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.
Purpose: The high fatality-to-case ratio of ovarian cancer is directly related to platinum resistance. Exportin-1 (XPO1) is a nuclear exporter that mediates nuclear export of multiple tumor suppressors. We investigated possible clinicopathologic correlations of XPO1 expression levels and evaluated the efficacy of XPO1 inhibition as a therapeutic strategy in platinum-sensitive and -resistant ovarian cancer.Experimental Design: XPO1 expression levels were analyzed to define clinicopathologic correlates using both TCGA/GEO datasets and tissue microarrays (TMA). The effect of XPO1 inhibition, using the small-molecule inhibitors KPT-185 and KPT-330 (selinexor) alone or in combination with a platinum agent on cell viability, apoptosis, and the transcriptome was tested in immortalized and patient-derived ovarian cancer cell lines (PDCL) and platinum-resistant mice (PDX). Seven patients with late-stage, recurrent, and heavily pretreated ovarian cancer were treated with an oral XPO1 inhibitor.Results: XPO1 RNA overexpression and protein nuclear localization were correlated with decreased survival and platinum resistance in ovarian cancer. Targeted XPO1 inhibition decreased cell viability and synergistically restored platinum sensitivity in both immortalized ovarian cancer cells and PDCL. The XPO1 inhibitor-mediated apoptosis occurred through both p53-dependent and p53-independent signaling pathways. Selinexor treatment, alone and in combination with platinum, markedly decreased tumor growth and prolonged survival in platinum-resistant PDX and mice. In selinexortreated patients, tumor growth was halted in 3 of 5 patients, including one with a partial response, and was safely tolerated by all.Conclusions: Taken together, these results provide evidence that XPO1 inhibition represents a new therapeutic strategy for overcoming platinum resistance in women with ovarian cancer.
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.
Background: Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung cancer. SCLC progression and treatment resistance involve epigenetic processes. However, links between SCLC DNA methylation and drug response remain unclear. We performed an epigenome-wide study of 66 human SCLC cell lines using the Illumina Infinium MethylationEPIC BeadChip array. Correlations of SCLC DNA methylation and gene expression with in vitro response to 526 antitumor agents were examined. Results: We found multiple significant correlations between DNA methylation and chemosensitivity. A potentially important association was observed for TREX1, which encodes the 3′ exonuclease I that serves as a STING antagonist in the regulation of a cytosolic DNA-sensing pathway. Increased methylation and low expression of TREX1 were associated with the sensitivity to Aurora kinase inhibitors AZD-1152, SCH-1473759, SNS-314, and TAK-901; the CDK inhibitor R-547; the Vertex ATR inhibitor Cpd 45; and the mitotic spindle disruptor vinorelbine. Compared with cell lines of other cancer types, TREX1 had low mRNA expression and increased upstream region methylation in SCLC, suggesting a possible relationship with SCLC sensitivity to Aurora kinase inhibitors. We also identified multiple additional correlations indicative of potential mechanisms of chemosensitivity. Methylation of the 3′UTR of CEP350 and MLPH, involved in centrosome machinery and microtubule tracking, respectively, was associated with response to Aurora kinase inhibitors and other agents. EPAS1 methylation was associated with response to Aurora kinase inhibitors, a PLK-1 inhibitor and a Bcl-2 inhibitor. KDM1A methylation was associated with PLK-1 inhibitors and a KSP inhibitor. Increased promoter methylation of SLFN11 was correlated with resistance to DNA damaging agents, as a result of low or no SLFN11 expression. The 5′ UTR of the epigenetic modifier EZH2 was associated with response to Aurora kinase inhibitors and a FGFR inhibitor. Methylation and expression of YAP1 were correlated with response to an mTOR inhibitor. Among nonneuroendocrine markers, EPHA2 was associated with response to Aurora kinase inhibitors and a PLK-1 inhibitor and CD151 with Bcl-2 inhibitors.
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 9 concentrations in triplicate with an exposure time of 96 hrs 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.
The formation and stability of protein-protein interfaces are of obvious biological importance. While a large body of literature exists describing the effect of osmolytes on protein folding, very few studies address the effect of osmolytes on protein association and binding. The plant lectin concanavalin A (ConA), which undergoes a reversible tetramer-to-dimer equilibrium as a function of pH, was used as a model system to investigate the influence of nine osmolytes on protein self-association. The stabilizing or destabilizing impacts of the osmolytes were evaluated from pH titrations combined with circular dichroism spectroscopy. Relative to the dimer, trimethylamine N-oxide, betaine, proline, sarcosine, sorbitol, sucrose, and trehalose all stabilized the ConA tetramer to varying extents. Glycerol had a negligible effect, and urea destabilized the tetramer. From multiple titrations in different osmolyte concentrations, an m-value (a thermodynamic parameter describing the change in the association free energy per molar of osmolyte) was determined for each osmolyte. Experimental m-values were compared with those calculated using two theoretical models. The Tanford transfer model, with transfer free energies determined by Bolen and co-workers, failed to accurately predict the m-values in most cases. A model developed by Record and co-workers, currently applicable only to urea, betaine, and proline, more accurately predicted our experimental m-values, but significant discrepancies remained. Further theoretical work is needed to develop a thermodynamic model to predict the effect of osmolytes on protein-protein interfaces, and further experimental work is needed to determine if there is a general stabilization by osmolytes of such interfaces.
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‐3β 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.
Genetic and functional complexity of bulk tumor has become evident through rapid advances in sequencing technologies. As a unique integrated approach to characterizing tumor heterogeneity, we demonstrate the multifaceted capabilities of a novel nanofluidic platform to enable single-cell phenotypic and genetic profiling of ovarian cancer patient-derived tumor cells. This approach has enabled increased resolution of tumor cell phenotypic and genetic heterogeneity, providing a better understanding of underlying biological drivers of the disease. A range of CA-125 expression levels is observed within cells from individuals, demonstrating clonal diversity consistent with other phenotypic data. Further, TP53 mutation analysis demonstrates a sub-population of cells exhibiting high mutation frequency that likely drives downstream growth kinetics and protein expression. Finally, genomic data is orthogonally used to address clonal heterogeneity across ovarian tumors when compared to bulk sequencing, illustrating the potential for single-cell sequencing data integrated with cellular functional and growth data toward future therapeutic intervention.
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