MiR-9-1 controls osteoblastic regulation of lymphopoiesis.

The highly conserved MicroRNA-9 (miR-9) family consists of three members. We discovered that miR-9-1 deletion reduced mature miR-9 expression, causing 43% of the mice to display smaller size and postweaning lethality. MiR-9-1-deficient mice with growth defects experienced severe lymphopenia, but other blood cells were unaffected. The lymphopenia wasn't due to defects in hematopoietic progenitors, as mutant bone marrow (BM) cells underwent normal lymphopoiesis after transplantation into wild-type recipients. Additionally, miR-9-1-deficient mice exhibited impaired osteoblastic bone formation, as mutant mesenchymal stem cells (MSCs) failed to differentiate into osteoblastic cells (OBs). RNA sequencing revealed reduced expression of master transcription factors for osteoblastic differentiation, Runt-related transcription factor 2 (Runx2) and Osterix (Osx), and genes related to collagen formation, extracellular matrix organization, and cell adhesion, in miR-9-1-deficient MSCs. Follistatin (Fst), an antagonist of bone morphogenetic proteins (BMPs), was found to be a direct target of miR-9-1. Its deficiency led to the up-regulation of Fst, inhibiting BMP signaling in MSCs, and reducing IL-7 and IGF-1. Thus, miR-9-1 controls osteoblastic regulation of lymphopoiesis by targeting the Fst/BMP/Smad signaling axis.

Plasma tissue-type plasminogen activator is associated with lipoprotein(a) and clinical outcomes in hospitalized patients with COVID-19.

Background: Patients with COVID-19 have a higher risk of thrombosis and thromboembolism, but the underlying mechanism(s) remain to be fully elucidated. In patients with COVID-19, high lipoprotein(a) (Lp(a)) is positively associated with the risk of ischemic heart disease. Lp(a), composed of an apoB-containing particle and apolipoprotein(a) (apo(a)), inhibits the key fibrinolytic enzyme, tissue-type plasminogen activator (tPA). However, whether the higher Lp(a) associates with lower tPA activity, the longitudinal changes of these parameters in hospitalized patients with COVID-19, and their correlation with clinical outcomes are unknown. Objectives: To assess if Lp(a) associates with lower tPA activity in COVID-19 patients, and how in COVID-19 populations Lp(a) and tPA change post infection. Methods: Endogenous tPA enzymatic activity, tPA or Lp(a) concentration were measured in plasma from hospitalized patients with and without COVID-19. The association between plasma tPA and adverse clinical outcomes was assessed. Results: In hospitalized patients with COVID-19, we found lower tPA enzymatic activity and higher plasma Lp(a) than that in non-COVID-19 controls. During hospitalization, Lp(a) increased and tPA activity decreased, which associates with mortality. Among those who survived, Lp(a) decreased and tPA enzymatic activity increased during recovery. In patients with COVID-19, tPA activity is inversely correlated with tPA concentrations, thus, in another larger COVID-19 cohort, we utilized plasma tPA concentration as a surrogate to inversely reflect tPA activity. The tPA concentration was positively associated with death, disease severity, plasma inflammatory, and prothrombotic markers, and with length of hospitalization among those who were discharged. Conclusion: High Lp(a) concentration provides a possible explanation for low endogenous tPA enzymatic activity, and poor clinical outcomes in patients with COVID-19.

Molecular characterization stratifies VQ myeloma cells into two clusters with distinct risk signatures and drug responses.

