Rene Delosh

Identification of CBX3 and ABCA5 as Putative Biomarkers for Tumor Stem Cells in Osteosarcoma

Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma.

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.

Abstract 5590: Screening of pediatric and adult sarcoma cell lines reveals novel patterns of sensitivity toward approved oncology drugs and investigational agents.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Sarcomas represent a heterogeneous group of neglected cancers with significant unmet medical need. To date, large-scale screening of compounds for activity against sarcomas has not been carried out in any systematic fashion to enable drug discovery. Our laboratory has compiled a panel of > 85 human pediatric and adult sarcoma cell lines, a tumor type not represented in the NCI60, and has carried out a high throughput screen of approximately 60 of these lines against the Approved Oncology Drugs (AOD) and Investigational Agents libraries, using inhibition of proliferation as an endpoint. The AOD library is comprised of 93 FDA-approved anticancer agents, and is available from The NCI/DTP Open Chemical Repository. The Investigational Agents library includes about 350 small molecules of interest. The compounds were screened for inhibition of cell proliferation in 9-point concentration response, starting at 10 uM and diluting by half-logs. The cells were cultured in 384-well plates for 24 hrs, at which time compounds were added with a final DMSO concentration of 0.25%. Viability was determined after 96 hrs of exposure using Alamar Blue. The % viability was determined relative to a vehicle-treated control, and this value was plotted relative to compound concentration to determine the EC50. The Ewing sarcoma lines (19) were unexpectedly more sensitive to pemetrexed, cabazitaxel, and floxuridine, and were, as expected, sensitive to topoisomerase 1 inhibitors relative to other sarcomas and to historical NCI60 data. Synovial sarcoma lines were sensitive to vinorelbine, vandetinib, and gemcitabine relative to other sarcomas. Further analyses and findings from the data will be presented. Funded by NCI Contract no. HHSN261200800001E. Citation Format: E. Michael August, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Michael Selby, Thomas Silvers, Joel Morris, Beverly A. Teicher. Screening of pediatric and adult sarcoma cell lines reveals novel patterns of sensitivity toward approved oncology drugs and investigational agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5590. doi:10.1158/1538-7445.AM2013-5590

Abstract 606: Similar 3D pharmacodynamic (3D-PD) responses of human tumor spheroids and xenografts to topoisomerase 1 inhibitor-induced DNA damage

Three-dimensional (3D) cultures have been proposed as higher fidelity models of in vivo tumors than 2D cultures. To determine whether this idea extends to drug pharmacodynamics, we examined whether 3D cultures of the human melanoma line A375 (grown as tumor spheroids) could replicate the timing and magnitude of the nuclear γH2AX response to the topoisomerase 1 inhibitor, topotecan observed in A375 tumor xenografts (Kinders et al, Clin Can Res 2010). The appearance of γH2AX-positive nuclear foci has been used as a biomarker for drug- and radiation-induced double-strand breaks in DNA; we reported previously that treatment of nu/nu mice harboring A375 xenografts with a single-dose of topotecan induced nuclear γH2AX foci in a dose- and time-dependent manner, which peaked 4 hours after drug administration. A375 spheroids were generated by seeding cells into ULA U-bottom plates; 4 hours of exposure to 0.1 μM topotecan elicited a γH2AX signal in these 3D cultures while cell viability and intracellular ATP levels remained unchanged, indicating a DNA damage repair response at the maximally tolerated topotecan concentration for human hematopoietic cells (Erickson-Miller et al, Can Chemo Pharmacol 2009). Extending drug exposure to 24 hours caused substantial loss of viable cells (calcein AM+) and 50% decline in ATP levels but no further increase in γH2AX. In contrast, the HT29 tumor line was refractory to topotecan in vivo, and exposing 3D cultures of HT-29 spheroids to 0.1 μM topotecan for 24 hours elicited a strong nuclear γH2AX biomarker response in only a small fraction of cells at the surface of the spheroids. Longer exposure durations or supra-pharmacological concentrations (1 μM) of topotecan were required to achieve a strong nuclear γH2AX response in HT-29 spheroids. These results support the hypothesis that the 3D pharmacodynamics (PD) of drug response is similar to PD drug response in vivo for the camptothecin class of topoisomerase 1 inhibitors. Funded by NCI Contract No. HHSN261200800001E. Citation Format: David M. Evans, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Michael Selby, Silvers Thomas, Robert Kinders, Tony Navas, Scott Lawrence, Anne Monks, Annamaria Rapisarda, Ralph E. Parchment, James H. Doroshow, Beverly Teicher. Similar 3D pharmacodynamic (3D-PD) responses of human tumor spheroids and xenografts to topoisomerase 1 inhibitor-induced DNA damage. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 606.

