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1
Horowitz, Netanel A., Elizabeth A. Blevins, Whitney M. Miller, Ashely R. Perry, Kathryn E. Talmage, Eric S. Mullins, Brett P. Monia, Jay L. Degen, and Joseph S. Palumbo. "Thrombin-Thrombomodulin Interactions Are An Important Determinant of Metastatic Potential." Blood 116, no. 21 (November 19, 2010): 822. http://dx.doi.org/10.1182/blood.v116.21.822.822.
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Abstract Abstract 822 A substantial body of evidence indicates that tumor cell-associated (i.e., tissue factor) and circulating hemostatic system components (i.e., thrombin, fibrinogen, platelets) play a cooperative role in supporting metastasis. However, the role of endothelial regulators of hemostasis in metastasis remains largely unexplored. Thrombomodulin (TM) is the endothelial thrombin receptor central to thrombin-mediated activation of protein C. To test the hypothesis that thrombin-TM interactions are an important determinant of metastasis, we used mice carrying the Glu387Pro mutation in thrombomodulin (TMPro) known to decrease thrombin affinity ∼100 fold and APC generation ∼1000 fold. TMPro/Pro and control mice were intravenously injected with either a low dose (3 × 104 cells/mouse) or high dose (3 × 105 cells/mouse) of Lewis lung carcinoma cells (LLC) in separate experiments. At the low cell dose, the majority of wild-type mice developed no discernable pulmonary metastases, while TMPro/Pro mice each developed ∼100 grossly apparent pulmonary metastases. At the high cell dose the outcome was possibly even more striking, with few metastatic foci apparent in wild-type mice and fully confluent surface metastases too numerable to count in TMPro/Pro mice. Histological analyses confirmed that lung tissue from TMPro/Pro mice had been largely replaced with tumor. The dramatic augmentation in metastasis observed in TMPro/Pro mice did not appear to be due to genotype dependent differences in tumor growth potential as LLC cells transplanted into the dorsal subcutis of TMPro/Pro and control mice grew at similar rates and were histologically indistinguishable. Rather, tumor cell fate analyses using 125I-radiolabeled LLC cells revealed that the imposition of the TMPro mutation dramatically improved the early survival of tumor cells in the lung. Twenty minutes after tumor cell injection >80% of the tumor cells were localized within the lungs regardless of animal genotypes, indicating that the TMPro mutation did not have a major impact on initial tumor cell adhesion/stabilization within the pulmonary vasculature. In contrast, 6 hours post-injection <20% of the initial inoculum remained in the lungs of control mice, while 75% remained in the lungs of TMPro/Pro mice. To determine whether the prometastatic phenotype conferred by the TMPro mutation is directly dependent on tumor cell-associated procoagulant function, TMPro/Pro and control mice were challenged with previously described fibrosarcoma cells genetically incapable of tissue factor (TF) expression, or fibrosarcoma cells in which TF expression had been genetically restored. The number of metastatic foci formed by TF-expressing cells was dramatically higher in TMPro/Pro mice relative to wild-type animals, whereas TF-deficient tumor cells were essentially incapable of forming metastases in mice of either genotype. Thus, the prometastatic effect of the TMPro mutation is contingent upon TF expression by the tumor cell. Depletion of circulating prothrombin levels to <5% of normal by an anti-sense oligonucleotide approach also profoundly limited the formation of metastatic foci in both TMPro/Pro and control mice, consistent with the conclusion that thrombin-TM interactions are a key determinant of metastasis. Taken together, these studies demonstrate for the first time that endothelial thrombin-thrombomodulin interactions strongly control metastatic potential and suggest that intervention at the level of thrombomodulin could represent a novel therapeutic strategy for preventing or treating metastatic disease. Disclosures: No relevant conflicts of interest to declare.
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Deng, Wei, Mengting Yang, Zhiwen Duan, Cheng Peng, Zhiming Xia, and Shuzhong Yuan. "Molecular basis of resistance to bensulfuron-methyl and cross-resistance patterns to ALS-inhibiting herbicides in Ludwigia prostrata." Weed Technology 35, no. 4 (June 21, 2021): 656–61. http://dx.doi.org/10.1017/wet.2021.47.
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AbstractLudwigia prostrata is a problematic weed in rice fields in China, where acetolactate synthase (ALS)-inhibiting herbicides (e.g., bensulfuron-methyl) are widely used for the management of broadleaf weeds. Recently, an L. prostrata biotype (JS-R) that failed to be controlled with ALS-inhibiting herbicides was found in Jiangsu Province, China. This study aims to determine the level and molecular mechanism of resistance to bensulfuron-methyl in this JS-R biotype and to evaluate its spectrum of cross-resistance to other ALS-inhibiting herbicides. The dose–response assays indicated that the JS-R L. prostrata biotype had evolved 21.2-fold resistance to bensulfuron-methyl compared with the susceptible biotype (JS-S). ALS gene sequencing revealed that a nucleotide mutation (CCA to TCA) at codon 197, resulting in a Pro-197-Ser mutation, was detected in the resistant plants. Moreover, while the JS-R biotype contained the Pro-197-Ser resistance mutation and showed cross-resistance to pyrazosulfuron-ethyl (12.0-fold), it was sensitive to penoxsulam, bispyribac-sodium, and imazethapyr, which may serve as alternative herbicides to control the resistant L. prostrata biotype. This is the first confirmation of an L. prostrata biotype resistant to bensulfuron-methyl due to a Pro-197-Ser resistance mutation in the ALS gene.
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Weijer, Sebastiaan, Catharina W. Wieland, Sandrine Florquin, and Tom van der Poll. "A thrombomodulin mutation that impairs activated protein C generation results in uncontrolled lung inflammation during murine tuberculosis." Blood 106, no. 8 (October 15, 2005): 2761–68. http://dx.doi.org/10.1182/blood-2004-12-4623.
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AbstractThrombomodulin (TM) plays an essential role in the generation of activated protein C (APC), a mediator with both anticoagulant and anti-inflammatory properties, and is preferentially expressed in lungs. To investigate the role of TM in the coagulant and inflammatory response in the lung during tuberculosis, mice with a mutation in the TM gene (Thbd), which results in a minimal capacity for APC generation (TMpro/pro mice), were intranasally infected with live virulent Mycobacterium tuberculosis. Whereas pulmonary tuberculosis was not associated with activation of coagulation in either wild-type or TMpro/pro mice, 5 weeks after infection TMpro/pro mice displayed an uncontrolled inflammatory response in their lungs, as reflected by higher lung weights, a diminished ability to form well-shaped granulomas, elevated levels of proinflammatory cytokines, and concurrently reduced concentrations of anti-inflammatory cytokines. During a 36-week follow-up after infection with a lower dose of M tuberculosis, 35% of TMpro/pro mice died from week 28 onward versus none of the wild-type mice, and the surviving TMpro/pro mice displayed increased lung inflammation accompanied by higher mycobacterial loads in liver and spleen. These data suggest that a TM mutation that impairs APC generation results in uncontrolled lung inflammation during tuberculosis.
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Alwarnaidu Vijayarajan, Vijaya Bhaskar, Patrick D. Forristal, Sarah K. Cook, David Schilder, Jimmy Staples, Michael Hennessy, and Susanne Barth. "First Detection and Characterization of Cross- and Multiple Resistance to Acetyl-CoA Carboxylase (ACCase)- and Acetolactate Synthase (ALS)-Inhibiting Herbicides in Black-Grass (Alopecurus myosuroides) and Italian Ryegrass (Lolium multiflorum) Populations from Ireland." Agriculture 11, no. 12 (December 14, 2021): 1272. http://dx.doi.org/10.3390/agriculture11121272.
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Understanding the resistance spectrum and underlying genetic mechanisms is critical for managing herbicide-resistant populations. In this study, resistance to acetyl CoA carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors was investigated in four suspected resistant populations of Alopecurus myosuroides (ALOMY-001 to ALOMY-004) and Lolium multiflorum (LOLMU-001 to LOLMU-004), collected from cereal production fields in Ireland. Glasshouse assays with three ALOMY-active herbicides [propaquizafop, cycloxydim (ACCase) and mesosulfuron + iodosulfuron (ALS)] or five LOLMU-active herbicides [pinoxaden, propaquizafop, cycloxydim (ACCase) and mesosulfuron + iodosulfuron, pyroxsulam (ALS)], and target-site resistance mechanism studies, based on pyrosequencing, were carried out in each of those populations. For A. myosuroides, Ile-1781-Leu ACCase mutation contributed to propaquizafop and cycloxydim resistance (shoot dry weight GR50 resistance factor (RF) = 7.5–35.5) in all ALOMY populations, and the independent Pro-197-Thr or Pro-197-Ser ALS mutation contributed to mesosulfuron + iodosulfuron resistance (RF = 3.6–6.6), in ALOMY-002 to ALOMY-004. Most of the analyzed plants for these mutations were homo/heterozygous combinations or only heterozygous. For L. multiflorum, phenotypic resistance to mesosulfuron + iodosulfuron (RF = 11.9–14.6) and pyroxsulam (RF = 2.3–3.1) was noted in all LOLMU populations, but the Pro-197-Gln or Pro-197-Leu ALS mutation (mostly in homozygous status) was identified in LOLMU-001, LOLMU-002 and LOLMU-004 only. Additionally, despite no known ACCase mutations in any LOLMU populations, LOLMU-002 survived pinoxaden and propaquizafop application (RF = 3.4 or 1.3), and LOLMU-003 survived pinoxaden (RF = 2.3), suggesting the possibility of non-target-site resistance mechanisms for ACCase and/or ALS resistance in these populations. Different resistance levels, as evidenced by a reduction in growth as dose increased above field rates in ALOMY and LOLMU, were due to variations in mutation rate and the level of heterozygosity, resulting in an overall resistance rating of low to moderate. This is the first study confirming cross- and multiple resistance to ACCase- and ALS-inhibiting herbicides, highlighting that resistance monitoring in A. myosuroides and L. multiflorum in Ireland is critical, and the adoption of integrated weed management strategies (chemical and non-chemical/cultural strategies) is essential.
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Takano, Hudson K., Rafael R. Mendes, Leonardo B. Scoz, Ramiro F. Lopez Ovejero, Jamil Constantin, Todd A. Gaines, Philip Westra, Franck E. Dayan, and Rubem S. Oliveira. "Proline-106 EPSPS Mutation Imparting Glyphosate Resistance in Goosegrass (Eleusine indica) Emerges in South America." Weed Science 67, no. 1 (December 18, 2018): 48–56. http://dx.doi.org/10.1017/wsc.2018.71.
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AbstractGlyphosate-resistant (GR) goosegrass [Eleusine indica(L.) Gaertn.] was recently identified in Brazil, but its resistance mechanism was unknown. This study elucidated the resistance mechanism in this species and developed a molecular marker for rapid detection of this target-site resistance trait. The resistance factor for the resistant biotype was 4.4-fold compared with the glyphosate-susceptible (GS) in greenhouse dose–response experiments. This was accompanied by a similar (4-fold) difference in the levels of in vitro andin plantashikimate accumulation in these biotypes. However, there was no difference in uptake, translocation, or metabolism of glyphosate between the GS and GR biotypes. Moreover, both biotypes showed similar values for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) copy number and transcription. Sequencing of a 330-bp fragment of theEPSPSgene identified a single-nucleotide polymorphism that led to a Pro-106-Ser amino acid substitution in the enzyme from the GR biotype. This mutation imparted a 3.8-fold increase in the amount of glyphosate required to inhibit 50% of EPSPS activity, confirming the role of this amino acid substitution in resistance to glyphosate. A quantitative PCR–based genotyping assay was developed for the rapid detection of resistant plants containing this Pro-106-Ser mutation.
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Pan, Lang, Haitao Gao, Han Wu, and Liyao Dong. "Molecular basis of multiple resistance to herbicides inhibiting acetyl-CoA carboxylase and acetolactate synthase in American sloughgrass (Beckmannia syzigachne) from China." Crop and Pasture Science 67, no. 11 (2016): 1208. http://dx.doi.org/10.1071/cp16109.
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American sloughgrass (Beckmannia syzigachne Steud.) is a problematic grass that is widely distributed in wheat and oilseed rape fields in China. The herbicides fenoxaprop-P-ethyl and mesosulfuron-methyl failed to control B. syzigachne JCWJ-R populations collected from a wheat field in Jiangsu Province. Dose-response experiments showed that JCWJ-R was resistant to the acetyl-CoA carboxylase (ACCase) inhibitors fenoxaprop-P-ethyl (33.8-fold), haloxyfop-R-methyl (12.7-fold), clethodim (7.8-fold) and pinoxaden (11.6-fold), and to the acetolactate synthase (ALS) inhibitors mesosulfuron-methyl (15.9-fold), pyroxsulam (17.6-fold), flucarbazone-Na (10.7-fold) and imazethapyr (7-fold). Resistance to ALS inhibitors was due to a Pro-197-Ser mutation in the ALS gene and resistance to ACCase inhibitors was due to an Ile-1781-Leu mutation in the ACCase gene. A derived cleaved amplified polymorphic sequence method was developed to detect the ALS mutation in B. syzigachne. This was combined with a previously established method to detect Ile-1781-Leu, and the mutation frequency and homozygous mutation rates in the JCWJ-R population were determined. The evolution of multiple resistance to ACCase and ALS inhibitors in this B. syzigachne population indicated that alternative methods should be developed to control resistant weeds.
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Lethagen, Stefan, Christina Isaksson, Charlotta Schaedel, and Lars Holmberg. "Von Willebrand’s Disease caused by Compound Heterozygosity for a Substitution Mutation (T1156M) in the D3 Domain of the Von Willebrand Factor and a Stop Mutation (Q2470X)." Thrombosis and Haemostasis 88, no. 09 (2002): 421–26. http://dx.doi.org/10.1055/s-0037-1613232.
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SummaryHereditary defects of the von Willebrand factor (VWF) gene cause von Willebrand’s disease (VWD) which shows great variability dependent on the nature and location of the mutation. We here describe the characteristics of a substitution of methionine for threonine 1156 in the D3 domain of the VWF, i.e. the domain involved in the intracellular multimerization of pro-VWF dimers. A VWD patient with severe symptoms was a compound heterozygote for the T1156M mutation and a null allele (Q2470X) on the other chromosome. This led to marked reduction of plasma VWF concentration to about 0.05 U/ml and an abnormality of VWF multimers as in type 2A VWD. Expression in vitro of the mutation demonstrated that 1156M-VWF is secreted from COS-7 cells in a much reduced amount and lacking large multimers. When coexpressed with normal VWF 1156M-VWF decreased the secretion of normal VWF in a dose-dependent manner, the secreted VWF showing all the multimers. Two relatives of the propositus were single heterozygotes for the T1156M mutation and were either asymptomatic or had the manifestations of mild type 1 VWD. The expression data and studies of platelet VWF indicate that the T1156M mutation results in intracellular retention of VWF rather than impaired synthesis. Three other members of the family were heterozygotes for the Q2470X mutation and demonstrated the variable expressivity of a null allele.
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Manshouri, Taghi, Zeev Estrov, Alfonso Quintas-Cardama, Jorge Cortes, Francis Giles, David Harris, Waldemar Priebe, Hagop Kantarjian, and Srdan Verstovsek. "WP1066 Inhibits Growth of Human Cells Carrying the JAK2 V617F Mutation." Blood 108, no. 11 (November 16, 2006): 4885. http://dx.doi.org/10.1182/blood.v108.11.4885.4885.
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Abstract Myeloproliferative disorders (MPDs) are characterized by proliferation of one or more myeloid cell lineages in bone marrow and peripheral blood, with relatively preserved differentiation. Recent discovery of a dominant gain-of-function mutation in the Janus kinase 2 (JAK2) gene in patients with MPDs, involving the substitution of valine for phenylalanine at position 617 of the JAK2 protein (JAK2 V617F), represents the first acquired somatic mutation in hematopoietic stem cells described in these disorders. This discovery has opened new avenues for the development of targeted therapies for MPDs. WP1066 is a small molecule, a member of a novel class of anticancer agents whose development was based upon the backbone of AG490, a tyrphostin with activity against JAK2 V617F-expressing cell lines but limited in vivo activity. We investigated the inhibitory activity of the WP1066 against the JAK2 V617F-mutant expressing erythroid leukemia HEL cell line and peripheral blood mononuclear cells from patients with polycythemia vera (PV). WP1066 significantly inhibited the phosphorylation of JAK2 and downstream signal transduction proteins STAT3, STAT5, and ERK1/2 in a dose- and time-dependent manner. It induced a time- and dose-dependent antiproliferative and pro-apoptotic effects (activation of caspase 3, release of cytochrome c, and cleavage of PARP) in the JAK2 V617F-bearing HEL cell line in the low micromolar range. Pretreatment of cells with pan-caspase inhibitor Z-VAD abolished WP1066-induced apoptosis. The expression of apoptosis related proteins bcl-2, bax, and XIAP, however, was not changed. More important, WP1066 was effective in inhibiting cell growth in clonogenic assays of mononuclear cells harboring the JAK2 V617F mutation obtained from peripheral blood of patients with PV. We conclude that WP1066 is active both in vitro and ex vivo against cells carrying the JAK2 V617F mutation and represents a solid candidate for the treatment of JAK2 V617V-expressing MPDs.
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Salgotra, Romesh, and Bhagirath Singh Chauhan. "The First Report of Target-Site Resistance to Glyphosate in Sweet Summer Grass (Moorochloa eruciformis)." Plants 10, no. 9 (September 11, 2021): 1885. http://dx.doi.org/10.3390/plants10091885.
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Sweet summer grass is a problematic weed in the central Queensland region of Australia. This study found glyphosate resistance in two biotypes (R1 and R2) of sweet summer grass. The level of resistance in these biotypes was greater than 8-fold. The glyphosate dose required to reduce dry matter by 50% (GR50) for the resistant populations varied from 1993 to 2100 g ha−1. A novel glyphosate resistance double point mutation in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene was identified for the first time in sweet summer grass. Multiple mutations, including multiple amino acid changes at the glyphosate target site, as well as mutations involving two nucleotide changes at a single amino acid codon, were observed. Both resistant biotypes exhibited a nucleotide change of CAA to ACA in codon 106, which predicts an amino acid change of proline to a threonine (Pro-106-Thr). In addition, the R1 biotype also possessed a mutation at codon 100, where a nucleotide substitution of T for G occurred (GCT to TCT), resulting in a substitution of serine for alanine (Ala-100-Ser). Understanding the molecular mechanism of glyphosate resistance will help to design effective management strategies to control invasive weeds.
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Gerakari, Maria, Nikolina Cheimona, Eleni Tani, Ilias Travlos, Demosthenis Chachalis, Donato Loddo, Solvejg Kopp Mathiassen, et al. "Biochemical and Rapid Molecular Analyses to Identify Glyphosate Resistance in Lolium spp." Agronomy 12, no. 1 (December 25, 2021): 40. http://dx.doi.org/10.3390/agronomy12010040.
