Transcription Factor NFE2L1 Decreases in Glomerulonephropathies after Podocyte Damage.

Podocyte cellular injury and detachment from glomerular capillaries constitute a critical factor contributing to kidney disease. Notably, transcription factors are instrumental in maintaining podocyte differentiation and homeostasis. This study explores the hitherto uninvestigated expression of Nuclear Factor Erythroid 2-related Factor 1 (NFE2L1) in podocytes. We evaluated the podocyte expression of NFE2L1, Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2), and NAD(P)H:quinone Oxidoreductase (NQO1) in 127 human glomerular disease biopsies using multiplexed immunofluorescence and image analysis. We found that both NFE2L1 and NQO1 expressions were significantly diminished across all observed renal diseases. Furthermore, we exposed human immortalized podocytes and ex vivo kidney slices to Puromycin Aminonucleoside (PAN) and characterized the NFE2L1 protein isoform expression. PAN treatment led to a reduction in the nuclear expression of NFE2L1 in ex vivo kidney slices and podocytes.

NUC-7738 regulates ß-catenin signalling resulting in reduced proliferation and self-renewal of AML cells.

Acute myeloid leukemia (AML) stem cells are required for the initiation and maintenance of the disease. Activation of the Wnt/ß-catenin pathway is required for the survival and development of AML leukaemia stem cells (LSCs) and therefore, targeting ß-catenin is a potential therapeutic strategy. NUC-7738, a phosphoramidate transformation of 3'-deoxyadenosine (3'-dA) monophosphate, is specifically designed to generate the active anti-cancer metabolite 3'-deoxyadenosine triphosphate (3'-dATP) intracellularly, bypassing key limitations of breakdown, transport, and activation. NUC-7738 is currently in a Phase I/II clinical study for the treatment of patients with advanced solid tumors. Protein expression and immunophenotypic profiling revealed that NUC-7738 caused apoptosis in AML cell lines through reducing PI3K-p110α, phosphorylated Akt (Ser473) and phosphorylated GSK3ß (Ser9) resulting in reduced ß-catenin, c-Myc and CD44 expression. NUC-7738 reduced ß-catenin nuclear expression in AML cells. NUC-7738 also decreased the percentage of CD34+ CD38- CD123+ (LSC-like cells) from 81% to 47% and reduced the total number and size of leukemic colonies. These results indicate that therapeutic targeting of the PI3K/Akt/GSK3ß axis can inhibit ß-catenin signalling, resulting in reduced clonogenicity and eventual apoptosis of AML cells.

Spatial Analysis of NQO1 in Non-Small Cell Lung Cancer Shows Its Expression Is Independent of NRF1 and NRF2 in the Tumor Microenvironment.

Nuclear factor erythroid 2-related factor 1 (NFE2L1, NRF1) and nuclear factor erythroid 2-related factor 2 (NFE2L2, NRF2) are distinct oxidative stress response transcription factors, both of which have been shown to perform cytoprotective functions, modulating cell stress response and homeostasis. NAD(P)H:quinone oxidoreductase (NQO1) is a mutual downstream antioxidant gene target that catalyzes the two-electron reduction of an array of substrates, protecting against reactive oxygen species (ROS) generation. NQO1 is upregulated in non-small cell lung cancer (NSCLC) and is proposed as a predictive biomarker and therapeutic target. Antioxidant protein expression of immune cells within the NSCLC tumor microenvironment (TME) remains undetermined and may affect immune cell effector functions and survival outcomes. Multiplex immunofluorescence was performed to examine the co-localization of NQO1, NRF1 and NRF2 within the tumor and TME of 162 chemotherapy-naïve, early-stage NSCLC patients treated by primary surgical resection. This study demonstrates that NQO1 protein expression is high in normal, tumor-adjacent tissue and that NQO1 expression varies depending on the cell type. Inter and intra-patient heterogenous NQO1 expression was observed in lung cancer. Co-expression analysis showed NQO1 is independent of NRF1 and NRF2 in tumors. Density-based co-expression analysis demonstrated NRF1 and NRF2 double-positive expression in cancer cells is associated with improved overall survival.

The Novel Nucleoside Analogue ProTide NUC-7738 Overcomes Cancer Resistance Mechanisms In Vitro and in a First-In-Human Phase I Clinical Trial.

