Type I Interferon Research Articles

Abstract 1356: The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation

Abstract VISTA, a predominantly myeloid immune checkpoint, promotes an immunosuppressive tumor microenvironment and has been associated with resistance to approved checkpoint therapies. While blockade of VISTA with antibodies has shown anti-tumor efficacy in preclinical models, the biological mechanisms that underlie this effect remain poorly understood. To investigate the mechanistic basis of anti-tumor responses, we performed single-cell RNA sequencing of tumor infiltrating leukocytes from the cell-derived murine colon carcinoma xenograft model, CT26, which is known to be sensitive to VISTA blockade. We used HMBD-002, a uniquely blocking but non-depleting clinical-stage IgG4 anti-VISTA antibody, to identify the key immune cells and pathways impacted by VISTA blockade in the absence of Fc-mediated cell depletion. The translatability of these findings was validated in a human PBMC mixed leukocyte reaction assay. Subsequently, targeted depletion or inhibition of the identified immune populations and pathways was performed to confirm their contribution to the anti-tumor efficacy of HMBD-002.The blockade of VISTA by HMBD-002 significantly increased CD8+ T cells in the CT26 tumors, promoting an activated cytotoxic Tc1 program without inducing exhaustion. Concurrently, a substantial increase in proinflammatory M1 tumor-associated macrophages (TAMs) was noted within treated tumors, while other immune cell populations remained unaffected. Among the signaling pathways, the type-I interferon (IFN) response exhibited a significant upregulation, particularly in TAMs, indicating a type-I IFN-driven M1 reprogramming by HMBD-002. Validation experiments further demonstrated that the depletion of TAMs or CD8+ T cells, but not CD4+ T cells, abrogated the efficacy of HMBD-002. Moreover, blocking the type-I IFN signaling effectively impeded HMBD-002-induced TAM reprogramming and subsequent anti-tumor efficacy, thus confirming the pivotal roles of CD8+ T cells, TAMs, and type-I IFN signaling in driving the anti-tumor response to VISTA blockade. In conclusion, VISTA blockade with HMBD-002 promotes an anti-tumor response by type-I IFN mediated reprogramming of TAMs and activation of cytotoxic CD8+ T cells. Citation Format: Bhushan Dharmadhikari, Olga Zharkova, Dipti Thakkar, Roberto Tirado-Magallanes, Konrad Paszkiewicz, Piers J. Ingram, Jerome D. Boyd-Kirkup. The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell 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 1356.

Abstract 3927: Metabolic inhibition of BATF2 dampens type-I interferon-mediated immune sensing of cancer

Abstract Background: The type-I interferon (IFN-I) system appeared before adaptive immunity during evolution as the primary defense system for metazoans. Initiating cancers frequently disable this first line of defense before establishing T-cell exhaustion programs. However, the molecules linking IFN-I, danger signals, and unique metabolic conditions in the tumor microenvironment (TME) remain to be discovered. Methods: We performed centered log-ratio transformation on the cluster composition data using the R-package ALDEx2 to compare the clusters in single-cell data sets. IFN-I signatures were quantified using qPCR and ELISA. ChIP was performed to examine the H3K27me3 epigenetic marker on the promoters of BATF2 and IFN-I genes in bone marrow-derived macrophage (BMDM). For the cigarette smoke carcinogen-induced head and neck squamous cell carcinoma (HNSCC) model, experimental mice were fed with water containing 50 μg/mL of 4-NQO for 16 weeks and then switched to regular feeding water. We started monitoring 10 weeks post-treatment for the number, location, and diameter of the lesions. TME analysis was performed using single-cell RNA-Seq and multispectral imaging. Results: We analyzed three large independent cohorts of patients and found that BATF2 strongly correlated with the immunogenicity of HNSCC, with a stronger correlation coefficient than STING. We generated Batf2−/− BMDM and found that Batf2 deficiency rendered cells insensitive to STING agonists. However, the dsRNA-induced IFN-I activation was not affected by Batf2 deletion. BATF2 interacted with STING, promoting its phosphorylation and oligomerization. Glutamine is enriched in the TME. We found that glutamine inhibited the BATF2-STING pathway by increasing a repressive epigenetic marker, H3K27me3, on the promoters of BATF2 and IFNB1. The expression of BATF2 in cancer cells resulted in IFN-I- and γδ T-cell-dependent tumor rejection. Single-cell immune profiling showed that Batf2-expressing tumors contained significantly expanded Cxcl10+ myeloid cells and γδ T-cells. Utilizing the 4-NQO-induced HNSCC model, we found that BATF2 limited suppressive myeloid cells and expanded effector cells in the TME, serving as a potent tumor suppressor. Conclusion: BATF2 is a glutamine-responsive tumor suppressor that limits tumor initiation by engaging the STING-γδ T-cell pathway. Acknowledgments: This work was supported by NIH grants R01 DE026728, U01 DE029255, R01 DE031951, and U01 DE033330 Citation Format: Wang Gong, Hülya Taner, Yuesong Wu, Zaiye Li, Wanqing Cheng, Christopher Donnelly, Felipe Nör, Yumin He, Zackary Fitzsimonds, Jianqian Li, Haitao Wen, Steven Chinn, Yuying Xie, James Moon, Yu Leo Lei. Metabolic inhibition of BATF2 dampens type-I interferon-mediated immune sensing of cancer [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 3927.

