bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hydroxamate Pan-HDAC Inhibitor LBH589 Depletes EZH2 and DNMT1, Partly through hsp90 Inhibition and Its Chaperone Association with DNMT1.
Authors: Fiskus Warren , Herger Bryan , Rao Rekha , Atadja Peter , Bhalla Kapil 
Published: October 2019

Abstract
The PRC (polycomb repressive complex) 2 is a multi-protein complex, which includes EZH2, SUZ12 and EED proteins. The PRC2 complex possesses histone methyltransferase (HMTase) activity mediated by the SET domain of EZH2, which methylates histone H3 on lysine (K)-27. We have previously reported that the treatment with the hydroxamate, pan-HDAC inhibitor LBH589 acetylates and inhibits the ATP binding and chaperone function of hsp90, as well as depletes the levels of EZH2, Suz12 and EED in AML cells (Blood106 (11) 2482, 2005). Recently, within the PRC2, EZH2 was shown to interact with and modulate the DNA methyltransferases DNMT1, DNMT3a and DNMT3b, which affects their binding to the EZH2-targeted gene promoters. Thus EZH2 may serve as a recruitment platform for DNA methyltransferases, thereby mechanistically linking the two epigenetic silencing systems, maintaining the chromatin in the silenced state and inhibiting gene expression. In the present studies we determined the effects of hydroxamate pan-HDAC inhibitor LBH589 on the interaction between the PRC2 proteins EZH2 and EED and DNMT1 in CML blast crisis (CML-BC) K562 and LAMA-84 cells. Treatment with ≥50 nM LBH589 for 24 hours disrupted the interaction between DNMT1 and Ezh2, as well as depleted the levels of DNMT1 in a dose and time dependent manner. LBH589 (≥50 nM) also disrupted the binding of DNMT1 to hsp90. This was associated with increased polyubiquitylation and accumulation of DNMT1 in the detergent insoluble fraction of the leukemia cells. Notably, similar findings were obtained following treatment with the hsp90 inhibitor 17-DMAG (250 nM). Taken together, these findings suggest that DNMT1 has chaperone association with hsp90, which is disrupted by LBH589. Additionally, RT-PCR analysis demonstrated that treatment with 100 nM LBH589 for 8 hours depleted the mRNA expression of DNMT1 in K562 cells, suggesting that both transcriptional and post-transcriptional mechanisms are involved in LBH589 mediated down regulation of DNMT1. Depletion of DNMT1 due to LBH589 treatment was associated with induction of JunB mRNA expression. This may be because JunB is repressed by DNA hypermethylation in CML-BC cells. A similar upregulation of JunB mRNA and protein in K562 cells also followed shRNA mediated depletion of DNMT1 (down to 40%). These findings indicate that treatment with pan-HDAC inhibitor LBH589 not only induces histone acetylation and depletes the levels of histone methyl transferase EZH2, but also down regulates DNMT1 through transcriptional mechanism and proteasome degradation. The latter effect may be due to LBH589 mediated acetylation and inhibition of chaperone function of hsp90, an effect also seen with treatment with 17-DMAG.
Constitutive Activation of Toll like Receptor Signaling in Adult T-Cell Leukemia Cells.
Authors: Mizobe Takamitsu , Tsukada Junichi , Higashi Takehiro , Mouri Fumihiko , Matsuura Ai , Tanaka Yoshiya 
Published: October 2019

Abstract
Human T-cell leukemia virus type I (HTLV-I) is etiologically associated with the development of an aggressive and fatal malignancy of CD4+ T lymphocytes called adult T-cell leukemia (ATL). Constitutive activation of nuclear factor-κB (NF-κB) is a common feature of ATL. Although the mechanism by which NF-κB is spontaneously activated in ATL cells still remains unclear, inhibition of NF-κB activity induces apoptosis, suggesting a central role of NF-κB in their proliferation. Toll like receptors (TLRs) are involved in innate cell activation by conserved structures expressed by microorganisms. Engagement of IL-1R or TLR with their cognate ligands causes an adaptor protein MyD88 to be recruited to the receptor complex, which in turn promotes its association with the IL-1R-associated kinase (IRAK) via an interaction between the respective death domains of each molecule. Several recent reports have indicated unique expression profiles of TLRs on different subsets of human T cells, and that some TLR ligands modulate the function of human T cells. We examined expression of the TLR mRNAs in primary ATL cells and ATL cell lines, MT2, MT4 and HUT102 by RT-PCR. Expression of TLR mRNAs, except of TLR7 and TLR8, was detected in all cell samples examined. We further demonstrated constitutive association of MyD88, an adaptor protein for the TLR signaling, with the IL-1R-associated kinase 1 (IRAK1) in ATL cell lines, MT2, MT4 and HUT102. In MT2 cells, constitutive activation of NF-κB and NF-IL6, but not Stat3 was significantly inhibited by expression of a dominant negative form of MyD88 protein (MyD88dn). Spontaneous transcriptional activation of IL-1α, IFN-γ and TNF-α gene promoters in MT2 cells was also suppressed by MyD88dn expression. MyD88dn inhibited cell proliferation and induced apoptosis of MT2 cells. In addition, overexpression of wild-type MyD88 and HTLV-I Tax induces synergistically transcriptional activity of NF-κB in 293T cells, showing interaction of Tax with MyD88. Thus, our results show a critical role of MyD88 in dysregulated gene activation and cell proliferation in HTLV-I-transformed T-cells, and further suggest the involvement of MyD88 in Tax-mediated intracellular signal transduction in HTLV-I-infected cells. Considering the fact that blocking NF-κB is a potential strategy to treat ATL, our argument raises a possibility that we may be able to find new treatment targets against ATL.
Imidazoacridinones Are Bifunctional Targeting Agents Active in Leukemia Cells.
Authors: Chau MyDoanh , Otake Yoko , Stuart Robert K. , Fernades Daniel J. , Ajami Alfred M. 
Published: October 2019

Abstract
C-1311 (Symadex™) is the lead compound in clinical development from a new series of agents, the imidazoacridinones. It was previously reported that C-1311 was solely a topoisomerase II (TOP2) cleavable complex inhibitor and DNA damaging agent. However, contemporary analyses of C-1311 point to alternative structural parallelisms with the molecular scaffolds now seen as common motifs in receptor tyrosine kinase (RTK) inhibitors. To investigate this mechanism of action, a kinase inhibitor screening program was initiated, first in silico by molecular similarity matching, and then in vitro against recombinant RTKs. C-1311 was identified to cluster with RTK inhibitors, especially when sunitinib is used as a molecular “seed” for pattern recognition. The statistical similarity coefficients fall in the 0.7–0.8 positive probability range, compared to 0.2–0.4 for the classical TOP2 inhibitors. C-1311 was found to selectively inhibit wild-type (WT) FLT3 and its D835Y mutant at nanomolar concentrations of 8–15 nM IC50. FGFR2 was also targeted at approximately 100-fold greater IC50 value of 1.2 μM. Inhibition of PDGFR, FGFR1, and KIT was observed at about 3–4 μM IC50; other prominent kinases had values of greater than 10 μM. As a part of the dual mechanism reexamination, TOP2 decatenation of kDNA, DNA relaxation, and competition assays were performed to assess C-1311 interaction with TOP2. Results confirmed that C-1311 is not a cleavable complex inhibitor when compared to etoposide, suggesting that it may not be an inducer of the MLL cleavage. Additionally, the IC50 of C-1311 from the decatenation assay is 44 μM compared to 3, 12, and 149 μM for mitoxantrone, daunorubicin, and merbarone, respectively. Cellular screens were performed to assess the potency of C-1311 in myeloid and lymphoid leukemia cell lines. C-1311 was equally effective in both WT FLT3 expressing (RS4;11) and mutant FLT3 expressing (MV-4–11) cell lines. The dual function of C-1311 may contribute to the potency in these cell lines. These findings warrant further investigation of C-1311 in yet unexplored indications, including other hematological malignancies.
Abnormalities in Glucose Uptake and Metabolism in Imatinib-Resistant Bcr-Abl Positive Cells.
Authors: Serkova Natalie J. , Kominsky Douglas J. , Brown Jaimi L. , Miljus Jelena , Boros Laszlo G. , Eckhardt Gail S. , Melo Junia V. 
Published: October 2019

Abstract
The development of imatinib resistance has become a significant therapeutic problem, in which the etiology appears to be multifactoral (Bcr-Abl mutation and/or overexpression, multidrug-resistance, protein overexpression, etc.) and poorly understood. As of today, there are no precise clinical criteria to predict the development of imatinib resistance in CML, other than relapse of the disease. However, there is evidence that the control of glucose-substrate flux is an important mechanism of the antiproliferative action of imatinib. Thus, imatinib-resistant gastrointestinal stromal (GIST) KIT-activated tumors reveal highly elevated glucose uptake in PET scans. Currently, there is no information about the changes in glucose cell metabolism during development of imatinib resistance in CML patients. We used nuclear magnetic resonance (NMR) spectroscopy and gas chromatography mass spectrometry (GC-MS) to assess 13C glucose uptake and metabolism (glycolysis, the TCA cycle, nucleic acid ribose synthesis) during imatinib treatment in cultured human CML cells with different sensitivity to imatinib. Our results demonstrate that imatinib-sensitive K562 and LAMA84 Bcr-Abl positive cells have decreased glucose uptake, decreased lactate production and improved oxidative TCA cycle upon imatinib treatment. The LAMA84-r (resistant to imatinib due to Bcr-Abl overexpression) and the K562-r (unknown mechanism of resistance) cells maintained high glycolytic metabolic phenotype with elevated glucose uptake and lactate production in the presence of imatinib (Figure 1). In addition, oxidative synthesis of RNA ribose from 13C-glucose via glucose-6-phosphate dehydrogenase was decreased and RNA synthesis via the non-oxidative transketolase pathway was increased in imatinib resistant cells. CML cells which exhibited an [oxidative/non-oxidative] flux ratio for nucleic acid ribose synthesis > 1, were sensitive to imatinib. The resistant K562-r and LAMA84-r exhibited the [oxidative/non-oxidative] flux ratio < 0.7. We correlated 13C glucose uptake and metabolism with intracellular expression and localization of GLUT-1 transporter protein in these cells. There was no difference in total protein load of GLUT-1 upon imatinib treatment between imatinib-sensitive and resistant cell lines. However, the changes in glucose uptake and metabolism were accompanied by intracellular trans-location of GLUT-1 from the plasma membrane to the cytoplasm in imatinib-sensitive treated cells, while GLUT-1 remained at the membrane surface in LAMA84-r and K562-r cells (Figure 1). In summary, elevated glucose uptake and non-oxidative glycolytic metabolic phenotype can be used as sensitive markers for imatinib resistance in Bcr-Abl positive cells with different origins of resistance.
Figure 1: Figure 1:.
VEGFR-1 (FLT-1), b1 Integrin and hERG K+ Channel Form a Macromolecular Signaling Complex in Acute Myeloid Leukemia Cells.
Authors: Arcangeli Annarosa , Pillozzi Serena , Brizzi Maria F. , Bernabei Pietro A. , Bartolozzi Benedetta , Caporale Roberto , Basile Venere , Boddi Vieri , Torre Eugenio , Pegoraro Luigi , Becchetti Andrea 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) is a heterogeneous group of clonal bone marrow (BM) malignancies, presenting a wide spectrum of morphologic, immunophenotypic, cytogenetic, and molecular features. There is general agreement that persistent detection of leukemia cells outside the BM microenvironment, especially when accompanied by infiltration into extramedullary sites, is a clear negative prognostic factor. Infiltration may also reduce leukemia responsiveness to induction chemotherapy. Great attention is thus being played to the molecular mechanisms that regulate acute leukemia cells exit from the BM. A crucial role is thought to be played by the angiogenic factors and angiogenesis-related signals, that operate inside the BM microenvironment.The persistence of circulating leukemia blasts and their engraftment into extramedullary sites are negative prognostic factors, in patients with acute leukemia. Leukemia cell exit from the bone marrow is regulated by angiogenic factors and in particular by the Vascular Endothelial Growth Factor and its receptor 1, FLT-1. We have studied the cross talk between
FLT-1, the human eag-related gene 1 (hERG1) K+ channels, integrin receptors, in acute myeloid leukemia (AML) cells.
