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Purpose: Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non–Hodgkin lymphomas with aggressive clinical behavior. We performed comprehensive miRNA profiling in PTCLs and corresponding normal CD4+ Th1/2 and TFH-like polarized subsets to elucidate the role of miRNAs in T-cell lymphomagenesis. Experimental Design: We used nCounter (NanoString Inc) for miRNA profiling and validated using Taqman qRT-PCR (Applied Biosystems, Inc). Normal CD4+ T cells were polarized into effector Th subsets using signature cytokines, and miRNA significance was revealed using functional experiments. Results: Effector Th subsets showed distinct miRNA expression with corresponding transcription factor expression (e.g., BCL6/miR-19b, -106, -30d, -26b, in IL21-polarized; GATA3/miR-155, miR-337 in Th2-polarized; and TBX21/miR-181a, -331-3p in Th1-polarized cells). Integration of miRNA signatures suggested activation of TCR and PI3K signaling in IL21-polarized cells, ERK signaling in Th1-polarized cells, and AKT–mTOR signaling in Th2-polarized cells, validated at protein level. In neoplastic counterparts, distinctive miRNAs were identified and confirmed in an independent cohort. Integrative miRNA–mRNA analysis identified a decrease in target transcript abundance leading to deregulation of sphingolipid and Wnt signaling and epigenetic dysregulation in angioimmunoblastic T-cell lymphoma (AITL), while ERK, MAPK, and cell cycle were identified in PTCL subsets, and decreased target transcript abundance was validated in an independent cohort. Elevated expression of miRNAs (miR-126-3p, miR-145-5p) in AITL was associated with poor clinical outcome. In silico and experimental validation suggest two targets (miR-126→ SIPR2 and miR-145 → ROCK1) resulting in reduced RhoA-GTPase activity and T–B-cell interaction. Conclusions: Unique miRNAs and deregulated oncogenic pathways are associated with PTCL subtypes. Upregulated miRNA-126-3p and miR-145-5p expression regulate RhoA-GTPase and inhibit T-cell migration, crucial for AITL pathobiology.

T-cell prolymphocytic leukemia (T-PLL) is a poor-prognostic neoplasm. Differentiation stage and immune-effector functions of the underlying tumor cell are insufficiently characterized. Constitutive activation of the T-cell leukemia 1A (TCL1A) oncogene distinguishes the (pre)leukemic cell from regular postthymic T cells. We assessed activation-response patterns of the T-PLL lymphocyte and interrogated the modulatory impact by TCL1A. Immunophenotypic and gene expression profiles revealed a unique spectrum of memory-type differentiation of T-PLL with predominant central-memory stages and frequent noncanonical patterns. Virtually all T-PLL expressed a T-cell receptor (TCR) and/or CD28-coreceptor without overrepresentation of specific TCR clonotypes. The highly activated leukemic cells also revealed losses of negative-regulatory TCR coreceptors (eg, CTLA4). TCR stimulation of T-PLL cells evoked higher-than-normal cell-cycle transition and profiles of cytokine release that resembled those of normal memory T cells. More activated phenotypes and higher TCL1A correlated with inferior clinical outcomes. TCL1A was linked to the marked resistance of T-PLL to activation- and FAS-induced cell death. Enforced TCL1A enhanced phospho-activation of TCR kinases, second-messenger generation, and JAK/STAT or NFAT transcriptional responses. This reduced the input thresholds for IL-2 secretion in a sensitizer-like fashion. Mice of TCL1A-initiated protracted T-PLL development resembled such features. When equipped with epitope-defined TCRs or chimeric antigen receptors, these Lckpr-hTCL1Atg T cells gained a leukemogenic growth advantage in scenarios of receptor stimulation. Overall, we propose a model of T-PLL pathogenesis in which TCL1A enhances TCR signals and drives the accumulation of death-resistant memory-type cells that use amplified low-level stimulatory input, and whose loss of negative coregulators additionally maintains their activated state. Treatment rationales are provided by combined interception in TCR and survival signaling.

Changes in species dominance may drive regime shifts because dominant biotic feedbacks reflect functional traits of a community. Changes in species dominance has been documented by a 25-year fish community dataset encompassing six reaches of the Upper Mississippi and Illinois Rivers. Specifically, common carp (Cyprinus carpio) abundance has declined across all reaches, whereas silver carp (Hypophthalmichthys molitrix) and bighead carp (Hypophthalmichthys nobilis) have increased in abundance in the southern three reaches. To test whether signals in the data were consistent with regime transitions, changes in body-size patterns and trends in functional biomass and variance of functional biomass of the fish community were assessed. I further explored biomass thresholds relative to transitions. Shifts in body-size aggregations and trends in functional biomass support hypotheses that transitions from common carp dominance to a more functional diverse community represent alternate regimes. Results indicate such transitions occurred in the early 2000s for the two most northern reaches, and that the third most northern reach is nearing this transition. In the southern reaches, results indicate that transitions from common carp dominance to silver and bighead carp dominance also represent alternate regimes. Regime transitions support biomass thresholds between 8000 and 10,000 g per unit of day electrofishing effort of common carp and between 20 and 2000 g per unit effort in silver and bighead carp. Consistencies across reaches with similar dominance patterns provide support that reinforcing feedbacks, unique to the functional attributes of the dominant species, occur at similar biomass levels. Building knowledge of such feedback mechanisms is imperative to effectively manage for desired regimes.

Extra-nodal natural killer (NK)/T-cell lymphoma, nasal type (ENKTCL) is a highly aggressive lymphoma, in which the tumor suppressor gene PRDM1 is frequently lost or inactivated. We employed two different CRISPR/Cas9 approaches to generate PRDM1-/- primary NK cells to study the role of this gene in NK-cell homeostasis. PRDM1-/- NK cells showed a marked increase in cloning efficiency, higher proliferation rate and less apoptosis compared with their wild-type counterparts. Gene expression profiling demonstrated a marked enrichment in pathways associated with proliferation, cell cycle, MYC, MYB and TCR/NK signaling in PRDM1-/- NK cells, but pathways associated with normal cellular functions including cytotoxic functions were downregulated, suggesting that the loss of PRDM1 shifted NK cells toward proliferation and survival rather than the performance of their normal functions. We were also able to further modify a PRDM1-deleted clone to introduce heterozygous deletions of common tumor suppressor genes in ENKTCL such as TP53, DDX3X, and PTPN6. We established an in vitro model to elucidate the major pathways through which PRDM1 mediates its homeostatic control of NK cells. This approach can be applied to the study of other relevant genetic lesions and oncogenic collaborations in lymphoma pathogenesis.

Transcriptomic, proteomic, and methylation aging clocks demonstrate that aging has a predictable preset program, while transcriptome trajectory turning points indicate that the 20–40 age range in humans is the likely stage at which the progressive loss of homeostatic control, and in turn aging, begins to have detrimental effects. Turning points in this age range overlapping with human aging clock genes revealed five candidates that we hypothesized could play a role in aging or age-related physiological decline. To examine these gene’s effects on lifespan and health-span, we utilized whole body and heart-specific gene knockdown of human orthologs in Drosophila melanogaster. Whole body lysyl oxidase like 2 (Loxl2), fz3, and Glo1 RNAi positively affected lifespan as did heart-specific Loxl2 knockdown. Loxl2 inhibition concurrently reduced age-related cardiac arrythmia and collagen (Pericardin) fiber width. Loxl2 binds several transcription factors in humans and RT-qPCR confirmed that a conserved transcriptional target CDH1 (Drosophila CadN2) has expression levels which correlate with Loxl2 reduction in Drosophila. These results point to conserved pathways and multiple mechanisms by which inhibition of Loxl2 can be beneficial to heart health and organismal aging.