Abstract 6628: Mapping the spatial and molecular determinants of macrophage heterogeneity in breast cancer

Abstract Tumor-infiltrating macrophages support critical steps in tumor progression, and their accumulation in the tumor microenvironment (TME) is associated with adverse outcomes and therapeutic resistance across human cancers. In the TME, macrophages adopt diverse phenotypic alterations, giving rise to heterogeneous immune activation states and induction of cell cycle. While the transcriptional profiles of these activation states are well-annotated across human cancers, the underlying signals that regulate macrophage heterogeneity and accumulation remain incompletely understood. Here, we leveraged a novel ex vivo organotypic TME (oTME) model of breast cancer, in vivo murine models, and human samples to map the determinants of functional heterogeneity of TME macrophages. We identified a subset of F4/80high Sca-1+ self-renewing macrophages maintained by type-I interferon (IFN) signaling and requiring physical contact with cancer-associated fibroblasts. We discovered that the contact-dependent self-renewal of TME macrophages is mediated via Notch4, and its inhibition abrogated tumor growth of breast and ovarian carcinomas in vivo, as well as lung dissemination in a PDX model of triple-negative breast cancer (TNBC). Through spatial multi-omic profiling of protein markers and transcriptomes, we found that the localization of macrophages further dictates functionally distinct but reversible phenotypes, regardless of their ontogeny. Whereas immune-stimulatory macrophages (CD11C+CD86+) populated the tumor epithelial nests, the stroma-associated macrophages (SAMs) were proliferative, immunosuppressive (Sca-1+CD206+PD-L1+), resistant to CSF-1R depletion, and associated with worse patient outcomes. Notably, following cessation of CSF-1R depletion, macrophages rebounded primarily to the SAM phenotype, which was associated with accelerated growth of mammary tumors. Our work reveals the spatial determinants of macrophage heterogeneity in breast cancer and highlights the disruption of macrophage self-renewal as a potential new therapeutic strategy. Citation Format: Nir Ben Chetrit, Xiang Niu, Jesus Sotelo, Ariel Swett, Vinagolu Rajasekhar, Maria Jiao, Caitlin Stewart, Priya Bhardwaj, Sanjay Kottapalli, Saravanan Ganesan, Pierre-Louis Loyher, Catherine Potenski, Kristy Brown, Neil M. Iyengar, Dilip D. Giri, Scott W. Lowe, John H. Healey, Frederic Geissmann, Irit Sagi, Johanna A. Joyce, Dan A. Landau. Mapping the spatial and molecular determinants of macrophage heterogeneity in breast cancer [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 6628.

Abstract 7481: The allosteric STING agonist CRD3874 is systemically tolerated demonstrates systemic tolerability

Abstract Background: The activation of STING by CDN bind site targeting ligands leads to the generation of Type I interferons as well as pyroptosis and autophagy of the cell expressing STING due to STING’s ability to transport protons across the endoplasmic reticulum. CDN site targeting STING agonists tested in the clinic thus far have shown limited systemic tolerability as well as a tendency to kill the very immune cells that they are designed to activate. CRD3874 is an allosteric small-molecule human STING agonist with a unique binding mode that generates robust Type I interferon responses while simultaneously blocking STINGs proton transport activity that is associated with pyroptosis and autophagy. Methods: Allosteric binding was established using radioligand binding assays. Anti-cancer activity of CRD3874-SI by the IV route was evaluated in human STING KI C57/BL6 mice (genOway, France). Inflammasome and autophagy markers were studies in human and mouse cells. Results: In contrast with competitive STING agonists, CRD3874 potentiates the binding of radiolabeled cGAMP to STING. Treatment of immune cells with CRD3874 or other STING agonists, led to dose dependent increases in Type1 interferons and other pro-inflammatory cytokines. However, unlike other STING agonists, activation of STING by CRD3874 did not lead to induction of autophagy or Inflammasome markers in cells or mice. Intravenous administration of CRD3874-SI in human STING knock-in mice caused tumor regression and survival benefit in multiple murine tumor models. The compound was well tolerated at high doses in the primate GLP study when administered intravenously and caused dose and exposure dependent increases in cytokines. This profile of retaining the high efficacy of a STING agonist while demonstrating systemic safety is unique to CRD3874-SI. Conclusions: CRD3874 is a systemically administered STING agonist with promising single agent activity and an excellent IV safety profile in NHP. An investigator sponsored FIH Phase 1 trial with CRD3874-SI has been initiated at Memorial Sloan Kettering, NY, in sarcoma, MCC patients under the supervision of Dr. Ciara Kelly (NCT06021626). Citation Format: Monali Banerjee, Sandip Middya, Ritesh Shrivastava, Anindita Middya, Nagaswamy Mane, Nidhi Rawat, Thanilsana Soram, Debjani Chakraborty, Rajib Ghosh, Rubeena R. Mansuri, Dharmendra Yadav, Anuj Gautam, Anuj Singh, Kavita Puniya, David Pryde, Sourav Basu, Fabiane Sonego, Gaelle H. Martin, Kader Thiam, Arjun Surya. The allosteric STING agonist CRD3874 is systemically tolerated demonstrates systemic tolerability [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 7481.

