-negative breast and non-breast tumors. METHODS: A fully virulent, tumor-targeted oncolytic Herpes simplex virus-1 (MSLN-THV) with a selectivity for mesothelin-expressing cancer cells was generated. Recombineering technology was used to replace an essential moiety of the viral glycoprotein D with antibody fragments derived from clinically validated MSLN monoclonal antibodies, and to allow IL12 cargo expression in infected cells. Panels of breast and female reproductive system cell lines were used to verify the oncolytic potential of the viral constructs. A platform for production of the retargeted viruses was developed in HEK 293 cells, providing stable expression of a suitable chimeric receptor. RESULTS: We demonstrated the selectivity of viral infection and cytotoxicity by MSLN-retargeted viruses in a panel of mesothelin-positive cancer cells, originating from breast and female reproductive system tumors. We also developed a second-generation oncolytic MSLN-THV, encoding IL12, to enhance the immunotherapeutic potential of the viral backbone. A non-tumor cell line expressing a chimeric MSLN/Nectin-1 receptor, de-sensitized from antiviral responses by genetic inactivation of the Stimulator of Interferon Genes (STING)-dependent pathway was engineered, to optimize viral yields. CONCLUSIONS: Our proof-of-concept study proposes MSLN-retargeted herpesviruses as potential cancer immunotherapeutics for assessments in preclinical models of MSLN-positive tumors, complementing the available panel of oncolytic viruses to HER2-negative breast tumors.

Novel Combinations of Human Immunomodulatory mAbs Lacking Cardiotoxic Effects for Therapy of TNBC.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer characterized by a higher mortality rate among breast cancer subtypes. Poly(ADP-ribose) polymerase (PARP) inhibitors are used in clinics to treat a subgroup of TNBC patients, but other targeted therapies are urgently needed. Programmed death-ligand 1 (PD-L1), involved in tumor immune escape, was recently identified as a target for TNBC; accordingly, the anti-PD-L1 monoclonal antibody (mAb), atezolizumab, has been approved by FDA in combination with Paclitaxel for the therapy of metastatic TNBC. Here, we tested novel combinations of fully human immunomodulatory mAbs, including anti-PD-L1 mAbs generated in our laboratory and atezolizumab, on TNBC and other tumor cell lines. We evaluated their anti-tumor efficacy when used as single agents or in combinatorial treatments with anti-CTLA-4 mAbs in in vitro co-cultures of hPBMCs with tumor cells, by measuring tumor cell lysis and IL-2 and IFNγ cytokines secretion by lymphocytes. In parallel, by using co-cultures of hPBMCs and cardiomyocytes, we analyzed the potential cardiotoxic adverse side effects of the same antibody treatments by measuring the cardiac cell lysis and the secretion of pro-inflammatory cytokines. We identified novel combinations of immunomodulatory mAbs endowed with more potent anti-cancer activity on TNBC and lower cardiotoxic side effects than the combination of atezolizumab and ipilimumab.

New viral vectors for infectious diseases and cancer.

Since the discovery in 1796 by Edward Jenner of vaccinia virus as a way to prevent and finally eradicate smallpox, the concept of using a virus to fight another virus has evolved into the current approaches of viral vectored genetic vaccines. In recent years, key improvements to the vaccinia virus leading to a safer version (Modified Vaccinia Ankara, MVA) and the discovery that some viruses can be used as carriers of heterologous genes encoding for pathological antigens of other infectious agents (the concept of 'viral vectors') has spurred a new wave of clinical research potentially providing for a solution for the long sought after vaccines against major diseases such as HIV, TB, RSV and Malaria, or emerging infectious diseases including those caused by filoviruses and coronaviruses. The unique ability of some of these viral vectors to stimulate the cellular arm of the immune response and, most importantly, T lymphocytes with cell killing activity, has also reawakened the interest toward developing therapeutic vaccines against chronic infectious diseases and cancer. To this end, existing vectors such as those based on Adenoviruses have been improved in immunogenicity and efficacy. Along the same line, new vectors that exploit viruses such as Vesicular Stomatitis Virus (VSV), Measles Virus (MV), Lymphocytic choriomeningitis virus (LCMV), cytomegalovirus (CMV), and Herpes Simplex Virus (HSV), have emerged. Furthermore, technological progress toward modifying their genome to render some of these vectors incompetent for replication has increased confidence toward their use in infant and elderly populations. Lastly, their production process being the same for every product has made viral vectored vaccines the technology of choice for rapid development of vaccines against emerging diseases and for 'personalised' cancer vaccines where there is an absolute need to reduce time to the patient from months to weeks or days. Here we review the recent developments in viral vector technologies, focusing on novel vectors based on primate derived Adenoviruses and Poxviruses, Rhabdoviruses, Paramixoviruses, Arenaviruses and Herpesviruses. We describe the rationale for, immunologic mechanisms involved in, and design of viral vectored gene vaccines under development and discuss the potential utility of these novel genetic vaccine approaches in eliciting protection against infectious diseases and cancer.

