bluuebunny/crossref_metadata_2025 huggingface dataset:
Myeloid Translocation Gene 16 (MTG16) Regulates Lymphopoiesis
Authors: Hunt Aubrey A , Steapleton Melissa Ann , Moreno Isabel , Hiebert Scott
Published: April 2020

The Myeloid Translocation Gene (MTG) family was first discovered through the (8;21) translocation that leads to acute myeloid leukemia. This translocation fuses nearly all of Myeloid Translocation Gene 8 (MTG8) to an N-terminal portion of Acute Myeloid Leukemia 1 (AML1), thus disrupting the normal function of MTG8 as a transcriptional co-repressor. Two other family members have since been identified: Myeloid Translocation Gene 16 (MTG16) and Myeloid Tumor Gene Related-1 (MTGR1), both of which are implicated in leukemogenesis. To examine the physiological roles of Mtg16, a target of the t(16;21) that produces Acute Myeloid Leukemia, we deleted it in mice. We found that deletion of Mtg16 perturbs both B-and T-cell development, resulting in a reduced number of peripheral lymphocytes confirmed by complete blood counts and flow cytometry analysis of spleen and lymph node populations. These mice also display a dramatic two-to-three fold decrease in thymic cellularity. Analysis of the bone marrow indicated that there was also loss of B220+ cells, suggesting that there was a general deficit in lymphopoiesis in the absence of Mtg16. Upon closer examination, we find several significant changes throughout the development of both B and T cells. B-cells accumulate in the earliest stages of development, with Pre-pro B increasing in proportion of total B cells after inactivation of Mtg16. This change occurs at the expense of more mature populations, as immature and mature B-cells decrease in percentage significantly. Mtg16 (−/−) thymocytes show slight but statistically significant decreases in Double Negative 1 (DN1) and Double Negative 2 (DN2) subpopulations. Thymocytes also accumulate in the most mature CD4+ and CD8+ subsets in the absence of Mtg16. Mtg16 thus regulates lymphopoiesis at multiple steps, but the most intriguing changes occur in stem and progenitor populations. In fact, the deletion of Mtg16 also results in an increase in myeloid lineages, particularly granulocytes and macrophages, suggesting that Mtg16 plays a role in myeloid vs. lymphoid lineage decisions. Indeed, the deficit in lymphopoiesis appears to be due to increased myeloid commitment and/or expansion of myeloid progenitor cells. Even when cultured in conditions favoring lymphopoiesis, Mtg16-null lineage-/Sca1+/c-Kit+ cells displayed a predisposition towards myeloid development. Because of its ability to interact with a number of different transcription factors that participate in lymphoid and myeloid cell fate decisions, Mtg16 may provide a key piece of the transcriptional machinery that regulates lineage commitment.
Production and Expression of Inflammatory Mediators in Leukocytes of Sickle Cell Anaemia Patients and Effects of Hydroxyurea Therapy on This Production
Authors: Lanaro Carolina , Franco-Penteado Carla Fernanda , Albuquerque Dulcinéia M , Saad Sara T.O. , Conran Nicola , Costa Fernando F.
