David M. Evans

Bromodomain and hedgehog pathway targets in small cell lung cancer.

Small cell lung cancer (SCLC) is an extremely aggressive cancer that frequently recurs. Twenty-three human SCLC lines were selected representing varied Myc status. Gene expression of lung cancer, stem-like, hedgehog pathway, and notch pathway genes were determined by RT(2)-PCR array and Exon 1.0 ST array. Etoposide and topotecan concentration response was examined. The IC50s for etoposide and topotecan ranged over nearly 3 logs upon 96 hrs exposure to the drugs. Myc status, TOP2A, TOP2B and TOP1 mRNA expression or topoisomerase 1 and topoisomerase 2 protein did not account for the range in the sensitivity to the drugs. γ-secretase inhibitors, RO429097 and PF-03084014, had little activity in the SCLC lines over ranges covering the clinical Cmax concentrations. MYC amplified lines tended to be more sensitive to the bromodomain inhibitor JQ1. The Smo antagonists, erismodegib and vismodegib and the Gli antagonists, HPI1 and SEN-450 had a trend toward greater sensitivity of the MYC amplified line. Recurrent SCLC is among the most recalcitrant cancers and drug development efforts in this cancer are a high priority.

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.

Abstract 5508: Bromodomain and hegdehog pathway targets in small cell lung cancer

Small cell lung cancer (SCLC) is an extremely aggressive cancer that frequently recurs after conventional cytotoxic chemotherapy. SCLC is a neuroendocrine lung malignancy for which there is no effective treatment that affects >200,000 people world-wide every year with a very high mortality rate ( -4-fold more sensitive to JQ1 than the lines without Myc amplification while the L-Myc over-expressing lines were >-3-fold more sensitive and the n-Myc over-expressing lines were >-9-fold more sensitive to JQ1 than the lines without Myc amplification. Concentration response experiments with the same 24 SCLC lines were carried out with the Hedgehog pathway inhibitors HPI1, SEN-450, LDE225 (erismodegib; clinical Cmax = 9 uM) and GDC0049 (vismodegib; clinical Cmax = 34 uM). The SCLC lines which over-express Myc were more sensitive to the 4 hedgehog pathway inhibitors than were the lines without Myc amplification. Although the 6 c-Myc over-expressing lines had higher levels of SMO gene expression than the other SCLC lines, they were not different in sensitivity to the SMO inhibitors than the other Myc over-expressing lines. Further studies of SCLC lines response to investigational agents are underway. Citation Format: Gurmeet Kaur, Russell A. Reinhart, Anne Monks, David Evans, Joel Morris, Beverly A. Teicher. Bromodomain and hegdehog pathway targets in small cell lung cancer. [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 5508. doi:10.1158/1538-7445.AM2014-5508

3D Cell Culture Models

In the 1960s, scientists searching for a cell-based system that would reflect the physiology and heterogeneity of solid tumors in a well-controlled experimental setting developed multicell spheroid cultures. Early studies focused on the biology, physiology, and drug and radiation response of spheroids. With the advent of high-throughput screening, the focus returned to improving the cell-based models. Plasticware and other tools were developed to allow high-throughput screening with spheroids. To improve cell-based models further, mixed-cell spheroids, including stromal components, fibroblasts, and endothelial cells, are being used in compound and drug testing. To move models closer to the patient, clinical specimens are being tested as tumor minceates. Malignant and other cells from the tumor tissue are treated as organoids to select drugs for specific patients. The field is moving toward freshly-prepared tumor cells from clinical specimens to allow rapid screening of drugs for personalized medicine in the treatment of cancer.

Abstract 348: Comparison of the response of the NCI60 NSCLC panel with the response of patient-derived NSCLC lines to approved and investigational agents

