Novel Small Molecule ROCK2 Inhibitor GNS-3595 Attenuates Pulmonary Fibrosis in Preclinical Studies.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that leads to respiratory decline due to scarring and thickening of lung tissues. Multiple pathways contribute to the fibrotic process in this disease, such as inflammation, epithelial to mesenchymal transition and oxidative stress. The RhoA/ROCK signaling pathway is a key regulator of profibrotic signaling, as it affects the organization of actin-myosin and the remodeling of the extracellular matrix. ROCK1/2, a downstream effector of RhoA, is overexpressed in IPF patients and is a promising target for IPF therapy. However, due to hypotensive side effects of ROCK1/2 inhibitors, selective ROCK2 compounds are being explored. In this study, we report the discovery of GNS-3595, a potent and selective ROCK2 inhibitor that has ~80-fold selectivity over ROCK1 at physiological concentrations of ATP. GNS-3595 effectively inhibited ROCK2-mediated phosphorylation of myosin light chain (p-MLC) and reduced the expression of fibrosis-related proteins, such as collagen, fibronectin, and alpha-smooth muscle actin (α-SMA) in various in vitro cellular models. GNS-3595 also prevented transforming growth factor beta (TGF-ß)-induced fibroblast-to-myofibroblast transition (FMT). Additionally, in a bleomycin-induced mouse model of pulmonary fibrosis, therapeutic exposure to GNS-3595, suppressed lung fibrosis, stabilized body weight loss, and prevented fibrosis-induced lung weight gain. Transcriptome and protein expression analysis from lung tissues showed that GNS-3595 can revert the fibrosis-related gene expression induced by bleomycin. These results indicate that GNS-3595 is a highly potent, selective, and orally active ROCK2 inhibitor with promising therapeutic efficacy against pulmonary fibrosis.

Combinatorial ixazomib and belinostat therapy induces NFE2L2-dependent apoptosis in Hodgkin and T-cell lymphoma.

Ixazomib activity and transcriptomic analyses previously established in T cell (TCL) and Hodgkin (HL) lymphoma models predicted synergistic activity for histone deacetylase (HDAC) inhibitory combination. In this present study, we determined the mechanistic basis for ixazomib combination with the HDAC inhibitor, belinostat, in HL and TCL cells lines (ixazomib-sensitive/resistant clones) and primary tumour cells. In ixazomib-treated TCL and HL cells, transient inhibition followed by full recovery of proteasomal activity observed was accompanied by induction of proteasomal gene expression with NFE2L2 (also termed NRF2) as a prominent upstream regulator. Downregulation of both NFE2L2 and proteasomal gene expression (validated by quantitative real time polymerase chain reaction) occurred with belinostat treatment in Jurkat and L428 cells. In addition, CRISPR/Cas9 mediated knockdown of NFE2L2 in Jurkat cells resulted in a significant decrease in cell viability with ixazomib compared with untreated control cells. Using transcriptomic and proteasomal activity evaluation of ixazomib, belinostat, or ixazomib + belinostat treated cells, we observed that NFE2L2, proteasome gene expression and functional recovery were abrogated by ixazomib + belinostat combination, resulting in synergistic drug activity in ixazomib-sensitive and -resistant cell lines and primary cells. Altogether, these results suggest that the synergistic activity of ixazomib + belinostat is mediated via inhibition NFE2L2-dependent proteasomal recovery and extended proteasomal inhibition culminating in increased cell death.

A network of conserved damage survival pathways revealed by a genomic RNAi screen.

Damage initiates a pleiotropic cellular response aimed at cellular survival when appropriate. To identify genes required for damage survival, we used a cell-based RNAi screen against the Drosophila genome and the alkylating agent methyl methanesulphonate (MMS). Similar studies performed in other model organisms report that damage response may involve pleiotropic cellular processes other than the central DNA repair components, yet an intuitive systems level view of the cellular components required for damage survival, their interrelationship, and contextual importance has been lacking. Further, by comparing data from different model organisms, identification of conserved and presumably core survival components should be forthcoming. We identified 307 genes, representing 13 signaling, metabolic, or enzymatic pathways, affecting cellular survival of MMS-induced damage. As expected, the majority of these pathways are involved in DNA repair; however, several pathways with more diverse biological functions were also identified, including the TOR pathway, transcription, translation, proteasome, glutathione synthesis, ATP synthesis, and Notch signaling, and these were equally important in damage survival. Comparison with genomic screen data from Saccharomyces cerevisiae revealed no overlap enrichment of individual genes between the species, but a conservation of the pathways. To demonstrate the functional conservation of pathways, five were tested in Drosophila and mouse cells, with each pathway responding to alkylation damage in both species. Using the protein interactome, a significant level of connectivity was observed between Drosophila MMS survival proteins, suggesting a higher order relationship. This connectivity was dramatically improved by incorporating the components of the 13 identified pathways within the network. Grouping proteins into "pathway nodes" qualitatively improved the interactome organization, revealing a highly organized "MMS survival network." We conclude that identification of pathways can facilitate comparative biology analysis when direct gene/orthologue comparisons fail. A biologically intuitive, highly interconnected MMS survival network was revealed after we incorporated pathway data in our interactome analysis.

