LRRC15+ myofibroblasts dictate the stromal setpoint to suppress tumour immunity.

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)1-3. However, the molecular signals that underlie the development of LRRC15+ cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFß receptor type 2 signalling in healthy dermatopontin+ universal fibroblasts is essential for the development of cancer-associated LRRC15+ myofibroblasts. This axis also predominantly drives fibroblast lineage diversity in human cancers. Using newly developed Lrrc15-diphtheria toxin receptor knock-in mice to selectively deplete LRRC15+ CAFs, we show that depletion of this population markedly reduces the total tumour fibroblast content. Moreover, the CAF composition is recalibrated towards universal fibroblasts. This relieves direct suppression of tumour-infiltrating CD8+ T cells to enhance their effector function and augments tumour regression in response to anti-PDL1 immune checkpoint blockade. Collectively, these findings demonstrate that TGFß-dependent LRRC15+ CAFs dictate the tumour-fibroblast setpoint to promote tumour growth. These cells also directly suppress CD8+ T cell function and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by reducing the population of pro-disease LRRC15+ myofibroblasts may improve patient survival and response to immunotherapy.

Diffusion tensor MRI is sensitive to fibrotic injury in a mouse model of oxalate-induced chronic kidney disease.

Chronic kidney disease (CKD) is characterized by inflammation and fibrosis in the kidney. Renal biopsies and estimated glomerular filtration rate (eGFR) remain the standard of care, but these endpoints have limitations in detecting the stage, progression, and spatial distribution of fibrotic pathology in the kidney. MRI diffusion tensor imaging (DTI) has emerged as a promising noninvasive technology to evaluate renal fibrosis in vivo both in clinical and preclinical studies. However, these imaging studies have not systematically identified fibrosis particularly deeper in the kidney where biopsy sampling is limited, or completed an extensive analysis of whole organ histology, blood biomarkers, and gene expression to evaluate the relative strengths and weaknesses of MRI for evaluating renal fibrosis. In this study, we performed DTI in the sodium oxalate mouse model of CKD. The DTI parameters fractional anisotropy, apparent diffusion coefficient, and axial diffusivity were compared between the control and oxalate groups with region of interest (ROI) analysis to determine changes in the cortex and medulla. In addition, voxel-based analysis (VBA) was implemented to systematically identify local regions of injury over the whole kidney. DTI parameters were found to be significantly different in the medulla by both ROI analysis and VBA, which also spatially matched with collagen III immunohistochemistry (IHC). The DTI parameters in this medullary region exhibited moderate to strong correlations with histology, blood biomarkers, hydroxyproline, and gene expression. Our results thus highlight the sensitivity of DTI to the heterogeneity of renal fibrosis and importance of whole kidney noninvasive imaging.NEW & NOTEWORTHY Chronic kidney disease (CKD) can be characterized by inflammation and fibrosis of the kidney. Although standard of care methods have been limited in scope, safety, and spatial distribution, MRI diffusion tensor imaging (DTI) has emerged as a promising noninvasive technology to evaluate renal fibrosis in vivo. In this study, we performed DTI in an oxalate mouse model of CKD to systematically identify local kidney injury. DTI parameters strongly correlated with histology, blood biomarkers, hydroxyproline, and gene expression.

Ubiquitin ligase COP1 coordinates transcriptional programs that control cell type specification in the developing mouse brain.

The E3 ubiquitin ligase CRL4COP1/DET1 is active in the absence of ERK signaling, modifying the transcription factors ETV1, ETV4, ETV5, and c-JUN with polyubiquitin that targets them for proteasomal degradation. Here we show that this posttranslational regulatory mechanism is active in neurons, with ETV5 and c-JUN accumulating within minutes of ERK activation. Mice with constitutive photomorphogenesis 1 (Cop1) deleted in neural stem cells showed abnormally elevated expression of ETV1, ETV4, ETV5, and c-JUN in the developing brain and spinal cord. Expression of c-JUN target genes Vimentin and Gfap was increased, whereas ETV5 and c-JUN both contributed to an expanded number of cells expressing genes associated with gliogenesis, including Olig1, Olig2, and Sox10. The mice had subtle morphological abnormalities in the cerebral cortex, hippocampus, and cerebellum by embryonic day 18 and died soon after birth. Elevated c-JUN, ETV5, and ETV1 contributed to the perinatal lethality, as several Cop1-deficient mice also lacking c-Jun and Etv5, or lacking Etv5 and heterozygous for Etv1, were viable.

