Frizzled 7 (FZD7), a Wnt receptor that activates canonical Wnt/β-catenin signaling, has been implicated in multiple cancers, including Wilms tumor (WT), the most common pediatric kidney malignancy. We previously identified FZD7 as a marker of the WT cancer stem cell population and a potential therapeutic target. To evaluate this, we generated a panel of monoclonal anti-FZD7 antibodies using epitope mapping of the receptor and assessed their functional activity in primary WT cells and xenograft models. Among the panel, clone 288.1 induced significant cell death in primary Wilms tumor cells and inhibited cell proliferation and migration. This effect correlated with canonical Wnt signaling inhibition, a reduction in activated β-catenin and downregulation of Wnt/β-catenin target genes concomitant with diminished Wilms tumor cancer stem cell (CSC) markers. In vivo, treatment with anti-FZD7-288.1 significantly inhibited WT xenograft growth, resulting in reduced tumor volume. These findings demonstrate that FZD7 is a critical driver of Wilms tumor progression and support antibody-mediated FZD7 blockade as a promising therapeutic strategy.

Background:The nephron progenitor cells generate approximately one million nephrons during human nephrogenesis. At 34-36 weeks of human genstation, silencing of the key kidney progenitor genes results in depletion of this progenitor pool, limiting the regeneration capacity of the mature kidney. Concurrently, the increasing incidence of end-stage kidney disease underscores the urgent need for innovative regenerative strategies. Methods:We employed lentiviral vectors to ectopically induce two key kidney progenitor genes OSR1 and SIX2 individually or together in primary human adult kidney (hAK) cells. We then analyzed the cellular and molecular consequences through morphological assessments, functional assays, in vivo transplantation studies, and comprehensive transcriptional profiling. Results:OSR1 and SIX2 induced distinct reprogramming processes with differential functional outcomes; SIX2 overexpression was found to maintain epithelial morphology while significantly enhancing proliferation and clonogenic efficiency. Transcriptionally, SIX2 established epithelialization and cell-cycle networks by downregulating proximal tubule markers while upregulating distal nephron markers and proliferation genes. In vivo, SIX2-expressing cells formed organized tubular structures with a distinct luminal architecture in a proof-of-concept model. In contrast, OSR1 overexpression was found to induce morphological changes and activate developmental morphogenetic pathways, including epithelial tube morphogenesis and canonical Wnt signaling; however, it did not enhance proliferation and showed minimal tubulogenic capacity in vivo. Unexpectedly, OSR1 overexpression led to malignant transformation in one clone and exhibited Wilms'-tumor-like features, including expression of kidney developmental markers (i.e., SIX2, NCAM1, and WT1) and blastemal phenotype. Conclusion:Our findings suggest that SIX2 overexpression in primary hAK cells functionally confers enhanced self-renewal and tubulogenic capacity while transcriptionally inducing a proximal-to-distal tubular cell diversion with maintained proliferative programs. In contrast, OSR1 activates the broader developmental morphogenetic networks but poses potential oncogenic risks. The malignant transformation observed with OSR1 overexpression provides insights into the potential cellular origins of Wilms' tumor and raises important safety considerations for regenerative medicine approaches involving developmental gene induction in adult kidney cells.

Objective RasGTPases are master regulators of multiple intracellular signaling cascades. Perturbation of this pathway has been implicated in the pathogenesis of rheumatoid arthritis (RA). In this study we aimed to define the therapeutic potential of a novel RasGTPases inhibitor, farnesylthiosalicylate (FTS), in the preclinical mouse model of collagen‐induced arthritis (CIA) and better delineate its immunomodulatory effects both ex vivo and in the mouse. Methods We analyzed in vitro the immunomodulatory effects of FTS on various CD4 + T‐cell functions such as activation, proliferation, T‐helper polarization, and production of proinflammatory cytokines. Using the CIA model, we further determined the efficacy of FTS to inhibit clinical, histopathologic, and diverse immunological outcomes of arthritis. Results FTS treatment of CD4 + T cells in vitro effectively targeted distinct kinases (extracellular signal‐regulated kinase 1/2, p38, protein kinase B/AKT, and mammalian target of rapamycin), the production of interleukin (IL)‐17A, IL‐22, and granulocyte‐macrophage colony‐stimulating factor, and Th17 polarization. FTS therapy in the mouse CIA model significantly reduced clinical disease severity and joint inflammation/damage by histology. Importantly, FTS suppressed the in vivo induction of splenic IL‐17 + IL‐22 + Th17 cells and the secretion of proinflammatory cytokines. The production of pathogenic autoantibodies and their abnormal hyposialylation was significantly attenuated by FTS therapy. Importantly, in vivo generation of collagen type‐II specific effector CD4 + T cells was likewise repressed by FTS therapy. Conclusion The RasGTPases inhibitor FTS attenuates the production of proinflammatory cytokines by in vitro–activated T cells and is a potent immunomodulatory compound in the CIA model, primarily targeting the generation of autoreactive Th17 cells and the production of autoantibodies and their subsequent pathogenic hyposialylation.

