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Despite recent advances in cancer immunotherapy, certain tumor types, such as Glioblastomas, are highly resistant due to their tumor microenvironment disabling the anti-tumor immune response. Here we show, by applying an in-silico multidimensional model integrating spatially resolved and single-cell gene expression data of 45,615 immune cells from 12 tumor samples, that a subset of Interleukin-10-releasing HMOX1 +  myeloid cells, spatially localizing to mesenchymal-like tumor regions, drive T-cell exhaustion and thus contribute to the immunosuppressive tumor microenvironment. These findings are validated using a human ex-vivo neocortical glioblastoma model inoculated with patient derived peripheral T-cells to simulate the immune compartment. This model recapitulates the dysfunctional transformation of tumor infiltrating T-cells. Inhibition of the JAK/STAT pathway rescues T-cell functionality both in our model and in-vivo, providing further evidence of IL-10 release being an important driving force of tumor immune escape. Our results thus show that integrative modelling of single cell and spatial transcriptomics data is a valuable tool to interrogate the tumor immune microenvironment and might contribute to the development of successful immunotherapies. The tumour microenvironment counteracts immune therapy in Glioblastomas. Authors show here, using spatially resolved and single cell transcriptomics, that dysfunctional T cells are induced by a myeloid cell subset via Interleukin-10 signalling, and inhibition of the downstream JAK/STAT pathway might restore glioblastoma immune therapy responsiveness.

Mutational inactivation of VHL is the earliest genetic event in the majority of clear cell renal cell carcinomas (ccRCC), leading to accumulation of the HIF-1α and HIF-2α transcription factors. While correlative studies of human ccRCC and functional studies using human ccRCC cell lines have implicated HIF-1α as an inhibitor and HIF-2α as a promoter of aggressive tumour behaviours, their roles in tumour onset have not been functionally addressed. Herein we show using an autochthonous ccRCC model that Hif1a is essential for tumour formation whereas Hif2a deletion has only minor effects on tumour initiation and growth. Both HIF-1α and HIF-2α are required for the clear cell phenotype. Transcriptomic and proteomic analyses reveal that HIF-1α regulates glycolysis while HIF-2α regulates genes associated with lipoprotein metabolism, ribosome biogenesis and E2F and MYC transcriptional activities. HIF-2α-deficient tumours are characterised by increased antigen presentation, interferon signalling and CD8 + T cell infiltration and activation. Single copy loss of HIF1A or high levels of HIF2A mRNA expression correlate with altered immune microenvironments in human ccRCC. These studies reveal an oncogenic role of HIF-1α in ccRCC initiation and suggest that alterations in the balance of HIF-1α and HIF-2α activities can affect different aspects of ccRCC biology and disease aggressiveness. Genetic inactivation of VHL leads to stabilization of HIF-1α/HIF-2α and is associated with clear cell renal cell carcinoma (ccRCC) initiation and progression. Using an autochthonous mouse model of ccRCC with Vhl deletion, here the authors show that HIF-1α is necessary for tumor formation, while HIF-2α deletion has only a moderate effect.

Oncogenic Ras mutations occur in various leukemias. It was unclear if, besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation-related effect of KRAS contributes to the disease. Here, we identify a functional link between oncogenic Kras G12D and NLRP3 inflammasome activation in murine and human cells. Mice expressing active Kras G12D in the hematopoietic system developed myeloproliferation and cytopenia, which is reversed in Kras G12D mice lacking NLRP3 in the hematopoietic system. Therapeutic IL-1-receptor blockade or NLRP3-inhibition reduces myeloproliferation and improves hematopoiesis. Mechanistically, Kras G12D -RAC1 activation induces reactive oxygen species (ROS) production causing NLRP3 inflammasome-activation. In agreement with our observations in mice, patient-derived myeloid leukemia cells exhibit KRAS/RAC1/ROS/NLRP3/IL-1β axis activity. Our findings indicate that oncogenic KRAS not only act via its canonical oncogenic driver function, but also enhances the activation of the pro-inflammatory RAC1/ROS/NLRP3/IL-1β axis. This paves the way for a therapeutic approach based on immune modulation via NLRP3 blockade in KRAS-mutant myeloid malignancies. Oncogenic Ras mutations are common drivers in myeloid leukemia. Here, the authors show in patient cells and in mice that oncogenic K-Ras activates NLRP3 inflammasome to drive myeloproliferation, which can be reversed by genetic or pharmacologic NLRP3 blockade.