Abstract 473: PVT1 exon 9 overexpression induces AR null phenotype and cellular reprogramming in prostate cancer

Abstract Plasmacytoma variant translocation 1 (PVT1) is an oncogenic long-noncoding RNA (lncRNA) gene located directly downstream of MYC on chromosome 8q24. PVT1 is an oncogene overexpressed in prostate cancer (PCa) and can promote oncogenesis by altering gene expression directly or acting as a noncoding RNA sponge. Beyond functioning as a lncRNA, PVT1 gene encodes six microRNAs that each have independent functions. Our group identified overexpression of PVT1 exon 9 in PCa samples derived from men of African Ancestry and overexpression may explain why this population group experiences more aggressive disease. We overexpressed PVT1 exon 9 in RWPE1 prostate epithelial cells (RWPE1_ex9) and found enhanced migration and invasion compared to empty vector (RWPE1_ev). Implantation of RWPE1_ex9 cells into mice led to the formation of tumors with the neuroendocrine PCa (NEPC) phenotype that was histopathologically confirmed. RNA-sequencing analysis of PVT1 exon 9 overexpression showed significant (p-value < 0.05) upregulation of 141 genes. The majority of pathways impacted by PVT1 exon 9 overexpression were interferon type 1 signaling (RSAD2, CMPK2), developmental NOTCH signaling (HES7), cargo-transport (STX16) and tumor microenvironment promoting factors (HS3ST3A1). We found loss of androgen receptor (AR) expression at mRNA and protein levels in RWPE1_ex9 cells, a hallmark of NEPC, which further supports our hypothesis that PVT1 exon 9 overexpression plays a role in NEPC development. Validation of RNA-sequencing confirmed upregulation of RSAD2, CMPK2, STX16, HES7 and HS3ST3A1 within RWPE1_ex9 cells compared to RWPE1_ev and RWPE1 wild-type, suggestive of cellular reprogramming of prostate epithelial cells. We further analyzed expression profile in a novel circulating tumor cell model of castration-resistant 22RV1 cells (C22OH) and found enhanced expression of PVT1 exon 9, suppression of AR, phosphorylation of AR serine residue 308 and enhanced CDK11 activity compared to the primary 22RV1 cell line T22OH suggestive of a cell-cycle mediated regulatory mechanism. We further found enhanced expression of chromogramin A in the C22OH cell line, a marker of NEPC. These results suggest PVT1 exon 9 overexpression may play a critical role in NEPC development from normal epithelial cells via cellular reprogramming as well as facilitating a transition to NEPC from a castration resistant phenotype. PVT1 exon 9 may be a useful predictive biomarker that can identify populations which are likely to have disease progression from castration resistance to NEPC and further highlights the need to identify therapeutic strategies to target PVT1 exon 9 overexpression. Citation Format: Rachel E. Sexton-Bonacci, Chinedum Udekwu, Baris Boylu, Olorunseun O. Ogunwobi. PVT1 exon 9 overexpression induces AR null phenotype and cellular reprogramming in prostate cancer [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 473.

Abstract 3279: MUC1 is necessary for the inflammatory memory response to osimertinib resistance in NSCLC cells

Abstract Emergence of resistance is an invariable outcome in cancer progression that may involve memory of cancer cells to treatment. The oncogenic MUC1-C protein confers pleotropic resistance of cancer cells to diverse cytotoxic and targeted agents by unclear mechanisms. We demonstrate that MUC1-C regulates the interferon (IFN) type I/II pathways in H1975 non-small cell lung cancer (NSCLC) cells selected for osimertinib resistance (H1975-OR). Targeting MUC1-C in H1975-OR cells suppressed STAT1 and inflammatory IFN signaling in association with reversing the osimertinib-resistant phenotype. Studies of H1975-OR cells selected for growth in the absence of osimertinib demonstrate that revertant H1975-RT cells retain MUC1-C-dependent memory necessary for recalling osimertinib resistance. Mechanistically, osimertinib treatment of H1975-RT cells activates the MUC1 gene at a (i) promoter-like signature (PLS) by MUC1-C and STAT1, and (ii) proximal enhancer-like signature (pELS) by PBAF and JUN/AP-1. Targeting MUC1-C in osimertinib-treated H1975-RT and MGH170 NSCLC MET-amplified cells isolated from a patient refractory to osimertinib treatment suppressed induction of STAT1 and downstream IFN-stimulated genes (ISGs) encoding MX1, OAS1 and ISG15. Targeting MUC1-C, STAT1, PBRM1/PBAF and JUN/AP-1 further confirmed their dependencies for reemergence of osimertinib resistance in H1975-RT and MGH170 cells. In support of these results, high MUC1-C protein expression analyzed by IHC is associated with shorter progression free survival for patients treated with osimertinib. These findings and the demonstration that MUC1-C is upregulated in NSCLC cells from patients refractory to osimertinib indicate that MUC1-C drives osimertinib resistance in NSCLC cells by promoting an inflammatory memory response. Citation Format: Naoki Haratake, Atrayee Bhattacharya, Ayako Nakashoji, Hiroki Ozawa, Mototsugu Shimokawa, Donald Kufe. MUC1 is necessary for the inflammatory memory response to osimertinib resistance in NSCLC cells [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 3279.

