bluuebunny/crossref_metadata_2025 huggingface dataset:
Blocking Adhesion of Sickle Erythrocytes to Endothelial P-Selectin Using an RNA Aptamer.
Authors: Nishimura Jun-ichi , Burnette Angela D. , Oney Sabah , Batchvarova Milena , Delahunty Martha , Zennadi Rahima , Sullenger Bruce A. , Telen Marilyn J.
Published: April 2020

Vaso-occlusive crises are a major clinical feature of sickle cell disease (SCD), and the adhesion of sickle erythrocytes (SS-RBCs) to vascular endothelium is crucial to the generation of vaso-occlusion. SS-RBCs express many adhesion molecules. Adhesive SS-RBCs bind to endothelial cell P-selectin and other adhesion molecules, as well as extracellular matrix proteins. Thrombin causes endothelial retraction with exposure of proadhesive extracellular matrix components as well as upregulation of endothelial expression of P-selectin. Other investigators have shown that SS-RBCs can bind to P-selectin, thus suggesting that P-selectin is a suitable target against which to develop reagents to prevent or treat vaso-occlusive crises in SCD. Aptamers are oligonucleotides that bind to molecular targets in a manner conceptually similar to antibodies and have been identified against a wide range of therapeutic targets. RNA aptamers that bind to human P-selectin, inhibit P-selectin binding to its ligand sialyl Lewis X, and inhibit neutrophil-platelet adhesion have previously been identified (Antisense Nucleic Acid Drug Dev,8:265,1998). We have now tested one of these high-affinity P-selectin aptamers (clone PF377: ACGCUCAACGAGCCAGGAACAUCGACGUCAGCAAACGCGAGCGCAACCAGUAACACC) for its ability to prevent cellular adhesion of SS-RBCs to endothelial cells. Aptamer clone PF377 bound P-selectin with a Kd of 6nM and a Bmax of 87%. To measure its anti-adhesion activity, an in vitro flow chamber assay was adopted. The anti-adhesion activity of aptamer clone PF377 was confirmed using primary human umbilical vein endothelial cells (HUVECs) treated with IL-13 (50ng/mL) for 48hrs, followed by stimulation with histamine (25μM for 12mins at RT) immediately prior to a flow chamber assay. Aptamer clone PF377 at 60nM had anti-adhesion activity similar to heparin (a previously identified inhibitor of SS-RBC adhesion to P-selectin) and 9E1 (an inhibitory antibody to P-selectin), whereas Bi 9.3t (a non-functional aptamer to thrombin, used as a negative control, 60nM) and AC1.2 (a non-inhibitory antibody to P-selectin), did not prevent adhesion (Figure). Normalized % inhibition by aptamer clone (PF377) at 60nM at 1 dynes/cm2 was 99% (Figure). In conclusion, we observed significant anti-adhesion activity of P-selectin aptamer clone PF377, and this aptamer is now suitable for modification to enhance stability and bioavailability. Since we previously reported strong anti-adhesion activity of integrin αvβ3 aptamer clone 17.16 (Blood,108:206a,2006), in vivo experiments in mice are currently employing intravital microscopy to measure anti-adhesion activity of aptamer clone 17.16, PF377, and both in combination. The development of combinatorial blocking aptamers against various adhesion molecules, including αvβ3 and P-selectin, represents a novel potential therapeutic option for patients with SCD.

Figure Figure
Prognostic Value of Immunophenotyping of Multiple Myeloma: A Study on 685 Patients Uniformly Treated with High-Dose Therapy.
Authors: Mateo Gema , Montalban Maria A. , Vidriales Maria-Belén , Lahuerta Juan J. , Mateos Maria-Victoria , Gutiérrez Norma C. , Rosiñol Laura , Montejano Laura , López-Berges Ma Consuelo , Bladé Joan , Martínez Rafael , de la Rubia Javier , Diaz-Mediavilla Joaquín , Sureda Anna , Ribera José M. , Ojanguren José M. , de Arriba Felipe , Palomera Luis , Terol Ma José , Gardella Santiago , Orfao Alberto , Miguel J.F. San
Published: April 2020

