Fast proliferating and slowly migrating non-small cell lung cancer cells are vulnerable to decitabine and retinoic acid combinatorial treatment.

Non-small cell lung cancer (NSCLC) patients are often elderly or unfit and thus cannot tolerate standard aggressive therapy regimes. In our study, we test the efficacy of the DNA-hypomethylating agent decitabine (DAC) in combination with all-trans retinoic acid (ATRA), which has been shown to possess little systemic adverse effects. Screening a broad panel of 56 NSCLC cell lines uncovered a decrease in cell viability after the combination treatment in 77% of the cell lines. Transcriptomics, proteomics, proliferation and migration profiling revealed that fast proliferating and slowly migrating cell lines were more sensitive to the drug combination. The comparison of mutational profiles found oncogenic KRAS mutations only in sensitive cells. Additionally, different cell lines showed a heterogeneous gene expression response to the treatment pointing to diverse mechanisms of action. Silencing KRAS, RIG-I or RARB partially reversed the sensitivity of KRAS-mutant NCI-H460 cells. To study resistance, we generated two NCI-H460 cell populations resistant to ATRA and DAC, which migrated faster and proliferated slower than the parental sensitive cells and showed signs of senescence. In summary, this comprehensive dataset uncovers a broad sensitivity of NSCLC cells to the combinatorial treatment with DAC and ATRA and indicates that migration and proliferation capacities correlate with and could thus serve as determinants for drug sensitivity in NSCLC.

CRISPRi screening identifies CASP8AP2 as an essential viability factor in lung cancer controlling tumor cell death via the AP-1 pathway.

Since lung cancer remains the leading cause of cancer death globally, there is an urgent demand for novel therapeutic targets. We carried out a CRISPR interference (CRISPRi) loss-of-function screen for human lung adenocarcinoma (LUAD) targeting 2098 deregulated genes using a customized algorithm to comprehensively probe the functionality of every resolvable transcriptional start site (TSS). CASP8AP2 was identified as the only hit that significantly affected the viability of all eight screened LUAD cell lines while the viability of non-transformed lung cells was only moderately impacted. Knockdown (KD) of CASP8AP2 induced both autophagy and apoptotic cell death pathways. Systematic expression profiling linked the AP-1 transcription factor to the CASP8AP2 KD-induced cancer cell death. Furthermore, inhibition of AP-1 reverted the CASP8AP2 silencing-induced phenotype. Overall, the tailored CRISPRi screen profiled the impact of over 2000 genes on the survival of eight LUAD cell lines and identified the CASP8AP2 - AP-1 axis mediating lung cancer viability.

Chimeric oligonucleotides combining guide RNA and single-stranded DNA repair template effectively induce precision gene editing.

The ability to precisely alter the genome holds immense potential for molecular biology, medicine and biotechnology. The development of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) into a genomic editing tool has vastly simplified genome engineering. Here, we explored the use of chemically synthesized chimeric oligonucleotides encoding a target-specific crRNA (CRISPR RNA) fused to a single-stranded DNA repair template for RNP-mediated precision genome editing. By generating three clinically relevant oncogenic driver mutations, two non-stop extension mutations, an FGFRi resistance mutation and a single nucleotide change, we demonstrate the ability of chimeric oligos to form RNPs and direct Cas9 to effectively induce genome editing. Further, we demonstrate that the polarity of the chimeric oligos is crucial: only chimeric oligos with the single-stranded DNA repair template fused to the 3'-end of the crRNA are functional for accurate editing, while templates fused to the 5'-end are ineffective. We also find that chimeras can perform editing with both symmetric and asymmetric single-stranded DNA repair templates. Depending on the target locus, the editing efficiency using chimeric RNPs is similar to or less than the efficiency of editing using the bipartite standard RNPs. Our results indicate that chimeric RNPs comprising RNA-DNA oligos formed from fusing the crRNA and DNA repair templates can successfully induce precise edits. While chimeric RNPs do not display an advantage over standard RNPs, they nonetheless represent a viable approach for one-molecule precision genome editing.

A pan-cancer analysis reveals nonstop extension mutations causing SMAD4 tumour suppressor degradation.

