Interferon Response Genes Research Articles

Abstract 4892: MMAE drives immunomodulatory changes in a preclinical xenograft model that are distinct from other clinical-stage ADC payloads

Abstract Monomethyl auristatin E (MMAE), the payload delivered by vedotin ADCs, exerts its cytotoxic effects via microtubule disruption and induction of ER stress, leading to apoptosis and tumor cell death. In addition, MMAE also has the ability to induce immunogenic cell death (ICD) and immune modulation in the tumor microenvironment (TME) (Liu, 2020; Gray, 2020). Other clinical-stage and approved ADCs incorporate payloads that also cause microtubule disruption (DM1, DM4), or that drive DNA damage through topoisomerase I inhibition (exatecan) as the primary mechanism of action (MOA). While ADCs with different linker payloads produce clinical benefit, their long-term impact on survival and how they pair with PD(L)1 agents may differ based on their ability to elicit immune modulation. In this study we have investigated how these payloads compare to MMAE in their ability to drive immune changes in the TME and describe how these changes could support anti-tumor immunity and improve outcomes in the clinic.Building upon an initial in vitro assessment in which we observed superior induction of ICD markers and underlying ER stress with an MMAE ADC, we utilized a B7H4-expressing MDA-MB-468 xenograft model of triple-negative breast cancer to further characterize the ICD properties of ADC payloads. Tumor-bearing mice were treated with a single dose of B7H4-targeted antibody conjugated to either MMAE, DM1, DM4 or exatecan. Tumors were harvested seven days after treatment and processed for immunohistochemistry (IHC) to assess changes in immune cell infiltration and analyzed by RNA-seq to determine alterations in gene expression. Payload-driven anti-tumor activity was observed with all ADC treatments, each leading to a similar reduction in tumor volume compared to vehicle or unconjugated antibody. IHC staining of tumor sections for F4/80, a mouse macrophage marker, revealed that of the microtubule-disrupting agents, only MMAE significantly increased F4/80+ macrophage infiltration in both the tumor nests and stroma. Increased F4/80+ macrophages in tumor nests and stroma was also observed as a result of B7H4-exatecan ADC treatment. RNA-seq analysis revealed a unique profile for the MMAE conjugate, with an increase in human transcripts encoding cytokine and type I interferon response genes. In summary, the anti-tumor activity of the B7H4-MMAE ADC resulted in recruitment of mouse innate immune cells to the xenograft tumors. Importantly, immune response genes related to ICD were increased following B7H4-MMAE treatment compared to DM1, DM4, and exatecan conjugates. Together, these data suggest that treatment with vedotin ADCs results in robust immunomodulatory changes in vivo that are distinct from other clinical ADC payloads. Citation Format: Michelle Ulrich, Kerry Klussman, John J. Gosink, Sean Allred, Kelly Hensley, Piper M. Treuting, Nikhil J. Parekh, Elizabeth E. Gray, Shyra J. Gardai. MMAE drives immunomodulatory changes in a preclinical xenograft model that are distinct from other clinical-stage ADC payloads. [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 4892.

Abstract 2123: Glycine decarboxylase regulates renal carcinoma progression via interferon stimulated gene factor 3-mediated pathway

Abstract Glycine decarboxylase (GLDC) is a mitochondrial enzyme that mediates the degradation of glycine as part of the glycine cleavage system; it is implicated as a tumor suppressor in hepatocellular carcinoma but as an oncogene in the lung cancer. Although GLDC expression in the kidney is second highest next to the liver, very little is known as to the role of GLDC in the kidney. Thus, this study was designed to determine the role of GLDC in the renal carcinoma. We found markedly increased expressions of GLDC in patients with renal cell carcinoma (RCC) classified as poor-risk group based on the Memorial Sloan Kettering Cancer Center prognostic model. In vitro studies revealed that cellular proliferation, colony formation, and migration were decreased in GLDC-deficient renal carcinoma cells while those were increased in GLDC-overexpressing renal carcinoma cells. In vivo xenograft studies with GLDC-deficient and -overexpressing cells also revealed that tumor sizes were noticeably decreased in GLDC-deficient cell-injected mice while those were increased in GLDC-overexpressing cell-injected mice. Mechanistically, we found GLDC regulates renal carcinoma progression via interferon (IFN) stimulated gene factor 3 (ISGF3)-mediated pathway. Poly-IC induced mRNA expression of IFN-α more strongly in GLDC-deficient cells. Expressions of IRF9 and STAT2 were increased in GLDC-deficient cells while those were decreased in GLDC-overexpressing cells. Interferon-responsive genes (IFIT1, IFI44, IFI44L, and IFI27) were also regulated by GLDC as evidenced by increased mRNA expressions in GLDC-deficient cells and increased mRNA expressions in GLDC-overexpressing cells. Knock-down of IRF9 and STAT2 in GLDC-deficient cells reversed decreased cellular proliferation, colony formation, and migration. In summary, the data in this study indicates that GLDC regulates renal carcinoma progression via ISFG3-medated pathway and lay a scientific foundation that could support a therapeutic strategy that targets GLDC for the treatment of renal carcinoma. Citation Format: Jin Young Kim, Mai Thi Tuyet Pham, Byung Hoon Kim, Ji Hae Seo, Sojin Shin, Eunyoung Ha. Glycine decarboxylase regulates renal carcinoma progression via interferon stimulated gene factor 3-mediated pathway [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 2123.

