Transcriptional Circuitry of NKX2-1 and SOX1 Defines an Unrecognized Lineage Subtype of Small-Cell Lung Cancer

Abstract

<b>Rationale:</b> The current molecular classification of small-cell lung cancer (SCLC) on the basis of the expression of four lineage transcription factors still leaves its major subtype SCLC-A as a heterogeneous group, necessitating more precise characterization of lineage subclasses. <b>Objectives:</b> To refine the current SCLC classification with epigenomic profiles and to identify features of the redefined SCLC subtypes. <b>Methods:</b> We performed unsupervised clustering of epigenomic profiles on 25 SCLC cell lines. Functional significance of NKX2-1 (NK2 homeobox 1) was evaluated by cell growth, apoptosis, and xenograft using clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-associated protein 9)-mediated deletion. NKX2-1-specific cistromic profiles were determined using chromatin immunoprecipitation followed by sequencing, and its functional transcriptional partners were determined using coimmunoprecipitation followed by mass spectrometry. <i>Rb1^flox/flox; Trp53^flox/flox</i> and <i>Rb1^flox/flox; Trp53^flox/flox; Nkx2-1^flox/flox</i> mouse models were engineered to explore the function of <i>Nkx2-1</i> in SCLC tumorigenesis. Epigenomic landscapes of six human SCLC specimens and 20 tumors from two mouse models were characterized. <b>Measurements and Main Results:</b> We identified two epigenomic subclusters of the major SCLC-A subtype: SCLC-Aα and SCLC-Aσ. SCLC-Aα was characterized by the presence of a super-enhancer at the <i>NKX2-1</i> locus, which was observed in human SCLC specimens and a murine SCLC model. We found that NKX2-1, a dual lung and neural lineage factor, is uniquely relevant in SCLC-Aα. In addition, we found that maintenance of this neural identity in SCLC-Aα is mediated by collaborative transcriptional activity with another neuronal transcriptional factor, SOX1 (SRY-box transcription factor 1). <b>Conclusions:</b> We comprehensively describe additional epigenomic heterogeneity of the major SCLC-A subtype and define the SCLC-Aα subtype by the core regulatory circuitry of NKX2-1 and SOX1 super-enhancers and their functional collaborations to maintain neuronal linage state.