Abstract 3187: Enhancing STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy

Abstract Background: Activating the innate immune pathway of stimulator of interferon genes (STING) can elicit potent anti-tumor immunity via the production of type-I interferons (IFN-I). However, cyclic-dinucleotide (CDN) STING agonists have undesirable pharmacological properties. Most CDNs in clinical development are administered intratumorally, thus precluding their utility against advanced cancer. Here, we report the development of a new nanoparticle system for the systemic delivery of CDN STING agonists with potent efficacy, favorable pharmaceutical properties, and acceptable safety profiles. Methods: Various metal ions were screened for synergy with CDNs for IFN-I response from bone marrow-derived dendritic cells. Lipid nanoparticles carrying CDN and manganese ions (termed SNP) were synthesized. Mice bearing syngenetic tumors (including CT26 and B16F10) as well as genetically engineered mouse models were treated by intravenous administration of SNP, followed by tumor monitoring and immune profiling of the tumor microenvironment. White New Zealand rabbits bearing VX2 tumors as well as healthy mongrel dogs were treated with SNP and evaluated. Lastly, human tumor biopsies derived from head and neck squamous cell carcinoma (HNSCC) patients were incubated with SNP or soluble STING agonists and analyzed for immune activation. Results: We identified that Mn2+ achieved strong synergy with CDN STING agonists for inducing IFN-I production. SNP carrying CDN and Mn2+ was dosed intravenously in mice, leading to robust IFN-I response, remodeling of the immunosuppressive tumor microenvironment, and expansion of anti-tumor CD8+ T cells. Intravenous SNP therapy resulted in robust anti-tumor efficacy in multiple murine tumor models, including CT26, B16F10, and a genetically engineered mouse model of MMTV-PyMT triple-negative breast cancer. We have also observed robust anti-tumor efficacy of SNP against VX2 squamous carcinomas in rabbits and have demonstrated the safety of intravenous SNP therapy in healthy dogs. Mechanistically, the efficacy of SNP therapy was dependent on the host STING expression but independent of the tumor STING expression. In addition, the anti-tumor efficacy of SNP was decreased in mice lacking Ifnar1 or Ifngr1 expression in the B16F10 melanoma model, showing the crucial effects of IFN-I and IFN-II responses. Moreover, SNP treatment led to robust IFN-I responses from fresh human tumor biopsies derived from HNSCC patients. Conclusions: SNP empowers highly effective systemic cancer immunotherapy via nanotechnology. It also underscores the potential of utilizing metal ions for immune-mediated disease treatment. Citation Format: Xingwu Zhou, Xiaoqi Sun, Wang Gong, Ziye Wan, Teresa Krieger-Burke, Vilma Yuzbasiyan-Gurkan, Yu Leo Lei, James Moon. Enhancing STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy [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 3187.

Abstract 3574: Cancer testis antigen expression correlates with stromal natural killer cell activation and longer overall survival in small bowel neuroendocrine tumors

Abstract Background: Patients with small bowel neuroendocrine tumors (SBNET) frequently present with metastatic disease and the efficacy of available therapies, including immune checkpoint blockade (ICB), is limited. Toward developing novel immunomodulatory strategies, we interrogated the tumor immune microenvironment of SBNETs using bulk transcriptional and digital spatial profiling (DSP). Methods: Patients with SBNET who underwent resection at MD Anderson Cancer Center from 2003-2016 were retrospectively analyzed. Overall survival (OS) was assessed using the Kaplan-Meier method and the Cox proportional hazards model was used for multivariate analysis (MVA). Bulk transcriptional profiling was performed using the Nanostring PanCancer-Immune Panel. DSP with whole human transcriptome was performed using PanCK to segment tumor and adjacent stroma; 88 distinct regions were analyzed using differential gene expression and Reactome pathway enrichment pipelines. Results: Resected SBNET from 42 patients were selected for transcriptional analysis. Unsupervised clustering of gene expression demonstrated dichotomization into high and low cancer testis antigen (CTA) expression. CTA-high patients (n=12) demonstrated elevated expression of type-I interferons (IFN), and CTAs such as PRAME, GAGE1 and MAGEA3 and had significantly longer OS compared to CTA-low patients (n=30; median OS not reached vs 1975 days, p=0.008). MVA controlling for age, sex, stage, and Ki-67% confirmed CTA-high status as an independent predictor of longer OS (hazard ratio [HR] 0.2, 95% confidence interval [CI] 0.05-0.83, p=0.014). A pilot DSP experiment on 4 CTA-high and 4 CTA-low tumors confirmed differential gene expression in the PanCK+ tumor compartment of these samples (R2=0.09, p=0.001). In particular, epigenetic modification genes (e.g., H2BC8, H4C8, H3C1, p<0.001) and epigenetic pathways (e.g., DNA methylation, histone acetylation) were enriched in CTA-high tumors. CTA-high tumors exhibited increased M2 macrophage presence (p=0.025) and increased NK cell activation within the PanCK- stromal compartment (p=0.001). Conclusions: High CTA and type-I IFN expression in resected SBNET identifies patients with longer survival, agnostic of stage or grade. While CTA expression has been implicated in tumor immunogenicity, this is the first work to identify a clinically relevant signal in SBNET. Increased IFN in CTA-high tumors, corroborated by increased stromal NK cell activation, suggests that enhanced immunogenicity may drive this survival difference. Spatial transcriptomic analysis reveals epigenetic differences in CTA-high tumors and highlights the potential for combination epigenetic modifiers and immunotherapy in future trials. Citation Format: Reed I. Ayabe, Yongwoo D. Seo, Brenda Melendez, Rossana Lazcano, Brittany C. Fields, Khalida Wani, Sarah Johnson, Manoj Chelvanambi, Courtney Hudgens, Sharia D. Hernandez, Nadim J. Ajami, Jennifer A. Wargo, Alexander J. Lazar, Daniel M. Halperin, Jeannelyn S. Estrella, Jessica E. Maxwell. Cancer testis antigen expression correlates with stromal natural killer cell activation and longer overall survival in small bowel neuroendocrine tumors [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 3574.

