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Inborn errors of immunity (IEIs) unveil regulatory pathways of human immunity. We describe a new IEI caused by mutations in the GTPase of the immune-associated protein 6 (GIMAP6) gene in patients with infections, lymphoproliferation, autoimmunity, and multiorgan vasculitis. Patients and Gimap6−/− mice show defects in autophagy, redox regulation, and polyunsaturated fatty acid (PUFA)–containing lipids. We find that GIMAP6 complexes with GABARAPL2 and GIMAP7 to regulate GTPase activity. Also, GIMAP6 is induced by IFN-γ and plays a critical role in antibacterial immunity. Finally, we observed that Gimap6−/− mice died prematurely from microangiopathic glomerulosclerosis most likely due to GIMAP6 deficiency in kidney endothelial cells.

The p53 transcription factor drives anti-proliferative gene expression programs in response to diverse stressors, including DNA damage and oncogenic signaling. Here, we seek to uncover new mechanisms through which p53 regulates gene expression using tandem affinity purification/mass spectrometry to identify p53-interacting proteins. This approach identified METTL3, an m(6)A RNA-methyltransferase complex (MTC) constituent, as a p53 interactor. We find that METTL3 promotes p53 protein stabilization and target gene expression in response to DNA damage and oncogenic signals, by both catalytic activity-dependent and independent mechanisms. METTL3 also enhances p53 tumor suppressor activity in in vivo mouse cancer models and human cancer cells. Notably, METTL3 only promotes tumor suppression in the context of intact p53. Analysis of human cancer genome data further supports the notion that the MTC reinforces p53 function in human cancer. Together, these studies reveal a fundamental role for METTL3 in amplifying p53 signaling in response to cellular stress.

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.

Cell lineage specification is accomplished by a concerted action of chromatin remodeling and tissue-specific transcription factors. However, the mechanisms that induce and maintain appropriate lineage-specific gene expression remain elusive. Here, we used an unbiased proteomics approach to characterize chromatin regulators that mediate the induction of neuronal cell fate. We found that Tip60 acetyltransferase is essential to establish neuronal cell identity partly via acetylation of the histone variant H2A.Z. Despite its tight correlation with gene expression and active chromatin, loss of H2A.Z acetylation had little effect on chromatin accessibility or transcription. Instead, loss of Tip60 and acetyl-H2A.Z interfered with H3K4me3 deposition and activation of a unique subset of silent, lineage-restricted genes characterized by a bivalent chromatin configuration at their promoters. Altogether, our results illuminate the mechanisms underlying bivalent chromatin activation and reveal that H2A.Z acetylation regulates neuronal fate specification by establishing epigenetic competence for bivalent gene activation and cell lineage transition.

Cutaneous T cell lymphomas (CTCL) are rare but aggressive cancers without effective treatments. While a subset of patients derive benefit from PD-1 blockade, there is a critically unmet need for predictive biomarkers of response. Herein, we perform CODEX multiplexed tissue imaging and RNA sequencing on 70 tumor regions from 14 advanced CTCL patients enrolled in a pembrolizumab clinical trial (NCT02243579). We find no differences in the frequencies of immune or tumor cells between responders and non-responders. Instead, we identify topographical differences between effector PD-1+ CD4+ T cells, tumor cells, and immunosuppressive Tregs, from which we derive a spatial biomarker, termed the SpatialScore, that correlates strongly with pembrolizumab response in CTCL. The SpatialScore coincides with differences in the functional immune state of the tumor microenvironment, T cell function, and tumor cell-specific chemokine recruitment and is validated using a simplified, clinically accessible tissue imaging platform. Collectively, these results provide a paradigm for investigating the spatial balance of effector and suppressive T cell activity and broadly leveraging this biomarker approach to inform the clinical use of immunotherapies., PD-1 blockade is effective for only a subset of patients with cutaneous T cell lymphomas. Here, the authors report a spatial biomarker that uses immune and cancer cell topography to predict response to PD-1 blockade in this disease.