Canonical hedgehog signaling augments tumor angiogenesis by induction of VEGF-A in stromal perivascular cells.

Hedgehog (Hh) signaling is critical to the patterning and development of a variety of organ systems, and both ligand-dependent and ligand-independent Hh pathway activation are known to promote tumorigenesis. Recent studies have shown that in tumors promoted by Hh ligands, activation occurs within the stromal microenvironment. Testing whether ligand-driven Hh signaling promotes tumor angiogenesis, we found that Hh antagonism reduced the vascular density of Hh-producing LS180 and SW480 xenografts. In addition, ectopic expression of sonic hedgehog in low-Hh-expressing DLD-1 xenografts increased tumor vascular density, augmented angiogenesis, and was associated with canonical Hh signaling within perivascular tumor stromal cells. To better understand the molecular mechanisms underlying Hh-mediated tumor angiogenesis, we established an Hh-sensitive angiogenesis coculture assay and found that fibroblast cell lines derived from a variety of human tissues were Hh responsive and promoted angiogenesis in vitro through a secreted paracrine signal(s). Affymetrix array analyses of cultured fibroblasts identified VEGF-A, hepatocyte growth factor, and PDGF-C as candidate secreted proangiogenic factors induced by Hh stimulation. Expression studies of xenografts and angiogenesis assays using combinations of Hh and VEGF-A inhibitors showed that it is primarily Hh-induced VEGF-A that promotes angiogenesis in vitro and augments tumor-derived VEGF to promote angiogenesis in vivo.

In situ validation of an intestinal stem cell signature in colorectal cancer.

OBJECTIVE: Wnt/Tcf, Lgr5, Ascl2 and/or Bmi1 signalling is believed to define the mouse intestinal stem cell niche(s) from which adenomas arise. The aim of this study was to determine the relevance of these putative intestinal stem cell markers to human colorectal cancer. DESIGN: 19 putative intestinal stem cell markers, including Ascl2 and Lgr5, were identified from published data and an evaluation of a human colorectal gene expression database. Associations between these genes were assessed by isotopic in situ hybridisation (ISH) in 57 colorectal adenocarcinomas. Multiplex fluorescent ISH and chromogenic non-isotopic ISH were performed to confirm expression patterns. The prognostic significance of Lgr5 was assessed in 891 colorectal adenocarcinomas. RESULTS: Ascl2 and Lgr5 were expressed in 85% and 74% of cancers respectively, and expression was positively correlated (p=0.003). Expression of Bmi1 was observed in 47% of cancers but was very weak in 98% of cases with expression. Both Ascl2 and/or Lgr5 were positively correlated with the majority of genes in the signature but neither was correlated with Cdk6, Gpx2, Olfm4 or Tnfrsf19. Lgr5 did not have prognostic significance. CONCLUSION: These data suggest that 74-85% of colorectal cancers express a Lgr5/Ascl2 associated signature and support the hypothesis that they derive from Lgr5(+)/Ascl2(+) crypt stem cells, not Bmi1(+) stem cells. However, Olfm4 was not found to be a useful marker of Lgr5(+) cells in normal colon or tumours. In this large series, Lgr5 expression is not associated with increased tumour aggressiveness, as might be expected from a cancer stem cell marker.

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis.

The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.

Selective PROTAC-mediated degradation of SMARCA2 is efficacious in SMARCA4 mutant cancers.

