ParSE-seq: A Calibrated Multiplexed Assay to Facilitate the Clinical Classification of Putative Splice-altering Variants.

Background: Interpreting the clinical significance of putative splice-altering variants outside 2-base pair canonical splice sites remains difficult without functional studies. Methods: We developed Parallel Splice Effect Sequencing (ParSE-seq), a multiplexed minigene-based assay, to test variant effects on RNA splicing quantified by high-throughput sequencing. We studied variants in SCN5A, an arrhythmia-associated gene which encodes the major cardiac voltage-gated sodium channel. We used the computational tool SpliceAI to prioritize exonic and intronic candidate splice variants, and ClinVar to select benign and pathogenic control variants. We generated a pool of 284 barcoded minigene plasmids, transfected them into Human Embryonic Kidney (HEK293) cells and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), sequenced the resulting pools of splicing products, and calibrated the assay to the American College of Medical Genetics and Genomics scheme. Variants were interpreted using the calibrated functional data, and experimental data were compared to SpliceAI predictions. We further studied some splice-altering missense variants by cDNA-based automated patch clamping (APC) in HEK cells and assessed splicing and sodium channel function in CRISPR-edited iPSC-CMs. Results: ParSE-seq revealed the splicing effect of 224 SCN5A variants in iPSC-CMs and 244 variants in HEK293 cells. The scores between the cell types were highly correlated (R2=0.84). In iPSCs, the assay had concordant scores for 21/22 benign/likely benign and 24/25 pathogenic/likely pathogenic control variants from ClinVar. 43/112 exonic variants and 35/70 intronic variants with determinate scores disrupted splicing. 11 of 42 variants of uncertain significance were reclassified, and 29 of 34 variants with conflicting interpretations were reclassified using the functional data. SpliceAI computational predictions correlated well with experimental data (AUC = 0.96). We identified 20 unique SCN5A missense variants that disrupted splicing, and 2 clinically observed splice-altering missense variants of uncertain significance had normal function when tested with the cDNA-based APC assay. A splice-altering intronic variant detected by ParSE-seq, c.1891-5C>G, also disrupted splicing and sodium current when introduced into iPSC-CMs at the endogenous locus by CRISPR editing. Conclusions: ParSE-seq is a calibrated, multiplexed, high-throughput assay to facilitate the classification of candidate splice-altering variants.

Multicenter clinical and functional evidence reclassifies a recurrent noncanonical filamin C splice-altering variant.

BACKGROUND: Truncating variants in filamin C (FLNC) can cause arrhythmogenic cardiomyopathy (ACM) through haploinsufficiency. Noncanonical splice-altering variants may contribute to this phenotype. OBJECTIVE: The purpose of this study was to investigate the clinical and functional consequences of a recurrent FLNC intronic variant of uncertain significance (VUS), c.970-4A>G. METHODS: Clinical data in 9 variant heterozygotes from 4 kindreds were obtained from 5 tertiary health care centers. We used in silico predictors and functional studies with peripheral blood and patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Isolated RNA was studied by reverse transcription polymerase chain reaction. iPSC-CMs were further characterized at baseline and after nonsense-mediated decay (NMD) inhibition, using quantitative polymerase chain reaction (qPCR), RNA-sequencing, and cellular electrophysiology. American College of Medical Genetics and Genomics (ACMG) criteria were used to adjudicate variant pathogenicity. RESULTS: Variant heterozygotes displayed a spectrum of disease phenotypes, spanning from mild ventricular dysfunction with palpitations to severe ventricular arrhythmias requiring device shocks or progressive cardiomyopathy requiring heart transplantation. Consistent with in silico predictors, the c.970-4A>G FLNC variant activated a cryptic splice acceptor site, introducing a 3-bp insertion containing a premature termination codon. NMD inhibition upregulated aberrantly spliced transcripts by qPCR and RNA-sequencing. Patch clamp studies revealed irregular spontaneous action potentials, increased action potential duration, and increased sodium late current in proband-derived iPSC-CMs. These findings fulfilled multiple ACMG criteria for pathogenicity. CONCLUSION: Clinical, in silico, and functional evidence support the prediction that the intronic c.970-4A>G VUS disrupts splicing and drives ACM, enabling reclassification from VUS to pathogenic.

Exposure time versus cytotoxicity for anticancer agents.