SUMMARYThe p53 transcription factor, encoded by the most frequently mutated gene in human cancer, plays a critical role in tissue homeostasis in response to stress signals. The mechanisms through which p53 promotes downstream tumor suppressive gene expression programs remain, however, only superficially understood. Here, we used tandem affinity purification and mass spectrometry to reveal new components of the p53 response. This approach uncovered Mettl3, a component of the m6A RNA methyltransferase complex (MTC), as a p53-interacting protein. Analysis of Mettl3- deficient cells revealed that Mettl3 promotes p53 protein stabilization and target gene expression in response to DNA damage. Mettl3 acts in part by competing with the p53 negative regulator, Mdm2, for binding to the p53 transactivation domains to promote methyltransferase-independent stabilization of p53. In addition, Mettl3 relies on its catalytic activity to augment p53 responses, with p53 recruiting Mettl3 to p53 target genes to co-transcriptionally direct m6A modification of p53 pathway transcripts to enhance their expression. Mettl3 also promotes p53 activity downstream of oncogenic signals in vivo, in both allograft and autochthonous lung adenocarcinoma models, suggesting cooperative action of p53 and Mettl3 in tumor suppression. Accordingly, we found in diverse human cancers that mutations in MTC components perturb expression of p53 target genes and that MTC mutations are mutually exclusive with TP53 mutations, suggesting that the MTC enhances the p53 transcriptional program in human cancer. Together, these studies reveal a fundamental role for Mettl3 in amplifying p53 signaling through protein stabilization and epitranscriptome regulation.

The PBX1 homeodomain transcription factor is converted by t(1;19) chromosomal translocations in acute leukemia into the chimeric E2A-PBX1 oncoprotein. Fusion with E2A confers potent transcriptional activation and constitutive nuclear localization, bypassing the need for dimerization with protein partners that normally stabilize and regulate import of PBX1 into the nucleus, but the mechanisms underlying its oncogenic activation are incompletely defined. We demonstrate here that E2A-PBX1 self-associates through the PBX1 PBC-B domain of the chimeric protein to form higher-order oligomers in t(1;19) human leukemia cells, and that this property is required for oncogenic activity. Structural and functional studies indicate that self-association facilitates the binding of E2A-PBX1 to DNA. Mutants unable to self-associate are transformation defective, however their oncogenic activity is rescued by the synthetic oligomerization domain of FKBP, which confers conditional transformation properties on E2A-PBX1. In contrast to self-association, PBX1 protein domains that mediate interactions with HOX DNA-binding partners are dispensable. These studies suggest that oligomeric self-association may compensate for the inability of monomeric E2A-PBX1 to stably bind DNA and circumvents protein interactions that otherwise modulate PBX1 stability, nuclear localization, DNA binding, and transcriptional activity. The unique dependence on self-association for E2A-PBX1 oncogenic activity suggests potential approaches for mechanism-based targeted therapies.

LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the cancer state that stems from Lkb1 deficiency can be reverted remains unknown. To identify the processes governed by LKB1 in vivo, we generated an allele which enables Lkb1 inactivation at tumor initiation and subsequent Lkb1 restoration in established tumors. Restoration of Lkb1 in oncogenic KRAS-driven lung tumors suppressed proliferation and led to tumor stasis. Lkb1 restoration activated targets of C/EBP transcription factors and drove neoplastic cells from a progenitor-like state to a less proliferative alveolar type II cell-like state. We show that C/EBP transcription factors govern a subset of genes that are induced by LKB1 and depend upon NKX2-1. We also demonstrate that a defining factor of the alveolar type II lineage, C/EBPα, constrains oncogenic KRAS-driven lung tumor growth in vivo. Thus, this key tumor suppressor regulates lineage-specific transcription factors, thereby constraining lung tumor development through enforced differentiation.

A thorough understanding of complex spatial host-disease interactions in situ is necessary in order to develop effective preventative measures and therapeutic strategies. Here, we developed Protein And Nucleic acid IN situ Imaging (PANINI) and coupled it with Multiplexed Ion Beam Imaging (MIBI) to sensitively and simultaneously quantify DNA, RNA, and protein levels within the microenvironments of tissue compartments. The PANINI-MIBI approach was used to measure over 30 parameters simultaneously across large sections of archival lymphoid tissues from non-human primates that were healthy or infected with simian immunodeficiency virus (SIV), a model that accurately recapitulates human immunodeficiency virus infection (HIV). This enabled multiplexed dissection of cellular phenotypes, functional markers, viral DNA integration events, and viral RNA transcripts as resulting from viral infection. The results demonstrated immune coordination from an unexpected upregulation of IL10 in B cells in response to SIV infection that correlated with macrophage M2 polarization, thus conditioning a potential immunosuppressive environment that allows for viral production. This multiplexed imaging strategy also allowed characterization of the coordinated microenvironment around latently or actively infected cells to provide mechanistic insights into the process of viral latency. The spatial multi-modal framework presented here is applicable to deciphering tissue responses in other infectious diseases and tumor biology.