Published: April 2020

Leukocytosis is frequently observed in sickle cell disease (SCD) in the absence of bacterial infection. An elevated baseline leukocyte count is associated with an increased risk of early death and leukocytes play a significant role in the initiation of vaso-occlusive events. Inflammation, cell adhesion to vascular endothelium, and subsequent endothelial injury appear to contribute to sickle cell anemia (SCA) vaso-occlusion. Furthermore, blood levels of inflammatory and anti-inflammatory cytokines are reported to be elevated (TNF-α, IL-6, IL-10, GM-CSF), both in steady state and during crisis, but reports have been conflicting and a conclusive role for these molecules in the disease remains to be established. Furthermore, the effect of hydroxyurea therapy (HU) on the release of inflammatory mediators is not understood. The aim of this study was to determine plasma levels and leukocyte gene expressions of inflammatory mediators in healthy controls (n=30), steady-state SCA patients (n=45) and SCA patients on HU therapy (n=24). qRT-PCR analysis was use to examine gene expression and ELISA protein production. TNF-α, IL-8 and PGE2 plasma levels were significantly higher in the plasma of steady-state SCA individuals, when compared to control individuals (2.95 ± 0.4 pg/ml; 16.5 ± 2.5 pg/ml; 5.7 ± 0.6 pg/ml; 128.3 ± 12.2 pg/ml vs 1.43 ± 0.2 pg/ml, 88.5 ± 5.9 pg/ml, P=0.006; P<0.0001; P=0.012, respectively). HU therapy significantly reversed augmented TNF-α (1.6 ± 0.2 pg/ml, P=0.006) and, interestingly, increased plasma anti-inflammatory IL-10 (P<0.05). IL-10, IFN-γ, COX-2 and iNOS gene expressions were unaltered in SCA mononuclear cells (MC), however gene expressions of TNF-α, IL-8 and the protective enzyme, heme oxygenase-1 (HO-1), were significantly higher compared to healthy controls (0.46 ± 0.01; 0.08 ± 0.02; 0.21 ± 0.05 vs 0.18 ± 0.04; 0.02 ± 0.005; 0.035 ± 0.008; respectively, P<0.02). HU therapy was not associated with significantly altered SCA MC inflammatory gene expression, although COX-2 mRNA expression was decreased (0.11 ± 0.05; 0.37 ± 0.12, SCAHU and SCA, respectively; P<0.05). In SCA neutrophils, gene expressions of IL-8, IFN-γ, iNOS and HO-1 were significantly higher compared to those of control subjects (0.32 ± 0.07; 0.69 ± 0.19; 0.19 ± 0.06; 0.33 ± 0.09, P=0.02, P=0.025, P<0.05; P=0.027, respectively). Patients on HU therapy demonstrated lower iNOS and higher IL-10 neutrophil gene expressions compared to SCA not on HU therapy (0.038 ± 0.03; 0.72 ± 0.13, P<0.05; P<0.05, respectively). Taken together, data suggest that alterations in the gene expressions and productions of a number of pro-and anti-inflammatory mediators are present in SCA and knowledge of these pathways may be important for identifying novel drug targets for the disease.
Expression Levels of CD11b, CD18, CD32, CD62L (L-Selectin) and CD62P (P-Selectin) and Its Role in Sickle Cell Anemia Inflammatory State
Authors: Cerqueira Bruno Antônio Veloso , Boas Wendell Vilas , Seixas Magda Oliveira , Damasceno Elder Trindade , Souza Cyntia Cajado , Reis Mitermayer Galvão , Goncalves Marilda Souza
Published: April 2020

Vascular occlusions (VOC) underlie most of the acute and chronic sickle cell anemia (SCA) clinical complications and have been correlated to soluble adhesion molecules up regulation and leukocyte activation. A sequential process involving adhesion through selectins and integrins governs leukocyte recruitment to activated endothelium and to sickle red blood cells (RBC), resulting in heterotypic aggregation and VOC. The chronic hemolysis is the major cause of oxidative stress and it can induce transcriptions factors involved in the recruitment of adherent leukocytes in venules. In this work, we assessed the inflammatory potential of leukocytes in venous blood samples by examining cell surface antigens expression by flow cytometry, activation state and its association with hemolytic state in SCA patients group. The study was approved by the FIOCRUZ ethical committee and informed consents were signed by patients or official responsible. Leukocytes were obtained from 22 SCA patients and 22 healthy controls after RBC lyses and labeled with monoclonal anti-CD11b, anti-CD18, anti-CD32, anti-CD62L (L-Selectin) and anti-CD62P (P-Selectin). Leukocyte activation was stimulated by LPS. Statistical analyses were performed using SPSS version 9.0. The basal expression levels on leukocytes cell surface antigens from patients were not different from the control group. However, the CD62L expression levels were associated to C-reactive protein (CRP) higher levels and decrease of fetal hemoglobin in SCA patients (p=0.012). The SCA patients had higher CRP levels when compared to reference levels. Moreover, the data showed a co-expression of CD11b with CD18 (p<0.0001) and CD62P (p=0.011).The platelet count was positively correlated to CD11b expression (p=0.016) and the alanine transaminase high levels with the CD62P expression (p=0.012). Our results demonstrate a leukocyte chronic activation state by expression of CD62L related to CRP higher levels. Interestingly, the platelets could be activated by the indirect activation of CD62P by CD11b, participating of the inflammation state presents at vaso-occlusive events. It seems that the stress oxidative generated by the hemolytic state can contribute to endothelial dysfunction and vaso-occlusive events by CD62L activation in SCA. Fetal hemoglobin is a prognostic factor for several sickle cell complications and we showed that it can ameliorate the CD62L expression on leukocytes, decreasing the chronic inflammatory state of this disease. CD62L serves to as a homing receptor for naïve T lymphocytes and dendritic cells to lymph nodes, mediating the biding of T cells to high endothelial venules, in this view, this can be important marker to inflammatory state and vascular complications in SCA.
