Ras Signaling Inhibitors Attenuate Disease in Adjuvant-Induced Arthritis via Targeting Pathogenic Antigen-Specific Th17-Type Cells

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

A Human Integrin-α3 Mutation Confers Major Renal Developmental Defects

<div><p>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 <i>ITGA3</i> were shown to cause congenital nephrotic syndrome, epidermolysis bullosa and interstitial lung disease, otherwise termed NEP syndrome (<b>N</b>ephrotic syndrome, <b>E</b>pidermolysis bullosa and <b>P</b>ulmonary disease). Herein, we performed histological and molecular analysis on the kidneys of a single patient from the initial cohort harboring an <i>ITGA3</i> mutation, to illuminate the role of <i>ITGA3</i> 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. <i>OCT4</i> and <i>PAX8</i>) 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 <i>ITGA3</i> to the list of CAKUT (congenital anomalies of the kidney and urinary tract)-causing genes.</p></div

Clinical and histological features of the index patient.

<p>(A) A pedigree presenting the patient, born to healthy consanguineous parents, as the only affected child among nine siblings. (B) Renal ultrasound examination, demonstrating a small hyper-echogenic left kidney and an enlarged right kidney. (C) H&E staining of the patient's right kidney demonstrating a typical nephrotic syndrome phenotype including global sclerosis and mesangial proliferation. (D) The patient's left kidney presents histology consistent with renal hypodysplasia including the presence of cartilage, stroma and renal lesions of nephrotic syndrome similar to those observed in the right kidney.</p

Immuno-localization and interrogation of global gene expression of the patient's kidney.

<p>(A) Immunohistochemical staining for integrin α3 reveals a widespread expression pattern in the developing human fetal kidney (hFK), with localization to early duct precursors, ureteric buds and their differentiated derivatives and basement membrane of assembled fetal glomeruli. Integrin α3 expression was absent in the patient's kidneys. (B) Heat-map comparison of gene expression profile between the patient's kidney (PK) and an age matched control (CK) kidney. Unsupervised hierarchical clustering demonstrates that the PK is more similar genetically to human fetal kidney (hFK) than to the human adult kidney counterpart (hAK). (C) Microarray expression analysis of selected genes demonstrated altered expression in the PK of genes crucial for normal nephron formation, including the Wnt and TGFβ signaling pathways, early developmental genes and renal differentiation genes.</p

Wilms’ Tumor Blastemal Stem Cells Dedifferentiate to Propagate the Tumor Bulk

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

In Vivo Expansion of Cancer Stemness Affords Novel Cancer Stem Cell Targets: Malignant Rhabdoid Tumor as an Example

Summary: 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. : Golan et al. demonstrate that long-term propagation of human MRT xenografts robustly enriches for cancer stem cell frequency. This was exploited in turn for the identification of potential therapeutic targets in MRT such as lysyl oxidase and disclosed a platform to identify CSC targets in other rare pediatric tumors for which novel therapeutics are sought. Keywords: stem cells, cancer stem cells, PDX, MRT, targeted therapy, ALDH1, LOX inhibitio