Background: Shiga-toxin producing Escherichia coli (STEC) O26:H11 is the second most common cause of severe diarrhea and hemolytic uremic syndrome worldwide. The implementation of whole genome sequencing (WGS) enhances the detection and in-depth characterization of these non-O157 STEC strains. The aim of this study was to compare WGS to phenotypic serotyping and pulse field gel electrophoresis (PFGE) for characterization of STECO26 strains following a zoonotic outbreak from cattle to humans. Methods and results: This study evaluated seven E. coli strains; two strains isolated from two children with gastrointestinal symptoms and five strains from five calves suspected as the source of infection. Six of these isolates were serotyped phenotypically and by WGS as E. coli O26:H11 while one bovine isolate could be serotyped only by WGS as E. coli O182:H25. Stx1 was detected in two human- and two bovine-isolates using PCR and WGS. Using WGS, all four STECO26 isolates belong to sequence type (ST) 21 while the two stx1 negative E. coli O26 were ST29. All four STECO26 isolates were indistinguishable by PFGE. However, the data generated by WGS linked the two human STECO26 isolates to only one bovine STECO26 strain by having identical high-quality single nucleotide polymorphisms (hqSNPs) and identical virulence factor profiles while the remaining bovine STECO26 isolate differed by 7 hqSNPs and lacked virulence factor toxB. Conclusions: These data demonstrated that WGS provided significant information beyond traditional epidemiological tools allowing for comprehensive characterization of the STEC. Using this approach, WGS was able to identify the specific source of infection in this study. Keywords: Whole genome sequencing, Shiga toxin, E. coli O26, Public health

Most cellular processes descend into failure during aging. While a large collection of longevity pathways has been identified in the past decades, the mechanism for age-related decline of cellular homeostasis and organelle function remains largely unsolved. It is known that many organelles undergo structural and functional changes during normal aging, which significantly contributes to the decline of tissue function at old ages. Since recent studies have revealed an emerging role of organelles as regulatory hubs in maintaining cellular homeostasis, understanding of organelle aging will provide important insights into the cellular basis of organismal aging. Here we review current progress on the characterization of age-dependent structural and functional alterations in the more well-studied organelles, as well as the known mechanisms governing organelle aging in model organisms, with a special focus on the fruit fly Drosophila melanogaster.

Understanding how gene flow influences adaptive divergence is important for predicting adaptive responses. Theoretical studies suggest that when gene flow is high, clustering of adaptive genes in fewer genomic regions would protect adaptive alleles from among-population recombination and thus be selected for, but few studies have tested this hypothesis with empirical data. Here, we used RADseq to generate genomic data for six fish species with contrasting life histories from six reaches of the Upper Mississippi River System, USA. We then conducted genome scans for genomic islands of divergence to examine the distribution of adaptive loci and investigated whether these loci were found in inversions. We found that gene flow varied among species, and adaptive loci were clustered more tightly in species with higher gene flow. For example, the two species with the highest overall FST (0.03 - 0.07) and therefore lowest gene flow showed little evidence of clusters of adaptive loci, with adaptive loci spread uniformly across the genome. In contrast, nearly all adaptive loci in the species with the lowest FST (0.0004) were found in a single large putative inversion. Two other species with intermediate gene flow (FST ~ 0.004) also showed clustered genomic architectures, with most islands of divergence clustered on a few chromosomes. These results provide important empirical evidence to support the hypothesis that increasingly clustered architectures of local adaptation are associated with high gene flow. Our study utilized a unique system with species spanning a large gradient of life histories to highlight the importance of gene flow in shaping adaptive divergence.

The spectrin cytoskeleton has been shown to be critical in diverse processes such as axon development and degeneration, myoblast fusion, and spermatogenesis. Spectrin can be modulated in a tissue specific manner through junctional protein complexes, however, it has not been shown that long noncoding RNAs (lncRNAs) interact with and modulate spectrin. Here, we provide evidence of a lncRNA CR45362 that interacts with α-Spectrin, is required for spermatid nuclear bundling during Drosophila spermatogenesis. We observed that CR45362 showed high expression in the cyst cells at the basal testis, and CRISPR-mediated knockout of CR45362 led to sterile male, unbundled spermatid nuclei, and disrupted actin cones. Through chromatin isolation by RNA precipitation—mass spectrometry (ChIRP-MS), we identified actin-spectrin cytoskeletal components physically interact with the lncRNA CR45362. Genetic screening on identified cytoskeletal factors revealed that cyst cell-specific knockdown of α-Spectrin phenocopied CR45362 mutants and resulted in spermatid nuclear bundle defects. Consistently, CR45362 knockout disrupted the co-localization of α-Spectrin and spermatid nuclear bundles in the head cyst cells at the basal testis. Thus, we uncovered a novel lncRNA CR45362 that interacts with α-Spectrin to stabilize spermatid nuclear bundles during spermatid maturation.

International audience; Peripheral T cell lymphomas (PTCLs) represent a significant unmet medical need with dismal clinical outcomes. The T cell receptor (TCR) is emerging as a key driver of T lymphocyte transformation. However, the role of chronic TCR activation in lymphomagenesis and in lymphoma cell survival is still poorly understood. Using a mouse model, we report that chronic TCR stimulation drove T cell lymphomagenesis, whereas TCR signaling did not contribute to PTCL survival. The combination of kinome, transcriptome, and epigenome analyses of mouse PTCLs revealed a NK cell-like reprogramming of PTCL cells with expression of NK receptors (NKRs) and downstream signaling molecules such as Tyrobp and SYK. Activating NKRs were functional in PTCLs and dependent on SYK activity. In vivo blockade of NKR signaling prolonged mouse survival, demonstrating the addiction of PTCLs to NKRs and downstream SYK/mTOR activity for their survival. We studied a large collection of human primary samples and identified several PTCLs recapitulating the phenotype described in this model by their expression of SYK and the NKR, suggesting a similar mechanism of lymphomagenesis and establishing a rationale for clinical studies targeting such molecules.

Detecting nuisance species at low abundance or in newly established areas is critical to developing pest management strategies. Due to their sensitivity to disturbance and erratic jumping behavior, Silver Carp Hypophthalmichthys molitrix can be difficult to collect with traditional sampling methods. We compared catch per unit effort (CPUE) of all species from a Long Term Resource Monitoring (LTRM) electrofishing protocol to an experimental electrofishing technique designed to minimize Silver Carp evasion through tactical boat maneuvering and selective application of power. Differences in CPUE between electrofishing methods were detected for 2 of 41 species collected across 2 years of sampling at 20 sites along the Illinois River. The mean catch rate of Silver Carp using the experimental technique was 2.2 times the mean catch rate of the LTRM electrofishing technique; the increased capture efficiency at low relative abundance emphasizes the utility of this method for early detection. The experiment...

The transcription factor foxo is a known regulator of lifespan extension and tissue homeostasis. It has been linked to the maintenance of neuronal processes across many species and has been shown to promote youthful characteristics by regulating cytoskeletal flexibility and synaptic plasticity at the neuromuscular junction (NMJ). However, the role of foxo in aging neuromuscular junction function has yet to be determined. We profiled adult Drosophila foxo- null mutant abdominal ventral longitudinal muscles and found that young mutants exhibited morphological profiles similar to those of aged wild-type flies, such as larger bouton areas and shorter terminal branches. We also observed changes to the axonal cytoskeleton and an accumulation of late endosomes in foxo null mutants and motor neuron-specific foxo knockdown flies, similar to those of aged wild-types. Motor neuron-specific overexpression of foxo can delay age-dependent changes to NMJ morphology, suggesting foxo is responsible for maintaining NMJ integrity during aging. Through genetic screening, we identify several downstream factors mediated through foxo-regulated NMJ homeostasis, including genes involved in the MAPK pathway. Interestingly, the phosphorylation of p38 was increased in the motor neuron-specific foxo knockdown flies, suggesting foxo acts as a suppressor of p38/MAPK activation. Our work reveals that foxo is a key regulator for NMJ homeostasis, and it may maintain NMJ integrity by repressing MAPK signaling.