Prognostic Impact of Mutations in the Nucleophosmin (NPM1) and of the FMS-Related Tyrosine Kinase 3 (FLT3) Genes in Elderly Patients with Acute Myeloid Leukemia (AML) Treated with Intensive Chemotherapy
Authors: Etienne Anne , Borel Cecile , Reutenauer Sarah , Olschwang Sylviane , Mozziconacci Marie-Joelle , Delabesse Eric , Demur Cecile , Prebet Thomas , Huguet Francoise , Charbonnier Aude , Recher Christian , Vey Norbert
Published: April 2020

AML is a heterogeneous disease and the actual most reliable prognostic factor is the karyotype. Mutations of NPM1 and FLT3 constitute the most frequent molecular genetic alterations in patients with AML. Their prognostic impact is now well recognized, especially in young patients with intermediate prognosis karyotype. However few studies have focused on elderly patients. We retrospectively studied the prevalence of NPM1 mutation and FLT3 internal tandem duplications (ITD) and its association with complete remission (CR) and survival in 86 patients aged 61 years or older treated between 1996 and 2007 for acute non promyelocytic leukemia with intermediate karyotype in the Institut Paoli-Calmettes, Marseille, and the university hospital of Toulouse. All patients received intensive induction chemotherapy (“3+7” regimen). Median age was 70 years (range, 61–79). The median follow-up of surviving patients was 34 months. 44 patients (51%) had NPM1 mutation and 21 had FLT3-ITD (24%). In the NPM1 mutated group, there were significantly more: female patients, absence of antecedent hematologic disorder, de novo AML, and low CD34 expression. In the FLT3-ITD group, there were significantly more: female patients, increased white blood cell counts and peripheral blood blasts. Overall CR rate was 67%; median disease free survival (DFS) and overall survival (OS) were 11 and 10 months, respectively. CR rate was negatively associated with a poor performans status and a high score previously described including assessment of comorbidities (Etienne et al., Cancer2007; 109(7):1376–83). CR rate was 57% versus 72% for patients with FLT3-ITD compared to patients with wild-type FLT3 (p 0.2), and 64% versus 71% for the NPM1 mutated group compared with the NPM1 non-mutated group (p 0.5). Median OS was 6 months versus 12 months for patients with FLT3-ITD mutation versus wild type patients (p = 0.04). Median OS was 9 months for patients with NPM1 mutation and did not differ from non-mutated patients. No significant difference in term of CR rate and OS was found between the 29 patients carrying NPM1 mutation without FLT3-ITD and the 57 other patients without NPM1 mutation or with FLT3-ITD. Median DFS was 21 months versus 9 months for these two groups, respectively (p 0.2). These results confirm the prognostic impact of FLT3-ITD in our series of old patients with AML. Unlike young patients, NPM1 mutated elderly patients have a similar outcome than NPM1 wild type patients. The absence of prognostic impact of NPM1 mutation without FLT3-ITD has to be validated on larger prospective cohort. FLT3 status should be taken into account for treatment choices in elderly patients.
