USP9X inhibition promotes radiation-induced apoptosis in non-small cell lung cancer cells expressing mid-to-high MCL1

Abstract

Background and Purpose: Radiotherapy (RT) is vital for the treatment of locally advanced non-small cell lung cancer (NSCLC), yet its delivery is limited by tolerances of adjacent organs. We sought therefore to identify and characterize gene targets whose inhibition may improve RT. Materials and Methods: Whole genome pooled shRNA cytotoxicity screens were performed in A549 and NCI-H460 using a retroviral library of 74,705 sequences. Cells were propagated with or without daily radiation Monday–Friday. Radiosensitization by top differential dropout hits was assessed by clonogenic assays. Apoptosis was assessed using a caspase 3/7 cell-based activity assay and by annexin V-FITC and PI staining. MCL1 expression was assessed by qPCR and Western blotting. Results: USP9X, a deubiquitinase, was a top hit among druggable gene products. WP1130, a small molecule USP9X inhibitor, showed synergistic cytotoxicity with IR. MCL1, an anti-apoptotic protein deubiquitinated by USP9X, decreased with USP9X inhibition and IR. This was accompanied by increases in caspase 3/7 activity and apoptosis. In a panel of NSCLC lines, MCL1 and USP9X protein and gene expression levels were highly correlated. Lines showing high levels of MCL1 expression were the most sensitive to USP9X inhibition. Conclusions: These data support the use of MCL1 expression as a predictive biomarker for USP9X inhibitors in NSCLC therapy.

Keywords:

• apoptosis
• lung cancer
• MCL1
• radiosensitizing agents
• RNAi screen
• USP9X

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website.

Funding

DK is supported by the American Society for Radiation Oncology (ASTRO) Junior Faculty Career Research Training Award, a Joint Center for Radiation Therapy Foundation Grant, a Dana-Farber/Harvard Cancer Center SPORE Developmental Research Project Award in Lung Cancer Research and the National Cancer Institute of the National Institutes of Health under Award Number K08CA172354.