Metastasis is the major cause of cancer-associated death. Partial activation of the epithelial-to-mesenchymal transition program (partial EMT) was considered a major driver of tumour progression from initiation to metastasis. However, the role of EMT in promoting metastasis has recently been challenged, in particular concerning effects of the Snail and Twist EMT transcription factors (EMT-TFs) in pancreatic cancer. In contrast, we show here that in the same pancreatic cancer model, driven by Pdx1-cre-mediated activation of mutant Kras and p53 (KPC model), the EMT-TF Zeb1 is a key factor for the formation of precursor lesions, invasion and notably metastasis. Depletion of Zeb1 suppresses stemness, colonization capacity and in particular phenotypic/metabolic plasticity of tumour cells, probably causing the observed in vivo effects. Accordingly, we conclude that different EMT-TFs have complementary subfunctions in driving pancreatic tumour metastasis. Therapeutic strategies should consider these potential specificities of EMT-TFs to target these factors simultaneously. Adding to the recent debate on the role of epithelial–mesenchymal transition (EMT) in cancer cell invasion and metastasis, Brabletz and colleagues show that the EMT-inducing transcription factor Zeb1 drives pancreatic tumorigenesis and metastasis.

The transcription factor TCF7L2 is indispensable for intestinal tissue homeostasis where it transmits mitogenic Wnt/β-Catenin signals in stem and progenitor cells, from which intestinal tumors arise. Yet, TCF7L2 belongs to the most frequently mutated genes in colorectal cancer (CRC), and tumor-suppressive functions of TCF7L2 were proposed. This apparent paradox warrants to clarify the role of TCF7L2 in colorectal carcinogenesis. Here, we investigated TCF7L2 dependence/independence of CRC cells and the cellular and molecular consequences of TCF7L2 loss-of-function. By genome editing we achieved complete TCF7L2 inactivation in several CRC cell lines without loss of viability, showing that CRC cells have widely lost the strict requirement for TCF7L2. TCF7L2 deficiency impaired G1/S progression, reminiscent of the physiological role of TCF7L2. In addition, TCF7L2-negative cells exhibited morphological changes, enhanced migration, invasion, and collagen adhesion, albeit the severity of the phenotypic alterations manifested in a cell-line-specific fashion. To provide a molecular framework for the observed cellular changes, we performed global transcriptome profiling and identified gene-regulatory networks in which TCF7L2 positively regulates the proto-oncogene MYC, while repressing the cell cycle inhibitors CDKN2C/CDKN2D. Consistent with its function in curbing cell motility and invasion, TCF7L2 directly suppresses the pro-metastatic transcription factor RUNX2 and impinges on the expression of cell adhesion molecules. Altogether, we conclude that the proliferation-stimulating activity of TCF7L2 persists in CRC cells. In addition, TCF7L2 acts as invasion suppressor. Despite its negative impact on cell cycle progression, TCF7L2 loss-of-function may thereby increase malignancy, which could explain why TCF7L2 is mutated in a sizeable fraction of colorectal tumors.

