Interferon Regulatory Factor Research Articles

Large B-cell Lymphoma With Interferon Regulatory Factor 4 Rearrangement Presenting as a Primary Central Nervous System Lymphoma in the Brain

Large B-cell Lymphoma With Interferon Regulatory Factor 4 Rearrangement Presenting as a Primary Central Nervous System Lymphoma in the Brain

Fludarabine attenuates inflammation and dysregulated autophagy in alveolar macrophages via inhibition of STAT1/IRF1 pathway

BackgroundAcute lung injury (ALI), including its most severe form, acute respiratory distress syndrome (ARDS), is a common cause of acute hypoxemic respiratory failure. Although its clinical characteristics have been well characterized, the relevant mechanism remains unclear. An imbalance in autophagy leads to alveolar remodeling and triggers the pathogenesis of ARDS. In this study, we assessed the therapeutic efficacy of the STAT1 inhibitor fludarabine (Fluda) in ALI. C57BL6 mice were exposed to lipopolysaccharide (LPS), and their lung tissues were analyzed via next-generation transcriptome sequencing.ResultsWestern blotting revealed that interferon regulatory factor 1 (IRF1) was highly expressed and STAT1 was phosphorylated following LPS exposure. Fluda significantly decreased the protein expression of STAT1/IRF1 and inhibited the alveolar infiltration of neutrophils and macrophages. Nitric oxide (NO), inducible nitric oxide synthase, tumor necrosis factor-α (TNF-α), interferon-γ, and interleukin-6 (IL-6) release was decreased in the lungs of mice and RAW264.7 macrophages following Fluda treatment. In LPS-induced GFP-LC3 transgenic mice treated with Fluda, the counts of LC3-expressing neutrophils and macrophages in bronchoalveolar (BAL) fluid were significantly decreased. Furthermore, Fluda decreased LC3 and p62 protein expression, thereby inhibiting the release of NO, IL-6, and TNF-α in BAL. In RAW264.7 cells, the inhibition of STAT1/IRF1 by Fluda decreased LPS-induced ERK and NF-κB p65 phosphorylation.ConclusionsThe inhibition of STAT1/IRF1 by Fluda plays a pivotal role in modulating dysregulated autophagy by suppressing the MAPK and NF-κB p65 pathways in ALI.

IFITMs exhibit antiviral activity against Chikungunya and Zika virus infection via the alteration of TLRs and RLRs signaling pathways

Chikungunya virus (CHIKV) poses a significant challenge as there are currently no targeted antiviral drugs or vaccines to combat this infection. Here, we demonstrate that interferon-induced transmembrane proteins (IFITMs), including IFITM1, IFITM2, and IFITM3, which are interferon-stimulated genes (ISGs), inhibit CHIKV infection in human skin fibroblasts. Overexpression of IFITMs in cells restricts viral infection, whereas knockdown of IFITMs enhances viral infection. IFITMs overexpression causes a substantial upregulation of antiviral genes, namely TLR3, TLR7, TLR8, and TLR9, and their downstream signaling molecules such as TRADD, IRAK1, TRAF6, and MAP3K7, involved in TLRs signaling pathways. Furthermore, the DHX58 gene encoding the LGP2 protein, a negative regulator of RIG-I in RLRs signaling pathways, was downregulated in the overexpressed cells. Transcription factors including interferon regulatory factors (IRF) 3/5/7, which are downstream signaling components of both TLR and RLR signaling pathways, were also upregulated, resulting in enhanced IFNs signaling. IFITMs not only inhibits the early and late stages of viral infection but can also alter the antiviral innate-immune response to restrict CHIKV infection in human skin fibroblasts. Additionally, IFITMs exhibit their antiviral activity against Zika virus (ZIKV). Altogether, these results show the broad-spectrum antiviral property of IFITMs against arboviruses in foreskin cells.

Neutrophil Extracellular Traps-Associated RNA Impedes CD4+ Treg Differentiation by TLR7-IRF7 Axis in Ankylosing Spondylitis.

