Immune Escape Mechanisms Research Articles

Genomic landscape and distinct molecular subtypes of primary testicular lymphoma.

Primary testicular lymphoma (PTL) is a rare lymphoma predominantly occurring in the elderly male population. It is characterized by a limited response to treatment and a heightened tendency towards relapse. Histologically, approximately 90% of PTL cases are classified as diffuse large B-cell lymphomas (DLBCL). Genetic features of PTL were delineated in a limited scope within several independent studies. Some of the articles which analyzed the genetic characterization of DLBCL have incorporated PTL samples, but these have been constrained by small sample sizes. In addition, there have been an absence of independent molecular typing studies of PTL. This report summarizes the common mutational features, copy number variations (CNVs) and molecular typing of PTL patients, based on whole-exome sequencing (WES) conducted on a cohort of 25 PTL patients. Among them, HLA, CDKN2A and MYD88 had a high mutation frequency. In addition, we found two core mutational characteristics in PTL including mutation in genes linked to genomic instability (TP53 and CDKN2A) and mutation in immune-related genes (HLA, MYD88, CD79B). We performed molecular typing of 25 PTL patients into C1 subtype with predominantly TP53 mutations and C2 subtype with predominantly HLA mutations. Notably, mutations in the TP53 gene predicted a poor outcome in most types of lymphomas. However, the C1 subtype, dominated by TP53 mutations, had a better prognosis compared to the C2 subtype in PTL. C2 subtype exhibited a worse prognosis, aligning with our finding that the mechanism of immune escape in PTL was primarily the deletions of HLA rather than PD-L1/PD-L2 alterations, a contrast to other DLBCLs. Moreover, we calculated the tumor mutation burden (TMB) and identified that TMB can predict prognosis and recurrence rate in PTL. Our study underscores the significance of molecular typing in PTL based on mutational characteristics, which plays a crucial role in prognostication and guiding therapeutic strategies for patients.

Exploring the immune escape mechanisms in gastric cancer patients based on the deep AI algorithms and single-cell sequencing analysis.

Gastric cancer is a prevalent and deadly malignancy, and the response to immunotherapy varies among patients. This study aimed to develop a prognostic model for gastric cancer patients and investigate immune escape mechanisms using deep machine learning and single-cell sequencing analysis. Data from public databases were analysed, and a prediction model was constructed using 101 algorithms. The high-AIDPS group, characterized by increased AIDPS expression, exhibited worse survival, genomic variations and immune cell infiltration. These patients also showed immunotherapy tolerance. Treatment strategies targeting the high-AIDPS group identified three potential drugs. Additionally, distinct cluster groups and upregulated AIDPS-associated genes were observed in gastric adenocarcinoma cell lines. Inhibition of GHRL expression suppressed cancer cell activity, inhibited M2 polarization in macrophages and reduced invasiveness. Overall, AIDPS plays a critical role in gastric cancer prognosis, genomic variations, immune cell infiltration and immunotherapy response, and targeting GHRL expression holds promise for personalized treatment. These findings contribute to improved clinical management in gastric cancer.

Downregulation of lncRNA MIR17HG reduced tumorigenicity and Treg-mediated immune escape of non-small-cell lung cancer cells through targeting the miR-17-5p/RUNX3 axis.

Long noncoding RNA MIR17HG was involved with the progression of non-small-cell lung cancer (NSCLC), but specific mechanisms of MIR17HG-mediated immune escape of NSCLC cells were still unknown. The present study investigated the function of MIR17HG on regulatory T cell (Treg)-mediated immune escape and the underlying mechanisms in NSCLC. Expression of MIR17HG and miR-17-5p in NSCLC tissue samples were detected using quantitative real-time PCR (qRT-PCR). A549 and H1299 cells were transfected with sh-MIR17HG, miR-17-5p inhibitor, or sh-MIR17HG + miR-17-5p inhibitor, followed by cocultured with Tregs. Cell proliferation was measured using 5-ethynyl-20-deoxyuridine (Edu) staining assay and cell counting kit-8 (CCK-8) assay. Flow cytometry was used for determining positive numbers of FOXP3+CD4+/CD25+/CD8+ Tregs. Through subcutaneous injection with transfected A549 cells, a xenograft nude mouse model was established. Weights and volumes of xenograft tumors were evaluated. Additionally, the expressions of immune-related factors includingtransforming growth factor beta (TGF-β), vascular endothelial growth factor A (VEGF-A), interleukin-10 (IL-10), IL-4, and interferon-gamma (IFN-γ) in cultured cells, were evaluated by enzyme-linked immunosorbent assay and western blot analysis. Then, miR-17-5p was decreased and MIR17HG was enhanced in both NSCLC tissues and cell lines. MIR17HG knockdown significantly suppressed cell proliferation, tumorigenicity, and immune capacity of Tregs in A549 and H1299 cells, whereas sh-MIR17HG significantly reduced expression levels of VEGF-A, TGF-β, IL-4, and IL-10 but promoted the IFN-γ level in vitro and in vivo. Moreover, downregulation of miR-17-5p significantly reversed the effects of sh-MIR17HG. Additionally, we identified that runt- related transcription factor 3 (RUNX3) was a target of miR-17-5p, and sh-MIR17HG and miR-17-5p mimics downregulated RUNX3 expression. In conclusion, downregulation of MIR17HG suppresses tumorigenicity and Treg-mediated immune escape in NSCLC through downregulating the miR-17-5p/RUNX3 axis, indicating that this axis contains potential biomarkers for NSCLC.

