Inhibition Of Antigen Presentation Research Articles

Abstract LB403: Suppression of antigen presentation in pediatric rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma that is diagnosed based on the tumor cell's resemblance to embryonic or poorly differentiated skeletal muscle progenitors. Multiple immunotherapy clinical trials have found no evidence of immune reactivity against RMS, which has been attributed to the relatively low tumor mutation burden in these cancers. However, we made an unexpected observation that many patient RMS tumors had a near absence of MHC-I expression, a key requisite for anti-tumor CD8+ T lymphocytes and many immunotherapies. In this study, we explore the etiology of low antigen presentation in RMS and systematically evaluate therapeutic interventions that sensitize RMS to immunotherapy. Methods: Surface MHC-I expression was evaluated on pediatric tumor cell lines (n=65) from five major pediatric tumor types. RNA-seq, whole exome and genome sequencing data were analyzed from RMS tumors (total of n=247) and iPSC models of skeletal muscle development (PMID: 32011235). Epigentic drugs targeting DNA methylation (decitabine), EZH2 (tazemetostat), Class I/IV HDACs (mocetinostat), and LSD1 (GSK-LSD1) were used at sub-cytotoxic doses on RMS cell lines (n=10) and analyzed for surface MHC-I expression with flow cytometry and gene expression. T cell cytotoxicity assays were performed using engineered human T cells expressing an optimized TCR which recognizes an HLA*A2:01 restricted peptide from PRAME. Results: RMS cell line models exhibited a range of MHC-I expression with most having low or undetectable MHC-I expression, similar to that of a known MHC-I negative tumor type MYCN-amplified neuroblastoma. Exome sequencing of RMS tumors showed no evidence of loss-of-function alterations in essential antigen processing and presentation (APP) pathway genes. Gene expression profiling by RNAseq and western blotting, however, revealed an absence of core APP genes in many RMS tumors and cell lines. We uncovered that during a specific window of embryonic myogenesis, APP is suppressed and that RMS tumors mimicking this state of development show the deepest loss of APP. Multiple classes of epigenetic drugs were evaluated to rescue APP in RMS. Treatment of RMS using a DNA demethylating agent or HDAC inhibitor exhibited the strongest MHC-I restoration across RMS models with no changes observed using LSD1 or EZH2 inhibitors. As a proof-of-concept, we demonstrate that targeting the RMS antigen PRAME using TCR therapy is enhanced by the inclusion of epigenetic therapy. Conclusions and Significance: We revealed that epigenetic suppression of APP is a new hallmark of RMS and normal embryonic skeletal muscle development. Pharmacological reversal of APP pathway downregulation could be achieved using drugs targeting DNA methylation or class I/IV HDACs, and enhanced T cell cytotoxicity against RMS when used in combination. Overall, these findings may explain the historical lack of RMS responsiveness to immunotherapy and unlocks new immunotherapy strategies against RMS that rely on targeting MHC-I antigens. Citation Format: David Milewski, Hsien-Chao Chou, Vineela Gangalapudi, Meijie Tian, Yong Kim, Young Song, Jun Wei, Javed Khan. Suppression of antigen presentation in pediatric rhabdomyosarcoma [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 LB403.

Sequence signatures within the genome of SARS-CoV-2 can be used to predict host source.

We conducted an in silico analysis to better understand the potential factors impacting host adaptation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in white-tailed deer, humans, and mink due to the strong evidence of sustained transmission within these hosts. Classification models trained on single nucleotide and amino acid differences between samples effectively identified white-tailed deer-, human-, and mink-derived SARS-CoV-2. For example, the balanced accuracy score of Extremely Randomized Trees classifiers was 0.984 ± 0.006. Eighty-eight commonly identified predictive mutations are found at sites under strong positive and negative selective pressure. A large fraction of sites under selection (86.9%) or identified by machine learning (87.1%) are found in genes other than the spike. Some locations encoded by these gene regions are predicted to be B- and T-cell epitopes or are implicated in modulating the immune response suggesting that host adaptation may involve the evasion of the host immune system, modulation of the class-I major-histocompatibility complex, and the diminished recognition of immune epitopes by CD8+ T cells. Our selection and machine learning analysis also identified that silent mutations, such as C7303T and C9430T, play an important role in discriminating deer-derived samples across multiple clades. Finally, our investigation into the origin of the B.1.641 lineage from white-tailed deer in Canada discovered an additional human sequence from Michigan related to the B.1.641 lineage sampled near the emergence of this lineage. These findings demonstrate that machine-learning approaches can be used in combination with evolutionary genomics to identify factors possibly involved in the cross-species transmission of viruses and the emergence of novel viral lineages.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible virus capable of infecting and establishing itself in human and wildlife populations, such as white-tailed deer. This fact highlights the importance of developing novel ways to identify genetic factors that contribute to its spread and adaptation to new host species. This is especially important since these populations can serve as reservoirs that potentially facilitate the re-introduction of new variants into human populations. In this study, we apply machine learning and phylogenetic methods to uncover biomarkers of SARS-CoV-2 adaptation in mink and white-tailed deer. We find evidence demonstrating that both non-synonymous and silent mutations can be used to differentiate animal-derived sequences from human-derived ones and each other. This evidence also suggests that host adaptation involves the evasion of the immune system and the suppression of antigen presentation. Finally, the methods developed here are general and can be used to investigate host adaptation in viruses other than SARS-CoV-2.

