ALBERT

Description: Ultra-high frequency (UHF) space-based (SB) synthetic aperture radar (SAR) can suffer from the degrading effects of a scintillating ionosphere which modulates both the phase and the amplitude of the radar signal. In this paper, we use Global Navigation Satellite System (GNSS) signals to synthesize an L-band SAR point spread function (PSF). The process of transforming the GNSS signal to the equivalent SAR PSF is described. The synthesized PSF is used to explore the possibility of using a phase correction determined from a point target in a SAR image to correct the ionospheric degradation. GNSS data recorded on equatorial Ascension Island during scintillation events are used to test the feasibility of this approach by applying a phase correction to one GNSS receiver from another located along a magnetic east–west baseline. The peak-to-sidelobe-ratios of the synthesized L-band SAR point spread functions before and after the correction are compared and it is shown that this approach improves the L-band PSF over distances of ~3000 m in the ionosphere, corresponding to 6000 m on the ground.

Description: With support from the NASA sounding rocket program, the Air Force Research Laboratory (AFRL) launched two sounding rockets in the Kwajalein Atoll, Marshall Islands in May 2013 known as the Metal Oxide Space Cloud (MOSC) experiment. The rockets released samarium metal vapor at preselected altitudes in the lower F-region that ionized forming a plasma cloud. Data from ALTAIR incoherent scatter radar and high frequency (HF) radio links have been analyzed to understand the impacts of the artificial ionization on radio wave propagation. The HF radio wave ray-tracing toolbox PHaRLAP along with ionospheric models constrained by electron density profiles measured with the ALTAIR radar have been used to successfully model the effects of the cloud on HF propagation. Up to three new propagation paths were created by the artificial plasma injections. Observations and modeling confirm that the small amounts of ionized material injected in the lower-F region resulted in significant changes to the natural HF propagation environment.

Description: Key PointsAEB071 demonstrates preclinical in vitro and in vivo activity against CLL independent of survival signaling and stromal cell protection. AEB071 can either inhibit or activate the WNT pathway emphasizing the importance of pharmacodynamic monitoring in its development.

Description: Sickle Cell Disease (SCD) is an autosomal recessive disorder that affects thousands of people across the United States. Although it is not a disease exclusive to the African American population, it is most common among persons from this ethnic group with an estimated 1 out of every 500 individuals affected. The disease is caused by a point mutation in the sixth codon of the beta-globin gene which is found on chromosome 11. This missense mutation involves the replacement of the hydrophilic glutamic acid with a hydrophobic valine that affects the structure of the b-GLOBIN protein leading to altered erythrocyte elasticity and oxygen carrying capacity. As a result, less oxygen is delivered to the tissue and the deformed, rigid cells often become trapped in the microvasculature causing local tissue ischemia, inflammation and the pain crisis that is characteristic of the disease. Early research into the understanding of this devastating disease has shown that a modulator of SCD severity is the persistence of fetal hemoglobin (HbF or γ-GLOBIN). Indeed SCD patients carrying a genetic mutation leading to hereditary persistence of HbF are able to maintain a near crisis-free life. This is so because HbF is made up of 2 alpha and 2 gamma subunits and lacks the beta subunits found in adult hemoglobin. As a result, HbF lacks the ability to sickle when there are significant changes in the oxygen levels. Several genetic, biochemical and clinical observations indicate that even modest induction of γ-GLOBIN may be sufficient to ameliorate the SCD phenotype. In the past decade efforts have focused on identifying regulatory complexes that modulate fetal-γ-GLOBIN gene expression that could be targeted for pharmacological intervention to improve and to monitor the outcome of this incurable disease. Physiologic transcriptional silencing of the γ-GLOBIN locus during development could be reversed for therapeutic benefit. To date, compounds such as 5-aza-2-deoxycytidine, hydroxyurea, short-chain fatty acids, and histone deacetylase (HDAC) inhibitors, have been used to increase HbF, however, effects are variable and mechanism of action is poorly understood. Thus, it is imperative that additional therapies be developed to enhance HbF expression in patients not responding well to the drugs available on the market. Recent work has identified key components of the repressor complex that silences fetal-γ-GLOBIN including protein arginine methyltransferase 5 enzyme (PRMT5) and histone deacetylase protein 1 and 2 (HDAC1/2). Studies are ongoing to characterize the regulatory complex that is recruited to the γ-GLOBIN gene promoter and develop a lead optimized PRMT5 inhibitor drug for clinical translation. Pretreatment of the erythroleukemia cell line K562 with three of our newly developed PRMT5 inhibitors (CMP5, HLCL7 and CMP12) leads to re-expression of g-GLOBIN gene product HbF at mRNA and protein levels in a time and dose dependent fashion. As PRMT5, LSD1 and HDAC1/2 have been shown to be all involved in the regulation of expression of HbF gene we further evaluated the effect of specific inhibitors targeting each of these proteins alone or in combination. Our data strongly indicate that while each agent alone led to re-expression of HbF, combination treatments led to an enhanced protein expression. Studies are ongoing to caracterize the regulatory complex that is recruited to the γ-GLOBIN gene promoter and develop a lead optimized PRMT5 inhibitor drug for clinical translation. We believe that our results will be the key for developing of more effective and specific therapies for this incurable disease. Disclosures: No relevant conflicts of interest to declare.

