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Summary: Interactions between cells in the tumor microenvironment (TME) shape cancer progression and patient prognosis. To gain insights into how the TME influences cancer outcomes, we derive gene expression signatures indicative of signaling between stromal fibroblasts and cancer cells, and demonstrate their prognostic significance in multiple and independent squamous cell carcinoma cohorts. By leveraging information within the signatures, we discover that the HB-EGF/EGFR/MAPK axis represents a hub of tumor-stroma crosstalk, promoting the expression of CSF2 and LIF and favoring the recruitment of macrophages. Together, these analyses demonstrate the utility of our approach for interrogating the extent and consequences of TME crosstalk.

Rapid intestinal regeneration after injury is critical to maintain barrier integrity and homeostasis, but must be tightly controlled to prevent tumorigenesis. Here they show that the aryl hydrocarbon receptor is required to terminate the regenerative response after wound healing.

The synthesis of serine from glucose is a key metabolic pathway supporting cellular proliferation in healthy and malignant cells. Despite this, the role that this aspect of metabolism plays in germinal center biology and pathology is not known. Here, we performed a comprehensive characterization of the role of the serine synthesis pathway in germinal center B cells and lymphomas derived from these cells. We demonstrate that upregulation of a functional serine synthesis pathway is a metabolic hallmark of B cell activation and the germinal center reaction. Inhibition of phosphoglycerate dehydrogenase (PHGDH), the first and rate-limiting enzyme in this pathway, led to defective germinal formation and impaired high-affinity antibody production. In addition, overexpression of enzymes involved in serine synthesis was a characteristic of germinal center B cell–derived lymphomas, with high levels of expression being predictive of reduced overall survival in diffuse large B cell lymphoma. Inhibition of PHGDH induced apoptosis in lymphoma cells, reducing disease progression. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in lymphoma.

GM-CSF is involved in control over M. tuberculosis infection. Here the authors show that GM-CSF reduces type 1 interferon driven neutrophil recruitment, NETosis and bacterial growth in the lungs of infected mice, and provide evidence that this NETosis occurs in infected humans who are not responsive to antibiotic therapy.

The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease.

The authors present an extensive profile of host transcriptional respones to a diverse group of pathogens and allergens. In doing so, they identify TH1, type I IFN, TH17, and TH2 responses, that underlie each immune response in both the blood and lung, which represents a global profile of host-pathogen immune responses.

WNK1, a kinase that controls kidney salt homeostasis, also regulates adhesion and migration in CD4+ T cells. Wnk1 is highly expressed in thymocytes, and since migration is important for thymocyte maturation, we investigated a role for WNK1 in mouse thymocyte development. We find that WNK1 is required for the transition of double negative (DN) thymocytes through the β-selection checkpoint and subsequent proliferation and differentiation into double positive (DP) thymocytes. Furthermore, we show that WNK1 negatively regulates LFA1-mediated adhesion and positively regulates CXCL12-induced migration in DN thymocytes. Despite this, migration defects of WNK1-deficient thymocytes do not account for the developmental arrest. Instead, we show that in DN thymocytes WNK1 transduces pre-TCR signals via OXSR1 and STK39 kinases, and the SLC12A2 ion co-transporter that are required for post-transcriptional upregulation of MYC and subsequent proliferation and differentiation into DP thymocytes. Thus, a pathway regulating ion homeostasis is a critical regulator of thymocyte development.

Summary: Loss of p53 function contributes to the development of many cancers. While cell-autonomous consequences of p53 mutation have been studied extensively, the role of p53 in regulating the anti-tumor immune response is still poorly understood. Here, we show that loss of p53 in cancer cells modulates the tumor-immune landscape to circumvent immune destruction. Deletion of p53 promotes the recruitment and instruction of suppressive myeloid CD11b+ cells, in part through increased expression of CXCR3/CCR2-associated chemokines and macrophage colony-stimulating factor (M-CSF), and attenuates the CD4+ T helper 1 (Th1) and CD8+ T cell responses in vivo. p53-null tumors also show an accumulation of suppressive regulatory T (Treg) cells. Finally, we show that two key drivers of tumorigenesis, activation of KRAS and deletion of p53, cooperate to promote immune tolerance. : TP53 is one of the most frequently mutated genes in cancer; however, its significance in controlling tumor-immune crosstalk is not fully understood. Blagih et al. highlight a key role for tumor-associated loss of p53, a common oncogenic event, in regulating myeloid and T cell responses. Keywords: p53, Kras, tumor, myeloid cells, T cell response

Conventional dendritic cells (cDCs) are thought to descend from a DC precursor downstream of the common myeloid progenitor (CMP). However, a mouse lymphoid-primed multipotent progenitor has been shown to generate cDCs following a DC-specific developmental pathway independent of monocyte and granulocyte poiesis. Similarly, here we show that, in humans, a large fraction of multipotent lymphoid early progenitors (MLPs) gives rise to cDCs, in particular the subset known as cDC1, identified by co-expression of DNGR-1 (CLEC9A) and CD141 (BDCA-3). Single-cell analysis indicates that over one-third of MLPs have the potential to efficiently generate cDCs. cDC1s generated from CMPs or MLPs do not exhibit differences in transcriptome or phenotype. These results demonstrate an early imprinting of the cDC lineage in human hematopoiesis and highlight the plasticity of developmental pathways giving rise to human DCs.