ZNF689 deficiency promotes intratumor heterogeneity and immunotherapy resistance in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive disease characterized by remarkable intratumor heterogeneity (ITH), which poses therapeutic challenges. However, the clinical relevance and key determinant of ITH in TNBC are poorly understood. Here, we comprehensively characterized ITH levels using multi-omics data across our center's cohort (n = 260), The Cancer Genome Atlas cohort (n = 134), and four immunotherapy-treated cohorts (n = 109). Our results revealed that high ITH was associated with poor patient survival and immunotherapy resistance. Importantly, we identified zinc finger protein 689 (ZNF689) deficiency as a crucial determinant of ITH formation. Mechanistically, the ZNF689-TRIM28 complex was found to directly bind to the promoter of long interspersed element-1 (LINE-1), inducing H3K9me3-mediated transcriptional silencing. ZNF689 deficiency reactivated LINE-1 retrotransposition to exacerbate genomic instability, which fostered ITH. Single-cell RNA sequencing, spatially resolved transcriptomics and flow cytometry analysis confirmed that ZNF689 deficiency-induced ITH inhibited antigen presentation and T-cell activation, conferring immunotherapy resistance. Pharmacological inhibition of LINE-1 significantly reduced ITH, enhanced antitumor immunity, and eventually sensitized ZNF689-deficient tumors to immunotherapy in vivo. Consistently, ZNF689 expression positively correlated with favorable prognosis and immunotherapy response in clinical samples. Altogether, our study uncovers a previously unrecognized mechanism underlying ZNF689 deficiency-induced ITH and suggests LINE-1 inhibition combined with immunotherapy as a novel treatment strategy for TNBC.

Hydroxychloroquine exacerbates imiquimod-induced psoriasis-like dermatitis through stimulating overexpression of IL-6 in keratinocytes

Objective Hydroxychloroquine (HCQ) is a US Food and Drug Administration (FDA)-approved treatment for systemic lupus erythematosus (SLE) through inhibition of antigen presentation and subsequent reduction in T cell activation. Psoriasis relapse after antimalarial therapy have been reported in up to 18% of patients with psoriasis. Here, we explored the role of HCQ on exacerbating dermatitis utilizing an imiquimod (IMQ)-induced psoriasis-like dermatitis mouse model. Methods Thirty-six C57BL/6 female mice were divided into six groups: wild-type control, IMQ-Only, pre-treat HCQ (30 mg/kg and 60 mg/kg HCQ), and co-treat HCQ with IMQ (30 mg/kg and 60 mg/kg HCQ). Besides control, all were topically treated with IMQ for 5 days. Pharmacological effects and mechanisms of HCQ were assessed by clinical severity of dermatitis, histopathology, and flow cytometry. HaCaT cells were co-treated with both HCQ and recombinant IL-17A, followed by the detection of proinflammatory cytokine expression and gene profiles through enzyme-linked immunosorbent assay and next-generation sequencing. Results In the pre-treated and co-treated HCQ groups, skin redness and scaling were significantly increased compared to the IMQ-Only group, and Th17 cell expression was also upregulated. Acanthosis and CD11b+IL23+ dendritic cell (DC) infiltration were observed in the HCQ treatment group. IL-6 overexpression was detected in both the HaCaT cells and skin from the experimental mice. Psoriasis-related genes were regulated after being co-treated with HCQ and recombinant IL-17A in HaCaT cells. Conclusions HCQ exacerbates psoriasis-like skin inflammation by increasing the expression of IL-6, stimulating DC infiltration, and promoting Th17 expression in the microenvironment of the skin. Key messages This study provided possible mechanisms for inducing psoriasis during HCQ treatment through an animal model.

Abstract A026: Administration of preoperative ketorolac prevents osteosarcoma recurrence by promoting T cell memory and inhibiting immunosuppression

Abstract Introduction: Surgery induces an inflammatory response that is important for wound healing, but can also promote the survival of cancer cells and future tumor recurrence. Surgical injury activates the cyclooxygenase pathway and the production of downstream mediators such as prostaglandin E2 (PGE2) which has tumor-promoting and immunosuppressive activity. PGE2 stimulates regulatory T cells (Tregs), inhibits antigen presentation, and suppresses natural killer cells. Using a murine model of metastatic osteosarcoma, we investigated if preoperative blockade of this inflammatory cascade with ketorolac, a nonsteroidal anti-inflammatory drug, can boost anticancer T cell immunity. We also assessed if ketorolac had a synergistic effect when administered with a Programmed cell death protein-1 (PD-1) inhibitor. Methods: C3H/HeJ mice were inoculated with LM8 metastatic osteosarcoma cells. Tumors were resected at 50-150 mm3 and 2 hours before surgery, mice received an intraperitoneal injection containing either PBS, ketorolac, or ketorolac and antibodies to PD-1. All mice who survived beyond 100 days were considered long-term survivors and rechallenged with LM8 cells to evaluate immunological memory. Peripheral blood and spleen were collected and processed to perform flow cytometric analysis and single-cell sequencing of immune cell populations. Results: Following resection of primary tumors, 24 out of 25 control mice succumbed to lung metastasis by day 36 after resection while 4 out of 25 mice administered preoperative ketorolac exhibited long-term survival (defined as >90 days after tumor resection). All long-term survivors who received preoperative ketorolac were resistant to a rechallenge with LM8 cells. There was no difference in survival between mice who did and did not receive PD-1 inhibition. Flow cytometry analysis showed that the ketorolac-treated mice had increased T cell activity with a higher percent of effector and effector memory CD4+ and CD8+ T cells. In addition, a lower percent of myeloid derived suppressor cells (MDSCs) and Tregs were found in the spleens of mice treated with preoperative ketorolac. Single cell sequencing revealed that ketorolac-treated mice exhibited an expansion in B cells, specifically follicular B cells that expressed genes related to antigen presentation and cell proliferation. Conclusions: Administration of preoperative ketorolac in mice with osteosarcoma resulted in long-term survival after tumor resection and prevention of tumor recurrence after secondary challenge. Further, treatment with ketorolac reduced two cell populations of immunosuppressive significance, while enhancing immune cell memory. Thus, targeting surgery-induced inflammation with preoperative ketorolac should be further explored as a strategy to bolster a tumor-specific, T cell mediated immune response. Citation Format: Rebecca Pankove, Ramya Ganesan, Swati S Bhasin, Marina Michaud, Manoj Bhasin, Vikas P Sukhatme. Administration of preoperative ketorolac prevents osteosarcoma recurrence by promoting T cell memory and inhibiting immunosuppression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A026.

