MYC Overexpression Drives Immune Evasion in Hepatocellular Carcinoma That Is Reversible through Restoration of Proinflammatory Macrophages

Abstract

Cancers evade immune surveillance, which can be reversed through immune-checkpoint therapy in a small subset of cases. Here, we report that the MYC oncogene suppresses innate immune surveillance and drives resistance to immunotherapy. In 33 different human cancers, MYC genomic amplification and overexpression increased immune-checkpoint expression, predicted nonresponsiveness to immune-checkpoint blockade, and was associated with both Th2-like immune profile and reduced CD8 T-cell infiltration. MYC transcriptionally suppressed innate immunity and MHCI-mediated antigen presentation, which in turn impeded T-cell response. Combined, but not individual, blockade of PDL1 and CTLA4 could reverse MYC-driven immune suppression by leading to the recruitment of proinflammatory antigen-presenting macrophages with increased CD40 and MHCII expression. Depletion of macrophages abrogated the antineoplastic effects of PDL1 and CTLA4 blockade in MYC-driven hepatocellular carcinoma (HCC). Hence, MYC is a predictor of immune-checkpoint responsiveness and a key driver of immune evasion through the suppression of proinflammatory macrophages. The immune evasion induced by MYC in HCC can be overcome by combined PDL1 and CTLA4 blockade.

Significance: Macrophage-mediated immune evasion is a therapeutic vulnerability of MYC-driven cancers, which has implications for prioritizing MYC-driven hepatocellular carcinoma for combination immunotherapy.

Figure 1. The MYC Oncogene Drives Human Tumorigenesis Associated with Immune Evasion

A. TumorMap rendering of the Pan-Cancer-Atlas of 33 cancers; tumors are defined by different tissues of origin in the first box. Second box shows the prevalence of MYC genomic amplification. Epithelial tumors with high rates of MYC amplification are annotated. Third box shows the gene expression of a signature derived from MYC amplification. (LAML- Acute Myeloid Leukemia, ACC- Adrenocortical carcinoma, BLCA- Bladder Urothelial Carcinoma, LGG- Brain Lower Grade Glioma, BRCA- Breast invasive carcinoma, CESC- Cervical squamous cell carcinoma and endocervical adenocarcinoma, CHOL- Cholangiocarcinoma, LCML- Chronic Myelogenous Leukemia, COAD- Colon adenocarcinoma, ESCA- Esophageal carcinoma, GBM- Glioblastoma multiforme, HNSC- Head and Neck squamous cell carcinoma, KICH- Kidney Chromophobe, KIRC- Kidney renal clear cell carcinoma, KIRP- Kidney renal papillary cell carcinoma, LIHC- Liver hepatocellular carcinoma, LUAD- Lung adenocarcinoma, LUSC- Lung squamous cell carcinoma, DLBC- Lymphoid Neoplasm Diffuse Large B-cell Lymphoma, MESO- Mesothelioma, OV- Ovarian serous cystadenocarcinoma, PAAD- Pancreatic adenocarcinoma, PCPG- Pheochromocytoma and Paraganglioma, PRAD- Prostate adenocarcinoma, READ- Rectum adenocarcinoma, SARC- Sarcoma, SKCM- Skin Cutaneous Melanoma, STAD- Stomach adenocarcinoma, TGCT- Testicular Germ Cell Tumors, THYM- Thymoma, THCA- Thyroid carcinoma, UCS- Uterine Carcinosarcoma, UCEC- Uterine Corpus Endometrial Carcinoma, UVM- Uveal Melanoma). B. Overall and progression-free survival in patients with MYC amplification and MYC overexpression (top and bottom quartiles). C. Correlation of MYC expression with gene expression of immune checkpoints PDL1 and CTLA4 (top) and with gene signature of immune checkpoint blockade (ICB) (Jiang et al. 2018 (28); bottom). D. Expression of gene signatures associated with T helper cells 1 (Th1), Th17 and Th2 compared between tumors with (1) and without (0) MYC amplification. E. Expression of gene signatures associated with CD8 T cells, NK cells and monocytes compared between tumors with (1) and without (0) MYC amplification. F. Immune deconvolution analysis showing comparison of abundance of different immune subsets between tumors with (1) and without (0) MYC amplification. G. Immunohistochemistry showing CD8 T-cell infiltration in representative HCC tumors from the TCGA cohort with (1, n=20) and without MYC amplification (0, n=8). *p<0.05, ***p<0.0001.

