Immunotherapy Of Human Cancer Research Articles

Metformin Suppresses Both PD-L1 Expression in Cancer Cells and Cancer-Induced PD-1 Expression in Immune Cells to Promote Antitumor Immunity.

Metformin, a drug prescribed for patients with type 2 diabetes, has potential efficacy in enhancing antitumor immunity; however, the detailed underlying mechanisms remain to be elucidated. Therefore, we aimed to identify the inhibitory molecular mechanisms of metformin on programmed death ligand 1 (PD-L1) expression in cancer cells and programmed death 1 (PD-1) expression in immune cells. We employed a luciferase reporter assay, quantitative real-time PCR, immunoblotting analysis, immunoprecipitation and ubiquitylation assays, and a natural killer (NK) cell-mediated tumor cell cytotoxicity assay. A mouse xenograft tumor model was used to evaluate the effect of metformin on tumor growth, followed by flow-cytometric analysis using tumor-derived single-cell suspensions. Metformin decreased AKT-mediated β-catenin S552 phosphorylation and subsequent β-catenin transactivation in an adenosine monophosphate-activated protein kinase (AMPK) activation-dependent manner, resulting in reduced CD274 (encoding PD-L1) transcription in cancer cells. Tumor-derived soluble factors enhanced PD-1 protein stability in NK and T cells via dissociation of PD-1 from ubiquitin E3 ligases and reducing PD-1 polyubiquitylation. Metformin inhibited the tumor-derived soluble factor-reduced binding of PD-1 to E3 ligases and PD-1 polyubiquitylation, resulting in PD-1 protein downregulation in an AMPK activation-dependent manner. These inhibitory effects of metformin on both PD-L1 and PD-1 expression ameliorated cancer-reduced cytotoxic activity of immune cells in vitro and decreased tumor immune evasion and growth in vivo. Metformin blocks both PD-L1 and PD-1 within the tumor microenvironment. This study provided a mechanistic insight into the efficacy of metformin in improving immunotherapy in human cancer.

Abstract 2821: Development of therapeutic monoclonal antibodies against DKK1 peptide-HLA-A2 complex to treat human cancers

Abstract Immunotherapy is a promising option for cancer treatment. Targeted immunotherapy with monoclonal antibodies (mAbs) is an effective and safe method for the treatment of malignancies. In recent years, efforts have been made to identify potential therapeutic mAbs by defining alternative or novel target antigens. We reported that Dickkopf-1 (DKK1) is a good target for immunotherapy of human cancers based on its wide expression in different cancers but not in normal tissues. As DKK1 is a secreted protein, mAbs directly binding to DKK1 may have limited effects on cancer cells in vivo. Our previous studies demonstrated that DKK1 peptide (such as P20 and P66v, which bind with HLA-A*0201 (HLA-A2) molecule) specific cytotoxic T cells specifically kill myeloma and other cancer cells that express DKK1 and HLA-A2, but not HLA-A2+ normal cells, indicating that DKK1+ tumor cells naturally express these peptides, in the context of HLA-A2 molecules, on their surface. To develop cancer therapeutic antibodies, DKK1 peptide P20-HLA-A2 monomer was synthesized and used to immunize mice. Hybridomas secreting mAbs recognizing cell surface-expressed DKKl P20-HLA-A2 complex (DKK1-A2) were obtained and analyzed. In this study, we generated novel (DKK1-A2) mAbs recognizing DKK1 (P20) peptide in the context of human HLA-A*0201 (HLA-A2) molecules (DKK1-A2 complexes) that are naturally expressed by HLA-A2+DKK1+ cancer cells. These mAbs bound to HLA-A2+DKK1+ hematologic (myeloma, lymphoma, and leukemia) and solid (breast, pancreatic, and prostate) cancer cells and directly induced apoptosis in the cancer cells by activating the caspase-9 cascade. DKK1-A2 mAbs also effectively lysed cancer cells in vitro by mediating CDC and ADCC activities. Furthermore, DKK1-A2 mAbs were therapeutic against established hematologic and solid cancers in their xenograft mouse models. To enhance the therapeutic efficacy of DKK1-A2 mAbs, we investigated the effect of combination treatment of DKK1-A2 mAbs with lenalidomide. Our results showed that there is an additive therapeutic effect between DKK1-A2 mAbs and lenalidomide in vivo, underscoring a potential clinical development strategy for combining DKK1-A2 mAb with lenalidomide to treat cancer patients. As DKK1 is not detected in most human tissues, DKK1-A2 mAbs did not bind to or kill HLA-A2+ blood cells in vitro nor caused tissue damage in vivo in tumor-free or tumor-bearing HLA-A2-transgenic mice. Thus, our study suggests that DKK1-A2 mAbs may be a promising therapeutic agent to treat human cancers and warrants further preclinic studies to determine their translatability to clinic. Citation Format: Jianfei Qian, Wang Qiang, Liuling Xiao, Wei Xiong, Miao Xian, Chuanchao Zhang, Yabo Li, Qing Yi. Development of therapeutic monoclonal antibodies against DKK1 peptide-HLA-A2 complex to treat human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2821.