Activating mutations in the Vav guanine nucleotide exchange factor 1 (VAV1) gene are reported in various subtypes of mature T-cell neoplasms (TCN). However, oncogenic activities associated with VAV1 mutations in TCN remain unclear. To define them, we established transgenic mice expressing VAV1 mutants cloned from human TCN. Although we observed no tumors in these mice for up to a year, tumors did develop in comparably-aged mice on a p53-null background (p53-/- VAV1-Tg), and p53-/- VAV1-Tg mice died with shorter latencies than did p53-null (p53-/-) mice. Notably, various TCN with tendency of maturation developed in p53-/- VAV1-Tg mice, while p53-/- mice exhibited only immature TCN. Mature TCN in p53-/- VAV1-Tg mice mimicked human peripheral T-cell lymphoma (PTCL)-GATA3 and exhibited features of type2 T helper (TH2) cells. Phenotypes seen following transplantation of either p53-/- VAV1 or p53-/- tumor cells into nude mice were comparable, indicating cell-autonomous tumor-initiating capacity. Whole transcriptome analysis (WTA) showed enrichment of multiple Myc-related pathways in TCN from p53-/- VAV1-Tg mice relative to p53-/- or wild-type T cells. Remarkably, amplification of Myc locus were found recurrently in TCN of p53-/- VAV1-Tg mice. Finally, treatment of nude mice transplanted with p53-/- VAV1-Tg tumor cells with JQ1, a bromodomain inhibitor, which targets the Myc pathway, prolonged survival of mice. We conclude that VAV1 mutations function in malignant transformation of T cells in vivo and that VAV1-mutant expressing mice could provide an efficient tool for screening new therapeutic targets in TCN harboring these mutations. We conclude that VAV1 mutations function in malignant transformation of T cells in vivo and that VAV1-mutant expressing mice could provide an efficient tool for screening new therapeutic targets in TCN harboring these mutations.

Despite the hypothesized benefits to age-0 Scaphirhynchus sturgeon, the role that slow, shallow water plays during early life history remains uncertain. Although several studies have reported the depths and velocities observed at age-0 sturgeon capture sites, conditions at larger spatial scales beyond the immediate capture location may also be important. Therefore, the specific objectives of this study were to (a) compare catch among large reaches (26–37 km in length) that varied in the availability of water

Angioimmunoblastic T-cell lymphoma (AITL), the most frequent subtype of peripheral T-cell lymphoma (PTCL), is a neoplasm with characteristics of mature T follicular helper (TFH) cells. We and others have identified frequent (~75%) inactivating mutations in the TET2 (Ten-Eleven Translocation-2) gene in AITL. TET2 belongs to a 3 member family of TET dioxygenases that catalyze DNA demethylation by oxidation of 5-methyl-cytosine (5-mC) to 5-hydroxymethyl-cytosine (5-hmC) and further oxidative cytosine products. Thus, loss of function (LOF) of TET2 will cause aberrant genome hypermethylation and reduction in 5-hmC. Studies of the variant allele fraction (VAF) of TET2 mutants suggest that this mutation is a founding abnormality in AITL. However, how TET2 loss promotes the development of AITL is still unclear. To study LOF of TET2 in CD4 T-cell lymphomagenesis without the noise generated by other mutations in an established lymphoma, we generated a human TET2 knock-out (KO) CD4 T-cell model using CRISPR/Cas9 technology, which allows us to perform functional genomic studies by directly editing genes at their genomic loci. Whole transcriptome sequencing and single-cell transcriptome sequencing were used to study the cell evolution after KO. We generated multiple TET2 KO primary CD4 T-cell models using two different CRISPR/Cas9 methods. The first approach used the plasmid PX458-a, which expresses green fluorescent protein (GFP) fused Cas9 and guide RNA-a targeting TET2 exon 6, to electroporate CD4 T-cell from healthy donor F25. The second approach used homologous DNA repair (HDR) mediated knock-in (KI) of tandem GFP gene and a SV40 transcription stop signal to terminate TET2 expression at exon 3. Cas9/sgRNA-e RNP complex, along with a long DNA template (about 1.6 kb), was electroporated into CD4 T-cells from two healthy donors, F25 and M40. GFP-positive cells were sorted by FACS after electroporation and were considered to be edited cells. Edited CD4 T-cells were cultured in vitro with 50 U/ml IL-2, and stimulated regularly (every 7~10 days) with 1:1 ratio of anti-CD3/CD28 T activator beads. TET2 KO in these cells was confirmed by qRT-PCR, Sanger sequencing and Western blotting. Compared with wild-type (WT) CD4 T-cells under the same culture conditions, a lower level of 5-hmC in TET2 KO cells was observed, indicating successful editing of TET2. Compared to WT cells, KO cells had a higher growth rate, due to a lower apoptosis rate and a higher proliferation rate, by Annexin V staining, EdU staining, and MTS experiments. The growth of KO cells or WT cells was still dependent on IL-2 and T activator beads stimulation. All batches of KO cells, generated by different guide RNAs or from different donors, showed a much longer life span than WT cells, which usually lived for 3~4 months, but KO cells can keep proliferating longer than one year. We also performed TCR analysis on these cell samples. Both WT and KO cells demonstrated oligoclonality when examined at Day 40 (40D, early stage) and TET2 KO cells showed a dominant clone by Day 90 (90D, late stage). We performed single-cell transcriptome analysis on M40 KO vs. WT cells, at 40D and 90D. KO90D cells had a low TCR diversity with the dominant population representing ~88% of cells (TRAV9-2,TRBV5-1). From single-cell transcriptome analysis, cell clustering profiles were very distinctive in these 4 cell populations analyzed (Figure 1A) and these clusters had unique gene expression profiles (Figure 1B). Cluster 6 was prominent in KO90D but almost absent in WT90D, whereas the reverse was true for clusters 1 and 5. From pathway analysis, KO90D cells showed a higher expression of signatures associated with proliferation, cell cycle and chemokine signaling and lower histidine and tryptophan metabolism signatures. Sanger sequencing showed a 79 bp indel in addition to the GFP KI allele in KO90D cells, demonstrated the homozygous deletion of TET2 on these cells. Similar results were observed in F25 TET2 KO cells by plasmid PX458-a. This indicated the selection of homozygously deleted TET2 cells in long-term culture. However, clonal evolution is highly dynamic and a minor clone in KO40D cells may become the dominant clone in KO90D cells. Comparison of the 5-mC and 5-hmC profiles between KO and WT cells are being conducted to elucidate epigenetic alterations that are associated with the functional alterations and predisposition to AITL lymphomagenesis. Figure Disclosures No relevant conflicts of interest to declare.

The activated B cell (ABC-like) subtype of diffuse large B cell lymphoma (DLBCL) is characterized by chronic activation of signaling initiated by immunoglobulin μ (IgM). By analyzing the DNA copy number profiles of 1000 DLBCL tumors, we identified gains of 18q21.2 as the most frequent genetic alteration in ABC-like DLBCL. Using integrative analysis of matched gene expression profiling data, we found that the TCF4 (E2-2) transcription factor gene was the target of these alterations. Overexpression of TCF4 in ABC-like DLBCL cell lines led to its occupancy on immunoglobulin (IGHM) and MYC gene enhancers and increased expression of these genes at the transcript and protein levels. Inhibition of TCF4 activity with dominant-negative constructs was synthetically lethal to ABC-like DLBCL cell lines harboring TCF4 DNA copy gains, highlighting these gains as an attractive potential therapeutic target. Furthermore, the TCF4 gene was one of the top BRD4-regulated genes in DLBCL cell lines. BET proteolysis-targeting chimera (PROTAC) ARV771 extinguished TCF4, MYC, and IgM expression and killed ABC-like DLBCL cells in vitro. In DLBCL xenograft models, ARV771 treatment reduced tumor growth and prolonged survival. This work highlights a genetic mechanism for promoting immunoglobulin signaling in ABC-like DLBCL and provides a functional rationale for the use of BET inhibitors in this disease.