Background: The Ras family of GTPases plays an important role in signaling nodes downstream to T cell antigen receptor (TCR) and CD28, potentially lowering the threshold for TCR activation by autoantigens [1]. Somatic mutation in NRAS or KRAS may cause a rare autoimmune disorder coupled with abnormal expansion of lymphocytes. T cells from Rheumatoid Arthritis (RA) patients show excessive activation of Ras/MEK/ERK pathway. The small molecule Farnesylthiosalicylic acid (FTS) interferes with the interaction between RasGTPases and their prenyl-binding chaperones to inhibit proper plasma membrane localization and effective downstream signaling. Previous studies in the Lewis rat adjuvant induced arthritis show that FTS attenuates arthritis development and that the inhibition of pathogenic Th17-type cells is a central mechanism of action of this compound [2]. Objectives: To further study the therapeutic efficacy and molecular mechanisms that mediate the immunomodulatory effects of FTS in DBA/1 mouse collagen type-II induced arthritis (CIA) the pre-clinical model. Methods: Arthritis was induced in 8-10 week old male DBA/1 mice by immunization with collagen type-II (CII) and complete Freund's adjuvant. Animals were treated semi-prophylactically with daily oral FTS (100 mg/kg); weekly i.p. injection of MTX (0.5 mg/kg); or daily 0.5% CMC vehicle solution (control treatment). Arthritis severity was graded daily by a validated clinical score (0-16 scale), starting at disease onset till study termination. In addition, multiple relevant immunological and molecular biomarkers were analyzed. Results: We found that the clinical scores of mice in the FTS and MTX arms was significantly reduced (by ∼80%, area under curve) compared to the control arm. Accordingly, FTS therapy significantly reduced joint pathology scores for inflammation, pannus formation, bone resorption, and cartilage damage. FTS also significantly inhibited the production of pathogenic anti-CII autoantibodies, anti–citrullinated peptide antibodies, and notably the de-sialylation of these autoantibodies as compared to control mice (Figure 1). The analysis of the effect of FTS on the T cell response to CII immunization, revealed strong attenuation of IL-22, IL-17, IL-9, GM-CSF, TNF, and IFN-gamma producing pro-inflammatory CD4+ Th cells. Importantly, we found that in vitro FTS treatment during TCR-stimulation (anti-CD3/CD28 mAbs) significantly inhibited the ensuing phosphorylation of multiple critical MAP kinases such as Erk, Akt, p38, and mTOR. Conclusion: We determined in the preclinical CIA model that FTS, a first-in-class oral Ras-GTPases inhibitor, is a potent immune modulator, via the inhibition of TCR/CD28/Ras-dependent activation of critical MAPKs, consequently attenuating the generation of pro-inflammatory autoreactive T cells. References [1] Singh K, et al. (2012) K-RAS GTPase- and B-RAF kinase-mediated T-cell tolerance defects in rheumatoid arthritis. Proc Natl Acad Sci USA109(25):E1629-1637. [2] Zayoud M, et al. (2017) Ras Signaling Inhibitors Attenuate Disease in Adjuvant-Induced Arthritis via Targeting Pathogenic Antigen-Specific Th17-Type Cells. Frontiers in immunology8:799. Acknowledgement: This work was supported by the U.S Department of Defense Grant number AMRMC W81XWH-14-1-0609/10 Disclosure of Interests: None declared

