bluuebunny/crossref_metadata_2025 huggingface dataset:
Erythrocyte Adhesion and Phosphatidylserine Exposure in HbSC Disease: Baseline Data from the CHAMPS Study
Authors: Telen Marilyn J. , Delahunty Martha , Snyder Cathie , Wang Winfred C.
Published: April 2020

Red blood cells (RBCs) in sickle cell anemia are characterized by increased adhesion to laminin and endothelial cells, due both to increased adhesion receptor expression as well as receptor activation. However, these RBC properties have not been comprehensively evaluated in HbSC disease. In the on-going multi-center CHAMPS trial, all subjects have a diagnosis of HbSC disease and have had at least one significant vaso-occlusive event in the previous 12 months (but none in the previous 4 weeks). All subjects are at least 5 years of age (median 13.4 yr, range = 5.2–53.3 yr) at enrollment. Subjects receive hydroxyurea (HU) and/or magnesium pidolate (Mg) (or their placebos); endpoints include the effect of study drugs on erythrocyte density and RBC-endothelial interactions. To characterize the adhesive properties of RBCs at baseline in these patients, we performed the following analyses on blood samples shipped to a central lab at Duke University from 11 participating sites: adhesion to laminin at 1 dyne/cm2 shear stress; adhesion to endothelial cells after epinephrine (epi) stimulation of RBCs at shear stress of 1 dyne/cm2; and phosphatidylserine exposure, as measured by mean fluorescence intensity after binding of fluoresceinated annexin V. Most subjects had two pre-treatment specimens analyzed. Analysis of the first 83 samples (47 patients) showed that a mean of 1.64% (± 0.15% SEM) of circulating HbSC RBCs expressed phosphatidylserine, or a little less than half as many as in HbSS RBC samples (4.12% positive ± .91% SEM, n = 29) tested as controls (HbSC vs HbSS p=.0009). However HbSC RBCs were also different from HbAA RBCs (1.076% positive, n = 5), and all three sets of values were significantly different by ANOVA (p = .0017) (Figure). The two baseline visits for HbSC subjects resulted in nearly identical means of 1.64% and 1.65%, respectively. Adhesion of HbSC red cells to laminin at baseline was quite variable among patients, with a mean of 36.1 adherent cells/mm2 (range 2–256). This was slightly but not significantly lower than the values obtained using HbSS control RBCs (mean 58.8 cells/mm2, range 0.2–258). After epi stimulation of RBCs, 9.6% of cells adhered to endothelial monolayers at 1 dyne/cm2, whereas 14.8% of HbSS RBCs were adherent (p = .186). HbAA RBCs showed minimal adhesion in both assays (< 5 cells/mm2 and <3%, respectively). Finally, no correlation was found by linear regression analysis between phosphatidylserine exposure and either adhesion to laminin or adhesion to endothelial cells. In summary, HbSC RBCs show appreciable adhesion to both laminin and endothelial cells, as well as phosphatidylserine exposure. While in general these abnormalities are less pronounced than for HbSS RBCs, they are still well outside the normal range and provide potential targets for therapeutic intervention.

Figure Figure
Statins Inhibit the Function of Human Antigen-Presenting Cells
Authors: Shimabukuro-Vornhagen Alexander , Liebig Tanja , Zoghi Shahram , Wendtner Clemens M , von Bergwelt-Baildon Michael S
Published: April 2020

Statins are lipid-lowering drugs that reduce cholesterol production by inhibition of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway. It recently has been shown that statins, apart from their lipid-lowering action, have pleiotropic effects on the immune system. They possess immunomodulatory and anti-inflammatory activity. Our analysis of gene expression data comparing resting B cells with CD40-activated B cells, which are potent antigen-presenting cells (APC), showed that following CD40-activation expression of the enzymes in the mevalonate pathway was up-regulated. These results together with the known ability of some statins to directly interfere with the interaction of lymphocyte function-associated antigen 1 (LFA-1), an adhesion molecule involved in activation of APCs, with its ligands provided the rational for our investigation of the effects of HMG-CoA reductase inhibition on the function of human APCs. Addition of simvastatin led to inhibition of the proliferation and adhesion of B cells activated by via CD40 or TLR9. Likewise, the expression of MHC class II (HLA-DR) and of the costimulatory molecules CD80 and CD86 was reduced. The antigen presenting capacity of simvastatin-treated B cells was reduced as was shown by a reduction of T cell proliferation induced by allogeneic simvastatin-treated B cells. Simvastatin inhibited activation of resting B cells as well as already activated B cells. The inhibition was dose-dependent and doses as low as 10nM Simvastatin led to signification reduction of B cell activation. We observed similar effects on human dendritic cells. Interestingly, the concentration of simvastatin required for inhibition of T cell proliferation in response to stimulation by CD3/CD28-coated beads was 1–2 logs higher than for B cells. The inhibitory action of statins was only partially dependent on the mevalonate pathway since addition of excess mevalonate did not fully restore B cell activation. Our findings of the effects of statins on human APCs in conjunction with the observation made by others that preemptive treatment with a HMG-CoA reductase inhibitor protected mice from graft-versus-host-disease (GVHD) without affecting the graft-versus-leukemia effect provide the rational for further testing of statins in patients undergoing allogeneic stem cell transplantion. Since APCs are required for the development of acute GVHD the modulation of antigen presentation by DCs and B cells might be an important mechanism of action of statins. The exquisite sensitivity of APCs to statin-mediated inhibition could synergize with the T cell-directed immunosuppression of conventional immunosuppressants. In summary, statins represent a promising new immunomodulatory drug for the treatment immune-mediated diseases such as GVHD and autoimmune disease.
Soluble CD14 Protects Human Lymphocytes from Apoptosis
Authors: Naparstek Elizabeth , Sredni Benjamin , Zigman Eti , Senyor Gali , Tartakovsky Boris
Published: April 2020

CD14, a 56 Kd glycoprotein, typically present on myeloid cells, has been traditionally associated with innate immunity and pattern recognition. Recently its membrane bound form has been shown to be involved in apoptosis, as a tethering receptor for apoptotic cells on the surface of phagocytes-in this case with the purpose of removing apoptotic cells, and also as a surface molecule involved in protection from apoptosis of monocytes, neutrophils and recently on enterocytes, challenged with LPS.

Our aim was to evaluate the possible involvement of the soluble CD14 in the apoptotic pathway of human lymphocytes.

Methods: Freshly obtained human peripheral blood lymphocytes were cultured in vitro with gliotoxin, an apoptotic inducer. Human recombinant CD14 was added to the culture at physiological concentrations (10μg/ml-0.5 μg/ml) and apoptosis was assessed by cell membrane integrity using 7AAD, mitochondrial membrane potential by DiOC6(3) and cytoplasm shrinkage by cell size scatter analysis.

Results: Using DiOC6(3) we were able to show that human lymphocytes cultured in the presence of gliotoxin contained 63.8%±21 apoptotic cells, as opposed to 12.2%±11.5 in control cultures. Addition of recombinant human CD14 at a concentration of 10 mg/ml neutralized the apoptotic effect of gliotoxin back to 20.2%±10 (p<0.003). This inhibitory effect was blocked by CD14-specific monoclonal antibodies, but not by control antibodies. We then identified and synthesized the fragment within the CD14 molecule that was responsible for this apoptosis protective effect, and demonstrated its comparable protective efficacy in vitro as shown in figure 1. The figure clearly reveals that this specific peptide, as opposed to the scrambled peptide, protected the lymphocytes form apoptosis, similarly to the full CD14 protein. Same results were obtained using 7AAD and cytoplasm shrinkage.

Conclusion: Our data thus suggest that circulating CD14 may play an important role in the prevention of apoptosis of lymphocytes and perhaps of other cells.

Figure Figure
Suppression of Interferon Regulatory Factor 8 Modulates Toll Like Receptor 2 and Toll Like Receptor 7/8 Induced Dendritic Cell Activation
Authors: Werth Daniela , Bringmann Anita , Brauer Katharina , von Schwarzenberg Karin , Held Stefanie , Brossart Peter
Published: April 2020

Interferon regulatory factor 8 (IRF-8) is a member of the IRF family of transcription factors, which are stimulated through interferon mediated pathways. In mice, IRF-8 seems to play an essential role in the development and maturation of dendritic cells (DCs). However, very limited knowledge is available about the potential role of IRF-8 in the human system. To bridge this gap we analyzed function and activation of human monocyte-derived dendritic cells (mDCs) lacking IRF-8 expression. To knockdown IRF-8 protein levels, we electroporated mDCs with different siRNAs against IRF-8. Additionally, we stimulated the electroporated mDCs with the Toll like receptor (TLR) 2 ligand Pam3Cys or the TLR 7/8 ligand R848. IRF-8 knockdown in mDCs was verified constantly by Western Blot analysis using an anti-IRF-8 antibody. We found that IRF-8 knockdown clearly diminished the expression of the human lymphocyte antigen molecules HLA-ABC and HLA-DR in Pam3Cys and R848 stimulated mDCs. To gain functional data, we performed ELISAs to determine cytokine and chemokine secretion. The electroporation of mDCs with IRF-8 specific siRNA resulted in profound inhibition of secretion of the cytokines IL-6, IL-12 and TNF-a as well as the chemokines MIP-1a (CCL3), MCP-1 (CCL2) and RANTES (CCL5). To get additional information on IRF-8 function in human mDCs, the regulation of signal transduction pathways was determined by Western Blot analysis. The suppression of IRF-8 diminished the nuclear translocation of the NF-kB family member’s c-Rel and RelB as well as PU.1 and IRF-3 in activated mDCs. In addition, we showed that the suppression of IRF-8 caused a reduced phosphorylation of ERK and JNK, but had no effect on the expression of STAT3. In summary, the knockdown of IRF-8 reduced the capability of mDCs to develop appropriate phenotype and functions in response to activating stimuli. Our results indicate that these effects are mediated via the ERK, NF-kB and PU.1 signalling pathways. IRF-8 plays an important role in the activation and function of human mDCs.
Enhanced B Cell Activation in the Absence of CD81
Authors: Sanyal Mrinmoy , Fernandez Rosemary , Levy Shoshana
Published: April 2020

CD81 is a component of the CD19/CD21 coreceptor complex in B cells. This tetraspanin molecule was previously shown to enable membrane reorganization in B cells responding to complement-bound antigens. Here we stimulated B cells via their B cell receptor (BCR) and demonstrate that Cd81−/− B cells fluxed higher intracellular free calcium ion along with increased phosphorylation of PLCγ2 and Syk. The stimulated Cd81−/− B cells also proliferated faster and secreted higher amounts of antibodies. Moreover, activation of the TLR4 pathway in Cd81−/− B cells induced increased proliferation and antibody secretion. Furthermore, Cd81−/− mice mounted a significantly higher immune response to T-cell independent antigens than their wildtype counterparts. Finally, analysis of Cd81−/− B cells that were generated by bone marrow transplantation into Rag1−/− mice confirmed a cell intrinsic hyperactive phenotype. Taken together, these results indicate that CD81 plays a negative role in B cell activation in vitro and in vivo.
Cyclin D3 Is Required for the Germinal Center Reaction
Authors: Peled Jonathan U. , Yu J. Jessica , Ding Beibei Belinda , Shaknovich Rita , Sicinski Piotr , Scharff Matthew D. , Ye B. Hilda
Published: April 2020

Germinal Centers (GC) of secondary lymphoid tissues are critical to mounting a high-affinity humoral immune response. B cells within the GC undergo rapid clonal expansion and selection while diversifying their antibody genes through class switch recombination and somatic hypermutation. Although it is generally believed that GC B cells employ a unique proliferative program to accommodate these processes, very little is known about how the GC-associated cell cycle is orchestrated. The D-type cyclins are important regulators of the G1 phase of the cell cycle and are the ultimate targets of many mitogenic and oncogenic stimuli. The Cyclin D3 gene is rearranged and over-expressed in certain mature B cell malignancies, and its overexpression has been reported to predict poor clinical outcome in patients with diffuse large B cell lymphoma. It has been observed that during their development, B cells switch from expressing cyclin D2 to cyclin D3 when they are recruited into the GC response. It is unclear, however, whether this switch simply reflects a change in the transcription factors that govern cyclin expression or serves a biological mandate. Here we report that mice deficient in cyclin D3 are profoundly impaired in their ability to form GCs as measured by immunohistochemistry and flow cytometry. Production of antigen-specific antibodies and affinity maturation, as ascertained by ELISA, are concomitantly reduced in these animals. These phenotypes can be at least partially explained by a significant block in the G1-phase of the cell cycle of GC B cells in vivo. Interestingly, this block in the G1-S transition is observed despite an apparent compensatory increase in cyclin D2 expression. In addition, naive B cells activated in vitro by either LPS or LPS and IL-4 display only minor changes in cell-cycle profile, suggesting that a specific requirement for cyclin D3 is unique to GC B cells. We also find moderately reduced Bcl6 mRNA expression in both naïve and GC B cells from the cyclin D3 knockout mice. Since Bcl6 is a master regulator of the GC response, decreased activity of this transcriptional repressor may further contribute to the severity of the GC phenotype. This is the first demonstration that cyclin D3 plays a unique role during the GC response in that it is required for its optimal structure and function. In addition to expanding appreciation for the cell type- and tissue-specific functions of the three D-type cyclin molecules, our findings have implications for understanding the role of Cyclin D3 in human B cell lymphomas.
Sulforaphane and PEITC Augment Activity of Conventional and Novel Anti-Myeloma Drugs
Authors: Jakubikova Jana , Sedlak Jan , Klippel Steffen , Leiba Merav , Tai Yu-Tzu , Hideshima Teru , Podar Klaus , Chauhan Dharminder , Raje Noopur , Richardson Paul G. , Anderson Kenneth C. , Mitsiades Constantine S.
Published: April 2020

Isothiocyanates (ITCs) represent a family of phytochemicals found in cruciferous vegetables. Several epidemiological studies indicated that high intake of diet-derived ITCs may provide chemopreventive effect associated with a reduced risk of renal, prostate, pancreatic and colorectal carcinoma. ITCs have also been reported to enhance the chemosensitivity of diverse types of tumor cells. We therefore evaluated the cytotoxic effect of ITC, sulforaphane (SFN) and phenylethyl isothiocyanate (PEITC) on a panel of human MM cell lines, including cells resistant to doxorubicin (RPMI-Dox40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5), and dexamethasone (MM.1R, OPM1 and OPM2); as well as cells with low sensitivity to thalidomide derivatives (RPMI 8226-S) and sensitive cell lines (MM.1S). We evaluated the anti-MM activity of these compounds using both MTT and flow cytometric assays. Our results suggest that all tested MM cell lines are susceptible to the cytotoxic effect of both ITCs at concentrations in the same order of magnitude as those achieved in vivo by dietary consumption of cruciferous vegetables. PEITC (IC50 of 3.5–8.2 μM) was more potent than SFN (IC50 of 5–15 mM) at 48 h. ITCs induce apoptotic death of MM cells, evidenced by Annexin V-FITC staining, increased sub-G1 population measured by flow cytometry, and cleavage of PARP and caspase-3 by western blot analysis, ITCs also induced G2/M cell cycle arrest and depletion of mitochondrial potential in JC-1 probed MM cell lines. Multiplex analysis of phoshorylation signaling pathways, using the Luminex system and confirmed by conventional western blot analysis, revealed that PEITC at 2hrs triggers MAPK activation (MEK1 (4-fold), ERK1/2 (4.4-fold), JNK (25.4-fold) and p38MAPK (6-fold)) at 2h, which likely are stress responses of cells to PEITC treatment. SFN induced less pronounced but a more sustained MAPK activation (up to 24 h) than PEITC. Concentration-dependent increases in phosphorylation of GSK3α/β, c-jun, p70S6 kinase, and p90RSK were also observed at early time-points after ITCs-treatment. In contrast, decreased phoshorylation of Akt was observed in MM cells treated with SFN at 2 h and PEITC at 24 h. Chou-Talalay analysis of the effects of combinations of ITCs with anti-MM drugs (dexamethasone, melphalan and bortezomib) revealed all combinations to have synergistic MM cytotoxicity. Importantly, ITCs treatment, both alone and in combination with the aforementioned agents, also significantly suppressed proliferation of CFSE-labeled MM cell lines co-cultured with the human bone stromal cell line HS-5. These results indicate that SFN and PEITC suppress survival and proliferation of MM cells, both alone and in combination, and suggest their therapeutic potential in MM.
Arsenic Trioxide Plus ABT-737 Is Synergistic to Induce Apoptosis in AML Cells by Repression of Mcl-1 Protein and Inactivation of Bcl-2 Protein
Authors: Xia Lijuan , Wang Rui , Qian Hao , Gabrilove Janice , Waxman Samuel , Jing Yongkui
Published: April 2020

Arsenic trioxide as a single agent induces remission in acute promyelocytic leukemia (APL) patients with minimal toxicity but not in other types of acute myeloid leukemia (AML). In vitro, APL as compared to AML cells are more sensitive to arsenic trioxide-induced apoptosis. Arsenic trioxide-induced apoptosis in APL cells results from activation of a mitochondria-mediated pathway. The Bcl-2 antiapoptotic protein family members Bcl-2, Bcl-XL, Bfl-1 and Mcl-1 block mitochondria-mediated apoptosis. In studies of several AML cell lines, we detected high levels of Bcl-2 and Mcl-1 protein, but lower or no expression of Bcl-XL and Bfl-1. Arsenic trioxide treatment decreased the levels of Mcl-1 without inducing apoptosis in AML cells suggesting that Bcl-2 would be a key factor causing arsenic trioxide resistance. We therefore hypothesize that inhibitors of Bcl-2 might restore arsenic trioxide-induced programmed cell death. In this study, the apoptotic effects of arsenic trioxide, ABT-737 (a potent Bcl-2 inhibitor) and their combination were investigated in NB4, HL-60, U937 and K562 cells. Arsenic trioxide at 1–2 μM induced apoptosis only in NB4 cells but decreased the levels of Mcl-1 in all of the four cell lines. ABT-737 at concentrations lower than 5 μM induced apoptosis in NB4, HL-60 and U937 cells but not in K562 cells which had undetectable Bcl-2 levels. Arsenic trioxide (2 μM) plus ABT-737 (0.05–0.5 μM) synergistically induced apoptosis in HL-60 and U937 but not in K562 cells as determined by PARP cleavage and Annexin V staining. Our data suggest that inhibition of Mcl-1 expression by arsenic trioxide and inactivation of Bcl-2 activity by ABT-737 leads to the synergistic apoptosis observed with this combination and is the basis for a novel treatment for AML.
Hyperglycemia Promotes in Vitro All Blast Resistance to Steroid Induced Blast Lysis
Authors: Papadakis Vassilios , Tsitsikas Konstantinos , Vasdekis Vassilios , Parcharidou Agapi , Paisiou Anna , Papargyri Sofia , Polychronopoulou Sophia
Published: April 2020

Clinical patient observations led to the hypothesis of increased ALL blast resistance in hyperglycemia (HG) states in pediatric ALL patients, either with preexisting diabetes mellitus (DM) or with steroid induced diabetes (SID). To evaluate the above, the chromometric MTT (methyl thiazole tetrazolium) assay was used Kaspers GJ et al, Leuk Res1995;19:175–81.Ficoll separated nucleated bone marrow cells from newly diagnosed pediatric ALL patients were cultured in usual conditions (NL) and in higher glucose levels (233–295 mg/dl), with the addition of titrated prednisolone (PDN) concentrations. The PDN concentration that lyses 50 % of the cultured blast cells (LD50) is the measure of PDN drug sensitivity. PDN concentrations of 0.02–2.0, 2.1–150, above 150 μg/uL indicate high, intermediate and low blast sensitivity to PDN, respectively. Hence, patient blasts with LD50 at >150 μg/uL are resistant to PDN. The MTT assay results have been related to patient outcome (remission induction and event free survival, Pieters R et al Pediatr Oncol1994;22:299–308). Twelve newly diagnosed pediatric patients, with median age 6.4 years (range, 1.7 to 16.5 years) with common/CD10+ (N=8), pre-B (N=2), early pro-B (N=1) and maturing B (N=1) ALL were studied. All patients exhibited >80% ALL blast BM infiltration. Sensitivity (LD50 < 150 μg/uL) in both NL and HG conditions was evident for 2 patients, while resistance to both conditions was seen in 3 patients. Patients resistant at NL conditions were resistant in HG, too. In contrast, 7 of 12 patients sensitive to PDN in NL conditions proved resistant in HG conditions (glucose 233–295 mg/dL). All 9 sensitive in NL conditions patients (in HG conditions 2 sensitive and 7 resistant) exhibited a median ×800 fold increase of the MTT LD50 measured (range 6–29.000 fold). For all patients (N=12) median MTT LD50 under NL conditions was 0.15 μg/uL and in HG conditions 167.5 μg/uL (× 116.6 fold increase). This change was statistically significant (Mc Nemar’s test, p=0,016) and this change was not simply related to the change in glucose concentration. These findings of greatly increased ALL blast resistance in hyperglycemia environment, can support the notions given earlier (Weiser MA et al, Cancer2004;100:1179–85, Pieter’s R EHA 2008) that hyperglycemia by itself can promote PDN resistance. Given that steroids is a mainstay in ALL treatment and that steroid induced hyperglycemia is a common occurrence, blood glucose monitoring and hyperglycemia correction appears to be of paramount significance in delivering an effective ALL treatment.
PRT060318, a Novel Syk Inhibitor, Prevents Heparin-Induced Thrombocytopenia in a Transgenic Mouse Model
Authors: Reilly Michael P. , Sinha Uma , Andre Pierrette , Taylor Scott M. , Pak Yvonne , DeGuzman Francis R. , Nanda Nisha , Pandey Anjali , McKenzie Steven E.
Published: April 2020

Heparin-induced thrombocytopenia (HIT), in which patients develop antibodies to complexes formed by heparin and platelet factor 4 (PF4), is the most frequent drug-induced immune thrombocytopenia. Extensive studies in vitro and our previous studies in vivo using a transgenic mouse model of HIT have shown that antibodies reactive with heparin-PF4 complexes lead to FcgRIIa receptor-mediated platelet activation. In this study we investigated whether PRT060318 (PRT318), a novel Syk inhibitor, prevents HIT antibody-mediated platelet activation both in vitro and in vivo. PRT318 at concentrations of 0.3 to 3 μM completely inhibited HIT immune complex (IC)-induced aggregation in both human and transgenic mouse platelets. In the absence of the inhibitor, HIT IC-induced final aggregation was 50–60%. At concentrations of PRT318 less than 0.1 μM, or in the presence of vehicle only, there was no inhibition of aggregation. Aggregation was not inhibited by PRT318 at any concentration when platelets were stimulated by ADP (5–20 μM final concentration). We also show that PRT318 prevents HIT IC-induced thrombocytopenia in vivo using a transgenic mouse HIT model. All mice were treated with KKO, a mouse monoclonal HIT antibody. On days 1 to 4 following antibody injection, the experimental group (n = 13) received orally dosed PRT318 (30 mg/kg body weight) twice a day by gavage while the control group (n = 11) was similarly treated with vehicle only (water). Both experimental and control mice were injected with heparin (1600 U/kg body weight, SQ, once daily). Nadir platelet counts of PRT318-treated mice were significantly higher than control mice (89.8 ± 1.1% of baseline vs. 48.8 ± 6.7%; p = 0.00003). The PRT318 concentration, 2 hrs post dose, in mouse plasma from treated mice was measured as 7.1 μM, consistent with the concentration which blocked FcgRIIa-mediated platelet activation in vitro. These studies demonstrate that Syk inhibitor PRT318 is an active agent in HIT.

Figure Figure
Dynamic Contrast-Enhanced Magnetic Resonance Imaging Identifies a Subgroup of Patients with Asymptomatic Monoclonal Plasma Cell Disease and Pathological Microcirculation
Authors: Hillengass Jens , Zechmann Christian M , Bäuerle Tobias , Heiss Christiane , Benner Axel , Neben Kai , Ho Anthony D. , Goldschmidt Hartmut , Delorme Stefan , Moehler Thomas M.
Published: April 2020

The aim of the present study was to investigate whether dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) allows visualization of a change in microcirculation between healthy controls on the one side and early/advanced stages of plasma cell disease on the other. We examined a group of 222 individuals consisting of 60 patients with monoclonal gammopathy of undetermined significance (MGUS), 65 patients with asymptomatic multiple myeloma (aMM), 75 patients with newly diagnosed symptomatic MM (sMM) and 22 healthy controls with DCE-MRI of the lumbar spine. A continuous increase in the microcirculation parameter amplitude A reflecting vascular volume was detected from normal controls over MGUS and asymptomatic to symptomatic MM. Significant differences were found between controls and aMM (p = 0.03), controls and sMM (p = 0.001) and between asymptomatic and symptomatic MM (p = 0.02) respectively. While diffuse microcirculation patterns were found in healthy controls as well as MGUS and MM, a pattern with focal hot spots was exclusively detected in 42.6 % of sMM and in 3 patients with MGUS and 3 patients with aMM. Patients with MGUS and aMM with increased microcirculation patterns showed a significantly higher bone marrow plasmocytosis compared to patients with a low microcirculation pattern. Our investigations substantiate by means of a non invasive assessment of bone marrow microcirculation the concept of an angiogenic switch from early plasma cell disorders to symptomatic MM. Pathological DCE-MRI findings can be identified and correlate with an adverse prognostic parameter.
Inhibition of eIF2α Dephosphorylation Maximizes Bortezomib Efficiency and Eliminates Quiescent Multiple Myeloma Cells Surviving Therapy
Authors: Schewe Denis M , Aguirre-Ghiso Julio A
Published: April 2020

The proteasome inhibitor Bortezomib (Velcade®) effectively eradicates multiple myeloma (MM) cells, partly by activating endoplasmic reticulum (ER) stress apoptotic signaling. However, MM recurrences in Bortezomib-treated patients are invariable. We have shown that ER stress signaling can also induce growth arrest and survival in cancer cells. Thus, we hypothesized that Bortezomib therapy could induce quiescence and survival of residual MM cells, contributing to disease recurrence. Here we report that in MM cells, proteasome inhibition with MG-132 or Bortezomib results in a surviving cell fraction that enters a prolonged quiescent state (G0-G1 arrest). Mechanism analysis revealed that in Bortezomib-surviving quiescent cells eIF2α phosphorylation and induction of the ER stress pro-apoptotic gene, GADD153, were attenuated. In contrast, p38 and the ER-chaperone BiP/Grp78 were persistently activated and induced, respectively. The Bortezomib-surviving quiescent fraction could be eradicated by a simultaneous or sequential combination therapy with Salubrinal, an inhibitor of GADD34-PP1C phosphatase complex, and in consequence, eIF2α dephosphorylation. This effect was mimicked by expression of a phospho-mimetic eIF2α-S51D mutant. Our data indicate that Bortezomib can induce growth arrest in therapy-surviving MM cells and that attenuation of eIF2α phosphorylation and GADD153 induction contribute to this survival. Most importantly, this ability of MM cells to evade Bortezomib toxicity can be prevented by inhibiting eIF2a dephosphorylation. Thus, strategies that maintain eIF2α in a hyper-phosphorylated state may be a novel therapeutic approach to maximize Bortezomib-induced apoptosis and reduce recurrences in this type of cancer.
Parathyroid Hormones (PTH) Serum Variations Are Associated with Bortezomib Response in Multiple Myeloma Patients
Authors: Zangari Maurizio , Pappas Lisa , Zhan Fenghuang , kumar Naveen Sanath , Cavallo Federica , Suva Larry J , Tricot Guido , Esseltine Dixie-Lee , Yaccoby Shmuel
Published: April 2020

A correlation between increase in bone markers (alkaline phosphatase (ALP) and response to bortezomib in patients with multiple myeloma (MM) has been previously described. We now report results from a prospective study examining the relationship of serum PTH variation with skeletal effects and myeloma response to bortezomib treatment.

Methods: Single agent bortezomib (1.3 mg/m2 patients 1–10; 1mg/m2 patient 11–20), was administered to patients with relapsed/refractory MM on days 1, 4, 8 and 11 on a 21 day interval for a total of 3 cycles; patients were not allowed to receive concurrent bisphosphonates or any other anti-myeloma drugs during the study period. Dynamic indices of bone turnover were prospectively evaluated by high-resolution microCT. Architectural parameters such as bone volume/total volume (BVTV), trabecular number (TbN), and thickness (Tb.Th) was obtained. PTH along with bone markers (osteocalcin, calcium, magnesium, phosphorus and serum creatinine) were measured on days 1, 4, 8, 11 before and after each bortezomib dose and every 4 hours thereafter, daily for the other days of the treatment cycle.

Results: Seventeen patients were enrolled in the study with a median age of 63 years, 41 % were male and 3/4 of the patients previously failed high-dose chemotherapy. Histomorphometric microCT comparative analysis was completed (baseline and post-treatment) in 7 of the 17 patients enrolled in the trial. Baseline BV/TV values ranged from 13% to 90%. After 3 cycles of bortezomib treatment a statistically significant increase in BV/TV was recorded in 6 of 7 patients (P<0.02).

Median serum osteocalcin values increased from 1.95 ng/ml at baseline to 7.16 ng/ml at the end of the third bortezomib cycle. Serial PTH levels were measured in all enrolled patients. Median PTH variation across cycle of the entire group of patients is shown in Fig. 1A. Comparing patients who achieved at least a PR (according to the European Group for Blood and Marrow Transplant criteria) versus the others, median PTH levels separate after the second Bortezomib injections and were statistically higher in responding patients on days 11 (p=0.04), 21 (p=0.04), and 33 (p=0.04) (Fig. 1b). No significant changes from baseline in PTH levels were observed in control patients (≤PR). No serum differences in calcium, magnesium, phosphorus, and creatinine levels were recorded between responders and non-responder individuals.

Conclusion: This Phase II study is the first to show that response to bortezomib in multiple myeloma treated patients is associated with changes in serum PTH level concentration, which appears to be independent from calcium metabolic regulation.

Figure Figure
A Phase I Study of XL019, a Selective JAK2 Inhibitor, in Patients with Polycythemia Vera
Authors: Paquette Ronald , Sokol Lubomir , Shah Neil P. , Silver Richard T. , List Alan F. , Clary Douglas O. , Bui Lynne A. , Talpaz Moshe
Published: April 2020

More than 98% of patients with polycythemia vera (PV) carry the activating JAK2 mutation V617F, which may play a role in the pathogenesis of PV by promoting hematopoetic cell proliferation and survival. PV is characterized by an increased red blood cell mass and is generally accompanied by increased myeloid and megakaryocytic production. Current therapy consists of supportive care measures including aspirin, phlebotomy, interferon and hydroxyurea to reduce the risk of complications of the disease such as thrombotic events. Such palliative therapy is often unsatisfactory due to side effects and the persistent susceptibility of the patients to development of thrombosis, and the failure to prevent transformation to myelofibrosis or acute leukemia. XL019 potently and selectively inhibits JAK2 (Ki of 2 nM); relative to other JAK family kinases (JAK1 Ki of 134 nM, JAK3 Ki of 195 nM and TYK2 Ki of 344 nM). Cellular selectivity of XL019 was confirmed in primary human cell assays; while EPO-stimulated pSTAT5 in primary erythroid cells was quite sensitive to XL019 treatment (IC50 of 64 nM), stimulation of T-cells with IL-2 or B-cells with IL-4 or IL-6 gave pSTAT IC50s of >800 nM in each case. Potent effects on JAK2-STAT signaling were also demonstrated in single-dose pharmacodynamic studies in xenograft tumors, including HEL92.1.7, CFPAC-1 and DU 145. XL019 is being evaluated in a Phase 1 dose escalation study in subjects with PV who have failed or are intolerant of standard therapies defined as follows: symptomatic splenomegaly in spite of current therapy, thrombocytosis with a platelet count >450K/μl, or non-controlled constitutional symptoms such as pruritus, night sweats, or weight loss. The primary objectives of this study are to determine the safety and tolerability of XL019 when administered orally either once daily, or every Monday, Wednesday, and Friday in 28-day cycles (starting doses 10 mg QD and 25 mg QMWF). Secondary objectives include determination of the pharmacokinetics and pharmacodynamics of XL019, and to evaluate clinical response using the following endpoints:

hematologic response; time to hematologic response; duration of hematologic response; phlebotomy independence; change in spleen and/or liver size; change in bone marrow morphology; impact on quality of life (QOL); and impact on functional exercise capacity.

Pharmacodynamic assessments for this study include JAK2 V617F allele burden, erythropoietin-independent colony formation, changes in cytokine levels and JAK2 signaling pathways in peripheral blood mononuclear cells, and changes in bone marrow histology. Our initial findings regarding the tolerability, pharmacokinetics, pharmacodynamics, and preliminary clinical activity of XL019 in PV patients will be presented.
CD74 Is a Regulator of Fas-Mediated Apoptotic Signaling
Authors: Berkova Zuzana , Wang Shu , Wise Jillian , Wang Suizhao , Tao Ronghua , Maeng Hoyoung , Hawke David , Kwak Larry W. , Samaniego Felipe
Published: April 2020

Resistance to Fas-mediated apoptosis in hematopoietic cancers interferes with the efficacy of currently available chemotherapy. Our prior studies of human herpesvirus 8 oncoprotein K1 showed that K1 binds to Fas and interferes with activation of Fas-mediated apoptotic signaling (Wang, W, et al, Blood2007; 109:5455–62). Herpesvirus proteins often mimic host proteins or their functions, we thus searched for cellular proteins associated with inactive Fas in order to identify potential regulators of Fas signaling. We identified CD74, the invariant light chain of the major histocompatibility class II complex, associated exclusively with inactive/activation resistant Fas in B-cell lymphoma-derived BJAB cell line. Interestingly, overexpression of CD74 has been previously reported in ~ 90% of hematopoietic cancers and derived cell lines (Stein, R, et al., Ciln Cancer Res.2007; 13: 5556s–63s), as well as in ~ 80% of non-small-cell lung carcinoma (Ioachim, H.L., Am J Surg Pathol.1996; 20: 64–71). The role of CD74 in carcinogenesis was thus suspected. Through siRNA suppression of CD74 expression in BJAB cells we have determined that CD74 positive cells are more resistant to agonistic antibody CH-11-induced Fas-mediated apoptosis then the CD74 siRNA transfected cells (38 ± 7.8 % vs. 54 ± 9.8 %; P = 0.045). In addition, a challenge with agonistic anti-Fas antibody showed a significant survival advantage of the mice expressing CD74 in their livers over the vector-transfected mice (83% vs. 0 %; P < 0.016). We have mapped domain of CD74 required for its association with Fas and for significant protection of mice to a membrane-proximal extracellular region of CD74. Treatment of BJAB cells for 24 hours with humanized anti-CD74 antibody (hLL1 + crosslinking antibody), FasL-Fc, or anti-Fas antibody CH-11 induced apoptosis in 19%, 25% and 13% of cells, respectively. Combination of hLL1+crosslinking antibody with FasL-Fc or CH-11 increased apoptosis to ~ 45% of cells in both cases (P < 0.01), while combination of FasL-Fc or CH-11 with hLL1 + irrelevant antibody had no significant effect on the extent of apoptosis. Our results support the idea of an endogenous regulatory system of Fas-mediated apoptosis that utilizes transmembrane proteins interacting with Fas. We anticipate that specific blocking of the CD74-Fas interaction will sensitize CD74 overexpressing cancer cells to Fas-mediated apoptosis and thus will lead to a more effective chemotherapy for hematopoietic cancers.
Rig-I Activates Expression of Interferon-Stimulated Genes (ISGs) and Inhibits the Proliferation of Acute Myeloid Leukemia Cells
Authors: Zhang Nan-Nan , Chen Lei , Zhang Wu , Li Xian-Yang , Jiang Lin-Jia , Ding Fei , Zhang Hong-Xin , Chen Sai-Juan , Wang Zhu-Gang , Chen Zhu , Zhu Jiang
Published: April 2020

Acute promyelocytic leukemia (APL) is initiated by the formation of PML/RARα oncogenic fusion protein, a potent transcriptional repressor. Retinoid acid (RA) at pharmacological dosage can physically bind to the PML/RARα protein, ushering in the unfolding of downstream programs normally regulated by the wild type RARα. However, through what particular regulatory pathways RA inhibits APL malignant hematopoiesis has remained largely obscured. Rig-I is one of the genes whose mRNA levels were highly up-regulated, along with all-trans-RA (ATRA)-induced terminal granulocytic differentiation of APL cell line NB4 cells in vitro. Based on the analysis of a Rig-I−/− mouse model, recently we have reported a critical regulatory role of Rig-I in normal granulopoiesis. To understand the functional contribution of Rig-I induction in RA-mediated leukemia cell differentiation, we converted a pair of previously reported Rig-I RNAi-duplex sequences into a miR30a-based small hairpin-encoding sequence, which was expressed under the CMV enhancer/promoter within a lentiviral vector. As expected, Rig-I shRNAmir30 infection induced a significant knockdown of Rig-I protein level, and accordingly its delivery into HL-60 cells partially inhibited ATRA-induced granulocytic differentiation, growth inhibition/cell cycle arrest and apoptosis induction, suggesting that Rig-I upregulation participates in RA-induced granulocytic differentiation of acute myeloid leukemia cells. In order to investigate the effect of Rig-I induction on the proliferation of APL cells in vivo, we transduced PML/RARα-harboring leukemic cells with vector or Rig-I-expressing retrovirus, and then transplanted these cells into the syngeneic mice. The vector-transduced APL cells readily expanded in vivo, but the proliferation of Rig-I-transduced cells was apparently prohibited. Moreover, we found that the forced expression of Rig-I induced the expression of numerous ISGs in APL cells, which was recapitulated by the transduction of the C terminal part of Rig-I, but not by the N terminal part. In line with this, during the in vitro short-term culture post-IFNγ or IFNα stimulation, Stat1 phosphorylation at p701 in Rig-I−/− granulocytes was significantly inhibited. In parallel, the induction of multiple ISGs by IFNs was also significantly impaired. In conclusion, our findings indicate that the Rig-I induction inhibited APL reconstitution potentially through up-regulating a number of ISGs via regulating Stat1Tyr701 phosphorylation.
The D816V Mutation of C-Kit Circumvents a Requirement for Src Family Kinases in C-Kit Mediated Signal Tranduction and Transformation
Authors: Sun Jianmin , Pedersen Malin , Rönnstrand Lars
Published: April 2020

