Infection-induced peripheral mitochondria fission drives ER encapsulations and inter-mitochondria contacts that rescue bioenergetics.

The dynamic regulation of mitochondria shape via fission and fusion is critical for cellular responses to stimuli. In homeostatic cells, two modes of mitochondrial fission, midzone and peripheral, provide a decision fork between either proliferation or clearance of mitochondria. However, the relationship between specific mitochondria shapes and functions remains unclear in many biological contexts. While commonly associated with decreased bioenergetics, fragmented mitochondria paradoxically exhibit elevated respiration in several disease states, including infection with the prevalent pathogen human cytomegalovirus (HCMV) and metastatic melanoma. Here, incorporating super-resolution microscopy with mass spectrometry and metabolic assays, we use HCMV infection to establish a molecular mechanism for maintaining respiration within a fragmented mitochondria population. We establish that HCMV induces fragmentation through peripheral mitochondrial fission coupled with suppression of mitochondria fusion. Unlike uninfected cells, the progeny of peripheral fission enter mitochondria-ER encapsulations (MENCs) where they are protected from degradation and bioenergetically stabilized during infection. MENCs also stabilize pro-viral inter-mitochondria contacts (IMCs), which electrochemically link mitochondria and promote respiration. Demonstrating a broader relevance, we show that the fragmented mitochondria within metastatic melanoma cells also form MENCs. Our findings establish a mechanism where mitochondria fragmentation can promote increased respiration, a feature relevant in the context of human diseases.

Malignant skin neoplasms in goats in Sicily, Italy: clinical, virological and pathological investigations.

Neoplasms in small ruminants are considered uncommon and their reported incidence is variable. The aims of this investigation were to characterize malignant skin neoplasms in adult goats reared in Sicily, Italy, and to evaluate potential correlations between gross and histopathology features of the tumours and signalment, tumour location and/or viral infections. A total of 75 malignant skin masses were examined. In selected animals with perineal masses (n = 28) virological and serological investigations on tissues and blood were also conducted. According to the histological features, the lesions were classified as 67 squamous cell carcinomas (SCCs) (of which 65 were located in the perineum), six melanomas and two fibrosarcomas. In three cases, neoplasms at the base of the horn were associated with nasal polyps. Among the selected perineal SCCs, papillomaviruses (PVs), caprine herpesvirus 1 and parapoxvirus were not detected on polymerase chain reaction or on serological examination. However, further investigation on a larger sample size is required to evaluate the potential role of PVs in the pathogenesis of skin tumours in goats.

Molecular mechanisms of human papilloma virus related skin cancers: A review.

The human papillomavirus (HPV) belongs to the Papillomaviridae family of viruses which includes small, double-stranded DNA viral agents. Approximately 90% of HPV infections occur asymptomatically and resolve spontaneously. However, infection with high-risk viral strains can lead to the development of preneoplastic lesions, with an increased propensity to become cancerous. The location of these malignancies includes the oral cavity, cervix, vagina, anus, and vulva, among others. The role of HPV in carcinogenesis has already been demonstrated for the aforementioned neoplasia. However, regarding skin malignancies, the mechanisms that pinpoint the role played by HPV in their initiation and progression still elude our sight. Until now, the only fully understood mechanism of viral cutaneous oncogenesis is that of human herpes virus 8 infection in Kaposi sarcoma. In the case of HPV infection, however, most data focus on the role that beta strains exhibit in the oncogenesis of cutaneous squamous cell carcinoma (cSCC), along with ultraviolet radiation (UVR) and other environmental or genetic factors. However, recent epidemiological investigations have highlighted that HPV could also trigger the onset of other non-melanocytic, for example, basal cell carcinoma (BCC), and/or melanocytic skin cancers, for example, melanoma. Herein, we provide an overview of the role played by HPV in benign and malignant skin lesions with a particular focus on the main epidemiological, pathophysiological, and molecular aspects delineating the involvement of HPV in skin cancers.

Neoadjuvant talimogene laherparepvec plus surgery versus surgery alone for resectable stage IIIB-IVM1a melanoma: a randomized, open-label, phase 2 trial.

