James Bean

MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib

In human lung adenocarcinomas harboring EGFR mutations, a second-site point mutation that substitutes methionine for threonine at position 790 (T790M) is associated with approximately half of cases of acquired resistance to the EGFR kinase inhibitors, gefitinib and erlotinib. To identify other potential mechanisms that contribute to disease progression, we used array-based comparative genomic hybridization (aCGH) to compare genomic profiles of EGFR mutant tumors from untreated patients with those from patients with acquired resistance. Among three loci demonstrating recurrent copy number alterations (CNAs) specific to the acquired resistance set, one contained the MET proto-oncogene. Collectively, analysis of tumor samples from multiple independent patient cohorts revealed that MET was amplified in tumors from 9 of 43 (21%) patients with acquired resistance but in only two tumors from 62 untreated patients (3%) (P = 0.007, Fishers Exact test). Among 10 resistant tumors from the nine patients with MET amplification, 4 also harbored the EGFRT790M mutation. We also found that an existing EGFR mutant lung adenocarcinoma cell line, NCI-H820, harbors MET amplification in addition to a drug-sensitive EGFR mutation and the T790M change. Growth inhibition studies demonstrate that these cells are resistant to both erlotinib and an irreversible EGFR inhibitor (CL-387,785) but sensitive to a multikinase inhibitor (XL880) with potent activity against MET. Taken together, these data suggest that MET amplification occurs independently of EGFRT790M mutations and that MET may be a clinically relevant therapeutic target for some patients with acquired resistance to gefitinib or erlotinib.

The effects of molecular noise and size control on variability in the budding yeast cell cycle

Molecular noise in gene expression can generate substantial variability in protein concentration. However, its effect on the precision of a natural eukaryotic circuit such as the control of cell cycle remains unclear. We use single-cell imaging of fluorescently labelled budding yeast to measure times from division to budding (G1) and from budding to the next division. The variability in G1 decreases with the square root of the ploidy through a 1N/2N/4N ploidy series, consistent with simple stochastic models for molecular noise. Also, increasing the gene dosage of G1 cyclins decreases the variability in G1. A new single-cell reporter for cell protein content allows us to determine the contribution to temporal G1 variability of deterministic size control (that is, smaller cells extending G1). Cell size control contributes significantly to G1 variability in daughter cells but not in mother cells. However, even in daughters, size-independent noise is the largest quantitative contributor to G1 variability. Exit of the transcriptional repressor Whi5 from the nucleus partitions G1 into two temporally uncorrelated and functionally distinct steps. The first step, which depends on the G1 cyclin gene CLN3, corresponds to noisy size control that extends G1 in small daughters, but is of negligible duration in mothers. The second step, whose variability decreases with increasing CLN2 gene dosage, is similar in mothers and daughters. This analysis decomposes the regulatory dynamics of the Start transition into two independent modules, a size sensing module and a timing module, each of which is predominantly controlled by a different G1 cyclin.

Specific EGFR Mutations Predict Treatment Outcome of Stage IIIB/IV Patients With Chemotherapy-Naive Non–Small-Cell Lung Cancer Receiving First-Line Gefitinib Monotherapy

PURPOSE To explore predictive factors for time to treatment failure (TTF) in chemotherapy-naive non-small-cell lung cancer (NSCLC) patients receiving gefitinib treatment. PATIENTS AND METHODS We designed a phase II study to test gefitinib antitumor efficacy in advanced-stage, chemotherapy-naive NSCLC patients. Patients were treated with gefitinib 250 mg/d. Tumor assessments were performed every 2 months. Responding or stable patients were treated until progression or unacceptable toxicity. All scans were reviewed independently. EGFR exons 18-21 sequence, K-ras exon 2 sequence, and MET gene copy numbers were examined in available samples. Clinical or molecular predictors of TTF were examined by multivariate analysis. RESULTS One hundred six patients were enrolled. Ninety patients had tumor samples for biomarker tests. Overall response rate was 50.9% (95% CI, 41.4% to 60.4%). Median TTF was 5.5 months, and median overall survival (OS) was 22.4 months. The response rate and median TTF of the patients with exon 19 deletion (n = 20) were 95.0% and 8.9 months, for exon 21 L858R mutation (n = 23) were 73.9% and 9.1 month, and for other types of EGFR mutations (N = 12) were 16.7% and 2.3 months, respectively. In multivariate analysis, the presence of EGFR deletion exon 19 or L858R EGFR mutations in adenocarcinoma patients predicted longer TTF. High copy number of MET seemed to correlate with shorter TTF in patients with gefitinib-sensitive activating EGFR mutations. CONCLUSION In this prospective study, EGFR exon 19 deletion or L858R mutations in adenocarcinoma were the best predictors for longer TTF in stage IIIB/IV chemotherapy-naive NSCLC patients receiving first-line gefitinib monotherapy.