Figure 2:. MYC causes Reversible Transcriptional Repression of Antitumor Immune Responses

A. Heat map shows similarity between MYC-driven murine HCC (MYC-On, n=3 vs. MYC-Off, n=3) and 11 human HCC transcriptome data sets including the TCGA cohort; the top 10 upregulated and downregulated genes with enrichment z-scores across the different data sets are shown. Bar plot at the bottom shows log of p-value of similarity between the individual cohorts and murine MYC-HCC. B. Heatmap shows z-scores of enrichment of canonical pathways and biological functions in murine MYC-HCC transcriptome and the human HCC immune signatures of exhausted PD1+ CD8 T cells(31) and inactivated 41BB-ve PD1+ T cells in human HCC(30). C. Gene signature derived from murine MYC HCC used to classify human HCC in the TCGA cohort to identify MYC-driven human HCCs (created using Biorender.com). D. Human HCCs enriched in mMYC^On signature show MYC molecular pathway activation and strong association with MYC gene amplification. E. Human HCCs with high expression of mMYC^On signature showed enrichment of a gene program of T cell exclusion and resistance to anti-PD1 checkpoint therapy (32), and upregulation of CTLA4. F. PCA plot comparing the transcriptional profile of a transgenic model of HCC overexpressing wild-type MYC (MYC^On, n=16), a mutant MYC defective in Miz1 binding (V394D) (MYC^Vd, n=11) and tumors upon MYC inactivation for a short time point of 16 hours (MYC^Off, n=8)(36) to identify MYC/Miz-1 repressed genes. G. Heatmap shows genes which are reversibly repressed by MYC in the MHC protein complex, complement system and cytokines. H. Immunohistochemistry shows the abundance of macrophages (F4/80+) by day 1 and CD4 T-cell recruitment by day 4 of MYC inactivation in HCC. I. Immune deconvolution analysis showing comparison of abundance of different immune subsets in murine MYC HCC upon MYC inactivation. **p<0.01, ***p<0.0001.

Figure 3.. Combined Anti- PDL1 and Anti-CTLA4 delays tumor progression in MYC-HCC

A. Experimental scheme of MYC-HCC treatment with IgG control (n=5), PDL1 antibody (n=5), CTLA4 antibody (n=5), or their combination (n=5) (created using Biorender.com). B. Weekly MRI showing tumor progression in representative MYC-HCC mice in the 4 treatment groups (n=5 each group). C. Quantification of volumetric tumor measurement using MRI of MYC-HCC mice in the 4 treatment groups (n=5 each group). D. End-of-treatment gross appearance, histology of representative MYC-HCC mice in the 4 treatment groups (n=5 each group). E. Quantification of liver tumor burden at end-of-treatment of MYC-HCC mice in the 4 treatment groups (n=5 each group). **p<0.01, ***p<0.0001

Figure 4.. Safety profile of combined Anti- PDL1 and Anti-CTLA in MYC-HCC

A. Body weight and liver tests at end-of-treatment of MYC-HCC bearing mice treated with either IgG control (n=5) or PDL1 antibody (n=5) or CTLA4 antibody (n=5) or their combination (n=5). B. Peripheral white cell counts at end-of-treatment of MYC-HCC bearing mice treated with either IgG control (n=5) or PDL1 antibody (n=5) or CTLA4 antibody (n=5) or their combination (n=5). C. Histology and CD8 T cell infiltration in normal surrounding liver and colon tissue of mice treated with the 4 treatment groups. *p<0.05, **p<0.01.

