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One mechanism by which lymphoid malignancies resist standard apoptosis-intending (cytotoxic) treatments is genetic attenuation of the p53/p16-CDKN2A apoptosis axis. Depletion of the epigenetic protein DNA methyltransferase 1 (DNMT1) using the deoxycytidine analog decitabine is a validated approach to cytoreduce malignancy independent of p53/p16. In vivo decitabine activity, however, is restricted by rapid catabolism by cytidine deaminase (CDA). We, therefore, combined decitabine with the CDA-inhibitor tetrahydrouridine and conducted a pilot clinical trial in patients with relapsed lymphoid malignancies: the doses of tetrahydrouridine/decitabine used (∼10/0.2 mg/kg orally (PO) 2×/week) were selected for the molecular pharmacodynamic objective of non-cytotoxic, S-phase dependent, DNMT1-depletion, guided by previous Phase 1 studies. Patients with relapsed/refractory B- or T-cell malignancies (n = 7) were treated for up to 18 weeks. Neutropenia without concurrent thrombocytopenia is an expected toxicity of DNMT1-depletion and occurred in all patients (Grade 3/4). Subjective and objective clinical improvements occurred in 4 of 7 patients, but these responses were lost upon treatment interruptions and reductions to manage neutropenia. We thus performed parallel experiments in a preclinical in vivo model of lymphoma to identify regimen refinements that might sustain DNMT1-targeting in malignant cells but limit neutropenia. We found that timed-alternation of decitabine with the related molecule 5-azacytidine, and combination with inhibitors of CDA and de novo pyrimidine synthesis could leverage feedback responses of pyrimidine metabolism to substantially increase lymphoma cytoreduction but with less neutropenia. In sum, regimen innovations beyond incorporation of a CDA-inhibitor are needed to sustain decitabine DNMT1-targeting and efficacy against chemo-resistant lymphoid malignancy. Such potential solutions were explored in preclinical in vivo studies.

The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.

Background/aim Chemoresistance is a major consequence of multicycle chemotherapy and can be attributed to constitutive activation of pro-survival signaling pathways. Nitric oxide is a ubiquitous signaling molecule which has been shown to inhibit several pathways involved with survival signaling in cancer cells. We have previously demonstrated the anti-tumor activity of a nitric oxide-donor, nitrosylcobalamin (NO-Cbl), mediated by increased expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. We also demonstrated that a functional Apo2L/TRAIL receptor is necessary for the induction of cell death by NO-Cbl and the Apo2L/TRAIL death receptor DR4 (TRAIL R1) is S-nitrosylated. The aim of the study was to examine the effects of nitric oxide (NO) on nuclear factor kappa B (NF-κB) and determine whether nitric oxide could sensitize drug-resistant melanomas to Apo2L/TRAIL via inhibition of NF-κB or inhibitor kappa B kinase (IKK). Materials and methods Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibroblast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by the TUNEL assay. The activation status of NF-κB was established by assaying luciferase reporter activity, the phosphorylation status of IκBα, and in vitro IKK activity. Results NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL, but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. NO-Cbl suppressed Apo2L/TRAIL- and TNF-α-mediated activation of a transfected NF-κB-driven luciferase reporter. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of IκBα. Conclusion NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of nitric oxide to inhibit NF-κB and potentiate the effects of chemotherapeutic agents, such as Apo2L/TRAIL, represents a promising anti-cancer combination based on recent clinical investigations of anti-TRAIL antibodies for cancer treatment strategies.

Previously, we reported apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), has potent anti-melanoma activity. We used DNA microarray and bioinformatics to interrogate gene expression profiles of tumors from apoA-I expressing (A-I Tg+/–) versus apoA-I-null (A-I KO) animals to gain insights into mechanisms of apoA-I tumor protection. Differential expression analyses of 11 distinct tumors per group with > 1.2-fold cut-off and a false discovery rate adjusted p < 0.05, identified 176 significant transcripts (71 upregulated and 105 downregulated in A-I Tg+/– versus A-I KO group). Bioinformatic analyses identified the mevalonate and de novo serine/glycine synthesis pathways as potential targets for apoA-I anti-tumor activity. Relative to A-I KO, day 7 B16F10L melanoma tumor homografts from A-I Tg+/– exhibited reduced expression of mevalonate-5-pyrophosphate decarboxylase (Mvd), a key enzyme targeted in cancer therapy, along with a number of key genes in the sterol synthesis arm of the mevalonate pathway. Phosphoglycerate dehydrogenase (Phgdh), the first enzyme branching off glycolysis into the de novo serine synthesis pathway, was the most repressed transcript in tumors from A-I Tg+/–. We validated our mouse tumor studies by comparing the significant transcripts with adverse tumor markers previously identified in human melanoma and found 45% concordance. Our findings suggest apoA-I targets the mevalonate and serine synthesis pathways in melanoma cells in vivo, thus providing anti-tumor metabolic effects by inhibiting the flux of biomolecular building blocks for macromolecule synthesis that drive rapid tumor growth.

Simple Summary Multiple myeloma (MM) is a cancer of antibody-producing plasma cells that remains incurable. These cells heavily depend on protein disulfide isomerase, PDIA1, for folding and structural integrity of antibodies and other secretory proteins to avoid unresolvable stress caused if they remain unfolded. High PDIA1 expression confers resistance to proteasome inhibitors and other stressors due to the gain in endoplasmic reticulum (ER) function, while maintaining or increasing vulnerability to PDIA1 inhibition. Here we report the identification and characterization of an orally bioavailable novel PDIA1 inhibitor CCF642-34 that is effective against multiple myeloma in pre-clinical models. PDIA1, the ER resident enzyme essential for the folding of disulfide bond-containing proteins, is upregulated in relapse and refractory myeloma. This increase in PDIA1 confers its sensitivity to CCF642-34, a structurally optimized PDIA1 inhibitor that induces apoptosis in myeloma cells but not in normal bone-marrow-derived CD34+ hematopoietic stem and progenitor cells. Abstract Multiple myeloma is a genetically complex hematologic neoplasia in which malignant plasma cells constantly operate at the maximum limit of their unfolded protein response (UPR) due to a high secretory burden of immunoglobulins and cytokines. The endoplasmic reticulum (ER) resident protein disulfide isomerase, PDIA1 is indispensable for maintaining structural integrity of cysteine-rich antibodies and cytokines that require accurate intramolecular disulfide bond arrangement. PDIA1 expression analysis from RNA-seq of multiple myeloma patients demonstrated an inverse relationship with survival in relapsed or refractory disease, supporting its critical role in myeloma persistence. Using a structure-guided medicinal chemistry approach, we developed a potent, orally bioavailable small molecule PDIA1 inhibitor CCF642-34. The inhibition of PDIA1 overwhelms the UPR in myeloma cells, resulting in their apoptotic cell death at doses that do not affect the normal CD34+ hematopoietic stem and progenitor cells. Bortezomib resistance leads to increased PDIA1 expression and thus CCF642-34 sensitivity, suggesting that proteasome inhibitor resistance leads to PDIA1 dependence for proteostasis and survival. CCF642-34 induces acute unresolvable UPR in myeloma cells, and oral treatment increased survival of mice in the syngeneic 5TGM1 model of myeloma. Results support development of CCF642-34 to selectively target the plasma cell program and overcome the treatment-refractory state in myeloma.