B-cell non-Hodgkin’s lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL such as diffuse large B-cell lymphoma (DLBCL) have been comprehensively interrogated at the genomic level. But rarer subtypes such as mantle cell lymphoma (MCL) remain sparsely characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth; including DLBCL, MCL, follicular lymphoma (FL), and Burkitt lymphoma (BL). We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumor from each subtype, including the frequent genetic deregulation of the ubiquitin proteasome system (UPS). In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.

Peripheral T-cell lymphoma (PTCL) is a group of complex clinicopathological entities, often associated with an aggressive clinical course. Angioimmunoblastic T-cell lymphoma (AITL) and PTCL-not otherwise specified (PTCL-NOS) are the 2 most frequent categories, accounting for >50% of PTCLs. Gene expression profiling (GEP) defined molecular signatures for AITL and delineated biological and prognostic subgroups within PTCL-NOS (PTCL-GATA3 and PTCL-TBX21). Genomic copy number (CN) analysis and targeted sequencing of these molecular subgroups revealed unique CN abnormalities (CNAs) and oncogenic pathways, indicating distinct oncogenic evolution. PTCL-GATA3 exhibited greater genomic complexity that was characterized by frequent loss or mutation of tumor suppressor genes targeting the CDKN2A/B-TP53 axis and PTEN-PI3K pathways. Co-occurring gains/amplifications of STAT3 and MYC occurred in PTCL-GATA3. Several CNAs, in particular loss of CDKN2A, exhibited prognostic significance in PTCL-NOS as a single entity and in the PTCL-GATA3 subgroup. The PTCL-TBX21 subgroup had fewer CNAs, primarily targeting cytotoxic effector genes, and was enriched in mutations of genes regulating DNA methylation. CNAs affecting metabolic processes regulating RNA/protein degradation and T-cell receptor signaling were common in both subgroups. AITL showed lower genomic complexity compared with other PTCL entities, with frequent co-occurring gains of chromosome 5 (chr5) and chr21 that were significantly associated with IDH2R172 mutation. CN losses were enriched in genes regulating PI3K–AKT–mTOR signaling in cases without IDH2 mutation. Overall, we demonstrated that novel GEP-defined PTCL subgroups likely evolve by distinct genetic pathways and provided biological rationale for therapies that may be investigated in future clinical trials.

LB-100 is an experimental cancer therapeutic with cytotoxic activity against cancer cells in culture and antitumor activity in animals. The first phase I trial (NCT01837667) evaluating LB-100 recently concluded that safety and efficacy parameters are favorable for further clinical testing. Although LB-100 is widely reported as a specific inhibitor of serine/threonine phosphatase 2A (PP2AC/PPP2CA:PPP2CB), we could find no experimental evidence in the published literature demonstrating the specific engagement of LB-100 with PP2A in vitro, in cultured cells, or in animals. Rather, the premise for LB-100 targeting PP2AC is derived from studies that measure phosphate released from a phosphopeptide (K-R-pT-I-R-R) or inferred from the ability of LB-100 to mimic activity previously reported to result from the inhibition of PP2AC by other means. PP2AC and PPP5C share a common catalytic mechanism. Here, we demonstrate that the phosphopeptide used to ascribe LB-100 specificity for PP2A is also a substrate for PPP5C. Inhibition assays using purified enzymes demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C. The structure of PPP5C cocrystallized with LB-100 was solved to a resolution of 1.65Å, revealing that the 7-oxabicyclo[2.2.1]heptane-2,3-dicarbonyl moiety coordinates with the metal ions and key residues that are conserved in both PP2AC and PPP5C. Cell-based studies revealed some known actions of LB-100 are mimicked by the genetic disruption of PPP5C. These data demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C and suggest that the observed antitumor activity might be due to an additive effect achieved by suppressing both PP2A and PPP5C.

The threat posed by bigheaded carps (Hypophthalmichthys spp.) to novel ecosystems has focused efforts on preventing further range expansion; upstream progression in the Illinois River is a major concern due to its connection with the uninvaded Great Lakes. In addition to an electric barrier system, commercial harvest of silver carp (H. molitrix) and bighead carp (H. nobilis) in the upper river is intended to reduce propagule pressure and prevent range expansion. To quantify demographics and evaluate harvest efficacy, the upper river was sampled between 2012 and 2015 using mobile hydroacoustic methods. Reach-specific densities, size structures and species compositions varied interannually but the advancing population was characterized longitudinally as small-bodied, silver carp-dominated at the highest densities downstream, shifting to large-bodied, bighead carp-dominated at the low-density population front. The use of hydroacoustic sampling for harvest evaluation was validated in backwater lakes; there was a significant positive correlation between density estimates and the corresponding harvest catch-per-unit-effort of bigheaded carps. Localized densities of bigheaded carps were reduced by up to 64.4 % immediately post-harvest but generally rebounded within weeks. However, annual sampling of the entire upper river indicated that density of bigheaded carps decreased by over 40 % (between 2012 and 2013) and subsequently remained stable (between 2013 and 2014). The annual harvest of bigheaded carps increased during this period (from 45,192 to 102,453 individuals), in years of contrasting discharge conditions. At this spatiotemporal scale, harvest appears to have contributed to initial reduction, and subsequent maintenance of, bigheaded carps density levels, but discharge likely plays an important role (e.g., through immigration) in determining the extent of its impact. Mobile hydroacoustic sampling enabled robust quantification of the population over varying spatial scales and density gradients, highlighting the potential of this approach as an assessment tool for invasive fishes in riverine environments.

Follicular lymphoma (FL) is typically an indolent disease, but 30-40% of FL cases transform into an aggressive lymphoma (tFL) with a poor prognosis. To identify the genetic changes that drive this transformation, we sequenced the exomes of 12 cases with paired FL and tFL biopsies and identified 45 recurrently mutated genes in the FL-tFL data set and 39 in the tFL cases. We selected 496 genes of potential importance in transformation and sequenced them in 23 additional tFL cases. Integration of the mutation data with copy-number abnormality (CNA) data provided complementary information. We found recurrent mutations of miR-142, which has not been previously been reported to be mutated in FL/tFL. The genes most frequently mutated in tFL included KMT2D (MLL2), CREBBP, EZH2, BCL2 and MEF2B. Many recurrently mutated genes are involved in epigenetic regulation, the Janus-activated kinase-signal transducer and activator of transcription (STAT) or the nuclear factor-κB pathways, immune surveillance and cell cycle regulation or are TFs involved in B-cell development. Of particular interest are mutations and CNAs affecting S1P-activated pathways through S1PR1 or S1PR2, which likely regulate lymphoma cell migration and survival outside of follicles. Our custom gene enrichment panel provides high depth of coverage for the study of clonal evolution or divergence.Leukemia advance online publication, 8 July 2016; doi:10.1038/leu.2016.175.

Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of mature T-cell neoplasms with a poor prognosis. Recently, mutations in TET2 and other epigenetic modifiers as well as RHOA have been identified in these diseases, particularly in angioimmunoblastic T-cell lymphoma (AITL). CD28 is the major co-stimulatory receptor in T-cells which, upon binding ligand, induces sustained T-cell proliferation and cytokine production when combined with T-cell receptor stimulation. We have identified recurrent mutations in CD28 in PTCLs. Two residues – D124 and T195 – were recurrently mutated in 11.3% of cases of AITL and in one case of PTCL, not otherwise specified (PTCL-NOS). Surface plasmon resonance analysis of mutations at these residues with predicted differential partner interactions showed increased affinity for ligand CD86 (residue D124) and increased affinity for intracellular adaptor proteins GRB2 and GADS/GRAP2 (residue T195). Molecular modeling studies on each of these mutations suggested how these mutants result in increased affinities. We found increased transcription of the CD28-responsive genes CD226 and TNFA in cells expressing the T195P mutant in response to CD3 and CD86 co-stimulation and increased downstream activation of NF-κB by both D124V and T195P mutants, suggesting a potential therapeutic target in CD28-mutated PTCLs.

