AP-1 Complex Research Articles

Discovery and evaluation of HW161023 as a potent and orally active AAK1 inhibitor

AAK1, also known as AP2-associated protein kinase 1, is an enzyme that belongs to the family of serine/threonine protein kinases. It regulates the assembly and disassembly of clathrin-coated pits and thereby protein endocytosis, by phosphorylating the μ2 subunit of the AP2 complex, which is a key component of clathrin-coated vesicles. LX9211 is currently the only selective small molecule AAK1 inhibitor at the clinical trial stage for diabetic peripheral neuropathic pain, which was found to be safe and well tolerated in healthy participants in phase I clinical trials. The present manuscript described a series of fused-ring derivatives as a novel class of potent AAK1 inhibitors, resulting in the discovery of compound 5, namely HW161023, which showed high inhibitory potency against AAK1 enzyme and satisfactory oral pharmacokinetic profile with weaker HepG2 cell toxicity and hERG inhibition than LX9211.

Masks of Albinism: Clinical Spectrum of Hermansky–Pudlak Syndrome

Hermansky–Pudlak syndrome (HPS) is a rare disease inherited in the autosomal recessive mode, including 11 clinical genetic subtypes. They are associated with impaired function of the BLOC protein complex (Biogenesis of Lysosome-related Organelles Complexes), and the subunits of the AP-3 complex (adaptor protein complex). Each has its own clinical features, but they are all characterized by albinism, bleeding disorder, and visual abnormalities. Eleven patients from eight unrelated families with an incoming diagnosis of albinism were examined and novel and previously described genetic variants in HPS1, HPS6, and BLOC1S6 genes (types HPS1, HPS6, and HPS9) were found. To determine the optimal therapy and recommendations for further follow up, it is necessary to consider the entire clinical spectrum and genetic polymorphism of the disease. An interdisciplinary approach, combined with the use of non-routine diagnostic techniques such as RNA analysis, is essential for achieving accurate diagnoses in certain complex cases.

The scaffold protein IQGAP1 promotes reorientation of epithelial cell polarity at the two-cell stage for cystogenesis.

A single epithelial cell embedded in extracellular matrix (ECM) can proliferate to form an apical lumen-harboring cyst, whose formation is a fundamental step in epithelial organ development. At an early two-cell stage after cell division, the cell doublet typically displays "inverted" polarity, with apical and basolateral proteins being located to the ECM-facing and cell-cell-contacting plasma membranes, respectively. Correct cystogenesis requires polarity reorientation, a process containing apical protein endocytosis from the ECM-abutting periphery and subsequent apical vesicle delivery to a cell-cell contact site for lumen formation. Here, we show that downstream of the ECM-signal-transducer β1-integrin, Rac1, and its effector IQGAP1 promote apical protein endocytosis, contributing to polarity reorientation of mammalian epithelial Madin-Darby canine kidney (MDCK) cells at a later two-cell stage in three-dimensional culture. Rac1-GTP facilitates IQGAP1 interaction with the Rac-specific activator Tiam1, which also contributes to the endocytosis and enhances the effect of IQGAP1. These findings suggest that Tiam1 and IQGAP1 form a positive feedback loop to activate Rac1. With Rac1-GTP, IQGAP1 also binds to AP2α, an adaptor protein subunit for clathrin-mediated endocytosis; depletion of the AP2 complex impairs apical protein endocytosis in MDCK doublets. Thus, Rac1 likely participates in polarity reorientation at the two-cell stage via its interaction with IQGAP1.

AP-1 contributes to endosomal targeting of ubiquitin ligase RNF13 via a secondary and novel non-canonical binding motif.

Cellular trafficking between organelles is typically assured by short motifs that contact carrier proteins to transport them to their destination. Ubiquitin E3 ligase RING finger protein 13 (RNF13), a regulator of proliferation, apoptosis, and protein trafficking, localizes to endolysosomal compartments through the binding of a dileucine motif to clathrin adaptor protein complex AP-3. Mutations within this motif reduce the ability of RNF13 to interact with AP-3. Here, our study shows the discovery of a glutamine-based motif that resembles a tyrosine-based motif within RNF13's C-terminal region that binds to the clathrin adaptor protein complex AP-1, notably without a functional interaction with AP-3. Using biochemical, molecular, and cellular approaches in HeLa cells, our study demonstrates that a RNF13 dileucine variant uses an AP-1-dependent pathway to be exported from the Golgi towards the endosomal compartment. Overall, this study provides mechanistic insights into the alternate route used by variant of RNF13's dileucine sorting motif.

