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The 90 kDa Ribosomal S6 Kinase (RSK) drives cell proliferation and survival in cancers, although its oncogenic mechanism has not been well characterized. Phosphorylated level of RSK (T573) was increased in acute myeloid leukemia (AML) patients and associated with poor survival. To examine the role of RSK in AML, we analyzed apoptosis and the cell cycle profile following treatment with BI-D1870, a potent inhibitor of RSK. BI-D1870 treatment increased the G2/M population and induced apoptosis in AML cell lines and patient AML cells. Characterization of mitotic phases showed that the metaphase/anaphase transition was significantly inhibited by BI-D1870. BI-D1870 treatment impeded the association of activator CDC20 with APC/C, but increased binding of inhibitor MAD2 to CDC20, preventing mitotic exit. Moreover, the inactivation of spindle assembly checkpoint or MAD2 knockdown released cells from BI-D1870-induced metaphase arrest. Therefore, we investigated whether BI-D1870 potentiates the anti-leukemic activity of vincristine by targeting mitotic exit. Combination treatment of BI-D1870 and vincristine synergistically increased mitotic arrest and apoptosis in acute leukemia cells. These data show that BI-D1870 induces apoptosis of AML cells alone and in combination with vincristine through blocking mitotic exit, providing a novel approach to overcoming vincristine resistance in AML cells.

A molecular systems architecture is presented for acute myeloid leukemia (AML) to provide a framework for organizing the complexity of biomolecular interactions. AML is a multifactorial disease resulting from impaired differentiation and increased proliferation of hematopoietic precursor cells involving genetic mutations, signaling pathways related to the cancer cell genetics, and molecular interactions between the cancer cell and the tumor microenvironment, including endothelial cells, fibroblasts, myeloid-derived suppressor cells, bone marrow stromal cells, and immune cells (e.g., T-regs, T-helper 1 cells, T-helper 17 cells, T-effector cells, natural killer cells, and dendritic cells). This molecular systems architecture provides a layered understanding of intra- and inter-cellular interactions in the AML cancer cell and the cells in the stromal microenvironment. The molecular systems architecture may be utilized for target identification and the discovery of single and combination therapeutics and strategies to treat AML.

Dasatinib is a multi-tyrosine kinase inhibitor approved for treatment of Ph+ acute lymphoblastic leukemia (ALL), but its efficacy is limited by resistance. Recent preclinical studies suggest that dasatinib may be a candidate therapy in additional ALL subtypes including pre-BCR+ ALL. Here we utilized shRNA library screening and global transcriptomic analysis to identify several novel genes and pathways that may enhance dasatinib efficacy or mitigate potential resistance in human pre-BCR+ ALL. Depletion of the transcriptional coactivator CBP increased dasatinib sensitivity by downregulating transcription of the pre-BCR signaling pathway previously associated with dasatinib sensitivity. Acquired resistance was due, in part, to upregulation of alternative pathways including WNT through a mechanism, suggesting transcriptional plasticity. Small molecules that disrupt CBP interactions with the CREB KID domain or β-catenin showed promising preclinical efficacy in combination with dasatinib. These findings highlight novel modulators of sensitivity to targeted therapies in human pre-BCR+ ALL, which can be reversed by small-molecule inhibitors. They also identify promising therapeutic approaches to ameliorate dasatinib sensitivity and prevent resistance in ALL. Significance: These findings reveal mechanisms that modulate sensitivity to dasatinib and suggest therapeutic strategies to improve the outcome of patients with acute lymphoblastic leukemia. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/22/6497/F1.large.jpg. Cancer Res; 78(22); 6497–508. ©2018 AACR.

To examine the ability of different elements of the Self-Feeding Assessment Tool for Elderly with Dementia (SFED) to predict mortality risk in nursing home residents.Data from 387 residents in five nursing homes for the elderly in Japan were obtained using a baseline survey. This measure's ability to predict mortality risk was examined over a two-year observation period. Participants and Measurement: Demographic information (sex, age, height, weight, medical history) on 387 initial participants was gathered. A total of 10 individuals were excluded from the analysis because of the inability to eat by mouth at baseline, while 36 were excluded owing to missing mortality data during the observation period. The resulting 341 residents were divided into a death group or survival group according to whether they were still alive after two-year observation period. In addition to basic information and the SFED, the baseline survey included the Barthel Index (BI), Clinical Dementia Rating (CDR), and Mini Nutritional Assessment-Short Form (MNA®-SF). The ability of SFED to predict time-to-event mortality was examined using Cox proportional hazards regression analysis, including other measures associated with mortality as confounding variables.In total, 129 participants (37.8%) died during the observation period, and their mean SFED score was significantly lower than that of surviving ones (11.1 ± 6.7 vs. 15.0 ± 5.6, P0.001). SFED score was significantly associated with two-year mortality in the Cox proportional hazards regression analysis after adjusting for sex, age, medical history, BI, CDR, and MNA®-SF (hazard ratio = 0.941, 95% confidence interval = 0.898-0.985, P = 0.010). Additionally, three SFED categories were significantly associated with mortality risk: movement ("able to eat without dropping food"), concentration ("able to maintain attention to meal"), and safety ("able to swallow without choking, with no change in vocal quality after eating").Self-feeding ability as measured by SFED score was associated with long-term mortality in elderly living in nursing homes. Accordingly, adjusting feeding assistance based on regular SFED-based assessments may help maintain self-feeding ability and enhance quality of life in this population, as well as providing evidence for end-of-life care options and greatly improving care quality.

Aims We aim to determine if Pseudomonas fluorescens is a viable biological control for Erwinia tracheiphila within the insect vector, Acalymma vittatum. Methods and results Pseudomonas fluorescens secreted fluorescein and inhibited growth of E. tracheiphila in disc diffusion assays. To determine if this antagonism was conserved within the insect vector, we performed in vivo assays by orally injecting beetles with bacterial treatments and fluorescent in situ hybridization to determine bacterial presence within the alimentary canal. Conclusions Pseudomonas fluorescens inhibited the growth of E. tracheiphila on a nutrient-limiting medium. In situ experiments demonstrated that P. fluorescens is maintained within the alimentary canal of the beetle for at least 4 days, and co-occurred with E. tracheiphila. When beetles were first presented with Pseudomonas and then challenged with E. tracheiphila, E. tracheiphila was not recovered via FISH after 4 days. These data suggest that P. fluorescens has potential as a biological control agent to limit E. tracheiphila within the insect vector. Significance and impact of the study This is a novel approach for controlling E. tracheiphila that has the potential to decrease reliance on insecticides, providing a safer environment for pollinators and growers.