Taenia solium cysticerci's extracellular vesicles Attenuate the AKT/mTORC1 pathway for Alleviating DSS-induced colitis in a murine model.

The excretory-secretory proteome plays a pivotal role in both intercellular communication during disease progression and immune escape mechanisms of various pathogens including cestode parasites like Taenia solium. The cysticerci of T. solium causes infection in the central nervous system known as neurocysticercosis (NCC), which affects a significant population in developing countries. Extracellular vesicles (EVs) are 30-150-nm-sized particles and constitute a significant part of the secretome. However, the role of EV in NCC pathogenesis remains undetermined. Here, for the first time, we report that EV from T. solium larvae is abundant in metabolites that can negatively regulate PI3K/AKT pathway, efficiently internalized by macrophages to induce AKT and mTOR degradation through auto-lysosomal route with a prominent increase in the ubiquitination of both proteins. This results in less ROS production and diminished bacterial killing capability among EV-treated macrophages. Due to this, both macro-autophagy and caspase-linked apoptosis are upregulated, with a reduction of the autophagy substrate sequestome 1. In summary, we report that T. solium EV from viable cysts attenuates the AKT-mTOR pathway thereby promoting apoptosis in macrophages, and this may exert immunosuppression during an early viable stage of the parasite in NCC, which is primarily asymptomatic. Further investigation on EV-mediated immune suppression revealed that the EV can protect the mice from DSS-induced colitis and improve colon architecture. These findings shed light on the previously unknown role of T. solium EV and the therapeutic role of their immune suppression potential.

Integrating Single-cell and Bulk RNA Sequencing Reveals Stemness Phenotype Associated with Clinical Outcomes and Potential Immune Evasion Mechanisms in Hepatocellular Carcinoma

Aims: Bulk and single-cell RNA sequencing data were analyzed to explore the association of stemness phenotype with dysfunctional anti-tumor immunity and its impact on clinical outcomes of primary and relapse HCC. Background: The stemness phenotype is gradually acquired during cancer progression; however, it remains unclear the effect of stemness phenotype on recurrence and clinical outcomes in hepatocellular carcinoma (HCC). Methods: The stemness index (mRNAsi) calculated by a one-class logistic regression algorithm in multiple HCC cohorts was defined as the stemness phenotype of the patient. Using single-cell profiling in primary or early-relapse HCC, cell stemness phenotypes were evaluated by developmental potential. Differential analysis of stemness phenotype, gene expression and interactions between primary and recurrent samples revealed the underlying immune evasion mechanisms. Results: A strong correlation was discovered between mRNAsi and clinical outcomes in patient with HCC. The high and low mRNAsi groups had distinct tumor immune microenvironments. Cellular stemness phenotype varied by cell type. Moreover, compared with primary tumors, early-relapse tumors had increased stemness of dendritic cells and tumor cells and reduced stemness of T cells and B cells. Moreover, in relapse tumors, CD8+ T cells displayed a low stemness state, with a high exhausted state, unlike the high stemness state observed in primary HCC. Conclusions: The comprehensive characterization of the HCC stemness phenotype provides insights into the clinical outcomes and immune escape mechanisms associated with recurrence.

PD-1/PD-L1 pathway: Current research in breast cancer.

