DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria.

Mitochondrial DNA (mtDNA) has been reported to contain 5-methylcytosine (5mC) at CpG dinucleotides, as in the nuclear genome, but neither the mechanism generating mtDNA methylation nor its functional significance is known. We now report the presence of 5-hydroxymethylcytosine (5hmC) as well as 5mC in mammalian mtDNA, suggesting that previous studies underestimated the level of cytosine modification in this genome. DNA methyltransferase 1 (DNMT1) translocates to the mitochondria, driven by a mitochondrial targeting sequence located immediately upstream of the commonly accepted translational start site. This targeting sequence is conserved across mammals, and the encoded peptide directs a heterologous protein to the mitochondria. DNMT1 is the only member of the three known catalytically active DNA methyltransferases targeted to the mitochondrion. Mitochondrial DNMT1 (mtDNMT1) binds to mtDNA, proving the presence of mtDNMT1 in the mitochondrial matrix. mtDNMT1 expression is up-regulated by NRF1 and PGC1α, transcription factors that activate expression of nuclear-encoded mitochondrial genes in response to hypoxia, and by loss of p53, a tumor suppressor known to regulate mitochondrial metabolism. Altered mtDNMT1 expression asymmetrically affects expression of transcripts from the heavy and light strands of mtDNA. Hence, mtDNMT1 appears to be responsible for mtDNA cytosine methylation, from which 5hmC is presumed to be derived, and its expression is controlled by factors that regulate mitochondrial function.

Immunoblot Analysis from Single Cells Using Milo™ Single-Cell Western Platform.

In this new era of precision medicine, characterization of single-cell subpopulations to better understand disease etiology is paramount. It is thus an opportune time to explore techniques that allow molecular analysis of single cells and to better understand the basis of pathogenesis of diseases like cancer. Single-cell western blotting is one such method that allows analysis of single cells at the protein level. In contrast to traditional western blotting, which relies heavily on bulk analysis of lysates generated from tissues and is often indicative of the population average, this technique allows analysis of lysates from single-cell subpopulations thereby providing a glimpse into cell heterogeneity. The method entails the use of a chip containing 30 µm thick photoactivated polyacrylamide gel spotted with nearly 6400 microwells. Single cells loaded on the chip are captured in the microwells by passive gravity and are then lysed and electrophoresed using the MILO™ single-cell western platform. This method forgoes the use of transfer of proteins on a PVDF and a nitrocellulose membrane, as performed in traditional western blotting, and all other steps including probing of primary and fluorescent secondary antibodies against the protein of interest are performed directly on the chip. The proteins of interest can then be visualized by scanning a chip with the use of a microarray scanner. The entire procedure can be performed in as less as 4-6 h, and thus this method provides several advantages over traditional western blotting.

CLIP-170S is a microtubule +TIP variant that confers resistance to taxanes by impairing drug-target engagement.

Taxanes are widely used cancer chemotherapeutics. However, intrinsic resistance limits their efficacy without any actionable resistance mechanism. We have discovered a microtubule (MT) plus-end-binding CLIP-170 protein variant, hereafter CLIP-170S, which we found enriched in taxane-resistant cell lines and patient samples. CLIP-170S lacks the first Cap-Gly motif, forms longer comets, and impairs taxane access to its MT luminal binding site. CLIP-170S knockdown reversed taxane resistance in cells and xenografts, whereas its re-expression led to resistance, suggesting causation. Using a computational approach in conjunction with the connectivity map, we unexpectedly discovered that Imatinib was predicted to reverse CLIP-170S-mediated taxane resistance. Indeed, Imatinib treatment selectively depleted CLIP-170S, thus completely reversing taxane resistance. Other RTK inhibitors also depleted CLIP-170S, suggesting a class effect. Herein, we identify CLIP-170S as a clinically prevalent variant that confers taxane resistance, whereas the discovery of Imatinib as a CLIP-170S inhibitor provides novel therapeutic opportunities for future trials.

Microtubule Engagement with Taxane Is Altered in Taxane-Resistant Gastric Cancer.