The receptor tyrosine kinase c-Kit plays a critical role in hematopoiesis and gain-of-function mutations of the receptor are frequently seen in several malignancies, including acute myeloid leukemia (AML), mastocytoma and sinonasal NK/T cell lymphomas. The most common mutation of c-Kit in these disorders is a substitution of the aspartic acid residue in position 816 to a valine (D816V), leading to constitutive activation of the receptor. In this study we aimed to investigate the role of Src family kinases in c-Kit/D816V signaling. Src family kinases are necessary for the phosphorylation of wild-type c-Kit as well as for activation of downstream signaling pathways including receptor ubiquitination and the Ras/MEK/Erk pathway. Tyrosine 568 of c-Kit, that is important for Src activation by wild-type c-Kit, was mutated to phenylalanine in both wild-type c-Kit and c-Kit/D816V and stably transfected into the hematopoietic cell line Ba/F3. Our data demonstrate that, unlike wild-type c-Kit, the phosphorylation of c-Kit/D816V is not dependent on Src family kinases. In addition we found that neither receptor ubiquitination nor Erk activation by c-Kit/D816V required activation of Src family kinases. In vitro kinase assay using synthetic peptides revealed that c-Kit/D816V had an altered substrate specificity resembling Src and Abl tyrosine kinases. The serine/threonine kinases Akt and Erk play important roles in cell survival and proliferation mediated by receptor tyrosine kinases. We could show constitutive activation of both PI3-kinase pathway and Erk in Ba/F3 cells expressing c-Kit/D816V, although ligand stimulation induced even stronger activation. We further present evidence that, in contrast to wild-type c-Kit, Src family kinases are dispensible for c-Kit/D816V cell survival. Taken together, we demonstrate that the signal transduction pathways mediated by c-Kit/D816V are markedly different from those activated by wild-type c-Kit and that altered substrate substrate specificity of c-Kit circumvents a need for Src family kinases in signaling of growth and survival, thereby contributing to the transforming potential of c-Kit/D816V.
Leukemia Induced by Altered TRK-Signaling Is Sensitive to Rapamycin Treatment in a Preclinical Model
Authors: Rhein Mathias , Yang Min , Kaever Volkhard , Meyer Johann , Baum Christopher , Li Zhixiong
Published: April 2020

Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) are high affinity NT-receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. Recently, we and others have obtained evidence for potential involvement of this receptor system in leukemia. In a prospective study involving 94 adult patients, we demonstrated for the first time cell surface expression of the three TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK receptor was expressed in 55% of the analyzed cases (Li Z et al., ASH 2007). Altered TRK signaling efficiently transformed murine hematopoietic stem/progenitor cells (Meyer J et al., Leukemia 2007; Li Z et al., ASH 2007). We observed constitutive activation of mammalian target of rapamycin (mTOR) both in murine and human leukemic cells. Murine leukemic cells induced by altered TRK signaling were very sensitive to rapamycin treatment in vitro. We next tested the therapeutic effect of rapamycin on altered TRK-induced leukemia in a mouse model (C57Bl/6J). Leukemic cells isolated from #483 mouse transplanted with primary hematopoietic stem/progenitor cells modified with deltaTrkA, an active mutant of TRKA isolated from a patient with acute myeloid leukemia, grew factor-independently. Treatment of #483 cells with rapamycin (10–50nM) induced apoptosis and showed a dose-dependent growth inhibition (up to 100%) in colony forming assay. Consistently, mTOR was strongly dephosphorylated. In pilot studies, we found that

transplantation of 106 #483 cells into recipients constituted with sublethal irradiation (7.5Gy) gave rise to leukemia development in all animals after a latency of 8 weeks; Daily i.p. injection of 2mg/kg rapamycin in healthy animals mediated a high level of rapamycin in whole blood (around 50ng/ml).

Thus, after irradiation, 20 animals were transplanted with 106 #483 cells and randomized in two groups. One group was treated daily with rapamycin 2mg/kg i.p., the other group received only carrier (placebo). The treatment begun 3 weeks after transplantation and continued until the last animal succumbed to leukemia. Rapamycin treatment significantly prolonged the survival of animals compared with control group (mean survival 48.5 and 32 days, respectively, P=0.0087) (Figure 1). Concentration of rapamycin in whole blood at the time of end point analysis ranged 26–151ng/ml. Our findings suggest that mTOR plays an important role in leukemogenesis induced by altered TRK signaling. TRKs and mTOR might serve as therapeutic targets in acute leukemia.

Figure 1 Kaplan-Meier estimate of survival of animals after treatment. Figure 1. Kaplan-Meier estimate of survival of animals after treatment.
Functional Genomics in Zebrafish Permits Rapid Characterization of Novel Platelet Membrane Proteins
Authors: Salles Isabelle I , O’Connor Marie N , Watkins Nicholas A. , Walker Adam , Garner Stephen F , Jones Chris I , Macauley Iain , Steward Michael , Zwaginga Jaap Jan , Bray Sarah L , Cvejic Ana , Dudbridge Frank , de Bono Bernard , Deckmyn Hans , Stemple Derek L. , Ouwehand Willem
Published: April 2020

In this study we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell the megakaryocyte, together with nucleated blood cell elements, endothelial cells and erythroblasts, identified novel platelet membrane proteins with hitherto unknown role in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of five of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet Glycoprotein (GP) IIb and the coagulation protein Factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of four of the five novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.