Pleuropulmonary blastoma (PPB) is a rare pediatric lung neoplasm that recapitulates developmental pathways of early embryonic lungs. As lung development proceeds with highly regulated mesenchymal-epithelial interactions, a DICER1 mutation in PPB generates a faulty lung differentiation program with resultant biphasic tumors composed of a primitive epithelial and mesenchymal stroma with early progenitor blastomatous cells. Deciphering of PPB progression has been hampered by the difficulty of culturing PPB cells, and specifically progenitor blastomatous cells. Here, we show that in contrast with in-vitro culture, establishment of PPB patient-derived xenograft (PDX) in NOD-SCID mice selects for highly proliferating progenitor blastoma overexpressing critical regulators of lung development and multiple imprinted genes. These stem-like tumors were sequentially interrogated by gene profiling to show a FGF module that is activated alongside Neural cell adhesion molecule 1 (NCAM1). Targeting the progenitor blastoma and these transitions with an anti-NCAM1 immunoconjugate (Lorvotuzumab mertansine) inhibited tumor growth and progression providing new paradigms for PPB therapeutics. Altogether, our novel in-vivo PPB xenograft model allowed us to enrich for highly proliferating stem-like cells and to identify FGFR and NCAM1 as two key players that can serve as therapeutic targets in this poorly understood and aggressive disease.

Cancer stem cell (CSC) identification relies on transplantation assays of cell subpopulations sorted from fresh tumor samples. Here, we attempt to bypass limitations of abundant tumor source and predetermined immune selection by in vivo propagating patient-derived xenografts (PDX) from human malignant rhabdoid tumor (MRT), a rare and lethal pediatric neoplasm, to an advanced state in which most cells behave as CSCs. Stemness is then probed by comparative transcriptomics of serial PDXs generating a gene signature of epithelial to mesenchymal transition, invasion/motility, metastasis, and self-renewal, pinpointing putative MRT CSC markers. The relevance of these putative CSC molecules is analyzed by sorting tumorigenic fractions from early-passaged PDX according to one such molecule, deciphering expression in archived primary tumors, and testing the effects of CSC molecule inhibition on MRT growth. Using this platform, we identify ALDH1 and lysyl oxidase (LOX) as relevant targets and provide a larger framework for target and drug discovery in rare pediatric cancers.

Cancer stem cells (CSC) form a specific population within the tumor that has been shown to have self-renewal and differentiation properties, increased ability to migrate and form metastases, and increased resistance to chemotherapy. Consequently, even a small number of cells remaining after therapy can repopulate the tumor and cause recurrence of the disease. CSCs in Wilms tumor, a pediatric renal cancer, were previously shown to be characterized by neural cell adhesion molecule (NCAM) expression. Therefore, NCAM provides a specific biomarker through which the CSC population in this tumor can be targeted. We have recently developed an NCAM-targeted nanosized conjugate of paclitaxel bound to a biodegradable polyglutamic acid polymer. In this work, we examined the ability of the conjugate to inhibit Wilms tumor by targeting the NCAM-expressing CSCs. Results show that the conjugate selectively depleted the CSC population of the tumors and effectively inhibited tumor growth without causing toxicity. We propose that the NCAM-targeted conjugate could be an effective therapeutic for Wilms tumor. Mol Cancer Ther; 16(11); 2462–72. ©2017 AACR.

The Ras family of GTPases plays an important role in signaling nodes downstream to T cell receptor and CD28 activation, potentially lowering the threshold for T-cell receptor activation by autoantigens. Somatic mutation in NRAS or KRAS may cause a rare autoimmune disorder coupled with abnormal expansion of lymphocytes. T cells from rheumatoid arthritis (RA) patients show excessive activation of Ras/MEK/ERK pathway. The small molecule farnesylthiosalicylic acid (FTS) interferes with the interaction between Ras GTPases and their prenyl-binding chaperones to inhibit proper plasma membrane localization. In the present study, we tested the therapeutic and immunomodulatory effects of FTS and its derivative 5-fluoro-FTS (F-FTS) in the rat adjuvant-induced arthritis model (AIA). We show that AIA severity was significantly reduced by oral FTS and F-FTS treatment compared to vehicle control treatment. FTS was as effective as the mainstay anti-rheumatic drug methotrexate, and combining the two drugs significantly increased efficacy compared to each drug alone. We also discovered that FTS therapy inhibited both the CFA-driven in vivo induction of Th17 and IL-17/IFN-γ producing "double positive" as well as the upregulation of serum levels of the Th17-associated cytokines IL-17A and IL-22. By gene microarray analysis of effector CD4+ T cells from CFA-immunized rats, re-stimulated in vitro with the mycobacterium tuberculosis heat-shock protein 65 (Bhsp65), we determined that FTS abrogated the Bhsp65-induced transcription of a large list of genes (e.g., Il17a/f, Il22, Ifng, Csf2, Lta, and Il1a). The functional enrichment bioinformatics analysis showed significant overlap with predefined gene sets related to inflammation, immune system processes and autoimmunity. In conclusion, FTS and F-FTS display broad immunomodulatory effects in AIA with inhibition of the Th17-type response to a dominant arthritogenic antigen. Hence, targeting Ras signal-transduction cascade is a potential novel therapeutic approach for RA.

Ras-GTPases are molecular switches that regulate key cellular processes, such as proliferation, differentiation, apoptosis, and motility. In T cells, Ras-family GTPases (e.g. K/N-Ras) are crucial for proper TCR-dependent activation following antigen recognition. Defective Ras GTPases signaling has been associated with T cell anergy, and accordingly increased expression of active Ras was shown to reverse anergy and to restore IL-2 production. Importantly, T cells from patients with Rheumatoid Arthritis (RA) display augmented activation of the Ras/Raf/MEK/ERK1/2 signaling pathway, and accordingly overexpression of active K-RAS in normal CD4+ T cells has been shown to promote T cells reactivity to relevant autoantigen in RA. Thus, Ras GTPases appear to be a promising molecular target for inhibiting T cell activation in RA. Based on an innovative concept Kloog (the partnering PI) and colleagues discovered a potent non-toxic inhibitor of Ras, Farnesylthiosalicylic acid (FTS). This small molecule does not belong to the class of farnesyltransferase inhibitors (FTIs) that failed in clinical trials. It interferes with the interactions between Ras and distinct prenyl-binding chaperone proteins that are vital for the proper plasma membrane (PM) localization and signaling dynamics of RasGTPases, and indeed FTS dislodges the classical H/N/K-Ras GTPases from the PM and inhibits their effective downstream signaling. In multiple preclinical animal studies it has been shown that FTS effectively inhibited in vivo tumor growth of oncogenic K/N-Ras-dependent cancers. Thus, in collaboration with Concordia Pharmaceuticals Inc., FTS was developed into and oral drug, Salirasib®. The drug has been already tested in the clinic for the treatment of cancers with oncogenic mutations in KRAS and NRAS. No dose-limiting toxicities or major adverse events were reported during Salirasib® treatment, in phase I clinical trials of patients with advanced pancreatic cancer, hematolological malignancies (NCT00867230; M.D. Anderson Cancer Center, TX), and in phase II clinical study in non-small-cell lung cancer patients (NCT00531401; Memorial Sloan Kettering Cancer Center, NY). Thus, Salirasib® is the only available successful Ras GTPases inhibitor that reached Phase II clinical trials, and moreover received an orphan drug designation by the FDA for the treatment of pancreatic cancer. Importantly, we have extensively studied the effects of FTS and related derivatives (e.g., FTSAmide and FTS-methoxymethylester), in multiple pre-clinical animal models of autoimmune inflammation, such as: experimental autoimmune encephalomyelitis; Type-1 diabetes; colitis and others. More recently, our preliminary studies in the adjuvant-induced arthritis (AIA) rat model − a classical animal model for RA − imply that FTS attenuates disease manifestation, as assessed by: clinical scores; MRI imaging; histopathology; and serum levels of pro-inflammatory cytokines. Thus, our working hypothesis is that Salirasib® has a good potential to be a “silver bullet” drug for RA and other T cell-dependent autoimmune disorders. Our objectives are to test further this hypothesis in the AIA model as well as in another established animal model of RA, the collagen type-II induced arthritis (CIA) in DBA/1 (H-2q) mice. In parallel we will study in vitro, the effects of FTS and its different newer derivatives on a wide range of T cell functions and signaling networks implicated in RA. For the therapeutic treatment protocol, we will be administer FTS orally by gavage, once daily, starting immediately after disease initiation. Multiple modalities will be used to assess joint inflammation/damage and the immune response, as follows: arthritis clinical scores; MRI scans, micro-CT; histopathology examination by a blinded pathologist; serum cytokine profiles; T cell subset analysis (e.g., Foxp3+ Treg, Th1, Th17, etc.) by polychromatic flow cytometry. Additionally, we will analyzed the activation of different Ras downstream effectors and relevant cellular programs by Western blotting, Affymetrix GeneChip® whole-transcript arrays, and quantitative real-time PCR analysis. The proposed project is highly relevant to the FY13 PRMRP topic area of Rheumatoid Arthritis (RA). The short-term impact of our research will be an improved understanding of the role of Ras GTPases in shaping, tuning, and regulating the autoimmune T cell response, and the effects of Ras inhibitors on the pathogenesis of the inflammatory arthritis in two relevant preclinical models of RA. We envision that the long-term impact of our proposed research plan will be the introduction of a new class of synthetic drugs, orally available small molecule Rasinhibitors such as Salirasib®, to advance the clinical management of RA patients with conceivably fewer side effects and reduced healthcare system costs compared to biologic drugs.

Background Ras-GTPases are vital for normal T cell activation, and downstream effectors of Ras include the MEK/ERK, PI3-kinase/AKT, mTOR/p70S6-kinase, and NF-kB pathways. Somatic mutations in NRAS cause an autoimmune lymphoproliferative disorder and T cells from Rheumatoid Arthritis (RA) patients exhibit perturbation of the Ras/MEK/ERK pathway. The small molecule Farnesylthiosalicylic acid (FTS) inhibits the interaction between Ras-GTPases and prenyl-binding chaperones vital for proper plasma membrane localization and downstream signaling [1]. Previous pre-clinical studies suggest that FTS has an immunomodulatory effect in various animal models of autoimmunity [2]. Objectives To test in the Lewis rat adjuvant induced arthritis (AIA) and in the DBA/1 mouse collagen type-II induced arthritis (CIA) models the therapeutic immunomodulatory effect of FTS alone or combined with methotrexate (MTX). Methods Arthritis was induced in 8–12 week old male Lewis rats by complete Freund9s adjuvant (CFA) injection and in male DBA/1 mice by collagen type-II (CII) immunization. Animals were treated prophylactically with once daily oral FTS (100 mg/kg); weekly i.p injection of MTX (0.5 mg/kg), oral FTS combined with MTX, or daily oral vehicle solution (0.5% carboxy methyl cellulose; CMC). Arthritis severity was scored daily from disease onset until study termination. In addition, we measured multiple disease- and drug-related immunological/molecular biomarkers. Results AIA severity was significantly reduced by FTS treatment compared to CMC controls (Figure 1A, P<0.001). Combining FTS and low dose MTX significantly increased its therapeutic efficacy compared to each drug alone (Figure 1A, P<0.05). FTS or FTS+MTX treatment also suppressed the upsurge in serum IL-17 and CRP compared to ailing controls. Global gene expression analysis of relevant splenic CD4+ T cells revealed that FTS is a potent inhibitor of pro-inflammatory and TH17 related gene networks. Next, our data from the mouse CIA model show that the therapeutic efficacy of FTS was non-inferior to MTX and it significantly reduced arthritis severity compared to controls (Figure 2, P<0.001). Importantly, FTS significantly inhibited the production of pathogenic anti-CII autoantibodies and upregulation of serum IL-6 and IL-17A compared to control arthritic mice. The in depth, multiplex, analysis of the effect of FTS on the T cell cytokine response to CII, revealed strong suppression of IL-22, IL-17, IL-9, GM-CSF and TNF production. Noteworthy, FTS therapy positively correlated with reduced Ras-GTP, p-ERK and p-AKT levels in splenic lymphocytes (drug related biomarkers). Conclusions FTS, a first-in-class oral selective Ras-GTPases inhibitor, exhibits a potent immunomodulatory effect in two classical murine model of arthritis, coupled with the inhibition of the TH17 response to relevant arthritogenic-antigens. Thus, Ras-signaling-blockade is a promising novel therapeutic approach for RA. References Kloog Y, Cox AD. Prenyl-binding domains: potential targets for Ras inhibitors and anti-cancer drugs. Semin Cancer Biol. 2004 Aug; 14(4):253–261. Mor A, Aizman E, Chapman J, Kloog Y. Immunomodulatory properties of farnesoids: the new steroids? Curr Med Chem. 2013; 20(10):1218–1224. Disclosure of Interest None declared

Angiomyolipoma (AML), the most common benign renal tumor, can result in severe morbidity from hemorrhage and renal failure. While mTORC1 activation is involved in its growth, mTORC1 inhibitors fail to eradicate AML, highlighting the need for new therapies. Moreover, the identity of the AML cell of origin is obscure. AML research, however, is hampered by the lack of in vivo models. Here, we establish a human AML‐xenograft (Xn) model in mice, recapitulating AML at the histological and molecular levels. Microarray analysis demonstrated tumor growth in vivo to involve robust PPARγ‐pathway activation. Similarly, immunostaining revealed strong PPARγ expression in human AML specimens. Accordingly, we demonstrate that while PPARγ agonism accelerates AML growth, PPARγ antagonism is inhibitory, strongly suppressing AML proliferation and tumor‐initiating capacity, via a TGFB‐mediated inhibition of PDGFB and CTGF. Finally, we show striking similarity between AML cell lines and mesenchymal stem cells (MSCs) in terms of antigen and gene expression and differentiation potential. Altogether, we establish the first in vivo human AML model, which provides evidence that AML may originate in a PPARγ‐activated renal MSC lineage that is skewed toward adipocytes and smooth muscle and away from osteoblasts, and uncover PPARγ as a regulator of AML growth, which could serve as an attractive therapeutic target.§ Angiomyolipoma (AML) research is hampered by the lack of an animal model. In a novel in vivo human AML model, PPARG inhibition is identified as a potential therapy and the AML cell of origin as a resident MSC/pericyte skewed toward adipogenic differentiation. Angiomyolipoma (AML) research is hampered by the lack of an animal model. In a novel in vivo human AML model, PPARG inhibition is identified as a potential therapy and the AML cell of origin as a resident MSC/pericyte skewed toward adipogenic differentiation.

Adenosine deaminase acting on RNA (ADAR) 1 is the master editor of the transcriptome, catalyzing the conversion of adenosine to inosine (A-to-I). RNA transcripts fold into a variety of secondary structures including long intramolecular RNA duplexes that are the major substrate of ADAR1. Most A-to-I editing sites occur within RNA duplexes formed by complementary pairing of inverted retrotransposable elements interspersed within noncoding regions of transcripts. This catalytic activity of ADAR1 most likely prevents the abnormal activation of cytosolic nucleic acid sensors by self-dsRNAs. Homozygous disruption of mouse Adar is embryonic lethal due to a toxic type-I interferons response and correspondingly biallelic missense mutations in human ADAR1 cause a severe congenital interferonopathy. Here, we report that Cd19-Cre-mediated Adar gene ablation in the mouse causes a significant defect in the final stages of B cell development with an almost complete absence of newly formed immature and CD23+ mature recirculating B cells in the BM. Adar ablation in pre-B cells induced upregulation of typical interferon-stimulated genes (ISGs) and apoptosis upon further maturation. ADAR1 deficiency also inhibited the in vitro, IL-7-mediated, differentiation of BM-derived B cell precursors. In summary, ADAR1 is required, non-redundantly, for normal B lymphopoiesis in the BM and peripheral maintenance.

When assembling a nephron during development a multipotent stem cell pool becomes restricted as differentiation ensues. A faulty differentiation arrest in this process leads to transformation and initiation of a Wilms’ tumor. Mapping these transitions with respective surface markers affords accessibility to specific cell subpopulations. NCAM1 and CD133 have been previously suggested to mark human renal progenitor populations. Herein, using cell sorting, RNA sequencing, in vitro studies with serum-free media and in vivo xenotransplantation we demonstrate a sequential map that links human kidney development and tumorigenesis; In nephrogenesis, NCAM1 + CD133 − marks SIX2 + multipotent renal stem cells transiting to NCAM1 + CD133 + differentiating segment-specific SIX2 − epithelial progenitors and NCAM1 − CD133 + differentiated nephron cells. In tumorigenesis, NCAM1 + CD133 − marks SIX2 + blastema that includes the ALDH1 + WT cancer stem/initiating cells, while NCAM1 + CD133 + and NCAM1 − CD133 + specifying early and late epithelial differentiation, are severely restricted in tumor initiation capacity and tumor self-renewal. Thus, negative selection for CD133 is required for defining NCAM1 + nephron stem cells in normal and malignant nephrogenesis.

An open question remains in cancer stem cell (CSC) biology whether CSCs are by definition at the top of the differentiation hierarchy of the tumor. Wilms' tumor (WT), composed of blastema and differentiated renal elements resembling the nephrogenic zone of the developing kidney, is a valuable model for studying this question because early kidney differentiation is well characterized. WT neural cell adhesion molecule 1-positive (NCAM1+) aldehyde dehydrogenase 1-positive (ALDH1+) CSCs have been recently isolated and shown to harbor early renal progenitor traits. Herein, by generating pure blastema WT xenografts, composed solely of cells expressing the renal developmental markers SIX2 and NCAM1, we surprisingly show that sorted ALDH1+ WT CSCs do not correspond to earliest renal stem cells. Rather, gene expression and proteomic comparative analyses disclose a cell type skewed more toward epithelial differentiation than the bulk of the blastema. Thus, WT CSCs are likely to dedifferentiate to propagate WT blastema.

The development of the mammalian kidney is a highly complex process dependent upon the interplay of various cell types, secreted morphogens, and the extra-cellular matrix (ECM). Although integrins are the most important receptors for ECM proteins and are ubiquitously expressed during kidney development, mice lacking expression of integrin α3 (Itga3) do not demonstrate a reduced number of nephrons, but mostly a disorganized GBM (glomerular basement membrane) leading to proteinuria. Thus, ITGA3 is considered mostly a passive GBM stabilizer and not an active player in nephrogenesis. Recently, mutations in the human ITGA3 were shown to cause congenital nephrotic syndrome, epidermolysis bullosa and interstitial lung disease, otherwise termed NEP syndrome (Nephrotic syndrome, Epidermolysis bullosa and Pulmonary disease). Herein, we performed histological and molecular analysis on the kidneys of a single patient from the initial cohort harboring an ITGA3 mutation, to illuminate the role of ITGA3 in human renal development. We show the patient to harbor a unique phenotype at birth, including severe unilateral renal hypodysplasia. Interrogation of global gene expression in the hypodysplastic kidney versus three controls (fetal, child and adult kidneys) revealed perturbed expression in several renal developmental pathways implicated in hypodysplasia, including the Wnt, BMP (bone morphogenetic protein) and TGF (transforming growth factor) pathways. Moreover, the affected kidney showed upregulation of early embryonic genes (e.g. OCT4 and PAX8) concomitant with downregulated kidney differentiation markers, implying a defect in proper renal differentiation. In conclusion, we show for the first time that ITGA3 is not merely a passive anchor for renal ECM proteins, as predicted by mouse models. Instead, our results may suggest it plays a central role in the interplay of cells, morphogens and ECM, required for proper nephrogenesis, thus adding ITGA3 to the list of CAKUT (congenital anomalies of the kidney and urinary tract)-causing genes.

The nephron is composed of a monolayer of epithelial cells that make up its various compartments. In development, these cells begin as mesenchyme. NCAM1, abundant in the mesenchyme and early nephron lineage, ceases to express in mature kidney epithelia. We show that, once placed in culture and released from quiescence, adult human kidney epithelial cells (hKEpCs), uniformly positive for CD24/CD133, re-express NCAM1 in a specific cell subset that attains a stem/progenitor state. Immunosorted NCAM1(+) cells overexpressed early nephron progenitor markers (PAX2, SALL1, SIX2, WT1) and acquired a mesenchymal fate, indicated by high vimentim and reduced E-cadherin levels. Gene expression and microarray analysis disclosed both a proximal tubular origin of these cells and molecules regulating epithelial-mesenchymal transition. NCAM1(+) cells generated clonal progeny when cultured in the presence of fetal kidney conditioned medium, differentiated along mesenchymal lineages but retained the unique propensity to generate epithelial kidney spheres and produce epithelial renal tissue on single-cell grafting in chick CAM and mouse. Depletion of NCAM1(+) cells from hKEpCs abrogated stemness traits in vitro. Eliminating these cells during the regenerative response that follows glycerol-induced acute tubular necrosis worsened peak renal injury in vivo. Thus, higher clone-forming and developmental capacities characterize a distinct subset of adult kidney-derived cells. The ability to influence an endogenous regenerative response via NCAM1 targeting may lead to novel therapeutics for renal diseases.

Objective: Interleukin (IL)-1 alpha and IL-1 beta are products of macrophages, endothelial cells and vascular smooth muscle cells; moreover, each of these cell types is affected by the pro-inflammatory properties of both IL-1's. Whereas several studies demonstrate the proatherogenic properties of IL-1 beta, the role of IL-1 alpha in atherogenesis remains unclear. We assessed whether IL-1 alpha and IL-1 beta from tissue resident vascular cells or emigrating bone marrow-derived cells promote the development of atherosclerosis in apoE-/- mice and determined the effect of selective macrophage IL-1 alpha or IL-1 beta deficiency on degradation of LDL and cytokine production.Methods: We generated strains of double knock-out (KO) mice (apoE-/-/IL-1 alpha-/- and apoE-/-/IL-1 beta-/-) and created chimeras consisting of apoE-/- mice reconstituted with bone marrow-derived cells from apoE-/-/IL-1+/+, apoE-/-/IL-1 alpha-/- and apoE-/-/IL-1 beta-/-.Results: The areas of aortic sinus lesions were lower in either double KO mice compared to solely apoE-/- mice, despite higher non-HDL cholesterol levels. Importantly, selective deficiency of IL-1 alpha or IL-1 beta in bone marrow-derived cells inhibited atherogenesis to the same extent as in double KO mice without affecting plasma lipids. Aortic sinus lesions in apoE-/- mice transplanted with IL-1 beta-/- or IL-1 alpha-/- cells were 32% and 52% lower, respectively, than in IL-1+/+ transplanted mice. Ex vivo, isolated IL-1 alpha-/- macrophages from atherosclerotic mice degraded LDL and secreted IL-6, TNF alpha and IL-12 similarly to IL-1+/+ macrophages; however, IL-1 alpha deficient macrophages secreted reduced levels of IL-1 beta (-50%) and 2-3-fold higher levels of the anti-inflammatory cytokine IL-10.Conclusion: We show for the first time that it is IL-1 alpha from bone marrow-derived cells that accelerates atherogenesis in apoE-deficient mice rather than constitutive IL-1 alpha in vascular cells, possibly by increasing the inflammatory cytokine profile of macrophages. (C) 2011 Elsevier Inc. All rights reserved.