Scaffold-mediated switching of lymphoma metabolism in culture.

BACKGROUND: Diffuse large B cell lymphoma (DLBCL) is an aggressive subtype of non-Hodgkin lymphoma (NHL) and accounts for about a third of all NHL cases. A significant proportion (~40%) of treated DLBCL patients develop refractory or relapsed disease due to drug resistance which can be attributed to metabolomic and genetic variations amongst diverse DLBCL subtypes. An assay platform that reproduces metabolic patterns of DLBCL in vivo could serve as a useful model for DLBCL. METHODS: This report investigated metabolic functions in 2D and 3D cell cultures using parental and drug-resistant DLBCL cell lines as compared to patient biopsy tissue. RESULTS: A 3D culture model controlled the proliferation of parental and drug-resistant DLBCL cell lines, SUDHL-10, SUDHL-10 RR (rituximab resistant), and SUDHL-10 OR (obinutuzumab resistant), as well as retained differential sensitivity to CHOP. The results from metabolic profiling and isotope tracer studies with D-glucose-13C6 indicated metabolic switching in 3D culture when compared with a 2D environment. Analysis of DLBCL patient tumor tissue revealed that the metabolic changes in 3D grown cells were shifted towards that of clinical specimens. CONCLUSION: 3D culture restrained DLBCL cell line growth and modulated metabolic pathways that trend towards the biological characteristics of patient tumors. Counter-intuitively, this research thereby contends that 3D matrices can be a tool to control tumor function towards a slower growing and metabolically dormant state that better reflects in vivo tumor physiology.

An Analysis of Transcriptomic Burden Identifies Biological Progression Roadmaps for Hematological Malignancies and Solid Tumors.

Biological paths of tumor progression are difficult to predict without time-series data. Using median shift and abacus transformation in the analysis of RNA sequencing data sets, natural patient stratifications were found based on their transcriptomic burden (TcB). Using gene-behavior analysis, TcB groups were evaluated further to discover biological courses of tumor progression. We found that solid tumors and hematological malignancies (n = 4179) share conserved biological patterns, and biological network complexity decreases at increasing TcB levels. An analysis of gene expression datasets including pediatric leukemia patients revealed TcB patterns with biological directionality and survival implications. A prospective interventional study with PI3K targeted therapy in canine lymphomas proved that directional biological responses are dynamic. To conclude, TcB-enriched biological mechanisms detected the existence of biological trajectories within tumors. Using this prognostic informative novel informatics method, which can be applied to tumor transcriptomes and progressive diseases inspires the design of progression-specific therapeutic approaches.

Perspectives on mechanistic implications of ROS inducers for targeting viral infections.

In this perspective, we propose to leverage reactive oxygen species (ROS) induction as a potential therapeutic measure against viral infections. Our rationale for targeting RNA viral infections by pro-oxidants is routed on the mechanistic hypothesis that ROS based treatment paradigm could impair RNA integrity faster than the other macromolecules. Though antiviral drugs with antioxidant properties confer potential abilities for preventing viral entry, those with pro-oxidant properties could induce the degradation of nascent viral RNA within the host cells, as RNAs are highly prone to ROS mediated degradation than DNA/proteins. We have previously established that Plumbagin is a highly potent ROS inducer, which acts through shifting of the host redox potential. Besides, it has been reported that Plumbagin treatment has the potential for interrupting viral RNA replication within the host cells. Since the on-going Corona Virus Disease - 2019 (COVID-19) global pandemic mediated by Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) exhibits high infectivity, the development of appropriate antiviral therapeutic strategies remains to be an urgent unmet race against time. Therefore, additional experimental validation is warranted to determine the appropriateness of repurposable drug candidates, possibly ROS inducers, for fighting the pandemic which could lead to saving many lives from being lost to COVID-19.

