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Background Overexpression of epidermal growth factor receptor (EGFR), and downstream pathway activation appears to be a common oncogenic driver in the majority of head and neck squamous cell cancers (HNSCCs); yet targeting EGFR for the treatment of HNSCC has met with limited success. Apart from the anti-EGFR antibody cetuximab, no small molecule EGFR/tyrosine kinase inhibitors (TKIs) have progressed to routine clinical use. The aim of this study was to determine factors contributing to the lack of response to TKIs and identify alternative therapeutic vulnerabilities. Methods Genomic and transcriptomic sequencing, high-throughput compound screens, overexpression and siRNA knockdown, western blot, in vivo xenograft studies. Findings We derived three pairs of isogenic gefitinib (TKI)-sensitive and resistant patient-derived HNSCC cell lines. Genomic sequencing of gefitinib-resistant cell lines identified a lack of activating and resistance-associated EGFR mutations. Instead, transcriptomic sequencing showed upregulated EMT gene signature in the gefitinib-resistant cells with a corresponding increase in their migratory phenotype. Additionally, the resistant cell displayed reduced growth rate. Surprisingly, while gefitinib-resistant cells were independent of EGFR for survival, they nonetheless displayed activation of downstream ERK and AKT signalling. High-throughput screening (HTS) of druggable, small molecule libraries revealed that the gefitinib-resistant cells were particularly sensitive to inhibitors of genes involved in cell cycle and mitosis, such as Aurora kinase inhibitors (AKIs), cyclin-dependent kinase (CDK) inhibitors, and microtubule inhibitors. Notably our results showed that in the EGFR inhibited state, Aurora kinases are essential for cell survival. Interpretation Our study demonstrates that in the absence of activating EGFR mutations, HNSCCs may gain resistance to gefitinib through decreased cell proliferation, which makes them exceptionally vulnerable to cell-cycle inhibitors. Funding Agency for Science, Technology, and Research (A*STAR), National Medical Research Council (NMRC), and the National Institutes of Health (NIH)/National Cancer Institute (NCI).

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., EGFR mutant lung adenocarcinoma (LUAD) exhibit diverse clinical outcomes in response to targeted therapies. Here the authors show that these LUADs involve a complex genomic landscape with high intratumor heterogeneity, providing insights into the evolutionary trajectory of oncogene-driven LUAD and potential mediators of EGFR TKI resistance.

BACKGROUND The comeasurement of both genomic and transcriptomic signatures in single cells is of fundamental importance to accurately assess how the genetic information correlates with the transcriptomic phenotype. However, existing technologies have low throughput and laborious work flows. METHODS We developed a new method for concurrent sequencing of the transcriptome and targeted genomic regions (CORTAD-seq) within the same single cell on an automated microfluidic platform. The method was compatible with the downstream library preparation, allowing easy integration into existing next-generation sequencing work flows. We incorporated a single-cell bioinformatics pipeline for transcriptome and mutation analysis. RESULTS As proof of principle, we applied CORTAD-seq to lung cancer cell lines to dissect the cellular consequences of mutations that result in resistance to targeted therapy. We obtained a mean detection of 6000 expressed genes and an exonic rate of 50%. The targeted DNA-sequencing data achieved a 97.8% detection rate for mutations and allowed for the identification of copy number variations and haplotype construction. We detected expression signatures of tyrosine kinase inhibitor (TKI) resistance, epidermal growth factor receptor (EGFR) amplification, and expansion of the T790M mutation among resistant cells. We also identified characteristics for TKI resistance that were independent of EGFR T790M, indicating that other alterations are required for resistance in this context. CONCLUSIONS CORTAD-seq allows assessment of the interconnection between genetic and transcriptomic changes in single cells. It is operated on an automated, commercially available single-cell isolation platform, making its implementation straightforward.

