Improper data practices erode the quality of global ecological databases and impede the progress of ecological research.

The scientific community has entered an era of big data. However, with big data comes big responsibilities, and best practices for how data are contributed to databases have not kept pace with the collection, aggregation, and analysis of big data. Here, we rigorously assess the quantity of data for specific leaf area (SLA) available within the largest and most frequently used global plant trait database, the TRY Plant Trait Database, exploring how much of the data were applicable (i.e., original, representative, logical, and comparable) and traceable (i.e., published, cited, and consistent). Over three-quarters of the SLA data in TRY either lacked applicability or traceability, leaving only 22.9% of the original data usable compared with the 64.9% typically deemed usable by standard data cleaning protocols. The remaining usable data differed markedly from the original for many species, which led to altered interpretation of ecological analyses. Though the data we consider here make up only 4.5% of SLA data within TRY, similar issues of applicability and traceability likely apply to SLA data for other species as well as other commonly measured, uploaded, and downloaded plant traits. We end with suggested steps forward for global ecological databases, including suggestions for both uploaders to and curators of databases with the hope that, through addressing the issues raised here, we can increase data quality and integrity within the ecological community.

ASCL1-ERK1/2 Axis: ASCL1 restrains ERK1/2 via the dual specificity phosphatase DUSP6 to promote survival of a subset of neuroendocrine lung cancers.

The transcription factor achaete-scute complex homolog 1 (ASCL1) is a lineage oncogene that is central for the growth and survival of small cell lung cancers (SCLC) and neuroendocrine non-small cell lung cancers (NSCLC-NE) that express it. Targeting ASCL1, or its downstream pathways, remains a challenge. However, a potential clue to overcoming this challenage has been information that SCLC and NSCLC-NE that express ASCL1 exhibit extremely low ERK1/2 activity, and efforts to increase ERK1/2 activity lead to inhibition of SCLC growth and surival. Of course, this is in dramatic contrast to the majority of NSCLCs where high activity of the ERK pathway plays a major role in cancer pathogenesis. A major knowledge gap is defining the mechanism(s) underlying the low ERK1/2 activity in SCLC, determining if ERK1/2 activity and ASCL1 function are inter-related, and if manipulating ERK1/2 activity provides a new therapeutic strategy for SCLC. We first found that expression of ERK signaling and ASCL1 have an inverse relationship in NE lung cancers: knocking down ASCL1 in SCLCs and NE-NSCLCs increased active ERK1/2, while inhibition of residual SCLC/NSCLC-NE ERK1/2 activity with a MEK inhibitor increased ASCL1 expression. To determine the effects of ERK activity on expression of other genes, we obtained RNA-seq from ASCL1-expressing lung tumor cells treated with an ERK pathway MEK inhibitor and identified down-regulated genes (such as SPRY4, ETV5, DUSP6, SPRED1) that potentially could influence SCLC/NSCLC-NE tumor cell survival. This led us to discover that genes regulated by MEK inhibition suppress ERK activation and CHIP-seq demonstrated these are bound by ASCL1. In addition, SPRY4, DUSP6, SPRED1 are known suppressors of the ERK1/2 pathway, while ETV5 regulates DUSP6. Survival of NE lung tumors was inhibited by activation of ERK1/2 and a subset of ASCL1-high NE lung tumors expressed DUSP6. Because the dual specificity phosphatase 6 (DUSP6) is an ERK1/2-selective phosphatase that inactivates these kinases and has a pharmacologic inhibitor, we focused mechanistic studies on DUSP6. These studies showed: Inhibition of DUSP6 increased active ERK1/2, which accumulated in the nucleus; pharmacologic and genetic inhibition of DUSP6 affected proliferation and survival of ASCL1-high NE lung cancers; and that knockout of DUSP6 "cured" some SCLCs while in others resistance rapidly developed indicating a bypass mechanism was activated. Thus, our findings fill this knowledge gap and indicate that combined expression of ASCL1, DUSP6 and low phospho-ERK1/2 identify some neuroendocrine lung cancers for which DUSP6 may be a therapeutic target.

A Framework for Pediatric Health Care Providers to Promote Active Play in Nature for Children.

