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Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood,initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however,the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome,RNA-seq),Colony forming assay and xenograft studies,we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones,both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells,JMML-PCs are not always restricted to this compartment,highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML,and the identity of JMML-PCs,and provides robust models to monitor the disease and test novel therapeutic approaches. View Publication

BACKGROUND Vascularization is important for the clinical application of tissue engineered products. Both adipose-derived stem cells (ASCs) and surgical prefabrication can be used to induce angiogenesis in scaffolds. Our aim was to compare the angiogenic potential of ASC-seeded scaffolds combined with scaffold prefabrication with that of non-seeded,non-prefabricated scaffolds. METHODS For prefabrication,functional blood vessels were introduced into the scaffold using a flow-through pedicle system. ASCs were isolated from rat fat deposits. Three-dimensional-printed cylindrical poly-$\epsilon$-caprolactone scaffolds were fabricated by fused deposition modelling. Three groups,each containing six rats,were investigated by using non-seeded,ASC-seeded,and osteogenic induced ASC-seeded scaffolds. In each group,one rat was implanted with two scaffolds in the inguinal region. On the right side,a scaffold was implanted subcutaneously around the inferior epigastric vessels (classic prefabrication group). On the left side,the inferior epigastric vessels were placed inside the prefabricated scaffold in the flow-through pedicle system (flow-through prefabrication group). The vessel density and vascular architecture were examined histopathologically and by $\mu$CT imaging,respectively,at 2 months after implantation. RESULTS The mean vessel densities were 10- and 5-fold higher in the ASC-seeded and osteogenic induced ASC-seeded scaffolds with flow-through prefabrication,respectively,than in the non-seeded classic prefabricated group (p {\textless} 0.001). $\mu$CT imaging revealed functional vessels within the scaffold. CONCLUSION ASC-seeded scaffolds with prefabrication showed significantly improved scaffold vasculogenesis and could be useful for application to tissue engineering products in the clinical settings. View Publication

The objective of the study was to identify the mechanism of action for a radiation mitigator of the gastrointestinal (GI) acute radiation syndrome (ARS),identified in an unbiased high-throughput screen. We used mice irradiated with a lethal dose of radiation and treated with daily injections of the radiation mitigator 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine to study its effects on key pathways involved in intestinal stem cell (ISC) maintenance. RNASeq,quantitative reverse transcriptase-polymerase chain reaction,and immunohistochemistry were performed to identify pathways engaged after drug treatment. Target validation was performed with competition assays,reporter cells,and in silico docking. 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine activates Hedgehog signaling by binding to the transmembrane domain of Smoothened,thereby expanding the ISC pool,increasing the number of regenerating crypts and preventing the GI-ARS. We conclude that Smoothened is a target for radiation mitigation in the small intestine that could be explored for use in radiation accidents as well as to mitigate normal tissue toxicity during and after radiotherapy of the abdomen. View Publication

Increased levels of intestinal bile acids (BAs) are a risk factor for colorectal cancer (CRC). Here,we show that the convergence of dietary factors (high-fat diet) and dysregulated WNT signaling (APC mutation) alters BA profiles to drive malignant transformations in Lgr5-expressing (Lgr5+) cancer stem cells and promote an adenoma-to-adenocarcinoma progression. Mechanistically,we show that BAs that antagonize intestinal farnesoid X receptor (FXR) function,including tauro-$\beta$-muricholic acid (T-$\beta$MCA) and deoxycholic acid (DCA),induce proliferation and DNA damage in Lgr5+ cells. Conversely,selective activation of intestinal FXR can restrict abnormal Lgr5+ cell growth and curtail CRC progression. This unexpected role for FXR in coordinating intestinal self-renewal with BA levels implicates FXR as a potential therapeutic target for CRC. View Publication

Foxp3+ Regulatory T cells (Tregs) are pivotal for the maintenance of tolerance. Alterations in their number and/or function have been proposed to occur in the autoimmune-prone non-obese diabetic (NOD) mouse. Comparing the frequencies and absolute numbers of CD4+Foxp3+CD25+ Tregs among 4 to 6-week old NOD,B6,and BALB/c mice,we observed differences in counts and Foxp3 expression in Tregs from secondary lymphoid organs,but not in the thymus. Upon TCR and IL-2 stimulation,NOD Tregs showed lower responses than Tregs from B6 and BALB/c mice. Indeed,NOD Tregs responded with less proliferation and with smaller increments in the expression of CD25,LAP-1,CD39,PD-1,PD-L1,and LAG-3,when in vitro cultured for 3 days with anti-CD3/CD28 in the absence or presence of IL-2,Tregs from NOD mice showed to be highly dependent on IL-2 to maintain Foxp3 expression. Moreover,NOD Tregs become producers of IL-17 and INF-gamma more easily than Tregs from the other strains. In addition,NOD Tregs showed lower responsiveness to IL-2,with significantly reduced levels of pSTAT5,even at high IL-2 doses,with respect to B6 and BALB/c Tregs. Interestingly,NOD Tregs exhibit differences in the expression of SOCS3,GRAIL,and OTUB1 when compared with Tregs from B6 and BALB/c mice. Both,at steady state conditions and also after activation,Tregs from NOD mice showed increased levels of OTUB1 and low levels of GRAIL. In addition,NOD Tregs had differences in the expression of ubiquitin related molecules that play a role in the maintenance of Foxp3 cellular pools. Indeed,significantly higher STUB1/USP7 ratios were detected in NOD Tregs,both at basal conditions and after stimulation,compared to in B6 and BALB/c Tregs. Moreover,the addition of a proteasome inhibitor to cell cultures,conferred NOD Tregs the ability to retain Foxp3 expression. Herein,we provide evidence indicating a differential expression of SOCS3,GRAIL,and STUB1/USP7 in Tregs from NOD mice,factors known to be involved in IL-2R signaling and to affect Foxp3 stability. These findings add to the current knowledge of the immunobiology of Tregs and may be related to the known insufficiency of Tregs from NOD mice to maintain self-tolerance. View Publication

BACKGROUND {\&} AIMS The nuclear receptor subfamily 0 group B member 2 (NR0B2,also called SHP) is expressed at high levels in the liver and intestine. Postprandial fibroblast growth factor 19 (human FGF19,mouse FGF15) signaling increases the transcriptional activity of SHP. We studied the functions of SHP and FGF19 in the intestines of mice,including their regulation of expression of the cholesterol transporter NPC1L1 )NPC1-like intracellular cholesterol transporter 1) and cholesterol absorption. METHODS We performed histologic and biochemical analyses of intestinal tissues from C57BL/6 and SHP-knockout mice and performed RNA-sequencing analyses to identify genes regulated by SHP. The effects of fasting and refeeding on intestinal expression of NPC1L1 were examined in C57BL/6,SHP-knockout,and FGF15-knockout mice. Mice were given FGF19 daily for 1 week; fractional cholesterol absorption,cholesterol and bile acid (BA) levels,and composition of BAs were measured. Intestinal organoids were generated from C57BL/6 and SHP-knockout mice,and cholesterol uptake was measured. Luciferase reporter assays were performed with HT29 cells. RESULTS We found that the genes that regulate lipid and ion transport in intestine,including NPC1L1,were up-regulated and that cholesterol absorption was increased in SHP-knockout mice compared with C57BL/6 mice. Expression of NPC1L1 was reduced in C57BL/6 mice after refeeding after fasting but not in SHP-knockout or FGF15-knockout mice. SHP-knockout mice had altered BA composition compared with C57BL/6 mice. FGF19 injection reduced expression of NPC1L1,decreased cholesterol absorption,and increased levels of hydrophilic BAs,including tauro-$\alpha$- and -$\beta$-muricholic acids; these changes were not observed in SHP-knockout mice. SREBF2 (sterol regulatory element binding transcription factor 2),which regulates cholesterol,activated transcription of NPC1L1. FGF19 signaling led to phosphorylation of SHP,which inhibited SREBF2 activity. CONCLUSIONS Postprandial FGF19 and SHP inhibit SREBF2,which leads to repression of intestinal NPC1L1 expression and cholesterol absorption. Strategies to increase FGF19 signaling to activate SHP might be developed for treatment of hypercholesterolemia. View Publication

BACKGROUND The combination of serum $\beta$2-microglubulin and albumin levels is highly prognostic in multiple myeloma (MM),defined as the International Staging System (ISS). Recurrent genomic abnormalities present in myeloma cells also have a strong prognostic power. This study aimed to assess,in a routine diagnostic setting,whether genomic aberrations can be used to identify sub-groups in ISS staging,as this system does not incorporate intrinsic myeloma cell variability at the molecular level. MATERIALS AND METHODS A prospective population-based study of 123 patients newly diagnosed with MM with ISS staging were included for karyotyping,interphase nuclei fluorescence in situ hybridization (iFISH) and oligo-based array comparative genomic hybridization (oaCGH) analyses. RESULTS Clonal abnormalities were identified in 27{\%} of analyses by karyotyping,in 83{\%} by iFISH,and in 99{\%} by oaCGH analysis. ISS staging combined with oaCGH aberrations identified ISS sub-groups. CONCLUSION oaCGH analysis is a valuable asset in detecting prognostically relevant genomic abnormalities. The combination of oaCGH data with ISS staging might help define new sub-groups in MM. View Publication

In addition to antigen-driven signals,T cells need co-stimulatory signals for robust activation. Several receptors,including members of the tumor necrosis factor receptor superfamily (TNFRSF),can deliver co-stimulatory signals to T cells. Thioredoxin interacting protein (TXNIP) is an important inhibitor of glucose uptake and cell proliferation,but it is unknown how TXNIP is regulated in T cells. The aim of this study was to determine expression levels and regulation of TXNIP in human T cells. We found that na{\{i}}ve T cells express high levels of TXNIP and that treatment of blood samples with TNF results in rapid down-regulation of TXNIP in the T cells. TNF-induced TXNIP down-regulation correlated with increased glucose uptake. Furthermore we found that density gradient centrifugation (DGC) induced down-regulation of TXNIP. We demonstrate that DGC induced TNF production that paralleled the TXNIP down-regulation. Treatment of blood with toll-like receptor (TLR) ligands induced TNF production and TXNIP down-regulation suggesting that damage-associated molecular patterns (DAMPs) such as endogenous TLR ligands released during DGC play a role in DGC-induced TXNIP down-regulation. Finally we demonstrate that TNF-induced TXNIP down-regulation is dependent on caspase activity and is caused by caspase-mediated cleavage of TXNIP." View Publication

BACKGROUND Upregulation of RNA polymerase (Pol) III products,including tRNAs and 5S rRNA,in tumor cells leads to enhanced protein synthesis and tumor formation,making it a potential target for cancer treatment. In this study,we evaluated the inhibition of Pol III transcription by triptolide and the anti-cancer effect of this drug in colorectal tumorigenesis. METHODS The effect of triptolide on colorectal cancer development was assessed in colorectal cancer mouse models,3D organoids,and cultured cells. Colorectal cancer cells were treated with triptolide. Pol III transcription was measured by real-time quantitative polymerase chain reaction (PCR). The formation of TFIIIB,a multi-subunit transcription factor for Pol III,was determined by chromatin immunoprecipitation (ChIP),co-immunoprecipitation (Co-IP),and fluorescence resonance energy transfer (FRET). RESULTS Triptolide reduced both tumor number and tumor size in adenomatous polyposis coli (Apc) mutated (ApcMin/+) mice as well as AOM/DSS-induced mice. Moreover,triptolide effectively inhibited colorectal cancer cell proliferation,colony formation,and organoid growth in vitro,which was associated with decreased Pol III target genes. Mechanistically,triptolide treatment blocked TBP/Brf1interaction,leading to the reduced formation of TFIIIB at the promoters of tRNAs and 5S rRNA. CONCLUSIONS Together,our data suggest that inhibition of Pol III transcription with existing drugs such as triptolide provides a new avenue for developing novel therapies for colorectal cancer. View Publication

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner,thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation,differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34+ human hematopoietic stem cells confirmed that expression of miR-130a inhibits monocyte differentiation by interfering with the expression of key transcription factors HOXA10,IRF8,KLF4,MAFB and PU-1. The data obtained in this study highlight that the correct modulation of miR-130a is necessary for normal differentiation to occur and confirming that deregulation of this miRNA might underlie the differentiation block occurring in AML. View Publication

Cyclic dinucleotides are second messengers in the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway,which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and inhibitor of nuclear factor-$\kappa$B (I$\kappa$B) kinase (IKK)/nuclear factor-$\kappa$B (NF$\kappa$B) signaling cascades. In this work,we describe an enzymatic preparation of 2'-5',3'-5'-cyclic dinucleotides (2'3'CDNs) with use of cyclic GMP-AMP synthases (cGAS) from human,mouse,and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide-5'-triphosphate (NTP) analogues and use them to prepare 33 2'3'CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell-based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next,we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect on monocytes. Additionally,we report X-ray crystal structures of two new CDNs bound to STING protein and discuss structure-activity relationship by using quantum and molecular mechanical (QM/MM) computational modeling. View Publication

Environmental factors contribute to Type 1 diabetes (T1D) susceptibility. The gut microbiome,which includes bacteria,viruses,and fungi,contributes to this environmental influence,and can induce immunological changes. The gut viral component of the microbiome,related to T1D has mostly focused on coxsackieviruses and rotavirus. The role of norovirus,another common enteric virus,in susceptibility to T1D was hitherto unknown. Norovirus is highly infectious and encountered by many children. We studied the mouse norovirus 4 (MNV4),related to human noroviruses,in the Non-obese diabetic (NOD) mouse model,to determine its role in influencing susceptibility to T1D. We infected MNV-free NOD mice with MNV4 by exposing the mice to MNV4-positive bedding from an endemically-infected mouse colony to mimic a natural infection. Control MNV-free NOD mice were exposed to MNV-free bedding from the same colony. Interestingly,MNV4 infection protected NOD mice from the development of T1D and was associated with an expansion of Tregs and reduced proinflammatory T cells. We also found MNV4 significantly modified the gut commensal bacteria composition,promoting increased $\alpha$-diversity and Firmicutes/Bacteroidetes ratio. To elucidate whether T1D protection was directly related to MNV4,or indirectly through modulating gut microbiota,we colonized germ-free (GF) NOD mice with the MNV4-containing or non-MNV4-containing viral filtrate,isolated from filtered fecal material. We found that MNV4 induced significant changes in mucosal immunity,including altered Tuft cell markers,cytokine secretion,antiviral immune signaling markers,and the concentration of mucosal antibodies. Systemically,MNV4-infection altered the immune cells including B cell subsets,macrophages and T cells,and especially induced an increase in Treg number and function. Furthermore,in vitro primary exposure of the norovirus filtrate to na{\{i}}ve splenocytes identified significant increases in the proportion of activated and CTLA4-expressing Tregs. Our data provide novel knowledge that norovirus can protect NOD mice from T1D development by inducing the expansion of Tregs and reducing inflammatory T cells. Our study also highlights the importance of distinguishing the mucosal immunity mediated by bacteria from that by enteric viruses." View Publication