High-throughput screening identifies aclacinomycin as a radiosensitizer of EGFR-mutant non-small cell lung cancer

Abstract

The endoplasmic reticulum (ER) provides a specialized environment for the folding and modification of trans-membrane proteins, including receptor tyrosine kinases (RTKs), which are vital for the growth and survival of malignancies. To identify compounds which disrupt the function of the ER and thus could potentially impair cancer cell survival signaling, we adapted a set of glycosylation-sensitive luciferase reporters for the development and optimization of a cell-based high-throughput screen (HTS). Secondary screens for false-positive luciferase activation and tertiary lectin-based and biochemical analyses were also devised for compound triage. Through a pilot screen of 2802 compounds from the National Cancer Institute (NCI) chemical libraries, we identified aclacinomycin (Acm) as a compound that preferentially affects ER function. We report that Acm reduces plasma membrane expression of glycoproteins including epidermal growth factor receptor (EGFR) and Met but does not inhibit N-linked glycosylation or generalized protein translation. Fluorescence microscopy co-localization experiments were also performed and demonstrated Acm accumulation in the ER in further support of the overall HTS design. The consequences of Acm treatment on cell survival were analyzed through clonogenic survival analysis. Consistent with the reduction of EGFR levels, pretreatment with Acm sensitizes the EGFR-mutant non-small cell lung cancer (NSCLC) cell lines HCC827 and HCC2935 to ionizing radiation and did not affect the sensitivity of the RTK-independent and KRAS-mutant A549 NSCLC cell line. Thus, Acm and similar compounds targeting the ER may represent a novel approach for radiosensitizing tumor cells dependent on RTK function.

Figure 1

Optimization of a bioluminescent HTS to detect changes in N-linked glycosylation. (A) Schematic demonstrating the principle for signal detection using the ERLucT reporter vector: Luciferase is translated into the ER, glycosylated, and inactivated. However, when ER function is disrupted, the luciferase retains bioluminescent activity. (B) DMSO concentration-activity relationship in the D54-ERLucT cell line; representative experiment showing average of eight wells per DMSO concentration. (C) Cell density optimization for 96-well plate format using 1 µM of the GlcNac-phosphotransferase inhibitor Tn; representative experiment showing average of eight wells per treatment. (D) Dose response of Tn under optimized conditions. Data represent average of eight wells per Tn dose. Tn activates luciferase activity with a half-maximal effective concentration of 40 nM.

Figure 2

HTS of NCI compound libraries. (A) Primary screens of 2802 compounds from the NCI Diversity, Mechanistic, and Natural Compound libraries were screened at 10 µM using the D54-ERLucT cell line. The activity of each compound relative to Tn is plotted. (B) Secondary screen of 117 compounds in the D54-LucT cell line, a false-positive screen for nonspecific luciferase activators. Y-axis represents the ratio of primary/secondary screen activity (ERLucT/LucT ratio). Acm is circled in A and B.

Figure 3

Acm does not prevent N-linked glycosylation. (A) Western blot analysis of luciferase size and quantity following incubation with 10 µM of compounds identified in the HTS (top panel). Parallel lysates were also depleted of glycosylated proteins through precipitation with the lectin Con A agarose. Tn was used as a positive control. (B) Activity of Acm (10 µM) in cell-free luciferase assays compared to resveratrol, a known luciferase inhibitor and false positive in cell-based luciferase assays. Data represent average of three independent measurements for each treatment. (C) Lectin-positive growth selection assays. MTT assays were performed in 96-well plates for wild-type CHO and the glycosylation-defective Lec15 CHO cell line with combinations of 2 µg/ml of the mannosidase inhibitor Sw and 12.5 µg/ml Con A (left panel). Data represent an average of three wells per treatment. D54 cells were grown under identical conditions with or without 25 nM Acm and treated with Sw, Con A, or a combination of the two (middle panel). Data represent three wells per treatment. Using the same methods, D54 cells were incubated with increasing concentrations of Con A either with or without Acm pretreatment (right panel). (D) FACE was performed to determine the levels and composition of LLOs extracted from D54 cultures pretreated with 10 µM of each compound for 6 hours. Tn (1 µM) was used as a positive control. The mature 14-carbohydrate LLO (Glc₃Man₉GlcNAc₂) is identified with an arrowhead. An oligosaccharide ladder (left) and a glucose-4 standard (right) provide molecular weight standards.

Figure 4

Acm reduces RTK protein levels. (A) D54 cells were treated with 10 µM Acm (A) or 1 µM Tn (T) for 24 hours, and protein levels of EGFR, Met, and Grp78 were evaluated with Western blots. (B) The effects of Acm A on RTK protein levels were analyzed in the HCC827 cell line using identical conditions. Blots are representative of three independent experiments. HSP70 was used as a loading control.

Figure 5

Localization of Acm in NSCLC. HCC2935 and HCC827 were treated with or without 10 µM Acm for 24 hours followed by incubation with ER-Tracker Blue-White DPX dye to observe co-localization. Cells were observed using an EVOS-FL digital inverted microscope, and merged images were created using the EVOS-FL on-board software. Images were captured using a x40 objective. Scale bar represents 100 µM.

Figure 6

Acm radiosensitizes EGFR-mutant lung cancer cell lines. Clonogenic survival analysis was performed on NSCLC cell lines harboring EGFR kinase domain mutations [(A) HCC827 and (B) HCC2935] or a KRAS mutation [(C) A549]. Cells were pretreated with (triangles) or without (circles) 500 nM Acm for 24 hours. Points represent the average of three experiments performed in triplicate (HCC-2935 and HCC-827) or two experiments performed in triplicate (A549), and error bars represent the SEM.