Positive and negative emotion are associated with generalized transcriptional activation in immune cells

Alterations in immune system gene expression have been implicated in psychopathology, but it remains unclear whether similar associations occur for intraindividual variations in emotion. The present study examined whether positive emotion and negative emotion were related to expression of pro-inflammatory and antiviral genes in circulating leukocytes from a community sample of 90 adolescents (Mage = 16.3 years, SD = 0.7; 51.1% female). Adolescents reported their positive emotion and negative emotion and provided blood samples twice, five weeks apart. Using a multilevel analytic framework, we found that within-individual increases in positive emotion were associated with reduced expression of both pro-inflammatory and Type I interferon (IFN) response genes, even after adjusting for demographic and biological covariates, and for leukocyte subset abundance. By contrast, increases in negative emotion were related to higher expression of pro-inflammatory and Type I IFN genes. When tested in the same model, only associations with positive emotion emerged as significant, and increases in overall emotional valence were associated with both lower pro-inflammatory and antiviral gene expression. These results are distinct from the previously observed Conserved Transcriptional Response to Adversity (CTRA) gene regulation pattern characterized by reciprocal changes in pro-inflammatory and antiviral gene expression and may reflect alterations in generalized immunologic activation. These findings highlight one biological pathway by which emotion may potentially impact health and physiological function in the context of the immune system, and future studies can investigate whether fostering positive emotion may promote adolescent health through changes in the immune system.

SARS-CoV-2 restructures host chromatin architecture.

Some viruses restructure host chromatin, influencing gene expression, with implications for disease outcome. Whether this occurs for SARS-CoV-2, the virus causing COVID-19, is largely unknown. Here we characterized the 3D genome and epigenome of human cells after SARS-CoV-2 infection, finding widespread host chromatin restructuring that features widespread compartment A weakening, A-B mixing, reduced intra-TAD contacts and decreased H3K27ac euchromatin modification levels. Such changes were not found following common-cold-virus HCoV-OC43 infection. Intriguingly, the cohesin complex was notably depleted from intra-TAD regions, indicating that SARS-CoV-2 disrupts cohesin loop extrusion. These altered 3D genome/epigenome structures correlated with transcriptional suppression of interferon response genes by the virus, while increased H3K4me3 was found in the promoters of pro-inflammatory genes highly induced during severe COVID-19. These findings show that SARS-CoV-2 acutely rewires host chromatin, facilitating future studies of the long-term epigenomic impacts of its infection.

DNA methyltransferase inhibition induces dynamic gene expression changes in lung CD4+ T cells of neonatal mice with E. coli pneumonia

Bacterial pulmonary infections are a major cause of morbidity and mortality in neonates, with less severity in older children. Previous studies demonstrated that the DNA of CD4+ T cells in the mouse lung, whose primary responsibility is to coordinate the immune response to foreign pathogens, is differentially methylated in neonates compared with juveniles. Nevertheless, the effect of this differential DNA methylation on CD4+ T cell gene expression and response to infection remains unclear. Here we treated E. coli-infected neonatal (4-day-old) and juvenile (13-day-old) mice with decitabine (DAC), a DNA methyltransferase inhibitor with broad-spectrum DNA demethylating activity, and performed simultaneous genome-wide DNA methylation and transcriptional profiling on lung CD4+ T cells. We show that juvenile and neonatal mice experienced differential demethylation in response to DAC treatment, with larger methylation differences observed in neonates. By cross-filtering differentially expressed genes between juveniles and neonates with those sites that were demethylated in neonates, we find that interferon-responsive genes such as Ifit1 are the most down-regulated methylation-sensitive genes in neonatal mice. DAC treatment shifted neonatal lung CD4+ T cells toward a gene expression program similar to that of juveniles. Following lung infection with E. coli, lung CD4+ T cells in neonatal mice exhibit epigenetic repression of important host defense pathways, which are activated by inhibition of DNA methyltransferase activity to resemble a more mature profile.