An 800 compound screen with the NCI60 cell lines and 5 patient-derived NSCLC lines (PD NSCLC), was conducted at 9 concentrations and included the FDA approved oncology agents and an investigational agents library. The screen was conducted using 384-well monolayer cultures, an exposure time of 72 hrs and compound concentrations from 1 nM to 10 uM. CellTiter-Glo was used to measure viability as an endpoint. The NCI60 NSCLC panel consists of 9 cell lines: A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460 and NCI-H522. The response of these lines was compared with the response of 5 PD NSCLC lines and 60 SCLC lines. While both sets of NSCLC lines had similar responses to the majority of compounds, there were some marked differences. Differences in response of the NCI60 NSCLC lines and the PD NSCLC lines were notable upon exposure to tubulin fragmenters, KSP/EG5 inhibitors, GAR transformylase inhibitors, Polo-like kinase-1 inhibitors (PLK1), MEK inhibitors, and IAP inhibitors. The NCI60 NSCLC lines were more sensitive to the tubulin fragmenters than were the PD NSCLC and the SCLC lines. Verubulin had a mean GI 50 in NCI60 NSCLC of 0.0037 uM and 0.0042 uM in SCLC versus 0.047uM in the PD NSCLC lines. The NCI60 NSCLC lines were more sensitive to the KSP inhibitor, ARRY-520 (mean GI 50 0.0049 uM) than the SCLC lines (0.028 uM) and the PD NSCLC lines (0.26 uM). The NCI60 NSCLC lines (mean GI 50 0.019 uM) and the SCLC lines (mean GI50 0.04 uM) were more sensitive to PLK1 inhibitors such as BI-2536 than the PD NSCLC lines (0.13 uM). However, the NCI60 NSCLC lines (mean GI 50 0.8 uM) and the SCLC lines (mean GI 50 0.134 uM) were less sensitive than the PD NSCLC lines (mean GI 50 0.025uM) to GAR transformylase inhibitors such as pelitrexol. The NCI60 NSCLC lines (mean GI 50 6.3 uM) and SCLC lines (mean GI 50 10 uM) were also less responsive to IAP inhibitors, than were PD NSCLC lines (mean GI 50 1.6 uM). A heterogeneous response to the MEK inhibitors such as cobimetinib (GDC-0973) was observed with the SCLC lines (mean GI 50 8.9 uM) versus 1.15 uM in the NCI60 NSCLC lines and 0.56 uM in the PD NSCLC lines. PD NSCLC lines exhibit some interesting differences in response from established lung cancer lines upon in vitro exposure to anticancer agents and thus add to our knowledge and understanding of NSCLC and help inform discovery efforts and/or clinical development of therapeutics in this disease. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261200800001E. Citation Format: Beverly A. Teicher, David Evans, Thomas Silvers, Michael Selby, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Joel Morris, Gurmeet Kaur, James Doroshow. Comparison of the response of the NCI60 NSCLC panel with the response of patient-derived NSCLC lines to approved and investigational agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 348. doi:10.1158/1538-7445.AM2017-348

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‐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.

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 5178: Determinants of response to temozolomide in an exceptionally sensitive patient derived model

Application of precision medicine to cancer treatment utilizes cutting-edge genomic sequencing techniques to identify specific mutations in tumors that can be matched to targeted therapies designed to treat those abnormalities. To complement NCI-MPACT, an ongoing molecular profiling-based clinical trial (NCT01827384), we used cell lines developed from several patient-derived xenograft (PDX) models to examine response to, and potential biomarkers for, the regimens of the 4-arm MPACT trial: veliparib (VLP)/temozolomide (TMZ), AZD1775/carboplatin, everolimus and trametinib. In vitro sensitivity of the PDX-derived cell lines to clinically achievable concentrations of these MPACT drugs (combinations and single agents) was examined in classic 2D cultures (monolayer on plastic) and 3D cultures (spheroids generated in ultra-low attachment culture plates). Responses in 2D and 3D cultures were similar after 7 days of drug exposure. Moreover, adding VLP (1.7μM or 5μM) in combination with TMZ did not enhance TMZ cytotoxicity. A bladder cancer cell line developed from a PDX model (BLX) showed exceptional sensitivity to TMZ (IC50 ∼ 3-5μM) compared to a lung cancer cell line (also produced from a PDX; LUX) which was insensitive (IC50 >40 μM). Loss of MGMT expression in glioma and possibly in colorectal carcinoma is considered a predictive biomarker for response to alkylating agents, such as dacarbazine and TMZ. Indeed, we could not detect MGMT protein expression in BLX, while MGMT was present at high levels in LUX. To elucidate unique determinants of BLX hypersensitivity to TMZ beyond MGMT expression, we examined DNA damage responses elicited in this model. Under both 2D and 3D conditions, exposure to TMZ (13 and 40μM) for various times (4, 8, 12, 24, 48, 72 and 96 hrs) induced γH2AX after 24 hr, while PARP1 cleavage was induced as early as 48 hrs after drug addition indicating the onset of apoptosis. TMZ activated ATR and ATM signaling pathways especially at the later time points, paralleling the pharmacodynamics of PARP cleavage. Moreover, concordant with the sensitivity profiles, signaling activation in 2D and 3D conditions was similar. In contrast, none of these pathways were activated in the TMZ non-responsive LUX model. Cell line models developed from PDXs with intermediate MGMT expression are being evaluated. A key to success of personalized medicine in oncology will be the identification of genomic determinants that predict which individual cases will show exceptional responses to particular treatments, and our data suggest that PDX cell line models may be valuable for elucidating molecular and genetic characteristics of response to specific drugs and for the identification of predictive biomarkers. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Lara H. el Touny, John Connelly, Curtis Hose, Anne Monks, Mark W. Burkett, Erik Harris, Rene’ M. Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Michael Selby, Thomas Silvers, David Evans, Dianne Newton, Luke Stockwin, Melinda Hollingshead, Ralph Parchment, James H. Doroshow, Beverly Teicher, Annamaria Rapisarda. Determinants of response to temozolomide in an exceptionally sensitive patient derived model. [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 5178.

Abstract 5406: Osteosarcoma cell lines response to approved and investigational anticancer agents and statins along with gene and microRNA expression

Background: Sarcomas are rare heterogeneous cancers with significant unmet medical need. Approved and Investigational compounds were tested against 64 human sarcoma cell lines. Gene expression was determined using Affymetrix Exon ST1 arrays. Eleven of 64 human sarcoma lines were osteosarcoma. Methods: 64 adult and pediatric sarcoma lines were screened against the approved and investigational agents. Compounds were screened in 9-pt concentration response (0.001-10 μM). The cells were cultured in 384-well plates overnight, and then compounds were added (DMSO, 0.25%). Viability was determined after 96h using Alamar Blue. In separate experiments, the response of the osteosarcoma lines to 9 statins was assessed at 9 concentrations with a 96h exposure time and CellTiter-Glo® as endpoint. Concentration response curves and IC50′s were determined from an average of 3 experiments. Gene expression was determined by the Exon ST1 array. MicroRNA expression was determined using the NanoString human miRNA probe set on the nCounter® Prep Station followed by immobilization in the cartridge for data collection. Results: The genes highly differentially expressed by the osteosarcoma lines, RUNX2, CDC42EP3, CGNT1, EHD4 and EST1, were compared with simvastatin (NSC633782) sensitivity. The response of the osteosarcoma lines to 9 statins showed that pitavastatin (NSC779706) was most effective with IC50 ranging from 0.2 - 5 μM. The SAOS-2 line was most responsive to the statins. Overall, the synthetic statins (fluvastatin, pitavastatin and atorvastatin) were more effective against the osteosarcoma cell lines than the fermentation-derived statins. Among the fermentation-derived statins, simvastatin had the most similarity in potency and pattern to the synthetic statins. All ten osteosarcoma cell lines tested were resistant to pravastatin, rosvustatin, and bestatin (IC50>10μM). ERBB4 was highly expressed in 3 of 11 osteosarcoma lines. These 3 lines were the most sensitive to 13 EGFR inhibitors tested. SAOS-2 cells express high FGFR2 and KIT, CHA59 cells express high FGFR3 and SK-ES-1 cells highly express KIT. Eighteen approved and investigational broad spectrum kinase inhibitors and selective FGFR, KIT and MET inhibitors were tested. The most sensitive osteosarcoma lines were SK-ES-1, CHA59, HOS, and Hu09 while SA-OS-2 was less sensitive. MYC, MYCL1, and MYCN gene expression indicated that SAOS-2, OHS, CHA59 and SK-ES-1 express high MYC. OHS, SK-ES-1, CHA59, HOS and Hu09 cells were sensitive to the two bromodomain inhibitors tested, JQ1 and GSK-1210151A, while SAOS-2 was less sensitive. The SJSA-1 line expresses high GLI1, PTCH1 and PTCH2. Five SMO inhibitors were tested and only the Hu09 cells responded. Conclusions: Testing investigational agents in a panel of sarcoma cell lines along with molecular characterization may help identify new drugs worthy of clinical trial in sarcoma. Citation Format: Gurmeet Kaur, Eric Polley, Joel Morris, Thomas Silvers, Michael Selby, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Anne Monks, Annamaria Rapisarda, David Evans, Beverly A. Teicher. Osteosarcoma cell lines response to approved and investigational anticancer agents and statins along with gene and microRNA expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5406. doi:10.1158/1538-7445.AM2015-5406