Microbial ligand-independent regulation of lymphopoiesis by NOD1.

Aberrant differentiation of progenitor cells in the hematopoietic system is known to severely impact host immune responsiveness. Here we demonstrate that NOD1, a cytosolic innate sensor of bacterial peptidoglycan, also functions in murine hematopoietic cells as a major regulator of both the generation and differentiation of lymphoid progenitors as well as peripheral T lymphocyte homeostasis. We further show that NOD1 mediates these functions by facilitating STAT5 signaling downstream of hematopoietic cytokines. In steady-state, loss of NOD1 resulted in a modest but significant decrease in numbers of mature T, B and natural killer cells. During systemic protozoan infection this defect was markedly enhanced, leading to host mortality. Lack of functional NOD1 also impaired T cell-dependent anti-tumor immunity while preventing colitis. These findings reveal that, in addition to its classical role as a bacterial ligand receptor, NOD1 plays an important function in regulating adaptive immunity through interaction with a major host cytokine signaling pathway.

Targeting EPHA2 with Kinase Inhibitors in Colorectal Cancer.

The ephrin type-A 2 receptor tyrosine kinase (EPHA2) is involved in the development and progression of various cancer types, including colorectal cancer (CRC). There is also evidence that EPHA2 plays a key role in the development of resistance to the endothelial growth factor receptor (EGFR) monoclonal antibody Cetuximab used clinically in CRC. Despite the promising pharmacological potential of EPHA2, only a handful of specific inhibitors are currently available. In this concept paper, general strategies for EPHA2 inhibition with molecules of low molecular weight (small molecules) are described. Furthermore, available examples of inhibiting EPHA2 in CRC using small molecules are summarized, highlighting the potential of this approach.

Optimization of the Lead Compound NVP-BHG712 as a Colorectal Cancer Inhibitor.

The ephrin type-A receptor 2 (EPHA2) kinase belongs to the largest family of receptor tyrosine kinases. There are several indications of an involvement of EPHA2 in the development of infectious diseases and cancer. Despite pharmacological potential, EPHA2 is an under-examined target protein. In this study, we synthesized a series of derivatives of the inhibitor NVP-BHG712 and triazine-based compounds. These compounds were evaluated to determine their potential as kinase inhibitors of EPHA2, including elucidation of their binding mode (X-ray crystallography), affinity (microscale thermophoresis), and selectivity (Kinobeads assay). Eight inhibitors showed affinities in the low-nanomolar regime (KD <10 nM). Testing in up to seven colon cancer cell lines that express EPHA2 reveals that several derivatives feature promising effects for the control of human colon carcinoma. Thus, we have developed a set of powerful tool compounds for fundamental new research on the interplay of EPH receptors in a cellular context.

Antisense transcription from lentiviral gene targeting linked to an integrated stress response in colorectal cancer cells.

Advances in gene therapy research have resulted in the successful development of new therapies for clinical use. Here, we explored a gene targeting approach to deplete ephrinB2 from colorectal cancer cells using an inducible lentiviral vector. EphrinB2, a transmembrane ephrin ligand, promotes colorectal cancer cell growth and viability and predicts poor patient survival when expressed at high levels in colorectal cancer tissues. We discovered that lentiviral vector integration and expression in the host DNA frequently drive divergent host gene transcription, generating antisense reads coupled with splicing events and generation of chimeric vector/host transcripts. Antisense transcription of host DNA was linked to development of an integrated stress response and cell death. Despite recent successes, off-target effects remain a concern in genetic medicine. Our results provide evidence that divergent gene transcription is a previously unrecognized off-target effect of lentiviral vector integration with built-in properties for regulation of gene expression.

Inactivation of axon guidance molecule netrin-1 in human colorectal cancer by an epigenetic mechanism.

Netrin-1, the protein product of the NTN1 gene, is an axon guidance molecule implicated in regulation of cell survival and tumorigenesis. Expression of the netrin-1 receptors deleted in colorectal cancer (DCC) and uncoordinated 5 homolog (UNC5H) is frequently silenced in colorectal cancer (CRC) by either loss of heterozygosity or epigenetic mechanisms. However, netrin-1 expression and regulation in CRC are mostly unknown. Here, we report that NTN1 expression is significantly reduced in most CRC tissues compared to the adjacent normal intestinal mucosa, and that NTN1 DNA methylation is significantly higher in CRCs (24.6%) than in the adjacent normal intestinal mucosa (4.0%). In 6 CRC cell lines, NTN1 expression is low. Treatment with 5-Aza-2'-deoxycytidine increased expression of NTN1 in CRC cell lines, indicating that DNA methylation represses NTN1 transcription in CRCs. NTN1 DNA hypermethylation was significantly associated with advanced CRC disease. Median netrin-1 serum levels were significantly decreased in CRC patients (330.1 pg/mL) compared with normal individuals (438.6 pg/mL). Our results suggest that netrin-1 is a candidate biomarker for CRC.