Talimogene laherparepvec (T-VEC) is a herpes simplex virus type 1-based intralesional oncolytic immunotherapy approved for the treatment of unresectable melanoma. The present, ongoing study aimed to estimate the treatment effect of neoadjuvant T-VEC on recurrence-free survival (RFS) of patients with advanced resectable melanoma. An open-label, phase 2 trial (NCT02211131) was conducted in 150 patients with resectable stage IIIB-IVM1a melanoma who were randomized to receive T-VEC followed by surgery (arm 1, n = 76) or surgery alone (arm 2, n = 74). The primary endpoint was a 2-year RFS in the intention-to-treat population. Secondary and exploratory endpoints included overall survival (OS), pathological complete response (pCR), safety and biomarker analyses. The 2-year RFS was 29.5% in arm 1 and 16.5% in arm 2 (overall hazard ratio (HR) = 0.75, 80% confidence interval (CI) = 0.58-0.96). The 2-year OS was 88.9% for arm 1 and 77.4% for arm 2 (overall HR = 0.49, 80% CI = 0.30-0.79). The RFS and OS differences between arms persisted at 3 years. In arm 1, 17.1% achieved a pCR. Increased CD8+ density correlated with clinical outcomes in an exploratory analysis. Arm 1 adverse events were consistent with previous reports for T-VEC. The present study met its primary endpoint and estimated a 25% reduction in the risk of disease recurrence for neoadjuvant T-VEC plus surgery versus upfront surgery for patients with resectable stage IIIB-IVM1a melanoma.

Evaluation of TTV replication as a biomarker of immune checkpoint inhibitors efficacy in melanoma patients.

Torque Teno Virus (TTV) is a small, non-enveloped, single-stranded and circular DNA virus that infects the majority of the population worldwide. Increased levels of plasma TTV viral load have been observed in various situations of immune deficiency or dysregulation, and several studies have suggested that TTV levels may be inversely correlated with immune competence. The measurement of TTV viremia by qPCR has been proposed as a potential biomarker for the follow-up of functional immune competence in immunosuppressed individuals, particularly hematopoietic stem cell transplant recipients. We hypothesized that TTV viral load could be used as a prognostic marker of immune checkpoint inhibitor (ICI) efficacy, and therefore investigated the TTV viral load in melanoma patients treated with nivolumab or pembrolizumab before and after 6 months of treatment. In the present study, TTV viral load was not different in melanoma patients before anti-PD-1 introduction compared to healthy volunteers, was not modified by ICI treatment and did not allowed to distinguish patients with treatment-sensitive tumor from patients with treatment-resistant tumor.

Clinical Responses of Oncolytic Coxsackievirus A21 (V937) in Patients With Unresectable Melanoma.

PURPOSE: We evaluated the activity of intratumoral Coxsackievirus A21 (V937) in 57 patients with unresectable stage IIIC or IV melanoma. PATIENTS AND METHODS: In this multicenter, open-label, phase II study, patients received up to a total V937 dose of 3 × 108 TCID50 (50% tissue culture infectious dose) in a maximum 4.0-mL volume by intratumoral injection. Ten sets of V937 injections were administered between days 1 and 127 (NCT01227551). Patients who had stable disease or were responding could continue treatment in an extension study (NCT01636882). Response and progression status were based on contrast-enhanced computed tomography, magnetic resonance imaging, or caliper measurement and were categorized using immune-related Response Evaluation Criteria in Solid Tumors (irRECIST). Other evaluations included monitoring of adverse events and serum levels of V937 and anti-V937 antibody titers. The primary efficacy end point was 6-month progression-free survival (PFS) rate per irRECIST. RESULTS: The primary efficacy end point, 6-month PFS rate per irRECIST, was 38.6% (95% CI, 26.0 to 52.4). Durable response rate (partial or complete response for ≥ 6 months) was 21.1% per irRECIST. Best overall response rate (complete plus partial response) was 38.6% (unconfirmed) and 28.1% (confirmed) per irRECIST. Regression of melanoma was observed in noninjected lesions. Based on Kaplan-Meier estimation, 12-month PFS was 32.9% (95% CI, 19.5 to 46.9) per irRECIST and 12-month overall survival was 75.4% (95% CI, 62.1 to 84.7). No treatment-related grade ≥ 3 adverse events occurred. Viral RNA was detected in serum within 30 minutes of administration. Neutralizing antibody titers increased to > 1:16 in all patients after day 22, without effect on clinical or immunologic response. CONCLUSION: V937 was well tolerated and warrants further investigation for treatment of patients with unresectable melanoma. Studies of combination approaches with V937 and immune checkpoint inhibitors are ongoing.