Disruption of polyunsaturated fatty acid biosynthesis drives STING-dependent acute myeloid leukemia cell maturation and death

The role of polyunsaturated fatty acid (PUFA) biosynthesis in acute myeloid leukemia (AML) remains largely undefined. A comparative expression analysis of 35 genes encoding fatty acid biosynthesis enzymes showed that fatty acid desaturase 1 (FADS1) was highly expressed across multiple AML subtypes relative to healthy controls and that elevated FADS1 expression correlates with worse overall AML patient survival. Functionally, shRNA-mediated inhibition of FADS1 reduced AML cell growth invitro and significantly delayed leukemia onset in an AML mouse model. AML cell lines depleted of FADS1 arrested in the G1/S-phase of the cell cycle, acquired characteristics of myeloid maturation and subsequently died. To understand the molecular consequences of FADS1 inhibition, a combination of mass spectrometry-based analysis of complex lipids and gene expression analysis (RNA-seq) was performed. FADS1 inhibition caused AML cells to exhibit significant lipidomic remodeling, including depletion of PUFAs from the phospholipids, phosphatidylserine, and phosphatidylethanolamine. These lipidomic alterations were accompanied by an increase induction of inflammatory and stimulator of interferon genes (STING)-mediated type-1 interferon signaling. Remarkably, genetic deletion of STING largely prevented the AML cell maturation and death phenotypes mediated by FADS1 inhibition. Highlighting the therapeutic implications of these findings, pharmacological blockade of PUFA biosynthesis reduced patient-derived AML cell numbers exvivo but not that of healthy donor cells. Similarly, STING agonism attenuated patient-derived-AML survival; however, STING activation also reduced healthy granulocyte numbers. Collectively, these data unveil a previously unrecognized importance of PUFA biosynthesis in leukemogenesis and that imbalances in PUFA metabolism can drive STING-mediated AML maturation and death.

Abstract 6733: Multiomics analysis of impact of CD8+ and other cell populations in STING agonist treated ex vivo mesothelioma samples

Abstract Background: Collective evidence highlights that agonism of the host STING pathway plays a critical role in eliciting robust and durable anti-tumor immunity. Dazostinag (TAK-676) is a novel systemically delivered STING agonist that ignites the innate immune system and mobilizes adaptive immunity. Preclinical studies with dazostinag demonstrated a strong, persistent CD8+ T cell anti-tumor response driven by innate immune derived type I IFN and pro-inflammatory cytokines. The role of CD8+ T cells in response to dazostinag was further supported by a previously demonstrated correlation between response and CD8+ T cell frequency in a patient-derived organotypic tumor spheroids (PDOTS) model of mesothelioma. Within the current work, we sought to determine the extent to which response to dazostinag in mesothelioma depends on CD8+ T cells versus the contribution of other immune cells. Methods: Surgical mesothelioma cases were collected along with clinicopathological variables at Brigham and Women’s Hospital under an IRB-approved protocol. PDOTS were generated as described previously. Ex vivo response was determined by fluorescent life/dead imaging, and orthogonally evaluated by multiplexed cytokine array, immunofluorescence, and flow cytometry; subset of tumors was studied using single cell RNA sequencing (scRNAseq). Results: Dazostinag-driven tumor cell killing was evaluated in 20 mesothelioma PDOTS. 11 samples were characterized as responders (R) with a cut-off of >30% reduction in live cell area; 9 as non-responders (NR). In 12 out of 20 PDOTS, CD8 neutralization effect was assessed: four were TAK-676 NR, and out of 8 R 4 had at least 20% killing abrogated by aCD8. Effect did not depend on the baseline amount of CD8+ T cells in the sample. scRNAseq analysis showed that R and NR T cells have different activation/exhaustion phenotypes and varying levels of interferon stimulated genes (ISG) in response to STING agonism. Other cell populations emerged as candidates for dazostinag resistance: NR cases carried remodeled immunosuppressive myeloid cells or matrix metalloproteinases-expressing fibroblasts. In general, myeloid cell abundance at baseline characterized by flow had a trend in negatively correlating with dazostinag response. Intrinsic expression of type I interferons shown by multiplexed cytokine array was linked to dazostinag sensitivity (R vs. NR IFN-β p=0.028; IFNα2a p=0.016) and can potentially be used as a predictor for response. Conclusion: We evaluated immune modulations in mesothelioma after treatment with a systemic STING agonist dazostinag using human tumor ex vivo culture. Innate interferon type I expression was higher in responding samples. TAK-676 efficacy was partially reduced in absence of effector lymphocytes, however other cells, such as immunosuppressive myeloid cells and fibroblasts, can have a negative impact on dazostinag response in mesothelioma. Citation Format: Elena Ivanova, Sung R. Park, Ari P. Zlota, Nathaniel Spicer, Minh Ha, Sophie Kivlehan, Iliana Gjeci, Simona Innocenti, Lauren Zasadil, Patrick Lizotte, Michael Y. Tolstorukov, Kai Ding, Adnan O. Abu-Yousif, Jeffrey Raizer, Vicky A. Appleman, Raphael Bueno, David A. Barbie, Cloud P. Paweletz. Multiomics analysis of impact of CD8+ and other cell populations in STING agonist treated ex vivo mesothelioma samples [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 6733.