Akkermansia muciniphila and herbal medicine in immune-related diseases: current evidence and future perspectives

Akkermansia muciniphila is considered the “paradigm for next-generation beneficial microorganisms” and has been reported to help alleviat immune-related diseases. Evidence shows that herbal medicine can treat disease by regulating the abundance of A. muciniphila. Recent studies have revealed a link between A. muciniphila and immune-related diseases. Here, we systematically reviewed the association between A. muciniphila, herbal medicine, and immune-related diseases (including inflammatory bowel disease, human immunodeficiency virus, cancer immunotherapy, and immune-related liver injury). We also summarize the potential mechanisms of action of A. muciniphila and offer perspectives for future studies.

Mutation Characteristics of the MYC Proximal Network and its Implications for Immunotherapy in Human Cancer

Mutation Characteristics of the MYC Proximal Network and its Implications for Immunotherapy in Human Cancer

Development of therapeutic monoclonal antibodies against DKK1 peptide-HLA-A2 complex to treat human cancers

BackgroundTargeted immunotherapy with monoclonal antibodies (mAbs) is an effective and safe method for the treatment of malignancies. Development of mAbs with improved cytotoxicity, targeting new and known tumor-associated antigens, therefore...

Characteristics of adaptive immune effectors involved in the secondary xenogeneic immune response to human melanoma cells

Introduction. Current approaches are being developed for adoptive cancer therapy using T-cells genetically modified with T-cell receptors (TCRs) with specificity for tumor antigens. The complexities of identifying antigen-specific TCRs in a patient’s repertoire and selecting therapeutic receptors necessitate the development of experimental strategies for generating tumor-specific T cells. One of such approaches could be the xenogeneic immunization of mice with human tumor cells. It seems plausible that the T cell repertoire stimulated by xenogeneic vaccination could be a source of TCRs suitable for adoptive cancer immunotherapy.Aim. To assess the prospects for using xenogeneic immunizations to generate tumor-specific memory T cells and identify their TCRs suitable for adoptive immunotherapy, we studied the dynamics of the secondary xenogeneic response in a model of induction of an immune response in mice to human melanoma cells.Materials and methods. Mice were immunized with human melanoma cells, and 45 days later, they were re-challenged with the immunizing tumor. The dynamics of the development of the secondary immune response in vivo and the composition of the involved effectors of adaptive immunity were analyzed by flow cytometry. The proliferation of lymphocytes from immune mice in response to human melanoma cells was evaluated in in vitro culture.Results. The secondary xenogeneic response was characterized by a more intense accumulation of T cells and the rapid development of the effector phase at the injection site of human melanoma. This correlated with an enhanced in vitro proliferative response of lymphocytes from immune animals to xenoantigens of the immunizing tumor. CD4+ and CD8+ memory T cells contributed equally to the development of a secondary response to human melanoma cells expressing HLA class I and II molecules. When only HLA class I was expressed on the cells of the immunizing xenogeneic tumor, CD8+ memory cells were formed, which dominated the secondary immune response.Conclusion. Our findings confirmed the formation of a specific immunological memory for xenoantigens during xenogeneic immunization. This suggests the possibility of generating xenogeneic TCRs specific for human tumor antigens, which opens up opportunities to developing approaches for screening among them for receptor variants suited for adoptive immunotherapy of human cancers.

An association between ATP7B expression and human cancer prognosis and immunotherapy: a pan-cancer perspective

BackgroundATP7B is a copper-transporting protein that contributes to the chemo-resistance of human cancer cells. It remains unclear what the molecular mechanisms behind ATP7B are in cancer, as well as its role in human pan-cancer studies.MethodsOur study evaluated the differential expression of ATP7B in cancer and paracancerous tissues based on RNA sequencing data from the GTEx and TCGA. Kaplan–Meier and Cox proportional hazards regressions were used to estimate prognostic factors associated with ATP7B.The correlations between the expression of ATP7B and immune cell infiltration, tumor mutation burden, microsatellite instability and immune checkpoint molecules were analyzed. Co-expression networks and mutations in ATP7B were analyzed using the web tools. An analysis of ATP7B expression difference on drug sensitivity on tumor cells was performed using the CTRP, GDSC and CMap database.ResultsATP7B expression differed significantly between cancerous and paracancerous tissues. The abnormal expression of ATP7B was linked to prognosis in LGG and KIRC. Infiltration of immune cells, tumor mutation burden, microsatellite instability and immunomodulators had all been linked to certain types of cancer. Cancer cells exhibited a correlation between ATP7B expression and drug sensitivity.ConclusionATP7B might be an immunotherapeutic and prognostic biomarker based on its involvement in cancer occurrence and development.