Abstract 605: Building better cell models for screening oncology compounds

The NCI60 screen is a valuable compound screening service offered to researchers for 20+ years. The screen allows the submission of small molecules, biologics and natural products for testing in the NCI60 cell line panel (60 cell lines covering 9 major tumor types). Sophisticated tools such as COMPARE allow stratification of compounds based on pattern of cell line response and these tools elucidated several compounds with differential specificity for a disease type (e.g. breast cancer over melanoma). Since inception the NCI60 screen has been performed in 96-well plates on monolayer cultures of cells. SulfoRhodamine B was used to measure cell viability after a 48h exposure to the compounds at either a single concentration or 5 point concentration response assay. However, recent work has suggested that cells in 3D culture may respond differently to compounds than in 2D. 3D culture systems produce tumor cell spheroids that exhibit features seen in vivo including greater cell-cell contact, and gradients of nutrients and oxygen between the periphery and the center of the spheroid. We sought to optimize the NCI60 cells for use in a 3D spheroid model that would be suitable for screening compounds. By varying NCI60 cell plating density and incubation times, we optimized the formation of spheroids of a given diameter (500um) in Ultra Low Attachment (ULA) plates. Spheroids were imaged using a high content imaging plate reader and classified into 4 categories based on their apparent morphologies. Comparisons were made in drug sensitivity between 2D and 3D model systems using the NCI60 and cells derived from PDX samples. Data on the optimal cell densities of the NCI60 lines used for spheroid formation as well as concentration response effects of select agents compared in 2D and 3D models are presented. These data will allow others to rapidly utilize these 3D models to determine the differential drug sensitivity of select cell types. Funded by NCI Contract No. HHSN261200800001E. Citation Format: David M. Evans, Michael Selby, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Thomas Silvers, Ralph E. Parchment, Beverly Teicher. Building better cell models for screening oncology compounds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 605.

Abstract 5450: Screening more than 60 human SCLC lines with approved and investigational agents indicates complex patterns of response: Identification of HSP90 and HDACs as potential targets

Small cell lung cancer (SCLC) is an aggressive cancer with a 5-year survival rate of 500 compounds against > 60 well characterized SCLC cell lines in culture. Cells were exposed to 101 Approved Oncology Drugs (AOD) and 433 Investigational Agents. By evaluating compounds at 9 concentrations (10uM to 1.5nM), using cell viability 96h post drug exposure as the endpoint (Cell Titer Glo), we were able to obtain concentration response curves and IC50 values for the compounds and rank activity of these agents. In parallel studies, using Affymetrix exon ST1 arrays and miRNA profiling, we examined these same cell lines for differences in gene expression patterns that may correlate with sensitivity or resistance to select agents in the screen. The Myc oncogene (cMyc, LMyc, or nMyc) is over-expressed in about 40% of SCLC lines. Surprisingly, early analyses of the SCLC screen data have been unable to correlate high Myc expression with response to drugs or investigational agents (there is a trend with bromodomain inhibitors). The large majority of SCLC lines were sensitive to compounds against select target classes (e.g. HSP90 inhibitors and HDAC inhibitors). Examining HSP70 and HSP90 levels by Western blot suggested a slight reduction in HSP90 levels in cells resistant to the HSP90 inhibitor Ganetespib with no change in HSP70 levels. Some inhibitors against these targets showed broader cell activity than others. We are currently determining whether such differential drug sensitivity is correlated with specific changes in gene expression patterns in these cells. Further data analyses from the screen combined with genomic profiling of the cells will be presented. Funded by NCI Contract No. HHSN261200800001E. Citation Format: David M. Evans, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Michael Selby, Thomas Silvers, John Connelly, Anne Monks, Eric Polley, Gurmeet Kaur, Joel Morris, Beverly Teicher. Screening more than 60 human SCLC lines with approved and investigational agents indicates complex patterns of response: Identification of HSP90 and HDACs as potential targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5450. doi:10.1158/1538-7445.AM2014-5450

Abstract C106: Screening small cell lung cancer lines against approved oncology drugs and investigational agents elucidates patterns of sensitivity.