Abstract PD4-03: PD4-03 Pelareorep primes the tumor for checkpoint inhibition therapy by activating the interferon-gamma signaling pathway and tumor inflammation signature in early breast cancer patients - results of the AWARE-1 trial

Abstract Background The status of the tumor microenvironment (TME) can profoundly affect the response to immune-based therapies for the treatment of cancer. Results previously reported from the AWARE-1 study in early breast cancer patients demonstrated that pelareorep (pela, an oncolytic reovirus), alone or in combination with checkpoint inhibitor (CPI) therapy, modified the inflammatory state of the TME. We also showed that many of pela’s effects on the TME were enhanced by the addition of checkpoint blockade. Here, we report the effect of treatment with pela on selected molecular markers associated with enhanced anti-tumor immunity. Methods Newly diagnosed HR+/HER2- early BC patients were enrolled into two cohorts: Cohort 1 (C1): pela + letrozole (n=10); and Cohort 2 (C2): pela + letrozole + atezolizumab (n=10). Pela was intravenously administered on days 1, 2 and 8, 9, and atezolizumab was given on day 3. For this analysis, tumor biopsies (FFPE samples) collected pre-treatment (D1) and on days 3 (D3, prior to the atezolizumab administration) were examined by GeoMx digital spatial profiling (DSP, using Nanostring’s Cancer Transcriptome Atlas [CTA]). Moreover, the expression of 770 immune-related genes was analyzed using a specific immune panel (n=20). Gene Set Enrichment Analysis (GSEA) (version 4.1.0) was used to assess pela-induced activation pathways. Results GeoMx DSP showed that pela therapy significantly activated IFN-gamma signaling and associated interferon response genes from D1 to D3 (Normalized Enrichment Score [NES] = 3.3, p-values < 0.02) in the cytokeratin-positive subset of the tumor samples. GSEA of the immune dataset (730 immune genes + 30 housekeeping genes) from the whole tissue also showed a significant upregulation of IFN-gamma signaling pathway genes (FDR < 25%, p-value< 0.001). Increases were also observed in genes reported to be associated with an enhanced tumor inflammatory signal (TIS) including PD-L1, IDO1, HLA-E and STAT1 (p-values < 0.005). Conclusions These results demonstrate that treatment with pela alters the TME to induce and enhance anti-tumor immunity. This enhancement of anti-tumor immunity may potentiate the TME for CPI therapy. Citation Format: Houra Loghmani, Joaquín Gavilá, Luis Manso, Matt Coffey, Richard Trauger, Fernando Salvador, Tomás Pascual, Aleix Prat, Thomas Heineman. PD4-03 Pelareorep primes the tumor for checkpoint inhibition therapy by activating the interferon-gamma signaling pathway and tumor inflammation signature in early breast cancer patients - results of the AWARE-1 trial [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 PD4-03.

Abstract P2-24-02: Analyzing susceptibility to macrophage-activating immunotherapy bexmarilimab in breast cancer by using patient-derived tumor explants

Abstract The immunosuppressive tumor microenvironment governed by tumor-associated macrophages (TAMs) remains a major obstacle to effective cancer immunotherapy. A novel humanized antibody bexmarilimab, targeting the scavenger receptor Clever-1 on TAMs, has shown clinical benefit in ~40% of patients with late-stage ER+ breast cancer (MATINS; NCT03733990). To increase response rates, a comprehensive understanding on how TAM phenotype and the tumor immune landscape affect bexmarilimab-induced immune activation is needed. Single-cell RNA sequencing analysis of breast tumor macrophages shows high phenotypic variation across pathological subtypes and Clever-1 mRNA (STAB1) expression on various immunosuppressive IL4I1+ and TREM2+ monocyte-derived TAMs. STAB1 expressing macrophages were located in both lymphocyte rich and excluded areas in the tumor stroma based on digital spatial profiling with GeoMx. Due to this diversity, we studied bexmarilimab mode-of-action in breast cancer patient-derived tumor explant cultures (PDEC) by RNA sequencing and cytokine profiling. Our results show that bexmarilimab induced TNFα and CXCL10 secretion in 30-40% of the treated explants. In depth analysis of the responsive PDECs revealed a lower baseline expression of interferon responsive genes in their tumor microenvironment. Correspondingly, in vitro assays with primary human macrophages showed that chronic IFNγ priming abolished bexmarilimab-induced immune activation, suggesting potential use of bexmarilimab in patients with immunologically cold breast tumors. Citation Format: Jenna H. Rannikko, Reetta Virtakoivu, Akira Takeda, Pia Boström, Ilkka Koskivuo, Petri Bono, Maija Hollmén. Analyzing susceptibility to macrophage-activating immunotherapy bexmarilimab in breast cancer by using patient-derived tumor explants [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 P2-24-02.