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Lolium spp. are troublesome weeds mainly found in winter cereal crops worldwide, including Europe. In recent years resistant mechanisms have been evolved to several important herbicides. In this study we investigated the mechanisms responsible for conferring glyphosate resistance in some Lolium spp. populations. A holistic approach was used, based on dose-response experiments, determination of shikimic acid concentration in plant leaf tissue, as well as molecular analyses. More specifically, in three Lolium spp. populations the existence of a mutation in the Pro-106 codon of the 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS) gene was investigated as well as the relative transcript levels of four ABC-transporter genes were monitored at three time points after glyphosate application. The results demonstrated that glyphosate resistance is a multifactor phenomenon. Relative transcript levels of the ABC-transporter genes were abundant at very early time points after glyphosate treatments. Dose-response experiments and shikimate analyses were in accordance with the findings of the quantitative PCR (qPCR) analyses. We suggest that relative expression ratio of ABC-transporter genes can be a useful tool to rapidly identify Lolium spp. populations resistant to glyphosate.
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Yi, Sha, Yan Chen, Lu Wen, Benping Zhang, Jing He, Guohui Cui, Lijing Yang, et al. "Deguelin, Selective Silencing of the NPM1 Mutant Protein, Induces Differentiation and Potentiates Apoptosis in Acute Myeloid Leukemia Cells Carrying NPM1 Mutation." Blood 120, no. 21 (November 16, 2012): 2435. http://dx.doi.org/10.1182/blood.v120.21.2435.2435.
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Abstract Abstract 2435 Acute myeloid leukemia (AML) carrying nucleophosmin (NPM1) gene mutations, leading to aberrant cytoplasmic expression of the nucleolar protein NPM1, accounts for about one-third of adult AML and shows distinctive biological and clinical features. In spite of the relatively good prognosis of NPM1-mutated AML, there are still cases that show poorer outcome, especially those associated with FLT3-ITD mutation and elderly patient population. Therefore new therapeutic strategies need to be explored. Deguelin, a rotenoid isolated from several plant species including Mondulea sericea (Leguminosae), exhibits significant inhibitory effects and induce apoptosis in a variety of cancer cell lines in vitro and in vivo. Researches have been revealed that NPM1 wild type (NPM-wt) and NPM1 mutant protein are potential therapeutic targets for AML cells harboring NPM1 mutations, however, isotype-specific inhibitors remain to be developed. Our previous study found deguelin induced apoptosis on Jurkat cells by disrupting NPM1 expression. Thus, in the present report we investigate the effects and molecular mechanisms of deguelin on AML NPMc+ cells, with focus on the NPM1 protein. Deguelin exerted dose-dependent dural effects on cell line OCI-AML3 carrying NPM1 mutation but not in the OCIM2 cell line (not harboring NPM1 gene mutation). Deguelin strongly induced OCI-AML3 cell apoptosis as shown by annexin V-fluoroisothyocyanate analysis (from 6.8% to 65.9%, 48 h at 32 μM), while apoptosis was minimal in OCIM2 cells (from 2.2% to 11.7%, 48h at 32 μM), which was further confirmed by a western blot assay to evaluate the activation of caspase-3 and the cleavage of PARP. Because of the pivotal role of NPM1 in AML cell survival and proliferation, we explored whether the above effects of deguelin were mediated by interfering with NPM1. Western blot analysis using specific antibodies showed marked downregulation of the leukemic NPM1 mutant protein upon deguelin treatment (0, 4 and 8μM, 48h), while NPM1-wt protein levels reminded unchanged. In addition, western blot and caspase activity assay revealed that the pro-apoptosis effect of deguelin was associated with caspase-6 and −8 activations, which might be caused by NPM mutation protein downregulation. In nontoxic concentration, morphologic features of granulocytic/monocytic differentiation were evident after 8 days treatment with dose of deguelin of 2μM in OCI-AML3 cells whereas no changes were found in OCIM2 cells. A more precise evaluation of cell differentiation by detecting CD11b (from 11.5% to 43.0%, P<0.01) and CD14 (from 1.2% to 11.1%, P<0.05) indicated that the proportion of the differentiation antigens increased in deguelin-treated cells. In accordance with the above results, NPM1 mutant protein was significant downregulated in OCI-AML3 cells even in nontoxic concentration of deguelin (from 0 to 2 μM, at 8 days), accompanied by p53, p21 and the 30kD band of C/EBPα decreasing, while there is no change in the protein level of NPM-wt, p53, p21 and C/EBPα in OCIM2 cells. Moreover, si-NPM-mut was used to further confirm the above results. Si-NPM-mut inhibited OCI-AML3 cell proliferation (inhibition rate 30% vs. control), induced differentiation (CD11b, from 4.2% to 19.2%) and caused significant decrease in the expression of pro-caspase-8, p21, p53 and the 30kD bands of C/EBPα compared with si-NC group, in consistent with the deguelin treatment in nontoxic concentration with long time duration, while it did not affect the levels of pro-caspase-6. Taken together, our results suggest that deguelin is a potent in vitro inhibitor of NPM1 mutation protein which provides the molecular basis for its anti-leukemia activities in NPMc+ AML cells, including suppression of proliferation, induction of apoptosis and differentiation. Disclosures: No relevant conflicts of interest to declare.
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Wolz, Lucie, Günter Krause, and Gerhard Scherer. "The Comet Assay with MCL-5 Cells as an Indicator of Genotoxic Treatment with Chemicals and Cigarette Smoke Condensate." Alternatives to Laboratory Animals 30, no. 3 (May 2002): 331–39. http://dx.doi.org/10.1177/026119290203000311.
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The metabolically competent human lymphoblastoid cell line MCL-5 was treated with a panel of mutagens to assess the induction of DNA damage. Treatment effects were observed by monitoring cell proliferation and by single-cell gel electrophoresis (SCGE). The direct-acting mutagens benzo[ a]pyrene-7,8-diol 9, 10-epoxide (BPDE) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), as well as pro-mutagens requiring metabolic activation, i.e. benzo[a]pyrene (BaP), 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyri-dine (PhIP), 4- N-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and cigarette-smoke condensate (CSC), were assayed by SCGE. Assay schemes were adapted for the MCL-5 cell line and for low levels of strand break induction, by inclusion of the DNA synthesis inhibitors cytosine arabinoside and hydroxyurea, and by extending the electrophoresis time. For all mutagens tested, dose-dependent increases of median and average tail moment values among 50 nucleoids per slide were observed. The determining factors for selecting the treatment doses for mutation-induction experiments were the solubility of BaP and PhIP in the exposure medium, and the cytotoxicity exhibited by BPDE, MNNG and CSC. Induction of DNA strand breaks was obtained at mutagen concentrations permitting sufficient cell proliferation, except in the case of MNNG.
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Zhao, Ning, Yaling Bi, Cuixia Wu, Dandan Wang, Ludan You, Weitang Liu, and Jinxin Wang. "Cross-resistance to acetolactate synthase (ALS) inhibitors associated with different mutations in Japanese foxtail (Alopecurus japonicus)." Weed Science 67, no. 4 (May 31, 2019): 389–96. http://dx.doi.org/10.1017/wsc.2019.15.
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AbstractJapanese foxtail (Alopecurus japonicus Steud.) is an invasive grass weed that severely threatens the production of wheat (Triticum aestivum L.) and canola (Brassica napus L.) crops in eastern Asia. Mesosulfuron-methyl is a highly efficient acetolactate synthase (ALS)-inhibiting herbicide widely used for control of this species in China. However, in recent years, some A. japonicus populations have evolved resistance to mesosulfuron-methyl by different amino acid substitutions (AASs) within the ALS gene. In the current study, 11 populations of A. japonicus were collected from Anhui Province, China, where the wheat fields were severely infested with this weed. Based on single-dose screening, eight of these populations evolved resistance to mesosulfuron-methyl, and gene sequencing revealed three AASs located in codon 197 or 574 of the ALS gene in the different resistant populations. Subsequently, three typical populations, AH-1, AH-4, and AH-10 with Trp-574-Leu, Pro-197-Thr, and Pro-197-Ser mutations, respectively, in ALS genes were selected to characterize their cross-resistance patterns to ALS inhibitors. Compared with the susceptible population AH-S, AH-1 showed broad-spectrum cross-resistance to sulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidines (TPs), and sulfonyl-aminocarbonyl-triazolinones (SCTs); whereas AH-4 and AH-10 were resistant to SUs, TPs, and SCTs but sensitive to IMIs. Moreover, all three resistant populations were sensitive to both photosystem II inhibitor isoproturon and 4-hydroxyphenylpyruvate dioxygenase inhibitor QYM201 (1-(2-chloro-3-(3-cyclopropyl-5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-6-(trifluoromethyl)phenyl)piperidin-2-one). Based on the current state of knowledge, this study is the first report of A. japonicus evolving cross-resistance to ALS-inhibiting herbicides due to a Pro-197-Ser mutation in the ALS gene.
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Lund, Troy C., Michelle L. Carter, Ashley C. Kramer, Bruce R. Blazar, and Julie A. Ross. "Mutant tp53 Causes a Gain of Function Increase in Sensitivity to Reactive Oxygen Species in Erythroid Precursors." Blood 124, no. 21 (December 6, 2014): 2668. http://dx.doi.org/10.1182/blood.v124.21.2668.2668.
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Abstract TP53 is mutated in approximated 10 – 15% of cases of leukemia and myelodysplastic syndrome. It is recently appreciated that oxidative stress plays a role in the dysplastic processes that drives these diseases. Animal modeling of oxidative stress has been a challenge as many of the central genes in the oxidative stress response pathway are necessary for life and knockout animals die as embryos due to overwhelming oxidative stress. The zebrafish model allows pro-oxidant exposure early in hematopoietic development, and gata1DsRed1 transgenic animals allow clear identification of erythroid precursors. We capitalized on these advantages to interrogate the effects of oxidative stress on erythroid precursors. The gata1DsRed1 erythroid precursors showed a dose-responsive increase in reactive oxygen species (ROS) generation after naphthol exposure. Pro-oxidant exposure significantly up-regulated several anti-oxidant genes including: hypoxia-inducible factor (hif1a), nuclear factor (erythroid derived)-like 2 (nrf2), ferritin heavy chain (fth1a), thioredoxin (txn), and heme oxygenase 1 (hmox1); (p < 0.05) shown by qRT-PCR. In silico promoter analysis of these genes revealed several tp53 binding sites within 4 kb upstream of the first exon in each gene. Pro-oxidant exposure was able to induce tp53 expression 3-fold over baseline by qRT-PCR. We next took advantage of the tp53 mutant line tp53M214K/M241K which has a mutation in the DNA binding region of tp53 rendering it non-functional. We found that tp53M214K/M124K fish were highly sensitive to pro-oxidant exposure with 80% of embryos showing severe to moderate anemia and cardiac edema after 72 hours of exposure to naphthol (versus 25% in wild-type control animals, n = 100/group; p < 0.001). There was a 3-fold decrease in the number of hemoglobin producing cells in naphthol treated tp53M214K/M124K animals as shown by o-dianisidine staining (versus control animals, n = 10/group; p < 0.01). A dose-response between the amount of pro-oxidant exposure and severity of anemia/edema also existed as determined by correlation of pro-oxidant concentration to an edema severity scale. We next measured the amount of ROS generated after naphthol exposure using CellROX detection assays and found that tp53M214K/M124K animals showed a 5 – 10-fold increase in ROS generated compared to wild-type (n = 30/group, p < 0.01). This increased ROS was maintained even in the heterozygous states of tp53M214K/wt suggesting a gain-of-function phenomenon. ROS generation could be completely reversed by treatment with the anti-oxidant n-acetylcysteine. When we completely abrogated mutant tp53 by morpholino knockdown, ROS generation and hemolysis were significantly decreased, providing further evidence that the ROS generation is a gain-of-function phenomenon of mutated tp53. Finally, uncoupling of mitochondrial respiration using oligomycin also decreased ROS generation by 50% (p < 0.01) implicating a dysregulatory process ongoing in mitochondria. In addition to the recently appreciated increased tumorigenicity caused by mutated tp53, our data suggest that cells harboring mutant tp53 also have increased ROS generation and increased sensitivity with pro-oxidant exposure. Understanding the mechanisms by which the anti-oxidant response is regulated will allow us to potentially find more effective druggable targets to treat the oxidative stress that accompanies the dysplastic process. Disclosures No relevant conflicts of interest to declare.
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Rühl, Heiko, Christina Berens, Sara Reda, Franziska Isabelle Winterhagen, Jens Müller, Johannes Oldenburg, Peter Hanfland, and Bernd Pötzsch. "The Variable Clinical Expressivity of Factor V Leiden, but Not of Prothrombin G20210A, Correlates with the Extent of the APC Response to In Vivo Thrombin Formation." Blood 132, Supplement 1 (November 29, 2018): 1221. http://dx.doi.org/10.1182/blood-2018-99-115949.
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Abstract Background: The factor V Leiden mutation (FVL) and the prothrombin G20210A mutation (PRO) are the most frequent thrombophilic mutations among Caucasians. Both mutations show a highly variable expressivity, ranging from a lifelong asymptomatic course to severe venous thromboembolism (VTE). Recently, we have shown increased generation rates of thrombin and activated protein C (APC) in response to in vivo thrombin formation in asymptomatic FVL carriers (VTE-). In vivo thrombin formation was triggered by low-dose administration of recombinant activated factor VII (rFVIIa). Aim of the present study was to extend this stress test approach to carriers of PRO and to FVL carriers with a history of VTE (VTE+). Methods: The study population consisted of 29 FVL carriers (thereof 25 heterozygotes: 13 VTE- and 12 VTE+) and 21 PRO carriers (thereof 19 heterozygotes: 12 VTE- and 7 VTE+). None of the subjects was under anticoagulant treatment at time of analysis. The control group consisted of 13 healthy volunteers tested negative for FVL and PRO. Blood samples were collected immediately before and during a period of 8 hours following injection of 15 µg/kg rFVIIa. Plasma levels of free thrombin and APC were quantified using oligonucleotide-based enzyme capture assays (OECAs). Prothrombin activation fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and D-dimer were measured additionally. Results: Injections of rFVIIa were well-tolerated by all subjects and median D-dimer levels remained within the reference range in all groups. Compared with the controls, peak plasma levels of F1+2 and TAT were significantly higher after rFVIIa injection in FVL carriers (p=.009 and p=1.0∙10-4, respectively), and in PRO carriers (p=.045 and p=6.0·10-4, respectively), but they did not differ between the FVL and PRO cohorts, and between the VTE+ and VTE- subgroups. Median plasma concentrations of thrombin were below the limit of detection at baseline, and rFVIIa did not induce statistically significant changes of thrombin levels in any group. APC increased to peak levels 30 minutes after rFVIIa injection, from 0.63 (0.54-1.16) to 3.14 (2.55-3.55) pmol/L (p=.017) in the controls, and from 1.39 (1.14-1.86) to 7.86 (5.55-11.55) pmol/L (p<1.0∙10-4) in the FVL group. In PRO carriers, plasma levels of APC increased from 1.07 (0.86-1.23) pmol/L at baseline to a peak of 5.86 (4.44-6.41) pmol/L 1 hour after rFVIIa injection p=4.0·10-4). Peak plasma levels of APC were higher in both FVL carriers and PRO carriers in comparison to the controls (p<1.0∙10-4 and p=4.0∙10-4, respectively), and lower in PRO carriers compared with those in FVL carriers (p=.003). With peak levels of 8.11 (7.59-11.77) vs. 5.62 (4.94-6.49) pmol/L, APC was significantly higher in the VTE- subgroup than in the VTE+ subgroup (p=.004) in heterozygous FVL carriers, while in heterozygous PRO carriers, APC peak levels did not differ between the VTE- subgroup and the VTE+ subgroup (Figure 1). Conclusion: The APC response to in vivo thrombin formation is significantly lower in symptomatic than in asymptomatic FVL carriers, implying that higher APC levels protect FVL carriers from thrombosis development. This hypothesized effect appears to be specific for FVL as prothrombin G20210A carriers do not show differences in the APC response related to VTE history. Further studies are warranted to identify the factors that modulate the APC response in FVL carriers. Disclosures Rühl: Swedish Orphan Biovitrum: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Shire: Research Funding; Grifols: Research Funding; CSL-Behring: Research Funding. Berens:Pfizer: Research Funding; Sanofi Genzyme: Research Funding; Shire: Research Funding; CSL-Behring: Research Funding; Biotest: Research Funding. Winterhagen:Swedish Orphan Biovitrum: Consultancy, Research Funding. Müller:Swedish Orphan Biovitrum: Consultancy, Research Funding. Oldenburg:Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Octapharma: Consultancy, Honoraria, Research Funding; Novo Nordisk: Consultancy, Honoraria; Grifols: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Chugai: Consultancy, Honoraria; Biotest: Consultancy, Honoraria; Biogen Idec: Consultancy, Honoraria; Bayer: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria, Research Funding; Swedish Orphan Biovitrum: Consultancy, Honoraria.
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Downes, Charlotte EJ, Barbara J. McClure, Jacqueline Rehn, James Breen, John B. Bruning, David T. Yeung, and Deborah L. White. "Acquired Mutations within the JAK2 Kinase Domain Confer Resistance to JAK Inhibitors in an in Vitro model of a High-Risk Acute Lymphoblastic Leukemia." Blood 136, Supplement 1 (November 5, 2020): 5–6. http://dx.doi.org/10.1182/blood-2020-133491.
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Introduction Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of ALL associated with high relapse rates and poor survival. Rearrangements of Janus kinase 2 (JAK2r) are present in approximately 5% and 14% of pediatric and young adult Ph-like ALL cases respectively. The resultant JAK2 gene fusions drive leukemogenesis through constitutive activation of the JAK/STAT signaling pathway and are associated with very poor outcomes in patients with Ph-like ALL. All JAK inhibitors in clinical development are type I inhibitors, which bind in the ATP-binding site of JAK2. A phase II clinical trial is currently assessing the only FDA-approved JAK1/2 inhibitor, ruxolitinib in high-risk B-cell ALL cases harboring JAK2 alterations. The development of treatment resistance to targeted inhibitors in other diseases is well documented and often results in disease relapse. Elucidating mechanisms of ruxolitinib resistance in JAK2r ALL will inform approaches to monitor the emergence of resistance in ongoing clinical trials and enable the development of therapeutic strategies to overcome or avert resistance. Methods JAK2r B-ALL was modelled in the pro-B cell line, Ba/F3, by expressing the high-risk B-ALL fusion, ATF7IP-JAK2. Ruxolitinib resistance in three independent ATF7IP-JAK2 Ba/F3 cell lines was achieved following dose escalation to a clinically relevant dose of 1 μM ruxolitinib. Sanger sequencing of the RT-PCR amplified JAK2 fusion revealed each resistant line had acquired a different mutation within the JAK2 kinase domain. Therapeutic sensitives were assessed by staining with Fixable Aqua Dead Cell Stain (Invitrogen) and Annexin V, and analysis by flow cytometry. Alterations in signaling pathways were determined using phosphoflow cytometry and western blot analysis. Computational modelling of acquired JAK2 mutations and subsequent influence on ruxolitinib binding was performed using ICM-Pro (Molsoft L.C.C.). Results In addition to the identification of two known ruxolitinib resistant mutations, JAK2 p.Y931C and p.L983F, a novel p.G993A mutation was identified. All mutations localized to the ATP/ruxolitinib binding site and conferred resistance to multiple type-I JAK inhibitors, including ruxolitinib, BMS-911543, and AZD-1480 (Table 1). JAK2 p.G993A ATF7IP-JAK2 Ba/F3 cells were also resistant to the type-II JAK inhibitor, CHZ-868, which binds in an allosteric site of JAK2 in addition to the ATP-binding site. Ruxolitinib resistance correlated with sustained downstream STAT5 activation in the presence of 1 μM ruxolitinib compared with non-mutant ATF7IP-JAK2 Ba/F3 cells. Intracellular phosphoflow cytometry of ruxolitinib-resistant ATF7IP-JAK2 Ba/F3 cells confirmed constitutive activation of JAK/STAT signaling in the presence of 50 nM ruxolitinib, in contrast to non-mutant ATF7IP-JAK2 Ba/F3 cells. Computational modelling suggested that JAK2 p.L983F (Fig. 1D) sterically hinders ruxolitinib binding, while JAK2 p.Y931C may reduce ruxolitinib binding affinity by disruption of a critical hydrogen-bond (Fig. 1B). The novel JAK2 p.G993A mutation is predicted to alter DFG-loop dynamics by stabilizing the JAK2 activation loop (Fig1C). Conclusions This study demonstrates that the JAK2 ATP-binding site is susceptible to JAK inhibitor resistant mutations following ruxolitinib exposure in the setting of JAK2r ALL, highlighting the importance of monitoring the emergence of mutations within this region. In addition to previously described mutations we identified a novel JAK2 p.G993A mutation that conferred resistance to both type-I and type-II JAK inhibitors. The JAK2 p.G993A mutation was postulated to modulate the stability of a conserved domain. Understanding mechanisms of ruxolitinib resistance, as modelled here, has the potential to inform future drug design and the development therapeutic strategies for this high-risk cohort. Disclosures White: Amgen: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding.