PURPOSE: Nucleoside analogues form the backbone of many therapeutic regimens in oncology and require the presence of intracellular enzymes for their activation. A ProTide is comprised of a nucleoside fused to a protective phosphoramidate cap. ProTides are easily incorporated into cells whereupon the cap is cleaved and a preactivated nucleoside released. 3'-Deoxyadenosine (3'-dA) is a naturally occurring adenosine analogue with established anticancer activity in vitro but limited bioavailability due to its rapid in vivo deamination by the circulating enzyme adenosine deaminase, poor uptake into cells, and reliance on adenosine kinase for its activation. In order to overcome these limitations, 3'-dA was chemically modified to create the novel ProTide NUC-7738. EXPERIMENTAL DESIGN: We describe the synthesis of NUC-7738. We determine the IC50 of NUC-7738 using pharmacokinetics (PK) and conduct genome-wide analyses to identify its mechanism of action using different cancer model systems. We validate these findings in patients with cancer. RESULTS: We show that NUC-7738 overcomes the cancer resistance mechanisms that limit the activity of 3'-dA and that its activation is dependent on ProTide cleavage by the enzyme histidine triad nucleotide-binding protein 1. PK and tumor samples obtained from the ongoing first-in-human phase I clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective proapoptotic agent in cancer cells with effects on the NF-κB pathway. CONCLUSIONS: Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and supports its further clinical evaluation as a novel cancer treatment within the growing pantheon of anticancer ProTides.

YAP Translocation Precedes Cytoskeletal Rearrangement in Podocyte Stress Response: A Podometric Investigation of Diabetic Nephropathy.

Podocyte loss plays a pivotal role in the pathogenesis of glomerular disease. However, the mechanisms underlying podocyte damage and loss remain poorly understood. Although detachment of viable cells has been documented in experimental Diabetic Nephropathy, correlations between reduced podocyte density and disease severity have not yet been established. YAP, a mechanosensing protein, has recently been shown to correlate with glomerular disease progression, however, the underlying mechanism has yet to be fully elucidated. In this study, we sought to document podocyte density in Diabetic Nephropathy using an amended podometric methodology, and to investigate the interplay between YAP and cytoskeletal integrity during podocyte injury. Podocyte density was quantified using TLE4 and GLEPP1 multiplexed immunofluorescence. Fourteen Diabetic Nephropathy cases were analyzed for both podocyte density and cytoplasmic translocation of YAP via automated image analysis. We demonstrate a significant decrease in podocyte density in Grade III/IV cases (124.5 per 106 µm3) relative to Grade I/II cases (226 per 106 µm3) (Student's t-test, p < 0.001), and further show that YAP translocation precedes cytoskeletal rearrangement following injury. Based on these findings we hypothesize that a significant decrease in podocyte density in late grade Diabetic Nephropathy may be explained by early cytoplasmic translocation of YAP.

Tissue-Specific Immunopathology in Fatal COVID-19.

Rationale: In life-threatening coronavirus disease (COVID-19), corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or an independent immunopathologic process is unknown.Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses and the relationships among viral presence, inflammation, and organ injury.Methods: Tissue was acquired from 11 detailed postmortem examinations. SARS-CoV-2 organotropism was mapped by using multiplex PCR and sequencing, with cellular resolution achieved by in situ viral S (spike) protein detection. Histologic evidence of inflammation was quantified from 37 anatomic sites, and the pulmonary immune response was characterized by using multiplex immunofluorescence.Measurements and Main Results: Multiple aberrant immune responses in fatal COVID-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein between or within tissues. An arteritis was identified in the lung, which was further characterized as a monocyte/myeloid-rich vasculitis, and occurred together with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticuloendothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues.Conclusions: Tissue-specific immunopathology occurs in COVID-19, implicating a significant component of the immune-mediated, virus-independent immunopathologic process as a primary mechanism in severe disease. Our data highlight novel immunopathologic mechanisms and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma-cell responses as well as promote pathogen tolerance in COVID-19.

Podocyte injury elicits loss and recovery of cellular forces.

In the healthy kidney, specialized cells called podocytes form a sophisticated blood filtration apparatus that allows excretion of wastes and excess fluid from the blood while preventing loss of proteins such as albumin. To operate effectively, this filter is under substantial hydrostatic mechanical pressure. Given their function, it is expected that the ability to apply mechanical force is crucial to the survival of podocytes. However, to date, podocyte mechanobiology remains poorly understood, largely because of a lack of experimental data on the forces involved. We perform quantitative, continuous, nondisruptive, and high-resolution measurements of the forces exerted by differentiated podocytes in real time using a recently introduced functional imaging modality for continuous force mapping. Using an accepted model for podocyte injury, we find that injured podocytes experience near-complete loss of cellular force transmission but that this loss of force is reversible under certain conditions. The observed changes in force correlate with F-actin rearrangement and reduced expression of podocyte-specific proteins. By introducing robust and high-throughput mechanical phenotyping and by demonstrating the significance of mechanical forces in podocyte injury, this research paves the way to a new level of understanding of the kidney. In addition, in an advance over established force mapping techniques, we integrate cellular force measurements with immunofluorescence and perform continuous long-term force measurements of a cell population. Hence, our approach has general applicability to a wide range of biomedical questions involving mechanical forces.