Correction: Inhibition of the Growth Factor MDK/Midkine by a Novel Small Molecule Compound to Treat Non-Small Cell Lung Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0071093.].

FOXA2 Cooperates with Mutant KRAS to Drive Invasive Mucinous Adenocarcinoma of the Lung.

The endoderm-lineage transcription factor FOXA2 has been shown to inhibit lung tumorigenesis in in vitro and xenograft studies using lung cancer cell lines. However, FOXA2 expression in primary lung tumors does not correlate with an improved patient survival rate, and the functional role of FOXA2 in primary lung tumors remains elusive. To understand the role of FOXA2 in primary lung tumors in vivo, here, we conditionally induced the expression of FOXA2 along with either of the two major lung cancer oncogenes, EGFRL858R or KRASG12D, in the lung epithelium of transgenic mice. Notably, FOXA2 suppressed autochthonous lung tumor development driven by EGFRL858R, whereas FOXA2 promoted tumor growth driven by KRASG12D. Importantly, FOXA2 expression along with KRASG12D produced invasive mucinous adenocarcinoma (IMA) of the lung, a fatal mucus-producing lung cancer comprising approximately 5% of human lung cancer cases. In the mouse model in vivo and human lung cancer cells in vitro, FOXA2 activated a gene regulatory network involved in the key mucous transcription factor SPDEF and upregulated MUC5AC, whose expression is critical for inducing IMA. Coexpression of FOXA2 with mutant KRAS synergistically induced MUC5AC expression compared with that induced by FOXA2 alone. ChIP-seq combined with CRISPR interference indicated that FOXA2 bound directly to the enhancer region of MUC5AC and induced the H3K27ac enhancer mark. Furthermore, FOXA2 was found to be highly expressed in primary tumors of human IMA. Collectively, this study reveals that FOXA2 is not only a biomarker but also a driver for IMA in the presence of a KRAS mutation. SIGNIFICANCE: FOXA2 expression combined with mutant KRAS drives invasive mucinous adenocarcinoma of the lung by synergistically promoting a mucous transcriptional program, suggesting strategies for targeting this lung cancer type that lacks effective therapies.

CRISPRi links COVID-19 GWAS loci to LZTFL1 and RAVER1.

BACKGROUND: To identify host genetic variants (SNPs) associated with COVID-19 disease severity, a number of genome-wide association studies (GWAS) have been conducted. Since most of the identified variants are located at non-coding regions, such variants are presumed to affect the expression of neighbouring genes, thereby influencing COVID-19 disease severity. However, it remains largely unknown which genes are influenced by such COVID-19 GWAS loci. METHODS: CRISPRi (interference)-mediated gene expression analysis was performed to identify genes functionally regulated by COVID-19 GWAS loci by targeting regions near the loci (SNPs) in lung epithelial cell lines. The expression of CRISPRi-identified genes was investigated using COVID-19-contracted human and monkey lung single-nucleus/cell (sn/sc) RNA-seq datasets. FINDINGS: CRISPRi analysis indicated that a region near rs11385942 at chromosome 3p21.31 (locus of highest significance with COVID-19 disease severity at intron 5 of LZTFL1) significantly affected the expression of LZTFL1 (P<0.05), an airway cilia regulator. A region near rs74956615 at chromosome 19p13.2 (locus located at the 3' untranslated exonic region of RAVER1), which is associated with critical illness in COVID-19, affected the expression of RAVER1 (P<0.05), a coactivator of MDA5 (IFIH1), which induces antiviral response genes, including ICAM1. The sn/scRNA-seq datasets indicated that the MDA5/RAVER1-ICAM1 pathway was activated in lung epithelial cells of COVID-19-resistant monkeys but not those of COVID-19-succumbed humans. INTERPRETATION: Patients with risk alleles of rs11385942 and rs74956615 may be susceptible to critical illness in COVID-19 in part through weakened airway viral clearance via LZTFL1-mediated ciliogenesis and diminished antiviral immune response via the MDA5/RAVER1 pathway, respectively. FUNDING: NIH.

CRISPRi-mediated functional analysis of NKX2-1-binding sites in the lung.

The transcription factor NKX2-1/TTF-1 is involved in lung pathophysiology, including breathing, innate defense and tumorigenesis. To understand the mechanism by which NKX2-1 regulates genes involved in such pathophysiology, we have previously performed ChIP-seq and identified genome-wide NKX2-1-binding sites, which revealed that NKX2-1 binds to not only proximal promoter regions but also multiple intra- and inter-genic regions of the genes regulated by NKX2-1. However, the roles of such regions, especially non-proximal ones, bound by NKX2-1 have not yet been determined. Here, using CRISPRi (CRISPR/dCas9-KRAB), we scrutinize the functional roles of 19 regions/sites bound by NKX2-1, which are located in genes involved in breathing and innate defense (SFTPB, LAMP3, SFTPA1, SFTPA2) and lung tumorigenesis (MYBPH, LMO3, CD274/PD-L1). Notably, the CRISPRi approach reveals that a portion of NKX2-1-binding sites are functionally indispensable while the rest are dispensable for the expression of the genes, indicating that functional roles of NKX2-1-binding sites are unequally yoked.