Multiple myeloma (MM) is a cancer of malignant plasma cells in the bone marrow and extramedullary sites. We previously characterized a VQ model for human high-risk MM. The various VQ lines display different disease phenotypes and survival rates, suggesting significant intra-model variation. Here, we use whole-exome sequencing and copy number variation (CNV) analysis coupled with RNA-Seq to stratify the VQ lines into corresponding clusters: Group A cells had monosomy chromosome (chr) 5 and overexpressed genes and pathways associated with sensitivity to bortezomib (Btz) treatment in human MM patients. By contrast, Group B VQ cells carried recurrent amplification (Amp) of chr3 and displayed high-risk MM features, including downregulation of Fam46c, upregulation of cancer growth pathways associated with functional high-risk MM, and expression of Amp1q and high-risk UAMS-70 and EMC-92 gene signatures. Consistently, in sharp contrast to Group A VQ cells that showed short-term response to Btz, Group B VQ cells were de novo resistant to Btz in vivo. Our study highlights Group B VQ lines as highly representative of the human MM subset with ultrahigh risk.

CARD19, a Novel Regulator of the TAK1/NF-κB Pathway in Self-Reactive B Cells.

The caspase recruitment domain family member (CARD)11-Bcl10-Malt1 signalosome controls TGF-ß-activated kinase 1 (TAK1) activation and regulates BCR-induced NF-κB activation. In this study, we discovered that CARD19 interacted with TAK1 and inhibited TAB2-mediated TAK1 ubiquitination and activation. Although CARD19 deficiency in mice did not affect B cell development, it enhanced clonal deletion, receptor editing, and anergy of self-reactive B cells, and it reduced autoantibody production. Mechanistically, CARD19 deficiency increased BCR/TAK1-mediated NF-κB activation, leading to increased expression of transcription factors Egr2/3, as well as the E3 ubiquitin ligases c-Cbl/Cbl-b, which are known inducers of B cell tolerance in self-reactive B cells. RNA sequencing analysis revealed that although CARD19 deficiency did not affect the overall Ag-induced gene expression in naive B cells, it suppressed BCR signaling and increased hyporesponsiveness of self-reactive B cells. As a result, CARD19 deficiency prevented Bm12-induced experimental systemic lupus erythematosus. In summary, CARD19 negatively regulates BCR/TAK1-induced NF-κB activation and its deficiency increases Egr2/3 and c-Cbl/Cbl-b expression in self-reactive B cells, thereby enhancing B cell tolerance.

Cloned antibodies from patients with HIT provide new clues to HIT pathogenesis.

Heparin-induced thrombocytopenia (HIT) is a serious adverse drug reaction characterized by antibodies that recognize platelet factor 4/heparin complexes (PF4/H) and activate platelets to create a prothrombotic state. Although a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset also makes antibodies that are platelet activating (PA). A close correlation between PA antibodies and the likelihood of experiencing HIT has been demonstrated in clinical studies, but how PA (presumptively pathogenic) and nonactivating (NA) (presumptively benign) antibodies differ from each other at the molecular level is unknown. To address this issue, we cloned 7 PA and 47 NA PF4/H-binding antibodies from 6 patients with HIT and characterized their structural and functional properties. Findings showed that PA clones differed significantly from NA clones in possessing 1 of 2 heavy chain complementarity-determining region 3 (HCDR3) motifs, RX1-2R/KX1-2R/H (RKH) and YYYYY (Y5), in an unusually long complementarity-determining region 3 (≥20 residues). Mutagenic studies showed that modification of either motif in PA clones reduced or abolished their PA activity and that appropriate amino acid substitutions in HCDR3 of NA clones can cause them to become PA. Repertoire sequencing showed that the frequency of peripheral blood IgG+ B cells possessing RKH or Y5 was significantly higher in patients with HIT than in patients without HIT given heparin, indicating expansion of B cells possessing RKH or Y5 in HIT. These findings imply that antibodies possessing RKH or Y5 are relevant to HIT pathogenesis and suggest new approaches to diagnosis and treatment of this condition.

Differential roles of BAF and PBAF subunits, Arid1b and Arid2, in MLL-AF9 leukemogenesis.

The Switch/Sugar Non-Fermenting (SWI/SNF) nucleosome remodeling complexes play important roles in normal development and in the development of various cancers. Core subunits of the SWI/SNF complexes have been shown to have oncogenic roles in acute myeloid leukemia. However, the roles of the unique targeting subunits, including that of Arid2 and Arid1b, in AML leukemogenesis are not well understood. Here, we used conditional knockout mouse models to elucidate their role in MLL-AF9 leukemogenesis. We uncovered that Arid2 has dual roles; enhancing leukemogenesis when deleted during leukemia initiation and yet is required during leukemia maintenance. Whereas, deleting Arid1b in either phase promotes leukemogenesis. Our integrated analyses of transcriptomics and genomic binding data showed that, globally, Arid2 and Arid1b regulate largely distinct sets of genes at different disease stages, respectively, and in comparison, to each other. Amongst the most highly dysregulated transcription factors upon their loss, Arid2 and Arid1b converged on the regulation of Etv4/Etv5, albeit in an opposing manner while also regulating distinct TFs including Gata2,Tcf4, Six4, Irf4 and Hmgn3. Our data demonstrate the differential roles of SWI/SNF subunits in AML leukemogenesis and emphasize that cellular context and disease stage are key in determining their functions during this process.

LncRNA00492 is required for marginal zone B-cell development.

B-cell development undergoes a series of steps from the bone marrow to the secondary lymphoid organs. A defect in B-cell development can lead to immunodeficiency or malignant disorders, such as leukaemia or lymphoma. Long non-coding RNAs have been reported to act as important regulators of many pathological processes. However, very little is known regarding the role of lncRNAs during B-cell development and the regulation of their expression. In this study, we explored the expression and role of lncRNA Gme00492 in B-cell development. We observed that lnc00492 was highly expressed in B-cell development and primarily expressed in the nucleus. Lnc00492-deficient mice had fewer marginal zone B cells in the spleen, likely due to a developmental block. Importantly, lnc00492 interacts with CTBP1 and targets it for ubiquitination and degradation during B-cell development, whereas the transcriptional corepressor factor CTBP1 plays a critical role in Notch2 signalling. Thus, we identified a novel regulatory axis between lnc00492 and CTBP1 in B cells, suggesting that lnc00492 is essential for marginal zone B-cell development.

Immune cells surveil aberrantly sialylated O-glycans on megakaryocytes to regulate platelet count.

Immune thrombocytopenia (ITP) is a platelet disorder. Pediatric and adult ITP have been associated with sialic acid alterations, but the pathophysiology of ITP remains elusive, and ITP is often a diagnosis of exclusion. Our analysis of pediatric ITP plasma samples showed increased anti-Thomsen-Friedenreich antigen (TF antigen) antibody representation, suggesting increased exposure of the typically sialylated and cryptic TF antigen in these patients. The O-glycan sialyltransferase St3gal1 adds sialic acid specifically on the TF antigen. To understand if TF antigen exposure associates with thrombocytopenia, we generated a mouse model with targeted deletion of St3gal1 in megakaryocytes (MK) (St3gal1MK-/-). TF antigen exposure was restricted to MKs and resulted in thrombocytopenia. Deletion of Jak3 in St3gal1MK-/- mice normalized platelet counts implicating involvement of immune cells. Interferon-producing Siglec H-positive bone marrow (BM) immune cells engaged with O-glycan sialic acid moieties to regulate type I interferon secretion and platelet release (thrombopoiesis), as evidenced by partially normalized platelet count following inhibition of interferon and Siglec H receptors. Single-cell RNA-sequencing determined that TF antigen exposure by MKs primed St3gal1MK-/- BM immune cells to release type I interferon. Single-cell RNA-sequencing further revealed a new population of immune cells with a plasmacytoid dendritic cell-like signature and concomitant upregulation of the immunoglobulin rearrangement gene transcripts Igkc and Ighm, suggesting additional immune regulatory mechanisms. Thus, aberrant TF antigen moieties, often found in pathological conditions, regulate immune cells and thrombopoiesis in the BM, leading to reduced platelet count.