Traditional treatments of small-cell lung cancer (SCLC) with cisplatin, a standard-of-care therapy, spare the tumor-initiating cells (TIC) that mediate drug resistance. Here we report a novel therapeutic strategy that preferentially targets TICs in SCLC, in which cisplatin is combined with CBL0137, an inhibitor of the histone chaperone facilitates chromatin transcription (FACT), which is highly expressed in TICs. Combination of cisplatin and CBL0137 killed patient-derived and murine SCLC cell lines synergistically. In response to CBL0137 alone, TICs were more sensitive than non-TICs, in part, because CBL0137 increased expression of the tumor suppressor NOTCH1 by abrogating the binding of negative regulator SP3 to the NOTCH1 promoter, and in part because treatment decreased the high expression of stem cell transcription factors. The combination of cisplatin and CBL0137 greatly reduced the growth of a patient-derived xenograft in mice and also the growth of a syngeneic mouse SCLC tumor. Thus, CBL0137 can be a highly effective drug against SCLC, especially in combination with cisplatin. Significance: These findings reveal a novel therapeutic regimen for SCLC, combining cisplatin with an inhibitor that preferentially targets tumor-initiating cells. Cancer Res; 78(9); 2396–406. ©2018 AACR.

INTRODUCTION Pathogenic T cells cause many diseases including most autoimmune diseases and graft-versus-host disease (GVHD). Selectively targeting these pathogenic T cells while sparing the normal T cells and other tissues is a "holy grail" of therapeutics development in modern clinical immunology. So far, pan-immunosuppressive drugs such as corticosteroids are used to treat these patients, with limited efficacy and severe adverse effects. It is also well-established that these pathogenic T cells, whether auto- or alloreactive, proliferate following antigen recognition to cause tissue damage while the other normal T cells remain quiescent. Selectively targeting the proliferating T cells could be an effective strategy to develop new drugs for diseases mediated by the pathogenic T cells. Antibody-drug conjugates (ADCs) are developed by conjugating a potent toxin onto a monoclonal antibody (mAb) specific for a cancer cell surface antigen. Monomethyl auristatin E (MMAE), a synthetic mitotic toxin, is the payload in several FDA-approved ADCs, and it kills the actively dividing cancer cells by blocking the polymerization of tubulin, rapidly inducing apoptosis. These cancer cells and pathogenic T cells have one feature in common-both of them are actively proliferating, thus, this ADC approach proven successful in cancer treatment could be re-purposed to selectively kill pathogenic T cells for the treatment of T cell-mediated diseases. CD6 is a cell surface glycoprotein that is expressed at high levels on all T cells except Treg cells, a small portion of B cells and many human NK cells. CD6 is not detectable on other tissue cells, making it a highly specific target candidate for T cells. We have generated CD6 knockout mice and CD6 humanized mice, and developed anti-CD6 mAbs that treat mouse models T cell-mediated diseases. A CD6-targeted ADC (CD6-ADC) thus might be effective for treating T cell-mediated diseases by selectively eliminating the proliferating pathogenic T cells. METHODS A CD6-ADC was developed by conjugating a latent form of MMAE onto the high-affinity anti-human CD6 mAb. Its potency of selectively killing pathogenic T cells was evaluated in an antigen-specific T cell recall assay with BrdU-incorporation followed by flow cytometric analyses. To determine the toxicity of the CD6-ADC on normal quiescent T cells, naïve CD6 humanized mice were treated with the CD6-ADC or non-binding control ADC (0.5 mg/kg) by intraperitoneal injection, then numbers of circulating T cells were monitored by flow cytometry daily. To evaluate the efficacy of CD6-ADC in treating T cell-mediated autoimmune diseases, the same dose (0.5 mg/kg) of CD6-ADC, or the parental anti-CD6 mAb, or the control ADC was used to treat a model of autoimmune uveitis, and disease severities were assessed by various ocular imaging techniques including indirect ophthalmoscopy, confocal scanning laser ophthalmoscopy and optical coherence tomography. To examine the potential of the CD6-ADC in treating GVHD, the same dose (0.5mg/kg) of CD6-ADC or control ADC was tested in a model of GVHD induced in NSG mice after adoptive transfer of human PBMC. GVHD severities were assessed by flow cytometric analyses of circulating human T cells and by histopathological analyses of different tissues. RESULTS: The CD6-ADC selectively killed antigen-specific proliferating T cells in vitro. Treating naive CD6-humanized mice with this CD6-ADC (0.5 mg/kg) did not significantly eliminate normal T cells in vivo. Furthermore, systemic delivery of the same dose (0.5 mg/kg) of CD6-ADC, but not the anti-CD6 mAb alone nor the control IgG effectively reduced retinal inflammation in a preclinical model of autoimmune uveitis. The same dose of CD6-ADC, but not the control ADC, also effectively depleted activated xenogeneic T cells and prevented the development of GVHD in NSG mice after the adoptive transfer of human PBMC. CONCLUSION: These data indicate that this CD6-ADC holds promise as a new drug for treating diseases in which T cells are integrally involved in the pathogeneses such as GVHD. Figure 1 Figure 1. Disclosures Lin: Takeda Pharma: Consultancy, Research Funding.

Castration-resistant prostate cancer (CRPC) is defined by tumor microenvironment heterogeneity affecting intrinsic cellular mechanisms including dysregulated androgen signaling, aerobic glycolysis (Warburg effect), and aberrant activation of transcription factors including androgen receptor (AR) and c-Myc. Using in vitro, in vivo, and animal models, we find a direct correlation between miR-644a downregulation and dysregulation of essential cellular processes. MiR-644a downregulated expression of diverse tumor microenvironment drivers including c-Myc, AR coregulators, and antiapoptosis factors Bcl-xl and Bcl2. Moreover, miR-644a modulates epithelial–mesenchymal transition (EMT) by directly targeting EMT-promoting factors ZEB1, cdk6, and Snail. Finally, miR-644a expression suppresses the Warburg effect by direct targeting of c-Myc, Akt, IGF1R, and GAPDH expression. RNA sequencing analysis revealed an analogous downregulation of these factors in animal tumor xenografts. These data demonstrate miR-644a mediated fine-tuning of oncogenesis, stimulating pathways and resultant potentiation of enzalutamide therapy in CRPC patients. Significance: This study demonstrates that miR-644a therapeutically influences the CRPC tumor microenvironment by suppressing androgen signaling and additional genes involved in metabolism, proliferation, Warburg effect, and EMT, to potentiate the enzalutamide therapy.

Compact fuel cells are one logical progression for portable power in comparison to batteries since fuel cells have much higher energy densities. However, fuel cells have not fully entered the portable power market as they require a readily available hydrogen feed. Microplasmas offer a way to produce hydrogen portably. Microplasma reactors developed from standard semiconductor fabrication techniques, specifically microhollow cathode discharge (MHCD) devices, have been tested by our group in reforming various hydrocarbons. Experiments with these reactors were successful in showing the feasibility of a portable microplasma fuel reformer, but they regularly had short lifetimes ( SEM imaging has shown the formation and re-deposition of silicon and silicon dioxide on the inner walls of the microchannel. The thickness of this layer has been found to be directly linked to device lifetime. Input electrical power as well as flowrate has also shown to be related to this deposition and overall device lifetime. This failure analysis study enables us to improve the design of the microplasma reactor and further advance the development of a portable fuel reformer-fuel cell system.

Pulmonary hypertension (PH) is a frequently under recognized complication of myelofibrosis (MF). The pathophysiology of PH in MF is unknown and no definitive therapies have been established. We studied 15 patients with MF-associated PH and compared their echocardiographic and PH relevant biomarkers (nitric oxide (NO), N-terminal pro-hormone of brain natriuretic peptide (NT-pro BNP), von Willebrand antigen (vWB), ristocetin-cofactor activity (RCA) and uric acid (UA)) pre- and post-ruxolitinib treatment. Ruxolitinib decreased the plasma levels of NT-pro BNP (73%; P=0.043), UA (60%), vWB (86%) and RCA (73%; P=0.036). Improvements in echocardiographic findings were also seen in 66% of patients (P=0.022). Furthermore, marked increase in NO compared with baseline (69.75 vs 40.1 picomolar (pM); P=0.001) was observed post-ruxolitinib therapy, whereas no changes were noted with conventional therapies. Treatment with ruxolitinib also resulted in the reduction of key cytokines (tumor necrosis factor alpha, interleukin-4 (IL-4), IL-6 and IL-8) and induction of interferon-gamma. Animal studies further supported the role of ruxolitinib in the induction of NO levels. In conclusion, aberrant Janus kinase (JAK)-signal transducer and activator of transcription signaling in MF may mediate PH through dysregulation of NO and cytokine levels, which can be restored by therapy with JAK inhibitors suggesting that inhibition of this pathway is a novel target for the management of patients with PH.