Our objective was to investigate the role of neutrophil extracellular traps (NETs) in the pathogenesis of inflammatory disorders in ankylosing spondylitis (AS). Local and circulating NETs levels were determined by immunofluorescence (IF) and myeloperoxidase(MPO)-DNA quantification in both patients with AS and AS model SKG mice. Flow cytometry (FCM) was performed to detect the effect of NETs on CD4+ subpopulation differentiation. The therapeutic effects of the neutrophil elastase inhibitor sivelestat (SVT) and the peptidylarginine deiminase4 (PAD4) inhibitor CI-amidine were evaluated in SKG mice. The localization of NETs and their ability to impede CD4+ Treg cell differentiation were evaluated via IF, FCM, and Western blotting. RNA sequencing and specific inhibitors were used to clarify the detailed mechanism by which NETs inhibit CD4+ Treg differentiation. The NETs levels were elevated locally and systemically in both patients with AS and SKG mice, which impeded the differentiation of CD4+ Treg cells. Blocking NETs formation via SVT or CI-amidine restored the CD4+ Treg ratio and subsequently alleviated inflammation in SKG mice. NETs were internalized by CD4+ T cells, and their associated RNA activated the Toll-like receptor 7 (TLR7)-interferon regulatory factor 7 (IRF-7) axis, which then inhibited Treg differentiation. Inhibiting CD4+ T cells endocytosis, removing the bound RNA component, or blocking the TLR7-IRF-7 axis abrogated the negative effect of NETs on CD4+ Treg differentiation. Elevated NETs impeded CD4+ Treg differentiation by activating the TLR7-IRF-7 axis via their associated RNA in AS, and targeting NETs may be a novel treatment strategy for AS and related inflammatory disorders.

Disruption of the novel nested gene Aff3ir mediates disturbed flow-induced atherosclerosis in mice

Disturbed shear stress-induced endothelial atherogenic responses are pivotal in the initiation and progression of atherosclerosis, contributing to the uneven distribution of atherosclerotic lesions. This study investigates the role of Aff3ir-ORF2, a novel nested gene variant, in disturbed flow-induced endothelial cell activation and atherosclerosis. We demonstrate that disturbed shear stress significantly reduces Aff3ir-ORF2 expression in athero-prone regions. Using three distinct mouse models with manipulated Aff3ir-ORF2 expression, we demonstrate that Aff3ir-ORF2 exerts potent anti-inflammatory and anti-atherosclerotic effects in Apoe-/- mice. RNA sequencing revealed that interferon regulatory factor 5 (Irf5), a key regulator of inflammatory processes, mediates inflammatory responses associated with Aff3ir-ORF2 deficiency. Aff3ir-ORF2 interacts with Irf5, promoting its retention in the cytoplasm, thereby inhibiting the Irf5-dependent inflammatory pathways. Notably, Irf5 knockdown in Aff3ir-ORF2 deficient mice almost completely rescues the aggravated atherosclerotic phenotype. Moreover, endothelial-specific Aff3ir-ORF2 supplementation using the CRISPR/Cas9 system significantly ameliorated endothelial activation and atherosclerosis. These findings elucidate a novel role for Aff3ir-ORF2 in mitigating endothelial inflammation and atherosclerosis by acting as an inhibitor of Irf5, highlighting its potential as a valuable therapeutic approach for treating atherosclerosis.

Disruption of the novel nested gene Aff3ir mediates disturbed flow-induced atherosclerosis in mice.

Disturbed shear stress-induced endothelial atherogenic responses are pivotal in the initiation and progression of atherosclerosis, contributing to the uneven distribution of atherosclerotic lesions. This study investigates the role of Aff3ir-ORF2, a novel nested gene variant, in disturbed flow-induced endothelial cell activation and atherosclerosis. We demonstrate that disturbed shear stress significantly reduces Aff3ir-ORF2 expression in athero-prone regions. Using three distinct mouse models with manipulated Aff3ir-ORF2 expression, we demonstrate that Aff3ir-ORF2 exerts potent anti-inflammatory and anti-atherosclerotic effects in Apoe-/- mice. RNA sequencing revealed that interferon regulatory factor 5 (Irf5), a key regulator of inflammatory processes, mediates inflammatory responses associated with Aff3ir-ORF2 deficiency. Aff3ir-ORF2 interacts with Irf5, promoting its retention in the cytoplasm, thereby inhibiting the Irf5-dependent inflammatory pathways. Notably, Irf5 knockdown in Aff3ir-ORF2 deficient mice almost completely rescues the aggravated atherosclerotic phenotype. Moreover, endothelial-specific Aff3ir-ORF2 supplementation using the CRISPR/Cas9 system significantly ameliorated endothelial activation and atherosclerosis. These findings elucidate a novel role for Aff3ir-ORF2 in mitigating endothelial inflammation and atherosclerosis by acting as an inhibitor of Irf5, highlighting its potential as a valuable therapeutic approach for treating atherosclerosis.

IHNV induced miR-19-3p modulates immune response of rainbow trout (Oncorhynchus mykiss) by targeting DHX58-dependent RLR signaling pathway.

IHNV induced miR-19-3p modulates immune response of rainbow trout (Oncorhynchus mykiss) by targeting DHX58-dependent RLR signaling pathway.

Identification of a type I IFN- and IRF-inducible enhancer in the 5'-UTR intron of MAVS in large yellow croaker Larimichthys crocea.

Identification of a type I IFN- and IRF-inducible enhancer in the 5'-UTR intron of MAVS in large yellow croaker Larimichthys crocea.

STING regulates porphyromonas gingivalis lipopolysaccharide-induced pyroptosis and inflammatory response through the NF-κB/NLRP3 signaling pathway in human gingival fibroblasts.

STING regulates porphyromonas gingivalis lipopolysaccharide-induced pyroptosis and inflammatory response through the NF-κB/NLRP3 signaling pathway in human gingival fibroblasts.

Shexiang Baoxin Pill alleviates atherosclerosis by inhibiting macrophage-mediated inflammation via suppressing KMT5A mediated Irf7 transcription.

Shexiang Baoxin Pill alleviates atherosclerosis by inhibiting macrophage-mediated inflammation via suppressing KMT5A mediated Irf7 transcription.

Targeting CTP synthetase 1 to restore interferon induction and impede nucleotide synthesis in SARS-CoV-2 infection.

Despite the global impact caused by the most recent SARS-CoV-2 pandemic, our knowledge of the molecular underpinnings of its highly infectious nature remains incomplete. We report here that SARS-CoV-2 exploits cellular CTP synthetase 1 (CTPS1) to promote CTP synthesis and suppress interferon (IFN) induction. In addition to catalyzing CTP synthesis, CTPS1 also deamidates interferon regulatory factor 3 (IRF3) to dampen interferon induction. Screening a SARS-CoV-2 expression library, we identified several viral proteins that interact with CTPS1. Functional analyses demonstrate that ORF8 and Nsp8 activate CTPS1 to deamidate IRF3 and negate IFN induction, whereas ORF7b and ORF8 activate CTPS1 to promote CTP synthesis. These results highlight CTPS1 as a signaling node that integrates cellular metabolism and innate immune response. Indeed, small-molecule inhibitors of CTPS1 deplete CTP and boost IFN induction in SARS-CoV-2-infected cells, thus effectively impeding SARS-CoV-2 replication and pathogenesis in mouse models. Our work uncovers an intricate mechanism by which a viral pathogen couples immune evasion to metabolic activation to fuel viral replication. Inhibition of the cellular CTPS1 offers an attractive means to develop antiviral therapy against highly mutagenic viruses.IMPORTANCEOur understanding of the underpinnings of highly infectious SARS-CoV-2 is rudimentary at best. We report here that SARS-CoV-2 activates CTPS1 to promote CTP synthesis and suppress IFN induction, thus coupling immune evasion to activated nucleotide synthesis. Inhibition of the key metabolic enzyme not only depletes the nucleotide pool but also boosts host antiviral defense, thereby impeding SARS-CoV-2 replication. Targeting cellular enzymes presents a strategy to counter the rapidly evolving SARS-CoV-2 variants.

Identification and validation of programmed cell death related biomarkers for the treatment and prevention COVID-19

Purpose Programmed cell death (PCD) plays a key role in the progression of coronavirus disease 2019 (COVID-19). However, PCD-relevant biomarkers have not been fully discovered. The aim of this study was to explore the PCD-relevant biomarkers for the treatment and prevention of COVID-19. Methods Bioinformatic analyses were performed to explore the clinical relevant PCD genes with differential expression (DE) in COVID-19 compared with matched controls. PPI network was used for hub genes screening and machine learning methods were employed for filtering feature genes. The biomarker genes were screened by Venn diagram. The correlations between biomarkers with clinical features and immune microenvironment were further explored. Biomarker validation was performed in clinical samples by real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). Results In total, 118 clinically relevant and PCD associated differential expressed genes (DEGs) were screened, which were mainly related with apoptosis related pathways, among which six biomarkers (Cyclin B1 (CCNB1), cyclin-dependent kinase 1 (CDK1), interferon regulatory factor 4 (IRF4), lipoteichoic acid (LTA), matrix metallopeptidase 9 (MMP9) and Oncostatin M (OSM)) were identified. The excellent or good diagnostic performance of biomarkers was determined by receiver operating characteristic (ROC) curve analysis. The biomarkers showed diverse correlations with clinical indicators, such as age, sex and Intensive Care Unit (ICU) admission. Total 14 types of immune cells exerted differential infiltration between COVID-19 and controls. Biomarkers were correlated with immune cells at varying levels. COVID-19 was classified in three clusters, which showed differential expression of biomarker genes and significant associations with clinical information, such as sex, age and ICU admission. The DEGs of biomarkers were determined in COVID-19 patients relative to controls. Conclusion The six biomarkers (CCNB1, CDK1, IRF4, LTA, MMP9 and OSM) can be served as the biomarkers for the treatment and prevention of COVID-19.

13C and 15N resonance assignments of the DNA binding domain of interferon regulatory factor-3.

The Interferon Regulatory Factor (IRF) family of transcription factors is well known for its anti-viral activity in vertebrates. The IRF family comprises nine members (IRF1-9) which have the ability to induce the Interferon beta (IFNβ) promotor. The IRF3 and IRF7 are the key family members involved in the production of type I and type III IFN. IRF3 and IRF7 both comprise of a DNA binding domain (DBD) which binds to its cognate interferon responsive element (IRE) on its target gene promoters. Here, we report near complete backbone and partial side-chain resonance assignments of the DBD domain of the IRF3 subunit of the IRF family. The predicted secondary structure using the backbone chemical shifts largely conforms with that obtained from the crystal structure, with the TALOS-N predicted secondary structures showing slightly elongated β-strands.

A Quinoxaline Derivative as a New Therapeutic Agent for Sepsis through Suppression of TLR4 Signaling Pathways.

Sepsis is a severe systemic inflammatory syndrome and one of the leading causes of global morbidity and mortality. Preclinical studies have identified several quinoxaline-based compounds with anti-inflammatory properties, but their effects in sepsis have not been investigated. This study aimed to identify a quinoxaline derivative with anti-inflammatory properties in sepsis. Examining the inflammatory response of primary mouse macrophages to Lipopolysaccharides (LPS) revealed that 2-methoxy-N-(3-quinoxalin-2-ylphenyl)benzamide (2-MQB) is a promising molecule. It suppressed the production of several inflammatory cytokines, including Interleukin-1β (IL-1β), IL-6, IL-12p70, Interferon-γ (IFN-γ), IFN-β, and Tumor necrosis factor-α (TNF-α). Importantly, 2-MQB inhibited the transcriptional activities of Toll-like receptor 4 (TLR4) signaling pathways, including Nuclear factor-κB (NF-κB) and Interferon regulatory factor 3 (IRF3). This was accompanied by lower expression of TLR4, Myeloid differentiation primary response 88 (MyD88), TIR Domain-containing adaptor molecule 1 (Trif), and TNF Receptor-associated factor 3 (Traf3). Additionally, 2-MQB selectively reduced the expression of genes encoding CD80, CD86, and Programmed death-ligand 1 (PD-L1). In vivo, 2-MQB improved mice survival, mitigated tissue damage in the spleen, kidney, and lung, and reduced pro-inflammatory cytokine levels in both LPS-induced endotoxin shock and Cecal ligation and puncture (CLP) models. Notably, 2-MQB decreased the numbers of CD4+ and CD8+ T cells in the spleen and inhibited TLR4 signaling pathways in LPS-induced endotoxemia. In conclusion, these results introduce the quinoxaline derivative 2-MQB as a potential therapeutic agent for sepsis by inhibiting TLR4 signaling pathways, paving the way for future clinical applications.