Acquired Resistance to Epidermal Growth Factor Receptor Kinase Inhibitors Associated with a Novel T854A Mutation in a Patient with EGFR-Mutant Lung Adenocarcinoma

Purpose: Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene are associated with sensitivity of lung adenocarcinomas to the EGFR tyrosine kinase inhibitors, gefitinib and erlotinib. Acquired drug resistance is frequently associated with a secondary somatic mutation that leads to the substitution of methionine for threonine at position 790 (T790M). We aimed to identify additional second-site alterations associated with acquired resistance. Experimental Design: Tumor samples were obtained from 48 patients with acquired resistance. Tumor cell DNA was analyzed for EGFR kinase domain mutations. Molecular analyses were then done to characterize the biological properties of a novel mutant EGFR allele. Results: A previously unreported mutation in exon 21 of EGFR, which leads to substitution of alanine for threonine at position 854 (T854A), was identified in one patient with a drug-sensitive EGFR L858R–mutant lung adenocarcinoma after long-term treatment with tyrosine kinase inhibitors. The T854A mutation was not detected in a pretreatment tumor sample. The crystal structure analyses of EGFR suggest that the T854 side chain is within contact distance of gefitinib and erlotinib. Surrogate kinase assays show that the EGFR T854A mutation abrogates the inhibition of tyrosine phosphorylation by erlotinib. Such resistance seems to be overcome by a new irreversible dual EGFR/HER2 inhibitor, BIBW 2992. Conclusions: The T854A mutation is the second reported second-site acquired resistance mutation that is within contact distance of gefitinib and erlotinib. These data suggest that acquired resistance to ATP-mimetic EGFR kinase inhibitors may often be associated with amino acid substitutions that alter drug contact residues in the EGFR ATP-binding pocket.

Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA

RbpA and CarD are essential transcription regulators in mycobacteria. Mechanistic analyses of promoter open complex (RPo) formation establish that RbpA and CarD cooperatively stimulate formation of an intermediate (RP2) leading to RPo; formation of RP2 is likely a bottleneck step at the majority of mycobacterial promoters. Once RPo forms, CarD also disfavors its isomerization back to RP2. We determined a 2.76 Å-resolution crystal structure of a mycobacterial transcription initiation complex (TIC) with RbpA as well as a CarD/RbpA/TIC model. Both CarD and RbpA bind near the upstream edge of the −10 element where they likely facilitate DNA bending and impede transcription bubble collapse. In vivo studies demonstrate the essential role of RbpA, show the effects of RbpA truncations on transcription and cell physiology, and indicate additional functions for RbpA not evident in vitro. This work provides a framework to understand the control of mycobacterial transcription by RbpA and CarD. DOI: http://dx.doi.org/10.7554/eLife.22520.001

Antibiotic treatment for Tuberculosis induces a profound dysbiosis of the microbiome that persists long after therapy is completed

Mycobacterium tuberculosis, the cause of Tuberculosis (TB), infects one third of the world’s population and causes substantial mortality worldwide. In its shortest format, treatment of TB requires six months of multidrug therapy with a mixture of broad spectrum and mycobacterial specific antibiotics, and treatment of multidrug resistant TB is longer. The widespread use of this regimen makes this one of the largest exposures of humans to antimicrobials, yet the effects of TB treatment on intestinal microbiome composition and long-term stability are unknown. We compared the microbiome composition, assessed by both 16S rDNA and metagenomic DNA sequencing, of TB cases during antimycobacterial treatment and following cure by 6 months of antibiotics. TB treatment does not perturb overall diversity, but nonetheless dramatically depletes multiple immunologically significant commensal bacteria. The microbiomic perturbation of TB therapy can persist for at least 1.2 years, indicating that the effects of TB treatment are long lasting. These results demonstrate that TB treatment has dramatic effects on the intestinal microbiome and highlight unexpected durable consequences of treatment for the world’s most common infection on human ecology.

Altered Transcriptional Control Networks with Trans-Differentiation of Isogenic Mutant-KRas NSCLC Models.

Background: The capacity of cancer cells to undergo epithelial mesenchymal trans-differentiation has been implicated as a factor driving metastasis, through the acquisition of enhanced migratory/invasive cell programs and the engagement of anti-apoptotic mechanisms promoting drug and radiation resistance. Our aim was to define molecular signaling changes associated with mesenchymal trans-differentiation in two KRas mutant NSCLC models. We focused on central transcription and epigenetic regulators predicted to be important for mesenchymal cell survival. Experimental design: We have modeled trans-differentiation and cancer stemness in inducible isogenic mutant-KRas H358 and A549 non-small cell lung cell backgrounds. As expected, our models show mesenchymal-like tumor cells acquire novel mechanisms of cellular signaling not apparent in their epithelial counterparts. We employed large-scale quantitative phosphoproteomic, proteomic, protein–protein interaction, RNA-Seq, and network function prediction approaches to dissect the molecular events associated with the establishment and maintenance of the mesenchymal state. Results: Gene-set enrichment and pathway prediction indicated BMI1, KDM5B, RUNX2, MYC/MAX, NFκB, LEF1, and HIF1 target networks were significantly enriched in the trans-differentiation of H358 and A549 NSCLC models. Physical overlaps between multiple networks implicate NR4A1 as an overlapping control between TCF and NFκB pathways. Enrichment correlations also indicated marked decrease in cell cycling, which occurred early in the EMT process. RNA abundance time course studies also indicated early expression of epigenetic and chromatin regulators within 8–24 h, including CITED4, RUNX3, CMBX1, and SIRT4. Conclusion: Multiple transcription and epigenetic pathways where altered between epithelial and mesenchymal tumor cell states, notably the polycomb repressive complex-1, HP1γ, and BAF/Swi-Snf. Network analysis suggests redundancy in the activation and inhibition of pathway regulators, notably factors controlling epithelial cell state. Through large-scale transcriptional and epigenetic cell reprograming, mesenchymal trans-differentiation can promote diversification of signaling networks potentially important in resistance to cancer therapies.

