Genomic landscape of lung adenocarcinoma in East Asians.

Lung cancer is the world's leading cause of cancer death and shows strong ancestry disparities. By sequencing and assembling a large genomic and transcriptomic dataset of lung adenocarcinoma (LUAD) in individuals of East Asian ancestry (EAS; n = 305), we found that East Asian LUADs had more stable genomes characterized by fewer mutations and fewer copy number alterations than LUADs from individuals of European ancestry. This difference is much stronger in smokers as compared to nonsmokers. Transcriptomic clustering identified a new EAS-specific LUAD subgroup with a less complex genomic profile and upregulated immune-related genes, allowing the possibility of immunotherapy-based approaches. Integrative analysis across clinical and molecular features showed the importance of molecular phenotypes in patient prognostic stratification. EAS LUADs had better prediction accuracy than those of European ancestry, potentially due to their less complex genomic architecture. This study elucidated a comprehensive genomic landscape of EAS LUADs and highlighted important ancestry differences between the two cohorts.

Ascorbate and Iron Are Required for the Specification and Long-Term Self-Renewal of Human Skeletal Mesenchymal Stromal Cells.

The effects of ascorbate on adult cell fate specification remain largely unknown. Using our stepwise and chemically defined system to derive lateral mesoderm progenitors from human pluripotent stem cells (hPSCs), we found that ascorbate increased the expression of mesenchymal stromal cell (MSC) markers, purity of MSCs, the long-term self-renewal and osteochondrogenic capacity of hPSC-MSCs in vitro. Moreover, ascorbate promoted MSC specification in an iron-dependent fashion, but not in a redox-dependent manner. Further studies revealed that iron synergized with ascorbate to regulate hPSC-MSC histone methylation, promote their long-term self-renewal, and increase their osteochondrogenic capacity. We found that one of the histone demethylases affected by ascorbate, KDM4B, was necessary to promote the specification of hPSC-MSCs. This mechanistic understanding led to the metabolic optimization of hPSC-MSCs with an extended lifespan in vitro and the ability to fully repair cartilage defects upon transplantation in vivo. Our results highlight the importance of ascorbate and iron metabolism in adult human cell fate specification.

A Near-Infrared Probe Tracks and Treats Lung Tumor Initiating Cells by Targeting HMOX2.

Tumor initiating cells (TIC) are resistant to conventional anticancer therapy and associated with metastasis and relapse in cancer. Although various TIC markers and their antibodies have been proposed, it is limited to the use of antibodies for in vivo imaging or treatment of TIC. In this study, we discovered heme oxygenase 2 (HMOX2) as a novel biomarker for TIC and developed a selective small molecule probe TiNIR (tumor initiating cell probe with near infrared). TiNIR detects and enriches the functionally active TIC in human lung tumors, and through the photoacoustic property, TiNIR also visualizes lung TIC in the patient-derived xenograft (PDX) model. Furthermore, we demonstrate that TiNIR inhibits tumor growth by blocking the function of HMOX2, resulting in significantly increased survival rates of the cancer model mice. The novel therapeutic target HMOX2 and its fluorescent ligand TiNIR will open a new path for the molecular level of lung TIC diagnosis and treatment.

Methionine is a metabolic dependency of tumor-initiating cells.

Understanding cellular metabolism holds immense potential for developing new classes of therapeutics that target metabolic pathways in cancer. Metabolic pathways are altered in bulk neoplastic cells in comparison to normal tissues. However, carcinoma cells within tumors are heterogeneous, and tumor-initiating cells (TICs) are important therapeutic targets that have remained metabolically uncharacterized. To understand their metabolic alterations, we performed metabolomics and metabolite tracing analyses, which revealed that TICs have highly elevated methionine cycle activity and transmethylation rates that are driven by MAT2A. High methionine cycle activity causes methionine consumption to far outstrip its regeneration, leading to addiction to exogenous methionine. Pharmacological inhibition of the methionine cycle, even transiently, is sufficient to cripple the tumor-initiating capability of these cells. Methionine cycle flux specifically influences the epigenetic state of cancer cells and drives tumor initiation. Methionine cycle enzymes are also enriched in other tumor types, and MAT2A expression impinges upon the sensitivity of certain cancer cells to therapeutic inhibition.