The Role of NCS1 in Immunotherapy and Prognosis of Human Cancer.

The Neural Calcium Sensor1 (NCS1) is a crucial protein that binds to Ca2+ and is believed to play a role in regulating tumor invasion and cell proliferation. However, the role of NCS1 in immune infiltration and cancer prognosis is still unknown. Our study aimed to explore the expression profile, immune infiltration pattern, prognostic value, biological function, and potential compounds targeting NCS1 using public databases. High expression of NCS1 was detected by immune histochemical staining in LIHC (Liver hepatocellular carcinoma), BRCA (Breast invasive carcinoma), KIRC (Kidney renal clear cell carcinoma), and SKCM (Skin Cutaneous Melanoma). The expression of NCS1 in cancer was determined by TCGA (The Cancer Genome Atlas Program), GTEx (The Genotype-Tissue Expression), the Kaplan-Meier plotter, GEO (Gene Expression Omnibus), GEPIA2.0 (Gene Expression Profiling Interactive Analysis 2.0), HPA (The Human Protein Atlas), UALCAN, TIMER2.0, TISIDB, Metascape, Drugbank, chEMBL, and ICSDB databases. NCS1 has genomic mutations as well as aberrant DNA methylation in multiple cancers compared to normal tissues. Also, NCS1 was significantly different in the immune microenvironment, tumor mutational burden (TMB), microsatellite instability (MSI), and immune infiltrate-associated cells in different cancers, which could be used for the typing of immune and molecular subtypes of cancer and the presence of immune checkpoint resistance in several cancers. Univariate regression analysis, multivariate regression analysis, and gene enrichment analysis to construct prognostic models revealed that NCS1 is involved in immune regulation and can be used as a prognostic biomarker for SKCM, LIHC, BRCA, COAD, and KIRC. These results provide clues from a bioinformatic perspective and highlight the importance of NCS1 in a variety of cancers.

Cancer immunotherapy with enveloped self-amplifying mRNA CARG-2020 that modulates IL-12, IL-17 and PD-L1 pathways to prevent tumor recurrence

Targeting multiple immune mechanisms may overcome therapy resistance and further improve cancer immunotherapy for humans. Here, we describe the application of virus-like vesicles (VLV) for delivery of three immunomodulators alone and in combination, as a promising approach for cancer immunotherapy. VLV vectors were designed to deliver single chain interleukin (IL)-12, short-hairpin RNA (shRNA) targeting programmed death ligand 1 (PD-L1), and a dominant-negative form of IL-17 receptor A (dn-IL17RA) as a single payload or as a combination payload. Intralesional delivery of the VLV vector expressing IL-12 alone, as well as the trivalent vector (designated CARG-2020) eradicated large established tumors. However, only CARG-2020 prevented tumor recurrence and provided long-term survival benefit to the tumor-bearing mice, indicating a benefit of the combined immunomodulation. The abscopal effects of CARG-2020 on the non-injected contralateral tumors, as well as protection from the tumor cell re-challenge, suggest immune-mediated mechanism of protection and establishment of immunological memory. Mechanistically, CARG-2020 potently activates Th1 immune mechanisms and inhibits expression of genes related to T cell exhaustion and cancer-promoting inflammation. The ability of CARG-2020 to prevent tumor recurrence and to provide survival benefit makes it a promising candidate for its development for human cancer immunotherapy.

Transient regulatory-T-cell interruption promotes skin-resident memory T cells mediated tumor protection

Most cancer immunotherapy approaches aim to stimulate cytotoxic CD8+ T lymphocytes to reject tumor cells. Due to the tumor-mediated suppressive micro-environment, of which the major contributor is regulatory T cells (Tregs), promising preclinical approaches were disappointing in clinical settings. Our recent study demonstrated that transient interruption of Tregs could induce CD8+ T cell responses to reject tumors in an animal model. The long-term tumor protective effect has yet not to be investigated. In this study, mice with Treg depletion rejected tumors and were rechallenged to study anti-tumor memory immune responses. The effects of major immune cell subsets on tumor protection were explored. Finally, we demonstrate that transient depletion of Tregs during primary tumor challenge can result in long-lasting protection against the tumor rechallenge. Skin-resident memory T cells (sTRM) were major factors in rejecting rechallenged tumors even when peripheral T cells were deficient. These findings highlight a promising strategy for empowering tissue-resident memory T cells for cancer prevention and immunotherapy in humans by interrupting Tregs.

