Targeted long-read sequencing of the Ewing sarcoma 6p25.1 susceptibility locus identifies germline-somatic interactions with EWSR1-FLI1 binding.

Ewing sarcoma (EwS) is a rare bone and soft tissue malignancy driven by chromosomal translocations encoding chimeric transcription factors, such as EWSR1-FLI1, that bind GGAA motifs forming novel enhancers that alter nearby expression. We propose that germline microsatellite variation at the 6p25.1 EwS susceptibility locus could impact downstream gene expression and EwS biology. We performed targeted long-read sequencing of EwS blood DNA to characterize variation and genomic features important for EWSR1-FLI1 binding. We identified 50 microsatellite alleles at 6p25.1 and observed that EwS-affected individuals had longer alleles (>135 bp) with more GGAA repeats. The 6p25.1 GGAA microsatellite showed chromatin features of an EWSR1-FLI1 enhancer and regulated expression of RREB1, a transcription factor associated with RAS/MAPK signaling. RREB1 knockdown reduced proliferation and clonogenic potential and reduced expression of cell cycle and DNA replication genes. Our integrative analysis at 6p25.1 details increased binding of longer GGAA microsatellite alleles with acquired EWSR-FLI1 to promote Ewing sarcomagenesis by RREB1-mediated proliferation.

Germline-somatic JAK2 interactions are associated with clonal expansion in myelofibrosis.

Myelofibrosis is a rare myeloproliferative neoplasm (MPN) with high risk for progression to acute myeloid leukemia. Our integrated genomic analysis of up to 933 myelofibrosis cases identifies 6 germline susceptibility loci, 4 of which overlap with previously identified MPN loci. Virtual karyotyping identifies high frequencies of mosaic chromosomal alterations (mCAs), with enrichment at myelofibrosis GWAS susceptibility loci and recurrently somatically mutated MPN genes (e.g., JAK2). We replicate prior MPN associations showing germline variation at the 9p24.1 risk haplotype confers elevated risk of acquiring JAK2V617F mutations, demonstrating with long-read sequencing that this relationship occurs in cis. We also describe recurrent 9p24.1 large mCAs that selectively retained JAK2V617F mutations. Germline variation associated with longer telomeres is associated with increased myelofibrosis risk. Myelofibrosis cases with high-frequency JAK2 mCAs have marked reductions in measured telomere length - suggesting a relationship between telomere biology and myelofibrosis clonal expansion. Our results advance understanding of the germline-somatic interaction at JAK2 and implicate mCAs involving JAK2 as strong promoters of clonal expansion of those mutated clones.

Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter.

Capillary endothelial cells of the human blood-brain barrier (BBB) express high levels of P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2). However, little information is available regarding ATP-binding cassette transporters expressed at the zebrafish BBB, which has emerged as a potential model system. We report the characterization and tissue localization of two genes that are similar to ABCB1, zebrafish abcb4 and abcb5. When stably expressed in HEK293 cells, both Abcb4 and Abcb5 conferred resistance to P-gp substrates; however, Abcb5 poorly transported doxorubicin and mitoxantrone compared to zebrafish Abcb4. Additionally, Abcb5 did not transport the fluorescent P-gp probes BODIPY-ethylenediamine or LDS 751, while they were transported by Abcb4. High-throughput screening of 90 human P-gp substrates confirmed that Abcb4 has an overlapping substrate specificity profile with P-gp. In the brain vasculature, RNAscope probes for abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. The abcb4 probe also colocalized with claudin-5 in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, potentially indicating different functions. The data suggest that zebrafish Abcb4 functionally phenocopies P-gp and that the zebrafish may serve as a model to study the role of P-gp at the BBB.

Incident disease associations with mosaic chromosomal alterations on autosomes, X and Y chromosomes: insights from a phenome-wide association study in the UK Biobank.

BACKGROUND: Mosaic chromosomal alterations (mCAs) are large chromosomal gains, losses and copy-neutral losses of heterozygosity (LOH) in peripheral leukocytes. While many individuals with detectable mCAs have no notable adverse outcomes, mCA-associated gene dosage alterations as well as clonal expansion of mutated leukocyte clones could increase susceptibility to disease. RESULTS: We performed a phenome-wide association study (PheWAS) using existing data from 482,396 UK Biobank (UKBB) participants to investigate potential associations between mCAs and incident disease. Of the 1290 ICD codes we examined, our adjusted analysis identified a total of 50 incident disease outcomes associated with mCAs at PheWAS significance levels. We observed striking differences in the diseases associated with each type of alteration, with autosomal mCAs most associated with increased hematologic malignancies, incident infections and possibly cancer therapy-related conditions. Alterations of chromosome X were associated with increased lymphoid leukemia risk and, mCAs of chromosome Y were linked to potential reduced metabolic disease risk. CONCLUSIONS: Our findings demonstrate that a wide range of diseases are potential sequelae of mCAs and highlight the critical importance of careful covariate adjustment in mCA disease association studies.