The mammalian SWItch/Sucrose Non-Fermentable (SWI/SNF) helicase SMARCA4 is frequently mutated in cancer and inactivation results in a cellular dependence on its paralog, SMARCA2, thus making SMARCA2 an attractive synthetic lethal target. However, published data indicates that achieving a high degree of selective SMARCA2 inhibition is likely essential to afford an acceptable therapeutic index, and realizing this objective is challenging due to the homology with the SMARCA4 paralog. Herein we report the discovery of a potent and selective SMARCA2 proteolysis-targeting chimera molecule (PROTAC), A947. Selective SMARCA2 degradation is achieved in the absence of selective SMARCA2/4 PROTAC binding and translates to potent in vitro growth inhibition and in vivo efficacy in SMARCA4 mutant models, compared to wild type models. Global ubiquitin mapping and proteome profiling reveal no unexpected off-target degradation related to A947 treatment. Our study thus highlights the ability to transform a non-selective SMARCA2/4-binding ligand into a selective and efficacious in vivo SMARCA2-targeting PROTAC, and thereby provides a potential new therapeutic opportunity for patients whose tumors contain SMARCA4 mutations.

The peptide symporter SLC15a4 is essential for the development of systemic lupus erythematosus in murine models.

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease representing a serious unmet medical need. The disease is associated with the loss of self-tolerance and exaggerated B cell activation, resulting in autoantibody production and the formation of immune complexes that accumulate in the kidney, causing glomerulonephritis. TLR7, an important mediator of the innate immune response, drives the expression of type-1 interferon (IFN), which leads to expression of type-1 IFN induced genes and aggravates lupus pathology. Because the lysosomal peptide symporter slc15a4 is critically required for type-1 interferon production by pDC, and for certain B cell functions in response to TLR7 and TLR9 signals, we considered it as a potential target for pharmacological intervention in SLE. We deleted the slc15a4 gene in C57BL/6, NZB, and NZW mice and found that pristane-challenged slc15a4-/- mice in the C57BL/6 background and lupus prone slc15a4-/- NZB/W F1 mice were both completely protected from lupus like disease. In the NZB/W F1 model, protection persisted even when disease development was accelerated with an adenovirus encoding IFNα, emphasizing a broad role of slc15a4 in disease initiation. Our results establish a non-redundant function of slc15a4 in regulating both innate and adaptive components of the immune response in SLE pathobiology and suggest that it may be an attractive drug target.

Anti-LYPD1/CD3 T-Cell-Dependent Bispecific Antibody for the Treatment of Ovarian Cancer.

Ovarian cancer is a diverse class of tumors with very few effective treatment options and suboptimal response rates in early clinical studies using immunotherapies. Here we describe LY6/PLAUR domain containing 1 (LYPD1) as a novel target for therapeutic antibodies for the treatment of ovarian cancer. LYPD1 is broadly expressed in both primary and metastatic ovarian cancer with ∼70% prevalence in the serous cancer subset. Bispecific antibodies targeting CD3 on T cells and a tumor antigen on cancer cells have demonstrated significant clinical activity in hematologic cancers. We have developed an anti-LYPD1/CD3 T-cell-dependent bispecific antibody (TDB) to redirect T-cell responses to LYPD1 expressing ovarian cancer. Here we characterize the nonclinical pharmacology of anti-LYPD1/CD3 TDB and show induction of a robust polyclonal T-cell activation and target dependent killing of LYPD1 expressing ovarian cancer cells resulting in efficient in vivo antitumor responses in PBMC reconstituted immune-deficient mice and human CD3 transgenic mouse models. Anti-LYPD1/CD3 TDB is generally well tolerated at high-dose levels in mice, a pharmacologically relevant species, and showed no evidence of toxicity or damage to LYPD1 expressing tissues.

NRG1 is a critical regulator of differentiation in TP63-driven squamous cell carcinoma.