Nonstop or stop-loss mutations convert a stop into a sense codon, resulting in translation into the 3' untranslated region as a nonstop extension mutation to the next in-frame stop codon or as a readthrough mutation into the poly-A tail. Nonstop mutations have been characterized in hereditary diseases, but not in cancer genetics. In a pan-cancer analysis, we curated and analysed 3,412 nonstop mutations from 62 tumour entities, generating a comprehensive database at http://NonStopDB.dkfz.de. Six different nonstop extension mutations affected the tumour suppressor SMAD4, extending its carboxy terminus by 40 amino acids. These caused rapid degradation of the SMAD4 mutants via the ubiquitin-proteasome system. A hydrophobic degron signal sequence of ten amino acids within the carboxy-terminal extension was required to induce complete loss of the SMAD4 protein. Thus, we discovered that nonstop mutations can be functionally important in cancer and characterize their loss-of-function impact on the tumour suppressor SMAD4.

LINC00261 and the Adjacent Gene FOXA2 Are Epithelial Markers and Are Suppressed during Lung Cancer Tumorigenesis and Progression.

Lung cancer continues to be the leading cause of cancer-related deaths worldwide, with little improvement in patient survival rates in the past decade. Long non-coding RNAs (lncRNAs) are gaining importance as possible biomarkers with prognostic potential. By large-scale data mining, we identified LINC00261 as a lncRNA which was significantly downregulated in lung cancer. Low expression of LINC00261 was associated with recurrence and poor patient survival in lung adenocarcinoma. Moreover, the gene pair of LINC00261 and its neighbor FOXA2 were significantly co-regulated. LINC00261 as well as FOXA2 negatively correlated with markers for epithelial-to-mesenchymal transition (EMT) and were suppressed by the EMT inducer TGFß. Hierarchical clustering of gene expression data from lung cancer cell lines could further verify the association of high LINC00261/FOXA2 expression to an epithelial gene signature. Furthermore, higher expression of the LINC00261/FOXA2 locus was associated with lung cancer cell lines with lower migratory capacity. All these data establish LINC00261 and FOXA2 as an epithelial-specific marker pair, downregulated during EMT and lung cancer progression, and associated with lower cell migration potential in lung cancer cells.

Challenges of CRISPR/Cas9 applications for long non-coding RNA genes.

The CRISPR/Cas9 system provides a revolutionary genome editing tool for all areas of molecular biology. In long non-coding RNA (lncRNA) research, the Cas9 nuclease can delete lncRNA genes or introduce RNA-destabilizing elements into their locus. The nuclease-deficient dCas9 mutant retains its RNA-dependent DNA-binding activity and can modulate gene expression when fused to transcriptional repressor or activator domains. Here, we systematically analyze whether CRISPR approaches are suitable to target lncRNAs. Many lncRNAs are derived from bidirectional promoters or overlap with promoters or bodies of sense or antisense genes. In a genome-wide analysis, we find only 38% of 15929 lncRNA loci are safely amenable to CRISPR applications while almost two-thirds of lncRNA loci are at risk to inadvertently deregulate neighboring genes. CRISPR- but not siPOOL or Antisense Oligo (ASO)-mediated targeting of lncRNAs NOP14-AS1, LOC389641, MNX1-AS1 or HOTAIR also affects their respective neighboring genes. Frequently overlooked, the same restrictions may apply to mRNAs. For example, the tumor suppressor TP53 and its head-to-head neighbor WRAP53 are jointly affected by the same sgRNAs but not siPOOLs. Hence, despite the advantages of CRISPR/Cas9 to modulate expression bidirectionally and in cis, approaches based on ASOs or siPOOLs may be the better choice to target specifically the transcript from complex loci.

Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9.

Systematic identification of noncoding regulatory elements has, to date, mainly relied on large-scale reporter assays that do not reproduce endogenous conditions. We present two distinct CRISPR-Cas9 genetic screens to identify and characterize functional enhancers in their native context. Our strategy is to target Cas9 to transcription factor binding sites in enhancer regions. We identified several functional enhancer elements and characterized the role of two of them in mediating p53 (TP53) and ERα (ESR1) gene regulation. Moreover, we show that a genomic CRISPR-Cas9 tiling screen can precisely map functional domains within enhancer elements. Our approach expands the utility of CRISPR-Cas9 to elucidate the functions of the noncoding genome.