Immune checkpoint inhibitor monotherapy is sufficient to promote microenvironmental normalization via the type Iinterferon pathway in PD-L1-expressing head and neck cancer.

Immune checkpoint blockers (ICBs) targeting programmed cell death protein 1 (PD-1) have been proven to be an effective first-line therapy against programmed cell death 1 ligand 1 (PD-L1; also known as CD274 molecule)-expressing head and neck squamous cell carcinoma (HNSCC) in recent KEYNOTE-048 trial. However, associated changes in the tumor microenvironment (TME) and underlying mechanisms remain elusive. Oral tumors in C57/BL6 mice were induced by administering 7,12-dimethylbenzanthracene into the buccal mucosa. Single-cell suspension was isolated from tumor tissue; proliferating cells were injected subcutaneously into the left flank of mice to establish Ajou oral cancer (AOC) cell lines. Subsequently, a syngeneic PD-L1-expressing HNSCC model was developed by injecting AOC cells into the buccal or tongue area. The model recapitulated human HNSCC molecular features and showed reliable in vivo tumorigenicity with significant PD-L1 expression. ICB monotherapy induced global changes in the TME, including vascular normalization. Furthermore, the antitumor effect of ICB monotherapy was superior to those of other therapeutic agents, including cisplatin and inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2). The ICB-induced antitumorigenicity and TME normalization were alleviated by blocking the type I interferon pathway. In summary, ICB monotherapy is sufficient to induce TME normalization in the syngeneic model; the type I interferon pathway is indispensable in realizing the effects of ICBs. Furthermore, these results explain the underlying mechanism of the efficacy of ICB monotherapy against PD-L1-expressing HNSCC in the KEYNOTE-048 trial.

CYTOKINE PROFILE RESPONSE IN PATIENTS WITH FRACTURES OF LONG BONES DURING COVID-19

Introduction: The immune system swiftly responds to the onslaught of trauma and the infectious agents it triggers. However, the mechanisms of action of the COVID-19 virus remain unidentified to date. Distinctive alterations that occur in the immune system of trauma victims with COVID-19, setting them apart from changes observed in other diseases. This underscores the specificity of traumatic disease within this patient with COVID-19.
 The study aims to assess the response of humoral immunity markers in patients with fractures of long bones and COVID-19 and to explore their role in early diagnosing infectious complications in this particular group of patients.
 Materials and methods: To accomplish the study objectives, we assembled a retrospective dataset comprising 289 cases of skeletal bone fractures treated at the Kyiv City Clinical Hospital of Emergency Medical Care from March, 2020 to February, 2021, meeting the inclusion criteria for the study. The entire dataset consisted of 289 cases of skeletal bone fractures, categorized into two groups: the main group and the control group.
 Results: On the first day of treatment, an increase in some pro-inflammatory cytokines (IL-6, IL-1β and IL-4) with a normal level of TNF-α, normal values of IFN-γ, IFN-α and a sharp decrease in the anti-inflammatory cytokine IL was observed among the patients of the main group -10. On the third day of the treatment, there was a significant increase in all pro-inflammatory cytokines (IL-6, IL-1β, IL-4 and TNF-α), both types of interferons, and an even sharper decrease in the anti-inflammatory cytokine IL-10. On the tenth day, the growth in the level of pro-inflammatory cytokines was noted, among which the level of TNF-α was the leader, a subnormal level of interferons and anti-inflammatory interleukin 10 was observed. Conclusions: The dynamics of pro-inflammatory cytokines in patients with long bone fractures against the background of COVID-19 tended to increase and had a maximum level on the third day of treatment. Anti-inflammatory cytokines demonstrate a tendency to depression and reache the reference value only on the 10th day of the study.