Immunotherapy has shown encouraging outcomes in breast cancer (BC) treatment in recent years. The programmed cell death ligand 1 (PD-L1) transmembrane protein is suggested to function as a co-inhibitory factor in the immune response, where it collaborates with programmed cell death protein 1 (PD-1) to stimulate apoptosis, suppress cytokine release from PD-1 positive cells, and limit the growth of PD-1 positive cells. Furthermore, in many malignancies, PD-L1 reduces the immune system's response to neoplastic cells. These observations suggest that the PD-1/PD-L1 axis plays a vital role in cancer therapy and the regulation of cancer immune escape mechanisms. This review aimed to provide an overview of the functions of PD-1 and PD-L1 in BC cancer therapy. This research design is a literature review. The style is a traditional review on topics or variables relating to the PD-1/PD-L1 pathway. A literature search was carried out using three online databases. The search using the keywords yielded a total of 248 studies. Each result was filtered again according to the inclusion and exclusion criteria, resulting in a final total of 4 studies to be included in the literature review. The combination of PD-1/PD-L1 is essential for many malignancies. According to the evidence presented, this combination presents both an opportunity and a challenge in cancer treatment. Since many solid cancers, especially BC, express high levels of PD-1/PD-L1, cancer treatment mainly involves targeted therapies.

Analysis of the Immunogenicity of African Swine Fever F317L Protein and Screening of T Cell Epitopes.

The African swine fever virus (ASFV) encodes numerous proteins characterized by complex immune escape mechanisms. At present, the structure and function of these proteins, including the F317L protein, have yet to be fully elucidated. In this study, we examined the immunogenicity of the F317L protein. Mice were subcutaneously immunized with the F317L protein using initial and subsequent booster doses, and, at the 28th day post-treatment, we assessed the humoral and cellular immune responses of mice. The F317L protein stimulated production of specific antibodies and activated humoral immune responses. In addition, F317L stimulated the production of large amounts of IFN-γ by splenic lymphocytes, thereby activating cellular immune responses. Using informatics technology, we predicted and synthesized 29 F317L protein T cell epitopes, which were screened using IFN-γ ELISpot. Among these, the F25 (246SRRSLVNPWT255) peptide was identified as having a stronger stimulatory effect than the full-length protein. Collectively, our findings revealed that the ASFV F317L protein can stimulate both strong humoral and cellular immunity in mice, and that the F25 (246SRRSLVNPWT255) peptide may be a potential active T cell epitope. These findings will provide a reference for further in-depth studies of the F317L protein and screening of antigenic epitopes.

Engineering immune-evasive allogeneic cellular immunotherapies.

Allogeneic cellular immunotherapies hold a great promise for cancer treatment owing to their potential cost-effectiveness, scalability and on-demand availability. However, immune rejection of adoptively transferred allogeneic T and natural killer (NK) cells is a substantial obstacle to achieving clinical responses that are comparable to responses obtained with current autologous chimeric antigen receptor T cell therapies. In this Perspective, we discuss strategies to confer cell-intrinsic, immune-evasive properties to allogeneic T cellsand NK cells in order to prevent or delay their immune rejection, thereby widening the therapeutic window. We discuss how common viral and cancer immune escape mechanisms can serve as a blueprint for improving the persistence of off-the-shelf allogeneic cell therapies. The prospects of harnessing genome editing and synthetic biology to design cell-based precision immunotherapies extend beyond programming target specificities and require careful consideration of innate and adaptive responses in the recipient that may curtail the biodistribution, in vivo expansion and persistence of cellular therapeutics.

Prediction of Prospective Mutational Landscape of SARS-CoV-2 Spike ssRNA and Evolutionary Basis of Its Host Interaction.

Booster doses are crucial against severe COVID-19, as rapid virus mutations and variant emergence prolong the pandemic crisis. The virus's quick evolution, short generation-time, and adaptive changes impact virulence and evolvability, helping predictions about variant of concerns' (VOCs') landscapes. Here, in this study, we used a new computational algorithm, to predict the mutational pattern in SARS-CoV-2 ssRNA, proteomics, structural identification, mutation stability, and functional correlation, as well as immune escape mechanisms. Interestingly, the sequence diversity of SARS Coronavirus-2 has demonstrated a predominance of G- > A and C- > U substitutions. The best validation statistics are explored here in seven homologous models of the expected mutant SARS-CoV-2 spike ssRNA and employed for hACE2 and IgG interactions. The interactome profile of SARS-CoV-2 spike with hACE2 and IgG revealed a strong correlation between phylogeny and divergence time. The systematic adaptation of SARS-CoV-2 spike ssRNA influences infectivity and immune escape. Data suggest higher propensity of Adenine rich sequence promotes MHC system avoidance, preferred by A-rich codons. Phylogenetic data revealed the evolution of SARS-CoV-2 lineages' epidemiology. Our findings may unveil processes governing the genesis of immune-resistant variants, prompting a critical reassessment of the coronavirus mutation rate and exploration of hypotheses beyond mechanical aspects.