Oncogenic Integration of Nucleotide Metabolism via Fatty Acid Synthase in Non-Hodgkin Lymphoma.

Metabolic dysfunctions enabling increased nucleotide biosynthesis are necessary for supporting malignant proliferation. Our investigations indicate that upregulation of fatty acid synthase (FASN) and de novo lipogenesis, commonly observed in many cancers, are associated with nucleotide metabolic dysfunction in lymphoma. The results from our experiments showed that ribonucleotide and deoxyribonucleotide pool depletion, suppression of global RNA/DNA synthesis, and cell cycle inhibition occurred in the presence of FASN inhibition. Subsequently, we observed that FASN inhibition caused metabolic blockade in the rate-limiting step of the oxidative branch of the pentose phosphate pathway (oxPPP) catalyzed by phosphogluconate dehydrogenase (PGDH). Furthermore, we determined that FASN inhibitor treatment resulted in NADPH accumulation and inhibition of PGDH enzyme activity. NADPH is a cofactor utilized by FASN, also a known allosteric inhibitor of PGDH. Through cell-free enzyme assays consisting of FASN and PGDH, we delineated that the PGDH-catalyzed ribulose-5-phosphate synthesis is enhanced in the presence of FASN and is suppressed by increasing concentrations of NADPH. Additionally, we observed that FASN and PGDH were colocalized in the cytosol. The results from these experiments led us to conclude that NADP-NADPH turnover and the reciprocal stimulation of FASN and PGDH catalysis are involved in promoting oxPPP and nucleotide biosynthesis in lymphoma. Finally, a transcriptomic analysis of non-Hodgkin's lymphoma (n = 624) revealed the increased expression of genes associated with metabolic functions interlinked with oxPPP, while the expression of genes participating in oxPPP remained unaltered. Together we conclude that FASN-PGDH enzymatic interactions are involved in enabling oxPPP and nucleotide metabolic dysfunction in lymphoma tumors.

Interaction kinetics with transcriptomic and secretory responses of CD19-CAR natural killer-cell therapy in CD20 resistant non-hodgkin lymphoma.

We investigated the cytolytic and mechanistic activity of anti-CD19 chimeric antigen receptor natural killer (CD19.CAR.NK92) therapy in lymphoma cell lines (diffuse large B-cell, follicular, and Burkitt lymphoma), including rituximab- and obinutuzumab-resistant cells, patient-derived cells, and a human xenograft model. CD19.CAR.NK92 therapy significantly increased cytolytic activity at E:T ratios (1:1-10:1) via LDH release and prominent induction of apoptosis in all cell lines, including in anti-CD20 resistant lymphoma cells. The kinetics of CD19.CAR.NK92 cell death measured via droplet-based single cell microfluidics analysis showed that most lymphoma cells were killed by single contact, with anti-CD20 resistant cell lines requiring significantly longer contact duration with NK cells. In addition, systems biology transcriptomic analyses of flow-sorted lymphoma cells co-cultured with CD19.CAR.NK92 revealed conserved activation of IFNγ signaling, execution of apoptosis, ligand binding, and immunoregulatory and chemokine signaling pathways. Furthermore, a 92-plex cytokine panel analysis showed increased secretion of granzymes, increased secretion of FASL, CCL3, and IL10 in anti-CD20 resistant SUDHL4 cells with induction of genes relevant to mTOR and G2/M checkpoint activation, which were noted in all anti-CD20 resistant cells co-cultured with CD19.CAR.NK92 cells. Collectively, CD19.CAR.NK92 was associated with potent anti-lymphoma activity across a host of sensitive and resistant lymphoma cells that involved distinct immuno-biologic mechanisms of cell death.

Microfluidic assembly of hydrogel-based immunogenic tumor spheroids for evaluation of anticancer therapies and biomarker release.

Diffuse large B cell lymphoma (DLBCL), the most common subtype of Non-Hodgkin lymphoma, exhibits pathologic heterogeneity and a dynamic immunogenic tumor microenvironment (TME). However, the lack of preclinical in vitro models of DLBCL TME hinders optimal therapeutic screening. This study describes the development of an integrated droplet microfluidics-based platform for high-throughput generation of immunogenic DLBCL spheroids. The spheroids consist of three cell types (cancer, fibroblast and lymphocytes) in a novel hydrogel combination of alginate and puramatrix, which promoted cell adhesion and aggregation. This system facilitates dynamic analysis of cellular interaction, proliferation and therapeutic efficacy via spatiotemporal monitoring and secretome profiling. The immunomodulatory drug lenalidomide had direct anti-proliferative effect on activated B-cell like DLBCL spheroids and reduced several cytokines and other markers (e.g., CCL2, CCL3, CCL4, CD137 and ANG-1 levels) compared with untreated spheroids. Collectively, this novel spheroid platform will enable high-throughput anti-cancer therapeutic screening in a semi-automated manner.