Bee viral ecology is a fascinating emerging area of research: viruses exert a range of effects on their hosts, exacerbate impacts of other environmental stressors, and, importantly, are readily shared across multiple bee species in a community. However, our understanding of bee viral communities is limited, as it is primarily derived from studies of North American and European Apis mellifera populations. Here, we examined viruses in populations of A. mellifera and 11 other bee species from 9 countries, across 4 continents and Oceania. We developed a novel pipeline to rapidly and inexpensively screen for bee viruses. This pipeline includes purification of encapsulated RNA/DNA viruses, sequence-independent amplification, high throughput sequencing, integrated assembly of contigs, and filtering to identify contigs specifically corresponding to viral sequences. We identified sequences for (+)ssRNA, (−)ssRNA, dsRNA, and ssDNA viruses. Overall, we found 127 contigs corresponding to novel viruses (i.e. previously not observed in bees), with 27 represented by >0.1% of the reads in a given sample, and 7 contained an RdRp or replicase sequence which could be used for robust phylogenetic analysis. This study provides a sequence-independent pipeline for viral metagenomics analysis, and greatly expands our understanding of the diversity of viruses found in bee communities.

The 9vHPV vaccine was efficacious, immunogenic, and well-tolerated in Asian participants from 2 international, randomized clinical trials. Data support 9vHPV vaccination programs in Asia., Background A 9-valent human papillomavirus-6/11/16/18/31/33/45/52/58 (9vHPV) vaccine extends coverage to 5 next most common oncogenic types (31/33/45/52/58) in cervical cancer versus quadrivalent HPV (qHPV) vaccine. We describe efficacy, immunogenicity, and safety in Asian participants (India, Hong Kong, South Korea, Japan, Taiwan, and Thailand) from 2 international studies: a randomized, double-blinded, qHPV vaccine-controlled efficacy study (young women aged 16–26 years; NCT00543543; Study 001); and an immunogenicity study (girls and boys aged 9–15 years; NCT00943722; Study 002). Methods Participants (N = 2519) were vaccinated at day 1 and months 2 and 6. Gynecological samples (Study 001 only) and serum were collected for HPV DNA and antibody assessments, respectively. Injection-site and systemic adverse events (AEs) were monitored. Data were analyzed by country and vaccination group. Results 9vHPV vaccine prevented HPV-31/33/45/52/58–related persistent infection with 90.4%–100% efficacy across included countries. At month 7, ≥97.9% of participants seroconverted for each HPV type. Injection-site AEs occurred in 77.7%–83.1% and 81.9%–87.5% of qHPV and 9vHPV vaccine recipients in Study 001, respectively, and 62.4%–85.7% of girls/boys in Study 002; most were mild to moderate. Conclusions The 9vHPV vaccine is efficacious, immunogenic, and well tolerated in Asian participants. Data support 9vHPV vaccination programs in Asia. Clinical Trials Registration NCT00543543; NCT00943722.

Simple Summary To investigate whether pediatric and adult chronic myeloid leukemia (CML) have unique molecular characteristics, we studied the transcriptomic signature of pediatric and adult CML cells using high-throughput RNA sequencing. We identified differentially expressed genes and pathways unique to pediatric CML cells compared to adult CML cells. The Rho pathway was significantly dysregulated in pediatric CML cells compared to adult CML cells, suggesting the potential importance in the pathogenesis of pediatric CML. Our study is the first to compare transcriptome profiles of CML across different age groups. A better understanding of the biology of CML across different ages may inform future treatment approaches. Abstract Children with chronic myeloid leukemia (CML) tend to present with higher white blood counts and larger spleens than adults with CML, suggesting that the biology of pediatric and adult CML may differ. To investigate whether pediatric and adult CML have unique molecular characteristics, we studied the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy pediatric and adult CD34+ control cells. Using high-throughput RNA sequencing, we found 567 genes (207 up- and 360 downregulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. Directly comparing pediatric and adult CML CD34+ cells, 398 genes (258 up- and 140 downregulated), including many in the Rho pathway, were differentially expressed in pediatric CML CD34+ cells. Using RT-qPCR to verify differentially expressed genes, VAV2 and ARHGAP27 were significantly upregulated in adult CML CD34+ cells compared to pediatric CML CD34+ cells. NCF1, CYBB, and S100A8 were upregulated in adult CML CD34+ cells but not in pediatric CML CD34+ cells, compared to healthy controls. In contrast, DLC1 was significantly upregulated in pediatric CML CD34+ cells but not in adult CML CD34+ cells, compared to healthy controls. These results demonstrate unique molecular characteristics of pediatric CML, such as dysregulation of the Rho pathway, which may contribute to clinical differences between pediatric and adult patients.

Chronic myeloid leukemia (CML) accounts for 2-9% of leukemias in children and adolescents, and occurs with much greater frequency in adults. Compared to adults, children with CML tend to present with higher white blood cell counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML are due to unique molecular characteristics that differ from adult CML. To test this hypothesis, we compared the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy age-matched CD34+ cells. CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric healthy (n=10), and adult healthy (n=10) bone marrow samples. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes. Four comparisons were performed: (1) pediatric CML vs pediatric healthy, (2) adult CML vs adult healthy, (3) pediatric CML vs adult CML, and (4) pediatric healthy vs adult healthy. A False Discovery Rate of ≤ .05 and absolute log2 fold-change > 1 was used to define differentially expressed genes (DEGs) in each comparison. To identify potentially unique pathways based on DEG, pathway over-representation was calculated with either goana from the limma package or clueGO. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients. Median WBC counts were 273,000 and 143,000 in pediatric and adult patients respectively. A total of 1352 genes were differentially expressed in either adult or pediatric CML CD34+ cells compared to healthy CD34+ cells, 174 of which were expressed similarly in pediatric and adult CML CD34+ cells (54 up- and 120 down-regulated). There were 746 differentially expressed genes (325 up- and 421 down-regulated) in adult CML CD34+ cells compared to adult healthy CD34+ cells, and 432 differentially expressed genes (156 up- and 276 down-regulated) in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. In direct comparison of pediatric and adult CML CD34+ cells, 446 genes (270 up and 176 down) were dysregulated in pediatric CML CD34+ cells. Pathway analysis showed that Rho signaling pathway was downregulated in pediatric CML CD34+ cells and several genes in Rho pathway were uniquely dysregulated. ARHGAP27 and VAV2 were significantly upregulated in adult CML CD34+ cells by 3.7-fold (p=0.0453) and 11-fold (p=0.0072), respectively, compared to pediatric CML CD34+ cells. In addition, several genes involved in the NADPH oxidase pathway, one of the best-characterized Rho GTPase-regulated systems, were differently expressed in CML. NCF1, CYBB, and S100A8 were significantly upregulated in adult CML CD34+ cells by 4-fold (p=0.0045), 3.26-fold (p These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the molecular biology of CML across the ages will provide new insights into the pathogenesis of pediatric CML and potentially inform future treatment decisions. Disclosures Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria. Hijiya: Novartis: Consultancy; Stemline Therapeutics: Consultancy.