In-utero exposure to zidovudine-containing antiretroviral therapy and clonal hematopoiesis in HIV-exposed uninfected newborns.

OBJECTIVE: Zidovudine (ZDV) has been extensively used in pregnant women to prevent vertical transmission of HIV but few studies have evaluated potential mutagenic effects of ZDV during fetal development. DESIGN: Our study investigated clonal hematopoiesis in HIV-exposed uninfected (HEU) newborns, 94 of whom were ZDV-exposed and 91 antiretroviral therapy (ART)-unexposed and matched for potential confounding factors. METHODS: Utilizing high depth sequencing and genotyping arrays, we comprehensively examined blood samples collected during the first week after birth for potential clonal hematopoiesis associated with fetal ZDV exposure, including clonal single nucleotide variants (SNVs), small insertions and deletions (indels), and large structural copy number or copy neutral alterations. RESULTS: We observed no statistically significant difference in the number of SNVs and indels per person in ZDV-exposed children (adjusted ratio [95% confidence interval, CI] for expected number of mutations = 0.79 [0.50--1.22], P = 0.3), and no difference in the number of large structural alterations. Mutations in common clonal hematopoiesis driver genes were not found in the study population. Mutational signature analyses on SNVs detected no novel signatures unique to the ZDV-exposed children and the mutational profiles were similar between the two groups. CONCLUSION: Our results suggest that clonal hematopoiesis at levels detectable in our study is not strongly influenced by in-utero ZDV exposure; however, additional follow-up studies are needed to further evaluate the safety and potential long-term impacts of in-utero ZDV exposure in HEU children as well as better investigate genomic aberrations occurring late in pregnancy.

Identification of Activators of Human Fumarate Hydratase by Quantitative High-Throughput Screening.

Fumarate hydratase (FH) is a metabolic enzyme that is part of the Krebs cycle and reversibly catalyzes the hydration of fumarate to malate. Mutations of the FH gene have been associated with fumarate hydratase deficiency (FHD), hereditary leiomyomatosis and renal cell cancer (HLRCC), and other diseases. Currently, there are no high-quality small-molecule probes for studying human FH. To address this, we developed a quantitative high-throughput screening (qHTS) FH assay and screened a total of 57,037 compounds from in-house libraries in dose-response. While no inhibitors of FH were confirmed, a series of phenyl-pyrrolo-pyrimidine-diones were identified as activators of human FH. These compounds were not substrates of FH, were inactive in a malate dehydrogenase counterscreen, and showed no detectable reduction-oxidation activity. The binding of two compounds from the series to human FH was confirmed by microscale thermophoresis. The low hit rate in this screening campaign confirmed that FH is a "tough target" to modulate, and the small-molecule activators of human FH reported here may serve as a starting point for further optimization and development into cellular probes of human FH and potential drug candidates.

Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening.

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes, and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed the cell-based cytotoxicity of nearly 10,000 compounds in the National Institutes of Health, National Center for Advancing Translational Sciences annotated libraries and more than 100,000 compounds in a diversity library against four normal cell lines (HEK 293, NIH 3T3, CRL-7250, and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity, and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitute a valuable resource for the scientific community and provide insight into the extent of cytotoxic compounds in screening libraries, allowing for the identification and avoidance of compounds with cytotoxicity during high-throughput screening campaigns.

A High-Throughput Screen of a Library of Therapeutics Identifies Cytotoxic Substrates of P-glycoprotein.

The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to limit both brain penetration and oral bioavailability of many chemotherapy drugs. Although US Food and Drug Administration guidelines require that potential interactions of investigational drugs with P-gp be explored, often this information does not enter the literature. In response, we developed a high-throughput screen to identify substrates of P-gp from a series of chemical libraries, testing a total of 10,804 compounds, most of which have known mechanisms of action. We used the CellTiter-Glo viability assay to test library compounds against parental KB-3-1 human cervical adenocarcinoma cells and the colchicine-selected subline KB-8-5-11 that overexpresses P-gp. KB-8-5-11 cells were also tested in the presence of a P-gp inhibitor (tariquidar) to assess reversibility of transporter-mediated resistance. Of the tested compounds, a total of 90 P-gp substrates were identified, including 55 newly identified compounds. Substrates were confirmed using an orthogonal killing assay against human embryonic kidney-293 cells overexpressing P-gp. We confirmed that AT7159 (cyclin-dependent kinase inhibitor), AT9283, (Janus kinase 2/3 inhibitor), ispinesib (kinesin spindle protein inhibitor), gedatolisib (PKI-587, phosphoinositide 3-kinase/mammalian target of rampamycin inhibitor), GSK-690693 (AKT inhibitor), and KW-2478 (heat-shock protein 90 inhibitor) were substrates. In addition, we assessed direct ATPase stimulation. ABCG2 was also found to confer high levels of resistance to AT9283, GSK-690693, and gedatolisib, whereas ispinesib, AT7519, and KW-2478 were weaker substrates. Combinations of P-gp substrates and inhibitors were assessed to demonstrate on-target synergistic cell killing. These data identified compounds whose oral bioavailability or brain penetration may be affected by P-gp. SIGNIFICANCE STATEMENT: The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to be expressed at barrier sites, where it acts to limit oral bioavailability and brain penetration of substrates. In order to identify novel compounds that are transported by P-gp, we developed a high-throughput screen using the KB-3-1 cancer cell line and its colchicine-selected subline KB-8-5-11. We screened the Mechanism Interrogation Plate (MIPE) library, the National Center for Advancing Translational Science (NCATS) pharmaceutical collection (NPC), the NCATS Pharmacologically Active Chemical Toolbox (NPACT), and a kinase inhibitor library comprising 977 compounds, for a total of 10,804 compounds. Of the 10,804 compounds screened, a total of 90 substrates were identified of which 55 were novel. P-gp expression may adversely affect the oral bioavailability or brain penetration of these compounds.