Small cell lung cancer (SCLC) is an aggressive cancer with a 5-year survival rate of 75 human SCLC lines and is screening approximately 60 (well characterized) lines against 103 agents from the Approved Oncology Drugs (AOD) library) and 420 agents from the Investigational Agents library. Compounds are evaluated at 9 concentrations (10uM to 1.5nM) in comparison with vehicle controls using cell viability 96h post drug exposure as the endpoint - allowing a direct comparison between the IC50, anticipated compound molecular target and the breadth of activity across the cell lines. These cells represent a broad coverage of those available as representatives of SCLC with an array of growth behaviors. Cell lines grow as adherent cultures, in single cell suspension, in suspension as spheres and /or clumps, or mixed cultures. Despite these varied phenotypes, the screen was robust (Z’ score > 0.5) in all of the lines studied to date. Interestingly, SHP77 (an unusual undifferentiated large cell morphological variant with biochemical properties of SCLC) consistently showed lower sensitivity to the drugs than other cell lines tested. Trends emerging from the early data indicate that: 1) Comparing compound activity (IC50 value) by cell line and clustering the compounds based on their reported targets, we could readily observe the target classes that produced the greatest cell growth inhibition across the widest set of the SCLC cell lines; and 2) from these data it appears that discrete HDAC inhibitors and HSP90 inhibitors produce potent growth inhibition across a wide array of SCLC lines (including SHP77). We are expanding the number of SCLC lines under study. Each cell line will be examined for gene expression patterns, miRNA expression patterns and sensitivity to the approved and investigational agents (IC50). These data will allow correlations to be made regarding sensitivity of the cells to agents with discrete cellular targets versus the genomic background in these cells. Data from the drug screens combined with genomic profiling of the cells may allow clinical trials to select therapeutic agents to maximize benefit in patients presenting with SCLC. Further analyses and findings from the data will be presented. Funded by NCI Contract No. HHSN261200800001E. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C106. Citation Format: David M. Evans, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Michael Selby, Thomas Silvers, Anne Monks, Gurmeet Kaur, Joel Morris, Beverly A. Teicher. Screening small cell lung cancer lines against approved oncology drugs and investigational agents elucidates patterns of sensitivity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C106.

Abstract C103: Sarcoma cell line sensitivity towards approved oncology drugs and investigational agents identifies distinct patterns of response which can be interrogated with associated gene expression.

Sarcomas represent a heterogeneous group of cancers with significant unmet medical needs. We have examined the response of 64 sarcoma cell lines to treatment with 103 approved oncology drugs (available from The NCI/DTP Open Chemical Repository) and 420 agents in the investigational agents library, using inhibition of proliferation as an endpoint. Cells were exposed to compounds at varying concentrations (10μM to 1.5nM) for 96 h and the effect of compound on cell viability was monitored using Alamar Blue. From curve fitting algorithms we determined the IC50 values of each agent on the cell lines. Adult sarcomas comprise 23% of this sarcoma panel and have a different spectrum of sensitivities to selected agents. They were generally more chemoresistant than the pediatric lines, although they are marginally more sensitive to MEK inhibitors. Synovial tumor cell lines were an exception, being sensitive to several classes including dasatinib and Bcr-abl inhibitors. Ewings tumors tend to be the most sensitive pediatric group responding to Parp-1 and IGF-1R inhibitors. Overall, gene, and to a lesser extent, miRNA profiles from the adult sarcomas were more similar to the profiles of normal, non-tumor cells, than the pediatric tumors, and this lack of genotypic divergence may underlie the insensitive phenotype observed in the sarcoma panel. From an analysis of sensitivity clustering, IGF-1R inhibitors (8), cluster with some of the AKT (8) and ALK (8) inhibitors. None of the cell lines have the EM4L-ALK translocation, thus we investigated the genes associated with sensitivity to these three mechanisms. Dendrograms identified a close relationship between the IGF-1R and AKT inhibitors, based on the gene expression patterns, while the ALK inhibitors were quite distinct, substantiating known pathways of IGFR-1R signaling through AKT, but unrelated to ALK. Genes associated with ALK sensitivity included several from gluconeogenesis, and potential activation of the MYC response. In contrast, genes associated with AKT and IGF1R sensitivity are focused around FOXO1 transcription factor. Interestingly, the PAX-FOXO1 gene fusion is a hallmark of the aggressive alveolar rhabdomyosarcoma which are more sensitive to these agents than embryonal rhabdomyosarcoma. Thus the combination of drug sensitivity data, together with the gene and miRNA profiles may allow correlations in treatment efficacy that may point to new avenues for clinical development in sarcoma. Funded by NCI Contract No. HHSN261200800001E. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C103. Citation Format: Anne Monks, David Evans, Thomas Silvers, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Michael Selby, John Connelly, Annamaria Rapisarda, Mark Kunkel, Joel Morris, Kazimierz Wrzeszczynski, Eric Polley, Beverley Teicher. Sarcoma cell line sensitivity towards approved oncology drugs and investigational agents identifies distinct patterns of response which can be interrogated with associated gene expression. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C103.