Retargeted and Multi-cytokine-Armed Herpes Virus Is a Potent Cancer Endovaccine for Local and Systemic Anti-tumor Treatment.

Oncolytic viruses (OVs) are novel anti-tumor agents with the ability to selectively infect and kill tumor cells while sparing normal tissue. Beyond tumor cytolysis, OVs are capable of priming an anti-tumor immune response via lysis and cross-presentation of locally expressed endogenous tumor antigens, acting as an "endovaccine." The effectiveness of OVs, similar to other immunotherapies, can be hampered by an immunosuppressive tumor microenvironment. In this study, we modified a previously generated oncolytic herpes simplex virus (oHSV) retargeted to the human HER2 (hHER2) tumor molecule and encoding murine interleukin-12 (mIL-12), by insertion of a second immunomodulatory molecule, murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), to maximize therapeutic efficacy. We assessed the efficacy of this double-armed virus (R-123) compared to singly expressing GM-CSF and IL-12 oHSVs in tumor-bearing mice. While monotherapies were poorly effective, combination with α-PD1 enhanced the anti-tumor response, with the highest efficacy of 100% response rate achieved by the combination of R-123 and α-PD1. Efficacy was T cell-dependent, and the induced immunity was long lasting and able to reject a second contralateral tumor. Importantly, systemic delivery of R-123 combined with α-PD1 was effective in inhibiting the development of tumor metastasis. As such, this approach could have a significant therapeutic impact paving the way for further development of this platform in cancer immunotherapy.

Integrity of the Antiviral STING-mediated DNA Sensing in Tumor Cells Is Required to Sustain the Immunotherapeutic Efficacy of Herpes Simplex Oncolytic Virus.

The dichotomic contribution of cancer cell lysis and tumor immunogenicity is considered essential for effective oncovirotherapy, suggesting that the innate antiviral immune response is a hurdle for efficacy of oncolytic viruses. However, emerging evidence is resizing this view. By sensing cytosolic DNA, the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) axis can both counteract viral spread and contribute to the elicitation of adaptive immunity via type I interferon responses. In this paper, we analyzed the tumor-resident function of Sting-mediated DNA sensing in a combined approach of oncovirotherapy and PD-1 immune checkpoint blockade, in an immunocompetent murine model. While supporting increased lytic potential by oncolytic HER2-retargeted HSV-1 in vitro and in vivo, Sting-knockout tumors showed molecular signatures of an immunosuppressive tumor microenvironment. These signatures were correspondingly associated with ineffectiveness of the combination therapy in a model of established tumors. Results suggest that the impairment in antiviral response of Sting-knockout tumors, while favoring viral replication, is not able to elicit an adequate immunotherapeutic effect, due to lack of immunogenic cell death and the inability of Sting-knockout cancer cells to promote anti-tumor adaptive immune responses. Accordingly, we propose that antiviral, tumor-resident Sting provides fundamental contributions to immunotherapeutic efficacy of oncolytic viruses.

CA IX Stabilizes Intracellular pH to Maintain Metabolic Reprogramming and Proliferation in Hypoxia.