Invasive bigheaded carps, genus Hypophthalmichthys, are spreading throughout the Mississippi River basin. To explore the efficacy of a consumer-based market (i.e., invasivorism) to manage them, we developed a conceptual model and evaluated three harvest approaches&mdash, direct contracted removal, volume-based incentives (&ldquo, fisher-side&rdquo, control), and set-quota harvest (&ldquo, market-side&rdquo, control). We quantified the efficacy of these approaches and potential population impact in the Illinois River. Contracted removal was effective for suppressing small populations at the edge of the range but cannot support a market. &ldquo, Fisher-side&rdquo, removals totaled 225,372 kg in one year. However, participation was low, perhaps due to reporting requirements for fishers. The &ldquo, set-quota approach removed &gt, 1.3 million kg of bigheaded carp in less than 6 months. Larger, older fish were disproportionately harvested, which may hinder the ability to suppress population growth. Total density declined in one river reach, and harvest may reduce upstream movement toward the invasion fronts. With sufficient market demand, harvest may control bigheaded carp. However, lack of processing infrastructure and supply chain bottlenecks could constrain harvest, particularly at low commodity prices. Given the geographical scale of this invasion and complicated harvest logistics, concerns about economic dependence on invasivorism that encourage stock enhancement are likely unmerited.

Purpose: To identify tumor suppressor genes epigenetically silenced by promoter hypermethylation in extranodal natural killer cell lymphoma (NKCL). Experimental Design: Promoter methylation was analyzed with global and locus-specific methylation assays in NKCL cases and NK cell lines. Gene expression profiles were used to identify genes for which aberrant promoter methylation was associated with transcriptional silencing. Selected DNA methylations were validated by RRBS, pyrosequencing, or q-MSP. Decitabine treatment was performed to evaluate reactivation of methylated genes. The tumor suppressor effect of silenced genes was evaluated functionally by reintroducing them into NK cell lines. Results: We observed significant promoter hypermethylation in most NKCL samples compared with normal NK cells. Correlation of global promoter methylation with gene expression profiles identified 95 genes with strong evidence for being silenced because of promoter methylation, including BCL2L11 (BIM), DAPK1, PTPN6 (SHP1), TET2, SOCS6, and ASNS. Known tumor suppressor genes were significantly overrepresented in this set of genes. Decitabine treatment of NK cell lines was associated with reexpression of all 10 selected methylated and silenced genes. Ectopic expression of frequently silenced BIM in two BIM-nonexpressing NK cell lines led to increased apoptosis and eventual elimination of BIM-transduced cells. It also sensitized these cell lines to chemotherapy-induced apoptosis. Similarly, reintroduction of SOCS6 significantly inhibited growth in SOCS6-nonexpressing NK cell lines. NK cell lines lacking ASNS expression showed increased sensitivity to treatment with l-asparaginase. Reintroduction of ASNS reduced drug sensitivity. Conclusion: Promoter region hypermethylation is frequent in NKCL, and aberrantly methylated genes are pathologically and clinically significant. Clin Cancer Res; 21(7); 1699–711. ©2015 AACR.

Transcriptional coordination is a vital process contributing to metabolic homeostasis. As one of the key nodes in the metabolic network, the forkhead transcription factor FOXO has been shown to interact with diverse transcription co-factors and integrate signals from multiple pathways to control metabolism, oxidative stress response, and cell cycle. Recently, insulin/FOXO signaling has been implicated in the regulation of insect development via the interaction with insect hormones, such as ecdysone and juvenile hormone. In this study, we identified an interaction between Drosophila FOXO (dFOXO) and the zinc finger transcription factor Kruppel homolog 1 (Kr-h1), one of the key players in juvenile hormone signaling. We found that Kr-h1 mutants show delayed larval development and altered lipid metabolism, in particular induced lipolysis upon starvation. Notably, Kr-h1 physically and genetically interacts with dFOXO in vitro and in vivo to regulate the transcriptional activation of insulin receptor (InR) and adipose lipase brummer (bmm). The transcriptional co-regulation by Kr-h1 and dFOXO may represent a broad mechanism by which Kruppel-like factors integrate with insulin signaling to maintain metabolic homeostasis and coordinate organism growth.

The activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by the chronic activation of signaling initiated by immunoglobulin-μ (IgM). By analyzing DNA copy profiles of 1,000 DLBCLs, we identified gains of 18q21.2 as the most frequent genetic alteration in ABC-like DLBCL. We show that these alterations target the TCF4 (E2-2) transcription factor, and that over-expression of TCF4 leads to its occupancy on immunoglobulin gene enhancers and increased expression of IgM at the transcript and protein level. The TCF4 gene is one of the top BRD4-regulated genes in DLBCL. Using a BET proteolysis-targeting chimera (PROTAC) we show that TCF4 and IgM expression can be extinguished, and ABC-like DLBCL cells can be killed in vitro and in vivo. This highlights a novel genetic mechanism for promoting immunoglobulin signaling in ABC-like DLBCL and provides a functional rationale for the use of BET inhibitors in this disease.

Transcriptional coordination is a vital process contributing to metabolic homeostasis. As one of the key nodes in the metabolic network, the forkhead transcription factor FOXO has been shown to interact with diverse transcription co-factors and integrate signals from multiple pathways to control metabolism, oxidative stress response, and cell cycle. Recently, insulin/FOXO signaling has been implicated in the regulation of insect development via the interaction with insect hormones, such as ecdysone and juvenile hormone. In this study, we identified an interaction between dFOXO and the zinc finger transcription factor Kruppel homolog 1 (Kr-h1), one of the key players in juvenile hormone signaling in Drosophila. We found that Kr-h1 mutants have reduced triglyceride storage, decreased insulin signaling and delayed larval development. Notably, Kr-h1 physically and genetically interacts with dFOXO in vitro and in vivo to regulate the transcriptional activation of adipose lipase brummer (bmm). The transcriptional co-regulation by Kr-h1 and dFOXO may represent a broad mechanism by which Kruppel-like factors integrate with insulin signaling to maintain metabolic homeostasis and coordinate organism growth.

The oncogene Mdmx is overexpressed in many human malignancies, and together with Mdm2, negatively regulates the p53 tumor suppressor. However, a p53-independent function of Mdmx that impacts genome stability has been described, but this function is not well understood. In the present study, we determined that of the thirteen different cancer types evaluated, 6–90% of those that had elevated levels of Mdmx had concurrent inactivation (mutated or deleted) of p53. We show elevated levels of Mdmx inhibited double-strand DNA break repair and induced chromosome and chromatid breaks independent of p53, leading to genome instability. Mdmx impaired early DNA damage response signaling, such as phosphorylation of the serine/threonine-glutamine motif, mediated by the ATM kinase. Moreover, we identified Mdmx associated with Nbs1 of the Mre11-Rad50-Nbs1 (MRN) DNA repair complex, and this association increased upon DNA damage and was detected at chromatin. Elevated Mdmx levels also increased cellular transformation in a p53-independent manner. Unexpectedly, all Mdmx-mediated phenotypes also occurred in cells lacking Mdm2 and were independent of the Mdm2-binding domain (RING) of Mdmx. Therefore, Mdmx-mediated inhibition of the DNA damage response resulted in delayed DNA repair and increased genome instability and transformation independent of p53 and Mdm2. Our results reveal a novel p53- and Mdm2-independent oncogenic function of Mdmx that provides new insight into the many cancers that overexpress Mdmx.