Diamond Blackfan Anemia (DBA) is associated with a hypoproliferative anemia, congenital abnormalities, and cancer. The disease typically presents within the first year of life with the majority of patients carrying mutations in one of at least 17 ribosomal proteins, with RPS19 being the most common. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions, chronic effects from long term corticosteroid use, or complications from stem cell transplantation. The serine threonine kinase Nemo-like Kinase (NLK) is an atypical member of the MAP kinase family of enzymes and has been shown to be chronically hyper-activated in RPS19- and RPL11-haploinsufficient murine and human models of DBA, as well as in erythroid progenitors from DBA patients. In RPS19-insufficient human hematopoietic stem and progenitor cells, genetic silencing of NLK by shRNA increased erythroid expansion by 220.3% (SD = 6.6%), indicating that aberrant NLK activation may contribute to the pathogenesis of the disease and is a potential target for DBA therapy. A number of clinically approved or advanced compounds have been developed to inhibit MAP kinases with various degrees of cross reactivity among its family members. We therefore screened a number of compounds that inhibit NLK as an off-target and found that these NLK inhibitors improved erythroid expansion in DBA models. Of these inhibitors, OTS-167 performed optimally, improving erythropoiesis by 2-fold at 300nM, with an EC50 of 146nM. Previous studies of OTS-167 in xenograft models of neuroblastoma for one month did not result in neutropenia, suggesting very little to no toxicity to myeloid cells. The goal in treating DBA patients with NLK inhibitors is to sufficiently raise the hemoglobin to prevent the need for chronic red cell transfusions or treatment with steroids. Our results suggest that pharmacologic inhibition of NLK is a potential approach to treat patients with DBA. We are currently investigating other NLK inhibitors in preclinical models for future clinical application. Disclosures Glader: Agios: Consultancy.

Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is typically associated with mutations in one of at least 20 ribosomal genes and is associated with anemia, congenital abnormalities, and cancer predisposition. The current treatment for DBA is associated with toxicities including iron overload from repeated transfusions, immune suppression from chronic corticosteroid therapy or consequences from stem cell transplantation. Developing new therapies for DBA remains a challenge, since it is a rare disease and the connection between faulty ribosomes and defects in erythropoiesis is still being explored. As such, repurposing existing, approved drugs is one approach to find new ways to treat this disease. We recently identified that Nemo-like Kinase (NLK) is hyper-activated and contributes to disease pathogenesis in the erythroid progenitors of patients with DBA, irrespective of the genetic mutation. NLK is an atypical member of the MAPK family of kinases. Due to a high degree of conservation, kinase inhibitors that specifically target NLK are not currently available, although several small molecules inhibit NLK as an off-target. We are actively pursuing a number of these compounds as potential therapies for DBA. In addition to known kinase inhibitors, we have examined the active components of widely available nutritional supplements. Although rigorous clinical evaluation is required, these supplements are well tolerated and offer an alternative or a complement to conventional drug therapy for DBA. Ginsenoside Rb1, an active component of ginseng, increases erythropoiesis in in vitro models of DBA (2.6-fold) at 50mM (p=0.048) with an EC 50 of 2.3mM. Importantly, ginsenoside Rb1 does not impact healthy erythropoiesis or other hematopoietic lineages and is nontoxic to normal cells. Our results demonstrate that ginsenoside Rb1 does not inhibit NLK kinase activity directly, but rather induces a microRNA (miR-208) that binds to NLK mRNA leading to degradation of transcript by 35.9% (p=0.007) before the protein can be translated. Another nutritional supplement that has been indicated to improve erythropoiesis (in DBA and non-diseased models) and is currently in clinical trials is the amino acid leucine. In DBA progenitors, 5mM leucine increases erythropoiesis from 8.8 to 16.3% of healthy controls. Leucine acts by stimulating the activity of mTORC1 but similar to erythropoiesis, mTORC1 activity is only stimulated from 26.4 to 57.2% of controls in DBA progenitors. Our results demonstrate that leucine does not impact NLK expression or activity directly. Aberrantly activated NLK phosphorylates and inhibits the activation of mTORC1 (target of leucine). The suppression of NLK by 50mM ginsenoside Rb1 restored mTORC1 activity to basal (94.7% of non-diseased control) but also restored leucine sensitivity (from 57.2 to 88.1% of non-diseased controls). Importantly, combining 5mM leucine and 50mM ginsenoside Rb1 increased erythropoiesis from 1.9-fold and 2.6-fold when used alone, to 8.9-fold (or 78.3% of healthy controls) together in our in vitro models of DBA (CI=0.31). Ginseng and leucine offer promising alternatives to steroids and other immunosuppressive drugs for DBA patients. The goal of these studies is to raise the hemoglobin in DBA patients to avoid the need for red cell transfusions. As nutritional supplements are widely available and well tolerated, this class of compounds provides alternatives to currently approved drugs to treat DBA. Disclosures Glader: Agios: Consultancy.

Juvenile myelomonocytic leukemia (JMML) is a deadly pediatric cancer characterized by excessive accumulation of monocytes/macrophages with myelodysplastic features and splenomegaly. Hematopoietic stem cell transplantation (HSCT) is the only curative option, but relapse occurs in ~50% of the patients. Mutations in the RAS pathway are the major drivers and DNA hypermethylation is associated with severe clinical phenotype. DNA methylation is linked to histone modifications in normal cell development and cancer pathogenesis. Gene expression levels are programmed by histone modifications. However, a comprehensive characterization of histone modifications in JMML is yet to be elucidated. It was reported that the CD34+ hematopoietic stem and progenitors cells (HSPCs) possess leukemia initiation potential (Caye et al., 2015; Krombholz et al., 2016; Louka et al., 2021; Yoshimi et al., 2017). We hypothesized that dysregulated histone modifications contribute to leukemogenesis in JMML. To this goal, we investigated epigenetic landscape at the single-cell level in JMML HSPCs using cytometry by time-of-flight (EpiTOF). We isolated HSPCs from the spleens of JMML patients with PTPN11 mutations which were collected at diagnosis, before treatment (provided by the European Working Groups of Myelodysplastic Syndromes). The CD34+ cells from JMML Spleens (n=5) and Bone Marrow (BM n=1) alongside healthy controls (cord blood CB n=5; BM n=2) were stained with antibodies against 15 immunophenotypic and 46 key histone markers and subsequently analyzed on a mass cytometer (Fluidigm, Helios). We found that a group of key histone markers were significantly downregulated in JMML in comparison to healthy CBs, including H4K16ac, H3K23ac, H3K56ac, H3K36me1, H3K9me1 and H3K27me2 (Fig.1). Dimensionality reduction analyses showed a global reduction in histone acetylation in all JMML spleen samples (~70%) when compared to CBs (average >95%). Our study revealed significant dysregulation of key histone markers and their respective modifiers (histone modifying enzymes) within the leukemogenic CD34+ compartment (Fig.2). Our findings demonstrated an aberrant epigenetic regulation in JMML patient samples with concurrent loss of histone acetylation and some histone methylation markers, which have been reported as hallmarks of other cancers. Further characterization of the epigenetic landscape in JMML was performed using transposase-accessible chromatin with high-throughput sequencing (bulk ATAC-seq). Notably, ATAC-seq data revealed significantly lower expression of key histone acetyltranferases (HAT), such as Kat6b and Kat8 (Fig.2) , which specifically regulate acetylation of H3K23 and H4K16 respectively. Additionally, an increased activity of histone deacetylase, HDAC9, was also observed in these samples (Fig.2). Collectively, our data suggests aberrant activities of histone modifying enzymes (HMEs) in JMML HSPCs (Fig.2), with both increased HDAC and decreased HAT activities observed in our ATAC-data. This finding is concurrent with our EpiTOF findings of global loss of histone acetylation in JMML (Fig.1A). It is known that histone modifications are easier to reverse when compared to DNA methylation, and we believe targeted inhibition of specific HDACs that we have found to be upregulated in JMML (such as HDAC9) might hold great therapeutic potential. Additionally, investigation into the impact of altered chromatin structure of JMML HSPCs on gene expression is currently ongoing using single-cell RNA-seq. In conclusion, our study identified a novel mechanism of epigenetic dysregulation in putative JMML leukemia initiating cells related to imbalanced activities of histone modifying enzymes which could potentially represent future therapeutic targets. Figure 1 Figure 1. Disclosures Bertaina: AdicetBio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees.

Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion. To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs. SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype. SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis. HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes. While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. Disclosures No relevant conflicts of interest to declare.

Blood cell development is regulated through intrinsic gene regulation and local factors including the microenvironment and cytokines. The differentiation of hematopoietic stem and progenitor cells (HSPCs) into mature erythrocytes is dependent on these cytokines binding to and stimulating their cognate receptors and the signaling cascades they initiate. Many of these pathways include kinases that can diversify signals by phosphorylating multiple substrates and amplify signals by phosphorylating multiple copies of each substrate. Indeed, synthesis of many of these cytokines is regulated by a number of signaling pathways including phosphoinositide 3-kinase (PI3K)-, extracellular signal related kinases (ERK)-, and p38 kinase-dependent pathways. Therefore, kinases act both upstream and downstream of the erythropoiesis-regulating cytokines. While many of the cytokines are well characterized, the nuanced members of the network of kinases responsible for appropriate induction of, and response to, these cytokines remains poorly defined. Here, we will examine the kinase signaling cascades required for erythropoiesis and emphasize the importance, complexity, enormous amount remaining to be characterized, and therapeutic potential that will accompany our comprehensive understanding of the erythroid kinome in both healthy and diseased states.

Nemo-like kinase (NLK) is a member of the mitogen-activated protein kinase family of kinases and shares a highly conserved kinase domain with other mitogen-activated protein kinase family members. The activation of NLK contributes to the pathogenesis of Diamond–Blackfan anemia (DBA), reducing c-myb expression and mechanistic target of rapamycin activity, and is therefore a potential therapeutic target. Unlike other anemias, the hematopoietic effects of DBA are largely restricted to the erythroid lineage. Mutations in ribosomal genes induce ribosomal insufficiency and reduced protein translation, dramatically impacting early erythropoiesis in the bone marrow of patients with DBA. We sought to identify compounds that suppress NLK and increases erythropoiesis in ribosomal insufficiency. We report that the active component of ginseng, ginsenoside Rb1, suppresses NLK expression and improves erythropoiesis in in vitro models of DBA. Ginsenoside Rb1–mediated suppression of NLK occurs through the upregulation of miR-208, which binds to the 3′-UTR of NLK mRNA and targets it for degradation. We also compare ginsenoside Rb1–mediated upregulation of miR-208 with metformin-mediated upregulation of miR-26. We conclude that targeting NLK expression through miRNA binding of the unique 3′-UTR is a viable alternative to the challenges of developing small-molecule inhibitors to target the highly conserved kinase domain of this specific kinase.

The operating range of Francis turbines is limited at full-load conditions by the formation of a cavitation vortex rope that may enter self-oscillations under certain conditions. This induces severe pressure pulsations in the entire system, as well as output power swings putting at risk the integrity of the electro-mechanical components. The understanding of the underlying physical mechanisms and the prediction of the stability of hydropower units at full-load conditions are therefore crucial to ensure a safe extension of their operating range. In the present paper, the dynamic behaviour of a stable cavitation vortex rope at full load is investigated by high-speed visualizations while the test rig is excited at its first and second hydroacoustic eigenfrequencies. It is first demonstrated that the cavitation volume and the pressure in the draft tube are more likely to oscillate at the first eigenfrequency, in agreement with the observations of self-excited oscillations at the first eigenfrequency of the cavitation vortex rope during unstable full-load conditions. In addition, it is observed that the amplitude of both the cavitation volume and pressure fluctuations in the draft tube reach a limit value when the amplitude of the excitation is further increased. Further investigations will determine if this behaviour can be generalized to any full-load conditions and will focus on the determination of the hydro-acoustic parameters of the draft tube cavitation flow based on the behaviour of the vortex rope during forced oscillations.

The most accurate calculation of nitrate concentration from the ultraviolet (UV) absorption spectrum of seawater requires that the absorption signal due to bromide in seawater be removed before nitrate concentrations are computed. Recent work suggests that the UV absorption spectrum of bromide in seawater has a pressure dependence. Neglect of this signal could add a bias when nitrate concentrations at high pressure are computed from UV measurements. Laboratory tests were conducted to determine the pressure dependence of the bromide absorption in seawater. Our results confirm the existence of a pressure coefficient in the bromide spectrum. The percentage change in bromide molar absorptivity is wavelength and temperature independent. The effect of pressure on the absorptivity of sea salt (ESW), which is dominated by bromide ion, can therefore be calculated as ESW pressure=ESW 1 dbar∗ (1 – 0.0 26 ∗ Pressure (dbar)/1000) The correction amounts to an error of around 0.95 μM nitrate at 1000 dbar. The pressure correction should be used in the calculations of nitrate concentrations from UV absorption spectra at high pressures.

The Southern Ocean Carbon and Climate Observations and Modeling (SOCCOM) program has begun deploying a large array of biogeochemical sensors on profiling floats in the Southern Ocean. As of February 2016, 86 floats have been deployed. Here the focus is on 56 floats with quality-controlled and adjusted data that have been in the water at least 6 months. The floats carry oxygen, nitrate, pH, chlorophyll fluorescence, and optical backscatter sensors. The raw data generated by these sensors can suffer from inaccurate initial calibrations and from sensor drift over time. Procedures to correct the data are defined. The initial accuracy of the adjusted concentrations is assessed by comparing the corrected data to laboratory measurements made on samples collected by a hydrographic cast with a rosette sampler at the float deployment station. The long-term accuracy of the corrected data is compared to the GLODAPv2 data set whenever a float made a profile within 20 km of a GLODAPv2 station. Based on these assessments, the fleet average oxygen data are accurate to 1 +/- 1%, nitrate to within 0.5 +/- 0.5 mu mol kg(-1), and pH to 0.005 +/- 0.007, where the error limit is 1 standard deviation of the fleet data. The bio-optical measurements of chlorophyll fluorescence and optical backscatter are used to estimate chlorophyll a and particulate organic carbon concentration. The particulate organic carbon concentrations inferred from optical backscatter appear accurate to with 35 mg C m(-3) or 20%, whichever is larger. Factors affecting the accuracy of the estimated chlorophyll a concentrations are evaluated.