Pharmacokinetics and drug-likeness of anti-cancer traditional Chinese medicine: molecular docking and molecular dynamics simulation study

MCM7 (Minichromosome Maintenance Complex Component 7) is a component of the DNA replication licensing factor, which controls DNA replication. The MCM7 protein is linked to tumor cell proliferation and has a function in the development of several human cancers. Several types of cancer may be treated by inhibiting the protein, as it is strongly produced throughout this process. Significantly, Traditional Chinese Medicine (TCM), which has a long history of clinical adjuvant use against cancer, is rapidly gaining traction as a valuable medical resource for the development of novel cancer therapies, including immunotherapy. Therefore, the goal of the research was to find small molecular therapeutic candidates against the MCM7 protein that may be used to treat human cancers. A computational-based virtual screening of 36,000 natural TCM libraries is carried out for this goal using a molecular docking and dynamic simulation technique. Thereby, ∼8 novel potent compounds i.e., ZINC85542762, ZINC95911541, ZINC85542617, ZINC85542646, ZINC85592446, ZINC85568676, ZINC85531303, and ZINC95914464 were successfully shortlisted, each having the capacity to penetrate the cell as potent inhibitors for MCM7 to curb this disorder. These selected compounds were found to have high binding affinities compared to the reference (AGS compound) i.e. < −11.0 kcal/mol. ADMET and pharmacological properties showed that none of these 8 compounds poses any toxic property (carcinogenicity) and have anti-metastatic, and anticancer activity. Additionally, MD simulations were run to assess the compounds’ stability and dynamic behavior with the MCM7 complex for about 100 ns. Finally, ZINC95914464, ZINC95911541, ZINC85568676, ZINC85592446, ZINC85531303, and ZINC85542646 are identified as highly stable within the complex throughout the 100 ns simulations. Moreover, the results of binding free energy suggested that the selected virtual hits significantly bind to the MCM7 which implied these compounds may act as a potential MCM7 inhibitor. However, in vitro testing protocols are required to further support these results. Further, assessment through various lab-based trial methods can assist with deciding the action of the compound that will give options in contrast to human cancer immunotherapy. Communicated by Ramaswamy H. Sarma

Towards a better understanding of human iNKT cell subpopulations for improved clinical outcomes.

Invariant natural killer T (iNKT) cells are a unique T lymphocyte population expressing semi-invariant T cell receptors (TCRs) that recognise lipid antigens presented by CD1d. iNKT cells exhibit potent anti-tumour activity through direct killing mechanisms and indirectly through triggering the activation of other anti-tumour immune cells. Because of their ability to induce potent anti-tumour responses, particularly when activated by the strong iNKT agonist αGalCer, they have been the subject of intense research to harness iNKT cell-targeted immunotherapies for cancer treatment. However, despite potent anti-tumour efficacy in pre-clinical models, the translation of iNKT cell immunotherapy into human cancer patients has been less successful. This review provides an overview of iNKT cell biology and why they are of interest within the context of cancer immunology. We focus on the iNKT anti-tumour response, the seminal studies that first reported iNKT cytotoxicity, their anti-tumour mechanisms, and the various described subsets within the iNKT cell repertoire. Finally, we discuss several barriers to the successful utilisation of iNKT cells in human cancer immunotherapy, what is required for a better understanding of human iNKT cells, and the future perspectives facilitating their exploitation for improved clinical outcomes.

Abstract 6636: Metformin suppresses tumor immune evasion through downregulation of PD-L1 expression in cancer cells and PD-1 expression in NK cells and T cells

Abstract Up-regulation of programmed cell death 1 (PD-1) receptor on T cells and its ligand (PD-L1) on cancer cells is reported in various tumor microenvironment, and the interaction between PD-L1 and PD-1 inhibits the cytotoxic activity of NK cells and T cells, allowing cancer cells to evade immune destruction. Extensive studies suggested that metformin, the most widely used first-line drug for treatment of type 2 diabetes, had a potential efficacy of enhancing anti-tumor immune response. However, the detail mechanisms underlying the efficacy are unknown. Here, we showed that metformin decreases AKT-mediated β-catenin S552 phosphorylation and β-catenin transactivation in an AMPK activation-dependent manner, resulting in reduced CD274 transcription in cancer cells. In the cocultures of cancer cells with NK cells or T cells, cancer cells induced PD-1 protein stability and expression in the NK cells and T cells, these PD-1 upregulation was also observed in infiltrated NK cells and T cells in vivo mouse tumors. Metformin reduced the half-life and expression of PD-1 in an AMPK activation-dependent manner in vitro and in vivo. These inhibitory roles of metformin in PD-L1 expression in cancer cells and in cancer cell-induced PD-1 expression in NK cells and T cells led to ameliorated the cancer cell-reduced cytotoxic activity of NK cells and T cells and inhibited tumor growth in vivo models. These findings suggest that metformin may act as both PD-L1 and PD-1 blockades within tumor microenvironment, providing a mechanistic insight for the efficacy of metformin to improve immunotherapy in human cancer. Citation Format: Su Hwan Park, Jong-Ho Lee. Metformin suppresses tumor immune evasion through downregulation of PD-L1 expression in cancer cells and PD-1 expression in NK cells and T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6636.

