Abstract
BackgroundSmall cell lung cancer (SCLC) is a more aggressive subtype of lung cancer that often results in rapid tumor growth, early metastasis, and acquired therapeutic resistance. Consequently, such phenotypical characteristics of SCLC set limitations on viable procedural options, making it difficult to develop both screenings and effective treatments. In this study, we examine a novel mechanistic insight in SCLC cells that could potentially provide a more sensitive therapeutic alternative for SCLC patients.MethodsBiochemistry studies, including size exclusion chromatography, mass spectrometry, and western blot analysis, were conducted to determine the protein-protein interaction between additional sex combs-like protein 3 (ASXL3) and bromodomain-containing protein 4 (BRD4). Genomic studies, including chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing, and genome-wide analysis, were performed in both human and mouse SCLC cells to determine the dynamic relationship between BRD4/ASXL3/BAP1 epigenetic axis in chromatin binding and its effects on transcriptional activity.ResultsWe report a critical link between BAP1 complex and BRD4, which is bridged by the physical interaction between ASXL3 and BRD4 in an SCLC subtype (SCLC-A), which expresses a high level of ASCL1. We further showed that ASXL3 functions as an adaptor protein, which directly interacts with BRD4’s extra-terminal (ET) domain via a novel BRD4 binding motif (BBM), and maintains chromatin occupancy of BRD4 to active enhancers. Genetic depletion of ASXL3 results in a genome-wide reduction of histone H3K27Ac levels and BRD4-dependent gene expression in SCLC. Pharmacologically induced inhibition with BET-specific chemical degrader (dBET6) selectively inhibits cell proliferation of a subtype of SCLC that is characterized with high expression of ASXL3.ConclusionsCollectively, this study provides a mechanistic insight into the oncogenic function of BRD4/ASXL3/BAP1 epigenetic axis at active chromatin enhancers in SCLC-A subtype, as well as a potential new therapeutic option that could become more effective in treating SCLC patients with a biomarker of ASXL3-highly expressed SCLC cells.
Highlights
Small cell lung cancer (SCLC) is a more aggressive subtype of lung cancer that often results in rapid tumor growth, early metastasis, and acquired therapeutic resistance
Among all of the genes that were positively correlated with additional sex combs-like protein 3 (ASXL3) expression, we found that the expression of achaete-scute family bHLH transcription factor 1 (ASCL1), a lineage oncogenic transcription factor in SCLC [37], was enriched approximately 400 times as much in ASXL3-high group SCLC cell lines compared to ASXL3low group SCLC cell lines (Fig. 1e and Additional file 2: Figure S1C) and vice versa (Additional file 2: Figure S1D)
HSP90 was used as an internal control, n = 3. h Whole-cell lysates were used for western blot with BRD2, BRD3, bromodomain-containing protein 4 (BRD4), ASXL3, JMJD6, NSD3, and CHD4 antibodies in human SCLC cell line NCI-H1963 treated with different concentrations of dBET6 for 8 h; HSP90 was used as an internal control, n = 3. i 8 different human SCLC cell lines NCI-H748, NCI-H1882, NCI-H1963, NCI-H209, NCI-H1436, NCI-H1105, NCI-H889, and NCI-H2171 were treated with different concentrations of dBET6 for 72 h
Summary
Small cell lung cancer (SCLC) is a more aggressive subtype of lung cancer that often results in rapid tumor growth, early metastasis, and acquired therapeutic resistance. Such phenotypical characteristics of SCLC set limitations on viable procedural options, making it difficult to develop both screenings and effective treatments. BAP1 complex functions by binding to both promoters and enhancers at the chromatin level [4] and acts as a general transcriptional activator via its deubiquitinase activity on histone H2A monoubiquitination in drosophila [11]. The enzymatic activity of BAP1 is required for the malignant progression of some cancer types [12, 13], which express high levels of RNF2 [14]
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