Targeting metabolism of breast cancer and its implications in T cell immunotherapy.

Breast cancer is a prominent health issue amongst women around the world. Immunotherapies including tumor targeted antibodies, adoptive T cell therapy, vaccines, and immune checkpoint blockers have rejuvenated the clinical management of breast cancer, but the prognosis of patients remains dismal. Metabolic reprogramming and immune escape are two important mechanisms supporting the progression of breast cancer. The deprivation uptake of nutrients (such as glucose, amino acid, and lipid) by breast cancer cells has a significant impact on tumor growth and microenvironment remodeling. In recent years, in-depth researches on the mechanism of metabolic reprogramming and immune escape have been extensively conducted, and targeting metabolic reprogramming has been proposed as a new therapeutic strategy for breast cancer. This article reviews the abnormal metabolism of breast cancer cells and its impact on the anti-tumor activity of T cells, and further explores the possibility of targeting metabolism as a therapeutic strategy for breast cancer.

Combination of IFN-gamma with STING agonist and PD-1 immune checkpoint blockade: a potential immunotherapy for gastric cancer.

Suppression of the cGAS-STING pathway is an immune escape mechanism in cancer cells. The critical role of this pathway in gastric cancer (GC) is not fully understood. Herein, we evaluated the effect of the interferon-gamma (IFN-gamma), STING agonist, PD-1 immune checkpoint blockade, and their combination on the cGAS-STING pathway in GC. Expression of cGAS and STING in tumor tissue samples and adjacent normal tissue (ANT) biopsies of fifty new GC patients was evaluated by quantitative real-time PCR (qRT-PCR). Moreover, cGAS and STING expression levels were examined in Peripheral Blood Mononuclear Cells (PBMC) samples of forty GC patients and twenty-five healthy subjects. The apoptosis rate of cancer cells was analyzed by Annexin V-FITC/PI. Cell proliferation was measured by the BrdU assay. Also, IFN-β levels were evaluated in the supernatants of the treated groups. The cGAS expression was decreased in patients with distant metastasis. Co-cultures treated with IFN-gamma showed an elevated level of cGAS and STING expressions in PBMC and cancer cells. The rate of apoptosis increased in all the treatment groups. In addition, the rate of proliferation in PBMCs increased in different treated groups. The main role of PBMCs in cytotoxicity was determined by a comparative analysis of the viability of cells treated with all treatments, both with and without PBMCs. The production of IFN-β was elevated in all treated groups. The current study suggests that a combination therapy using IFN-gamma, STING agonist, and anti-PD-1 antibody can provide a promising approach to the treatment of GC.

The role of fibrinogen-like protein 1 in immune escape and tumor growth mechanism of Warthin’s tumor

Background: One of the benign tumors of the salivary glands is Warthin’s Tumor (WT), which consists of cystic bilayer papillary epithelial cells accompanied by the presence of a lymphoid stroma. Several cases have been reported to turn malignant. One of the markers developed to identify tumor proteins is fibrinogen-like protein 1 (FGL1). Along with lymphocyte-activation gene 3 (LAG-3), FGL1 establishes an immune checkpoint pathway that plays a role in the mechanism of tumor immune escape. Not much has been reported regarding FGL1 expression in WT, but some studies have reported that its expression is associated with tumor growth. Purpose: This study aims to analyze the location of FGL1 expression in WT and its relation to the mechanism of tumor immune release through the interaction between FGL1 and LAG-3. Methods: Cases of WT (n = 11) and breast cancer (n = 1) were used as positive controls. All cases were stained with hematoxylin-eosin and Recombinant Anti-FGL1 antibody. The FGL1 expression was observed in the cell membrane, cytoplasm, and lymphoid stroma. The results are presented in the form of figures. Results: All cases of WT expressed FGL1 in the cell membrane, cytoplasm, and lymphoid stroma. Its expression in the cell membrane and cytoplasm is possibly related to the process of tumorigenesis and the increasing size of the lesion. Additionally, its expression is seen in the lymphoid stroma, which is closely related to immune escape by inhibiting lymphocytes against tumor cells. Conclusion: Warthin’s tumor cells express FGL1, and this expression plays a role in tumor immune escape mechanisms and tumor growth.