Silencing of Mcl-1 overcomes resistance of melanoma cells against TRAIL-armed oncolytic adenovirus by enhancement of apoptosis.

Arming of oncolytic viruses with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown as a viable approach to increase the antitumor efficacy in melanoma. However, melanoma cells may be partially or completely resistant to TRAIL or develop TRAIL resistance, thus counteracting the antitumor efficiency of TRAIL-armed oncolytic viruses. Recently, we found that TRAIL resistance in melanoma cells can be overcome by inhibition of antiapoptotic Bcl-2 protein myeloid cell leukemia 1 (Mcl-1). Here, we investigated whether the cytotoxicity of AdV-TRAIL, an oncolytic adenovirus, which expresses TRAIL after induction by doxycycline (Dox), can be improved in melanoma cells by silencing of Mcl-1. Two melanoma cell lines, the TRAIL-resistant MeWo and the TRAIL-sensitive Mel-HO were investigated. Treatment of both cell lines with AdV-TRAIL resulted in a decrease of cell viability, which was caused by an increase of apoptosis and necrosis. The proapoptotic effects were dependent on induction of TRAIL by Dox and were more pronounced in Mel-HO than in MeWo cells. SiRNA-mediated silencing of Mcl-1 resulted in a further significant decrease of cell viability and a further increase of apoptosis and necrosis in AdV-TRAIL-infected MeWo and Mel-HO cells. However, while in absolute terms, the effects were more pronounced in Mel-HO cells, in relative terms, they were stronger in MeWo cells. These results show that silencing of Mcl-1 represents a suitable approach to increase the cytotoxicity of a TRAIL-armed oncolytic adenovirus in melanoma cells. KEY MESSAGES: • Cytotoxicity of TRAIL-expressing adenovirus can be enhanced by silencing of Mcl-1. • The effect occurs in TRAIL-sensitive and TRAIL-resistant melanoma cells. • Increase of apoptosis is the main mechanism induced by Mcl-1 silencing.

Talimogene laherparepvec upregulates immune-cell populations in non-injected lesions: findings from a phase II, multicenter, open-label study in patients with stage IIIB-IVM1c melanoma.

BACKGROUND: Talimogene laherparepvec (T-VEC), an oncolytic virus, was designed to selectively replicate in and lyse tumor cells, releasing tumor-derived antigen to stimulate a tumor-specific immune response. METHODS: In this phase II study in patients with unresectable stage IIIB-IV melanoma, we evaluated non-injected lesions to establish whether baseline or change in intratumoral CD8+ T-cell density (determined using immunohistochemistry) correlated with T-VEC clinical response. RESULTS: Of 112 enrolled patients, 111 received ≥1 dose of T-VEC. After a median follow-up of 108.0 weeks, objective/complete response rates were 28%/14% in the overall population and 32%/18% in patients with stage IIIB-IVM1a disease. No unexpected toxicity occurred. Baseline and week 6 change from baseline CD8+ T-cell density results were available for 91 and 65 patients, respectively. Neither baseline nor change in CD8+ T-cell density correlated with objective response rate, changes in tumor burden, duration of response or durable response rate. However, a 2.4-fold median increase in CD8+ T-cell density in non-injected lesions from baseline to week 6 was observed. In exploratory analyses, multiparameter immunofluorescence showed that after treatment there was an increase in the proportion of infiltrating CD8+ T-cells expressing granzyme B and checkpoint markers (programmed death-1, programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen-4) in non-injected lesions, together with an increase in helper T-cells. Consistent with T-cell infiltrate, we observed an increase in the adaptive resistance marker PD-L1 in non-injected lesions. CONCLUSIONS: This study indicates that T-VEC induces systemic immune activity and alters the tumor microenvironment in a way that will likely enhance the effects of other immunotherapy agents in combination therapy. TRIAL REGISTRATION NUMBER: NCT02366195.

Impact of COVID-19 pandemic on eye cancer care in United Kingdom.

