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The development of cancer therapies may be improved by the discovery of tumor-specific molecular dependencies. The requisite tools include genetic and chemical perturbations, each with its strengths and limitations. Chemical perturbations can be readily applied to primary cancer samples at large scale, but mechanistic understanding of hits and further pharmaceutical development is often complicated by the fact that a chemical compound has affinities to multiple proteins. To computationally infer specific molecular dependencies of individual cancers from their ex vivo drug sensitivity profiles, we developed a mathematical model that deconvolutes these data using measurements of protein-drug affinity profiles. Through integrating a drug-kinase profiling dataset and several drug response datasets, our method, DepInfeR, correctly identified known protein kinase dependencies, including the EGFR dependence of HER2+ breast cancer cell lines, the FLT3 dependence of acute myeloid leukemia (AML) with FLT3 -ITD mutations and the differential dependencies on the B-cell receptor pathway in the two major subtypes of chronic lymphocytic leukemia (CLL). Furthermore, our method uncovered new subgroup-specific dependencies, including a previously unreported dependence of high-risk CLL on Checkpoint kinase 1 (CHEK1). The method also produced a detailed map of the kinase dependencies in a heterogeneous set of 117 CLL samples. The ability to deconvolute polypharmacological phenotypes into underlying causal molecular dependencies should increase the utility of high-throughput drug response assays for functional precision oncology.

A second generation of prophylactic human papillomavirus (HPV) vaccines based on the minor capsid protein L2 has entered clinical trials as promising alternative to meet the gaps left out by the current vaccines concerning type-restricted protection, high costs and low penetrance in immunization programs of lowand middle-income countries. Most of the serological assays available to assess anti-HPV humoral responses are, however, not well suited for measuring vaccine-induced anti-L2 antibody responses. In this work, we have advanced our automated, purely add-on High-Throughput Pseudovirion-Based Neutralization Assay (HT-PBNA) in an L2-oriented approach for measuring antibody-mediated neutralization of HPV types 6/16/18/31/33/52/58. With the optimized settings, we observed 24- to 120-fold higher sensitivity for detection of neutralizing Ab to the L2 protein of HPV6, HPV16, HPV18, and HPV31, compared to the standard HT-PBNA. Alternatively, we have also developed a highly sensitive, cell-free, colorimetric L2-peptide capture ELISA for which the results were strongly concordant with those of the advanced neutralization assay, named HT-fc-PBNA. These two high-throughput scalable assays represent attractive approaches to determine antibody-based correlates of protection for the HPV L2 vaccines that are to come.

A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA) with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV) 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV) of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA) based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R(2) = 0.7) was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.

Nonsense‐mediated RNA decay (NMD) is an RNA‐based quality control mechanism that eliminates transcripts bearing premature translation termination codons (PTC). Approximately, one‐third of all inherited disorders and some forms of cancer are caused by nonsense or frame shift mutations that introduce PTCs, and NMD can modulate the clinical phenotype of these diseases. 5‐azacytidine is an analogue of the naturally occurring pyrimidine nucleoside cytidine, which is approved for the treatment of myelodysplastic syndrome and myeloid leukemia. Here, we reveal that 5‐azacytidine inhibits NMD in a dose‐dependent fashion specifically upregulating the expression of both PTC‐containing mutant and cellular NMD targets. Moreover, this activity of 5‐azacytidine depends on the induction of MYC expression, thus providing a link between the effect of this drug and one of the key cellular pathways that are known to affect NMD activity. Furthermore, the effective concentration of 5‐azacytidine in cells corresponds to drug levels used in patients, qualifying 5‐azacytidine as a candidate drug that could potentially be repurposed for the treatment of Mendelian and acquired genetic diseases that are caused by PTC mutations. Synopsis The clinically approved drug 5‐azacytidine inhibits nonsense‐mediated decay (NMD) through a MYC‐dependent mechanism. This supports its repurposing to treat Mendelian or acquired genetic diseases that may benefit from NMD inhibition. 5‐azacytidine is medically licensed and has been used for the treatment of some forms of leukemia for many years. Nonsense‐mediated decay (NMD) can be inhibited by 5‐azacytidine via a MYC‐dependent mechanism at concentrations that correspond to drug levels in patients. 5‐azacytidine might thus be repurposed for the treatment of Mendelian or acquired genetic diseases that may benefit from an inhibition of NMD. Graphical Abstract The clinically approved drug 5‐azacytidine inhibits nonsense‐mediated decay (NMD) through a MYC‐dependent mechanism. This supports its repurposing to treat Mendelian or acquired genetic diseases that may benefit from NMD inhibition.