Stromal Interferon Regulatory Factor 3 Can Antagonize Human Papillomavirus Replication by Supporting Epithelial-to-Mesenchymal Transition

Stromal Interferon Regulatory Factor 3 Can Antagonize Human Papillomavirus Replication by Supporting Epithelial-to-Mesenchymal Transition

Oxaliplatin accelerates immunogenic cell death by activating the cGAS/STING/TBK1/IRF5 pathway in gastric cancer.

Immunogenic cell death is a tumor cell death involving both innate and adaptive immune responses. Given the published findings that oxaliplatin causes the secretion of high mobility group box 1 (HMGB1) from cancer cells, which is necessary for the initiation of immunogenic cell death, we investigated whether oxaliplatin plays an anticancer role in gastric cancer by inducing immunogenic cell death and further explored its mechanism. We found that oxaliplatin inhibited viability and induced pyroptosis, immunogenic cell death, the production of reactive oxygen species, mitochondrial permeability transition pore (mPTP) opening, and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis activation in gastric cancer cells. Suppressing mPTP opening (cyclosporine treatment), depleting mitochondrial DNA (mtDNA; ethidium bromide treatment), or STING downregulation (H151 or si-STING treatment) reversed cGAS/STING pathway activation and the increased immunogenic cell death induced by oxaliplatin in MKN-45 and AGS cells. Moreover, oxaliplatin induced immunogenic cell death via activating the cGAS/STING/TANK-binding kinase 1 (TBK1; also known as serine/threonine-protein kinase TBK1)/interferon regulatory factor 5 (IRF5) pathway. In conclusion, oxaliplatin treatment could induce immunogenic cell death and mPTP opening and activate the cGAS/STING/TBK1/IRF5 pathway in gastric cancer cells.

Abstract 903: Mechanisms mediating hypoxic memory of type I IFN signaling suppression and implications for immune evasion in luminal breast cancer cells

Abstract Solid tumors often contain patches of hypoxic regions that yield aggressive, pro-metastatic phenotypes. Prevention and understanding of metastases is critical for the future of breast cancer research, as metastatic cancer is extremely difficult to manage and treat. Recent work in our lab has shown that long-term hypoxia suppresses type I interferon (IFN) signaling in breast cancer cells. Even upon reoxygenation, these gene expression changes are maintained, indicative of “hypoxic memory.” Breast cancer cells that have disseminated from the primary tumor as circulating tumor cells with this “post-hypoxic” memory phenotype show enhanced metastatic potential. IFNs typically boost and enhance immune cell activity through interferon-stimulated genes (ISGs); thus, hypoxic suppression of IFN signaling yields an immunosuppressive tumor microenvironment. More research is needed to understand how ISGs are downregulated in hypoxia and how those changes in gene expression contribute to immunosuppressive ability of hypoxic and post-hypoxic cells. Interferon-inducible 44-like (IFI44L) is an ISG that has been associated with tumor-suppressive properties, and correlated with presence of tumor-infiltrating lymphocytes in other cancers, but its role in breast cancer has not been studied. We found that IFI44L is significantly suppressed in breast cancer cells in hypoxia and maintained after reoxygenation as a hypoxic memory. To try to understand possible mechanisms of hypoxic suppression, we overexpressed interferon regulatory factors (IRFs), which are positive regulators of the type I IFN pathway. However, this did not rescue hypoxic suppression and memory of IFI44L, suggesting that IRFs are not the sole regulator of IFN and ISG expression in hypoxia. Additional experiments are currently ongoing in the lab to address other possible mechanisms of hypoxic suppression and memory of IFN signaling. Additionally, co-culture experiments of breast cancer cells with knockdown or overexpression of IFI44L with different immune cell populations will shed light on the role of IFI44L in immune interactions in the tumor and in circulation. To better understand implications of maintained IFI44L suppression, we will also examine immune cell activity and metastatic potential using in vivo mouse studies. We expect to see less immune cell effect and enhanced metastasis with IFI44L knockdown and reversed phenotypes with overexpression of IFI44L. Understanding mechanisms of immune evasion mediated via IFI44L can lead to strategies for targeting hypoxia-mediated tumorigenesis and metastasis. Citation Format: Rebecca Marker, Aidan Moriarty, Remi Klotz, Min Yu. Mechanisms mediating hypoxic memory of type I IFN signaling suppression and implications for immune evasion in luminal breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 903.