Squamous cell carcinomas (SCCs) account for the majority of cancer mortalities. Although TP63 is an established lineage-survival oncogene in SCCs, therapeutic strategies have not been developed to target TP63 or it's downstream effectors. In this study we demonstrate that TP63 directly regulates NRG1 expression in human SCC cell lines and that NRG1 is a critical component of the TP63 transcriptional program. Notably, we show that squamous tumors are dependent NRG1 signaling in vivo, in both genetically engineered mouse models and human xenograft models, and demonstrate that inhibition of NRG1 induces keratinization and terminal squamous differentiation of tumor cells, blocking proliferation and inhibiting tumor growth. Together, our findings identify a lineage-specific function of NRG1 in SCCs of diverse anatomic origin.

Effects of src-deficiency on the expression of in vivo toxicity of TCDD in a strain of c-src knockout mice procured through six generations of backcrossings to C57BL/6 mice.

The effect of TCDD was studied in c-src-deficient C57BL6-src(tm1sor) (N6 src -/- and -/+) mice, and their wild-type littermate mice (N6 src +/+). The former was created from the original strain of B6, 129-src(tm1sor) mice through six generations of backcrossings with C57BL6 mice. The results of a high dose TCDD toxicity tests in male mice indicated that N6 src-/+ mice were significantly less responsive to the toxic action of TCDD (115 microg/kg single i.p. injection) than N6 src+/+ mice in terms of reduced % body weight gain, the increase in the liver to body weight ratio, and the decrease in the adipose tissue to liver weight ratio and in the weight of pancreas. To understand the cause for these differential effects of TCDD we studied TCDD-induced changes in several biochemical parameters at day 10 and found that most drastically affected ones were glycogen depletion and phosphoenolpyruvate carboxykinase (PEPCK) downregulation. In addition, the degree of triglyceride accumulation in liver was less pronounced in N6-/+ than in N6+/+ mice. These findings suggest that the absence of c-src expression indeed affects the development of selected, TCDD-induced toxic endpoints that are related to wasting syndrome.

Distribution of polychlorinated biphenyls and chlorinated pesticide residues in trout in the Sierra Nevada.

Organochlorine compounds are known to be atmospherically transported to long distances from their original sources. To understand the influence of California's Sierra Nevada range on the air transport and subsequent distribution pattern of some of these residues within the range, we have chosen salmonid fish as an indicator species. Fish were collected from 10 locations throughout the northern and central Sierra Nevada and polychlorinated biphenyl (PCB), toxaphene, chlordane, and DDT [1,1,1-trichloro, 2,2'-bis (p-chlorophenyl) ethane] residues in muscle tissue were analyzed. Rainbow trout (Oncorhynchus mykiss) were found in all sampling locations, and therefore analyses mainly focused on this species. When similar-sized rainbow trout samples from several similar oligotrophic, high-altitude lakes and streams were compared, it became apparent that altitude is one of the factors affecting the residual levels of PCB (r(2) = 0.882), but not for total DDT, toxaphene, or chlordane in trout. Analysis of correlations among these four organochlorine compound residue groups indicated that there are modest correlations in patterns of distribution between chlordane vs. toxaphene (r(2) = 0.345), and chlordane vs. total DDT (r(2) = 0.239), but toxaphene residues are not correlated with PCB or total DDT. In view of significant correlation to the altitude it is concluded that PCB residue in rainbow trout is a good monitoring tool for studying the effect of high-altitude mountain ranges on the long-range transport and distribution of those persistent pollutants.

Estrogenic action of ß-HCH through activation of c-Neu in MCF-7 breast carcinoma cells.

ß-HCH is known to be a poor agonist for the estrogen receptor (ER), and yet it has been shown to act like an estrogen in stimulating foci formation in MCF-7 cells. We investigated the reason for such an action of ß-HCH, using a rat prolactin-luciferase reporter system transfected to MCF-7 cells. We found that the presence of c-Neu (erbB2), ER and ERE is needed for ß-HCH to act estrogenic at the transcription activation level in this cell line. We compared the action of ß-HCH to that of EGF which is known to act estrogenic without being an agonist for ER in this cell and found that their action patterns are quite similar, the only difference being that the former action is blocked by an antibody against c-Neu and the latter by both c-Neu and EGF receptor antibody. We concluded that ß-HCH's estrogenic action in this cell model is mediated through "ligand-independent activation of ER pathway".