CRISPRi-mediated functional analysis of lung disease-associated loci at non-coding regions.

Genome-wide association studies have identified lung disease-associated loci; however, the functions of such loci are not well understood in part because the majority of such loci are located at non-coding regions. Hi-C, ChIP-seq and eQTL data predict potential roles (e.g. enhancer) of such loci; however, they do not elucidate the molecular function. To determine whether these loci function as gene-regulatory regions, CRISPR interference (CRISPRi; CRISPR/dCas9-KRAB) has been recently used. Here, we applied CRISPRi along with Hi-C, ChIP-seq and eQTL to determine the functional roles of loci established as highly associated with asthma, cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Notably, Hi-C, ChIP-seq and eQTL predicted that non-coding regions located at chromosome 19q13 or chromosome 17q21 harboring single-nucleotide polymorphisms (SNPs) linked to asthma/CF/COPD and chromosome 11p15 harboring an SNP linked to IPF interact with nearby genes and function as enhancers; however, CRISPRi indicated that the regions with rs1800469, rs2241712, rs12603332 and rs35705950, but not others, regulate the expression of nearby genes (single or multiple genes). These data indicate that CRISPRi is useful to precisely determine the roles of non-coding regions harboring lung disease-associated loci as to whether they function as gene-regulatory regions at a genomic level.

An EGFR ligand promotes EGFR-mutant but not KRAS-mutant lung cancer in vivo.

EGFR ligands (e.g., EGF and TGFA) have been shown to be clinically associated with poor survival in lung cancer. Since TGFA itself initiates autochthonous tumors in liver, breast, and pancreas but not in the lung in transgenic mice in vivo, it would appear that an EGFR ligand may not initiate but rather promote lung cancer. However, it has not been proven in vivo whether lung cancer is promoted by an EGFR ligand. Using transgenic mouse models conditionally expressing EGFRL858R or KrasG12D with TGFA (an EGFR ligand) in lung epithelium, we determined that TGFA promoted the growth of EGFRL858R-lung tumors in airway regions but not that of KrasG12D-lung tumors. Analysis of TCGA datasets identified ΔNp63 and AGR2 as potential key tumor-promoting regulators, which were highly induced in the TGFA-induced EGFRL858R-lung tumors. The expression of AGR2 was positively correlated with the expression of TGFA in human EGFR-mutant lung adenocarcinomas. The expression of TGFA in human EGFR-mutant lung adenocarcinomas but not in the EGFR wild-type lung adenocarcinoma was associated with poor survival. These results suggest that targeting EGFR ligands may benefit patients who carry EGFR-mutant lung tumors but will not benefit patients with KRAS-mutant lung tumors.

Gene signature driving invasive mucinous adenocarcinoma of the lung.

Though invasive mucinous adenocarcinoma of the lung (IMA) is pathologically distinctive, the molecular mechanism driving IMA is not well understood, which hampers efforts to identify therapeutic targets. Here, by analyzing gene expression profiles of human and mouse IMA, we identified a Mucinous Lung Tumor Signature of 143 genes, which was unexpectedly enriched in mucin-producing gastrointestinal, pancreatic, and breast cancers. The signature genes included transcription factors FOXA3, SPDEF, HNF4A, mucins MUC5AC, MUC5B, MUC3, and an inhibitory immune checkpoint VTCN1/B7-H4 (but not PD-L1/B7-H1). Importantly, induction of FOXA3 or SPDEF along with mutant KRAS in lung epithelium was sufficient to develop benign or malignant mucinous lung tumors, respectively, in transgenic mice. FOXA3 and SPDEF induced MUC5AC and MUC5B, while HNF4A induced MUC3 in human mucinous lung cancer cells harboring a KRAS mutation. ChIP-seq combined with CRISPR/Cas9 determined that upstream enhancer regions of the mucin genes MUC5AC and MUC5B, which were bound by SPDEF, were required for the expression of the mucin genes. Here, we report the molecular signature and gene regulatory network driving mucinous lung tumors.

Inhibition of the growth factor MDK/midkine by a novel small molecule compound to treat non-small cell lung cancer.

Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic KRAS mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.