AIM: Time outdoors and contact with nature are positively associated with a broad range of children's health outcomes. Pediatricians are uniquely positioned to promote active play in nature (APN) but may face challenges to do so during well child visits. The objective of this study was to understand barriers to children's APN, before and during the COVID-19 pandemic, and how health care providers could promote APN. METHODS: Focus groups were conducted with 14 pediatric providers and interviews with 14 parents (7 in English, 7 in Spanish) of children ages 3 to 10 on public insurance. Dedoose was used for coding and content analysis. We contextualized this work within the WHO's Commission on Social Determinants of Health conceptual framework. RESULTS: Parents mentioned a range of material circumstances (time, finances, family circumstances, access to safe outdoor play spaces and age-appropriate activities) and behavioral/psychosocial factors (previous experiences in nature, safety, and weather concerns), many of which were exacerbated by the pandemic, that serve as barriers to children's APN. Providers said they were motivated to talk to families about children's APN but mentioned barriers to this conversation such as time, other pressing priorities for the visit, and lack of resources to give families. CONCLUSIONS: Many pre-pandemic barriers to APN were exacerbated by the COVID-19 pandemic. Well-child visits may be an effective setting to discuss the benefits of APN during and beyond the pandemic, and there is a need for contextually appropriate resources for pediatric providers and families.

AXL targeting restores PD-1 blockade sensitivity of STK11/LKB1 mutant NSCLC through expansion of TCF1+ CD8 T cells.

Mutations in STK11/LKB1 in non-small cell lung cancer (NSCLC) are associated with poor patient responses to immune checkpoint blockade (ICB), and introduction of a Stk11/Lkb1 (L) mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 loss (KP) resulted in an ICB refractory syngeneic KPL tumor. Mechanistically this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype recapitulated in human STK11/LKB1 mutant NSCLCs. Systemic inhibition of Axl results in increased type I interferon secretion from dendritic cells that expanded tumor-associated TCF1+PD-1+CD8 T cells, restoring therapeutic response to PD-1 ICB in KPL tumors. This was observed in syngeneic immunocompetent mouse models and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts. NSCLC-affected individuals with identified STK11/LKB1 mutations receiving bemcentinib and pembrolizumab demonstrated objective clinical response to combination therapy. We conclude that AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC.

Lentiviral-Driven Discovery of Cancer Drug Resistance Mutations.

Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). Here we optimized and leveraged this property to identify drug resistance mutations, developing a technique we term LentiMutate. This technique was validated by identifying clinically relevant EGFR resistance mutations, then applied to two additional clinical anticancer drugs: imatinib, a BCR-ABL inhibitor, and AMG 510, a KRAS G12C inhibitor. Novel deletions in BCR-ABL1 conferred resistance to imatinib. In KRAS-G12C or wild-type KRAS, point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations conferred resistance to AMG 510. LentiMutate should prove highly valuable for clinical and preclinical cancer-drug development. SIGNIFICANCE: LentiMutate can evaluate a drug's on-target activity and can nominate resistance mutations before they occur in patients, which could accelerate and refine drug development to increase the survival of patients with cancer.

EGFR inhibition triggers an adaptive response by co-opting antiviral signaling pathways in lung cancer.

EGFR inhibition is an effective treatment in the minority of non-small cell lung cancer (NSCLC) cases harboring EGFR-activating mutations, but not in EGFR wild type (EGFRwt) tumors. Here, we demonstrate that EGFR inhibition triggers an antiviral defense pathway in NSCLC. Inhibiting mutant EGFR triggers Type I IFN-I upregulation via a RIG-I-TBK1-IRF3 pathway. The ubiquitin ligase TRIM32 associates with TBK1 upon EGFR inhibition, and is required for K63-linked ubiquitination and TBK1 activation. Inhibiting EGFRwt upregulates interferons via an NF-κB-dependent pathway. Inhibition of IFN signaling enhances EGFR-TKI sensitivity in EGFR mutant NSCLC and renders EGFRwt/KRAS mutant NSCLC sensitive to EGFR inhibition in xenograft and immunocompetent mouse models. Furthermore, NSCLC tumors with decreased IFN-I expression are more responsive to EGFR TKI treatment. We propose that IFN-I signaling is a major determinant of EGFR-TKI sensitivity in NSCLC and that a combination of EGFR TKI plus IFN-neutralizing antibody could be useful in most NSCLC patients.

eIF5B drives integrated stress response-dependent translation of PD-L1 in lung cancer.