Rapid Vacuolar Sorting of the Borate Transporter BOR1 Requires the Adaptor Protein Complex AP-4 in Arabidopsis.

Plants maintain nutrient homeostasis by controlling the activities and abundance of nutrient transporters. In Arabidopsis thaliana, the borate (B) transporter BOR1 plays a role in the efficient translocation of B under low-B conditions. BOR1 undergoes polyubiquitination in the presence of sufficient B and is then transported to the vacuole via multivesicular bodies (MVBs) to prevent B accumulation in tissues at a toxic level. A previous study indicated that BOR1 physically interacts with µ subunits of adaptor protein complexes AP-3 and AP-4, both involved in vacuolar sorting pathways. In this study, we investigated the roles of AP-3 and AP-4 subunits in BOR1 trafficking in Arabidopsis. The lack of AP-3 subunits did not affect either vacuolar sorting or polar localization of BOR1-GFP, whereas the absence of AP-4 subunits resulted in a delay in high-B-induced vacuolar sorting without affecting polar localization. Super-resolution microscopy revealed a rapid sorting of BOR1-GFP into AP-4-positive spots in the trans-Golgi network (TGN) upon high-B supply. These results indicate that AP-4 is involved in sequestration of ubiquitinated BOR1 into a TGN-specific subdomain "vacuolar-trafficking zone," and is required for efficient sorting to MVB and vacuole. Our findings elucidate the rapid vacuolar sorting process facilitated by AP-4 in plant nutrient transporters.

The AP-1 adaptor complex is essential for intracellular trafficking of the ORF2 capsid protein and assembly of Hepatitis E virus

Although the Hepatitis E virus (HEV) is an emerging global health burden, little is known about its interaction with the host cell. HEV genome encodes three proteins including the ORF2 capsid protein that is produced in different forms, the ORF2i protein which is the structural component of viral particles, and the ORF2g/c proteins which are massively secreted but are not associated with infectious material. We recently demonstrated that the endocytic recycling compartment (ERC) is hijacked by HEV to serve as a viral factory. However, host determinants involved in the subcellular shuttling of viral proteins to viral factories are unknown. Here, we demonstrate that the AP-1 adaptor complex plays a pivotal role in the targeting of ORF2i protein to viral factories. This complex belongs to the family of adaptor proteins that are involved in vesicular transport between the trans-Golgi network and early/recycling endosomes. An interplay between the AP-1 complex and viral protein(s) has been described for several viral lifecycles. In the present study, we demonstrated that the ORF2i protein colocalizes and interacts with the AP-1 adaptor complex in HEV-producing or infected cells. We showed that silencing or drug-inhibition of the AP-1 complex prevents ORF2i protein localization in viral factories and reduces viral production in hepatocytes. Modeling of the ORF2i/AP-1 complex also revealed that the S domain of ORF2i likely interacts with the σ1 subunit of AP-1 complex. Hence, our study identified for the first time a host factor involved in addressing HEV proteins (i.e. ORF2i protein) to viral factories.

The WDR11 complex is a receptor for acidic-cluster-containing cargo proteins

Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e.,"cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11-FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.