Description: Acute Myeloid Leukemia (AML) is a heterogeneous disease that is characterized by an accumulation of neoplastic myeloid precursor cells in the bone marrow. Recently, multiple agents targeting AML associated mutations in FLT3, IDH1 and IDH2 have been developed. However, a majority of AML patients lack these mutations. Therefore, development of a novel therapeutic approach broadly relevant to AML would be attractive. The clonal capacity of AML cells is maintained by leukemic initiating cells (LICs), which possess self-renewal capabilities and are resistant against cytotoxic combination chemotherapy. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in the generation of NAD+, which is an important enzymatic cofactor and serves as a metabolite required for cellular respiration. LICs show a higher energy turnover rate than normal cells and are heavily reliant on oxidative phosphorylation. This suggests that energy generation processes, such as NAD+ biosynthesis, are critically required in myeloid malignancies. Thus, targeting the regeneration of NAD+ offers an attractive alternative therapeutic strategy in AML. Unlike many targeted therapies that are limited to one genetic subtype of AML, targeting regeneration of NAD+ via NAMPT inhibition could be relevant to a much broader patient population based upon metabolic differences between tumor and normal cells. We show that inhibition of NAMPT using the agent KPT-9274 induces loss of glycolytic and mitochondrial activity, more specifically a depleted total spare reserve capacity (p

Description: BACKGROUND: Mature circulating red blood cells, though devoid of a nucleus, have been shown to contain an abundance of miRNAs. Further, it has been shown that sickle cell patient-derived RBCs have a dramatic difference in miRNA content than normal RBCs. Given that a range of miRNAs are involved in the regulation of immunity, including the release of inflammatory mediators, we hypothesize that miRNAs enriched in circulating red blood cells function to prolong the inflammatory state in sickle cell disease. Further, we hypothesize that these miRNAs can be used as biomarkers for use in the clinic to predict crisis and differentiate acute versus chronic pain. Exploring this miRNA enrichment in circulating red blood cells in sickle cell patients will provide practical insight for the inflammation state and will inform characteristics of patients who may need greater care in the clinic. METHODS: Twenty steady state patients were recruited and categorized according to their chronic pain status and crisis frequency per year. Whole blood was drawn during routine visits to the OSU Wexner Medical Center Hematology Clinic. Additionally, whole blood was drawn from five patients either in acute pain crisis (recruited prior to crisis) or within a few days of crisis. Samples were subject to double gradient centrifugation and red cells were resuspended in Trizol and cryopreserved. MiRNAs were isolated from red cell Trizol suspensions using a commercial isolation kit (QIAGEN Cat#217004). Isolated miRNAs were then subject to a NanoString Human miR (v3) expression assay. Differential expression analysis was conducted to compare miRNAs with at least 1.5 fold difference (p = 0.05) between steady state and acute crisis. Target prediction and GO ontology analysis was performed for statistically significant miRNAs using DIANA Tools mirPath v3. Follow-up qPCRs were performed using TaqMan Advanced miRNA cDNA Synthesis Kit (Cat#A28007) and TaqMan Advanced miRNA Assays (Cat#A25576) to validate the decreased expression of miRNAs. Additional qPCRs were performed using TaqMan Gene Expression Assays (Cat#4331182) to investigate mRNA regulatory effects of significant miRNAs in the total red cell population. Western blots were also performed to investigate regulatory effects of these miRNAs at the protein level. RESULTS & CONCLUSION: Comparison of RBC miRNA profiles from patients during acute crisis to those in steady state shows several significantly decreased (〉1.5 fold) miRNAs in crisis. Among these miRs we have found previously uncharacterized miRNAs, hsa-miR-2116-5p and hsa-miR-302d-3p. DIANA tools miRNA analysis software predicts these miRNAs to be involved in regulation of cell-to-cell adhesion pathways through gene transcripts such as Protocadherin Beta 6 (PCDHB6) and Neural Cell Adhesion Molecule 2 (NCAM2). Interestingly, inspection of miRNA predicted targets that fall under significant GO terms also predicts several individual miRNAs to regulate inflammatory response and nociceptive signaling gene transcripts like A20 (TNFAIP3) and Cathepsin S (CTSS). Validation of these miRNAs was performed via qPCR for 5 out of the 6 significantly decreased miRNAs. Of the 5 miRNAs tested, hsa-miR-2116-5p, hsa-miR-302d-3p, and hsa-miR-1246 were validated as having decreased expression in acute crisis patients compared to steady state. qPCRs were then performed to probe for miRNA based regulation of top predicted target mRNA transcripts. Both CTSS and TNFAIP3 showed increased expression of mRNA transcripts in acute crisis patient red cells as compared to steady state. Next, western blot analysis was performed on red cell protein lysate. Interestingly, this analysis revealed a pattern in activated CTSS expression that was independent of acute crisis. Steady state patients reporting chronic pain showed increased activated CTSS compared to those without chronic pain. Activated CTSS was not found in red cell lysates from three normal, non-SCD donors. Taken together, these results suggest that red blood cells may play a larger role in inflammation and pain responses in sickle cell disease than previously thought. Further these results suggest activated CTSS as a potential biomarker for differentiating chronic pain in patients. Follow-up studies are underway to further stratify and investigate these findings. Disclosures Desai: University of Pittsburgh: Research Funding; Ironwood: Other: Adjudication Committee; NIH: Research Funding; FDA: Research Funding; Selexy/Novartis: Research Funding; Pfizer: Research Funding.