Identification of Circulating Serum Multi-MicroRNA Signatures in Human DLBCL Models.

There remains a need to identify new sensitive diagnostic and predictive blood-based platforms in lymphoma. We previously discovered a novel circulating microRNA (miRNA) signature in a Smurf2-deficient mouse model that spontaneously develops diffuse large B-cell lymphoma (DLBCL). Herein, we investigated this 10-miRNA signature (miR-15a, let-7c, let-7b, miR-27a, miR-10b, miR-18a, miR-497, miR-130a, miR24, and miR-155) in human lymphoma cell lines, mice engrafted with patient-derived xenografts (PDXs), and DLBCL patient serum samples leveraging systems biology analyses and droplet digital PCR (ddPCR) technology. Overall, 90% of the miRNAs were enriched in PDX DLBCL models and human lymphoma cell lines. Circulating miRNAs from the serum of 86 DLBCL patients were significantly increased compared with healthy controls and had similar patterns to the murine models. Strikingly, miRNAs were identified up to 27-fold higher levels in the serum of PDX-bearing mice and human patients compared with lymphoma cell lysates, suggesting a concentration of these factors over time within sera. Using cut-points from recursive partitioning analysis, we derived a 5-miRNA signature (let-7b, let-7c, miR-18a, miR-24, and miR-15a) with a classification rate of 91% for serum from patients with DLBCL versus normal controls. In addition, higher levels of circulating let-7b miRNA were associated with more advanced stage disease (i.e., III-IV vs. I-II) in DLBCL patients and higher levels of miR-27a and miR-24 were associated with MYC rearrangement. Taken together, circulating multi-miRNAs were readily detectable in pre-clinical cell line and human lymphoma models as well as in DLBCL patients where they appeared to distinguish clinico-pathologic subtypes and disease features.

An analysis of normalization methods for Drosophila RNAi genomic screens and development of a robust validation scheme.

Genome-wide RNA interference (RNAi) screening allows investigation of the role of individual genes in a process of choice. Most RNAi screens identify a large number of genes with a continuous gradient in the assessed phenotype. Screeners must decide whether to examine genes with the most robust phenotype or the full gradient of genes that cause an effect and how to identify candidate genes. The authors have used RNAi in Drosophila cells to examine viability in a 384-well plate format and compare 2 screens, untreated control and treatment. They compare multiple normalization methods, which take advantage of different features within the data, including quantile normalization, background subtraction, scaling, cellHTS2 (Boutros et al. 2006), and interquartile range measurement. Considering the false-positive potential that arises from RNAi technology, a robust validation method was designed for the purpose of gene selection for future investigations. In a retrospective analysis, the authors describe the use of validation data to evaluate each normalization method. Although no method worked ideally, a combination of 2 methods, background subtraction followed by quantile normalization and cellHTS2, at different thresholds, captures the most dependable and diverse candidate genes. Thresholds are suggested depending on whether a few candidate genes are desired or a more extensive systems-level analysis is sought. The normalization approaches and experimental design to perform validation experiments are likely to apply to those high-throughput screening systems attempting to identify genes for systems-level analysis.

Correction: Comparative oncology DNA sequencing of canine T cell lymphoma via human hotspot panel.

[This corrects the article DOI: 10.18632/oncotarget.25209.].

Comparative oncology DNA sequencing of canine T cell lymphoma via human hotspot panel.