Baricitinib in Combination with High-Dose Dexamethasone As First-Line Treatment for Newly Diagnosed Immune Thrombocytopenia: A Prospective, Multicenter, Randomized Trial

Introduction: High-dose dexamethasone is among the standard initial therapeutic options for patients with newly diagnosed immune thrombocytopenia (ITP). Although most patients achieve initial remission, optimal first-line therapy remains a challenge due to the low rates of sustained response and the high incidence of corticosteroid-dependency. Antiplatelet autoantibodies and splenic macrophages play central roles in the pathogenesis of ITP. Baricitinib, a Janus-associated kinase 1/2 (JAK1/2) inhibitor, can inhibit antigen presentation by innate immune cells and subsequent T-cell activation while also alleviating B-cell abnormalities and antibody production. The results of our single-arm pilot study suggested that baricitinib might increase the rate of durable response in patients with steroid-resistant or relapsed ITP (NCT05446831, presented at EHA2023, P1594). Accordingly, we further conducted a randomized, controlled trial to evaluate the efficacy and safety of baricitinib plus high-dose dexamethasone as first-line therapy for adulthood ITP. Methods: This multicenter, open-label, randomized, controlled, phase 2 trial was conducted in 10 different tertiary medical centers in China. Adult patients with newly diagnosed, treatment-naïve, confirmed primary ITP who had a platelet count <30×10⁹/L, or a platelet count <50×10⁹/L with clinically significant bleeding, were eligible for this study. Patients were randomly assigned at a 1:1 ratio to receive baricitinib (2 mg daily for 6 consecutive months) plus high-dose dexamethasone (40 mg daily for 4 consecutive days, repeated in the case of lack of response by Day 10) or high-dose dexamethasone alone. The primary endpoint was durable response at the 6-month follow-up. Efficacy endpoints were analyzed in the intention-to-treat population. Safety profiles were analyzed in all patients receiving at least one dose of study medications. The study protocol was approved by the ethics committee or institutional review board of each participating center and was registered with ClinicalTrials.gov (NCT 05932524). Results: A total of 172 patients were screened, of which 132 patients were eligible for this study. Patients were randomly assigned to receive baricitinib plus high-dose dexamethasone (n=66) or high-dose dexamethasone monotherapy (n=66). Four patients did not receive allocated treatment, leaving 128 patients in the safety analysis set. At the 6-month follow-up, a significantly higher proportion of participants in the baricitinib plus high-dose dexamethasone group (42/66, 63.6%) than in the high-dose dexamethasone monotherapy group (25/66, 37.9%) achieved durable response (OR 2.870, 95% CI 1.416-5.816; p=0.003). Initial response was achieved by 52 (78.8%) patients in the combination group and 47 (71.2%) patients in the monotherapy group. No significant difference was observed in the median time from initial treatment to response between the two groups. Complete response (at any time during the treatment) was achieved by 40 (60.6%) patients in the combination group and 35 (53.0%) patients in the monotherapy group. Patients responding to treatment in the combination group showed significantly longer durations of response than responders in the monotherapy group. Patients in both groups reported significantly improved health-related quality of life. The most common adverse events were infections (15/64, 23.4%) and insomnia (12/64, 18.8%) in the combination group, and insomnia (10/64, 15.6%) and anxiety or mood disorders (9/64, 14.1%) in the monotherapy group. No grade 4 or worse adverse events or treatment-related deaths were reported in either group. No thrombotic events, major adverse cardiovascular events (MACE), treatment-related bleeding or anemia were observed. Treatment discontinuation due to an adverse event was reported in 2 (3.1%) patients in the combination group and 3 (4.7%) patients in the monotherapy group. Conclusions: This is the first study reporting JAK inhibition as a first-line therapy for ITP. Baricitinib at 2 mg daily plus high-dose dexamethasone was well tolerated in patients with newly diagnosed ITP and might contribute to achieving a higher rate of durable response.

Inhibitory effect of adenosine on adaptive antitumor immunity and intervention strategies.