PURPOSE: Time is a critical factor in drug action. The duration of inhibition of the target or residence time of the drug molecule on the target often guides drug scheduling. METHODS: The effects of time on the concentration-dependent cytotoxicity of approved and investigational agents [300 compounds] were examined in the NCI60 cell line panel in 2D at 2, 3, 7 and in 3D 11 days. RESULTS: There was a moderate positive linear relationship between data from the 2-day NCI60 screen and the 3-, 7- and 11-day and a strong positive linear relationship between 3-, 7- and 11-day luminescence screen IC50s by Pearson correlation analysis. Cell growth inhibition by agents selective for a specific cell cycle phase plateaued when susceptible cells were growth inhibited or killed. As time increased the depth of cell growth inhibition increased without change in the IC50. DNA interactive agents had decreasing IC50s with increasing exposure time. Epigenetic agents required longer exposure times; several were only cytotoxic after 11 days' exposure. For HDAC inhibitors, time had little or no effect on concentration response. There were potency differences amongst the three BET bromodomain inhibitors tested, and an exposure duration effect. The PARP inhibitors, rucaparib, niraparib, and veliparib reached IC50s < 10 µM in some cell lines after 11 days. CONCLUSIONS: The results suggest that variations in compound exposure time may reflect either mechanism of action or compound chemical half-life. The activity of slow-acting compounds may optimally be assessed in spheroid models that can be monitored over prolonged incubation times.

Small cell lung carcinoma cell line screen of etoposide/carboplatin plus a third agent.

The SCLC combination screen examined a 9-point concentration response of 180 third agents, alone and in combination with etoposide/carboplatin. The predominant effect of adding a third agent to etoposide/carboplatin was additivity. Less than additive effects occurred frequently in SCLC lines sensitive to etoposide/carboplatin. In SCLC lines with little or no response to etoposide/carboplatin, greater than additive SCLC killing occurred over the entire spectrum of SCLC lines but never occurred in all SCLC lines. Exposing SCLC lines to tubulin-targeted agents (paclitaxel or vinorelbine) simultaneously with etoposide/carboplatin resulted primarily in less than additive cell killing. As single agents, nuclear kinase inhibitors including Aurora kinase inhibitors, Kinesin Spindle Protein/EG5 inhibitors, and Polo-like kinase-1 inhibitors were potent cytotoxic agents in SCLC lines; however, simultaneous exposure of the SCLC lines to these agents along with etoposide/carboplatin, generally, resulted in less than additive cell killing. Several classes of agents enhanced the cytotoxicity of etoposide/carboplatin toward the SCLC lines. Exposure of the SCLC lines to the MDM2 inhibitor JNJ-27291199 produced enhanced killing in 80% of the SCLC lines. Chk-1 inhibitors such as rabusertib increased the cytotoxicity of etoposide/carboplatin to the SCLC lines in an additive to greater than additive manner. The combination of GSK-3ß inhibitor LY-2090314 with etoposide/carboplatin increased killing in approximately 40% of the SCLC lines. Exposure to the BET bromodomain inhibitor MK-8628 increased the SCLC cell killing by etoposide/carboplatin in 20-25% of the SCLC lines. Only 10-15% of the SCLC lines had an increased response to etoposide/carboplatin when simultaneously exposed to the PARP inhibitor talazoparib.

3D Models of the NCI60 Cell Lines for Screening Oncology Compounds.

The NCI60 cell line panel screen includes 60 human tumor cell lines derived from nine tumor types that has been used over the past 20+ years to screen small molecules, biologics, and natural products for activity. Cells in monolayer culture in 96-well plates are exposed to compounds for 48 h, and Sulforhodamine B is used to determine cell viability. Data analysis tools such as COMPARE allow classification of compounds based on the pattern of cell line response. However, many compounds highly active in monolayer cell culture fail to show efficacy in vivo. Therefore, we explored 3D culture of the NCI60 panel as a strategy to improve the predictive accuracy of the screen. 3D cultures more closely resemble tumors than monolayer cultures with tighter cell-cell contact and nutrient and oxygen gradients between the periphery and the center. We optimized the NCI60 cell line panel for generating 3D spheroids of a prespecified diameter (300-500 µm) in ultra-low attachment (ULA) plates. Spheroids were classified into four categories based on imaging, and concentration response of select agents in 2D and 3D models is presented.

Small Cell Lung Cancer Screen of Oncology Drugs, Investigational Agents, and Gene and microRNA Expression.

BACKGROUND: Small cell lung carcinoma (SCLC) is an aggressive, recalcitrant cancer, often metastatic at diagnosis and unresponsive to chemotherapy upon recurrence, thus it is challenging to treat. METHODS: Sixty-three human SCLC lines and three NSCLC lines were screened for response to 103 US Food and Drug Administration-approved oncology agents and 423 investigational agents. The investigational agents library was a diverse set of small molecules that included multiple compounds targeting the same molecular entity. The compounds were screened in triplicate at nine concentrations with a 96-hour exposure time using an ATP Lite endpoint. Gene expression was assessed by exon array, and microRNA expression was derived by direct digital detection. Activity across the SCLC lines was associated with molecular characteristics using pair-wise Pearson correlations. RESULTS: Results are presented for inhibitors of targets: BCL2, PARP1, mTOR, IGF1R, KSP/Eg5, PLK-1, AURK, and FGFR1. A relational map identified compounds with similar patterns of response. Unsupervised microRNA clustering resulted in three distinct SCLC subgroups. Associating drug response with micro-RNA expression indicated that lines most sensitive to etoposide and topotecan expressed high miR-200c-3p and low miR-140-5p and miR-9-5p. The BCL-2/BCL-XL inhibitors produced similar response patterns. Sensitivity to ABT-737 correlated with higher ASCL1 and BCL2. Several classes of compounds targeting nuclear proteins regulating mitosis produced a response pattern distinct from the etoposide response pattern. CONCLUSIONS: Agents targeting nuclear kinases appear to be effective in SCLC lines. Confirmation of SCLC line findings in xenografts is needed. The drug and compound response, gene expression, and microRNA expression data are publicly available at http://sclccelllines.cancer.gov.

Sarcoma Cell Line Screen of Oncology Drugs and Investigational Agents Identifies Patterns Associated with Gene and microRNA Expression.

The diversity in sarcoma phenotype and genotype make treatment of this family of diseases exceptionally challenging. Sixty-three human adult and pediatric sarcoma lines were screened with 100 FDA-approved oncology agents and 345 investigational agents. The investigational agents' library enabled comparison of several compounds targeting the same molecular entity allowing comparison of target specificity and heterogeneity of cell line response. Gene expression was derived from exon array data and microRNA expression was derived from direct digital detection assays. The compounds were screened against each cell line at nine concentrations in triplicate with an exposure time of 96 hours using Alamar blue as the endpoint. Results are presented for inhibitors of the following targets: aurora kinase, IGF-1R, MEK, BET bromodomain, and PARP1. Chemical structures, IC50 heat maps, concentration response curves, gene expression, and miR expression heat maps are presented for selected examples. In addition, two cases of exceptional responders are presented. The drug and compound response, gene expression, and microRNA expression data are publicly available at http://sarcoma.cancer.gov. These data provide a unique resource to the cancer research community.

A high-throughput fluorescence-anisotropy screen that identifies small molecule inhibitors of the DNA binding of B-ZIP transcription factors.

We have developed a high-throughput fluorescence anisotropy screen, using a 384-well format, to identify small molecules that disrupt the DNA binding of B-ZIP proteins. Binding of a B-ZIP dimer to fluorescently labeled DNA can be monitored by fluorescence anisotropy. We screened the National Cancer Institute diversity set of 1990 compounds to identify small molecules that disrupt the B-ZIP|DNA complex of CREB, C/EBPbeta, VBP, and AP-1 (FOS|JUND) bound to their cognate DNA sequence. We identified 21 compounds that inhibited the DNA binding of at least one B-ZIP protein, and 12 representative compounds were grouped depending on whether they displaced ethidium bromide from DNA. Of the 6 compounds that did not displace ethidium bromide, 2 also inhibited B-ZIP binding to DNA in a secondary electrophoretic mobility shift assay screen with some specificity. Thermal stability monitored by circular dichroism spectroscopy demonstrated that both compounds bound the basic region of the B-ZIP motif. NSC13778 preferentially binds C/EBPalpha 1000-fold better than it binds C/EBPbeta. Chimeric proteins combining C/EBPalpha and C/EBPbeta mapped the binding of NSC13778 to three amino acids immediately N terminal of the leucine zipper of C/EBPalpha. These experiments suggest that the DNA binding of B-ZIP transcription factors is a potential target for clinical intervention.