Trafficking of adhesion and aggregation-modulating proteins during the early stages of Dictyostelium development

The social amoeba Dictyostelium discoideum has been studied for close to a century to better understand conserved cellular and developmental processes. The life cycle of this model eukaryote is composed of a unicellular growth phase and a multicellular developmental phase that is induced by starvation. When starved, individual cells undergo chemotactic aggregation to form multicellular mounds that develop into slugs. Terminal differentiation of cells within slugs forms fruiting bodies, each composed of a stalk that supports a mass of viable spores that germinate and restart the life cycle when nutrients become available. Calcium-dependent cell adhesion protein A (CadA) and countin (CtnA) are two proteins that regulate adhesion and aggregation, respectively, during the early stages of D. discoideum development. While the functions of these proteins have been well-studied, the mechanisms regulating their trafficking are not fully understood. In this study, we reveal pathways and cellular components that regulate the intracellular and extracellular amounts of CadA and CtnA during aggregation. During growth and starvation, CtnA localizes to cytoplasmic vesicles and punctae. We show that CtnA is glycosylated and this post-translational modification is required for its secretion. Upon autophagy induction, a signal peptide for secretion facilitates the release of CtnA from cells via a pathway involving the μ subunit of the AP3 complex (Apm3) and the WASP and SCAR homolog, WshA. Additionally, CtnA secretion is negatively regulated by the D. discoideum orthologs of the human non-selective cation channel mucolipin-1 (Mcln) and sorting receptor sortilin (Sort1). As for CadA, it localizes to the cell periphery in growth-phase and starved cells. The intracellular and extracellular amounts of CadA are modulated by autophagy genes (atg1, atg9), Apm3, WshA, and Mcln. We integrate these data with previously published findings to generate a comprehensive model summarizing the trafficking of CadA and CtnA in D. discoideum. Overall, this study enhances our understanding of protein trafficking during D. discoideum aggregation, and more broadly, provides insight into the multiple pathways that regulate protein trafficking and secretion in all eukaryotes.

LRK-1/LRRK2 and AP-3 regulate trafficking of synaptic vesicle precursors through active zone protein SYD-2/Liprin-α.

Synaptic vesicle proteins (SVps) are transported by the motor UNC-104/KIF1A. We show that SVps travel in heterogeneous carriers in C. elegans neuronal processes, with some SVp carriers co-transporting lysosomal proteins (SV-lysosomes). LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 play a critical role in the sorting of SVps and lysosomal proteins away from each other at the SV-lysosomal intermediate trafficking compartment. Both SVp carriers lacking lysosomal proteins and SV-lysosomes are dependent on the motor UNC-104/KIF1A for their transport. In lrk-1 mutants, both SVp carriers and SV-lysosomes can travel in axons in the absence of UNC-104, suggesting that LRK-1 plays an important role to enable UNC-104 dependent transport of synaptic vesicle proteins. Additionally, LRK-1 acts upstream of the AP-3 complex and regulates its membrane localization. In the absence of the AP-3 complex, the SV-lysosomes become more dependent on the UNC-104-SYD-2/Liprin-α complex for their transport. Therefore, SYD-2 acts to link upstream trafficking events with the transport of SVps likely through its interaction with the motor UNC-104. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. SYD-2 acts in concert with AP complexes to ensure polarized trafficking & transport of SVps.

Elucidation of cellular signaling mechanism involved in Vibrio cholerae chitin-binding protein GbpA mediated IL-8 secretion in the intestinal cells

BackgroundVibrio cholerae N-acetylglucosamine-binding protein (GbpA) is a four-domain, secretory colonization factor which is essential for chitin utilization in the environment, as well as in adherence to intestinal cells. GbpA is also involved in inducing intestinal inflammation by enhancing mucin and interleukin-8 secretion. The underlying cell signaling mechanism involved in the induction of the pro-inflammatory response and IL-8 secretion has yet to be deciphered in detail. MethodsHerein, the process through which GbpA triggers the induction of IL-8 in intestinal cells was investigated by examining the role of GbpA in intestinal cell line HT 29. ResultsGbpA, specifically through the fourth domain, forms a binding connection with Toll-like receptor 2 (TLR2) and additionally, recruits TLR1 along with CD14 within a lipid raft micro-domain to initiate the signaling pathway. Notably, disruption of this micro-domain complex resulted in a reduction in IL-8 secretion. The lipid raft association served as the catalyst that invoked a downstream cellular inflammatory signaling pathway. This cascade involved the activation of various MAP kinases and NFκB and assembly of the AP-1 complex. This coordinated activation of signaling molecules eventually leads to enhanced IL-8 transcription via increased promoter activity. These findings suggested that GbpA is a crucial protein in V. cholerae, capable of inciting a pro-inflammatory response during infection by orchestrating the formation of the GbpA-TLR1/2-CD14 lipid raft complex. Activation of AP-1 and NFκB in the nucleus eventually enhanced IL-8 transcription through increased promoter activity. ConclusionsCollectively, these findings indicated that GbpA plays a pivotal role within V. cholerae by triggering a pro-inflammatory response during infection. This response is instrumented by the formation of the GbpA-TLR1/2-CD14 lipid raft complex.

Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

The endothelial glycocalyx, located at the luminal surface of the endothelium, plays an important role in the regulation of leukocyte adhesion, vascular permeability, and vascular homeostasis. Endomucin (EMCN), a component of the endothelial glycocalyx, is a mucin-like transmembrane glycoprotein selectively expressed by venous and capillary endothelium. We have previously shown that knockdown of EMCN impairs retinal vascular development in vivo and vascular endothelial growth factor 165 isoform (VEGF165)-induced cell migration, proliferation, and tube formation by human retinal endothelial cells in vitro and that EMCN is essential for VEGF165-stimulated clathrin-mediated endocytosis and signaling of VEGF receptor 2 (VEGFR2). Clathrin-mediated endocytosis is an essential step in receptor signaling and is of paramount importance for a number of receptors for growth factors involved in angiogenesis. In this study, we further investigated the molecular mechanism underlying EMCN’s involvement in the regulation of VEGF-induced endocytosis. In addition, we examined the specificity of EMCN's role in angiogenesis-related cell surface receptor tyrosine kinase endocytosis and signaling. We identified that EMCN interacts with AP2 complex, which is essential for clathrin-mediated endocytosis. Lack of EMCN did not affect clathrin recruitment to the AP2 complex following VEGF stimulation, but it is necessary for the interaction between VEGFR2 and the AP2 complex during endocytosis. EMCN does not inhibit VEGFR1 and FGFR1 internalization or their downstream activities since EMCN interacts with VEGFR2 but not VEGFR1 or FGFR1. Additionally, EMCN also regulates VEGF121-induced VEGFR2 phosphorylation and internalization.Graphical

Abstract LB288: Reducing the effect of cellular stress in murine oral tumors with pharmacological blockade of β-catenin/CBP activity

Abstract Oral squamous cell carcinoma (OSCC), a primary subtype of head and neck squamous cell carcinoma (HNSCC), is a complex malignancy comprising multiple anatomical sites. OSCC ranks among the deadliest cancers globally, with a 5-year survival rate of ~65%. In previous studies, we have shown that pharmacological blockade of Wnt/β-catenin/CBP activity with small molecule inhibitors effectively abolished oncogenic cell phenotypes in OSCC. However, the underlying mechanisms promoting changes in OSCC cell identities remain unknown. To address this knowledge gap, we used an immunocompetent mouse model of OSCC induced by a tobacco-derived carcinogen, 4-nitroquinoline-1-oxide (4NQO), to interrogate cell states by single-cell RNA sequencing (scRNAseq) of tongue tissues from healthy mice (n=2), 4NQO-derived mouse tongue OSCC (n=2), and from 4NQO-derived mouse tongue tissues treated with an inhibitor of β-catenin/CBP (n=4), and generated a high-quality dataset comprising ~50K cells across all conditions. We performed multiple analyses of the generated data to catalogue the cell type repertoire and its changes among conditions. We observed significant changes in cellular composition between the 4NQO-induced and inhibitor-treated groups. The proportion of epithelial cells decreased upon treatment consistent with greatly diminished tumor volumes, while endothelial and fibroblast populations increased compared to the 4NQO control group. Epithelial sub-typing using known markers revealed a decrease in basal cancer stem-like cells (Krt5+, Krt14+) concomitant with an increase in cycling cells (Top2a+, Cdc20+). In addition, we identified a decrease in a stress cell phenotype associated with the AP-1 complex (Jun+, Fos+). The latter subgroup exhibited a positive enrichment for the “stress” module (n=100 genes) derived from human HNSCC patients described by Puram et al. To further validate the relationship between the stress subtype with β-catenin/CBP activity and its relation to the AP-1 complex, we projected the Puram stress module and the down-regulated signature from the β-catenin/CBP inhibitor-treated group compared with the 4NQO-control group onto independent cohorts of human subjects with HPV-negative HNSCC. We used both the bulk TCGA RNA-seq subset (n=367 patients) and a previously published scRNAseq dataset from Choi et al., specifically profiling the epithelial compartment (3K cells, n = 16 patients). We observed highly significant positive association of the stress program and β-catenin/CBP activity in both datasets (pearson ρ = 0.74 and 0.70, respectively), indicating that inhibition of β-catenin/CBP activity reduces the stress response. Our results suggest that mitigation of tumorigenic profiles in 4NQO-induced tumors upon inhibition of β-catenin/CBP signaling in OSCC may serve as an effective treatment strategy for the benefit of human patients and shed further light into the molecular mechanisms driving treatment response. Citation Format: Mohammed Muzamil Khan, Eric Reed, Lina Kroehling, Kenichi Nomoto, Junji Matsui, Manish Bais, Xaralabos Varelas, Maria Kukuruzinska, Stefano Monti. Reducing the effect of cellular stress in murine oral tumors with pharmacological blockade of β-catenin/CBP activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB288.

