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Poxviruses are large enveloped viruses that replicate exclusively in the cytoplasm. Like all viruses, their replication cycle begins with virion adsorption to the cell surface. Unlike most other viral families, however, no unique poxviral receptor has ever been identified. In the absence of a unique receptor, poxviruses are instead thought to adhere to the cell surface primarily through electrostatic interactions between the positively charged viral envelope proteins and the negatively charged sulfate groups on cellular glycosaminoglycans (GAGs). While these negatively charged GAGs are an integral part of all eukaryotic membranes, their specific expression and sulfation patterns differ between cell types. Critically, while poxviral binding has been extensively studied using virally centered genetic strategies, the impact of cell-intrinsic changes to GAG charge has never been examined. Here we show that loss of heparin sulfation, accomplished by deleting the enzyme N-Deacetylase and N-Sulfotransferase-1 (NDST1) which is essential for GAG sulfation, significantly reduces the binding affinity of both vaccinia and myxoma viruses to the cell surface. Strikingly, however, while this lowered binding affinity inhibits the subsequent spread of myxoma virus, it actually enhances the overall spread of vaccinia by generating more diffuse regions of infection. These data indicate that cell-intrinsic GAG sulfation plays a major role in poxviral infection, however, this role varies significantly between different members of the poxviridae.

Multiple myeloma (MM) is a clonal malignancy of plasma cells that is newly diagnosed in ~30,000 patients in the US each year. While recently developed therapies have improved the prognosis for MM patients, relapse rates remain unacceptably high. To overcome this challenge, researchers have begun to investigate the therapeutic potential of oncolytic viruses as a novel treatment option for MM. Preclinical work with these viruses has demonstrated that their infection can be highly specific for MM cells and results in impressive therapeutic efficacy in a variety of preclinical models. This has led to the recent initiation of several human trials. This review summarizes the current state of oncolytic therapy as a therapeutic option for MM and highlights a variety of areas that need to be addressed as the field moves forward.

Surgical removal of tumors remains a front-line therapy for many types of cancer. However, this treatment often fails to eradicate disease due to either recurrence of the original tumor or development of distant micrometastases. To address these challenges, patients are often given non-curative treatments presurgery with the intent of improving surgical outcomes. These treatments, collectively known as neoadjuvant therapies, have traditionally focused on the presurgical use of chemotherapeutics. Recently, however, a variety of immunotherapies have also been identified as potentially effective in the neoadjuvant setting. One of these immunotherapies is oncolytic virotherapy, whose clinical use has exploded with the Food and Drug Administration approval of Talimogene Laherparepvec. This review summarizes both the preclinical and clinical literature examining the use of oncolytic virotherapy in the neoadjuvant setting for different types of cancers and discusses some of the major questions that still need to be addressed in order for this unique use of immunotherapy to become clinically viable.

BackgroundArginine (Arg) is a semiessential amino acid whose bioavailability is required for the in vitro replication of several oncolytic viruses. In vivo, Arg bioavailability is regulated by a combination of dietary intake, protein catabolism, and limited biosynthesis through portions of the urea cycle. Interestingly, despite the importance of bioavailable Arg to support cellular proliferation, many forms of cancer are functionally auxotrophic for this amino acid due to the epigenetic silencing of argininosuccinate synthetase 1 (ASS1), an enzyme responsible for the conversion of citrulline and aspartate into the Arg precursor argininosuccinate. The impact of this silencing on oncolytic virotherapy (OV), however, has never been examined.MethodsTo address this gap in knowledge, we generated tumor cells lacking ASS1 and examined how loss of this enzyme impacted the in vivo replication and therapeutic efficacy of oncolytic myxoma virus (MYXV). We also generated a series of recombinant MYXV constructs expressing exogenous ASS1 to evaluate the therapeutic benefit of virally reconstituting Arg biosynthesis in ASS1−/− tumors.ResultsOur results show that the in vitro replication of oncolytic MYXV is dependent on the presence of bioavailable Arg. This dependence can be overcome by the addition of the metabolic precursor citrulline, however, this rescue requires expression of ASS1. Because of this, tumors formed from functionally ASS1−/− cells display significantly reduced MYXV replication as well as poorer therapeutic responses. Critically, both defects could be partially rescued by expressing exogenous ASS1 from recombinant oncolytic MYXVs.ConclusionsThese results demonstrate that intratumoral defects to Arg metabolism can serve as a novel barrier to virally induced immunotherapy and that the exogenous expression of ASS1 can improve the efficacy of OV in Arg-auxotrophic tumors.