In vivo, we treated CRC xenografted SCID-beige mice with a human equivalent statin dose, which reduced MACC1 expression, tumour burden and metastasis formation (day 24; control vs. statin treatment p < .0001, Figure S2 and Figure 2). [...]at a molecular/mechanistic level, we provide experimental evidence that statins act, at least partly, by inhibiting transcription of the tumour-promoting and metastasis-inducing MACC1 gene. [...]we extended the RWE results by utilizing a clinical research platform (TriNetX) to access a large, international cohort of anonymized electronic health record (EHR) data (aggregate statistics only). [...]our study revealed strong evidence for cancer-preventive effects of statins in a large trans-Atlantic cohort, comprised of long-term statin users.

Reactivation of fetal hemoglobin expression alleviates the symptoms associated with β‐globinopathies, severe hereditary diseases with significant global health implications due to their high morbidity and mortality rates. The symptoms emerge following the postnatal transition from fetal‐to‐adult hemoglobin expression. Extensive research has focused on inducing the expression of the fetal γ‐globin subunit to reverse this switch and ameliorate these symptoms. Despite decades of research, only one compound, hydroxyurea, found its way to the clinic as an inducer of fetal hemoglobin. Unfortunately, its efficacy varies among patients, highlighting the need for more effective treatments. Erythroid cell lines have been instrumental in the pursuit of both pharmacological and genetic ways to reverse the postnatal hemoglobin switch. Here, we describe the first endogenously tagged fetal hemoglobin reporter cell line based on the adult erythroid progenitor cell line HUDEP2. Utilizing CRISPR‐Cas9‐mediated knock‐in, a bioluminescent tag was integrated at the HBG1 gene. Subsequent extensive characterization confirmed that the resulting reporter cell line closely mirrors the HUDEP2 characteristics and that the cells report fetal hemoglobin induction with high sensitivity and specificity. This novel reporter cell line is therefore highly suitable for evaluating genetic and pharmacologic strategies to induce fetal hemoglobin. Furthermore, it provides an assay compatible with high‐throughput drug screening, exemplified by the identification of a cluster of known fetal hemoglobin inducers in a pilot study. This new tool is made available to the research community, with the aspiration that it will accelerate the search for safer and more effective strategies to reverse the hemoglobin switch.

Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation.

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.

Colorectal cancer (CRC) metastasis continues to account for a substantial proportion of cancer-related deaths worldwide. Calcium-binding protein S100A4 is a known executor of CRC metastasis. S100A4 has been correlated to metastasis formation in the past, and therefore pharmaceutical intervention reduces the metastatic phenotype. Herein, a high-throughput screen (HTS) of 105,600 compounds from the EMBL screening library using an S100A4 promoter-driven luciferase construct transfected into HCT116 cells identified novel compounds for S100A4 transcriptional inhibition. The most promising inhibitors identified were tested for S100A4 transcriptional inhibition, their impact on wound healing, migration, proliferation and viability of cancer cells. Subsequently, the leading candidate E12 was tested in a xenograft mouse model (HCT116/CMVp- Luc). After several testing rounds, E12 a 2-(4-fluorobenzenesulfonamido)benzamide-based compound showed the strongest inhibition of S100A4 expression at mRNA (EC < 1 µM; 48 h) and protein level and concomitant restriction of metastatic abilities in two CRC cell lines with a tolerable viability reduction. , a reduction in metastasis formation was demonstrated, displayed by reduced overall bioluminescence of tumors and human satellite DNA in the liver of treated mice. This study exhibited E12's promising potential for S100A4 targeted metastasis inhibition therapy to improve the outcome of metastasized CRC patients.

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.