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TGF-β ligands suppress growth yet can paradoxically and potently promote cancer invasion and metastasis depending on downstream pathway mutational context, such as loss of Mothers against decapentaplegic homolog 4 ( Smad4 ). Here, we characterised phenotypes and associated gene expression signatures in conditional murine intestinal adenoma with and without Smad4 . Conditional Lgr5 -CreER T2 activation in Apc fl/fl Smad4 fl/fl mice resulted in homozygote floxed alleles (Apc Δ/Δ Smad4 Δ/Δ ) and adenoma formation. The adenoma phenotype was discordant, with reduced small intestinal adenoma burden yet development of large non-metastatic caecal adenoma with nuclear localisation of phospho-Smad2/3. Derived Apc Δ/Δ Smad4 Δ/Δ adenoma organoids resisted TGF-β1 dose dependent growth arrest and cell death (IC 50 534 pM) compared to Apc Δ/Δ Smad4 +/+ (IC 50 24 pM). TGF-β1 (390 pM) altered adenoma bulk mRNA expression most significantly for Id1 low and Spp1 high in Apc Δ/Δ Smad4 Δ/Δ . Single cell RNAseq of caecal adenoma identified expansion of Lgr5 low , Pak3 high and Id1 low progenitor populations in Apc Δ/Δ Smad4 Δ/Δ . Of the 76 Smad4 and TGF-β1 dependent genes identified in Apc fl/fl Smad4 fl/fl adenoma organoids, only 7 human equivalent genes were differentially expressed in SMAD4 mutated colorectal cancer (TCGA cohorts), including ID1 low . SMAD4 low , ID1 low SPP1 high and PAK3 high all correlated with poorer survival. Murine adenoma identified Smad4 dependent gene expression signatures that require further evaluation as functional biomarker classifiers of SMAD4 mutated cancer subtypes.

Breast cancer, particularly triple-negative breast cancer (TNBC), evades the body’s immune defences, in part by cultivating an immunosuppressive tumour microenvironment. Here, we show that suppressing local steroidogenesis can augment anti-tumour immunity against TNBC. Through targeted metabolomics of steroids coupled with immunohistochemistry, we profiled the existence of immunosuppressive steroids in TNBC patient tumours and discerned the steroidogenic activity in immune-infiltrating regions. In mouse, genetic inhibition of immune cell steroidogenesis restricted TNBC tumour progression with a significant reduction in immunosuppressive components such as tumour associated macrophages. Steroidogenesis inhibition appears to bolster anti-tumour immune responses in dendritic and T cells by impeding glucocorticoid signalling. Undertaking metabolic modelling of the single-cell transcriptomics and targeted tumour-steroidomics, we pinpointed the predominant steroidogenic cells. Inhibiting steroidogenesis pharmacologically using a identified drug, posaconazole, curtailed tumour expansion in a humanised TNBC mouse model. This investigation paves the way for targeting steroidogenesis and its signalling pathways in breast cancer affected by immune-steroid maladaptation. Researchers show inhibiting immune cell steroidogenesis enhances anti-tumour immunity in triple-negative breast cancer. Posaconazole showed promise in a humanised mouse model.

Genetic variations underlying susceptibility to complex autoimmune and allergic diseases are concentrated within noncoding regulatory elements termed enhancers 1 . The functions of a large majority of disease-associated enhancers are unknown, in part owing to their distance from the genes they regulate, a lack of understanding of the cell types in which they operate, and our inability to recapitulate the biology of immune diseases in vitro. Here, using shared synteny to guide loss-of-function analysis of homologues of human enhancers in mice, we show that the prominent autoimmune and allergic disease risk locus at chromosome 11q13.5 2 – 7 contains a distal enhancer that is functional in CD4 + regulatory T (T reg ) cells and required for T reg -mediated suppression of colitis. The enhancer recruits the transcription factors STAT5 and NF-κB to mediate signal-driven expression of Lrrc32 , which encodes the protein glycoprotein A repetitions predominant (GARP). Whereas disruption of the Lrrc32 gene results in early lethality, mice lacking the enhancer are viable but lack GARP expression in Foxp3 + T reg cells, which are unable to control colitis in a cell-transfer model of the disease. In human T reg cells, the enhancer forms conformational interactions with the promoter of LRRC32 and enhancer risk variants are associated with reduced histone acetylation and GARP expression. Finally, functional fine-mapping of 11q13.5 using CRISPR-activation (CRISPRa) identifies a CRISPRa-responsive element in the vicinity of risk variant rs11236797 capable of driving GARP expression. These findings provide a mechanistic basis for association of the 11q13.5 risk locus with immune-mediated diseases and identify GARP as a potential target in their therapy. Shared synteny guides loss-of-function analysis of human enhancer homologues in mice, identifying a distal enhancer at the autoimmune and allergic disease risk locus at chromosome 11q13.5 whose function in regulatory T cells provides a mechanistic basis for its role in disease.

