A RIPK1-specific PROTAC degrader achieves potent antitumor activity by enhancing immunogenic cell death.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies.

The pruritus- and TH2-associated cytokine IL-31 promotes growth of sensory nerves.

BACKGROUND: Pruritus is a cardinal symptom of atopic dermatitis, and an increased cutaneous sensory network is thought to contribute to pruritus. Although the immune cell-IL-31-neuron axis has been implicated in severe pruritus during atopic skin inflammation, IL-31's neuropoietic potential remains elusive. OBJECTIVE: We sought to analyze the IL-31-related transcriptome in sensory neurons and to investigate whether IL-31 promotes sensory nerve fiber outgrowth. METHODS: In vitro primary sensory neuron culture systems were subjected to whole-transcriptome sequencing, ingenuity pathway analysis, immunofluorescence, and nerve elongation, as well as branching assays after IL-31 stimulation. In vivo we investigated the cutaneous sensory neuronal network in wild-type, Il31-transgenic, and IL-31 pump-equipped mice. RESULTS: Transgenic Il31 overexpression and subcutaneously delivered IL-31 induced an increase in the cutaneous nerve fiber density in lesional skin in vivo. Transcriptional profiling of IL-31-activated dorsal root ganglia neurons revealed enrichment for genes promoting nervous system development and neuronal outgrowth and negatively regulating cell death. Moreover, the growth cones of primary small-diameter dorsal root ganglia neurons showed abundant IL-31 receptor α expression. Indeed, IL-31 selectively promoted nerve fiber extension only in small-diameter neurons. Signal transducer and activator of transcription 3 phosphorylation mediated IL-31-induced neuronal outgrowth, and pharmacologic inhibition of signal transducer and activator of transcription 3 completely abolished this effect. In contrast, transient receptor potential cation channel vanilloid subtype 1 channels were dispensable for IL-31-induced neuronal sprouting. CONCLUSIONS: The pruritus- and TH2-associated novel cytokine IL-31 induces a distinct transcriptional program in sensory neurons, leading to nerve elongation and branching both in vitro and in vivo. This finding might help us understand the clinical observation that patients with atopic dermatitis experience increased sensitivity to minimal stimuli inducing sustained itch.

SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response.

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population.

Identifying high-confidence capture Hi-C interactions using CHiCANE.

The ability to identify regulatory interactions that mediate gene expression changes through distal elements, such as risk loci, is transforming our understanding of how genomes are spatially organized and regulated. Capture Hi-C (CHi-C) is a powerful tool to delineate such regulatory interactions. However, primary analysis and downstream interpretation of CHi-C profiles remains challenging and relies on disparate tools with ad-hoc input/output formats and specific assumptions for statistical modeling. Here we present a data processing and interaction calling toolkit (CHiCANE), specialized for the analysis and meaningful interpretation of CHi-C assays. In this protocol, we demonstrate applications of CHiCANE to region capture Hi-C (rCHi-C) and promoter capture Hi-C (pCHi-C) libraries, followed by quality assessment of interaction peaks, as well as downstream analysis specific to rCHi-C and pCHi-C to aid functional interpretation. For a typical rCHi-C/pCHi-C dataset this protocol takes up to 3 d for users with a moderate understanding of R programming and statistical concepts, although this is dependent on dataset size and compute power available. CHiCANE is freely available at https://cran.r-project.org/web/packages/chicane .

3D Functional Genomics Screens Identify CREBBP as a Targetable Driver in Aggressive Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo-like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population. SIGNIFICANCE: This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors.

SOX11 promotes epithelial/mesenchymal hybrid state and alters tropism of invasive breast cancer cells.

SOX11 is an embryonic mammary epithelial marker that is normally silenced prior to birth. High SOX11 levels in breast tumours are significantly associated with distant metastasis and poor outcome in breast cancer patients. Here, we show that SOX11 confers distinct features to ER-negative DCIS.com breast cancer cells, leading to populations enriched with highly plastic hybrid epithelial/mesenchymal cells, which display invasive features and alterations in metastatic tropism when xenografted into mice. We found that SOX11+DCIS tumour cells metastasize to brain and bone at greater frequency and to lungs at lower frequency compared to cells with lower SOX11 levels. High levels of SOX11 leads to the expression of markers associated with mesenchymal state and embryonic cellular phenotypes. Our results suggest that SOX11 may be a potential biomarker for breast tumours with elevated risk of developing metastases and may require more aggressive therapies.

Combining BRAF inhibition with oncolytic herpes simplex virus enhances the immune-mediated antitumor therapy of BRAF-mutant thyroid cancer.