BRAFV600E confers poor prognosis and is associated with a distinct subtype of colorectal cancer (CRC). Little is known, however, about the genetic events driving the initiation and progression of BRAFV600E mutant CRCs. Recent genetic analyses of CRCs indicate that BRAFV600E often coexists with alterations in the WNT- and p53 pathways, but their cooperation remains ill-defined. Therefore, we systematically compared small and large intestinal organoids from mice harboring conditional BraffloxV600E, Trp53LSL-R172H, and/or Apcflox/flox alleles. Using these isogenic models, we observe tissue-specific differences toward sudden BRAFV600E expression, which can be attributed to different ERK-pathway ground states in small and large intestinal crypts. BRAFV600E alone causes transient proliferation and suppresses epithelial organization, followed by organoid disintegration. Moreover, BRAFV600E induces a fetal-like dedifferentiation transcriptional program in colonic organoids, which resembles human BRAFV600E-driven CRC. Co-expression of p53R172H delays organoid disintegration, confers anchorage-independent growth, and induces invasive properties. Interestingly, p53R172H cooperates with BRAFV600E to modulate the abundance of transcripts linked to carcinogenesis, in particular within colonic organoids. Remarkably, WNT-pathway activation by Apc deletion fully protects organoids against BRAFV600E-induced disintegration and confers growth/niche factor independence. Still, Apc-deficient BRAFV600E-mutant organoids remain sensitive toward the MEK inhibitor trametinib, albeit p53R172H confers partial resistance against this clinically relevant compound. In summary, our systematic comparison of the response of small and large intestinal organoids to oncogenic alterations suggests colonic organoids to be better suited to model the human situation. In addition, our work on BRAF-, p53-, and WNT-pathway mutations provides new insights into their cooperation and for the design of targeted therapies.

Transforming growth factor beta (TGFβ) superfamily signaling is a prime inducer of epithelial-mesenchymal transitions (EMT) that foster cancer cell invasion and metastasis, a major cause of cancer-related deaths. Yet, TGFβ signaling is frequently inactivated in human tumor entities including colorectal cancer (CRC) and pancreatic adenocarcinoma (PAAD) with a high proportion of mutations incapacitating SMAD4 , which codes for a transcription factor (TF) central to canonical TGFβ and bone morphogenetic protein (BMP) signaling. Beyond its role in initiating EMT, SMAD4 was reported to crucially contribute to subsequent gene regulatory events during EMT execution. It is therefore widely assumed that SMAD4 -mutant ( SMAD4 mut ) cancer cells are unable to undergo EMT. Here, we scrutinized this notion and probed for potential SMAD4-independent EMT execution using SMAD4 mut CRC cell lines. We show that SMAD4 mut cells exhibit morphological changes, become invasive, and regulate EMT marker genes upon induction of the EMT-TF SNAIL1. Furthermore, SNAIL1-induced EMT in SMAD4 mut cells was found to be entirely independent of TGFβ/BMP receptor activity. Global assessment of the SNAIL1-dependent transcriptome confirmed the manifestation of an EMT gene regulatory program in SMAD4 mut cells highly related to established EMT signatures. Finally, analyses of human tumor transcriptomes showed that SMAD4 mutations are not underrepresented in mesenchymal tumor samples and that expression patterns of EMT-associated genes are similar in SMAD4 mut and SMAD4 wild-type ( SMAD4 wt ) cases. Altogether, our findings suggest that alternative TFs take over the gene regulatory functions of SMAD4 downstream of EMT-TFs, arguing for considerable plasticity of gene regulatory networks operating in EMT execution. Further, they establish that EMT is not categorically precluded in SMAD4 mut tumors, which is relevant for their diagnostic and therapeutic evaluation.

