bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeting MEK/MAPK Signal Transduction Module Potentiates Arsenic Trioxide (ATO)-Induced Apoptosis in Multiple Myeloma Cells through Multiple Signaling Pathways.
Authors: Lunghi Paolo , Giuliani Nicola , Mazzera Laura , Morandi Francesca , Salvatore Luigi , Mangoni Marcellina , Rizzoli Vittorio , Bonati Antonio 
Published: April 2020

Abstract
Multiple Myeloma (MM) cells are extremely resistant to apoptosis and currently new potential drug combinations are under investigation. We have shown that the combined treatment with the MEK1/2 inhibitor PD184352 (PD) and Arsenic Trioxide (ATO) resulted in the synergistic (Combination Index &lt;1.0) induction of apoptosis in 7 human myeloma cell lines (HMCLs: XG1, XG6, OPM2, JJN3, RPMI, H929, Sultan) analyzed, irrespective of their p53 status. The combined treatment was also a highly potent inducer of apoptosis and mitochondrial damage in the majority of the primary multiple myeloma (MM) cell samples ex vivo analyzed at different disease stage (9 out of 12). Growth factors, IL-6 or insulin-like growth factor 1 (IGF-1), or a co-culture system with bone marrow stromal cells (BMSCs) failed to confer resistance to this combination regimen. The combination PD/ATO had a minimal effect on normal B cells in vitro. By investigating the molecular mechanisms involved in MM cells PD/ATO-induced apoptosis, we found that co-treatment with PD strikingly elevated the (DR4+DR5)/(DcR1+DcR2) tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors ratio, caspase-8 activation, Bid fragmentation, mitochondrial depolarization and caspase-9 activation of ATO-treated HMCLs that do not have a functional p53 pathway. In HMCLs carrying a functional p53 pathway, the treatment with PD greatly enhanced the ATO-induced p53 accumulation (two fold increase) and p73, a p53 paralogue, cooperated with p53 in the pro-apoptotic p53/p73 target genes up regulation, caspase-9, -3 activation and apoptosis induction; in these HMCLs the selective down-regulation of p53 or p73 demonstrated that both have a biological relevance in PD/ATO-induced caspase-3 activation, PARP fragmentation and apoptosis. In HMCLs carrying a functional p53 the extrinsic caspase-8 mediated pathway was partially activated by PD/ATO treatment. We also demonstrated that, in MM cells carrying or not a functional p53 pathway, the combined treatment PD/ATO increased the level of the pro-apoptotic Bim (PD-mediated) and decreased its neutralizing anti-apoptotic protein Mcl-1 (ATO-mediated). The selective down-regulation of Bim significantly diminished caspase-8/-9/-3 cleavage/activation, PARP fragmentation and apoptosis of PD/ATO-treated MM cells, thereby indicating that Bim can play an important role not only in the intrinsic mitochondrial programmed cell death but also in the extrinsic caspase-8 mediated pathway. Accordingly, a physical interaction between Bim and DR4/DR5 TRAIL receptors in PD/ATO-treated MM cells carrying a non functional p53 was found by coimmunoprecipitation and Western blot studies. Our experiments have enlightened some relevant mechanisms that explain the apoptotic response of myeloma cells to ATO plus MEK inhibitor combination.
Lineage Distribution of JAK2 Exon12 Mutations and JAK2-V617F in Patients with Polycythemia Vera
Authors: Li Sai , Kralovics Robert , De Libero Gennaro , Tichelli Andre , Skoda Radek C. 