Genetic variations underlying susceptibility to complex autoimmune and allergic diseases are concentrated within noncoding regulatory elements termed enhancers.sup.1. The functions of a large majority of disease-associated enhancers are unknown, in part owing to their distance from the genes they regulate, a lack of understanding of the cell types in which they operate, and our inability to recapitulate the biology of immune diseases in vitro. Here, using shared synteny to guide loss-of-function analysis of homologues of human enhancers in mice, we show that the prominent autoimmune and allergic disease risk locus at chromosome 11q13.5.sup.2-7 contains a distal enhancer that is functional in CD4.sup.+ regulatory T (T.sub.reg) cells and required for T.sub.reg-mediated suppression of colitis. The enhancer recruits the transcription factors STAT5 and NF-[kappa]B to mediate signal-driven expression of Lrrc32, which encodes the protein glycoprotein A repetitions predominant (GARP). Whereas disruption of the Lrrc32 gene results in early lethality, mice lacking the enhancer are viable but lack GARP expression in Foxp3.sup.+ T.sub.reg cells, which are unable to control colitis in a cell-transfer model of the disease. In human T.sub.reg cells, the enhancer forms conformational interactions with the promoter of LRRC32 and enhancer risk variants are associated with reduced histone acetylation and GARP expression. Finally, functional fine-mapping of 11q13.5 using CRISPR-activation (CRISPRa) identifies a CRISPRa-responsive element in the vicinity of risk variant rs11236797 capable of driving GARP expression. These findings provide a mechanistic basis for association of the 11q13.5 risk locus with immune-mediated diseases and identify GARP as a potential target in their therapy.

Whereas effector CD4 + and CD8 + T cells promote immune activation and can drive clearance of infections and cancer, CD4 + regulatory T (T reg ) cells suppress their function, contributing to both immune homeostasis and cancer immunosuppression. The transcription factor BACH2 functions as a pervasive regulator of T cell differentiation, promoting development of CD4 + T reg cells and suppressing the effector functions of multiple effector T cell (T eff ) lineages. Here, we report the development of a stable cell-based bioluminescence assay of the transcription factor activity of BACH2. Tetracycline-inducible BACH2 expression resulted in suppression of phorbol 12-myristate 13-acetate (PMA)/ionomycin-driven activation of a luciferase reporter containing BACH2/AP-1 target sequences from the mouse Ifng  + 18k enhancer. BACH2 expression repressed the luciferase signal in a dose-dependent manner but this activity was abolished at high levels of AP-1 signalling, suggesting contextual regulation of AP-1 driven gene expression by BACH2. Finally, using the reporter assay developed, we find that the histone deacetylase 3 (HDAC3)-selective inhibitor, RGFP966, inhibits BACH2-mediated repression of signal-driven luciferase expression. In addition to enabling mechanistic studies, this cell-based reporter may enable identification of small molecule agonists or antagonists of BACH2 function for drug development.

BackgroundCo-targeting of PD1 and LAG3 on T cells has shown superior efficacy vs. PD1 monotherapy and is now an FDA-approved combinatorial regimen in melanoma. We recently reported that PD1/LAG3 synergized to drive CD8 T cell exhaustion (TEX) in cancer, and features of the CD8 TEXprogram are well-established. However, recent evidence suggests that intratumor CD4 T conventional cells (Tconv) exhibit an exhausted-like profile that correlates with worse outcomes, although the defining features, significance, and regulatory mechanisms of CD4 TEX remain unclear. Further, little is known regarding mechanistic impact of PD1/LAG3 on CD4 Tconv. Given the dynamic ability of CD4 Tconv to exert direct cytotoxicity and also provide help, better understanding of CD4 TEX may be of particular importance for improving cancer immunotherapy. We hypothesize that PD1/LAG3 exert differential yet synergistic effects on CD4 Tconv to drive their exhaustion and impaired anti-tumor responses.MethodsWe utilized B16-F10 murine melanoma together with adoptive transfers of T cell receptor transgenic CD4 T cells specific to tyrosinase-related protein-1 (Trp1), a melanocyte differentiation antigen expressed by both normal melanocytes and melanoma, crossed to Pdcd1 -/- and/or Lag3 -/- mice. Trp1 CD4 T cells were adoptively transferred into tumor-bearing WT C57BL/6 mice. CD8 depletion antibodies were administered to assess direct vs. helper roles. WT tumor-bearing mice were also given anti-PD1/anti-LAG3 therapy and CD4 Tconv phenotype assessed.ResultsWe found that tumor-infiltrating Trp1 CD4 T cells upregulated PD1, LAG3, TOX, and CD39, suggestive of TEX phenotype. Interestingly, as little as 10,000 Trp1Pdcd1 -/- CD4 T cells were sufficient to clear established tumors. In the absence of CD8 T cells, higher doses (up to 200,000) of Trp1Pdcd1 -/- CD4 T cells had a cytotoxic effect on tumor growth, whereas the effects were minimized when fewer cells were transferred, suggesting that CD8 T cells may also be required for Trp1 CD4 anti-tumor efficacy at suboptimal doses. Further, PD1/LAG3 therapeutic blockade appears to modulate CD4 Tconv phenotype.ConclusionsOur preliminary data suggests that PD1 may play a crucial role in restraining antigen-specific CD4 responses. Further, tumor-specific CD4 T cells may leverage both direct cytotoxicity and CD8 help for anti-tumor effects. Ongoing work is focused on assessing effects of CD4 T cell-specific PD1/LAG3 loss on tumor growth and cytotoxic/helper functions using Trp1Pdcd1 -/- /Lag3 -/- mice as well as CRISPR-mediated deletion. Future plans include quad-transfers to study cell-intrinsic effects of PD1/LAG3 across the same microenvironment. Collectively, these studies aim to enhance mechanistic understanding of CD4+ TEX and inform novel immunotherapeutic strategies.