Immunomodulators of pathogens frequently affect multiple cellular targets, thus preventing recognition by different immune cells. For instance, the K5 modulator of immune recognition (MIR2) from Kaposi sarcoma-associated herpesvirus prevents activation of cytotoxic T cells, natural killer cells, and natural killer T cells by downregulating major histocompatibility complex (MHC) class I molecules, the MHC-like molecule CD1, the cell adhesion molecules ICAM-1 and PECAM, and the co-stimulatory molecule B7.2. K5 belongs to a family of viral- and cellular-membrane-spanning RING ubiquitin ligases. While a limited number of transmembrane proteins have been shown to be targeted for degradation by this family, it is unknown whether additional targets exist. We now describe a quantitative proteomics approach to identify novel targets of this protein family. Using stable isotope labeling by amino acids, we compared the proteome of plasma, Golgi, and endoplasmic reticulum membranes in the presence and absence of K5. Mass spectrometric protein identification revealed four proteins that were consistently underrepresented in the plasma membrane of K5 expression cells: MHC I (as expected), bone marrow stromal antigen 2 (BST-2, CD316), activated leukocyte cell adhesion molecule (ALCAM, CD166) and Syntaxin-4. Downregulation of each of these proteins was independently confirmed by immunoblotting with specific antibodies. We further demonstrate that ALCAM is a bona fide target of both K5 and the myxomavirus homolog M153R. Upon exiting the endoplasmic reticulum, ALCAM is ubiquitinated in the presence of wild-type, but not RING-deficient or acidic motif-deficient, K5, and is targeted for lysosomal degradation via the multivesicular body pathway. Since ALCAM is the ligand for CD6, a member of the immunological synapse of T cells, its removal by viral immune modulators implies a role for CD6 in the recognition of pathogens by T cells. The unbiased global proteome analysis therefore revealed novel immunomodulatory functions of pathogen proteins.

Adoptive T cell transfer therapy (ACT) using tumor infiltrating lymphocytes or lymphocytes redirected with antigen receptors (CAR or TCR) has revolutionized the field of cancer immunotherapy. Although CAR T cell therapy mediates robust responses in patients with hematological malignancies, this approach has been less effective for treating patients with solid tumors. Additionally, toxicities post T cell infusion highlight the need for safer ACT protocols. Current protocols traditionally expand T lymphocytes isolated from patient tumors or from peripheral blood to large magnitudes in the presence of high dose IL-2 prior to infusion. Unfortunately, this expansion protocol differentiates T cells to a full effector or terminal phenotype , consequently reducing their long-term survival and antitumor effectiveness . Post-infusion, T cells face further obstacles limiting their persistence and function within the suppressive tumor microenvironment. Therapeutic manipulation of T cells with common γ chain cytokines, which are critical growth factors for T cells, may be the key to bypass such immunological hurdles. Herein, we discuss the primary functions of the common γ chain cytokines impacting T cell survival and memory and then elaborate on how these distinct cytokines have been used to augment T cell-based cancer immunotherapy.

Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins.

Immuno-oncology has revolutionized cancer treatment by mobilizing the immune system to eliminate tumors. Although immune checkpoint inhibitors and T cell therapies have mediated durable responses in hematologic malignancies and select solid tumors, most patients still relapse or fail to respond. To overcome these limitations, novel, next-generation immune constructs, including bispecific-cell therapy combinations, armored cells, tethered cytokines, immune-stimulatory antibody conjugates, and in vivo gene editing, are being developed to enhance specificity, persistence, and immune activation. This Society for Immunotherapy of Cancer roadmap highlights emerging technologies that integrate, redirect, or potentiate immune response. We examine advances in construct design, strategies for clinical translation, and opportunities for combinatorial approaches. By addressing translational barriers and real-world challenges, we outline how innovative engineering can unlock a new era of safe, durable, and accessible immunotherapies. Realizing this potential will require coordinated efforts from researchers, clinicians, industry leaders, and policymakers to deliver curative outcomes to patients worldwide.

Cancer has become one of the most critical health issues of modern times. To overcome the ineffectiveness of current treatment options, research is being done to explore new therapeutic modalities. One such novel treatment is oncolytic virotherapy (OV) which uses tumor tropic viruses to specifically target and kill malignant cells. While OV has shown significant promise in recent clinical trials, the therapeutic use of viruses poses a number of unique challenges. In particular, obtaining effective viral spread throughout the tumor microenvironment remains problematic. Previous work has suggested this can be overcome by forcing oncolytic viruses to induce syncytia formation. In the current work, we generated a series of recombinant myxoma viruses expressing exogenous fusion proteins from other viral genomes and examined their therapeutic potential in vitro and in vivo. Similar to previous studies, we observed that the expression of these fusion proteins during myxoma infection induced the formation of multinucleated syncytia which increased viral spread and lytic potential compared to non-fusogenic controls. Contrary to expectations, however, the treatment of established tumors with these viruses resulted in decreased therapeutic efficacy which corresponded with reduced viral persistence. These findings indicate that enhanced viral spread caused by syncytia formation can actually reduce the efficacy of OV and supports a number of previous works suggesting that the in vitro properties of viruses frequently fail to predict their in vivo efficacy.

Blockade of the programmed cell death protein 1 (PD1) pathway is clinically effective against human cancers. Although multiple types of malignancies have been shown to respond to PD1 agents, only a small percentage of patients typically benefit from this treatment. In addition, PD1 therapy often causes serious immune-related adverse events. A recent study demonstrated that local, intra-tumoral, administration of modified oncolytic myxoma virus which expresses a truncated version of the PD1 protein resulted in both increased efficacy and reduced toxicity in a clinically relevant melanoma model.