Publisher Correction: Methionine is a metabolic dependency of tumor-initiating cells.

In the version of this article originally published, there is an error in Fig. 5a. Originally, 'MAT2A' appeared between 'Methionine' and 'Homocysteine'. 'MAT2A' should have been 'MTR'. The error has been corrected in the PDF and HTML versions of this article.

Global H3.3 dynamic deposition defines its bimodal role in cell fate transition.

H3.3 is a histone variant, which is deposited on genebodies and regulatory elements, by Hira, marking active transcription. Moreover, H3.3 is deposited on heterochromatin by Atrx/Daxx complex. The exact role of H3.3 in cell fate transition remains elusive. Here, we investigate the dynamic changes in the deposition of the histone variant H3.3 during cellular reprogramming. H3.3 maintains the identities of the parental cells during reprogramming as its removal at early time-point enhances the efficiency of the process. We find that H3.3 plays a similar role in transdifferentiation to hematopoietic progenitors and neuronal differentiation from embryonic stem cells. Contrastingly, H3.3 deposition on genes associated with the newly reprogrammed lineage is essential as its depletion at the later phase abolishes the process. Mechanistically, H3.3 deposition by Hira, and its K4 and K36 modifications are central to the role of H3.3 in cell fate conversion. Finally, H3.3 safeguards fibroblast lineage by regulating Mapk cascade and collagen synthesis.

A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells.

How are closely related lineages, including liver, pancreas, and intestines, diversified from a common endodermal origin? Here, we apply principles learned from developmental biology to rapidly reconstitute liver progenitors from human pluripotent stem cells (hPSCs). Mapping the formation of multiple endodermal lineages revealed how alternate endodermal fates (e.g., pancreas and intestines) are restricted during liver commitment. Human liver fate was encoded by combinations of inductive and repressive extracellular signals at different doses. However, these signaling combinations were temporally re-interpreted: cellular competence to respond to retinoid, WNT, TGF-ß, and other signals sharply changed within 24 hr. Consequently, temporally dynamic manipulation of extracellular signals was imperative to suppress the production of unwanted cell fates across six consecutive developmental junctures. This efficiently generated 94.1% ± 7.35% TBX3+HNF4A+ human liver bud progenitors and 81.5% ± 3.2% FAH+ hepatocyte-like cells by days 6 and 18 of hPSC differentiation, respectively; the latter improved short-term survival in the Fah-/-Rag2-/-Il2rg-/- mouse model of liver failure.

Identification of Tumor Initiating Cells with a Small-Molecule Fluorescent Probe by Using Vimentin as a Biomarker.

Tumor initiating cells (TICs) have been implicated in clinical relapse and metastasis of a variety of epithelial cancers, including lung cancer. While efforts toward the development of specific probes for TIC detection and targeting are ongoing, a universal TIC probe has yet to be developed. We report the first TIC-specific fluorescent chemical probe, TiY, with identification of the molecular target as vimentin, a marker for epithelial-to-mesenchymal transition (EMT). TiY selectively stains TICs over differentiated tumor cells or normal cells, and facilitates the visualization and enrichment of functionally active TICs from patient tumors. At high concentration, TiY also shows anti-TIC activity with low toxicity to non-TICs. With the unexplored target vimentin, TiY shows potential as a first universal probe for TIC detection in different cancers.

Elucidating the genomic architecture of Asian EGFR-mutant lung adenocarcinoma through multi-region exome sequencing.