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Margot, Nicolas, Laurie Vanderveen, Vidula Naik, Renee Ram, PC Parvangada, Ross Martin, Martin Rhee, and Christian Callebaut. "Phenotypic resistance to lenacapavir and monotherapy efficacy in a proof-of-concept clinical study." Journal of Antimicrobial Chemotherapy 77, no. 4 (January 13, 2022): 989–95. http://dx.doi.org/10.1093/jac/dkab503.
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Abstract Background Lenacapavir in vitro resistance selections identified seven mutations in HIV-1 capsid protein (CA) associated with reduced susceptibility. Objectives To analyse lenacapavir activity against lenacapavir-associated resistance mutations in multiple assays. We also report Day 10 resistance analyses conducted in a Phase 1b study of lenacapavir (Study 4072) in people with HIV (PWH). Methods Mutations were inserted in a proviral DNA clone by site-directed mutagenesis, and viruses (n = 12) were generated by transfection. Sequences were used to generate single-cycle (SC) test vectors that were evaluated in a Gag-Pro assay, and replicative viruses were tested in a multicycle (MC) MT-2 assay to determine lenacapavir susceptibility. Study 4072 was a Phase 1b, double-blinded, placebo-controlled, dose-ranging, randomized study of lenacapavir in untreated PWH. Participants received a single dose of lenacapavir (up to 750 mg) or placebo (10 day monotherapy). CA resistance was characterized using genotypic and/or phenotypic assays. Results Lenacapavir susceptibility in the SC assay showed an inverse relationship between replication capacity and resistance. In Study 4072, all 29 participants receiving lenacapavir showed a robust virological response with no rebound. At baseline, no participant had resistance mutations to lenacapavir, and all had WT susceptibility to lenacapavir. Post-monotherapy analyses revealed the emergence of CA mutation Q67H at Day 10 in two participants. Conclusions In vitro assays confirmed that increased resistance to lenacapavir was associated with decreased replication capacity of mutant viruses. In the clinical study no pre-existing lenacapavir resistance was detected. Emergence of Q67H occurred at exposures below the dose used in current Phase 2/3 studies. These results support development of lenacapavir as an antiretroviral agent.
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Kishida, Yuya, Yuho Najima, Yuki Otsuka, Kenya Toma, Atsushi Wada, Hiroto Adachi, Ryosuke Konuma, et al. "Post-Transplant Maintenance Treatment with Ponatinib for Philadelphia Chromosome Positive Leukemia." Blood 134, Supplement_1 (November 13, 2019): 5694. http://dx.doi.org/10.1182/blood-2019-128031.
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[Background] Philadelphia chromosome positive (Ph+) leukemia is characterized by highly proliferative nature and clone instability that evokes the emergence of mutated clones, including BCR-ABL1 T315I mutated clone. Established evidence on the use of tyrosine kinase inhibitors (TKIs) after allogeneic hematopoietic stem cell transplantation (HSCT) is still lacking. The use of second-generation TKIs as a maintenance treatment after HSCT has been studied, and it is expected that their use would improve the prognosis by suppressing recurrence. The advent of ponatinib (PON), a potent inhibitor of tyrosine kinase including T315I mutated BCR-ABL1, is expected to improve clinical outcome of Ph+leukemia. However, there are few reports of a maintenance treatment using PON after HSCT. [Methods] We retrospectively reviewed data of 13 patients (pts) who received PON for Ph+leukemia after HSCT while in hematological complete remission (CR) between April 1, 2016 and July 15, 2019. Prophylactic treatment (Pro) was defined as post-transplant administration of PON while in minimal residual disease (MRD) negative CR. Pre-emptive treatment (Pre) was defined as starting PON when the bcr-abl transcript was detected by either quantitative or nested qualitative PCR after HSCT. ABL1 mutation was analyzed through the direct sequencing method. Adverse events were evaluated according to the Common Terminology Criteria for Adverse Events version 5.0. Overall survival (OS) was estimated using Kaplan-Meier method. Non-relapse mortality (NRM) and cumulative incidence of hematological relapse (CIR) were calculated using Gray's test. This study protocol was approved by the ethics committee of Tokyo Metropolitan Komagome Hospital. [Results] Underlying diseases were Ph+ALL in 8 pts (5 in CR, 3 in non-CR at HSCT), CML in 5 (all in second chronic phase). ABL1 mutations were analyzed in 12 pts and T315I mutation was detected in 4 pts with Ph+ALL and 2 with CML. Furthermore, compound mutations (CMs) in BCR-ABL1 were detected in 4 pts before HSCT. PON was used in 6 only after HSCT, and in 7 both before and after HSCT. During the median observation after HSCT of 584 days (range, 116-1,110) for survivors, no vascular occlusion event occurred. With regard to adverse events (AEs), grade 3 AEs occurred in 2 pts (15.4%) and no grade 4 AE was observed. Two had liver dysfunction and one of them discontinued PON due to grade 3 abnormalities in liver function tests. One suffered from grade 3 thrombocytopenia. Four had skin rashes lower than grade 3 that were indistinguishable from skin graft-versus-host disease, and all of them resolved through topical steroid therapy. Of all, 6 were in Pro group and 7 were in Pre group. The initial dose of PON was median 15mg (range 45mg/twice a week - 15mg/day) in Pro and median 30mg (range, 15-45mg) in Pre. The median days from HSCT to the start of PON was 107 days (range, 32-174) in Pro and 208 days (range, 50-364) in Pre. The median duration of PON treatment was 297 days (range, 20-699) in Pro and 188 days (range, 5-608) in Pre. At final observation in Pro group, 2 pts relapsed and died during the salvage therapy, 1 pt discontinued PON due to hepatic adverse event, and 3 pts were still on PON. Meanwhile, in Pre group, 5 pts achieved MRD negative CR after PON administration (1 pt also received donor lymphocyte infusion and stop PON due to liver dysfunction, 1 discontinued PON by the patient's request, and 3 of them were still on PON). One pt with CM relapsed but achieved CR through salvage therapy and 1 pt with low performance status (KPS 60) died at home of unknown cause six days after taking PON 30mg daily. For all the 13 pts receiving PON maintenance therapy, OS was 74.6% (95%CI; 39.8-91.1), CIR was 23.1% (95%CI; 5.1-48.5), and NRM was 7.7% (95%CI; 0.4-30.6) at 1 year after transplant (Figure 1). Two out of 4 pts with CMs (V299L/F317L and E255K/T315I/F317L) remains in MRD negative CR. The other 2 with CMs (E255K/T315I and D276G/T315I) had progressed to hematological relapse, suggesting the resistance to PON. In contrast, only one out of 9 without CMs relapsed on PON treatment. [Conclusion] Our results suggested that post-transplant maintenance treatment using PON was tolerable in the majority of patients with Ph+leukemia, although the optimal dose or the initiation strategy (Pre or Pro) are still undetermined. Furthermore, some patients with T315I-inclusive CMs seemed to be resistant to PON. The longer observation in a larger cohort is warranted. Disclosures No relevant conflicts of interest to declare.
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Gutierrez, Martin, Giuseppe Giaccone, Stephen V. Liu, Zhonglin Hao, Christie Hilton, James M. Hinson, Everardus Otto Orlemans, and Alexander E. Drilon. "A phase 1B study of SNX-5422 plus carboplatin (C) and paclitaxel (P) in patients with advanced non-small-cell lung cancer (NSCLC)." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): e20622-e20622. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e20622.
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e20622 Background: SNX-5422 is an orally bioavailable pro-drug of SNX-2112, a highly potent and selective heat shock protein (Hsp) 90 inhibitor with synergy with platinum and paclitaxel. In this study, SNX-5422 + C/P followed by SNX-5422 maintenance therapy was examined in patients (pts) with NSCLC. Preliminary study results from the first 12 pts established the maximum tolerated dose (MTD) for SNX-5422 as 100 mg/m2 for the combination, with one G3 dose limiting toxicity (DLT) of diarrhea. The updated efficacy and safety results following enrollment completion (N = 20) are presented. Methods: Eligible pts had NSCLC (EGFR wild-type or non-sensitizing mutation) and ≤1 prior line of chemotherapy. Pts received C (AUC 5) and P (175 mg/m2) every 3 weeks for up to 6 cycles and SNX-5422 every other day (starting at 50 mg/m2) for 21 of 28 days, with a standard 3+3 dose escalation design during the combination, followed by SNX-5422 (100 mg/m2 every other day) monotherapy for maintenance until disease progression. Results: Twenty pts with NSCLC (pathology assessment: 18 adenocarcinoma, 2 squamous cell carcinoma) were enrolled (mean age, 59 yrs), with 19 evaluable for efficacy. Best response was partial response in 37% (7/19) of pts, stable disease in 58% (11/19) of pts, and progressive disease in 5% (1/19) of pts. Twelve of the 19 pts (63%) continued maintenance SNX-5422 monotherapy, and 7 of the 12 (58%) received therapy for ≥8 cycles. Four pts had G3 DLTs with combined therapy (considered possibly related to ≥1 agent): diarrhea (1 pt discontinued), increased alanine aminotransferase/aspartate aminotransferase (ALT/AST) (1), nausea, vomiting, and diarrhea (1), and peripheral sensory neuropathy (1). The most common adverse events considered possibly related to the combination were diarrhea (n = 14), nausea (n = 10), fatigue (n = 8), alopecia (n = 5), and vomiting (n = 5), and the majority of these events were G1/2. Conclusions: SNX-5422 added to C/P was well-tolerated at doses up to 100 mg/m2, and preliminary efficacy of this combination in NSCLC warrants further study. Clinical trial information: NCT01892046.
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Sarno, Samantha, Jamie Shaw, Edward Spooner, Jianguo Ma, Ann Clark, Charles Dumontet, and Angela Romanelli. "The Novel Aurora Kinase Inhibitor AS703569 Shows Potent Anti-Tumor Activity in Acute Myeloid Leukemia (AML)." Blood 110, no. 11 (November 16, 2007): 915. http://dx.doi.org/10.1182/blood.v110.11.915.915.
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Abstract Aurora kinases have been implicated in the onset of several human cancers. They are overexpressed in different tumor types including colon, breast, and pancreatic cancers, as well as in leukemias. AS703569 (formerly known as R763) is an orally bioavailable inhibitor of all three Aurora kinase isoforms (A, B and C), and is currently in Phase I clinical studies for both solid tumors and hematological malignancies. AS703569 also exhibits potent inhibition of the receptor tyrosine kinase FLT3. FLT3 is postulated to play an important role in the pathogenesis of AML. Somatic mutations and internal tandem duplications in FLT3 resulting in constitutive kinase activation are the most commonly acquired genomic abnormalities found in this disease. The potential therapeutic benefits of AS703569 as a dual Aurora kinase/FLT3 inhibitor is being explored in AML. The anti-proliferative effect of AS703569 has been tested in a panel of AML cell lines harboring different genetic defects. All cell lines were potently inhibited by AS703569 with IC50s between 0.7nM and 1 μM. The MV4-11 cell line possessing the FLT3 ITD was the most sensitive. Examination of cell cycle profiles demonstrated that AS703569 induced a block in the G2/M phase with a subsequent accumulation of cells in sub G (indicative of apoptosis). Dose-dependent induction of endoreduplication was observed in a subset of the cell lines tested. The pro-apoptotic role for the compound was further supported by the observation that increasing concentrations of AS703569 led to a significant increase in Caspase-3 cleavage and Annexin-V staining. AS703569 treatment led to a dose-dependent inhibition of FLT3 and Histone H3 phosphorylation, suggesting that modulation of both FLT3 and Aurora kinase B are implicated in the observed anti-proliferative, pro-apoptotic activity of AS703569 in these cell lines. To extend our investigation from cell lines to primary patient samples, AML blasts isolated from 15 patients were exposed ex vivo to increasing concentrations of AS703569 or Ara-C. In every case, AS703569 induced a dose-dependent decrease in viability that was more potent than Ara-C. We also tested the anti-tumor activity of AS703569 in two leukemia xenograft mouse models: MV4-11 and HL-60. In the MV4-11 model, tumors were grown to different sizes: 230, 750, and 1100 mm3, and in all three experimental conditions, tumor regression was seen after one dose (75 mg/kg) of AS703569. We found that a weekly dosing schedule is best tolerated by mice and reproducibly shows potent anti-tumor activity. Consistent with our in vitro data, AS703569 was less potent in the HL-60 xenograft model where the FLT3 mutation is absent. In this model, AS703569 only partially inhibited tumor growth (data not shown). Taken together, our data suggest that AS703569 shows particularly promising therapeutic activity in AML, especially cases presenting FLT3 mutations.
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Kutny, Matthew A., Steven J. Collins, Keith Loeb, Roland B. Walter, and Soheil Meshinchi. "All-Trans-Retinoic Acid (ATRA) Causes Extensive Differentiation In the NPM Mutant, Non-APL Leukemic Cell Line OCI-AML3." Blood 116, no. 21 (November 19, 2010): 3305. http://dx.doi.org/10.1182/blood.v116.21.3305.3305.
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Abstract Abstract 3305 The differentiating agent ATRA has been used successfully in the treatment of acute promyelocytic leukemia (APL). By comparison, non-APL AML has not shown similar sensitivity to ATRA induced differentiation. Recent data has suggested that a subset of de novo AML patients with nucleophosmin (NPM1) mutations may benefit from addition of ATRA to conventional therapy. The NPM1 gene has several functions affecting cell cycle proliferation including regulation of ribosome biogenesis and centrosome duplication and it acts as a histone chaperone. Mutation of the NPM1 gene leads to differentiation arrest contributing to AML pathogenesis. We hypothesized that leukemia cells with NPM1 mutations could be induced to undergo differentiation. We tested this hypothesis with the NPM1 mutant AML cell line OCI-AML3 and compared the results to identical assays using the AML cell line HL-60 which has been previously well documented to differentiate in response to ATRA therapy. OCI-AML3 and HL-60 cell lines were treated for 5 days with control media and four ATRA doses including 0.2 μM, 1 μM, 5 μM, and 25 μM. Cell viability was assessed by flow cytometry. Compared to the control condition, OCI-AML3 cells treated with the lowest dose of ATRA (0.2 μM) had a live cell count 21.6% of the control. HL-60 cells treated at even the highest ATRA dose (25 uM) had a live cell count 79.3% of the control. Due to the sensitivity of OCI-AML3 cells to the toxic effects of ATRA, the experiment was repeated with lower doses of ATRA including 0.001 μM, 0.01 μM and 0.1 μM. At the lowest dose of ATRA (0.001 μM), OCI-AML3 cells demonstrated a cell viability of 49% with further decrease to 26% at 0.1 μM dose of ATRA. At similar ATRA doses, cell viability for HL-60 cells was 91% and 85%, respectively (see table 1). Table 1: Cell viability as a percent of control cells after 5 days of treatment at three different doses of ATRA in OCI-AML3 and HL-60 cell lines. Cell Line: ATRA 0.001 μM ATRA 0.01 μM ATRA 0.1 μM OCI-AML3 49% 33% 26% HL-60 91% 91% 85% We subsequently determined the time course of changes in cell growth and the extent of differentiation at each point was determined by morphologic assessment. Both cell lines were treated with ATRA at doses of 0.001 μM, 0.01 μM, 0.1 μM, and 1 μM for a total of 4 days. Each day viable cell number was determined. In contrast to the HL-60 cells which had continued growth in lower ATRA doses, OCI-AML3 cells demonstrated exquisite sensitivity to growth arrest at the lowest doses of ATRA. Cell morphology was assessed daily with modified Wright-Giemsa staining of cells. Cells were examined for signs of myeloid differentiation including decrease in nuclear to cytoplasmic (N/C) ratio, nuclear segmentation, and cytoplasmic granules and vacuoles. At the lowest dose of ATRA (0.001 μM), after 4 days of exposure, significant number of OCI-AML3 cells demonstrated morphologic evidence of differentiation. At this ATRA dose and exposure interval, HL-60 cells showed no evidence of differentiation. At an ATRA dose of 1 μM (considered a standard dose used for differentiation of HL-60 cells), the OCI-AML3 cells showed differentiation changes as early as day 2 with nuclear segmentation and decreased N/C ratio while HL-60 cells did not show any change at this time point. After 4 days of ATRA exposure, most OCI-AML3 cells showed segmented nuclei and vacuolated cytoplasm, whereas HL-60 cells showed less distinct signs of differentiation with some cytoplasm granules and cup shaped nuclei. This data suggests that leukemic cells with NPM mutations may be susceptible to the pro-differentiating properties of ATRA. Further substantiation of this data with primary human specimens may ultimately provide the rationale for a novel therapeutic option using ATRA-based differentiation therapy for subsets of non-APL AML. Disclosures: No relevant conflicts of interest to declare.
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Robertson, S. A., R. J. Skinner, and A. S. Care. "267. Interleukin-10 inhibits TNFα synthesis and protects against LPS-induced miscarriage and preterm labour." Reproduction, Fertility and Development 17, no. 9 (2005): 109. http://dx.doi.org/10.1071/srb05abs267.
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The immune-deviating and anti-inflammatory cytokine interleukin-10 (IL-10) is expressed throughout pregnancy in the decidual and placental tissues. Mice with a null mutation in the IL-10 gene mice are fertile with no reduction in litter size, although fetal growth trajectories and placental structure are altered. IL-10 is known to terminate inflammatory responses and to limit inflammation-induced tissue pathology by inhibiting macrophage synthesis of tumor necrosis factor-α (TNFα). To investigate the anti-inflammatory role of IL-10 in pregnancy, the susceptibility of null mutant mice to low dose LPS-induced miscarriage and preterm labour has been evaluated. When IL-10 null mutant C57Bl/6 (IL-10–/–) and control (IL-10+/+) mice were given low dose E.coli LPS on d10 of pregnancy, IL-10 deficiency was associated with greater fetal loss with fewer mated IL-10–/– mice carrying viable fetuses at day 18 and increased rate of fetal resorption. In mice treated with LPS on day 17, preterm delivery within 24 h occurred in a higher proportion of IL-10–/– mice than IL-10+/+ mice. LPS induced very high and sustained TNFα and IL-6 content in serum, uterine and placental tissue in IL-10–/– mice, associated with upregulated mRNA expression of both cytokines in gestational tissues. These data show that IL-10 modulates placental resistance to inflammatory stimuli by down-regulating expression of the pro-inflammatory cytokines TNFα and IL-6. We conclude that IL-10 has a dual role in pregnancy, acting to regulate placental morphogenesis and fetal growth trajectory, and to protect against inflammation-induced miscarriage and preterm labour.