Reusable Single Cell for Iterative Epigenomic Analyses.

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses. In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell. Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.

Bone marrow niches in myelodysplastic syndromes.

Genetic and epigenetic lesions within hematopoietic cell populations drive diverse hematological malignancies. Myelodysplastic syndromes (MDS) are a group of myeloid neoplasms affecting the hematopoietic stem cells characterized by recurrent genetic abnormalities, myelodysplasia (a pathological definition of abnormal bone marrow structure), ineffective hematopoiesis resulting in blood cytopenia, and a propensity to evolve into acute myelogenous leukemia. Although there is evidence that the accumulation of a set of genetic mutations is an essential event in MDS, there is an increased appreciation of the contribution of specific microenvironments, niches, in the pathogenesis of MDS and response to treatment. In physiologic hematopoiesis, niches are critical functional units that maintain hematopoietic stem and progenitor cells and regulate their maturation into mature blood cells. In MDS and other hematological malignancies, altered bone marrow niches can promote the survival and expansion of mutant hematopoietic clones and provide a shield from therapy. In this review, we focus on our understanding of the composition and function of hematopoietic niches and their role in the evolution of myeloid malignancies, with an emphasis on MDS.

Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth.

The tyrosine phosphatase SHP2 is oncogenic in cancers driven by receptor-tyrosine-kinases, and SHP2 inhibition reduces tumor growth. Here, we report that SHP2 is an essential promoter of endothelial cell survival and growth in the remodeling tumor vasculature. Using genetic and chemical approaches to inhibit SHP2 activity in endothelial cells, we show that SHP2 inhibits pro-apoptotic STAT3 and stimulates proliferative ERK1/2 signaling. Systemic SHP2 inhibition in mice bearing tumor types selected for SHP2-independent tumor cell growth promotes degeneration of the tumor vasculature and blood extravasation; reduces tumor vascularity and blood perfusion; and increases tumor necrosis. Reduction of tumor growth ensues, independent of SHP2 targeting in the tumor cells, blocking immune checkpoints, or recruiting macrophages. We also show that inhibiting the Angiopoietin/TIE2/AKT cascade magnifies the vascular and anti-tumor effects of SHP2 inhibition by blocking tumor endothelial AKT signaling, not a target of SHP2. Since the SHP2 and Ang2/TIE2 pathways are active in vascular endothelial cells of human melanoma and colon carcinoma, SHP2 inhibitors alone or with Ang2/TIE2 inhibitors hold promise to effectively target the tumor endothelium.

Iterative epigenomic analyses in the same single cell.

Gene expression in individual cells is epigenetically regulated by DNA modifications, histone modifications, transcription factors, and other DNA-binding proteins. It has been shown that multiple histone modifications can predict gene expression and reflect future responses of bulk cells to extracellular cues. However, the predictive ability of epigenomic analysis is still limited for mechanistic research at a single cell level. To overcome this limitation, it would be useful to acquire reliable signals from multiple epigenetic marks in the same single cell. Here, we propose a new approach and a new method for analysis of several components of the epigenome in the same single cell. The new method allows reanalysis of the same single cell. We found that reanalysis of the same single cell is feasible, provides confirmation of the epigenetic signals, and allows application of statistical analysis to identify reproduced reads using data sets generated only from the single cell. Reanalysis of the same single cell is also useful to acquire multiple epigenetic marks from the same single cells. The method can acquire at least five epigenetic marks: H3K27ac, H3K27me3, mediator complex subunit 1, a DNA modification, and a DNA-interacting protein. We can predict active signaling pathways in K562 single cells using the epigenetic data and confirm that the predicted results strongly correlate with actual active signaling pathways identified by RNA-seq results. These results suggest that the new method provides mechanistic insights for cellular phenotypes through multilayered epigenome analysis in the same single cells.