The tumors with a microenvironment characterized by a lack of immune cell infiltration, referred as "cold tumors", typically exhibit low responsiveness to immune therapy. Targeting the factors contributing to "cold tumors" formation and converting them into "hot tumors" would be a novel strategy for improving the efficacy of immunotherapy. Adenosine, a hydrolysis product of ATP, accumulates with a significantly higher concentration in the tumor microenvironments compared to normal tissue and exerts inhibitory effects on tumor-specific adaptive immunity. Tumor cells, dendritic cells, macrophages, and T cells express abundant adenosine receptors on their surfaces. The binding of adenosine to these receptors initiates downstream signaling pathways that suppress tumor antigen presentation and immune cell activation, consequently dampening adaptive immune responses against tumors. Adenosine downregulates the expression of major histocompatibility complex class Ⅱ and co-stimulatory factors on dendritic cells and macrophages, thereby inhibiting antigen presentation to T cells. Adenosine also inhibits ligand-receptor binding and transmembrane signaling on T cells, concomitantly suppressing the secretion of anti-tumor cytokines and impairing T cell activation. Furthermore, adenosine hinders effector T cell trafficking to tumor sites and infiltration by inhibiting chemokine secretion and KCa3.1 channels. Additionally, adenosine promotes the secretion of immunosuppressive cytokines, increases immune checkpoint protein expression, and enhances the activity of immunosuppressive cells, collectively curbing cytotoxic T cell-mediated tumor cell killing. Given the immunosuppressive role of adenosine in adaptive antitumor immunity, several inhibitors targeting adenosine generation or adenosine receptor blockade are currently in preclinical or clinical development with the aim of enhancing the effectiveness of other immunotherapies. This review provides an overview of inhibitory effects of adenosine on adaptive antitumor immunity, elucidating the molecular mechanisms involved, and summarizes the latest advances in the application of adenosine inhibition strategies in anticancer immunotherapy.

Global inhibition of Ly-49/MHC-I interactions by anti-MHC-I augments innate and adaptive immunity against cancer metastasis.

Abstract We have identified an pan-anti-MHC-I mAb (M1/42), which blocks the interaction of NK cell inhibitory receptors (Ly-49 antigens) with MHC-I, but does not inhibit antigen presentation by MHC-I. Administration of M1/42 in vivo markedly activated IFNg-producing NK cells. NK cell-derived IFNg stimulated APC to produce IL-12/-15/-18 cytokine cascades that further drove the proliferation of NK cells and memory phenotype (MP) T cells. M1/42 treatments profoundly augmented innate and adaptive immunity against PD-1-sensitive and -resistant transplanted tumors. Given that NK cells are critical to control of cancer metastases, we evaluated the effects of M1/42 therapy in several metastatic cancer models. M1/42 treatments successfully constrained B16F10 lung metastasis by enhancing CD8 +T cell infiltration and inducing melanoma-specific MP CD8 +T cell expansion and activation. In contrast, treatment with conventional checkpoint inhibitors (anti-PD-1/PD-L1, anti-CTLA-4 and anti-NKG2A) had no therapeutic effect. scRNAseq analysis of B16F10 lung melanoma tumor infiltrating lymphocytes revealed that M1/42 treatment significantly enhanced the expression of multiple genes associated with NK cell proliferation and activation. M1/42 also enhanced Th1-type innate and adaptive immune responses, and greatly restricted liver and lung metastasis produced by pancreatic adenocarcinoma cell line via intrasplenic or intravenous injection. Thus, the global inhibition of Ly-49/MHC-I interactions by M1/42 results in activation of a potent innate and adaptive immune response that restrains the growth of both transplanted tumors and cancer metastasis produced by intrasplenic or intravenous injection of tumor cells. This work was supported by the Intramural Research Program of NIAID, NIH.

Abstract A10: Targeting MHC-I antigen presentation for cancer immune evasion in acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is a clonal hematopoietic neoplasm, accounting for ~80% of acute leukemia cases in adults. The poor clinical outcome of AML patients (~27% five-year overall survival) is largely due to the inefficiency and toxicity of standard therapies. In the past decade, immunotherapies or cellular therapies (CAR-T/TCR-T cells) have achieved FDA approvals in certain types of solid tumors and leukemia, but currently there is no approved immunotherapy in AML. One potential reason is that AML actively inhibits antigen presentation (AP) to reduce its immunogenicity, thus dampening the efficacies of immunotherapies. This growing consensus is supported by the clinical evidence that AML exhibits low levels of AP machinery at diagnosis and even lower when relapsed from conventional chemotherapies. Besides, better outcomes could be achieved when combining immune checkpoint blockade with neo-antigens-inducing hypomethylating agents, highlighting the importance of AP in AML. We thus hypothesize that targeting tumor-endogenous AP suppressors will enhance AML immunogenicity and benefit immunotherapies. Recent studies have suggested that autophagy or soluble protein PCSK9 can mediate MHC-I degradation or disrupt MHC-I recycling, respectively. Transcription/epigenetic repressors TRAF3 and EZH2, as well as thymidylate synthase, were identified as MHC-I inhibitors. However, their specificity in MHC-I modulation and precise roles in regulating tumor antigenic peptide-MHC-I complexes (pMHC-I) are still unclear. Hence, there is an increasing need to identify AML-specific AP regulation mechanisms. To map such mechanisms, we performed pMHC-I-guided CRISPR screens in both human and mouse AML cell lines. Through these screens, we constructed positive and negative regulatory networks of pMHC-I modulation and compared the role of these novel regulators in the simultaneous modulation of MHC-I expression. This is the first-in-class systematic identification of AP regulators in AML. Among these negative AP regulators, we are particularly interested in the interferon regulatory factor 2 binding protein 2 (IRF2BP2), an AML-specific transcriptional regulator that is highly expressed in AML compared with normal hematopoietic stem and progenitor cells (HSPCs). Notably, IRF2BP2 expression negatively correlates with major histocompatibility complex class I (MHC-I) expression, interferon (IFN) response signatures, and T cell activity in AML patients. Ablation of IRF2BP2 enhanced AP in AML and facilitated T cell-mediated elimination of AML. Moreover, IRF2BP2 depletion synergized with IFN treatment to further boost AP at both transcriptional and protein levels. Our findings reveal a new class of tumor-associated immune-evasion mechanisms that target AP, with potential application as therapeutic targets for next-generation cancer immunotherapies. Citation Format: Xufeng Chen, Qiao Lu, Hua Zhou, Jia Liu, Bettina Nadorp, Audrey Lasry, Zhengxi Sun, Jiangyan Zhang, Michael Cammer, Kun Wang, Zoe Ciantra, Jia You, Qianjin Guo, Hongbing Zhang, Debrup Sengupta, Ahmad Boukhris, Cheng Liu, Peter Cresswell, Patricia L. M. Dahia, Jun Wang, Iannis Aifantis. Targeting MHC-I antigen presentation for cancer immune evasion in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A10.