EGFR-mutant lung adenocarcinomas (LUAD) display diverse clinical trajectories and are characterized by rapid but short-lived responses to EGFR tyrosine kinase inhibitors (TKIs). Through sequencing of 79 spatially distinct regions from 16 early stage tumors, we show that despite low mutation burdens, EGFR-mutant Asian LUADs unexpectedly exhibit a complex genomic landscape with frequent and early whole-genome doubling, aneuploidy, and high clonal diversity. Multiple truncal alterations, including TP53 mutations and loss of CDKN2A and RB1, converge on cell cycle dysregulation, with late sector-specific high-amplitude amplifications and deletions that potentially beget drug resistant clones. We highlight the association between genomic architecture and clinical phenotypes, such as co-occurring truncal drivers and primary TKI resistance. Through comparative analysis with published smoking-related LUAD, we postulate that the high intra-tumor heterogeneity observed in Asian EGFR-mutant LUAD may be contributed by an early dominant driver, genomic instability, and low background mutation rates.

Induced-Decay of Glycine Decarboxylase Transcripts as an Anticancer Therapeutic Strategy for Non-Small-Cell Lung Carcinoma.

Self-renewing tumor-initiating cells (TICs) are thought to be responsible for tumor recurrence and chemo-resistance. Glycine decarboxylase, encoded by the GLDC gene, is reported to be overexpressed in TIC-enriched primary non-small-cell lung carcinoma (NSCLC). GLDC is a component of the mitochondrial glycine cleavage system, and its high expression is required for growth and tumorigenic capacity. Currently, there are no therapeutic agents against GLDC. As a therapeutic strategy, we have designed and tested splicing-modulating steric hindrance antisense oligonucleotides (shAONs) that efficiently induce exon skipping (half maximal inhibitory concentration [IC50] at 3.5-7 nM), disrupt the open reading frame (ORF) of GLDC transcript (predisposing it for nonsense-mediated decay), halt cell proliferation, and prevent colony formation in both A549 cells and TIC-enriched NSCLC tumor sphere cells (TS32). One candidate shAON causes 60% inhibition of tumor growth in mice transplanted with TS32. Thus, our shAONs candidates can effectively inhibit the expression of NSCLC-associated metabolic enzyme GLDC and may have promising therapeutic implications.

Isolation and 3D expansion of multipotent Sox9+ mouse lung progenitors.

Multiple adult tissues are maintained by stem cells of restricted developmental potential which can only form a subset of lineages within the tissue. For instance, the two adult lung epithelial compartments (airways and alveoli) are separately maintained by distinct lineage-restricted stem cells. A challenge has been to obtain multipotent stem cells and/or progenitors that can generate all epithelial cell types of a given tissue. Here we show that mouse Sox9+ multipotent embryonic lung progenitors can be isolated and expanded long term in 3D culture. Cultured Sox9+ progenitors transcriptionally resemble their in vivo counterparts and generate both airway and alveolar cell types in vitro. Sox9+ progenitors that were transplanted into injured adult mouse lungs differentiated into all major airway and alveolar lineages in vivo in a region-appropriate fashion. We propose that a single expandable embryonic lung progenitor population with broader developmental competence may eventually be used as an alternative for region-restricted adult tissue stem cells in regenerative medicine.

Molecular characterization of circulating colorectal tumor cells defines genetic signatures for individualized cancer care.

Studies on circulating tumor cells (CTCs) have largely focused on platform development and CTC enumeration rather than on the genomic characterization of CTCs. To address this, we performed targeted sequencing of CTCs of colorectal cancer patients and compared the mutations with the matched primary tumors. We collected preoperative blood and matched primary tumor samples from 48 colorectal cancer patients. CTCs were isolated using a label-free microfiltration device on a silicon microsieve. Upon whole genome amplification, we performed amplicon-based targeted sequencing on a panel of 39 druggable and frequently mutated genes on both CTCs and fresh-frozen tumor samples. We developed an analysis pipeline to minimize false-positive detection of somatic mutations in amplified DNA. In 60% of the CTC-enriched blood samples, we detected primary tumor matching mutations. We found a significant positive correlation between the allele frequencies of somatic mutations detected in CTCs and abnormal CEA serum level. Strikingly, we found driver mutations and amplifications in cancer and druggable genes such as APC, KRAS, TP53, ERBB3, FBXW7 and ERBB2. In addition, we found that CTCs carried mutation signatures that resembled the signatures of their primary tumors. Cumulatively, our study defined genetic signatures and somatic mutation frequency of colorectal CTCs. The identification of druggable mutations in CTCs of preoperative colorectal cancer patients could lead to more timely and focused therapeutic interventions.