Analysis of the antigenic profile of malignant cells has shown to be a valuable tool for the diagnosis and the identification of prognostic markers in haematological malignancies. By contrast, in multiple myeloma (MM), immunophenotyping is rarely used in clinical practice. Moreover, conflicting results have been reported probably because they were based on short series of heterogeneously treated MM patients. In the present study, we have prospectively analysed the prognostic impact of antigenic markers, assessed by multiparametric flow cytometry, in a series of 685 newly diagnosed MM patients that were uniformly treated according to the GEM-2000 protocol. The results show that three antigenic markers (CD19, CD28 and CD117) afford prognostic information. The expression of both CD19 and CD28 as well as the absence of CD117 were associated with a significantly shorter progression free-survival (PFS) and overall survival (OS). Interestingly, the CD28 expression correlated with t(14;16) and Del(17p), while CD117 negative cases were associated with t(4;14) and Del(13q) as well as several other disease characteristics. The simultaneous assessment of CD28 and CD117 antigens, allowed stratification of myeloma patients into three risk categories:

poor risk (CD28+CD117−), intermediate (either both markers negative or both positive), and good risk (CD28−CD117+), with PFS rates of 30, 37 and 45 months, respectively (p=0.01), and OS of 45, 68 and not reached, respectively (p=0.0001).

To the best of our knowledge, this is the first prospective analysis in which the prognostic impact of a relatively high number of antigenic markers has been simultaneously analysed in a large series of uniformly treated patients.
Myeloma International Staging System Retains Its Prognostic Value at Disease Relapse.
Authors: Verma Rajanshu , Kumar Shaji , Lacy Martha Q. , Dispenzieri Angela , Hayman Suzanne , Greipp Philip R. , Rajkumar S. Vincent , Gertz Morie A.
Published: April 2020

Background: The international staging system is a simple, but powerful staging system that is based on two simple and easily available laboratory measurements. The system was developed and validated on a large group pf patients from across the world and has become the standard for patients with newly diagnosed myeloma. While it’s prognostic value is clear in patients with newly diagnosed myeloma, its value at relapse has not been explored.

Methods: We examined a uniform cohort of patients relapsing after an autologous stem cell transplant to assess the utility of ISS determined at the time of documented relapse. Relapse was defined using standard EBMTR response criteria. Beta-2 microglobulin and albumin values from within 30 days of the date of relapse were extracted from the medical records. ISS was determined based on the published cut offs.

Results: Of the 389 patients evaluated, 132 had values available for B2M and 276 patients had the serum albumin available. Only 131 patients had both the data available and the ISS stage could be determined. Of these patients, 64 patients (49%) were ISS stage I, 50 (38%) were stage II and 17 (13%) were stage III. An albumin < 3.5 mg/dL was prognostic for overall survival post relapse with a median survival of 10.5 months for those with albumin < 3.5 mg/dL compared to 26.3 months for the rest of the group (P < 0.0001). Similarly the median OS from relapse was 15 months for those with B2M > 3.5 mg/dL compared to 23.3 months for those with lower B2M (P = 0.014). The ISS stage predicted overall survival for this group of patients with the OS from relapse estimated at 27.3 mos, 17.8 mos and 12.3 months for ISS stage I, II and III respectively.

Conclusions: In a uniform group of patients relapsing after autologous stem cell transplantation, the B2M and albumin maintain their prognostic value and allows the use of the International Staging System for determining prognosis. This supports the use of ISS stage in relapsed trials and will allow comparisons across studies. Results should be validated in a larger dataset that will allow comparisons to other known prognostic factors.

Figure Figure
C/EBPβ Is a Critical Factor for Proliferation and Apoptosis in MM Cells by Controlling Transcription Factors Like IRF-4, PAX5 and Blimp1.
Authors: Pal Rekha , Janz Martin , Galson Deborah , Lentzsch Suzanne
Published: April 2020