Abstract 6736: Suppression of antigen presentation is a hallmark of pediatric rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue tumor in children and young adults, accounting for approximately 350-400 new cases annually in the US. These tumors are diagnosed based on their expression of skeletal muscle lineage markers. Clinical trials testing conventional immunotherapies such as cancer vaccines or immune checkpoint blockade (ICB) therapy have found no activity in RMS, although the root causes of failure are largely unexplored. A recent study by our group found that RMS tumors have a lack of immune infiltrate, consistent with previous reports. However, we made an unexpected observation that RMS tumors had a near absence of antigen processing and presentation (APP) gene expression, a key requisite for anti-tumor CD8+ T lymphocytes. In this study, we investigate the root causes of the lack of APP in RMS and evaluate implications for immunotherapy. Methods: Publicly available DNA sequencing datasets were used to evaluate APP gene mutations in RMS tumor samples. RNAseq from reporter iPSC cell lines demarcating specific stages of skeletal muscle differentiation were used to evaluate APP gene expression during myogenesis. Surface MHC class I expression was evaluated using flow cytometry against a panel of pediatric sarcoma cell lines. RMS PDX and cell line APP protein levels were measured by western blot. Stimulation of APP in RMS cell lines was tested using IFNγ or sub-cytotoxic doses of different epigenetic therapeutics (DNMTi, HDACi, EZH2i) before analyzing surface MHC class I expression by flow cytometry. Results: RNAseq analysis of the differentiation states across the normal human skeletal muscle lineage revealed a myoblastic cell state which transiently downregulates the APP pathway. RNA expression of HLA-A,B,C genes were lower in RMS than any other pediatric extracranial solid tumor histology studied. RMS cell lines and PDX models also displayed low APP pathway gene expression by western blot and flow cytometry, with FOXO1 fusion positive RMS having a near absence of expression. RMS tumor exome sequencing data from RMS patients showed a lack of enrichment of APP gene mutations. Instead, we found that APP could be induced in nearly all samples tested using IFNγ or epigenetic stimulation with Decitabine, Entinostat, or Tazemetostat. Conclusions: We uncovered a previously unappreciated lack of antigen presentation in RMS which phenocopies myoblasts in normal skeletal muscle development. The downregulation of MHC class I antigen presentation is not due to APP pathway mutations but rather a partially reversible downregulation mirroring this specific stage of normal skeletal muscle development. Our findings suggest that pharmacological reversal of the APP pathway downregulation may enhance immunotherapies targeting MHC class I antigens in RMS. Citation Format: David Milewski, Meijie Tian, Yong Kim, Jun Wei, Javed Khan. Suppression of antigen presentation is a hallmark of pediatric rhabdomyosarcoma [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 6736.

Abstract 954: PIK3CA mutation induces immunogenicity and increases the immune checkpoint inhibitor response in urothelial carcinoma

Abstract Background: Immune checkpoint inhibitor (ICI) has been widely used for advanced urothelial carcinoma (mUC) globally. However, little is known about the potential predictive factors of the ICI response. The study aims to identify a potential biomarker to predict ICI response in UC. Material and methods: We performed targeted next generation sequencing (NGS) of pre-treatment tumor Formalin-fixed paraffin-embedded (FFPE) samples by using ACTOnco® comprehensive genomic panel (ACT Genomics, Co. Ltd., Taiwan) to target all coding exons of 440 cancer-associated genes. We retrospective analyzed a total of 70 patients with mUC underwent at least one cycle of ICI with or without chemotherapy at Kaohsiung Chang Gung Memorial Hospital between April 2016 and November 2019. Patients were defined as responders (CR/PR) and non-responders (SD/PD) according to the RECIST evaluation. TMB was estimated by calculating all coding exons variants, including somatic non-synonymous, frameshift and splice site variants. Results: A total of 55 UC tumor samples passed QC process were analyzed. The median age was 67, and 74% of all patients were in ICI monotherapy cohort. The most common genetic alterations were TP53 (66%), KMT2D (42%) and MUC16 (32%). In ICI monotherapy group, responders had a higher TMB than non-responders (median 19.5 vs 6.5 mt/Mb; p = 0.004). Patients with high TMB on ICI monotherapy had a significantly longer PFS (14.6 vs 2.9 months; p = 0.003) and OS (NR vs 11.0 months; p = 0.01) than TMB low patients. We found that PIK3CA (32% vs 3%), UBR5 (23% vs 0%), PRKDC (32% vs 10%) were the top 3 enriched mutations between responders and non-responders. All PIK3CA mutation were hot-spot mutation, and tumor harbored PIK3CA mutation had a median high TMB than PIK3CA wild type in UC, NSCLC, breast and colorectal cancer. By analyzing tumor and immune microenvironment by TISIDB platform, UC with PIK3CA mutation was highly associated with increasing MHC molecules expression (HLA-A, HLA-B, HLA-C, B2M) in UC and gastric cancer. In vitro study, we confirmed anti-PDL1 with PBMC had a synergistic cytotoxicity in PIK3CA mutated cell lines (BFTC-909, AGS and DLD-1), but not in PIK3CA wild type cell lines (T24, RT4, UMUC-14, N87 and SW837). Knocking-down PIK3CA expression diminished the cytotoxic activity of anti-PDL1 with PBMC in BFTC-909 and AGS cells. PIK3CA down-regulation inhibits antigen presentation machinery process. We confirmed that knockout PIK3CA gene function by CRISPR/Cas9 decreased MHC1 and B2M molecules expression, and showing modest resistance to ICI than mice with preserved PIK3CA function. Conclusion: UC with PIK3CA missense mutation exhibits a higher immunogenicity through enhancing MHC class I and B2M expression. PIK3CA missense mutation is a potential predictive marker for ICI response in UC. Citation Format: Harvey Yu-Li Su, Ling-Yi Xiao, Shih-Yu Huang, Li-Chung Chang, Yi-Hua Chen, Hao-Lun Luo, Chia-Ling Wu. PIK3CA mutation induces immunogenicity and increases the immune checkpoint inhibitor response in urothelial carcinoma [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 954.