Published: April 2020

Abstract
Mutations in exon 12 of the JAK2 gene have been identified in a minority of patients with myeloproliferative disorders (MPD) and are associated with a selective increase in erythropoiesis resulting in polycythemia vera (PV) or idiopathic erythrocytosis. We compared the lineage distribution of JAK2 mutations in the peripheral blood of 8 PV patients with mutations in exon 12 and 21 PV patients with the JAK2-V617F mutation. Five different exon 12 mutations were observed in the 8 patients studied. Peripheral blood cells were fractionated to obtain granulocytes, platelets and mononuclear cells, which were further separated by FACS into T cells, B cells, NK cells and monocytes. Using a sensitive and quantitative assay to assess the percentages of chromosomes carrying exon 12 mutations, we detected exon 12 mutations in purified granulocytes, monocytes and platelets of all patients studied. A similar distribution was also found for the JAK2-V617F mutation. Exon 12 mutation was absent in sorted lymphoid cells of 5/8 patients, in 2/8 patients only NK cells were positive and in 1/8 patients also B cells carried the mutation. Exon 12 mutations were absent in T cells of all patients studied and JAK2-V617F was present in T cells of only 1/21 patients. Thus, inter-individual differences are notable in the involvement of lymphoid lineages for both exon 12 and JAK2-V617F mutations. To determine the presence of the JAK2 mutations in the erythroid lineage, we performed colony assays in methylcellulose, picked single erythroid colonies grown in the presence or absence of Epo and determined the allelic ratios for each individual colony. In 4/8 patients an exon 12 mutation (E543-D544del) was present in all EECs examined and similarly, in 8/12 patients the JAK2-V617F mutation was found in all EECs. In the remaining patients, we detected some EECs with only the wild type JAK2, suggesting that additional clonal events may also be present in some patients with exon 12 mutations. Interestingly, one patient carried exon 12 and JAK2-V617F mutations. None of the erythroid colonies in this patient carried both mutations simultaneously, indicating that the exon 12 mutation and JAK2-V617F represent two separate clones. One patient displayed erythroid colonies homozygous for the exon 12 mutation and an allelic ratio greater that 50% in granulocytes, indicating that progression to homozygosity can occur in some patients. The lineage distributions of exon 12 mutations and JAK2-V617F are similar and do not explain why exon 12 mutations are associated solely with PV phenotype, whereas JAK2-V617F can also cause essential thrombocythemia or primary myelofibrosis.
Kras

G12D
Expression in Hematopoietic Stem/Progenitor Cells Initiates T Cell Acute Lymphoblastic Leukemia/Lymphoma.
Authors: Dail Monique , Li Qing , Lauchle Jennifer , McDaniel Andrew , Kim Doris , Akagi Keiko , Wolff Linda , Jenkins Nancy , Copeland Neil , Shannon Kevin 
Published: April 2020

Abstract
T cell acute lymphoblastic leukemia/lymphoma (T-ALL/L) is characterized by clonal expansion of immature T lymphocytes that demonstrate extensive proliferation and tissue infiltration. RAS proto-oncogenes are mutated in ∼10% of human T-ALL/Ls, and elevated levels of active Ras•GTP have been obsereved in 50% of these cancers, suggesting that Ras signaling is deregulatd by multiple genetic mechanisims (Clin. Cancer Res.6, 1804, 2000). We are performing retroviral insertional mutagenesis in Mx1-Cre, KrasG12D mutant mice, that have a myeloproliferative disorder (PNAS101, 597, 2004) to identify genes that cooperate in vivo with hyperactive Ras in the genesis of hematologic malignancies. Unexpectedly, we observe a high incidence of T-ALL/L that is both transplantable and immunphenotypically resembles T-ALL/Ls found in human patients. We have cloned retroviral integration sites from 25 tumors to identify genetic lesions that cooperate with KrasG12D in lymphomagenesis and have identified IKZF1 and AHI1 as common insertion sites in 26% and 15% of tumors, repectively. IKZF1 encodes a zinc finger transcription factor that is required for murine lymphocyte development and is deregulated in human T-ALL/L and AHI1 encodes a scaffolding protein previously implicated in both murine and human leukemias. Furthermore, these T-ALL/Ls frequently aquire mutations in the NOTCH1 gene that are independent of retroviral integration. Interestingly, KrasG12D T-ALL/Ls have variable activation of the MAP Kinase pathway. We have screened cell lines generated from these T-ALL/Ls for sensitivity to γ-secretase and MEK inhibitors and found that biochemical activation predicts drug sensitivity and results in a therapeutic index. We are using multiparameter flow cytometry in conjunction with proliferation assays to idenitfy additional biochemical targets important for tumor proliferation and survival. Our studies, which are in agreement with studies of human T-ALL/L, implicate hyperactive Ras in T cell tumorigenesis and provides a platform for identifying novel cooperating genes and for testing novel therapeutic strategies.
Adaptor Protein Lnk Negatively Regulates Mutant MPL and JAK2 Alleles Associated with Myeloproliferative Disorders
Authors: Gery Sigal , Gueller Saskia , Sohn Julia , Nabavinouri Shayne , Leiter Amanda , Koeffler H. Phillip 
Published: April 2020

Abstract
Activating mutations in the cytokine receptor/JAK2 signaling axis are found at high frequency in myeloproliferative disorders (MPD). Lnk, an SH2-containing adaptor protein, is a negative regulator of several hematopoietic cytokine receptors including MPL and EpoR. Here, we assessed whether Lnk can attenuate the activity of mutant MPLW515L, JAK2V617F and JAK2K539L found in MPD patients. Lnk overexpression in Ba/F3-MPLW515L cells inhibited cytokine-independent growth, while suppression of Lnk in UT7-MPLW515L cells enhanced proliferation. Lnk-mediated growth inhibition was associated with downregulation of JAK/STAT, MAPK and PI3K signaling pathways. Similarly, Lnk inhibited cytokine-independent growth conferred by JAK2V617F and JAK2K539L in Ba/F3-EpoR cells. Following thrombopoietin stimulation, Lnk became tyrosyl-phosphorylated and associated with activated wild-type (WT) MPL and MPLW515L at the plasma membrane of Ba/F3 cells. An SH2 mutant Lnk (R392E) failed to bind and inhibit WT MPL and MPLW515L, demonstrating that the SH2 domain is essential for Lnk down-modulation of the receptors. The Lnk-MPL interaction was also detected with endogenously expressed proteins from cultured bone marrow cells. A series of C-terminally truncated Lnk constructs were used to determine which Lnk regions are required for Lnk inhibition of JAK2. Experiments in 293T cells indicated that Lnk SH2 domain binds to phosphorylated JAK2. In addition, other Lnk regions associated with non-phosphorylated JAK2, and these interactions were critical for Lnk inhibition of JAK2V617F and JAK2K539L constitutive activation. Our data suggest a model wherein Lnk downregulation of the receptor/JAK2 signaling involves two mechanisms; one is inhibition of the cytokine receptor utilizing JAK2, the second is direct suppression of JAK2 kinase activity. Furthermore, while the receptor mediated inhibition requires the SH2 domain, direct inhibition of JAK2 mutants, V617F and K539L, relies on other Lnk domains. Further elucidating the molecular mechanisms underlying Lnk inhibition of signaling pathways abnormally activated by oncogenic alleles, will provide insight into the pathogenesis of MPD and may have therapeutic value.
Erythropoiesis in Polycythemia Vera Is Hyper-Proliferative and Has Accelerated Differentiation during In Vitro Erythroid Expansion and Is Associated with Higher Expressions of cMYB and EPOR at Early Erythroid Progenitors
Authors: Bruchova Hana , Yoon Donghoon , Agarwal Archana , Otahalova Eva , Kim Hyojin , Prchal Josef T. 
