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1
Kocabas, F., S. N. Eren, M. Uslu, and M. Y. Yuksel. "P69Identification of cardiogenic and hematopoietic MEIS Inhibitors." Cardiovascular Research 114, suppl_1 (April 1, 2018): S18—S19. http://dx.doi.org/10.1093/cvr/cvy060.033.
2
GİRGİN, Birkan, Medine KARADAĞ-ALPASLAN, and Fatih KOCABAŞ. "Oncogenic and tumor suppressor function of MEIS and associated factors." TURKISH JOURNAL OF BIOLOGY 44, no. 6 (December 14, 2020): 328–55. http://dx.doi.org/10.3906/biy-2006-25.
Анотація:
MEIS proteins are historically associated with tumorigenesis, metastasis, and invasion in cancer. MEIS and associated PBX-HOX proteins may act as tumor suppressors or oncogenes in different cellular settings. Their expressions tend to be misregulated in various cancers. Bioinformatic analyses have suggested their upregulation in leukemia/lymphoma, thymoma, pancreas, glioma, and glioblastoma, and downregulation in cervical, uterine, rectum, and colon cancers. However, every cancer type includes, at least, a subtype with high MEIS expression. In addition, studies have highlighted that MEIS proteins and associated factors may function as diagnostic or therapeutic biomarkers for various diseases. Herein, MEIS proteins and associated factors in tumorigenesis are discussed with recent discoveries in addition to how they could be modulated by noncoding RNAs or newly developed small-molecule MEIS inhibitors.
3
Barbosa, Karina, Anagha Deshpande, Ping Xiang, Bo-Rui Chen, Adam Brown, Neil Robertson, Younguk Sun, et al. "High-Density Domain-Focused CRISPR Screens Reveal Epigenetic Regulators of Hox/Meis Gene Expression in Acute Myeloid Leukemia." Blood 136, Supplement 1 (November 5, 2020): 2–3. http://dx.doi.org/10.1182/blood-2020-141412.
Анотація:
The aberrant and constitutive activation of the HOXA cluster genes and the their-co-factor MEIS1 (HOX/MEIS) is a recurrent feature in several types of myeloid and lymphoid leukemias. Aberrant HOX/MEIS expression has been shown to drive limitless leukemia stem cell self-renewal and is therefore an attractive target for therapy in acute myeloid leukemia (AML). However, since HOX/MEIS genes encode DNA-binding transcription factors, small molecules targeting these proteins directly are lacking. Furthermore, targeting the HOX/MEIS network is complicated by the fact that these genes are coordinately regulated and have redundant functions in sustaining leukemic self-renewal. One way of therapeutically targeting aberrant HOX/MEIS transcription is the identification and pharmacologic inhibition of upstream chromatin regulators that coordinately modulate their expression. In order to identify such chromatin regulators, we made use of an endogenous GFP reporter knocked-in to the MEIS1 locus in the high HOX/MEIS-expressing U937 human AML cell line. Using this system, we first performed a high-throughput flow-cytometry-based small-molecule inhibitor screen with a library of 261 compounds targeting epigenetic regulators. In our screen, the most potent hits that reproducibly showed >50% MEIS1-GFP inhibition were small molecules that targeted DOT1L, the histone methyltransferase. DOT1L inhibitors have already been well-characterized as HOX/MEIS regulators and most epigenetic regulators are not targeted by existing compound libraries. Therefore, we decided to use a genetic screening approach to more extensively interrogate the landscape of epigenetic regulators of HOX/MEIS expression in AML. For this, we designed a custom computational pipeline and built a CRISPR library of 10,000 sgRNAs targeting functionally conserved protein domains of all catalogued chromatin modulatory proteins (> 600 proteins - 5 sgRNAs per conserved domain). This list of epigenetic regulators included histone modifying enzymes, chromatin readers, nucleosome remodelers, adaptor proteins and proteins involved in DNA and RNA modifications, as well as other transcriptional regulators. Using this comprehensive, domain-focused CRISPR library, we conducted a phenotypic enrichment screen. Specifically, we used flow cytometry to purify the top 20% GFP-MEIS1 (high) and bottom 20% GFP-MEIS1 (low) expressing cells and identified sgRNAs that were enriched particularly in the GFP-MEIS1 -low vs -high fraction using next generation sequencing. Given the extent and complexity of the CRISPR library, our approach uncovered members of six distinct chromatin modifying complexes as MEIS1 regulators (MAGeCKFlute pipeline, 2 SD > mean) and we could validate > 10 of these hits as bonafide regulators of MEIS1 as well as HOXA genes. We also demonstrated their essentiality for the proliferation of HOX-driven AML cells using arrayed sgRNA competition assays. These validated hits included several known as well as novel chromatin readers and writers amenable to small-molecule targeting. We focused our attention on the KAT7/JADE3 complex and the casein kinase 2 (CK2) family that we validated as potent and selective regulators of HOX/MEIS expression in AML cells. Our studies demonstrated that genetic depletion of components of the KAT7 complex or of the CK2 family could reverse HOX/MEIS activation in human AML cells, leading to a progressive loss of proliferative potential. Importantly, the use of the clinical-grade CK2 inhibitor CX4945 (Silmitasertib) caused a concentration-dependent down-regulation of HOX/MEIS expression in models of HOX-driven AML, leading to significant anti-leukemia effects. Our study provides a framework for the multiplexed identification of actionable dependencies targeting therapeutically recalcitrant oncogenic networks in cancer. Specifically for AML, since Silmitasertib is in Phase 2 trials for treatment of other cancers, our studies may solve the long-standing problem of targeting leukemia stem cells in AML potentially overcoming therapy refractoriness in this devastating disease. Disclosures No relevant conflicts of interest to declare.
4
Bisaillon, Richard, Eva Schmidt, Anne-Sophie Guenier, and Guy Sauvageau. "Identification of MEIS-PBX inhibitors as potential anti-leukemic agents using a high-throughput bret-based assay." Experimental Hematology 41, no. 8 (August 2013): S51. http://dx.doi.org/10.1016/j.exphem.2013.05.201.
5
Castellet, Helena, Guillermo Ramil López, Ana Garrido, Alícia Artigas-Baleri, Marta Pratcorona, Olga Salamero, Albert Cortés-Bullich, et al. "Ubtf tandem Duplications Define a Novel Subtype of Acute Myeloid Leukemia Associated with Younger Age, WT1 Mutations and HOXA9 Marked Overexpression." Blood 142, Supplement 1 (November 28, 2023): 6045. http://dx.doi.org/10.1182/blood-2023-186064.
Анотація:
Background Upstream Binding Transcription Factor ( UBTF) gene plays an important role in ribosomal RNA transcription. UBTF tandem duplications (TD) have been recently described as a recurrent genetic lesion in acute myeloid leukemia (AML). UBTF-TDs appear to be mutually exclusive with other class-defining lesions in AML and display a unique comutational signature. UBTF-mutated AML affects predominantly younger patients and is associated with trisomy 8, internal tandem duplication of FLT3 ( FLT3-ITD), mutations in WT1 and poor prognosis. Moreover, HOX and MEIS genes seem to be overexpressed in AML with UBTF-TD similarly to NPM1-mutated ( NPM1m) or KMT2A-rearragend ( KMT2Ar) AML, suggesting potential sensitivity to menin inhibitors. In this study we sought to characterize UBTF-TDs in our AML cohort. Methods We studied 456 adult patients (median age 54 years; range 18-70) diagnosed with de novo AML and included in AML-12 protocol from CETLAM group. All patients from the 2012-2017 period (n=403) were consecutively studied and we also selected 53 cases of WT1-mutated AML from the 2017-2020 period, given the strong association with UBTF-TD. Screening for UBTF-TD was performed on bone marrow (BM) or peripheral blood (PB) genomic DNA samples. Exon 13 of UBTF gene was amplified and analysed by Polymerase chain reaction (PCR) and subsequent fragment length analysis by capillary electrophoresis. Mutated samples were confirmed through Sanger sequencing. Further, RNA from 9 UBTF-TD cases was available and HOXA9 and MEIS1 gene expression analysis was performed by Real-Time Polymerase Chain Reaction (RT-qPCR). Clinical and biological data were obtained from CETLAM registry. Results UBTF-TD was found in 13 patients from our cohort whose characteristics are detailed in Table 1. UBTF-TDs range from 45 base pairs (bp) to 365 bp, being the 48 bp the most recurring size. As described, although small UBTF-TDs are in frame insertions, the largest ones are not multiple of 3. Notably, most UBTF-TDs presented small insertions and deletions among the duplication. Presence of UBTF-TD was associated with younger age (36 vs 55 years; p<0.001) and BM dysplastic changes (80% of U BTFMUT). UBTF-TD was particularly frequent (8.9% of all cases) in patients below 40 years, compared to other lesions as CEBPA mutations (1.1%). Eleven (85%) U BTFMUT patients presented intermediate cytogenetics, being trisomy 8 (5), normal karyotype (3) and 9q deletion (2) the most recurrent findings. Two cases of adverse karyotype with MECOM-rearrangements were found. As previously described, there was a strong association between UBTF-TD and of FLT3-ITD (38.5%). Other recurrent genetic lesions in AML like NPM1 and CEBPA mutations or core-binding factor (CBF) rearrangements were exclusive of UBTF wild type patients. Further, we found that UBTFMUT leukemias showed marked overexpression of HOXA9 compared with other AML subtypes (22.1-fold vs 5.0-fold; p<0.001; Fig. 1) and similar to NPM1m AML (22.3-fold). On the other hand, MEIS expression did not differ significantly among groups. Conclusions In our cohort of adult AML patients, UBTF-TDs are found in 1.8% of cases and are especially frequent in younger individuals in absence of other class-defining genetic lesions. The presence of UBTF-TD is strongly associated with cytomorphological dysplasia and co-occurrence of FLT3-ITD and WT1 mutations, while NPM1 and CEBPA mutations and CBF rearrangements appear to be mutually exclusive. HOXA9 upregulation could be a leukemogenic event in UBTF-TD AML, as described in NPM1-m and KMT2A-r AML, suggesting potential utility of menin inhibitors in this subset of patients. Screening for UBTF-TD is going to be incorporated in our molecular diagnostic panel for new AML cases.
6
Breitinger, Constanze, Emanuel Maethner, Maria-Paz Garcia-Cuellar, Alexandra Schambony, Kirsten Schilling, Klaus D. Fischer, and Robert K. Slany. "Hox Genes Regulate Rac1 Activity Through Control of Vav2 expression." Blood 118, no. 21 (November 18, 2011): 60. http://dx.doi.org/10.1182/blood.v118.21.60.60.
Анотація:
Abstract Abstract 60 Next to their function in embryogenesis the clustered Hox-homeobox genes are also master regulators of hematopoietic differentiation and development. Perturbed HOX expression can be found in a significant percentage of acute leukemia. Recently we have shown that several members of the HOXA cluster transform primary hematopoietic cells and induce leukemia in vivo [1]. In an attempt to define Hox downstream targets important for leukemogenesis we determined the gene expression pattern in hematopoietic precursor cells transformed by conditional HOXA1 and HOXA9 derivatives. Interestingly, amongst the several hundred genes controlled by each HOX protein individually we could identify a significant overlap of genes that responded to both. This group included c-Myb that has been shown before to be important for Hox-mediated leukemogenesis indicating that this approach is able to identify a candidate set of genes important for transformation by HOX proteins. Within this collection the gene for the guanine exchange factor Vav2 caught our interest because it had been originally identified in a screen for bona fide oncogenes. Moreover it is known that Vav2 controls activity of the small Rho-type GTPase Rac1 that in turn is an important mediator of (leukemic) stem cell mobility and engraftment. ChIP experiments confirmed HOXA9 binding in the putative vav2 promoter region. In addition a luciferase reporter placed under control of the vav2 promoter showed Hox-responsive behavior indicating that vav2 is a direct Hox-target. Concomitant with vav2-levels also Rac1 activity was under strict control of Hox factors. This was true also for HOX+Meis double transformed cells. To investigate the importance of vav2 for Hox-mediated leukemogenesis hematopoietic cells from vav2 knock-out mice were investigated in detail. vav2−/−cells could be transformed by HOXA9 (+Meis) in vitro with identical efficiency as their wild-type counterparts. Colony formation in replating assays, the capability to induce outgrowth of permanent myeloid precursor lines and the differentiation state of these lines did not differ significantly amongst the wt and vav2−/− samples. However, no active Rac1 could be detected in vav2−/− cell lines transformed by HOXA9. Importantly, retroviral re-expression of vav2 could rescue part of this defect. Obviously, Vav2 constitutes the major GEF controlling Rac1 activity in myeloid precursors despite the presence of the homologous proteins Vav1 and Vav3. Because Rac1 has been implicated in surface receptor recycling and in stem-cell homing we checked the level of the SDF1 “homing-cytokine” receptor CxCR4 (CD184) on Hox-transformed wt and vav2−/−cells and could confirm a complete absence of CD184 on the latter. Again reintroduction of vav2 rescued part of this phenotype. Transplantation experiments are underway and preliminary results indicate that loss of vav2 is accompanied by an impaired ability for extramedullary hemopoiesis, an import determinant of leukemia aggressiveness. Our results explain how Hox genes that are highly expressed in stem- and precursor cells can regulate the important process of stem cell homing. Rac1 inhibitors are known and these substances have been tested [2] in mice with special success on MLL fusion induced leukemias that are a paradigma for Hox-induced leukemogenesis. Therefore our results do not only provide the molecular framework for this observation but they are also able to identify a potential patient collective that may benefit from pharmacological Rac1 inhibition. Disclosures: No relevant conflicts of interest to declare.
