The WNK1-ERK5 route plays a pathophysiological role in ovarian cancer and limits therapeutic efficacy of trametinib.

BACKGROUND: The dismal prognosis of advanced ovarian cancer calls for the development of novel therapies to improve disease outcome. In this regard, we set out to discover new molecular entities and to assess the preclinical effectiveness of their targeting. METHODS: Cell lines, mice and human ovarian cancer samples were used. Proteome profiling of human phosphokinases, in silico genomic analyses, genetic (shRNA and CRISPR/Cas9) and pharmacological strategies as well as an ex vivo human preclinical model were performed. RESULTS: We identified WNK1 as a highly phosphorylated protein in ovarian cancer and found that its activation or high expression had a negative impact on patients' survival. Genomic analyses showed amplification of WNK1 in human ovarian tumours. Mechanistically, we demonstrate that WNK1 exerted its action through the MEK5-ERK5 signalling module in ovarian cancer. Loss of function, genetic or pharmacological experiments, demonstrated anti-proliferative and anti-tumoural effects of the targeting of the WNK1-MEK5-ERK5 route. Additional studies showed that this pathway modulated the anti-tumoural properties of the MEK1/2 inhibitor trametinib. Thus, treatment with trametinib activated the WNK1-MEK5-ERK5 route, raising the possibility that this effect may limit the therapeutic benefit of ERK1/2 targeting in ovarian cancer. Moreover, in different experimental settings, including an ex vivo patient-derived model consisting of ovarian cancer cells cultured with autologous patient sera, we show that inhibition of WNK1 or MEK5 increased the anti-proliferative and anti-tumour efficacy of trametinib. CONCLUSIONS: The present study uncovers the participation of WNK1-MEK5-ERK5 axis in ovarian cancer pathophysiology, opening the possibility of acting on this pathway with therapeutic purposes. Another important finding of the present study was the activation of that signalling axis by trametinib, bypassing the anti-tumoural efficacy of this drug. That fact should be considered in the context of the use of trametinib in ovarian cancer.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

Reduction of Metastasis via Epigenetic Modulation in a Murine Model of Metastatic Triple Negative Breast Cancer (TNBC).

This study investigates the effects of a dual selective Class I histone deacetylase (HDAC)/lysine-specific histone demethylase 1A (LSD1) inhibitor known as 4SC-202 (Domatinostat) on tumor growth and metastasis in a highly metastatic murine model of Triple Negative Breast Cancer (TNBC). 4SC-202 is cytotoxic and cytostatic to the TNBC murine cell line 4T1 and the human TNBC cell line MDA-MB-231; the drug does not kill the normal breast epithelial cell line MCF10A. Furthermore, 4SC-202 reduces cancer cell migration. In vivo studies conducted in the syngeneic 4T1 model, which closely mimics human TNBC in terms of sites of metastasis, reveal reduced tumor burden and lung metastasis. The mechanism of action of 4SC-202 may involve effects on cancer stem cells (CSC) which can self-renew and form metastatic lesions. Approximately 5% of the total 4T1 cell population grown in three-dimensional scaffolds had a distinct CD44high/CD24low CSC profile which decreased after treatment. Bulk transcriptome (RNA) sequencing analyses of 4T1 tumors reveal changes in metastasis-related pathways in 4SC-202-treated tumors, including changes to expression levels of genes implicated in cell migration and cell motility. In summary, 4SC-202 treatment of tumors from a highly metastatic murine model of TNBC reduces metastasis and warrants further preclinical studies.

Bioengineering the Oxygen-Deprived Tumor Microenvironment Within a Three-Dimensional Platform for Studying Tumor-Immune Interactions.

Oxygen deprivation within tumors is one of the most prevalent causes of resilient cancer cell survival and increased immune evasion in breast cancer (BCa). Current in vitro models do not adequately mimic physiological oxygen levels relevant to breast tissue and its tumor-immune interactions. In this study, we propose an approach to engineer a three-dimensional (3D) model (named 3D engineered oxygen, 3D-O) that supports the growth of BCa cells and generates physio- and pathophysiological oxygen levels to understand the role of oxygen availability in tumor-immune interactions. BCa cells (MDA-MB-231 and MCF-7) were embedded into plasma-derived 3D-O scaffolds that reflected physio- and pathophysiological oxygen levels relevant to the healthy and cancerous breast tissue. BCa cells grown within 3D-O scaffolds were analyzed by flow cytometry, confocal imaging, immunohistochemistry/immunofluorescence for cell proliferation, extracellular matrix protein expression, and alterations in immune evasive outcomes. Exosome secretion from 3D-O scaffolds were evaluated using the NanoSight particle analyzer. Peripheral blood mononuclear cells were incorporated on the top of 3D-O scaffolds and the difference in tumor-infiltrating capabilities as a result of different oxygen content were assessed by flow cytometry and confocal imaging. Lastly, hypoxia and Programmed death-ligand 1 (PD-L1) inhibition were validated as targets to sensitize BCa cells in order to overcome immune evasion. Low oxygen-induced adaptations within 3D-O scaffolds validated known tumor hypoxia characteristics such as reduced BCa cell proliferation, increased extracellular matrix protein expression, increased extracellular vesicle secretion and enhanced immune surface marker expression on BCa cells. We further demonstrated that low oxygen in 3D-O scaffolds significantly influence immune infiltration. CD8+ T cell infiltration was impaired under pathophysiological oxygen levels and we were also able to establish that hypoxia and PD-L1 inhibition re-sensitized BCa cells to cytotoxic CD8+ T cells. Bioengineering the oxygen-deprived BCa tumor microenvironment in our engineered 3D-O physiological and tumorous scaffolds supported known intra-tumoral hypoxia characteristics allowing the study of the role of oxygen availability in tumor-immune interactions. The 3D-O model could serve as a promising platform for the evaluation of immunological events and as a drug-screening platform tool to overcome hypoxia-driven immune evasion.

