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.

Lymphocyte Classification from Hoechst Stained Slides with Deep Learning.

Multiplex immunofluorescence and immunohistochemistry benefit patients by allowing cancer pathologists to identify proteins expressed on the surface of cells. This enables cell classification, better understanding of the tumour microenvironment, and more accurate diagnoses, prognoses, and tailored immunotherapy based on the immune status of individual patients. However, these techniques are expensive. They are time consuming processes which require complex staining and imaging techniques by expert technicians. Hoechst staining is far cheaper and easier to perform, but is not typically used as it binds to DNA rather than to the proteins targeted by immunofluorescence techniques. In this work we show that through the use of deep learning it is possible to identify an immune cell subtype without immunofluorescence. We train a deep convolutional neural network to identify cells expressing the T lymphocyte marker CD3 from Hoechst 33342 stained tissue only. CD3 expressing cells are often used in key prognostic metrics such as assessment of immune cell infiltration, and by identifying them without the need for costly immunofluorescence, we present a promising new approach to cheaper prediction and improvement of patient outcomes. We also show that by using deep learning interpretability techniques, we can gain insight into the previously unknown morphological features which make this possible.

Use of High-Plex Data Reveals Novel Insights into the Tumour Microenvironment of Clear Cell Renal Cell Carcinoma.

Although immune checkpoint inhibitors (ICIs) have significantly improved the oncological outcomes, about one-third of patients affected by clear cell renal cell carcinoma (ccRCC) still experience recurrence. Current prognostic algorithms, such as the Leibovich score (LS), rely on morphological features manually assessed by pathologists and are therefore subject to bias. Moreover, these tools do not consider the heterogeneous molecular milieu present in the Tumour Microenvironment (TME), which may have prognostic value. We systematically developed a semi-automated method to investigate 62 markers and their combinations in 150 primary ccRCCs using Multiplex Immunofluorescence (mIF), NanoString GeoMx® Digital Spatial Profiling (DSP) and Artificial Intelligence (AI)-assisted image analysis in order to find novel prognostic signatures and investigate their spatial relationship. We found that coexpression of cancer stem cell (CSC) and epithelial-to-mesenchymal transition (EMT) markers such as OCT4 and ZEB1 are indicative of poor outcome. OCT4 and the immune markers CD8, CD34, and CD163 significantly stratified patients at intermediate LS. Furthermore, augmenting the LS with OCT4 and CD34 improved patient stratification by outcome. Our results support the hypothesis that combining molecular markers has prognostic value and can be integrated with morphological features to improve risk stratification and personalised therapy. To conclude, GeoMx® DSP and AI image analysis are complementary tools providing high multiplexing capability required to investigate the TME of ccRCC, while reducing observer bias.

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.

Computerized Image Analysis of Tumor Cell Nuclear Morphology Can Improve Patient Selection for Clinical Trials in Localized Clear Cell Renal Cell Carcinoma.

BACKGROUND: Clinicopathological scores are used to predict the likelihood of recurrence-free survival for patients with clear cell renal cell carcinoma (ccRCC) after surgery. These are fallible, particularly in the middle range. This inevitably means that a significant proportion of ccRCC patients who will not develop recurrent disease enroll into clinical trials. As an exemplar of using digital pathology, we sought to improve the predictive power of "recurrence free" designation in localized ccRCC patients, by precise measurement of ccRCC nuclear morphological features using computational image analysis, thereby replacing manual nuclear grade assessment. MATERIALS AND METHODS: TNM 8 UICC pathological stage pT1-pT3 ccRCC cases were recruited in Scotland and in Singapore. A Leibovich score (LS) was calculated. Definiens Tissue studio® (Definiens GmbH, Munich) image analysis platform was used to measure tumor nuclear morphological features in digitized hematoxylin and eosin (H&E) images. RESULTS: Replacing human-defined nuclear grade with computer-defined mean perimeter generated a modified Leibovich algorithm, improved overall specificity 0.86 from 0.76 in the training cohort. The greatest increase in specificity was seen in LS 5 and 6, which went from 0 to 0.57 and 0.40, respectively. The modified Leibovich algorithm increased the specificity from 0.84 to 0.94 in the validation cohort. CONCLUSIONS: CcRCC nuclear mean perimeter, measured by computational image analysis, together with tumor stage and size, node status and necrosis improved the accuracy of predicting recurrence-free in the localized ccRCC patients. This finding was validated in an ethnically different Singaporean cohort, despite the different H and E staining protocol and scanner used. This may be a useful patient selection tool for recruitment to multicenter studies, preventing some patients from receiving unnecessary additional treatment while reducing the number of patients required to achieve adequate power within neoadjuvant and adjuvant clinical studies.

