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Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells2–6. CRISPR–Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.Optimized conditions for ribonucleoprotein delivery of Cas9–sgRNA complexes enables precise and efficient gene editing to restore fetal hemoglobin expression in sickle cell disease patient-derived HSCs

Capture and isolation of flowing cells and particulates from body fluids has enormous implications in diagnosis, monitoring, and drug testing, yet monovalent adhesion molecules used for this purpose result in inefficient cell capture and difficulty in retrieving the captured cells. Inspired by marine creatures that present long tentacles containing multiple adhesive domains to effectively capture flowing food particulates, we developed a platform approach to capture and isolate cells using a 3D DNA network comprising repeating adhesive aptamer domains that extend over tens of micrometers into the solution. The DNA network was synthesized from a microfluidic surface by rolling circle amplification where critical parameters, including DNA graft density, length, and sequence, could readily be tailored. Using an aptamer that binds to protein tyrosine kinase-7 (PTK7) that is overexpressed on many human cancer cells, we demonstrate that the 3D DNA network significantly enhances the capture efficiency of lymphoblast CCRF-CEM cells over monovalent aptamers and antibodies, yet maintains a high purity of the captured cells. When incorporated in a herringbone microfluidic device, the 3D DNA network not only possessed significantly higher capture efficiency than monovalent aptamers and antibodies, but also outperformed previously reported cell-capture microfluidic devices at high flow rates. This work suggests that 3D DNA networks may have broad implications for detection and isolation of cells and other bioparticles.

Cyclophilin A, secreted by bone marrow endothelial cells, acts as a chemotactic factor for myeloma cells, which helps explain their homing to the bone marrow and suggests a potential new therapeutic strategy. B cell malignancies frequently colonize the bone marrow. The mechanisms responsible for this preferential homing are incompletely understood. Here we studied multiple myeloma (MM) as a model of a terminally differentiated B cell malignancy that selectively colonizes the bone marrow. We found that extracellular CyPA (eCyPA), secreted by bone marrow endothelial cells (BMECs), promoted the colonization and proliferation of MM cells in an in vivo scaffold system via binding to its receptor, CD147, on MM cells. The expression and secretion of eCyPA by BMECs was enhanced by BCL9, a Wnt–β-catenin transcriptional coactivator that is selectively expressed by these cells. eCyPA levels were higher in bone marrow serum than in peripheral blood in individuals with MM, and eCyPA-CD147 blockade suppressed MM colonization and tumor growth in the in vivo scaffold system. eCyPA also promoted the migration of chronic lymphocytic leukemia and lymphoplasmacytic lymphoma cells, two other B cell malignancies that colonize the bone marrow and express CD147. These findings suggest that eCyPA-CD147 signaling promotes the bone marrow homing of B cell malignancies and offer a compelling rationale for exploring this axis as a therapeutic target for these malignancies.

Lymphoblastic leukemia/lymphoma, a neoplasm of precursor B or T lineage lymphoid cells, usually involves the bone marrow and peripheral blood, and may involve nodal and/or extranodal sites. The diagnosis is based on morphologic assessment, immunophenotypic analysis, usually by flow cytometry, and genetic analysis, including cytogenetics and FISH analysis, as well as molecular diagnostic analysis. This review will focus on the flow cytometric immunophenotypic findings in B- and T-lymphoblastic leukemia/lymphoma, which include expressions of early B or T cell markers, low-level expressions of CD45, as well as expressions of terminal deoxynucleotidyl transferase (TdT), and, in many cases, stem/progenitor cell marker CD34.

Clinical uncertainty exists regarding which assay should be designated as the standard monitoring coagulation test for intravenous unfractionated heparin (UFH). Several studies have compared the use of activated partial thromboplastin time (aPTT) and antifactor-Xa (anti-Xa) and have come out with varying results. The correlation between these 2 tests varied, markedly from strong to weak. Some have demonstrated that monitoring with anti-Xa heparin assay leads to fewer dose adjustments, resulting in fewer laboratory tests, while others have not. In the current study, we evaluated the correlation between aPTT and anti-Xa values to guide clinical management of UFH, with the intention to develop a new correlation nomogram.

A case of hypothyroidism with a very high requirement for thyroxine replacement was found to be due to production of type 3 iodothyronine deiodinase (D3) by a gastrointestinal stromal tumor (GIST). Of other patients with this tumor type, 82% had D3-producing tumors. A 51-year-old man was found to have a large abdominal tumor during elective inguinal hernia repair. Given the absence of disseminated disease, a partial gastrectomy and transverse colectomy with en bloc resection of the primary tumor were performed with curative intent. Pathological examination led to the diagnosis of a GIST. Two years later, computed tomographic surveillance revealed liver lesions that were confirmed on biopsy as metastatic GIST. This finding prompted the initiation of treatment with imatinib mesylate (which was discontinued after 10 months owing to disease progression) and then sorafenib (which was discontinued after 5 months owing to disease progression). . . .