Abstract GS5-05: ZNF689 deficiency promotes intratumor heterogeneity and resistance to immune checkpoint blockade in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive disease characterized by remarkable intratumor heterogeneity (ITH), which poses a significant therapeutic challenge. However, the key determinants and underlying mechanisms of ITH in TNBC remain to be fully elucidated. Methods: We used multi-omics data from our cohort (n = 260) and The Cancer Genome Atlas (n = 134) cohort to comprehensively characterize ITH at the genetic and histologic levels. Transcriptomic differences between high ITH and low ITH tumors were compared to identify the core genes contributing ITH in TNBC. Xenograft models were used to examine the role of key determinants in TNBC ITH. The molecular mechanism was investigated by mass spectrometry, coimmunoprecipitation, pull-down, RNA-seq, long interspersed element-1 (LINE-1) reporter, ATAC-seq, luciferase reporter assays, chromatin immunoprecipitation, flow cytometry and coculture assay. Results: We found that high ITH was associated with poor patient survival and immune checkpoint blockade (ICB) resistance, which were validated in four independent ICB-treated trials. Further analysis indicated zinc finger protein 689 (ZNF689) deficiency as an important determinant of TNBC ITH. Mechanistically, the ZNF689-TRIM28 complex directly bound to the promoter of LINE-1, inducing H3K9me3-mediated transcriptional silencing. ZNF689 deficiency reactivated LINE-1 retrotransposition to exacerbate genomic instability, which promoted ITH. ZNF689 deficiency-induced ITH inhibited antigen presentation and CD8+ T cell infiltration, leading to ICB resistance. Pharmacological inhibition of LINE-1 retrotransposition reduced ITH, augmented antitumor immunity, and eventually sensitized ZNF689-deficient tumors to ICB. Consistently, ZNF689 expression positively correlated with favorable prognosis and ICB responsiveness in clinical samples. Conclusions: Our study uncovers a new mechanism underlying ZNF689 deficiency-induced ITH and suggests LINE-1 inhibition combined with ICB as a novel treatment strategy in TNBC. Citation Format: Li-Ping Ge, Xi Jin, Gen-Hong Di, Yi-Zhou Jiang, Zhi-Ming Shao. ZNF689 deficiency promotes intratumor heterogeneity and resistance to immune checkpoint blockade in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS5-05.

Vitamin D and Omega-3 (Fatty Acid) Supplementation in Pregnancy for the Primary Prevention of Food Allergy in Children-Literature Review.

During the last decades the prevalence of food allergy (FA), an adverse immune response to a specific food antigen, has risen, with negative effects on the quality of life (QoL) of many children and their families. The pathogenesis of FA is complex, involving both genetic and environmental factors. SPINK5, STAT6, HLA and FOXP3 are some of the genes that are reported to be implicated in FA development. Regarding environmental factors, particular interest has been focused on modification of the dietary habits of pregnant women for the primary prevention of FA. Specifically, Vitamin D and omega-3 (Ω-3) fatty acid supplementation during pregnancy may influence the development of FA in the offspring. Vitamin D is a hormone with various actions, including mediation of the immune system, reducing the production of inflammatory cytokines and promoting tolerance. Vitamin D deficiency in pregnancy suppresses T-regulatory cells in the fetus, and Vitamin D supplementation might protect against FA development. Dietary Ω-3 fatty acids are found mainly in fish and vegetable oils. They are beneficial for human health, playing a role in the immune system as anti-inflammatory agents, and providing cell membrane stabilization with inhibition of antigen presentation. It is documented that maternal supplementation with Ω-3 during pregnancy may protect from allergic sensitization in the children. The aim of this literature review was to explore the potential preventive role of maternal supplementation during pregnancy with Vitamin D and Ω-3 in the development of FA in the offspring. With the prevalence of FA rising, all the possible protective mechanisms and measures for FA prevention need to be explored, starting with those that can be modified.

Single-Cell Profiling Comparisons of Tumor Microenvironment between Primary Advanced Lung Adenocarcinomas and Brain Metastases and Machine Learning Algorithms in Predicting Immunotherapeutic Responses.