Published: April 2020

Abstract
Erythroid differentiation is a dynamic process leading to the production of mature red blood cells. Even small variations in this process may result in severe disease phenotype. To study this process, we used a three-phase erythroid expansion system to expand homogeneous erythroid progenitors (EPs) from peripheral blood mononuclear cells (PB-MNCs) (Bruchova H. et al, 2007, Exp. Hematology, in press). We then characterized the expanded EPs from polycythemia vera (PV) patients and healthy donors at various points of maturation comparing cell proliferation and differentiation stage. EPs from PV patients outgrew controls up to day 14 (∼12 fold for PV and ∼4 fold for control compared to day 1). Differentiation was analyzed using both FACS analysis (with CD71/CD235a staining) and morphological evaluation (Wright-Giemsa staining), and demonstrated a more rapid differentiation of PV EPs when compared to controls up to day 14. We then evaluated apoptosis/cell cycle analysis by propidium iodide staining. Although PV EPs contained larger S phase population (45%) than controls (34%) at day 11, the apoptosis proportion of PV EPs was increased ∼2 fold to control from day 14. To understand the molecular mechanism of these differences between PV and controls, we analyzed the gene expression of several known regulators in erythropoiesis - BCL2, EPOR, cMYB, p27. Two transcripts (EPOR and cMYB) showed unique profiles on PV EPs. The EPOR transcript increased earlier in PV; i.e. from day 7 until day 21 and reached a plateau at day 11, compared to day 9 until day 19 and plateau at day 14 in controls. In addition, PV EPs contained higher levels of EPOR transcripts than control on most of timepoints. Interestingly, cMYB, which is known to augment early progenitor proliferation, was highly expressed from day 7 in PV, through day 11. Control EPs also expressed cMYB from day 9 through day 11; however, cMYB levels from any stages of control EPs were markedly lower than PV EPs at day 7. In this study, we demonstrate that PV erythropoiesis has unique features of hyperproliferation and an accelerated differentiation. These features are associated with earlier and higher expressions of cMYB and EPOR at the early stage of erythropoiesis.
A Novel Small-Molecule Approach To Inhibit Jak2 Tyrosine Kinase Signaling.
Authors: Kapuria Vaibhav , Bartholomeusz Geoffrey , Kong Ling-Yuan , Bornmann William , Peng Zhenghong , Pal Ashutosh , Maxwell David , Talpaz Moshe , Donato Nicholas 
Published: April 2020

Abstract
Jak kinases are non-receptor protein tyrosine kinases that play a pivotal role in cytokine/growth factor signaling through phosphorylation of specific proteins such as the Stat molecules. Activated Stats translocate to the nucleus where they mediate transcription of several target genes involved in cell cycle progression and survival (Bcl-xL, cyclin D1, c-myc, survivin. Many tumors have highly activated Stats that are associated with aberrant Jak2 regulation and recent studies have shown that activating mutations in Jak2 (V617F) play a key role in many myeloproliferative disorders such as polycythemia vera and essential thrombocythemia. Jak inhibitors may be useful in treating many diseases with aberrant Jak2/Stat signaling. The most commonly used inhibitor of Jak2 is the tyrphostin AG490, which inhibits Stat3 activation by preventing its tyrosine phosphorylation. However AG490 has limited in vivo efficacy and must be administered at high concentrations (&gt;50 μM) for anti-tumor effects. We describe here a new class of compounds, termed degrasyns, that block Jak2 mediated activation of Stat3 in intact cells at high nM to low μM concentrations. Degrasyns (WP1130/CP2005) did not directly inhibit Jak2 tyrosine kinase activity but suppressed Stat3 activation by reducing the cytoplasmic levels of Jak2. Degrasyn-mediated Jak2 down-regulation was rapid (complete in 2 hrs) and not inhibited by proteasomal, lysosomal, or serine/threonine protease inhibitors. Biochemical studies and confocal microscopy show that degrasyn induces translocation of Jak2 from the plasma membrane/cytosolic fraction into the cytoskeletal fraction and this altered partitioning of Jak2 was associated with loss of cytokine-mediated Stat activation by degrasyn. Jak2 translocation was associated with tyrosine phosphorylation of specific proteins which complex with Jak2. Lyn kinase in the cytoskeletal fraction was highly activated by degrasyn in multiple hematopoetic tumors (multiple myeloma, mantle cell lymphoma, leukemias). Jak2 translocation and Stat inhibition by degrasyn is mechanistically distinct from “classical” Jak2 inhibitors and is not associated with a translocation of other kinases or cytokine signaling molecules in the Jak2 cascade (IL-6R, gp130, Lyn, Btk, Hck, Akt, PI-3K, Erk, Src, Jak1). Degrasyn induces cytoskeletal translocation of both wild-type and mutant (V617F) Jak2 and was associated with induction of apoptosis in HEL cells expressing the Jak2 V617F mutation. These results suggest that degrasyn suppresses Jak/Stat signaling through a unique mechanism involving translocation of Jak2 into a signal transduction incompetent compartment and may be used to investigate a novel form of Stat suppression. Degrasyn may also have anti-tumor effects on cells with aberrant activation of Jak/Stat signaling.
PTP1B, Distinctively Expressed in GCB-Like and ABC-Like DLBCL, Is a New Regulator of IL-4-STAT6 Intracellular Signaling.
Authors: Lu XiaoQing , Malumbres Raquel , Tiganis Tony , Lossos Izidore S. 
Published: April 2020

Abstract
Gene expression profiling studies sub-classified diffuse large B-cell lymphomas (DLBCL) into two clinically distinct types: Germinal Center B cell (GCB)-like and Activated B-cell (ABC)-like tumors, characterized by long and short survival, respectively. We have recently reported distinct responsiveness of GCB-like and ABC-like DLBCL cell lines to IL-4 stimulation. In ABC-like lymphoma cells, STAT6 signaling is ameliorated due to enhanced STAT6 dephosphorylation resulting in non-inducibility of IL-4 target genes in these tumors. (Lu et al: Blood2005;105:2924). Furthermore, we have demonstrated that IL-4 may increase doxorubicin and complement-mediated rituxmab cellular cytotoxicity in GCB-like cell-lines, but protects the ABC-like DLBCL. At least some of the observed differences were attributed to the differential expression of TC-PTP - a new STAT6 nuclear protein tyrosine phosphatase (PTP) (Lu et al MCB2007;27:2166). PTP1B, another PTP highly expressed in ABC-like DLBCL, exhibits a 71% amino acid identity in the catalytic domain with TC-PTP. In contrast to TC-PTP that is mainly located in nuclei, PTP1B localizes to the endoplasmic reticulum. Herein, we demonstrate that PTP1B may serve as a physiological cytoplasmic PTP of phosphorylated STAT6. Over-expression of PTP1B leads to STAT6 dephosphorylation while PTP1B knock down augment STAT6 phosphorylation. We demonstrate interactions between endogenous PTP1B and STAT6 and delineate the STAT6 and PTP1B protein domains responsible for the interaction: the catalytic domain of PTP1B and the C terminal transactivation domain of STAT6. Furthermore, we demonstrate that PTP1B may also dephosphorylate JAK1 and JAK3. However, by using the PTK inhibitor, staurosporine, we show that the PTP1B effect on STAT6 is direct. We confirm a potential regulatory role of PTP1B on IL-4-STAT6 signaling in a cellular context by demonstrating that over-expression of PTP1B ameliorates IL-4-induced STAT6 reporter luciferase activity and IL-4 induced expression of its target genes. Furthermore, we delineate a new negative regulatory loop of IL-4-JAK-STAT6 signaling. We demonstrate that IL-4 induces PTP1B mRNA and enhances PTP1B protein stability. The effects of IL-4 on PTP1B mRNA are STAT6 independent and are mediated by PIK3 intracellular pathway. These observations further highlight the complexity of the intracellular regulation of cytokine-STAT signaling and may have important implications for both neoplastic and inflammatory processes. In summary, our findings identify STAT6 as a new target of PTP1B and suggest that distinct PTP expression profiles may contribute to different biological characteristics of GCB-like and ABC-like DLBCL.
Prognostic Utility of Extranodal Disease Involvement in Diffuse Large B-Cell Lymphoma in Patients Treated with R-CHOP.
Authors: Hui David , Donaldson Jane , Hoskins Paul , Klasa Richard , Savage Kerry J. , Shenkier Tamara , Gascoyne Randy D. , Connors Joseph M. , Sehn Laurie 
Published: April 2020

Abstract
Background: Extranodal status is one of the five key factors in the International Prognostic Index (IPI) for diffuse large B-cell lymphoma (DLBCL). We have re-examined the prognostic utility of extranodal involvement in DLBCL patients by determining whether the number and location of extranodal sites correlate with survival, particularly in the post-rituximab era.
Methods: Using the Lymphoid Cancer Database of the British Columbia Cancer Agency, all patients with a diagnosis of DLBCL were included between January 1979 and May 2006 except those who were less than age 18 at diagnosis, HIV positive, presented with disease in the testicular or central nervous system, had discordant indolent lymphoma at or before diagnosis, or did not receive any chemotherapy. Any degree of extranodal involvement was recorded as positive for extranodal disease and all sites of involvement were documented.
Results: 1803 patients were identified with the following characteristics: median age 62 (range 19–93), male 58%, stage III/IV 49%, elevated LDH 51% and performance status ≥2 34%. Of the 1268 patients treated without rituximab, 358 (28%) had only nodal disease, 532 (42%) had 1 extranodal site and 378 (30%) had ≥2 extranodal sites. Among 535 patients who received rituximab, 163 (30%), 217 (41%) and 155 (29%) had 0, 1 and ≥2 extranodal sites, respectively. Of the 37 coded extranodal sites, the most commonly involved sites included soft tissue (16.2%), bone (11.3%), bone marrow (9.1%), pleura (9.1%), head and neck soft tissue (8.2%), stomach (8.7%), intestine (8.2%), lung (7.8%) and liver (5.8%). In multivariate analysis that included other IPI variables, extranodal involvement as defined in the IPI (≥2 sites) was not a significant prognostic factor in our cohort (with or without rituximab, see Table 1). However, the presence of any extranodal disease was found to correlate with a poorer overall survival in patients treated with rituximab (p=0.03). When specific extranodal sites were examined in the rituximab group, bone marrow (p&lt;0.001), liver (p=0.03) and pleura (p=0.05) involvement correlated with inferior overall survival in univariate analysis, but only bone marrow involvement (HR=1.9, p=0.02) remained significant in multivariate analysis.
Conclusions: In the post-rituximab era, extranodal involvement remains a significant prognostic factor, although not as traditionally defined in the IPI, but rather refined as the presence of any extranodal disease involvement. Furthermore, bone marrow involvement represents the only extranodal site associated with a poorer overall survival independent of the IPI.
Table 1: Prognostic Significance of Extranodal Disease Involvement for Overall Survival
Prognostic Impact of Tumor Infiltrating FOXP3 Positive Regulatory T Cells in Diffuse Large B-Cell Lymphoma at Diagnosis.
Authors: Kwak Jae-Yong , Lee Na-Ri , Song Eun-Kee , Jang Kyu Yun , Choi Ha Na , Moon Woo Sung , Kwon KeunSang , Lee Ju-Hyung , Yim Chang-Yeol 
Published: April 2020

Abstract
Tumor infiltrating immune cells play an important role in host immune reactions against diffuse large B-cell lymphoma (DLBCL). In the present study, we have identified a subset of tumor infiltrating FOXP3-positive regulatory T cells (Tregs) in the initial DLBCL biopsy specimens and evaluated its prognostic significance. Ninety-six patients with DLBCL were retrospectively evaluated. Expression pattern of FOXP3 protein was examined using standard immunohistochemistry in paraffin-embedded tissue samples. The median overall survival (OS) was 28 months. Compared to the others, the patients with higher percentage of FOXP3-positive Tregs in the initial tumor biopsy, showed a significantly longer OS (p=0.003). When prognostic factors were evaluated in a multivariate model, the international prognostic index and the percentage of infiltrating FOXP3-positive Tregs in the initial biopsy were shown to be independent predictors of OS. In conclusion, the presence of increased percentage of FOXP3-positive Tregs in DLBCL predicts a better prognosis.
Figure Figure