The innate immune system is a two-edged sword; it is absolutely required for host defense against infection but, uncontrolled, can trigger a plethora of inflammatory diseases. Here we used systems biology approaches to predict and validate a gene regulatory network involving a dynamic interplay between the transcription factors NF-κB, C/EBPδ, and ATF3 that controls inflammatory responses. We mathematically modeled transcriptional regulation of Il6 and Cebpd genes and experimentally validated the prediction that the combination of an initiator (NF-κB), an amplifier (C/EBPδ) and an attenuator (ATF3) forms a regulatory circuit that discriminates between transient and persistent Toll-like receptor 4-induced signals. Our results suggest a mechanism that enables the innate immune system to detect the duration of infection and to respond appropriately.

Summary Mutations in MECP2, encoding the epigenetic regulator methyl-CpG-binding protein 2, are the predominant cause of Rett syndrome, a disease characterized by both neurological symptoms and systemic abnormalities. Microglial dysfunction is thought to contribute to disease pathogenesis, and here we found microglia become activated and subsequently lost with disease progression in Mecp2-null mice. Mecp2 was found to be expressed in peripheral macrophage and monocyte populations, several of which also became depleted in Mecp2-null mice. RNA-seq revealed increased expression of glucocorticoid- and hypoxia-induced transcripts in Mecp2-null microglia and peritoneal macrophages. Furthermore, Mecp2 was found to regulate inflammatory gene transcription in response to TNF stimulation. Postnatal re-expression of Mecp2 using Cx3cr1creER increased the lifespan of otherwise Mecp2-null mice. These data suggest Mecp2 regulates microglia and macrophage responsiveness to environmental stimuli to promote homeostasis. Dysfunction of tissue-resident macrophages may contribute to the systemic pathologies observed in Rett syndrome.

Although TLR5 regulates the innate immune response to bacterial flagellin, it is unclear whether its function is essential during in vivo murine infections. To examine this question, we challenged Tlr5−/− mice transurethrally with Escherichia coli. At 2 days postinfection, wild-type mice exhibited increased inflammation of the bladder in comparison to Tlr5−/− mice. By day 5 postinfection, Tlr5−/− mice had significantly more bacteria in the bladders and kidneys in comparison to wild-type mice and showed increased inflammation in both organs. In addition, flagellin induced high levels of cytokine and chemokine expression in the bladder that was dependent on TLR5. Together, these data represent the first evidence that TLR5 regulates the innate immune response in the urinary tract and is essential for an effective murine in vivo immune response to an extracellular pathogen.

Antiviral responses must be tightly regulated to rapidly defend against infection while minimizing inflammatory damage. Type 1 interferons (IFN-I) are crucial mediators of antiviral responses1 and their transcription is regulated by a variety of transcription factors2; principal amongst these is the family of interferon regulatory factors (IRFs)3. The IRF gene regulatory networks are complex and contain multiple feedback loops. The tools of systems biology are well suited to elucidate the complex interactions that give rise to precise coordination of the interferon response. Here we have used an unbiased systems approach to predict that a member of the forkhead family of transcription factors, FOXO3, is a negative regulator of a subset of antiviral genes. This prediction was validated using macrophages isolated from Foxo3-null mice. Genome-wide location analysis combined with gene deletion studies identified the Irf7 gene as a critical target of FOXO3. FOXO3 was identified as a negative regulator of Irf7 transcription and we have further demonstrated that FOXO3, IRF7 and IFN-I form a coherent feed-forward regulatory circuit. Our data suggest that the FOXO3-IRF7 regulatory circuit represents a novel mechanism for establishing the requisite set points in the interferon pathway that balances the beneficial effects and deleterious sequelae of the antiviral response.