Objectives:The primary aims of the study were to examine the effect of resveratrol on skeletal muscle SIRT1 expression and energy expenditure in subjects with Type 2 diabetes mellitus (T2DM).Background:Animal and in vivo studies indicate that resveratrol increases SIRT1 expression that stimulates PGC1α activity. Subsequent upregulation of AMPK and GLUT4 expression are associated with improved insulin sensitivity in peripheral tissues.Methods:Ten subjects with T2DM were randomized in a double-blind fashion to receive 3g resveratrol or placebo daily for 12 weeks. Secondary outcomes include measures of AMPK, p-AMPK and GLUT4 expression levels, energy expenditure, physical activity levels, distribution of abdominal adipose tissue and skeletal muscle fiber type composition, body weight, HbA1c, plasma lipid subfraction, adiponectin levels, and insulin sensitivity.Results:There was a significant increase in both SIRT1 expression (2.01 vs. 0.86 arbitrary units [AU], p = .016) and p-AMPK to AMPK expression ratio (2.04 vs. 0.79 AU, p = .032) in the resveratrol group compared with the placebo group. Although the percentage of absolute change (8.6 vs. –13.9%, p = .033) and percentage of predicted resting metabolic rate (RMR; 7.8 vs. –13.9%, p = .013) were increased following resveratrol, there was a significant reduction in average daily activity (–38 vs. 43.2%, p = .028) and step counts (–39.5 vs. 11.8%, p = .047) when compared with placebo.Conclusions:In patients with T2DM, treatment with resveratrol regulates energy expenditure through increased skeletal muscle SIRT1 and AMPK expression. These findings indicate that resveratrol may have beneficial exercise-mimetic effects in patients with T2DM.

Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy.

Resistance to therapy is one of the major causes of cancer-associated death. While a number of mechanisms for the development of resistance to epidermal growth factor receptor, EGFR-targeting drugs in lung cancer have been described, most genomic and transcriptomic studies have focused on analyzing bulk samples that can only provide an average measurement over the entire mixture of cell populations of a tumor. With this approach, it is difficult to resolve cell-to-cell variability of drug resistance within a heterogeneous tumor. In order to accurately describe and eventually delineate the underlying causes of cancer progression, it requires the analysis of single cells on both, the transcriptomic and the genomic level. Only the co-detection of mutations and expression features in individual cells allows to define the connection between the two. We therefore aimed to establish a new methodology for concurrent evaluation of transcriptomic and genomic features within the same single cell on the Fluidigm C1 system. We applied this protocol to an EGFR-mutant lung cancer cell line, PC9, that has developed resistance to tyrosine kinase inhibitor, TKI treatment after prolonged exposure to sublethal doses of Gefitinib. We amplified cDNA and selected genomic EGFR regions of the TKI responsive and resistant PC9 clones from a total of ≈300 single cells. We used our in-house single cell’s analysis pipeline for parallel processing of transcriptome and variants detection. In addition, a novel algorithm, NODES was used to normalize the single cell RNAseq data and detect differentially expressed genes. The differential transcriptome and emergence of the EGFR T790M resistance mutation in relation to the TKI response were evaluated. We observed up-regulation of receptor tyrosine kinase AXL in the resistant cell lines. This observation is in agreement with previous findings that AXL is the key player that promotes TKI resistance in lung cancer. In addition, we found up-regulation of other genes that have not been previously described. Cumulatively, this method allows us to dissect the underpinnings of the TKI resistance mechanism and enables us to identify biomarker for specific cellular features that are connected with resistance and thereby with lung cancer patient’s response to TKI treatment. Citation Format: Say Li Kong, Huipeng Li, Dave Ruff, Joyce An Yi Tai, Elise T. Courtois, Huay Mei Poh, Dawn Pingxi Lau, Audrey Ann Liew, Gek San Tan, Tony Kiat Hon Lim, Daniel Shao Weng Tan, Shyam Prabhakar, Axel M. Hillmer. Transcriptome differences in tyrosine kinase inhibitor-resistant clones of EGFR-mutant lung cancer using a new microfluidic assay for concurrent single-cell RNA and targeted DNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3153. doi:10.1158/1538-7445.AM2017-3153

Background: Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation. Methodology/Principal Findings: Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12–1275 CTCs/ml; Lung cancer samples: 10–1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples. Conclusions/Significance: We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis., Singapore-MIT Alliance for Research and Technology