To better characterize the heterogeneity of multiple myeloma (MM), we profiled plasma cells (PCs) and their B cell lymphopoiesis in the BM samples from patients with monoclonal gammopathy of undetermined significance, smoldering MM, and active MM by mass cytometry (CyTOF) analysis. Characterization of intra- and interneoplastic heterogeneity of malignant plasmablasts and PCs revealed overexpression of the MM SET domain (MMSET), Notch-1, and CD47. Variations in upregulation of B cell signaling regulators (IFN regulatory factor 4 [IRF-4], CXCR4, B cell lymphoma 6 [Bcl-6], c-Myc, myeloid differentiation primary response protein 88 [MYD88], and spliced X box-binding protein 1 [sXBP-1]) and aberrant markers (CD319, CD269, CD200, CD117, CD56, and CD28) were associated with different clinical outcomes in clonal PC subsets. In addition, prognosis was related to heterogeneity in subclonal expression of stemness markers, including neuroepithelial stem cell protein (Nestin), SRY-box transcription factor 2 (Sox2), Krüppel-like factor 4 (KLF-4), and Nanog. Furthermore, we have defined significantly elevated levels of MMSET, MYD88, c-Myc, CD243, Notch-1, and CD47 from hematopoietic stem cells to PCs in myeloma B cell lymphopoiesis, noted even in premalignant conditions, with variably modulated expression of B cell development regulators, including IRF-4, Bcl-2, Bcl-6, and sXBP-1; aberrant PC markers (such as CD52, CD44, CD200, CD81, CD269, CD117, and CXCR4); and stemness-controlling regulators, including Nanog, KLF-4, octamer-binding transcription factor 3/4 (Oct3/4), Sox2, and retinoic acid receptor α2 (RARα2). This study provides the rationale for precise molecular profiling of patients with MM by CyTOF technology to define disease heterogeneity and prognosis.

Novel drug discoveries have shifted the treatment paradigms of most hematological malignancies, including multiple myeloma (MM). However, this plasma cell malignancy remains incurable, and novel therapies are therefore urgently needed. Whole-genome transcriptome analyses in a large cohort of MM patients demonstrated that alterations in pre-mRNA splicing (AS) are frequent in MM. This manuscript describes approaches to identify disease-specific alterations in MM and proposes RNA-based therapeutic strategies to eradicate such alterations. As a “proof of concept”, we examined the causes of aberrant HMMR (Hyaluronan-mediated motility receptor) splicing in MM. We identified clusters of single nucleotide variations (SNVs) in the HMMR transcript where the altered splicing took place. Using bioinformatics tools, we predicted SNVs and splicing factors that potentially contribute to aberrant HMMR splicing. Based on bioinformatic analyses and validation studies, we provided the rationale for RNA-based therapeutic strategies to selectively inhibit altered HMMR splicing in MM. Since splicing is a hallmark of many cancers, strategies described herein for target identification and the design of RNA-based therapeutics that inhibit gene splicing can be applied not only to other genes in MM but also more broadly to other hematological malignancies and solid tumors as well.

To evaluate CD200 expression in B-cell proliferative disorders. We analyzed 180 recent specimens of B-cell neoplasms for CD200 expression by flow cytometric immunophenotypic analysis, which is better able to assess relative intensity of staining than immunohistochemical staining. We found that hairy cell leukemia exhibits a high level of staining for CD200 in comparison to other B-cell lymphoproliferative disorders, including hairy cell leukemia-variant (HCL-V), marginal zone lymphoma, and lymphoplasmacytic lymphoma. We confirmed this observation by semiquantitative immunohistochemical staining. Assessment of the CD200 expression level is helpful to distinguish HCL from HCL-V and other B-cell lymphoproliferative disorders and in the differential diagnosis of B-cell neoplasms in general.

Tissue remodeling with fibrosis is a predominant pathophysiological mechanism of many human diseases. Systemic sclerosis is a rare, often lethal, disorder of unknown etiology manifested by dermal fibrosis (scleroderma) and excessive connective tissue deposition in internal organs. Currently, there are no available antifibrotic therapeutics, a reflection of our lack of understanding of this process. Animal models of scleroderma are useful tools to dissect the transcription factors and cytokines that govern fibrosis. A disproportionate increase of type 2 cytokines, like TGF-β and IL-4, more than type 1 cytokines, like IFN-γ, is thought to underlie the pathogenesis of scleroderma. In this study, we show that mice deficient in the transcription factor T-box expressed in T cells (T-bet), a master regulator of type 1 immunity, display increased sensitivity to bleomycin-induced dermal sclerosis. Despite the well-established role of T-bet in adaptive immunity, we also show that RAG2⁻/⁻ mice, which lack T and B cells, are vulnerable to bleomycin-induced scleroderma and that RAG2/T-bet double-deficient mice maintain the increased sensitivity to bleomycin observed in T-bet⁻/⁻ mice. Furthermore, overexpression of T-bet in T cells does not affect the induction of skin sclerosis in this model. Lastly, we show that IL-13 is the profibrotic cytokine regulated by T-bet in this model. Together, we conclude that T-bet serves as a repressor of dermal sclerosis through an IL-13-dependent pathway in innate immune cells. T-bet, and its transcriptional network, represent an attractive target for the treatment of systemic sclerosis and other fibrosing disorders.