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Development of targeted cancer therapy requires a thorough understanding of mechanisms of tumorigenesis as well as mechanisms of action of therapeutics. This is challenging because by the time patients are diagnosed with cancer,early events of tumorigenesis have already taken place. Similarly,development of cancer immunotherapies is hampered by a lack of appropriate small animal models with autologous human tumor and immune system. In this article,we report the development of a mouse model of human acute myeloid leukemia (AML) with autologous immune system for studying early events of human leukemogenesis and testing the efficacy of immunotherapeutics. To develop such a model,human hematopoietic stem/progenitor cells (HSPC) are transduced with lentiviruses expressing a mutated form of nucleophosmin (NPM1),referred to as NPM1c. Following engraftment into immunodeficient mice,transduced HSPCs give rise to human myeloid leukemia,whereas untransduced HSPCs give rise to human immune cells in the same mice. The de novo AML,with CD123+ leukemic stem or initiating cells (LSC),resembles NPM1c+ AML from patients. Transcriptional analysis of LSC and leukemic cells confirms similarity of the de novo leukemia generated in mice with patient leukemia and suggests Myc as a co-operating factor in NPM1c-driven leukemogenesis. We show that a bispecific conjugate that binds both CD3 and CD123 eliminates CD123+ LSCs in a T cell-dependent manner both in vivo and in vitro. These results demonstrate the utility of the NPM1c+ AML model with an autologous immune system for studying early events of human leukemogenesis and for evaluating efficacy and mechanism of immunotherapeutics. View Publication

Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM),a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation,we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast,in ML-DS,clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model,we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically,genetically,and transcriptionally mirrored ML-DS. View Publication

TET enzymes are dioxygenases that promote DNA demethylation by oxidizing the methyl group of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Here,we report a close correspondence between 5hmC-marked regions,chromatin accessibility and enhancer activity in B cells,and a strong enrichment for consensus binding motifs for basic region-leucine zipper (bZIP) transcription factors at TET-responsive genomic regions. Functionally,Tet2 and Tet3 regulate class switch recombination (CSR) in murine B cells by enhancing expression of Aicda,which encodes the activation-induced cytidine deaminase (AID) enzyme essential for CSR. TET enzymes deposit 5hmC,facilitate DNA demethylation,and maintain chromatin accessibility at two TET-responsive enhancer elements,TetE1 and TetE2,located within a superenhancer in the Aicda locus. Our data identify the bZIP transcription factor,ATF-like (BATF) as a key transcription factor involved in TET-dependent Aicda expression. 5hmC is not deposited at TetE1 in activated Batf-deficient B cells,indicating that BATF facilitates TET recruitment to this Aicda enhancer. Our study emphasizes the importance of TET enzymes for bolstering AID expression and highlights 5hmC as an epigenetic mark that captures enhancer dynamics during cell activation. 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

The subset of regulatory T (Treg) cells,with its specific transcription Foxp3,is a unique cell type for the maintenance of immune homeostasis by controlling effector T (Teff) cell responses. Although it is common that a defect in Treg cells with Treg/Teff disorder causes autoimmune diseases; however,the precise mechanisms are not thoroughly revealed. Here,we report that miR-34a could attenuate human and murine Foxp3 gene expression via targeting their 3' untranslated regions (3' UTR). The human miR-34a,increased in peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) patients,displayed a positive correlation with some serum markers of inflammation including rheumatoid factor (RF),anti-streptolysin antibody (ASO),erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) as well as Th17 signature gene RORgammat,but inversely correlated with the mRNA expression levels of FOXP3. In addition,murine miR-34a levels were downregulated in TGF-beta-induced Treg cells but upregulated in Th17 cells induced in vitro compared to activated CD4+ T cells. It has also been demonstrated that elevated miR-34a disrupting Treg/Th17 balance in vivo contributed to the progress of pathogenesis of collagen induced arthritis (CIA) mice. Furthermore,IL-6 and TNF-alpha were responsible for the upregulation of miR-34a and downregulation of Foxp3,which was reverted by the addition of NF-kappaB/p65 inhibitor BAY11-7082,thus indicating that NF-kappaB/p65 inhibited Foxp3 expression in an miR-34a-dependent manner. Finally,IL-6 or TNF-alpha-activated p65 could bind to the miR-34a promotor and enhance its activity,resulting in upregulation of its transcription. Taken together,we show that NF-kappaB activated by inflammatory cytokines,such as IL-6 and TNF-alpha,ameliorates Foxp3 levels via regulating miR-34a expression,which provides a new mechanistic and therapeutic insight into the ongoing of autoimmune diseases. View Publication

Rheumatoid Arthritis (RA) causes chronic inflammation of joints. The cytokines TNFalpha and IFNgamma are central players in RA,however their source has not been fully elucidated. Natural Killer (NK) cells are best known for their role in elimination of viral-infected and transformed cells,and they secrete pro-inflammatory cytokines. NK cells are present in the synovial fluids (SFs) of RA patients and are considered to be important in bone destruction. However,the phenotype and function of NK cells in the SFs of patients with erosive deformative RA (DRA) versus non-deformative RA (NDRA) is poorly characterized. Here we characterize the NK cell populations present in the blood and SFs of DRA and NDRA patients. We demonstrate that a distinct population of activated synovial fluid NK (sfNK) cells constitutes a large proportion of immune cells found in the SFs of DRA patients. We discovered that although sfNK cells in both DRA and NDRA patients have similar phenotypes,they function differently. The DRA sfNK secrete more TNFalpha and IFNgamma upon exposure to IL-2 and IL-15. Consequently,we suggest that sfNK cells may be a marker for more severely destructive RA disease. View Publication

We introduce machine learning (ML) to perform classification and quantitation of images of nuclei from human blood neutrophils. Here we assessed the use of convolutional neural networks (CNNs) using free,open source software to accurately quantitate neutrophil NETosis,a recently discovered process involved in multiple human diseases. CNNs achieved {\textgreater}94{\%} in performance accuracy in differentiating NETotic from non-NETotic cells and vastly facilitated dose-response analysis and screening of the NETotic response in neutrophils from patients. Using only features learned from nuclear morphology,CNNs can distinguish between NETosis and necrosis and between distinct NETosis signaling pathways,making them a precise tool for NETosis detection. Furthermore,by using CNNs and tools to determine object dispersion,we uncovered differences in NETotic nuclei clustering between major NETosis pathways that is useful in understanding NETosis signaling events. Our study also shows that neutrophils from patients with sickle cell disease were unresponsive to one of two major NETosis pathways. Thus,we demonstrate the design,performance,and implementation of ML tools for rapid quantitative and qualitative cell analysis in basic science. View Publication

Despite the clinical success of cancer immunotherapies,the majority of patients fail to respond or develop resistance through disruption of pathways that promote neo-antigen presentation on MHC I molecules. Here,we conducted a series of unbiased,genome-wide CRISPR/Cas9 screens to identify genes that limit natural killer (NK) cell anti-tumor activity. We identified that genes associated with antigen presentation and/or interferon-$\gamma$ (IFN-$\gamma$) signaling protect tumor cells from NK cell killing. Indeed,Jak1-deficient melanoma cells were sensitized to NK cell killing through attenuated NK cell-derived IFN-$\gamma$-driven transcriptional events that regulate MHC I expression. Importantly,tumor cells that became resistant to T cell killing through enrichment of MHC I-deficient clones were highly sensitive to NK cell killing. Taken together,we reveal the genes targeted by tumor cells to drive checkpoint blockade resistance but simultaneously increase their vulnerability to NK cells,unveiling NK cell-based immunotherapies as a strategy to antagonize tumor immune escape. View Publication

Recurrent oncogenic mutations of MyD88 have been identified in a variety of lymphoid malignancies. Gain-of-function mutations of MyD88 constitutively activate downstream NF-$\kappa$B signaling pathways,resulting in increased cellular proliferation and survival. However,whether MyD88 activity can be aberrantly regulated in MyD88-wild-type lymphoid malignancies remains poorly understood. SPOP is an adaptor protein of CUL3-based E3 ubiquitin ligase complex and frequently mutated genes in prostate and endometrial cancers. In this study,we reveal that SPOP binds to and induces the nondegradative ubiquitination of MyD88 by recognizing an atypical SPOP-binding motif in MyD88. This modification blocks Myddosome assembly and downstream NF-$\kappa$B activation. SPOP is mutated in a subset of lymphoid malignancies,including diffuse large B-cell lymphoma (DLBCL). Lymphoid malignancies-associated SPOP mutants exhibited impaired binding to MyD88 and suppression of NF-$\kappa$B activation. The DLBCL-associated,SPOP-binding defective mutants of MyD88 escaped from SPOP-mediated ubiquitination,and their effect on NF-$\kappa$B activation is stronger than that of wild-type MyD88. Moreover,SPOP suppresses DLBCL cell growth in vitro and tumor xenograft in vivo by inhibiting the MyD88/NF-$\kappa$B signaling. Therefore,SPOP acts as a tumor suppressor in DLBCL. Mutations in the SPOP-MyD88 binding interface may disrupt the SPOP-MyD88 regulatory axis and promote aberrant MyD88/NF-$\kappa$B activation and cell growth in DLCBL. View Publication

Our previous studies have demonstrated that a previously unrecognized role of CFTR in hematopoiesis and acute leukemia. Here,we show that CFTR inhibitor CFTR-inh172 possesses ability to inhibit human T-cell acute lymphoblastic leukemia cells. In detail,CFTR-inh172 inhibited cell proliferation,promoted apoptosis and arrested the cell cycle in human T-cell acute lymphoblastic leukemia cell CCRF-CEM,JURKAT and MOLT-4. Furthermore,transcriptome analysis reveals that CFTR-inh172 induces significant alteration of gene expression related to apoptosis and proliferation. These findings demonstrate the potential of CFTR inhibitor CFTR-inh172 in human T-cell acute lymphoblastic leukemia treatment. View Publication

Despite the key role that antibodies play in protection,the cellular processes mediating the acquisition of humoral immunity against malaria are not fully understood. Using an infection model of severe malaria,we find that germinal center (GC) B cells upregulate the transcription factor T-bet during infection. Molecular and cellular analyses reveal that T-bet in B cells is required not only for IgG2c switching but also favors commitment of B cells to the dark zone of the GC. T-bet was found to regulate the expression of Rgs13 and CXCR3,both of which contribute to the impaired GC polarization observed in the absence of T-bet,resulting in reduced IghV gene mutations and lower antibody avidity. These results demonstrate that T-bet modulates GC dynamics,thereby promoting the differentiation of B cells with increased affinity for antigen. View Publication