Evaluation of the dual mTOR/PI3K inhibitors Gedatolisib (PF-05212384) and PF-04691502 against ovarian cancer xenograft models.

This study investigated the antitumour effects of two dual mTOR/PI3K inhibitors, gedatolisib (WYE-129587/PKI-587/PF-05212384) and PF-04691502 against a panel of six human patient derived ovarian cancer xenograft models. Both dual mTOR/PI3K inhibitors demonstrated antitumour activity against all xenografts tested. The compounds produced tumour stasis during the treatment period and upon cessation of treatment, tumours re-grew. In several models, there was an initial rapid reduction of tumour volume over the first week of treatment before tumour stasis. No toxicity was observed during treatment. Biomarker studies were conducted in two xenograft models; phospho-S6 (Ser235/236) expression (as a readout of mTOR activity) was reduced over the treatment period in the responding xenograft but expression increased to control (no treatment) levels on cessation of treatment. Phospho-AKT (Ser473) expression (as a readout of PI3K) was inhibited by both drugs but less markedly so than phospho-S6 expression. Initial tumour volume reduction on treatment and regrowth rate after treatment cessation was associated with phospho-S6/total S6 expression ratio. Both drugs produced apoptosis but minimally influenced markers of proliferation (Ki67, phospho-histone H3). These results indicate that mTOR/PI3K inhibition can produce broad spectrum tumour growth stasis in ovarian cancer xenograft models during continuous chronic treatment and this is associated with apoptosis.

Genome-scale CRISPR/Cas9 screen determines factors modulating sensitivity to ProTide NUC-1031.

Gemcitabine is a fluoropyrimidine analogue that is used as a mainstay of chemotherapy treatment for pancreatic and ovarian cancers, amongst others. Despite its widespread use, gemcitabine achieves responses in less than 10% of patients with metastatic pancreatic cancer and has a very limited impact on overall survival due to intrinsic and acquired resistance. NUC-1031 (Acelarin), a phosphoramidate transformation of gemcitabine, was the first anti-cancer ProTide to enter the clinic. We find it displays important in vitro cytotoxicity differences to gemcitabine, and a genome-wide CRISPR/Cas9 genetic screening approach identified only the pyrimidine metabolism pathway as modifying cancer cell sensitivity to NUC-1031. Low deoxycytidine kinase expression in tumour biopsies from patients treated with gemcitabine, assessed by immunostaining and image analysis, correlates with a poor prognosis, but there is no such correlation in tumour biopsies from a Phase I cohort treated with NUC-1031.

The role of HDAC2 in chromatin remodelling and response to chemotherapy in ovarian cancer.

Chromatin undergoes structural changes in response to extracellular and environmental signals. We observed changes in nuclear morphology in cancer tissue biopsied after chemotherapy and hypothesised that these DNA damage-induced changes are mediated by histone deacetylases (HDACs). Nuclear morphological changes in cell lines (PE01 and PE04 models) and a xenograft model (OV1002) were measured in response to platinum chemotherapy by image analysis of nuclear texture. HDAC2 expression increased in PEO1 cells treated with cisplatin at 24h, which was accompanied by increased expression of heterochromatin protein 1 (HP1). HDAC2 and HP1 expression were also increased after carboplatin treatment in the OV1002 carboplatin-sensitive xenograft model but not in the insensitive HOX424 model. Expression of DNA damage response pathways (pBRCA1, γH2AX, pATM, pATR) showed time-dependent changes after cisplatin treatment. HDAC2 knockdown by siRNA reduced HP1 expression, induced DNA double strand breaks (DSB) measured by γH2AX, and interfered with the activation of DNA damage response induced by cisplatin. Furthermore, HDAC2 depletion affected γH2AX foci formation, cell cycle distribution, and apoptosis triggered by cisplatin, and was additive to the inhibitory effect of cisplatin in cell lines. By inhibiting expression of HDAC2, reversible alterations in chromatin patterns during cisplatin treatment were observed. These results demonstrate quantifiable alterations in nuclear morphology after chemotherapy, and implicate HDAC2 in higher order chromatin changes and cellular DNA damage responses in ovarian cancer cells in vitro and in vivo.

