Anand D. Jeyasekharan

PARP1-dependent recruitment of KDM4D histone demethylase to DNA damage sites promotes double-strand break repair

Significance Sophisticated DNA damage repair mechanisms are required to fix DNA lesions and preserve the integrity of the genome. This manuscript provides characterization of KDM4D role in promoting the repair of double-strand breaks (DSBs). Our findings show that KDM4D lysine demethylase is swiftly recruited to DNA breakage sites via its C-terminal region in a PARP1-dependent manner. Further, we have uncovered an exciting function of KDM4D in regulating the association of the DNA damage response master kinase, ATM, with chromatin, thus explaining the defective phosphorylation of ATM substrates found in KDM4D-depleted cells. Altogether, this study advances our understanding of the molecular mechanisms that regulate the repair of DSBs, a critical pathway that is essential for maintaining genome integrity. Members of the lysine (K)-specific demethylase 4 (KDM4) A–D family of histone demethylases are dysregulated in several types of cancer. Here, we reveal a previously unrecognized role of KDM4D in the DNA damage response (DDR). We show that the C-terminal region of KDM4D mediates its rapid recruitment to DNA damage sites. Interestingly, this recruitment is independent of the DDR sensor ataxia telangiectasia mutated (ATM), but dependent on poly (ADP-ribose) polymerase 1 (PARP1), which ADP ribosylates KDM4D after damage. We demonstrate that KDM4D is required for efficient phosphorylation of a subset of ATM substrates. We note that KDM4D depletion impairs the DNA damage-induced association of ATM with chromatin, explaining its effect on ATM substrate phosphorylation. Consistent with an upstream role in DDR, KDM4D knockdown disrupts the damage-induced recombinase Rad51 and tumor protein P53 binding protein foci formation. Consequently, the integrity of homology-directed repair and nonhomologous end joining of DNA breaks is impaired in KDM4D-deficient cells. Altogether, our findings implicate KDM4D in DDR, furthering the links between the cancer-relevant networks of epigenetic regulation and genome stability.

LSD1 Overexpression Is Associated with Poor Prognosis in Basal-Like Breast Cancer, and Sensitivity to PARP Inhibition

LSD1, a lysine-specific histone demethylase, is overexpressed in several types of cancers and linked to poor outcomes. In breast cancer, the significance of LSD1 overexpression is not clear. We have performed an in silico analysis to assess the relationship of LSD1 expression to clinical outcome. We demonstrate that LSD1 overexpression is a poor prognostic factor in breast cancer, especially in basal-like breast cancer, a subtype of breast cancer with aggressive clinical features. This link is also observed in samples of triple negative breast cancer. Interestingly, we note that overexpression of LSD1 correlates with down-regulation of BRCA1 in triple negative breast cancer. This phenomenon is also observed in in vitro models of basal-like breast cancer, and is associated with an increased sensitivity to PARP inhibitors. We propose therefore that high expression levels of the demethylase LSD1 is a potential prognostic factor of poor outcome in basal-like breast cancer, and that PARP inhibition may be a therapeutic strategy of interest in this poor prognostic subtype with overexpression of LSD1.

EBV-associated primary nodal T/NK-cell lymphoma shows distinct molecular signature and copy number changes.

The molecular biology of primary nodal T- and NK-cell lymphoma and its relationship with extranodal NK/T-cell lymphoma, nasal type is poorly understood. In this study, we assessed the relationship between nodal and extranodal Epstein-Barr virus-positive T/NK-cell lymphomas using gene expression profiling and copy number aberration analyses. We performed gene expression profiling and copy number aberration analysis on 66 cases of Epstein-Barr virus-associated T/NK-cell lymphoma from nodal and extranodal sites, and correlated the molecular signatures with clinicopathological features. Three distinct molecular clusters were identified with one enriched for nodal presentation and loss of 14q11.2 (TCRA loci). T/NK-cell lymphomas with a nodal presentation (nodal-group) were significantly associated with older age, lack of nasal involvement, and T-cell lineage compared to those with an extranodal presentation (extranodal-group). On multivariate analysis, nodal presentation was an independent factor associated with short survival. Comparing the molecular signatures of the nodal and extranodal groups it was seen that the former was characterized by upregulation of PD-L1 and T-cell-related genes, including CD2 and CD8, and downregulation of CD56, consistent with the CD8+/CD56-immunophenotype. PD-L1 and CD2 protein expression levels were validated using multiplexed immunofluorescence. Interestingly, nodal group lymphomas were associated with 14q11.2 loss which correlated with loss of TCR loci and T-cell origin. Overall, our results suggest that T/NK-cell lymphoma with nodal presentation is distinct and deserves to be classified separately from T/NK-cell lymphoma with extranodal presentation. Upregulation of PD-L1 indicates that it may be possible to use anti-PD1 immunotherapy in this distinctive entity. In addition, loss of 14q11.2 may be a potentially useful diagnostic marker of T-cell lineage.