Description: Acute Myeloid Leukemia (AML) is the most common adult acute leukemia and is characterized by numerous driver mutations and/or cytogenetic rearrangements that promote disruption of stem cell/early myeloid progenitor differentiation, apoptosis, and proliferation. Identification of both personalized targets specific to a mutation or genomic abnormality and also global ubiquitous tumor-related targets relevant to AML represents a high priority to improve therapy. p21 protein (Cdc42/Rac)-activated kinase 4 (PAK4) is involved in disease progression for several solid tumors but its expression and contribution to disease pathogenesis in AML has not been examined. Since multiple cellular pathways important in AML (e.g., RAS and Wnt/β-catenin) are regulated by PAKs, we hypothesized PAK4 (and other family members) could represent an attractive pharmacologic target. We first evaluated the expression of PAK4 in AML cell lines and patient blasts (55 patients) using RNA sequencing and Western blot confirmation. This demonstrated PAK4 to be abundantly expressed at the mRNA and protein level in virtually all the analyzed samples. We then tested the in vitro effects of the PAK4 allosteric modulators (PAMs) KPT-9274 (clinical candidate) and KPT-9331 (tool compound) on AML cell lines. These included MV4-11, HL-60, THP-1 and Kasumi-1. Cell lines were treated for 24, 48 and 72 with PAMs KPT-9274 and KPT-9331 at dosages ranging from 1nM to 10uM. Proliferation was measured by MTS assay. All cell lines showed a dose-and time- dependent decrease in cell proliferation with IC50 ranging from 0.14 to 0.28 µM for both compounds. Cell lines with low protein and mRNA expression of PAK4, such as HL-60, were sensitive to PAM treatment suggesting possible alternative targets of these agents. To determine the effect of PAMs on apoptosis, MV4-11 and THP-1 cell lines were treated with KPT-9331 at IC50 concentration (~0.25 µM) and cell death was measured via Annexin-V/PI flow cytometric analysis after 24, 48h and 72h treatment. KPT-9331 induced a time dependent increase in apoptosis in both cell lines. In MV4-11 cells, KPT-9331 caused cell cycle arrest and inhibition of proliferation after 24hrs. We also tested the effect of PAMs in primary AML cells. Patient samples cocultured with a human stromal cell lines were treated with PAMs for 96 hours. IC50 values ranged from 0.14 - 0.19 µM. A dose dependent decrease in proliferation following PAM treatment was observed in all the five analyzed samples irrespective of genetic subtype. PAMs treatment for 48hrs using a whole blood viability assay from normal donors showed no significant cytotoxic effect on T and NK cells, but modest toxicity to normal B cells. Normal hematopoietic colony forming cell assays are being initiated and will be presented. We next utilized a human AML leukemia xenograft model with MV4-11 cells to assess the in vivo activity of KPT-9274. Mice were dosed once daily via oral gavage with KPT-9274 (150 mg/kg) or vehicle control. KPT-9274 dramatically inhibited tumor growth, prevented invasion of MV4-11 cells, and improved overall survival with all mice (n=7) being alive (median not reached) at day 49 of experiment as compared to 1 out of 7 vehicle-treated mice (median survival 36 days) being alive at this time. In summary, KPT-9274 demonstrates promising activity in pre-clinical AML models and warrants further investigation in this disease. Ongoing efforts include validating the specificity of the reported target in AML (versus alternative targets), in vivo exploration in primary human AML xenograft models, and understanding the effects of this compound on normal hematopoiesis and function. Disclosures Baloglu: Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Blum:Gilead Sciences: Research Funding. Byrd:Acerta Pharma BV: Research Funding.