T-cell lymphoma (TCL) is an uncommon and aggressive form of human cancer. Lymphoma is the most common hematopoietic tumor in canines (companion animals), with TCL representing approximately 30% of diagnoses. Collectively, the canine is an appealing model for cancer research given the spontaneous occurrence of cancer, intact immune system, and phytogenetic proximity to humans. We sought to establish mutational congruence of the canine with known human TCL mutations in order to identify potential actionable oncogenic pathways. Following pathologic confirmation, DNA was sequenced in 16 canine TCL (cTCL) cases using a custom Human Cancer Hotspot Panel of 68 genes commonly mutated in human TCL. Sequencing identified 4,527,638 total reads with average length of 229 bases containing 346 unique variants and 1,474 total variants; each sample had an average of 92 variants. Among these, there were 258 germline and 32 somatic variants. Among the 32 somatic variants there were 8 missense variants, 1 splice junction variant and the remaining were intron or synonymous variants. A frequency of 4 somatic mutations per sample were noted with >7 mutations detected in MET, KDR, STK11 and BRAF. Expression of these associated proteins were also detected via Western blot analyses. In addition, Sanger sequencing confirmed three variants of high quality (MYC, MET, and TP53 missense mutation). Taken together, the mutational spectrum and protein analyses showed mutations in signaling pathways similar to human TCL and also identified novel mutations that may serve as drug targets as well as potential biomarkers.

Dynamic Analysis of Human Natural Killer Cell Response at Single-Cell Resolution in B-Cell Non-Hodgkin Lymphoma.

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level.

Proteasomal Inhibition by Ixazomib Induces CHK1 and MYC-Dependent Cell Death in T-cell and Hodgkin Lymphoma.

Proteasome-regulated NF-κB has been shown to be important for cell survival in T-cell lymphoma and Hodgkin lymphoma models. Several new small-molecule proteasome inhibitors are under various stages of active preclinical and clinical development. We completed a comprehensive preclinical examination of the efficacy and associated biologic effects of a second-generation proteasome inhibitor, ixazomib, in T-cell lymphoma and Hodgkin lymphoma cells and in vivo SCID mouse models. We demonstrated that ixazomib induced potent cell death in all cell lines at clinically achievable concentrations. In addition, it significantly inhibited tumor growth and improved survival in T-cell lymphoma and Hodgkin lymphoma human lymphoma xenograft models. Through global transcriptome analyses, proteasomal inhibition showed conserved overlap in downregulation of cell cycle, chromatin modification, and DNA repair processes in ixazomib-sensitive lymphoma cells. The predicted activity for tumor suppressors and oncogenes, the impact on "hallmarks of cancer," and the analysis of key significant genes from global transcriptome analysis for ixazomib strongly favored tumor inhibition via downregulation of MYC and CHK1, its target genes. Furthermore, in ixazomib-treated lymphoma cells, we identified that CHK1 was involved in the regulation of MYC expression through chromatin modification involving histone H3 acetylation via chromatin immunoprecipitation. Finally, using pharmacologic and RNA silencing of CHK1 or the associated MYC-related mechanism, we demonstrated synergistic cell death in combination with antiproteasome therapy. Altogether, ixazomib significantly downregulates MYC and induces potent cell death in T-cell lymphoma and Hodgkin lymphoma, and we identified that combinatorial therapy with anti-CHK1 treatment represents a rational and novel therapeutic approach. Cancer Res; 76(11); 3319-31. ©2016 AACR.

Altered expression of cyclins and cdks in premature infant baboon model of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature infants, which results in substantial morbidity. The pathophysiology of BPD includes oxidant injury, baro/volutrauma, and disordered lung repair. As lung development, differentiation, and repair require cell division, we hypothesized dysregulation of the cell cycle in oxygen exposure of premature infants that may contribute to the evolution of BPD. In this investigation, we studied the expression of cyclins and cyclin-dependent kinases (cdks) that regulate transition from G1 and G2 phases of the cell cycle. We report here that expression of cyclin D1, cyclin E, and cyclin A is modulated in premature baboons in respiratory distress. In addition, the expression of cdk1 or cdk4 was also modulated in these premature animals. The phosphorylation of retinoblastoma protein was progressively decreased in 125-day animals and in 140-day animals exposed to 6 or 14 days of PRN oxygen. These results indicate that due to altered cyclin and cdk expression, the repair of injured epithelium may proceed in a disordered manner that is characteristic of BPD. Thus, altered cell cycle regulation may be an important factor in the evolution of BPD.

