bioRxiv preprint doi: https://doi.org/10.1101/329664; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. MTAP loss correlates with an immunosuppressive profile in GBM and its substrate MTA stimulates alternative macrophage polarization Landon J. Hansen1,2,3, Rui Yang1,2, Karolina Woroniecka1,2, Lee Chen1,2, Hai Yan1,2, Yiping He 1,2,* From the 1The Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC, USA; 2Department of Pathology, Duke University Medical Center, Durham, NC, USA; 3Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA; 4Department of Neurosurgery, Duke University Medical Center, Durham, NC, USA *Corresponding Author: Yiping He, PhD, 203 Research Drive, Medical Science Research Building 1, Room 199A Durham, NC, USA 27710 Phone: (919) 684-4760 E-mail: [email protected] Keywords: MTAP, GBM, macrophages, M2, MTA, adenosine ABSTRACT INTRODUCTION Glioblastoma (GBM) is a lethal brain cancer known for Immunotherapy possesses enormous potential for treating its potent immunosuppressive effects. Loss of cancer and has reshaped the way we understand and treat Methylthioadenosine Phosphorylase (MTAP) expression, certain cancer types (1,2). Despite recent progress, via gene deletion or epigenetic silencing, is one of the however, the promise of immunotherapy-based most common alterations in GBM. Here, we show that approaches for treating brain tumors, in particular high MTAP loss in GBM cells is correlated with differential grade glioblastoma (GBM), remains to be fully realized expression of immune regulatory genes. In silico analysis (3-5). GBM is the most common and lethal brain tumor, of gene expression profiles in GBM samples revealed that with a dismal median survival of 12-15 months from the low MTAP expression is correlated with reduced time of diagnosis (6). It is also a cancer characterized by proportions of γδT cells, fewer activated CD4 cells, and its immune-suppressive nature. It has been well- an increased proportion of M2 macrophages. Using in established that GBM cells actively employ multiple vitro macrophage models, we found that strategies to escape immune surveillance and to create an methylthioadenosine (MTA), the metabolite that immunosuppressive microenvironment (7-9). As such, to accumulates as a result of MTAP loss in GBM cells, fully harness the power of immunotherapy for GBM promotes the immunosuppressive alternative activation requires better understanding and more effective (M2) of macrophages. We show that this effect of MTA strategies for countering the tumors’ immunosuppressive on macrophages is independent of IL4/IL3 signaling, is effects. mediated by the adenosine A2B receptor, and can be Recent genomic studies have provided insights pharmacologically reversed. This study suggests that into the molecular mechanisms of GBM pathogenesis, MTAP loss in GBM cells contributes to the revealing the most commonly mutated genes in tumor immunosuppressive microenvironment, and that MTAP cells (10,11). Gliomas can be classified based on their status should be a factor for consideration in genetic alterations and gene expression profiles into understanding GBM immune states and devising subtypes that predict tumor characteristics and patient immunotherapy-based approaches for treating MTAP- prognosis (12). Further studies, in both glioma and other null GBM. types of cancer, have associated genetic alterations with tumor cells’ evasion of immune surveillance and manipulation of the immune microenvironment, bioRxiv preprint doi: https://doi.org/10.1101/329664; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. providing new rationale for tailoring treatment based on distinct from the actions of adenosine. Finally, we show cancer cells’ genetic composition (13-15). As an example, that the MTA-induced alternative macrophage activation recent discoveries have linked IDH1 mutations in glioma can be pharmacologically reversed. These results provide to immune evasion through interference with immune a basis for blocking adenosine A2B receptor signaling as a activation pathways, providing new opportunities for strategy to potentiate immunotherapy targeting MTAP- devising highly specific immunological treatments (16- null GBMs. 19). Thus, identifying additional cancer-specific mutations that confer a similar immune escape/ RESULTS suppression-based advantage to glioma cells will likely MTAP loss in GBM cells is associated with an lead to new rationale for immunotherapeutic designs. immunosuppressive gene expression profile One of the most common genetic alterations in We established patient-derived GBM cell lines, GBM, occurring