Myelodysplastic syndromes (MDS) ultimately will progress to a higher-risk form or acute myeloid leukemia. This evolution is accompanied by acquisition of genetic hits following ancestral mutations, with further subclonal diversification of the clonal architecture. Conceptually, therapeutic approaches targeting ancestral hits have the potential to eradicate MDS at early stages of ontogenesis. Founder SF3B1 mutations are frequent in MDS and therefore represent rational targets for drug development. During our drug discovery efforts we identified an existing drug that selectively inhibits the growth of SF3B1 mutant (SF3B1MT) cells. We consequently examined the effects of known compounds possibly arresting clonal expansion of SF3B1MT MDS cells. For our studies, we generated CRISPR/Cas9 knock-in human cells stably expressing the recurrent SF3B1 K700E mutation and a chromophore-tagged reporter (mRFP). Because of the high frequency of SF3B1 mutations and their effects on the splicing of a multitude of genes, we hypothesized that some of the corresponding downstream effects could be effectively targeted. Using luminescent cell viability assays and flow cytometry analysis, we screened a 3,000 compound library for selective sensitivity against isogenic SF3B1MT cells. A subset of this library contained 23 calcium2+ channel blockers (CCBs) of which one specific dihydropirimidine (DHP) showed the highest inhibitory activity against SF3B1MT cells. Several studies point towards a role of calcium signaling in MDS. Proteins active in calcium metabolism (GPR68, calpain, calpastatin) are involved in modulating sensitivity to drugs used in MDS (e.g., lenalidomide). Divalent substrates including Fe2+ may use CCs and it's plausible that our DHP can inhibit iron overload by blocking Fe2+ trafficking through L-type CCs. In this regard, CCBs have been shown to stimulate the activity of adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters. Furthermore, it is known that the SF3B1 mutations reduce the expression of the iron transporter ABCB7, leading to increased iron accumulation. All of this led us to further evaluate the effects of DHP in vitro and in vivo. DHP had higher growth inhibitory activity against SF3B1MT cells when tested in vitro using 8 serial concentrations in half-log dilutions for a period of 3 days (20% and 60% growth inhibition at 1 and 3μM). Mixed competitive experiment of K700EmRFP and WTGFP cells treated with increased doses of DHP (1, 3, 5, 10, 20, 50 μM) reduced the competitiveness of K700EmRFP over the time inducing a 3-fold reduction at 50μM. K700EmRFP cells expressed half the amount of ABCB7 mRNA compared to WTGFP cells by RT-PCR. Therapeutic index provided by DHP was determined in vivo. Bone marrow cells of B6. Gt(ROSA)26Sortm1Sor/J (CD45.2) donors were transplanted in pre-lethally irradiated B6.SJL-PtprcaPepcb/BoyJ (CD45.1) recipients. Two weeks after transplantation, average engraftment (measured as percentage of CD45.2(+) donor cells) was 96% ± 0.02. DHP (10 mg/Kg) was orally administered. No decrease in the proportion of CD45.2(+) donor cells was seen post-treatment compared to pre-treatment (96±0.01 % vs. 96%±0.009). Similarly no change in the proportion of CD45.2(+) donor cells was observed in competitive repopulation experiments when B6. Gt(ROSA)26Sortm1Sor/J cells were mixed 1:1 with transgenic Sf3b1+/K700E cells (40%±0.13 vs. 44%±0.02). In contrast, preliminary experiments showed that DHP had an effect on reducing Sf3b1+/K700E allele burden in two chimeric mice (to 21% and 24%) compared to pre-treatment. DHP is structurally unique in comparison to other DHP-based CCBs; in that it possess a -CH20-CH2CH2NH2 moiety linked directly to the DHP scaffold that may in itself provide opportunities or modes for Fe2+ chelation as well as its L-type CCB activity. Our observations and structural analyses therefore provide impetus to explore this feature to possibly improve the drug's efficacy. In sum, we have demonstrated that the clonal growth of cells carrying SF3B1 mutations might be suppressed using the known L-type CCB DHP. This might represent a novel modulator of ATPase activity of ABC transporters in SF3B1MT expressing low ABCB7 levels. Disclosures Sekeres: Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

SF3B1 mutations disrupt normal pre-mRNA splicing to cause disease. Drugs inhibiting the interaction between the SF3b complex and RNA or agents degrading auxiliary splicing factors are being tested as new avenues for targeted therapy in myeloid neoplasia (MN) with SF3B1 mutations. Here we describe the ability of small molecules to restore altered RNA processes in SF3B1MT MN. We previously reported (Visconte, ASH 2018) the identification of the small molecule 4-pyridyl-2-anilinothiazole (PAT) which showed growth inhibition of CRISPR/Cas9 SF3B1+/K700E cells and primary SF3B1MT cells. PAT did not influence the growth of other cell models without (THP1, MOLM13FLT3, OCIAML3DNMT3A, SIGM5TET2/DNMT3A, K562PHF6) and with other splicing factor mutations (K562U2AF1, K562LUC7L2). We now describe data from medicinal chemistry, transcriptome, and in vivo studies to advance drug development for SF3B1MT MN. SAR studies focused on logical and systematic modifications of PAT, e.g., i) replacement of the 2,4-disubstituted thiazole spacer ring with other heteroatom containing rings (5,6,7 membered aromatic or aliphatic ring structures); ii) alternative linking groups for the NH linker of the aniline of the tail region (sulfonamide, amide, substituted amine linkers); iii) alternative substituted aromatic and aliphatic ring structures for the phenyl head region substituent, led us to identify permissive sites for further chemical optimization. For example, a 4-chlorophenyl analog demonstrated activity [IC50, 3μM] similar to PAT. Competitive repopulation assays of bone marrow (BM) cells from dual reporters (ACTBtdTomato; EGFP) B6.GtROSA26 mixed with BM cells from conditional knock-in Sf3b1+/K700E mice injected in pre-lethally irradiated B6.SJL-PtprcaPepcb/BoyJ (CD45.1) recipients (n=18) were used as a preclinical murine model. This model then allowed i) demonstration of drug efficacy in reducing the competitiveness of SF3B1MT cells and ii) evaluation of therapeutic index in normal hematopoiesis. Post-transplant recovery, recipients of B6.GtROSA26 cells underwent PAT treatment (10 mg/Kg/IP/5 days weekly) for a period of 6 weeks without showing any signs of distress or drug intolerance (drop in blood count, weight loss, abdominal swelling, liver or kidney toxicity). Two weeks after transplantation, donor Sf3b1+/K700E cells had an engraftment capability similar to that of donor B6.GtROSA26 cells (83.6 ± 4 vs. 86.4 ± 2.4) when transplanted as a sole graft in CD45.1 recipients. PAT reduced almost half the percentage of Sf3b1+/K700E donor cells at 6 weeks of treatment (47.4%) vs. pre-treatment (83.6%). In mixed (1:1) BM transplants, Sf3b1+/K700E cellshad a repopulative disadvantage against competitors B6.GtROSA26 contributing for 16% of the marrow reconstitution. Similar to single graft transplants, PAT decreased the percentage of Sf3b1+/K700E cells at 6 weeks vs. pre-treatment (average, 6% vs. 16%) in chimeras. Consistent with the lack of toxicity of PAT treatment B6.GtROSA26 cells in chimeras were not affected by PAT and gradually repopulated the host (post-treatment, 80% vs. pre-treatment, 64%). Subsequently, we focused our efforts identifying important genes known to be dysregulated in MDS that were mostly influenced by drug treatment and minimally affected in normal cells. Our approach was based on the analyses of genes linked to erythropoiesis (a key hallmark of low-risk MDS). In normal hematopoiesis TGF-β signaling inhibits terminal erythroid maturation. Out of 13,775 genes, 5% (664/13,775) were found differentially expressed between CRISPR/Cas9 SF3B1+/K700E and parental cells of which 60% of these genes were significantly up-regulated and 40% down-regulated. Pathway analysis showed that the expression levels of SMAD family of genes and GDF factors changed significantly upon drug treatment. SMAD7 mRNA levels are 3-fold lower in MDS CD34+ cells (n=159) compared to the ones of healthy subjects (n=17) (GEO accession GSE58831) leading to TGF-β over activation. PAT treatment normalized SMAD7 expression levels in CRISPR/Cas9 SF3B1+/K700E cells by 3-fold while reducing the levels of GDF11. In summary, we have identified new drug entities that are modulators of transcriptomic changes which decrease the competitiveness of SF3B1MT cells. These results suggest combination therapies with current TGF-β pathway inhibitors. Disclosures Advani: Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding. Kelly:Novartis, Bayer, Janssen, Pharmacyclics, Celgene, Astrazeneca, Seattle Genetics: Honoraria, Speakers Bureau; Takeda: Research Funding; Genentech, Verastem: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