Tumor hypoxia represents a severe microenvironmental stress that is frequently associated with acidosis. Cancer cells respond to these stresses with changes in gene expression that promote survival at least in part through pH regulation and metabolic reprogramming. Hypoxia-induced carbonic anhydrase IX (CA IX) plays a critical adaptive role in response to hypoxic and acidic environments by catalytically hydrating extracellular CO2 to produce bicarbonate for buffering intracellular pH (pHi). We used proteome-wide profiling to study the cellular response to transient CA IX knockdown in hypoxia and found a decrease in the levels of key glycolytic enzymes and lactate dehydrogenase A (LDHA). Interestingly, the activity of LDH was also decreased as demonstrated by native in-gel activity assay. These changes led to a significant reduction in glycolytic flux and extracellular lactate levels in cancer cells in vitro, contributing to a decrease in proliferation. Interestingly, addition of the alternative LDH substrate alpha-ketobutyrate restored LDHA activity, extracellular acidification, pHi, and cellular proliferation. These results indicate that in the absence of CA IX, reduction of pHi disrupts LDHA activity and hinders the cellular capacity to regenerate NAD+ and secrete protons to the extracellular space. Hypoxia-induced CA IX therefore mediates adaptation to microenvironmental hypoxia and acidosis directly, by enzymatically converting extracellular CO2 to bicarbonate, and indirectly, by maintaining glycolysis-permissive intracellular milieu.

Isolation of Two Novel Human Anti-CTLA-4 mAbs with Intriguing Biological Properties on Tumor and NK Cells.

The cytotoxic T lymphocyte-antigen 4 (CTLA-4) has been considered an IC exclusively expressed on T cells, where it counteracts the co-stimulatory CD28 receptor, by competing for its binding to CD-80 and CD-86. We recently found that it is expressed also on tumor and NK cells, suggesting other possible unknown roles of CTLA-4. To shed light on these novel aspects of CTLA-4, we used Ipilimumab, the first FDA approved human antibody targeting CTLA-4, in parallel studies with two novel human mAbs we isolated by using an efficient phage display selection strategy on live activated lymphocytes and purified mouse and human CTLA-4. The selection for cross-reactive mAbs was guaranteed by a high throughput sequencing to identify the sequences commonly enriched by two parallel pannings on human and mouse CTLA-4. Two isolated antibodies were found to bind with high affinity to both human and mouse CTLA-4 and lymphocytes, showing nanomolar or sub-nanomolar Kd values. They were able to kill Treg cells by ADCC, and to activate both human and mouse PBMCs, by strongly increasing cytokines secretion. Interestingly, they activated NK cells, exhibited cytotoxicity against cancer cells by inducing ADCC and inhibited tumor cell growth by affecting CTLA-4 downstream pathways in a similar fashion to CD-80 and CD-86 ligands and differently from Ipilimumab. Moreover, the novel mAbs showed a reduced ability to interfere in the binding of CD-80 ligands to CTLA-4 on T cells with respect to Ipilimumab, suggesting that they could allow for anti-tumor effects without the irAEs associated with the potent antagonistic activity of Ipilimumab.

Replicative conditioning of Herpes simplex type 1 virus by Survivin promoter, combined to ERBB2 retargeting, improves tumour cell-restricted oncolysis.

Oncolytic virotherapy is emerging as a promising therapeutic option for solid tumours. Several oncolytic vectors in clinical testing are based on attenuated viruses; thus, efforts are being taken to develop a new repertoire of oncolytic viruses, based on virulent viral genomes. This possibility, however, raises concerns dealing with the safety features of the virulent phenotypes. We generated a double regulated Herpes simplex type-1 virus (HSV-1), in which tumour cell restricted replicative potential was combined to selective entry via ERBB2 receptor retargeting. The transcriptional control of the viral alpha4 gene encoding for the infected cell protein-4 (ICP4) by the cellular Survivin/BIRC5 promoter conferred a tumour cell-restricted replicative potential to a virulent HSV-1 genome. The combination of the additional ERBB2 retargeting further improved the selectivity for tumour cells, conferring to the double regulated virus a very limited ability to infect and propagate in non-cancerous cells. Accordingly, a suitable replicative and cytotoxic potential was maintained in tumour cell lines, allowing the double regulated virus to synergize in vivo with immune checkpoint (anti-PD-1) blockade in immunocompetent mice. Thus, restricting the replicative spectrum and tropism of virulent HSV-1 genomes by combination of conditional replication and retargeting provides an improved safety, does not alter the oncolytic strength, and is exploitable for its therapeutic potential with immune checkpoint blockade in cancer.