Abstract 2899: PARG inhibition provokes a DNA damage-dependent innate immune reaction that enhances ICI-driven anti-tumor immunity

Abstract Poly (ADP-Ribose) glycohydrolase (PARG) is an emerging oncology drug target with a unique role in the resolution of DNA damage repair and DNA replication fork restart through hydrolysis of Poly (ADP-ribose) (PAR) chains. IDE161 is a potent, orally bioavailable, small molecule that directly binds the active site of PARG to impede PAR chain hydrolysis and exhibits robust anti-tumor activity in preclinical models associated with replication stress. Molecular profiling of baseline cell states across 257 tumor cell lines indicated innate immune signaling was strongly associated with responsiveness to IDE161. Moreover, induction of interferon regulatory factor (IRF) activity was the dominant gene regulation program detected in tumors in response to IDE161 exposure in vivo. Given the body of evidence indicating replication stress can lead to a type 1 interferon response via nucleic acid sensing, we speculated that IDE161-induced innate immune signaling is a consequence of cytosolic exposure to DNA repair intermediates including RNA: DNA heteroduplexes produced by failed resolution of stalled DNA replication forks. Consistent with this, IDE161-dependent cancer cell death was preceded by transcription-replication conflicts, replication catastrophe, innate immune pathway activation, and IFN-β production and secretion. To evaluate the potential for innate immune priming of an adaptive immune reaction, IDE161/immune checkpoint inhibitor (ICI) combination activity was assessed in a syngeneic mouse tumor model reported to be partially responsive to PARG inhibition. The combination of IDE161 with RPM1-14, an antibody directed against the mouse-programmed cell death protein 1 (PD-1) receptor, showed greater anti-tumor activity than either agent alone and altered the frequency of tumor-infiltrating lymphocytes towards an immune-activated phenotype. Moreover, T-cell receptor sequencing of clinical PBMC specimens from IDE161 monotherapy patients revealed clonal expansion in the T-cell receptor repertoires on treatment as compared to baseline. These cumulative observations suggest that IDE161 can induce an innate-immune-pathway-mediated priming of an antitumor adaptive immune response, and thus may deliver both tumor autonomous and nonautonomous therapeutic benefits. This relationship is currently under evaluation with IDE161/pembrolizumab combination therapy In patients with advanced endometrial cancer who have previously progressed on anti-PD-1 therapy (NCT05787587) Citation Format: Reeja Maskey, Megan Conway, Pei Xin Lim, Amber C. Donahue, Emily Fitz, Vidhya Nagarajan, Arjun A. Rao, Zabrisky Roland, Natalie Bresnahan, David Trinh, Ivan Shabalin, Kelly Trego, Jonathon Ryan, Rebeca M. Choy, Peter Teriete, Christian Frey, Yuchen Bai, Paul A. Barsanti, Claire Neilan, Mike White, Diana M. Munoz. PARG inhibition provokes a DNA damage-dependent innate immune reaction that enhances ICI-driven anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2899.

Abstract 6170: SPHK2 modulates the tumor-intrinsic response to STING agonism in triple-negative breast cancer cells