Motivation: Histone acetylation (HAc) is associated with open chromatin, and HAc has been shown to facilitate transcription factor (TF) binding in mammalian cells. In the innate immune system context, epigenetic studies strongly implicate HAc in the transcriptional response of activated macrophages. We hypothesized that using data from large-scale sequencing of a HAc chromatin immunoprecipitation assay (ChIP-Seq) would improve the performance of computational prediction of binding locations of TFs mediating the response to a signaling event, namely, macrophage activation.Results: We tested this hypothesis using a multi-evidence approach for predicting binding sites. As a training/test dataset, we used ChIP-Seq-derived TF binding site locations for five TFs in activated murine macrophages. Our model combined TF binding site motif scanning with evidence from sequence-based sources and from HAc ChIP-Seq data, using a weighted sum of thresholded scores. We find that using HAc data significantly improves the performance of motif-based TF binding site prediction. Furthermore, we find that within regions of high HAc, local minima of the HAc ChIP-Seq signal are particularly strongly correlated with TF binding locations. Our model, using motif scanning and HAc local minima, improves the sensitivity for TF binding site prediction by ∼50% over a model based on motif scanning alone, at a false positive rate cutoff of 0.01.Availability: The data and software source code for model training and validation are freely available online at http://magnet.systemsbiology.net/hac.Contact: aderem@systemsbiology.org; ishmulevich@systemsbiology.orgSupplementary information: Supplementary data are available at Bioinformatics online.

Pancreatic ductal adenocarcinoma (PDAC) is classified into two key subtypes, classical and basal, with basal PDAC predicting worse survival. Using in vitro drug assays, genetic manipulation experiments, and in vivo drug studies in human patient-derived xenografts (PDXs) of PDAC, we found that basal PDACs were uniquely sensitive to transcriptional inhibition by targeting cyclin-dependent kinase 7 (CDK7) and CDK9, and this sensitivity was recapitulated in the basal subtype of breast cancer. We showed in cell lines, PDXs, and publicly available patient datasets that basal PDAC was characterized by inactivation of the integrated stress response (ISR), which leads to a higher rate of global mRNA translation. Moreover, we identified the histone deacetylase sirtuin 6 (SIRT6) as a critical regulator of a constitutively active ISR. Using expression analysis, polysome sequencing, immunofluorescence, and cycloheximide chase experiments, we found that SIRT6 regulated protein stability by binding activating transcription factor 4 (ATF4) in nuclear speckles and protecting it from proteasomal degradation. In human PDAC cell lines and organoids as well as in murine PDAC genetically engineered mouse models where SIRT6 was deleted or down-regulated, we demonstrated that SIRT6 loss both defined the basal PDAC subtype and led to reduced ATF4 protein stability and a nonfunctional ISR, causing a marked vulnerability to CDK7 and CDK9 inhibitors. Thus, we have uncovered an important mechanism regulating a stress-induced transcriptional program that may be exploited with targeted therapies in particularly aggressive PDAC.

Pancreatic Ductal Adenocarcinoma (PDA) can be characterized by two distinct transcriptional subtypes: Classical and Basal-like. The basal PDA subtype is more aggressive and has the worst overall survival. Previous work has shown that Sirtuin 6 histone deacetylase (SIRT6) acts as a tumor suppressor and cooperates with oncogenic KRAS in GEMM models of PDA. Our study identifies SIRT6 as a biomarker for basal PDA and investigates the underlying sensitivity of basal PDA to inhibition of transcriptional cyclin dependent kinases (CDKs). Through analysis of data from hundreds of human PDA tumors, we identified that low SIRT6 expression correlates with basal PDA while the converse is true for classical PDA. Using many in vitro drug assays, genetic manipulation experiments, and in vivo drug studies in human patient-derived xenografts of PDA, we also discovered that basal PDA is uniquely sensitive to the covalent CDK7/12/13 inhibitor THZ1 as well as other inhibitors of transcriptional CDKs. Importantly, removal of SIRT6 can sensitize SIRT6 high/classical PDA to THZ1. Further investigation identified that SIRT6 low/basal PDA has an inactivated Integrated Stress Response (ISR), which is primarily driven by ATF4. To determine how loss of SIRT6 leads to an inability to activate the ISR, we have integrated use of human PDA cell lines, and organoids as well as murine PDA GEMMs where SIRT6 has been deleted or downregulated. We uncovered that SIRT6 regulates the ISR through control of ATF4 translation, specifically through a 3’ UTR open reading frame (uORF) dependent mechanism. To determine whether ISR activation could protect basal PDA cells from transcriptional CDK inhibition, we manipulated expression of ISR pathway components. We found that activation of the ISR in SIRT6 low/basal PDA reduces sensitivity to THZ1, while inactivation of the ISR sensitizes SIRT6 high/classical PDA to this inhibitor. Interestingly, both basal and classical PDA exhibit increased phosphorylation of eIF2a upon inhibition of transcriptional CDKs, which typically indicates activation of the ISR in response to stress. However, this activation of the ISR remains incomplete since ATF4 translation is not upregulated in basal PDA in the absence of SIRT6. Therefore, we discovered that loss of SIRT6 sensitizes basal PDA to inhibition of transcriptional CDKs through an inability to launch a functional ISR response. Furthermore, we have uncovered an important biomarker which controls a stress-induced transcriptional program that may be exploited with targeted therapies in a particularly aggressive PDA subtype. Citation Format: Jessica Gianopulos, Nithya Kartha, Zachary Schrank, Stephanie Dobersch, Sarah Cavender, Bryan Kynnap, Adrianne Wallace-Povirk, Cynthia Wladyka, Juan Santana, Jaeseung C. Kim, Angela Yu, Caroline Bridgwater, Kathrin Fuchs, Sarah Dysinger, Aaron Lampano, Faiyaz Notta, David Price, Andrew Hsieh, Sunil Hingorani, Sita Kugel. Sirtuin 6 histone deacetylase is required for the integrated stress response and resistance to inhibition of transcriptional cyclin dependent kinases in Pancreatic Ductal Adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B075.

