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Figure S6. TCGA LUAD methylation at the LINC00261 locus. A. Individual CpGs are denoted by their Illumina 450K designation. Scale = 0 (no methylation) to 1 (complete methylation). Hg19-based coordinates for each probe are listed below the cg ascension number. Distance from the LINC00261 Transcription Start Site (TSS) is also shown. "+" is downstream of TSS (within the gene), "-" is upstream of TSS. B. RNA expression levels of DNMT enzymes in normal human alveolar epithelial cells (AEC) and available 16 LUAD cancer cell lines (LUAD). Dashed line denotes RPKM cutoff of 3, the commonly accepted cutoff for baseline expression. ** = p value 0.0076, unpaired T Test. N=3 for AEC, 16 for LUAD.

Lung cancer is the leading cause of cancer-related death in the United States. Long noncoding RNAs (lncRNA) are a class of regulatory molecules whose role in lung carcinogenesis is poorly understood. In this study, we profiled lncRNA expression in lung adenocarcinoma (LUAD) cell lines, compared their expression with that of purified alveolar epithelial type II cells (the purported cell of origin for LUAD), cross-referenced these with lncRNAs altered in the primary human tumors, and interrogated for lncRNAs whose expression correlated with patient survival. We identified LINC00261, a lncRNA with unknown function in LUAD, adjacent to the pioneering transcription factor FOXA2. Loss of LINC00261 was observed in multiple tumor types, including liver, breast, and gastric cancer. Reintroduction of LINC00261 into human LUAD cell lines inhibited cell migration and slowed proliferation by inducing G2–M cell-cycle arrest, while upregulating DNA damage pathway genes and inducing phosphorylation-mediated activation of components of the DNA damage pathway. FOXA2 was able to induce LINC00261 expression, and the entire locus underwent hypermethylation in LUAD, leading to loss of expression. We have thus identified an epigenetically deregulated lncRNA, whose loss of expression in LUAD promotes the malignant phenotype and blocks activation of the DNA damage machinery, predisposing lung cells to cancer development.Significance:These findings identify LINC00261 as a tumor suppressor that blocks cellular proliferation by activating the DNA damage response and suggest that epigenetic therapy to inhibit DNA methylation may enhance treatment of LUAD.

Supplemental Table T1. List of Primers for LINC00261 detection and cloning. Supplemental Table T2. Differentially expressed lncRNAs in LUAD versus primary AEC. Supplemental Table T3. Differentially expressed genes in H522-CMV-LINC00261 vs CMV-NEO control cells.

Lung cancer is the leading cause of cancer-related death in the United States. Long noncoding RNAs (lncRNA) are a class of regulatory molecules whose role in lung carcinogenesis is poorly understood. In this study, we profiled lncRNA expression in lung adenocarcinoma (LUAD) cell lines, compared their expression with that of purified alveolar epithelial type II cells (the purported cell of origin for LUAD), cross-referenced these with lncRNAs altered in the primary human tumors, and interrogated for lncRNAs whose expression correlated with patient survival. We identified LINC00261, a lncRNA with unknown function in LUAD, adjacent to the pioneering transcription factor FOXA2. Loss of LINC00261 was observed in multiple tumor types, including liver, breast, and gastric cancer. Reintroduction of LINC00261 into human LUAD cell lines inhibited cell migration and slowed proliferation by inducing G2–M cell-cycle arrest, while upregulating DNA damage pathway genes and inducing phosphorylation-mediated activation of components of the DNA damage pathway. FOXA2 was able to induce LINC00261 expression, and the entire locus underwent hypermethylation in LUAD, leading to loss of expression. We have thus identified an epigenetically deregulated lncRNA, whose loss of expression in LUAD promotes the malignant phenotype and blocks activation of the DNA damage machinery, predisposing lung cells to cancer development. Significance: These findings identify LINC00261 as a tumor suppressor that blocks cellular proliferation by activating the DNA damage response and suggest that epigenetic therapy to inhibit DNA methylation may enhance treatment of LUAD.

SUMMARY Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3′ UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recently generated nuclear factor κB (NF-κB) reporter line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen and discover a number of previously unidentified positive and negative regulators of the NF-κB pathway. We unravel complexities of the TNF signaling cascade, showing that it can function in an autocrine manner in macrophages to negatively regulate the pathway. Utilizing a single complex library design, we are capable of interrogating various aspects of macrophage biology, thus generating a resource for future studies., Graphical Abstract, In Brief Covarrubias et al. screen ~21,000 targets, generating a resource guide of genes required for macrophage viability as well as previously unidentified positive and negative regulators of NF-κB signaling. They identify regulatory elements within essential genes and show that membrane-bound TNF primarily functions in macrophages in an autocrine fashion to negatively regulate inflammation.