Tutorial: design, production and testing of oncolytic viruses for cancer immunotherapy.

Oncolytic viruses (OVs) represent a novel class of cancer immunotherapy agents that preferentially infect and kill cancer cells and promote protective antitumor immunity. Furthermore, OVs can be used in combination with established or upcoming immunotherapeutic agents, especially immune checkpoint inhibitors, to efficiently target a wide range of malignancies. The development of OV-based therapy involves three major steps before clinical evaluation: design, production and preclinical testing. OVs can be designed as natural or engineered strains and subsequently selected for their ability to kill a broad spectrum of cancer cells rather than normal, healthy cells. OV selection is further influenced by multiple factors, such as the availability of a specific viral platform, cancer cell permissivity, the need for genetic engineering to render the virus non-pathogenic and/or more effective and logistical considerations around the use of OVs within the laboratory or clinical setting. Selected OVs are then produced and tested for their anticancer potential by using syngeneic, xenograft or humanized preclinical models wherein immunocompromised and immunocompetent setups are used to elucidate their direct oncolytic ability as well as indirect immunotherapeutic potential in vivo. Finally, OVs demonstrating the desired anticancer potential progress toward translation in patients with cancer. This tutorial provides guidelines for the design, production and preclinical testing of OVs, emphasizing considerations specific to OV technology that determine their clinical utility as cancer immunotherapy agents.

Genome-guided discovery of cancer therapeutic targets.

The success of precision oncology-which aims to match the right therapies to the right patients based on molecular status-is predicated on a robust pipeline of molecular targets against which therapies can be developed. Recent advances in genomics and functional genetics have enabled the unbiased discovery of novel molecular targets at scale. We summarize the promise and challenges in integrating genomic and functional genetic landscapes of cancer to establish the next generation of cancer targets.

A genetic basis for cancer sex differences revealed in Xp11 translocation renal cell carcinoma.

Xp11 translocation renal cell carcinoma (tRCC) is a female-predominant kidney cancer driven by translocations between the TFE3 gene on chromosome Xp11.2 and partner genes located on either chrX or on autosomes. The rearrangement processes that underlie TFE3 fusions, and whether they are linked to the female sex bias of this cancer, are largely unexplored. Moreover, whether oncogenic TFE3 fusions arise from both the active and inactive X chromosomes in females remains unknown. Here we address these questions by haplotype-specific analyses of whole-genome sequences of 29 tRCC samples from 15 patients and by re-analysis of 145 published tRCC whole-exome sequences. We show that TFE3 fusions universally arise as reciprocal translocations with minimal DNA loss or insertion at paired break ends. Strikingly, we observe a near exact 2:1 female:male ratio in TFE3 fusions arising via X:autosomal translocation (but not via X inversion), which accounts for the female predominance of tRCC. This 2:1 ratio is at least partially attributable to oncogenic fusions involving the inactive X chromosome and is accompanied by partial re-activation of silenced chrX genes on the rearranged chromosome. Our results highlight how somatic alterations involving the X chromosome place unique constraints on tumor initiation and exemplify how genetic rearrangements of the sex chromosomes can underlie cancer sex differences.

How splicing confers treatment resistance in prostate cancer.

A splice variant of the androgen receptor that drives prostate cancer resistance translocates into the nucleus using a different mechanism from the full-length receptor and exhibits distinct molecular properties once inside.

NAD+ depletion enhances reovirus-induced oncolysis in multiple myeloma.

Cancer cell energy metabolism plays an important role in dictating the efficacy of oncolysis by oncolytic viruses. To understand the role of multiple myeloma metabolism in reovirus oncolysis, we performed semi-targeted mass spectrometry-based metabolomics on 12 multiple myeloma cell lines and revealed a negative correlation between NAD+ levels and susceptibility to oncolysis. Likewise, a negative correlation was observed between the activity of the rate-limiting NAD+ synthesis enzyme NAMPT and oncolysis. Indeed, depletion of NAD+ levels by pharmacological inhibition of NAMPT using FK866 sensitized several myeloma cell lines to reovirus-induced killing. The myelomas that were most sensitive to this combination therapy expressed a functional p53 and had a metabolic and transcriptomic profile favoring mitochondrial metabolism over glycolysis, with the highest synergistic effect in KMS12 cells. Mechanistically, U-13C-labeled glucose flux, extracellular flux analysis, multiplex proteomics, and cell death assays revealed that the reovirus + FK866 combination caused mitochondrial dysfunction and energy depletion, leading to enhanced autophagic cell death in KMS12 cells. Finally, the combination of reovirus and NAD+ depletion achieved greater antitumor effects in KMS12 tumors in vivo and patient-derived CD138+ multiple myeloma cells. These findings identify NAD+ depletion as a potential combinatorial strategy to enhance the efficacy of oncolytic virus-based therapies in multiple myeloma.

