Abstract 204: Notch2NL is a novel regulator of radiation sensitivity in non-small cell lung cancer and a positive regulator of MYC activity

Abstract

Abstract Background: A gain-of-function genetic screen identified NOTCH2 N-terminal like (NOTCH2NL) as a candidate novel gene involved in radiation resistance in non-small cell lung cancer (NSCLC). NOTCH2NL is located on chromosome position 1q21.1 which was incorrectly aligned in previous builds of the reference human genome sequence. NOTCH2NL was derived from a segmental duplication event in NOTCH2 during the evolutionary divergence between humans and non-human primates, forming three Notch2NL proteins: Notch2NL-A, Notch2NL-B and Notch2NL-C. Consequently, the only species whose genomes contain NOTCH2NL are modern and ancient humans. Notch2NL-B is a secreted protein and has been shown to promote Notch signalling in cortical stem cell progenitors via inhibition of inhibitory cis-DLL1/NOTCH signalling. Methods: Stable overexpression of NOTCH2NLB and secreted Notch2NL-B protein were validated by RT-qPCR and Western Blotting. The effects of NOTCH2NLB overexpression; transfer of Notch2NL-B protein from conditioned media onto wild-type cell lines; and siRNA knockdown of NOTCH1-4 transmembrane receptors on Notch signalling were quantified by a Notch luciferase reporter assay, RT-qPCR of classical NOTCH target genes and cancer stem cell marker (CSC) and RNA-Seq. Clonogenic cell survival assays determined the impact of NOTCH2NLB overexpression on radiation response. Cell proliferation was measured using the IncuCyte Zoom system. shRNA stable cell lines to knockdown NOTCH2NL were generated to mitigate the NOTCH2NLB overexpression phenotype we observed. RNA-Seq data from the first 100 patients from the TRACERx lung cancer study was used to quantify hallmark gene expression signatures and NOTCH2NLB expression. Results: NOTCH2NLB overexpression upregulates CSL-dependent Notch reporter luciferase activity; confers a radiation-resistant phenotype and impacts proliferation rate in NSCLC cell lines. Furthermore, we demonstrate that NOTCH2NLB overexpression and secreted Notch2NL-B protein transfer increase expression of classical NOTCH target genes (c-MYC, HES1 and HEY1) and CSC marker, ALDH1A1. Analysis of RNA-Seq data from the Lung TRACERx cohort study shows that NOTCH2NL is commonly expressed in NSCLC. NOTCH2NL expression positively correlates with notch hallmark gene expression signature in squamous (R=0.59, p=5.6e-9) and non-squamous carcinoma (R=0.51, p=4.7e-10). Interestingly, NOTCH2NL expression also positively correlated with the myc hallmark gene expression signature in squamous cell carcinoma (R=0.45, p=2.4e-5). Conclusions: Notch2NL is a novel determinant of radiation therapy response and impacts canonical Notch signalling in lung cancer. It is commonly expressed in NSCLC and can act in a paracrine manner as a secreted protein. Notch2NL, through canonical notch signalling, can regulate MYC expression and increase activity in the MYC pathway. Citation Format: Simranpreet Kaur Summan, Barbara Chow, Rhona Millar, Su Kit Chew, Philip East, Robert Hynds, Carlos Martinez Ruiz, Eva Gronroos, Mariam Jamal-Hanjani, Kevin Litchfield, Nicholas McGranahan, Nnennaya Kanu, Charles Swanton, Crispin Thomas Hiley. Notch2NL is a novel regulator of radiation sensitivity in non-small cell lung cancer and a positive regulator of MYC activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 204.