7
Jarocha, Danuta Jadwiga, Paul Gadue, Wei Tong, Robert C. Newton, and Mortimer Poncz. "Janus Kinase (Jak) 1 Inhibition Affects Both Megakaryopoiesis and Thrombopoiesis." Blood 132, Supplement 1 (November 29, 2018): 2559. http://dx.doi.org/10.1182/blood-2018-99-115407.
Анотація:
Abstract JAK inhibitors are being developed to treat inflammatory, myeloproliferative and neoplastic disorders. Murine and human studies have demonstrated an essential role for JAK2 in the proliferation of hematopoietic stem/progenitor cells (HSPC) and multiple hematopoietic lineages, including erythrocytes and megakaryocytes, while Jak1 murine studies have shown a role in HSPC proliferation and myelopoiesis, but not in megakaryopoiesis. Patients enrolled in clinical studies of INCB052793, which selectively binds to JAK1, have shown thrombocytopenia occurring within 2 weeks. The aim of this study was to elucidate the basis for thrombocytopenia associated with this JAK1 inhibitor, in comparison to INCB026115, which inhibits JAK2 more so than Jak1 (Jak2/1). Knowing the precise mechanism by which Jak inhibitors induce thrombocytopenia may lead to therapeutic strategies limiting side effects, while preserving intended clinical application. We tested a broad concentration range of each of these Jak1 and Jak2/1 inhibitors from IC50 (40 and 30nM, respectively) to IC90 (400 and 300nM) to 10xIC90 (4 and 3 µM) on mobilized progenitor-derived CD34+ cells incubated 12-14 days under semisolid and under liquid conditions, focusing on effects on megakaryocyte (Meg) and platelet production. At IC90, the Jak1-selective inhibitor limited large Meg colony number to 47±8% of untreated control in semisolid growth conditions. Under similar concentrations in liquid growth conditions, the number of Megs seen was 45±8% of the untreated controls, but with a 139±17% higher level of ≥8N Megs. Agonist response of mature Megs to thrombin was not compromised. Total number of healthy, in vitro-released, platelet-like particles (PLPs) collected from Jak1-exposed cultures at Day 12 was reduced to 57±14% of the control, and similar to the decrease in Meg yield. At a similar level of inhibition, the Jak2/1 inhibitor was more robust at inhibiting megakaryopoiesis. At IC90, the Jak2/1 inhibitor fully inhibited development of large Meg colonies and reduced the number of small colonies to 43±14% of untreated control. Under liquid growth conditions, the number of Megs seen at Day 12 was 20±9% of the untreated controls, but with 132±28% higher % of ≥8N Megs. Agonist response of mature Megs was not compromised. Total number of healthy PLPs collected at Day 12 was insignificantly different despite much lower Meg yield. More detailed Jak2/1 inhibitor cultures analysis revealed enhanced Meg apoptosis by 209±61% at Day 7, and accelerated maturation as indicated by a 2-fold and 3-fold mpl receptor level at Days 7 and 11 and 321±217% higher number of Megs >2N at Day 7. As opposite to what might be expected, thrombopoiesis appeared not to be impaired by the Jak2/1 inhibitor. Inhibitor-treated Megs released similar or higher number of platelets per Meg as untreated controls upon their infusion into immunocompromized NSG mice, with similar high levels of young, thiazole orange-positive, low apoptotic, Annexin-V+ platelets. Baseline released platelet CD62p expression and PAC1 binding prior to agonist exposure were similar and increased to the same extent after thrombin (0.1-10U/ml) stimulation. In contrast, Jak1 inhibitor-treated Megs had ~50% lower number of released human platelets upon infusion into NSG mice although the released platelets were healthy and responsive to agonists. In summary, our results shed significant insight into the mechanisms of Jak1 inhibitor-associated thrombocytopenia observed in patients. We show that thrombocytopenia post the Jak2/1 inhibitor INCB026115 is due to impaired megakaryopoiesis with intact thrombopoiesis and functional, released platelets. In contrast, thrombocytopenia post the Jak1 inhibitor INCB052793 is a result of combined impairment of both megakaryopoiesis and thrombopoiesis, although the released platelets appear intact. The exact pathways blocked by the Jak1 inhibitor important for thrombopoiesis remain to be defined. Also, as liver hepatocytes together with bone marrow stromal cells are a source of thrombopoietin (TPO), and Jak1 and Jak2 are known to be involved in regulation of TPO production, studies to check the influence of Jak inhibitors on TPO production from both hepatocytes and marrow stromal cells are needed to fully understand the influence of Jak inhibitors on megakaryopoiesis/thrombopoiesis. Disclosures Jarocha: Incyte Corporation: Consultancy, Research Funding. Gadue:Incyte Corporation: Consultancy, Research Funding. Tong:Incyte Corporation: Consultancy, Research Funding. Newton:Incyte Research Institute: Employment, Equity Ownership. Poncz:Incyte Corporation: Consultancy, Research Funding.
8
Haneline, Laura S., Khadijeh R. Bijangi-Vishehsaraei, Adam Werne, Kristina Wilson McKenzie, Susanna Davis, Hidenori Ichijo та Mohammad R. Saadatzadeh. "TNF-α Hypersensitivity of Fanconi Anemia Type C Deficient Cells Is Dependent on the Apoptosis Signal-Regulating Kinase 1 Signaling Pathway." Blood 104, № 11 (16 листопада 2004): 724. http://dx.doi.org/10.1182/blood.v104.11.724.724.
Анотація:
Abstract Fanconi anemia (FA) is a chromosomal instability disorder characterized by a progressive bone marrow (BM) aplasia. Previous studies from our lab and others suggest that enhanced oxidant- and inhibitory cytokine-mediated apoptosis of hematopoietic stem/progenitor cells are important mechanisms in the pathogenesis of BM failure in FA. Recently, we showed that oxidant-induced apoptosis in Fancc −/− murine embryonic fibroblasts (MEFs) was mediated through hyperactivation of the redox-dependent protein, apoptosis signal-regulating kinase 1 (ASK1, Saadatzadeh et al, JBC 2004). Here, we tested whether alterations in ASK1 signaling also participate in the pro-apoptotic phenotype of primary Fancc −/− MEFs and progenitors in response to the inhibitory cytokine, TNF-α. To determine whether Fancc −/− MEFs exhibit altered ASK1 signaling after TNF-α treatment, we examined ASK1 activity using in vitro kinase assays. These studies demonstrated a 2–3 fold increase in TNF-α-induced ASK1 activity in Fancc −/− MEFs compared to WT (n=5). Consistent with ASK1 hyperactivation, we observed an increase in activation of the downstream stress kinases, c-jun N-terminal kinase and p38, in Fancc −/− MEFs compared to WT (n=5). Utilizing a dominant negative ASK1 cDNA to decrease ASK1 activity, we determined that TNF-α-induced apoptosis in Fancc −/− MEFs was ASK1 dependent (n=3, p<0.05). In addition, TNF-α-induced apoptosis in Fancc −/− MEFs was completely blocked by both antioxidants (selenomethionine and N-acetylcysteine, n=3, p<0.001) and the p38 inhibitor SB 203580 (n=6, p<0.001). To examine whether ASK1 has a critical role in the hypersensitivity of Fancc −/− progenitors to TNF-α-induced apoptosis, we crossed ASK1 knockout mice with Fancc mice to generate the following four experimental genotypes; WT, Fancc +/+;ASK1 −/−, Fancc −/−;ASK1 +/+, and Fancc −/−;ASK1 −/−. BM cellularity and progenitors/femur were similar between all genotypes (n=3). Interestingly, TNF-α hypersensitivity of Fancc −/− progenitors was restored to WT levels when ASK1 was deleted (n=3, p<0.05), suggesting that TNF-α-induced hypersensitivity of Fancc −/− progenitors is mediated through ASK1, similar to Fancc −/− MEFs. In addition, TNF-α-induced apoptosis in Fancc −/− progenitors was restored to WT levels after culturing with the p38 inhibitor SB 203580 (n=6, p<0.005). Since ASK1 activity is directly regulated by the cellular redox state, these data support the idea that aberrant redox regulation may be involved in the observed pro-apoptotic phenotype of Fancc −/− cells after exposure to inhibitory cytokines such as TNF-α. Collectively, these data suggest that inhibiting the ASK1 apoptotic pathway in Fancc −/− cells via antioxidants or small molecule inhibitors may protect Fancc −/− hematopoeitic stem/progenitors from an apoptotic fate and delay BM failure.
9
Wang, Wei, Peng Li, Annette Schettino, Zhe Peng, and Maureen McLeod. "Characterization of Functional Regions in the Schizosaccharomyces pombe mei3 Developmental Activator." Genetics 150, no. 3 (November 1, 1998): 1007–18. http://dx.doi.org/10.1093/genetics/150.3.1007.
Анотація:
Abstract The Schizosaccharomyces pombe mei3+ gene is expressed only in diploid cells undergoing meiosis. Ectopic expression of mei3+ in haploid cells causes meiotic catastrophe. Mei3 is an inhibitor of Ran1/Pat1 kinase and contains a nine-amino-acid motif, Mei3-RKDIII, that resembles two regions in the Ste11 substrate for Ran1/Pat1. Substitution of serine for Arg-81 within Mei3-RKDIII transforms the inhibitor into a substrate for Ran1/Pat1. Thus, it is likely that Mei3-RKDIII defines a pseudosubstrate sequence. In this study, we constructed a series of mei3 deletion mutations and assayed each for activity. This analysis indicates that the carboxy-terminal domain of Mei3 is sufficient for function in vivo. Alanine-scanning mutagenesis identifies critical residues within the inhibitory domain. Two mutations, SM1 and SM8, fail to cause meiotic catastrophe. The SM1 mutation contains alterations of amino acid residues in Mei3-RKDIII. Recombinant SM1 protein exhibits reduced ability to inhibit Ran1/Pat1 kinase in vitro and interacts inefficiently with the kinase in a two-hybrid assay. The SM8 protein binds to Ran1/Pat1 in a two-hybrid assay but fails to inhibit Ran1/Pat1 substrate phosphorylation in vitro. These findings provide evidence that Mei3-RKDIII defines a Ran1/Pat1-binding site that is necessary but not sufficient for inhibition of the kinase. Using fusions to green fluorescent protein, the cellular localization of Ran1 and Mei3 was examined in living cells. Ran1 is concentrated in the nucleus. Mei3 is also enriched in the nucleus and, consistent with the genetic and biochemical results, the inhibitory domain of Mei3 is sufficient for nuclear localization.
10
KAYA, Mustafa Oğuzhan. "Out-of-mind Inhibitors Of Human Serum Paraoxonase 1 (PON1): An In Vitro study." Middle East Journal of Science 3, no. 1 (August 26, 2017): 59–77. http://dx.doi.org/10.23884/mejs.2017.3.1.07.
11
Agarwal, Archana, Gregory Russell Pond, Catherine Curran, Amin Nassar, Pier Vitale Nuzzo, Vivek Kumar, Bradley Alexander McGregor, et al. "Impact of concurrent medications on outcomes with PD1/PD-L1 inhibitors for metastatic urothelial carcinoma." Journal of Clinical Oncology 37, no. 7_suppl (March 1, 2019): 435. http://dx.doi.org/10.1200/jco.2019.37.7_suppl.435.
Анотація:
435 Background: The impact of concurrent medications (meds) on outcomes with PD1/PD-L1 inhibitors in metastatic urothelial carcinoma (mUC) is unclear. We investigated whether candidate concurrent meds (NSAIDS [N], metformin [M], antibiotics [A], statins [S] and corticosteroids [C]) have an association with outcomes in mUC patients (pts) receiving a PD1/PD-L1 inhibitor. We hypothesized that A and C compromise outcomes, while N, M and S improve outcomes. Methods: Data from mUC pts who received PD1/PD-L1 inhibitors at the Dana-Farber Cancer Institute (DFCI) was obtained. The concurrent medication was required to be administered within 1 month before starting to anytime during PD1/PD-L1 inhibitor therapy. A Cox regression analysis was done to study the association of variables with response and survival. Results: Data was available for 101 pts with mUC who received atezolizumab [n = 52], pembrolizumab [n = 39], nivolumab [n = 9] and durvalumab [n = 1]. Prior platinum had been administered in 74 pts (73.2%), 25 were chemonaive (24.8%) and prior therapy status was unknown in 2 pts (2%). The concurrent meds were N (n = 30), M (n = 7), A (n = 26), S (n = 33) and C (n = 12). The median survival was 57.9 weeks. Response was seen in 26 pts [25.7%]. A was associated with a lower probability of response (11.5%) than those not on A (30.7%), and worse survival (HR = 1.93, 95% CI 1.93 – 3.42, P = 0.024). Pts who received neither A nor C, one of them or both had a response rate (RR) of 30.6%, 20% and 0%, and median survival of 65.3, 53.1 and 14.9 weeks, respectively (HR = 3.02, 95% CI = 1.34-6.83, p = 0.027). Pts who did not receive N, M and S (n = 52) exhibited a median OS of 39.6 weeks, while those who received ≥1 of these meds (n = 49) exhibited a median survival of 160.3 weeks (p = NS). The study is limited by the retrospective design and modest sample size. Conclusions: In this hypothesis-generating study, concurrent antibiotics or corticosteroids compromised outcomes in mUC pts receiving a PD1/PD-L1 inhibitor and receiving both further compromised outcomes. The numerically higher survival with concurrent N, M or S did not attain statistical significance, but requires further study in larger datasets.