CEACAM6 is upregulated by Helicobacter pylori CagA and is a biomarker for early gastric cancer

Early detection of gastric cancers saves lives, but remains a diagnostic challenge. In this study, we aimed to identify cell-surface biomarkers of early gastric cancer. We hypothesized that a subset of plasma membrane proteins induced by the Helicobacter pylori oncoprotein CagA will be retained in early gastric cancers through non-oncogene addiction. An inducible system for expression of CagA was used to identify differentially upregulated membrane protein transcripts in vitro. The top hits were then analyzed in gene expression datasets comparing transcriptome of gastric cancer with normal tissue, to focus on markers retained in cancer. Among the transcripts enriched upon CagA induction in vitro, a significant elevation of CEACAM6 was noted in gene expression datasets of gastric cancer. We used quantitative digital immunohistochemistry to measure CEACAM6 protein levels in tissue microarrays of gastric cancer. We demonstrate an increase in CEACAM6 in early gastric cancers, when compared to matched normal tissue, with an AUC of 0.83 for diagnostic validity. Finally, we show that a fluorescently conjugated CEACAM6 antibody binds avidly to freshly resected gastric cancer xenograft samples and can be detected by endoscopy in real time. Together, these results suggest that CEACAM6 upregulation is a cell surface response to H. pylori CagA, and is retained in early gastric cancers. They highlight a novel link between CEACAM6 expression and CagA in gastric cancer, and suggest CEACAM6 to be a promising biomarker to aid with the fluorescent endoscopic diagnosis of early neoplastic lesions in the stomach.

Combination of vaccine-strain measles and mumps virus synergistically kills a wide range of human hematological cancer cells: Special focus on acute myeloid leukemia

Through combining vaccine-derived measles and mumps viruses (MM), we efficiently targeted a wide range of hematopoietic cancer cell lines. MM synergistically killed many cell lines including acute myeloid leukemia (AML) cell lines. Further investigation suggested that enhanced oncolytic effect of MM was due to increased apoptosis induction. In an U937 xenograft AML mouse model, MM displayed greater tumor suppression and prolonged survival. Furthermore, MM efficiently killed blasts from 16 out of 20 AML patients and elicited more efficient killing effect on 11 patients when co-administered with Ara-C. Our results demonstrate that MM is a promising therapeutic candidate for hematological malignancies.

Quantitative Analysis of a Multiplexed Immunofluorescence Panel in T-Cell Lymphoma:

Immunohistochemistry (IHC) provides clinically useful information on protein expression in cancer cells. However, quantification of colocalizing signals using conventional IHC and visual scores is challenging. Here we describe the application of quantitative immunofluorescence in angioimmunoblastic T-cell lymphoma (AITL), a peripheral T-cell lymphoma characterized by cellular heterogeneity that impedes IHC interpretation and quantification. A multiplexed immunofluorescence (IF) panel comprising T- and B-lymphocyte markers along with T-follicular helper (TFH) markers was validated for appropriate cellular localization in sections of benign tonsillar tissue and tested in two samples of AITL, using a Vectra microscope for spectral imaging and InForm software for analysis. We measured the percentage positivity of the TFH markers, BCL6 and PD1, in AITL CD4-positive cells to be approximately 26% and 45%, with 12% coexpressing both markers. The pattern is similar to CD4 cells within the germinal center of normal tonsils and clearly distinct from extragerminal CD4 cells. This study demonstrates the feasibility of automated and quantitative imaging of a multiplexed panel of cellular markers in formalin-fixed, paraffin-embedded tissue sections of a cellularly heterogenous lymphoma. Multiplexed IF allows the simultaneous scoring of markers in malignant and immune cell populations and could potentially increase accuracy for establishment of diagnostic thresholds.