Brain metastasis (BM) occurs commonly in patients with lung adenocarcinomas. Limited evidence indicates safety and efficacy of immunotherapy for this metastatic tumor, though immune checkpoint blockade has become the front-line treatment for primary advanced non-small cell lung cancer. We aim to comprehensively compare tumor microenvironments (TME) between primary tumors (PT) and BM at single-cell resolution. Single-cell RNA transcriptomics from tumor samples of PT (N = 23) and BM (N = 16) and bulk sequencing data were analyzed to explore potential differences in immunotherapeutic efficacy between PT and BM of lung adenocarcinomas. Multiple machine learning algorithms were used to develop and validate models that predict responses to immunotherapy using the external cohorts. We found obviously less infiltration of immune cells in BM than PT, characterized specifically by deletion of anti-cancer CD8+ Trm cells and more dysfunctional CD8+ Tem cells in BM tumors. Meanwhile, macrophages and dendritic cells within BM demonstrated more pro-tumoral and anti-inflammatory effects, represented by distinct distribution and function of SPP1+ and C1Qs+ tumor-associated microphages, and inhibited antigen presentation capacity and HLA-I gene expression, respectively. Besides, we also found the lack of inflammatory-like CAFs and enrichment of pericytes within BM tumors, which may be critical factors in shaping inhibitory TME. Cell communication analysis further revealed mechanisms of the immunosuppressive effects associated with the activation of some unfavorable pathways, such as TGFβ signaling, highlighting the important roles of stromal cells in the anti-inflammatory microenvironment, especially specific pericytes. Furthermore, pericyte-related genes were identified to optimally predict immunotherapeutic responses by machine learning models with great predictive performance. Overall, various factors contribute to the immunosuppressive TME within BM tumors, represented by the lack of critical anti-cancer immune cells. Meanwhile, pericytes may help shape the TME and targeting the associated mechanisms may enhance immunotherapy efficacy for BM tumors in patients with lung adenocarcinomas.

Nipocalimab’s Selective Targeting of FcRn and IgG Clearance Preserves Key Immune Functions

Objective To characterize the effect of nipocalimab, a fully human, effectorless IgG1 anti-neonatal Fc receptor (FcRn) monoclonal antibody on immune function. Background Nipocalimab binds to FcRn with high affinity which prevents IgG recycling, leading to reduced serum levels of total IgGs, including pathogenic IgG autoantibodies. Rapid, sustained lowering of IgG was observed in the phase 2 VIVACITY study in generalized myasthenia gravis (gMG) and in phase 1 healthy volunteers. In gMG patients, nipocalimab induced rapid and sustained lowering of anti-AChR autoantibodies and MG-ADL scores, but no serious adverse events including clinically significant infections. Design/Methods Nipocalimab was evaluated extensively in vitro and in nonhuman primate-based chronic toxicology studies to evaluate selectivity, tolerability, safety and immunopharmacology. Safety, tolerability and immune-focused assessments in clinical phase 1 and Phase 2 MG studies were also completed (NCT02828046, NCT03772587). Results Nipocalimab binds specifically in vitro to FcRn without activation of effector function or inhibition of antigen presentation. In nonhuman primates administered up to 300 mg/kg nipocalimab QW for up to 6 months, sustained lowering of IgG was observed without adverse effects. Immunotoxicology identified no effect on immune cell phenotypes; CD8 T cell, NK or innate cell functions; T-dependent neoantigen IgM responses. Neoantigen IgG production was observed, but with lowered peak IgG titers consistent with the anticipated increase in IgG clearance with nipocalimab. In clinical studies, nipocalimab demonstrated a reproducible selective decrease in total serum IgG, including all subclasses of IgG, with no effect on IgM, IgA, IgE, CH50, C3, C4, inflammatory cytokines or acute phase proteins including, C-reactive protein (CRP). Conclusions These data suggest that nipocalimab can selectively lower IgG and IgG autoantibodies while preserving cellular immunity, complete IgM response and IgG production after neoantigen challenge. Overall, nipocalimab's selective effect on IgG recycling provides a mechanistic rationale for potentially decreased infection risk despite substantial IgG lowering.

SELECTIVE TARGETING OF FCRN AND IGG CLEARANCE BY NIPOCALIMAB PRESERVES KEY IMMUNE FUNCTIONS

Autoantibody disorders are caused by immune-system-produced antibodies that can damage the body's tissues or organs. In the case of warm autoimmune hemolytic anemia (wAIHA), pathogenic autoantibodies lead to the destruction of red blood cells. There is an unmet need for innovative targeted therapies that address the underlying pathogenesis of wAIHA in a safe and efficacious way. Nipocalimab is a fully human, aglycosylated, effectorless IgG1 monoclonal antibody that blocks the IgG binding site on the neonatal Fc receptor (FcRn) which prevents IgG recycling, leading to reduced serum levels of total IgGs, including pathogenic IgG autoantibodies. Nipocalimab is in clinical development for the treatment of multiple IgG-mediated autoantibody diseases, including wAIHA (NCT04119050). Rapid, sustained lowering of IgG was observed in the phase 2 VIVACITY study in generalized myasthenia gravis (gMG) and in phase 1 healthy volunteers. In gMG patients, nipocalimab induced rapid and sustained lowering of anti-AChR autoantibodies and MG-ADL scores, but no serious adverse events including clinically significant infections. Here, we characterize the effect of nipocalimab on immune function. Nipocalimab was evaluated extensively in vitro and in nonhuman primate-based chronic toxicology studies to evaluate selectivity, tolerability, safety and immunopharmacology. Safety, tolerability and immune-focused assessments in clinical phase 1 and Phase 2 MG studies were also completed (NCT02828046, NCT03772587). Nipocalimab binds specifically in vitro to FcRn without activation of effector function or inhibition of antigen presentation. In nonhuman primates administered up to 300 mg/kg nipocalimab QW for up to 6 months, sustained lowering of IgG was observed without adverse effects. Immunotoxicology identified no effect on immune cell phenotypes; CD8 T cell, NK or innate cell functions; T-dependent neoantigen IgM responses. Neoantigen IgG production was observed, but with lowered peak IgG titers consistent with the anticipated increase in IgG clearance with nipocalimab. In clinical studies, nipocalimab demonstrated a reproducible selective decrease in total serum IgG, including all subclasses of IgG, with no effect on IgM, IgA, IgE, CH50, C3, C4, inflammatory cytokines or acute phase proteins including, C-reactive protein (CRP). These data suggest that nipocalimab can selectively lower IgG and IgG autoantibodies while preserving cellular immunity, complete IgM response and IgG production after neoantigen challenge. Overall, nipocalimab's selective effect on IgG recycling provides a mechanistic rationale for potentially decreased infection risk despite substantial IgG lowering. LEL *, FY *, CJB *, JB *, RX *, AMBP are employed by the Janssen Pharmaceutical Companies of Johnson & Johnson and may hold stock or stock options in Johnson & Johnson; ST * is an employee of Orna Therapeutics; SK * is an employee of Checkmate Pharmaceuticals; JD * is an employee of Faze Medicines; WA * is an employee of Kisbee Therapeutics. *Former employees of Momenta Pharmaceuticals which was acquired by Janssen Pharmaceutical Companies of Johnson & Johnson.