Mucosal-associated invariant and γδ T cell subsets respond to initial Mycobacterium tuberculosis infection

Innate immune responses that control early Mtb infection are poorly understood, but understanding these responses may inform vaccination and immunotherapy strategies. Innate T cells that respond to conserved bacterial ligands such as mucosal-associated invariant T (MAIT) and γδ T cells are prime candidates to mediate these early innate responses but have not been examined in subjects who have been recently exposed to Mtb. We recruited a cohort living in the same household with an active tuberculosis (TB) case and examined the abundance and functional phenotypes of 3 innate T cell populations reactive to M. tuberculosis: γδ T, invariant NK T (iNKT), and MAIT cells. Both MAIT and γδ T cells from subjects with Mtb exposure display ex vivo phenotypes consistent with recent activation. However, both MAIT and γδ T cell subsets have distinct response profiles, with CD4+ MAIT and γδ T cells accumulating after infection. Examination of exposed but uninfected contacts demonstrates that resistance to initial infection is accompanied by robust MAIT cell CD25 expression and granzyme B production coupled with a depressed CD69 and IFNγ response. Finally, we demonstrate that MAIT cell abundance and function correlate with the abundance of specific gut microbes, suggesting that responses to initial infection may be modulated by the intestinal microbiome.

Blood transcriptomic markers of Mycobacterium tuberculosis load in sputum

BACKGROUND Peripheral blood transcriptome signatures that distinguish active pulmonary tuberculosis (TB) from control groups have been reported, but correlations of these signatures with sputum mycobacterial load are incompletely defined. METHODS We assessed the performance of published TB transcriptomic signatures in Haiti, and identified transcriptomic biomarkers of TB bacterial load in sputum as measured by Xpert® MTB/RIF molecular testing. People in Port au Prince, Haiti, with untreated pulmonary TB (n = 51) formed the study cohort: 19 people with low and 32 with high sputum Mycobacterium tuberculosis load. Peripheral whole blood transcriptomes were generated using RNA sequencing. RESULTS Twenty of the differentially expressed transcripts in TB vs. no TB were differentially expressed in people with low vs. high sputum mycobacterial loads. The difference between low and high bacterial load groups was independent of radiographic severity. In a published data set of transcriptomic response to anti-tuberculosis treatment, this 20-gene subset was more treatment-responsive at 6 months than the full active TB signature. CONCLUSION We identified genes whose transcript levels in the blood distinguish active TB with high vs. low M. tuberculosis loads in the sputum. These transcripts may reveal mechanisms of mycobacterial control of M. tuberculosis during active infection, as well as identifying potential biomarkers for bacterial response to anti-tuberculosis treatment.

Mass Spectrometric Identification of Urinary Biomarkers of Pulmonary Tuberculosis

Background Tuberculosis (TB) is the leading infectious cause of death worldwide. A major barrier to control of the pandemic is a lack of clinical biomarkers with the ability to distinguish active TB from healthy and sick controls and potential for development into point-of-care diagnostics. Methods We conducted a prospective case control study to identify candidate urine-based diagnostic biomarkers of active pulmonary TB (discovery cohort) and obtained a separate blinded “validation” cohort of confirmed cases of active pulmonary TB and controls with non-tuberculous pulmonary disease for validation. Clean-catch urine samples were collected and analyzed using high performance liquid chromatography-coupled time-of-flight mass spectrometry. Results We discovered ten molecules from the discovery cohort with receiver-operator characteristic (ROC) area-under-the-curve (AUC) values >85%. These 10 molecules also significantly decreased after 60 days of treatment in a subset of 20 participants followed over time. Of these, a specific combination of diacetylspermine, neopterin, sialic acid, and N-acetylhexosamine exhibited ROC AUCs >80% in a blinded validation cohort of participants with active TB and non-tuberculous pulmonary disease. Conclusion Urinary levels of diacetylspermine, neopterin, sialic acid, and N-acetylhexosamine distinguished patients with tuberculosis from healthy controls and patients with non-tuberculous pulmonary diseases, providing a potential noninvasive biosignature of active TB. Funding This study was funded by Weill Cornell Medicine, the National Institute of Allergy and Infectious Diseases, the Clinical and Translational Science Center at Weill Cornell, the NIH Fogarty International Center grants, and the NIH Tuberculosis Research Unit (Tri-I TBRU).