Increased genomic instability and reshaping of tissue microenvironment underlie oncogenic properties of Arid1a mutations.

Oncogenic mutations accumulating in many chromatin-associated proteins have been identified in different tumor types. With a mutation rate from 10 to 57%, ARID1A has been widely considered a tumor suppressor gene. However, whether this role is mainly due to its transcriptional-related activities or its ability to preserve genome integrity is still a matter of intense debate. Here, we show that ARID1A is largely dispensable for preserving enhancer-dependent transcriptional regulation, being ARID1B sufficient and required to compensate for ARID1A loss. We provide in vivo evidence that ARID1A is mainly required to preserve genomic integrity in adult tissues. ARID1A loss primarily results in DNA damage accumulation, interferon type I response activation, and chronic inflammation leading to tumor formation. Our data suggest that in healthy tissues, the increased genomic instability that follows ARID1A mutations and the selective pressure imposed by the microenvironment might result in the emergence of aggressive, possibly immune-resistant, tumors.

Epigenetic control over the cell-intrinsic immune response antagonizes self-renewal in acute myeloid leukemia

AbstractEpigenetic modulation of the cell-intrinsic immune response holds promise as a therapeutic approach for leukemia. However, current strategies designed for transcriptional activation of endogenous transposons and subsequent interferon type-I (IFN-I) response, show limited clinical efficacy. Histone lysine methylation is an epigenetic signature in IFN-I response associated with suppression of IFN-I and IFN-stimulated genes, suggesting histone demethylation as key mechanism of reactivation. In this study, we unveil the histone demethylase PHF8 as a direct initiator and regulator of cell-intrinsic immune response in acute myeloid leukemia (AML). Site-specific phosphorylation of PHF8 orchestrates epigenetic changes that upregulate cytosolic RNA sensors, particularly the TRIM25-RIG-I-IFIT5 axis, thereby triggering the cellular IFN-I response-differentiation-apoptosis network. This signaling cascade largely counteracts differentiation block and growth of human AML cells across various disease subtypes in vitro and in vivo. Through proteome analysis of over 200 primary AML bone marrow samples, we identify a distinct PHF8/IFN-I signature in half of the patient population, without significant associations with known clinically or genetically defined AML subgroups. This profile was absent in healthy CD34+ hematopoietic progenitor cells, suggesting therapeutic applicability in a large fraction of patients with AML. Pharmacological support of PHF8 phosphorylation significantly impairs the growth in samples from patients with primary AML. These findings provide novel opportunities for harnessing the cell-intrinsic immune response in the development of immunotherapeutic strategies against AML.

STAT3 protects hematopoietic stem cells by preventing activation of a deleterious autocrine type-I interferon response.

Hematopoietic stem and progenitor cells (HSPCs) maintain blood-forming and immune activity, yet intrinsic regulators of HSPCs remain elusive. STAT3 function in HSPCs has been difficult to dissect as Stat3-deficiency in the hematopoietic compartment induces systemic inflammation, which can impact HSPC activity. Here, we developed mixed bone marrow (BM) chimeric mice with inducible Stat3 deletion in 20% of the hematopoietic compartment to avoid systemic inflammation. Stat3-deficient HSPCs were significantly impaired in reconstitution ability following primary or secondary bone marrow transplantation, indicating hematopoietic stem cell (HSC) defects. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells (LSKs) revealed aberrant activation of cell cycle, p53, and interferon (IFN) pathways in Stat3-deficient HSPCs. Stat3-deficient LSKs accumulated γH2AX and showed increased expression of DNA sensors and type-I IFN (IFN-I), while treatment with A151-ODN inhibited expression of IFN-I and IFN-responsive genes. Further, the blockade of IFN-I receptor signaling suppressed aberrant cell cycling, STAT1 activation, and nuclear p53 accumulation. Collectively, our results show that STAT3 inhibits a deleterious autocrine IFN response in HSCs to maintain long-term HSC function. These data signify the importance of ensuring therapeutic STAT3 inhibitors are targeted specifically to diseased cells to avoid off-target loss of healthy HSPCs.

Anti-histone and anti-nucleosome rather than anti-dsDNA antibodies associate with IFN-induced biomarkers in Sudanese and Swedish Systemic Lupus Erythematosus patients.