Innate and adaptive immune evasion by dengue virus.

Dengue is a mosquito-borne disease which causes significant public health concerns in tropical and subtropical countries. Dengue virus (DENV) has evolved various strategies to manipulate the innate immune responses of the host such as ‘hiding’ in the ultrastructure of the host, interfering with the signaling pathway through RNA modifications, inhibiting type 1 IFN production, as well as inhibiting STAT1 phosphorylation. DENV is also able to evade the adaptive immune responses of the host through antigenic variation, antigen-dependent enhancement (ADE), partial maturation of prM proteins, and inhibition of antigen presentation. miRNAs are important regulators of both innate and adaptive immunity and they have been shown to play important roles in DENV replication and pathogenesis. This makes them suitable candidates for the development of anti-dengue therapeutics. This review discusses the various strategies employed by DENV to evade innate and adaptive immunity. The role of miRNAs and DENV non-structural proteins (NS) are promising targets for the development of anti-dengue therapeutics.

Discovery of the First Selective Nanomolar Inhibitors of ERAP2 by Kinetic Target‐Guided Synthesis

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a key enzyme involved in the trimming of antigenic peptides presented by Major Histocompatibility Complex class I. It is a target of growing interest for the treatment of autoimmune diseases and in cancer immunotherapy. However, the discovery of potent and selective ERAP2 inhibitors is highly challenging. Herein, we have used kinetic target-guided synthesis (KTGS) to identify such inhibitors. Co-crystallization experiments revealed the binding mode of three different inhibitors with increasing potency and selectivity over related enzymes. Selected analogues engage ERAP2 in cells and inhibit antigen presentation in a cellular context. 4 d (BDM88951) displays favorable in vitro ADME properties and in vivo exposure. In summary, KTGS allowed the discovery of the first nanomolar and selective highly promising ERAP2 inhibitors that pave the way of the exploration of the biological roles of this enzyme and provide lead compounds for drug discovery efforts.

Discovery of the First Selective Nanomolar Inhibitors of ERAP2 by Kinetic Target-Guided Synthesis.

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a key enzyme involved in the trimming of antigenic peptides presented by Major Histocompatibility Complex class I. It is a target of growing interest for the treatment of autoimmune diseases and in cancer immunotherapy. However, the discovery of potent and selective ERAP2 inhibitors is highly challenging. Herein, we have used kinetic target-guided synthesis (KTGS) to identify such inhibitors. Co-crystallization experiments revealed the binding mode of three different inhibitors with increasing potency and selectivity over related enzymes. Selected analogues engage ERAP2 in cells and inhibit antigen presentation in a cellular context. 4 d (BDM88951) displays favorable in vitro ADME properties and in vivo exposure. In summary, KTGS allowed the discovery of the first nanomolar and selective highly promising ERAP2 inhibitors that pave the way of the exploration of the biological roles of this enzyme and provide lead compounds for drug discovery efforts.

Enhancing the Immunogenicity of Vaccinia Virus.

The conventional live smallpox vaccine based on the vaccinia virus (VACV) cannot be widely used today because it is highly reactogenic. Therefore, there is a demand for designing VACV variants possessing enhanced immunogenicity, making it possible to reduce the vaccine dose and, therefore, significantly eliminate the pathogenic effect of the VACV on the body. In this study, we analyzed the development of the humoral and T cell-mediated immune responses elicited by immunizing mice with low-dose VACV variants carrying the mutant A34R gene (which increases production of extracellular virions) or the deleted A35R gene (whose protein product inhibits antigen presentation by the major histocompatibility complex class II). The VACV LIVP strain, which is used as a smallpox vaccine in Russia, and its recombinant variants LIVP-A34R*, LIVP-dA35R, and LIVP-A34R*-dA35R, were compared upon intradermal immunization of BALB/c mice at a dose of 104 pfu/animal. The strongest T cell-mediated immunity was detected in mice infected with the LIVP-A34R*-dA35R virus. The parental LIVP strain induced a significantly lower antibody level compared to the strains carrying the modified A34R and A35R genes. Simultaneous modification of the A34R gene and deletion of the A35R gene in VACV LIVP synergistically enhanced the immunogenic properties of the LIVP-A34R*-dA35R virus.