Background & Aims: The identification of the cellular and molecular pathways that mediate the development of non-alcoholic steatohepatitis is of crucial importance. Cytokines produced by liver-resident and infiltrating inflammatory cells, play a pivotal role in liver inflammation. The role of the proinflammatory cytokines IL-1 alpha and IL-1 beta in steatohepatitis remains elusive.Methods: We employed IL-1 alpha and IL-1 beta-deficient mice and transplanted marrow cells to study the role of liver-resident and bone marrow-derived IL-1 in steatosis and its progression to steatohepatitis.Results: Atherogenic diet-induced steatohepatitis in wild-type mice was associated with 16 and 4.6 fold-elevations in mRNA levels of hepatic IL-1 alpha and IL-1 beta, respectively. In mice deficient in either IL-1 alpha or IL-1 beta the transformation of steatosis to steatohepatitis and liver fibrosis was markedly reduced. This protective effect in IL-1 alpha-deficient mice was noted despite increased liver cholesterol levels. Deficiency of IL-1 alpha markedly reduced plasma serum amyloid A and steady-state levels of mRNA coding for inflammatory genes (P-selectin, CXCL1, IL-6, and TNF alpha) as well as pro-fibrotic genes (MMP-9 and Collagen) and particularly a 50% decrease in TGF beta levels (p = 0.004). IL-1 alpha mRNA levels were two-folds lower in IL-1 beta-deficient mice, and IL-1 beta transcripts were three-folds lower in IL-1 alpha-deficient compared to wild-type mice. Hepatic cell derived IL-1 alpha rather than from recruited bone marrow-derived cells was required for steatohepatitis development.Conclusions: These data demonstrate the critical role of IL-1 alpha and IL-1 beta in the transformation of steatosis to steatohepatitis and liver fibrosis in hypercholesterolemic mice. Therefore, the potential of neutralizing IL-1 alpha and/or IL-1 beta to inhibit the development of steatohepatitis should be explored. (C) 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

: Ras-GTPases are molecular switches that regulate key cellular processes, such as proliferation, differentiation, apoptosis, and motility. In T cells, Ras-family GTPases (e.g. K/N-Ras) are crucialfor proper TCR-dependent activation following antigen recognition. Defective Ras GTPasessignaling has been associated with T cell anergy, and accordingly increased expression of activeRas was shown to reverse anergy and to restore IL-2 production. Importantly, T cells frompatients with Rheumatoid Arthritis (RA) display augmented activation of theRas/Raf/MEK/ERK1/2 signaling pathway, and accordingly overexpression of active K-RAS innormal CD4+ T cells has been shown to promote T cells reactivity to relevant autoantigen in RA.Thus, Ras GTPases appear to be a promising molecular target for inhibiting T cell activation inRA. Based on an innovative concept Kloog (the partnering PI) and colleagues discovered apotent non-toxic inhibitor of Ras, Farnesylthiosalicylic acid (FTS). This small molecule does notbelong to the class of farnesyl transferase inhibitors (FTIs) that failed in clinical trials. Itinterferes with the interactions between Ras and distinct prenyl-binding chaperone proteins thatare vital for the proper plasma membrane (PM) localization and signaling dynamics of Ras-GTPases, and indeed FTS dislodges the classical H/N/K-Ras GTPases from the PM and inhibitstheir effective downstream signaling. In multiple preclinical animal studies it has been shownthat FTS effectively inhibited in vivo tumor growth of oncogenic K/N-Ras-dependent cancers.Thus, in collaboration with Concordia Pharmaceuticals Inc., FTS was developed into and oral drug, Salirasib (registered).