in approximately 50% of all cases, is the verified MTAP status, and used the affymetrix gene homozygous deletion or epigenetic silencing of expression microarray to characterize their global gene methylthioadenosine phosphorylase (MTAP) (10,20). We expression profile (L. Hansen, et al., submitted). Analysis have recently demonstrated that MTAP deletion is of differentially regulated genes (unpaired ANOVA associated with increased tumorigenesis and with comparing MTAP WT vs MTAP null cell lines) revealed shortened disease-free survival in GBM patients (Hansen, a list of downregulated inflammatory pathways, including et al, submitted). MTAP is a metabolic enzyme that the KEGG pathway of antigen processing and functions in the salvage pathway of adenine and presentation (Fig. 1A). One group of genes common to all methionine, and loss of MTAP results in the accumulation these pathways are the human leukocyte antigen (HLA) of its direct metabolite substrate, methylthioadenosine genes, which were found to be consistently (MTA), in both intracellular and environmental downregulated in tumor cells that lack MTAP (Fig 1B). compartments (21-23). MTA is known to be functionally We compiled a gene set with all HLA genes included in active within cells as an inhibitor of methyltransferases the gene expression microarray (Supporting data Table 2) (22,23). This metabolite has also been shown to suppress and performed gene set enrichment analysis (GSEA) as cell proliferation via different, cellular context-dependent previously described ( 32) to examine the correlation of mechanisms, including targeting the Akt signal pathway this set of genes with MTAP status. GSEA confirmed that and interfering with intracellular protein methylation in T MTAP null cells displayed markedly lower expression of cells (24), or acting through adenosine receptors on the HLA genes, including HLA Class I and both α and β cell surface of melanoma cell lines (25). Studies on chains of HLA class II (Fig. 1C). Consistent with our mechanism of pathogen-induced host inflammatory finding of differentially expressed inflammatory responses have linked MTA to downregulation of TNFα pathways, GSEA using gene sets of inflammatory production by macrophages through engaging adenosine cytokines showed that MTAP null cells had reduced receptors (26), and have revealed a role of MTA in expression of inflammatory cytokines compared to MTAP controlling host inflammation response, such that MTA WT cells (Fig. 1D). Examination of individual genes has been used as an immunosuppressive drug for treating showed that while several regulators of the adaptive colitis, liver inflammation, brain inflammation and immune response were affected (IL2, CTLA4, and CD44), autoimmunity in animal models (27-29). Lastly, MTA has most differentially expressed genes were associated with been shown to be elevated in the blood of septic patients the innate immune response, including those that regulate (30) and in the urine of children with severe combined monocyte/macrophage proliferation, differentiation and immunodeficiency (SCID) (31), further supporting its activation, such as CSF1, IL13, IL34, IL37, ALOX5, and role in regulating the immune response. toll-like receptor related genes, TLR4, TIRAP, and LY96 In this study, we investigated the link between (Fig. 1E). Collectively, these results illustrate that MTAP MTAP loss in GBM cells and the GBM expression correlates with numerous immune-regulatory microenvironment. We show that MTAP expression genes in a manner suggesting that loss or reduction of correlates with the expression of genes regulating innate MTAP expression contributes to the immune-suppressive or adaptive immune response in both experimental cell nature of GBM cells. models and in GBM samples. We reveal that in GBM tissues, low expression level of MTAP is associated with Loss of MTAP expression is associated with an altered immune cell populations indicative of a more immunosuppressive molecular profile in GBM immunosuppressive context. In particular, we provide To evaluate the relevance of the above in vitro evidence that MTAP loss-induced MTA accumulation findings to GBM, we took advantage of The Cancer stimulates M2 alternative macrophage activation. We Genome Atlas (TCGA) large gene expression dataset illustrate that this effect of MTA on macrophages is (33,34) to test the link between MTAP expression and the independent of IL4/IL3 signaling, is mediated by the immune status of GBM tumor samples. First, we analyzed adenosine A2B receptor and STAT3 signaling, and is expression of individual