Engineered antibody cytokine chimera synergizes with DNA-launched nanoparticle vaccines to potentiate melanoma suppression in vivo.

Cancer immunotherapy has demonstrated great promise with several checkpoint inhibitors being approved as the first-line therapy for some types of cancer, and new engineered cytokines such as Neo2/15 now being evaluated in many studies. In this work, we designed antibody-cytokine chimera (ACC) scaffolding cytokine mimetics on a full-length tumor-specific antibody. We characterized the pharmacokinetic (PK) and pharmacodynamic (PD) properties of first-generation ACC TA99-Neo2/15, which synergized with DLnano-vaccines to suppress in vivo melanoma proliferation and induced significant systemic cytokine activation. A novel second-generation ACC TA99-HL2-KOA1, with retained IL-2Rβ/γ binding and attenuated but preserved IL-2Rα binding, induced lower systemic cytokine activation with non-inferior protection in murine tumor studies. Transcriptomic analyses demonstrated an upregulation of Type I interferon responsive genes, particularly ISG15, in dendritic cells, macrophages and monocytes following TA99-HL2-KOA1 treatment. Characterization of additional ACCs in combination with cancer vaccines will likely be an important area of research for treating melanoma and other types of cancer.

TET1 and TDG Suppress Inflammatory Response in Intestinal Tumorigenesis: Implications for Colorectal Tumors With the CpG Island Methylator Phenotype

Aberrant DNA methylation is frequent in colorectal cancer (CRC), but underlying mechanisms and pathologic consequences are poorly understood. We disrupted active DNA demethylation genes Tet1 and/or Tdg from ApcMin mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas. There were increased numbers of small intestinal adenomas in ApcMin mice expressing the TdgN151A allele, whereas Tet1-deficient and Tet1/TdgN151A-double heterozygous ApcMin colonic adenomas were larger with features of erosion and invasion. We detected reduction in global DNA hypomethylation in colonic adenomas from Tet1- and Tdg-mutant ApcMin mice and hypermethylation of CpG islands in Tet1-mutant ApcMin adenomas. Up-regulation of inflammatory, immune, and interferon response genes was present in Tet1- and Tdg-mutant colonic adenomas compared to control ApcMin adenomas. This up-regulation was also seen in murine colonic organoids and human CRC lines infected with lentiviruses expressing TET1 or TDG short hairpin RNA. A 127-gene inflammatory signature separated colonic adenocarcinomas into 4 groups, closely aligned with their microsatellite or chromosomal instability and characterized by different levels of DNA methylation and DNMT1 expression that anticorrelated with TET1 expression. Tumors with the CpG island methylator phenotype (CIMP) had concerted high DNMT1/low TET1 expression. TET1 or TDG knockdown in CRC lines enhanced killing by natural killer cells. Our findings reveal a novel epigenetic regulation, linked to the type of genomic instability, by which TET1/TDG-mediated DNA demethylation decreases methylation levels and inflammatory/interferon/immune responses. CIMP in CRC is triggered by an imbalance of methylating activities over demethylating activities. These mice represent a model of CIMP CRC.

Butyrate limits the replication of porcine epidemic diarrhea virus in intestine epithelial cells by enhancing GPR43-mediated IFN-III production.

Porcine epidemic diarrhea virus (PEDV) is a threat to the health of newborn piglets and has a significant impact on the swine industry. Short-chain fatty acids (SCFAs) are gut microbial metabolites that regulate intestinal function through different mechanisms to enhance the intestinal barrier and immune function. In this study, we aimed to determine whether butyrate displayed a better effect than other SCFAs on limiting PEDV replication in porcine intestinal epithelial cells. Mechanistically, butyrate treatment activated the interferon (IFN) response and interferon-stimulated gene (ISG) expression. Further experiments showed that inhibition of GPR43 (free fatty acid receptor 2) in intestinal epithelial cells increased virus infection and reduced antiviral effects through IFN λ response. Our findings revealed that butyrate exerts its antiviral effects by inducing GPR43-mediated IFN production in intestinal epithelial cells.

