ALBERT

Description: Multiple myeloma (MM) is an invariably fatal form of cancer characterized by clonal proliferation of malignant plasma cells in the bone marrow. The canonical Wnt signaling pathway is activated in MM cells through constitutively active β-catenin, a messenger molecule relevant to growth, survival, and migration of MM cells. The identification of a number of small molecular compounds, such as PKF115–584, which disrupt the interaction of the transcriptionally active β-catenin/TCF protein complex, provides valuable new therapeutic tools to target an alternative pathway in MM independent of the proteasome. Here we evaluated the transcriptional, proteomic, signaling changes, and biological sequelae associated with the inhibition of Wnt signaling in MM by PKF115–584. The compound blocks expression of Wnt target genes and induces cytotoxicity in both patient MM cells and MM cell lines without a significant effect in normal plasma cells. In xenograft models of human MM, PKF115–584 inhibits tumor growth and prolongs survival. Taken together, these data demonstrate the efficacy of disrupting the β-catenin/TCF transcriptional complex to exploit tumor dependence on Wnt signaling as a therapeutic approach in the treatment of MM.

Description: The use of water-saving irrigation techniques has been encouraged in rice fields in response to irrigation water scarcity. Straw return is an important means of straw reuse. However, the environmental impact of this technology, e.g., nitrogen leaching loss, must be further explored. A two-year (2017–2018) experiment was conducted to investigate the vertical migration and leaching of nitrogen in paddy fields under water-saving and straw return conditions. Treatments included traditional flood irrigation (FI) and two water-saving irrigation regimes: rain-catching and controlled irrigation (RC-CI) and drought planting with straw mulching (DP-SM). RC-CI and DP-SM both significantly decreased the irrigation input compared with FI. RC-CI increased the rice yield by 8.23%~12.26%, while DP-SM decreased it by 8.98%~15.24% compared with FI. NH4+-N was the main form of the nitrogen leaching loss in percolation water, occupying 49.06%~50.97% of TN leaching losses. The NH4+-N and TN concentration showed a decreasing trend from top to bottom in soil water of 0~54 cm depth, while the concentration of NO3−-N presented the opposite behavior. The TN and NH4+-N concentrations in percolation water of RC-CI during most of the rice growth stage were the highest among treatments in both years, and DP-SM showed a trend of decreasing TN and NH4+-N concentrations. The NO3−-N concentrations in percolation water showed a regular pattern of DP-SM 〉 RC-CI 〉 FI during most of the rice growth stage. RC-CI and DP-SM remarkably reduced the amount of N leaching losses compared to FI as a result of the significant decrease of percolation water volumes. The tillering and jointing-booting stages were the two critical periods of N leaching (accounted for 74.85%~86.26% of N leaching losses). Great promotion potential of RC-CI and DP-SM exists in the lower reaches of the Yangtze River, China, and DP-SM needs to be further optimized.

Description: Energy & Fuels DOI: 10.1021/acs.energyfuels.5b02024

Description: To our knowledge, little attention has been paid to the sexual function of health workers. The aim of the present study was to assess the frequency of sexual dysfunction among Chinese doctors. In addition, the risk factors for sexual dysfunction were analyzed. This was a questionnaire-based multicenter cross-sectional survey performed in five large academic medical centers in China. A total of 539 male doctors, 547 female doctors were evaluated. We analyzed doctors' demographic characteristics, quality of life, sexual function and attitudes towards sexual problems. Chinese doctors are at high risk of sexual dysfunction and poor health. The prevalence of sexual dysfunction appears to increase with age, and is associated with various social and demographic factors including monthly income, physical exercise, working hours, night shift and health-related quality. The quality of life and sexual health of doctors deserves special attention as a significant public health concern. Alleviating work pressure, increasing income, improving quality of life and therapeutic sexual problems should be considered comprehensively.

Description: Abstract 1786 Poster Board I-812 Background Multiple Myeloma (MM) is characterized by a clonal proliferation of antibody producing malignant plasma cells. Complete or partial monoallelic deletion of chromosome 13, is commonly observed in tumor cells of patients with monoclonal gammopathy of unknown significance and in over 50% of MM patients, as well as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. Recurrent loss of a minimal common region (MCR) of 10 megabases at 13q14, in MM and CLL suggests the MCR harbors a tumor suppressor gene(s) (TSG) with biological and clinical relevance. Within this MCR resides the Ret Finger Protein 2 (RFP2) encoding gene, which produce an E3 ubiquitin ligase located in the endoplasmic reticulum (ER). Because of its copy number-dependent expression, its strong and unique promoter, and its associated inferior survival with reduced expression in MM, RFP2 represents a candidate TSG. Nevertheless, its role and targets have not yet been established. Here we describe a functional analysis of RFP2 in MM cells. Methods: The MMS1 MM cell line lacks chromosome 13 deletion. To study the effects of loss of RFP2 in this line we used the PLKO- GFP lentiviral vector to stably transduce a RFP2 shRNA. Flow cytometer selected cell lines exhibit significantly reduced expression of RFP2 relative transduced shRNA controls or to the parental line. Cell growth rate was measured using trypan blue counting, soft agar colony formation and thymidine incorporation. Cell cycle analysis and apoptosis were measured by flow cytometry after staining with PI or Annexin-V PE and 7AAD, respectively. Intracellular signal modulation was demonstrated by Western blotting. Results At day six post transduction, 75-95% of MMS1 cells were GFP positive. RFP2 downregulation induced an impairment of cell growth with a G2 phase arrest and a profound apoptosis (over 50% at day six as compared with less than 15% of controls). This effect was mediated through ER stress evidenced by upregulation of p-eIF2a and Bip, and the induction of Caspase-8, 9 and 3 cleavage. RFP2 complementation did not produce by itself a significant growth promoting effect, but was able to rescue the knockdown-induced growth retardation. The above described presence of ER stress, combined with the previous reports that RFP2 has E3 ubiquitin ligase activity prompted us to assess total protein ubiquitination. Concordant with its effects on ER stress, RFP2 downregulation was associated with significantly higher levels of poly-ubiquitinated proteins. Subsequently, we were able to document a significant reduction (60% inhibition) in 20S proteasome activity in RFP2 down regulated cells. Proteasome inhibition by RFP2 down regulation was confirmed in other MM cell lines and was partially abrogated by restoring RFP2 levels by overexpression. Importantly, RFP2 down regulated cells were more sensitive to bortezomib; indeed proteasome inhibition was synergistic with RFP2 downregulation in MM cells. The above results prompted us to study the mechanism whereby RFP2 impacts survival and proliferation of MM cells. Inhibition of the NF-kappa-B (NFκB) pathway is a hallmark of proteasome-related growth retardation and apoptosis and is a key pathway in MM. We show that NFkB luciferase reporter assay was associated with significant activity reduction with RFP2 downregulation. To define the mechanism of this process, we examined the level of NFkB related proteins in nuclear and cytoplasmic fractions. Interestingly, the most prominent effect observed in RFP2 down regulated cells was increased levels of IkBá in the nucleus. Altogether, these results support our supposition that the effects of RFP2 downregulation are mediated through an inhibition of the NFkB pathway that is associated with increased nuclear IkBa as well as a decrease in 20S proteasome activity. Conclusions RFP2 is a gene mapping to a deletion hotspot at 13q14 and reduced RNA expression is associated with poor survival in MM. Functional studies revealed that shRNA mediated knockdown of RFP2 in MM causes growth retardation and apoptosis, mediated by ER stress and a G2 arrest, mediated by proteasome inhibition and reduced NFkB activity. Although RFP2 did not prove itself to be a tumor suppressor gene in our studies, targeting RFP2 may represent a novel therapeutic approach in MM and other lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Multiple myeloma (MM) is a cancer of plasma cells with complex molecular characteristics that evolves from monoclonal gammopathy of undetermined significance, a highly prevalent premalignant condition. MM is the second most frequent hematologic cancer in the United States, and it remains incurable, thereby highlighting the need for new therapeutic approaches, particularly those targeting common molecular pathways involved in disease progression and maintenance, shared across different MM subtypes. Here we report that Wnt/β-catenin is one such pathway. We document the involvement of β-catenin in cell-cycle regulation, proliferation, and invasion contributing to enhanced proliferative and metastatic properties of MM. The pleiotropic effects of β-catenin in MM correlate with its transcriptional function, and we demonstrate regulation of a novel target gene, Aurora kinase A, implicating β-catenin in G2/M regulation. β-catenin and Aurora kinase A are present in most MM but not in normal plasma cells and are expressed in a pattern that parallels progression from monoclonal gammopathy of undetermined significance to MM. Our data provide evidence for a novel functional link between β-catenin and Aurora kinase A, underscoring a critical role of these pathways in MM disease progression.

Description: In multiple myeloma (MM), bone marrow-derived endothelial progenitor cells (EPCs) contribute to tumor neoangiogenesis and their levels covary with tumor mass and prognosis. Recent X-chromosome inactivation studies in female patients showed that, similar to tumor cells, EPCs are clonally restricted in MM. Genomic profiling of MM using high-resolution array comparative genomic hybridization (aCGH) has been previously utilized to mine the genome and find clinical correlates in MM patients. In this study, clonotypic aspects of bone marrow-derived EPCs and MM cells were investigated using aCGH and expression profiling analysis. Confluent EPCs were outgrown from bone marrow aspirates by adherence to laminin. EPCs were 〉98% vWF/CD133/KDR+ and 50% CD38+. For aCGH and for gene expression profiling, genomic DNA and total RNA from EPCs and MM cells were hybridized to human oligonucleotide arrays (Agilent Technologies) and human cDNA microarrays (Affymetrix), respectively. High resolution aCGH with segmentation analysis showed that EPCs and MM cells in one of ten cases share identical patterns of chromosomal gains and losses, while another 5 cases shared multiple focal copy number alterations (CNAs) including gains and losses. The genomes of EPCs and MM cells additionally displayed exclusive CNAs, but these were far fewer in EPCs than in MM cells. In 3 patients, EPCs harbored a common 0.6Mb deletion at 1q21 not shared by MM cells. Pertinent genes in this region that could affect proliferation and tumor suppression include N2N, NBPF10, and TXNIP. Validation studies of aCGH findings by other methods are ongoing. Gene expression profiling showed decreased expression of 1q21 region genes (e.g., calgranulin C and lamin A/C). A genome-wide comparison of patients’ MM cells and EPCs, which is focused on their shared genetic characteristics, will be presented.

Description: Abstract 1963 Poster Board I-986 Background: Multiple Myeloma (MM), a cancer of plasma cells is characterized by frequent chromosomal alterations. Deletion of chromosome 13, especially band 13q14, is commonly observed in early stages of MM, suggesting the presence of tumor suppressor genes within this region. When studied in the context of CLL, the miR 15a and 16-1 cluster was associated with a distinct miR signature and clinical outcome. Over-expression of miR16 caused induction of apoptosis and downregulation of the anti apoptotic gene BCL2 in a megakaryocytic leukemia cell line and induced growth arrest in MM cells. Nonetheless, being lost in CLL, MM, MCL and LPL, their functional role has not been studied using a loss-of-function approach in any of these lymphoid malignancies. Here, we describe the generation of an in vivo system for the long term, stable knockdown of miR 15a/ 16-1 expression in myeloma cells to recapitulate the conditions seen in chromosome 13q14 deleted MM. Methods: Using lentiviral vectors to stably express a competitive sponge miR16 inhibitor we set up a system to functionally validate the role of microRNA 15a/16-1 cluster. Pure populations of lentivirally transduced MM cell lines were sorted by flow cytometry using GFP marker. Decreased miRs 15a and 16 expression levels were assessed by Northern blot and R-luciferase reporter system. Cell growth rate was measured using trypan blue counting, and thymidine incorporation. Cell cycle analysis was measured by flow cytometry after staining with PI. Intracellular signal modulation was demonstrated by Western blotting. RNA from MM cell lines expressing the control sponge or sponge16 were hybridized on an Affymetrix U133A 2.0 array chip, and validated using quantitative real time PCR. Xenograft murine models were performed using the stable MM cell lines injected into 6-week old NOD.CB17-PrkdcSCID/J irradiated mice. Results: Selected stable miR knockdown MM cell lines exhibited significantly reduced expression of miRs15a/16-1 as assessd by both by mRNA levels and miR luciferase reporter assays. The knockdown cells showed a significant increase in growth rates (1.5-2 fold) compared to control cells, as measured by viable cell counts and proliferation by thymidine incorporation in vitro. Importantly, miR16 inhibition decreased animal survival in a xenograft model of MM by increasing tumor load, invasiveness and host angiogenesis. To further delineate the role of miR15a/16 in MM and to gain additional insight into the possible target genes regulated by this cluster, we performed gene expression-profiling analysis in controls and miR16 deficient MMS1 and RPMI cell lines. Since our sponge system produces downregulation of the miRs, we focused on the upregulated probes. Expression profiling analysis of miR16 deficient cells identified a surprisingly large number of downstream target-genes such as FGFR1, PI3KCa, MDM4, VEGFa, as well as secondary affected genes such as JUN and Jag1. These results were verified both at the mRNA level and the protein level, as well as in other MM cell lines. Moreover, we were able to show that these knockdown cells were partially addicted to some of these pathways using specific drug inhibitors. We further validated designated genes as direct miR16 targets by showing binding sites within the conserved 3' UTR and also within the mRNA coding region, thus indicating that the miRs may have many more possible targets than anticipated by conventional prediction methods. Conclusions: Using this loss-of-function system, which mimics the pleiotropic chronic effects of microRNA loss at the 13q chromosomal deletion, provides a valuable tool to investigate their function as tumor suppressor genes in MM pathogenesis, affecting multiple targets, and may represent a novel potential for therapeutic targeting in MM and other lymphoid malignancies. Disclosures: Munshi: Seattle Genetics, Inc.: Research Funding.