Abstract LB092: Novel tumor gene expression signatures predictive of outcome in large B cell lymphoma treated with CAR T cell therapy (axicabtagene ciloleucel)

Abstract We leveraged ZUMA-7, the first and largest, phase III randomized CAR-T cell clinical study of relapsed/refractory large B cell lymphoma (LBCL) in the 2nd line setting, to discover tumor gene expression (GE) signatures associated with outcome to anti-CD19 CAR-T cell therapy, Axicabtagene Ciloleucel (axi-cel). We performed GE profiling on pre-treatment LBCL tumor samples from 134 axi-cel-treated (safety dataset) patients using IO-360 Nanostring panel (769 genes). Multivariate analysis with penalized Cox models was conducted to identify genes whose expression (transcripts) associated with event-free survival (EFS), progression-free survival (PFS), and duration of response (DOR), leading to identification of two predictive GE signatures. For technical validation, the predictive value of these signatures was reproduced in a subset of ZUMA-7 samples profiled by RNA-seq (n=124). High levels (> median) of a favorable 6-transcript GE signature (6-GES) composed of CD19, CD45RA, CCL22, KLRK1, SOX11, and SIGLEC5 positively correlated with EFS (HR: 0.27, 95% CI: 0.16–0.44; P=1.82 × 10−8) and PFS (HR: 0.27, 95% CI: 0.16–0.46; P=1.35 × 10−7) in patients treated with axi-cel. The 6-GES may capture lymphomas with abundant adhesion molecules, a relatively low inflammation, and abundant expression of the targeted antigen (CD19). Conversely, high levels of an unfavorable 17-transcript GE signature (17-GES; CD45RO, BCL2, IL-18R1, TNFSF4 [OX40L], KLRB1 [CD161], KIR3DL2, ITGB8, DUSP5, GPC4, PSMB5, RPS6KB1, SERPINA9, NBN, GLUD1, ESR1, ARID1A, and SLC16A1) negatively correlated with Axi-cel EFS (HR: 6.19, 95% CI: 3.60–10.65; P=1.51 × 10−13) and PFS (HR: 7.58, 95% CI: 4.16–13.81; P=2.70 × 10−14). The 17-GES signature is consistent with a high level of immune infiltration and inflammation paralleled by the activation of immune-escape mechanisms, such as the upregulation of anti-apoptotic genes. Notably, the 17-GES was elevated at progression after axi-cel treatment (n=18). These signatures did not associate with outcome to 2nd-line SOC from ZUMA-7 (n = 122 for nanostring and n = 125 for ZUMA-7 RNAseq datasets, respectively), nor with outcome to 1st -line R-CHOP from two online datasets, indicating their predictive rather than prognostic value. In conclusion, our transcriptomic analysis of ZUMA-7 dataset identified novel GE signatures predictive of outcome with axi-cel. These GE signatures could support risk-stratification of LBCL patients. Citation Format: Yuan Tian, Justin Budka, Frederick L. Locke, Jason R. Westin, Christina To, Shilpa Shahani, Davide Bedognetti, Rhine R. Shen, Jorge Andrade, Simone Filosto. Novel tumor gene expression signatures predictive of outcome in large B cell lymphoma treated with CAR T cell therapy (axicabtagene ciloleucel) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB092.

Immune Response and Metastasis-Links between the Metastasis Driver MACC1 and Cancer Immune Escape Strategies.

Metastasis remains the most critical factor limiting patient survival and the most challenging part of cancer-targeted therapy. Identifying the causal drivers of metastasis and characterizing their properties in various key aspects of cancer biology is essential for the development of novel metastasis-targeting approaches. Metastasis-associated in colon cancer 1 (MACC1) is a prognostic and predictive biomarker that is now recognized in more than 20 cancer entities. Although MACC1 can already be linked with many hallmarks of cancer, one key process-the facilitation of immune evasion-remains poorly understood. In this review, we explore the direct and indirect links between MACC1 and the mechanisms of immune escape. Therein, we highlight the signaling pathways and secreted factors influenced by MACC1 as well as their effects on the infiltration and anti-tumor function of immune cells.

A single-cell atlas enables mapping of homeostatic cellular shifts in the adult human breast

Here we use single-cell RNA sequencing to compile a human breast cell atlas assembled from 55 donors that had undergone reduction mammoplasties or risk reduction mastectomies. From more than 800,000 cells we identified 41 cell subclusters across the epithelial, immune and stromal compartments. The contribution of these different clusters varied according to the natural history of the tissue. Age, parity and germline mutations, known to modulate the risk of developing breast cancer, affected the homeostatic cellular state of the breast in different ways. We found that immune cells from BRCA1 or BRCA2 carriers had a distinct gene expression signature indicative of potential immune exhaustion, which was validated by immunohistochemistry. This suggests that immune-escape mechanisms could manifest in non-cancerous tissues very early during tumor initiation. This atlas is a rich resource that can be used to inform novel approaches for early detection and prevention of breast cancer.