Special Issue: Quantitative Imaging in Life Sciences and Biomedical Research

Seeing is believing. We have evolved with a reliance on our visual system to understand the world around us. This is evident in the vital role of imaging in major discoveries in medicine and biological sciences throughout the past few centuries. But have we reached the limit of what we can learn from simple visual inspection of images? The human eye and brain are good at recognizing patterns, but not as adept at detecting subtle differences in intensity. As we move into an era of big data and high-content experiments, there is also a limit to how many images can be reliably assessed manually in a defined timeframe. Enter quantitative imaging, where the focus is on removing the tedious aspects of imaging and image analysis, enabling scientists to devote time and energy toward using these techniques to answer important biological and medical questions. Quantitative imaging also addresses a key issue of reproducibility in life sciences research. Selected representative images are no longer considered adequate proof of a discovery, because these run the risk of observer bias. The development of algorithms to quantify images has transformed the way microscopic and macroscopic imaging studies are performed and reported, by allowing assessment and quantification from a larger set of samples. Enabling these advances for the use of imaging in medicine and life sciences research requires technological advances in several fields. The quantitative imaging revolution affects multiple steps of the experimental process, requiring advances in technology to facilitate imaging, capture high-content images, and analyze them. In this special issue, SLAS Technology shares examples of studies that provide novel insight into different steps of the quantitative imaging process. Mella et al. report the development of a novel imagingbased biosensor for high-throughput assays of G protein–coupled receptor (GPCR) signaling, with a specific focus on discriminating Ca+-mediated and cyclic adenosine monophosphate (cAMP)-mediated signaling. This is an advance in development of biological tools that aid the process of screening, which may aid the identification of novel GPCR ligands and regulators of signaling in this clinically relevant set of targets. Li et al. focus on the nature of cell culture systems used in high-throughput imaging assays and the development of a novel co-culture system of immortalized endothelial and mesenchymal cell lines to evaluate angiogenesis. Such a system allows the evaluation of compounds that may influence angiogenesis, a key process in development and cancer. Lee et al. take the step of developing robust cell culture systems a step further by addressing the issue of high-throughput screening in three-dimensional (3D) cell culture. The behavior of cells in two-dimensional (2D) cell cultures is known to be different from that in 3D cell culture, which is ostensibly a closer reflection of 3D multicellular organisms. They describe the differences in cell viability screening using 2D and 3D cell culture systems, suggesting a higher accuracy of measurements in 3D culture systems. Given the notoriously poor correlation between in vitro findings and in vivo findings for cytostatic and cytotoxic drugs in oncology, such advances are likely to help bridge the bench-to-bedside gap. Leary et al. further address this issue by describing ratiometric fluorescent assays of a nuclear dye that can be measured by confocal imaging to enable screening approaches and biological experiments in 3D cell culture systems. Although single stains are advantageous in their simplicity, they may not capture adequate information in a high-throughput imaging screening assay or experiment. Rose et al. share a combination of unrelated cellular stains that can be used in quantitative microscopy experiments to facilitate drug screening by increasing the number of composite cellular outcomes that are measured. The proof in the pudding, when it comes to translational research, lies in the ability to quantitatively assess images that are obtained from human samples of disease and health. Ng et al. describe a novel assay to quantitate markers of follicular T-helper cells, within a T-cell lymphoma, using a digital pathology platform. This allows the identification of angio-immunoblastic T-cell lymphoma, a T-lymphoma subtype that is challenging to diagnose for inexperienced histopathologists and has potential clinical diagnostic applicability. Beyond microscopic imaging, quantitation in medicine is vital for radiological images. Wang et al. describe a marker-free 3D tool based on diffusion-weighted magnetic resonance imaging (MRI) to discriminate healthy from fibrotic livers in humans. Quantitative approaches with incorporation of artificial intelligence tools are likely to transform the use of radiological 768816 JLAXXX10.1177/2472630318768816SLAS TECHNOLOGY: Translating Life Sciences InnovationJeyasekharan editorial2018

The Genomics and Molecular Biology of Natural Killer/T-Cell Lymphoma: Opportunities for Translation

Extranodal NK/T-cell lymphoma, nasal type (ENKTL), is an aggressive malignancy with a poor prognosis. While the introduction of L-asparaginase in the treatment of this disease has significantly improved the prognosis, the outcome of patients relapsing after asparaginase-based chemotherapy, which occurs in up to 50% of patients with disseminated disease, remains dismal. There is hence an urgent need for effective targeted therapy especially in the relapsed/refractory setting. Gene expression profiling studies have provided new perspectives on the molecular biology, ontogeny and classification of ENKTL and further identified dysregulated signaling pathways such as Janus associated kinase (/Signal Transducer and activation of transcription (JAK/STAT), Platelet derived growth factor (PDGF), Aurora Kinase and NF-κB, which are under evaluation as therapeutic targets. Copy number analyses have highlighted potential tumor suppressor genes such as PR Domain Zinc Finger Protein 1 (PRDM1) and protein tyrosine phosphatase kappa (PTPRK) while next generation sequencing studies have identified recurrently mutated genes in pro-survival and anti-apoptotic pathways. The discovery of epigenetic dysregulation and aberrant microRNA activity has broadened our understanding of the biology of ENKTL. Importantly, immunotherapy via Programmed Cell Death -1 (PD-1) and Programmed Cell Death Ligand1 (PD-L1) checkpoint signaling inhibition is emerging as an attractive therapeutic strategy in ENKTL. Herein, we present an overview of the molecular biology and genomic landscape of ENKTL with a focus on the most promising translational opportunities.