Background: We have developed a novel gene therapy platform based on fusogen biology that allows targeted delivery of CAR transgenes to “resting” T cells through systemic administration of a CD8-targeted paramyxovirus-based viral vector (VV). Therapies utilizing autologous CAR T cells have demonstrated success using the murine CD19-specific scFv, FMC63. These murine sequences have the potential to incite T cell and antibody responses against the CAR, which may play a role in CAR T cell elimination. This scenario increases in likelihood in the context of in vivo CAR delivery, where immunosuppression via lymphodepletion is not required. To reduce the potential for CAR-specific immunogenicity, we developed a novel, fully human CD19-specific CAR for use with our in vivo delivery platform. Methods: For efficacy-based screening of human CD19 binders, healthy donor T cells were transduced with VSV-g pseudotyped lentivirus containing second-generation fully human CD19 CARs. FMC63 CARs were produced in a similar fashion and used as benchmark controls for initial characterization studies. CD8-targeted fusogen was generated through targeted mutation to ablate binding to its native receptor. This “blinded” fusogen was subsequently engineered to display a novel scFv that is specific for human CD8α. VV encoding either FMC63 or human CD19 CARs were pseudotyped with CD8-specific fusogen to produce CD8-targeted fusosomes. CD19 CAR efficacy was analyzed in vitro via luciferase assays against NALM-6 or CD19KO NALM-6 tumor lines. Systemic NALM-6 in vivo tumor models (IV injection) were performed in NSG mice. Results: Here we show, our fully human CD19 CAR is comparable to FMC63 CAR in vitro using both short-term luciferase cytotoxicity and long-term Incucyte tumor killing assays. Furthermore, human CD19 CAR T cells demonstrate similar levels of tumor control NALM-6 tumor bearing mice treated with ex vivo generated CD19 CAR+ cells (Mean AUC [n = 3 donors]: FMC63 = 1.26e4, huCD19 = 4.86e4). Our lead human CD19 CAR, like FMC63, lacked killing and cytokine production when cultured with CD19KO NALM-6 tumor cells, demonstrating CD19 antigen specificity. Furthermore, CD8-targeted in vivo delivery of our fully human CD19 CAR showed reduction in NALM-6 tumor load similar to FMC63 CAR (Mean AUC [n = 2 donors]: FMC63 = 1.59e5, huCD19 = 5.06e4). Summary: In vivo delivery of our fully human CD19 CAR with CD8-targeted fusosomes affords tumor clearance comparable to FMC63-CAR in mice bearing NALM-6 tumors. We believe this fully human CD19 CAR has the potential to reduce the risk of immunogenicity compared to a CD19 CAR with the murine scFv FMC63 potentially allowing increased expansion and persistence of CD19 CAR T cells following in vivo delivery in patients. Citation Format: Jeremy M. Kinder, Christie Ciarlo, Chanel Athena Estrada, Neal van Hoeven, Vandana Chaturvedi, Garrett Zipp, Aaron Lampano, Adam J. Johnson, Kutlu Elpek, Terry J. Fry, Aaron E. Foster. Development of a novel, fully human anti-CD19 chimeric antigen receptor for in vivo delivery via CD8-targeted fusosome [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 2735.