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Crews, Leslie A., Larissa Balaian, Heather Leu, Nathaniel Delos Santos, Angela C. Court, Anil Sadarangani, Maria A. Zipeto, et al. "RNA Splicing Modulation Impairs Acute Myeloid Leukemia Stem Cell Maintenance." Blood 126, no. 23 (December 3, 2015): 567. http://dx.doi.org/10.1182/blood.v126.23.567.567.
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Abstract Introduction Disease relapse is the leading cause of death in secondary AML (sAML), which evolves from antecedent hematologic disorders like myelodysplastic syndrome (MDS) or myeloproliferative neoplasms (MPNs) or following exposure to chemotherapy. Persistence of therapy-resistant leukemia stem cells (LSC) harboring enhanced survival and self-renewal capacity has been linked to high relapse rates in sAML. Previously, we showed that missplicing of a stem cell regulatory gene, GSK3 b, and splice isoform switching favoring pro-survival BCL2 family isoform expression promoted generation of therapy-resistant LSC (Abrahamsson et al PNAS 2009; Goff et al Cell Stem Cell 2013). However, whether aberrant pre-mRNA splicing promotes sAML LSC generation, in the absence of mutation, and if pharmacological splicing modulation impairs LSC maintenance, in a mutation-independent manner, has not been elucidated. Methods and Results Comparative RNA-sequencing and gene set enrichment analyses revealed significant alterations in splicing factor gene expression in purified progenitors from untreated sAML compared with normal samples. In addition, using an isoform-specific alignment algorithm, we established a sAML LSC splice isoform expression signature that identified increased expression of select transcripts, e.g. CD82 and PTK2B. Thus, we investigated the LSC inhibitory efficacy of a stable, potent splicing modulatory agent, 17S -FD-895, in humanized AML LSC stromal co-culture and primagraft assays. Notably, there was a dose-dependent reduction in AML LSC (n=4) survival and self-renewal after in vitro 17S -FD-895 treatment, with a favorable therapeutic index compared to normal controls (n=3, p<0.01). Splicing reporter activity and PCR analyses revealed rapid and potent 17S -FD-895-induced alterations in splicing, promoting pro-apoptotic isoform expression and intron inclusion in the stem cell regulatory gene MCL1. Also, 17S -FD-895 restored normal expression patterns of PTK2B, and MCL1-L/S and BLC2-L/S expression ratios. Flow cytometric analyses in AML LSC primagraft models treated with 17S -FD-895 (5-10 mg/kg delivered intravenously in 3 doses over 2 weeks) revealed a decrease in human stem (CD45+ CD34+ CD38- Lin-, 68% reduction in the spleens of the 10 mg/kg group versus vehicle controls, n=5 mice per group, p<0.05) and progenitor (CD45+ CD34+ CD38+ Lin-, 80% reduction to nearly zero in the spleens of the 10 mg/kg group versus vehicle controls, p=0.08) cell frequencies. Furthermore, MCL1-L/S and BCL2-L/S expression ratios were significantly reduced in LSC-enriched fractions from 17S -FD-895-treated mice compared to vehicle controls. Consistent with a reduction in functional LSC burden after 17S -FD-895 treatment, subsequent serial transplantation studies showed a 47-65% reduction in leukemic burden in the hematopoietic tissues of recipients of CD34+ cells from mice in the 10 mg/kg treatment group versus vehicle controls (n=5 mice per group, p<0.05). Conclusions Here we demonstrate that a potent and stable splicing modulatory agent, 17S -FD-895, normalized sAML-specific splice isoform expression patterns as well as MCL1-L/S and BLC2-L/S ratios. Moreover, pharmacologic splicing modulation reduced AML LSC survival and self-renewal in a dose-dependent manner in both in vitro and in vivo models with a favorable therapeutic index. Further evaluation of this compound as a splicing-targeted single agent or combined with standard of care therapy may reduce or eradicate LSC burden in therapy-resistant sAML. In addition, LSC-specific splice isoforms may represent important biomarkers that could be developed as companion diagnostics for splicing-targeted therapies in sAML and other recalcitrant malignancies. Disclosures Jamieson: Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.
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Lund, Troy C., Michelle L. Carter, Ashley C. Kramer, Nardina Nash, and Bruce R. Blazar. "The Oxidative Stress Response In Erythroid Precursors Is Mediated By tp53." Blood 122, no. 21 (November 15, 2013): 7. http://dx.doi.org/10.1182/blood.v122.21.7.7.
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Abstract Oxidative stress plays a key role in acute and especially chronic anemia as well red blood cell storage. Recent findings suggest that both erythroid precursors as well as mature red cells have increased sensitively to oxidative stress. Modeling oxidative stress in animals has been a challenge as many of the central genes in the oxidative stress response pathway are necessary for life and knockout animals die of overwhelming oxidative stress early in life. The zebrafish model allows pro-oxidant exposure early in hematopoietic development, and gata1DsRed1 transgenic animals allow clear identification of erythroid precursors. We capitalized on these advantages of the zebrafish to interrogate the effects of oxidative stress on erythroid precursors. After 72 hours of exposure to the strong pro-oxidant naphthol at 10 – 30 µg, embryonic zebrafish up-regulated several anti-oxidant genes including: hypoxia-inducible factor (hif1a), nuclear factor (erythroid derived)-like 2 (nrf2), ferritin heavy chain (fth1a), thioredoxin (txn), and heme oxygenase 1 (hmox1); 1.5, 2.3, 2.5, and 3.0-fold respectively (p < 0.05) as shown by qRT-PCR. To understand if a common pathway was driving anti-oxidant gene expression, we performed an in silico promoter analysis of these genes and discovered several tp53 binding sites within 4 kb upstream of the first exon in each gene. We showed naphthol was able to induce tp53 expression 3-fold over baseline by qRT-PCR. We next took advantage of the tp53 mutant line tp53M214K which has a mutation in the DNA binding region of tp53 rendering it non-functional similar to a complete knockout. We found that tp53M214K fish were highly sensitive to pro-oxidant exposure with 80% of embryos showing severe to moderate anemia and cardiac edema after 72 hours of exposure to naphthol (versus 25% in wild-type control animals, n = 100/group; p < 0.001). There was also a 3-fold decrease in the number of hemoglobin staining cells in naphthol treated tp53M214K animals as shown by o-dianisidine staining (versus control animals, n = 10/group; p < 0.01). A dose-response between the amount of pro-oxidant exposure and severity of anemia/edema also existed as determined by correlation of pro-oxidant concentration to an edema severity scale. We next measured the amount of reactive oxygen species (ROS) generated after naphthol exposure using CellROX detection assays and found that tp53M214K animals showed a doubling in ROS generated compared to wild-type (n = 30/group, p < 0.01) in whole animals. To disable tp53 by an alternative manner, we employed a known tp53 inhibitor, pifithrin, to inactivate tp53. Exposure of animals to pifithrin simultaneous with naphthol recapitulated the finding that inhibition of tp53 increased sensitivity to ROS as 90% of exposed embryos displayed moderate to severe anemia induced cardiac edema (versus 30% in controls, n = 100/group; p < 0.01). Although pifithrin combined with naphthol exposure caused no increase in ROS above that seen with naphthol alone. Our hypothesis is that the anemic phenotype is largely caused by hemolysis in erythroid precursors. The gata1DsRed1 zebrafish has labeled erythroid precursors, and using our experimental system we were able to specifically measure a dose responsive induction of ROS in erythroid precursors after naphthol exposure. Furthermore, gata1DsRed1 animals harboring tp53M214K showed a 20-fold increase in ROS after naphthol exposure (versus tp53+/+ animals, n = 10/group; p < 0.01). Accompanying the increase ROS is apoptosis of erythroid precursors as shown by flow cytometry for gata1DsRed1 cells. In conclusion, we show that amongst the many functions of tp53, providing an anti-oxidant response is also a mechanism though which erythroid precursors metabolize ROS after exposure to pro-oxidants. Understanding the mechanisms by which the anti-oxidant response is regulated will allow us to potentially find more effective drug-able targets to treat the oxidative stress that accompanies acute and chronic anemia’s. Disclosures: No relevant conflicts of interest to declare.
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Jimbo, Keisuke, Toshiaki Okuno, Ryuichi Ohgaki, Kou Nishikubo, Yuri Kitamura, Yumiko Sakurai, Lili Quan, et al. "A novel mutation in the SLCO2A1 gene, encoding a prostaglandin transporter, induces chronic enteropathy." PLOS ONE 15, no. 11 (November 9, 2020): e0241869. http://dx.doi.org/10.1371/journal.pone.0241869.
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Chronic enteropathy associated with SLCO2A1 gene (CEAS) is caused by loss-of-function mutations in SLCO2A1, which encodes a prostaglandin (PG) transporter. In this study, we report a sibling case of CEAS with a novel pathogenic variant of the SLCO2A1 gene. Compound heterozygous variants in SLCO2A1 were identified in an 8-year-old boy and 12-year-old girl, and multiple chronic nonspecific ulcers were observed in the patients using capsule endoscopy. The splice site mutation (c.940 + 1G>A) of the paternal allele was previously reported to be pathogenic, whereas the missense variant (c.1688T>C) of the maternal allele was novel and had not yet been reported. The affected residue (p.Leu563Pro) is located in the 11th transmembrane domain (helix 11) of SLCO2A1. Because SLCO2A1 mediates the uptake and clearance of PGs, the urinary PG metabolites were measured by liquid chromatography coupled to tandem mass spectrometry. The urinary tetranor-prostaglandin E metabolite levels in the patients were significantly higher than those in unaffected individuals. We established cell lines with doxycycline-inducible expression of wild type SLCO2A1 (WT-SLCO2A1) and the L563P mutant. Immunofluorescence staining showed that WT-SLCO2A1 and the L563P mutant were dominantly expressed on the plasma membranes of these cells. Cells expressing WT-SLCO2A1 exhibited time- and dose-dependent uptake of PGE2, while the mutant did not show any uptake activity. Residue L563 is very close to the putative substrate-binding site in SLCO2A1, R561 in helix 11. However, in a molecular model of SLCO2A1, the side chain of L563 projected outside of helix 11, indicating that L563 is likely not directly involved in substrate binding. Instead, the substitution of Pro may twist the helix and impair the transporter function. In summary, we identified a novel pathogenic variant of SLCO2A1 that caused loss-of-function and induced CEAS.
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Chan, Mable, Anders Leung, Bryan D. Griffin, Robert Vendramelli, Nikesh Tailor, Kevin Tierney, Jonathan Audet, and Darwyn Kobasa. "Generation and Characterization of a Mouse-Adapted Makona Variant of Ebola Virus." Viruses 11, no. 11 (October 26, 2019): 987. http://dx.doi.org/10.3390/v11110987.
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Ebola virus (EBOV) is a zoonotic pathogen that poses a significant threat to public health, causing sporadic yet devastating outbreaks that have the potential to spread worldwide, as demonstrated during the 2013–2016 West African outbreak. Mouse models of infection are important tools for the development of therapeutics and vaccines. Exposure of immunocompetent mice to clinical isolates of EBOV is nonlethal; consequently, EBOV requires prior adaptation in mice to cause lethal disease. Until now, the only immunocompetent EBOV mouse model was based on the Mayinga variant, which was isolated in 1976. Here, we generated a novel mouse-adapted (MA)-EBOV based on the 2014 Makona isolate by inserting EBOV/Mayinga-MA mutations into the EBOV/Makona genome, followed by serial passaging of the rescued virus in suckling mice. The resulting EBOV/Makona-MA causes lethal disease in adult immunocompetent mice within 6 to 9 days and has a lethal dose (LD50) of 0.004 plaque forming units (PFU). Two additional mutations emerged after mouse-adaptation in the viral nucleoprotein (NP) and membrane-associated protein VP24. Using reverse genetics, we found the VP24 mutation to be critical for EBOV/Makona-MA virulence. EBOV/Makona-MA infected mice that presented with viremia, high viral burden in organs, increased release of pro-inflammatory cytokines/chemokines, and lymphopenia. Our mouse model will help advance pre-clinical development of countermeasures against contemporary EBOV variants.
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Chen, Brandon, Wong Pamela, Daniel Auclair, Jonathan J. Keats, Paul Secrist, Justin Cidado, Adriana E. Tron, et al. "Myeloma Patient-Derived MCL1 Point Mutations Can Influence MCL1-Inhibitor Function." Blood 132, Supplement 1 (November 29, 2018): 951. http://dx.doi.org/10.1182/blood-2018-99-113444.
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Abstract Multiple myeloma is a malignancy of long-lived plasma cells of the bone marrow that is rarely curable. Thus, despite recent advances in the development of new therapies, additional approaches are required. We investigated potential molecular vulnerabilities in the BCL2 family. Using the MMRF CoMMpass (NCT0145429) study (IA13), we determined the frequency of nonsynonymous coding mutations in the BCL2 family. Analysis of baseline samples from 982 patients revealed that mutations in the BCL2 family are relatively rare events. No mutations were observed in the 3 pro-apoptotic effector genes BAX, BAK1 and BOK. Similarly, in the BH3-only genes, mutations were rare with no mutations in BCL2L11 (BIM), BAD, BID, HRK and BMF and only single mutations in BBC3 (PUMA) and PMAIP1 (NOXA). In the anti-apoptotic BCL2 genes, mutations were also rare with no mutations in BCL2, BCL2L1 (BCLX) and BCL2L10 (BCLB). A single sample had a mutation in BCL2A1 (A1) while 2 samples had mutations in BCL2L2 (BCLW). Interestingly MCL1 was mutated in 10 baseline samples (1.02%) and the frequency of the mutations in these samples was high (median 0.391, range 0.066-0.531). Therefore, we further investigated the functional consequences of the MCL1 mutant alleles. Of the 10 mutations detected, 1 was in the N-terminal region (G32R) and 4 were in the PEST domain in the N-terminal half of MCL1 that is associated with regulating protein stability (V140I, P142S, E149Q and E173K). An additional mutation was found in an uncharacterized region between the PEST and BH1 domains (L186F). We focused on the 4 mutations that lie within or near the functional BH1 (V249L and L267V) and within the BH3 (N223S, and R214Q) domains. Wild type (WT) MCL1 and the four mutant MCL1 constructs were introduced into a murine B-ALL cell line that has endogenous murine MCL1 flanked with LoxP sites and confirmed expression by western blot analysis. Human MCL1 can replace murine MCL1 in this cell model, therefore we are determining if the myeloma-derived mutants of MCL1 can complement loss of mouse Mcl1 and will report on these findings. However, we began to functionally characterize these mutations by taking advantage of an anomaly in the development of inhibitors of human MCL1. To date, no inhibitor developed against human MCL1 is as effective against murine Mcl1. Thus, differences in the activity of these inhibitors reflects changes in dependence on the introduced human MCL1. We treated cells with increasing concentrations of the MCL1 inhibitor S63845 and measured cell death (Annexin V/PI) at 24 hours. As expected, cells where the empty vector was introduced were highly resistant to S63845-induced cell death (less than 20% at 1000 nM) while cells expressing the human WT MCL1 were significantly more susceptible to the MCL1 inhibitor across a concentration range of 100 to 1000 nM. The V249L, N223S and R214Q mutations mimicked the sensitivity of the WT MCL1 suggesting they did not alter MCL1 function in the cells. In contrast the L267V mutation resulted in a dose curve that was more similar to the empty vector control suggesting this mutation either resulted in loss of function or in an MCL1 molecule that could not be inhibited by the drug. Since drug binding can stabilize MCL1 by competing for E3 ligase binding, we determined the effect of S63845 on MCL1 protein levels. We found that with all the mutants, S63845 dramatically increased human MCL1 protein expression ruling out lack of drug binding. We next performed MCL1 co-immunoprecipitation assays, and found that BIM release correlated with S63845 sensitivity. In addition to not releasing BIM, the L267V did not effectively release NOXA and BAK after S63845 treatment. Taken together, the L267V mutation does not prevent the binding of S63845 to free MCL1, rather it blocks the ability of drug to displace pro-apoptotic proteins required to induce cell death. We then tested another MCL-1 inhibitor in clinical trials, AZD-5991. The L267V mutation was completely resistant to AZD-5991-induced apoptosis despite evidence of drug binding. Interestingly the other 3 mutations also resulted in diminished killing activity when compared to cells expressing the WT MCL1, suggesting that these mutations may also influence drug function. Together these data suggest that myeloma-derived mutations in MCL1 may not necessarily influence MCL1 function, however they could alter responses to an emerging class of inhibitors where 3 drugs are currently in clinical trials. Disclosures Secrist: AstraZeneca: Employment. Cidado:AstraZeneca: Employment, Equity Ownership. Tron:AstraZeneca: Employment. Lonial:Amgen: Research Funding. Boise:Abbvie: Consultancy; AstraZeneca: Honoraria.
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Klimczak, Aleksandra, Agnieszka Zimna, Agnieszka Malcher, Urszula Kozlowska, Katarzyna Futoma, Jaroslaw Czarnota, Pawel Kemnitz, Anna Bryl, and Maciej Kurpisz. "Co-Transplantation of Bone Marrow-MSCs and Myogenic Stem/Progenitor Cells from Adult Donors Improves Muscle Function of Patients with Duchenne Muscular Dystrophy." Cells 9, no. 5 (April 30, 2020): 1119. http://dx.doi.org/10.3390/cells9051119.
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Duchenne muscular dystrophy (DMD) is a genetic disorder associated with a progressive deficiency of dystrophin that leads to skeletal muscle degeneration. In this study, we tested the hypothesis that a co-transplantation of two stem/progenitor cell populations, namely bone marrow-derived mesenchymal stem cells (BM-MSCs) and skeletal muscle-derived stem/progenitor cells (SM-SPCs), directly into the dystrophic muscle can improve the skeletal muscle function of DMD patients. Three patients diagnosed with DMD, confirmed by the dystrophin gene mutation, were enrolled into a study approved by the local Bioethics Committee (no. 79/2015). Stem/progenitor cells collected from bone marrow and skeletal muscles of related healthy donors, based on HLA matched antigens, were expanded in a closed MC3 cell culture system. A simultaneous co-transplantation of BM-MSCs and SM-SPCs was performed directly into the biceps brachii (two patients) and gastrocnemius (one patient). During a six-month follow-up, the patients were examined with electromyography (EMG) and monitored for blood kinase creatine level. Muscle biopsies were examined with histology and assessed for dystrophin at the mRNA and protein level. A panel of 27 cytokines was analysed with multiplex ELISA. We did not observe any adverse effects after the intramuscular administration of cells. The efficacy of BM-MSC and SM-SPC application was confirmed through an EMG assessment by an increase in motor unit parameters, especially in terms of duration, amplitude range, area, and size index. The beneficial effect of cellular therapy was confirmed by a decrease in creatine kinase levels and a normalised profile of pro-inflammatory cytokines. BM-MSCs may support the pro-regenerative potential of SM-SPCs thanks to their trophic, paracrine, and immunomodulatory activity. Both applied cell populations may fuse with degenerating skeletal muscle fibres in situ, facilitating skeletal muscle recovery. However, further studies are required to optimise the dose and timing of stem/progenitor cell delivery.
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Sharma, Rachna, Samuel Tesfay, Farol L. Tomson, Rajani P. Kanteti, V. K. Viswanathan, and Gail Hecht. "Balance of bacterial pro- and anti-inflammatory mediators dictates net effect of enteropathogenicEscherichia colion intestinal epithelial cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 290, no. 4 (April 2006): G685—G694. http://dx.doi.org/10.1152/ajpgi.00404.2005.
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Enteropathogenic Escherichia coli (EPEC) virulence requires a type III secretion system (TTSS) to deliver effector molecules in host cells. Although the TTSS is crucial to EPEC pathogenesis, its function in EPEC-induced inflammation is not known. The aim of this study was to investigate the role of the TTSS in EPEC-induced inflammation. HT-29 intestinal epithelial cells were infected with wild-type (WT) EPEC or select mutant strains or exposed to corresponding filter-sterilized supernatants (SN), and interleukin-8 (IL-8) secretion was determined by ELISA. EPEC SN stimulated significantly greater IL-8 production than EPEC organisms. Flagellin, as well as a TTSS-independent >50-kDa nonflagellin protein, was found to significantly contribute to this response. Dose-response studies showed that increasing concentrations of WT SN proportionally increased IL-8, whereas increasing multiplicity of infection of EPEC inversely correlated with IL-8 secretion, suggesting that EPEC dampens this host response. Infection with Δ escN (nonfunctional TTSS) markedly increased IL-8 compared with WT, indicating that a functional TTSS is required for this anti-inflammatory property; complementation of escN restored the attenuated response. Mutation of espB also enhanced the IL-8 response, and complementation returned IL-8 to near WT levels, suggesting involvement of this effector. The anti-inflammatory effect extends to both bacterial and host-derived proinflammatory stimuli, since prior infection with EPEC suppressed the IL-8 response to tumor necrosis factor-α, IL-1β, and enterohemorrhagic E. coli flagellin. These findings indicate that EPEC-induced inflammation is a balance between pro- and anti-inflammatory proteins; extracellular factors, including flagellin and an unidentified TTSS-independent, >50-kDa protein, trigger inflammation while intracellular TTSS-dependent factors, including EspB, attenuate this response.
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Cultrera, Dorina, Raimondo De Cristofaro, Paola Giordano, Silvia Linari, Silvia Macchi, Renato Marino, Angelo Claudio Molinari, et al. "Identification of the Profile of the Patients with Hemophilia B Eligible for Treatment with Nonacog Alfa Once-Weekly." Reports — Medical Cases, Images, and Videos 3, no. 1 (January 27, 2020): 3. http://dx.doi.org/10.3390/reports3010003.
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This study aimed to identify the characteristics of patients with hemophilia B eligible for once-weekly treatment with Nonacog alfa. Methods: A survey was conducted in 14 Hemophilia (HCs) of Italy. These centers were given a questionnaire consisting of ten closed multiple-choice questions. The centers were asked: (a) the percentages of their hemophilia B (HB) patients undergoing replacement therapy, “On-demand”, or weekly prophylaxis, (b) the criteria guiding the monitoring of patients, the advantages according to the age of patients, and (c) the obstacles to prophylaxis. The percentage of patients receiving “On-demand” (OD) treatment or continuous prophylaxis (prophy) differed depending on patient age and the severity of the disease. Only 57% of HCs provided “On-demand” therapy to the mild HB patients, about 93% to moderate ones, of whom 43% on prophylaxis. About 78% of patients <6 years old, were on treatment in 9 out of 14 HCs, by prophylaxis 66.7% and 33.3% by On-demand. In the 6–18 age group, 90.1% of HCs treated HB patients with prophylaxis, 42.8% in the 18–30 age range. On-demand treatment was the therapy of choice in 61.5% of HCs for patients aged 30–65 years. In total, 64% of the HCs assigned the maximum score to bleeding frequency, especially in the <6 and 6–18 age groups. Bleeding severity was also taken into significant consideration, particularly in subjects up to 30 years old. The scores regarding venous access were distributed relatively evenly throughout all age groups. The majority of the centers attributed a medium-high score to treatment compliance, especially in the 6–65 age range. In actuality, 55% of HCs attributed pro-thrombotic comorbidity a low score in the 18–30 age group, whereas 81% gave pro-hemorrhagic comorbidity a high rating in patients aged >65 years old. Many centers assigned a medium-high score to the baseline concentration of FIX level at diagnosis in all age groups. Most HCs attributed a medium-high score to type of genetic mutation in the younger age groups. As for socio-cultural barriers and quality of life, the majority of respondents gave a medium-high score in all age groups. For periodic monitoring of patients receiving continuous prophylaxis, 59% of the centers reported using clinical assessment. With regard to prophylaxis administration method, the majority of hemophiliacs were given infusions twice weekly, while as regards to the dose of FIX concentrate delivered, 50% of the centers reported administering prophylaxis once-weekly at a dose ranging from 5–100 IU/kg in 10–50% of HB patients. Thus, 93% of the centers reported using a dose of 25–50 IU/kg for twice-weekly prophylaxis in 6–100% of the patients. The majority of centers (86%) believe that, in a program of early primary prevention, once-weekly treatment with nonacog alfa may represent an alternative strategy to dose escalation. The results show that patients with mild hemophilia, with functional musculoskeletal status and difficulties with venous access, are candidates for once-weekly prophylaxis with nonacog alfa. For such patients, this regimen can improve treatment compliance and quality of life.
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Mueller, Christian, Nibedita Gupta, Felix C. Saalfeld, Rolf Findeisen, Nadine Rudolph, Steffen Klamt, Sandra Heise, Fred Schaper, and Thomas Fischer. "Expression of JAK2-V617F Kinase in Myeloid Progenitors and Proerythroblasts Induces Differential Patterns of Hypersensitivity in Activation of Key Signaling Nodes upon Incubation with Low-Dose, Physiologic EPO Concentrations." Blood 124, no. 21 (December 6, 2014): 4573. http://dx.doi.org/10.1182/blood.v124.21.4573.4573.
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Abstract Introduction: Chronic myeloproliferative neoplasms (MPNs), including Polycythemia vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are a spectrum of clonal hematological disorders. An acquired somatic mutation of the JAK2-gene (JAK2V617F) drives clonal expansion of hematopoietic progenitor cells in 90-95% in cases of PV and in approximately 40-50% in ET and PMF patients. This mutation induces an aberrant cellular signaling affecting the activation of several downstream protein cascades of cytokine receptors and substrates of the JAK2 protein. This constitutive activation leads to deregulated proliferation and differentiation of one or several myeloid lineages. Studies have shown that progenitor cells derived from JAK2V617F-positive patients are characterized by hypersensitivity to cytokines, e.g. erythropoietin (EPO), leading to an increased proliferation and disturbed differentiation under low –dose cytokine treatment. Methods: Murine 32D progenitor cells and I-11 proerythroblasts were transduced with retroviral vectors expressing cDNAs of the erythropoietin receptor and JAK2WT/JAK2V617F. Stably transduced cell lines were treated with increasing concentrations of EPO. To study the kinetics within the EPO signaling network, cell lines were starved for 4h and then treated with increasing doses of EPO (1 – 10 IU EPO for 32D, 0.5 – 3 IU EPO for I-11) for 1h (32D cells) and for 30min (I-11 cells). Phosphorylation of STAT3/STAT5, JAK2, Erk1/2, Akt and PLCg1, respectively were detected by immunoblot analysis. Densitometric analysis of immunoblot-signals (phospho-signals adjusted to total protein signals) was performed to quantitatively determine differences in phosphorylation of the main molecular pathways of EPO signaling. Results: Treatment with low physiologic doses of EPO resulted in an enhanced cell proliferation in JAK2V617F expressing cells compared to JAK2WT. However, this effect leveled off upon EPO concentrations >0.75 IU/ml. To investigate the molecular mechanisms of this hypersensitive status, we quantitatively monitored dose- and time-dependent phosphorylation of signaling proteins, which play a major role in the EPO signaling network, including STAT3/STAT5, Erk1/2, Akt and PLCg1. In unstimulated JAK2V617F mutated 32D and I-11 cells, we identified constitutive activation of these key signaling regulators. Moreover, we could demonstrate that in JAK2V617F expressing cells EPO dependent activation of these key signaling molecules is significantly more sensitive to low EPO concentrations as compared to the situation in JAK2WT expressing cells. Thus, we detected a higher peak of phosphorylation/activation of STAT3/STAT4, Erk1/2, Akt and PLCg1 in low-dose treated JAK2V617F cells. In addition, in JAK2V617F positive cells, three patterns of signaling kinetics were observed: 1. Left shift of activation curve and higher maximum of activation which is overcome by high EPO concentrations (e.g. phospho-JAK2); 2. Left shift of activation curve and higher maximum of activation which cannot be overcome by high EPO concentrations (e.g. phospho-STAT5); 3. Pattern exhibiting minor differences only (e.g. phospho-ERK1/2). We hypothesize that this is due to differential activation of feedback and feedforward loops. Quantitative and qualitative modelling is currently being performed to identify the molecular mechanisms involved. Along this line, we identified the docking protein Grb2-associated-binding protein1 (Gab1) as a member of EPO-dependent proteins. Gab1 plays a major role in co-activation of MAPK- and PI3K-pathway. Thus, for the first time we demonstrate constitutive activation of Gab1 in JAK2V617F mutated cells. Conclusions: We here demonstrate that hypersensitivity in proliferation of JAK2V617F mutated progenitor cells and proerythroblasts is molecularly corroborated by differential sensitivity of the pro-proliferative signaling network. We identified at least four different signaling pathways, which show higher sensitivity for activation in JAK2V617F mutated cells compared wild type cells. Hypersensitivity to low-dose EPO treatment on a molecular level may explain some of the biological features observed in JAK2V617F positive MPNs and may offer novel targets for therapeutic intervention. Disclosures No relevant conflicts of interest to declare.
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Gionfriddo, Ilaria, Federica Mezzasoma, Federica Cecchetti, Roberta Rossi, Francesca Strozzini, Francesco Di Raimondo, Valentina Pettirossi, Brunangelo Falini, and Maria Paola Martelli. "Histone Deacetylase Inhibitors Induce Cell Growth Inhibition and Apoptosis in NPM1-Mutated AML Cells: A Possible Role for Epigenetic Therapies in AML Carrying NPM1 Gene Mutations." Blood 118, no. 21 (November 18, 2011): 2621. http://dx.doi.org/10.1182/blood.v118.21.2621.2621.
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Abstract Abstract 2621 Acute myeloid leukemia (AML) carrying nucleophosmin (NPM1) gene mutations account for about one-third of adult AML, show distinctive biological and clinical features and have been included as a provisional entity in the 2008 World Health Organization (WHO) classification of myeloid neoplasms. In spite of the relatively good prognosis of NPM1-mutated AML, there are still cases that show poorer outcome, especially those associated with FLT3-ITD mutation and elderly patient population. Therefore new therapeutic strategies need to be explored. As for other types of cancer, evidence is emerging that, besides the genomic DNA alterations, epigenetic dysregulation may play a role in AML pathogenesis. We investigated the effects of sodium butyrate, a short-chain fatty acid which has long been known to be a histone deacetylase inhibitor (HDACi) able to induce maturation in normal and tumor cells, in cellular models of NPM1-mutated AML in vitro: i) the OCI/AML3 cell line, previously identified as a human AML cell line carrying cytoplasmic mutated NPM1 in the absence of FLT3-ITD; ii) primary AML cells originated from a patient with NPM1-mutated AML bearing FLT3-ITD mutation (MONT1) and propagated as cell line in NOD/SCID mice; and iii) primary AML cells from NPM1-mutated AML patients at diagnosis. In either cell lines or patients' primary AML cells carrying NPM1 mutation, but not in the U937 or OCI/AML2 cell lines (not harboring NPM1 gene mutation) used as control, growth arrest, cell cycle arrest (G0-G1 phase) and pro-apoptotic effects were evident after 24 hrs and marked after 48 hrs of treatment with doses of drug of 0.5–1 mM. No signs of differentiation were evident at morphological and flow cytometric examinations of treated cells. Western blot analysis with specific antibodies showed that levels of either NPM1 mutant or wild-type protein did not appear significantly affected by treatment with sodium butyrate. Interestingly, induction of apoptosis was associated with marked activation of caspase-8, suggesting involvement of the death cell receptors pathway. Indeed, flow cytometric analysis showed 2-fold increased expression of TRAIL-receptor DR5 upon drug treatment at 48 hrs. Moreover, concomitant treatment with a specific caspase-8 inhibitor prevented sodium butyrate induced-cell growth arrest and markedly reduced apoptosis in OCI/AML3 cell line. Derivatives of butyrate, which are capable of acting as HDAC inhibitors, are currently being investigated in clinical trials; preliminary results suggest their potential applicability in cancer treatment. Here we investigated the effect of panobinostat (LBH589), a pan HDACi, in human AML cells lines and show results similar to those observed upon sodium butyrate treatment in OCI/AML3 cells. In particular, we show that panobinostat induces dose-dependent cell growth arrest, with G0-G1 block, associated with p21 protein induction, and apoptosis, associated with TRAIL-receptor DR5 upregulation and activation of caspase 8, in AML cells harboring NPM1 gene mutation. However, unlike what was observed with sodium butyrate, panobinostat induced also dowregulation of NPM1 mutant (and to a lesser extent, wild type) protein. Mechanisms underlying this phenomenon are under investigation. In particular, the possible role of acetylation of the heat shock protein Hsp90 (as a consequence of panobinostat-mediated HDAC6 inhibition) with disruption of its chaperone functions followed by instability of its client proteins, including cytoplasmic NPM1, is explored. Disclosures: Falini: Xenomics: Patents & Royalties.
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Preston, Christopher, Fleur C. Dolman, and Peter Boutsalis. "Multiple Resistance to Acetohydroxyacid Synthase–Inhibiting and Auxinic Herbicides in a Population of Oriental Mustard (Sisymbrium orientale)." Weed Science 61, no. 2 (June 2013): 185–92. http://dx.doi.org/10.1614/ws-d-12-00117.1.
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A population of oriental mustard from Port Broughton in South Australia was reported as not being controlled by 2,4-D. Dose response experiments determined this population was resistant to both 2,4-D and MCPA, requiring greater than 20 times more herbicide for equivalent control compared to a known susceptible population (from Roseworthy, South Australia) and a population resistant only to the acetohydroxyacid synthase (AHAS)-inhibiting herbicides (from Tumby Bay, South Australia). The Port Broughton population was also found to be resistant to three chemical groups that inhibit AHAS; however, the level of resistance was lower than the known acetolactate synthase–resistant population from Tumby Bay. Herbicides from other modes of action were able to control the Port Broughton population. Assays of isolated AHAS from the Port Broughton population showed high levels of resistance to the sulfonylurea and sulfonamide herbicide groups, but not to the imidazolinone herbicides. A single nucleotide change in the AHAS gene that predicted a Pro to Ser substitution at position 197 in the protein was identified in the Port Broughton population. This population of oriental mustard has evolved multiple resistance to AHAS-inhibiting herbicides (AHAS inhibitors) and auxinic herbicides, through a mutation in AHAS and a second nontarget-site mechanism. Whether the same mechanism provides resistance to both AHAS inhibitors and auxinic herbicides remains to be determined. Multiple resistance to auxinic herbicides and AHAS inhibitors in the Port Broughton population will make control of this population more difficult.
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Nakka, Sridevi, Curtis R. Thompson, Dallas E. Peterson, and Mithila Jugulam. "Target Site–Based and Non–Target Site Based Resistance to ALS Inhibitors in Palmer Amaranth (Amaranthus palmeri)." Weed Science 65, no. 6 (September 20, 2017): 681–89. http://dx.doi.org/10.1017/wsc.2017.43.
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Resistance to acetolactate synthase (ALS)-inhibitor herbicides due to continuous and repeated selection is widespread in many troublesome weed species, including Palmer amaranth, throughout the United States. The objective of this research was to investigate the physiological and molecular basis of resistance to ALS inhibitors in a chlorsulfuron-resistant Palmer amaranth population (KSR). Our results indicate that the KSR population exhibits a high level of resistance to chlorsulfuron compared with two known susceptible populations, MSS and KSS from Mississippi and Kansas, respectively. MSS is highly susceptible to chlorsulfuron, whereas KSS is moderately sensitive. Dose–response analysis revealed that KSR was more than 275-fold more resistant compared with KSS. Nucleotide sequence analysis of theALSgene from the plants that survived chlorsulfuron treatment revealed the possibility of evolution of both target site–based and non–target site based resistance to ALS inhibitors in the KSR population. The most common mutation (Pro-197-Ser) in theALSgene associated with resistance to the sulfonylureas in many weed species was found only in 30% of the KSR population. A preliminary malathion study showed that the remaining 70% of resistant plants might have cytochrome P450–mediated non–target site resistance. This is the first report elucidating the mechanism of resistance to ALS inhibitors in Palmer amaranth from Kansas. Presence of both target site– and non–target site based mechanisms of resistance limits the herbicide options to manage Palmer amaranth in cropping systems.
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Dumbrava, Ecaterina Elena, Amit Mahipal, Xin Gao, Geoffrey Shapiro, Jason S. Starr, Parminder Singh, Muhammad Furqan, et al. "Phase 1/2 study of eprenetapopt (APR-246) in combination with pembrolizumab in patients with solid tumor malignancies." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): TPS3161. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.tps3161.
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TPS3161 Background: The p53 pathway has been implicated in antitumor immunity, including antigen presentation and T-cell proliferation. Loss of p53 function can increase resistance to immunotherapy across many tumor types. Eprenetapopt (eprenet) is a small molecule that stabilizes the folded structure of p53, resulting in activation of mutant p53 and stabilization of wild-type (WT) p53. It also targets the cellular redox homeostasis, resulting in induction of apoptosis in tumor cells. In vivo, mice carrying supernumerary copies of the TP53 gene harbor a pro-inflammatory tumor microenvironment, an effect recapitulated in TP53 normal-copy mice treated with eprenetapopt. Combining eprenetapopt and anti-PD1 or anti-CTLA4 therapy resulted in enhanced tumor growth inhibition and improved survival in TP53 WT mice inoculated with B16 melanoma and MC38 colon adenocarcinoma cells . Based on these results, we hypothesized that eprenet-induced p53 stabilization may augment response to immunotherapy. To test this hypothesis, we are conducting a phase 1b/2 study of eprenet in combination with pembrolizumab (eprenet+pembro) in pts with solid tumors. Methods: The primary objectives are to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) and to assess the safety and tolerability of eprenet+pembro in pts with advanced solid tumors. The secondary objectives are to estimate the anti-tumor activity and to describe the pharmacokinetics of the combination. Exploratory objectives include assessing predictive and pharmacodynamic markers of response. The study includes a safety lead-in with a 3+3 dose de-escalation design for pts with advanced solid tumors with known tumor TP53 mutation status ( TP53 WT is acceptable) (max 18 pts), followed by expansion cohorts in pts with NSCLC, gastric/GEJ and urothelial cancer (max 100 pts). In expansion, pts with urothelial and gastric cancers must be naïve to anti-PD-1/ L1 therapy. Eprenet is given IV once daily on Days 1–4 while pembro is administered on Day 3 of each 21-day cycle. The RP2D of eprenet+pembro is considered the dose at which ≤ 1 of 6 pts in a cohort has a dose-limiting toxicity (DLT). Primary endpoints are occurrence of DLTs, adverse events (AEs) and serious AEs with eprenet+pembro. Key secondary endpoints are best objective response, progression free survival and overall survival. Exploratory endpoints include gene mutations by next generation sequencing (including TP53), mRNA expression, multiplex immunohistochemistry and transcriptomics, multiplex flow cytometry on peripheral blood mononuclear cells and cytokines in serum. Continuous monitoring of toxicity will be conducted. The trial opened in May 2020 and is actively enrolling patients. Clinical trial information: NCT04383938.