Impact of the SARS-CoV-2 Spike Protein on the Innate Immune System: A Review.

The Spike protein enables the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by binding to multiple receptors, including the angiotensin-converting enzyme 2 (ACE2). Scientific studies also indicate that Spike is involved in severe forms of coronavirus disease 2019 (COVID-19), "long-haul COVID diseases" - also known as "long COVID syndromes" or "post-acute sequelae of SARS-CoV-2 infection" (PACS) - or, recently, in adverse reactions to lipid nanoparticle-messenger ribonucleic acid (mRNA) vaccines or other anti-COVID19 products. Numerous mutations, notably within the subunit 1 of Spike (S1), prevent neutralization by antibodies, but more generally, the virus has developed numerous strategies to avoid immune system surveillance, especially type-I interferons (IFN-I). Meanwhile, a "hyperinflammatory" state, named "cytokine storm,"sets in. However, what role does the Spike protein play in the immune escape mechanisms? Can its inflammatory activities affect IFN-I? Does Spike block IFN-I or hijack them for the virus benefits? What are the other potential consequences? This article was written to provide an up-to-date and more general overview of the impact of the Spike protein on the innate immune system and its effectors at the molecular level.

Abstract 66: A new mechanism of tumor immune escape: TCF19 promotes the development of novel terminally exhausted T cells through E2F and MYC transcription family

Abstract Introduction: ICB (immune checkpoint blocking) has been a promising therapy in cancer treatment, but there are still some patients do not response to this therapy. And it is well known that the majority cells reactivated to ICB are TCF1+ precursor exhausted T cells. Thus, studying the formation and heterogeneity of exhausted T cells are very necessary for improving the efficacy of ICB therapy. In this research, we identified a new population as TCF19+PD1+TIM3+ exhausted CD8+ T cells, which blocked the responds to ICB therapy. Methods: We collected single cell sequencing data, including colorectal cancer, gastric cancer, melanoma, and lung cancer, and drew tumor immune cells map through cluster analysis. Then using bioinformatics tools CytoTRACE. T and CCAT. A to calculate the differentiation of exhausted T cell populations, which discovered a new subset of highly proliferative and poorly differentiated terminal exhausted T cells. Subsequently, the existence of this group of cells was verified through in vitro experiments, and their formation mechanism and correlation with immunotherapy were further studied. Results: We utilized CytoTRACE.T and CCAT.A to predict differentiation states of terminally exhausted T cells. Then GEO and KEGG pathway gene set enrichment analysis were used. After analyzing, we found out that there was a subset of cells in terminally exhausted T cells defined as TCF19+PD1+TIM3+ Tex cells, which presented low differentiated and high proliferative potential.Then we performed multiple immunofluorescence staining and flow cytometry to verified the existence of TCF19+PD1+TIM3+ Tex cells in vitro. Clarifing the generation of these cell subset through regulating the expression of E2F and MYC transcription factor families.By collecting pathological sections of patients with immunotherapy information, we found that the amount of TCF19+PD1+TIM3+ Tex cells was inversely proportional to the efficacy of ICB, through immunofluorescence staining and immunohistochemical analysis as well as organoid models cocultured with T cells. Conclusion: In this research, we found a new subpopulation of terminal Tex cells, which showed high proliferation and low differentiation. For cells, TCF19 mediated the generation of stemness terminal Tex cells may through regulating the expression of E2F and MYC. Finally, we discovered that the existence of TCF19+PD1+TIM3+ Tex cells can predict the efficacy of ICB, that the more TCF19+PD1+TIM3+ Tex cells contained, the less efficacy of ICB in patients.This finding may provide a new research direction for immunotherapy. Thus, more attention should be paid to this new cell subset. Citation Format: Fuhui Wang, Nan Dong, Changhua Zhang. A new mechanism of tumor immune escape: TCF19 promotes the development of novel terminally exhausted T cells through E2F and MYC transcription family [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 66.