Vasculopathy and Coagulopathy Associated with SARS-CoV-2 Infection.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has resulted in > 500,000 deaths worldwide, including > 125,000 deaths in the U.S. since its emergence in late December 2019 and June 2020. Neither curative anti-viral drugs nor a protective vaccine is currently available for the treatment and prevention of COVID-19. Recently, new clinical syndromes associated with coagulopathy and vasculopathy have emerged as a cause of sudden death and other serious clinical manifestations in younger patients infected with SARS-CoV-2 infection. Angiotensin converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 and other coronaviruses, is a transmembrane protein expressed by lung alveolar epithelial cells, enterocytes, and vascular endothelial cells, whose physiologic role is to induce the maturation of angiotensin I to generate angiotensin 1-7, a peptide hormone that controls vasoconstriction and blood pressure. In this review, we provide the general context of the molecular and cellular mechanisms of SARS-CoV-2 infection with a focus on endothelial cells, describe the vasculopathy and coagulopathy syndromes in patients with SARS-CoV-2, and outline current understanding of the underlying mechanistic aspects.

Viral interleukin-6 encoded by an oncogenic virus promotes angiogenesis and cellular transformation by enhancing STAT3-mediated epigenetic silencing of caveolin 1.

Kaposi's sarcoma (KS) caused by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) is a highly angiogenic and invasive vascular tumor and the most common AIDS-associated cancer. KSHV-encoded viral interleukin-6 (vIL-6) is implicated in the development of KSHV-induced malignancies; however, the mechanisms underlying vIL-6-induced angiogenesis and tumorigenesis remain undefined. Here, we show that vIL-6 promotes angiogenesis, cell proliferation, and invasion by downregulating caveolin 1 (CAV1) that plays a pivotal and versatile role in multiple cancer-associated processes. Mechanistically, vIL-6 signaling led to the phosphorylation and acetylation of STAT3 that targeted DNA methyltransferase 1 (DNMT1) in a sequential manner. Specifically, the vIL-6-induced phosphorylated form of STAT3 transcriptionally activated DNMT1 expression. Furthermore, vIL-6-induced acetylated form of STAT3 interacted with DNMT1 to form a transcription factor complex that bound to and methylated the CAV1 promoter, leading to CAV1 expression silencing. In fact, downregulation of CAV1 expression resulted in the activation of AKT signaling, promoting cell invasion, and growth transformation induced by KSHV. Finally, genetic deletion of vIL-6 from the KSHV genome abolished KSHV-induced cellular transformation and impaired angiogenesis. Our results reveal that vIL-6 epigenetically silences CAV1 expression to promote angiogenesis and tumorigenesis by regulating the formation of STAT3-DNMT1 complex. These novel findings define a mechanism by which KSHV inhibits the CAV1 pathway and establish the scientific basis for targeting this pathway to treat KSHV-associated cancers.

Identification of Eph receptor signaling as a regulator of autophagy and a therapeutic target in colorectal carcinoma.

Advanced colorectal carcinoma is currently incurable, and new therapies are urgently needed. We report that phosphotyrosine-dependent Eph receptor signaling sustains colorectal carcinoma cell survival, thereby uncovering a survival pathway active in colorectal carcinoma cells. We find that genetic and biochemical inhibition of Eph tyrosine kinase activity or depletion of the Eph ligand EphrinB2 reproducibly induces colorectal carcinoma cell death by autophagy. Spautin and 3-methyladenine, inhibitors of early steps in the autophagic pathway, significantly reduce autophagy-mediated cell death that follows inhibition of phosphotyrosine-dependent Eph signaling in colorectal cancer cells. A small-molecule inhibitor of the Eph kinase, NVP-BHG712 or its regioisomer NVP-Iso, reduces human colorectal cancer cell growth in vitro and tumor growth in mice. Colorectal cancers express the EphrinB ligand and its Eph receptors at significantly higher levels than numerous other cancer types, supporting Eph signaling inhibition as a potential new strategy for the broad treatment of colorectal carcinoma.

DLC1 deficiency and YAP signaling drive endothelial cell contact inhibition of growth and tumorigenesis.

Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene deleted in many cancers, including angiosarcoma, an aggressive malignancy of endothelial cell derivation. DLC1-deficiency in primary endothelial cells causes the loss of cell contact inhibition of growth through incompletely defined mechanisms. We report that DLC1 is a regulator of YAP, a transcriptional coactivator of proliferation-promoting and tumor-promoting genes; when confluent, active/nuclear YAP was significantly more abundant in DLC1-deficient endothelial cells compared with control cells. We also found that YAP is a required effector of the loss of cell contact inhibition of growth manifested by DLC1-deficient endothelial cells, as the silencing of YAP prevents this loss. Consistently, human angiosarcomas specimens contained a significantly greater proportion of DLC1- tumor cells with nuclear YAP compared with the DLC1+ normal cells in the adjacent tissue. Verteporfin, an inhibitor of YAP, significantly reduced angiosarcoma growth in mice. These results identify YAP as a previously unrecognized effector of DLC1 deficiency-associated loss of cell contact growth inhibition in endothelial cells and a potential therapeutic target in angiosarcoma.