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas and approximately 30% of PTCLs are designated as not-otherwise specified (PTCL-NOS). Gene expression profiling (GEP) identified molecular classifiers for PTCL entities and identified 2 novel biological subgroups within PTCL-NOS (PTCL-GATA3 and PTCL-TBX21), associated with T-cell differentiation subsets. To further investigate molecular oncogenesis, we performed microRNA expression profiling (miR-EP) in several molecular subtypes of PTCL including angioimmunoblastic T-cell lymphoma (AITL), PTCL-GATA3 and PTCL-TBX21 using formalin fixed paraffin embedded tissues. We also performed miR-EP of normal T-cell subsets polarized to represent different differentiation stages (TFH, TH1 and TH2). We performed miR-EP on 102 PTCL cases using either quantitative real time PCR (ABI, Biosystem) or ultra-sensitive direct miRNA counting (nCounter, NanoString). Corresponding GEP (mRNA) were available for 67 PTCL cases. Normal T-cells were polarized in-vitro with different cytokine milieu and examined by flow cytometry. We observed distinct miRNA profiles, with miRNA being uniquely expressed in TFH polarized cells (miR-26a-5p, miR-17-5p, miR-30d-5p, miR-22-3p, miR-222-3p, miR-142-3p, let-7i-5p and miR-29b-3p). In contrast, the TH1 lineage was enriched for expression of miR-155-5p, miR-146a-5p, miR-1246, miR-93-5p, miR-16-5p, miR-21-5p, miR-363-3p, miR-1260a, miR-186-5p, miR-148a-3p and miR-579-3p, whereas TH2 polarized cells expressed miR-181a-5p, let-7a-5p, miR-191-5p, miR-15b-5p, let-7d-5p, let-7b-5p, miR-140-5p, miR-98-5p, miR-423-5p and miR-630. Several of these miRNA expressed in the T-cells subsets showed corresponding expression in their respective PTCL entity such as miR-142-3p, let7i-5p, miR-21-5p and miR-29b-3p with AITL, miR-146-5p, miR-155-5p and miR-16-5p in PTCL-TBX21 and miR-181a-5p, miR-630 and let7a-5p in PTCL-GATA3. We also performed the MiRNA Enrichment Analysis and Annotation (miEAA) for miRNA signatures and observed an enrichment of miRNA regulating epigenetic modifications in TFH cells (p=0.028), whereas TH1 showed an enrichment of miRNA regulating IFN-g signaling (p=0.0024), and miRNA signatures in TH2 showed negative regulation of TGF-b signaling (p=0.023). Supervised analysis (p=0.05) of the miRNA profiles identified significant association of miR-126, miR-145, and let-7c-5p with AITL, when compared to other PTCLs. Similarly, miR-92a, miR-25, miR-636, miR-210, miR-222 and miR-491-5p significantly associated with PTCL-GATA3 and miRNA 126-3p, 145-5p, miR-26a-5p and miR-34a-5p associated with PTCL-TBX21. The miEAA for tumor miRNA signatures revealed enrichment of miRNAs regulating histone methylation (h3 k4 methylation) and chemokine receptor signaling in AITL, whereas miRNA regulating T-cell receptor were enriched in PTCL-TBX21 and TP53 signaling pathway in PTCL-GATA3. We validated the expression of miR-126 in AITL by qRT-PCR and also observed its increased expression in IL21 stimulated CD4+ T-cells. Ectopic expression of miR-126 resulted in a ~3 fold increased expression in T-cell lines and led to reduced proliferation and increased apoptosis with expression of T-cell exhaustion makers PD1 and TIM3. Computational algorithmic programs identified relevant biological targets of miR-126, including p85/PIK3R2, S1PR2 and DNMT3A that were further validated in-vitro. We observed an inverse correlation of miR-126 expression with S1PR2 expression (r=-0.64). S1PR2 is a crucial G protein-coupled receptor regulating B and T-cell migration in the germinal center (GC) reaction. Migration assays demonstrated significant decreases in T to B-cell migration, when B-cells (Raji) were co-cultured with Jurkat cells with ectopic expression of miR-126. With the GC reaction holding an important role in AITL, we investigated the biological significance of miRNA-126 in the context of the AITL microenvironment. High expression of miRNA-126 significantly associated with inferior survival in AITL (p=0.008) and significant differences in tumor microenvironment signatures. We identified distinct miRNA signatures for AITL and molecular subgroups of PTCL-NOS. Furthermore, elevated expression of miR-126 may contribute to the dysregulation and the homing of TFH cells in GC reaction through S1PR2 and warrants further mechanistic investigation. Disclosures No relevant conflicts of interest to declare.

Introduction: Transformed nodular lymphocyte predominant Hodgkin lymphoma (tNLPHL) with a typical diffuse large B-cell lymphoma (DLBCL) pattern is rare and not well studied by genomic analysis. We employed next generation sequencing and copy number analysis (CNA) to examine the pathogenesis of these tumors. Methods: We identified 19 cases of tNLPHL with DLBCL morphology and sheet-like growth from three institutions. NLPHL preceded transformation in 5 patients and was concurrent with transformation in 11. All cases of tNLPHL were sequenced using a targeted sequencing panel of 356 genes that included commonly mutated genes associated with lymphoma. We had 8 cases with matched germline DNA. We also performed CNA using Oncoscan on 18 cases of tNLPHL. Library preparation with paired end 100 bp sequencing and 6-10 million reads/case was performed on an Illumina HiSeq 2500. Fisher's exact test was used to compare the role of mutations in tNLPHL to three large series of de novo DLBCL. Results: The CNA showed frequent gains in REL and loss of CDKN2A. Mutation analysis showed frequent mutations of genes associated with the PI3K pathway such as SGK1 (26%), ZFP36L1 (16%), PIK3R1 (11%), and IL7R (11%), the NF-kB pathway such as CARD11 (21%), JUNB (21%), BCL10 (11%), NFKBIA (11%), TNFAIP3 (11%), histone/DNA modification such as KMT2D (26%), EP300 (21%), TET2 (11%), TET3 (11%), and the NOTCH pathway such as NOTCH2 (16%), NOTCH1 (1 case), CTBP2 (11%). Mutations in genes involved in immune surveillance and TP53 abnormalities were infrequent. Compared to de novo DLBCL, mutations in IL7R (10.5% vs 0.6%, p=0.03), JUNB (21% vs 4.2%, p=0.01), and SMARCAL1 (11% vs 0%, p=0.01) were significantly higher in tNLPHL than in germinal center B-cell (GCB) subtype of DLBCL. Conclusion: The mutational spectrum of tNLPHL resembles the DLBCL Cluster 4 of Chapuy et al (Nat Med, 2018), which were primarily GCB-DLBCL with frequent mutations in the PI3K pathway (SGK1), NF-kB pathway (CARD11, JUNB), and histone modification. The mutational spectrum is also distinctive in having frequent mutations that are not often seen together in DLBCL, such as TET2, JUNB and NOTCH2. Distinct from transformed follicular lymphoma, TP53 abnormalities and mutations affecting immune surveillance are uncommonly observed. This study provides new insights into the biology of tNLPHL and may highlight potential targets for therapy in the future. Disclosures Herrera: Adaptive Biotechnologies: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; AstraZeneca: Research Funding; Merck: Consultancy, Research Funding; Genentech, Inc.: Consultancy, Research Funding; Pharmacyclics: Research Funding; Immune Design: Research Funding; Kite Pharma: Consultancy, Research Funding.