BACKGROUND: The aggressive clinical behavior of poorly differentiated and anaplastic thyroid cancers (PDTC and ATC) has proven challenging to treat, and survival beyond a few months from diagnosis is rare. Although 30%-60% of these tumors contain mutations in the BRAF gene, inhibitors designed specifically to target oncogenic BRAF have shown limited and only short-lasting therapeutic benefits as single agents, thus highlighting the need for improved treatment strategies, including novel combinations. METHODS: Using a BRAFV600E-driven mouse model of ATC, we investigated the therapeutic efficacy of the combination of BRAF inhibition and oncolytic herpes simplex virus (oHSV). Analyses of samples from tumor-bearing mice were performed to immunologically characterize the effects of different treatments. These immune data were used to inform the incorporation of immune checkpoint inhibitors into triple combination therapies. RESULTS: We characterized the immune landscape in vivo following BRAF inhibitor treatment and detected only modest immune changes. We, therefore, hypothesized that the addition of oncolytic virotherapy to BRAF inhibition in thyroid cancer would create a more favorable tumor immune microenvironment, boost the inflammatory status of tumors and improve BRAF inhibitor therapy. First, we showed that thyroid cancer cells were susceptible to infection with oHSV and that this process was associated with activation of the immune tumor microenvironment in vivo. Next, we showed improved therapeutic responses when combining oHSV and BRAF inhibition in vivo, although no synergistic effects were seen in vitro, further confirming that the dominant effect of oHSV in this context was likely immune-mediated. Importantly, both gene and protein expression data revealed an increase in activation of T cells and natural killer (NK) cells in the tumor in combination-treated samples. The benefit of combination oHSV and BRAF inhibitor therapy was abrogated when T cells or NK cells were depleted in vivo. In addition, we showed upregulation of PD-L1 and CTLA-4 following combined treatment and demonstrated that blockade of the PD-1/PD-L1 axis or CTLA-4 further improved combination therapy. CONCLUSIONS: The combination of oHSV and BRAF inhibition significantly improved survival in a mouse model of ATC by enhancing immune-mediated antitumor effects, and triple combination therapies, including either PD-1 or CTLA-4 blockade, further improved therapy.

PARP inhibition enhances tumor cell-intrinsic immunity in ERCC1-deficient non-small cell lung cancer.

The cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway detects cytosolic DNA to activate innate immune responses. Poly(ADP-ribose) polymerase inhibitors (PARPi) selectively target cancer cells with DNA repair deficiencies such as those caused by BRCA1 mutations or ERCC1 defects. Using isogenic cell lines and patient-derived samples, we showed that ERCC1-defective non-small cell lung cancer (NSCLC) cells exhibit an enhanced type I IFN transcriptomic signature and that low ERCC1 expression correlates with increased lymphocytic infiltration. We demonstrated that clinical PARPi, including olaparib and rucaparib, have cell-autonomous immunomodulatory properties in ERCC1-defective NSCLC and BRCA1-defective triple-negative breast cancer (TNBC) cells. Mechanistically, PARPi generated cytoplasmic chromatin fragments with characteristics of micronuclei; these were found to activate cGAS/STING, downstream type I IFN signaling, and CCL5 secretion. Importantly, these effects were suppressed in PARP1-null TNBC cells, suggesting that this phenotype resulted from an on-target effect of PARPi on PARP1. PARPi also potentiated IFN-γ-induced PD-L1 expression in NSCLC cell lines and in fresh patient tumor cells; this effect was enhanced in ERCC1-deficient contexts. Our data provide a preclinical rationale for using PARPi as immunomodulatory agents in appropriately molecularly selected populations.

Microbe-host interplay in atopic dermatitis and psoriasis.

Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here we perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analysed by 16S amplicon or whole genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. We find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and associated with a disease relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism and immune activation. In contrast, psoriasis is characterized by co-occurring communities of microbes with weak associations with disease related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis.

Anti-Folate Receptor-α IgE but not IgG Recruits Macrophages to Attack Tumors via TNFα/MCP-1 Signaling.

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen-specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcε receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα+ and CD80+ macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127-41. ©2017 AACR.

Detection of composite communities in multiplex biological networks.

The detection of community structure is a widely accepted means of investigating the principles governing biological systems. Recent efforts are exploring ways in which multiple data sources can be integrated to generate a more comprehensive model of cellular interactions, leading to the detection of more biologically relevant communities. In this work, we propose a mathematical programming model to cluster multiplex biological networks, i.e. multiple network slices, each with a different interaction type, to determine a single representative partition of composite communities. Our method, known as SimMod, is evaluated through its application to yeast networks of physical, genetic and co-expression interactions. A comparative analysis involving partitions of the individual networks, partitions of aggregated networks and partitions generated by similar methods from the literature highlights the ability of SimMod to identify functionally enriched modules. It is further shown that SimMod offers enhanced results when compared to existing approaches without the need to train on known cellular interactions.

The PRINTS database: a fine-grained protein sequence annotation and analysis resource--its status in 2012.

The PRINTS database, now in its 21st year, houses a collection of diagnostic protein family 'fingerprints'. Fingerprints are groups of conserved motifs, evident in multiple sequence alignments, whose unique inter-relationships provide distinctive signatures for particular protein families and structural/functional domains. As such, they may be used to assign uncharacterized sequences to known families, and hence to infer tentative functional, structural and/or evolutionary relationships. The February 2012 release (version 42.0) includes 2156 fingerprints, encoding 12 444 individual motifs, covering a range of globular and membrane proteins, modular polypeptides and so on. Here, we report the current status of the database, and introduce a number of recent developments that help both to render a variety of our annotation and analysis tools easier to use and to make them more widely available. Database URL: www.bioinf.manchester.ac.uk/dbbrowser/PRINTS/.