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Altman, Jessica K., Heather Glaser, Amanda Redig, Antonella Sassano, Martin S. Tallman, and Leonidas C. Platanias. "Targeting the Mnk Pathway Enhances Chemotherapy-Induced Antileukemic Responses." Blood 112, no. 11 (November 16, 2008): 1617. http://dx.doi.org/10.1182/blood.v112.11.1617.1617.
Анотація:
Abstract Mnk kinases (mitogen-activated protein kinase [MAPK]-interacting kinases) are downstream effectors of Map kinase pathways, including the MEK/Erk and the p38 Map kinase signaling cascades. We have previously shown that Mnk kinases and the p38 Map kinase pathway are activated in a negative feedback regulatory manner during treatment of cells with arsenic trioxide, and that molecular or pharmacological inhibition of their activation enhances arsenic trioxide-dependent apoptosis and antileukemic responses (J Biol Chem. 283:12034–42, 2008, and Cancer Res. 66:6763–71, 2006). We examined the activation status of Mnk kinases in response to treatment of AML cells with chemotherapy and the function of these kinases in the generation of antileukemic responses. The human AML cell lines U937, K562, or MM6 were treated with cytarabine, in the presence or absence of a commercially available pharmacological Mnk-1 inhibitor (Calbiochem); and the phosphorylation of Mnk and its downstream effector, eIF4E, were assessed. Treatment with cytarabine increased phosphorylation of Mnk and eIF4E. The cytarabine-dependent eIF4E phosphorylation was blocked when human leukemia cell lines were treated with the pharmacologic Mnk inhibitor, indicating that Mnk regulates eIF4E activity. Such phosphorylation was also found to be defective in Mnk1/Mnk2 double knockout mouse embryonic fibroblast (MEF) cells, as compared to wild-type MEFs. Importantly, cytarabine-induced apoptosis was strongly enhanced in Mnk1−/− Mnk2−/− MEFs, as compared to Mnk1+/+Mnk2+/+ MEFs. To define the role of Mnk kinases in the generation of chemotherapy-induced antileukemic responses, human leukemia cell lines and bone marrow or peripheral blood mononuclear cells from patients with AML were used in clonogenic assays in methylcelluose to determine the effects of Mnk inhibition in the cytarabine-mediated leukemic progenitor (CFU-L) growth. The Mnk inhibitor potentiated the inhibitory effects of cytarabine on U937-derived CFU-L colonies and bone marrow or peripheral blood-derived CFU-L from 3 patients with AML. Interestingly, combinations of the Mnk inhibitor with the mTOR inhibitor, rapamycin, also resulted in more pronounced inhibitory effects on CFU-L colony formation than each agent alone. Altogether, these findings demonstrate that the Mnk pathway is activated during treatment of AML cells with cytarabine and that such activation occurs in a negative feedback regulatory manner to counteract the antileukemic effects of cytarabine. They also raise the possibility that targeting Mnk kinases may provide a novel approach to enhance the effects of chemotherapy on AML cells in vitro and possibly in vivo.
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Waters, Andrew Michael, Wen-Hsuan Chang, Clint Stalnecker, Cole Edwards, Runying Yang, Craig M. Goodwin, Adrienne D. Cox, and Channing J. Der. "Abstract 1075: Identification of resistance mechanisms to direct KRAS inhibition in pancreatic cancer." Cancer Research 83, no. 7_Supplement (April 4, 2023): 1075. http://dx.doi.org/10.1158/1538-7445.am2023-1075.
Анотація:
Abstract KRAS mutations occur in 95% of pancreatic ductal adenocarcinomas (PDAC) and are a well-validated driver of PDAC growth. Therefore, anti-KRAS therapies are expected to make a significant impact on the treatment of this deadly cancer, where there are currently no effective targeted therapies. Supporting this premise, early clinical trial results with KRASG12C inhibitors have shown promising disease control rates (84-100%) in KRASG12C-mutant PDAC. Despite these observations, two key issues limit the impact of KRASG12C inhibitors in PDAC. First, KRASG12C(OFF) mutations comprise less than 2% of KRAS mutations in PDAC. Second, patients initially responsive to KRASG12C inhibitors invariably relapse due to treatment-induced resistance. To begin qualifying KRAS inhibitors that target KRAS mutations more frequently found in PDAC, we characterized a RAS inhibitor that targets the multiple KRAS mutations as well as wild-type RAS proteins. We evaluated the impact of this inhibitor on RAS signaling and anti-proliferative activity in KRAS-mutant pancreatic cancer human cell lines and in a panel of RASless MEFs (ras-null mouse embryo fibroblasts) stably expressing exogenous RAS mutant proteins that are commonly found in PDAC. To identify genetic mechanisms of resistance to KRAS inhibition in pancreatic cancer, we applied CRISPR-Cas9 loss-of-function screens to KRASG12C-, KRASG12D-, KRASG12R-, and KRASQ61H-mutant PDAC cells treated with KRAS inhibitors to identify genes that modulate KRAS inhibitor anti-proliferative activity. We identified expected and novel mechanisms of resistance, including those that have been observed in patients treated with KRASG12C inhibitors. Citation Format: Andrew Michael Waters, Wen-Hsuan Chang, Clint Stalnecker, Cole Edwards, Runying Yang, Craig M. Goodwin, Adrienne D. Cox, Channing J. Der. Identification of resistance mechanisms to direct KRAS inhibition in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1075.
14
Mattos, Daphne R., Xuemei Wan, Jeffrey D. Serrill, Minh H. Nguyen, Ian R. Humphreys, Benoit Viollet, Amos B. Smith, Kerry L. McPhail, and Jane E. Ishmael. "The Marine-Derived Macrolactone Mandelalide A Is an Indirect Activator of AMPK." Marine Drugs 20, no. 7 (June 27, 2022): 418. http://dx.doi.org/10.3390/md20070418.
Анотація:
The mandelalides are complex macrolactone natural products with distinct macrocycle motifs and a bioactivity profile that is heavily influenced by compound glycosylation. Mandelalides A and B are direct inhibitors of mitochondrial ATP synthase (complex V) and therefore more toxic to mammalian cells with an oxidative metabolic phenotype. To provide further insight into the pharmacology of the mandelalides, we studied the AMP-activated protein kinase (AMPK) energy stress pathway and report that mandelalide A is an indirect activator of AMPK. Wild-type mouse embryonic fibroblasts (MEFs) and representative human non-small cell lung cancer (NSCLC) cells showed statistically significant increases in phospho-AMPK (Thr172) and phospho-ACC (Ser79) in response to mandelalide A. Mandelalide L, which also harbors an A-type macrocycle, induced similar increases in phospho-AMPK (Thr172) and phospho-ACC (Ser79) in U87-MG glioblastoma cells. In contrast, MEFs co-treated with an AMPK inhibitor (dorsomorphin), AMPKα-null MEFs, or NSCLC cells lacking liver kinase B1 (LKB1) lacked this activity. Mandelalide A was significantly more cytotoxic to AMPKα-null MEFs than wild-type cells, suggesting that AMPK activation serves as a protective response to mandelalide-induced depletion of cellular ATP. However, LKB1 status alone was not predictive of the antiproliferative effects of mandelalide A against NSCLC cells. When EGFR status was considered, erlotinib and mandelalide A showed strong cytotoxic synergy in combination against erlotinib-resistant 11-18 NSCLC cells but not against erlotinib-sensitive PC-9 cells. Finally, prolonged exposures rendered mandelalide A, a potent and efficacious cytotoxin, against a panel of human glioblastoma cell types regardless of the underlying metabolic phenotype of the cell. These results add biological relevance to the mandelalide series and provide the basis for their further pre-clinical evaluation as ATP synthase inhibitors and secondary activators of AMPK.
15
Delgado, Irene, Alejandra C. López-Delgado, Alberto Roselló-Díez, Giovanna Giovinazzo, Vanessa Cadenas, Laura Fernández-de-Manuel, Fátima Sánchez-Cabo, Matthew J. Anderson, Mark Lewandoski, and Miguel Torres. "Proximo-distal positional information encoded by an Fgf-regulated gradient of homeodomain transcription factors in the vertebrate limb." Science Advances 6, no. 23 (June 2020): eaaz0742. http://dx.doi.org/10.1126/sciadv.aaz0742.
Анотація:
The positional information theory proposes that a coordinate system provides information to embryonic cells about their position and orientation along a patterning axis. Cells interpret this information to produce the appropriate pattern. During development, morphogens and interpreter transcription factors provide this information. We report a gradient of Meis homeodomain transcription factors along the mouse limb bud proximo-distal (PD) axis antiparallel to and shaped by the inhibitory action of distal fibroblast growth factor (FGF). Elimination of Meis results in premature limb distalization and HoxA expression, proximalization of PD segmental borders, and phocomelia. Our results show that Meis transcription factors interpret FGF signaling to convey positional information along the limb bud PD axis. These findings establish a new model for the generation of PD identities in the vertebrate limb and provide a molecular basis for the interpretation of FGF signal gradients during axial patterning.
16
Schevzov, Galina, Anthony J. Kee, Bin Wang, Vanessa B. Sequeira, Jeff Hook, Jason D. Coombes, Christine A. Lucas, et al. "Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments." Molecular Biology of the Cell 26, no. 13 (July 2015): 2475–90. http://dx.doi.org/10.1091/mbc.e14-10-1453.
Анотація:
ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor–stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells.
17
Ihlefeld, Katja, Ralf Frederik Claas, Alexander Koch, Josef M. Pfeilschifter, and Dagmar Meyer zu Heringdorf. "Evidence for a link between histone deacetylation and Ca2+ homoeostasis in sphingosine-1-phosphate lyase-deficient fibroblasts." Biochemical Journal 447, no. 3 (October 5, 2012): 457–64. http://dx.doi.org/10.1042/bj20120811.
Анотація:
Embryonic fibroblasts from S1P (sphingosine-1-phosphate) lyase-deficient mice [Sgpl1−/− MEFs (mouse embryonic fibroblasts)] are characterized by intracellular accumulation of S1P, elevated cytosolic [Ca2+]i and enhanced Ca2+ storage. Since S1P, produced by sphingosine kinase 2 in the nucleus of MCF-7 cells, inhibited HDACs (histone deacetylases) [Hait, Allegood, Maceyka, Strub, Harikumar, Singh, Luo, Marmorstein, Kordula, Milstein et al. (2009) Science 325, 1254–1257], in the present study we analysed whether S1P accumulated in the nuclei of S1P lyase-deficient MEFs and caused HDAC inhibition. Interestingly, nuclear concentrations of S1P were disproportionally elevated in Sgpl1−/− MEFs. HDAC activity was reduced, acetylation of histone 3-Lys9 was increased and the HDAC-regulated gene p21 cyclin-dependent kinase inhibitor was up-regulated in these cells. Furthermore, the expression of HDAC1 and HDAC3 was reduced in Sgpl1−/− MEFs. In wild-type MEFs, acetylation of histone 3-Lys9 was increased by the S1P lyase inhibitor 4-deoxypyridoxine. The non-specific HDAC inhibitor trichostatin A elevated basal [Ca2+]i and enhanced Ca2+ storage, whereas the HDAC1/2/3 inhibitor MGCD0103 elevated basal [Ca2+]i without influence on Ca2+ storage in wild-type MEFs. Overexpression of HDAC1 or HDAC2 reduced the elevated basal [Ca2+]i in Sgpl1−/− MEFs. Taken together, S1P lyase-deficiency was associated with elevated nuclear S1P levels, reduced HDAC activity and down-regulation of HDAC isoenzymes. The decreased HDAC activity in turn contributed to the dysregulation of Ca2+ homoeostasis, particularly to the elevated basal [Ca2+]i, in Sgpl1−/− MEFs.
18
Hamaguchi, Yukiyoshi, Steven K. Juhn та Yasuo Sakakura. "Protease Inhibitors in Middle Ear Effusions from Experimental Otitis Media with Effusion: Kinetics of α1-Antitrypsin and α2-Macroglobulin Levels". Annals of Otology, Rhinology & Laryngology 98, № 4 (квітень 1989): 287–92. http://dx.doi.org/10.1177/000348948909800410.
Анотація:
Alpha 1-antitrypsin (α1-AT) and alpha 2-macroglobulin (α2-M) were measured by both immunochemical and functional assays in paired plasma and middle ear effusions (MEEs) from experimental otitis media models (serous otitis media [SOM], purulent otitis media [POM], and SOM + POM). The MEE/plasma ratio of α1-AT in SOM was significantly higher than that in POM (p< .01) because of the high α1-AT level in POM plasma. The ratio of both antitryptic activity and trypsin-binding activity in POM was significantly higher than that in SOM + POM (p< .01, < .05), and significantly lower than that in SOM (p< .01). The majority of both inhibitors in SOM exist as the free state, and the reaction between proteases and inhibitors in POM and SOM + POM is more active than that in SOM. It is concluded that both α1-AT and α2-M appear to play an important role in the protection of middle ear mucosa by forming protease-inhibitor complexes to reduce proteolytic damage in POM and SOM + POM models.
19
Kundu, Mondira, Chia-Ying Yang, Kelly McCastlain, Junmin Wu, Ji Zhang, Tullia Lindsten, Paul Ney, and Craig B. Thompson. "Hsp90 regulates Ulk1-mediated autophagic clearance of mitochondria." Blood 112, no. 11 (November 16, 2008): 3458. http://dx.doi.org/10.1182/blood.v112.11.3458.3458.
Анотація:
Abstract Recent studies have suggested that the serine-threonine kinase Ulk1, a mammalian homologue of the yeast autophagy regulator, Atg1, plays an important role in mitochondrial clearance during reticulocyte maturation. In order to gain insight into the regulation and activity of Ulk1, we used an unbiased proteomics approach to identify Ulk1-interacting proteins. Here, we demonstrate that Ulk1 interacts with heat shock protein 90 (Hsp90) and Cdc37, and is among a growing list of proteins whose steady state levels are regulated by this kinase-specific chaperone complex. Treatment of murine embryonic fibroblasts (MEFs) with the HSP90 inhibitor, 17AAG, inhibits the AKT/Tor pathway and induces autophagy despite the decreased steady state levels of Ulk1. By contrast, Ulk1-deficient MEFs are defective in autophagy induction, as measured by LC3 puncta formation (but not LC3 conversion) and ultrastructural analysis. Exposure of terminally differentiating erythroid cells to HSP90 inhibitors prevents the normal accumulation of Ulk1 protein and results in impaired autophagic clearance of organelles, similar to the effect of Ulk1 gene deletion. These findings highlight the importance of Ulk1 protein levels in terminal erythroid maturation and induction of autophagy and its regulation by the Hsp90/Cdc37 chaperone complex.
20
Weddle, Carly, and Paul Burridge. "Abstract A070: Examining patient-specific responses to PARP inhibitors in a novel, human induced pluripotent stem cell-based model of breast cancer." Cancer Research 84, no. 3_Supplement_1 (February 1, 2024): A070. http://dx.doi.org/10.1158/1538-7445.advbc23-a070.
Анотація:
Abstract Patient-derived human induced pluripotent stem cells (hiPSCs) are a powerful tool for disease modeling because hiPSCs provide a scalable source of cells that retain patient-specific genomic variants. Recently, somatic cell reprogramming has been applied to modeling cancer: cancer cells are reprogrammed into hiPSCs, then re-differentiated into the relevant cancer cell lineage. hiPSCs derived from cancer cells retain tumor- and subclone-specific genomic variants and drug sensitivities, providing a unique tool to deconstruct the contributions of tumor heterogeneity to drug response. While hiPSCs have been shown to model various hematological malignancies, no study to date has examined the potential of hiPSCs to model breast cancer. We demonstrate for the first time that primary human breast cancer cells can be reprogrammed into hiPSCs by transiently overexpressing canonical reprogramming factors. Using an optimized reprogramming methodology, we generated a biobank of >200 hiPSC lines from 10 breast tumor samples, including hormone receptor positive, HER2 positive, and triple negative breast tumors. Breast tumor-derived hiPSCs retain patient-specific oncogenic variants. Additionally, we present a protocol to differentiate hiPSCs into mammary epithelial cells (hiPSC-MECs) and mammary-like organoids for in vitro disease modeling. hiPSC-MECs are proliferative and exhibit a gene expression profile that closely resembles primary MECs. Finally, we demonstrate that this breast tumor model can be used to assess patient-specific responses to PARP inhibitors. We show that hiPSC-MECs derived from BRCA1 and BRCA2 mutant tumors are deficient in homology-directed repair and are more sensitive to PARP inhibitor treatment. This work establishes the foundation of hiPSCs as a novel disease modeling platform for breast cancer and highlights the potential of tumor-derived hiPSCs to predict patient and subclone-specific drug responses. Citation Format: Carly Weddle, Paul Burridge. Examining patient-specific responses to PARP inhibitors in a novel, human induced pluripotent stem cell-based model of breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A070.
21
Yu, Lanlan, Baohui Wang, Xurui Sun, and Jian Song. "Synthesis and properties of a MEAS quadripolymer scale inhibitor." Desalination and Water Treatment 52, no. 10-12 (May 16, 2013): 1865–71. http://dx.doi.org/10.1080/19443994.2013.792014.
22
Renaldo, Kyle, Shelby Sloan, JiHyun Chung, Lindsay Courtney, Konstantin Shilo, William Kisseberth, and Robert A. Baiocchi. "Exploring PRMT5 As a Potential Therapeutic Target in Canine Lymphomas." Blood 132, Supplement 1 (November 29, 2018): 4182. http://dx.doi.org/10.1182/blood-2018-99-119115.
Анотація:
Abstract Non-Hodgkin lymphoma (NHL) represents approximately 4 percent of all cancer diagnoses in the United States, with diffuse large B-cell lymphoma (DLBCL) accounting for approximately 40 percent of all new cases. There are numerous histologic (GC, NGC) and genetic (double hit/expressor) subtypes of DLBCL and the overall outcome of patients who receive standard therapy is heterogeneous. Lymphoma is also a common, malignancy in dogs, and while initial responses to combination chemotherapy show clinical responses, remission time is short and cures rare. Protein arginine methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase (PRMT) enzyme capable of driving symmetric demethylation of arginine residues on histones (H3(me2)R8; H4(me2)R3) and other proteins such as P53, and the NFkB subunit p65. PRMT5 is overexpressed and dysregulated in both solid and hematologic tumors and plays a key role in the driver activity of MYC, CYCLIND1 and NOTCH in lymphoma models. Experimental therapeutic strategies aimed at targeting PRMT5 activity have led to numerous, highly selective inhibitors that are currently being translated into the clinic with several clinical trials underway or in development. Here, we characterized patterns of PRMT5 expression and correlated these with histologic subtype in canine lymphoma tissue microarrays (TMAs, n=337 lymphoma specimens). We characterized expression of PRMT5 and its symmetric dimethylation histone marks in three canine lymphoma-derived cell lines, CLBL-1, 17-71, and OSW. Treatment of PRMT5 with a highly selective small molecule PRMT5 inhibitor (CMP220) was performed to determine the dose-dependent effects (1nM-10uM range) on proliferation (MTS assay), viability (annexin V/PI flow cytometry) and biomarkers of PRMT5 activity (SDMA, ADMA, histone symmetric demethylation). CMP220 showed PRMT5 inhibitory activity that was highly selective in a methyltransferase screening assay with 37 enzymes/complexes. TMAs of all histologic subtypes of lymphoma showed over-expression of PRMT5 in 96% of specimens. Canine DLBCL showed variable over-expression in the cytoplasmic compartment (48.8% strong, 50.0% weak, n = 165) compared to negative or weak staining in normal and hyperplastic lymph nodes (n = 40). Nuclear staining was observed primarily in lymphomas of T cell lineage (PTCL, and pre T lymphoblastic lymphomas). Primary and cell line lymphoma samples showed PRMT5 over expression by Western blot and RT-PCR. PRMT5 inhibition showed a dose-dependent decrease in symmetric dimethylarginine (SDMA) and symmetric demethylation of histone H4 arginine-3 (H4(me2s)R3) with no effect on asymmetric dimethylarginine (ADMA). Interestingly, in contrast to PRMT5 inhibition in human lymphomas, symmetric demethylation of histone H3 arginine-8 (H3(me2s)R8) showed no change with PRMT5 inhibition. The PRMT5 small molecule inhibitor CMP220 inhibited growth of CLBL-1 (IC50 of 123.2 nM at 6d) and 17-71 (IC50 of 100 nM at 6d). The OSW PTCL cell line showed profound resistance to PRMT5 inhibition with little cell death observed at 10 uM. Ex-vivo canine patient samples showed suppression of growth in a time and dose-dependent fashion. We have demonstrated that PRMT5 is expressed in canine B cell primary lymphomas and cell lines and that PRMT5 inhibition leads to growth suppression and induction of apoptosis. We are currently exploring genome-wide recruitment of PRMT5 on chromatin and examining chromatin and whole transcriptome changes that occur in canine lymphoma cell lines treated with PRMT5 inhibitors. This data provides justification for incorporating the canine lymphoma model into the preclinical development of PRMT5 inhibitors. Disclosures No relevant conflicts of interest to declare.
23
Alexander, D. L., L. G. Ganem, P. Fernandez-Salguero, F. Gonzalez, and C. R. Jefcoate. "Aryl-hydrocarbon receptor is an inhibitory regulator of lipid synthesis and of commitment to adipogenesis." Journal of Cell Science 111, no. 22 (November 15, 1998): 3311–22. http://dx.doi.org/10.1242/jcs.111.22.3311.
Анотація:
The aryl-hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological effects of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). In mouse embryo fibroblasts, TCDD activates expression of multiple genes, including CYP1B1, the predominant cytochrome P450 expressed in these cells. Here, we analyze constitutive functions of the AhR in primary mouse embryo fibroblasts (MEFs) and spontaneously immortalized MEF cell lines derived from wild-type (WT) C57BL/6 mice and also from congenic mice with a targeted disruption of the AhR gene (AhR-/-). After multiple passages, primary MEFs exhibit spontaneous differentiation, growth cessation and senescence. Eventually, colonies of immortalized MEFs arise to provide clonal lines. The senescent phase occurs much earlier for AhR-/- MEFs, while immortalization is substantially delayed. Comparison of AhR-/- and WT MEFs also indicates that constitutive AhR activity is required for basal expression of CYP1B1 and suppresses lipogenesis in subconfluent cultures. Primary WT and AhR-/- MEFs and the corresponding lines undergo adipogenesis when treated at confluence with the appropriate hormonal inducers. Addition of TCDD before or concurrent with hormonal induction suppressed PPAR gamma mRNA and adipogenesis, as measured by lipid accumulation, glycerol phosphate dehydrogenase activity and stearoyl CoA desaturase type 1 mRNA expression. This effect of TCDD treatment was absent in AhR-/- MEFs, establishing the role of AhR in hormone-induced adipogenesis. Such hormonal activation of confluent MEFs and preadipocytes results in a limited proliferative expansion followed by irreversible growth arrest. TCDD-treated MEFs undergo the mitotic expansion but fail to exit the cell cycle. In AhR-/- MEFs, there is no such effect of TCDD. These findings implicate the AhR as a constitutive inhibitor of triglyceride synthesis, and as an early regulator of adipocyte differentiation. AhR interference with cell-cycle arrest in differentiation may be linked to the increased rate of senescence.
24
Lambert, Michele P., Yuhuan Wang, M. Anna Kowalska, and Mortimer Poncz. "Negative Paracrine Effect of Platelet Factor 4 on Megakaryopoiesis Occurs through Lipoprotein Related Protein Receptor-1 on Megakaryocytes." Blood 110, no. 11 (November 16, 2007): 97. http://dx.doi.org/10.1182/blood.v110.11.97.97.
Анотація:
Abstract Platelet Factor 4 (PF4) is a paracrine inhibitor of megakaryopoiesis in vivo, with platelet counts inversely related to platelet PF4 levels in mice. Duration of thrombocytopenia after chemotherapy-induced thrombocytopenia (CIT) was directly related to platelet PF4 levels. Defining the molecular basis by which PF4 has this effect may have clinical implications in patients with CIT. Based on a recent observation that PF4 could activate endothelial cells through the Lipoprotein Related Protein Receptor-1 (LRP-1), we investigated whether a similar receptor might be involved in the negative paracrine effect of PF4 on megakaryopoiesis. We found that the addition of receptor-associated protein (RAP), a general inhibitor of members of the LRP family of receptors by binding to the alpha subunit, could block the inhibitory affect on in vitro megakaryocyte (MEG) colony number per plated marrow cells in a specific and dose-dependent fashion. Thus, at 0.2 μM RAP could prevent ∼50% of the inhibitory affect of the addition of 20 μg/mL of recombinant PF4 and at 0.4 μM RAP, the inhibitory effect of the added PF4 was completely abolished (p = 0.01 of no RAP vs. 0.4 μM RAP). Since a number of LRP receptors are inhibited by RAP, we repeated these studies using a specific anti-LRP-1 monoclonal antibody that blocks LRP-1 activation by binding to its alpha subunit (American Diagnostic). We have previously shown that transgenic mice overexpressing human PF4 by 6-fold have a 50% decrease in MEG-containing colonies in the above colony assay relative to WT littermates due to increase release of PF4 from developing megakaryocytes. We now found that the number of colonies derived from these high PF4 marrows increased to that seen in WT littermates when 25 μg/mL of the anti-LRP-1 antibody was included (p = 0.01). Since there are no prior studies showing that LRP-1 is expressed on human and murine bone marrow and murine fetal liver-derived MEGs, we performed RT-PCR on the appropriate RNAs as well as flow cytometry for the presence of LRP-1. RT-PCR of MEG RNA shows that LRP-1 message is present in MEGS. By flow cytometry, LRP-1 is clearly present on both human and murine MEGs. These studies support that PF4 inhibits megakaryopoiesis through LRP-1 on developing megakaryocytes. Studies were also done on the megakaryocyte-erythroid progenitor cell line G1ME. These cells do not express LRP-1, but when stimulated to differentiate, LRP-1 is detectable by flow cytometry on maturing megakaryocytes, suggesting that PF4 exerts its negative paracrine effect on developing MEGS and not on earlier progenitor cells. Consistent with this finding, we observed that in mice with different levels of platelet PF4, MEG content in the marrow is inversely related to platelet PF4 levels, but the size and ploidy are not correlated to PF4 content. Thus, we believe that we have defined the receptor system involved as well as the target cell of the observed inhibitory effect of PF4 on megakaryopoiesis. Such studies on the mechanism by which PF4 exerts this negative paracrine effect should lead to a more complete understanding of the regulation of megakaryopoiesis and may offer novel strategies to intervene in clinical thrombocytopenia, especially CIT.
25
Khan, Husain Yar, Sahar Bannoura, Hirva Mamdani, Amro Aboukameel, Yousef Mzannar, Yiwei Li, Mohammad Najeeb Al-Hallak, et al. "Abstract 5315: Anti-tumor activity of KRASG12C inhibitors is enhanced when combined with Cdc42 effector p21-activated kinase 4 targeting agents." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5315. http://dx.doi.org/10.1158/1538-7445.am2022-5315.
