Airway injury induces alveolar epithelial and mesenchymal responses mediated by macrophages.

Acute injury in the airways or the lung activates local progenitors and stimulates changes in cell-cell interactions to restore homeostasis, but it is not appreciated how more distant niches are impacted. We utilized mouse models of airway-specific epithelial injury to examine secondary tissue-wide alveolar, immune, and mesenchymal responses. Single-cell transcriptomics and in vivo validation revealed transient, tissue-wide proliferation of alveolar type 2 (AT2) progenitor cells after club cell-specific ablation. The AT2 cell proliferative response was reliant on alveolar macrophages (AMs) via upregulation of Spp1 which encodes the secreted factor Osteopontin. A previously uncharacterized mesenchymal population we termed Mesenchymal Airway/Adventitial Niche Cell 2 (MANC2) also exhibited dynamic changes in abundance and a pro-fibrotic transcriptional signature after club cell ablation in an AM-dependent manner. Overall, these results demonstrate that acute airway damage can trigger distal lung responses including altered cell-cell interactions that may contribute to potential vulnerabilities for further dysregulation and disease.

Age-associated H3K9me2 loss alters the regenerative equilibrium between murine lung alveolar and bronchiolar progenitors.

The lung contains multiple progenitor cell types, but how their responses are choreographed during injury repair and whether this changes with age is poorly understood. We report that histone H3 lysine 9 di-methylation (H3K9me2), mediated by the methyltransferase G9a, regulates the dynamics of distal lung epithelial progenitor cells and that this regulation deteriorates with age. In aged mouse lungs, H3K9me2 loss coincided with fewer alveolar type 2 (AT2) cell progenitors and reduced alveolar regeneration but increased the frequency and activity of multipotent bronchioalveolar stem cells (BASCs) and bronchiolar progenitor club cells. H3K9me2 depletion in young mice decreased AT2 progenitor activity and impaired alveolar injury repair. Conversely, H3K9me2 depletion increased chromatin accessibility of bronchiolar cell genes, increased BASC frequency, and accelerated bronchiolar cell injury repair. These findings indicate that during aging, the epigenetic regulation that coordinates lung progenitor cells' regenerative responses becomes dysregulated, aiding our understanding of age-related susceptibility to lung disease.

Lung Cancer Stem Cells and Their Clinical Implications.

It is now widely accepted that stem cells exist in various cancers, including lung cancer, which are referred to as cancer stem cells (CSCs). CSCs are defined in this context as the subset of tumor cells with the ability to form tumors in serial transplantation and cloning assays and form tumors at metastatic sites. Mouse models of lung cancer have shown that lung CSCs reside in niches that are essential for the maintenance of stemness, plasticity, enable antitumor immune evasion, and provide metastatic potential. Similar to normal lung stem cells, Notch, Wnt, and the Hedgehog signaling cascades have been recruited by the CSCs to regulate stemness and also provide therapy-driven resistance in lung cancer. Compounds targeting ß-catenin and Sonic hedgehog (Shh) activity have shown promising anti-CSC activity in preclinical murine models of lung cancer. Understanding CSCs and their niches in lung cancer can answer fundamental questions pertaining to tumor maintenance and associated immune regulation and escape that appear important in the quest to develop novel lung cancer therapies and enhance sensitivity to currently approved chemo-, targeted-, and immune therapeutics.

Organoids Model Transcriptional Hallmarks of Oncogenic KRAS Activation in Lung Epithelial Progenitor Cells.

Mutant KRAS is a common driver in epithelial cancers. Nevertheless, molecular changes occurring early after activation of oncogenic KRAS in epithelial cells remain poorly understood. We compared transcriptional changes at single-cell resolution after KRAS activation in four sample sets. In addition to patient samples and genetically engineered mouse models, we developed organoid systems from primary mouse and human induced pluripotent stem cell-derived lung epithelial cells to model early-stage lung adenocarcinoma. In all four settings, alveolar epithelial progenitor (AT2) cells expressing oncogenic KRAS had reduced expression of mature lineage identity genes. These findings demonstrate the utility of our in vitro organoid approaches for uncovering the early consequences of oncogenic KRAS expression. This resource provides an extensive collection of datasets and describes organoid tools to study the transcriptional and proteomic changes that distinguish normal epithelial progenitor cells from early-stage lung cancer, facilitating the search for targets for KRAS-driven tumors.

Author Correction: EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors.

We wish to correct two mutations in Supplementary Table 4 of this Letter. The NCI-H460 cell line was annotated as being mutant for TP53. NCI-H460 has been verified to be TP53 wild type by several sources1. The NCI-H2009 cell line was annotated as being mutant for PIK3CA. As annotated by COSMIC (ref. 24 of the original Letter) and CCLE (ref. 25 of the original Letter), the NCI-H2009 cell line has a mutation in PIK3C3, rather than PIK3CA. The cell line is wild type for PIK3CA. The Supplementary Information of this Amendment contains the corrected Supplementary Table 4. These errors do not affect our conclusions. The original Letter has not been corrected.