Inhibition of fibroblast growth factor 19 reduces tumor growth by modulating beta-catenin signaling.

Fibroblast growth factors (FGF) play important roles in development, angiogenesis, and cancer. FGF19 uniquely binds to FGF receptor 4 (FGFR4). Our previous study has shown that FGF19 transgenic tumors have an activated Wnt-pathway phenotype. Wnt signaling is implicated in initiating or promoting FGF signaling in various cell types and organs. In this study, we examined whether FGF19 or inhibition of FGF19 affects the beta-catenin signaling pathway using human colon cancer cell lines (HCT116, Colo201). Our results show that FGF19 increases tyrosine phosphorylation of beta-catenin and causes loss of beta-catenin-E-cadherin binding. FGF19 increases p-GSK3beta and active beta-catenin levels and anti-FGF19 antibody (1A6) treatment abrogates this effect of FGF19. Anti-FGF19 antibody treatment increases S33/S37/T41 phosphorylation and ubiquitination of beta-catenin. Ion-trap mass spectrometric analysis confirmed that 1A6 increases phosphorylation of beta-catenin in the NH(2) terminus. Using HCT116-paired beta-catenin knockout cells, we show that FGF19 induces TCF/LEF reporter activity in parental (WT/Delta45) and in WT/--but not in mutant (-/Delta45) cells, and that inhibition of endogenous FGF19 reduces this reporter activity, indicating that wild-type beta-catenin is accessible for modulation. FGFR4 knockdown using inducible short hairpin RNA significantly reduces the colony-forming ability in vitro and tumor growth in vivo. Although cleaved caspase-3 immunoreactivity remains unchanged, the number of ki67-positive nuclei is reduced in FGFR4 knockdown tumor xenograft tissues. Consistent with the reduced beta-catenin activation, Taqman analyses show that FGF19/FGFR4 inhibition reduced beta-catenin target gene (cyclin D1, CD44, c-jun, Cox-2, UPAR) expression. These findings highlight that FGF19/FGFR4 cross-talk with beta-catenin and that pathway intervention reduces tumor growth.

Correlation between the high expression of C/EBPbeta protein in F442A cells and their relative resistance to antiadipogenic action of TCDD in comparison to 3T3-L1 cells.

We compared the ability of two clonally derived murine preadipocyte cell lines, 3T3-L1(L1) and 3T3-F442A (F442A), to differentiate after treatment by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and found that the former cell line was clearly suppressed by TCDD but the latter was not. It was initially postulated that the easiest way to explain the lack of response to TCDD in F442A cells could be an alteration in aryl hydrocarbon receptor (AhR) functionality. This hypothesis was tested first, but no differences were found in the levels or functions of AhR. To find an alternate explanation for such a differential effect of TCDD, we tested the action of several diagnostic agents on the process of adipocyte differentiation of these two cells. No differences were found between these two lines of cells in the susceptibility to the antiadipogenic action of 12-0-tetradecanoylphorbol-13-acetate (TPA), or to TNFalpha, indicating that the basic biochemical components engaged in the antiadipogenic actions of these agents in these two cell lines are similar. In contrast, F442A cells were found to be more resistant to the antiadipogenic action of EGF or TGFbeta than L1 cells which were tested side by side. Based on the knowledge that TNFalpha preferentially affects C/EBPalpha and that TGFbeta specifically controls C/EBPbeta and delta in their antiadipogenic action, we hypothesized that the major cause for the differential response of these two similar cell lines could be the insensitivity of C/EBPbeta and/or delta of F442A cells to the action of TCDD. We could obtain supporting data for this hypothesis, showing that in F442A cells, the level of C/EBPbeta is already high even before the addition of adipocyte differentiation factors and that TCDD did not cause any significant changes in the titer of C/EBPbeta.