Differential roles of type I interferon signaling in tumor versus host cells in experimental glioma models

Despite multimodal treatment approaches including surgery, radiotherapy and chemotherapy, the median survival for patients with glioblastoma remains in the range of one year and thus poor. Type I interferons (IFN) are involved in immune responses to viral infection and exhibit anti-tumor activity in certain cancers.Here we explored the biological relevance of constitutive type I IFN signaling in murine glioma models in vitro and in vivo. CT-2A, GL-261, SMA-497, SMA-540 and SMA-560 murine glioma cells expressed IFN type I receptors IFNAR1 and IFNAR2 and were responsive to exogenous IFN stimulation. CRISPR/Cas9-mediated deletion of IFNAR1 decreased the baseline expression of type I IFN response genes in GL-261 cells, but neither in CT-2A nor in SMA-560 cells. IFNAR1 deletion slowed growth in GL-261 and SMA-560, but not in CT-2A cells. However, only the growth of IFNAR1-depleted GL-261 tumors and not that of SMA-560 tumors was delayed in vivo upon orthotopic tumor cell implantation into syngeneic mice. This survival gain was no longer detected when the IFNAR1-depleted GL-261 cells were inoculated into IFNAR1-deficient mice. Altogether these data suggest that constitutive type I IFN signaling in gliomas may be pro-tumorigenic, but only in a microenvironment that is proficient for type I IFN signaling in the host.

Zika virus alters osteogenic lineage progression of human mesenchymal stromal cells.

Arboviruses target bone forming osteoblasts and perturb bone remodeling via paracrine factors. We previously reported that Zika virus (ZIKV) infection of early-stage human mesenchymal stromal cells (MSCs) inhibited the osteogenic lineage commitment of MSCs. To understand the physiological interplay between bone development and ZIKV pathogenesis, we employed a primary in vitro model to examine the biological responses of MSCs to ZIKV infection at different stages of osteogenesis. Precommitted MSCs were infected at the late stage of osteogenic stimulation (Day 7) with ZIKV (multiplicity of infectionof 5). We observe that MSCs infected at the late stage of differentiation are highly susceptible to ZIKV infection similar to previous observations with early stage infected MSCs (Day 0). However, in contrast to ZIKV infection at the early stage of differentiation, infection at a later stage significantly elevates the key osteogenic markers and calcium content. Comparative RNA sequencing (RNA-seq) of early and late stage infected MSCs reveals that ZIKV infection alters the mRNA transcriptome during osteogenic induction of MSCs (1251 genes). ZIKV infection provokes a robust antiviral response at both stages of osteogenic differentiation as reflected by the upregulation of interferon responsive genes (n > 140). ZIKV infection enhances the expression of immune-related genes in early stage MSCs while increasing cell cycle genes in late stage MSCs. Remarkably, ZIKA infection in early stage MSCs also activates lipid metabolism-related pathways. In conclusion, ZIKV infection has differentiation stage-dependent effects on MSCs and this mechanistic understanding may permit the development of new therapeutic or preventative measures for bone-related effects of ZIKV infection.

Histone Deacetylase Inhibitor Panobinostat Benefits the Therapeutic Efficacy of Oncolytic Herpes Simplex Virus Combined with PD-1/PD-L1 Blocking in Glioma and Squamous Cell Carcinoma Models.

Combination therapy has been widely explored for oncolytic virus (OV), as it can be met with tumor resistance. The HDAC inhibitor (HDACi) panobinostat is a potent pan-deacetylase inhibitor which blocks multiple cancer-related pathways and reverses epigenetic events in cancer progression. In this study, oncolytic activity in vitro and antitumor therapeutic efficacy in vivo when combined with oHSV and panobinostat were investigated. (1) Treatment with panobinostat enhanced oHSV propagation and cytotoxicity in human glioma A172 and squamous cell carcinoma SCC9 cells. (2) Combined treatment with oHSV and panobinostat enhanced virus replication mediated by the transcriptional downregulation of IFN-β- and IFN-responsive antiviral genes in human glioma A172 and squamous cell carcinoma SCC9 cells. (3) Panobinostat treatment induced upregulation of PD-L1 expression in both glioma and squamous cell carcinoma cells. (4) A significantly enhanced therapeutic efficacy was shown in vivo for the murine glioma CT-2A and squamous cell carcinoma SCC7 models when treated with a combination of oHSV, including PD-1/PD-L1 blockade and HDAC inhibition. Consequently, these data provide some new clues for the clinical development of combination therapy with OVs, epigenetic modifiers, and checkpoint blockades for glioma and squamous cell carcinoma.

Breast cancer cells survive chemotherapy by activating targetable immune-modulatory programs characterized by PD-L1 or CD80.