In SLE, anti-dsDNA can co-occur with autoantibodies against other chromatin components, like histones and nucleosomes. These antibodies induce type-1 interferon production, a hallmark of SLE. We measured antinuclear antibody (ANA) sub-specificities and investigated their associations to inflammatory biomarkers including interferon-regulated chemokines. We included 93 Sudanese and 480 Swedish SLE patients and matched controls (N = 104 + 192). Autoantibodies targeting ANA-subspecificites: dsDNA, Sm, Sm/U1RNPcomplex, U1RNP, SSA/Ro52, SSA/Ro60, SSB/La, ribosomal P, PCNA and histones were quantified in all subjects, anti-nucleosome only in the Swedish patients, with a bead-based multiplex immunoassay. Levels of 72 plasma biomarkers were determined with Proximity Extension Assay technique or ELISA. Among Sudanese patients, the investigated antibodies significantly associated with 9/72 biomarkers. Anti-histone antibodies showed the strongest positive correlations with MCP-3 and S100A12 as well as with interferon I-inducible factors MCP-1 and CXCL10. Anti-dsDNA antibodies associated with CXCL10 and S100A12, but in multivariate analyses, unlike anti-histone, associations lost significance.Among Swedish patients, MCP-1, CXCL10, SA100A12 also demonstrated stronger associations to anti-histone and anti-nucleosome antibodies, compared with anti-dsDNA and other ANA sub-specificities. In multiple regression models, anti-histone/nucleosome retained the strongest associations. When excluding anti-histone or anti-nucleosome positive patients, the associations between MCP-1/CXCL10 and anti-dsDNA were lost. In contrast, when excluding anti-dsDNA positive patients, associations with anti-histone and anti-nucleosome remained significant. In two cohorts of different ethnical origin, autoantibodies targeting chromatin correlate stronger with IFN-induced inflammatory biomarkers than anti-dsDNA or other ANA sub-specificities. Our results suggest that anti-histone/nucleosome autoantibodies may be main drivers of type-1 interferon activity in SLE.

Tumor-necrosis factor α-rich environment alters type-I interferon response to viral stimuli in patients with juvenile idiopathic arthritis by altering myeloid dendritic cell phenotype

The balance between the tumor-necrosis factor α (TNFα) and type-I interferon (T1IFN) pathways is crucial for proper immune function. Dysregulation of either pathway can contribute to autoimmune diseases development. Even though TNFα blockade has shown promising results in various autoimmune diseases, the effect on the balance between TNFα and T1IFN is elusive. We used targeted anti-TNFα therapies in juvenile idiopathic arthritis (JIA) as an experimental approach to study the cross-regulation between TNFα and type-I IFN. We found that TNFα-rich environment affected viral defense through the attenuation of T1IFN responses and affected the phenotype and distribution of myeloid dendritic cells, which are engaged in early viral infections. Anti-TNFα therapy normalized the observed deviations in JIA patients. We hypothesize that the inadequate immune response caused by a high TNFα environment could be projected to more frequent or lengthy viral infections and possibly play a role in the process of JIA disease development.

Antiviral responses induced by Tdap-IPV vaccination are associated with persistent humoral immunity to Bordetella pertussis

Many countries continue to experience pertussis epidemics despite widespread vaccination. Waning protection after booster vaccination has highlighted the need for a better understanding of the immunological factors that promote durable protection. Here we apply systems vaccinology to investigate antibody responses in adolescents in the Netherlands (N = 14; NL) and the United Kingdom (N = 12; UK) receiving a tetanus-diphtheria-acellular pertussis-inactivated poliovirus (Tdap-IPV) vaccine. We report that early antiviral and interferon gene expression signatures in blood correlate to persistence of pertussis-specific antibody responses. Single-cell analyses of the innate response identified monocytes and myeloid dendritic cells (MoDC) as principal responders that upregulate antiviral gene expression and type-I interferon cytokine production. With public data, we show that Tdap vaccination stimulates significantly lower antiviral/type-I interferon responses than Tdap-IPV, suggesting that IPV may promote antiviral gene expression. Subsequent in vitro stimulation experiments demonstrate TLR-dependent, IPV-specific activation of the pro-inflammatory p38 MAP kinase pathway in MoDCs. Together, our data provide insights into the molecular host response to pertussis booster vaccination and demonstrate that IPV enhances innate immune activity associated with persistent, pertussis-specific antibody responses.

Malaria blood stage infection suppresses liver stage infection via host-induced interferons but not hepcidin

Malaria-causing Plasmodium parasites first replicate as liver stages (LS), which then seed symptomatic blood stage (BS) infection. Emerging evidence suggests that these stages impact each other via perturbation of host responses, and this influences the outcome of natural infection. We sought to understand whether the parasite stage interplay would affect live-attenuated whole parasite vaccination, since the efficacy of whole parasite vaccines strongly correlates with their extend of development in the liver. We thus investigated the impact of BS infection on LS development of genetically attenuated and wildtype parasites in female rodent malaria models and observed that for both, LS infection suffered severe suppression during concurrent BS infection. Strikingly and in contrast to previously published studies, we find that the BS-induced iron-regulating hormone hepcidin is not mediating suppression of LS development. Instead, we demonstrate that BS-induced host interferons are the main mediators of LS developmental suppression. The type of interferon involved depended on the BS-causing parasite species. Our study provides important mechanistic insights into the BS-mediated suppression of LS development. This has direct implications for understanding the outcomes of live-attenuated Plasmodium parasite vaccination in malaria-endemic areas and might impact the epidemiology of natural malaria infection.

Abstract B081: Type-I interferon (IFN-I) signaling following aurora kinase A (AURKA) inhibition is modulated by tumor p53 status

Abstract B081: Type-I interferon (IFN-I) signaling following aurora kinase A (AURKA) inhibition is modulated by tumor p53 status