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Cantilena, Caroline R., Xin Zhao, Sachiko Kajigaya, Neil Dunavin, Xin Tian, Stephen A. Strickland, Bipin N. Savani, et al. "Activity of the Telomerase Inhibitor GRN163L (Imetelstat) on Acute Myeloblastic Leukemia Blasts Is Enhanced By DNA Methyltransferase Inhibitors Irrespective of TERT Promoter Methylation Status." Blood 126, no. 23 (December 3, 2015): 1267. http://dx.doi.org/10.1182/blood.v126.23.1267.1267.
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Abstract Introduction. The high telomerase activity in leukemia cells protects them from proliferation arrest, senescence, and apoptosis and may be driven by mutation or epigenetic alteration in the telomerase promoter. However, the mechanism of telomerase regulation and potential therapeutic application of telomerase inhibition in leukemia are not fully understood. We evaluated epigenetic methylation patterns in the telomerase promoter region in myeloid cell lines and primary acute myeloid leukemia (AML) blasts. These epigenetic patterns may serve as a biomarker for sensitivity to DNA methyltransferase (DNMT) inhibitors and have prognostic significance. We also studied whether the telomerase inhibitor GRN163L (imetelstat)can favorably combine with the DNMT inhibitor 5-Azacytidine (5-Aza) to target poor prognosis leukemias. Methods. We developed a pyrosequencing-based methylation assay to screen methylation profiles of the proximal promoter and partial exon 1 of the human telomerase reverse transcriptase (hTERT pro/Ex1) region in primary leukemic cells and various cell lines.We used a chemosensitivity assay to determine specific killing of primary leukemia and cell lines by imetelstat. An inert mismatched oligonucleotide (Geron Corporation, Menlo Park, CA, USA) was used to control for specific inhibition of the telomerase active site. Cells were cultured for 48 hours with either active imetelstat or the inert control at varying concentrations, stained with annexin-V and propidium iodide, and then analyzed by flow cytometry to measure cell viability, apoptosis, and necrosis. Results. The hTERT pro/Ex1 region was highly methylated in cell lines, relative to de novo primary leukemic cells. Primary leukemic cells showed significantly different methylation profiles and hypermethylation status correlated to poor survival of AML patients. Three commercially available leukemia cell lines (K562, Ramos, THP-1), two primary leukemia-derived cell lines (AML1, CML1), and CD34+ blasts isolated from primary leukemia in six different AML patients with varying degrees of hTERT pro/Ex1 region methylation were tested. Imetelstat showed dose dependent cytotoxicity to both myeloid leukemia cell lines and primary leukemic blasts. Cell toxicity was telomerase specific since the inert control had no or minimal toxicity at the half inhibitory concentration (IC50) of imetelstat between 10-40 µM. Higher methylation status of the hTERT pro/Ex1 region was significantly associated with increased resistance to imetelstat in leukemia cell lines (Figure 1A). However, no correlation was found in primary leukemic blasts. Pretreatment of leukemia cell lines with 5-Aza for 24 hours prior to imetelstat exposure was associated with a decrease in viability from 0.78±0.01 to 0.54±0.01 at a concentration of 10µM of imetelstat (Figure 1B). 5-Aza alone had no effect on the leukemic cell lines' viability. Conclusion. High risk primary leukemias are susceptible to killing by the telomerase inhibitor irrespective of the degree of methylation of the hTERT pro/Ex1 region. Furthermore, demethylating agents can enhance the activity of the telomerase inhibitor, imetelstat. These findings suggest that combination therapy of imetelstat and DNMT inhibitors may have synergistic anti-leukemic efficacy in high risk AML patients. Disclosures Strickland: Amgen: Other: Advisory Board Particpation; Boehringer-Ingelheim: Other: Advisory Board Particpation; Daiichi-Sankyo: Other: Advisory Board Particpation; Sunesis Pharmaceuticals: Other: Steering Committee and Advisory Board Participation; Alexion Pharmaceuticals: Other: Advisory Board Particpation. Rezvani:Pharmacyclics: Research Funding. Townsley:Novartis: Research Funding; GSK: Research Funding.
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Yadav, Satya Prakash, Mohammed Ramzan, Yogi Raj Chopra, Nivedita Dhingra, and Anupam Sachdeva. "Acquired Whim Syndrome: A Possible New Side Effect Of Dasatinib!" Blood 122, no. 21 (November 15, 2013): 4933. http://dx.doi.org/10.1182/blood.v122.21.4933.4933.
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Abstract Introduction WHIM (Warts, Hypogammaglobulinemia, Infections and Myelokathexis) syndrome is a hereditary disorder caused by activating mutation in CXCR4 receptor. CXCR4 receptor is expressed in many cells including myeloid and lymphoid cells. Dasatanib is aTKI inhibitor and very effective drug in treating Ph+ve acute lymphoblastic leukaemia (Ph+ALL). Dasatinib has many off target effects which are not completely known as yet. Dasatinib effect on CXCR4 is not well described. In Chronic myeloid leukaemia it causes reacquisition CXCR4 activation leading to hiding of myeloid leukaemia cells inside the bone marrow. However some work has been done on effects of Dasatinib on mature neutrophil indicates adhesion defect and inhibition of pro-inflammatory effect. Here we describe two children who developed acquired WHIM syndrome like features while on Dasatinib therapy. Methods Two girls aged 9 and 11 year old with Ph+ALL were treated initially as per BFM95 protocol and Imatinib 375 mg/m2 daily. Both went in remission and later achieved complete molecular remission. However during maintenance therapy first patient at 19 months from diagnosis and second at 12 months from diagnosis started having rising value of quantitative BCR-ABL values. As both children had no siblings and no matched unrelated donor was available so in both Imatinib was stopped and after taking informed consent of parents Dasatinib was started at a dose of 100/mg/m2/day in two divided doses and maintenance therapy of oral 6-Mercaptopurine (6-MP) and Methotrexate (MTX) was continued. They both achieved reduction in BCR-ABL quantitative PCR after 3 months and molecular remission at 6 and 9 months respectively after starting Dasatinib. Results During therapy with dasatinib and oral 6-MP and MTX both children started having recurrent respiratory tract infections with neutropenia and warts appeared on face and body. Serum immunoglobulin G levels were lower than 200 mg/dl in both. We felt that these symptoms resembled WHIM syndrome and possibly could be due to Dasatinib. So we gave Intravenous immunoglobulin to both the children and IV antibiotics and stopped both Dasatinib and chemotherapy for a week. Neutropenia recovered within 72 hr after stopping of dasatinib. However, we did not perform bone marrow examination in both the cases to prove myelokathexis. Both children did well when Dasatinib was restarted at a lower dose of 50 mg/m2/day once daily and oral 6-MP and MTX. Warts disappeared over next 4-6 months and did not have any further episodes of fever or febrile neutropenia needing IV antibiotics. Their repeat serum immunoglobulin G levels stayed normal over next 6-12 months. Conclusion Both our children on dasatinib developed WHIM syndrome like features which resolved on reducing dose of Dasatinib. Dasatinib possibly mediates this side effect through CXCR4 receptor or downstream pathway. More data are needed to prove this association. Disclosures: No relevant conflicts of interest to declare.
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Mahoney, Denis J., David L. Jordan, Nilda Roma-Burgos, Katherine M. Jennings, Ramon G. Leon, Matthew C. Vann, Wesley J. Everman, and Charles W. Cahoon. "Susceptibility of Palmer amaranth (Amaranthus palmeri) to herbicides in accessions collected from the North Carolina Coastal Plain." Weed Science 68, no. 6 (September 1, 2020): 582–93. http://dx.doi.org/10.1017/wsc.2020.67.
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AbstractPalmer amaranth (Amaranthus palmeri S. Watson) populations resistant to acetolactate synthase (ALS)-inhibiting herbicides and glyphosate are fairly common throughout the state of North Carolina (NC). This has led farm managers to rely more heavily on herbicides with other sites of action (SOA) for A. palmeri control, especially protoporphyrinogen oxidase and glutamine synthetase inhibitors. In the fall of 2016, seeds from A. palmeri populations were collected from the NC Coastal Plain, the state’s most prominent agricultural region. In separate experiments, plants with 2 to 4 leaves from the 110 populations were treated with field use rates of glyphosate, glufosinate-ammonium, fomesafen, mesotrione, or thifensulfuron-methyl. Percent visible control and survival were evaluated 3 wk after treatment. Survival frequencies were highest following glyphosate (99%) or thifensulfuron-methyl (96%) treatment. Known mutations conferring resistance to ALS inhibitors were found in populations surviving thifensulfuron-methyl application (Ala-122-Ser, Pro-197-Ser, Trp-574-Leu, and/or Ser-653-Asn), in addition to a new mutation (Ala-282-Asp) that requires further investigation. Forty-two populations had survivors after mesotrione application, with one population having 17% survival. Four populations survived fomesafen treatment, while none survived glufosinate. Dose–response studies showed an increase in fomesafen needed to kill 50% of two populations (LD50); however, these rates were far below the field use rate (less than 5 g ha−1). In two populations following mesotrione dose–response studies, a 2.4- to 3.3-fold increase was noted, with LD90 values approaching the field use rate (72.8 and 89.8 g ha−1). Screening of the progeny of individuals surviving mesotrione confirmed the presence of resistance alleles, as there were a higher number of survivors at the 1X rate compared with the parent population, confirming resistance to mesotrione. These data suggest A. palmeri resistant to chemistries other than glyphosate and thifensulfuron-methyl are present in NC, which highlights the need for weed management approaches to mitigate the evolution and spread of herbicide-resistant populations.
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Pepper, Chris J., Hani Y. Osman, Saman Hewamana, Elisabeth J. Walsby, Alan K. Burnett, and Steven Knapper. "L-Gossypol Inhibits NF- κB, Down Regulates Mcl-1 and Induces Apoptosis of Primary Acute Myeloid Leukaemia Cells." Blood 114, no. 22 (November 20, 2009): 4813. http://dx.doi.org/10.1182/blood.v114.22.4813.4813.
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Abstract Abstract 4813 Standard treatments for acute myeloid leukaemia (AML) result in a median survival of approximately 1 year. There is now a realisation that in order to significantly improve outcomes in this disease more targeted therapies that take account of the specific biology of the tumour cell are required. L-Gossypol is a polyphenolic oil cotton seed extract that has anti-tumour activity against a range of haematological malignancies but has never been evaluated in AML cells. It is known to act as a BH3-mimetic, binding to the BH3 pocket of anti-apoptotic proteins and displacing pro-death partners to induce apoptosis. However, knowledge of the molecular events that underpin its downstream effects is limited. In this study we analysed the in vitro effects of L-Gossypol in 50 primary AML samples in order to determine its efficacy and mode of action. Apoptosis was induced in all the samples tested in a dose- and time-dependent manner as evidenced by increased Annexin V / propidium iodide labelling and the activation of caspase-9 and caspase-3. The median LD50 value (the concentration of drug required to kill 50% of the cells) was 27.5μM ± 18.3μM. There was no association between LD50 and age, sex, presenting white cell count, FLT3 mutation status or karyotype. Mechanistically, L-gossypol decreased the DNA binding activity of the NF-κB subunit, Rel A, in a concentration-dependent manner; this inhibition was evident after only 4 hours and preceded the induction of apoptosis. Furthermore, treatment with L-Gossypol inhibited the transcription of the NF-κB-regulated genes CFLAR, BCL2, BIRC5 and MCL1 in the same timeframe. Finally, studies of Mcl-1 protein expression showed down regulation in a dose-dependent manner but this was only apparent after 8 hours exposure to L-Gossypol. Taken together, our data demonstrate that L-Gossypol works, at least in part, through the inhibition of NF-κB and our data provides a rationale for clinical investigations of this agent in AML patients. Disclosures: No relevant conflicts of interest to declare.
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Stuebig, Thomas, Christine Wolschke, Haefaa Alchalby, Francis Ayuk, Marion Heinzelmann, Ioanna Triviai, Anita Badbaran, York Hildebrandt, and Nicolaus Kroeger. "The Pan- JAK Inhibitor Ruxolitinib Impairs T-Cell Activation, Cytokine Production and Proliferation In Vivo and In Vitro." Blood 122, no. 21 (November 15, 2013): 2001. http://dx.doi.org/10.1182/blood.v122.21.2001.2001.
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Abstract Myelofibrosis (MF) is a clonal myeloproliferative neoplasm, in which the JAK2-V617F mutation is frequently observed. The appearance in up to 50% of the cases makes the JAK2 mutation attractive as therapeutical target. In 2012 Ruxolitinib (Ruxo) a pan-JAK inhibitor was approved for the treatment of MF and showed efficacy in disease treatment, irrespectively of the JAK2V617 mutation status. Currently allogeneic stem cell transplantation (allo SCT) remains the only curative treatment option for MF. To further improve transplant outcome in MF reduction of spleen size and constitutional symptoms prior transplantation is a reasonable target. Harnessing graft versus myelofibrosis post transplantation by immune-modulating drugs may help to reduce the risk of relapse. Ruxolitinib may be used as pre- and post-transplantation drug to improve transplant outcome. However the impact of Ruxolitinib on the immune system, especially on T-cells, is poorly understood. Here we investigated the effects of Ruxolitinib on T-cells in vivo and in vitro. T-cells from healthy donors were isolated by magnetic cell sorting to pan CD3+, CD4+ and CD8+ fraction. All three different cell subsets were cultured with different dosages of Ruxolitinib (100, 250, 500, 750nM and 1µM) for additional 48h. Thereafter cells were analysed for cell growth, cell death, RNA expression, immune phenotype. Additionally, immune profiles of 9 patients were analysed for the changes of the T-cell compartment during the treatment with Ruxolitinib over a period of 3 weeks T –cells from healthy donors showed a dosage dependent impairment in the proliferation capacity compared to non-treated control cells (4.1x106 CD3 cells /ml vs. 1.9x106 CD3 cells /ml, p<0.05), additionally KI67 expression was reduced from 48% in control cells to 12% in 100nM treated CD3 cells and 9% in 500nM treated CD3 cells, p<0.05. Strikingly apoptotic cell death increased from 11% in control cells to 43% and 48% in 100nM and 500nM Ruxo treated cells, p<0.03. Analysing the immune phenotype of Ruxo treated CD3, CD4 and CD8 cells we found a significant reduction in the expression of activation marker like CD25 and HLA-DR (38% vs. 6% and 4.5% respectively, p<0.05 and 63% vs. 47% and 40% respectively, p<0.05). Furthermore, we found that the effector cells, marked by CCR7/CD45RA expression, decreased in the CD8 compartment from 22% to 10.5% and 7.8% respectively, p<0.05. When analysing regulatory T-cells we also observed a decrease in a dose dependent manner (4% vs. 1.2% and 0.8%, p=0.05). While control Treg showed a KI67 expression of >60%, Ruxo (100nM) treated T-reg did not expressed KI67. Likewise to CD8 effector cells and Tregs we found a decrease in pro-inflammatory TH1 and TH17 cells in vitro (27% vs. 14% and 12% for TH1 cells and 6% vs. 4% and 4% for TH17 cells). Next, we analysed mRNA expression and found that pro-inflammatory cytokines like IL23, IL18, IL7 were down regulated after Ruxo treatment. To in contrast to pro- inflammatory cytokines, p53 and cell cycle inhibitor of the cip/waf locus showed to be up regulated in CD3 and CD4 cells suggesting that the observed increase in apoptosis in T-cells is mediated by p53. We next investigated the impact of Ruxolitinib on T-cells in patients. Therefore we analysed the blood of patients treated with Ruxolitinib in weekly intervals. Likewise to in vitro CD3 cells showed a decrease which turned to be significant after two and three weeks of treatment (1560/µl vs. 688/µl and 410/µl, p<0.05), this was mainly through the reduction of CD8+ T-cells (630/µl before treatment vs. 250/µl at week 2 and 200/µl at week 3, p <0.05). We also observed a decrease of CD3+/ HLA-DR+ (as activation marker) from 355/µl before to 130/µl and 70/µl however this did not reached statistical significance. The same was found for Tregs in vivo (5.6% vs. 2.3% and 1.9%, respectively). These data argue that treatment of T-cells by Ruxolitinib impairs their proliferation capacity by inducing apoptosis through an up regulation of p53. This increase of cell death applies all analysed T-cell compartments, and thereby may explains why Ruxo treated T-cells were less able to show a pro-inflammatory as well as regulatory phenotype. Disclosures: No relevant conflicts of interest to declare.
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Schittenhelm, Marcus M., Figen Akmut, Barbara Illing, Julia Frey, Katja Schuster, Abhijit Ramachandran, Lothar Kanz, and Kerstin M. Kampa-Schittenhelm. "Gain-of-Function KIT Mutations Sensitize the Mutant Isoform to the Type I Tyrosine Kinase Inhibitor Crenolanib: A Rationale for the Therapeutic Use in Systemic Mastocytosis (SM) and Core Binding Factor Leukemias (CBFL)." Blood 124, no. 21 (December 6, 2014): 2230. http://dx.doi.org/10.1182/blood.v124.21.2230.2230.