Increased apoptosis and expression of p21 and p53 in premature infant baboon model of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a major complication of premature infants who receive prolonged ventilatory support. The pathophysiology of BPD involves oxidant injury, baro/volutrauma, and disordered lung repair. Exposure of premature lung that is poorly adapted for air breathing (>3% oxygen in fetal lung) to a higher concentration of oxygen can cause significant oxidant injury. Cell growth and differentiation of the developing lung require selective and ordered cell division. As hyperoxia can increase the expression of cell-cycle checkpoints that can cause growth arrest of lung cells, in this report we examined the expression of checkpoint proteins p53 and p21 in a premature infant the baboon model of BPD. Additionally, we also determined whether enhanced apoptosis occurs in baboon BPD model. We have shown that p53 and p21 expression are increased in 125-day as well as 140-day premature baboons with BPD. We also demonstrate increased apoptosis in lung tissue of premature baboons with BPD. These results demonstrate that cell growth inhibition is a likely factor in the evolution of BPD. Additionally, lung cells may undergo increased apoptosis that can impair the repair process in the postventilatory recovery period.

Redox-cycling of anthracyclines by thioredoxin system: increased superoxide generation and DNA damage.

Anthracyclines such as doxorubicin and daunomycin undergo bioreductive activation by redox-cycling, and this is associated with generation of reactive oxygen species. Toxicity of anthracyclines is attributed to DNA intercalation by an anthracycline semiquinone radical that is generated via redox-cycling. Flavoprotein enzymes catalyze the bioreductive activation of anthracyclines. Thioredoxin reductase (TR), which is also a flavoprotein enzyme, participates in bioreductive activation of anthracyclines. In the present study we showed that addition of E. coli thioredoxin (Trx) enhances the rate of superoxide production by E. coli TR in the presence of anthracyclines. The superoxide generated in this redox-cycling process induced DNA damage as determined by an in vitro plasmid DNA damage assay. In addition, Trx-SH enhanced the activity of cyto-chrome P450 reductase and the redox-cycling of anthracyclines independently of NADPH. Furthermore,when A549 cells were incubated with E. coli Trx followed by doxorubicin treatment, increased levels of ROS generation were observed. Taken together, these results show a novel property of the Trx system in bioreductive activation of anthracyclines.

The novel organic arsenical darinaparsin induces MAPK-mediated and SHP1-dependent cell death in T-cell lymphoma and Hodgkin lymphoma cells and human xenograft models.

PURPOSE: Darinaparsin (Zio-101) is a novel organic arsenical compound with encouraging clinical activity in relapsed/refractory T-cell lymphoma (TCL) and Hodgkin lymphoma (HL); however, little is known about its mechanism of action. EXPERIMENTAL DESIGN: TCL cell lines (Jurkat, Hut78, and HH) and HL cell lines (L428, L540, and L1236) were examined for in vitro cell death by MTT assay and Annexin V-based flow cytometry. Jurkat and L540-derived xenografts in SCID mice were examined for in vivo tumor inhibition and survival. Biologic effects of darinaparsin on the MAPK pathway were investigated using pharmacologic inhibitors, RNAi and transient transfection for overexpression for SHP1 and MEK. RESULTS: Darinaparsin treatment resulted in time- and dose-dependent cytotoxicity and apoptosis in all TCL and HL cell lines. In addition, darinaparsin had more rapid, higher, and sustained intracellular arsenic levels compared with arsenic trioxide via mass spectrometry. In vivo experiments with Jurkat (TCL) and L540 (HL)-derived lymphoma xenografts showed significant inhibition of tumor growth and improved survival in darinaparsin-treated SCID mice. Biologically, darinaparsin caused phosphorylation of ERK (and relevant downstream substrates) primarily by decreasing the inhibitory SHP1 phosphatase and coimmunoprecipitation showed significant ERK/SHP1 interaction. Furthermore, ERK shRNA knockdown or constitutive overexpression of SHP1 resulted in increased apoptosis, whereas cotreatment with pharmacologic MEK inhibitors resulted in synergistic cell death. Conversely, SHP1 blockade (via pharmacologic inhibition or RNAi) and MEK constitutive activation decreased darinaparsin-related cell death. CONCLUSIONS: Altogether, these data show that darinaparsin is highly active in HL and TCL and its activity is dependent primarily on MAPK mechanisms.

Identification of genes required for damage survival using a cell-based RNAi screen against the Drosophila genome.

Exposure to DNA-damaging agents invokes biological responses necessary for damage recovery and cell survival. Despite the presence of intact DNA repair pathways, lack of certain other biological pathways has been shown to sensitize cells to DNA-damaging agents' exposure. It is likely that following DNA damage a complex interplay between DNA repair pathways and other biological pathways might be required to ensure cell survival. In this chapter, we describe a high-throughput method for the identification of genes essential for cell survival following DNA damage by using a cell-based assay to measure viability in combination with an RNA interference-based genome-wide screening experiment.