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Published on: March 27, 2020
CRISPR Screen of Druggable Targets in Small Cell Lung Cancer Identified ATM Inhibitor (AZD1390) as a Radiosensitizer
Xiaozhuo Ran1, Bell Xi Wu2, Mary Shi1
1Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
Purpose:
Small cell lung cancer (SCLC) is an aggressive and lethal form of lung cancer and the overall 5-year survival (OS) for patients is a dismal 7%. Radiation therapy (RT) provides some benefit for selected patients with SCLC but could be improved with radiosensitizing agents. In this study, we identified novel radiosensitizers for SCLC by a CRISPR-Cas9 screen and evaluated the efficacy of ATM inhibitor AZD1390 as a radiosensitizer of SCLC.
Methods And Materials:
We transduced the SCLC cell line SBC5 with a custom CRISPR sgRNA library focused on druggable gene targets and treated cells with RT. Cells collected at multiple timepoints were subjected to next-generation sequencing. We determined radiosensitization both in vitro with cell lines assessed by short-term viability and clonogenic assays, and in vivo mouse models by tumor growth delay. Pharmacodynamic effects of AZD1390 were quantified by ATM-Ser1981 phosphorylation, and RT-induced DNA damage by comet assay.
Results:
Using a CRISPR dropout screen, we identified multiple radiosensitizing genes for SCLC at various timepoints with ATM as a top determinant gene for radiosensitivity. Validation by ATM knockout (KO) demonstrated increased radiosensitivity by short-term viability assay (dose modification factor [DMF]50 = 3.25-3.73 in SBC5 ATM-KO) and clonogenic assays (DMF37 1.25-1.65 in SBC5 ATM-KO). ATM inhibition by AZD1390 effectively abrogated ATM Ser1981 phosphorylation in SCLC cell lines and increased RT-induced DNA damage. AZD1390 synergistically increased the radiosensitivity of SCLC cell lines (cell viability assay: SBC5 DMF37 = 2.19, SHP77 DMF37 = 1.56, H446 DMF37 = 3.27, KP1 DMF37 = 1.65 at 100nM; clonogenic assay: SBC5 DMF37 = 4.23, H1048 DMF37 = 1.91), and in vivo murine syngeneic, KP1, and patient-derived xenograft (PDX) models, JHU-LX108 and JHU-LX33.
Conclusions:
In this study, we demonstrated that genetically and pharmacologically (AZD1390) inhibiting ATM markedly enhanced RT against SCLC, providing a novel pharmacologically tractable radiosensitizing strategy for patients with SCLC.
Insights
ATM inhibition with AZD1390 enhances radiation therapy for small cell lung cancer (SCLC). This study identified ATM as a key radiosensitizer, offering a new treatment strategy for SCLC patients.
Area of Science:
- Oncology
- Cancer Biology
- Genetics
Background:
- Small cell lung cancer (SCLC) has a poor prognosis with a 5-year survival rate of 7%.
- Radiation therapy (RT) offers benefits for SCLC but can be improved with radiosensitizing agents.
- Identifying novel radiosensitizers is crucial for enhancing SCLC treatment efficacy.
Purpose of the Study:
- To identify novel radiosensitizers for SCLC using a CRISPR-Cas9 screen.
- To evaluate the efficacy of the ATM inhibitor AZD1390 as a radiosensitizer for SCLC.
- To investigate the role of ATM in SCLC radiosensitivity.
Main Methods:
- Conducted a CRISPR-Cas9 dropout screen in SCLC cell lines treated with RT.
- Validated radiosensitizing genes, including ATM, using knockout models.
- Assessed radiosensitization in vitro via viability and clonogenic assays.
- Evaluated AZD1390's pharmacodynamic effects and its impact on RT-induced DNA damage.
- Tested efficacy in vivo using mouse models of SCLC.
Main Results:
- ATM was identified as a key determinant of radiosensitivity in SCLC.
- ATM knockout significantly increased SCLC radiosensitivity in vitro.
- AZD1390 inhibited ATM phosphorylation and enhanced RT-induced DNA damage.
- AZD1390 synergistically increased SCLC radiosensitivity across multiple cell lines and in vivo models.
Conclusions:
- Genetic and pharmacological inhibition of ATM markedly enhances RT efficacy in SCLC.
- AZD1390 represents a novel and pharmacologically tractable radiosensitizing strategy for SCLC.
- Targeting ATM offers a promising approach to improve outcomes for SCLC patients receiving radiation therapy.
