ALBERT

Description: Analytical Chemistry DOI: 10.1021/ac200662s

Description: Potential damages to existing tunnels represent a major concern for constructing deep excavations in urban areas. The uncertainty of subsurface conditions and the nonlinear interactions between multiple agents (e.g., soils, excavation support structures, and tunnel structures) make the prediction of the response of tunnel induced by adjacent excavations a rather difficult and complex task. This paper proposes an initiative to solve this problem by using process-based modelling, where information generated from the interaction processes between soils, structures, and excavation activities is utilized to gradually reduce uncertainty related to soil properties and to learn the interaction patterns through machine learning techniques. To illustrate such a concept, this paper presents a simple process-based model consisting of artificial neural network (ANN) module, inverse modelling module, and mechanistic module. The ANN module is trained to learn and recognize the patterns of the complex interactions between excavation deformations, its geometries and support structures, and soil properties. The inverse modelling module enables a gradual reduction of uncertainty associated with soil characterizations by accumulating field observations during the construction processes. Based on the inputs provided by the former two modules, the mechanistic module computes the response of tunnel. The effectiveness of the proposed process-based model is evaluated against high-fidelity numerical simulations and field measurements. These evaluations suggest that the strategy of combining artificial intelligence techniques with information generated during interaction processes can represent a promising approach to solve complex engineering problems in conventional industries.

Description: Follicular dendritic cells (FDCs), an essential component of the lymph node microenvironment, regulate and support B-lymphocyte differentiation, survival, and lymphoma progression. Here, we demonstrate that adhesion of mantle cell lymphoma and other non-Hodgkin lymphoma cells to FDCs reduces cell apoptosis and is associated with decreased levels of the proapoptotic protein, Bim. Bim down-regulation is posttranscriptionally regulated via up-regulation of microRNA-181a (miR-181a). miR-181a overexpression decreases, whereas miR-181a inhibition increases Bim levels by directly targeting Bim. Furthermore, we found that cell adhesion–up-regulated miR-181a contributes to FDC-mediated cell survival through Bim down-regulation, implicating miR-181a as an upstream effector of the Bim-apoptosis signaling pathway. miR-181a inhibition and Bim upregulation significantly suppressed FDC-mediated protection against apoptosis in lymphoma cell lines and primary lymphoma cells. Thus, FDCs protect B-cell lymphoma cells against apoptosis, in part through activation of a miR-181a–dependent mechanism involving down-regulation of Bim expression. We demonstrate, for the first time, that cell-cell contact controls tumor cell survival and apoptosis via microRNA in mantle cell and other non-Hodgkin lymphomas. Regulation of microRNAs by B-cell–FDC interaction may support B-cell survival, representing a novel molecular mechanism for cell adhesion–mediated drug resistance and a potential therapeutic target in B-cell lymphomas.

Description: Abstract 883 Specific expression of Bruton's tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in regulating osteoclastogenesis. Although Btk is critical in B cell maturation and myeloid function, it has not been characterized in plasma cell malignancies including multiple myeloma (MM) and Waldenström Macroglobulinemia (WM). We here investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) microenvironment, as well as on MM and WM cancer cells. We further define molecular targets of Btk signaling cascade in OCs and MM in the BM milieu. In CD14+ OC precursor cells, RANKL and M-CSF stimulate phosphorylation of Btk in a time-dependent fashion; conversely, PCI-32765 abrogates RANKL/M-CSF-induced activation of Btk and downstream PLCγ2. Importantly, PCI-32765 decreased number of multinucleated OC (〉3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and the secretion of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It increased size of OCs and number of nuclei per OC, with significantly defective bone resorption activity as evidenced by diminished pit formation on dentine slices. Moreover, lack of effect of Dexamethasone on OC activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 significantly reduced cytokine and chemokine secretion from OC cultures, including MIP1α, MIP1β, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1–0.48 nM). It potently decreased IL-6, SDF-1, MIP1α, MIP1β, and M-CSF in CD138-negative cell cultures from active MM patients, associated with decreased TRAP staining in a dose-dependent manner. In MM and WM cells, immunoblotting analysis confirmed a higher Btk expression in CD138+ cells from majority of MM patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated a higher expression of Btk and its downstream signaling components in WM cells than in CD19+ normal bone marrow cells. PCI-32765 significantly inhibits SDF-1-induced adhesion and migration of MM cells. It further blocked cytokine expression (MIP1a, MIP-1β) at mRNA level in MM and WM tumor cells, correlated with inhibition of Btk-mediated pPLCγ2, pERK and NF-kB activation. Importantly, PCI-32765 inhibited growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs in IL-6-dependent INA6 and ANBL6 MM cells. Furthermore, myeloma stem-like cells express Btk and PCI-32765 (10–100 nM) blocks their abilities to form colonies from MM patients (n=5). In contrast, PCI-32765 has no adverse effects on Btk-negative BMSCs and OBs, as well as Btk-expressing dendritic cells. Finally, oral administration of PCI-32765 (12 mg/kg) in mice significantly suppresses MM cell growth (p〈 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results provide compelling evidence to target Btk in the BM microenvironment against MM and WM., strongly supporting clinical trials of PCI-32765 to improve patient outcome in MM and WM. Disclosures: Chang: Pharmacyclics Inc: Employment. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership. Elias:Pharmacyclics Inc: Consultancy. Treon:Millennium: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Genomic instability is not only a hallmark of cancer, but potentially a primary mechanism for its occurrence. DNA repair mechanisms play a protective role during DNA damage induced by both normal metabolic activities and environmental factors such as reactive oxygen species (ROS), UV light and γ-irradiation. Genes related to DNA repair are usually considered as tumor suppressors. However, incomplete repair may induce severe genomic instability, leading eventually to transformation. Apurinic/apyrimidinic endonuclease 1 (APEX1), a gene involved in DNA repair with an important role in the base excision repair pathway, leads to transformation of normal cells in vitro. To investigate the role of APEX1 in tumor initiation in vivo, we generated a novel transgenic zebrafish model to overexpress APEX1 in fish. Specifically, pDestTol2A2_ubi:loxP-EGFP-loxP-APEX1-mCherry plasmid was injected into single cell embryos derived from the TP53 mutant line Tp53M214K/M214K to generate a stable conditional inducible transgenic zebrafish line: Tg:APEX1fl/- mCherry Tp53M214K/M214K. To activate APEX1 expression in vivo, this line was mated with Tg:HSP70-Cre+/+ fish. Compound zebrafish Tg: APEX1fl/- mCherry,Tp53+/M214K ,HSP70-Cre+/- carrying a Cre-activatable APEX1 knock-in allele were heated at 24hpf, and induction of APEX1 expression was monitored by downstream reporter - mCherry expression. Ten to twelve months post-fertilization, Tg:APEX1fl/- mCherry,Tp53+/M214K ,HSP70-Cre+/- fish developed abnormal swelling. Flow cytometry analysis of fish kidney marrow and peripheral blood showed dramatically increased precursor populations in scatter analysis. Histopathologic analysis showed that multiple organs were infiltrated with malignant lymphoblastic cells. None of the control fish Tg: GFP,Tp53+/M214K ,HSP70-Cre+/- developed tumors during their life span. Zebrafish with T-ALL have heterozygous Tp53+/M214K background, but the expression of p21, mdm2 and bax in Tp53+/M214K fish is exactly the same as in Tp53+/+ fish; and Tp53+/M214K zebrafish themselves have not developed tumors during their life span. RNA from lymphoblastic cells was evaluated by qRT-PCR and showed increased expression of CD3, LCK and Tal indicating a T-cell acute lymphoblastic leukemia/lymphoma (T-ALL). We have performed whole genomic DNA sequencing in extracted DNA from fish tumor cells and compared it with their normal counterpart and observed multiple copy number changes and mutations. We have now begun to see the development of other tumors in other organs including the eye and the testis. To uncover the molecular mechanism of tumorigenesis induced by APEX1, we have performed Mass Spectrometry analysis on APEX1 pulled down from 293T and AG08498 cells ectopically expressing APEX1. Besides verified binding proteins, such as PCNA, we also identified Ku70 and Ku80 binding to APEX1 directly. Further immunoflurescent staining and confocal microscopy of 293T cells also found co-localization of APEX1 and Ku70/Ku80. Those two proteins initiate Non-Homologous End Joining (NHEJ) repair and start the error-prone double strand repair and DNA damage. These results indicate that excessive repair activity may induce DNA damage and genomic instability. In summary, this is the first demonstration where overexpression of a DNA repair gene is responsible for induction of genomic instability leading to malignant transformation. It provides new insight into the process of tumorigenesis and development of both therapy as well as preventive strategies. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Description: Abstract 3121 MicroRNAs (miR)-15a and miR-16-1 are known to act as tumor suppressors. Expression of these miRNAs inhibits cell proliferation, promotes apoptosis of cancer cells, and suppresses tumorigenicity by targeting multiple oncogenes. Loss or down-regulation of these miRNAs has been reported in a variety of hematopoietic and solid tumors. Our recent study demonstrated that miR-15a/16-1 are down-regulated in the majority of patients with mantle cell lymphoma (MCL, Zhao et al., Blood. 2010 1;115(13):2630-9). However, the mechanism responsible for miR-15a/16 suppression was unknown. Here, we have investigated mechanism of miR-15a/16-1 repression and its transcriptional and epigenetic regulation by Myc and histone deacetylase (HDAC) in MCL. Over-expression of Myc protein was detected in all MCL cell lines, and miR-15a/16 and DLEU-2 mRNA were significantly up-regulated in Mino and Jeko-1 cells when Myc was depleted by c-Myc siRNA. Treatment with pan HDAC inhibitor, SAHA and specific HDAC-3 shRNA resulted in increase of miR15a/16 expression. Co-immunoprecipitation study showed that c-Myc interacted with HDAC3. Moreover, chromatin immunoprecipitation (ChIP)demonstrated that both Myc and HDAC3 co-localized to the two promoters of miR-15a/16 cluster gene, DLEU-2. Luciferase reporter assay confirmed that both c-Myc and HDAC3 inhibited transcriptional activity of two E-box (Myc-binding)-specific DLEU-2 promoters. Computational analysis and mutagenesis study further supported direct association of Myc with miR-15a/16 promoter resulting in HDAC3-mediated transcriptional repression. These findings highlight an important role for HDAC-3 in Myc-driven lymphoma cell proliferation and reveal novel transcriptional and posttranscriptional mechanisms in Myc-mediated malignant transformation in MCL. Disclosures: No relevant conflicts of interest to declare.

Description: We previously reported that telomerase activity is elevated in multiple myeloma (MM), and its inhibition induces telomere shortening and growth arrest in cancer cells. We have now gone on to study the role of telomerase in DNA break repair and genome maintenance in MM cells. To demonstrate the role of telomerase in DNA break repair: 1) We used g-H2AX staining (marker for DNA breaks) and comet assay, a gel-based technique for detection of DNA breaks in individual cells, and observed that telomerase inhibition leads to significantly increased DNA breaks in MM cells; 2) We have confirmed the repair and re-circularization of a linearized plasmid by telomerase in MM cell extracts; and 3) Demonstrated increased genomic instability, especially deletions, upon telomerase inhibition in MM cells. This does not necessarily suggest role of telomerase in DNA repair as telomerase inhibition with attrition of telomeres can also lead to increased instability. To confirm the direct role of telomerase in DNA repair in MM, we now present the evidence and mechanism of DNA break repair by telomerase by demonstrating: 1) The presence of “TTAGGG” repeats at non-telomeric sites at higher frequency in cancer vs normal cells; and 2) Decline in “TTAGGG” insertions at non-telomeric sites in MM cells following suppression of telomerase. To evaluate rare telomeric insertions in the cancer genome, we created libraries of genomic DNA fragments enriched for “TTAGGG” sequences from primary MM and matching normal PBMCs derived from the same patient. The libraries were sequenced using Illumina platform and reads containing 4 or more telomeric repeats were filtered for further analysis. Telomeric insertion sites were located from unique genomic sequences immediately following TTAGGG at one end of each read. By subtracting telomeric insertions detected in normal cells, from MM cells of same patent, we identified 94 unique loci with telomeric insertion in the primary MM cells. To investigate if telomerase inserts new “TTAGGG” repeats within cancer genome following DNA breaks, UV-treated RPMI cells were incubated with and without telomerase inhibitor for 4 days, cultured without telomerase inhibition for another 6 days, harvested and DNA libraries prepared and enriched for telomeric fragments and subjected to sequencing. DNA from cells preserved before UV treatment (day 0) was used as baseline control and their telomeric insertions were subtracted from UV-treated control and telomerase-inhibited cells. Following induction of DNA breaks by UV, 21 and 3 new telomeric insertions were detected in control and telomerase-inhibited MM cells, respectively, indicating 86% reduction of telomeric insertions within MM cell genome upon telomerase inhibition. Analyses of flanking sequences indicated that 71% of the new telomeric insertions in the UV-treated control cells occurred at sites which did not have any pre-existing “TTAGGG” repeats. Similarly in primary MM cells, 67%, 29% and 4% of the new insertions were observed at positions containing 0, 1 and 2 copies of “TTAGGG” repeats, respectively, indicating that telomerase could use both telomeric as well as non-telomeric DNA as substrate for interstitial telomeric sequence insertions. Evaluation of a few telomeric insertions by Q-PCR confirmed the sequencing data. For an insertion on chr16 (q24.1), a 9.2-fold increase in telomeric signal in UV-treated control relative to background (day 0) cells was observed, whereas the same locus in telomerase-inhibited sample showed near background amplification. We also looked for somatic telomere insertions in 55 largely untreated patients with Waldenström’s macroglobulinemia for whom whole genome sequencing data was available. The absolute number of telomere insertions correlated with the number of somatic structural variants (translocation, inversions, and large deletions) per genome (tau = 0.3 p=0.001) indicating a possible role in DNA double stranded break repair. Thus telomerase contributes to survival of MM and other cancer cells, not only by preventing telomere attrition, but also the repair of DNA breaks which involves the insertion of telomeric repeats within genome. Inhibition of telomerase therefore, may increase the efficacy of chemotherapeutic agents targeting DNA repair. Evaluating interstitial telomeric insertion pattern in cancer could also be a potentially useful tool to study tumor progression or evolution upon treatment. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3853 B-cell differentiation is tightly regulated by synchronized suppression and/or induction of specific transcription factors. Among them, B-cell lymphoma 6 (BCL6) and PRDM1 are considered to be master regulators for germinal center formation and terminal B-cell differentiation. Dysregulation of BCL6 and PRDM1 also have been associated with lymphomagenesis. Their regulation still need further study especially at the posttranscriptional level. Here, by using co-culture system and whole genomic microRNA microarray profiling, we show for the first time that direct B lymphoma cell-stroma cell contact between follicular dendritic cells and B-lymphocytes could induce upregulation of miR-30 family and downregulation of miR-9 and let-7 family. In silico analysis showed that miR-30s can target genes BCL6 and miR-9/let7 can target PRDM1 with direct binding sites in 3`UTR region of their mRNAs. The microarray data can be proved by microRNA specific Q-RT-PCR. Specifically, by both gain of function and loss of function studies, we functionally verified that FDCs Regulate Expression of BCL6 and PRDM1 via Cell-Cell Direct Contact induced correlated microRNA dysregulation. To further validate the direct interaction between BCL6 and miR-30, we constructed luciferase reporters containing the BCL6 3`-UTR that included miR-30 binding sites and a mutant 3`-UTR harboring mutations in the “seed pairing” sequences of the miR-30 binding site. Co-transfection of miR-30 and reporter construct into cells significantly decreased luciferase activity in wild-type but not in mutant BCL6-3`-UTR transfected cells, supporting the role of miR-30 family in the regulation of BCL6 expression. BCL6 and PRDM1 and their regulation miRNAs, let-7 and miR-30, also can be validated in primary normal B-lymphocytes and lymphoma cells by using our co-culture system. Dysregulation of BCL6 and PRDM1 is often associated with lymphomagenesis. We firstly identified that BCL6 is the direct target of miR-30 family and also verified PRDM1 is the target of miR-9, and let-7 in our system. Our studies provide a novel mechanism of post-transcriptional regulation of BCL6 and PRDM1 by several microRNAs. In the context of micro-environment, it provides a clue for germinal center B-cell differentiation as well as B-cell lymphomas progression regulated by lymphocyte cell-stroma cell contact through microRNAs. Disclosures: No relevant conflicts of interest to declare.

Description: The polarization of naïve CD4+ T cells may initiate multiple reactions in immune system. The balance between Th1 and Th2 cells is critical for innate and acquired immune reactions. But the exact mechanism of its polarization is still unclear. IL-4 is specifically produced by Th2 cells, and regulates Th2 differentiation. Once IL-4 binds to IL-4 receptor (IL-4R), the Th2 polarization signal is activated by phosphorylation of STAT6 (recruited by IL-4Rα), its relocation to nucleus, activation of STAT6 downstream genes (gata3, il-4 and il-4rα etc) and consequent Th2 polarization. CD44 an important T cell activation and T helper cell differentiation gene participates in the regulation of Th1 and Th2 differentiation. Furthermore, CD44 variant isoforms produced by alternative RNA splicing, have different physiological and pathological functions including tumor metastasis, drug resistance and anti-apoptosis effect in tumor cells. Here we report hitherto unknown specific CD44 variant isoforms involved in T helper cell differentiation and functional regulator of CD4+ T cell polarization. We developed various PCR primer sets able to distinguish different CD44 isoforms in human and mouse Th2 cells. We found higher expression of CD44 variant 4 (CD44v4) and CD44v5 in both human and mouse Th2 cells compared with Th1 cells, indicating their role in Th2 cell differentiation. In order to investigate the role of CD44v4 and CD44v5 in Th2 cells polarization, we treated human naïve CD4+ T cells with CD44v4 or CD44v5 antibody separately for 3 days in polarizing condition. We observed that CD44v5 antibody treatment dramatically decreased the level of phospho-JAK1 and pSTAT6 compared to control cells treated with same amount of normal mouse IgG. At the same time, the expression of GATA3 detected by western blot and the secretion of IL-4 measured by ELISA decreased. Notably, the phosphorylation of STAT1 in Th1 cells was not inhibited by CD44v5 blocking. There is a significant decrease in GATA3 and IL4 expression with CD44v5 antibody but not in CD44v4 antibody treated group, which indicated that Th2 polarization was mainly influenced by CD44v5. In order to verify our finding, CD44v5 specific siRNA were used and we observed similar result in CD4+ T cells. Interestingly, we found that the degradation of IL-4Rα increased after treatment of Th2 cells with CD44v5 antibody compared with control group. Using confocal microscopy of single cell, we observed that CD44v5 co-localized with IL-4Rα. Importantly, CD44v5 antibody treatment could interrupt the CD44v5 and IL-4Rα interaction and also the co-localization of T cell receptor (TCR). On Th1 cells, we didn't find the co-localization between IFNgR and CD44v5. The IFN-γ secretion in Th1 cells were not influenced by either CD44v5 blocking or CD44v5-deficient CD4+ T cells indicating that CD44v5 only influenced the differentiation of Th2 cell, but not Th1 cells. In conclusion, CD44v5 plays an important role in naive T cell differentiates into Th2 cells. We hypothesis that CD44v5 can bind to IL-4Rα through CD44v5 variant domain and stabilize the IL-4Rα, blocking CD44v5 induced IL-4R degradation and reduce the Th2 cell differentiation. CD44v5 antibody treatment inhibiting Th2 differentiation without affecting Th1 development provides a potential novel immuno-therapy target. Disclosures No relevant conflicts of interest to declare.