The development and maturation of plasma cells is dictated by multiple interacting transcription factors (TFs). C/EBPb (NF-IL6) is a TF regulated by IL-6 and has profound effects on the regulation of growth, survival and differentiation of B-cells. Mice deficient in C/EBPb show impaired generation of B lymphocytes suggesting that C/EBPb plays an important role in B lymphopoiesis. In this study we delineated the effect of C/EBPb on transcription factors critical for myeloma cell proliferation by over-expressing and inhibiting C/EBPb in myeloma cells. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226 and H929 were transiently transfected with GFP, C/EBPb (pcNF-IL6), and truncated C/EBPb with a deletion of the internal spII-spII fragment [pcmNF-IL6(Dspl)] by using Bio-Rad Gene Pulser Xcell, followed by G418 selection. A pool of transfected cells was selected and subjected to thymidine incorporation, flow cytometry and western blot analysis. We found that transfection of a truncated form of C/EBPb induced a down-regulation of C/EBPb in MM cell lines (MM.1S, RPMI-8226 and H929) as measured by western blot. Down-regulation of C/EBPβ significantly inhibited proliferation and induced apoptosis of MM cell lines analyzed by annexin V-FITC/PI staining. This was accompanied by a complete down-regulation of the anti-apoptotic protein BCL-2. Further, inhibition of C/EBPb completely decreased IRF-4 expression. In contrast, over-expression of C/EBPb increased protein levels of IRF-4 suggesting that IRF-4 is under control of C/EBPb. IRF-4, which was over-expressed in all our tested MM cells lines, is an essential TF for the generation of plasma cells by regulating TFs like Blimp-1 and PAX-5, which are critical for plasma cell differentiation. Our studies showed that down-regulation of IRF-4 resulted in a complete abrogation of Blimp-1 and PAX-5 suggesting that the expression of these factors is C/EBPb/IRF-4 dependent. In conclusion, our data indicate that C/EBPb is an important key regulator for survival and growth of MM cells. We show for the first time that C/EBPb is a critical regulator upstream of IRF-4. Down-regulation of the C/EBPb and consequently IRF-4 results in complete disruption of the network of TFs necessary for MM growth and survival. Targeting C/EBPb may provide a novel therapeutic approach in the treatment of MM.
AKT Dependent and AKT Independent Multiple Myeloma: Heterogeneity in Tumor Cell Signaling.
Authors: Zöllinger Angela , Stühmer Thorsten , Chatterjee Manik , Bargou Ralf C.
Published: April 2020

The PI3K/Akt pathway has been reported to critically contribute to survival and malignant growth of multiple myeloma (MM). Because most of these data are based on pharmacologic inhibition it is not clear if the effects are due to Akt inhibition or off-target effects. Furthermore, the Akt family of kinases consists of three highly homologous isoforms, that may, nonetheless, display individual functional properties. We therefore conducted siRNA experiments to evaluate if any single isoform posesses a special role for the viability of MM cells. This was complemented with extensive analyses into the functional and signaling properties of the Akt pathway in primary MM cells (n = 30). Our knock-down experiments revealed that in MM.1S, an MM cell line with constitutive phospho-Akt signaling, Akt1 and Akt2 both contributed to MM cell survival whereas Akt3 seemed to be of less relevance. Conversely, survival of MM cell line AMO-1 which has no constitutive phospho-Akt signal was completely unaffected. Treatment of these MM cell lines with the Akt1 and Akt2 specific inhibitor Akti-1/2 showed that this drug totally abolished the phospho-Akt signal in MM.1S at a concentration of 10 microM. Again, MM.1S cells underwent apoptosis whereas AMO-1 cells were resistant. Next, we analyzed Akt signaling in a large panel of primary MM samples. Phosphorylated Akt was determined with intracellular staining and flow cytometry analysis in primary tumor samples and could be detected in about 60% of MM cases. This constitutive signal could be blocked with Akti-1/2 in the presence and absence of bone marrow stromal cells. Pharmacologic inhibition of Akt led to strong induction of cell death in 46% of primary MM samples, whereas the rest was largely resistant to Akt inhibition. The samples sensitive to Akt inhibition were mostly identical to those that displayed a constitutive phospho-Akt signal. Of interest, there was no correlation between Akt dependence and mutational inactivation of PTEN. Further inhibition of other signaling cascades implicated in growth and survival of MM cells, such as the MAPK or STAT3 pathways, had only minor additional effects on tumor cell viability of samples resistant to Akt inhibition. Our analysis indicates substantial heterogeneity in MM cells that defines Akt dependent and Akt independent MM subgroups. Akt1 and Akt2 proved relevant for the survival of subsets of MM cell lines and primary samples. Taken together, this is the first comprehensive functional and molecular signaling analysis of primary MM samples which led to the identification of novel functionally defined myeloma subgroups.
S-2209, a Novel, Non-Boronic Proteasome Inhibitor, Induces Apoptosis and Cell Growth Arrest in Multiple Myeloma Cells.
Authors: Baumann Philipp , Mueller Karin , Mandl-Weber Sonja , Ostermann Helmut , Schmidmaier Ralf , Oduncu Fuat , Leban Johann , Doblhofer Robert , Ammendola Astrid , Baumgartner Roland
Published: April 2020

Purpose: Multiple Myeloma (MM) is still an incurable disease. Patients become resistant to cytotoxic drugs and die of disease progression. Bortezomib is the first approved member of a new class of antineoplastic agents, the proteasome inhibitors. It has synergistic effects with genotoxic drugs and steroids in vitro and in vivo. However, single agent activity in humans is only moderate and specific toxicity (e.g. neurotoxicity) often limits its clinical use. Further proteasome inhibitors need to be developed to optimize this promising treatment option.

Methods: The new proteasome inhibitor S-2209 was characterized by several assays. Inhibition of the chymotryptic activity of the human 20S proteasome was determined with the in-vivo protease inhibition assay. Additionally, proteasome inhibition was determined in isolated PBMCs from S2209-pretreated wistar rats. Inhibition of NFκB activity was determined using a NFκB reporter gene assay. Cell growth rates of MM cells (OPM-2, U266, RPMI-8226 and NCI-H929) were measured with the WST-1 assay. Induction of apoptosis was shown by flow cytometry after staining with annexin-V-FITC and propidium iodide. Intracellular signal modulation was demonstrated by western blotting. Toxicity of the substance was tested in male wistar rats.

Results: The proteasome inhibition assay revealed an IC50 at ∼220nM. The NFκB inhibition assay using an A549-NFκB-SEAP transfected cell line showed an EC50 of 0.9μM. Upon incubation with S-2209, cell growth as well as cell proliferation in MM cell lines was significantly inhibited (IC50 100nM – 600nM). Furthermore, the incubation with S-2209 resulted in strong induction of apoptosis in all four MM cell lines even at nanomolar concentrations (IC50 at ∼300nm) as well as primary cells. Western blotting revealed caspase-3 cleavage and upregulon of p53 and increased phosphorylation of IκB. No induction of apoptosis was detected in PBMCs from healthy humans. Despite the administration of 5, 10 or 15mg/kg/day in wistar rats, no toxicity with respect to body weight, hepatic enzymes (ALAT ASAT, ALP), creatinin or hemoglobin was seen. Proteasome inhibition in white blood cells isolated from the treated rats was higher in the S-2209 treated animals than in control animals treated with 0.1mg/kg/d bortezomib (89% vs. 70% respectively).

Conclusions: The proteasome inhibitor S-2209 inhibitis MM cell growth and induces apoptosis. This is accompanied by a strong inhibition of proteasome and of the NFκB activity. Because S-2209 shows a favourable toxicity profile in vivo, further clinical development of this promising drug is urgently needed.
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

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 <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

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

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

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

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

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 (>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

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

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<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

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
Polymorphisms in One-Carbon Metabolism Genes and Overall Survival in Diffuse Large B-Cell Lymphoma (DLBCL).
Authors: Cerhan James R. , Maurer Matthew J. , Hartge Patricia , Chanock Stephen J. , Habermann Thomas M. , Cozen Wendy , Davis Scott , Lynch Charles F. , Severson Richard K. , Rothman Nathaniel , Morton Lindsay M. , Wang Sophia S.
Published: April 2020

Background. Intracellular one-carbon transfer reactions are essential for nucleotide synthesis and methylation of biologic compounds including DNA. Previous studies have linked genetic variants in one-carbon metabolism genes with risk of developing NHL, but little is known regarding the impact of these variants on disease outcome. We evaluated the hypothesis that inherited genetic variation in genes involved in one-carbon metabolism is associated with overall survival in DLBCL.

Methods. We genotyped 30 single nucleotide polymorphisms (SNPs) from 18 candidate one-carbon metabolism genes in 215 DLBCL patients who participated in a population-based case-control study conducted from 1998–2000 using the SEER (Surveillance, Epidemiology and End Results) cancer registries in the Detroit, Iowa, Los Angeles and Seattle. Stage, B-symptoms, first course of therapy, date of last follow-up and vital status through early 2005 were obtained from cancer registry files. Cox proportional hazards analysis was used to estimate hazard ratios (HR) and corresponding 95% confidence intervals for the association between individual SNPs and overall survival, adjusting for age, demographic and clinical factors. We also used parallel modeling strategies to identify the best summary multi-SNP risk score to predict survival.

Results. The median age at diagnosis was 57 years (range, 20–74), and 50 (23%) of the patients died during follow-up, with a median follow-up of 57 months (range, 31–78 months) for surviving patients. After adjusting for demographic and clinical variables, SNPs in SHMT1 (rs1979276; HRCT/TT=2.47, 1.31–4.67), BHMT (rs585800; HRAT/TT=2.02, 1.16–3.54), and TCN1 (rs526934; HRTT=1.86, 1.04–3.33) were the strongest and most robust predictors of survival. A summary score of the number of deleterious genotypes (0–3) from these three genes was strongly associated with survival (p=7.9 x 10−5) after accounting for demographic and clinical variables (HR=2.58 per deleterious genotype, 95% CI 1.75–3.80). A risk score combining the three SNPs with clinical and demographic variables (score of 0 to 5) was even more strongly associated with survival (p=1.4 x 10−13); the Kaplan Meier survival curves are shown in the Figure. In a time-dependent ROC analysis, the combined risk score had a concordance index of 0.75 at 5 years of follow-up (95% CI 0.69–0.81).