While EGFR tyrosine kinase inhibitors (TKIs) can elicit significant tumor shrinkage in the clinic, drug resistance inevitably ensues, likely due to a persistent subpopulation. T790M mutation is a common resistance mechanism found in 60% of tumors progressing on TKI. The aim of this study is to uncover novel mechanisms of gefitnib-resistant (GR) NSCLC by selecting TKI naïve PC9 single cell clones (SCC) differing in stemness and vulnerabilities to gefitinib (G), and inducing resistance through sublethal drug exposures. A high throughput drug screen was performed in 4 SCC to discover alternate therapies to target T790M negative GR clones. 231 SCC of PC9 were isolated and confirmed to be T790M negative (0.3% sensitivity) using Agena SABER assay. All clones were characterized by their vulnerability to G (viable after 48 hour exposure to 0.1uM) and ALDH1 activity using the ALDEFLUOR assay and flow cytometry. Four “diverse” clones (stratified according to ALDH1 and sensitivity to TKI) were selected to generate GR cell lines. Agglomerative hierarchical clustering of differentially expressed genes revealed that the clone exhibiting extreme low survival and ALDH1 signature clusters significantly apart from the rest. Subsequent iterations of this experiment (in total 5 per clone) revealed unique (and mostly stochastic) patterns of acquiring T790M across all clones. GR clones were then subjected to mutational analysis using the Ion AmpliSeq™ Colon and Lung Cancer Panel v1, and showed that 11/20 (55%) PC9 SCC GR lines have acquired the T790M mutation. T790M positive (AF range: 2.4 - 41.0%) GR lines were sensitive to Afatinib and WZ4002, a 2nd and 3rd generation EGFR TKI, with EGFR1086 phosphorylation being completely absent in all non-responding lines. T790M negative GR lines further acquired NRAS Q61K, G12D mutation and KRAS Q22K mutation. A high throughput drug screen with kinase inhibitor library from Selleck Chemicals (n = 273 kinases, n = 349 anticancer library) confirmed the selectivity of MEK inhibitors, as well as identified a subset of T790M-ve lines that are sensitive to Aurora kinase inhibitors. Our study highlights the potential for pEGFR to complement T790M status as a selection biomarker, particularly where low tumor cell concentration may yield false negative results. Taken together, we have uncovered potential therapeutically tractable subsets of T790M negative EGFR TKI resistant cell lines. pEGFR may identify a subset of T790M negative tumors that remain driven by and may be an alternate selection biomarker to 2nd/3rd gen TKI. Combinational approaches with MEK and aurora kinase inhibitors could be further explored in T790M negative/ pEGFR negative tumors. Citation Format: Dawn Pingxi Lau, Shivaji Rikka, Hui Sun Leong, Gek San Tan, Eleni G Christodoulou, Sai Sakktee Krisna, Shen Yon Toh, Xue Lin Kwang, Fui Teen Chong, Audrey Ann Liew, Tony Kiat Hon Lim, DasGupta Ramanuj, N Gopalakrishna Iyer, Daniel Shao-Weng Tan. Clonal heterogeneity of EGFR TKI-resistant NSCLC revealed through single-cell dilutional cloning and high-throughput functional screens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2109.

Background: Adiponectin is the most abundant circulating adipokine in human that regulates insulin actions. Association of adiponectin gene variations with type 2 diabetes (T2DM) has been reported albeit predominantly in non-Asian populations. Additionally, proof of variant functionality beyond statistical association is often unavailable. We studied six common (minor allele frequency ?0.05) adiponectin single nucleotide polymorphisms (SNPs) in Singaporean Chinese adults with follow-up functional genetic experiments. Methods: In a case-control study (N=588), genotyping of six common adiponectin haplotype tagging SNPs [-3964A>G(rs822396), +45T>G(rs2241766), 276C>A(rs1501299), 973G>A(rs3774262), 4551G>C(rs1063539) and 5852G>A(rs6444175)] was performed using Taqman genotyping assay. Allele-dependent differential efficiency of mRNA expression was tested with quantitative real time PCR using human subcutaneous and omental adipose tissues. Results: Distributions of genotypes for all SNPs among controls were consistent with Hardy-Weinberg Equilibrium. Single locus, genotyped-based analysis suggested borderline significant (P=0.07) association between an exon- 2 coding-synonymous +45T>G(rs2241766) and T2DM. We demonstrated that the relative mRNA expression of adiponectin gene was ~80% lower among carriers of minor G allele in human subcutaneous adipose tissue (N=43, p G(rs2241766) and T2DM among Singaporean Chinese adults. Functional experiments in both human subcutaneous and omental adipose tissue suggested that polymorphisms in +45T>G(rs2241766) may be associated with differential allelic expression.