Lineage commitment pathways epigenetically oppose oncogenic Gαq/11-YAP signaling in dormant disseminated uveal melanoma.

The mechanisms driving late relapse in uveal melanoma (UM) patients remains a medical mystery and major challenge. Clinically it is inferred that UM disseminated cancer cells (DCCs) persist asymptomatic for years-to-decades mainly in the liver before they manifest as symptomatic metastasis. Here we reveal using Gαq/11 mut /BAP wt human uveal melanoma models and human UM metastatic samples, that the neural crest lineage commitment nuclear receptor NR2F1 is a key regulator of spontaneous UM DCC dormancy in the liver. Using a quiescence reporter, RNA-seq and multiplex imaging we revealed that rare dormant UM DCCs upregulate NR2F1 expression and genes related to neural crest programs while repressing gene related to cell cycle progression. Gain and loss of function assays showed that NR2F1 silences YAP1/TEAD1 transcription downstream of Gαq/11 signaling and that NR2F1 expression can also be repressed by YAP1. YAP1 expression is repressed by NR2F1 binding to its promoter and changing the histone H3 tail activation marks to repress YAP1 transcription. In vivo CRISPR KO of NR2F1 led dormant UM DCCs to awaken and initiate relentless liver metastatic growth. Cut&Run and bulk RNA sequencing further confirmed that NR2F1 epigenetically stimulates neuron axon guidance and neural lineage programs, and it globally represses gene expression linked to G-protein signaling to drive dormancy. Pharmacological inhibition of Gαq/11 mut signaling resulted in NR2F1 upregulation and robust UM growth arrest, which was also achieved using a novel NR2F1 agonist. Our work sheds light on the molecular underpinnings of UM dormancy revealing that transcriptional programs driven by NR2F1 epigenetically short-circuit Gαq/11 signaling to its downstream target YAP1. Highlights: Quiescent solitary uveal melanoma (UM) DCCs in the liver up- and down-regulate neural crest and cell cycle progression programs, respectively.NR2F1 drives solitary UM DCC dormancy by antagonizing the Gαq/11-YAP1 pathway; small molecule Gαq/11 inhibition restores NR2F1 expression and quiescence. NR2F1 short-circuits oncogenic YAP1 and G-protein signaling via a chromatin remodeling program. Loss of function of NR2F1 in dormant UM DCCs leads to aggressive liver metastasis.

SOX10 Loss Sensitizes Melanoma Cells to Cytokine-Mediated Inflammatory Cell Death.

The transcription factor, SOX10, plays an important role in the differentiation of neural crest precursors to the melanocytic lineage. Malignant transformation of melanocytes leads to the development of melanoma, and SOX10 promotes melanoma cell proliferation and tumor formation. SOX10 expression in melanomas is heterogeneous, and loss of SOX10 causes a phenotypic switch toward an invasive, mesenchymal-like cell state and therapy resistance; hence, strategies to target SOX10-deficient cells are an active area of investigation. The impact of cell state and SOX10 expression on antitumor immunity is not well understood but will likely have important implications for immunotherapeutic interventions. To this end, we tested whether SOX10 status affects the response to CD8+ T cell-mediated killing and T cell-secreted cytokines, TNFα and IFNγ, which are critical effectors in the cytotoxic killing of cancer cells. We observed that genetic ablation of SOX10 rendered melanoma cells more sensitive to CD8+ T cell-mediated killing and cell death induction by either TNFα or IFNγ. Cytokine-mediated cell death in SOX10-deficient cells was associated with features of caspase-dependent pyroptosis, an inflammatory form of cell death that has the potential to increase immune responses. IMPLICATIONS: These data support a role for SOX10 expression altering the response to T cell-mediated cell death and contribute to a broader understanding of the interaction between immune cells and melanoma cells.

Kinome profiling identifies MARK3 and STK10 as potential therapeutic targets in uveal melanoma.