Genetic variations underlying susceptibility to complex autoimmune and allergic diseases are concentrated within noncoding regulatory elements termed enhancers . The functions of a large majority of disease-associated enhancers are unknown, in part owing to their distance from the genes they regulate, a lack of understanding of the cell types in which they operate, and our inability to recapitulate the biology of immune diseases in vitro. Here, using shared synteny to guide loss-of-function analysis of homologues of human enhancers in mice, we show that the prominent autoimmune and allergic disease risk locus at chromosome 11q13.5 contains a distal enhancer that is functional in CD4 regulatory T (T ) cells and required for T -mediated suppression of colitis. The enhancer recruits the transcription factors STAT5 and NF-κB to mediate signal-driven expression of Lrrc32, which encodes the protein glycoprotein A repetitions predominant (GARP). Whereas disruption of the Lrrc32 gene results in early lethality, mice lacking the enhancer are viable but lack GARP expression in Foxp3 T cells, which are unable to control colitis in a cell-transfer model of the disease. In human T cells, the enhancer forms conformational interactions with the promoter of LRRC32 and enhancer risk variants are associated with reduced histone acetylation and GARP expression. Finally, functional fine-mapping of 11q13.5 using CRISPR-activation (CRISPRa) identifies a CRISPRa-responsive element in the vicinity of risk variant rs11236797 capable of driving GARP expression. These findings provide a mechanistic basis for association of the 11q13.5 risk locus with immune-mediated diseases and identify GARP as a potential target in their therapy.

TGF-β ligand activation suppresses cell growth yet can paradoxically and potently promote cancer invasion and metastasis depending on downstream pathway mutational context. Here, we evaluated the basis of this observation in conditional murine intestinal adenoma models with and without loss of Mothers against decapentaplegic homolog 4 (Smad4), with the aim of identifying TGF-β-BMP-SMAD4 pathway dependent gene expression biomarkers for translational application. Conditional Lgr5-CreERT2 activation in Apcfl/flSmad4fl/flresulted in adenoma formation with recombined homozygote floxed alleles (ApcΔ/ΔSmad4Δ/Δ). The adenoma phenotype was discordant, with a reduced small intestinal adenoma burden yet development of large non-metastatic caecal adenoma with nuclear localisation of phospho-Smad2/3. Derived ApcΔ/ΔSmad4Δ/Δ adenoma organoids resisted TGF-β1 dose dependent growth arrest and cell death (IC50 534pM) compared to ApcΔ/ΔSmad4+/+ (IC 24pM). TGF-β1 (390pM) modified adenoma mRNA expression (bulk RNA-Seq) most significantly for Id1low and Spp1high in ApcΔ/ΔSmad4Δ/Δ. Single cell RNAseq of caecal adenoma identified expansion of Lgr5low, Pak3high and Id1low progenitor populations in ApcΔ/ΔSmad4Δ/Δ. Of the 76 Smad4 and TGF-β1 dependent genes identified in adenoma organoids, 7 human equivalent genes were also significantly differentially expressed in colorectal cancer, including ID1low, SPP1high and PAK3high that also correlated with poorer survival (TCGA cohorts). Murine conditional models identified Smad4 loss of function mRNA expression biomarkers that require further evaluation as functional classifiers of colorectal cancer subtypes.

Immune checkpoint inhibitors (ICI), such as anti-PD-1, have revolutionized cancer treatment, but they are only effective for a minority of patients. The gut microbiome plays a crucial role in modulating immunotherapy treatment responses, and previous studies correlated lipopolysaccharide (LPS)-producing gut microbes with poorer prognosis. However, LPS from diverse bacterial species have activities ranging from immunostimulatory to inhibitory. By functionally analyzing fecal metagenomes from 112 melanoma patients prior to anti-PD-1 therapy, we found that a subset of LPS-producing bacteria encoding immunostimulatory hexa-acylated LPS was enriched in the microbiomes of clinical responders. We confirmed robust activation of the NF-kB pathway by hexa-acylated LPS in vitro, and this activation was significantly inhibited by penta-acylated LPS in a dose-dependent manner. Importantly, oral administration of hexa-acylated LPS augmented anti-PD-1-mediated anti-tumor immunity in an in vivo mouse model of cancer immunotherapy. Microbiome hexa-acylated LPS may therefore represent an accessible predictor and potential enhancer of clinical anti- PD-1 immunotherapy responses. Functional rather than taxonomic profiling of patient gut microbiomes reveals hexa-acylated LPS as a novel biomarker of responsiveness and a targetable pathway for enhancing responses to anti-PD-1, informing future studies and current patient treatment.