Evaluation of carbonic anhydrase IX as a therapeutic target for inhibition of breast cancer invasion and metastasis using a series of in vitro breast cancer models.

Triple negative, resistant or metastatic disease are major factors in breast cancer mortality, warranting novel approaches. Carbonic anhydrase IX (CAIX) is implicated in survival, migration and invasion of breast cancer cells and inhibition provides an innovative therapeutic strategy. The efficacy of 5 novel ureido-substituted sulfamate CAIX inhibitors were assessed in increasingly complex breast cancer models, including cell lines in normoxia and hypoxia, 3D spheroids and an ex-vivo explant model utilizing fresh biopsy tissue from different breast cancer subtypes. CAIX expression was evaluated in a tissue microarray (TMA) of 92 paired lymph node and primary breast cancers and 2 inhibitors were appraised in vivo using MDA-MB-231 xenografts. FC11409B, FC9398A, FC9403, FC9396A and S4 decreased cell proliferation and migration and inhibited 3D spheroid invasion. S4, FC9398A and FC9403A inhibited or prevented invasion into collagen. FC9403A significantly reversed established invasion whilst FC9398A and DTP348 reduced xenograft growth. TMA analysis showed increased CAIX expression in triple negative cancers. These data establish CAIX inhibition as a relevant therapeutic goal in breast cancer, targeting the migratory, invasive, and metastatic potential of this disease. The use of biopsy tissue suggests efficacy against breast cancer subtypes, and should provide a useful tool in drug testing against invasive cancers.

Relationship between differentially expressed mRNA and mRNA-protein correlations in a xenograft model system.

Differential mRNA expression studies implicitly assume that changes in mRNA expression have biological meaning, most likely mediated by corresponding changes in protein levels. Yet studies into mRNA-protein correspondence have shown notoriously poor correlation between mRNA and protein expression levels, creating concern for inferences from only mRNA expression data. However, none of these studies have examined in particular differentially expressed mRNA. Here, we examined this question in an ovarian cancer xenograft model. We measured protein and mRNA expression for twenty-nine genes in four drug-treatment conditions and in untreated controls. We identified mRNAs differentially expressed between drug-treated xenografts and controls, then analysed mRNA-protein expression correlation across a five-point time-course within each of the four experimental conditions. We evaluated correlations between mRNAs and their protein products for mRNAs differentially expressed within an experimental condition compared to those that are not. We found that differentially expressed mRNAs correlate significantly better with their protein product than non-differentially expressed mRNAs. This result increases confidence for the use of differential mRNA expression for biological discovery in this system, as well as providing optimism for the usefulness of inferences from mRNA expression in general.

Systems biology reveals new strategies for personalizing cancer medicine and confirms the role of PTEN in resistance to trastuzumab.

Resistance to targeted cancer therapies such as trastuzumab is a frequent clinical problem not solely because of insufficient expression of HER2 receptor but also because of the overriding activation states of cell signaling pathways. Systems biology approaches lend themselves to rapid in silico testing of factors, which may confer resistance to targeted therapies. Inthis study, we aimed to develop a new kinetic model that could be interrogated to predict resistance to receptor tyrosine kinase (RTK) inhibitor therapies and directly test predictions in vitro and in clinical samples. The new mathematical model included RTK inhibitor antibody binding, HER2/HER3 dimerization and inhibition, AKT/mitogen-activated protein kinase cross-talk, and the regulatory properties of PTEN. The model was parameterized using quantitative phosphoprotein expression data from cancer cell lines using reverse-phase protein microarrays. Quantitative PTEN protein expression was found to be the key determinant of resistance to anti-HER2 therapy in silico, which was predictive of unseen experiments in vitro using the PTEN inhibitor bp(V). When measured in cancer cell lines, PTEN expression predicts sensitivity to anti-HER2 therapy; furthermore, this quantitative measurement is more predictive of response (relative risk, 3.0; 95% confidence interval, 1.6-5.5; P < 0.0001) than other pathway components taken in isolation and when tested by multivariate analysis in a cohort of 122 breast cancers treated with trastuzumab. For the first time, a systems biology approach has successfully been used to stratify patients for personalized therapy in cancer and is further compelling evidence that PTEN, appropriately measured in the clinical setting, refines clinical decision making in patients treated with anti-HER2 therapies.