Abstract 5784: Spatial clustering reveals immune hub interaction with reservoir of stem-like CD8 T cells and predicts immunotherapy response in lung cancer patients

Abstract Background: Immunologic tumor control involves coordination of multiple cell types, but the organization of these interactions in human tumors is poorly understood. We recently reported that immunogenic tumors contain spatially-localized multicellular immunity hubs characterized by expression of interferon-stimulated genes, including CXCL10/CXCL11 (T cell attracting chemokines), and the presence of IFNG+ T cells. This suggested a positive feedback loop where T cell-derived IFNγ stimulates production of CXCR3 ligands, thereby attracting more T cells. However, we did not know (1) whether these hubs predict response to immunotherapy and (2) how hubs intersect with the various CD8 T cell states which play central roles in anti-tumor immunity. Methods: To understand the composition of this immunity hub and its potential association with immunotherapy response, we performed multiplex RNA FISH to visualize hub components in NSCLC tissue from 68 patients prior to PD1-blockade. Cells were segmented and phenotyped automatically. We additionally imaged serial sections with a second panel for markers of CD8 T cell state. We then computationally registered sequential images, integrating our panels. To identify hubs in an unbiased manner, we employed kmeans clustering. Results: We found that the presence of the immunity hub is predictive of subsequent response to PD1-blockade. Image registration revealed that hubs are enriched for CD8 T cells in multiple states, confirming their role as key sites of anti-tumor T cell activity. Furthermore, immunity hubs in responders contained more activated CD8 T cells and IFNG+ cells than those in nonresponders. To determine how hub heterogeneity may influence hub functions, we subclustered hubs by phenotypic composition. This analysis uncovered a ‘hybrid hub’ subclass that spatially overlaps with structures containing stem-like TCF7+ CD8 T cells, resembling the interfollicular zone of lymph nodes. The presence of a single hybrid hub was strongly associated with RECIST response (p = 0.0005), found in 85% of responders and only 24% of non-responders. Hybrid hubs also showed a striking association with patient PFS (p = 0.0014). Using ultra-highplex RNA ISH technologies, we further explored the hybrid hubs and found they are enriched in macrophages that express CXCR3 ligands. Conclusions: Our study provides insight into the multicellular networks that underlie anti-tumor immunity. Immunity hubs are predictive of response to immunotherapy in human lung cancer and organize intratumoral CD8 T cell activity. Moreover, hybrid hubs may represent an active intratumoral niche for tumor-specific stem-like T cells that sustain anti-tumor immunity. These multicellular networks are excellent candidates for biomarker development and targets for immunotherapy. Citation Format: Jonathan Chen, Linda Nieman, Maxwell Spurrell, Justin Gainor, Nir Hacohen. Spatial clustering reveals immune hub interaction with reservoir of stem-like CD8 T cells and predicts immunotherapy response in lung cancer patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5784.

Abstract 2964: Targeting tumor-associated macrophages to enhance anti-tumor immunity with the Dectin-2 agonistic antibody BDC-3042

Abstract Tumor-associated macrophages (TAMs) are the largest immune cell population in many cancers and play a key role in establishing the immunosuppressive tumor microenvironment (TME) that enables tumor progression. However, TAMs are phenotypically plastic and have the potential to be reprogrammed into immunostimulatory cells that enhance innate and adaptive anti-tumor immunity. To this end, we developed BDC-3042, an agonistic antibody targeting an immune-activating receptor expressed on TAMs known as Dectin-2 (CLEC6A). Dectin-2 is a C-type lectin receptor (CLR) known best for its role in pathogen recognition and induction of protective immune responses against fungi and other microbes. We previously demonstrated that Dectin-2 agonism with natural ligands stimulates pro-inflammatory cytokine secretion and antigen presentation by TAMs, resulting in robust CD8+ T cell-mediated anti-tumor immunity in syngeneic mouse models. Here we present our preclinical studies demonstrating the therapeutic potential of the Dectin-2 agonistic antibody, BDC-3042, as a novel TAM-directed immunotherapy for diverse human cancers. BDC-3042 exhibits strong binding to Dectin-2-expressing macrophages generated in vitro and to primary human TAMs from a range of solid tumor types. In contrast, BDC-3042 binds weakly to peripheral monocytes and minimally to other immune cells in blood and tumor tissues. Macrophages exposed to cytokines and growth factors commonly found in the TME exhibit increased Dectin-2 expression and BDC-3042 binding. Treatment with BDC-3042 activates in vitro-generated macrophages and primary TAMs to produce an array of pro-inflammatory cytokines and chemokines associated with anti-tumor immunity. Consistent with low target expression, BDC-3042 elicits little to no activation of peripheral monocytes or cells in whole blood. BDC-3042 activity is dependent on both Dectin-2 and FcγRs, as indicated through studies utilizing Fc variants with enhanced or attenuated effector function. In mice with humanized immune systems, BDC-3042 elicits activation of TAMs, as evidenced by modulation of key activation markers. Systemically administered BDC-3042 mediates anti-tumor efficacy as a monotherapy, and combination with checkpoint blockade therapy enhances anti-tumor efficacy. The data presented demonstrate the therapeutic potential of targeting Dectin-2 expressed by TAMs with the agonistic antibody BDC-3042 as a novel pan-cancer approach for myeloid cell-directed tumor immunotherapy. Citation Format: Justin A. Kenkel, Fang Xiao, Po Y. Ho, Jess L. Nolin, Rishali K. Gadkari, Laughing Bear Torrez, David T. Omstead, Katelynn A. McEachin, Jason Ptacek, Rachel Grgich, Cecelia I. Pearson, Stefan Chun, Cindy L. Kreder, Karla A. Henning, Han K. Kim, Lu Xu, Steven J. Chapin, Michael N. Alonso, Shelley E. Ackerman. Targeting tumor-associated macrophages to enhance anti-tumor immunity with the Dectin-2 agonistic antibody BDC-3042 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2964.