ABT-348 [1-(4-(4-amino-7-(1-(2-hydroxyethyl)-1H-pyrazol-4-yl)thieno[3,2-c]pyridin-3-yl)phenyl)-3-(3-fluorophenyl)urea] is a novel ATP-competitive multitargeted kinase inhibitor with nanomolar potency (IC(50)) for inhibiting binding and cellular autophosphorylation of Aurora B (7 and 13 nM), C (1 and 13 nM), and A (120 and 189 nM). Cellular activity against Aurora B is reflected by inhibition of phosphorylation of histone H3, induction of polyploidy, and inhibition of proliferation of a variety of leukemia, lymphoma, and solid tumor cell lines (IC(50) = 0.3-21 nM). In vivo inhibition of Aurora B was confirmed in an engrafted leukemia model by observing a decrease in phosphorylation of histone H3 that persisted in a dose-dependent manner for 8 h and correlated with plasma concentration of ABT-348. Evaluation of ABT-348 across a panel of 128 kinases revealed additional potent binding activity (K(i) < 30 nM) against vascular endothelial growth factor receptor (VEGFR)/platelet-derived growth factor receptor (PDGFR) families and the Src family of cytoplasmic tyrosine kinases. VEGFR/PDGFR binding activity correlated with inhibition of autophosphorylation in cells and inhibition of vascular endothelial growth factor (VEGF)-stimulated endothelial cell proliferation (IC(50) ≤ 0.3 nM). Evidence of on-target activity in vivo was provided by the potency for blocking VEGF-mediated vascular permeability and inducing plasma placental growth factor. Activity against the Src kinase family was evident in antiproliferative activity against BCR-ABL chronic myeloid leukemia cells and cells expressing the gleevec-resistant BCR-ABL T315I mutation. On the basis of its unique spectrum of activity, ABT-348 was evaluated and found effective in representative solid tumor [HT1080 and pancreatic carcinoma (MiaPaCa), tumor stasis] and hematological malignancy (RS4;11, regression) xenografts. These results provide the rationale for clinical assessment of ABT-348 as a therapeutic agent in the treatment of cancer.

BCL6 encodes a transcriptional repressor that is essential for the germinal center (GC) reaction and important in lymphomagenesis. Although its promoter has been well studied, little is known concerning its possible regulation by more distal elements. To gain such information, we mapped critical histone modifications associated with active transcription within BCL6 as well as far upstream sequences at nucleosomal resolution in B-cell lines and in normal naive and GC B cells. Promoter-associated and intronic CpG islands (CGIs) in BCL6 showed a reciprocal pattern of histone modifications. Gene expression correlated with a paradoxical loss from the intronic CGI of histone H3 lysine-4 trimethylation, normally associated with transcription, suggesting that the intronic CGI may interfere with transcription. In an ∼110-kb region extending 150–260 kb upstream of BCL6 , highly active histone modifications were present only in normal GC B cells and a GC B-cell line; this region overlaps with an alternative breakpoint region for chromosomal translocations and contains a GC-specific noncoding RNA gene. By chromosome conformation capture, we determined that the BCL6 promoter interacts with this distant upstream region. It is likely that transcriptional enhancers in this region activate BCL6 and overcome strong autorepression in GC B cells.

Apoptosis is a highly regulated process of programmed cell death essential for normal physiology. Dysregulation of apoptosis contributes to the development and progression of various diseases, including cancer, neurodegenerative disorders, and chronic heart failure. Quantitative noninvasive imaging of apoptosis in preclinical models would allow for dynamic longitudinal screening of compounds and facilitates a more rapid determination of therapeutic efficacy. In this study, we report the in vivo characterization of Z-DEVD-aminoluciferin, a modified firefly luciferase substrate that in apoptotic cells is cleaved by caspase-3 to liberate aminoluciferin, which can be consumed by luciferase to generate a luminescent signal. In two oncology models, namely SKOV3-luc and MDA-MB-231-luc-LN, at 24, 48, and 72 h after treatment with docetaxel, animals were injected with Z-DEVD-aminoluciferin and bioluminescent images were acquired. Significantly more light was detected at 24 (P

A mark–recapture field study was conducted to determine fish passage at 5 concrete box culverts and 5 low-water crossings (concrete slabs vented by culverts) as well as 10 control sites (below a natural riffle) in Flint Hills streams of northeastern Kansas. Additionally, we tested the upstream passage of four fish species native to Great Plains streams (Topeka shiner Notropis topeka, green sunfish Lepomis cyanellus, red shiner Cyprinella lutrensis, and southern redbelly dace Phoxinus erythrogaster) through three simulated crossing designs (box culverts, round corrugated culverts, and natural rock riffles) at water velocities of 0.1 to 1.1 m/s in an experimental stream. The field study indicated that cyprinids were twice as likely to move upstream of box culverts than low-water crossings and 1.4 times as likely to move upstream of control reaches than any crossing type. The best models indicated that the proportion of cyprinids that moved upstream increased with decreased culvert slope and length,...

Small molecule inhibitors of PARP-1 have been pursued by various organizations as potential therapeutic agents either capable of sensitizing cytotoxic treatments or acting as stand-alone agents to combat cancer. As one of the strategies to expand our portfolio of PARP-1 inhibitors, we pursued unsaturated heterocycles to replace the saturated cyclic amine derivatives appended to the benzimidazole core. Not only did a variety of these new generation compounds maintain high enzymatic potency, many of them also displayed robust cellular activity. For example, the enzymatic IC(50) and cellular EC(50) values were as low as 1 nM or below. Compounds 24 (EC(50) = 3.7 nM) and 44 (EC(50) = 7.8 nM), featuring an oxadiazole and a pyridine moiety, respectively, demonstrated balanced potency and PK profiles. In addition, these two molecules exhibited potent oral in vivo efficacy in potentiating the cytotoxic agent temozolomide in a B16F10 murine melanoma model.

The oncoprotein murine double minute 2 (Mdm2) is frequently overexpressed in many types of human malignancies. Although Mdm2 has an essential role in negatively regulating the p53 tumor suppressor, it also has less well characterized p53-independent functions that influence pathways that are crucial for controlling tumorigenesis. In addition to the impact Mdm2 has on p53-independent apoptosis, mounting evidence is linking increased Mdm2 levels to altered cell-cycle regulation, DNA replication and DNA repair leading to loss of genome stability. Mdm2 involvement in pathways that influence chromosome stability and cell death, distinct from its role in the p53 pathway, strengthens the position of Mdm2 as a desirable therapeutic target for the treatment of human cancers.

Mdm2 is a critical negative regulator of the p53 tumor suppressor and is frequently overexpressed in human cancers. However, reports, including our own studies, suggest that Mdm2 has both p53-dependent and p53-independent functions that contribute to genomic instability and transformation when deregulated. We recently elucidated a p53-independent role for Mdm2 in the regulation of the DNA double-strand break repair response, genomic stability, and transformation through interaction with Nbs1, a member of the Mre11/Rad50/Nbs1 DNA double-strand break repair complex. In light of these findings, targeting Mdm2 in human malignancies may have effects other than activating p53. [Cancer Res 2009;69(5):1697–701]

AZD1152 is a highly selective Aurora B kinase inhibitor currently undergoing Phase I and II clinical evaluation in patients with acute myelogenous leukemia and advanced solid malignancies. We have established two AZD1152-resistant cell lines from SW620 colon and MiaPaCa pancreatic carcinoma lines, which are >100-fold resistant to the active metabolite of AZD1152, AZD1152 HQPA and interestingly, cross-resistant to the pan-Aurora kinase inhibitor, VX-680/MK0457. Using whole-genome microarray analysis and comparative genomic hybridization, we were able to identify MDR1 and BCRP as the causative genes that underlie AZD1152 HQPA-resistance in these models. Furthermore, the upregulation of either of these genes is sufficient to render in vivo tumor growth insensitive to AZD1152. Finally, the upregulation of MDR1 or BCRP is predictive of tumor cell sensitivity to this agent, both in vitro and in vivo. The data provide a genetic basis for resistance to Aurora kinase inhibitors, which could be utilized to predict clinical response to therapy.