Cathepsin D (CTSD) is a lysosomal protease and a marker of poor prognosis in breast cancer. However, the cells responsible for this association and the function of CTSD in cancer are still incompletely understood. By using a conditional CTSD knockout mouse crossed to the transgenic MMTV-PyMT breast cancer model we demonstrate that CTSD deficiency in the mammary epithelium, but not in myeloid cells, blocked tumor development in a cell-autonomous manner. We show that lack of CTSD impaired mechanistic Target of Rapamycin Complex 1 (mTORC1) signaling and induced reversible cellular quiescence. In line, CTSD-deficient tumors started to grow with a two-month delay and quiescent Ctsd -/- tumor cells re-started proliferation upon long-term culture. This was accompanied by rewiring of oncogenic gene expression and signaling pathways, while mTORC1 signaling remained permanently disabled in CTSD-deficient cells. Together, these studies reveal a tumor cell-autonomous effect of CTSD deficiency, and establish a pivotal role of this protease in the cellular response to oncogenic stimuli. The lysosomal aspartic protease Cathepsin D (CTSD) is associated with breast cancer progression. Here the authors show that selective inactivation of CTSD in mammary epithelium delays tumor onset due to impaired mTORC1 signaling, but resumes malignant growth due to compensatory oncogenic pathways

The inflammatory response after myocardial infarction (MI) is a precisely regulated process that greatly affects subsequent remodeling. Here, we show that basophil granulocytes infiltrated infarcted murine hearts, with a peak occurring between days 3 and 7. Antibody-mediated and genetic depletion of basophils deteriorated cardiac function and resulted in enhanced scar thinning after MI. Mechanistically, we found that basophil depletion was associated with a shift from reparative Ly6Clo macrophages toward increased numbers of inflammatory Ly6Chi monocytes in the infarcted myocardium. Restoration of basophils in basophil-deficient mice by adoptive transfer reversed this proinflammatory phenotype. Cellular alterations in the absence of basophils were accompanied by lower cardiac levels of IL-4 and IL-13, two major cytokines secreted by basophils. Mice with basophil-specific IL-4/IL-13 deficiency exhibited a similarly altered myeloid response with an increased fraction of Ly6Chi monocytes and aggravated cardiac function after MI. In contrast, IL-4 induction in basophils via administration of the glycoprotein IPSE/α-1 led to improved post-MI healing. These results in mice were corroborated by the finding that initially low counts of blood basophils in patients with acute MI were associated with a worse cardiac outcome after 1 year, characterized by a larger scar size. In conclusion, we show that basophils promoted tissue repair after MI by increasing cardiac IL-4 and IL-13 levels.

Phenotypic conversion of tumor cells through epithelial-mesenchymal transition (EMT) requires massive gene expression changes. How these are brought about is not clear. Here we examined the impact of the EMT master regulator SNAIL1 on the FOXA family of transcription factors which are distinguished by their particular competence to induce chromatin reorganization for the activation of transcriptional enhancer elements. We show that the expression of SNAIL1 and FOXA genes is anticorrelated in transcriptomes of colorectal tumors and cell lines. In cellular EMT models, ectopically expressed Snail1 directly represses FOXA1 and triggers downregulation of all FOXA family members, suggesting that loss of FOXA expression promotes EMT. Indeed, cells with CRISPR/Cas9-induced FOXA-deficiency acquire mesenchymal characteristics. Furthermore, ChIP-seq data analysis of FOXA chromosomal distribution in relation to chromatin structural features which characterize distinct states of transcriptional activity, revealed preferential localization of FOXA factors to transcriptional enhancers at signature genes that distinguish epithelial from mesenchymal colon tumors. To validate the significance of this association, we investigated the impact of FOXA factors on structure and function of enhancers at the CDH1, CDX2 and EPHB3 genes. FOXA-deficiency and expression of dominant negative FOXA2 led to chromatin condensation at these enhancer elements. Site-directed mutagenesis of FOXA binding sites in reporter gene constructs and by genome-editing in situ impaired enhancer activity and completely abolished the active chromatin state of the EPHB3 enhancer. Conversely, expression of FOXA factors in cells with inactive CDX2 and EPHB3 enhancers led to chromatin opening and de novo deposition of the H3K4me1 and H3K27ac marks. These findings establish the pioneer function of FOXA factors at enhancer regions of epithelial genes and demonstrate their essential role in maintaining enhancer structure and function. Thus, by repressing FOXA family members, SNAIL1 targets transcription factors at strategically important positions in gene-regulatory hierarchies, which may facilitate transcriptional reprogramming during EMT.