Forkhead box M1 (FOXM1) belongs to the forkhead/winged-helix family of transcription factors and regulates a network of proliferation-associated genes. Its abnormal upregulation has been shown to be a key driver of cancer progression and an initiating factor in oncogenesis. FOXM1 is also highly expressed in stem/progenitor cells and inhibits their differentiation, suggesting that FOXM1 plays a role in the maintenance of multipotency. However, the exact molecular mechanisms by which FOXM1 regulates human stem/progenitor cells are still uncharacterized. To understand the role of FOXM1 in normal hematopoiesis, human cord blood CD34+ cells were transduced with FOXM1 short hairpin ribonucleic acid (shRNA) lentivirus. Knockdown of FOXM1 resulted in a 2-fold increase in erythroid cells compared to myeloid cells. Additionally, knockdown of FOXM1 increased bromodeoxyuridine (BrdU) incorporation in erythroid cells, suggesting greater proliferation of erythroid progenitors. We also observed that the defective phosphorylation of FOXM1 by checkpoint kinase 2 (CHK2) or cyclin-dependent kinases 1/2 (CDK1/2) increased the erythroid population in a manner similar to knockdown of FOXM1. Finally, we found that an inhibitor of FOXM1, forkhead domain inhibitor-6 (FDI-6), increased red blood cell numbers through increased proliferation of erythroid precursors. Overall, our data suggest a novel function of FOXM1 in normal human hematopoiesis.

Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10-15% of pediatric myeloid leukemias and 2-9% of all pediatric leukemias. There are several unique characteristics of CML diagnosed in children, adolescents, and young adults, compared to adults. They present with higher white blood counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML patients are due to unique molecular characteristics that differ from adult CML patients. To test this hypothesis, we studied the transcriptomic signature of pediatric CD34+ CML cells compared to adult CML and normal age-matched bone marrow CD34+ cells. Methods CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric normal (n=10) and adult normal (n=10) bone marrow samples. Total RNA was isolated from cells, and cDNA libraries were generated. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values (TMM) from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes (LogFC). We performed three comparisons: (1) Pediatric CML vs Normal, (2) Adult CML vs Normal, and (3) Pediatric CML vs Adult CML. A False Discovery Rate (FDR) of £ .05 and absolute log2 fold-change > 1 was used to define differentially expressed genes in each comparison. Over-representation analysis was used to identify potentially unique pathways based on differentially expressed genes. Clinical and demographic features at diagnosis were extracted for pediatric and adult CML patients and compared using Fisher's exact test (categorical variables) or Wilcoxon rank sum test (continuous variables). Results Pediatric patients were diagnosed with CML at a median of 11 years (interquartile range (IQR): 10-14) compared to 54 years (IQR: 33-62) for adult patients. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients, whereas the white blood cell count and phase at diagnosis did not differ. We found 606 genes (210 up- and 396 down-regulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric normal controls. Interestingly, transcriptional regulators involved in blood cell differentiation including GATA1, TAL1, and KLF1 were differentially enriched in pediatric CML. In comparing adult CML patients to normal adult CD34+ cells, we found 920 genes (379 up- and 541 down-regulated) differentially expressed. Among all dysregulated genes we identified (1352 genes), 174 genes (54 up- and 120-down-regulated) overlapped when comparing pediatric and adult CML patients. Significantly enriched pathways in both adult and pediatric CML cells included PI3K/AKT signaling, MAPK signaling, and Notch/Wnt signaling, which have been previously reported. We found 437 unique genes that were dysregulated only in pediatric CML (270 up- and 167 down-regulated). Notch/Wnt signaling and Rho signaling pathways were significantly enriched. DLC1, a tumor suppressor gene that encodes a RhoGTPase-activating protein, has been known to be downregulated in solid tumors and hematologic malignancies. Interestingly, our data showed that DLC1 is significantly upregulated by 3-fold (p=0.0238) in pediatric CML, but not adult CML CD34+ cells. In addition, we observed that ABR, an inducer of C/EBPa that encodes an activator of RhoGEF and GTPase, was significantly downregulated by 2-fold (p=0.0119) in pediatric but not in adult CML CD34+ cells. Conclusion These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the particular biology of pediatric CML might impact the treatment of those patients in the future. Disclosures Gotlib: Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: co-chair of the Study Steering Committee and Research Funding; Blueprint Medicines Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair of the Response Adjudication Committee and Research Funding, Research Funding.

Background:After accumulation of evidences, it is recognized that inhibition of IL-6 signaling is one of the most established strategies for rheumatoid arthritis (RA) treatment. Tocilizumab (TCZ), an anti-IL6 receptor monoclonal antibody, is the pioneer which blocks IL-6 signaling by preventing IL-6 from binding to both membrane-bound and soluble receptors. Tofacitinib (TOF) inhibits Janus kinase (JAK) 1, JAK3 and, to a lesser extent, JAK2. Recently, Baricitinib (BAR), JAK 1 and JAK2 selective kinase inhibitor, were also approved to treat RA. These JAK inhibitors are known to inhibit cytokine signaling including interleukin (IL)-6. It is very important for clinicians to know whether these treatments affect common biological processes or disparate, because it will provide a rationale for switching each other if one of these treatments resulted in lack of efficacy.Objectives:To compare the gene-expression modification profiles among TOF, BAR and TCZ treatments.Methods:Total of 38 RA cases were analyzed, including TOF (n=15: 6-20mg/d), BAR (n=10: 2-4mg/d) and TCZ (n=13: 8mg/kg/4w or 163mg/2w) treatment groups. Peripheral blood was drawn at just before (pre) and 3 months after (post) these treatments. Total RNAs were then extracted with using PAXgene miRNA kit. After constructing single-stranded, strand-specific libraries, multiplex sequencing was done. After quantifying the expressions of transcripts, differentially expressed genes (DEGs) were selected by paired comparison (post vs. pre), setting thresholds at 2-fold change up/down and less than P=0.05 in paired T-test. And then, hierarchical clustering analysis and enrichment analysis using gene ontology (GO) terms were performed.Results:From the comparison of post- vs. pre-treatment of TOF, BAR and TCZ, the 120 (up-regulated=25/down-regulated=95), 62 (up=20/down=42) and 193 (up=54/ down=139) genes were selected as DEGs respectively. It seems to be discrete depending on the treatment, because overlapped genes were only 1.0% in up-regulated and 5.7% in down-regulated genes. The hierarchical clustering with expression profiles of these DEGs showed major 4 clusters. 92.3% of TCZ and 70% of BAR cases were segregated into 1stand 3rdclusters respectively, while those of TOF cases fell into 2ndand 4thclusters. Disparate GO terms were enriched in each DEGs group. For example, genes relevant to viral defense including ‘response to type I interferon (IFN)’ were suppressed in TOF group. Meanwhile, down regulation of genes involved in phosphorylation process including ‘IL-7 signaling’ seemed to be significant in BAR group. It is noteworthy that terms related to wound healing such as ‘platelet activation’ were enriched in the down-regulated genes of TCZ group.Conclusion:It is speculated that the downstream biological cascade for TOF, BAR and TCZ treatment might be shared, as IL-6 signaling is mediated by JAK1/JAK2/TYK2 activation. However, the influence of these treatments over the transcriptome in the peripheral blood seems to be disparate. Enrichment analysis using GO terms also indicated that different biological processes were involved in the effect of each treatment. Our findings will support a rationale for switching each other if one of these treatments resulted in lack of efficacy. An increased risk of herpes zoster by a treatment with JAK inhibitors has been well recognized. It makes sense because IFN signaling is also mediated by JAK/STAT pathway. On the other hand, we have experienced a case with exacerbation of skin ulcer during TCZ treatment despite the activity of RA was absolutely under control. It is accounted for by the suppression of genes involved in wound healing after TCZ treatment.Disclosure of Interests: :Yoshinobu Koyama Grant/research support from: Eli-Lilly and Mochida., Speakers bureau: BMS, Ayumi, Chugai, Ono, Mitsubishi Tanabe, Abbvie and Eisai., Yoshiharu Sato: None declared, Moe Sakamoto: None declared, Hiroshi Iijima: None declared, Toshie Higuchi: None declared