Breast cancer cells must avoid intrinsic and extrinsic cell death to relapse following chemotherapy. Entering senescence enables survival from mitotic catastrophe, apoptosis and nutrient deprivation, but mechanisms of immune evasion are poorly understood. Here we show that breast tumors surviving chemotherapy activate complex programs of immune modulation. Characterization of residual disease revealed distinct tumor cell populations. The first population was characterized by interferon response genes, typified by Cd274, whose expression required chemotherapy to enhance chromatin accessibility, enabling recruitment of IRF1 transcription factor. A second population was characterized by p53 signaling, typified by CD80 expression. Treating mammary tumors with chemotherapy followed by targeting the PD-L1 and/or CD80 axes resulted in marked accumulation of T cells and improved response; however, even combination strategies failed to fully eradicate tumors in the majority of cases. Our findings reveal the challenge of eliminating residual disease populated by senescent cells expressing redundant immune inhibitory pathways and highlight the need for rational immune targeting strategies.

DNA damage independent inhibition of NF-kB transcription by anthracyclines.

Anthracyclines are among the most used and effective anticancer drugs. Their activity has been attributed to DNA double-strand breaks resulting from topoisomerase II poisoning and to eviction of histones from select sites in the genome. Here we show that the extensively used anthracyclines Doxorubicin, Daunorubicin and Epirubicin decrease the transcription of nuclear factor kappa B (NF-κB)-dependent gene targets, but not interferon responsive genes in primary mouse (Mus musculus) macrophages. Using an NMR-based structural approach, we demonstrate that anthracyclines disturb the complexes formed between the NF-kB subunit RelA and its DNA binding sites. The anthracycline variants Aclarubicin, Doxorubicinone and the newly developed Dimethyl-doxorubicin, which share anticancer properties with the other anthracyclines but do not induce DNA damage, also suppressed inflammation, thus uncoupling DNA damage from the effects on inflammation. These findings have implications for anticancer therapy and for the development of novel anti-inflammatory drugs with limited side effects for life-threatening conditions such as sepsis.

IFITM3 regulates fibrinogen endocytosis and platelet reactivity in nonviral sepsis.

Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We determined whether nonviral sepsis induces differential platelet gene expression and reactivity. Nonviral sepsis upregulated IFN-induced transmembrane protein 3 (IFITM3), an IFN-responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we determined whether IFITM3 promoted endocytosis of α-granule proteins. IFN stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm-/- mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacted with clathrin and αIIb and altered their plasma membrane localization into lipid rafts. In vivo IFN administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm-/- mice were completely rescued from IFN-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on IFN-α and IFITMs. Platelets from patients with nonviral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.

Abstract B007: Endogenous retrotransposable elements induction in SMARCA4-deficient ovarian cancer is associated with immunomodulatory changes in the tumor microenvironment

Abstract Introduction: Ovarian cancer (OC) is the most lethal gynecologic cancer, and there is an urgent need to develop new therapies. Patient responses to immune checkpoint blockade have overall been disappointing, which is likely due to the immunosuppressive tumor microenvironment (TME) of most OCs that may be in part under the influence of epigenetic regulation. There is evidence that OCs that harbor loss-of-function mutations in the SWI/SNF chromatin remodeling complex catalytic subunit, SMARCA4, may be more immunogenic and amenable to immunotherapy, however the mechanisms by which this occurs is unclear. Proposed mechanisms include de-methylation of normally silenced noncoding elements. The aim of our study is to investigate the epigenetic changes of retrotransposable elements (RTEs) induced by SMARCA4 mutations that modulate the TME. Experimental Procedures: Stable isogenic SMARCA4-knockout (KO) murine (ID8) and human (OAW28) OC models were generated using CRISPR-Cas9 lentiviral transduction and clonal selection. Transcriptomic analyses of these cells were performed using RNA sequencing using ribosomal depletion protocol and validated using qRT-PCR. Moreover, differentially expressed coding genes in RNA sequencing were identified according to fold change between KO and control cells of RPKM (Reads Per Kilobase of transcript, per Million mapped reads) values. Gene Ontology enrichment analysis was then performed to identify the most altered pathways. Results: CRISPR-Cas9 KO of SMARCA4 in OC cells led to an increased fraction of reads coming from repetitive elements (repeatome). Within the repeatome, short interspersed nuclear elements (SINEs) have increased fraction of mapped reads in KO compared to wildtype OAW28 cells. Quantification analyses of known repeats demonstrate significant upregulation of endogenous retroviruses (ERVs) in KO compared to control ID8 cells. Certain RTEs known to be associated with viral mimicry and increased immunogenicity were upregulated in KO cells. Top 10 upregulated genes were significantly enriched in pathways related to interferon and viral responses. Further quantification analyses demonstrated that the dsRNA sensing pathway was activated in SMARCA4-deficient OCs leading to downstream increase in interferon-response genes. Knockdown of dsRNA sensor mitochondrial antiviral signaling protein (MAVS) downregulates the SMARCA4 loss-induced interferon-response activation. Conclusion: De-repression of normally silenced RTEs and subsequent increased immunogenicity of SMARCA4-deficient OCs suggest a potential new immunomodulatory biomarker for targeted therapy investigation. A mechanistic understanding of the role of SMARCA4 in OCs enhances our knowledge of the epigenetic processes that govern immune-stimulating responses in the TME. Studies are underway to explore combination epigenetic therapy and immunotherapy in these cancers. Citation Format: Melica N. Brodeur, Siyu Sun, Yingjie Zhu, Benjamin Greenbaum, Britta Weigelt, Dmitriy Zamarin. Endogenous retrotransposable elements induction in SMARCA4-deficient ovarian cancer is associated with immunomodulatory changes in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B007.