The COVID-19 pandemic has had an unprecedented impact on the National Health Service in United Kingdom. The UK Ocular Oncology Services evaluated the impact on the adult eye cancer care in the UK. All four adult Ocular Oncology centres participated in a multicentre retrospective review comparing uveal melanoma referral patterns and treatments in a 4-month period during the national lockdown and first wave of the COVID-19 pandemic in 2020 with corresponding periods in previous 2 years. During the national lockdown, referral numbers and confirmed uveal melanoma cases reduced considerably, equalling to ~120 fewer diagnosed uveal melanoma cases compared to previous 2 years. Contrary to the recent trend, increased caseloads of enucleation and stereotactic radiosurgery (p > 0.05), in comparison to fewer proton beam therapy (p < 0.05), were performed. In the 4-month period following lockdown, there was a surge in clinical activities with more advanced diseases (p < 0.05) presenting to the services. As the COVID-19 pandemic continues to mount pressure and reveal its hidden impact on the eye cancer care, it is imperative for the Ocular Oncology Services to plan recovery strategies and innovative ways of working.

The N-terminus of varicella-zoster virus glycoprotein B has a functional role in fusion.

Varicella-zoster virus (VZV) is a medically important alphaherpesvirus that induces fusion of the virion envelope and the cell membrane during entry, and between cells to form polykaryocytes within infected tissues during pathogenesis. All members of the Herpesviridae, including VZV, have a conserved core fusion complex composed of glycoproteins, gB, gH and gL. The ectodomain of the primary fusogen, gB, has five domains, DI-V, of which DI contains the fusion loops needed for fusion function. We recently demonstrated that DIV is critical for fusion initiation, which was revealed by a 2.8Å structure of a VZV neutralizing mAb, 93k, bound to gB and mutagenesis of the gB-93k interface. To further assess the mechanism of mAb 93k neutralization, the binding site of a non-neutralizing mAb to gB, SG2, was compared to mAb 93k using single particle cryogenic electron microscopy (cryo-EM). The gB-SG2 interface partially overlapped with that of gB-93k but, unlike mAb 93k, mAb SG2 did not interact with the gB N-terminus, suggesting a potential role for the gB N-terminus in membrane fusion. The gB ectodomain structure in the absence of antibody was defined at near atomic resolution by single particle cryo-EM (3.9Å) of native, full-length gB purified from infected cells and by X-ray crystallography (2.4Å) of the transiently expressed ectodomain. Both structures revealed that the VZV gB N-terminus (aa72-114) was flexible based on the absence of visible structures in the cryo-EM or X-ray crystallography data but the presence of gB N-terminal peptides were confirmed by mass spectrometry. Notably, N-terminal residues 109KSQD112 were predicted to form a small α-helix and alanine substitution of these residues abolished cell-cell fusion in a virus-free assay. Importantly, transferring the 109AAAA112 mutation into the VZV genome significantly impaired viral propagation. These data establish a functional role for the gB N-terminus in membrane fusion broadly relevant to the Herpesviridae.

Macrophage polarization contributes to the efficacy of an oncolytic HSV-1 targeting human uveal melanoma in a murine xenograft model.

Uveal melanoma (UM), the most common primary malignant tumor of the eye in adults, is difficult-to-treat. UM has a relatively high mortality secondary to distant metastasis and poor overall survival with existing therapies. The oncolytic virus herpes simplex virus type-1 (HSV-1) has been approved for clinical use in melanoma. This double-stranded DNA virus was suspected to directly activate lysis specifically in neoplastic cells. We tested the antitumor efficacy of recombinant oncolytic HSV-1-EGFP (oHSV-EGFP) in UM and characterized the local and systemic antitumor innate immune response in a murine xenograft model. We first determined the efficacy of the oncolytic virus in 92.1, MUM2B and MP41 cell lines. In murine xenograft models, oHSV-EGFP reduced intraocular tumors as well as systemic subcutaneous tumors. A significant change in cytokines was observed in viral infected cells, especially a rise in IFNγ. In vivo analyses showed increased anti-tumorigenic M1 macrophages and decreased pro-tumorigenic M2 macrophages in peripheral blood, and intraocular and distant tumors after intravitreal viral treatment. Increased infiltration of natural killer cells and mature dendritic cells was also detected after viral treatment. In addition, no virus was detected in major organs after the treatment. Our data support that oHSV-EGFP is effective, neoplasm specific, immune active and safe, providing possible clinical translatable options to treat ocular and metastatic UM.