Summary: Signal transduction pathways stimulated by secreted growth factors are tightly regulated at multiple levels between the cell surface and the nucleus. The trafficking of cell surface receptors is emerging as a key step for regulating appropriate cellular responses, with perturbations in this process contributing to human diseases, including cancer. For receptors recognizing ligands of the transforming growth factor β (TGF-β) family, little is known about how trafficking is regulated or how this shapes signaling dynamics. Here, using whole genome small interfering RNA (siRNA) screens, we have identified the ESCRT (endosomal sorting complex required for transport) machinery as a crucial determinant of signal duration. Downregulation of ESCRT components increases the outputs of TGF-β signaling and sensitizes cells to low doses of ligand in their microenvironment. This sensitization drives an epithelial-to-mesenchymal transition (EMT) in response to low doses of ligand, and we demonstrate a link between downregulation of the ESCRT machinery and cancer survival. : Miller et al. demonstrate, using whole genome siRNA screening, that TGF-β receptors are targeted for degradation by the ESCRT machinery. Inhibiting ESCRT components upregulates long-term TGF-β signaling and enhances functional outputs of the pathway to sensitize cells to low levels of ligand in the micro-environment. Keywords: epithelial-to-mesenchymal transition, ESCRT machinery, receptor trafficking, signaling dynamics, SMAD2, TGF-β

Cockayne syndrome (CS) is a severe neurodevelopmental disorder characterized by growth abnormalities, premature aging, and photosensitivity. Mutation of Cockayne syndrome B (CSB) affects neuronal gene expression and differentiation, so we attempted to bypass its function by expressing downstream target genes. Intriguingly, ectopic expression of Synaptotagmin 9 (SYT9), a key component of the machinery controlling neurotrophin release, bypasses the need for CSB in neuritogenesis. Importantly, brain-derived neurotrophic factor (BDNF), a neurotrophin implicated in neuronal differentiation and synaptic modulation, and pharmacological mimics such as 7,8-dihydroxyflavone and amitriptyline can compensate for CSB deficiency in cell models of neuronal differentiation as well. SYT9 and BDNF are downregulated in CS patient brain tissue, further indicating that sub-optimal neurotrophin signaling underlies neurological defects in CS. In addition to shedding light on cellular mechanisms underlying CS and pointing to future avenues for pharmacological intervention, these data suggest an important role for SYT9 in neuronal differentiation.

During metastatic colonization, tumor cells must establish a favorable microenvironment or niche that will sustain their growth. However, both the temporal and molecular details of this process remain poorly understood. Here, we found that metastatic initiating cells (MICs) exhibit a high capacity for lung fibroblast activation as a result of Thrombospondin 2 (THBS2) expression. Importantly, inhibiting the mesenchymal phenotype of MICs by blocking the epithelial-to-mesenchymal transition (EMT)-associated kinase AXL reduces THBS2 secretion, niche-activating ability, and, consequently, metastatic competence. Subsequently, disseminated metastatic cells revert to an AXL-negative, more epithelial phenotype to proliferate and decrease the phosphorylation levels of TGF-β-dependent SMAD2-3 in favor of BMP/SMAD1-5 signaling. Remarkably, newly activated fibroblasts promote this transition. In summary, our data reveal a crosstalk between cancer cells and their microenvironment whereby the EMT status initially triggers and then is regulated by niche activation during metastatic colonization.

Damage-associated molecular patterns (DAMPs) are molecules exposed or released by dead cells that trigger or modulate immunity and tissue repair. In vertebrates, the cytoskeletal component F-actin is a DAMP specifically recognised by DNGR-1, an innate immune receptor. Previously we suggested that actin is also a DAMP in Drosophila melanogaster by inducing STAT-dependent genes (Srinivasan et al., 2016). Here, we revise that conclusion and report that α-actinin is far more potent than actin at inducing the same STAT response and can be found in trace amounts in actin preparations. Recombinant expression of actin or α-actinin in bacteria demonstrated that only α-actinin could drive the expression of STAT target genes in Drosophila. The response to injected α-actinin required the same signalling cascade that we had identified in our previous work using actin preparations. Taken together, these data indicate that α-actinin rather than actin drives STAT activation when injected into Drosophila.

The best characterized signaling pathway downstream of transforming growth factor β (TGF-β) is through SMAD2 and SMAD3. However, TGF-β also induces phosphorylation of SMAD1 and SMAD5, but the mechanism of this phosphorylation and its functional relevance is not known. Here, we show that TGF-β-induced SMAD1/5 phosphorylation requires members of two classes of type I receptor, TGFBR1 and ACVR1, and establish a new paradigm for receptor activation where TGFBR1 phosphorylates and activates ACVR1, which phosphorylates SMAD1/5. We demonstrate the biological significance of this pathway by showing that approximately a quarter of the TGF-β-induced transcriptome depends on SMAD1/5 signaling, with major early transcriptional targets being the ID genes. Finally, we show that TGF-β-induced epithelial-to-mesenchymal transition requires signaling via both the SMAD3 and SMAD1/5 pathways, with SMAD1/5 signaling being essential to induce ID1. Therefore, combinatorial signaling via both SMAD pathways is essential for the full TGF-β-induced transcriptional program and physiological responses.