Protein Corona around Gold Nanorods as a Drug Carrier for Multimodal Cancer Therapy.

A single nanodevice based on gold nanorods (NRs) coloaded with a photosensitizer, Chlorin e6 (Ce6), and a chemotherapeutic, Doxorubicin (Dox), on its endogenously formed human serum (HS) protein corona, i.e., NR-HS-Ce6-Dox was developed with the aim of performing multimodal cancer therapy: photodynamic (PDT), photothermal (PTT) and chemotherapy (CTX) simultaneously upon irradiation with a single 665 nm laser. Here, the excitation of NRs and Ce6 resulted in photothermal ablation (PTT), and production of reactive oxygen species (ROS) to kill Cal 27 oral squamous cell carcinoma (OSCC) cells by oxidative stress (PDT) respectively, while the laser-triggered release of Dox intercalated into the DNA of cancer cells to result in DNA damage and cell death (CTX). High laser-triggered Dox release efficiency of 71.5% and strong plasmonic enhancement of ROS production by Ce6 (4.8-fold increase compared to free Ce6) was observed. Uptake of both Ce6 and Dox by Cal 27 cells was greatly enhanced, with 3.3 and 52 times higher intracellular Dox and Ce6 fluorescence observed, respectively, 6 h after dosing with NR-HS-Ce6-Dox compared to free drugs. The simultaneous trimodal therapy achieved a near complete eradication of cancer cells (98.7% cell death) with an extremely low dose of 15 pM NR-HS-Ce6-Dox loaded with just 1.26 nM Ce6 and 12.5 nM Dox due to strong synergistic enhancement in cancer cell kill compared to individual therapies performed separately. No dark toxicities were observed. These drug concentrations were far lower than any previously reported in vitro, thus eliminating any potential systemic toxicity of these agents.

Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors.

Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into 'induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors.

Cyclin A2 regulates erythrocyte morphology and numbers.

Cyclin A2 is an essential gene for development and in haematopoietic stem cells and therefore its functions in definitive erythropoiesis have not been investigated. We have ablated cyclin A2 in committed erythroid progenitors in vivo using erythropoietin receptor promoter-driven Cre, which revealed its critical role in regulating erythrocyte morphology and numbers. Erythroid-specific cyclin A2 knockout mice are viable but displayed increased mean erythrocyte volume and reduced erythrocyte counts, as well as increased frequency of erythrocytes containing Howell-Jolly bodies. Erythroblasts lacking cyclin A2 displayed defective enucleation, resulting in reduced production of enucleated erythrocytes and increased frequencies of erythrocytes containing nuclear remnants. Deletion of the Cdk inhibitor p27Kip1 but not Cdk2, ameliorated the erythroid defects resulting from deficiency of cyclin A2, confirming the critical role of cyclin A2/Cdk activity in erythroid development. Loss of cyclin A2 in bone marrow cells in semisolid culture prevented the formation of BFU-E but not CFU-E colonies, uncovering its essential role in BFU-E function. Our data unveils the critical functions of cyclin A2 in regulating mammalian erythropoiesis.

Targeted BMI1 inhibition impairs tumor growth in lung adenocarcinomas with low CEBPα expression.