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Abstract Activating mutations of the class III receptor tyrosine kinases FLT3 and KIT are associated with certain human neoplasms, including hematologic malignancies, i.e. the majority of patients with systemic mast cell disorders (KIT) and subsets of patients with acute myelogenous leukemia (FLT3 and KIT). Crenolanib is a potent selective FLT3 inhibitor with high efficacy against internal tandem dupliction mutations (ITD) – but also secondary kinase domain mutations conferring resistance towards other TKI. Interestingly, crenolanib does not target the wildtype KIT isoform, which is believed to reduce clinical side effects such as prolonged myelosuppression observed with other TKI. Clinical studies are currently enrolling. We now show that gain-of-function mutations of KIT, including codon D816 alterations as the most prevalent mutation in SM and CBFL, sensitize the mutant isoform to crenolanib. Several mast cell and leukemia cell lines harboring autoactivating KIT or FLT3 isoforms were treated with crenolanib in dose dilution series (MOLM14, MV4;11, HMC1.1/1.2, p815). To minimize cell-type specific off-target effects, an isogenic cell model was established. The murine pro B-cell line Ba/F3 was retrovirally transduced with either a FLT3 ITD or a KIT D816 isoform. Apoptosis induction was analyzed by annexin V-based assays. FLT3/KIT tyrosine phosphorylation was assessed by western immunoblots. As previously described, the FLT3 ITD positive cell line MOLM14 revealed high sensitivity towards crenolanib with IC50s in the lowest nanomolar range. We also confirmed high sensitivity towards crenolanib ex vivo in the low nanomolar range in a native sample of a heavily pretreated patient. This patient relapsed with FLT3 ITD positive leukemia harboring a secondary D835H mutation in a subclone. Interestingly, leukemia cells in the relapse situation were much more oncogene-addicted than cells at primary diagnosis, which is in line with previous findings by others. Due to the structural homology of FLT3 D835 and KIT D816 mutations, we extended our studies to mutant-KIT mastocytosis and leukemia cell models and confirm clinically relevant antiproliferative as well as proapoptotic sensitivities towards crenolanib: for HMC mastocytosis cells harboring a KIT V560G and/or a D816V mutation, potent induction of apoptosis was observed with IC50s of 100-250nM. The murine p815 mastocytosis cell line (harboring a D814Y mutation corresponding to D816Y in humans) demonstrated a proapoptotic effect of crenolanib with an IC50 of 60 nM. Treatment of corresponding KIT or FLT3 isoform-transduced Ba/F3 cells confirmed similar IC50s in the leukemia cell lines. Parental Ba/F3cells did not show any sensitivity towards crenolanib up to concentrations of 1000 nM. Additionally, potent dephosphorylation at 100 nM of KIT D816V in Ba/F3 and HMC cells after exposure to crenolanib confirmed mutant-KIT as a target of the drug. Evaluation of a broader range of native mast cell and leukemia patient samples as well as additional leukemia cell lines and isogenic Ba/F3 KIT or FLT3 transfectants is ongoing. First results demonstrate activity of crenolanib in native cells of a subset of patient samples with SM or CBFL treated ex vivo. Even more, combination of crenolanib with anthracyclines revealed additive to superadditive proapoptotic effects. Moreover, combination of crenolanib with cladribine, a hallmark agent in the treatment of systemic mastocytosis, resulted in potent induction of apoptosis already at doses that did not display any proapoptotic effects when administered as single agents, thereby providing a rationale for combinatorial therapeutic approaches. In summary, crenolanib is effective against the KIT D816V isoform associated with several hematologic malignancies. Notably, while not as effective towards mutant-KIT compared to the FLT3 ITD isoform, the observed estimated IC50 of crenolanib is well in the range of achievable plasma concentrations and in the range of the potent KIT inhibitor dasatinib, which is successfully under clinical investigation in CBFL. Our data provide a rationale to test crenolanib as a potent inhibitor of mutant-KIT isoforms in KIT-associated neoplasms. Disclosures Schuster: AROG Pharmaceuticals: Employment. Ramachandran:AROG: Employment.
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Liu, Wen Jun, Xiang Ju Wang, David C. Clark, Mario Lobigs, Roy A. Hall, and Alexander A. Khromykh. "A Single Amino Acid Substitution in the West Nile Virus Nonstructural Protein NS2A Disables Its Ability To Inhibit Alpha/Beta Interferon Induction and Attenuates Virus Virulence in Mice." Journal of Virology 80, no. 5 (March 1, 2006): 2396–404. http://dx.doi.org/10.1128/jvi.80.5.2396-2404.2006.
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ABSTRACT Alpha/beta interferons (IFN-α/β) are key mediators of the innate immune response against viral infection. The ability of viruses to circumvent IFN-α/β responses plays a crucial role in determining the outcome of infection. In a previous study using subgenomic replicons of the Kunjin subtype of West Nile virus (WNVKUN), we demonstrated that the nonstructural protein NS2A is a major inhibitor of IFN-β promoter-driven transcription and that a single amino acid substitution in NS2A (Ala30 to Pro [A30P]) dramatically reduced its inhibitory effect (W. J. Liu, H. B. Chen, X. J. Wang, H. Huang, and A. A. Khromykh, J. Virol. 78:12225-12235). Here we show that incorporation of the A30P mutation into the WNVKUN genome results in a mutant virus which elicits more rapid induction and higher levels of synthesis of IFN-α/β in infected human A549 cells than that detected following wild-type WNVKUN infection. Consequently, replication of the WNVKUNNS2A/A30P mutant virus in these cells known to be high producers of IFN-α/β was abortive. In contrast, both the mutant and the wild-type WNVKUN produced similar-size plaques and replicated with similar efficiency in BHK cells which are known to be deficient in IFN-α/β production. The mutant virus was highly attenuated in neuroinvasiveness and also attenuated in neurovirulence in 3-week-old mice. Surprisingly, the mutant virus was also partially attenuated in IFN-α/βγ receptor knockout mice, suggesting that the A30P mutation may also play a role in more efficient activation of other antiviral pathways in addition to the IFN response. Immunization of wild-type mice with the mutant virus resulted in induction of an antibody response of similar magnitude to that observed in mice immunized with wild-type WNVKUN and gave complete protection against challenge with a lethal dose of the highly virulent New York 99 strain of WNV. The results confirm and extend our previous original findings on the role of the flavivirus NS2A protein in inhibition of a host antiviral response and demonstrate that the targeted disabling of a viral mechanism for evading the IFN response can be applied to the development of live attenuated flavivirus vaccine candidates.
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Nabinger, Sarah C., Xing Jun Li, Baskar Ramdas, Yantao He, Xian Zhang, Briana Richine, Joshua Bowling, et al. "The Protein Tyrosine Phosphatase, Shp2, Positively Contributes to FLT3-ITD-Induced Malignant Disease in Vivo and Co-Localizes with Nuclear Phospho-STAT5 in FLT3-ITD-Expressing Leukemic Cells." Blood 120, no. 21 (November 16, 2012): 2420. http://dx.doi.org/10.1182/blood.v120.21.2420.2420.
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Abstract Abstract 2420 Internal tandem duplications in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in individuals with acute myeloid leukemia (AML). Based on the finding that the protein tyrosine phosphatase, Shp2, interacts with WT FLT3 tyrosine (Y) 599, which is commonly duplicated in FLT3-ITDs, we hypothesized that increased recruitment of Shp2 to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and aberrant STAT5 activation. Co-immunoprecipitation studies demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Additionally, we found that genetic disruption of Ptpn11, the gene encoding Shp2, significantly reduced N51-FLT3-induced hematopoietic cell hyperproliferation and STAT5 hyperphosphorylation in vitro. To investigate these findings further, Lin- bone marrow cells from Shp2flox/flox;Mx1Cre+ animals were retrovirally transduced with N51-FLT3, sorted to homogeneity, and transplanted into lethally irradiated congenic recipients. Transplanted animals were treated with polyI:polyC to delete Shp2 or with phosphate buffered saline (PBS control) 4 – 6 weeks following transplantation, and animals were followed temporally. The majority of PBS-treated animals (16/18) died of hematologic malignancy. In contrast, animals with Shp2 deletion (polyI:polyC-treated, n=16) succumbed to malignant disease less frequently (10/16), demonstrated a significantly prolonged survival (p=0.024 by log-rank test), and had smaller spleen sizes compared to the PBS-treated animals. Notably, Y599 has been shown to recruit Shp2 to WT FLT3 and mutation of Y599 to phenylalanine (F) within WT FLT3 causes a reduction in FL-stimulated cell proliferation. Thus, we generated point mutants including N51-Y599F1 bearing the Y to F mutation at the first Y599 and N51-Y599F1/2 bearing Y to F mutation at both the first and duplicated Y599. Murine bone marrow low density mononuclear cells were transduced with each construct and subjected to 3H-thymidine incorporation and immunoblot for proliferation and STAT5 activation, respectively. While mutation of the first Y599 alone failed to reduce proliferation or STAT5 phosphorylation, mutation of both the first and duplicated Y599 significantly reduced cellular proliferation and phospho-STAT5 levels. To investigate molecular mechanisms underlying how constitutive association of Shp2 with STAT5 may promote FLT3-ITD-induced leukemogenesis, we utilized the human FLT3-ITD positive AML-derived cell line, MV411. While previous studies have demonstrated nuclear localization of Shp2 in AML samples, the role of nuclear Shp2 in leukemia has never been investigated. We utilized in situ immunofluorescence to examine nuclear distribution of Shp2 and potential co-localization with phospho-STAT5. Strong nuclear expression of Shp2 was observed in MV411 cells, and upon merging of images, nuclear Shp2 co-localized strongly with nuclear phospho-STAT5, suggesting that Shp2 may work with STAT5 within the nucleus to enhance gene expression promoting leukemogenesis. We chose to examine the BCL2L1 promoter, a STAT5-responsive promoter which regulates expression of the prosurvival protein, Bcl-XL. Using chromatin immunoprecipitation assays, we found Shp2 is present at functional interferon-g activation sites (GAS) within the BCL2L1 promoter. Furthermore, knockdown of Shp2 in MV411 cells resulted in reduced phospho-STAT5 levels and reduced BCL2L1 promoter-directed luciferase expression. Moreover, using a novel small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was significantly reduced in a dose-dependent manner. Our findings suggest that constitutive association of Shp2 with N51-FLT3 promotes hyperproliferation and that either genetic disruption of Shp2 expression or mutation of the Shp2 binding sites on N51-FLT3 significantly abrogates N51-FLT3-induced hyperproliferation, STAT5 hyperactivation, and N51-FLT3-induced hematologic malignancy in vivo. Furthermore, Shp2 and STAT5 appear to work functionally in the nucleus to promote STAT5-responsive, pro-leukemogenic gene expression. Collectively, these studies demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to AML. Disclosures: No relevant conflicts of interest to declare.
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Ayad, Ahmed, Sara Nofal, Crystal Jobson, Yuval Raizen, and Mona Lisa Alattar. "Symptomatic Hemoglobin New Mexico and a Case of Therapeutic Benefit with Acetazolamide." Blood 132, Supplement 1 (November 29, 2018): 4918. http://dx.doi.org/10.1182/blood-2018-99-119250.
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Abstract INTRODUCTION Inherited disorders due to rare structural Hb variants are not included in national, neonatal screening tests and therefore likely underdiagnosed. Hemoglobin New Mexico is one such rare variant with only one case ever reported in 1985 in conjunction heterozygosity for Hb S.1 This Hb variant involves a missense mutation of the β chain substituting the hydrophobic Proline β100 with the hydrophilic Arginine.1 We present here the second case of Hb New Mexico variant ever reported to our knowledge. CASE PRESENTATION An 18-year-old athletic, Latin American female initially presented with episodic chest pain, palpitations, fatigue, headaches, and dyspnea on exertion to the point that she could no longer exercise for one year. Work up revealed erythrocytosis persistent for >1 year with hemoglobin level of 17.3 g/dL, and hematocrit of 47.2 % (Hb went as high as 19g/dL) with normal erythropoietin level, hemoglobin electrophoresis, and JAK2 V617 with reflex to JAK3 exon 12-15 negative. She had extensive evaluation to rule out acquired causes including cardiac, pulmonary, and neurologic evaluation which was all normal. She underwent therapeutic phlebotomies with no clinical improvement over 3 months and symptoms worsened as well as new iron deficiency from phlebotomies so this was discontinued. Evaluation with p50 oxygen dissociation curve revealed high oxygen affinity. Further gene sequencing revealed heterozygous inheritance of Hb New Mexico with HbA: 54.5%, HbA2: 3.6%, and Hb New Mexico: 41.9%. Beta Globin Gene sequencing identified the following mutation: Beta 100, CCT>CGT, Pro>Arg. Her symptoms worsened as well as an episode of syncope but no new neurologic, cardiac, or pulmonary etiologies were identified and at the time Hg 18g/dL. A trial of low dose acetazolamide 125mg daily was given to the patient and her symptoms improved by a 6 week follow up with notable reduction of headache, fatigue, dyspnea, and chest pain. CONCLUSIONS Given the rarity of this hemoglobin variant in a patient with severe symptoms, data is limited. Acetazolamide, a carbonic anhydrase inhibitor often used for migraines, is known to illicit mild acidosis and therefore potentially reduce hemoglobin-oxygen affinity and perhaps allow for relief of our patient's symptoms. Further therapeutic evaluation is warranted but likely challenging given rarity of the condition. Disclosures No relevant conflicts of interest to declare.
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Liu, Ligen, Yuanmmei Zhai, Li Yang, Limin Zhao, Li Chang, Chunrong Yin, Yingting Lu, Xuewei Jiang, and Zhizhi Zhang. "Clinical Efficacy Analysis of the Low-Dose Decitabine in Combination with Cytarabine, All-Trans Retinoic Acid and Granulocyte Colony-Stimulating Factor for Patients with MDS-EB." Blood 138, Supplement 1 (November 5, 2021): 3685. http://dx.doi.org/10.1182/blood-2021-144631.
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Abstract Abstract Purpose: Treatment of patients with myelodysplastic syndromes with excess blasts (MDS-EB) remains a great challenge. In this study, we evaluated the tolerance and efficacy of the combination of low-dose decitabine, cytarabine, all-trans retinoic acid and granulocyte colony-stimulating factor (DLAAG) for MDS-EB patients. Methods: A total of 18 patients with MDS-EB were enrolled in this study and 1 patient who did not follow-up the bone marrow evaluation was excluded from analysis. This study is registered at ClinicalTrials.gov, NCT03356080.The DLAAG regimen consisted of subcutaneous injection of decitabine (0.1-0.2mg/kg, d1-3/w, lasted for 1-3 weeks), cytarabine (6-15mg/m 2,q12h,d1-10), oral all-trans retinoic acid (45mg/m 2/d, d4-6; 15mg/m 2/d, d7-20) and G-CSF (300ug, from d0 until neutrophil count recovery to 20×10 9 cells/L). Results: Patient characteristics A total of 18 patients (11 males, 7 females) with a median age of 61 years (range 45 to 88 years) were enrolled in our study. The clinical characteristics of the patients were shown in Table 1. Response and survival The clinical outcome and overall survival (OS) of patients were summarized in Table2 including 1 could not evaluable after the first protocol. As shown in Table3, after one course of DLAAG, the CR and OR rate was 55.56% and 77.78% respectively. In addition, 3 patients suffered the disease progression (PD) (n=3,16.67%). We found that those who achieved the bone marrow remission after DLAAG had significantly pro-longed OS than others (p=0.0001, Fig.1). The Kaplan-Meier analysis revealed the median overall survival for all patients enrolled was 15 months (Fig.2A), and the relapse-free survival (RFS) for patients achieving CR or CRm was 11 months (Fig.2B). Genetic mutations and response to DLAAG Paired samples (pre- and post-treatment) of the objects were tested for gene mutations which were recurrently mutated in myeloid malignancies, except 1 patient who was failure to perform bone marrow review. 2 patients (11.1%) had no genetic or cytogenetic abnormalities either at baseline or during therapy (they were tested at baseline and d28). Most patients (15 out of 18) had at least one somatic mutation. In this latter group, only 1 patient acquired specific mutation of IDH2 during the therapy (it was tested on d28, data was not shown), while all the other genes were mutated in patients at baseline. Collectively, the most frequently mutated genes at diagnosis were RUNX1 (n=5, 29.4%), ASXL1 (n=4, 23.5%), as well as the overexpression of WT1 (n=4, 23.5%), and the others with an incidence of more than 10% were TET2(n=3,17.6%),TP53 (n=2, 11.8%), EZH2 (n=2, 11.8%), STAG2 (n=2, 11.8%), SF3B1 (n=2, 11.8%) and U2AF1 (n=2, 11.8%).Genes less frequently mutated such as CEBPA,CUX1 et al. were not shown. Remarkably, the proportion of patients with the above gene mutations showing no significant increasing during therapy as compared to baseline, the clones of WT1/TET2/TP53/EZH2/STAG2 can decrease or even disappear(Fig.3A).Moreover, we noted the complete response in patients with ASXL1, TP53 mutations or WT1 overexpression despite the concomitant presence of complex cytogenetic at diagnosis, whereas patients with the EZH2 mutation were less likely to respond (Fig.3B). Subgroup analysis A subgroup analysis based on the 2016 WHO classification revealed an association of EB-I patients with longer OS(P=0.002) (Fig. 4A). On the other hand, based on the IPSS score our study revealed that the lower-risk group patients have better OS (p= 0.0054) (Fig. 4B).In addition to this, we also compared the OS in different groups of age and karyotype, but there were no statistical difference (Fig. 4C-D). Safety and Tolerability All patients who received the therapy were eligible for toxicity evaluation. Given the patient-population enrolled and treatment regimen, myelosuppression occurred in all patients (Table 4). The grade 3 to 4 hematological toxicities including neutropenia and thrombocytopenia were common after treatment. Febrile neutropenia occurred in 77.8% of the patients. Conclusion: This study suggested the novel combination of LD-DAC, Ara-C, ATRA and G-CSF might be an optimal introduction therapy for patients with MDS-EB, and this combination warrants further investigation in larger trials. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Toki, Tsutomu, Rika Kanezaki, Souichi Adachi, Hisanori Fujino, Gang Xu, Tomohiko Sato, Kahori Suzuki, Hisamichi Tauchi, Mikiya Endo, and Etsuro Ito. "The Essential Role of Stem Cell Factor/KIT Signaling in the Proliferation and Survival of Blast Cells from Transient Leukemia in Neonates with Down Syndrome." Blood 112, no. 11 (November 16, 2008): 1807. http://dx.doi.org/10.1182/blood.v112.11.1807.1807.
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Abstract Children with Down syndrome (DS) are predisposed to developing leukemia. A leukemoid reaction occurring uniquely in approximately 10% of newborn infants with DS referred to as transient leukemia (TL). This disorder, in most cases, resolves spontaneously within three months after birth. Of all TL patients approximately 20–30% develop myeloid leukemia (ML-DS) within four years. Although treatment with low dose cytarabine is effective in high-risk TL cases, about 20% of severe patients still suffer early death. Improved treatments for TL are necessary for a better long-term prognosis of DS patients. In this study, we demonstrate abundant KIT expression in all 13 TL patients examined, although no significant difference in expression levels was observed between TL and acute myeloid leukemia (AML). Stem cell factor (SCF) mRNA was expressed at extremely low levels in TL cells and there were no significant differences in SCF expression between TL and AML. SCF stimulated the proliferation of the TL cells from all five patients examined and treatment with the tyrosine kinase inhibitor imatinib suppressed the proliferation and KIT phosphorylation effectively in vitro. To investigate the signal cascade, we established the first SCF-dependent, ML-DS cell line, KPAM1. Withdrawal of SCF or treatment with imatinib induced apoptosis of KPAM1 cells. SCF activated the RAS/MAPK and PI3K/AKT pathways, followed by downregulation of the pro-apoptotic factor BIM and upregulation of the anti-apoptotic factor MCL1. Although we found novel missense mutations of KIT in two of 14 TL patients, functional analysis using KPAM1 cells showed that neither mutation led to KIT activation and neither reduced the cytotoxic effects of imatinib. These results suggest the essential role of SCF/KIT signaling in the proliferation of DS-related leukemia and the possibility of therapeutic benefits of KIT-targeting tyrosine kinase inhibitors for TL patients.