Damage-associated molecular patterns (DAMPs) are molecules released by dead cells that trigger sterile inflammation and, in vertebrates, adaptive immunity. Actin is a DAMP detected in mammals by the receptor, DNGR-1, expressed by dendritic cells (DCs). DNGR-1 is phosphorylated by Src-family kinases and recruits the tyrosine kinase Syk to promote DC cross-presentation of dead cell-associated antigens. Here we report that actin is also a DAMP in invertebrates that lack DCs and adaptive immunity. Administration of actin to Drosophila melanogaster triggers a response characterised by selective induction of STAT target genes in the fat body through the cytokine Upd3 and its JAK/STAT-coupled receptor, Domeless. Notably, this response requires signalling via Shark, the Drosophila orthologue of Syk, and Src42A, a Drosophila Src-family kinase, and is dependent on Nox activity. Thus, extracellular actin detection via a Src-family kinase-dependent cascade is an ancient means of detecting cell injury that precedes the evolution of adaptive immunity.

DUSP6/MKP3 is a dual-specific phosphatase that regulates extracellular regulated kinase ERK1/2 and ERK5 activity, with an increasingly recognized role as tumor suppressor. In silico studies from Gene expression Omnibus (GEO) and Cancer Genome atlas (TCGA) databases reveal poor prognosis in those Non-small cell lung cancer (NSCLC) patients with low expression levels of DUSP6. In agreement with these data, here we show that DUSP6 plays a major role in the regulation of cell migration, motility and tumor growth. We have found upregulation in the expression of several genes involved in epithelial to mesenchymal transition (EMT) in NSCLC-DUSP6 depleted cells. Data obtained in RNA-seq studies carried out in DUSP6 depleted cells identified EGFR, TGF-β and WNT signaling pathways and several genes such as VAV3, RUNXR2, LEF1, FGFR2 whose expression is upregulated in these cells and therefore affecting cellular functions such as integrin mediated cell adhesion, focal adhesion and motility. Furthermore, EGF signaling pathway is activated via ERK5 and not ERK1/2 and TGF-β via SMAD2/3 in DUSP6 depleted cells. In summary DUSP6 is a tumor suppressor in NSCLC and re-establishment of its expression may be a potential strategy to revert poor outcome in NSCLC patients.

Strong FOXP1 protein expression is a poor risk factor in diffuse large B-cell lymphoma and has been linked to an activated B-cell-like subtype, which preferentially expresses short FOXP1 (FOXP1S) proteins. However, both short isoform generation and function are incompletely understood. Here we prove by mass spectrometry and N-terminal antibody staining that FOXP1S proteins in activated B-cell-like diffuse large B-cell lymphoma are N-terminally truncated. Furthermore, a rare strongly FOXP1-expressing population of normal germinal center B cells lacking the N-terminus of the regular long protein (FOXP1L) was identified. Exon-targeted silencing and transcript analyses identified three alternate 5′ non-coding exons [FOXP1-Ex6b(s), FOXP1-Ex7b and FOXP1-Ex7c], downstream of at least two predicted promoters, giving rise to FOXP1S proteins. These were differentially controlled by B-cell activation and methylation, conserved in murine lymphoma cells, and significantly correlated with FOXP1S protein expression in primary diffuse large B-cell lymphoma samples. Alternatively spliced isoforms lacking exon 9 (e.g. isoform 3) did not encode FOXP1S, and an alternate long human FOXP1 protein (FOXP1AL) likely generated from a FOXP1-Ex6b(L) transcript was detected. The ratio of FOXP1L:FOXP1S isoforms correlated with differential expression of plasmacytic differentiation markers in U-2932 subpopulations, and altering this ratio was sufficient to modulate CD19 expression in diffuse large B-cell lymphoma cell lines. Thus, the activity of multiple alternate FOXP1 promoters to produce multiple protein isoforms is likely to regulate B-cell maturation.

Kidins220/ARMS is a transmembrane protein playing a crucial role in neuronal and cardiovascular development. Kidins220/ARMS is a downstream target of neurotrophin receptors and interacts with several signalling and trafficking factors. Through computational modelling, we found two potential sites for alternative splicing of Kidins220/ARMS. The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33. Here we describe the conserved occurrence of several Kidins220/ARMS splice isoforms at RNA and protein levels. Kidins220/ARMS splice isoforms display spatio-temporal regulation during development with distinct patterns in different neuronal populations. Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript. Remarkably, alternative terminal exon splicing generates Kidins220/ARMS variants with distinct cellular localisation: Kidins220/ARMS containing exon 32 is targeted to the plasma membrane and neurite tips, whereas Kidins220/ARMS without exon 33 mainly clusters the full-length protein in a perinuclear intracellular compartment in PC12 cells and primary neurons, leading to a change in neurotrophin receptor expression. Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development.

The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells.