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

Daniel C. Bennett, Jonathan Charest, Katrina Sebolt, Mark Lehrman, Alnawez Rehemtulla, Joseph N. Contessa

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

The endoplasmic reticulum (ER) provides a specialized environment for the folding and modification of transmembrane 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 KRASmutant 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.

Original languageEnglish (US)
Pages (from-to)382-391
Number of pages10
JournalTranslational Oncology
Volume6
Issue number3
DOIs
StatePublished - 2013

Fingerprint

Epidermal Growth Factor Receptor
Non-Small Cell Lung Carcinoma
Endoplasmic Reticulum
Receptor Protein-Tyrosine Kinases
Luciferases
Glycosylation
Cell Survival
Small Molecule Libraries
Cell Line
Neoplasms
National Cancer Institute (U.S.)
Triage
Membrane Glycoproteins
Protein Biosynthesis
Survival Analysis
Ionizing Radiation
Fluorescence Microscopy
Lectins
aclacinomycins
Cell Membrane

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

High-throughput screening identifies aclacinomycin as a radiosensitizer of EGFR-mutant non-small cell lung cancer. / Bennett, Daniel C.; Charest, Jonathan; Sebolt, Katrina; Lehrman, Mark; Rehemtulla, Alnawez; Contessa, Joseph N.

In: Translational Oncology, Vol. 6, No. 3, 2013, p. 382-391.

Research output: Contribution to journalArticle

Bennett, Daniel C. ; Charest, Jonathan ; Sebolt, Katrina ; Lehrman, Mark ; Rehemtulla, Alnawez ; Contessa, Joseph N. / High-throughput screening identifies aclacinomycin as a radiosensitizer of EGFR-mutant non-small cell lung cancer. In: Translational Oncology. 2013 ; Vol. 6, No. 3. pp. 382-391.
@article{98e2da7c97534d71845666891a362022,
title = "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 transmembrane 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 KRASmutant 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.",
author = "Bennett, {Daniel C.} and Jonathan Charest and Katrina Sebolt and Mark Lehrman and Alnawez Rehemtulla and Contessa, {Joseph N.}",
year = "2013",
doi = "10.1593/tlo.13232",
language = "English (US)",
volume = "6",
pages = "382--391",
journal = "Translational Oncology",
issn = "1936-5233",
publisher = "Neoplasia Press",
number = "3",

}

TY - JOUR

T1 - High-throughput screening identifies aclacinomycin as a radiosensitizer of EGFR-mutant non-small cell lung cancer

AU - Bennett, Daniel C.

AU - Charest, Jonathan

AU - Sebolt, Katrina

AU - Lehrman, Mark

AU - Rehemtulla, Alnawez

AU - Contessa, Joseph N.

PY - 2013

Y1 - 2013

N2 - The endoplasmic reticulum (ER) provides a specialized environment for the folding and modification of transmembrane 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 KRASmutant 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.

AB - The endoplasmic reticulum (ER) provides a specialized environment for the folding and modification of transmembrane 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 KRASmutant 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.

UR - http://www.scopus.com/inward/record.url?scp=84878723890&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84878723890&partnerID=8YFLogxK

U2 - 10.1593/tlo.13232

DO - 10.1593/tlo.13232

M3 - Article

C2 - 23730419

AN - SCOPUS:84878723890

VL - 6

SP - 382

EP - 391

JO - Translational Oncology

JF - Translational Oncology

SN - 1936-5233

IS - 3

ER -