Photodynamic therapy of melanoma with new, structurally similar, NIR-absorbing ruthenium (II) complexes promotes tumor growth control via distinct hallmarks of immunogenic cell death.

Cancer therapies that generate T cell-based anti-cancer immune responses are critical for clinical success and are favored over traditional therapies. One way to elicit T cell immune responses and generate long-lasting anti-cancer immunity is through induction of immunogenic cell death (ICD), a form of regulated cell death that promotes antigenicity and adjuvanticity within dying cells. Therefore, research in the last decade has focused on developing cancer therapies which stimulate ICD. Herein, we report novel photodynamic therapy (PDT) compounds with immunomodulatory and ICD inducing properties. PDT is a clinically approved, minimally invasive anti-cancer treatment option and has been extensively investigated for its tumor-destroying properties, lower side effects, and immune activation capabilities. In this study, we explore two structurally related ruthenium compounds, ML19B01 and ML19B02, that can be activated with near infrared light to elicit superior cytotoxic properties. In addition to its direct cell killing abilities, we investigated the effect of our PSs on immunological pathways upon activation. PDT treatment with ML19B01 and ML19B02 induced differential expression of reactive oxygen species, proinflammatory response-mediating genes, and heat shock proteins. Dying melanoma cells induced by ML19B01-PDT and ML19B02-PDT contained ICD hallmarks such as calreticulin, ATP, and HMGB1, initiated activation of antigen presenting cells, and were efficiently phagocytosed by bone marrow-derived dendritic cells. Most importantly, despite the distinct profiles of ICD hallmark inducing capacities, vaccination with both PDT-induced dying cancer cells established anti-tumor immunity that protected mice against subsequent challenge with melanoma cells.

After virus exposure, early bystander naïve CD8 T cell activation relies on NAD+ salvage metabolism.

CD8 T cells play a central role in antiviral immunity. Type I interferons are among the earliest responders after virus exposure and can cause extensive reprogramming and antigen-independent bystander activation of CD8 T cells. Although bystander activation of pre-existing memory CD8 T cells is known to play an important role in host defense and immunopathology, its impact on naïve CD8 T cells remains underappreciated. Here we report that exposure to reovirus, both in vitro or in vivo, promotes bystander activation of naïve CD8 T cells within 24 hours and that this distinct subtype of CD8 T cell displays an innate, antiviral, type I interferon sensitized signature. The induction of bystander naïve CD8 T cells is STAT1 dependent and regulated through nicotinamide phosphoribosyl transferase (NAMPT)-mediated enzymatic actions within NAD+ salvage metabolic biosynthesis. These findings identify a novel aspect of CD8 T cell activation following virus infection with implications for human health and physiology.

Immune Checkpoint Blockade Augments Changes Within Oncolytic Virus-induced Cancer MHC-I Peptidome, Creating Novel Antitumor CD8 T Cell Reactivities.

The combination cancer immunotherapies with oncolytic virus (OV) and immune checkpoint blockade (ICB) reinstate otherwise dysfunctional antitumor CD8 T cell responses. One major mechanism that aids such reinstatement of antitumor CD8 T cells involves the availability of new class I major histocompatibility complex (MHC-I)-bound tumor epitopes following therapeutic intervention. Thus, therapy-induced changes within the MHC-I peptidome hold the key to understanding the clinical implications for therapy-reinstated CD8 T cell responses. Here, using mass spectrometry-based immuno-affinity methods and tumor-bearing animals treated with OV and ICB (alone or in combination), we captured the therapy-induced alterations within the tumor MHC-I peptidome, which were then tested for their CD8 T cell response-stimulating activity. We found that the oncolytic reovirus monotherapy drives up- as well as downexpression of tumor MHC-I peptides in a cancer type and oncolysis susceptibility dependent manner. Interestingly, the combination of reovirus + ICB results in higher numbers of differentially expressed MHC-I-associated peptides (DEMHCPs) relative to either monotherapies. Most importantly, OV+ICB-driven DEMHCPs contain biologically active epitopes that stimulate interferon-gamma responses in cognate CD8 T cells, which may mediate clinically desired antitumor attack and cancer immunoediting. These findings highlight that the therapy-induced changes to the MHC-I peptidome contribute toward the reinstated antitumor CD8 T cell attack established following OV + ICB combination cancer immunotherapy.