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Adam, Jacqueline, Thomas Gentinetta, Svetlana Diditchenko, Alexander Schaub, Gregory J. Kato, Nathan Brinkman, and Adrian Zuercher. "Prevention of Heme-Induced Human Endothelial Cell Activation By Hemopexin in Vitro." Blood 136, Supplement 1 (November 5, 2020): 8. http://dx.doi.org/10.1182/blood-2020-140238.
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Hemoglobin (Hb) is one of the most abundant proteins in the human body. When red blood cells rupture, cell-free Hb may initiate adverse pathophysiological reactions. Pathophysiology triggered by cell-free Hb plays an important role in modifying the phenotype of sickle cell disease (SCD). SCD is caused by a single nucleotide mutation of the β-globin gene resulting in Hemoglobin-S (HbS) instead of the normal HbA found in healthy individuals. Polymerization of HbS shortens the lifespan of sickle red blood cells and promotes intra- and extravascular hemolysis. In cell-free Hb ferrous Hb (Fe2+) is oxidized into ferric Hb (Fe3+) promoting the dissociation and transfer of heme into lipid compartments where it triggers lipid peroxidation and generation of cytotoxic and pro-inflammatory reaction products. These processes promote endothelial cell activation and damage. The endogenous plasma protein hemopexin exhibits the highest binding affinity for heme and binds heme in a 1:1 binding ratio. Heme bound to hemopexin is rendered relatively non-reactive and is delivered safely to hepatocytes for endocytosis and degradation. To investigate the endothelial-protective function of hemopexin based on its ability to scavenge heme, we exposed human umbilical vein endothelial cells (HUVEC) in vitro to heme(NaOH) in the presence or absence of different hemopexin doses. As a read-out, different markers for endothelial cell activation were analyzed by either flow cytometry or multiplexed particle-based flow cytometry (Luminex). Briefly, confluent HUVEC were preincubated with hemopexin at different concentrations for 5 min before stimulation with heme(NaOH) for 25 min. Following stimulation cells were analyzed by flow cytometry for expression of membrane bound P-Selectin, a robust marker of endothelial cell activation. Alternatively, heme(NaOH) stimulation of hemopexin-preincubated HUVEC was conducted for 16 h and cell culture supernatants were analyzed by Luminex for three additional well-characterized plasma markers of endothelial cell activation: pro-inflammatory cytokine IL-8, cell adhesion molecule VCAM-1 and glycoprotein Von Willebrand factor (vWF). In the absence of hemopexin, heme(NaOH) consistently induced robust cell surface expression of P-Selectin and elevated levels of soluble IL-8, VCAM-1 and vWF. However, hemopexin completely blocked the stimulatory potential of heme as HUVEC exposed to pre-formed heme:hemopexin complexes showed unchanged P-Selectin expression levels compared to negative control samples. We found that hemopexin reduced heme(NaOH)-mediated P-selectin expression on HUVEC in a dose-dependent fashion. Once an equimolar ratio between heme and hemopexin was reached, P-selectin expression was abolished as shown in figure 1. In addition to P-Selectin, hemopexin also had a strong effect to reduce the heme-induced expression of IL-8, VCAM-1 and vWF to background levels. Thus, the presented data underlines on the one hand the stimulatory capacity of heme(NaOH) on endothelial cells and demonstrates on the other hand the potential of hemopexin to efficiently neutralize free heme. In a stoichiometric fashion, hemopexin potently prevents the pro-inflammatory effect of heme on endothelial cells. Hence, our study suggests a protective role of hemopexin for endothelial cells exposed to elevated levels of cell-free heme due to intravascular hemolysis. Additional experiments are required to elucidate the effect of hemopexin on the endothelium in more detail. Combined with our other lines of data, our results further support the investigation of hemopexin as a potential therapeutic agent in the treatment of sickle cell disease. Disclosures Adam: CSL Behring AG: Current Employment. Gentinetta:CSL Behring: Current Employment. Diditchenko:CSL Behring AG: Current Employment. Schaub:CSL Behring AG: Current Employment. Kato:CSL Behring AG: Current Employment. Brinkman:CSL Behring: Current Employment. Zuercher:CSL Behring AG: Current Employment.
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Trung, Ly Quoc, Luis J. Espinoza, Dao T. T. An, and Shinji Nakao. "Resveratrol Inhibits Myeloid Leukemia Cell Lines With JAK2V617F Via Both Inactivation Of The JAK/STAT Pathway and Upregulation Of The ERK Pathway." Blood 122, no. 21 (November 15, 2013): 3854. http://dx.doi.org/10.1182/blood.v122.21.3854.3854.
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Abstract Resveratrol, a naturally-occurring polyphenol that has been extensively studied for its anti-inflammatory, cardioprotective and anti-cancer activities, has shown potential anti-tumor activities against malignant NK cells via JAK2/STAT3 pathway inhibition (Trung, et al. PLoS One, 2012). The inhibitory effect of resveratrol on the JAK2/STAT3 pathway suggests that it may be effective for the treatment of myeloid malignancies that are characterized by excessive activation of this signaling pathway, including myeloproliferative neoplasms with the JAK2V617F mutation. To test this hypothesis, we examined the anti-proliferative effects of resveratrol on JAK2V617F mutant myeloid leukemia cell lines, HEL and SET-2. Resveratrol inhibited the proliferation of these JAK2V617F mutant cell lines in a dose-dependent manner in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide) assay, and its inhibitory effects were 3.3-8.4 times greater than those on other myeloid leukemia cell lines without the JAK2V617F mutation, including K562, THP-1 and TF-1. The inhibitory effects on JAK2V617F(+) cell lines by 50 µM of resveratrol were comparable to those induced by 1000 ng/ml of ruxolitinib, a selective JAK1/2 inhibitor. Incubation of different myeloid leukemia cell lines at a resveratrol concentration of 50 µM or at a concentration of 500 ng/ml of ruxolitinib for 24 hours induced apoptosis in 25.1 ± 2.2%/14.4 ± 1.1% of HEL, 23.7 ± 1.5%/19.1 ± 2.6% of SET-2, 6.9 ± 0.7% /0.34 ± 0.41% of K562, 4.9 ± 1.6%/1.9 ± 0.7% of THP-1 and 7.1 ± 1.0%/0.99 ± 0.58% of TF-1 cells (Fig. 1A). Resveratrol inhibited the proliferation of K562 cells transfected with JAK2V617F (K562JAK2V617F) 1.68-times more than it did wild-type K562 cells (K562JAK2WT cells), and induced apoptosis in 12.9 ± 1.6% of K562JAK2V617F and 6.5 ± 0.5% of K562JAK2WT cells. The level of apoptosis induced in K562JAK2V617F cells by resveratrol (6.3±1.5%) was comparable to that (8.2 ± 2.1%) induced by 500 ng/ml of ruxolitinib. Resveratrol inhibited the phosphorylation of JAK1, JAK2 and Tyk2, but did not affect the phosphorylation of JAK3, PTEN or Akt, or their downstream target proteins, including STAT3 and STAT5 (Fig. 1B). In contrast to the inhibition of ERK phosphorylation by ruxolitinib in JAK2V617F cells, resveratrol dramatically enhanced the phosphorylation of ERK (Fig. 1B), which is known to activate caspase-3. The inhibition of the phosphorylation of JAK2/STAT3 and the upregulation of ERK phosphorylation by resveratrol were also observed in K562JAK2V617F cells (Fig. 1B). Resveratrol also induced robust G1 cell cycle arrest of HEL and SET-2 cells, and led to the downregulation of the anti-apoptotic protein, Mcl-1, as well as the upregulation of the pro-apoptotic protein, Bim, both of which play important roles in prolonging the survival of JAK2V617F cells. Moreover, 25 µM of resveratrol augmented the apoptosis in HEL cells induced by 400 ng/ml of ruxolitinib, leading to 2.7 times more apoptosis than ruxolitinib alone in the annexin V assay. These data suggest that resveratrol exerts a potent anti-tumor effect on malignant myeloid cells with the JAK2V617F mutation via both the inactivation of the JAK/STAT pathway and the upregulation of the ERK pathway. Increased ERK phosphorylation has recently been shown to induce apoptosis in several types of cancer cells (Nguyen, et al. Int J Oncol, 2008; Mustafi, et al. PLoS One, 2010). Thus, resveratrol may have therapeutic potential against myeloproliferative neoplasms with the JAK2V617F mutation, and also against other myeloid malignancies where the JAK/STAT pathway plays an essential role in their development. Disclosures: No relevant conflicts of interest to declare.
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van der Meer, Laurens T., Esmé Waanders, Marloes Levers, Hanka Venselaar, Debbie Roeleveld, Joachim Boos, Claudia Lanvers-Kaminsky, et al. "A Germline Mutation in Cathepsin B in a Child with ALL Points towards a Key Role for This Enzyme in L-Asparaginase Pharmacokinetics." Blood 120, no. 21 (November 16, 2012): 2458. http://dx.doi.org/10.1182/blood.v120.21.2458.2458.
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Abstract Abstract 2458 The bacterially derived enzyme L-Asparaginase (ASNase) is a key component in the multidrug therapy regimens used worldwide to treat pediatric and adult patients with acute lymphoblastic leukemia (ALL), however little is known about the molecular mechanisms that control the pharmacokinetics of this therapeutic protein. As a result, many patients who receive a standardized dose either exceed or do not reach the desired serum concentration. While elevated serum levels are associated with an increase in treatment related morbidity, underexposure seriously compromises therapeutic benefits. In search of molecular factors that determine ASNase turnover in vivo, we investigated a patient with strongly aberrant clearance kinetics. This 3-year old female diagnosed with common ALL suffered from severe ASNase-induced adverse events upon treatment with ErwiniaSNase as a result of strongly elevated serum ASNase levels. Pharmacokinetics data showed a severely delayed ASNase clearance. As a result, serum ASNase levels accumulated to intolerable levels upon repeated administration of the drug. We isolated DNA from peripheral blood mononuclear cells and buccal cells of this patient and performed targeted sequencing on genes suggested to be involved in ASNase clearance. We identified a novel heterozygous mutation in the gene encoding Cathepsin B in the germline of this patient. The mutant allele shows a deletion of a single codon, leading to a deletion of a lysine residue in the C terminus of the protein. We generated an EBV LCL cell line from this patients which showed a 75% reduction in Cathepsin B activity, relative to controls, indicating that this heterozygous mutation has a profound effect on the total Cathepsin B activity. Cathepsin B is normally synthesized as a 37 kD pre-pro enzyme and is processed in a two step process into a mature 2-chain active form. During this process, the protein is transported to the lysosome where it exerts its primary function. Using a combination of biochemical and imaging experiments we show that the mutant Cathepsin B cannot be processed into the mature form and is retained in the endoplasmatic reticulum. ASNase degradation assays demonstrate that this mutant form of Cathepsin B shows a diminished protease activity towards both E.coli and Erwinia ASNase, consistent with the reduced clearance observed in our patient. Cathepsin B and other cellular proteases are either actively secreted or released into the serum as a result of cell lysis. Although we find a variable low but detectable activity of Cathepin B in serum samples, all tested preparations of ASNase were stable upon prolonged incubation in serum, suggesting that serum components are not contributing to ASNase clearance in vivo. Hence, we propose that cellular uptake and subsequent proteolytic degradation of ASNase is the primary mechanism of clearance. In conclusion, we have identified a mutation in protease Cathepsin B and provide evidence that this mutation results in a loss of protease function towards ASNase, which can explain the strongly delayed clearance of ASNase in the patient. Our data suggest that differences in Cathepsin B activity may contribute to the large inter-patient variability in ASNase pharmacokinetics. Furthermore, given the role of Cathepsin proteases in antigen presentation, Cathepsin B may not only provide a target for predicting or controlling ASNase clearance kinetics but inhibition of Cathepsin may also prevent or delay the formation of inhibitory antibodies. Disclosures: Boos: European Erwinase Providers (EUSAPharm): Speakers Bureau; Medac: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lanvers-Kaminsky:Medac: Speakers Bureau.
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Garcia, Jacqueline S., Haesook T. Kim, Jennifer Brock, Amy Han, Jeremy A. Ryan, Thelma Mashaka, Anthony G. Letai, et al. "A Phase 1 Dose-Escalation Study of Adding Venetoclax to a Reduced Intensity Conditioning (RIC) Regimen Prior to Allogeneic Hematopoietic Cell Transplantation for Patients with High Risk Myeloid Malignancies." Blood 134, Supplement_1 (November 13, 2019): 258. http://dx.doi.org/10.1182/blood-2019-126172.
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Background: Patients (pts) with myeloid malignancies characterized by high risk mutations or cytogenetics who undergo allogeneic hematopoietic cell transplantation (HCT) have poor outcome primarily due to risk of relapse. Strategies to limit relapse include post-transplant maintenance or novel conditioning regimens. The selective BCL-2 inhibitor venetoclax (VEN), a BH3 mimetic, promotes mitochondrial apoptosis in myeloblasts in the presence of cytotoxic stress and is associated with high response rates when combined with hypomethylating agents or low dose cytarabine in untreated elderly AML. We found that VEN could induce apoptotic priming in myeloblasts regardless of poor risk mutations such as TP53 by BH3 profiling, which measures the mitochondrial response to perturbation by a panel of BH3 domain peptides. We thus hypothesized that VEN would promote the anti-leukemic effect of conditioning chemotherapy and therefore reduce the risk of relapse without undue toxicity. Methods: The primary objective of this phase 1 study is to determine the safety of adding VEN to the RIC regimen of fludarabine and busulfex (FluBu2). This study uses a 3+3 design with dose expansion. Eligible pts included: AML in CR (adverse risk per ELN or secondary AML) or MDS (t-MDS; Int-2 or higher IPSS; TP53 mutation; or RAS pathway gene mutation) or MDS/MPN (including +8; chr 7 abnl; complex karyotype; or ASXL1 mutation) with up to 10% blasts at the time of transplant. Dose-limiting toxicity (DLT) was defined as any treatment-related death, failure to engraft (defined as absolute neutrophil count (ANC) ≥ 500/μL on 2 consecutive measurements), or any gr 4 non-heme toxicity or tumor lysis syndrome from day -8 until day +28. Conditioning chemotherapy consisted of fludarabine 30 mg/m2/d, days -5 to -2 and busulfex 0.8 mg/kg bid, days -5 to -2, followed by PBSC infusion on day 0. VEN is scheduled to maximize overlap with FluBu2. VEN dose levels (DL) are 200 mg on days -8 through -3 (DL1); 200 mg on days -8 through -2 (DL2); and 400 mg on days -8 through -2 (DL3). Flow cytometry-based BH3 profiling was performed on pre-VEN treatment bone marrow in pts with measurable residual myeloblasts. NGS using a 95-gene TruSeq panel was performed on pre-VEN treatment and day +100 marrow samples. Results: Nine pts (67% male, median age of 65 y (range 41-71)) have been treated including 3 AML (1 with -17 and 2 with mutations in TP53, RUNX1, and/or ASXL1), 5 MDS (all mutant TP53), and 1 CMML (with mutant ASXL1) in DL1-DL3. 8 donors were matched unrelated and 1 was a matched related. Donors were 44% male. No DLTs were observed at DL1-DL3; dose expansion at DL3 is ongoing. VEN-related toxicities included gr 1 diarrhea (n=3), gr 1 fatigue (n=1), and gr 1 nausea (n=2). Median time to ANC ≥ 500/μL was 16 days (range 13, 25; 1 pt did not nadir) and median donor-derived myeloid chimerism was 100% (range 97, 100) at day +28. Median time to platelet ≥ 20K/μL was 14 days (range 13, 18; 3 did not nadir). 3 developed gr 1 acute graft-versus host disease (GVHD) involving skin and gastric sites. In the 4 followed beyond day +100, 3 developed chronic GVHD (2 moderate, 1 severe) and 1 died from complications of acute GVHD of the gut (stage 3) on day +183. With a median follow-up time of 5 months (range 1.3, 7.8), 2 of 9 pts had disease progression and 1 died. Five out of 6 pts with available paired day +100 samples had no detectable mutation by NGS with >200X mean coverage at the later time point (Fig A). Pt 3 had persistent mutations in TP53 at day +100. Myeloblasts that are more primed require lower doses of [BIM] or [PUMA] peptide to induce cytochrome c release. BH3 profiling of residual myeloblasts on pre-VEN treatment marrow using pro-apoptotic BIM and PUMA peptides (Fig B-C) showed differential individual baseline priming status, which we will correlate with response. Conclusions: VEN at doses up to 400 mg can be safely added to RIC conditioning with FluBu2 resulting in a high molecular negativity rate at day +100 without impairing neutrophil engraftment. Additional correlative studies include targeted deep sequencing to better evaluate measurable residual disease for individual genes and BH3 profiling analysis of myeloblasts collected pre-VEN and post-VEN/pre-FluBu2 to evaluate for a VEN-induced increase in apoptotic priming. To further minimize disease relapse in these high risk pts, we amended the trial to include maintenance therapy with a combination of VEN and azacitidine. Figure Disclosures Garcia: Abbvie: Research Funding; Genentech: Research Funding. Ryan:Vivid Biosciences: Consultancy. Letai:Zeno Pharmaceuticals, Vivid Bioscience, Flash Therapeutics, Dialectic Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder or Advisory Board member; AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding. Lindsley:Takeda Pharmaceuticals: Consultancy; Medlmmune: Research Funding; Jazz Pharmaceuticals: Research Funding. Ho:Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy; Omeros Corporation: Membership on an entity's Board of Directors or advisory committees. Koreth:Cugene: Consultancy; Amgen: Consultancy; Equillium: Consultancy. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Galinsky:ABIM: Other: Member on specialty oncology board; AbbVie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Merus Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Steensma:Summer Road: Consultancy; Arrowhead: Equity Ownership; H3 Biosciences: Other: Research funding to institution, not investigator.; Onconova: Consultancy; Astex: Consultancy; Pfizer: Consultancy; Stemline: Consultancy; Aprea: Research Funding. Stone:AbbVie, Actinium, Agios, Argenx, Arog, Astellas, AstraZeneca, Biolinerx, Celgene, Cornerstone Biopharma, Fujifilm, Jazz Pharmaceuticals, Amgen, Ono, Orsenix, Otsuka, Merck, Novartis, Pfizer, Sumitomo, Trovagene: Consultancy; Argenx, Celgene, Takeda Oncology: Other: Data and Safety Monitoring Board/Committee: ; Novartis, Agios, Arog: Research Funding. Soiffer:Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Kiadis: Other: supervisory board; Mana therapeutic: Consultancy; Cugene: Consultancy; Jazz: Consultancy. Cutler:BiolineRx: Other: DSMB; Cellect: Other: DSMB; Jazz: Consultancy; BMS: Consultancy; Genentech: Consultancy; ElsaLys: Consultancy; Kalytera: Other: DSMB; Pharmacyclics: Consultancy; Fate Therapeutics: Consultancy; Incyte: Consultancy; Kadmon: Consultancy; Omeros: Consultancy. DeAngelo:Amgen, Autolus, Celgene, Forty-seven, Incyte, Jazzs, Pfizer, Shire, Takeda: Consultancy; Novartis: Consultancy, Research Funding; Abbvie: Research Funding; Glycomimetics: Research Funding; Blueprint: Consultancy, Research Funding. OffLabel Disclosure: Venetoclax is a BH3 mimetic and selective BCL-2 inhibitor that was added to standard conditioning chemotherapy (fludarabine and busulfan) on a clinical trial.