Scientific Resources

MALT1 paracaspase is central for lymphocyte antigen-dependent responses including NF-kappaB activation. We discovered nanomolar,selective allosteric inhibitors of MALT1 that bind by displacing the side chain of Trp580,locking the protease in an inactive conformation. Interestingly,we had previously identified a patient homozygous for a MALT1 Trp580-to-serine mutation who suffered from combined immunodeficiency. We show that the loss of tryptophan weakened interactions between the paracaspase and C-terminal immunoglobulin MALT1 domains resulting in protein instability,reduced protein levels and functions. Upon binding of allosteric inhibitors of increasing potency,we found proportionate increased stabilization of MALT1-W580S to reach that of wild-type MALT1. With restored levels of stable MALT1 protein,the most potent of the allosteric inhibitors rescued NF-kappaB and JNK signaling in patient lymphocytes. Following compound washout,MALT1 substrate cleavage was partly recovered. Thus,a molecular corrector rescues an enzyme deficiency by substituting for the mutated residue,inspiring new potential precision therapies to increase mutant enzyme activity in other deficiencies. View Publication

Human induced pluripotent stem cells (hiPSCs) are a valuable tool for investigating complex cellular and molecular events that occur in several human diseases. Importantly,the ability to differentiate hiPSCs into any human cell type provides a unique way for investigating disease mechanisms such as complex mental health diseases. The in vitro transformation of human lymphocytes into lymphoblasts (LCLs) using the Epstein-Barr virus (EBV) has been the main method for generating immortalized human cell lines for half a century. However,the derivation of iPSCs from LCLs has emerged as an alternative source from which these cell lines can be generated. We show that iPSCs derived from LCLs using the Sendai virus procedure can be successfully differentiated into cardiomyocytes,neurons,and myotubes that express neuron- and myocyte-specific markers. We further show that these cardiac and neuronal cells are functional and generate action potentials that are required for cell excitability. We conclude that the ability to differentiate LCLs into neurons and myocytes will increase the use of LCLs in the future as a potential source of cells for modelling a number of diseases. View Publication

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy,we achieve up to 20{\%} targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45{\%}) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum,our study provides specificity,toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl. View Publication

Acute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation,generating self-renewing leukemic stem cells (LSCs). Here,we show that the mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m6A transcripts that contribute to the overall integrity of LSC function,including the tumor necrosis factor receptor Tnfrsf2,whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly,YTHDF2 is not essential for normal HSC function,with YTHDF2 deficiency actually enhancing HSC activity. Thus,we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion. View Publication

Hematopoiesis provides an accessible system for studying the principles underlying cell-fate decisions in stem cells. Proposed models of hematopoiesis suggest that quantitative changes in lineage-specific transcription factors (LS-TFs) underlie cell-fate decisions. However,evidence for such models is lacking as TF levels are typically measured via RNA expression rather than by analyzing temporal changes in protein abundance. Here,we used single-cell mass cytometry and absolute quantification by mass spectrometry to capture the temporal dynamics of TF protein expression in individual cells during human erythropoiesis. We found that LS-TFs from alternate lineages are co-expressed,as proteins,in individual early progenitor cells and quantitative changes of LS-TFs occur gradually rather than abruptly to direct cell-fate decisions. Importantly,upregulation of a megakaryocytic TF in early progenitors is sufficient to deviate cells from an erythroid to a megakaryocyte trajectory,showing that quantitative changes in protein abundance of LS-TFs in progenitors can determine alternate cell fates. View Publication

