The Kinome of Human Alveolar Type II and Basal Cells, and Its Reprogramming in Lung Cancer.

The discovery of mutant tyrosine kinases as oncogenic drivers of lung adenocarcinomas has changed the basic understanding of lung cancer development and therapy. Yet, expressed kinases (kinome) in lung cancer progenitor cells, as well as whether kinase expression and the overall kinome changes or is reprogrammed upon transformation, is incompletely understood. We hypothesized that the kinome differs between lung cancer progenitor cells, alveolar type II cells (ATII), and basal cells (BC) and that their respective kinomes undergo distinct lineage-specific reprogramming to adenocarcinomas and squamous cell carcinomas upon transformation. We performed RNA sequencing on freshly isolated human ATII, BC, and lung cancer cell lines to define the kinome in nontransformed cells and transformed cells. Our studies identified a unique kinome for ATII and BC and changes in their kinome upon transformation to their respective carcinomas.

Regulation of toll-like receptor signaling by the SF3a mRNA splicing complex.

The innate immune response plays a key role in fighting infection by activating inflammation and stimulating the adaptive immune response. However, chronic activation of innate immunity can contribute to the pathogenesis of many diseases with an inflammatory component. Thus, various negatively acting factors turn off innate immunity subsequent to its activation to ensure that inflammation is self-limiting and to prevent inflammatory disease. These negatively acting pathways include the production of inhibitory acting alternate proteins encoded by alternative mRNA splice forms of genes in Toll-like receptor (TLR) signaling pathways. We previously found that the SF3a mRNA splicing complex was required for a robust innate immune response; SF3a acts to promote inflammation in part by inhibiting the production of a negatively acting splice form of the TLR signaling adaptor MyD88. Here we inhibit SF3a1 using RNAi and subsequently perform an RNAseq study to identify the full complement of genes and splicing events regulated by SF3a in murine macrophages. Surprisingly, in macrophages, SF3a has significant preference for mRNA splicing events within innate immune signaling pathways compared with other biological pathways, thereby affecting the splicing of specific genes in the TLR signaling pathway to modulate the innate immune response.

Bioinformatics-driven discovery of rational combination for overcoming EGFR-mutant lung cancer resistance to EGFR therapy.

MOTIVATION: Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death in the United States. Targeted tyrosine kinase inhibitors (TKIs) directed against the epidermal growth factor receptor (EGFR) have been widely and successfully used in treating NSCLC patients with activating EGFR mutations. Unfortunately, the duration of response is short-lived, and all patients eventually relapse by acquiring resistance mechanisms. RESULT: We performed an integrative systems biology approach to determine essential kinases that drive EGFR-TKI resistance in cancer cell lines. We used a series of bioinformatics methods to analyze and integrate the functional genetics screen and RNA-seq data to identify a set of kinases that are critical in survival and proliferation in these TKI-resistant lines. By connecting the essential kinases to compounds using a novel kinase connectivity map (K-Map), we identified and validated bosutinib as an effective compound that could inhibit proliferation and induce apoptosis in TKI-resistant lines. A rational combination of bosutinib and gefitinib showed additive and synergistic effects in cancer cell lines resistant to EGFR TKI alone. CONCLUSIONS: We have demonstrated a bioinformatics-driven discovery roadmap for drug repurposing and development in overcoming resistance in EGFR-mutant NSCLC, which could be generalized to other cancer types in the era of personalized medicine. AVAILABILITY AND IMPLEMENTATION: K-Map can be accessible at: http://tanlab.ucdenver.edu/kMap. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples.

Next-generation sequencing has proven an extremely effective technology for molecular counting applications where the number of sequence reads provides a digital readout for RNA-seq, ChIP-seq, Tn-seq and other applications. The extremely large number of sequence reads that can be obtained per run permits the analysis of increasingly complex samples. For lower complexity samples, however, a point of diminishing returns is reached when the number of counts per sequence results in oversampling with no increase in data quality. A solution to making next-generation sequencing as efficient and affordable as possible involves assaying multiple samples in a single run. Here, we report the successful 96-plexing of complex pools of DNA barcoded yeast mutants and show that such 'Bar-seq' assessment of these samples is comparable with data provided by barcode microarrays, the current benchmark for this application. The cost reduction and increased throughput permitted by highly multiplexed sequencing will greatly expand the scope of chemogenomics assays and, equally importantly, the approach is suitable for other sequence counting applications that could benefit from massive parallelization.

Semi-automated library preparation for high-throughput DNA sequencing platforms.

Next-generation sequencing platforms are powerful technologies, providing gigabases of genetic information in a single run. An important prerequisite for high-throughput DNA sequencing is the development of robust and cost-effective preprocessing protocols for DNA sample library construction. Here we report the development of a semi-automated sample preparation protocol to produce adaptor-ligated fragment libraries. Using a liquid-handling robot in conjunction with Carboxy Terminated Magnetic Beads, we labeled each library sample using a unique 6 bp DNA barcode, which allowed multiplex sample processing and sequencing of 32 libraries in a single run using Applied Biosystems' SOLiD sequencer. We applied our semi-automated pipeline to targeted medical resequencing of nuclear candidate genes in individuals affected by mitochondrial disorders. This novel method is capable of preparing as much as 32 DNA libraries in 2.01 days (8-hour workday) for emulsion PCR/high throughput DNA sequencing, increasing sample preparation production by 8-fold.

Draft Genome Sequence of Mycobacterium chelonae Type Strain ATCC 35752.

Mycobacterium chelonae is a rapidly growing opportunistic nontuberculous mycobacterial (NTM) species that causes infections in humans and other hosts. Here, we report the draft genome sequence of Mycobacterium chelonae type strain ATCC 35752, consisting of 4.89 Mbp, 63.96% G+C content, 4,489 protein-coding genes, 48 tRNAs, and 3 rRNA genes.

Genome Sequence of an Epidemic Isolate of Mycobacterium abscessus subsp. bolletii from Rio de Janeiro, Brazil.

Multiple isolates of Mycobacterium abscessus subsp. bolletii, collectively called BRA100, were associated with outbreaks of postsurgical skin infections across various regions of Brazil from 2003 to 2009. We announce the draft genome sequence of a newly sequenced BRA100 strain, M. abscessus subsp. bolletii CRM-0020, isolated from a patient in Rio de Janeiro, Brazil.