Cancer cells express high levels of PD-L1, a ligand of the PD-1 receptor on T cells, allowing tumors to suppress T cell activity. Clinical trials utilizing antibodies that disrupt the PD-1/PD-L1 checkpoint have yielded remarkable results, with anti-PD-1 immunotherapy approved as first-line therapy for lung cancer patients. We used CRISPR-based screening to identify regulators of PD-L1 in human lung cancer cells, revealing potent induction of PD-L1 upon disruption of heme biosynthesis. Impairment of heme production activates the integrated stress response (ISR), allowing bypass of inhibitory upstream open reading frames in the PD-L1 5' UTR, resulting in enhanced PD-L1 translation and suppression of anti-tumor immunity. We demonstrated that ISR-dependent PD-L1 translation requires the translation initiation factor eIF5B. eIF5B overexpression, which is frequent in lung adenocarcinomas and associated with poor prognosis, is sufficient to induce PD-L1. These findings illuminate mechanisms of immune checkpoint activation and identify targets for therapeutic intervention.

An in vivo functional genomics screen of nuclear receptors and their co-regulators identifies FOXA1 as an essential gene in lung tumorigenesis.

Using a mini-library of 1062 lentiviral shRNAs targeting 40 nuclear hormone receptors and 70 of their co-regulators, we searched for potential therapeutic targets that would be important during in vivo tumor growth using a parallel in vitro and in vivo shRNA screening strategy in the non-small cell lung cancer (NSCLC) line NCI-H1819. We identified 21 genes essential for in vitro growth, and nine genes specifically required for tumor survival in vivo, but not in vitro: NCOR2, FOXA1, HDAC1, RXRA, RORB, RARB, MTA2, ETV4, and NR1H2. We focused on FOXA1, since it lies within the most frequently amplified genomic region in lung adenocarcinomas. We found that 14q-amplification in NSCLC cell lines was a biomarker for FOXA1 dependency for both in vivo xenograft growth and colony formation, but not mass culture growth in vitro. FOXA1 knockdown identified genes involved in electron transport among the most differentially regulated, indicating FOXA1 loss may lead to a decrease in cellular respiration. In support of this, FOXA1 amplification was correlated with increased sensitivity to the complex I inhibitor phenformin. Integrative ChipSeq analyses reveal that FOXA1 functions in this genetic context may be at least partially independent of NKX2-1. Our findings are consistent with a neomorphic function for amplified FOXA1, driving an oncogenic transcriptional program. These data provide new insight into the functional consequences of FOXA1 amplification in lung adenocarcinomas, and identify new transcriptional networks for exploration of therapeutic vulnerabilities in this patient population.

SLC43A3 Is a Biomarker of Sensitivity to the Telomeric DNA Damage Mediator 6-Thio-2'-Deoxyguanosine.

Cell membrane transporters facilitate the passage of nucleobases and nucleosides for nucleotide synthesis and metabolism, and are important for the delivery of nucleoside analogues used in anticancer drug therapy. Here, we investigated if cell membrane transporters are involved in the cellular uptake of the nucleoside analogue DNA damage mediator 6-thio-2'-deoxyguanosine (6-thio-dG). A large panel of non-small cell lung cancer (NSCLC) cell lines (73 of 77) were sensitive to 6-thio-dG; only four NSCLC lines were resistant to 6-thio-dG. When analyzed by microarray and RNA sequencing, the resistant NSCLC cell lines clustered together, providing a molecular signature for patients that may not respond to 6-thio-dG. Significant downregulation of solute carrier family 43 A3 (SLC43A3), an equilibrative nucleobase transporter, was identified as a candidate in this molecular resistance signature. High levels of SLC43A3 mRNA predicted sensitivity to 6-thio-dG and therefore SLC43A3 could serve as a promising biomarker for 6-thio-dG sensitivity in patients with NSCLC. SIGNIFICANCE: These findings identify a biomarker of resistance to the telomeric DNA damage mediator 6-thio-2'-deoxyguanosine.

Metabolic Diversity in Human Non-Small Cell Lung Cancer Cells.

Intermediary metabolism in cancer cells is regulated by diverse cell-autonomous processes, including signal transduction and gene expression patterns, arising from specific oncogenotypes and cell lineages. Although it is well established that metabolic reprogramming is a hallmark of cancer, we lack a full view of the diversity of metabolic programs in cancer cells and an unbiased assessment of the associations between metabolic pathway preferences and other cell-autonomous processes. Here, we quantified metabolic features, mostly from the 13C enrichment of molecules from central carbon metabolism, in over 80 non-small cell lung cancer (NSCLC) cell lines cultured under identical conditions. Because these cell lines were extensively annotated for oncogenotype, gene expression, protein expression, and therapeutic sensitivity, the resulting database enables the user to uncover new relationships between metabolism and these orthogonal processes.

Elucidating synergistic dependencies in lung adenocarcinoma by proteome-wide signaling-network analysis.