Neuronal nuclear calcium signaling suppression of microglial reactivity is mediated by osteoprotegerin after traumatic brain injury

BackgroundTraumatic brain injury (TBI) is characterized by massive changes in neuronal excitation, from acute excitotoxicity to chronic hyper- or hypoexcitability. Nuclear calcium signaling pathways are involved in translating changes in synaptic inputs and neuronal activity into discrete transcriptional programs which not only affect neuronal survival and synaptic integrity, but also the crosstalk between neurons and glial cells. Here, we report the effects of blunting neuronal nuclear calcium signals in the context of TBI.MethodsWe used AAV vectors to express the genetically encoded and nuclear-targeted calcium buffer parvalbumin (PV.NLS.mCherry) or the calcium/calmodulin buffer CaMBP4.mCherry in neurons only. Upon TBI, the extent of neuroinflammation, neuronal death and synaptic loss were assessed by immunohistochemistry and targeted transcriptome analysis. Modulation of the overall level of neuronal activity was achieved by PSAM/PSEM chemogenetics targeted to parvalbumin interneurons. The functional impact of neuronal nuclear calcium buffering in TBI was assessed by quantification of spontaneous whisking.ResultsBuffering neuronal nuclear calcium unexpectedly resulted in a massive and long-lasting increase in the recruitment of reactive microglia to the injury site, which was characterized by a disease-associated and phagocytic phenotype. This effect was accompanied by a substantial surge in synaptic loss and significantly reduced whisking activity. Transcriptome analysis revealed a complex effect of TBI in the context of neuronal nuclear calcium buffering, with upregulation of complement factors, chemokines and interferon-response genes, as well as the downregulation of synaptic genes and epigenetic regulators compared to control conditions. Notably, nuclear calcium buffering led to a substantial loss in neuronal osteoprotegerin (OPG), whereas stimulation of neuronal firing induced OPG expression. Viral re-expression of OPG resulted in decreased microglial recruitment and synaptic loss. OPG upregulation was also observed in the CSF of human TBI patients, underscoring its translational value.ConclusionNeuronal nuclear calcium signals regulate the degree of microglial recruitment and reactivity upon TBI via, among others, osteoprotegerin signals. Our findings support a model whereby neuronal activity altered after TBI exerts a powerful impact on the neuroinflammatory cascade, which in turn contributes to the overall loss of synapses and functional impairment.

Investigating Gene Expression Signatures and Cell-Type Composition Changes in Sickle Cell Disease Using Whole Blood Transcriptomics