Abstract 5138: Systemic agonist anti-CD40 treatment delivers precise melanoma immunotherapy within established autoimmune landscapes

Abstract The overlapping signatures of type 1 interferons (IFN) and IFN-gamma (IFNg) has proven challenging in human autoimmunity and cancer immunotherapy, particularly for resistance to anti-programmed death 1 (anti-PD1) monotherapy. By contrast, agonist abs targeting of CD40 (anti-CD40) antibodies (abs) has clinically elicit anti-tumor immunity with limited autoimmune complications. Here, we explore the relationships between anti-PD1 or agonist CD40 responses in melanoma tumors from type 1 IFN alpha receptor (IFNAR)-sufficient and type 1 IFNAR-deficient lupus-prone mice that overexpress IFNg. To address this, RNA-sequencing (RNA-seq) data on tumors treated with either anti-PD1 or agonist anti-CD40 was evaluated for tumor objective responses compared to untreated controls. Cellular and canonical pathway identification from RNA-seq data were interrogated from the IPA library for significantly differential expressed genes (DEG). Variance patterns of the anti-PD1 and anti-CD40 monotherapies identified known resistance and response pathways highly concordant with IFN co-expression patterns in the RNA-sequencing data, respectively. IPA biomarker analysis for uniquely regulated genes showed that anti-PD1 induced only 59 DEG; while anti-CD40 induced 494 DEG including Il-27, and PR/SET Domain 1 (PRDM1), a transcription factor regulated by IL-27. Specifically, signatures of MDSC development (irf8, il10); and adaptive resistance (pdcd1, pdcd1lg2, and TNFRSF5/CD40)) were negatively associated with response to anti-P1 monotherapy. By contrast, signatures of IFN activation (stat1, Ifng, and Ifngr1), chemotaxis (Cxcl10, Il12b and Il-27), attractants (Sema3g and Sema4a), and antigen presentation MHC II (h2-Aa, h2-Ab1, and ciita) were associated positively with response to agonist anti-CD40 monotherapy. Importantly, anti-CD40 alone delivered anti-tumor immune responses in type 1 IFN receptor-deficient lupus-prone mice that overexpress IFNg (Ifnar-/-ARE-/-) mice suggesting synergy with therapies blocking type 1 IFN signaling. Thus, these findings indicate that the rational use of CD40 agonists abs provide a better therapeutic platform to deliver precise anti-melanoma responses in complex autoimmune landscapes. Citation Format: Julio C. Valencia, Michael Sanford, Howard A. Young. Systemic agonist anti-CD40 treatment delivers precise melanoma immunotherapy within established autoimmune landscapes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5138.

Abstract 5203: Naturally occurring colorectal cancer in rhesus macaques as model for human cancer immunotherapy: a first report on pembrolizumab ± radiotherapy