Emerging evidence points toward an intricate relationship between the pandemic of coronavirus disease 2019 (COVID-19) and diabetes. While preexisting diabetes is associated with severe COVID-19, it is unclear whether COVID-19 severity is a cause or consequence of diabetes. To mechanistically link COVID-19 to diabetes, we tested whether insulin-producing pancreatic β cells can be infected by SARS-CoV-2 and cause β cell depletion. We found that the SARS-CoV-2 receptor, ACE2, and related entry factors (TMPRSS2, NRP1, and TRFC) are expressed in β cells, with selectively high expression of NRP1. We discovered that SARS-CoV-2 infects human pancreatic β cells in patients who succumbed to COVID-19 and selectively infects human islet β cells in vitro. We demonstrated that SARS-CoV-2 infection attenuates pancreatic insulin levels and secretion and induces β cell apoptosis, each rescued by NRP1 inhibition. Phosphoproteomic pathway analysis of infected islets indicates apoptotic β cell signaling, similar to that observed in type 1 diabetes (T1D). In summary, our study shows SARS-CoV-2 can directly induce β cell killing., Graphical abstract, Diabetic patients are at risk for severe COVID-19, but the virus may further damage insulin-secreting β cells. Wu et al. found that patient β cells are virally infected and the highly expressed neuropilin-1 receptor is critical for viral entry, causing cell death and reduced insulin secretion, exacerbating diabetes in patients.

Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology.

Multiple G protein-coupled receptors (GPCRs) are expressed in pancreatic islet cells, but the majority have unknown functions. We observed specific GPCRs localized to primary cilia, a prominent signaling organelle, in pancreatic α and β cells. Loss of cilia disrupts β-cell endocrine function, but the molecular drivers are unknown. Using functional expression, we identified multiple GPCRs localized to cilia in mouse and human islet α and β cells, including FFAR4, PTGER4, ADRB2, KISS1R, and P2RY14. Free fatty acid receptor 4 (FFAR4) and prostaglandin E receptor 4 (PTGER4) agonists stimulate ciliary cAMP signaling and promote glucagon and insulin secretion by α- and β-cell lines and by mouse and human islets. Transport of GPCRs to primary cilia requires TULP3, whose knockdown in primary human and mouse islets relocalized ciliary FFAR4 and PTGER4 and impaired regulated glucagon or insulin secretion, without affecting ciliary structure. Our findings provide index evidence that regulated hormone secretion by islet α and β cells is controlled by ciliary GPCRs providing new targets for diabetes.

Anti-PD-1 immunotherapies have transformed cancer treatment, yet the determinants of clinical response are largely unknown. We performed CODEX multiplexed tissue imaging and RNA sequencing on 70 tumor regions from 14 advanced cutaneous T cell lymphoma (CTCL) patients enrolled in a clinical trial of pembrolizumab therapy. Clinical response was not associated with the frequency of tumor-infiltrating T cell subsets, but rather with striking differences in the spatial organization and functional immune state of the tumor microenvironment (TME). After treatment, pembrolizumab responders had a localized enrichment of tumor and CD4+ T cells, which coincided with immune activation and cytotoxic PD-1+ CD4+ T cells. In contrast, non-responders had a localized enrichment of Tregs pre- and post-treatment, consistent with a persistently immunosuppressed TME and exhausted PD-1+ CD4+ T cells. Integrating these findings by computing the physical distances between PD-1+ CD4+ T cells, tumor cells, and Tregs revealed a spatial biomarker predictive of pembrolizumab response. Finally, the chemokine CXCL13 was upregulated in tumor cells in responders post-treatment, suggesting that chemoattraction of PD-1+ CD4+ T cells towards tumor cells facilitates a positive outcome. Together, these data show that T cell topography reflects the balance of effector and suppressive activity within the TME and predicts clinical response to PD-1 blockade in CTCL.