A Pilot Study of Liposomal Doxorubicin Combined with Bevacizumab followed by Bevacizumab Monotherapy in Patients with Advanced Kaposi Sarcoma.

PURPOSE: VEGF-A is important in the pathogenesis of Kaposi sarcoma, and bevacizumab has a response rate of 31%. We explored the combination of bevacizumab with liposomal doxorubicin in patients with Kaposi sarcoma. PATIENTS AND METHODS: Patients with Kaposi sarcoma requiring systemic therapy were enrolled in one of two cohorts. Cohort 1 included patients with human immunodeficiency virus (HIV)-negative Kaposi sarcoma or with HIV-associated Kaposi sarcoma who would not be expected to respond to antiretroviral therapy (ART) alone (i.e., either stable or progressive Kaposi sarcoma on ART). Cohort 2 included all other patients with HIV-associated Kaposi sarcoma. Patients were treated with six cycles of liposomal doxorubicin with bevacizumab every 3 weeks followed by up to 11 cycles of bevacizumab alone. RESULTS: Sixteen patients were enrolled: 10 (two HIV negative) in cohort 1 and six in cohort 2. Fourteen patients had advanced disease (AIDS Clinical Trials Group T1). Overall response rate (complete and partial responses) was 56% [80% confidence interval (CI), 38%-74%] for all patients and were similar in the two cohorts. Median progression-free survival was 6.9 months (95% CI, 4.5 months-not estimable). Grade 3 and 4 adverse events attributed to therapy included hypertension (n = 5), neutropenia (n = 6), gastrointestinal hemorrhage (n = 1), and cerebral ischemia (n = 1). There was a significant decrease in VEGF-A levels from baseline to the end of six cycles of combination therapy. CONCLUSIONS: Pegylated liposomal doxorubicin in combination with bevacizumab has activity in advanced Kaposi sarcoma, but it is unclear whether the combination yields better outcomes than liposomal doxorubicin used alone.

Effects of DLC1 Deficiency on Endothelial Cell Contact Growth Inhibition and Angiosarcoma Progression.

Background: Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene frequently deleted in cancer. However, DLC1 is not known to be deleted in angiosarcoma, an aggressive malignancy of endothelial cell derivation. Additionally, the physiologic functions of DLC1 protein in endothelial cells are poorly defined. Methods: We investigated the effects of shRNA-induced DLC1 depletion in endothelial cells. Cell growth was measured by 3H thymidine incorporation, IncuCyte imaging, and population doublings; cell death by cell cycle analysis; gene expression by Affimetrix arrays and quantitative polymerase chain reaction; NF-κB activity by reporter assays; and protein levels by immunoblotting and immunofluorescence staining. We tested Tanespimycin/17-AAG and Fasudil treatment in groups of nine to 10 mice bearing ISOS-1 angiosarcoma. All statistical tests were two-sided. Results: We discovered that DLC1 is a critical regulator of cell contact inhibition of proliferation in endothelial cells, promoting statistically significant (P < .001) cell death when cells are confluent (mean [SD] % viability: control DLC1 = 15.6 [19.3]; shDLC1 = 73.4 [13.1]). This prosurvival phenotype of DLC1-depleted confluent endothelial cells is attributable to a statistically significant and sustained increase of NF-κB activity (day 5, P = .001; day 8, P = .03) associated with increased tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) signaling. Consistently, we found that DLC1 is statistically significantly reduced (P < .001 in 5 of 6) and TNFAIP3/A20 is statistically significantly increased (P < .001 in 2 of 3 and P = 0.02 in 1 of 3) in human angiosarcoma compared with normal adjacent endothelium. Treatment with the NF-κB inhibitor Tanespimycin/17-AAG statistically significantly reduced angiosarcoma tumor growth in mice (treatment tumor weight vs control, 0.50 [0.19] g vs 0.91 [0.21] g, P = .001 experiment 1; 0.66 [0.26] g vs 1.10 [0.31] g, P = .01 experiment 2). Conclusions: These results identify DLC1 as a previously unrecognized regulator of endothelial cell contact inhibition of proliferation that is depleted in angiosarcoma and support NF-κB targeting for the treatment of angiosarcoma where DLC1 is lost.

Ephrin ligands and Eph receptors contribution to hematopoiesis.