Human Plasma-Derived 3D Cultures Model Breast Cancer Treatment Responses and Predict Clinically Effective Drug Treatment Concentrations.

Lack of efficacy and a low overall success rate of phase I-II clinical trials are the most common failures when it comes to advancing cancer treatment. Current drug sensitivity screenings present several challenges including differences in cell growth rates, the inconsistent use of drug metrics, and the lack of translatability. Here, we present a patient-derived 3D culture model to overcome these limitations in breast cancer (BCa). The human plasma-derived 3D culture model (HuP3D) utilizes patient plasma as the matrix, where BCa cell lines and primary BCa biopsies were grown and screened for drug treatments. Several drug metrics were evaluated from relative cell count and growth rate curves. Correlations between HuP3D metrics, established preclinical models, and clinical effective concentrations in patients were determined. HuP3D efficiently supported the growth and expansion of BCa cell lines and primary breast cancer tumors as both organoids and single cells. Significant and strong correlations between clinical effective concentrations in patients were found for eight out of ten metrics for HuP3D, while a very poor positive correlation and a moderate correlation was found for 2D models and other 3D models, respectively. HuP3D is a feasible and efficacious platform for supporting the growth and expansion of BCa, allowing high-throughput drug screening and predicting clinically effective therapies better than current preclinical models.

Mimicking tumor hypoxia and tumor-immune interactions employing three-dimensional in vitro models.

The heterogeneous tumor microenvironment (TME) is highly complex and not entirely understood. These complex configurations lead to the generation of oxygen-deprived conditions within the tumor niche, which modulate several intrinsic TME elements to promote immunosuppressive outcomes. Decoding these communications is necessary for designing effective therapeutic strategies that can effectively reduce tumor-associated chemotherapy resistance by employing the inherent potential of the immune system.While classic two-dimensional in vitro research models reveal critical hypoxia-driven biochemical cues, three-dimensional (3D) cell culture models more accurately replicate the TME-immune manifestations. In this study, we review various 3D cell culture models currently being utilized to foster an oxygen-deprived TME, those that assess the dynamics associated with TME-immune cell penetrability within the tumor-like spatial structure, and discuss state of the art 3D systems that attempt recreating hypoxia-driven TME-immune outcomes. We also highlight the importance of integrating various hallmarks, which collectively might influence the functionality of these 3D models.This review strives to supplement perspectives to the quickly-evolving discipline that endeavors to mimic tumor hypoxia and tumor-immune interactions using 3D in vitro models.

Analysis of Dual Class I Histone Deacetylase and Lysine Demethylase Inhibitor Domatinostat (4SC-202) on Growth and Cellular and Genomic Landscape of Atypical Teratoid/Rhabdoid.

Central nervous system atypical teratoid/rhabdoid tumors (ATRTs) are rare and aggressive tumors with a very poor prognosis. Current treatments for ATRT include resection of the tumor, followed by systemic chemotherapy and radiation therapy, which have toxic side effects for young children. Gene expression analyses of human ATRTs and normal brain samples indicate that ATRTs have aberrant expression of epigenetic markers including class I histone deacetylases (HDAC's) and lysine demethylase (LSD1). Here, we investigate the effect of a small molecule epigenetic modulator known as Domatinostat (4SC-202), which inhibits both class I HDAC's and Lysine Demethylase (LSD1), on ATRT cell survival and single cell heterogeneity. Our findings suggest that 4SC-202 is both cytotoxic and cytostatic to ATRT in 2D and 3D scaffold cell culture models and may target cancer stem cells. Single-cell RNA sequencing data from ATRT-06 spheroids treated with 4SC-202 have a reduced population of cells overexpressing stem cell-related genes, including SOX2. Flow cytometry and immunofluorescence on 3D ATRT-06 scaffold models support these results suggesting that 4SC-202 reduces expression of cancer stem cell markers SOX2, CD133, and FOXM1. Drug-induced changes to the systems biology landscape are also explored by multi-omics enrichment analyses. In summary, our data indicate that 4SC-202 has both cytotoxic and cytostatic effects on ATRT, targets specific cell sub-populations, including those with cancer stem-like features, and is an important potential cancer therapeutic to be investigated in vivo.