We have developed a series of cyclic amine-containing benzimidazole carboxamide poly(ADP-ribose)polymerase (PARP) inhibitors, with good PARP-1 enzyme potency, as well as cellular potency. These efforts led to the identification of a lead preclinical candidate, 10b, 2-(1-propylpiperidin-4-yl)-1H-benzimidazole-4-carboxamide (A-620223). 10b displayed very good potency against both the PARP-1 enzyme with a Ki of 8 nM and in a whole cell assay with an EC50 of 3 nM. 10b is aqueous soluble, orally bioavailable across multiple species, and demonstrated good in vivo efficacy in a B16F10 subcutaneous murine melanoma model in combination with temozolomide (TMZ) and in an MX-1 breast xenograph model in combination with cisplatin.

This laboratory and others have shown that agents that inhibit the in vitro catalytic activity of methionine aminopeptidase-2 (MetAP2) are effective in blocking angiogenesis and tumor growth in preclinical models. However, these prototype MetAP2 inhibitors are clearly not optimized for therapeutic use in the clinic. We have discovered an orally active class of MetAP2 inhibitors, the anthranilic acid sulfonamides exemplified by A-800141, which is highly specific for MetAP2. This orally bioavailable inhibitor exhibits an antiangiogenesis effect and a broad anticancer activity in a variety of tumor xenografts including B cell lymphoma, neuroblastoma, and prostate and colon carcinomas, either as a single agent or in combination with cytotoxic agents. We also have developed a biomarker assay to evaluate in vivo MetAP2 inhibition in circulating mononuclear cells and in tumors. This biomarker assay is based on the N -terminal methionine status of the MetAP2-specific substrate GAPDH in these cells. In cell cultures in vitro , the sulfonamide MetAP2 inhibitor A-800141 caused the formation of GAPDH variants with an unprocessed N-terminal methionine. A-800141 blocked tumor growth and MetAP2 activity in a similar dose–response in mouse models, demonstrating the antitumor effects seen for A-800141 are causally connected to MetAP2 inhibition in vivo . The sulfonamide MetAP2 inhibitor and GAPDH biomarker in circulating leukocytes may be used for the development of a cancer treatment.

Tumor angiogenesis is mediated by KDR and other VEGFR and PDGFR kinases. Their inhibition presents an attractive approach for developing anticancer therapeutics. Here, we report a series of aminopyrazolopyridine ureas as potent VEGFR/PDGFR multitargeted kinase inhibitors. A number of compounds have been identified to be orally bioavailable and efficacious in the mouse edema model.

A series of 1,4-dihydroindeno[1,2-c]pyrazole compounds with a cyanopyridine moiety at the 3-position of the tricyclic pyrazole core was explored as potent CHK-1 inhibitors. The impact of substitutions at the 6 and/or 7-position of the core on pharmacokinetic properties was studied in detail. Compounds carrying a side chain with an ether linker at the 7-position and a terminal morpholino group, such as 29 and 30, exhibited much-improved oral biovailability in mice as compared to earlier generation inhibitors. These compounds also possessed desirable cellular activity in potentiating doxorubicin and will serve as valuable tool compounds for in vivo evaluation of CHK-1 inhibitors to sensitize DNA-damaging agents.

A novel series of 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-ones have been synthesized as potent and selective checkpoint kinase 1 (Chk1) inhibitors via structure-based design. Aided by protein X-ray crystallography, medicinal chemistry efforts led to the identification of compound 46d, with potent enzymatic activity against Chk1 kinase. While maintaining a low cytotoxicity of its own, compound 46d exhibited a strong ability to abrogate G2 arrest and increased the cytotoxicity of camptothecin by 19-fold against SW620 cells. Pharmacokinetic studies revealed that it had a moderate bioavailabilty of 20% in mice. Two important binding interactions between compound 46b and Chk1 kinase, revealed by X-ray cocrystal structure, were hydrogen bonds between the hinge region and the amide bond of the core structure and a hydrogen bond between the methoxy group and Lys38 of the protein.

A study on substitutions at the four open positions on the phenyl ring of the 1,4-dihydroindeno[1,2-c]pyrazoles as potent CHK-1 inhibitors is described. Bis-substitution at both the 6- and 7-positions led to inhibitors with IC(50) values below 0.3nM. The compound with the best overall activities (36) was able to potentiate the anti-proliferative effect of doxorubicin in HeLa cells by at least 47-fold. Physicochemical, metabolic, and pharmacokinetic properties of selected inhibitors are also disclosed.

The synthesis of a novel series of 1,4-dihydroindeno[1,2-c]pyrazoles with acetylene-type side chains is described. Optimization of those compounds as KDR kinase inhibitors identified 8, which displayed an oral activity in an estradiol-induced murine uterine edema model (ED50 = 3 mg/kg) superior to Sutent (ED50 = 9 mg/kg) and showed potent antitumor efficacy in an MX-1 human breast carcinoma xenograft tumor growth model (tumor growth inhibition = 90% at 25 mg/kg.day po). The compound was docked into a homology model of the homo-tetrameric pore domain of the hERG potassium channel to identify strategies to improve its cardiac safety profile. Systematic interruption of key binding interactions between 8 and Phe656, Tyr652, and Ser624 yielded 90, which only showed an IC50 of 11.6 microM in the hERG patch clamp assay. The selectivity profile for 8 and 90 revealed that both compounds are multitargeted receptor tyrosine kinase inhibitors with low nanomolar potencies against the members of the VEGFR and PDGFR kinase subfamilies.

Selenium (Se) was applied to perennial ryegrass (Lolium perenne) and subterranean clover (Trifolium subterranean) pasture as a fertilizer to determine the effect of Se form and concentration on Se accumulation in subsequent forage growth. Treatments were a no Se control, 0.6 kg/ ha Se as sodium selenate, and 0.6, 1.1, and 2.2 kg/ha Se as sodium selenite, all applied to pasture plots with low soil Se concentration in southwestern Oregon (n = 3 plots per treatment). The plots were protected from grazing by use of electric fence, and total forage DM production and Se concentrations were measured after the spring growing season in yr 1. Pastures were grazed by sheep over the fall growing season, but then were protected from spring grazing to enable sampling of residual forage Se concentrations during yr 2. Application of 0.6 kg/ ha selenate provided greater (P < 0.01) average forage Se content in yr 1 (8.44 ± 0.08 mg/kg) than all other treatments. Compared with the control (0.09 ± 0.06 mg/kg), the plots in the 0.6 and 2.2 kg/ ha selenite treatments contained greater

Angioimmunoblastic T-cell lymphoma (AITL) is a common subtype of peripheral T-cell lymphoma (PTCL) with a poor prognosis. We performed targeted resequencing on 92 cases of PTCL and identified frequent mutations affecting RHOA, TET2, DNMT3A, and isocitrate dehydrogenase 2 (IDH2). Although IDH2 mutations are largely confined to AITL, mutations of the other 3 can be found in other types of PTCL, although at lower frequencies. These findings indicate a key role of epigenetic regulation in the pathogenesis of AITL. However, the epigenetic alterations induced by these mutations and their role in AITL pathogenesis are still largely unknown. We correlated mutational status with gene expression and global DNA methylation changes in AITL. Strikingly, AITL cases with IDH2(R172) mutations demonstrated a distinct gene expression signature characterized by downregulation of genes associated with TH1 differentiation (eg, STAT1 and IFNG) and a striking enrichment of an interleukin 12-induced gene signature. Ectopic expression of IDH2(R172K) in the Jurkat cell line and CD4(+) T cells led to markedly increased levels of 2-hydroxyglutarate, histone-3 lysine methylation, and 5-methylcytosine and a decrease of 5-hydroxymethylcytosine. Correspondingly, clinical samples with IDH2 mutations displayed a prominent increase in H3K27me3 and DNA hypermethylation of gene promoters. Integrative analysis of gene expression and promoter methylation revealed recurrently hypermethylated genes involved in T-cell receptor signaling and T-cell differentiation that likely contribute to lymphomagenesis in AITL.