Neural crest-derived tumor neuroblastoma and melanoma share 1p13.2 as susceptibility locus that shows a long-range interaction with the SLC16A1 gene.

Neuroblastoma (NB) and malignant cutaneous melanoma (CMM) are neural crest cells (NCC)-derived tumors and may have a shared genetic basis, but this has not been investigated systematically by genome-wide association studies (GWAS). We took a three-staged approach to conduct cross-disease meta-analysis of GWAS for NB and CMM (2101 NB cases and 4202 controls; 12 874 CMM cases and 23 203 controls) to identify shared loci. Findings were replicated in 1403 NB cases and 1403 controls of European ancestry and in 636 NB, 508 CMM cases and 2066 controls of Italian origin. We found a cross-association at locus 1p13.2 (rs2153977, odds ratio = 0.91, P = 5.36 × 10-8). We also detected a suggestive (P < 10-7) NB-CMM cross-association at 2q37.1 with opposite effect on cancer risk. Pathway analysis of 110 NB-CMM risk loci with P < 10-4 demonstrated enrichment of biological processes such as cell migration, cell cycle, metabolism and immune response, which are essential of human NCC development, underlying both tumors. In vitro and in silico analyses indicated that the rs2153977-T protective allele, located in an NB and CMM enhancer, decreased expression of SLC16A1 via long-range loop formation and altered a T-box protein binding site. Upon depletion of SLC16A1, we observed a decrease of cellular proliferation and invasion in both NB and CMM cell lines, suggesting its role as oncogene. This is the largest study to date examining pleiotropy across two NC cell-derived tumors identifying 1p13.2 as common susceptibility locus for NB and CMM risk. We demonstrate that combining genome-wide association studies results across cancers with same origins can identify new loci common to neuroblastoma and melanoma arising from tissues which originate from neural crest cells. Our results also show 1p13.2 confer risk to neuroblastoma and melanoma by regulating SLC16A1.

A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair.

BRCA1 and BRCA2 are the genes most frequently associated with hereditary breast and ovarian cancer (HBOC). They are crucial for the maintenance of genome stability, particularly in the homologous recombination-mediated repair pathway of DNA double-strand breaks (HR-DSBR). Widespread BRCA1/2 next-generation sequencing (NGS) screening has revealed numerous variants of uncertain significance. Assessing the clinical significance of these variants is challenging, particularly regarding the clinical management of patients. Here, we report the functional characterization of the unclassified BRCA2 c.8299C > T variant, identified in a young breast cancer patient during BRCA1/2 NGS screening. This variant causes the change of Proline 2767 to Serine in the DNA binding domain (DBD) of the BRCA2 protein, necessary for the loading of RAD51 on ssDNA during the HR-DSBR. Our in silico analysis and 3D-structure modeling predicted that the p.Pro2767Ser substitution is likely to alter the BRCA2 DBD structure and function. Therefore, to evaluate the functional impact of the p.Pro2767Ser variant, we used a minigene encoding a truncated protein that contains the BRCA2 DBD and the nearby nuclear localization sequence. We found that the ectopically expressed truncated protein carrying the normal DBD, which retains the DNA binding function and lacks the central RAD51 binding domain, interferes with endogenous wild-type BRCA2 mediator functions in the HR-DSBR. We also demonstrated that the BRCA2 Pro2767Ser DBD is unable to compete with endogenous BRCA2 DNA binding, thereby suggesting that the p.Pro2767Ser substitution in the full-length protein causes the functional loss of BRCA2. Consequently, our data suggest that the p.Pro2767Ser variant should be considered pathogenic, thus supporting a revision of the ClinVar interpretation. Moreover, our experimental strategy could be a valid method with which to preliminarily evaluate the pathogenicity of the unclassified BRCA2 germline variants in the DBD and their risk of predisposing to HBOC.

Publisher Correction: Revealing membrane alteration in cells overexpressing CA IX and EGFR by Surface-Enhanced Raman Scattering.

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