Background Sarcopenia is a geriatric syndrome characterized by loss of muscle mass and muscle function. As the population ages, there is a growing worldwide interest in the intersection of sarcopenia and heart failure (HF). However, estimates of the prevalence of sarcopenia in HF vary widely because of difference in diagnostic criteria. Although the Asian Working Group of Sarcopenia (AWGS) has announced a consensus on the diagnostic criteria of sarcopenia in Asian people, the prevalence and prognostic impact of sarcopenia based on AWGS criteria in patients with HF remains unclear. Purpose The aim of this study was to investigate the prevalence and predictive value of sarcopenia identified according to AWGS definition in non-dependent elderly patients with HF. Methods This study was a prospective, single-center cohort study in Japan. We consecutively enrolled 274 patients, aged 65 years or older, hospitalized due to acute HF or acute exacerbation of chronic HF and who were able to walk at least 20 m at discharge. Patients with severe cognitive or psychiatric disorders were excluded. Patients with implantable cardiac pacemaker or cardioverter defibrillator were also excluded because skeletal muscle mass was estimated by using bioimpedance analysis. At hospital discharge, we collected data on age, gender, left ventricular ejection fraction, brain natriuretic peptide, estimate glomerular filtration rate, body mass index and sarcopenia. Sarcopenia was diagnosed according to the AWGS criteria: low skeletal muscle index ( Results In this study, a total of 199 patents (43.4%) fulfilled sarcopenia criteria at discharge. During follow-up, 57 patients (20.8%) readmitted for HF. Kaplan-Meier survival curves showed that patients with sarcopenia had significantly lower event-free survival than those without sarcopenia (Figure). After adjusting for other prognostic factors, sarcopenia was independently associated with HF rehospitalization (hazard ratio: 2.31, 95% confidence interval: 1.20–4.53). Conclusion Based on AWGS criteria, sarcopenia is highly prevalent even among non-dependent elderly HF patients, and is an independent strong predictor of rehospitalization for worsening HF. AWGS criteria for sarcopenia may be useful for risk prediction in HF.

This chapter describes the drought early warning program of Assessment and Information Services Center (AISC), including procedures and tools used, technical assistance, problems encountered with transferring the system to developing nations, and potential benefits. It outlines suggestions for improving the assessments and information flow. In 1979, National Oceanic and Atmospheric Administration's (NOAA) / National Environmental Satellite, Data and Information Service/AISC began developing its international climate impact assessment technology with support from the US Agency for International Development, Office of US Foreign Disaster Assistance. The NOAA Drought Early Warning System is based on three subsystems: agroclimatic indices, satellite assessment models, and crop yield forecast models. The training aspect of the AISC technology transfer program is intensive and considered unique. The AISC Drought Early Warning System can be improved in several ways to upgrade the quality, timeliness, and quantification of the assessments. These include techniques development, computer hardware, communications, and expansion of technical assistance.

The del(5q) myelodysplastic syndrome (MDS) is a distinct subtype of MDS, associated with deletion of the ribosomal protein S14 (RPS14) gene that results in macrocytic anemia. This study sought to identify novel targets for the treatment of patients with del(5q) MDS by performing an in vivo drug screen using an rps14-deficient zebrafish model. From this, we identified the secreted gelatinase matrix metalloproteinase 9 (MMP9). MMP9 inhibitors significantly improved the erythroid defect in rps14-deficient zebrafish. Similarly, treatment with MMP9 inhibitors increased the number ofcolony forming unit-erythroid colonies and the CD71+erythroid population from RPS14 knockdown human BMCD34+cells. Importantly, we found that MMP9 expression is upregulated in RPS14-deficient cells by monocyte chemoattractant protein 1. Double knockdown of MMP9 and RPS14 increased the CD71+population compared with RPS14 single knockdown, suggesting that increased expression of MMP9 contributes to the erythroid defect observed in RPS14-deficient cells. In addition, transforming growth factorβ(TGF-β) signaling is activated in RPS14 knockdown cells, and treatment with SB431542, a TGF-βinhibitor, improved the defective erythroid development of RPS14-deficient models. We found that recombinant MMP9 treatment decreases the CD71+population through increased SMAD2/3 phosphorylation, suggesting that MMP9 directly activates TGF-βsignaling in RPS14-deficient cells. Finally, we confirmed that MMP9 inhibitors reduce SMAD2/3 phosphorylation in RPS14-deficient cells to rescue the erythroid defect. In summary, these study results support a novel role for MMP9 in the pathogenesis of del(5q) MDS and the potential for the clinical use of MMP9 inhibitors in the treatment of patients with del(5q) MDS.