A thorough understanding of complex spatial host-disease interactions in situ is necessary in order to develop effective preventative measures and therapeutic strategies. Here, we developed Protein And Nucleic acid IN situ Imaging (PANINI) and coupled it with Multiplexed Ion Beam Imaging (MIBI) to sensitively and simultaneously quantify DNA, RNA, and protein levels within the microenvironments of tissue compartments. The PANINI-MIBI approach was used to measure over 30 parameters simultaneously across large sections of archival lymphoid tissues from non-human primates that were healthy or infected with simian immunodeficiency virus (SIV), a model that accurately recapitulates human immunodeficiency virus infection (HIV). This enabled multiplexed dissection of cellular phenotypes, functional markers, viral DNA integration events, and viral RNA transcripts as resulting from viral infection. The results demonstrated immune coordination from an unexpected upregulation of IL10 in B cells in response to SIV infection that correlated with macrophage M2 polarization, thus conditioning a potential immunosuppressive environment that allows for viral production. This multiplexed imaging strategy also allowed characterization of the coordinated microenvironment around latently or actively infected cells to provide mechanistic insights into the process of viral latency. The spatial multi-modal framework presented here is applicable to deciphering tissue responses in other infectious diseases and tumor biology.

SummaryRegeneration is the “holy grail” of tissue repair, but skin injury typically yields fibrotic, non-functional scars. Developing pro-regenerative therapies requires rigorous understanding of the molecular progression from injury to fibrosis or regeneration. Here, we report the divergent molecular events driving skin wound cells toward either scarring or regenerative fates. We profile scarring versus YAP inhibition-induced wound regeneration at the transcriptional (single-cell RNA-sequencing), protein (timsTOF proteomics), and tissue (extracellular matrix ultrastructural analysis) levels. Using cell surface barcoding, we integrate these data to reveal fibrotic and regenerative “molecular trajectories” of healing. We show that disrupting YAP mechanical signaling yields regenerative repair orchestrated by fibroblasts with activated Trps1 and Wnt signaling. Our findings serve as a multimodal map of wound regeneration and could have therapeutic implications for pathologic fibroses.

SummaryMultiple G protein coupled receptors (GPCRs) are expressed in pancreatic islet cells but the majority have unknown functions. We observe specific GPCRs localized to primary cilia, a prominent signaling organelle, in pancreatic α- and β-cells. Loss of cilia disrupts β-cell endocrine function, but the molecular drivers are unknown. Using functional expression, we identified multiple GPCRs localized to cilia in mouse and human islet α- and β-cells, including FFAR4, PTGER4, DRD5, ADRB2, KISS1R, and P2RY14. Free fatty acid receptor 4 (FFAR4) and prostaglandin E receptor 4 (PTGER4) agonists stimulate ciliary cAMP signaling and promote glucagon and insulin secretion by α- and β-cell lines, and by mouse and human islets. Transport of GPCRs to primary cilia requires TULP3, whose knockdown in primary human and mouse islets depleted ciliary FFAR4 and PTGER4, and impaired regulated glucagon or insulin secretion, without affecting ciliary structure. Our findings provide index evidence that regulated hormone secretion by islet α- and β-cells is regulated by ciliary GPCRs providing new targets for diabetes.

Regeneration is the holy grail of tissue repair, but skin injury typically yields fibrotic, non-functional scars. Developing pro-regenerative therapies requires rigorous understanding of the molecular progression from injury to fibrosis or regeneration. Here, we report the divergent molecular events driving skin wound cells toward scarring or regenerative fates. We profile scarring versus YAP-inhibition-induced wound regeneration at the transcriptional (single-cell RNA sequencing), protein (timsTOF proteomics), and tissue (extracellular matrix ultrastructural analysis) levels. Using cell-surface barcoding, we integrate these data to reveal fibrotic and regenerative "molecular trajectories" of healing. We show that disrupting YAP mechanotransduction yields regenerative repair by fibroblasts with activated Trps1 and Wnt signaling. Finally, via in vivo gene knockdown and overexpression in wounds, we identify Trps1 as a key regulatory gene that is necessary and partially sufficient for wound regeneration. Our findings serve as a multi-omic map of wound regeneration and could have therapeutic implications for pathologic fibroses.

Summary Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.

