Nathan T. Martin

ATM–Dependent MiR-335 Targets CtIP and Modulates the DNA Damage Response

ATM plays a critical role in cellular responses to DNA double-strand breaks (DSBs). We describe a new ATM–mediated DSB–induced DNA damage response pathway involving microRNA (miRNA): irradiation (IR)-induced DSBs activate ATM, which leads to the downregulation of miR-335, a miRNA that targets CtIP, which is an important trigger of DNA end resection in homologous recombination repair (HRR). We demonstrate that CREB is responsible for a large portion of miR-335 expression by binding to the promoter region of miR-335. CREB binding is greatly reduced after IR, corroborating with previous studies that IR-activated ATM phosphorylates CREB to reduce its transcription activity. Overexpression of miR-335 in HeLa cells resulted in reduced CtIP levels and post-IR colony survival and BRCA1 foci formation. Further, in two patient-derived lymphoblastoid cell lines with decreased post-IR colony survival, a “radiosensitive” phenotype, we demonstrated elevated miR-335 expression, reduced CtIP levels, and reduced BRCA1 foci formation. Colony survival, BRCA1 foci, and CtIP levels were partially rescued by miRNA antisense AMO-miR-335 treatment. Taken together, these findings strongly suggest that an ATM–dependent CREB–miR-335–CtIP axis influences the selection of HRR for repair of certain DSB lesions.

SMRT compounds abrogate cellular phenotypes of ataxia telangiectasia in neural derivatives of patient-specific hiPSCs

Ataxia telangiectasia is a devastating neurodegenerative disease caused primarily by loss of function mutations in ATM, a hierarchical DNA repair gene and tumour suppressor. So far, murine models of ataxia telangiectasia have failed to accurately recapitulate many aspects of the disease, most notably, the progressive cerebellar ataxia. Here we present a model of human ataxia telangiectasia using induced pluripotent stem cells, and show that small molecule read-through compounds, designed to induce read-through of mRNA around premature termination codons, restore ATM activity and improve the response to DNA damage. This platform allows for efficient screening of novel compounds, identification of target and off-target effects, and preclinical testing on relevant cell types for the pathogenic dissection and treatment of ataxia telangiectasia.

Comprehensive profiling of radiosensitive human cell lines with DNA damage response assays identifies the neutral comet assay as a potential surrogate for clonogenic survival.

In an effort to explore the possible causes of human radiosensitivity and identify more rapid assays for cellular radiosensitivity, we interrogated a set of assays that evaluate cellular functions involved in recognition and repair of DNA double-strand breaks: (1) neutral comet assay, (2) radiation-induced γ-H2AX focus formation, (3) the temporal kinetics of structural maintenance of chromosomes 1 phosphorylation, (4) intra-S-phase checkpoint integrity, and (5) mitochondrial respiration. We characterized a unique panel of 19 “radiosensitive” human lymphoblastoid cell lines from individuals with undiagnosed diseases suggestive of a DNA repair disorder. Radiosensitivity was defined by reduced cellular survival using a clonogenic survival assay. Each assay identified cell lines with defects in DNA damage response functions. The highest concordance rate observed, 89% (17/19), was between an abnormal neutral comet assay and reduced survival by the colony survival assay. Our data also suggested that the neutral comet assay would be a more rapid surrogate for analyzing DNA repair/processing disorders.

The Gold Standard Paradox in Digital Image Analysis: Manual Versus Automated Scoring as Ground Truth

CONTEXT - Novel therapeutics often target complex cellular mechanisms. Increasingly, quantitative methods like digital tissue image analysis (tIA) are required to evaluate correspondingly complex biomarkers to elucidate subtle phenotypes that can inform treatment decisions with these targeted therapies. These tIA systems need a gold standard, or reference method, to establish analytical validity. Conventional, subjective histopathologic scores assigned by an experienced pathologist are the gold standard in anatomic pathology and are an attractive reference method. The pathologists score can establish the ground truth to assess a tIA solutions analytical performance. The paradox of this validation strategy, however, is that tIA is often used to assist pathologists to score complex biomarkers because it is more objective and reproducible than manual evaluation alone by overcoming known biases in a humans visual evaluation of tissue, and because it can generate endpoints that cannot be generated by a human observer. OBJECTIVE - To discuss common visual and cognitive traps known in traditional pathology-based scoring paradigms that may impact characterization of tIA-assisted scoring accuracy, sensitivity, and specificity. DATA SOURCES - This manuscript reviews the current literature from the past decades available for traditional subjective pathology scoring paradigms and known cognitive and visual traps relevant to these scoring paradigms. CONCLUSIONS - Awareness of the gold standard paradox is necessary when using traditional pathologist scores to analytically validate a tIA tool because image analysis is used specifically to overcome known sources of bias in visual assessment of tissue sections.

Ziram, a pesticide associated with increased risk for Parkinson's disease, differentially affects the presynaptic function of aminergic and glutamatergic nerve terminals at the Drosophila neuromuscular junction

Multiple populations of aminergic neurons are affected in Parkinsons disease (PD), with serotonergic and noradrenergic loci responsible for some non-motor symptoms. Environmental toxins, such as the dithiocarbamate fungicide ziram, significantly increase the risk of developing PD and the attendant spectrum of both motor and non-motor symptoms. The mechanisms by which ziram and other environmental toxins increase the risk of PD, and the potential effects of these toxins on aminergic neurons, remain unclear. To determine the relative effects of ziram on the synaptic function of aminergic versus non-aminergic neurons, we used live-imaging at the Drosophila melanogaster larval neuromuscular junction (NMJ). In contrast to nearly all other studies of this model synapse, we imaged presynaptic function at both glutamatergic Type Ib and aminergic Type II boutons, the latter responsible for storage and release of octopamine, the invertebrate equivalent of noradrenalin. To quantify the kinetics of exo- and endo-cytosis, we employed an acid-sensitive form of GFP fused to the Drosophila vesicular monoamine transporter (DVMAT-pHluorin). Additional genetic probes were used to visualize intracellular calcium flux (GCaMP) and voltage changes (ArcLight). We find that at glutamatergic Type Ib terminals, exposure to ziram increases exocytosis and inhibits endocytosis. By contrast, at octopaminergic Type II terminals, ziram has no detectable effect on exocytosis and dramatically inhibits endocytosis. In contrast to other reports on the neuronal effects of ziram, these effects do not appear to result from perturbation of the Ubiquitin Proteasome System (UPS) or calcium homeostasis. Unexpectedly, ziram also caused spontaneous and synchronized bursts of calcium influx (measured by GCaMP) and electrical activity (measured by ArcLight) at aminergic Type II, but not glutamatergic Type Ib, nerve terminals. These events are sensitive to both tetrodotoxin and cadmium chloride, and thus appear to represent spontaneous depolarizations followed by calcium influx into Type II terminals. We speculate that the differential effects of ziram on Type II versus Type Ib terminals may be relevant to the specific sensitivity of aminergic neurons in PD, and suggest that changes in neuronal excitability could contribute to the increased risk for PD caused by exposure to ziram. We also suggest that the fly NMJ will be useful to explore the synaptic effects of other pesticides associated with an increased risk of PD.

Abstract 4042: Quantifying immune oncology markers across multiple tissue sections with digital image analysis

Immuno-oncology (IO) approaches requires an understanding of the types of immune cells present in the tumor microenvironment (TME) as well as the precise localization of these immune cells. However, the acquisition of spatial IO information is technically challenging, due to the requirement for multiplex labeling of immune cells and the need to categorize their location and biomarker content simultaneously. Additionally, the multiplex biomarker panel must be engineered in advance based on a priori assumptions about the correct marker combinations and their location (such as the tumor epithelial nests, TME, or specifically the tumor-stroma interface). This limits the ability to implement novel, scientifically driven assessments into an existing clinical trial which already has defined immunohistochemistry endpoints. To meet the needs of IO clinical trials, Flagship Biosciences has developed a proprietary image analysis platform, FACTS (Feature Analysis on Complementary Tissue Sections), to deduce both the necessary multiplex staining information and the spatial context of IO markers based on the utilization of existing monochrome or multiplex stained slides from a clinical trial. In this study, we demonstrate the utility of this approach to deliver biologically relevant endpoints important for IO clinical trials. First, we performed single IO biomarker staining for CD4, CD8 and FoxP3 in colorectal cancer patient samples and developed a novel image analysis approach that allowed for the accurate quantification of multiple IO markers within specific margins of the tumor microenvironment across multiple tissue sections. For each biomarker the positive cell populations were binned based on distance from the tumor-TME boundary (i.e.% positive cells within 100μM, 200μM or 500μM of the tumor-TME boundary). Next, we used computational alignment for evaluating the co-localization of multiple immune cell biomarkers from serial sections with single stains for CD4, CD8 or FoxP3. We demonstrate that the information extracted from multiple single stained serial sections can be used to generate co-localization information for multiple IO markers in a given patient sample. Lastly, we developed multiplex chromogenic assays for these same markers, analyzed the multiplex-stained slides with image analysis, and derived the same analysis endpoints for the multiplex slides to the digitally aligned individually labeled sections. The study found that similar interpretation of the inflammatory landscape was possible with multiplex-stained slides and digital alignment of monoplex-stained slides. In summary, this study demonstrated a novel image analysis approach that allows for quantification of IO markers utilizing clinically relevant wet assay technologies and existing IHC stained slides derived from an immune-oncology clinical trial. Citation Format: Ciara A. Martin, Joshua Black, Nathan T. Martin, Logan Cerkovnik, Jasmeet Bajwa, Kristin Wilson, Daniel Rudmann, Carsten Schnatwinkel, AJ Milici. Quantifying immune oncology markers across multiple tissue sections with digital image analysis. [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 4042.