TET2 is one of the most commonly mutated genes in myeloid neoplasia. Somatic TET2 mutations (TET2MT) cause complete or partial loss of enzymatic activity. TET2 (along with TET1/3) are Fe2+ and αKG-dependent DNA-dioxygenases that catalyze the oxidation of 5mC→5hmC→5fC→5caC. Ultimately, 5hmC generated by TET2-dioxygenase passively prevents maintenance methylation due to DNA methyltransferase's inability to recognize 5hmC. Alternatively, demethylation may also be a result of base excision repair of fC and caC. TET2MT can serve as therapeutic targets because they are often initiating lesions and present in a large fraction of patients. In this study, a comprehensive analysis of the configurations of TET2MT in myeloid neoplasia including MDS (n=1809) and AML (n=808), showed a remarkable exclusivity with 2-HG producing neomorphic IDH1/2MT (Fig.A). TET2 expression in 97 healthy and 909 MDS/MPN or AML patients from two independent studies showed that IDH1/2MT cases have significantly higher TET2 expression and were also mutually exclusive in cases with lower TET2 expression. Doxycycline inducible expression of IDH1MT led to profound growth inhibition of both a natural TET2MT cell line SIG-M5 and engineered TET2-/- K562, while the effect to parental K562 was mild (Fig.B-E). These observations suggest that mutual exclusivity of TET2MT and IDH1/2MT is due to synthetic lethality of TET2MT cell caused by 2-HG production, rather than redundancy of the consequences of IDH1/2MT and TET2MT. In TET2MT cell 2-HG further inhibit the residual TET-activity (TET1/3) and may cause synthetic lethality to cells with affected TET2 function. SIG-M5 cells expresses significant amount of TET3 while negligible levels of TET1. The reliance on relative compensation through residual TET3 activity has been confirmed in cells by inducible TET3 knockdown. We hypothesized that transient suppression of the residual DNA dioxygenase activity with inhibitors may selectively eliminate TET2-deficient clones. The known TET inhibitors 2-HG, N-oxalylglycine (NOG) and dimethyl methyl fumarate (DMF) lack specificity, pharmacologic properties and potency. Based on the results of in silico docking simulations, we designed and synthesized 16 aKG derivatives. Among them, TETi76 showed best inhibition effect in both TET activity and cell growth of TET2 low expressing cell. TETi76 binds to the α-KG co-factor site of TET2 that principally involves H1801, H1381 and S1898. These amino acids are conserved in all three TET enzymes. To test the in vitro efficacy and specificity of TETi, we used several human myeloid cell lines that harbor loss of function TET2 mutations or constitutively express low TET2 levels as well as bone marrow derived from Tet2+/+, Tet2+/- and Tet2-/- mice (Fig.F-G). Results showed that cells with low 5hmC level were more sensitive to TETi76 treatment. Specificity of TETi76 was further confirmed by RNAseq analyses of TETi76 treated K562, TET2-/- K562 and parental control cells. Moreover, TETi treatment did not appear to affect the function of α-KG-dependent histone dioxygenases. Mechanistically, treatment of SIG-M5 cells with TETi76 induced early and late stages of apoptotic cell death, a finding further confirmed by PARP1 and caspase-3 cleavage. RNAseq analyses of SIGM5 cells after treatment with TETi demonstrated a significant down-regulation of genes involved in transcription and peptide elongation, consistent with the consequences of TET inhibition. Interestingly, we also observed significant up-modulation of oxidative stress response pathway genes consistent with the inhibition of dioxygenases. In particular, TETi76 treatment induces 8-fold increase of oxidative stress sensor NQO1 a NRF2 target gene. To further probe the effects of TETi76 on TET2 deficient cells, Tet2MT/Tet2WT BM cells were co-cultured at fixed ratios to mimic the evolving Tet2MT clones. TETi76 effectively eliminated otherwise dominating Tet2MT cells (Fig.H). To determine the in vivo effects of TETi e.g., on elimination of Tet2MT clones, we performed bone marrow competitive reconstitution assays in PEP mice. TETi treatment selectively restricted the proliferative advantage of Tet2MT HSC compared to vehicle control where, as expected, TET2 mutant clones took over the WT cells. In clinical applications, TET inhibitors may constitute a new class of agents to be used in a targeted fashion in TET2 mutant neoplasia. Figure. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Abazeed:Bayer AG: Honoraria, Other: Travel Support, Research Funding; Siemens: Research Funding. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Microplasmas have become increasingly attractive for activating chemical reactions because of their advantages over conventional plasma technology and catalytic processes. Advantages of reduced power requirements, atmospheric pressure operation, portability, and the circumventing of catalyst issues have made microplasmas a fascinating alternative for hydrocarbon reforming. Through the course of previous work, these microplasma reactors have displayed process and plasma variability that make characterizing the microplasma reactor challenging. In the current study, 24 experiments were run using 16 different microplasma reactors, with carbon dioxide as the only reactant. Carbon dioxide decomposition was chosen because it provides a simple experimental reaction environment that eliminates the need for a carrier gas and reduces the number of undesirable products. Carbon dioxide decomposition also has potential application for greenhouse gas mitigation. For example, an array of microplasma devices on the exhaus...

Because of its tumor-suppressive effect, interferon-based therapy has been used for the treatment of melanoma. However, limited data are available regarding the antitumor effects of pegylated interferons, either alone or in combination with approved anticancer drugs. We report that treatment of human WM-266-4 melanoma cells with peginterferon beta-1a induced apoptotic markers. Additionally, peginterferon beta-1a significantly inhibited the growth of human SK-MEL-1, A-375, and WM-266-4 melanoma xenografts established in immunocompromised mice. Peginterferon beta-1a regressed large, established WM-266-4 xenografts in nude mice. Treatment of SK-MEL-1 tumor-bearing mice with a combination of peginterferon beta-1a and the MEK inhibitor PD325901 ((R)-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide) significantly improved tumor growth inhibition compared with either agent alone. Examination of the antitumor activity of peginterferon beta-1a in combination with approved anticancer drugs in breast and renal carcinomas revealed improved antitumor activity in these preclinical xenograft models, as did the combination of peginterferon beta-1a and bevacizumab in a colon carcinoma xenograft model.