ARHGAP36 is a Rho GTPase-activating protein (GAP) family member that contributes to spinal cord development and tumorigenesis. This multidomain protein is composed of N-terminal sequences that vary between splice isoforms, the GAP-like region, and a unique C-terminal domain, and previous studies have revealed an N-terminal arginine-rich region that promotes protein kinase A catalytic subunit (PKAcat) degradation and Gli transcription factor activation. Although these signaling mechanisms can account for at least some aspects of ARHGAP36-dependent physiology, the functional roles of other structural elements in ARHGAP36 are not well understood. To address this question, we have mapped the ARHGAP36 structure-activity landscape with both domain- and amino acid-level resolution. We observe that ARHGAP36-mediated Gli activation in cultured mammalian cells can be autoinhibited by isoform-specific N-terminal sequences, which also regulate ARHGAP36 trafficking and the subcellular targeting of PKAcat pools. ARHGAP36 autoinhibition is counteracted by the GAP-like and C-terminal domains, which promote protein trafficking to the plasma membrane and primary cilium, respectively. In addition, our studies provide evidence that the GAP-like domain conditionally suppresses the N-terminal arginine-rich region and regulates ARHGAP36 binding to several interactors, including the prolyl oligopeptidase-like protein PREPL and the E3 ubiquitin ligase PRAJA2. Thus, multiple domains within ARHGAP36 can modulate its activity state, subcellular localization, and protein binding, potentially providing a means for achieving tissue-specific ARHGAP36 functions.

During aging, the regenerative capacity of muscle stem cells (MuSCs) decreases, diminishing the ability of muscle to repair following injury. We performed a small molecule library screen and discovered that the proliferation and expansion of aged MuSCs is regulated by signal transduction pathways organized by the primary cilium, a cellular protrusion that serves as a sensitive sensory organelle. Abolishing MuSC cilia in vivo severely impaired injury-induced muscle regeneration. In aged muscle, a cell intrinsic defect in MuSC ciliation leading to impaired Hedgehog signaling was associated with the decrease in regenerative capacity. This deficit could be overcome by exogenous activation of Hedgehog signaling which promoted MuSC expansion, both in vitro and in vivo. Delivery of the small molecule Smoothened agonist (SAG) to muscles of aged mice restored regenerative capacity leading to increased strength post-injury. These findings provide fresh insights into the signaling dysfunction in aging and identify the ciliary Hedgehog signaling pathway as a potential therapeutic target to counter the loss of muscle regenerative capacity which accompanies aging.

During aging, the regenerative capacity of muscle stem cells (MuSCs) decreases, diminishing the ability of muscle to repair following injury. We found that the ability of MuSCs to regenerate is regulated by the primary cilium, a cellular protrusion that serves as a sensitive sensory organelle. Abolishing MuSC cilia inhibited MuSC proliferation in vitro and severely impaired injury-induced muscle regeneration in vivo. In aged muscle, a cell intrinsic defect in MuSC ciliation was associated with the decrease in regenerative capacity. Exogenous activation of Hedgehog signaling, known to be localized in the primary cilium, promoted MuSC expansion, both in vitro and in vivo. Delivery of the small molecule Smoothened agonist (SAG1.3) to muscles of aged mice restored regenerative capacity leading to increased strength post-injury. These findings provide fresh insights into the signaling dysfunction in aged MuSCs and identify the ciliary Hedgehog signaling pathway as a potential therapeutic target to counter the loss of muscle regenerative capacity which accompanies aging.

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication1. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D–CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors. AMBRA1 is the main regulator of the degradation of D-type cyclins, and loss of AMBRA1 promotes cell proliferation and tumour growth, and reduces the sensitivity of cancer cells to inhibition of CDK4 and CDK6.

The human genome encodes tens of thousands circular RNAs (circRNAs) with mostly unknown functions. Circular RNAs require internal ribosome entry sites (IRES) if they are to undergo translation without a 5' cap. Here, we develop a high-throughput screen to systematically discover RNA sequences that can direct circRNA translation in human cells. We identify more than 17,000 endogenous and synthetic sequences as candidate circRNA IRES. 18S rRNA complementarity and a structured RNA element positioned on the IRES are important for driving circRNA translation. Ribosome profiling and peptidomic analyses show extensive IRES-ribosome association, hundreds of circRNA-encoded proteins with tissue-specific distribution, and antigen presentation. We find that circFGFR1p, a protein encoded by circFGFR1 that is downregulated in cancer, functions as a negative regulator of FGFR1 oncoprotein to suppress cell growth during stress. Systematic identification of circRNA IRES elements may provide important links among circRNA regulation, biological function, and disease.

SummaryIn response to excess nutrients, white adipose tissue expands by both generating new adipocytes and by upregulating lipogenesis in existing adipocytes. Here, we performed a genome-wide functional genomics screen to identify regulators of adipogenesis in the mouse 3T3-L1 cell model. The pooled screening strategy utilized FACS to isolate populations based on lipid content by gating for fluorescence intensity of the lipophilic, green fluorescent BODIPY 493/503 dye. Additionally, this approach categorized if genes functioned during mitotic expansion or lipogenesis. Cellular mechanisms regulating the rates of protein translation and protein stability were found to be critical for adipogenesis and lipogenesis. These mechanisms were further supported by proteomic analyses, which demonstrated that many changes in protein abundance during 3T3-L1 adipogenesis were not driven by transcription. Within these themes, we illustrate that hypusination is critical for translating adipogenic inducers of mitotic expansion and that the neddylation/ubiquitin pathway modulates insulin sensitivity to regulate lipogenesis.

SummaryHematopoietic stem cells (HSCs) are responsible for the generation of blood and immune cells throughout life. They have the unique ability to self-renew and generate more HSCs or differentiate into a progenitor cell in response to cell-intrinsic and -extrinsic stimuli. The balance of HSC fate commitment is critical for a healthy blood supply. Imbalances during hematopoiesis, which are frequent in aging, can result in hematological malignancies and pre-malignancies as well as increase risk of atherosclerosis. Given the importance of HSCs and their progenitors, they have been extensively characterized in genomic and transcriptomic studies. However, an understanding of protein expression within the HSC compartment and more broadly throughout hematopoiesis remains poorly understood, and it has been widely reported that the correlation between mRNA and proteins is more complicated than previously thought. Previous mouse mass spectrometry studies have focused either specifically on stem and the first early progenitor or broadly across mixed populations of stem and progenitor cells, which do not allow for cell-type specific protein resolution across stages of differentiation. Mass cytometry has been employed to characterize transcription factor expression in human HSCs and progenitors but does not apply an unbiased discovery approach. New mass spectrometry technology now allows for deep proteomic coverage with no more than 200 ng of sample input. We report here a proteomics resource characterizing protein expression in mouse adult and aged HSCs, multipotent progenitors and oligopotent progenitors, 12 cell types in total. We validated differential expression by flow cytometry analysis and immunofluorescence staining. Additionally, we investigated the relationship between mRNA and protein levels of individual genes in HSCs compared to progenitors through RNA sequencing studies and identified two proteins that appear to be uniquely regulated in the HSC compartment, Cpin1 and Adnp. In summary, this resource provides proteomic coverage of adult and aged hematopoietic stem cells and their progenitors and reveals changes in protein abundance between cell types, with potential future implications in understanding mechanisms for stem-cell maintenance, niche interactions and fate determination.

Summary Adult mesenchymal stem cells, including preadipocytes, possess a cellular sensory organelle called the primary cilium. Ciliated preadipocytes abundantly populate perivascular compartments in fat and are activated by a high-fat diet. Here, we sought to understand whether preadipocytes use their cilia to sense and respond to external cues to remodel white adipose tissue. Abolishing preadipocyte cilia in mice severely impairs white adipose tissue expansion. We discover that TULP3-dependent ciliary localization of the omega-3 fatty acid receptor FFAR4/GPR120 promotes adipogenesis. FFAR4 agonists and ω-3 fatty acids, but not saturated fatty acids, trigger mitosis and adipogenesis by rapidly activating cAMP production inside cilia. Ciliary cAMP activates EPAC signaling, CTCF-dependent chromatin remodeling, and transcriptional activation of PPARγ and CEBPα to initiate adipogenesis. We propose that dietary ω-3 fatty acids selectively drive expansion of adipocyte numbers to produce new fat cells and store saturated fatty acids, enabling homeostasis of healthy fat tissue.