To understand drug combination effect, it is necessary to decipher the interactions between drug targets-many of which are signaling molecules. Previously, such signaling pathway models are largely based on the compilation of literature data from heterogeneous cellular contexts. Indeed, de novo reconstruction of signaling interactions from large-scale molecular profiling is still lagging, compared to similar efforts in transcriptional and protein-protein interaction networks. To address this challenge, we introduce a novel algorithm for the systematic inference of protein kinase pathways, and applied it to published mass spectrometry-based phosphotyrosine profile data from 250 lung adenocarcinoma (LUAD) samples. The resulting network includes 43 TKs and 415 inferred, LUAD-specific substrates, which were validated at >60% accuracy by SILAC assays, including "novel' substrates of the EGFR and c-MET TKs, which play a critical oncogenic role in lung cancer. This systematic, data-driven model supported drug response prediction on an individual sample basis, including accurate prediction and validation of synergistic EGFR and c-MET inhibitor activity in cells lacking mutations in either gene, thus contributing to current precision oncology efforts.

Telomerase-Mediated Strategy for Overcoming Non-Small Cell Lung Cancer Targeted Therapy and Chemotherapy Resistance.

Standard and targeted cancer therapies for late-stage cancer patients almost universally fail due to tumor heterogeneity/plasticity and intrinsic or acquired drug resistance. We used the telomerase substrate nucleoside precursor, 6-thio-2'-deoxyguanosine (6-thio-dG), to target telomerase-expressing non-small cell lung cancer cells resistant to EGFR-inhibitors and commonly used chemotherapy combinations. Colony formation assays, human xenografts as well as syngeneic and genetically engineered immune competent mouse models of lung cancer were used to test the effect of 6-thio-dG on targeted therapy- and chemotherapy-resistant lung cancer human cells and mouse models. We observed that erlotinib-, paclitaxel/carboplatin-, and gemcitabine/cisplatin-resistant cells were highly sensitive to 6-thio-dG in cell culture and in mouse models. 6-thio-dG, with a known mechanism of action, is a potential novel therapeutic approach to prolong disease control of therapy-resistant lung cancer patients with minimal toxicities.

Chemistry-First Approach for Nomination of Personalized Treatment in Lung Cancer.

Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.

TNF-driven adaptive response mediates resistance to EGFR inhibition in lung cancer.

Although aberrant EGFR signaling is widespread in cancer, EGFR inhibition is effective only in a subset of non-small cell lung cancer (NSCLC) with EGFR activating mutations. A majority of NSCLCs express EGFR wild type (EGFRwt) and do not respond to EGFR inhibition. TNF is a major mediator of inflammation-induced cancer. We find that a rapid increase in TNF level is a universal adaptive response to EGFR inhibition in NSCLC, regardless of EGFR status. EGFR signaling actively suppresses TNF mRNA levels by inducing expression of miR-21, resulting in decreased TNF mRNA stability. Conversely, EGFR inhibition results in loss of miR-21 and increased TNF mRNA stability. In addition, TNF-induced NF-κB activation leads to increased TNF transcription in a feed-forward loop. Inhibition of TNF signaling renders EGFRwt-expressing NSCLC cell lines and an EGFRwt patient-derived xenograft (PDX) model highly sensitive to EGFR inhibition. In EGFR-mutant oncogene-addicted cells, blocking TNF enhances the effectiveness of EGFR inhibition. EGFR plus TNF inhibition is also effective in NSCLC with acquired resistance to EGFR inhibition. We suggest concomitant EGFR and TNF inhibition as a potentially new treatment approach that could be beneficial for a majority of lung cancer patients.

A comprehensively characterized cell line panel highly representative of clinical ovarian high-grade serous carcinomas.

Recent literature suggests that most widely used ovarian cancer (OVCA) cell models do not recapitulate the molecular features of clinical tumors. To address this limitation, we generated 18 cell lines and 3 corresponding patient-derived xenografts predominantly from high-grade serous carcinoma (HGSOC) peritoneal effusions. Comprehensive genomic characterization and comparison of each model to its parental tumor demonstrated a high degree of molecular similarity. Our characterization included whole exome-sequencing and copy number profiling for cell lines, xenografts, and matched non-malignant tissues, and DNA methylation, gene expression, and spectral karyotyping for a subset of specimens. Compared to the Cancer Genome Atlas (TCGA), our models more closely resembled HGSOC than any other tumor type, justifying their validity as OVCA models. Our meticulously characterized models provide a crucial resource for the OVCA research community that will advance translational findings and ultimately lead to clinical applications.

SMARCA4-inactivating mutations increase sensitivity to Aurora kinase A inhibitor VX-680 in non-small cell lung cancers.