Human Endogenous Retrovirus K Rec forms a Regulatory Loop with MITF that Opposes the Progression of Melanoma to an Invasive Stage.

The HML2 subfamily of HERV-K (henceforth HERV-K) represents the most recently endogenized retrovirus in the human genome. While the products of certain HERV-K genomic copies are expressed in normal tissues, they are upregulated in several pathological conditions, including various tumors. It remains unclear whether HERV-K(HML2)-encoded products overexpressed in cancer contribute to disease progression or are merely by-products of tumorigenesis. Here, we focus on the regulatory activities of the Long Terminal Repeats (LTR5_Hs) of HERV-K and the potential role of the HERV-K-encoded Rec in melanoma. Our regulatory genomics analysis of LTR5_Hs loci indicates that Melanocyte Inducing Transcription Factor (MITF) (also known as binds to a canonical E-box motif (CA(C/T)GTG) within these elements in proliferative type of melanoma, and that depletion of MITF results in reduced HERV-K expression. In turn, experimentally depleting Rec in a proliferative melanoma cell line leads to lower mRNA levels of MITF and its predicted target genes. Furthermore, Rec knockdown leads to an upregulation of epithelial-to-mesenchymal associated genes and an enhanced invasion phenotype of proliferative melanoma cells. Together these results suggest the existence of a regulatory loop between MITF and Rec that may modulate the transition from proliferative to invasive stages of melanoma. Because HERV-K(HML2) elements are restricted to hominoid primates, these findings might explain certain species-specific features of melanoma progression and point to some limitations of animal models in melanoma studies.

Measles virus in cancer therapy.

Over the last years, the development of viruses to treat cancer patients has re-gained considerable attention. A genetically modified herpesvirus, Talimogene laherparepvec, has already been authorized for the treatment of melanoma patients. Also recombinant measles virus (MeV) is developed as an oncolytic virus. Because of its high genetic flexibility, a number of different MeV strains have been the basis for the generation of targeted, armed, or shielded viruses that are highly specific for a given tumor target, more effective, or protected against serum neutralization. Such MeV have been extensively tested in vitro and in vivo, whereby remarkable oncolytic potency is accompanied by safety also in non-human primates. Therefore, MeV has been introduced into 19 different clinical trials and has reached phase II against two different tumor entities, multiple myeloma and ovarian carcinoma. Remarkably, one patient with advanced stage myeloma experienced long-term remission after treatment, visualizing the potency of this approach.

Diffuse pneumonitis from coronavirus HKU1 on checkpoint inhibitor therapy.

BACKGROUND: Immune checkpoint inhibitors (ICIs) can produce specific immune-related adverse events including pneumonitis. The impact of ICI therapy on the severity of acute coronavirus infection symptomatology warrants further exploration. CASE PRESENTATION: We report a 65-year-old man diagnosed with stage IV melanoma who developed pulmonary and brain metastases and was treated with bilateral craniotomies followed by combined nivolumab and ipilimumab immunotherapy. He developed early-onset severe dyspnea associated with acute coronavirus HKU1 (non-COVID-19) infection, with diffuse pneumonitis evidenced by ground glass opacification on CT scan. He was treated with steroids leading to resolution of pneumonitis on repeat imaging, suggesting an exacerbated immune-mediated toxicity. CONCLUSION: We report the first case of a patient with melanoma with severe and reversible diffuse pneumonitis in association with coronavirus HKU1 following combined nivolumab and ipilimumab immunotherapy. Although we do not have data on the impact of ICI therapy on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) symptomatology, a possible interaction should be considered when deciding on dosing in patients with possible SARS-CoV-2 exposure or when evaluating patients with presumed ICI-related pneumonitis during the COVID-19 pandemic.

Expression of the COVID-19 receptor ACE2 in the human conjunctiva.