FLT-1, hERG1 and the b1 integrin were found to form a macromolecular signaling complex. The latter mostly recruited the hERG1B isoform of hERG1 channels, and its assembly was necessary for subsequent FLT-1 tyrosine phosphorylation and sustained AML cell migration. What is more, both effects were inhibited when hERG1 channel were specifically blocked. The formation of an FLT-1/hERG1/b1 complex was also observed in primary AML blasts, obtained from a population of human patients. The coexpression of FLT-1 and hERG1 conferred a pro-migratory phenotype to these AML blasts. Such a phenotype was also observed in vivo. Herg1 positive blasts were more efficient in invading the peripheral circulation and the extramedullary sites after engraftment into immunodeficient mice. Moreover, herg1 expression in leukemia patients correlated with a shorter overall survival. Overall, we conclude that hERG1 channels mediate the FLT-1 dependent AML cell migration and that their expression is a negative prognostic factor, in AML.
C-Terminal Deletion Mutation of CD20 in B-Cell Malignancy Is Related to a Risk of Relapse and Resistance to Chimeric Anti-CD20 Antibody Therapy.
Authors: Terui Yasuhito , Mishima Yuji , Sugimura Natsuhiko , Kojima Kiyotsugu , Kuniyoshi Ryoko , Rokudai Akiko , Mishima Yuko , Yokoyama Masahiro , Takeuchi Kengo , Hatake Kiyohiko 
Published: October 2019

Abstract
Introduction: Rituximab was the first chimeric antibody that is most frequently used for CD20-posistive B-cell lymphomas and gives better response and prognosis. However, resistance to rituximab is one of the important issues to be clarified in the increasing number of monoclonal antibody therapy, and we experienced the case whose lymphoma cells never expressed CD20 at the relapsed phase during rithuximab therapy. We investigated whether CD20 mutation is related to expression level of CD20, relapse or resistance to rituximab therapy, and prognosis.
Methods: We analyzed 50 patients with fresh or relapsed/ resistant B- cell lymphomas and DNA sequencing analysis of CD20 products from genomic PCR and RT-PCR was performed. CD20 mutants were subcloned by TA cloning, and tested CD20 expression after transfection to K562 cells.
RESULTS: Four types of CD20 mutations were found in 11 of 50 NHL patients (22.0%), which include C-terminal deletion(8.0%) extracellular domain (2.0%) transmembrane domain (2.0%) and early termination (10.0%). The group of C-terminal deletion mutations significantly showed lower CD20 expression (3.26, 95%CI = 0.09 to 6.89) than non-mutation (30.8, 95%CI = 22.4 to 39.2) (p < 0.05), whereas five patients with early termination mutation did not show significant difference of CD20 expression (19.5, 95%CI = 10.7 to 28.4) as compared with non-mutation (p > 0.05). Although there was no significant difference between the groups with non-mutation and C-terminal deletion mutation in CR rate (49% versus 25%, Fisher’s exact test; p = 0.6137), time to progression after rituximab therapy in C-terminal deletion mutation (7 months, 95%CI = 0 to 18 months) was significantly shorter than that in non-mutation (31 months, 95%CI = 18 to 44 months) by log-rank test (p = 0.0481).
Conclusion: The important mutations of CD20 gene related to shorter duration to progression disease after rituximab therapy were discovered. Especially, point mutations bearing during rituximab therapy should be examined at progression disease after partial remission.
Rituximab-Mediated Inhibition of the p38 MAPK and NF-κB Survival Pathways In Vitro and Validation in Nude Mice Bearing the Raji B-NHL Tumor.