Анотація:
Abstract Background: KRASG12C inhibitors sotorasib and adagrasib have shown impressive results in clinical studies. These inhibitors efficiently block KRAS downstream effectors such as RAF, MEK, and ERK/p-ERK. However, factors such as feedback reactivation or bypass of KRAS dependence are emerging, resulting in the need for rational combination approaches. KRASG12C inhibitors have limited impact on the parallel oncogenic signaling driven by PI3K/AKT, its regulator molecules such as the Rho GTPases, and their effectors. This signaling pathway can collectively promote p-ERK re-activation thereby blunting the efficacy of KRASG12C targeted drugs. The p21-activated kinase (PAK) family are crucial effectors of the Rho family of GTPases and act as regulatory switches that control important cellular processes including motility, proliferation, and survival. PAK4 is the main effector of Rho GTPase, cell division control protein 42 homolog (Cdc42), and has an established role in mutant RAS-dependent tumor metastasis. In this study, we rationally targeted two parallel signaling pathways by combining KRASG12C inhibitors with the PAK4 inhibitor KPT-9274 in KRASG12C mutant PDAC and NSCLC in vitro and in vivo models. Methods: We evaluated the cytotoxicity and molecular profile of KRASG12C inhibitors in combination with PAK4 inhibitor KPT-9274 as well as positive control PF-3758309 in KRASG12C mutant 2D and 3D cellular/spheroid models. The anti-tumor activity of the combinations was evaluated in KRASG12C mutant cell line-derived xenograft. Tumor volumes were assessed throughout the treatment period using an ultrasound scanner. Results: PAK4 inhibitor KPT-9274 synergized with both sotorasib and adagrasib yielding suppressed growth of KRASG12C mutant PDAC and NSCLC cells in 2D and 3D cultures, but not in KRASwt, KRASG12V, and KRASG12D cell lines (NCI RASless MEFs). In addition, the combination reduced the clonogenic potential of KRASG12C mutant cancer cells. PAK4-KRASG12C inhibitor combinations resulted in enhanced and prolonged inhibition of KRAS downstream effector pathways. In xenograft studies, oral administration of KPT-9274 at sub-optimal dose (100 mg/kg QD x 5 x 3 weeks) and sotorasib at one-fourth of MTD (25 mg/kg QD x 5 x 3 weeks) demonstrated remarkable inhibition of KRASG12C mutant MiaPaCa-2 tumor burden (p<0.001 single agents vs combination treatment). Residual tumor analysis showed a reduction in proliferation index (ki67), activation of pro-apoptotic caspases, and inhibition of mutant RAS effector signaling. Studies in additional KRASG12C xenograft and tumor explant models are ongoing. Conclusion: This is the first study showing that KRASG12C inhibitors can synergize with PAK4 inhibitors. This combination can simultaneously target direct KRAS downstream effectors and the parallel PI3K/AKT oncogenic signaling warranting further clinical investigations. Citation Format: Husain Yar Khan, Sahar Bannoura, Hirva Mamdani, Amro Aboukameel, Yousef Mzannar, Yiwei Li, Mohammad Najeeb Al-Hallak, Ibrahim Azar, Steve Kim, Rafic Beydoun, Ramzi Mohammad, Yosef Landesman, Yue Zhang, Erkan Baloglu, William Senapedis, Ammar Sukari, Misako Nagasaka, Anthony Shields, Asfar S. Azmi. Anti-tumor activity of KRASG12C inhibitors is enhanced when combined with Cdc42 effector p21-activated kinase 4 targeting agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5315.
26
Nigam, Aradhya, Walid K. Chatila, Gnana P. Krishnamoorthy, Alan L. Ho, James A. Fagin, Nikolaus D. Schultz, and Brian R. Untch. "Abstract 1181: PTEN loss-of-function mutations prevalent in HRAS-mutant cancers results in resistance to targeted therapy." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1181. http://dx.doi.org/10.1158/1538-7445.am2022-1181.
Анотація:
Abstract Background: The clinical development of farnesyltransferase inhibitors (FTIs) as targeted therapy for HRAS-mutant cancers has demonstrated mixed responses dependent on cancer type. Co-occurring mutations may affect tumor response, supported by previous studies demonstrating that NF1 mutations confer resistance to HRAS inhibition by the FTI tipifarnib in thyroid cancer mouse models. We aimed to determine if PI3K pathway activating mutations altered responses to targeted therapy in HRAS-mutant cancers. Methods: Targeted sequencing data from MSK-IMPACT cohort and DFCI-GENIE (Version 9.0) database was used to investigate co-mutations amongst HRAS-mutant cancers. Fisher’s exact test was used to determine co-altered mutations found predominantly in HRAS-mutant cancers relative to respective KRAS- and NRAS-mutant cancers. ‘RASless’ (KRASlox/HRASKO/NRASKO) mouse embryonic fibroblasts (MEFs) were obtained that in the presence of 600nM tamoxifen (4OHT) resulted in a KRAS knock-out. ‘Rasless’ MEFs were transfected with HRASG13R to create a system for testing sensitivity to FTIs in the presence or absence of WT KRAS, or with concurrent PTEN loss generated by CRISPR-Cas9 technology. Results: A greater proportion of HRAS-mutant cancers had co-altered mutations (48.8%) in genes encoding effectors in the MAPK, PI3K or RTK pathways compared to KRAS- and NRAS-mutant cancers (41.4% and 38.4%, respectively; p<0.05). PTEN mutations were more prevalent in HRAS-mutant NSCLC (21%) compared to KRAS- and NRAS-mutant NSCLC (1% and 2%, respectively; p<0.05). Non-transfected MEFs were sensitized to tipifarnib by introduction of a HRASG13R allele in non-4OHT (IC50: MEF= >3uM, HRASG13R = 324.7nM) and 4OHT (IC50: MEF= >3uM, HRASG13R= 0.62nM; p<0.001) conditions, indicating that WT KRAS confers a relative resistance to the inhibitory effects of the FTI on HRAS. PTEN loss-of-function mutations led to tipifarnib resistance in HRASG13R MEFs in the absence (IC50: >3uM; p<0.001) or presence of 4OHT (IC50: 213.6nM; p<0.001). Combined treatment of HRASG13R/PTEN MEFs with the PIK3CB-specific inhibitor AZD8186 and tipifarnib sensitized cells in non-4OHT (IC50- 100nM:100nM Tipifarnib:AZD8186) and 4OHT (IC50- 100nM:10nM Tipifarnib:AZD8186) conditions. Conclusions: Co-altered mutations of MAPK, PI3K or RTK effectors are found more commonly in HRAS than in KRAS or NRAS-mutant cancers. Co-alteration of PTEN preferentially associated with HRAS-mutations in NSCLC. Deletion of PTEN resulted in resistance to FTI targeted therapy in vitro. Co-altered mutations may predict sensitivity and resistance to FTIs and guide clinical trial design. Citation Format: Aradhya Nigam, Walid K. Chatila, Gnana P. Krishnamoorthy, Alan L. Ho, James A. Fagin, Nikolaus D. Schultz, Brian R. Untch. PTEN loss-of-function mutations prevalent in HRAS-mutant cancers results in resistance to targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1181.
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Zhou, Yang, Sahar Alimohamadi, Li Wang, Ziqing Liu, Joseph B. Wall, Chaoying Yin, Jiandong Liu, and Li Qian. "A Loss of Function Screen of Epigenetic Modifiers and Splicing Factors during Early Stage of Cardiac Reprogramming." Stem Cells International 2018 (2018): 1–14. http://dx.doi.org/10.1155/2018/3814747.
Анотація:
Direct reprogramming of cardiac fibroblasts (CFs) to induced cardiomyocytes (iCMs) is a newly emerged promising approach for cardiac regeneration, disease modeling, and drug discovery. However, its potential has been drastically limited due to the low reprogramming efficiency and largely unknown underlying molecular mechanisms. We have previously screened and identified epigenetic factors related to histone modification during iCM reprogramming. Here, we used shRNAs targeting an additional battery of epigenetic factors involved in chromatin remodeling and RNA splicing factors to further identify inhibitors and facilitators of direct cardiac reprogramming. Knockdown of RNA splicing factors Sf3a1 or Sf3b1 significantly reduced the percentage and total number of cardiac marker positive iCMs accompanied with generally repressed gene expression. Removal of another RNA splicing factor Zrsr2 promoted the acquisition of CM molecular features in CFs and mouse embryonic fibroblasts (MEFs) at both protein and mRNA levels. Moreover, a consistent increase of reprogramming efficiency was observed in CFs and MEFs treated with shRNAs targeting Bcor (component of BCOR complex superfamily) or Stag2 (component of cohesin complex). Our work thus reveals several additional epigenetic and splicing factors that are either inhibitory to or required for iCM reprogramming and highlights the importance of epigenetic regulation and RNA splicing process during cell fate conversion.
28
Tanajura, Luiz, Áurea Chaves, Andrea Abizaid, and José Costa Júnior. "Monotherapy with P2Y12 receptor inhibitors in patients treated by percutaneous coronary intervention." Journal of Transcatheter Interventions 29 (December 2, 2021): 1–9. http://dx.doi.org/10.31160/jotci202129a202106.
Анотація:
Since the mid-1990s, the dual antiplatelet therapy, consisting of the association between acetylsalicylic acid and a platelet P2Y12 receptor inhibitor, is the core of thrombosis prevention after coronary stent implantation, regardless of the models used. It is also used to prevent the occurrence of atherothrombotic events in the late phase after the intervention. The clinical presentation of coronary artery disease influences the duration of dual therapy, which tends to be longer in treated cases of acute coronary syndrome (usually for one year), when compared to cases of chronic coronary disease (often for up to 6 months). After this period, the P2Y12 inhibitor is usually discontinued, and monotherapy with aspirin is maintained. However, in the last two decades, it has been observed that prolonged use of two associated antiplatelet agents predisposes treated cases to bleeding complications, with potentially severe consequences – including increased mortality. Thus, alternatives that minimize this risk have been considered and evaluated, such as early discontinuation of acetylsalicylic acid (between 1 and 3 months after discharge), or the so-called monotherapy with P2Y12 inhibitors, aiming to reduce bleeding without compromising prevention of ischemic events. In the last decade, a series of randomized clinical trials evaluated this hypothesis, generally resulting in reduced bleeding complications, although not necessarily of those classified as major, with no significant increase in the most relevant cardiovascular events. This review discusses the main results of these clinical trials and their potential clinical implications for routine cardiology practice.
29
Dinchuk, Joseph E., Carolyn Cao, Fei Huang, Karen A. Reeves, Jeanne Wang, Fanny Myers, Glenn H. Cantor, et al. "Insulin Receptor (IR) Pathway Hyperactivity in IGF-IR Null Cells and Suppression of Downstream Growth Signaling Using the Dual IGF-IR/IR Inhibitor, BMS-754807." Endocrinology 151, no. 9 (July 7, 2010): 4123–32. http://dx.doi.org/10.1210/en.2010-0032.
Анотація:
The biology of IGF-IR/IR signaling was studied in normal mouse embryonic fibroblasts (MEFs) that were either wild type (wt), heterozygous (het), or null for the IGF-IR. The ability of IGF-I, IGF-II, or insulin to stimulate serum-starved MEFs was characterized by gene expression profiling and biochemical analyses for activation of downstream signals. Each genotypic group of MEFs exhibited distinct patterns of expression both while resting and in response to stimulation. The insulin receptor (IR) pathway in IGF-IR null MEFs was hypersensitive to insulin ligand stimulation resulting in greater AKT phosphorylation than in wt or het MEFs stimulated with the same ligand. Interestingly, the IR pathway hypersensitivity in IGF-IR null MEFs occurred with no observed changes in the levels of IR isoforms A or B. A new small molecule IGF-IR inhibitor (BMS-754807), having equipotent activity against both IGF-IR and IR, proved effective in suppressing both AKT and ERK phosphorylation from both the IGF-IR and IR pathways by all three ligands tested in wt, het, and null MEFs. The use of a dual IGF-IR/IR inhibitor addresses concerns about the use of growth inhibiting therapies directed against the IGF-IR receptor in certain cancers. Lastly, comparison of the antiproliferative effects (IC50s) of various compounds in wt vs. null MEFs demonstrates that genetically characterized MEFs provide a simple and inexpensive tool with which to define compounds as having mostly on-target or off-target IGF-IR activities because off-target compounds affect both wt and null MEFs equally.
30
Hamaguchi, Yukiyoshi, Yuichi Majima, Kenji Sakakura, and Yasuo Sakakura. "Lysosomal Thiol Proteases in Middle Ear Effusions." Annals of Otology, Rhinology & Laryngology 95, no. 3_suppl (May 1986): 9–12. http://dx.doi.org/10.1177/00034894860950s303.
Анотація:
Hydrolytic activity of lysosomal cathepsins B and H, and trypsin-like proteases in 115 middle ear effusions (MEEs, 40 serous and 75 mucoid) from chronic otitis media with effusion (OME) patients was measured and compared to that in plasma. The activity of both cathepsins in MEEs was significantly higher than that in plasma (p<0.01), and cathepsin B activity in mucoid MEEs was also significantly higher than that in serous MEEs (p<0.01). The activity of trypsin-like proteases was very weak in both MEEs and plasma. Profiles of various inhibitors indicated the qualitative difference of proteolytic enzymes between MEEs and plasma. Mucoid MEEs had significantly higher activity of thiol proteases than serous ones (p<0.01). Cathepsin B-like lysosomal thiol proteases, derived mainly from macrophages, could become a major proteolytic factor to perpetuate and amplify the inflammatory reaction of chronic OME.