Abstract Introduction: Rhesus macaques share extensive similarities with humans including immune cell populations, regulatory mechanisms, and targets for cancer immunotherapy but also naturally develop cancers at similar life stages. Hence, treating rhesus macaques with spontaneous tumors bears enormous potential as model for human cancer immunotherapy. Material and Methods: We identified 2 female rhesus macaques, 23.6y (CRC_1) and 20.3y (CRC_2) old with naturally occurring colorectal cancer by imaging and clinical assessments. We collected a pre-treatment tumor biopsy in CRC_1 prior to treatment with 8 Gy intensity-modulated radiotherapy (IMRT) to the proximal portion of the tumor and 2 mg/kg i.v. pembrolizumab. The second candidate (CRC_2) was not tumor biopsy-eligible due to anemia and was treated with pembrolizumab alone. We collected blood and peripheral lymph nodes (LNs) pre- and on-treatment and complete tissue collection was performed at the 3w time point including tumor-adjacent mesenteric LNs. Follow-up assessments include IHC (e.g. CD3, CD20, IBA1), flow cytometry, cytokine assessment in plasma, and transcriptomics of LN and tumor samples. Results and Discussions: Imaging revealed focal thickening of the proximal colonic wall with disrupted architecture in CRC_1 and focal colonic thickening and an enlarged adjacent LN in CRC_2. Both tumors displayed mismatch repair-deficiency (MLH1 &amp; PMS2 loss) and a drastic reduction in infiltrating CD3 cells in neoplastic areas compared to adjacent healthy colon. On treatment, tumor-associated LNs demonstrated a mild increase in CD20+ cells in the medullary sinuses of both patients and increased follicle numbers with normal follicular architecture in reference to peripheral LNs. In both patients there was an increased number of tertiary lymphoid structures (TLS) surrounding the tumors, predominately populated with CD20+ cells and with Ki67+ germinal centers, profoundly so in CRC_2. Moreover, IBA1+ cells abundantly infiltrated both tumors but showed a higher aggregation within the stromal elements of the neoplasms. A gradient of histologic differentiation was evident in CRC_1 and coincided with a reduction of TLS, IBA1+ cell abundance, and CD3+ cell infiltration in those areas which were poorly differentiated. Within the irradiated tumor of CRC_1, there were abundant apoptotic cells which stained positively with cleaved caspase 3 at the 3w time point. Circulating blood counts as preliminary peripheral read-out revealed a reduction in circulating lymphocytes in both patients beginning at 3d (CRC_1) resp. 6d (CRC_2) and a sharp increase in monocytes at 3d in unirradiated patient CRC_2. Conclusion: The immunological and tumoral response to pembrolizumab and radiotherapy matches the clinical response in humans and enables an unprecedented examination of the peripheral and local lymph node response. Citation Format: Simon Deycmar, George W. Schaaf, Brendan J. Johnson, J. Daniel Bourland, James D. Ververs, Christopher T. Whitlow, J. Mark Cline. Naturally occurring colorectal cancer in rhesus macaques as model for human cancer immunotherapy: a first report on pembrolizumab ± radiotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5203.

Pan-Cancer Analysis of Histone Methyltransferase KMT2D with Potential Implications for Prognosis and Immunotherapy in Human Cancer.

Pan-cancer analysis is an efficient tool to obtain a panoramic view of cancer- related genes and identify their oncogenic processes, facilitating the development of new therapeutic targets. Lysine methyltransferase 2D (KMT2D), acting as a major enhancer coactivator for mammalian cells, is one of the most frequently mutated genes across various cancer types and is considered an oncogene and a rationale for epigenetic therapeutic targets. This study was designed to explore the potential role of KMT2D in human cancer through a pan-cancer analysis. The expression of KMT2D was assessed in normal tissues and cell lines, and pancancers from The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE), and Genotype-Tissue Expression (GTE) datasets were used to explore its correlation with prognosis, immune cell infiltration, tumor mutation burden, microsatellite instability, and mismatch repair. KMT2D expression was heterogeneous across different cancer types. Increased KMT2D indicated a worse prognosis in adrenocortical carcinoma (ACC), brain lower-grade glioma (LGG), and mesothelioma (MESO), while patients with high KMT2D expression showed better outcomes in renal clear cell carcinoma (KIRC). Moreover, KMT2D expression was positively correlated with immune cell infiltration and negative tumor mutation burden in multiple cancers. In addition, a significant correlation between KMT2D and immune checkpoint-related genes or mismatch repair genes was identified. These findings support the hypothesis that KMT2D is not only a potential biomarker for prognosis and immunotherapy response prediction but also an essential immune regulator in human cancer.

Abstract A021: Clinical and molecular characterization of naturally-occurring colorectal cancer in rhesus macaques reveals mismatch repair deficiency driven by epigenetic silencing of MLH1