On board reforming of hydrocarbons for fuel cell feed has become an attractive research topic due to the low energy densities of batteries. The implementation of a microplasma as a means for reforming the liquid fuel methanol is explored in this work. Hydrocarbon reforming is commonly accomplished through catalysis, but catalysts have a number of limitations such as poisoning, coking, coarsening, long start-up times and excessive costs. Published studies have shown the viability of plasma reforming but none have succeeded in achieving suitable system efficiencies for portable applications. Non-thermal microplasmas are particularly attractive for reforming due to their extremely high electron and power densities and the scale of microplasma devices make them well suited for portable applications. This study describes experimental microplasma reactors reforming methanol. The reactors are based on the microhollow cathode discharge (MHCD) structure fabricated with microelectromechanical systems (MEMS) fabrication techniques. Through modeling the reaction for all five experiments, conversions within the microchannel were found to be nearly 100%. Despite the variations in the five experiments due to input electrical power, flow rate and concentration, the model was validated in each test. The experiments discussed in this work show the promise of a portable, non-thermal microplasma reformer that generates hydrogen for fuel cells for portable power.

Tumor extracellular matrix has an abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. Innovative design and development of safe and effective targeted contrast agents to these biomarkers would allow effective MR cancer molecular imaging with high spatial resolution. In this study, we synthesized a low molecular weight CLT1 peptide targeted Gd(III) chelate CLT1-dL-(Gd-DOTA)(4) specific to clotted plasma proteins in tumor stroma for cancer MR molecular imaging. CLT1-dL-(Gd-DOTA)(4) was synthesized by conjugating four Gd-DOTA monoamide chelates to a CLT1 peptide via generation 1 lysine dendrimer. The T(1) relaxivity of CLT1-dL-(Gd-DOTA)(4) was 40.4 mM(-1) s(-1) per molecule (10.1 mM(-1) s(-1) per Gd) at 37 °C and 1.5 T. Fluorescence imaging showed high binding specificity of CLT1 to orthotopic PC3 prostate tumor in mice. The contrast agent resulted in improved tumor contrast enhancement in male athymic nude mice bearing orthotopic PC3 prostate tumor xenograft at a dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for high-resolution MR molecular imaging of prostate tumor.

A novel approach to chemical process intensification based on microplasma reactor systems is presented. Microplasmas (plasmas generated in sub-mm volumes) possess a favorable reaction environment because of high densities of reactive ions, radicals, and electrons. Moreover, operation under atmospheric pressure and near-room temperature makes them convenient for implementation. Experiments utilized a microfabricated microplasma reactor to intensify the reforming of methanol into a hydrogen-rich product stream useful for energy applications such as combustion or fuel cells. A reaction model elaborates the key mechanisms and demonstrates that reaction rates in the microplasma environment surpass those of conventional plasma reactors. The outcome is that microplasma chemical processing is a viable approach for hydrocarbon reforming and merits consideration for other chemical reaction applications.

To study the effectiveness of a peptide targeted nanoglobular Gd-DOTA complexes for MR molecular imaging of prostate cancer in a mouse orthotopic PC-3 prostate cancer model.A CLT1 (CGLIIQKNEC) peptide-targeted generation 2 nanoglobular Gd-DOTA monoamide conjugate [CLT1-G2-(Gd-DOTA)] was used for imaging fibrin-fibronectin complexes in prostate tumor using a non-specific peptide KAREC modified conjugate, KAREC-G2-(Gd-DOTA) as a control. Cy5 conjugates of CLT1 and KAREC were synthesized for binding studies. Orthotopic PC-3 prostate tumors were established in the prostate of athymic male nude mice. MRI study was performed on a Bruker 7T small animal MRI system.CLT1 peptide showed specific binding in the prostate tumor with no binding in normal tissues. The control peptide had little binding in normal and tumor tissues. CLT1-G2-(Gd-DOTA) resulted in stronger contrast enhancement in tumor tissue than KAREC-G2-(Gd-DOTA). CLT1-G2-(Gd-DOTA) generated ~100% increase in contrast-to-noise ratio (CNR) in the tumor compared to precontrast CNR at 1 min post-injection, while KAREC-G2-(Gd-DOTA) resulted in 8% increase.CLT1-G2-(Gd-DOTA) is a promising molecular MRI contrast agent for fibrin-fibronectin complexes in tumor stroma. It has potential for diagnosis and assessing prognosis of malignant tumors with MRI.

Drug resistance is a major obstacle in cancer treatments and diminishes the clinical efficacy of biological, cytotoxic, or targeted therapeutics. Being an antiapoptotic mediator of chemoresistance in breast and lung cancer cells, MKP1 phosphatase might be targeted for overcoming chemoresistance and improving therapeutic efficacy. In this work, tyrosine phosphatase inhibitor-3 (TPI-3) was identified as a novel small molecule inhibitor of MKP1 and was capable of sensitizing tumors to bio- and chemotherapeutics in mice as a tolerated oral agent. Effective against recombinant MKP1, TPI-3 selectively increased MKP1 phosphosubstrates in Jurkat cells and induced cell death via apoptosis at nanomolar concentrations. TPI-3 also increased MKP1 phosphosubstrates in WM9 human melanoma cells and synergized with biotherapeutic IFNα2b in the growth inhibition of melanoma cells in vitro (combination index

There is an increasing evidence that long-term alcohol intake can promote damage to most of the body's major organs. However, regular consumption of a small-moderate amount of alcohol is often recommended as being beneficial to health and of concern is that the effect of ingesting commercially available alcohol products on steroid hormone synthesis under variable nutritional conditions has not been thoroughly investigated. Many individuals consume alcohol alone prior to a meal and the aim of the present study was to assess the effect of consuming a small-moderate amount of commercially available alcohol on the level of salivary cortisol and salivary dehydroepiandrosterone sulfate (DHEAS) before and after a meal. A total of 24 males aged 19–22 years participated in the current investigation. The experimental procedure required participants to fast for 6 h before being asked to ingest either 40 g alcohol in the form of red wine ( n = 8), low alcohol and high beer ( n = 8), white wine ( n = 8) or the equivalent amount of placebo over a 135-min period before consuming food for 45-min. The level of blood alcohol, salivary cortisol and salivary DHEAS was assessed upon arrival and then at regular 45-min intervals during the 180-min experimental period. The results showed that the consumption of alcohol and placebo can significantly lower the level of salivary cortisol. However, the effect of consuming a small-moderate amount of commercially available alcohol on the level of salivary DHEAS was dependent on the nutritional content of the beverage with red wine promoting no change, white wine promoting a significant decrease, and beer having a variable effect on salivary DHEAS concentration when compared to placebo. It was concluded that the effect of commercially available alcohol on the HPA axis is not the same for all alcohol products and both the nutritional status of participants and the nutritional content of the alcoholic beverage being administered should be taken into consideration when investigating the effect of alcohol on the HPA axis.

Metal-organic chemical vapour deposition (MOCVD) of various phases in PrO x system has been studied in relation with deposition temperature (450–750 °C) and oxygen partial pressure (0.027–100 Pa or 0.2–750 mTorr). Depositions were carried out by pulsed liquid injection MOCVD using Pr(thd) 3 (thd = 2,2,6,6-tetramethyl-3,5-heptanedionate) precursor dissolved in toluene or monoglyme. By varying deposition temperature and oxygen partial pressure amorphous films or various crystalline PrO x phases (Pr 2 O 3 , Pr 7 O 12 , Pr 6 O 11 ) and their mixtures can be grown. The pure crystalline Pr 2 O 3 phase grows only in a narrow range of partial oxygen pressure and temperature, while high oxygen pressure (40–100 Pa) always leads to the most stable Pr 6 O 11 phase. The influence of annealing under vacuum at 750 °C on film phase composition was also studied. Near 90% step coverage conformity was achieved for PrO x films on structured silicon substrates with aspect ratio 1:10. In air degradation of Pr 2 O 3 films with transformation to Pr(OH) 3 was observed in contrast to Pr 6 O 11 films.