Conclusion: Host genetic variation in the one-carbon metabolism genes SHMT1, BHMT, and TCN1, individually and particularly in combination, was associated with overall DLBCL survival after accounting for clinical and demographic factors, supporting a role for this pathway in disease progression. Future work should evaluate interactions of genes from this pathway with dietary nutrients and therapeutic agents in DLBCL prognosis.

Figure Figure
Inhibition of Ubiquitin-Proteasome Pathway Causes Growth Suppression and Induces Apoptosis in Diffuse Large B Cell Lymphoma.
Authors: Uddin Shahab , Hussain Azhar , Khan Asma , Bavi Prashant , Al-Kuraya Khawla
Published: April 2020

SKP2, an F-box protein targets cell cycle regulators including cycle-dependent kinase inhibitor p27KIP1 via ubiquitin-mediated degradation. SKP2 is frequently overexpressed in variety of cancer cells and has been implicated in oncogenesis, however its role in diffuse large B-cell lymphoma (DLBCL) has not been elucidated. Therefore, we investigated the role of SKP2 and its ubiquitin-proteasome pathway in DLBL using a panel of cell lines and clinical samples. MG132, a reversible and specific inhibitor of ubiquitin-proteasome pathway that acts through blocking ubiquitin-mediated proteolysis via binding to 20S and 26S proteasomes. Our study showed that treatment of DLBCL cell lines (SUDHL4, SUDHL5, SUDHL8 and OCILY19) with MG132 suppressed growth and induced apoptosis. Treatment of DLBCL cells with MG132 caused downregulation of SKP2, accumulation and stabilization of p27KIP1 leading to the apoptosis. Further downstream, MG132 induced activation of caspase-8 and cleavage of Bid subsequently leading to loss of mitochondrial membrane potential and release of cytochrome c from mitochondria into cytosol, resulting in activation of caspase-3 and cleavage of PARP. zVAD-fmk, a universal inhibitor of caspases prevented caspase-3 activation and abrogated apoptosis induced by MG132 treatment. Finally, using immunohistochemistry, SKP2 was detected in 94 (32.9%) of 286 DLBCL tumors. Furthermore, patients with high SKP2 expression were associated with high expression of Ki67, a proliferative marker. Altogether, these results suggest that SKP2 and ubiquitin-proteasome pathway may be a potential target for therapeutic intervention in diffuse large B-cell lymphoma.
Expression of Phosphorylated mTOR (p-mTOR) Correlates with Advanced Stage in Diffuse Large B-Cell Lymphomas.
Authors: Patel Raina A. , Ahmad Imran , Sheehan Christine E. , Ross Jeffrey S. , Wilner George D. , Hayner-Buchan Alida , Nazeer Tipu
Published: April 2020

Background: The mammalian target of rapamycin (mTOR), plays a major role in regulating cell growth. The activated form of mTOR (phosphorylated mTOR) mediates its effects through mitogen and nutrient dependant signal transduction that regulates mRNA translation. Levels of phosphorylated mTOR (p-mTOR) have been shown to correlate with cell proliferation and inhibition of the mTOR pathway has shown therapeutic promise in some tumors. Information on the role of p-mTOR in hematopoietic malignancies is limited. The aim of the current study is to evaluate the expression of p-mTOR in diffuse large B-cell lymphomas.

Design: Formalin-fixed paraffin-embedded tissue sections from 45 DLBCLs were immunostained by an automated method (Ventana Medical Systems, Inc., Tucson, AZ) using p-mTOR rabbit monoclonal antibody (IHC specific Ser2448/49F9; Cell Signaling Technology, Inc., Danvers, MA). Immunoreactivity (cytoplasmic with membranous accentuation) was semiquantitatively assessed based on p-mTOR stain intensity (none, weak, moderate, intense) and distribution (none, focal<10%, regional 11–50%, diffuse>50%). Staining was correlated with histologic and prognostic factors.

Results: Membranous/cytoplasmic p-mTOR immunoreactivity was present in 88% of DLBCLs and correlated with advanced tumor stage, with 100% advanced stage versus 73% low stage tumors expressing membranous/cytoplasmic p-mTOR protein (p=0.002). One hundred percent of cases with complete absence of p-mTOR immunoreactivity were low stage DLBCLs.

Conclusion: Membranous/cytoplasmic protein expression of p-mTOR correlates with advanced stage in DLBCL. Activated mTOR may play an important role in disease progression and provide a target for therapy in DLBCL. Further studies of the m-TOR pathway in hematopoietic malignancies appear warranted.