Don't Stop Re-healin'! Cancer as an Ongoing Stem Cell Affair.

Tumors have long been suspected of hijacking stem cell mechanisms used for tissue maintenance and repair. Ge et al. now show that skin tumors exhibit merged chromatin profiles from distinct stem cell lineages. This "lineage infidelity" recreates a state akin to transient wound repair that persists to maintain uncontrolled growth.

S phase block following MEC1ATR inactivation occurs without severe dNTP depletion.

Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by chromosome breakage and cell death during G2/M. The S phase arrest is proposed to stem from a defect in Mec1-mediated degradation of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), causing a severe depletion in cellular dNTP pools. Here, the casual link between the S phase arrest, Sml1, and dNTP-levels is examined using a temperature sensitive mec1 mutant. In addition to S phase arrest, thermal inactivation of Mec1 leads to constitutively high levels of Sml1 and an S phase arrest. Expression of a novel suppressor, GIS2, a conserved mRNA binding zinc finger protein, rescues the arrest without down-regulating Sml1 levels. The dNTP pool in mec1 is reduced by ∼17% and GIS2 expression restores it, but only partially, to ∼93% of a control. We infer that the permanent S phase block following Mec1 inactivation can be uncoupled from its role in Sml1 down-regulation. Furthermore, unexpectedly modest effects of mec1 and GIS2 on dNTP levels suggest that the S phase arrest is unlikely to result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to small changes in its availability.

EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors.

Non-small-cell lung cancer is the leading cause of cancer-related death worldwide. Chemotherapies such as the topoisomerase II (TopoII) inhibitor etoposide effectively reduce disease in a minority of patients with this cancer; therefore, alternative drug targets, including epigenetic enzymes, are under consideration for therapeutic intervention. A promising potential epigenetic target is the methyltransferase EZH2, which in the context of the polycomb repressive complex 2 (PRC2) is well known to tri-methylate histone H3 at lysine 27 (H3K27me3) and elicit gene silencing. Here we demonstrate that EZH2 inhibition has differential effects on the TopoII inhibitor response of non-small-cell lung cancers in vitro and in vivo. EGFR and BRG1 mutations are genetic biomarkers that predict enhanced sensitivity to TopoII inhibitor in response to EZH2 inhibition. BRG1 loss-of-function mutant tumours respond to EZH2 inhibition with increased S phase, anaphase bridging, apoptosis and TopoII inhibitor sensitivity. Conversely, EGFR and BRG1 wild-type tumours upregulate BRG1 in response to EZH2 inhibition and ultimately become more resistant to TopoII inhibitor. EGFR gain-of-function mutant tumours are also sensitive to dual EZH2 inhibition and TopoII inhibitor, because of genetic antagonism between EGFR and BRG1. These findings suggest an opportunity for precision medicine in the genetically complex disease of non-small-cell lung cancer.

Tumor-propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis.

Metastasis is the leading cause of morbidity for lung cancer patients. Here we demonstrate that murine tumor propagating cells (TPCs) with the markers Sca1 and CD24 are enriched for metastatic potential in orthotopic transplantation assays. CD24 knockdown decreased the metastatic potential of lung cancer cell lines resembling TPCs. In lung cancer patient data sets, metastatic spread and patient survival could be stratified with a murine lung TPC gene signature. The TPC signature was enriched for genes in the Hippo signaling pathway. Knockdown of the Hippo mediators Yap1 or Taz decreased in vitro cellular migration and transplantation of metastatic disease. Furthermore, constitutively active Yap was sufficient to drive lung tumor progression in vivo. These results demonstrate functional roles for two different pathways, CD24-dependent and Yap/Taz-dependent pathways, in lung tumor propagation and metastasis. This study demonstrates the utility of TPCs for identifying molecules contributing to metastatic lung cancer, potentially enabling the therapeutic targeting of this devastating disease.

Keeping chromatin quiet: how nucleosome remodeling restores heterochromatin after replication.

Disruption of chromatin organization during replication poses a major challenge to the maintenance and integrity of genome organization. It creates the need to accurately reconstruct the chromatin landscape following DNA duplication but there is little mechanistic understanding of how chromatin based modifications are restored on newly synthesized DNA. ATP-dependent chromatin remodeling activities serve multiple roles during replication and recent work underscores their requirement in the maintenance of proper chromatin organization. A new component of chromatin replication, the SWI/SNF-like chromatin remodeler SMARCAD1, acts at replication sites to facilitate deacetylation of newly assembled histones. Deacetylation is a pre-requisite for the restoration of epigenetic signatures in heterochromatin regions following replication. In this way, SMARCAD1, in concert with histone modifying activities and transcriptional repressors, reinforces epigenetic instructions to ensure that silenced loci are correctly perpetuated in each replication cycle. The emerging concept is that remodeling of nucleosomes is an early event imperative to promote the re-establishment of histone modifications following DNA replication.

Maintenance of silent chromatin through replication requires SWI/SNF-like chromatin remodeler SMARCAD1.

Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.