Abstract Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and emerge at similar life stages as in human patients, but the carcinogenesis and molecular background remain elusive, limiting the value of the model for human disease and cancer therapy. In order to better understand this naturally occurring model, we established a diagnostic and staging pipeline including imaging (18F-FDG PET, plain and contrast-enhanced CT), histopathology, and clinical assessments. We observed cancer symptoms and co-morbidities in our cohort (n=16, all Indian-origin, 11 females, mean age at arrival 20.2y) such as hypoalbuminemia, fecal occult blood, and microcytic anemia, as frequently described in human CRC patients. Pathologically, all cancers were right-sided, involving the proximal colon and/or ileocecocolic junction, and most were densely fibrotic, restricting the colonic lumen. Most cancers appeared with glandular morphology and some (18.8%) had mucinous components. Immunohistochemistry revealed loss of MLH1 and PMS2 in 100% of investigated CRCs, indicating mismatch repair deficiency which furthermore resulted in microsatellite instability (PCR &amp; fragment analysis). Whole exome sequencing revealed the close genetic relatedness to human CRCs, particularly exemplified by mutations affecting KRAS (37.5%, e.g., p.G12D), APC (31.3%), TP53 (18.8%, e.g. p.R175H), ARID1A (56.3%), and ALK (43.8%), as similarly annotated in the human COSMIC database. Nonetheless, somatic mutations do not explain the loss of MLH1 in the entire CRC cohort. Transcriptomics on the other hand revealed the transcriptional suppression of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRC in comparison to adjacent healthy colon. Moreover, comparison of differentially expressed gene sets of rhesus CRC with a human annotated database (IPA) confirmed the disease similarities observed clinically, genetically, and histopathologically. Subsequently, we investigated DNA methylation of the promoter region of MLH1 and retranslated markers for the CpG island methylator phenotype (CIMP) as described in human CRC. While only 56.3% of CRCs were considered CIMP positive (≥3/5 markers hypermethylated), 100% of investigated CRCs exhibited MLH1 promoter hypermethylation. As a result, epigenetic silencing is suggested to suppress MLH1 transcription, cause the loss of MLH1 protein, and drive mismatch repair deficiency and genomic instability in naturally occurring CRC in rhesus macaques. We therefore consider spontaneous, uninduced CRC in rhesus macaques, their treatment-naïve nature, and their unaltered immune competence an outstanding model for human disease and in particular for human cancer immunotherapy. Citation Format: Simon Deycmar, Brendan Johnson, Karina Ray, David Caudell Caudell, John Olson, Greg Dugan, W. Shane Sills, Declan Ryan, Christopher Whitlow, Kiran K. Solingapuram Sai, Betsy Ferguson, Benjamin Bimber, Cassandra Cullin, Brandy Dozier, Emily Romero, Francois Villinger, Armando Burgos, Jeff Chou, Bruno Gomes, Michael Hettich, Maurizio Ceppi, Jehad Charo, J. Mark Cline. Clinical and molecular characterization of naturally-occurring colorectal cancer in rhesus macaques reveals mismatch repair deficiency driven by epigenetic silencing of MLH1 [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A021.

Pan-cancer analysis of AIM2 inflammasomes with potential implications for immunotherapy in human cancer: A bulk omics research and singlecell sequencing validation.

BackgroundThe absent in melanoma 2 (AIM2) inflammasome is a multi-protein platform that recognizes aberrant cytoplasmic double-stranded DNA(dsDNA) and induces cytokine maturation, release, and pyroptosis. Some studies found that the AIM2 inflammasome was a double-edged sword in many cancers. However, there have been fewer studies on AIM2 inflammasomes in pan-cancer.MethodsGene expression was analyzed using The Cancer Genome Atlas (TCGA) database and The Genotype-Tissue Expression (GTEx) database. Immunohistochemistry (IHC) was used to validate the expression of the AIM2. We used the survival curve to explore the prognostic significance of the AIM2 inflammasomes in pan-cancer. Mutations and methylation of AIM2 inflammasome-related genes (AIM2i-RGs) were also comprehensively analyzed. Single sample gene set enrichment analysis was used to calculate the AIM2 inflammasomes score and explore the correlation of the AIM2 inflammasomes score with immune-related genes and immune infiltrations. The function of AIM2 inflammasomes in pan-cancer was analyzed at the single-cell level. Single-cell transcriptome sequencing (scRNA-seq) data was used to assess the activation state of the AIM2 inflammasomes in the tumor microenvironment.ResultsWe found that AIM2i-RGs were aberrantly expressed in tumors and were strongly associated with prognosis. In pan-cancer, the expression of AIM2i-RGs was positively associated with copy number variation and negatively associated with methylation. In AIM2i-RGs, missense mutations were the predominant type of single nucleotide polymorphism. Moreover, we found that the drugs dimethyloxallyl glycine (DMOG) and Z-LNle-CHO may be sensitive to the AIM2 inflammasomes. The AIM2 inflammasomes score was significantly and positively correlated with the tumor immunity score and the stroma score. In most tumors, the AIM2 inflammasomes score was significantly and positively correlated with CD8+ T cell abundance in the tumor microenvironment. Additionally, the AIM2 inflammasomes score was significantly correlated with immune checkpoint genes in pan-cancer as well as immune checkpoint therapy-related markers including tumor mutational burden (TMB), microsatellite instability(MSI), and tumor immune dysfunction and exclusion(TIDE). scRNA-seq analysis suggested that AIM2 inflammasomes differ significantly among different cells in the tumor microenvironment. IHC confirmed low expression of AIM2 in colorectal cancer.DiscussionAIM2 inflammasomes may be a new target for future tumor therapy It is likely involved in tumor development, and its high expression may serve as a predictor of tumor immunotherapy efficacy.