Authors: Vega Mario I. , Huerta-Yepez Sara , Martinez-Paniagua Melisa A. , Bonilla Cesar G. , Bonavida Benjamin 
Published: October 2019

Abstract
Treatment of B-NHL cell lines with rituximab inhibited the p38 MAPK and NF-κB pathways, resulting in chemosensitization to drug-induced apoptosis (Vega et al., 2004 Oncogene, 23:3530–40; Jazirehi et al., 2005 Cancer Res., 65:264–76). Chemosensitization was the direct result of inhibition by these pathways of the anti-apoptotic gene products Bcl-2/Bcl-xL. Cell signaling by rituximab in B-NHL patients has not been investigated and thus, we have examined an in vivo model bearing a tumor xenograft for validation. Balb/c nude mice were transplanted s.c. with the B-NHL cell line Raji; a group of mice was left untreated and another group was treated with rituximab (1000 μg) at days 5 and 10 following implantation of 8x106 tumor cells. The animals were monitored for tumor cell growth and sacrificed at day 30 and tumors were obtained for further analysis. Treatment with rituximab resulted in significant inhibition of tumor cell growth compared to control. Tumor tissues were examined by immunohistochemistry for phospho and non-phospho p38 MAPK and NF-κB (p50), Bcl-2 and Bcl-xL expression. Analysis of tumor-derived tissues from control mice revealed overexpression of phospho-p38 MAPK and NF-κB (p50) and strong nuclear localization of phospho-p50. In addition, there was overexpression of Bcl-2 and Bcl-xL. In contrast, tumor tissues derived from rituximab-treated mice demonstrated significant inhibition of phospho-p38 MAPK, phospho-p50-NF-κB and reduced nuclear localization of phospho-p50. In addition, Bcl-2 and Bcl-xL expression was also significantly reduced. These findings established for the first time, in a pre-clinical model, rituximab-mediated inhibition of the cell survival pathways mediated by p38 MAPK and NF-κB. In addition, the study also corroborates the role of Bcl-2 and Bcl-xL expression in resistance and their inhibition by rituximab. In a separate abstract, tumor tissues derived from many untreated B-NHL patients were analyzed by immunohistochemistry for the activation of both phospho-p38 MAPK and NF-κB and compared to biopsies derived from control individuals. Overall, this study suggests that the p38 MAPK and NF-κB survival pathways are targets for rituximab-mediated effects and also suggests that such targets can be used for intervention in cases of rituximab resistance.
The PIG-A Gene Is Hypermutable in Lymphoid Neoplasms.
Authors: DiTata Kimberly , Araten David J. 
Published: October 2019

Abstract
In neoplasms, the presence of chromosomal abnormalities and point mutations is suggestive of genomic instability, but could also be a consequence of selection. While genomic instability may increase the chances that a malignant population acquires adaptive mutations, extremely high mutation rates may not be compatible with cell survival. To investigate the role of genomic instability in lymphoid neoplasms, we have applied a new method for the quantification of the human mutation rate, using the PIG-A gene as a sentinel. PIG-A is essential for the biosynthesis of glycosylphosphatidylinositol (GPI) and is mutated in blood cells of patients with Paroxysmal Nocturnal Hemoglobinuria. A broad range of mutations can produce the GPI (−) phenotype, and because PIG-A is on the X-chromosome, the effect of a single mutation is detectable. Since a host of proteins require GPI for attachment to the cell surface, rare mutants are readily detected by flow cytometry. We have previously shown that PIG-A mutations arise spontaneously in normal donors, and we determined that the mutation rate in normal B cell lines ranges from 2 to 29 per 107 cell divisions. Here we analyzed cell lines derived from:
a transformed low grade lymphoma harboring a t(14;18) translocation; a mantle cell lymphoma harboring a t(11;14) translocation; a marginal zone lymphoma; and T cell ALL.
Cells were first stained with an antibody specific for CD59 (a representative GPI-linked protein) and pre-existing GPI (−) cells were eliminated from the population by flow cytometric sorting, by gating on the upper 50th percentile of the distribution curve. The collected GPI (+) cells were then returned to culture and the number of cell divisions (d) determined by cell counts. After 3–4 weeks, the frequency (f) of new mutants arising in culture was determined by flow cytometric analysis of a large number of cells (median 2.3 x 106). Cells were stained simultaneously with antibodies specific for at least 3 GPI-linked proteins (e.g. CD48, CD52, CD55, and CD59) as well as a transmembrane protein (e.g. HLA-DR or CD45) to identify live cells. FLAER and proaerolysin-- which bind to GPI-- were used to confirm the phenotype. The frequency of mutants was determined by the number of GPI(−) cells divided by the number of GPI(+) cells analyzed, and the mutation rate (μ) was calculated with the formula μ = f ÷ d. We demonstrated a high mutation rate in 3 out of the 4 cells lines: 1750 x 10−7 (transformed lymphoma), 335 x 10−7 (mantle cell lymphoma), 112 x 10−7 (T cell ALL). Of note, the mutation rate was normal (4 x 10−7) in the marginal zone lymphoma—consistent with this being an indolent neoplasm. These data support the hypothesis that an elevated mutation rate is part and parcel of aggressive neoplasms and demonstrate that a 2-log elevation in this parameter is compatible with cell survival. With this model, it may be possible to predict the development of mutations that confer chemotherapy drug resistance.