SARS-CoV-2 is assumed to use angiotensin-converting enzyme 2 (ACE2) and other auxiliary proteins for cell entry. Recent studies have described conjunctival congestion in 0.8% of patients with laboratory-confirmed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and there has been speculation that SARS-CoV-2 can be transmitted through the conjunctiva. However, it is currently unclear whether conjunctival epithelial cells express ACE2 and its cofactors. In this study, a total of 38 conjunctival samples from 38 patients, including 12 healthy conjunctivas, 12 melanomas, seven squamous cell carcinomas, and seven papilloma samples, were analyzed using high-throughput RNA sequencing to assess messenger RNA (mRNA) expression of the SARS-CoV-2 receptor ACE2 and its cofactors including TMPRSS2, ANPEP, DPP4, and ENPEP. ACE2 protein expression was assessed in eight healthy conjunctival samples using immunohistochemistry. Our results show that the SARS-CoV-2 receptor ACE2 is not substantially expressed in conjunctival samples on the mRNA (median: 0.0 transcripts per million [TPM], min: 0.0 TPM, max: 1.7 TPM) and protein levels. Similar results were obtained for the transcription of other auxiliary molecules. In conclusion, this study finds no evidence for a significant expression of ACE2 and its auxiliary mediators for cell entry in conjunctival samples, making conjunctival infection with SARS-CoV-2 via these mediators unlikely.

Natural hybridization reveals incompatible alleles that cause melanoma in swordtail fish.

The establishment of reproductive barriers between populations can fuel the evolution of new species. A genetic framework for this process posits that "incompatible" interactions between genes can evolve that result in reduced survival or reproduction in hybrids. However, progress has been slow in identifying individual genes that underlie hybrid incompatibilities. We used a combination of approaches to map the genes that drive the development of an incompatibility that causes melanoma in swordtail fish hybrids. One of the genes involved in this incompatibility also causes melanoma in hybrids between distantly related species. Moreover, this melanoma reduces survival in the wild, likely because of progressive degradation of the fin. This work identifies genes underlying a vertebrate hybrid incompatibility and provides a glimpse into the action of these genes in natural hybrid populations.

STING Sensing of Murine Cytomegalovirus Alters the Tumor Microenvironment to Promote Antitumor Immunity.

CMV has been proposed to play a role in cancer progression and invasiveness. However, CMV has been increasingly studied as a cancer vaccine vector, and multiple groups, including ours, have reported that the virus can drive antitumor immunity in certain models. Our previous work revealed that intratumoral injections of wild-type murine CMV (MCMV) into B16-F0 melanomas caused tumor growth delay in part by using a viral chemokine to recruit macrophages that were subsequently infected. We now show that MCMV acts as a STING agonist in the tumor. MCMV infection of tumors in STING-deficient mice resulted in normal recruitment of macrophages to the tumor, but poor recruitment of CD8+ T cells, reduced production of inflammatory cytokines and chemokines, and no delay in tumor growth. In vitro, expression of type I IFN was dependent on both STING and the type I IFNR. Moreover, type I IFN alone was sufficient to induce cytokine and chemokine production by macrophages and B16 tumor cells, suggesting that the major role for STING activation was to produce type I IFN. Critically, viral infection of wild-type macrophages alone was sufficient to restore tumor growth delay in STING-deficient animals. Overall, these data show that MCMV infection and sensing in tumor-associated macrophages through STING signaling is sufficient to promote antitumor immune responses in the B16-F0 melanoma model.

The Susceptibility of Human Melanoma Cells to Infection with the Leningrad-16 Vaccine Strain of Measles Virus.

Oncolytic viruses, including live attenuated measles virus (MV) vaccine strains, have recently been shown as promising therapeutic agents against human malignancies. In this study, the oncolytic potential of the attenuated vaccine strain Leningrad-16 (L-16) of MV was evaluated in a panel of human metastatic melanoma cell lines. The L-16 measles virus was shown to replicate within melanoma cells mediating direct cell killing of tumor cells, although all melanoma cell lines varied in regard to their ability to respond to L-16 MV infection, as revealed by the different pattern of the Interferon Stimulated Gene expression, cytokine release and mechanisms of cell death. Furthermore, the statistically significant L-16 measles virus related tumor growth inhibition was demonstrated in a melanoma xenograft model. Therefore, L-16 MV represents an appealing oncolytic platform for target delivery of therapeutic genes along with other attenuated measles virus strains.