Most uveal melanoma cases harbor activating mutations in either GNAQ or GNA11. Despite activation of the mitogen-activated protein kinase (MAPK) signaling pathway downstream of Gαq/11, there are no effective targeted kinase therapies for metastatic uveal melanoma. The human genome encodes numerous understudied kinases, also called the "dark kinome". Identifying additional kinases regulated by Gαq/11 may uncover novel therapeutic targets for uveal melanoma. In this study, we treated GNAQ-mutant uveal melanoma cell lines with a Gαq/11 inhibitor, YM-254890, and conducted a kinase signaling proteomic screen using multiplexed-kinase inhibitors followed by mass spectrometry. We observed downregulated expression and/or activity of 22 kinases. A custom siRNA screen targeting these kinases demonstrated that knockdown of microtubule affinity regulating kinase 3 (MARK3) and serine/threonine kinase 10 (STK10) significantly reduced uveal melanoma cell growth and decreased expression of cell cycle proteins. Additionally, knockdown of MARK3 but not STK10 decreased ERK1/2 phosphorylation. Analysis of RNA-sequencing and proteomic data showed that Gαq signaling regulates STK10 expression and MARK3 activity. Our findings suggest an involvement of STK10 and MARK3 in the Gαq/11 oncogenic pathway and prompt further investigation into the specific roles and targeting potential of these kinases in uveal melanoma.

Gene signature reveals decreased SOX10-dependent transcripts in malignant cells from immune checkpoint inhibitor-resistant cutaneous melanomas.

Evidence is mounting for cross-resistance between immune checkpoint and targeted kinase inhibitor therapies in cutaneous melanoma patients. Since the loss of the transcription factor, SOX10, causes tolerance to MAPK pathway inhibitors, we used bioinformatic techniques to determine if reduced SOX10 expression/activity is associated with immune checkpoint inhibitor resistance. We integrated SOX10 ChIP-seq, knockout RNA-seq, and knockdown ATAC-seq data from melanoma cell models to develop a robust SOX10 gene signature. We used computational methods to validate this signature as a measure of SOX10-dependent activity in independent single-cell and bulk RNA-seq SOX10 knockdown, cell line panel, and MAPK inhibitor drug-resistant datasets. Evaluation of patient single-cell RNA-seq data revealed lower levels of SOX10-dependent transcripts in immune checkpoint inhibitor-resistant tumors. Our results suggest that SOX10-deficient melanoma cells are associated with cross-resistance between targeted and immune checkpoint inhibitors and highlight the need to identify therapeutic strategies that target this subpopulation.

Co-Targeting FASN and mTOR Suppresses Uveal Melanoma Growth.

Uveal melanoma (UM) displays a high frequency of metastasis; however, effective therapies for metastatic UM are limited. Identifying unique metabolic features of UM may provide a potential targeting strategy. A lipid metabolism protein expression signature was induced in a normal choroidal melanocyte (NCM) line transduced with GNAQ (Q209L), a driver in UM growth and development. Consistently, UM cells expressed elevated levels of fatty acid synthase (FASN) compared to NCMs. FASN upregulation was associated with increased mammalian target of rapamycin (mTOR) activation and sterol regulatory element-binding protein 1 (SREBP1) levels. FASN and mTOR inhibitors alone significantly reduced UM cell growth. Concurrent inhibition of FASN and mTOR further reduced UM cell growth by promoting cell cycle arrest and inhibiting glucose utilization, TCA cycle metabolism, and de novo fatty acid biosynthesis. Our findings indicate that FASN is important for UM cell growth and co-inhibition of FASN and mTOR signaling may be considered for treatment of UM.

A Genome-Wide Screen Identifies PDPK1 as a Target to Enhance the Efficacy of MEK1/2 Inhibitors in NRAS Mutant Melanoma.

Melanomas frequently harbor activating NRAS mutations. However, limited advance has been made in developing targeted therapy options for patients with NRAS mutant melanoma. MEK inhibitors (MEKi) show modest efficacy in the clinic and their actions need to be optimized. In this study, we performed a genome-wide CRISPR-Cas9-based screen and demonstrated that loss of phosphoinositide-dependent kinase-1 (PDPK1) enhances the efficacy of MEKi. The synergistic effects of PDPK1 loss and MEKi was validated in NRAS mutant melanoma cell lines using pharmacologic and molecular approaches. Combined PDPK1 inhibitors (PDPK1i) with MEKi suppressed NRAS mutant xenograft growth and induced gasdermin E-associated pyroptosis. In an immune-competent allograft model, PDPK1i+MEKi increased the ratio of intratumoral CD8+ T cells, delayed tumor growth, and prolonged survival; the combination treatment was less effective against tumors in immune-deficient mice. These data suggest PDPK1i+MEKi as an efficient immunostimulatory strategy against NRAS mutant melanoma. SIGNIFICANCE: Targeting PDPK1 stimulates antitumor immunity and sensitizes NRAS mutant melanoma to MEK inhibition, providing rationale for the clinical development of a combinatorial approach for treating patients with melanoma.

Multi-omics Profiling Shows BAP1 Loss Is Associated with Upregulated Cell Adhesion Molecules in Uveal Melanoma.

BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene that is mutated in cancer, including uveal melanoma. Loss-of-function BAP1 mutations are associated with uveal melanoma metastasis and poor prognosis, but the mechanisms underlying these effects remain unclear. Upregulation of cell-cell adhesion proteins is involved with collective migration and metastatic seeding of cancer cells. Here, we show that BAP1 loss in uveal melanoma patient samples is associated with upregulated gene expression of multiple cell adhesion molecules (CAM), including E-cadherin (CDH1), cell adhesion molecule 1 (CADM1), and syndecan-2 (SDC2). Similar findings were observed in uveal melanoma cell lines and single-cell RNA-sequencing data from uveal melanoma patient samples. BAP1 reexpression in uveal melanoma cells reduced E-cadherin and CADM1 levels. Functionally, knockdown of E-cadherin decreased spheroid cluster formation and knockdown of CADM1 decreased growth of BAP1-mutant uveal melanoma cells. Together, our findings demonstrate that BAP1 regulates the expression of CAMs which may regulate metastatic traits. IMPLICATIONS: BAP1 mutations and increased metastasis may be due to upregulation of CAMs.

Targeting SOX10-deficient cells to reduce the dormant-invasive phenotype state in melanoma.

Cellular plasticity contributes to intra-tumoral heterogeneity and phenotype switching, which enable adaptation to metastatic microenvironments and resistance to therapies. Mechanisms underlying tumor cell plasticity remain poorly understood. SOX10, a neural crest lineage transcription factor, is heterogeneously expressed in melanomas. Loss of SOX10 reduces proliferation, leads to invasive properties, including the expression of mesenchymal genes and extracellular matrix, and promotes tolerance to BRAF and/or MEK inhibitors. We identify the class of cellular inhibitor of apoptosis protein-1/2 (cIAP1/2) inhibitors as inducing cell death selectively in SOX10-deficient cells. Targeted therapy selects for SOX10 knockout cells underscoring their drug tolerant properties. Combining cIAP1/2 inhibitor with BRAF/MEK inhibitors delays the onset of acquired resistance in melanomas in vivo. These data suggest that SOX10 mediates phenotypic switching in cutaneous melanoma to produce a targeted inhibitor tolerant state that is likely a prelude to the acquisition of resistance. Furthermore, we provide a therapeutic strategy to selectively eliminate SOX10-deficient cells.

Pyruvate dehydrogenase inactivation causes glycolytic phenotype in BAP1 mutant uveal melanoma.

Effective therapeutic options are still lacking for uveal melanoma (UM) patients who develop metastasis. Metastatic traits of UM are linked to BRCA1-associated protein 1 (BAP1) mutations. Cell metabolism is re-programmed in UM with BAP1 mutant UM, but the underlying mechanisms and opportunities for therapeutic intervention remain unclear. BAP1 mutant UM tumors have an elevated glycolytic gene expression signature, with increased expression of pyruvate dehydrogenase (PDH) complex and PDH kinase (PDHK1). Furthermore, BAP1 mutant UM cells showed higher levels of phosphorylated PDHK1 and PDH that was associated with an upregulated glycolytic profile compared to BAP1 wild-type UM cells. Suppressing PDHK1-PDH phosphorylation decreased glycolytic capacity and cell growth, and induced cell cycle arrest of BAP1 mutant UM cells. Our results suggest that PDHK1-PDH phosphorylation is a causative factor of glycolytic phenotypes found in BAP1 mutant UM and propose a therapeutic opportunity for BAP1 mutant UM patients.

The AMP-dependent kinase pathway is upregulated in BAP1 mutant uveal melanoma.

Metastatic uveal melanoma (UM) responds poorly to targeted therapies and immune checkpoint inhibitors. Loss of BRCA1-associated protein 1 (BAP1) via inactivating mutations in the BAP1 gene is associated with UM progression. Thus, molecular alterations caused by BAP1 dysfunction may be novel therapeutic targets for metastatic UM. Here, we found that phosphorylation of AMP-dependent kinase (AMPK) was elevated in BAP1-altered (or mutant) compared to BAP1-unaltered (or wild-type [WT]) UM tumors. As a readout of AMPK pathway activation, phosphorylation of an AMPK downstream effector, acetyl-CoA-carboxylase (ACC), was also elevated. BAP1 re-expression in BAP1-null UM cell lines decreased phospho-AMPK (pAMPK) and phospho-ACC (pACC) levels. AMPK phosphorylation is mediated by calcium/calmodulin dependent protein kinase kinase 2 (CaMKK2) and potentially liver kinase B1 (LKB1) in BAP1 mutant UM cells. Knockdown of AMPKα1/2 reduced the viability of BAP1 mutant UM cells, indicating a survival function of AMPK in BAP1 mutant UM. Our data suggest that the AMPK pathway is an important mechanism mediating the survival of BAP1 mutant UM. Targeting the AMPK pathway may be a novel therapeutic strategy for metastatic UM.

SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects.

Developmental factors may regulate the expression of immune modulatory proteins in cancer, linking embryonic development and cancer cell immune evasion. This is particularly relevant in melanoma because immune checkpoint inhibitors are commonly used in the clinic. SRY-box transcription factor 10 (SOX10) mediates neural crest development and is required for melanoma cell growth. In this study, we investigate immune-related targets of SOX10 and observe positive regulation of herpesvirus entry mediator (HVEM) and carcinoembryonic-antigen cell-adhesion molecule 1 (CEACAM1). Sox10 knockout reduces tumor growth in vivo, and this effect is exacerbated in immune-competent models. Modulation of CEACAM1 expression but not HVEM elicits modest effects on tumor growth. Importantly, Sox10 knockout effects on tumor growth are dependent, in part, on CD8+ T cells. Extending this analysis to samples from patients with cutaneous melanoma, we observe a negative correlation with SOX10 and immune-related pathways. These data demonstrate a role for SOX10 in regulating immune checkpoint protein expression and anti-tumor immunity in melanoma.

Roles of the BAP1 Tumor Suppressor in Cell Metabolism.

BRCA1-associated protein 1 (BAP1) is emerging as an intensively studied cancer-associated gene. Germline mutations in BAP1 lead to a cancer syndrome, and somatic loss is found in several cancer types. BAP1 encodes a deubiquitinase enzyme, which plays key roles in cell-cycle regulation, cell death, and differentiation. Recent studies have demonstrated that BAP1 is also involved in several aspects of cellular metabolism, including metabolic homeostasis, glucose utilization, control of ferroptosis, and stress response. A better knowledge of the metabolic roles of cancer-associated genes is important to understanding tumor initiation and progression, as well as highlighting potential therapeutic avenues. With this review, we summarize the current knowledge regarding BAP1-mediated regulation of metabolic activities that may support new strategies to treat BAP1-mutated cancers.

BAP1 mutant uveal melanoma is stratified by metabolic phenotypes with distinct vulnerability to metabolic inhibitors.

Cancer cell metabolism is a targetable vulnerability; however, a precise understanding of metabolic heterogeneity is required. Inactivating mutations in BRCA1-associated protein 1 (BAP1) are associated with metastasis in uveal melanoma (UM), the deadliest adult eye cancer. BAP1 functions in UM remain unclear. UM patient sample analysis divided BAP1 mutant UM tumors into two subgroups based on oxidative phosphorylation (OXPHOS) gene expression suggesting metabolic heterogeneity. Consistent with patient data, transcriptomic analysis of BAP1 mutant UM cell lines also showed OXPHOShigh or OXPHOSlow subgroups. Integrated RNA sequencing, metabolomics, and molecular analyses showed that OXPHOShigh BAP1 mutant UM cells utilize glycolytic and nucleotide biosynthesis pathways, whereas OXPHOSlow BAP1 mutant UM cells employ fatty acid oxidation. Furthermore, the two subgroups responded to different classes of metabolic suppressors. Our findings indicate that targeting cancer metabolism is a promising therapeutic option for BAP1 mutant UM; however, tailored approaches may be required due to metabolic heterogeneities.

Metabolic Adaptations to MEK and CDK4/6 Cotargeting in Uveal Melanoma.