Lung cancer is the most common cause of cancer deaths. The expression of the transcription factor C/EBPα (CCAAT/enhancer binding protein α) is frequently lost in non-small cell lung cancer, but the mechanisms by which C/EBPα suppresses tumor formation are not fully understood. In addition, no pharmacological therapy is available to specifically target C/EBPα expression. We discovered a subset of pulmonary adenocarcinoma patients in whom negative/low C/EBPα expression and positive expression of the oncogenic protein BMI1 (B lymphoma Mo-MLV insertion region 1 homolog) have prognostic value. We also generated a lung-specific mouse model of C/EBPα deletion that develops lung adenocarcinomas, which are prevented by Bmi1 haploinsufficiency. BMI1 activity is required for both tumor initiation and maintenance in the C/EBPα-null background, and pharmacological inhibition of BMI1 exhibits antitumor effects in both murine and human adenocarcinoma lines. Overall, we show that C/EBPα is a tumor suppressor in lung cancer and that BMI1 is required for the oncogenic process downstream of C/EBPα loss. Therefore, anti-BMI1 pharmacological inhibition may offer a therapeutic benefit for lung cancer patients with low expression of C/EBPα and high BMI1.

Direct Induction and Functional Maturation of Forebrain GABAergic Neurons from Human Pluripotent Stem Cells.

Gamma-aminobutyric acid (GABA)-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here, we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs) into GABAergic neurons (iGNs) with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6-8 weeks. Furthermore, in vitro, iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs). Upon transplantation into immunodeficient mice, human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together, our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons.

Tumor-derived circulating endothelial cell clusters in colorectal cancer.

Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

Tumour-initiating cell-specific miR-1246 and miR-1290 expression converge to promote non-small cell lung cancer progression.

The tumour-initiating cell (TIC) model accounts for phenotypic and functional heterogeneity among tumour cells. MicroRNAs (miRNAs) are regulatory molecules frequently aberrantly expressed in cancers, and may contribute towards tumour heterogeneity and TIC behaviour. More recent efforts have focused on miRNAs as diagnostic or therapeutic targets. Here, we identified the TIC-specific miRNAs, miR-1246 and miR-1290, as crucial drivers for tumour initiation and cancer progression in human non-small cell lung cancer. The loss of either miRNA impacted the tumour-initiating potential of TICs and their ability to metastasize. Longitudinal analyses of serum miR-1246 and miR-1290 levels across time correlate their circulating levels to the clinical response of lung cancer patients who were receiving ongoing anti-neoplastic therapies. Functionally, direct inhibition of either miRNA with locked nucleic acid administered systemically, can arrest the growth of established patient-derived xenograft tumours, thus indicating that these miRNAs are clinically useful as biomarkers for tracking disease progression and as therapeutic targets.

CD275-Independent IL-17-Producing T Follicular Helper-like Cells in Lymphopenic Autoimmune-Prone Mice.

T cells undergo homeostatic expansion and acquire an activated phenotype in lymphopenic microenvironments. Restoration of normal lymphocyte numbers typically re-establishes normal homeostasis, and proinflammatory cytokine production returns to baseline. Mice deficient in guanine nucleotide exchange factor RasGRP1 exhibit dysregulated homeostatic expansion, which manifests as lymphoproliferative disease with autoantibody production. Our previous work revealed that autoreactive B cells lacking RasGRP1 break tolerance early during development, as well as during germinal center responses, suggesting that T cell-independent and T cell-dependent mechanisms are responsible. Examination of whether a particular T cell subset is involved in the breach of B cell tolerance revealed increased Th17 cells in Rasgrp1-deficient mice relative to control mice. Rasgrp1-deficient mice lacking IL-17R had fewer germinal centers, and germinal centers that formed contained fewer autoreactive B cells, suggesting that IL-17 signaling is required for a break in B cell tolerance in germinal centers. Interestingly, a fraction of Th17 cells from Rasgrp1-deficient mice were CXCR5(+) and upregulated levels of CD278 coordinate with their appearance in germinal centers, all attributes of T follicular helper cells (Tfh17). To determine whether CD278-CD275 interactions were required for the development of Tfh17 cells and for autoantibody, Rasgrp1-deficient mice were crossed with CD275-deficient mice. Surprisingly, mice deficient in RasGRP1 and CD275 formed Tfh17 cells and germinal centers and produced similar titers of autoantibodies as mice deficient in only RasGRP1. Therefore, these studies suggest that requirements for Tfh cell development change in lymphopenia-associated autoimmune settings.