31
Dizaye, Kawa F., and Asmaa A. Ahmed. "Renoprotective Evaluations of Different Angiotensin Inhibitors on Diabetic Nephropathy in Rats." Middle East Journal of Internal Medicine 9, no. 3 (November 2016): 3–11. http://dx.doi.org/10.5742/meim.2016.92897.
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Mack, Fiona A., Jagruti H. Patel, Mangatt P. Biju, Volker H. Haase, and M. Celeste Simon. "Decreased Growth of Vhl−/− Fibrosarcomas Is Associated with Elevated Levels of Cyclin Kinase Inhibitors p21 and p27." Molecular and Cellular Biology 25, no. 11 (June 1, 2005): 4565–78. http://dx.doi.org/10.1128/mcb.25.11.4565-4578.2005.
Анотація:
ABSTRACT Inactivating mutations within the von Hippel-Lindau (VHL) tumor suppressor gene predispose patients to develop a variety of highly vascularized tumors. pVHL targets α subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF), a critical regulator of energy metabolism, angiogenesis, hematopoiesis, and oxygen (O2) delivery, for ubiquitin-mediated degradation in an O2-dependent manner. To investigate the role of Vhl in cellular proliferation and tumorigenesis, we utilized mouse embryonic fibroblasts (MEFs), a common tool for analyzing cell cycle regulation, and generated Vhl − / − MEF-derived fibrosarcomas. Surprisingly, growth of both Vhl − / − MEFs and fibrosarcomas was impaired, although tumor vascularity was increased. Decreased proliferation of Vhl − / − MEFs was correlated with an overexpression of cyclin kinase inhibitors (CKIs) p21 and p27. The transcription of p21 and p27 is inhibited by c-Myc; therefore, the induction of CKIs was attributed to the ability of HIF to antagonize c-Myc activity. Indeed, p21 mRNA levels were elevated under normoxia in Vhl − / − MEFs, while c-Myc transcriptional activity was markedly reduced. Gene silencing of HIF-1α by small interfering RNA reduced p21 and p27 protein and mRNA levels in Vhl − / − MEFs. The induction of p21 and p27, mediated by constitutive activation of the HIF pathway, provides a mechanism for the decreased proliferation rates of Vhl − / − MEFs and fibrosarcomas. These results demonstrate that a loss of pVHL can induce growth arrest in certain cells types, which suggests that additional genetic mutations are necessary for VHL-associated tumorigenesis.
33
Shim, Sungbo, Yujin Kim, Jongdae Shin, Jieun Kim, and Soochul Park. "Regulation of EphA8 Gene Expression by TALE Homeobox Transcription Factors during Development of the Mesencephalon." Molecular and Cellular Biology 27, no. 5 (December 18, 2006): 1614–30. http://dx.doi.org/10.1128/mcb.01429-06.
Анотація:
ABSTRACT The mouse ephA8 gene is expressed in a rostral-to-caudal gradient in the developing superior colliculus, and these EphA gradients may contribute to the proper development of the retinocollicular projection. Thus, it is of considerable interest to elucidate how the ephA8 gene expression is controlled by upstream regulators during the development of the mesencephalon. In this study, we employed in vivo expression analysis in transgenic mouse embryos to dissect the cis-acting DNA regulatory region, leading to the identification of a CGGTCA sequence critical for the ephA8 enhancer activity. Using this element as the target in a yeast one-hybrid system, we identified a Meis homeobox transcription factor. Significantly, DNA binding sites for Pbx, another TALE homeobox transcription factor, were also identified in the ephA8 enhancer region. Meis2 and Pbx1/2 are specifically expressed in the entire region of the dorsal mesencephalon, where specific colocalization of EphA8 and Meis is restricted to a subset of cells. Meis2 and Pbx2 synergistically bind the ephA8 regulatory sequence in vitro, and this interaction is critical for the transcriptional activation of a reporter construct bearing the ephA8 regulatory region in the presence of histone deacetylase inhibitor. More importantly, when expressed in the embryonic midbrain, the dominant-negative form of Meis down-regulates endogenous ephA8. Interestingly, we found that both Meis2 and Pbx2 are constitutively bound in the ephA8 regulatory region in the dorsal mesencephalon. These studies strongly suggest that Meis and Pbx homeobox transcription factors tightly associate with the ephA8 regulatory sequence and require an additional unidentified regulator to ensure the specific activation of ephA8.
34
Sun, Jun, Juan Kong, Yingli Duan, Frances L. Szeto, Anne Liao, James L. Madara та Yan Chun Li. "Increased NF-κB activity in fibroblasts lacking the vitamin D receptor". American Journal of Physiology-Endocrinology and Metabolism 291, № 2 (серпень 2006): E315—E322. http://dx.doi.org/10.1152/ajpendo.00590.2005.
Анотація:
1,25-Dihydroxyvitamin D [1,25(OH)2D3] is known to have anti-inflammatory activity; however, the molecular mechanism remains poorly defined. Here we show that the nuclear vitamin D receptor (VDR) is directly involved in the regulation of NF-κB activation, a pathway essential for inflammatory response. In mouse embryonic fibroblasts (MEFs) derived from VDR−/− mice, the basal level of κB inhibitor (IκB) α protein was markedly decreased compared with VDR+/− MEFs; however, degradation of IκBα and its phosphorylation in response to TNF-α treatment or Salmonella infection were not altered in VDR−/− cells, neither were the levels of IκB kinase-α and IκB kinase-β proteins. Consistent with IκBα reduction, p65 accumulation in the nucleus was markedly increased in unstimulated VDR−/− cells. In addition, the physical interaction between VDR and p65 was absent in VDR−/− MEFs, which may free p65 and increase its activity. Consequently, these alterations combined led to a marked increase in nuclear p65 DNA binding and NF-κB transcriptional activity; consistently, induction of IL-6 by TNF-α or IL-1β was much more robust in VDR−/− than in VDR+/− cells, indicating that VDR−/− cells are more susceptible to inflammatory stimulation. Therefore, cells lacking VDR appear to be more proinflammatory due to the intrinsic high NF-κB activity. The reduction of IκBα in VDR−/− MEFs may be partially explained by the lack of VDR-mediated stabilization of IκBα by 1,25(OH)2D3. This is supported by the observation that IκBα degradation induced by TNF-α was inhibited by 1,25(OH)2D3 in VDR+/− cells, but not in VDR−/− cells. Taken together, these data suggest that VDR plays an inhibitory role in the regulation of NF-κB activation.
35
Yerlikaya, Azmi, Scot R. Kimball та Bruce A. Stanley. "Phosphorylation of eIF2α in response to 26S proteasome inhibition is mediated by the haem-regulated inhibitor (HRI) kinase". Biochemical Journal 412, № 3 (28 травня 2008): 579–88. http://dx.doi.org/10.1042/bj20080324.
Анотація:
The present study demonstrates that even brief inhibition of degradation by the 26S proteasome inhibits global protein synthesis, mediated through increased phosphorylation of eIF2α (eukaryotic translational initiation factor 2α) by the HRI (haem-regulated inhibitor) kinase. Exposure of COS-7 cells to the proteasome inhibitor MG-132 (the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-leucinal) for 4 h resulted in a 55–60% decrease in protein synthesis rate compared with control cells. This repression of protein synthesis after treatment with MG-132 is not due to induction of apoptosis, which is known to occur after longer periods of 26S inhibition. Instead, we observed a significantly increased phosphorylation of eIF2α, which is known to repress global protein synthesis. In three MEF (mouse embryonic fibroblast) knockout cell lines lacking one of the four kinases known to phosphorylate eIF2α, increased phosphorylation of eIF2α still occurred after inhibition of the 26S proteasome. These three cell lines included a deletion of the PKR (double-stranded-RNA-dependent protein kinase); a deletion of the PERK (PKR-like endoplasmic reticulum resident kinase); or a deletion of the GCN2 (positive general control of transcription-2) kinase, indicating that none of these kinases was primarily responsible for the observed phosphorylation of eIF2α. In contrast, in a fourth MEF knockout cell line, HRI−/− cells lacking the HRI kinase failed to increase eIF2α phosphorylation upon proteasome inhibitor treatment (MG-132 or various doses of Bortezomib), indicating that the HRI kinase is the primary kinase activated by brief treatment of MEFs with 26S proteasome inhibitors.
36
Saadatzadeh, M. Reza, Khadijeh Bijangi-Vishehsaraei, and Laura S. Haneline. "The Role of Stress Activated Protein Kinases in Promoting Fanconi Anemia Hematopoietic Stem and Progenitor Cell Dysfunction." Blood 112, no. 11 (November 16, 2008): 1043. http://dx.doi.org/10.1182/blood.v112.11.1043.1043.
Анотація:
Abstract The underlying molecular mechanisms that promote bone marrow failure in Fanconi anemia (FA) are incompletely understood. Evidence from our lab and others suggest that enhanced oxidant and TNF-α mediated apoptosis of hematopoietic stem and progenitor cells is a major contributing factor. Previously, we showed that enhanced apoptosis of FA type C deficient (Fancc −/−) progenitors involves aberrant activation of the stress kinase, p38MAPK. Given the importance of c-Jun N-terminal kinase (JNK) in the stress response, we hypothesized that enhanced TNF-α-induced apoptosis in Fancc −/− cells would also involve altered JNK activation. The aims of the current study were to determine whether Fancc −/− cells exhibit altered JNK activation and to examine whether inhibition of JNK and/or p38MAPK kinase would enhance Fancc −/− hematopoietic stem cell (HSC) repopulating ability. In Fancc −/− murine embryonic fibroblasts (MEFs), TNF-α induced a 2-fold increase in JNK in vitro kinase activity compared to WT (n=4, p<0.05). Use of either a JNK inhibitor (n=3, p<0.003) or knockdown of JNK1/JNK2 expression using siRNAs (n=3, p<0.001) protected Fancc −/− MEFs from TNF-α induced apoptosis. Importantly, TNF-α induced apoptosis of Fancc −/− ckit+ low-density bone marrow cells was also restored to WT levels by culturing with a JNK inhibitor (n=3, p<0.01), and clonogenic hematopoietic progenitor assays demonstrated that JNK inhibition improved Fancc −/− colony formation in the presence of TNF-α (n=3, p<0.002). Taken together these data suggest that the predisposition of Fancc −/− MEFs and hematopoietic progenitors to TNF-α induced apoptosis involves JNK activation. Based on these data and previous studies demonstrating a role for p38MAPK in enhanced Fancc −/− progenitor apoptosis, competitive repopulation assays were conducted to examine whether culturing Fancc −/− cells with JNK and/or p38MAPK inhibitors would enhance HSC reconstituting function. Surprisingly, in two separate experiments Fancc −/− donor cells cultured with the JNK inhibitor had levels of donor chimerism that were equivalent to Fancc −/− donor cells cultured with the vehicle control. In contrast, culturing Fancc −/− cells with a p38MAPK inhibitor (SB203580) significantly increased repopulating ability compared to Fancc −/− cells cultured with vehicle control in two separate transplants (total n=12–14 recipients/transplant group, p<0.01). Interestingly, a similar increase in donor chimerism was observed in WT donor cells cultured with the p38MAPK inhibitor (total n=12–14 recipients/transplant group, p<0.002), supporting an integral role of p38MAPK in maintaining HSC function. The improvement in reconstitution was sustained over 12 months, and multilineage analysis revealed enhanced lymphoid and myeloid reconstitution, supporting an increase in HSC function with inhibition of p38MAPK. Twelve months post-transplantation all mice exhibited normal peripheral blood counts, BM and spleen histology. Taken together these data suggest that p38MAPK, but not JNK has a critical role in maintaining survival of WT and Fancc −/− reconstituting cells under conditions of stress.
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Ishida, Yoji, Syugo Kowata, Kazunori Murai, Tatsuo Oyake, and Shigeki Ito. "TAT/HDL Induces the Phospholylation of Cofilin in the Process of Platelet Production of Megakaryocytes." Blood 104, no. 11 (November 16, 2004): 2186. http://dx.doi.org/10.1182/blood.v104.11.2186.2186.