Frequent GNAQ and GNA11 mutations in uveal melanoma hyperactivate the MEK-ERK signaling pathway, leading to aberrant regulation of cyclin-dependent kinases (CDK) and cell-cycle progression. MEK inhibitors (MEKi) alone show poor efficacy in uveal melanoma, raising the question of whether downstream targets can be vertically inhibited to provide long-term benefit. CDK4/6 selective inhibitors are FDA-approved in patients with estrogen receptor (ER)-positive breast cancer in combination with ER antagonists/aromatase inhibitors. We determined the effects of MEKi plus CDK4/6 inhibitors (CDK4/6i) in uveal melanoma. In vitro, palbociclib, a CDK4/6i, enhanced the effects of MEKi via downregulation of cell-cycle proteins. In contrast, in vivo CDK4/6 inhibition alone led to cytostasis and was as effective as MEKi plus CDK4/6i treatment at delaying tumor growth. RNA sequencing revealed upregulation of the oxidative phosphorylation (OxPhos) pathway in both MEKi-resistant tumors and CDK4/6i-tolerant tumors. Furthermore, oxygen consumption rate was increased following MEKi + CDK4/6i treatment. IACS-010759, an OxPhos inhibitor, decreased uveal melanoma cell survival in combination with MEKi + CDK4/6i. These data highlight adaptive upregulation of OxPhos in response to MEKi + CDK4/6i treatment in uveal melanoma and suggest that suppression of this metabolic state may improve the efficacy of MEKi plus CDK4/6i combinations.

Targeting PHGDH Upregulation Reduces Glutathione Levels and Resensitizes Resistant NRAS-Mutant Melanoma to MAPK Kinase Inhibition.

Melanomas frequently harbor activating NRAS mutations leading to activation of MAPK kinase (MEK) and extracellular signal-regulated kinase 1/2 signaling; however, the clinical efficacy of inhibitors to this pathway is limited by resistance. Tumors rewire metabolic pathways in response to stress signals such as targeted inhibitors and drug resistance, but most therapy-resistant preclinical models are generated in conditions that lack physiological metabolism. We generated human NRAS-mutant melanoma xenografts that were resistant to the MEK inhibitor (MEKi) PD0325901 in vivo. MEKi-resistant cells showed cross-resistance to the structurally distinct MEKi trametinib and elevated extracellular signal-regulated kinase 1/2 phosphorylation and downstream signaling. Additionally, we observed upregulation of the serine synthesis pathway and PHGDH, a key enzyme in this pathway. Suppressing PHGDH in MEKi-resistant cells together with MEKi treatment decreased oxidative stress tolerance and cell proliferation. Together, our data suggest targeting PHGDH as a potential strategy in overcoming MEKi resistance.

FOXD3 Regulates VISTA Expression in Melanoma.

Immune checkpoint inhibitors have improved patient survival in melanoma, but the innate resistance of many patients necessitates the investigation of alternative immune targets. Many immune checkpoint proteins lack proper characterization, including V-domain Ig suppressor of T cell activation (VISTA). VISTA expression on immune cells can suppress T cell activity; however, few studies have investigated its expression and regulation in cancer cells. In this study, we observe that VISTA is expressed in melanoma patient samples and cell lines. Tumor cell-specific expression of VISTA promotes tumor onset in vivo, associated with increased intratumoral T regulatory cells, and enhanced PDL-1 expression on tumor-infiltrating macrophages. VISTA transcript levels are regulated by the stemness factor Forkhead box D3 (FOXD3). BRAF inhibition upregulates FOXD3 and reduces VISTA expression. Overall, this study demonstrates melanoma cell expression of VISTA and its regulation by FOXD3, contributing to the rationale for therapeutic strategies that combine targeted inhibitors with immune checkpoint blockade.

Targeting BRD/BET proteins inhibits adaptive kinome upregulation and enhances the effects of BRAF/MEK inhibitors in melanoma.

BACKGROUND: BRAF-mutant melanoma patients respond to BRAF inhibitors and MEK inhibitors (BRAFi/MEKi), but drug-tolerant cells persist, which may seed disease progression. Adaptive activation of receptor tyrosine kinases (RTKs) has been associated with melanoma cell drug tolerance following targeted therapy. While co-targeting individual RTKs can enhance the efficacy of BRAFi/MEKi effects, it remains unclear how to broadly target multiple RTKs to achieve more durable tumour growth inhibition. METHODS: The blockage of adaptive RTK responses by the new BET inhibitor (BETi), PLX51107, was measured by RPPA and Western blot. Melanoma growth was evaluated in vitro by colony assay and EdU staining, as well as in skin reconstructs, xenografts and PDX models following BRAFi, MEKi and/or PLX51107 treatment. RESULTS: Treatment with PLX51107 limited BRAFi/MEKi upregulation of ErbB3 and PDGFR-ß expression levels. Similar effects were observed following BRD2/4 depletion. In stage III melanoma patients, expression of BRD2/4 was strongly correlated with ErbB3. PLX51107 enhanced the effects of BRAFi/MEKi on inhibiting melanoma growth in vitro, in human skin reconstructs and in xenografts in vivo. Continuous triple drug combination treatment resulted in significant weight loss in mice, but intermittent BETi combined with continuous BRAFi/MEKi treatment was tolerable and improved durable tumour inhibition outcomes. CONCLUSIONS: Together, our data suggest that intermittent inhibition of BET proteins may improve the duration of responses following BRAFi/MEKi treatment in BRAF-mutant melanoma.