SARS-CoV-2 receptor binding domain-specific antibodies activate platelets with features resembling the pathogenic antibodies in heparin-induced thrombocytopenia.

Severe COVID-19 is associated with unprecedented thromboembolic complications. We found that hospitalized COVID-19 patients develop immunoglobulin Gs (IgGs) that recognize a complex consisting of platelet factor 4 and heparin similar to those developed in heparin-induced thrombocytopenia and thrombosis (HIT), however, independent of heparin exposure. These antibodies activate platelets in the presence of TLR9 stimuli, stimuli that are prominent in COVID-19. Strikingly, 4 out of 42 antibodies cloned from IgG1+ RBD-binding B cells could activate platelets. These antibodies possessed, in the heavy-chain complementarity-determining region 3, an RKH or Y5 motif that we recently described among platelet-activating antibodies cloned from HIT patients. RKH and Y5 motifs were prevalent among published RBD-specific antibodies, and 3 out of 6 such antibodies tested could activate platelets. Features of platelet activation by these antibodies resemble those by pathogenic HIT antibodies. B cells with an RKH or Y5 motif were robustly expanded in COVID-19 patients. Our study demonstrates that SARS-CoV-2 infection drives the development of a subset of RBD-specific antibodies that can activate platelets and have activation properties and structural features similar to those of the pathogenic HIT antibodies.

Nras Q61R/+ and Kras-/- cooperate to downregulate Rasgrp1 and promote lympho-myeloid leukemia in early T-cell precursors.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive subtype of T-cell ALL. Although genetic mutations hyperactivating cytokine receptor/Ras signaling are prevalent in ETP-ALL, it remains unknown how activated Ras signaling contributes to ETP-ALL. Here, we find that in addition to the frequent oncogenic RAS mutations, wild-type (WT) KRAS transcript level was significantly downregulated in human ETP-ALL cells. Similarly, loss of WT Kras in NrasQ61R/+ mice promoted hyperactivation of extracellular signal-regulated kinase (ERK) signaling, thymocyte hyperproliferation, and expansion of the ETP compartment. Kras-/-; NrasQ61R/+ mice developed early onset of T-cell malignancy that recapitulates many biological and molecular features of human ETP-ALL. Mechanistically, RNA-sequencing analysis and quantitative proteomics study identified that Rasgrp1, a Ras guanine nucleotide exchange factor, was greatly downregulated in mouse and human ETP-ALL. Unexpectedly, hyperactivated Nras/ERK signaling suppressed Rasgrp1 expression and reduced Rasgrp1 level led to increased ERK signaling, thereby establishing a positive feedback loop to augment Nras/ERK signaling and promote cell proliferation. Corroborating our cell line data, Rasgrp1 haploinsufficiency induced Rasgrp1 downregulation and increased phosphorylated ERK level and ETP expansion in NrasQ61R/+ mice. Our study identifies Rasgrp1 as a negative regulator of Ras/ERK signaling in oncogenic Nras-driven ETP-like leukemia.

Arid2 regulates hematopoietic stem cell differentiation in normal hematopoiesis.

The switch/sugar nonfermenting (SWI/SNF) family of chromatin remodeling complexes have been implicated in normal hematopoiesis. The ARID2 protein is a component of the polybromo-associated BAF (PBAF), one of the two main SWI/SNF complexes. In the current study, we used a conditional Arid2 knockout mouse model to determine its role in normal hematopoiesis. We found that the loss of Arid2 has no discernable effects on steady-state hematopoiesis, with the exception of a modest effect on erythropoiesis. On bone marrow transplantation, however, the loss of Arid2 affects HSC differentiation in a cell-autonomous manner, resulting in significant decreases in the ability to reconstitute the lymphoid lineage. Gene expression analysis of Arid2 knockout cells revealed enrichment of myeloid-biased multipotent progenitor (MPP) cell signatures, while the lymphoid-biased MPPs are enriched in the wild type, consistent with the observed phenotype. Moreover, Arid2 knockout cells revealed enrichment of inflammatory pathways with upregulation of TLR receptors, as well as downstream signaling cascade genes. Furthermore, under lymphocyte-biased growth conditions in vitro, Arid2 null bone marrow cells have significantly impaired proliferation, which decreased further on lipopolysaccharide stimulation. Overall, these data suggest that the loss of Arid2 impairs HSC differentiation ability, and this effect may be mediated through upregulation of inflammatory pathways.