Анотація:
Abstract In the previous ASH Meetings, we have reported that thrombin/antithrombin complex in association with high density lipoprotein (TAT/HDL) directly stimulated proplatelet formation (PPF) of human or murine megakaryocytes. In this study, we investigated the signal transduction pathways responsible for platelet production from megakaryocytes using human megakaryoblastic cell line (MEG). TAT/HDL stimulated MEGs to induce the many platelet-like particles (PP) around the cells in the serum free culture during 12~24 hrs (Fig. 1). The frequency of MEGs with PP increased with the increase of TAT/HDL in a dose dependent manner (0 μg/ml: 1.8±0.5%, 50 μg/ml: 30.8±4.5%, 100 μg/ml: 46.5±7.8%, 200 μg/ml: 57.8±8.3%, mean±SD, n=4). The frequency of MEGs with PP, expressed as a pecentage of the control, was significantly decreased after the incubation with TAT/HDL(150 μg/ml) and Y27632, a specific inhibitor for Rho kinase(ROCK), (Y27632 10 μM: 61.3±7.9% of the control (p<0.01), 100 μM: 12.5±5.9% of the control (p<0.01), mean±SD, n=4). MEGs were incubated with TAT/HDL (150 μg/ml) in serum free condition for 5, 10, 15, 30, 60, 90, 120, 150 and 180 min. MEGs were havested, washed twice and lysed by the lysis buffer (50mM Tris/HCl, 150 mM NaCl. 0.5% Nonidet P-40 pH 6.8 with protease inhibitors’ cocktail) for the Western blot analysis. Activated Rho (GTP-bound state Rho), measured by the Western blot using Rhotekin-Rho binding domain (RBD) beads and anti-Rho antibody, increased at 90, 120 and 150 min after the incubation. ROCK was co-precipitated with activated Rho using Rhotekin-RBD beads and detected by the Western blot analysis using anti-ROCK antibody at 90, 120 and 150 min. LIM kinase was immunoprecipitated with ROCK/anti-ROCK antibody and protein G sepharose. Western blot analysis using anti-phosphothreonine antibody revealed that LIM-kinase was phospholylated at 90, 120 and 150 min after the incubation. Phospholylated cofilin (Ser3) expression was detected by the Western blot using anti-phosphocofilin antibody at 60 min, peaked at 120 min and tapered at 150 min after the incubation (Fig.2). The intensity of the phospholylated cofilin (Ser3) band decreased in the incubation of MEGs with TAT/HDL and Y27632 (100 μM) at each time. These results strongly suggested the platelet-like particles production from MEGs, stimulated by TAT/HDL, was involved in the pathway from Rho activation, ROCK activation, LIM kinase phospholylation and cofilin phospholylation, based on the evidence that phospholylated cofilin showed the inhibition of actin depolymerization and the stabilization of actin filaments. Fig.1 (a) MEGs with no stimulation (b) MEGs with TAT/HDL (200 μg/ml) for 24h Fig.2 Expression of phospholylated cofilin (Ser3). Figure Figure Figure Figure
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Sealover, Nancy, Bridget Finniff, and Robert Kortum. "Abstract B033: Wild-type RAS signaling is an essential therapeutic target in RAS-mutated cancers." Molecular Cancer Research 21, no. 5_Supplement (May 1, 2023): B033. http://dx.doi.org/10.1158/1557-3125.ras23-b033.
Анотація:
Abstract Single-agent targeting of RAS-mutated cancers using RAF/MEK/ERK or PI3K/AKT pathway inhibitors is ineffective; blocking one pathway relieves negative feedback control of RTK signaling and hyperactivates parallel effector pathways. While combined MEK and PI3K inhibition is effective in preclinical models, toxicity of this combination prevents its clinical use. Alternative strategies for treating RAS-mutated cancers are essential. Therapeutics directly targeting mutated RAS proteins have the potential to spare normal cellular function thereby lessening overall toxicity. Unfortunately, direct RAS inhibition as a monotherapy does not fully block RAS effector signaling. RAS proteins show differential activation of RAF and PI3K pathways: KRAS potently activates RAF but poorly activates PI3K, whereas HRAS effectively activates PI3K but poorly activates RAF. Further, mutant RAS inhibition relieves negative feedback controls leading to rapid hyperactivation of RTK−WT RAS signaling. A more comprehensive understanding of the interplay between mutant RAS and RTK−WT RAS signaling is essential to developing rational therapeutic approaches to treat RAS-mutated cancers. We found that WT RAS enhanced mutant RAS-driven transformation by activating RAS effectors poorly engaged by mutated RAS. Further, inhibition of RAS effectors activated poorly by mutant RAS synergized with and limited resistance to mutant HRAS and KRAS inhibitors. The mutant HRAS inhibitor tipifarnib blocked PI3K signaling and synergized with MEK inhibitors in HRAS-mutated cancer cell lines; covalent KRASG12C inhibitors blocked MEK signaling and synergized with PI3K inhibitors in KRASG12C-mutated cell lines. Synergy between inhibitors of mutant RAS and RAS effectors was dependent on intact RTK/WT RAS signaling and was lost in both RASless or SOSless MEFs. These results should inform the design of clinical trials for patients with RAS-mutated cancers. Citation Format: Nancy Sealover, Bridget Finniff, Robert Kortum. Wild-type RAS signaling is an essential therapeutic target in RAS-mutated cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B033.
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Rubin, Elina, Agnese C. Pippione, Matthew Boyko, Giacomo Einaudi, Stefano Sainas, Massimo Collino, Carlo Cifani та ін. "A New NF-κB Inhibitor, MEDS-23, Reduces the Severity of Adverse Post-Ischemic Stroke Outcomes in Rats". Brain Sciences 12, № 1 (28 грудня 2021): 35. http://dx.doi.org/10.3390/brainsci12010035.
Анотація:
Aim: Nuclear factor kappa B (NF-κB) is known to play an important role in the inflammatory process which takes place after ischemic stroke. The major objective of the present study was to examine the effects of MEDS-23, a potent inhibitor of NF-κB, on clinical outcomes and brain inflammatory markers in post-ischemic stroke rats. Main methods: Initially, a Toxicity Experiment was performed to determine the appropriate dose of MEDS-23 for use in animals, as MEDS-23 was analyzed in vivo for the first time. We used the middle cerebral artery occlusion (MCAO) model for inducing ischemic stroke in rats. The effects of MEDS-23 (at 10 mg/kg, ip) on post-stroke outcomes (brain inflammation, fever, neurological deficits, mortality, and depression- and anxiety-like behaviours) was tested in several efficacy experiments. Key findings: MEDS-23 was found to be safe and significantly reduced the severity of some adverse post-stroke outcomes such as fever and neurological deficits. Moreover, MEDS-23 significantly decreased prostaglandin E2 levels in the hypothalamus and hippocampus of post-stroke rats, but did not prominently alter the levels of interleukin-6 and tumor necrosis factor-α. Significance: These results suggest that NF-κB inhibition is a potential therapeutic strategy for the treatment of ischemic stroke.
40
GRILL, Constance, Ferdous GHEYAS, Priya DAYANANTH, Weihong JIN, Wei DING, Ping QIU, Luquan WANG, Ronald J. DOLL, and Jessie M. ENGLISH. "Analysis of the ERK1,2 transcriptome in mammary epithelial cells." Biochemical Journal 381, no. 3 (July 27, 2004): 635–44. http://dx.doi.org/10.1042/bj20031688.
Анотація:
MAPK (mitogen-activated protein kinase) pathways constitute major regulators of cellular transcriptional programmes. We analysed the ERK1,2 (extracellular-signal-regulated kinase 1,2) transcriptome in a non-transformed MEC (mammary epithelial cell) line, MCF-12A, utilizing rAd MEK1EE, a recombinant adenovirus encoding constitutively active MEK1 (MAPK/ERK kinase 1). rAd MEK1EE infection induced morphological changes and DNA synthesis which were inhibited by the MEK1,2 inhibitor PD184352. Hierarchical clustering of data derived from seven time points over 24 h identified 430 and 305 co-ordinately up-regulated and down-regulated genes respectively. c-Myc binding sites were identified in the promoters of most of these up-regulated genes. A total of 46 candidate effectors of the Raf/MEK/ERK1,2 pathway in MECs were identified by comparing our dataset with previously reported Raf-1-regulated genes. These analyses led to the identification of a suite of growth factors co-ordinately induced by MEK1EE, including multiple ErbB ligands, vascular endothelial growth factor and PHRP (parathyroid hormone-related protein). PHRP is the primary mediator of humoral hypercalcaemia of malignancy, and has been implicated in metastasis to bone. We demonstrate that PHRP is secreted by MEK1EE-expressing cells. This secretion is inhibited by PD184352, but not by ErbB inhibitors. Our results suggest that, in addition to anti-proliferative properties, MEK1,2 inhibitors may be anti-angiogenic and possess therapeutic utility in the treatment of PHRP-positive tumours.
41
Tergaonkar, Vinay, Virginie Bottero, Masahito Ikawa, Qiutang Li та Inder M. Verma. "IκB Kinase-Independent IκBα Degradation Pathway: Functional NF-κB Activity and Implications for Cancer Therapy". Molecular and Cellular Biology 23, № 22 (15 листопада 2003): 8070–83. http://dx.doi.org/10.1128/mcb.23.22.8070-8083.2003.
Анотація:
ABSTRACT Antiapoptotic activity of NF-κB in tumors contributes to acquisition of resistance to chemotherapy. Degradation of IκB is a seminal step in activation of NF-κB. The IκB kinases, IKK1 and IKK2, have been implicated in both IκB degradation and subsequent modifications of NFκB. Using mouse embryo fibroblasts (MEFs) devoid of both IKK1 and IKK2 genes (IKK1/2−/−), we document a novel IκB degradation mechanism. We show that this degradation induced by a chemotherapeutic agent, doxorubicin (DoxR), does not require the classical serine 32 and 36 phosphorylation or the PEST domain of IκBα. Degradation of IκBα is partially blocked by phosphatidylinositol 3-kinase inhibitor LY294002 and is mediated by the proteasome. Free NF-κB generated by DoxR-induced IκB degradation in IKK1/2−/− cells is able to activate chromatin based NF-κB reporter gene and expression of the endogenous target gene, IκBα. These results also imply that modification of NF-κB by IKK1 or IKK2 either prior or subsequent to its release from IκB is not essential for NF-κB-mediated gene expression at least in response to DNA damage. In addition, DoxR-induced cell death in IKK1/2−/− MEFs is enhanced by simultaneous inhibition of NF-κB activation by blocking the proteasome activity. These results reveal an additional pathway of activating NF-κB during the course of anticancer therapy and provide a mechanistic basis for the observation that proteasome inhibitors could be used as adjuvants in chemotherapy.
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Tanajura, Luiz, Áurea Chaves, Rafaela Freitas, Ana Barbosa, Kelvyn Vital, and José Delamain. "Aspirin versus P2Y12 inhibitors for secondary prevention after percutaneous coronary intervention." Journal of Transcatheter Interventions 31 (December 14, 2023): 1–7. http://dx.doi.org/10.31160/jotci202331a202304.
Анотація:
Currently, percutaneous coronary intervention with a drug-eluting stent implantation is the main method of myocardial revascularization in tertiary care hospitals, regardless of the clinical presentation of coronary artery disease. It is well known that to be effective, it requires the use of a dual antiplatelet therapy, which is a combination of acetylsalicylic acid and a P2Y12 platelet receptor inhibitor, which plays a key role in preventing thromboses after endoprosthesis implantation and is also indicated to prevent atherothrombotic events in the late clinical course, regardless of the stent model used. After a variable period of time, depending on some factors, such as the clinical presentation of coronary artery disease and the type of stent implanted, this therapy is discontinued, and the main current guidelines recommend interrupting the P2Y12 receptor inhibitor and maintaining acetylsalicylic acid in the long term, as one of the main pharmacological measures for secondary prevention of atherosclerosis. However, recently, due to their greater antiplatelet potency and probable lower potential for significant bleeding, especially in the digestive tract, P2Y12 inhibitors have been considered a valid and attractive option as an antiplatelet agent for long-term use; but this alternative has not been endorsed by guidelines yet. This review discusses the details related to this important decision that must be made by cardiologists when discontinuing the different antithrombotic therapies initially used after percutaneous coronary intervention. In principle, the scarcity of conclusive and normative clinical studies, especially in the population treated by percutaneous intervention, means that acetylsalicylic acid is the only antiplatelet agent with class I indication for secondary prevention of atherosclerosis.
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Vanzyl, Erin J., Hadil Sayed, Alex B. Blackmore, Kayleigh R. C. Rick, Pasan Fernando, and Bruce C. McKay. "The spliceosome inhibitors isoginkgetin and pladienolide B induce ATF3-dependent cell death." PLOS ONE 15, no. 12 (December 28, 2020): e0224953. http://dx.doi.org/10.1371/journal.pone.0224953.
Анотація:
The spliceosome assembles on pre-mRNA in a stepwise manner through five successive pre-spliceosome complexes. The spliceosome functions to remove introns from pre-mRNAs to generate mature mRNAs that encode functional proteins. Many small molecule inhibitors of the spliceosome have been identified and they are cytotoxic. However, little is known about genetic determinants of cell sensitivity. Activating transcription factor 3 (ATF3) is a transcription factor that can stimulate apoptotic cell death in response to a variety of cellular stresses. Here, we used a genetic approach to determine if ATF3 was important in determining the sensitivity of mouse embryonic fibroblasts (MEFs) to two splicing inhibitors: pladienolide B (PB) and isoginkgetin (IGG), that target different pre-spliceosome complexes. Both compounds led to increased ATF3 expression and apoptosis in control MEFs while ATF3 null cells were significantly protected from the cytotoxic effects of these drugs. Similarly, ATF3 was induced in response to IGG and PB in the two human tumour cell lines tested while knockdown of ATF3 protected cells from both drugs. Taken together, ATF3 appears to contribute to the cytotoxicity elicited by these spliceosome inhibitors in both murine and human cells.
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Mukherjee, Kaustav, Bruce Futcher, and Janet Leatherwood. "mmi1 and rep2 mRNAs are novel RNA targets of the Mei2 RNA-binding protein during early meiosis in Schizosaccharomyces pombe." Open Biology 8, no. 9 (September 2018): 180110. http://dx.doi.org/10.1098/rsob.180110.
