FARE Special interest Group (SIG) Awards

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FARE2023 Scientific Interest Group (SIG) Awards

SIG Awards were introduced to the Fellows Award for Research Excellence (FARE) Competition in 2018 to further recognize and merit the work of FARE awardees, and to provide an opportunity for them to showcase their research to the wider scientific community. The SIG Award involves the selection of a winning abstract from the FARE Competition by participating SIGs that they deem to be of high scientific merit. These awardees are then invited to present their work at one of the SIG meetings.

Participating SIGs:

SIG Scholar Awards:

Woman Scientist Advisors (WSA)

FARE2023 SIG Awardees

Antibody Interest Group

Raisa A. Glabman, VMD, MSPH (NCI-CCR)

CNear-infrared photoimmunotherapy targeting cancer-associated fibroblasts in the tumor microenvironment

Cancer-associated fibroblasts (CAFs) are a major component of the tumor stroma and play an important role in promoting tumor growth, angiogenesis, metastasis, and resistance to immunotherapy. CAFs form both a physical and immunological barrier to cancer therapeutics, by production of extracellular matrix and immunosuppressive cytokines, which contribute to immune cell exclusion. Although many preclinical studies targeting CAFs have shown promising anti-tumor effects, no definitive treatment targeting tumor stroma has been established, and none have demonstrated clinical efficacy in human clinical trials. There is a critical need to gain a more thorough understanding of CAFs, particularly their tumor-promoting processes and immunosuppressive role in the tumor microenvironment. The goal of this study is to evaluate the therapeutic efficacy of intratumoral CAF depletion with near-infrared photoimmunotherapy (NIR-PIT). We hypothesized that the depletion of CAFs within the tumor microenvironment will restrict tumor growth by restoring anti-tumor immunity. Using NIR-PIT, a novel therapy employing a cell-specific monoclonal antibody conjugated to a photosensitizer, CAFs expressing fibroblast activation protein (FAP) can be locally depleted with NIR light exposure following anti-FAP antibody conjugate administration. We used an immune competent, spontaneous mouse mammary tumor model (MMTV-PyVT) with multiple co-occurring tumors. Single tumors reaching 100 mm3 in volume were selected for NIR exposure, and both treated and total untreated tumor volumes were measured until endpoint (2 cm in diameter). We observed a significant reduction of tumor volume in the anti-FAP NIR-PIT group compared with Ab alone or untreated control group. Additionally, distant tumor volumes (i.e. total untreated tumor burden) decreased significantly. Preliminary flow cytometric analysis indicated that intratumoral CD8+ T cells and NK cells became activated following anti-FAP NIR-PIT and expressed interferon-gamma as early as 30 minutes, up to at least 3 hours after anti-FAP NIR-PIT. Additional studies are needed to further characterize this immune response; however, our results suggest that CAFs can be locally depleted using NIR-PIT, and this depletion can enhance anti-tumor immunity, leading to tumor reduction.

Cancer Metabolism Interest Group

Mateus Prates Mori, Ph.D. (NHLBI)

Mitochondrial respiration creates a hypoxic nuclear environment

Introduction: The endosymbiotic theory of the mitochondrion postulates that O2 consuming prokaryotes hosted by primordial anaerobic eukaryotes provided protection against O2 toxicity by reducing it to H2O. We previously demonstrated that non-respiring cells (SCO2-/-) with disruption of the cytochrome c oxidase (COX) complex have increased oxidative DNA damage and diminished growth under normoxia while thriving under hypoxia. With recent advances in fluorescence lifetime microscopy (FLIM) showing that the mitochondrial compartment is hypoxic relative to cytosol, we hypothesized that the perinuclear distribution of mitochondria in cells may create a hypoxic nuclear core and affect genomic regulation. Methods: To test this concept, we constructed FLIM probes localized to the cytosol – myoglobin-mCherry (MB-mCherry) – or targeted either to the mitochondrion (mtMB-mCherry) or to the nucleus (nMB-mCherry). The FRET-induced changes in the lifetime of mCherry is dependent on the O2-bound or O2-free (oxy/deoxy) state of MB allowing intracellular PO2 measurements. HCT116 cells were transfected with these probes and FLIM performed under different ambient O2 levels to demonstrate the dependence of O2 measurements on its supply and consumption. Results: Immunofluorescence imaging confirmed the subcellular localization of the respective probes. mtMB-mCherry FLIM measurements were shorter than that of untargeted MB-mCherry, indicating that the mitochondrial compartment is more hypoxic than cytosol as previously reported, but notably, nuclear PO2 levels were similarly low as hypothesized. Pharmacologic inhibition of respiration abrogated the nuclear hypoxia while bypassing the inhibited complex and reconstituting O2 consumption by COX recovered the nuclear hypoxia. Genetic disruption of respiration (SCO2-/-) also resulted in the loss of mitochondrial and nuclear hypoxia with recovery upon rescue of respiration. FLIM measurements of intracellular hypoxia associated with respiration were confirmed by HIF-1α and differential gene expression related to histone methylation state. Conclusion: Our current study highlights the potential importance of nuclear O2 homeostasis, likely important for preventing oxidative DNA damage, aging and associated diseases such as cancer. It also links mitochondrial respiration with O2 sensing and response via HIF-1α and with other O2-dependent epigenetic processes.

Chemistry Interest Group

Pinaki Bhattacharjee, Ph.D. (NIAAA)

A novel class of peripherally restricted CB1 antagonists for treatment of Metabolic syndrome (MetS) disorders

The endocannabinoid system is involved in the regulation of many physiological and pathological processes related to the immune system and metabolism. Exogenous ligands that activate or antagonize the endocannabinoid receptors mainly CB1/CB2 thus have great value in treating obesity, fibrosis and other metabolic disorders. One of our objectives involves the design and biological evaluation of functional antagonists at the CB1 receptor. The rationale for this work comes from studies over the past two decades from various literature which show that global blockade of CB1R results in reduced food intake and alleviation of metabolic complications arising from obesity and insulin resistance. Based on this paradigm, CB1R inverse agonist Rimonabant became the first-in-class molecule approved in the EU as an anti-obesity drug. However, despite its pronounced therapeutic potential, it was withdrawn from the market in 2008 due to its adverse neuropsychiatric side effects. Literature precedence suggests that peripheral blockade of CB1 receptors retain many metabolic benefits without CNS side-effects. This has paved the way for renewed and yet untapped potential for selective, peripheral CB1 antagonism-based therapy in treating obesity, pulmonary fibrosis. Compounds with PSA > 100Å2 have a lower permeability into the CNS which intrigued us to design a new class of CB1R antagonists without neuropsychiatric liabilities and reduced lipophilicity. We have synthesized a new series of peripherally restricted antagonists of CB1R having racemic diaryl-pyridazine core flanked with sulfonamide moiety and tested in radioligand binding experiments. Several analogues had high CB1 binding affinities (Ki = 1-15 nM) validating the in-silico results. The binding affinity of these molecules for the CB1 receptor (KiPB75AU = 13 nM) inspired us to study its effect on downstream signaling. We analyzed whether the molecules were also able to activate CB1 receptors through β-arrestin in HEK293-CB1-β-arrestin Nomad cells. We found that the IC50 for one of our potent compounds PB-75AU was 0.2 nM for the ß‐arrestin assay (Z´= 0.54) and 0.8 nM for the cAMP assay (Z´=0.72). In an extension of this work, we synthesized several compounds showing dual CB1/iNOS inhibitor in the nanomolar range. In a nutshell, we identified several compounds having nanomolar affinity and selectivity for CB1R and act as functional antagonists at this receptor with potential for treating fibrosis, diabetes, and obesity.

Developmental Biology Scientific Interest Group

Maria Vega Sendino, Ph.D. (NCI-CCR)

The homeobox transcription factor DUXBL controls exit from totipotency

Totipotency is defined as the ability of a single cell to develop into a full organism. It is a functional property strictly associated with zygotes and 2-cell (2C) stage embryos in mice. Indeed, the transition from maternal to zygotic transcription, a process known as zygotic gene activation (ZGA) and characterized by a large transcriptional burst at the end of the 2C stage, seems to be the trigger for loss of totipotency. Interestingly, the process of ZGA confers a unique transcriptional profile to the 2C embryo characterized by the specific expression of the so-called 2C-associated genes and MERVL, a subfamily of transposable elements. Although the induction of the 2C-associated transcriptional program during ZGA is required, it also needs to be quickly silenced for development to proceed. Importantly, the molecular mechanisms involved in the silencing of the 2C transcriptional program remain mostly unknown. In this work, we uncovered the essential role of the homeobox transcription factor DUXBL during the exit from totipotency. To mimic ZGA in vitro we overexpressed the transcription factor DUX in embryonic stem cells (ESC). DUX is expressed exclusively in the 2C embryo, it has been involved in ZGA and its overexpression recapitulates most 2C-associated transcriptional features. We observed that DUX directly induced the expression of DUXBL. CUT&RUN analysis revealed that exogenous DUXBL gained accessibility to DUX-bound regions in DUX-induced ESC while it was unable to bind those regions in uninduced cells. Importantly, we detected increased induction of the 2C transcriptional program upon DUX expression in Duxbl-knockout ESC whereas overexpression of DUXBL was sufficient to impair 2C-associated transcription. Mechanistically, we determined by mass spectrometry analyses that DUXBL interacted with TRIM24 and TRIM33, two members of the tripartite motif superfamily involved in promoting gene silencing, and co-localized with them in foci upon DUX expression. Finally, to examine the role of DUXBL in vivo we knocked-down Duxbl by microinjecting morpholinos and/or siRNAs in mouse zygotes and let them develop. Downregulation of DUXBL in embryos led to 2C-stage arrest while control embryos progressed until the blastocyst stage. Our data suggest a model where DUXBL is expressed and recruited together with TRIM24/33 in DUX-expressing cells to silence the 2C-transcriptional program revealing an unexpected role for DUXBL in controlling the exit from totipotency.

Immunology Interest Group

Rachel Philips, Ph.D. (NIAMS)

STAT1 gain of function mutation impairs immune response to viral infections

Regulation of T cells by interferons and other cytokines, which act via STAT family transcription factors, is critical for host defense. Patients with STAT1 gain-of-function (GOF) mutations develop lifelong ailments, including chronic infections, autoimmunity, and cancer. A characteristic feature of this disorder is chronic mucocutaneous candidiasis (CMC), related to an exaggerated type 1/interferon (IFN)-gamma bias that antagonizes a type 3/Th17 response required for fungal clearance. However, this type 1/IFN-gamma bias does not explain why almost 40 percent of reported patients exhibit chronic and sometimes lethal viral infections. This observation is unexpected, as cells with STAT1-GOF mutations exhibit increased expression of interferon stimulated genes and patients show more IFN-gamma-producing CD4 T cells. This paradox emphasizes the critical need to understand basic principles of cytokine signaling, specifically how a STAT1-GOF mutation alters cytokine output. To determine the effect of a STAT1-GOF mutation on viral response, we generated a novel conditional knock-in mouse where expression of a common GOF STAT1 mutation, T385M, is Cre-dependent. Using high-dimentional flow cytometry (Cytek Aurora) and RNA-seq, we found that ubiquitous STAT1-T385M expression recapitulates observations found in patients (i.e. elevated STAT1 activation, increased gene expression of STAT1 regulated genes, susceptibility to CMC), as well as a strong type 1/IFN bias at steady state. Despite this type 1 bias, STAT1-GOF mice respond poorly to viral infections that are easily controlled by WT mice, such as MCMV and LCMV-Armstrong. In particular, STAT1-GOF mice exhibit an impaired NK and CD8 T cell effector response and develop a cytokine storm seven days after infection, independent of viral load. Unexpectedly, the impaired immune response and cytokine storm is cell extrinsic and not due to one cell type, as when STAT1-T385M expression is restricted to only T cells, B cells, macrophages, dendritic cells, or NK cells, GOF mice respond normally to viral infections. In fact, the development of a cytokine storm is due to impaired IFN-gamma production by liver NK cells, ILC1s, and iNKT cells early during infection, as WT mice treated with IFN-gamma-blocking antibodies also develop a cytokine storm and exhibit impaired lymphocyte response later during infection. These results reveal that STAT1 activity must be tuned for proper immune response to viral infections.

Matrix Biology Special Interest Group

Vinod Nadella, Ph.D. (NIAMS)

The extracellular matrix maintained by hypodermal macrophages via IGF1 is a niche for Staphylococcus aureus infection

Macrophages (Mac) are a heterogeneous population of immune cells with diverse functions in maintaining tissue homeostasis, under steady-state and inflammatory conditions. The skin is a multilayered tissue and an immunological interface that harbors multiple immune cells including Mac. While skin Mac have been extensively studied in mice, they have been mostly characterized in ear skin, which lacks the hypodermis (HD), the deepest cutaneous layer. As such, hypodermal macrophages (HD-Mac) have not been studied and their functions under homeostatic and pathogenic conditions remains largely unexplored. This is clinically relevant as HD is a common site for Staphylococcus aureus (SA) skin infection, in the form of cellulitis. Using flow-cytometry, bulk RNA-seq and single-cell RNA-seq, we uncovered two Mac subsets with distinct transcriptomes, in both dermis and HD. Both Mac subsets relied on colony stimulating factor 1 (CSF1) receptor signaling for their survival. Single-cell RNA-seq of non-immune cells identified fibroblasts (FB) as a major source of dermal and hypodermal CSF1 and revealed differentially expressed Tek (encoding Tie2) in FB as a potential driver for HD-specific ablation of CSF1. Tek-cre x Csf1-floxed mice (Csf1Tek) displayed a striking post-natal ablation of HD-Mac, associated with an alteration of the collagen network and increased accumulation of hyaluronic acid (HA). Ligand-receptor interaction studies identified candidate genes involved in hypodermal Mac-FB crosstalk. Of note, depletion of insulin-like growth factor 1 (IGF1) from Mac in Csf1r-cre x Igf1-floxed mice (Igf1Csf1r) recapitulated the alteration of the extracellular matrix (ECM) observed in absence of HD-Mac. Immuno-fluorescence microscopy revealed that HD-Mac from Igf1Csf1r mice expressed reduced levels of LYVE-1, a HA receptor, and in vitro HA-FITC uptake assay suggested that these Igf1Csf1r Mac displayed defective HA uptake. Interestingly, upon injecting SA into the HD, Csf1Tek and Igf1Csf1r mice displayed striking reduction of the cellulitis phenotype as compared to wildtype (WT) mice. Furthermore, pre-injection of WT mice with HA markedly attenuated the cellulitis phenotype, suggesting that HA deposition in the absence of Mac or Mac-derived IGF1 conferred host protection against SA. Thus, while HD-Mac critically maintained ECM homeostasis, this provided a niche for SA infection, highlighting implications for novel therapeutic strategies in SA soft-tissue infections.

Protein Trafficking and Organelle Dynamics Interest Group

Michael Fernandopulle (NINDS)

Michael Fernandopulle is an MD/PhD candidate at Feinberg School of Medicine (Northwestern University), the University of Cambridge, and the National Institutes of Health. He received his Sc.B. in Chemistry from Brown University. Michael is interested in the roles of organellar metabolism in cell biology, particularly in the context of neurological disease. He is currently investigating the roles of lysosomes in site-specific mRNA translation in neurons.

Proteomics Scientific Interest Group

Yilun Sun, Ph.D. (NCI-CCR)

PARP1 promotes the repair of DNA-protein crosslinks by recruiting the endonuclease FEN1

DNA-protein crosslinks (DPCs) are among the most ubiquitous and detrimental DNA lesions arising from exposure to metabolic stresses, drugs, or crosslinking agents such as formaldehyde (FA). FA is a cellular by-product of methanol metabolism, histone demethylation, lipid peroxidation as well as environmental pollutants. Failure to repair FA-induced DPCs blocks nearly all chromatin-based processes, leading to immunodeficiencies, neurodegeneration and cancer predisposition. Yet, it remains largely unknown how the cell repairs FA-induced DPCs. The study of DPC repair is impeded by our ignorance about the types of proteins crosslinked by FA due to lack of techniques to purify and identify the DPCs. To solve this problem, we designed a novel bioassay to isolate FA-induced DPCs using cesium chloride differential ultracentrifugation. The isolated DPCs are digested with micrococcal nuclease to release the crosslinked proteins before being subjected to HPLC-mass spectrometry (MS). Using this method, we revealed the proteome of FA-induced DPCs in human cells and found that the most abundant proteins that form DPCs are PARP1, topoisomerases I and II, RNA polymerase II and histones. We also found that these DPCs are modified by poly-ADP-ribosylation (PARylation), a post-translational modification catalyzed by PARP1. PARP1 acts a key DNA damage response effector that PARylates and recruits DNA repair proteins. It remains unknown whether PARP1 plays a role in DPC repair. We performed immunoprecipitation (IP) assays with anti-PAR antibody to isolate DNA repair proteins that are PARylated in response to FA for HPLC-MS and identified FEN1 (flap endonuclease 1), a nuclease with 5'-flap endonuclease and exonuclease activities, as the most PARylated DNA repair protein. We confirmed its PARylation by in vitro PARylation assay using recombinant FEN1 and PARP1. Silencing of FEN1 was found to significantly increase FA-induced DPCs, suggesting a key role of FEN1 for the DPC repair. Further, we observed that blocking PARylation using PARP inhibitor attenuated the FA-induced interaction of FEN1, topoisomerases and DPC-targeted protease SPRTN using proximity ligation assay, suggesting that PARylation is required for FEN1 to localize to the DPC sites. Taken together, our work not only reports a MS-based method to characterize FA-induced DPC adductomes but also identifies the unprecedented PARP1-FEN1 pathway as an important DPC repair mechanism to prevent genomic instability.

RNA Club

Priyanka Singh, Ph.D. (NICHD)

Ded1 helicase mediates reprogramming of translation conferred by TORC1 inhibition: ded1 mutations reverse the downregulation of growth-promoting mRNAs

Ded1 is an essential DEAD-box helicase in budding yeast that broadly stimulates translation by facilitating initiation of mRNAs burdened with structured 5’UTRs. Recently we showed that subunits of m7G cap-binding factor eIF4F, eIF4A and eIF4E, bind distinct segments of the Ded1 N-terminal domain (NTD) to promote Ded1 function, exceeding in importance binding of the Ded1-CTD to the eIF4G subunit. Kinase TORC1 stimulates translation initiation via multiple pathways in a manner inhibited by rapamycin (Rap). Published data suggest that Rap induces Ded1mediated eIF4G degradation and diminished translation and cell growth, which is rescued by eliminating the Ded1-CTD or mutating the Ded1 catalytic domain to confer Rap resistance (RapR). Other studies suggest that cell stress down-regulates translation by dissociating Ded1 from eIF4F and replacing it with the inhibitory DEAD helicase Dhh1. At odds with these findings, we found no evidence that Rap leads to reduced expression of eIF4G or reduced Ded1-eIF4G association in vivo. We also found that deleting the Ded1 NTD also confers RapR, suggesting that any mutation that reduces Ded1 function may diminish the translational consequences of TORC1 inhibition. By analyzing mutations in known Ded1 phosphorylation sites, we found that Ala, but not phosphomimetic Asp, substitutions of S5 and S12 confer RapR, suggesting that phosphorylation of the WT residues may promote Ded1 function. To elucidate the molecular mechanism of RapR conferred by Ded1 mutations, we conducted ribosome profiling of cells treated with Rap and harboring either WT Ded1 or variants lacking the NTD (ΔN) or CTD (ΔC). In WT cells Rap reprogramed translation efficiencies (TEs) of many mRNAs in a manner generally opposite of that observed in our previous studies of other ded1 catalytic mutants, suggesting that reducing Ded1 function may reverse the translational consequences of TORC1 inhibition. Supporting this idea, the Ded1 ΔN and ΔC mutations partially reversed both the increased and decreased TEs conferred by Rap in WT cells. Because this effect included the Rap induced down regulation of ribosomal protein (RP) expression, it can account for the enhanced growth conferred by ΔN and ΔC in Rap-treated cells. Together, our results indicate that Ded1 function is required for the translational reprogramming conferred by TORC1 inhibition, and that ded1 mutations blunt the downregulation of RP and other growth promoting mRNAs to confer rap resistance.

Systems Biology Special Interest Group

Shah Md Toufiqur Rahman, Ph.D. (NIA)

Ligand and tissue-specificity of NF-κB signaling in primary cells

Nuclear factor Kappa B (NF-κB) is a ubiquitously expressed transcription factor which regulates wide array of biological systems in virtually all cell types. While the involvement of NF-κB in immune and inflammatory processes has been more clearly defined, it is not well understood how immune and other cell-types differentiate and encode different pathogenic and secreted cytokine signals. Emerging literature suggest that the temporal pattern of NF-κB signaling dynamics might encode the pathogen-specific information and regulate corresponding gene expression. However, the existing data on this topic are mostly acquired from one subunit of NF-κB, p65, also known as RelA, in cell lines overexpressing a fluorescent fusion protein. To monitor the stimulus and tissue-specific signaling dynamics of both subunits of the canonical NF-κB system, we have generated a double knock-in reporter mouse model expressing two endogenous subunits of NF-κB: mEGFP-RelA and mScarlet-c-Rel. We utilize high-throughput live-cell fluorescence microscopy to simultaneously monitor signaling dynamics of both NF-κB subunits in primary mouse macrophages as well as in primary fibroblast. Quantitative analysis of cells isolated from bone marrow-derived macrophages (BMDMs) reveals ligand-specific temporal patterns of RelA and c-Rel signaling dynamics. For all six different TLR ligands used in this study, both the RelA and c-Rel show non-oscillatory responses where the RelA signaling occurs faster than c-Rel. However, following TNF-α stimulation, both NF-κB subunits show oscillatory responses (period ~2.5 hours) where RelA signaling is still faster and stronger than c-Rel. Live-cell imaging of primary fibroblast shows distinct signaling dynamic patterns than that observed in BMDM in response to six TLR ligands and TNF-α. Deeper analysis of single-cell RelA and c-Rel time-series data for different ligands and cell types reveals ligand and tissue-specific dynamic features which might be the coding language to convey specific information and enable appropriate responses. We are also expanding our live-cell microscopy and analyses to other tissue-resident macrophages such as in microglia and other cell types to understand how different cell types respond to different pathogenic invasions. The findings of this research will shed light on identifying how different tissues communicate and coordinate in response to various immunological assaults.

Transcription Scientific Interest Group

Bega Murray, MSc (NCI-CCR)

Single Cell CRISPR Screen of Malignant Peripheral Nerve Sheath Tumors Identifies SWI/SNF Genes of Regulatory Importance.

Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas of the peripheral nervous system. These tumors are typically therapeutically resistant, highlighting an urgent need to decipher the molecular development of this tumor type to advance novel therapeutic strategies. Genetic alterations of the polycomb repressive complex 2 (PRC2) occur in up to 80% of MPNST cases. In healthy cells, this complex dynamically competes with a transcriptional activating chromatin remodeler, SWI/SNF, to govern development and maintenance of cellular identity. However, little is known about how PRC2 mutation effects this regulatory balance in MPNST. In this study, we elucidated the role of SWI/SNF in MPNST and investigated the dependency of this function on PRC2 mutant status. A CRISPR knock-out (KO) screen combined with single cell RNA sequencing was used to target 44 components of SWI/SNF complexes. This screen highlighted the Double PHD Fingers 1 gene, DPF1, as an important transcriptional regulator in MPNST. A bulk RNA-seq CRISPR KO system confirmed these findings, where gene sets involved in neuronal development and regulatory processes showed decreased (p.adj<0.0005) enrichment in DPF1 KO cells. Further, DPF1 expression was identified as significantly elevated (p.val<0.005) in MPNST cell lines and tumors when compared to normal tissue controls. This data indicates a key role of DPF1 in MPNST cell growth and viability. Glycerol gradient sedimentation confirmed DPF1 incorporation into large SWI/SNF complexes in MPNST cells, rather than functioning as an independent protein. Co-immunoprecipitation highlighted the ability of DPF1 to interact with core components of the neuronal SWI/SNF complex in MPNST cells, suggesting this complex formation may enable the transcriptional regulatory role of DPF1 in MPNST. Finally, we showed DPF1 KO has a range of phenotypic effects in MPNST cells, including reducing growth and proliferation in 2D cell culture, 3D spheroid assays, as well as reducing the colony formation ability of these cells in a soft agar assay. This data highlights DPF1 as a unique vulnerability in MPNST cells. Interestingly, a PRC2 inducible system in MPNST cell lines found this DPF1 vulnerability is independent of the cellular PRC2 status, as expression and complex formation of DPF1 remains stable regardless of PRC2 function. Therefore, DPF1 may represent a novel therapeutic target in both PRC2 functional and mutant MPNST genotypes.

Virology Interest Group

Djalma Lima, Ph.D. (NIAID)

Endogenous retrovirus reactivation promotes allergic inflammation

Mammals co-evolved with a multitude of microorganisms that include the commensal microbiota and endogenous retroelements such as the endogenous retroviruses (ERVs) that comprise a substantial fraction of the mammalian genome. Recently, we showed that ERVs expression acts as endogenous adjuvants that prime the host for immune responses to the microbiota thereby controlling both tissue homeostasis and inflammation. ERVs have been also involved in the development of a range of diseases such as cancer, inflammatory disorders, and autoimmune diseases. How ERVs activation impact on the pathogenesis of allergic diseases remains unknown. Allergies are one of most common chronic health conditions in the world and constitute a significant cause of morbidity worldwide. To explore the possibility that ERVs could contribute to the etiology of these disorders, we employed a murine model of house dust mite (HDM)-induced allergic inflammation. HDM intranasal sensitization significantly upregulated the expression of ERVs in several innate cell types in the lung, including B cells, alveolar macrophages, eosinophils, and neutrophils. ERVs can be reverse transcribed into DNA that can, when accumulating within the cytoplasm, alert the innate immune system. Antiretroviral treatment (a cocktail of two reverse transcriptase inhibitors) profoundly inhibited innate cells accumulation within the lung following HDM-allergic sensitization, including eosinophils, a hallmark for type-2 inflammation. Antiretroviral treatment also significant decreased the number of B cells, ILCs and CD4+ T cells during HDM sensitization as well as their ability to produce immunoglobulins and the type 2/17 cytokines such as IL-9, IL-33, IL-5, IL-13 and IL-17A. This phenomenon was associated with an intense reduction of perivascular and peribronchial infiltrate of inflammatory cells, and mucus production by the goblet cells in the lung epithelium. Of particular interest, we also found that genetic ablation of Emv2 (a highly expressed ERV) was sufficient to impair both innate and adaptive Th2 immunity during HDM sensitization. Together this work reveals an unexpected role for endogenous retroelements in the control of allergic inflammation in the lung. Importantly, these findings provide a new understanding of the etiology of inflammatory disorders allowing for the potential development of tailored clinical interventions aimed at controlling the inflammatory processes triggered by allergens exposure.

Woman Scientist Advisors (WSA)

Seungmi Ryu, Ph.D. (NCATS)

Establishing a new human iPSC-derived cerebellar organoid model for Friedreich’s ataxia