Cytokines in oncolytic virotherapy.

Tumors represent a hostile environment for the effector cells of cancer immunosurveillance. Immunosuppressive receptors and soluble or membrane-bound ligands are abundantly exposed and released by malignant entities and their stromal accomplices. As a consequence, executioners of antitumor immunity inefficiently navigate across cancer tissues and fail to eliminate malignant targets. By inducing immunogenic cancer cell death, oncolytic viruses profoundly reshape the tumor microenvironment. They trigger the local spread of danger signals and tumor-associated (as well as viral) antigens, thus attracting antigen-presenting cells, promoting the activation and expansion of lymphocytic populations, facilitating their infiltration in the tumor bed, and reinvigorating cytotoxic immune activity. The present review recapitulates key chemokines, growth factors and other cytokines that orchestrate this ballet of antitumoral leukocytes upon oncolytic virotherapy.

Closely related reovirus lab strains induce opposite expression of RIG-I/IFN-dependent versus -independent host genes, via mechanisms of slow replication versus polymorphisms in dsRNA binding σ3 respectively.

The Dearing isolate of Mammalian orthoreovirus (T3D) is a prominent model of virus-host relationships and a candidate oncolytic virotherapy. Closely related laboratory strains of T3D, originating from the same ancestral T3D isolate, were recently found to exhibit significantly different oncolytic properties. Specifically, the T3DPL strain had faster replication kinetics in a panel of cancer cells and improved tumor regression in an in vivo melanoma model, relative to T3DTD. In this study, we discover that T3DPL and T3DTD also differentially activate host signalling pathways and downstream gene transcription. At equivalent infectious dose, T3DTD induces higher IRF3 phosphorylation and expression of type I IFNs and IFN-stimulated genes (ISGs) than T3DPL. Using mono-reassortants with intermediate replication kinetics and pharmacological inhibitors of reovirus replication, IFN responses were found to inversely correlate with kinetics of virus replication. In other words, slow-replicating T3D strains induce more IFN signalling than fast-replicating T3D strains. Paradoxically, during co-infections by T3DPL and T3DTD, there was still high IRF3 phosphorylation indicating a phenodominant effect by the slow-replicating T3DTD. Using silencing and knock-out of RIG-I to impede IFN, we found that IFN induction does not affect the first round of reovirus replication but does prevent cell-cell spread in a paracrine fashion. Accordingly, during co-infections, T3DPL continues to replicate robustly despite activation of IFN by T3DTD. Using gene expression analysis, we discovered that reovirus can also induce a subset of genes in a RIG-I and IFN-independent manner; these genes were induced more by T3DPL than T3DTD. Polymorphisms in reovirus σ3 viral protein were found to control activation of RIG-I/ IFN-independent genes. Altogether, the study reveals that single amino acid polymorphisms in reovirus genomes can have large impact on host gene expression, by both changing replication kinetics and by modifying viral protein activity, such that two closely related T3D strains can induce opposite cytokine landscapes.

Neuronal mitochondrial calcium uniporter deficiency exacerbates axonal injury and suppresses remyelination in mice subjected to experimental autoimmune encephalomyelitis.

High-capacity mitochondrial calcium (Ca2+) uptake by the mitochondrial Ca2+ uniporter (MCU) is strategically positioned to support the survival and remyelination of axons in multiple sclerosis (MS) by undocking mitochondria, buffering Ca2+ and elevating adenosine triphosphate (ATP) synthesis at metabolically stressed sites. Respiratory chain deficits in MS are proposed to metabolically compromise axon survival and remyelination by suppressing MCU activity. In support of this hypothesis, clinical scores, mitochondrial dysfunction, myelin loss, axon damage and inflammation were elevated while remyelination was blocked in neuronal MCU deficient (Thy1-MCU Def) mice relative to Thy1 controls subjected to experimental autoimmune encephalomyelitis (EAE). At the first sign of walking deficits, mitochondria in EAE/Thy1 axons showed signs of activation. By contrast, cytoskeletal damage, fragmented mitochondria and large autophagosomes were seen in EAE/Thy1-MCU Def axons. As EAE severity increased, EAE/Thy1 axons were filled with massively swollen mitochondria with damaged cristae while EAE/Thy1-MCU Def axons were riddled with late autophagosomes. ATP concentrations and mitochondrial gene expression were suppressed while calpain activity, autophagy-related gene mRNA levels and autophagosome marker (LC3) co-localization in Thy1-expressing neurons were elevated in the spinal cords of EAE/Thy1-MCU Def compared to EAE/Thy1 mice. These findings suggest that MCU inhibition contributes to axonal damage that drives MS progression.

Improving MHC-I Ligand Identification by Incorporating Targeted Searches of Mass Spectrometry Data.

Effective immunotherapies rely on specific activation of immune cells. Class I major histocompatibility complex (MHC-I) bound peptide ligands play a major role in dictating the specificity and activation of CD8+ T cells and hence are important in developing T cell-based immunotherapies. Mass spectrometry-based approaches are most commonly used for identifying these MHC-bound peptides, wherein the MS/MS spectra are compared against a reference proteome database. Unfortunately, the effectiveness of matching the immunopeptide MS/MS spectra to a reference proteome database is hindered by inflated search spaces attributed to a lack of enzyme restriction in searches. These large search spaces limit the efficiency with which MHC-I peptides are identified. Here, we describe the implementation of a targeted database search approach and accompanying tool, SpectMHC, that is based on a priori-predicted MHC-I peptides. We have previously shown that this targeted search strategy improved peptide identifications for both mouse and human MHC ligands by greater than two-fold and is superior to traditional "no enzyme" search of reference proteomes (Murphy et al. J Res Proteome 16:1806-1816, 2017).

Near-infrared absorbing Ru(ii) complexes act as immunoprotective photodynamic therapy (PDT) agents against aggressive melanoma.

Mounting evidence over the past 20 years suggests that photodynamic therapy (PDT), an anticancer modality known mostly as a local treatment, has the capacity to invoke a systemic antitumor immune response, leading to protection against tumor recurrence. For aggressive cancers such as melanoma, where chemotherapy and radiotherapy are ineffective, immunomodulating PDT as an adjuvant to surgery is of interest. Towards the development of specialized photosensitizers (PSs) for treating pigmented melanomas, nine new near-infrared (NIR) absorbing PSs based on a Ru(ii) tris-heteroleptic scaffold [Ru(NNN)(NN)(L)]Cl n , were explored. Compounds 2, 6, and 9 exhibited high potency toward melanoma cells, with visible EC50 values as low as 0.292-0.602 µM and PIs as high as 156-360. Single-micromolar phototoxicity was obtained with NIR-light (733 nm) with PIs up to 71. The common feature of these lead NIR PSs was an accessible low-energy triplet intraligand (3IL) excited state for high singlet oxygen (1O2) quantum yields (69-93%), which was only possible when the photosensitizing 3IL states were lower in energy than the lowest triplet metal-to-ligand charge transfer (3MLCT) excited states that typically govern Ru(ii) polypyridyl photophysics. PDT treatment with 2 elicited a pro-inflammatory response alongside immunogenic cell death in mouse B16F10 melanoma cells and proved safe for in vivo administration (maximum tolerated dose = 50 mg kg-1). Female and male mice vaccinated with B16F10 cells that were PDT-treated with 2 and challenged with live B16F10 cells exhibited 80 and 55% protection from tumor growth, respectively, leading to significantly improved survival and excellent hazard ratios of ≤0.2.

Discovery of immunogenic cell death-inducing ruthenium-based photosensitizers for anticancer photodynamic therapy.

We report a new class of ruthenium (Ru)-based photosensitizers that induce potent cytotoxicity in melanoma cells following activation with NIR light. In addition to the direct cytotoxic effect, this Ru-based photodynamic therapy induces immunogenic cell death in melanoma cells that can be therapeutically exploited to establish protective antitumor immunity.

ALDH1A3-regulated long non-coding RNA NRAD1 is a potential novel target for triple-negative breast tumors and cancer stem cells.

To discover novel therapeutic targets for triple-negative breast cancer (TNBC) and cancer stem cells (CSCs), we screened long non-coding RNAs (lncRNAs) most enriched in TNBCs for high expression in CSCs defined by high Aldefluor activity and associated with worse patient outcomes. This led to the identification of non-coding RNA in the aldehyde dehydrogenase 1 A pathway (NRAD1), also known as LINC00284. Targeting NRAD1 in TNBC tumors using antisense oligonucleotides reduced cell survival, tumor growth, and the number of cells with CSC characteristics. Expression of NRAD1 is regulated by an enzyme that causes Aldefluor activity in CSCs, aldehyde dehydrogenase 1A3 (ALDH1A3) and its product retinoic acid. Cellular fractionation revealed that NRAD1 is primarily nuclear localized, which suggested a potential function in gene regulation. This was confirmed by transcriptome profiling and chromatin isolation by RNA purification, followed by sequencing (ChIRP-seq), which demonstrated that NRAD1 has enriched chromatin interactions among the genes it regulates. Gene Ontology enrichment analysis revealed that NRAD1 regulates expression of genes involved in differentiation and catabolic processes. NRAD1 also contributes to gene expression changes induced by ALDH1A3; thereby, the induction of NRAD1 is a novel mechanism through which ALDH1A3 regulates gene expression. Together, these data identify lncRNA NRAD1 as a downstream effector of ALDH1A3, and a target for TNBCs and CSCs, with functions in cell survival and regulation of gene expression.

Enhancing Mass Spectrometry-Based MHC-I Peptide Identification Through a Targeted Database Search Approach.

MHC-bound peptide ligands dictate the activation and specificity of CD8+ T- cells-based and thus are important for devising T-cell immunotherapies. In recent times, advances in mass spectrometry (MS) have enabled the precise identification of these peptides, wherein MS/MS spectra are compared against a reference proteome. Unfortunately, matching immunopeptide MS/MS to reference proteome databases is hindered by inflated search spaces attributed to the number of matches that need to be considered due to a lack of enzyme restriction. These large search spaces limit the efficiency with which MHC-I peptides are identified. Here we offer a solution to this problem whereby we describe a targeted database search approach and accompanying tool SpectMHC that is based on a priori predicted MHC-I peptides (Murphy et al., J Proteome Res 16:1806-1816, 2017).

Inhibition of Pyruvate Dehydrogenase Kinase Enhances the Antitumor Efficacy of Oncolytic Reovirus.

Oncolytic viruses (OV) such as reovirus preferentially infect and kill cancer cells. Thus, the mechanisms that dictate the susceptibility of cancer cells to OV-induced cytotoxicity hold the key to their success in clinics. Here, we investigated whether cancer cell metabolism defines its susceptibility to OV and if OV-induced metabolic perturbations can be therapeutically targeted. Using mass spectrometry-based metabolomics and extracellular flux analysis on a panel of cancer cell lines with varying degrees of susceptibility to reovirus, we found that OV-induced changes in central energy metabolism, pyruvate metabolism, and oxidative stress correlate with their susceptibility to reovirus. In particular, reovirus infection accentuated Warburg-like metabolic perturbations in cell lines relatively resistant to oncolysis. These metabolic changes were facilitated by oxidative stress-induced inhibitory phosphorylation of pyruvate dehydrogenase (PDH) that impaired the routing of pyruvate into the tricarboxylic acid cycle and established a metabolic state unsupportive of OV replication. From the therapeutic perspective, reactivation of PDH in cancer cells that were weakly sensitive for reovirus, either through PDH kinase (PDK) inhibitors dichloroacetate and AZD7545 or short hairpin RNA-specific depletion of PDK1, enhanced the efficacy of reovirus-induced oncolysis in vitro and in vivo. These findings identify targeted metabolic reprogramming as a possible combination strategy to enhance the antitumor effects of OV in clinics. SIGNIFICANCE: This study proposes targeted metabolic reprogramming as a valid combinatorial strategy to enhance the translational efficacy of oncolytic virus-based cancer therapies.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/15/3824/F1.large.jpg.

Therapy-Induced MHC I Ligands Shape Neo-Antitumor CD8 T Cell Responses during Oncolytic Virus-Based Cancer Immunotherapy.

Oncolytic viruses (OVs), known for their cancer-killing characteristics, also overturn tumor-associated defects in antigen presentation through the MHC class I pathway and induce protective neo-antitumor CD8 T cell responses. Nonetheless, whether OVs shape the tumor MHC-I ligandome remains unknown. Here, we investigated if an OV induces the presentation of novel MHC I-bound tumor antigens (termed tumor MHC-I ligands). Using comparative mass spectrometry (MS)-based MHC-I ligandomics, we determined differential tumor MHC-I ligand expression following treatment with oncolytic reovirus in a murine ovarian cancer model. In vitro, we found that reovirus changes the tumor ligandome of cancer cells. Concurrent multiplexed quantitative proteomics revealed that the reovirus-induced changes in tumor MHC-I ligand presentation were mostly independent of their source proteins. In an in vivo model, tumor MHC-I ligands induced by reovirus were detectable not only in tumor tissues but also the spleens (a source of antigen-presenting cells) of tumor-bearing mice. Most importantly, therapy-induced MHC-I ligands stimulated antigen-specific IFNγ responses in antitumor CD8 T cells from mice treated with reovirus. These data show that therapy-induced MHC-I ligands may shape underlying neo-antitumor CD8 T cell responses. As such, they should be considered in strategies promoting the efficacy of OV-based cancer immunotherapies.

Improving MHC-I Ligand Identifications from LC-MS/MS Data by Incorporating Allelic Peptide Motifs.

MHC class I (MHC-I)-bound ligands play a pivotal role in CD8 T cell immunity and are hence of major interest in understanding and designing immunotherapies. One of the most commonly utilized approaches for detecting MHC ligands is LC-MS/MS. Unfortunately, the effectiveness of current algorithms to identify MHC ligands from LC-MS/MS data is limited because the search algorithms used were originally developed for proteomics approaches detecting tryptic peptides. Consequently, the analysis often results in inflated false discovery rate (FDR) statistics and an overall decrease in the number of peptides that pass FDR filters. Andreatta et al. describe a new scoring tool (MS-rescue) for peptides from MHC-I immunopeptidome datasets. MS-rescue incorporates the existence of MHC-I peptide motifs to rescore peptides from ligandome data. The tool is demonstrated here using peptides assigned from LC-MS/MS data with PEAKs software but can be deployed on data from any search algorithm. This new approach increased the number of peptides identified by up to 20-30% and promises to aid the discovery of novel MHC-I ligands with immunotherapeutic potential.

Multiplexed Relative Quantitation with Isobaric Tagging Mass Spectrometry Reveals Class I Major Histocompatibility Complex Ligand Dynamics in Response to Doxorubicin.

MHC-I peptides are intracellular-cleaved peptides, usually 8-11 amino acids in length, which are presented on the cell surface and facilitate CD8+ T cell responses. Despite the appreciation of CD8+ T-cell antitumor immune responses toward improvement in patient outcomes, the MHC-I peptide ligands that facilitate the response are poorly described. Along these same lines, although many therapies have been recognized for their ability to reinvigorate antitumor CD8+ T-cell responses, whether these therapies alter the MHC-I peptide repertoire has not been fully assessed due to the lack of quantitative strategies. We develop a multiplexing platform for screening therapy-induced MHC-I ligands by employing tandem mass tags (TMTs). We applied this approach to measuring responses to doxorubicin, which is known to promote antitumor CD8+ T-cell responses during its therapeutic administration in cancer patients. Using both in vitro and in vivo systems, we show successful relative quantitation of MHC-I ligands using TMT-based multiplexing and demonstrate that doxorubicin induces MHC-I peptide ligands that are largely derived from mitotic progression and cell-cycle proteins. This high-throughput MHC-I ligand discovery approach may enable further explorations to understand how small molecules and other therapies alter MHC-I ligand presentation that may be harnessed for CD8+ T-cell-based immunotherapies.