Hematopoietic stem and progenitor cells reside predominantly in the bone marrow. They supply billions of mature blood cells every day during life through maturation into multilineage progenitors and self-renewal. Newly produced mature cells serve to replenish the pool of circulating blood cells at the end of their life-span. These mature blood cells and a few hematopoietic progenitors normally exit the bone marrow through the sinusoidal vessels, a specialized venous vascular system that spreads throughout the bone marrow. Many signals regulate the coordinated mobilization of hematopoietic cells from the bone marrow to the circulation. In this review, we present recent advances on hematopoiesis and hematopoietic cell mobilization with a focus on the role of Ephrin ligands and their Eph receptors. These constitute a large family of transmembrane ligands and receptors that play critical roles in development and postnatally. New insights point to distinct roles of ephrin and Eph in different aspects of hematopoiesis.

Evidence for a Mesothelial Origin of Body Cavity Effusion Lymphomas.

Background: Primary effusion lymphoma (PEL) is a Kaposi's sarcoma herpes virus (KSHV)-induced lymphoma that typically arises in body cavities of HIV-infected patients. PEL cells are often co-infected with Epstein-Barr virus (EBV). "PEL-like" lymphoma is a KSHV-unrelated lymphoma that arises in body cavities of HIV-negative patients. "PEL-like" lymphoma is sometimes EBV positive. The derivation of PEL/"PEL-like" cells is unclear. Methods: Mesothelial cells were cultured from body cavity effusions of 23 patients. Cell proliferation, cytokine secretion, marker phenotypes, KSHV/EBV infection, and clonality were evaluated by standard methods. Gene expression was measured by quantitative polymerase chain reaction and immunoblotting. A mouse model of PEL (3 mice/group) was used to evaluate tumorigenicity. Results: We found that the mesothelia derived from six effusions of HIV-infected patients with PEL or other KSHV-associated diseases contained rare KSHV + or EBV + mesothelial cells. After extended culture (16-17 weeks), some mesothelial cells underwent a trans-differentiation process, generating lymphoid-type CD45 + /B220 + , CD5 + , CD27 + , CD43 + , CD11c + , and CD3 - cells resembling "B1-cells," most commonly found in mouse body cavities. These "B1-like" cells were short lived. However, long-term KSHV + EBV - and EBV + KSHV - clonal cell lines emerged from mesothelial cultures from two patients that were clonally distinct from the monoclonal or polyclonal B-cell populations found in the patients' original effusions. Conclusions: Mesothelial-to-lymphoid transformation is a newly identified in vitro process that generates "B1-like" cells and is associated with the emergence of long-lived KSHV or EBV-infected cell lines in KSHV-infected patients. These results identify mesothelial cultures as a source of PEL cells and lymphoid cells in humans.

Burkitt lymphoma expresses oncofetal chondroitin sulfate without being a reservoir for placental malaria sequestration.

Burkitt lymphoma (BL) is a malignant disease, which is frequently found in areas with holoendemic Plasmodium falciparum malaria. We have previously found that the VAR2CSA protein is present on malaria-infected erythrocytes and facilitates a highly specific binding to the placenta. ofCS is absent in other non-malignant tissues and thus VAR2CSA generally facilitates parasite sequestration and accumulation in pregnant women. In this study, we show that the specific receptor for VAR2CSA, the oncofetal chondroitin sulfate (ofCS), is likewise present in BL tissue and cell lines. We therefore explored whether ofCS in BL could act as anchor site for VAR2CSA-expressing infected erythrocytes. In contrast to the placenta, we found no evidence of in vivo sequestering of infected erythrocytes in the BL tissue. Furthermore, we found VAR2CSA-specific antibody titers in children with endemic BL to be lower than in control children from the same malaria endemic region. The abundant presence of ofCS in BL tissue and the absence of ofCS in non-malignant tissue encouraged us to examine whether recombinant VAR2CSA could be used to target BL. We confirmed the binding of VAR2CSA to BL-derived cells and showed that a VAR2CSA drug conjugate efficiently killed the BL-derived cell lines in vitro. These results identify ofCS as a novel therapeutic BL target and highlight how VAR2CSA could be used as a tool for the discovery of novel approaches for directing BL therapy.

Characterization of Semaphorin 6A-Mediated Effects on Angiogenesis Through Regulation of VEGF Signaling.

Angiogenesis identifies the process of endothelial cell sprouting and remodeling leading to the formation of new and functional blood vessels. Vascular expansion during development and in the adult mammal provides nutrients and oxygen to areas with increased need. Although many molecules and pathways have been identified as regulators of angiogenesis, aspects of this complex process remain unclear. Particularly undefined are the signals that orchestrate vessel survival and pruning once new blood vessels have sprouted. These poorly characterized aspects of angiogenesis need exploration. This chapter describes the experiments and methods enabling the characterization of Semaphorin 6A as a critical regulator of endothelial cell survival and vessel function.