Background Cancer has been considered a serious global health problem and a leading cause of morbidity and mortality worldwide. Despite recent advances in cancer therapy,treatments of advance stage cancers are mostly ineffective resulting in poor survival of patients. Recent evidences suggest that multipotent human mesenchymal stem cells (hMSCs) play important roles in growth and metastasis of several cancers by enhancing their engraftment and inducing tumor neovascularization. However,the effect of hMSCs on cancer cells is still controversial because there are also evidences demonstrating that hMSCs inhibited growth and metastasis of some cancers. Methods In this study,we investigated the effects of bioactive molecules released from bone marrow and gestational tissue-derived hMSCs on the proliferation of various human cancer cells,including C3A,HT29,A549,Saos-2,and U251. We also characterized the hMSC-derived factors that inhibit cancer cell proliferation by protein fractionation and mass spectrometry analysis. Results We herein make a direct comparison and show that the effects of hMSCs on cancer cell proliferation and migration depend on both hMSC sources and cancer cell types and cancer-derived bioactive molecules did not affect the cancer suppressive capacity of hMSCs. Moreover,hMSCs use distinct combination of bioactive molecules to suppress the proliferation of human hepatoblastoma and colorectal cancer cells. Using protein fractionation and mass spectrometry analysis,we have identified several novel hMSC-derived factors that might be able to suppress cancer cell proliferation. Conclusion We believe that the procedure developed in this study could be used to discover other therapeutically useful molecules released by various hMSC sources for a future in vivo study. View Publication

T helper 17 (Th17) cells are pathogenic in many inflammatory diseases,but also support the integrity of the intestinal barrier in a non-inflammatory manner. It is unclear what distinguishes inflammatory Th17 cells elicited by pathogens and tissue-resident homeostatic Th17 cells elicited by commensals. Here,we compared the characteristics of Th17 cells differentiating in response to commensal bacteria (SFB) to those differentiating in response to a pathogen (Citrobacter rodentium). Homeostatic Th17 cells exhibited little plasticity towards expression of inflammatory cytokines,were characterized by a metabolism typical of quiescent or memory T cells,and did not participate in inflammatory processes. In contrast,infection-induced Th17 cells showed extensive plasticity towards pro-inflammatory cytokines,disseminated widely into the periphery,and engaged aerobic glycolysis in addition to oxidative phosphorylation typical for inflammatory effector cells. These findings will help ensure that future therapies directed against inflammatory Th17 cells do not inadvertently damage the resident gut population. View Publication

Tissue inhibitor matrix metalloproteinase-1 (TIMP1) is one of four identified members of the TIMP family. We evaluated the role of TIMP1 in gastric cancer using human and mouse tissues along with gastric organoids and in vitro cell models. Using quantitative real-time RT-PCR,we detected significant overexpression of TIMP1 in the human gastric cancer samples,as compared to normal stomach samples (P {\textless} 0.01). We also detected overexpression of Timp1 in neoplastic gastric lesions of the Tff1-knockout (KO) mice,as compared to normal stomach tissues. Reconstitution of TFF1 in human gastric cancer cell lines led to a significant decrease in the mRNA expression level of TIMP1 (P {\textless} 0.05). In vitro analysis demonstrated that TIMP1 mRNA expression is induced by TNF-alpha and activation of NF-kappaB whereas inhibition of NF-kappaB using BAY11-7082 led to inhibition of NF-kappaB and downregulation of TIMP1. Western blot analysis confirmed the decrease in TIMP1 protein level following reconstitution of TFF1. By using immunofluorescence,we showed nuclear localization of NF-kappaB and expression of TIMP1 in gastric organoids established from the Tff1-KO stomach where reconstitution of Tff1 using recombinant protein led to a notable reduction in the expression of both NF-kappaB and TIMP1. Using EDU assay,as a measure of proliferating cells,we found that TIMP1 promotes cellular proliferation whereas TFF1 reconstitution leads to a significant decrease in cellular proliferation (P {\textless} 0.05). In summary,our findings demonstrate overexpression of TIMP1 in mouse and human gastric cancers through NF-kB-dependent mechanism. We also show that TFF1 suppresses NF-kappaB and inhibits TIMP1-mediated proliferative potential in gastric cancer. View Publication