The removal of carbon dioxide (CO2) from industrial emissions has become essential in the fight against climate change. In this study, we employed Zeolite 13X for the capture and recovery of CO2 in a flow through system where the adsorbent was subjected to five adsorption-desorption cycles. The influent stream contained 1.5% CO2 at standard conditions. The adsorbent bed was 1 in. in length and 1 in. 3/8 in dia., and was packed with 10 g of the zeolite. Temperature swing adsorption (TSA) was employed as the regeneration method through heating to approximately 135 °C with helium as the purge gas. The adsorbent capacity at 90% saturation was found to decrease from 78 to 60gCO2/kgZeolite13X after the fifth cycle. The CO2 capture ratio or the mass of CO2 adsorbed to the total mass that entered the system decreased from 63% to only 61% after the fifth cycle. The CO2 recovery efficiency ranged from 82 to 93% during desorption, and the CO2 relative recovery, i.e., CO2 desorbed for the nth cycle to CO2 adsorbed for the first cycle, ranged from 88 to 68%. The service life of the adsorbent was determined to be equal to eleven cycles at a useful capacity of 40gCO2/kgZeolite13X. © 2007 American Institute of Chemical Engineers AIChE J, 2007

Background— Ultrasound can increase tissue blood flow, in part, through the intravascular shear produced by oscillatory pressure fluctuations. We hypothesized that ultrasound-mediated increases in perfusion can be augmented by microbubble contrast agents that undergo ultrasound-mediated cavitation and sought to characterize the biological mediators. Methods and Results— Contrast ultrasound perfusion imaging of hindlimb skeletal muscle and femoral artery diameter measurement were performed in nonischemic mice after unilateral 10-minute exposure to intermittent ultrasound alone (mechanical index, 0.6 or 1.3) or ultrasound with lipid microbubbles (2×10 8 IV). Studies were also performed after inhibiting shear- or pressure-dependent vasodilator pathways, and in mice with hindlimb ischemia. Ultrasound alone produced a 2-fold increase ( P P P 2a receptors and epoxyeicosatrienoic acids had minimal effect. Limb nitric oxide production and muscle phospho-endothelial nitric oxide synthase increased in a stepwise fashion by ultrasound and ultrasound with microbubbles. In mice with unilateral hindlimb ischemia (40%–50% reduction in flow), ultrasound (mechanical index, 1.3) with microbubbles increased perfusion by 2-fold to a degree that was greater than the control nonischemic limb. Conclusions— Increases in muscle blood flow during high-power ultrasound are markedly amplified by the intravascular presence of microbubbles and can reverse tissue ischemia. These effects are most likely mediated by cavitation-related increases in shear and activation of endothelial nitric oxide synthase.

Preclinical studies had determined that tamoxifen and interferon-alpha2b (IFN-alpha2b) synergistically inhibited growth of both estrogen-receptor positive and negative murine tumor xenografts and had combined antiangiogenic effects and that tamoxifen potentiated IFN-stimulated gene (ISG) expression. A phase I trial in 26 patients was conducted using the combination to define tolerance and potentiation of ISG expression. IFN- alpha2b at a dose of 3 x 10(6) units/m(2) daily was given subcutaneously (s.c.), and tamoxifen was initiated as a loading dose of 150 mg/m(2) and then 60 mg/m(2) twice daily on day 8. At this initial dose, reduction of dose of IFN- alpha2b was required in 4 of 11 patients, primarily because of fatigue. Another group of patients was treated with an identical tamoxifen dose but with IFN-alpha2b reduced to 2 x 10(6)/m(2) U; this was better tolerated. As the projected serum tamoxifen level to reduplicate preclinical effects was 300 mg/m(2), dose escalation in a third cohort was undertaken; it had to be discontinued secondary to grade III or IV toxicity in 2 of 2 patients. Increases in products of transcriptionally regulated ISGs, beta (2)-microglobulin, neopterin, and ISG15 were assessed. All ISGs increased after IFN-alpha2b, but only ISG15 had a further significant rise after initiation of tamoxifen. Because at doses not limited by unacceptable toxicities, no marked potentiation of ISGs by tamoxifen could be identified, clinical evaluation of the combination was terminated.

Using beta-diketonate Ru precursors diluted in n-octane Ru and RuO"2 layers were grown on plane 200nm SiO"2/Si wafers by atomic vapor deposition (AVD^(R)). AVD^(R) was carried out in an AIXTRON Tricent^(R) system using liquid delivery of the ruthenium precursor. The deposited multi-crystalline smooth (RMS surface roughness

The remanent magnetization and the magnetic anisotropy energy of 5–16 monolayer (ML) epitaxial Fe films grown on GaAs(001) were quantitatively monitored in situ by superconducting quantum interference device magnetometry and ferromagnetic resonance as a function of oxygen exposure at 300 K. We find that at an oxygen dose of 10 L the perpendicular uniaxial anisotropy decreases by almost 50%, whereas the magnetization is decreased by 8% only in the case of a 5 ML Fe film. The fourfold anisotropy constant K4 and in-plane uniaxial anisotropy constant , however, remain unchanged. Low-energy electron diffraction spots of the Fe surface disappear after 6 L of oxygen, indicating a disordered Fe oxide growth. For exposures below 70 L we observe a reduction of the remanent magnetization irrespective of the film thickness. At exposures >70 L we find a faster decrease of the remanent magnetization for the thinner films which is attributed to the finite size effect and a thickness dependent surface roughness of the iron films, respectively. As evidenced by Auger electron spectroscopy and x-ray diffraction, the iron oxide is found to be FeO in the first stage of oxidation, transforming into γ-Fe2O3 at higher dosages (>1000 L).

IL-2 therapy results in 10–20% response rates in advanced renal cell carcinoma (RCC) via activating immune cells, in which the protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) is a key negative regulator. Based on finding that sodium stibogluconate (SSG) inhibited SHP-1, the anti-RCC potential and action mechanism of SSG and SSG/IL-2 in combination were investigated in a murine renal cancer model (Renca). Despite its failure to inhibit Renca cell proliferation in cultures, SSG induced 61% growth inhibition of Renca tumors in BALB/c mice coincident with an increase (2-fold) in tumor-infiltrating macrophages (Mφ). A combination of SSG and IL-2 was more effective in inhibiting tumor growth (91%) and inducing tumor-infiltrating Mφ (4-fold), whereas IL-2 alone had little effect. Mφ increases were also detected in the spleens of mice treated with SSG (3-fold) or SSG/IL-2 in combination (6-fold), suggesting a systemic Mφ expansion similar to those in SHP-deficient mice. T cell involvement in the anti-Renca tumor action of the combination was suggested by the observations that the treatment induced spleen IFN-γ T cells in BALB/c mice, but failed to inhibit Renca tumor growth in athymic nude mice and that SSG treatment of T cells in vitro increased production of IFN-γ capable of activating tumoricidal Mφ. The SSG and SSG/IL-2 combination treatments were tolerated in the mice. These results together demonstrate an anti-Renca tumor activity of SSG that was enhanced in combination with IL-2 and functions via a T cell-dependent mechanism with increased IFN-γ production and expansion/activation of Mφ. Our findings suggest that SSG might improve anti-RCC efficacy of IL-2 therapy by enhancing antitumor immunity.