Mutations in the SMARCA4/BRG1 gene resulting in complete loss of its protein (BRG1) occur frequently in non-small cell lung cancer (NSCLC) cells. Currently, no single therapeutic agent has been identified as synthetically lethal with SMARCA4/BRG1 loss. We identify AURKA activity as essential in NSCLC cells lacking SMARCA4/BRG1. In these cells, RNAi-mediated depletion or chemical inhibition of AURKA induces apoptosis and cell death in vitro and in xenograft mouse models. Disc large homologue-associated protein 5 (HURP/DLGAP5), required for AURKA-dependent, centrosome-independent mitotic spindle assembly is essential for the survival and proliferation of SMARCA4/BRG1 mutant but not of SMARCA4/BRG1 wild-type cells. AURKA inhibitors may provide a therapeutic strategy for biomarker-driven clinical studies to treat the NSCLCs harbouring SMARCA4/BRG1-inactivating mutations.

PROTOCADHERIN 7 Acts through SET and PP2A to Potentiate MAPK Signaling by EGFR and KRAS during Lung Tumorigenesis.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-associated deaths worldwide. Given the efficacy of membrane proteins as therapeutic targets in human malignancies, we examined cell-surface receptors that may act as drivers of lung tumorigenesis. Here, we report that the PROTOCADHERIN PCDH7 is overexpressed frequently in NSCLC tumors where this event is associated with poor clinical outcome. PCDH7 overexpression synergized with EGFR and KRAS to induce MAPK signaling and tumorigenesis. Conversely, PCDH7 depletion suppressed ERK activation, sensitized cells to MEK inhibitors, and reduced tumor growth. PCDH7 potentiated ERK signaling by facilitating interaction of protein phosphatase PP2A with its potent inhibitor, the SET oncoprotein. By establishing an oncogenic role for PCDH7 in lung tumorigenesis, our results provide a rationale to develop novel PCDH7 targeting therapies that act at the cell surface of NSCLC cells to compromise their growth. Cancer Res; 77(1); 187-97. ©2016 AACR.

Leveraging an NQO1 Bioactivatable Drug for Tumor-Selective Use of Poly(ADP-ribose) Polymerase Inhibitors.

Therapeutic drugs that block DNA repair, including poly(ADP-ribose) polymerase (PARP) inhibitors, fail due to lack of tumor-selectivity. When PARP inhibitors and ß-lapachone are combined, synergistic antitumor activity results from sustained NAD(P)H levels that refuel NQO1-dependent futile redox drug recycling. Significant oxygen-consumption-rate/reactive oxygen species cause dramatic DNA lesion increases that are not repaired due to PARP inhibition. In NQO1+ cancers, such as non-small-cell lung, pancreatic, and breast cancers, cell death mechanism switches from PARP1 hyperactivation-mediated programmed necrosis with ß-lapachone monotherapy to synergistic tumor-selective, caspase-dependent apoptosis with PARP inhibitors and ß-lapachone. Synergistic antitumor efficacy and prolonged survival were noted in human orthotopic pancreatic and non-small-cell lung xenograft models, expanding use and efficacy of PARP inhibitors for human cancer therapy.

Telomerase inhibitor imetelstat has preclinical activity across the spectrum of non-small cell lung cancer oncogenotypes in a telomere length dependent manner.

Telomerase was evaluated as a therapeutic oncotarget by studying the efficacy of the telomerase inhibitor imetelstat in non-small cell lung cancer (NSCLC) cell lines to determine the range of response phenotypes and identify potential biomarkers of response. A panel of 63 NSCLC cell lines was studied for telomere length and imetelstat efficacy in inhibiting colony formation and no correlation was found with patient characteristics, tumor histology, and oncogenotypes. While there was no overall correlation between imetelstat efficacy with initial telomere length (ranging from 1.5 to 20 kb), the quartile of NSCLC lines with the shortest telomeres was more sensitive than the quartile with the longest telomeres. Continuous long-term treatment with imetelstat resulted in sustained telomerase inhibition, progressive telomere shortening and eventual growth inhibition in a telomere-length dependent manner. Cessation of imetelstat therapy before growth inhibition was followed by telomere regrowth. Likewise, in vivo imetelstat treatment caused tumor xenograft growth inhibition in a telomere-length dependent manner. We conclude from these preclinical studies of telomerase as an oncotarget tested by imetelstat response that imetelstat has efficacy across the entire oncogenotype spectrum of NSCLC, continuous therapy is necessary to prevent telomere regrowth, and short telomeres appears to be the best treatment biomarker.