Анотація:
The RNA-binding protein Mei2 is crucial for meiosis in Schizosaccharomyces pombe. In mei2 mutants, pre-meiotic S-phase is blocked, along with meiosis. Mei2 binds a long non-coding RNA (lncRNA) called meiRNA, which is a ‘sponge RNA’ for the meiotic inhibitor protein Mmi1. The interaction between Mei2, meiRNA and Mmi1 protein is essential for meiosis. But mei2 mutants have stronger and different phenotypes than meiRNA mutants, since mei2Δ arrests before pre-meiotic S, while the meiRNA mutant arrests after pre-meiotic S but before meiosis. This suggests Mei2 may bind additional RNAs. To identify novel RNA targets of Mei2, which might explain how Mei2 regulates pre-meiotic S, we used RNA immunoprecipitation and cross-linking immunoprecipitation. In addition to meiRNA, we found the mRNAs for mmi1 (which encodes Mmi1) and for the S-phase transcription factor rep2 . There were also three other RNAs of uncertain relevance. We suggest that at meiotic initiation, Mei2 may sequester rep2 mRNA to help allow pre-meiotic S, and then may bind both meiRNA and mmi1 mRNA to inactivate Mmi1 at two levels, the protein level (as previously known), and also the mRNA level, allowing meiosis. We call Mei2–meiRNA a ‘double sponge’ (i.e. binding both an mRNA and its encoded protein).
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Greene, Teshell K., Li Zhai, Beatrice Razzo, Karen Vo, Denise E. Sabatino, Rodney M. Camire, Valder R. Arruda, and Mortimer Poncz. "Platelet Factor VIII-Induced Megakaryocyte Apoptosis: Implications for Hemophilia A Gene Therapy." Blood 120, no. 21 (November 16, 2012): 2051. http://dx.doi.org/10.1182/blood.v120.21.2051.2051.
Анотація:
Abstract Abstract 2051 Hemophilia A is an X-chromosome-linked bleeding disorder due to a deficiency in clotting factor (F) VIII, affecting ∼1:5000 males. Inhibitor development to circulating FVIII is not an uncommon complication for patients receiving protein replacement therapy. We had proposed that the ectopic expression of FVIII in platelet alpha-granules will be targeted to sites of vascular injury, and be protected from circulating FVIII inhibitors. We and others have confirmed that this platelet (p)FVIII is in fact effective in a number of hemostatic models and is protected from circulating inhibitors. However, pFVIII has different temporal and spatial availability from plasma FVIII that may underlie the limited efficacy of pFVIII observed in some hemostatic models using FVIIInull mice. No group has been able to achieve levels greater than the equivalent of ∼200 mU/ml of whole blood pFVIII levels using standard human (h) B-domainless FVIII (hBFVIII). We therefore sought to improve pFVIII efficacy by expressing higher levels and/or by using pFVIII species with increased activity. We tested canine (c) BFVIII because of its reported higher efficacy in vitro and in mice, and because it is secreted at 1.5–2 fold HIGHER levels than hBFVIII in cell lines. We found that the level of pcBFVIII protein in Megs was actually ∼3-fold LOWER than phBFVIII. In spite of this lower level, pcBFVIII corrected the bleeding diathesis in FVIIInull mice more effectively than phBFVIII. Using Megs from transgenic FVIIInull mice that expressed pcBFVIII or phBFVIII, we found that mRNA levels of the FVIIIs were comparable. However, compared to littermate pFVIIInull mice, FVIIInull mice that expressed either pFVIII had lower ploidy Megs and these Megs showed higher levels of apoptosis. This apoptosis was enhanced by increasing the pFVIII level in the Megs by exposure to sodium butyrate. These effects were more marked in the pcBFVIII- than phBFVIII-expressing Megs. We then tested whether this phenomenon was limited to murine Megs, but found that lentiviral expression of pBFVIII increased Meg apoptosis whether the Megs were derived from mice, canine or human marrow, and that in each, levels of pcBFVIII were ∼3-fold lower than phBFVIII. In vivo studies support these in vitro apoptotic effects of pFVIII on Megs: In transgenic mice, steady-state platelet counts were normal in pFVIII mice, but TPO levels were 4-times higher in the phBFVIII and 8-times higher in the pcBFVIII compared to littermate controls (p<0.02 and p<0.0002 for phBFVIII/FVIIInull and pcBFVIII/FVIIInull vs. FVIIInull, respectively). In FVIII lentiviral transfected marrow/bone marrow transplant (lenti/BMT) studies, platelet counts did not differ in recovering recipient mice to negative controls, but both pBFVIII-expressing recipient lenti/BMT mice showed a peak in recipient platelets contributing to the total platelet mass at 4 weeks that decreased over the following month. This was especially true for the pcBFVIII-expressing lenti/BMT mice wherein >12% of the platelets were recipient-derived at 4 weeks compared to <2% in mice receiving a control lentivirus-transfected marrow (p<0.002). Thus, these studies suggest that there is a limit as to how much pFVIII can be stored in platelets, so efforts should be focused on increasing pFVIII activity. As an example of this approach, we had identified an R1645H Furin/PACE cleavage site difference between hBFVIII and cBFVIII that increases the level of single-chain to two-chain FVIII released from cell lines. The more single-chain FVIII, the slower the loss of FVIII activity, and the more overall activity was seen. We introduced this R1645H substitution into phBFVIII Megs and show antigen levels of this phBFVIIIR1645H comparable to phBFVIII in vitro and in lenti/BMT-reconstituted FVIIInull mice. Excitingly, these phBFVIIIR1645H lenti/BMT FVIIInull mice demonstrated normalization of hemostasis in several hemostatic models. Since pFVIII tends to be released deep inside a growing thrombus from degranulating platelets with limited washout, a more stable version of FVIII such as FVIIIR1645H may be particularly efficacious as pFVIII, and we intend to pursue this hFVIII variant in larger animal studies as it may be especially useful for clinical gene therapy. Disclosures: No relevant conflicts of interest to declare.
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Kariya, Shin, Mitsuhiro Okano, Katsuya Aoji, Michiya Kosaka, Emiko Chikumoto, Hisashi Hattori, Koji Yuen, Shinji Nishioka, Keiko Nishioka, and Kazunori Nishizaki. "Role of Macrophage Migration Inhibitory Factor in Otitis Media with Effusion in Adults." Clinical Diagnostic Laboratory Immunology 10, no. 3 (May 2003): 417–22. http://dx.doi.org/10.1128/cdli.10.3.417-422.2003.
Анотація:
ABSTRACT Otitis media with effusion (OME) is one of the most common ear diseases. Bacterial endotoxins and several inflammatory cytokines appear to be involved in the pathogenesis of OME in children; however, little is known of the immunological aspects of the onset of OME in adults. We sought to determine the presence of macrophage migration inhibitory factor (MIF) as well as interleukin 1β (IL-1β), tumor necrosis factor alpha (TNF-α), RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), and endotoxin in middle ear effusions (MEEs) from adult patients with OME. In addition, the levels of MIF in MEEs from adults and children were compared. MEE was obtained from 95 adults and 11 children. The levels of MIF, IL-1β, TNF-α, and RANTES were determined by enzyme-linked immunosorbent assay, and the concentrations of endotoxin and total protein were determined by the Endospec assay and bicinchoninic acid assay, respectively. MIF was detected in 97.9% of the MEEs from adults, while endotoxin, IL-1β, TNF-α, and RANTES were detected in 96.8, 12.6, 5.3, and 43.9%, respectively. In addition, the level of MIF was significantly higher than those of endotoxin, IL-1β, and TNF-α. A positive correlation between the levels of MIF and endotoxin was observed. MIF and endotoxin were detected in 81.8 and 72.7%, respectively, of the MEEs from the children. The level of MIF was significantly higher in the children, and conversely that of endotoxin was significantly higher in the adults. These results suggest that the interaction between MIF and endotoxin may promote fluid collection in the middle ear, particularly in adults.
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ÇAKMAK, Reşit, Ercan ÇINAR, Eyüp BAŞARAN, and Mehmet BOĞA. "SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL EVALUATION OF ESTER DERIVATIVES OF 4-(DIETHYLAMINO)SALICYLALDEHYDE AS CHOLINESTERASE, AND TYROSINASE INHIBITORS." Middle East Journal of Science 7, no. 2 (December 30, 2021): 137–44. http://dx.doi.org/10.51477/mejs.947973.
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Luciana, Luciana, Firdha Senja Maelaningsih, and Diah Permata Sari. "PENGGUNAAN PROTON PUMP INHIBITOR PADA PASIEN RAWAT JALAN DI RSIA DHIA CIPUTAT TANGERANG SELATAN." Edu Masda Journal 6, no. 1 (April 18, 2022): 36. http://dx.doi.org/10.52118/edumasda.v6i1.151.
Анотація:
Proton Pump Inhibitor are widely used in the treatment of peptic acid transmitters. The mechanism of action involves the inhibition of the hk-adenosime study was to determine how much use of proton pump inhibtors in outpatients in RSIA Dhia in July-December 2017. This study is non experimental with retrorespective data collection and analyzed with descriptive quantitative, sample taken by simple random sampling method. The sample obtained was 86. The results showed that the use of proton pum inhibitors was used more by women with a percentage of 62,8%. The use of this drug is also widely used by patients aged 26-45 years with a percentage of 46,5%. The accurary of the dosage that is in accordance with the literature standard is 65%. The most common diagnosis in this tudy was dyspepsia with a result of 54,7%. And the concomitant drug that is often used is sucralfat with a yield of 12%. It is suggested to RSIA Dhia to improve the complemeteness of medical record data to facilitater search.ABSTRAKProton Pump Inhibitor banyak digunakan dalam pengobatan penyalit asam peptic. Mekanisme kerjanya melibatkan penghambatan enzim hk-adenosime trifosfate yang ada dalam sel pariental mukosa lambung. Tujuan penelitian ini adalah untuk mengetahui berapa banyak penggunaan Proton Pump Inhibitor pada pasien rawat jalan di RSIA Dhia. Penelitian ini bersifat non eksperimental dengan pengambilan data secara retrospektif dan dianalisa dengan deskriptif kuantitatif, sampel diambil dengan metode simple random sampling. Sampel yang didapat adalah 86. Hasil penelitian menunjukan bahwa penggunaan Proton Pump Inhibitor lebih banyak digunakan oleh perempuan dengan presentase 62,8%. Penggunaan obat ini juga banyak digunakan oleh pasien dengan rentang usia 26-45 tahun dengan presentase sebanyak 39,5%. Jenis obat Proton Pump Inhibitor yang sering digunakan adalah omeprazole dengan hasil presentase 46,5%. Ketepatan dosis yang sudah sesuai dengan standar literature adalah 65%. Diagnose yang paling banyak dalam penelitian ini adalah dyspepsia dengan hasil 54,7%. Dan obat penyertayang sering digunakan adalah sucralfat dengan hasil 12%. Kepada RSIA Dhia disarankan lebih meningkatkan kelengkapan data rekam medis agar memudahkan dalam penelitian.
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Sealover, Nancy E., and Robert L. Kortum. "Abstract 413: WT RAS signaling is an essential therapeutic target in RAS mutated cancers." Cancer Research 82, no. 12_Supplement (June 15, 2022): 413. http://dx.doi.org/10.1158/1538-7445.am2022-413.
Анотація:
Abstract Single-agent targeting of RAS-mutated cancers using RAF/MEK/ERK or PI3K/AKT pathway inhibitors is ineffective; blocking one pathway relieves negative feedback control of RTK signaling and hyperactivates parallel effector pathways. While combined MEK and PI3K inhibition is effective in preclinical models, toxicity of this combination prevents its clinical use. Alternative strategies for treating RAS-mutated cancers are essential. Therapeutics directly targeting mutated RAS proteins have the potential to spare normal cellular function thereby lessening overall toxicity. Unfortunately, direct RAS inhibition as a monotherapy does not fully block RAS effector signaling. RAS proteins show differential activation of RAF and PI3K pathways: KRAS potently activates RAF but poorly activates PI3K, whereas HRAS potently activates PI3K but poorly activates RAF. Further, mutant RAS inhibition relieves negative feedback controls leading to rapid hyperactivation of RTK−WT RAS signaling. A more comprehensive understanding of the interplay between mutant RAS and RTK−WT RAS signaling is essential to developing rational therapeutic approaches to treat RAS-mutated cancers. We found that WT RAS enhanced mutant RAS-driven transformation by activating RAS effectors poorly engaged by mutated RAS. Further, inhibition of RAS effectors activated poorly by mutant RAS synergized with and limited resistance to mutant HRAS and KRAS inhibitors. The mutant HRAS inhibitor tipifarnib blocked PI3K signaling and synergized with MEK inhibitors in HRAS-mutated cancer cell lines; covalent KRASG12C inhibitors blocked MEK signaling and synergized with PI3K inhibitors in KRASG12C-mutated cell lines. Synergy between inhibitors of mutant RAS and RAS effectors was dependent on intact RTK/WT RAS signaling and was lost in both RASless or SOSless MEFs. Upstream of RAS, the RASGEFs SOS1 and SOS2 showed unique and overlapping functions to promote mutant RAS-driven transformation. SOS1 was critical for mutant RAS (G12V) activation and SOS1 inhibition augmented the efficacy of mutant RAS inhibitors. RTK−SOS2−PI3K signaling protected cells from anoikis and mediated mutant KRAS-driven transformation. These results should inform the design of clinical trials for patients with RAS-mutated cancers. Citation Format: Nancy E. Sealover, Robert L. Kortum. WT RAS signaling is an essential therapeutic target in RAS mutated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 413.
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Minami, Kahori, Yukihiro Tambe, Ryosuke Watanabe, Takahiro Isono, Masataka Haneda, Ken-ichi Isobe, Toshiyuki Kobayashi, et al. "Suppression of Viral Replication by Stress-Inducible GADD34 Protein via the Mammalian Serine/Threonine Protein Kinase mTOR Pathway." Journal of Virology 81, no. 20 (August 1, 2007): 11106–15. http://dx.doi.org/10.1128/jvi.01063-07.
Анотація:
ABSTRACT GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2α nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense.