B20 Oncogene-Mediated ERK Signaling Suppresses Neuroendocrine Transcription Factors and Facilitates Cellular Transformation in Small-Cell Lung Cancer Through Chromatin Remodeling

Abstract

In contrast to non-small cell lung cancer (NSCLC), small-cell lung cancer (SCLC) rarely harbors gene alterations that activate signaling through the receptor tyrosine kinase/RAS/RAF/MEK/ERK pathway. In addition, EGFR protein expression is universally lost during histologic transformation from mutant EGFR-driven lung adenocarcinoma (LUAD) to SCLC that occurs in a subset of patients that develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs), despite the original EGFR mutations being maintained in the transformed tumor. Based on these observations, we hypothesized that signaling through mitogen-activated protein kinases (MAPKs) is detrimental to SCLC tumors and suppresses the neuroendocrine (NE) differentiation program that is a hallmark of this lung cancer subtype. To test this, we induced MAPK signaling through expression of two LUAD driver oncogenes, KRASG12V and EGFRL858R, and assessed the impact on the phenotype and signaling profiles of SCLC. KRASG12V or EGFRL858R was exogenously expressed in an inducible manner in three SCLC cell lines (H2107, H82, and H524). Effects were characterized through microscopy, growth assays, gene expression and chromatin profiling, and Western blots of master NE transcription factors including insulinoma-associated protein 1 (INSM1), POU class 3 homeobox 2 (BRN2), achaete-scute hologue 1 (ASCL1), and neurogenic differentiation factor 1 (NEUROD1). Induction of mutant KRAS or EGFR caused transition from suspension to adherent phenotype that was reversed by pharmacologic inhibition of both ERK and AKT. Moreover, whereas both oncogenes downregulated NE transcription factors, effects were more prominent after KRASG12V induction, reflecting the difference in degrees of phospho-ERK levels. Inhibition of ERK completely rescued the repression of NE factors by KRASG12V induction, and partial effects were observed through inhibition of the downstream effectors MSK/RSK. Notably, KRASG12V-mediated suppression of NE factors was restored by inhibition of the histone modifiers p300/CBP or KDM5A in a cell line-specific manner. ATAC-seq analyses are currently underway to examine the changes of chromatin accessibility after KRASG12V induction +/- inhibition of ERK, MSK/RSK, or p300/CBP. In SCLC, activation of ERK and AKT by mutant KRAS or EGFR causes phenotypic transition to a NSCLC-like state, and ERK is the central hub for the regulation of NE factors. Histone modifications by hyperactivated ERK play an important role in this process and are mediated via tumor-specific mechanisms. These findings provide a biologic basis for why SCLC lacks alterations in the MAPK pathway and shed light on the underlying mechanisms of histologic transversion of SCLC to and from NSCLC, which may play a role in TKI resistance.