Figure 5.. Dual targeting of PDL1 and CTLA4 Restores Macrophage-Mediated Anti-tumor immunity

A. Immunohistochemistry for CD4+ T cells, CD8+ T cells and F4/80+ macrophages in MYC-HCC bearing mice treated with IgG control (n=5), PDL1 antibody (n=5), CTLA4 antibody (n=5), or their combination (n=5). Boxplots show quantification of cell counts. B. Violin plot showing differentially expressed genes between MYC-HCC treated with combination therapy (n=3) versus control IgG (n=3). Macrophage-related genes are upregulated with treatment with anti PDL1+CTLA4. C. Pathway analysis of differentially expressed genes between MYC-HCC treated with combination therapy (n=3) versus control IgG (n=3). D. Enrichment of inflammatory signature of macrophages stimulated with lipopolysaccharide (LPS) in tumors treated with combination therapy (n=3) versus control IgG (n=3). E. Pathways analysis of meta-analysis comparing the transcriptional changes induced by the combination therapy (n=3) to those induced by anti PDL1 (n=3) or CTLA4 monotherapies (n=3).

Figure 6.. Mass cytometry reveals unique early immune response to dual targeting of PDL1 and CTLA4

A. Experimental scheme of MYC-HCC treatment with IgG control (n=3), PDL1 antibody (n=3), CTLA4 antibody (n=2), or their combination (n=4) for 1 week, followed by mass cytometry analysis (created using Biorender.com). B. tSNE plot grouped by major immune subsets identified in the tumor immune microenvironment. C. tSNE plot showing the visual representation of myeloid marker expression between MYC-HCC treated with IgG control (n=3), PDL1 antibody (n=3), CTLA4 antibody (n=2), or their combination (n=4) D. Boxplots shows quantification of CCR2+/Ly6^High M1-Like Macrophages, Ly6C expression in the macrophages, abundance of PDL1+/Ly6C^Low/CCR2^Low M2-Like Macrophages and myeloid derived suppressor cells (MDSCs) in MYC-HCC treated with IgG control (n=3), PDL1 antibody (n=3), CTLA4 antibody (n=2), or their combination (n=4) respectively. E. Boxplots show quantification of CD40 expression in different myeloid subsets in MYC-HCC treated with IgG control (n=3), PDL1 antibody (n=3), CTLA4 antibody (n=2), or their combination (n=4) respectively. F. Boxplots shows quantification of MHCII expression in CD39+ and PDL1+ M2-Like Macrophages in MYC-HCC treated with IgG control (n=3), PDL1 antibody (n=3), CTLA4 antibody (n=2), or their combination (n=4) respectively.

Figure 7.. Macrophages are Essential for the anti-tumor efficacy of Combined Immune Checkpoint Therapy in MYC-HCC

A. Experimental scheme of macrophage depletion in MYC-HCC followed by treatment with either IgG control (n=4) or dual PDL1 and CTLA4 antibodies (n=3) (created using Biorender.com). B. Quantification of tumor burden at end-of-treatment with either IgG control (n=4) or dual PDL1 and CTLA4 antibodies (n=3) in macrophage-depleted MYC-HCC mice. C. End-of-treatment gross appearance, histology and immunofluorescence for CD8 T cells in representative macrophage-depleted MYC-HCC-bearing mice treated with IgG control (n=4) or dual PDL1 and CTLA4 antibodies (n=3). D. Quantitation of CD8T cell infiltration in macrophage-depleted MYC-HCC-bearing mice treated with IgG control (n=4) or dual PDL1 and CTLA4 antibodies (n=3). E. Figurative representation of the mechanism of efficacy of combined PDL1 and CTLA4 therapy in MYC-HCC. MYC-driven cancers are immune evasive with MHCI repression and PDL1 overexpression on cancer cells and macrophages which in turn lead to T cell exhaustion. Treatment with CTLA4 and PDL1 inhibitors leads to repolarization of macrophages to the M1-like phenotype with increased expression of CD40 and MHCII, which leads to enhanced antigen presentation and robust T cell activation resulting in delayed tumor progression (created using Biorender.com).