Extracellular cold-inducible RNA-binding protein (CIRP) exaggerates inflammation and tissue injury in sepsis. Neutrophil extracellular traps (NETs) are released by activated neutrophils during sepsis. NETs contribute to pathogen clearance,but excessive NET formation (NETosis) causes inflammation and tissue damage. Peptidylarginine deiminase 4 (PAD4) is associated with NETosis by increasing histone citrullination and chromatin decondensation. We hypothesized that CIRP induces NETosis in the lungs during sepsis via upregulating PAD4 expression. Sepsis was induced in C57BL/6 wild-type (WT) and CIRP-/- mice by cecal ligation and puncture (CLP). After 20 h of CLP induction,NETs in the lungs of WT and CIRP-/- mice were quantified by flow cytometry by staining the single cell suspensions with MPO and CitH3 Abs. PAD4 expression in the lungs of WT and CIRP-/- mice after sepsis was assessed by Western blotting. In vitro effects of recombinant mouse (rm) CIRP for NETosis and PAD4 expression in the bone marrow-derived neutrophils (BMDN) were assessed by flow cytometry and Western blotting,respectively. After 20 h of CLP,NETosis in the lungs was significantly decreased in CIRP-/- mice compared to WT mice,which also correlated with the decreased PAD4 expression. Intratracheal administration of rmCIRP into WT mice significantly increased NETosis and PAD4 expression in the lungs compared to vehicle-injected mice. In vitro culture of BMDN with rmCIRP significantly increased NETosis and PAD4 expression compared to PBS-treated control. Fluorescence microscopy revealed typical web-like structures consistent with NETs in rmCIRP-treated BMDN. Thus,CIRP serves as a novel inducer of NETosis via PAD4 during sepsis. View Publication

Immune activation may underlie the pathogenesis of irritable bowel syndrome (IBS),but the evidence is conflicting. We examined whether peripheral CD4+ T-cells from IBS patients demonstrated immune activation and changes in cytokine production. To gain mechanistic insight,we examined whether immune activation correlated with psychological stress and changing symptoms over time. IBS patients (n = 29) and healthy volunteers (HV; n = 29) completed symptom and psychological questionnaires. IBS patients had a significant increase in CD4+ T-cells expressing the gut homing marker integrin beta7 (p = 0.023) and lymphoid marker CD62L (p = 0.026) compared to HV. Furthermore,phytohaemagglutinin stimulated CD4+ T-cells from IBS-D patients demonstrated increased TNFalpha secretion when compared to HV (p = 0.044). Increased psychological scores in IBS did not correlate with TNFalpha production,while stress hormones inhibited cytokine secretion from CD4+ T-cells of HV in vitro. IBS symptoms,but not markers of immune activation,decreased over time. CD4+ T-cells from IBS-D patients exhibit immune activation,but this did not appear to correlate with psychological stress measurements or changing symptoms over time. This could suggest that immune activation is a surrogate of an initial trigger and/or ongoing parallel peripheral mechanisms. View Publication

Various preclinical models have been developed to clarify the pathophysiology of prostate cancer (PCa). Traditional PCa cell lines from clinical metastatic lesions,as exemplified by DU-145,PC-3,and LNCaP cells,are useful tools to define mechanisms underlying tumorigenesis and drug resistance. Cell line-based experiments,however,have limitations for preclinical studies because those cells are basically adapted to 2-dimensional monolayer culture conditions,in which the majority of primary PCa cells cannot survive. Recent tissue engineering enables generation of PCa patient-derived xenografts (PDXs) from both primary and metastatic lesions. Compared with fresh PCa tissue transplantation in athymic mice,co-injection of PCa tissues with extracellular matrix in highly immunodeficient mice has remarkably improved the success rate of PDX generation. PDX models have advantages to appropriately recapitulate the molecular diversity,cellular heterogeneity,and histology of original patient tumors. In contrast to PDX models,patient-derived organoid and spheroid PCa models in 3-dimensional culture are more feasible tools for in vitro studies for retaining the characteristics of patient tumors. In this article,we review PCa preclinical model cell lines and their sublines,PDXs,and patient-derived organoid and spheroid models. These PCa models will be applied to the development of new strategies for cancer precision medicine. View Publication

Serrated adenocarcinoma,an alternative pathway for colorectal cancer (CRC) development,accounts for 15{\%}-30{\%} of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C zeta (PKCzeta) and PKClambda/iota was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKClambda/iota deficiency led to immunogenic cell death and the infiltration of CD8+ T cells,which repressed tumor initiation,PKCzeta loss impaired interferon and CD8+ T cell responses,which resulted in tumorigenesis. Combined treatment with a TGF-beta receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC. View Publication