Friedreich’s ataxia (FRDA), an inherited neurodegenerative disorder, is the most common form of hereditary ataxia in the United States affecting about 1 in every 50,000 people with no cure or effective treatment. It is caused by a GAA repeat expansion mutation in the mitochondrial FXN gene and leads to degenerative changes of the cerebellum, a particular brain region that controls movement and motor coordination. Most of our current understanding of the human cerebellum is derived from postmortem tissues and animal models. Over the past decade, human induced pluripotent stem cells (iPSCs) have emerged as a powerful platform for disease modeling. Here, we developed a novel 60-day organoid differentiation strategy that recapitulates hallmarks of human cerebellar development. We confirmed the generation of two primordial regions, the rhombic lip and cerebellar plate ventricular zone, from iPSC lines derived from healthy individuals and FRDA patients. Immunostaining and Western blot analysis confirmed that these specified regions can give rise to the main neuronal cell types of the cerebellum such as granule cells, interneurons, Purkinje cells, and Bergmann glia-like cells that guide migratory neuroblasts to their final destination. In comparison to healthy controls, FRDA cerebellar organoids showed disease-specific signatures such as reduction of the mitochondrial enzyme ACO2 and an increased rate of apoptotic cells. Further analyses at the ultrastructural level indicated abnormal mitochondrial morphologies in FRDA cerebellar organoids. Moreover, elevated levels of reactive oxygen species were detected by using a flow cytometry method. Notably, cerebellar organoids with long GAA repeats, which correlates with disease severity in the clinic, also showed a higher degree of mitochondrial abnormality in vitro. Currently, gene editing experiments using CRISPR-Cas9 are underway to correct FRDA mutations and to find out if disease phenotypes can be reversed. In summary, the newly established cerebellar organoid model provides new opportunities to investigate the fundamental pathophysiology of FRDA in the laboratory and examine innovative therapeutic approaches.

Brittany D. Lord, Ph.D., MS, MPH (NCI-CPFP)

Influence of neighborhood deprivation on DNA methylation in Black and White women with breast cancer

Background Chronic life stress is more prevalent in low socioeconomic status (SES) communities and has been shown to affect DNA methylation and the immune system. Yet, the biological processes that mediate the impact of SES on health to promote the development of chronic diseases like cancer remain poorly understood. Our study investigates whether neighborhood socioeconomic deprivation is linked to DNA methylation, leading to a pro-inflammatory immune microenvironment which supports cancer development, progression, and reduced survival. We are specifically interested in this relationship among Black and White women with breast cancer, largely due to the 40% increased mortality that Black women experience compared to White women. Study Design DNA was extracted from frozen breast tumor tissue from 129 Black and 85 White women in the NCI-Maryland Breast Cancer Cohort. Using zip codes, we geocoded the locations of our study participants and linked these data to Census tract-level socioeconomic deprivation using a Neighborhood Deprivation Index (NDI). DNA methylation data was acquired using the Illumina EPIC 850K array and a differentially methylated probe (DMP) analysis was done to identify gene regions of hypo- or hyper-methylation by NDI. We also used methylCIBERSORT to estimate immune subpopulation differences by race and neighborhood deprivation. Results From the DMP analysis, we identified 27 differentially methylated probes by NDI in all women in our study after adjusting for age and body mass index. 5 probes were hypermethylated in women with higher NDI and were largely correlated to gene body regions. 22 probes were hypomethylated in women with high NDI and spans across promotor, intergenic and exon gene regions. The most significantly hypermethylated probe by NDI corresponded to the PDLIM4 gene region, a tumor suppressor gene that has implications in breast and prostate cancer. Our methylCIBERSORT analysis showed that for Black women with aggressive cancer, neighborhood deprivation was significantly positively correlated with absolute immune cell scores, including significantly higher absolute scores for endothelial cells and eosinophils. Significance To date, little is known about how the epigenome translates neighborhood disadvantage into health disparities. Our findings give mechanistic insight into how socioeconomic position may affect cancerous mammary gland biology by altering DNA methylation patterns and immune cell responses.

Zoe E. Piccus, BS (NICHD)

Amyotrophic lateral sclerosis (ALS) is a fatal disease affecting motor neurons. Recently, patients were identified with de novo mutations in the gene SPTLC1, leading to an ALS onset as early as 3 years of age. SPTLC1 is a subunit of serine palmitoyltransferase (SPT), the rate-limiting enzyme of sphingolipid (SL) synthesis. SLs are an essential lipid class found in myelin and are thus enriched in the nervous system. The ALS-linked mutations occur within a transmembrane domain of SPT required for its negative regulation, and disrupting this region leads to unrestrained SL synthesis. These are the first metabolic mutations linked to ALS, and no animal models of this syndrome exist. Current work is focused on characterizing these mutations as a preclinical mouse model using histological and functional readouts and can be leveraged to study how high SL levels lead to degeneration.