Mutant BRAF and MEK Inhibitors Regulate the Tumor Immune Microenvironment via Pyroptosis.

Combinations of BRAF inhibitors and MEK inhibitors (BRAFi + MEKi) are FDA-approved to treat BRAF V600E/K-mutant melanoma. Efficacy of BRAFi + MEKi associates with cancer cell death and alterations in the tumor immune microenvironment; however, the links are poorly understood. We show that BRAFi + MEKi caused durable melanoma regression in an immune-mediated manner. BRAFi + MEKi treatment promoted cleavage of gasdermin E (GSDME) and release of HMGB1, markers of pyroptotic cell death. GSDME-deficient melanoma showed defective HMGB1 release, reduced tumor-associated T cell and activated dendritic cell infiltrates in response to BRAFi + MEKi, and more frequent tumor regrowth after drug removal. Importantly, BRAFi + MEKi-resistant disease lacked pyroptosis markers and showed decreased intratumoral T-cell infiltration but was sensitive to pyroptosis-inducing chemotherapy. These data implicate BRAFi + MEKi-induced pyroptosis in antitumor immune responses and highlight new therapeutic strategies for resistant melanoma. SIGNIFICANCE: Targeted inhibitors and immune checkpoint agents have advanced the care of patients with melanoma; however, detailed knowledge of the intersection between these two research areas is lacking. We describe a molecular mechanism of targeted inhibitor regulation of an immune-stimulatory form of cell death and provide a proof-of-principle salvage therapy concept for inhibitor-resistant melanoma.See related commentary by Smalley, p. 176.This article is highlighted in the In This Issue feature, p. 161.

Murine Cytomegalovirus Infection of Melanoma Lesions Delays Tumor Growth by Recruiting and Repolarizing Monocytic Phagocytes in the Tumor.

Cytomegalovirus (CMV) is a ubiquitous betaherpesvirus that infects many different cell types. Human CMV (HCMV) has been found in several solid tumors, and it has been hypothesized that it may promote cellular transformation or exacerbate tumor growth. Paradoxically, in some experimental situations, murine CMV (MCMV) infection delays tumor growth. We previously showed that wild-type MCMV delayed the growth of poorly immunogenic B16 melanomas via an undefined mechanism. Here, we show that MCMV delayed the growth of these immunologically "cold" tumors by recruiting and modulating tumor-associated macrophages. Depletion of monocytic phagocytes with clodronate completely prevented MCMV from delaying tumor growth. Mechanistically, our data suggest that MCMV recruits new macrophages to the tumor via the virus-encoded chemokine MCK2, and viruses lacking this chemokine were unable to delay tumor growth. Moreover, MCMV infection of macrophages drove them toward a proinflammatory (M1)-like state. Importantly, adaptive immune responses were also necessary for MCMV to delay tumor growth as the effect was substantially blunted in Rag-deficient animals. However, viral spread was not needed and a spread-defective MCMV strain was equally effective. In most mice, the antitumor effect of MCMV was transient. Although the recruited macrophages persisted, tumor regrowth correlated with a loss of viral activity in the tumor. However, an additional round of MCMV infection further delayed tumor growth, suggesting that tumor growth delay was dependent on active viral infection. Together, our results suggest that MCMV infection delayed the growth of an immunologically cold tumor by recruiting and modulating macrophages in order to promote anti-tumor immune responses.IMPORTANCE Cytomegalovirus (CMV) is an exciting new platform for vaccines and cancer therapy. Although CMV may delay tumor growth in some settings, there is also evidence that CMV may promote cancer development and progression. Thus, defining the impact of CMV on tumors is critical. Using a mouse model of melanoma, we previously found that murine CMV (MCMV) delayed tumor growth and activated tumor-specific immunity although the mechanism was unclear. We now show that MCMV delayed tumor growth through a mechanism that required monocytic phagocytes and a viral chemokine that recruited macrophages to the tumor. Furthermore, MCMV infection altered the functional state of macrophages. Although the effects of MCMV on tumor growth were transient, we found that repeated MCMV injections sustained the antitumor effect, suggesting that active viral infection was needed. Thus, MCMV altered tumor growth by actively recruiting macrophages to the tumor, where they were modulated to promote antitumor immunity.