Expression of NrasQ61R and MYC transgene in germinal center B cells induces a highly malignant multiple myeloma in mice.

NRAS Q61 mutations are prevalent in advanced/relapsed multiple myeloma (MM) and correlate with poor patient outcomes. Thus, we generated a novel MM model by conditionally activating expression of endogenous NrasQ61R and an MYC transgene in germinal center (GC) B cells (VQ mice). VQ mice developed a highly malignant MM characterized by a high proliferation index, hyperactivation of extracellular signal-regulated kinase and AKT signaling, impaired hematopoiesis, widespread extramedullary disease, bone lesions, kidney abnormalities, preserved programmed cell death protein 1 and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain immune-checkpoint pathways, and expression of human high-risk MM gene signatures. VQ MM mice recapitulate most of the biological and clinical features of human advanced/high-risk MM. These MM phenotypes are serially transplantable in syngeneic recipients. Two MM cell lines were also derived to facilitate future genetic manipulations. Combination therapies based on MEK inhibition significantly prolonged the survival of VQ mice with advanced-stage MM. Our study provides a strong rationale to develop MEK inhibition-based therapies for treating advanced/relapsed MM.

BAD inactivation exacerbates rheumatoid arthritis pathology by promoting survival of sublining macrophages.

The resistance of synovial sublining macrophages to apoptosis has a crucial role in joint inflammation and destruction in rheumatoid arthritis (RA). However, the underlying mechanism is incompletely understood. Here we report that inactivation of the pro-apoptotic BCL-2 family protein BAD is essential for survival of synovial sublining macrophage in RA. Genetic disruption of Bad leads to more severe joint inflammation and cartilage and bone damage with reduced apoptosis of synovial sublining macrophages in collagen-induced arthritis (CIA) and TNFα transgenic (TNF-Tg) mouse models. Conversely, Bad3SA/3SA mice, in which BAD can no longer be inactivated by phosphorylation, are protected from collagen-induced arthritis. Mechanistically, phosphorylation-mediated inactivation of BAD specifically protects synovial sublining macrophages from apoptosis in highly inflammatory environment of arthritic joints in CIA and TNF-Tg mice, and in patients with RA, thereby contributing to RA pathology. Our findings put forward a model in which inactivation of BAD confers the apoptosis resistance on synovial sublining macrophages, thereby contributing to the development of arthritis, suggesting that BAD may be a potential therapeutic target for RA.

Kras-Deficient T Cells Attenuate Graft-versus-Host Disease but Retain Graft-versus-Leukemia Activity.

Acute graft-versus-host disease (aGVHD) is one major serious complication that is induced by alloreactive donor T cells recognizing host Ags and limits the success of allogeneic hematopoietic stem cell transplantation. In the current studies, we identified a critical role of Kras in regulating alloreactive T cell function during aGVHD. Kras deletion in donor T cells dramatically reduced aGVHD mortality and severity in an MHC-mismatched allogeneic hematopoietic stem cell transplantation mouse model but largely maintained the antitumor capacity. Kras-deficient CD4 and CD8 T cells exhibited impaired TCR-induced activation of the ERK pathway. Kras deficiency altered TCR-induced gene expression profiles, including the reduced expression of various inflammatory cytokines and chemokines. Moreover, Kras deficiency inhibited IL-6-mediated Th17 cell differentiation and impaired IL-6-induced ERK activation and gene expression in CD4 T cells. These findings support Kras as a novel and effective therapeutic target for aGVHD.

Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation.

Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.