Objective: The protocol was designed to examine the biological effects and clinical activity of interferon-β in patients with platinum/taxane-resistant ovarian cancer. Methods: Patients with resistant ovarian and fallopian tube cancers and primary peritoneal carcinoma were treated with recombinant human interferon-β (Rebif®, Serono International) at doses ranging from 6 to 24 million international units (MIU)/day, based on their tolerance to therapy. Levels of IP-10, an interferon-inducible protein, were measured in the serum to evaluate the biological effects of the drug. Also, the peripheral blood mononuclear cells and serum were examined for the induction of previously described novel regulators of interferon-induced death. Results: Eighteen patients were treated, of whom 9 (50%) could be treated at the highest dose level (24 MIU). The major toxicities were fever, chills and fatigue. The median duration of therapy was 6 weeks (range 1–22). No objective responses were observed. IP-10 levels were significantly increased, compared with baseline, at 2, 4, and 6 weeks after initiation of therapy (p < 0.01). Conclusions: Recombinant human interferon-β produced a definite biological effect in the serum of treated patients, but this outcome was not translated into any clinically observable or meaningful impact on the disease process.

Interferons (IFNs), pleiotropic cytokines that regulate antiviral, antitumor, apoptotic, and cellular immune responses, were the first endogenous antiangiogenic regulators identified. In a species-specific manner, IFNs inhibit secretion of such angiogenic factors as basic fibroblast growth factor from tumor cells. The antiangiogenic activity of IFNs is enhanced when they are combined with other antiangiogenic agents, such as tamoxifen and thalidomide.

Cancer cell resistance limits the efficacy of IFNs. In this study, we show that sodium stibogluconate (SSG) and IFN-α synergized to overcome IFN-α resistance in various human cancer cell lines in culture and eradicated IFN-α-refractory WM9 human melanoma tumors in nude mice with no obvious toxicity. SSG enhanced IFN-α-induced Stat1 tyrosine phosphorylation, inactivated intracellular SHP-1 and SHP-2 that negatively regulate IFN signaling, and induced cellular protein tyrosine phosphorylation in cancer cell lines. These effects are consistent with inactivation of phosphatases as the basis of SSG anticancer activity. Characterization of SSG by chromatography revealed that only selective compounds in SSG were effective protein tyrosine phosphatase inhibitors. These observations suggest the potential of SSG as a clinically usable protein tyrosine phosphatase inhibitor in cancer treatment and provide insights for developing phosphatase-targeted therapeutics.

The interferon (IFN)-β and all-trans-retinoic acid combination suppresses tumor growth by inducing apoptosis in several tumor cell lines. A genetic technique permitted the isolation of human thioredoxin reductase (TR) as a critical regulator of IFN/all-trans-retinoic acid-induced cell death. Our recent studies have shown that TR1:thioredoxin 1-regulated cell death is effected in part through the activation of p53-dependent responses. To understand its death regulatory function, we have performed a mutational analysis of TR. Human TR1 has three major structural domains, the FAD binding domain, the NADPH binding domain, and an interface domain (ID). Here, we show that the deletion of the C-terminal interface domain results in a constitutive activation of TR-dependent death responses and promotes p53-dependent gene expression. TR mutant without the ID still retains its dependence on thioredoxin for promoting these responses. Thus, our data suggest that TR-ID acts as a regulatory domain.

In the silicon industry new materials and particularly, their deposition methods have become a fundamental integral part to enable next generations of highly integrated devices. Especially in the field of volatile and non-volatile memory applications as well as in integrated capacitor devices for high-frequency applications existing technologies generate an increasing demand for new advanced innovative solutions to further follow “Moore's law” in the near future. Amongst available film growth technologies, metal organic chemical vapor deposition (MOCVD) basically offers unique combination of composition controllability, high uniformity over large areas, high throughput, and last but not least a high degree of conformality over 3D structures. However, MOCVD of complex materials was in the past often confronted with an absence of appropriate metal organic precursors and MOCVD-tools. This work provides an overview of some of the latest achievements in the field of MOCVD of complex oxides and electrode materials using novel MOCVD deposition methods and tools. It proves that there now exist mature deposition methods with the high degree of reliability and reproducibility mandatory for production.

The nonamethylene bridge of the title compound shows high conformational mobility and an asymmetric structure in the crystal. Molecular mechanics (MM+) calculations reproduced well the crystal structure, which has the lowest energy conformation.

To investigate the function of mGBP-2, a member of the interferon (IFN)-induced guanylate-binding protein family of GTPases, NIH 3T3 fibroblasts were generated that constitutively expressed mGBP-2. mGBP-2 induced a faster growth rate, with the highest expressing clones showing approximately a 50% reduction in doubling time. mGBP-2-expressing cells also grew to higher density and exhibited partial loss of contact growth inhibition, as evidenced by the formation of foci in post-confluent cultures. In addition, mGBP-2-expressing cells showed decreased dependence on serum-derived growth factors. However, they did not lose the requirement for anchorage-dependent growth. Finally, NIH 3T3 cells expressing mGBP-2 formed tumors in athymic mice. An mGBP-2 protein carrying a point mutation (S52N) that reduced GTP binding failed to produce these phenotypes when expressed at the same levels as wild type. The additional finding that IFN-γ treatment of NIH 3T3 cells resulted in an increase in proliferation similar to that observed for mGBP-2 in the absence of other IFN-induced proteins suggests that mGBP-2 may indeed be important for these growth changes.

Platinum (Pt), ruthenium (Ru), and ruthenium dioxide (RuO 2 ) have been considered as possible candidates as electrodes for high permittivity and ferroelectric oxides. The films were grown by metal organic chemical vapor deposition (MOCVD). High purity Pt films with near bulk resistivities (11 w z cm) were deposited. The Pt, Ru and RuO 2 films were found to be continuous and smooth and exhibited excellent adhesion properties on the underlying substrates. The temperature dependent electrical conductivity of the Ru and RuO 2 films was metallic type. Room temperature resistivities as low as 18 and 30 w z cm were determined for Ru and RuO 2 , respectively.

Intratumoral Prostate cancer (PCa) heterogeneity is characterized by multiple intrinsic mechanisms including Androgen signaling, aerobic glycolysis, aberrant transcriptional activation of c-Myc and dysregulation of miRNAs. The ability of prostate cancer cells to alter growth and metabolism in a manner distinct from benign cells is a major concern in the treatment of castration-resistant prostate cancer. Therefore, targeting the diverse oncogenesis promoting pathways involved in PCa progression and resistance at the posttranscriptional level using micro RNAs (miRNAs) offers a novel therapeutic option. Using a miRNA mimic screen, we identified a human-specific miRNA miR-644a, which modulates expression of a diverse set of oncogenic pathways in PCa including androgen signaling, AKT/IGF-1R signaling, c-myc mediated signaling and EMT pathways. We have also demonstrated a potent tumor suppressive function of miR-644a in mouse prostate cancer xenograft model. miR-644a also sensitizes the PCa tumors to current therapeutic options including AR antagonist Enzalutamide by regulating the key modulators in development of drug resistance. In conclusion, our study demonstrates that miR-644a is a novel tumor suppressor miRNA regulating key prostate carcinogenesis promoting pathways. Citation Format: Jey Sabith Ebron, Jagjit Singh, Eswar Shankar, Crystal Weyman, Sanjay Gupta, Daniel J. Lindner, Girish C. Shukla. Fine-tuning the expression of heterogeneous network of genes involved in androgen signaling, aerobic glycolysis, apoptosis and epithelial-mesenchymal transition by microRNA-644a in prostate cancer potentiation of AR signaling therapy by miR-644a: Selective manipulation of the prostate cancer transcriptome by miR-644a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1470. doi:10.1158/1538-7445.AM2017-1470

