Abstract PR08: Functional characterization and evolutionary reconstruction of small cell lung cancer transformation of EGFR-mutant lung adenocarcinomas
Authors: Lee June-Koo , Park Seongyeol , Kim Joon , Kim Tae Min , Ju Young Seok 
Published: February 2019

Abstract
Purpose: Recently, we performed a whole-genome sequencing (WGS) study of serially acquired tumors and demonstrated that an early, complete inactivation of RB1 and TP53 is common among patients with EGFR-mutant lung adenocarcinoma (LADC) whose disease was transformed into small cell lung cancer (SCLC). To further understand this phenomenon, here we functionally characterized a patient-derived cell line of transformed SCLC and related in vitro models. We also deepened our genome analysis focusing on the complex rearrangements to probe their evolutionary paths in detail.
Method: mRNA sequencing was performed for SNU-2962A cells and their transcriptome was analyzed with published mRNA sequencing datasets of primary LADCs (n = 87), and SCLCs (n = 81). A cell viability screening of SNU-2962A cells was performed to understand their therapeutic vulnerability using a kinome-targeting siRNA library. We also characterized isogenic RB1/TP53-knockout LADC cell lines with EGFR mutation (PC9, HCC-827 and HCC-4006). In addition, we reconstructed complex genomic rearrangements and analyzed them in relationship with whole-genome duplication to investigate their time points during clonal evolution.
Results: The SNU-2962A cell line was established from the pleural effusion of the patient LC1. These cells exhibited adherent morphology in vitro and strongly expressed neuroendocrine markers. A hierarchical clustering of SNU-2962A cells with the published LADCs and SCLCs showed complete clustering with SCLCs. This cell line strongly expressed NEUROD1 and MYC, which was consistent with the variant-type SCLC. Kinome siRNA library screening showed a significantly reduced viability of SNU-2962A cells with knockdown of PI3K-MTOR pathway genes (PIK3CA or MTOR). Pharmacologic inhibition of this pathway using PI3K or AKT inhibitors showed moderate sensitivity. We tried to induce transformation into SCLCs using three different EGFR-mutant LADC cell lines by knockout of both RB1 and TP53 as well as knockin of PIK3CA E545K mutations using CRISPR, but we did not observe any meaningful evidence of neuroendocrine differentiation. We further analyzed our WGS data of 11 tumors from five patients, and found that many large-scaled genomic events such as whole-genome duplication, chromothripsis, or kataegis are frequently shared by LADCs and subsequent SCLCs, indicating their early occurrence. MYC amplification is frequently selected in transformed SCLCs. We also found a somatic deletion of BIM gene in LC3 patient, and this deletion was shared by the early LADC and the late SCLC. This may confer a dysfunctional apoptosis upon EGFR inhibition, which could predispose the early emergence of resistant tumor in this patient.
Conclusion: Although a complete inactivation of both RB1 and TP53 is necessary for transformation from LADC into SCLC, it is not sufficient. Phylogenic reconstruction of large structural variations in our WGS dataset further clarified the mutational complexity of the common ancestor clones.
This abstract is also being presented as Poster A23.
Citation Format: June-Koo Lee, Seongyeol Park, Joon Kim, Tae Min Kim, Young Seok Ju. Functional characterization and evolutionary reconstruction of small cell lung cancer transformation of EGFR-mutant lung adenocarcinomas [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR08.