Abstract Triple-negative breast cancer (TNBC) accounts for 10-15% of breast cancers and has a poorer prognosis than other breast cancer subtypes. TNBCs with robust immune infiltrates are known to have improved responses to chemotherapy, immunotherapy, and radiotherapy. Stimulator of interferon genes (STING) promotes both innate and adaptive anti-tumor immune cell infiltration through type I interferon production, and we and others have shown that its activation is associated with therapeutic sensitivity in TNBC. Repression of STING expression or activity are commonly observed in various cancer types, and this has been shown to attenuate responses to treatment. TNBCs overexpress sphingosine kinase 2 (SPHK2), which produces the bioactive lipid spingosine-1-phosphate (S1P). SPHK2-mediated S1P biosynthesis has been shown to suppress inflammatory responses in lung injury models via inhibition of STING. In this study, we sought to determine if SPHK2 attenuated STING activity in TNBC cells. We analyzed publicly available RNA-seq data in The Cancer Genome Atlas and found that TNBC patients with higher SPHK2 expression had significantly worse disease-free survival and overall survival. For in vitro studies, we stably overexpressed SPHK2 in two commonly studied TNBC cell lines with mid-range SPHK2 and STING protein expression: MDA-MB-231 and BT-549. STING was stimulated with the synthetic agonist diABZI, the endogenous agonist 2’3’-cGAMP, or with ionizing radiation. Phosphorylated interferon regulatory factor 3, a downstream event of STING activation, was decreased in SPHK2 overexpressing cells following diABZI stimulation. We measured type I interferon signaling by analyzing transcriptional activity of interferon-stimulated response elements (ISRE) using a luciferase reporter assay. SPHK2 overexpressing MDA-MB-231 and BT-549 cells had decreased transcriptional activity of ISRE compared to controls at baseline and after both diABZI and 2’3’-cGAMP treatment, suggesting that SPHK2 suppresses STING-dependent type I interferon production in TNBC. To test a therapeutically relevant model, cells were also exposed to photon radiation. Compared to control cells, SPHK2 overexpressing cells had decreased radiation induced ISRE activity. The FDA-approved SPHK2 inhibitor opaganib was able to augment diABZI-stimulated ISRE transcriptional activity in a dose-dependent manner. These data suggest that malignancy-associated SPHK2 overexpression may attenuate STING activation in TNBC, and that SPHK2 inhibition may be a therapeutically relevant and viable approach to augment anti-tumor immunity. The interactions between SPHK2 and the STING-interferon pathway warrants further study to develop potential therapeutic combinations to benefit patients with TNBC. Citation Format: Colette R. Worcester, Shane R. Stecklein. SPHK2 modulates the tumor-intrinsic response to STING agonism in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6170.

Abstract 2771: CRISPR/Cas9 essentiality screens identify stand-alone and IRF4-linked genetic vulnerabilities in melanoma cells

Melanomas have poor prognosis at the metastatic stage despite impressive progress in targeted- and immuno-therapies. Therefore, identification of novel critical genes involved in melanoma biology with targeting potential is desirable. We and others have previously demonstrated the transcription factor interferon regulatory factor 4 (IRF4) as a dependency factor in various lymphocyte-derived cancers. These findings align with the therapeutic efficacy attained by immunomodulatory drugs which indirectly target IRF4 expression in these cancers. IRF4 also takes part in pigmentation in melanocytes, and its overexpression is prevalent in melanomas. Recently, we showed that high expression of IRF4 is linked to dependency in melanoma cells and mortality in patients. Mechanistically, we demonstrated that IRF4 regulates epigenetic silencing factors involved in DNA and histone H3K27 methylation, and consequently, controls the expression of melanoma-critical tumor suppressor genes and key downstream oncogenic pathways. In order to identify novel genes that melanoma cells rely on for survival or proliferation, we performed CRISPR/Cas9 drop-out screens. One set of screens was aimed at identifying single individual genes necessary for cell fitness. The other set of screens was aimed at uncovering genes, when knocked-out, compromised cell fitness synthetically with IRF4 deficiency. For each set, we screened in replicate two publicly available sub-genomic guide RNA libraries, one targeting genes involved in gene expression and the other targeting genes involved in apoptosis and cancer processes. In order to detect drop-outs between the start and the end of the experiments, we analyzed samples quantitatively via next generation sequencing of guides and the MAGeCK algorithm. Depletion of guides targeting core essential genes verified the screens’ ability to detect dependency genes. We then defined all significant dependency genes in their respective screens. We observed that, in addition to core essential genes, several genes that are deemed druggable show up as essential for melanoma cell fitness, some of which not previously reported. Several additional genes show up only in IRF4-knock out state, suggesting synthetic dependency. We followed up with pathway enrichment analyses to identify the prominent cellular pathways with dependency genes. Here, we observed gene transcription and translation, and cell cycle/mitosis as pathways enriched for melanoma dependency genes, among others. In addition, RNA processing and protein degradation are among the pathways enriched for dependency genes in the IRF4-knockout setting. The dependency genes uncovered in these CRISPR/Cas9 drop-out screens are expected to pave the way for novel targeting approaches in melanoma by themselves or in combination with IRF4. Citation Format: Ulduz Sobhiafshar, N. C. Tolga Emre. CRISPR/Cas9 essentiality screens identify stand-alone and IRF4-linked genetic vulnerabilities in melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2771.