Abstract 6836: Single-cell RNA sequencing of FUS::DDIT3 myxoid/round cell liposarcoma delineates mechanisms of oncogenic progression and immune escape

Abstract Myxoid/Round Cell Liposarcoma (MRCL) is an understudied soft tissue sarcoma that typically affects young adults and accounts for 30% of all liposarcomas. MRCL is characterized by oncogenic DDIT3 fusions, with FUS::DDIT3 being present in over 90% of cases. However, the cellular origins and oncogenic mechanisms of MRCL remain incompletely defined. Here we report oncogene transcription, and tumor-immune interactions in a cohort of 5 patient tumors with matched normal tissue for which we sequenced the transcriptomes of approximately 45,000 single cells using 10X Genomics, and full-length transcripts in approximately 20,000 single cells via Oxford Nanopore in 2 of these patients. Our results reveal two distinct cancer cell populations: Early Cancer Cells (ECC) and Advanced Cancer Cells (ACC). These cells appear to derive from adipose stem progenitor cells (ASPC) and fall along an ASPC→ECC→ACC pseudo-time axis. Along this axis, a distinct pattern emerges where oncogenes tend to be upregulated and tumor suppressor genes (TSG) tend to be downregulated and these appear to be mutually exclusive. Comparing ECC to ASPC we see increased expression of HIPPO and PI3K pathway oncogenes, FGFR2 and IGF1R RTKs, and downregulation of the TSG CDKN2A and CDKN1A. ACC show upregulation of hedgehog, ErbB, WNT, HIPPO, PI3K, and MAPK signaling pathways compared to ECC. Full-length transcript sequencing at the single-cell level shows a consistent expression of the FUS::DDIT3 fusion transcript in both ECC and ACC populations, confirming that these populations are cancer cells. Furthermore, MRCL displays several immune escape mechanisms, including poor immune infiltration and a marked decline of HLA Class I expression along the pseudo-time axis from ECC to ACC, suggesting a complex interplay between oncogenic signaling and immune evasion. These results suggest that MRCL likely originates from ASPC and expression of FUS:DDIT3 oncoprotein induces early HIPPO, PI3K, FGFR2, and insulin signaling pathway expression in ECC with further hedgehog, ErbB, MAPK, and WNT pathway activation in ACC. This progressive activation in oncogenic signaling is associated with immune evasion. This is the first single-cell RNA seq analysis to detect the presence of the FUS::DDIT3 fusion in ECC and ACC significantly advancing our understanding of the mechanisms of MRCL oncogenesis, immune escape, and identifies potential novel therapeutic targets. Citation Format: Rodrigo Gularte-Mérida, Evan Seffar, George Li, Young-Mi Kim, Narasimhan P. Agaram, Nicholas Socci, Francisco Sanchez-Vega, Nikolaus Schultz, Samuel Singer. Single-cell RNA sequencing of FUS::DDIT3 myxoid/round cell liposarcoma delineates mechanisms of oncogenic progression and immune escape [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 6836.

Abstract 5369: Mapping a T cell-cold phenotype to the colorectal cancer genome to identify POFUT1 as a driver of CD8+ T cell inhibition

Abstract CD8+ T cells are a critical anti-cancer immune subset; however, most colorectal cancer (CRC) patients have inadequate levels of CD8+ T cells in their tumor microenvironment (TME), contributing to poor survival and increased CRC aggression. While current immune checkpoint inhibitors are able to elicit anti-cancer CD8+ T cell responses in some patients, a majority of patients are still nonresponsive, and have minimal levels of tumor-infiltrating CD8+ T cells. This effect is potentially due to still-unrecognized cancer-driven mechanisms of immunosuppression. To identify candidate genes with roles in CD8+ T cell exclusion, we used a T cell gene signature (Tsig) to quantify CD8+ T cell infiltration in tumors profiled by RNA-seq. Partial Spearman correlation adjusted for tumor purity was performed for all annotated genes (N=20501) in the TCGA CRC RNA-seq data set (N=373) to identify genes most inversely correlated with Tsig. A locus at chromosome 20q was identified as the region of the genome most inversely correlated with Tsig - both by copy number analysis and by RNA expression. Interestingly, this region is the most copy number-amplified region in CRC tumors, and the genes at this locus are among the most highly expressed genes in CRC tumors. Within this region, the amplified and overexpressed gene Protein O-Fucosyltransferase 1 (POFUT1) had the strongest inverse correlation to Tsig. POFUT1 function is essential to canonical NOTCH signaling, and CRC-expressed NOTCH is implicated in increased CRC secretion of immunosuppressive factors IL-6, TGF-β and VEGF, and reduced CD8+ T cell secretion of IFN-γ and IL-2. POFUT1 overexpressing (OE) CT26 and MC38 cell lines were generated, and confirmed by Western Blot to have elevated levels of both cleaved NOTCH1 (NICD1), and NOTCH pathway transcriptional target, HES1. Murine CD8+ T cells were stimulated in vitro with CD3/CD28 microbeads and IL-2 in conditioned medium (CM) from vector or POFUT1-OE lines. CD8+ T cells cultured in CM from POFUT1-OE lines had significantly lower levels of activation markers (CD45 (p=0.0022), CD8 (p=0.0095), CD25 (p=0.0069, CD69 (p=0.0099)), reduced proliferation (CFSE, p=0.1281), and generated less ATP (Cell-Titer Glo, p=0.0003) than CD8+ T cells cultured in vector CM. In pan-cancer correlational analyses, POFUT1 is in the top 5% of genes most inversely correlated with Tsig in 12/24 solid tumor types annotated in TCGA (SKCM, THCA, COAD, READ, LUSC, CESC, KIRC, STAD, HNSC, KIRP, LIHC, UCEC p<0.0001). POFUT1 is robustly negatively correlated with T cell infiltration in many cancer types. We observed a positive association in CRC between POFUT1 and NOTCH signaling, and observed that POFUT1 expression reduced CD8+ T cell activity, potentially through upregulation of extracellular factors (in progress). Our findings fit a model where POFUT1 amplification in CRC promotes CD8+ T cell dysfunction as a mechanism of immune escape. Citation Format: Calvin J. Wagner, Eric D. Routh, Raven Rutledge, Lance D. Miller. Mapping a T cell-cold phenotype to the colorectal cancer genome to identify POFUT1 as a driver of CD8+ T cell inhibition [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 5369.