Background:Pulmonary hypertension (PH) is prominent as a vascular involvement of systemic sclerosis (SSc), which remains a leading cause of death in spite of current best treatments. Recently, hemodynamic definition of PH was updated from mPAP≥25mmHg to mPAP>20mmHg and PVR≥3WU. Although new definition may improve the prognosis of PH associated with SSc by giving a chance to start management early, it may be insufficient as more than 2/3 of the pulmonary circulation is already impaired by the time of meeting the definition. Therefore, the ideal therapeutic intervention should be started at the subclinical stage of PH in SSc patients, but little is known about underlying pathological mechanisms at the stage. In this study, we investigate progression to exercise-induced PH (exPH)1), which is considered subclinical PH, in the prospective registry of high-risk population for developing PH associated with SSc.Objectives:To detect the gene expressions in peripheral blood involved in the progression of pulmonary vessel disease (PVD) at the subclinical stage of PH associated with SSc.Methods:Total of 180 patients who had not met PH criteria with Raynaud phenomenon, skin sclerosis or SSc-related autoantibody was registered. To detect the early PVD, exercise Doppler echocardiography (exDE) was carried out every 6 or 12 months for up to 6 years. The definition of exPH was maximum sPAP>40mmHg or increase in sPAP>20mmHg estimated by exDE during exercise. For gene expression analysis, total RNAs from whole peripheral blood cells were extracted by PAXgene system, and then multiplex sequencing was done. To identify candidate genes involved in the progression to exPH, random forest machine learning method was employed. Volcano plots, a scatter plots to visualize fold-changes and p-values of differentially expressed genes (DEGs) between exPH and others (exN), were also used for seeking the important genes for disease progression.Results:At the time of registration, 34.4% of patients met exPH criteria, and 15.6% of patients developed exPH during follow-up period (35.0±18.1 months). Expression of TNF gene was selected as the most useful genes to predict progression to exPH by random forest, and the accuracy of the model was about 87%. Volcano plots indicated that expressions of TMEM176A and TMEM176B were prominent (fold-change >2.4 and -log10 p-value >3.5) in exPH patients. The accuracy was improved to 90% if the expression of TNF and TMEMA/B were used for the prediction of progression to exPH. We found that statistically significant increase in the expression of TNF was eliminated at the time of fulfilling the exPH criteria, while increase in expressions of TMEM A/B were still kept.Conclusion:It was reported that TNFα drives pulmonary arterial hypertension by suppressing the BMP type-II receptor and altering NOTCH signalling2). Our findings suggest that TNFα plays important role only in the period of pre-exPH. On the other hand, increase in expressions of TMEM A/B were observed through the period of pre-exPH to post-exPH. It suggests that there are multiple phases before developing PH associated with SSc. It is very important to understand the phases for the precise treatment to arrest the progression of PVD.References:[1]R. Naeije et al., Am J resp and critical care med 187, 576-583 (2013). 2) LA. Hurst et al., Nat Commun. 13;8:14079 (2017).Disclosure of Interests:Yoshinobu Koyama Speakers bureau: Asahikasei, Ayumi, BMS, Mitsubishi Tanabe, Shin-nihon, Paid instructor for: Asahikasei, Asteras, BMS, Grant/research support from: Eli-Lilly, Yoshiharu Sato: None declared, Tatsuma Shoji: None declared, Soichiro Fuke: None declared, Takatsune Umayahara: None declared, Moe Sakamoto: None declared

Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella upon primary infection and zoster upon reactivation from latency in sensory ganglion neurons. The replication of herpesviruses requires manipulation of cell signaling pathways. Notably, CREB, a factor involved in the regulation of several cellular processes, is activated upon infection of T cells with VZV. Here, we report that VZV infection also induced CREB phosphorylation in fibroblasts and that XX-650-23, a newly identified inhibitor of the phosphorylated-CREB (pCREB) interaction with p300/CBP, restricted cell-cell spread of VZV in vitro . CREB phosphorylation did not require the viral open reading frame 47 (ORF47) and ORF66 kinases encoded by VZV. Evaluating the biological relevance of these observations during VZV infection of human skin xenografts in the SCID mouse model of VZV pathogenesis showed both that pCREB was upregulated in infected skin and that treatment with XX-650-23 reduced infectious-virus production and limited lesion formation compared to treatment with a vehicle control. Thus, processes of CREB activation and p300/CBP binding are important for VZV skin infection and may be targeted for antiviral drug development. IMPORTANCE Varicella-zoster virus (VZV) is a common pathogen that causes chicken pox and shingles. As with all herpesviruses, the infection is acquired for life, and the virus can periodically reactivate from latency. Although VZV infection is usually benign with few or no deleterious consequences, infection can be life threatening in immunocompromised patients. Otherwise healthy elderly individuals who develop zoster as a consequence of viral reactivation are at risk for postherpetic neuralgia (PHN), a painful and long-lasting complication. Current vaccines use a live attenuated virus that is usually safe but cannot be given to many immunodeficient patients and retains the capacity to establish latency and reactivate, causing zoster. Antiviral drugs are effective against severe VZV infections but have little impact on PHN. A better understanding of virus-host cell interactions is relevant for developing improved therapies to safely interfere with cellular processes that are crucial for VZV pathogenesis.

CREB binding protein (CBP) and p300 are critical regulators of hematopoiesis through both their transcriptional coactivator and acetyltransferase activities. Loss or mutation of CBP/p300 results in hematologic deficiencies in proliferation and differentiation as well as disruption of hematopoietic stem cell renewal and the microenvironment. Aberrant lysine acetylation mediated by CBP/p300 has recently been implicated in the genesis of multiple hematologic cancers. Understanding the effects of disrupting the acetyltransferase activity of CBP/p300 could pave the way for new therapeutic approaches to treat patients with these diseases.

Six profiling floats equipped with nitrate and oxygen sensors were deployed at Ocean Station P in the Gulf of Alaska. The resulting six calendar years and 10 float years of nitrate and oxygen data were used to determine an average annual cycle for net community production (NCP) in the top 35 m of the water column. NCP became positive in February as soon as the mixing activity in the surface layer began to weaken, but nearly 3 months before the traditionally defined mixed layer began to shoal from its winter time maximum. NCP displayed two maxima, one toward the end of May and another in August with a summertime minimum in June corresponding to the historical peak in mesozooplankton biomass. The average annual NCP was determined to be 1.5 ± 0.6 mol C m−2 yr−1 using nitrate and 1.5 ± 0.7 mol C m−2 yr−1 using oxygen. The results from oxygen data proved to be quite sensitive to the gas exchange model used as well as the accuracy of the oxygen measurement. Gas exchange models optimized for carbon dioxide flux generally ignore transport due to gas exchange through the injection of bubbles, and these models yield NCP values that are two to three time higher than the nitrate-based estimates. If nitrate and oxygen NCP rates are assumed to be related by the Redfield model, we show that the oxygen gas exchange model can be optimized by tuning the exchange terms to reproduce the nitrate NCP annual cycle.

The transcription factor CREB (cAMP Response Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell cycle, and survival pathways, which may represent a novel approach for AML therapy.