PURPOSE: Although taxane-based therapy is standard treatment for advanced gastric cancer, a majority of patients exhibit intrinsic resistance to taxanes. Here, we aim to identify the molecular basis of taxane resistance in gastric cancer. EXPERIMENTAL DESIGN: We performed a post hoc analysis of the TAX-325 clinical trial and molecular interrogation of gastric cancer cell lines to assess the benefit of docetaxel in diffuse (DIF-GC) versus intestinal (INT-GC) gastric cancer. We assessed drug-induced microtubule stabilization in gastric cancer cells and in biopsies of patients with gastric cancer treated with taxanes. We performed transcriptome analysis in taxane-treated gastric cancer cells and patients to identify molecular drivers of taxane resistance. RESULTS: Patients with DIF-GC did not derive a clinical benefit from taxane treatment suggesting intrinsic taxane resistance. DIF-GC cell lines displayed intrinsic resistance specific to taxanes because of impaired drug-induced microtubule stabilization, in the absence of tubulin mutations or decreased drug accumulation. Using taxane-treated gastric cancer patient biopsies, we demonstrated that absence of drug-target engagement was correlated with clinical taxane resistance. Taxane-sensitive cell lines displayed faster microtubule dynamics at baseline, implicating proteins that regulate cytoskeletal dynamics in intrinsic taxane resistance. Differential gene expression analysis of untreated and docetaxel-treated gastric cancer lines and patient samples identified kinesins to be associated with taxane sensitivity in vitro and in patient samples. CONCLUSIONS: Our data reveal that taxane resistance is more prevalent in patients with DIF-GC, support assessment of drug-target engagement as an early read-out of taxane clinical efficacy, and encourage the investigation of kinesins and other microtubule-associated proteins as potentially targetable mediators of taxane resistance in gastric cancer.

Multicenter Phase II Study of Cabazitaxel in Advanced Gastroesophageal Cancer: Association of HER2 Expression and M2-Like Tumor-Associated Macrophages with Patient Outcome.

PURPOSE: We examined cabazitaxel, a novel next-generation taxoid, in patients with metastatic gastric cancer in a multicenter phase II study. PATIENTS AND METHODS: Patients who have progressed on one or more prior therapies for locally advanced, unresectable, or metastatic disease were eligible, and prior taxane therapy was allowed. Taxane-naïve and pretreated cohorts were analyzed independently for efficacy. The primary endpoint for both cohorts was progression-free survival (PFS) using RECIST 1.1, using a Simon's two-stage design (10% significance and 80% power) for both cohorts. Comprehensive molecular annotation included whole exome and bulk RNA sequencing. RESULTS: Fifty-three patients enrolled in the taxane-naïve cohort (Arm A) and 23 patients in the prior-taxane cohort (Arm B), from January 8, 2013, to April 8, 2015: median age 61.7 years (range, 35.5-91.8 years), 66% male, 66% Caucasian. The most common adverse events included neutropenia (17% Arm A and 39% Arm B), fatigue/muscle weakness (13%), and hematuria (12%). In Arm A, the 3-month PFS rate was 28% [95% confidence interval (CI), 17%-42%] and did not meet the prespecified efficacy target. The 3-month PFS rate in Arm B was 35% (95% CI, 16%-57%) and surpassed its efficacy target. HER2 amplification or overexpression was associated with improved disease control (P = 0.003), PFS (P = 0.04), and overall survival (P = 0.002). An M2 macrophage signature was also associated with improved survival (P = 0.031). CONCLUSIONS: Cabazitaxel has modest activity in advanced gastric cancer, including in patients previously treated with taxanes. Her2 amplification/overexpression and M2 high macrophage signature are potential biomarkers for taxane efficacy that warrant further evaluation.

A Comparison of Homogenization vs. Enzymatic Lysis for Microbiome Profiling in Clinical Endoscopic Biopsy Tissue Samples.

Identification of the human microbiome has proven to be of utmost importance with the emerging role of bacteria in various physiological and pathological processes. High throughput sequencing strategies have evolved to assess the composition of the microbiome. To identify possible bias that may exist in the processing of tissue for whole genome sequencing (WGS), it is important to evaluate the extraction method on the overall microbial content and composition. Here we compare two different methods of extraction, homogenization vs. enzymatic lysis, on gastric, esophageal and colorectal biopsies and survey the microbial content and composition using WGS and quantitative PCR (qPCR). We examined total bacterial content using universal 16S rDNA qPCR as well as the abundance of three phyla (Actinobacter, Firmicutes, Bacteroidetes) and one genus (Fusobacterium). We found minimal differences between the two extraction methods in the overall community structure. Furthermore, based on our qPCR analysis, neither method demonstrated preferential extraction of any particular clade of bacteria, nor significantly altered the detection of Gram-positive or Gram-negative organisms. However, although the overall microbial composition remained very similar and the most prevalent bacteria could be detected effectively using either method, the precise community structure and microbial abundances between the two methods were different, primarily due to variations in detection of low abundance genus. We also demonstrate that the homogenization extraction method provides higher microbial DNA content and higher read counts from human tissue biopsy samples of the gastrointestinal tract.