Discovery of novel inhibitors of human galactokinase by virtual screening.

Classic Galactosemia is a potentially lethal autosomal recessive metabolic disorder caused by deficient galactose-1-phosphate uridyltransferase (GALT) that results in the buildup of galactose-1-phosphate (gal-1-p) in cells. Galactokinase (GALK1) is the enzyme responsible for converting galactose into gal-1-p. A pharmacological inhibitor of GALK1 is hypothesized to be therapeutic strategy for treating galactosemia by reducing production of gal-1-p. In this study, we report the discovery of novel series of GALK1 inhibitors by structure-based virtual screening (VS). Followed by an extensive structural modeling and binding mode analysis of the active compounds identified from quantitative high-throughput screen (qHTS), we developed an efficient pharmacophore-based VS approach and applied for a large-scale in silico database screening. Out of 230,000 compounds virtually screened, 350 compounds were cherry-picked based on multi-factor prioritization procedure, and 75 representing a diversity of chemotypes exhibited inhibitory activity in GALK1 biochemical assay. Furthermore, a phenylsulfonamide series with excellent in vitro ADME properties was selected for downstream characterization and demonstrated its ability to lower gal-1-p in primary patient fibroblasts. The compounds described herein should provide a starting point for further development of drug candidates for the GALK1 modulation in the Classic Galactosemia.

High-throughput screening with nucleosome substrate identifies small-molecule inhibitors of the human histone lysine methyltransferase NSD2.

The histone lysine methyltransferase nuclear receptor-binding SET domain protein 2 (NSD2, also known as WHSC1/MMSET) is an epigenetic modifier and is thought to play a driving role in oncogenesis. Both NSD2 overexpression and point mutations that increase its catalytic activity are associated with several human cancers. Although NSD2 is an attractive therapeutic target, no potent, selective, and bioactive small molecule inhibitors of NSD2 have been reported to date, possibly due to the challenges of developing high-throughput assays for NSD2. Here, to establish a platform for the discovery and development of selective NSD2 inhibitors, we optimized and implemented multiple assays. We performed quantitative high-throughput screening with full-length WT NSD2 and a nucleosome substrate against a diverse collection of bioactive small molecules comprising 16,251 compounds. We further interrogated 174 inhibitory compounds identified in the primary screen with orthogonal and counter assays and with activity assays based on the clinically relevant NSD2 variants E1099K and T1150A. We selected five confirmed inhibitors for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. We found that all five NSD2 inhibitors bind the catalytic SET domain and one exhibited apparent activity in cells, validating the workflow and providing a template for identifying selective NSD2 inhibitors. In summary, we have established a robust discovery pipeline for identifying potent NSD2 inhibitors from small-molecule libraries.

Assessing inhibitors of mutant isocitrate dehydrogenase using a suite of pre-clinical discovery assays.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that are mutated in a variety of cancers to confer a gain-of-function activity resulting in the accumulation of an oncometabolite, D-2-hydroxyglutarate (2-HG). Accumulation of 2-HG can result in epigenetic dysregulation and a block in cellular differentiation, suggesting these mutations play a role in neoplasia. Based on its potential as a cancer target, a number of small molecule inhibitors have been developed to specifically inhibit mutant forms of IDH (mIDH1 and mIDH2). We present a comprehensive suite of in vitro preclinical drug development assays that can be used as a tool-box to identify lead compounds for mIDH drug discovery programs, as well as what we believe is the most comprehensive publically available dataset on the top mIDH inhibitors. This involved biochemical, cell-based, and tier-one ADME techniques.

Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity.

Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z'-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ∼1 µM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development.