Despite intensive study, no drugs in clinical use specifically target KRAS-mutant tumors. Uncharacterized feedback pathways and unmapped compensatory pathways, including compensation among paralogs, hinder our ability to target Ras effector pathways, requiring a better catalogue of pathways upstream and downstream of Ras. We used tandem affinity purification of Kras, Hras and Nras, their activated alleles and key proteins with known regulatory (GEFs, GAPs) or effectors (Raf, RalGDS1, RIN1/2) in both 293 cells and A549 NSCLC cells to generate a high-confidence protein-protein interaction (PPI) network of 220 proteins showing 1,400 physical interactions. The network was used to design an sgRNA library (10 sgRNAs/gene) and screen Cas9-expressing A549 cells for strong growth dependencies. These data were then used to select 120 genes and construct a 2-gene tandem sgRNA library of highest relevance to the Ras pathway (with 60 control sgRNAs). This 2-gene sgRNA library was tested in A549 and H23 NSCLC lines for quantitative single and two gene-dependent quantitative changes in growth, showing 100s of strong synthetic lethals among 14K pairwise tests. These genetic interactions in conjunction with PPIs and TCGA data identify extensive coupling between Raf/MEK/ERK kinases, Ral and Rap GTPases, the Rap1GDS1 small GTPase controller, and RADIL cell adhesion pathways. The screen identified new candidate effector pathways for cell adhesion, Rap GTPase regulation, and protein processing, including new understudied Kras direct effectors RADIL, RGL1/2/3, and RIN1/2. Additional 20 x 20 custom libraries were screened in a broader panel of Kras-mutant versus other NSCLC lines. These screens revealed systematic Kras-dependent synthetic lethality among components of the MAP kinase pathway (ERK1/ERK2, ERK1/RAF1, MEK1/MEK2 etc.) and other interactions between the MAPK pathway and components of the Ral and Rap GTPase, RADIL cell adhesion pathways and RIN1-dependent macropinocytosis pathways. Using the recent Kras G12C inhibitor in H23 cells, we have validated that sgRNA knockouts of these Kras effector affect these specific, new pathways: cell adhesion via RADIL, growth signaling via Rap1GDS1 and RhoA, and macropinocytosis via the Rab5 GEF RIN1. Application of the Kras inhibitor ARS-853 shows much-reduced effects on specific Kras effector pathways in cells deleted for these specific effectors, showing these effectors are highly coupled to Kras. Our systematic data reveal new genetic vulnerabilities and target candidates with potential for new therapeutics. Citation Format: Marcus Kelly, Kyuho Han, Kaja Kostyrko, Nancie Mooney, Edwin Jeng, Janos Demeter, Alejandro Sweet-Cordero, Michael Bassik, Peter K. Jackson. Combined proteomic and genetic interaction mapping reveals new Ras pathway effectors and regulators [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B25.

Summary Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)—a collection of components characteristic of the CRC iTME. Enrichment of PD-1+CD4+ T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains., Graphical Abstract, Highlights • FFPE-CODEX multiplexed tissue imaging of 56 markers in 140 tissues of 35 CRC patients • Cellular neighborhoods reveal spatial organization of the tumor microenvironment • Altered organization of tumor and immune components in low- versus high-risk patients • Local enrichment of PD-1+CD4+ T cells correlates with survival in high-risk patients, A multiplexed tissue imaging and computational analysis framework applied to colorectal cancer allows interrogation of how spatial organization of the immune tumor microenvironment is linked to clinical outcomes.

The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is the authoritative community resource for the Saccharomyces cerevisiae reference genome sequence and its annotation. In recent years, we have moved toward increased representation of sequence variation and allelic differences within S. cerevisiae. The publication of numerous additional genomes has motivated the creation of new tools for their annotation and analysis. Here we present the Variant Viewer: a dynamic open-source web application for the visualization of genomic and proteomic differences. Multiple sequence alignments have been constructed across high quality genome sequences from 11 different S. cerevisiae strains and stored in the SGD. The alignments and summaries are encoded in JSON and used to create a two-tiered dynamic view of the budding yeast pan-genome, available at http://www.yeastgenome.org/variant-viewer.

E2F transcription factors are central regulators of cell division and cell fate decisions. E2F4 often represents the predominant E2F activity in cells. E2F4 is a transcriptional repressor implicated in cell cycle arrest and whose repressive activity depends on its interaction with members of the RB family. Here we show that E2F4 is important for the proliferation and the survival of mouse embryonic stem cells. In these cells, E2F4 acts in part as a transcriptional activator that promotes the expression of cell cycle genes. This role for E2F4 is independent of the RB family. Furthermore, E2F4 functionally interacts with chromatin regulators associated with gene activation and we observed decreased histone acetylation at the promoters of cell cycle genes and E2F targets upon loss of E2F4 in RB family-mutant cells. Taken together, our findings uncover a non-canonical role for E2F4 that provide insights into the biology of rapidly dividing cells., E2F transcription factors are regulators of cell division and cell fate decisions. Here the authors show that E2F4 is important for proliferation and survival of mouse ESCs, independent of the RB family, and that E2F4 interacts with chromatin regulators associated with gene activation.