The Wingless planar cell polarity pathway is essential for optimal activity-dependent synaptic plasticity.

From fly to man, the Wingless (Wg)/Wnt signaling molecule is essential for both the stability and plasticity of the nervous system. The Drosophila neuromuscular junction (NMJ) has proven to be a useful system for deciphering the role of Wg in directing activity-dependent synaptic plasticity (ADSP), which, in the motoneuron, has been shown to be dependent on both the canonical and the noncanonical calcium Wg pathways. Here we show that the noncanonical planar cell polarity (PCP) pathway is an essential component of the Wg signaling system controlling plasticity at the motoneuron synapse. We present evidence that disturbing the PCP pathway leads to a perturbation in ADSP. We first show that a PCP-specific allele of disheveled (dsh) affects the de novo synaptic structures produced during ADSP. We then show that the Rho GTPases downstream of Dsh in the PCP pathway are also involved in regulating the morphological changes that take place after repeated stimulation. Finally, we show that Jun kinase is essential for this phenomenon, whereas we found no indication of the involvement of the transcription factor complex AP1 (Jun/Fos). This work shows the involvement of the neuronal PCP signaling pathway in supporting ADSP. Because we find that AP1 mutants can perform ADSP adequately, we hypothesize that, upon Wg activation, the Rho GTPases and Jun kinase are involved locally at the synapse, in instructing cytoskeletal dynamics responsible for the appearance of the morphological changes occurring during ADSP.

HPV16 E7 modulates the cell surface expression of MET and CD109 via the AP2 complex

Multiple cellular pathways are affected by HPV E6 and E7 oncoproteins, including endocytic and cellular trafficking. HPV-16 E7 can target the adaptor protein (AP) complex, which contains proteins important during endocytosis transport. To further investigate the role of HPV E7 during this process, we analysed the expression of cell surface proteins in NIKS cells expressing HPV-16 E7. We show that different cell surface proteins are regulated by HPV-16 E7 via interaction with AP2. We observed that the expression of MET and CD109 membrane protein seems to be upregulated in cells expressing E7. Moreover, the interaction of MET and CD109 with AP2 proteins is disrupted by HPV-16 E7. In addition, in the absence of HPV-16 E7, there is a downregulation of the cell membrane expression of MET and CD109 in HPV-positive cell lines. These results expand our knowledge of the functions of E7 and open new potential cellular pathways affected by this oncoprotein.

Polarized transport requires AP-1-mediated recruitment of KIF13A and KIF13B at the trans-Golgi.