Abstract 2375: 23ME-01473, a novel anti-ULBP6/2/5 monoclonal antibody, reinvigorates anti-tumor NK cell function through NKG2D and FcγRIIIa activation

Abstract Using genome-wide association studies (GWAS) of the 23andMe genetic and healthy survey database across all phenotypes as part of our unique target discovery platform, we identified immuno-oncology (I/O)-related genetic variants as those with opposing directionality of effects on cancer and immunological phenotypes, which comprise the I/O signature. This I/O signature identified ULBP6 as a potential cancer therapeutic target. ULBP6 is a stress-induced cell surface ligand found on cancer cells, that binds to the activating receptor, NKG2D, on NK and T cells to induce tumor cell killing. However, as a mechanism of immune escape, tumors shed ULBP6 to produce a soluble form that attenuates NKG2D activation and is elevated in cancers. Given that the 2 allelic variants of ULBP6 have the highest binding affinity to NKG2D of all human NKG2D ligands, ULBP6 may be the most immunosuppressive soluble NKG2D ligand and a critical regulator of anti-tumor response. Accordingly, soluble ULBP6 exhibited significant suppression of IFNγ release and detectable cell surface NKG2D expression on healthy donor PBMCs using in vitro cell-based assays. Moreover, the overexpression of human ULBP6 in a genetically modified MC38 murine model resulted in decreased infiltration of NK, NKT, and CD8 T cells in vivo. As such, we developed a novel humanized monoclonal antibody, 23ME-01473, that binds with high affinity to ULBP6, and due to the high sequence homology, to ULBP2 and ULBP5, to block their interaction with NKG2D. By preventing the binding of soluble ULBP6 to NKG2D on PBMCs in vitro, 23ME-01473 restored NKG2D activation as measured by increased detectable cell surface NKG2D expression, IFNγ and granzyme B release, and tumor cell killing by PBMCs. To further leverage the binding of 23ME-01473 to ULBP6/2/5, 23ME-01473 was designed to have an afucosylated Fc domain that enhances its binding affinity to the activating Fc receptor, FcγRIIIa, on NK cells to induce ADCC against tumor cells. The combination of NKG2D and FcγRIIIa activation resulted in synergistic PBMC-mediated IFNγ secretion and enhanced tumor cell killing, compared to activation of either NKG2D or FcγRIIIa. Taken together, these results reveal ULBP6 as a novel cancer therapeutic target, and given the dual activation of NKG2D and FcγRIIIa, suggest the potential of 23ME-01473 to activate NK cells and elicit anti-tumor immunity. Citation Format: Joel Benjamin, Abigail Jarret, Shashank Bharill, Shruti Yadav, Dina Ayupova, Clifford Hom, Zahra Bahrami Dizicheh, I-Ling Chen, Anh Diep, Shi Shi, Caroline Bonnans, Danielle Kellar, Germaine Fuh, Maike Schmidt, Kimberline Gerrick, Patrick Koenig, Mauro Poggio. 23ME-01473, a novel anti-ULBP6/2/5 monoclonal antibody, reinvigorates anti-tumor NK cell function through NKG2D and FcγRIIIa activation [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 2375.