Background: Sickle cell disease (SCD) is a devastating Mendelian disease affecting millions of people worldwide. Although SCD is caused by a single-point mutation (Glu6Val) in the β-globin chain of hemoglobin, the clinical manifestations are heterogeneous and systemic, impacting the functions of multiple organs and leading to early mortality. Predicting disease severity and identifying the molecular mechanisms underlying disease pathology and progression remain a challenge. Objective: The goal of this work was to characterize the clinical heterogeneity associated with SCD using transcriptomics. Methods: We integrated publicly available bulk and single-cell RNA sequencing (scRNA-seq) data sets that included samples from pediatric to young adult patients and contained clinical phenotypes to identify molecular signatures of SCD severity and heterogeneity (Table). Our integrative transcriptomics analyses included the following: Identify cell-type clusters and derive novel progenitor signatures from an integrative analysis of the 2 independent scRNA-seq data sets capturing CD34+-enriched or unselected bone marrow (BM) cells from SCD and control samples.Calculate the relative proportions of cell types in whole blood by deconvolution using both the scRNA-seq-derived novel cell-type signatures (dtangle) and publicly available immune cell-type signatures (xCell, EPIC, quanTIseq).Identify signatures (genes, pathways, cell types) associated with disease severity and hemoglobin genotypes using various bioinformatics approaches, including differential gene expression and single-sample Gene Set Enrichment Analysis, among others, and examine relationships of enriched cell types and pathways (queried from MSigDB) in discovery followed by confirmation in validation data sets. Results: Cell-type clusters and gene signatures were generated by combining and integrating scRNA-seq data sets from BM SCD and control samples (Table). Using our fine-mapped cell-type clusters, we found an erythroid progenitor subclass to be depleted, whereas a lymphoid progenitor subclass expanded in SCD BM samples, increasing granularity of the cell types reported by Hua et al. Cell-type deconvolution of bulk transcriptome data using the combined scRNA-seq-derived signatures revealed enriched monocyte relative proportions in acute crisis compared with steady-state SCD samples (β=0.01, P=1.13×10−7) and enriched erythroblast relative proportions in SCD compared with control samples (β=0.02, P=1.10×10−12). Deconvolution using publicly available immune cell signatures showed significant enrichment of neutrophil and macrophage signatures in acute SCD samples (Padj<0.1). Analysis of bulk RNA-seq data indicated a set of highly correlated genes differentially expressed in steady-state SCD and associated with varied pathways, including red blood cell (RBC) morphology, hematopoiesis, anemia, and cholelithiasis. Higher pathway scores were seen in severe (HbSS) genotype samples than in milder (HbSC) genotype samples, consistent with known disease pathology and severity (Figure). Building on results from integrative analyses, we found that interferon and inflammatory response genes were enriched in acute SCD in both discovery and validation data sets. We found significant correlations between monocyte relative proportions and interferon signaling pathway scores, and between erythroblast relative proportions and the pathway scores affecting RBC production, morphology, and pathology and potential SCD comorbidities such as cholelithiasis (Figure). Conclusions: Our integrative scRNA-seq analysis led to robust cell cluster identification to improve deconvolution analysis of bulk transcriptomics data. We found elevated interferon and inflammatory pathway activity in acute SCD samples, consistent with findings in the literature (PMIDs: 34166491, 32764656). Furthermore, we found enriched neutrophils and monocyte signatures in acute SCD, suggesting their role in mediating the innate immune response. These findings provide motivation to characterize the role of neutrophils and monocytes in acute SCD crises. Our integrative transcriptomics analyses showed defective RBC production and maturation in SCD. Using the signatures from this work, we aim to elucidate the heterogeneity within SCD pathophysiology. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IMMU-11. NONO IS A NEW ADENOVIRUS SENSOR: IMPLICATION FOR VIROTHERAPY

Abstract The median survival for GBM patients is 15 months with a five-year survival rate of less than 10%. Current conventional and experimental treatments, including immune checkpoint blockade, have not improved outcomes in the vast majority of glioma patients. Oncolytic viruses (OVs) are designed to specifically infect and lyse cancer cells to elicit an anti-tumor immune response. In a clinical trial developed by the MDACC team, 20% of GBM patients treated with the oncolytic adenovirus Delta-24-RGD showed complete response and survival longer than three years (NCT00805376). Although these studies have shown that robust immune responses can be invoked using oncolytic virotherapy, the molecular mechanisms underlying OV-mediated anti-tumor immunity remain elusive. Exposure to viruses by innate immune sensors induces protective antiviral immunity. Using bulk RNA sequencing of cells infected with adenovirus we identified the main upstream regulators of the innate immune response including IFN gamma, Interferon responsive genes 3 and 7, and STAT3. However, RNAseq unexpectedly uncovered NONO (Non-POU Domain Containing Octamer Binding) as the most relevant upstream regulator. In agreement with the RNAseq data, Protein analyses demonstrated the upregulation of NONO after adenovirus infection. In addition, immunofluorescence and confocal microscopy examination of the infected cells showed the overexpression and translocation to the nucleus of NONO upon virus infection. We are the first laboratory that has identified NONO as a sensor of adenovirus infection, which may have great significance for the future development of oncolytic viruses. Importantly, we discovered the network of proteins that are upregulated after NONO activation and, finally, we have established that changes in the expression level and subcellular localization of NONO are coincident in time during the infection of cultured cells. Future work is focused on examining the immunomodulatory functions of NONO in adenoviral infected tumors and subsequent generation of NONO-resistant oncolytic adenovirus.