5q-syndrome is a distinct form of myelodysplastic syndrome (MDS) where a deletion on chromosome 5 is the underlying cause. MDS is characterized by bone marrow failures, including macrocytic anemia. Genetic mapping and studies using various models support the notion that ribosomal protein S14 (RPS14) is the candidate gene for the erythroid failure. Targeted disruption of RPS14 causes an increase in p53 activity and p53-mediated apoptosis, similar to what is observed with other ribosomal proteins. However, due to the higher risk for cancer development in patients with ribosome deficiency, targeting the p53 pathway is not a viable treatment option. To better understand the pathology of RPS14 deficiency in 5q-deletion, we generated a zebrafish model harboring a mutation in the RPS14 gene. This model mirrors the anemic phenotype seen in 5q-syndrome. Moreover, the anemia is due to a late-stage erythropoietic defect, where the erythropoietic defect is initially p53-independent and then becomes p53-dependent. Finally, we demonstrate the versatility of this model to test various pharmacological agents, such as RAP-011, L-leucine, and dexamethasone in order to identify molecules that can reverse the anemic phenotype.

(Purpose) Considering the dramatic increase in average life expectancy throughout the world, the management of super-elderly patients over 80 years old (SEP) with breast cancer has become a global issue. However, there have been few clinical trials for SEP until now. The reasons for this were a small population, unpredictable prognosis, a large number of non-cancer-related deaths and a lower function of multiple organs in SEP. Surgical treatment or post-operative treatment based on evidence of clinical trials for SEP has also not been unclear. We hypothesized that the outcome of SEP with breast cancer compared with other ages were similarly depended on the breast cancer subtypes. The aim of this study was to clarify the breast cancer related survival (BRS) rate and overall survival (OS) rate at 5years for SEP according to breast cancer subtype. (Methods) We retrospectively analyzed 407 patients over 80 years old at initial operation between April, 1994 and April 2015 from 4 institutions of Chiba Youth Breast Oncology Research Group. Overall, 366 patients with stage I to Stage IIIc were included. 41 patients with Stage 0 or IV were excluded in this study. We compared the clinical characteristics, OS and BRS rates among the breast cancer subtype: such as ER positive HER2 negative (ER group), ER negative HER2 negative (TN group), ER negative HER2 positive (HER2 group) and ER positive HER2 positive (ER/HER2 group). Univariate and multivariate analyses were performed to identify the factors of Tumor size, Lymph node, Ly, ER, HER2 and characteristics, associated with the OS and BRS. (Results) The median age of the 366 patients was 83 years (range 80-96 years).The median follow-up duration was 32 months (range, 2-120). During the follow-up period, 25 (9.4%) patients in the ER group, 19 (27.5%) in TN group, 4 (22.2%) in HER2 group and 2 (20.0%) patients in ER-HER2 group died. The 5 year OS and BRS rates were 89.2%, 97.1% in ER group, 64.6%, 81.2% in TN group, 61.5%,33.3% in HER2 group and 83.3%, 100% in ER-HER2 group, respectively. Univariate and multivariate analyses revealed that ER was one prognostic factor to OS and BRS. ER positive patients treatment with Aromatase inhibitor had significantly longer survival rates than treatment with Tamoxifen or no treatment (p=0.05). There were no significant differences in OS or BRS of TN patients according to the use of chemotherapy (n=7) versus non treatment (n=61). (Conclusions) The prognosis and clinical course of super elderly patients with breast cancer depended on subtype. Adjuvant therapy for ER group was one prognostic factor to OS and BRS. Citation Format: Nakamura R, Matsuzaki H, Sakamoto M, Suda K, Hayama S, Sangai T. The outcomes for super elderly patients over 80 years old after breast cancer surgery. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-07-15.

// Bryan Mitton 1 , Katie Hsu 1 , Ritika Dutta 1 , Bruce C. Tiu 1 , Nick Cox 2 , Kevin G. McLure 1 , Hee-Don Chae 1 , Mark Smith 2 , Elizabeth A. Eklund 3 , David E. Solow-Cordero 4, * , Kathleen M. Sakamoto 1, * 1 Department of Pediatrics, Stanford University, Stanford, CA, USA 2 Medicinal Chemistry Knowledge Center, Stanford ChEM-H, Stanford, CA, USA 3 Division of Hematology/Oncology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 4 High-Throughput Bioscience Center, Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA * These authors have contributed equally and share senior authorship Correspondence to: Kathleen M. Sakamoto, e-mail: kmsakamo@stanford.edu Keywords: small molecule screen, novel therapeutics, CREB Received: September 25, 2015 Accepted: January 13, 2016 Published: January 30, 2016 ABSTRACT The transcription factor CREB (cAMP Response Element Binding Protein) is an important determinant in the growth of Acute Myeloid Leukemia (AML) cells. CREB overexpression increases AML cell growth by driving the expression of key regulators of apoptosis and the cell cycle. Conversely, CREB knockdown inhibits proliferation and survival of AML cells but not normal hematopoietic cells. Thus, CREB represents a promising drug target for the treatment of AML, which carries a poor prognosis. In this study, we performed a high-throughput small molecule screen to identify compounds that disrupt CREB function in AML cells. We screened ~114,000 candidate compounds from Stanford University’s small molecule library, and identified 5 molecules that inhibit CREB function at micromolar concentrations, but are non-toxic to normal hematopoietic cells. This study suggests that targeting CREB function using small molecules could provide alternative approaches to treat AML.

Perspective on Diamond–Blackfan anemia: lessons from a rare congenital bone marrow failure syndrome

Ribosomal S6 Kinases (RSKs) include a family of serine/threonine kinases that regulate cell proliferation and survival. In humans, four RSK isoforms (RSK1-4) have been identified. Although they are 73-80% conserved in sequence homology, RSK isoforms are reported to have distinct functions. RSKs phosphorylate cyclic AMP response element binding protein (CREB) which is a regulator of hematopoietic proliferation and differentiation. RSK1 is hyperactivated in AML and is therefore a potential therapeutic target for acute myeloid leukemia (AML). Therefore, inhibition of RSK requires understanding of its role during normal hematopoiesis. Furthermore, the role of RSK isoforms during hematopoiesis and leukemogenesis has not been well characterized. To study the expression of RSK isoforms in hematopoiesis, we cultured human cord blood CD34+ (CBCD34+) hematopoietic stem and progenitor cells (HSPCs) in differentiating media containing SCF, Flt3L, TOP, IL-3, IL-6, GM-CSF, and EPO and performed qPCR with mRNA of the RSK isoforms at different stages of maturation up to 14 days. RPS6KA1 (RSK1) was expressed throughout hematopoiesis, but RPS6KA3 (RSK2) and RPS6KA2 (RSK3) showed higher expression by 2-fold (p To study the requirement of RSK isoforms during hematopoiesis, we knocked down RSK expression by transducing lentivirus expressing isoform-specific shRNAs or scramble controls into human CBCD34+ cells. On day 5 following transduction, cells were sorted and plated in methylcellulose media and assessed for the colony-forming activity of RSK knockdown in HSPCs. While RSK3 knockdown increased BFU-E colonies by 1.5-fold (p These data demonstrate that RSK1 and RSK3 exert opposite functions during erythroid and myeloid differentiation of HSPCs, suggesting a novel role for RSK isoforms as a determinant of early fate decisions of HSCs. Disclosures No relevant conflicts of interest to declare.