Neurons are polarized cells that require accurate membrane trafficking to maintain distinct protein complements at dendritic and axonal membranes. The Kinesin-3 family members KIF13A and KIF13B are thought to mediate dendrite-selective transport, but the mechanism by which they are recruited to polarized vesicles and the differences in the specific trafficking role of each KIF13 have not been defined. We performed live-cell imaging in cultured hippocampal neurons and found that KIF13A is a dedicated dendrite-selective kinesin. KIF13B confers two different transport modes, dendrite- and axon-selective transport. Both KIF13s are maintained at the trans-Golgi network by interactions with the heterotetrameric adaptor protein complex AP-1. Interference with KIF13 binding to AP-1 resulted in disruptions to both dendrite- and axon-selective trafficking. We propose that AP-1 is the molecular link between the sorting of polarized cargoes into vesicles and the recruitment of kinesins that confer polarized transport.

ANKRD1 is a mesenchymal-specific driver of cancer-associated fibroblast activation bridging androgen receptor loss to AP-1 activation

There are significant commonalities among several pathologies involving fibroblasts, ranging from auto-immune diseases to fibrosis and cancer. Early steps in cancer development and progression are closely linked to fibroblast senescence and transformation into tumor-promoting cancer-associated fibroblasts (CAFs), suppressed by the androgen receptor (AR). Here, we identify ANKRD1 as a mesenchymal-specific transcriptional coregulator under direct AR negative control in human dermal fibroblasts (HDFs) and a key driver of CAF conversion, independent of cellular senescence. ANKRD1 expression in CAFs is associated with poor survival in HNSCC, lung, and cervical SCC patients, and controls a specific gene expression program of myofibroblast CAFs (my-CAFs). ANKRD1 binds to the regulatory region of my-CAF effector genes in concert with AP-1 transcription factors, and promotes c-JUN and FOS association. Targeting ANKRD1 disrupts AP-1 complex formation, reverses CAF activation, and blocks the pro-tumorigenic properties of CAFs in an orthotopic skin cancer model. ANKRD1 thus represents a target for fibroblast-directed therapy in cancer and potentially beyond.

Size exclusion chromatography based proteomic and degradomic profiling of inflammasome-activated, murine bone marrow-derived dendritic cells highlights complex retention and release of cleavage products.

Coupling size exclusion chromatography (SEC) with mass spectrometry-based proteomics enables investigating protein complexes, with degradomic profiling providing deeper insights into complex-associated proteolytic processing and retaining of cleavage products. This study aims to map protein complex formation upon inflammasome activation in bone marrow-derived dendritic cells (BMDCs) from gasdermin D-deficient mice, focusing on proteolytic enzymes and truncated proteins in higher molecular weight complexes. Cultured BMDCs were primed with LPS and subsequently treated with nigericin or Val-boroPro (VbP). SEC-fractionated proteins were TMT-labelled and analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified 6862 proteins and 70 802 peptides, including 14 714 semi-tryptic peptides indicating elevated endogenous proteolytic processing. The sequence motif of numerous cleavage sites maps to caspase-like activity. Inflammasome activation was corroborated by elevated levels of apoptosis-associated speck-like protein containing a CARD (ASC) in higher molecular weight (MW) fractions and increased IL-1β levels in low MW fractions upon nigericin or VbP treatment. The majority of truncated cleavage products remained within their corresponding, higher MW protein complexes while caspase-specific cleavage products of Rho-associated protein kinase 1, gelsolin, and AP-2 complex subunit alpha-2 dissociated to lower MW fractions. SEC profiles identified 174 proteases, with cell surface proteases forming high MW complexes, including ADAMs and DPP4 but not MMP14. VbP treatment led to the accumulation of ISG15 in low MW fractions while RNA polymerase II coactivator p15 shifted to higher MW fractions. This study demonstrates that SEC-coupled proteomics and degradomic profiling offer unique insights into protein complex dynamics and proteolytic processes upon inflammasome activation.

Human and mouse neutrophils share core transcriptional programs in both homeostatic and inflamed contexts

Neutrophils are frequently studied in mouse models, but the extent to which findings translate to humans remains poorly defined. In an integrative analysis of 11 mouse and 13 human datasets, we find a strong correlation of neutrophil gene expression across species. In inflammation, neutrophils display substantial transcriptional diversity but share a core inflammation program. This program includes genes encoding IL-1 family members, CD14, IL-4R, CD69, and PD-L1. Chromatin accessibility of core inflammation genes increases in blood compared to bone marrow and further in tissue. Transcription factor enrichment analysis implicates members of the NF-κB family and AP-1 complex as important drivers, and HoxB8 neutrophils with JunB knockout show a reduced expression of core inflammation genes in resting and activated cells. In independent single-cell validation data, neutrophil activation by type I or type II interferon, G-CSF, and E. coli leads to upregulation in core inflammation genes. In COVID-19 patients, higher expression of core inflammation genes in neutrophils is associated with more severe disease. In vitro treatment with GM-CSF, LPS, and type II interferon induces surface protein upregulation of core inflammation members. Together, we demonstrate transcriptional conservation in neutrophils in homeostasis and identify a core inflammation program shared across heterogeneous inflammatory conditions.

STAG2 loss Induces HSC Programs By Modulating Accessibility of AP-1 Bound Enhancers

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are blood disorders characterized by clonal expansion of mutant hematopoietic stem and progenitor cells (HSPCs). Mutations in genes encoding the cohesin complex, including STAG2, RAD21, SMC3 and SMC1A, are frequent genetic drivers in MDS and AML. However, the mechanisms by which cohesin mutations lead to clonal expansion of HSPCs are not well understood. Cohesin is essential for sister chromatid cohesion, DNA damage repair, maintenance of chromatin architecture, and gene regulation. To investigate the impact of cohesin mutations on chromatin accessibility, we performed ATAC-Seq in a panel of six isogenic STAG2 wild type (WT) and knockout (KO) U937 leukemia cell lines. Among the total of 97,798 ATAC-Seq peaks, we identified significantly more STAG2 KO (8,859) versus WT (1,622) uniquely accessible chromatin sites, which were strongly enriched for intergenic regions. Integration of ChIP-Seq profiling of cohesin binding and histone modifications demonstrated cohesin binding and an increase in the active enhancer and promoter histone mark H3K27Ac in the STAG2 KO newly accessible chromatin, suggesting a direct role for cohesin in regulating the activity of such enhancers. Together, our data demonstrate that STAG2 KO-induced open chromatin may be enriched for novel STAG2 KO-specific enhancers. To further infer the regulatory function of these putative enhancers, we predicted binding of transcription factors (TFs) in ATAC-Seq-defined regions by performing TF footprinting using the Transcription factor Occupancy Prediction By Investigation of ATAC-seq Signal (TOBIAS) algorithm. We observed that binding of members of the AP-1 transcription family, including JUN FOS andATF, previously implicated in oncogenic transformation, was significantly enriched in the STAG2 KO cells (Figure 1A). Conversely, STAG2 KO cells lost CEBP TF binding in regions that lost chromatin accessibility. To identify potential regulatory targets of these TFs, we mapped TF footprints to annotated promoter regions and to enhancer-gene pairs determined by the Activity by Contact (ABC) model. We observed a strong correlation between predicted AP-1 binding and gene expression of putative regulated genes in STAG2 KO cells, further supporting the role of these newly accessible AP-1 bound sites as regulatory elements. To validate our findings using in vivo models of cohesin-mutant disease, we performed RNA-Seq and ATAC-Seq in HSPC isolated from a Tet2/Stag2 mouse model of MDS previously developed in our lab. We confirmed an increase in accessible chromatin and demonstrated a strong enrichment of predicted Ap-1 binding in intergenic regions, in agreement with our observations in the U937 model. Gene set enrichment analysis in the Tet2/Stag2-mutant versus WT HSPC revealed that putative targets of Ap-1 binding were enriched for hematopoietic stem cell gene signatures (Figure 1B) . This is consistent with our observation of clonal expansion of HSPCs in this model, and suggests a mechanism by which novel Ap-1 bound enhancers may facilitate expression of key genes involved in HSPC expansion and development of MDS/AML. To explore whether our findings were applicable to other published models of Stag2 mutant myeloid neoplasms, we performed TF footprinting using publicly available ATAC-Seq datasets generated in HSPC isolated from Stag2 and Runx1/Stag2 conditional knockout mice. We recapitulated increased chromatin accessibility and significant enrichment of cohesin-bound Ap-1 footprints in Stag2 mutant over WT conditions. Together, these data further validate conservation of STAG2 KO-induced AP-1 bound regulatory elements in human and mouse models of MDS and AML. In summary, we show that STAG2 loss leads to the establishment of a novel set of accessible intergenic regions with features of enhancer activity in vitro and in vivo. We identify multiple members of the AP-1 complex with enriched predicted binding at these putative enhancers, and demonstrate that AP-1 may drive expression of HSPC signatures in models of Stag2 mutant MDS in vivo. We illustrate here a potential role for the AP-1 complex in cohesin mutant myeloid malignancies, which we are currently functionally interrogating.

DAP10 Co-Stimulation Imparts Memory-like Features to CD5 Targeting Cord Blood Derived CAR-NK Cells

Relapsed/refractory T-cell malignancies have a particularly poor prognosis and novel therapies are direly needed. CD5 is a great candidate for adoptive cellular therapy to target T-cell malignancies since it is ubiquitously expressed on T cells with restricted expression on other hematopoietic cells. NK cells are an attractive platform for CAR engineering to target CD5 since, unlike T cells, they do not express CD5 on their surface, which eliminates the risk of fratricide. Another advantage of NK cells for CAR engineering is their safety profile; in contrast to T cells, they do not cause cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS) and they are not associated with graft-versus-host disease (GVHD) in the allogeneic setting, opening the potential for a completely off-the-shelf cellular product to be used at point of care. Therefore, we sought to develop CD5 targeting CAR-NK cells for the treatment of T-cell malignancies. Multiple studies have demonstrated that the choice of co-stimulatory domain in CAR-T cells influences their function, persistence and metabolic profile. In the field of CAR-NK cells, the first constructs tested in the clinic have incorporated CD28, a T cell specific co-stimulatory domain, borrowing from the design of CAR-T cells. There is a sparsity of data regarding the impact of co-stimulatory domains on the proliferation, transcriptomic and proteomic profile, polyfunctionality, metabolism and fitness of CAR-NK cells. In this study, we aimed to do so, by comprehensively studying the impact of co-stimulatory domains including some that are more relevant for NK cell biology, namely DNAX-activating Protein 10 (DAP10), a major adaptor protein and the exclusive signaling intermediate of NKG2D in human NK cells, DNAX-activating Protein 12 (DAP12), an important adaptor molecule that associates with multiple activating receptors (e.g. NKG2C, NKp44, activating KIRs) and NKG2D, one of the most potent NK cytotoxicity receptors which is essential for anti-tumor immunity. Our results show that CD5 CAR-NK cells with DAP10 co-stimulatory domain show enhanced cytotoxicity against CD5+ T-cell leukemia targets even after multiple tumor rechallenges in an Incucyte live-cell imaging assay. They also show augmented polyfunctionality compared to CD5 CAR-NK cells with other co-stimulatory domains in an Isoplexis single-cell secretome assay. Moreover, DAP10 co-stimulation endowed CD5 CAR-NK cells with enhanced metabolic fitness as evidenced by increased oxidative phosphorylation compared to other co-stimulatory molecules. At the epigenetic level, CD5 CAR-NK cells with DAP10 co-stimulation interrogated by sc-ATACseq show enrichment in AP-1 complex and BATF transcription factors related to memory formation and exhaustion resistance. This translates to better in vivo performance as CD5 CAR-NK cells with DAP10 co-stimulatory domain significantly improve tumor control and survival in an NSG mouse model of CD5+ T-cell leukemia (CCRF-CEM) and show evidence of recall response following tumor rechallenge. In conclusion, our data show that DAP10 co-stimulation induces epigenetic reprogramming of CD5 CAR-NK cells leading to enhanced cellular fitness and memory formation ensuing better anti-tumor potential. Based on these preclinical data, a Phase I/II clinical trial evaluating the safety and efficacy of CD5 CAR-NK cells for the treatment of CD5+ malignancies is in preparation.