Prostate cancer growth and proliferation depends on androgen signaling mediated by transactivation of Androgen Receptor (AR). Androgen ablation remains the mainstay therapy for treatment of the disease. However, despite androgen ablation, the disease relapses to more aggressive form known as castration-resistant prostate cancer (CRPC). Androgen Signaling Inhibitor such as Abiraterone Acetate and Enzalutamide are the most effective treatment methods currently being used to treat CRPC. However, more than one-third of CRPC patients develop resistance to these treatments, mostly due to the gain of function in the AR protein and increase in intratumoral dihydrotestosterone (DHT) synthesis. Intratumoral DHT synthesis from steroid precursors in tumors is augmented by up-regulation of enzymes of cholestrogenesis and steroidogenesis, such as HMG-CoA reductase, SCARB1, and 17βHSD respectively. Tumor-specific downregulation of microRNAs which regulate the AR and steroid biosynthesis has been implicated in tumor growth and resistance to therapeutics in CRPC. We are focusing on tumor suppressive role of miR-149-5p in PCa. We discovered that miR-149-5p expression was significantly lower in prostate cancer tissues compared to normal tissues. The mechanistic investigation revealed that miR-149-5p downregulates wild type and alternatively spliced variant of AR. It also down regulates the expression of SCARB1, HMGCS and HMG-CoA reductase, the proteins known to facilitate intratumoral DHT synthesis. Ectopic expression of miR-149-5p negatively regulated the expression of prostate-specific antigen, a downstream target of androgen signaling and it also inhibited invasion and proliferation of CRPC cells. Our findings indicate a significant role of miR-149-5p in regulating androgen signaling and possibly DHT synthesis in CRPC. This provides a strong rationale for further investigating the significance of miR-149-5p for generation of new therapeutic for CRPC. Citation Format: Savita Singh, Jey Sabith Ebron, Eswar Shankar, Sanjay Gupta, Daniel Lindner, Girish Shukla. Regulation of androgen signaling axis and tumor suppressive function of miR-149-5p in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2531. doi:10.1158/1538-7445.AM2017-2531

The FERM domain containing protein Kindlin-3 has been recognized as a major regulator of integrin function in hematopoietic cells, but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ∼7-fold elevation in Kindlin-3 mRNA compared with nonneoplastic tissue by quantitative polymerase chain reaction. Kindlin-3 overexpression in a breast cancer cell line increased primary tumor growth and lung metastasis by 2.5- and 3-fold, respectively, when implanted into mice compared with cells expressing vector alone. Mechanistically, the Kindlin-3-overexpressing cells displayed a 2.2-fold increase in vascular endothelial growth factor (VEGF) secretion and enhanced β1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor that promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production, and knockdown of β1 integrins diminished Twist and VEGF production by Kindlin-3-overexpressing cells, while nontargeting small interfering RNA had no effect on expression of these gene products. Thus, Kindlin-3 influences breast cancer progression by influencing the crosstalk between β1 integrins and Twist to increase VEGF production. This signaling cascade enhances breast cancer cell invasion and tumor angiogenesis and metastasis.—Sossey-Alaoui, K., Pluskota, E., Davuluri, G., Bialkowska, K., Das, M., Szpak, D., Lindner, D. J., Downs-Kelly, E., Thompson, C. L., Plow, E. F. Kindlin-3 enhances breast cancer progression and metastasis by activating Twist-mediated angiogenesis.

Das aus dem pentafulvenoiden Allen 1 leicht zugängliche, kinetisch stabilisierte 1,1′-Diethinylferrocen lässt sich durch oxidative Kupplung in das [4.4]Ferrocenophan 2 überführen. Diese Verbindung hat eine bemerkenswert symmetrische Struktur, die Elektronen sind über die Butadiinbrücken hinweg delokalisiert, und im Kristall liegt 2 in einer helical-chiralen Konformation vor.

Building butadiyne bridges: The kinetically stabilized 1,1′-diethynylferrocene, readily prepared from the pentafulvenoid allene 1, can be transformed into the [4.4]ferrocenophane 2 by oxidative coupling. This compound exhibits a remarkably symmetrical structure in which the electrons are delocalized through the butadiyne bridges; in the crystalline state 2 exists in a helical-chiral conformation.

We have prepared RuO 2 layers by metal organic chemical vapour deposition using liquid delivery source and by thermal evaporation of powder precursors. The films were prepared on silicon and r-plane cut sapphire substrates. We discuss thermodynamics of both types of MOCVD techniques. Liquid delivery source technique using diglyme solvent results in deposition of metallic Ru film with some traces of RuO 2 , while films prepared by thermal evaporation of powder precursors consist of pure RuO 2 phase. Thermal evaporation MOCVD grown RuO 2 films exhibit excellent electrical properties; room temperature resistivity of 30 μΩcm and residual resistivity ratio between 8 and 30.

Interferons (IFNs) regulate the expression of genes that mediate their antiviral, antitumor, and immunomodulatory actions. We have previously shown that IFN-beta suppresses growth of human ovarian carcinoma xenografts in vivo and induces apoptosis of ovarian carcinoma cells in vitro. To investigate mechanisms of IFN-beta-induced apoptosis we employed an antisense technical knockout approach to identify gene products that mediate cell death and have isolated several regulators of interferon-induced death (RIDs). In this investigation, we have characterized one of the RIDs, RID-2. Sequence analysis revealed that RID-2 was identical to human inositol hexakisphosphate kinase 2 (IP6K2). IP6K2 is post-transcriptionally induced by IFN-beta in ovarian carcinoma cells. A mutant IP6K2 with substitutions in the putative inositol phosphate binding domain abrogates IFN-beta-induced apoptosis. These studies identify a novel function for IP6K2 in cell growth regulation and apoptosis.

It was demonstrated by X-ray diffraction analysis that the reaction of 3,3-dimethyl-6-trifluoromethyl-2,3-dihydro-4-pyrone with hydroxylamine afforded 5-hydroxyamino-3-(2-hydroxy-1,1-dimethylethyl)-5-trifluoromethyl-Δ2-isoxaline.

MOCVD is a suitable technique for the synthesis of functional metal oxide thin films. In the present paper, injection-MOCVD was proved to be an efficient method, not only to grow single layers, but also heterostructures and superlattices of functional oxide materials. We report on the deposition of BaxSr1−xTiO3 (BST) films and BaTiO3/SrTiO3 superlattices [(BTO/STO)n] obtained by injection MOCVD. In a first step, BST films were deposited on various substrates such as LaAlO3(LAO)(012), SrTiO3:Nb(STO:Nb)(001) and Al2O3(0001), resulting in epitaxial films in the case of STO or LAO substrates and highly textured films in the case of Al2O3 substrates. The good crystallinity of the films obtained was evidenced using X-ray analysis. Concerning the dielectric properties of BST films on STO:Nb, the Tc values were found to be the same as those for bulk materials. In a next step the feasibility of (BTO/STO)n superlattices was studied. The multilayers obtained were epitaxially grown on LAO and STO:Nb substrates. XPS was used to check in detail the pattern repetition of the superlattice and proved the low interface roughness of 1 nm as well as the absence of carbon contamination in the film. The measured periodicity of these superlattices were found to be in good agreement with simulation experiments. Further superlattices have been obtained by combining ferroelectric, magnetic (CMR) and superconducting (HTS) oxides. Typical results of these superlattices will be reported.

New BaTiO 3 -SrTiO 3 (BTO-STO)-superlattices which may be interesting for future electronic applications have been investigated by X-ray photoelectron spectroscopy (XPS) depth profiling. At first XPS measuring conditions were optimized for that non-conducting and thin layer systems (21 nm double layer thickness) considering the practical instrumental limitations. Second a simulation of the sputtering process for the concrete experimental conditions were done by a dynamic T-DYN code. By comparison of experimental and simulated depth profiles the maximum sample roughness could be estimated to be in the range of 2 nm.