Identification of Novel Activities of Immunomodulatory Drugs: Cytoskeleton Reorganization through Modulation of Rho GTPases
Authors: Xu Yibing , Li JianWu , Ferguson Greg , Mercurio Frank , Khambatta Gody , Morrison Lisa , Lopez-Girona Antonia , Corral Laura G , Webb David , Bennett Brydon L. , Xie Weilin
Published: April 2020

Immunomodulatory drugs including the IMiDs®, lenalidomide, and pomalidomide represent a novel class of compounds that have both anti-cancer and anti-inflammatory properties. While many studies have demonstrated that IMiDs have broad in vitro and in vivo biological activities including antiangiogenesis, inhibition of TNFa expression, enhancement of antitumor immunity, and induction of IL-2 in T cells, the molecular mechanism through which these drugs exert their effects is largely undefined. In primary human monocytes, IMID1 selectively activated RhoA and Rac1, but not Cdc42 or Ras. Importantly, the activation of these GTPases occurred immediately following treatment with IMID1 in the absence of any costimulation. Consistent with the activation of Rho GTPases, we found that IMID1enhanced F-actin formation, stabilized microtubules, and increased monocyte cell migration, all of which were blocked by selective inhibitors of ROCK1, a downstream effector of RhoA or Rac1. Finally, we demonstrated that IMID1 was able to regulate the activity of Rho GTPases and formation of F-actin in primary human T cells similarly as it did in monocytes, and showed that the activation of RhoA was essential for IMID1-induced IL-2 expression in T cells. In conclusion, these studies demonstrate a novel and acute molecular activity from IMiDs mediated via Rho GTPases. Activation of Rho by IMiDs and the resulting effect on cytoskeletal reorganization may represent a critical mechanism by which IMiDs function as therapeutic immunomodulatory agents.
Crystal Structure of Von Willebrand Factor (VWF) A1 Domain in Complex with Aptamer ARC1172, An Inhibitor of VWF-Platelet Binding
Authors: Huang Ren-Huai , Fremont David , Tuley Elodee A , Diener John L , Schaub Robert G , Sadler J. Evan
Published: April 2020

ARC1779 is a modified 40-mer oligonucleotide aptamer that binds with high affinity to the VWF A1 domain and blocks VWF binding to platelet GPIb-alpha. When given intravenously to volunteers and assayed ex vivo, ARC1779 dose-dependently inhibits VWF activity and prolongs the PFA-100 closure time with EC90 values of 2–3 μg/ml. ARC1779 also inhibits carotid artery thrombosis induced by electrical injury in nonhuman primates. These properties suggest that ARC1779 may be useful to prevent or treat platelet-dependent thrombosis in acute coronary syndromes, percutaneous coronary intervention procedures, or thrombotic thrombocytopenic purpura. To characterize this aptamer-VWF interaction, we determined the crystal structure of a complex between recombinant VWF domain A1 (residues 497–705, mature subunit numbering) expressed in E. coli and ARC1172, a 41-mer DNA oligonucleotide with the sequence 5′-GGC GTG CAG TGC CTT CGG CCG TGC GGT GCC TCC GTC ACG CC-invdT-3′. ARC1779 was derived from ARC1172 by adding modifications to prevent digestion by nucleases, and both aptamers bind VWF A1 with the same affinity. Crystals of the complex belonged to space group P212121 and diffracted to 2.4 angstroms. The A1 domain (Figure) has essentially the same structure as observed previously in isolation or bound to GPIb-alpha, botrocetin, bitiscetin, or Fab NMC4. Therefore, aptamer binding does not substantially alter the conformation of the VWF A1 domain. ARC1172 consists of three short helical segments and three connecting loops. Nucleotides C32-C40 interact with two segments G23-G26 and G2-G6 to form a 9-basepair helix with an interruption in one strand. The interruption comprises residues C7-T22, which protrude to form a 3-basepair helix between G11-C13 and G17-C19. The loop of ARC1172 composed of nucleotides G26-C32 makes extensive contacts with VWF A1 residues in helix alpha-4 (yellow) and alpha-5 (orange) and loop betaD-alpha4. For example, the side chains (shown in Figure) of Q625, Q628, R632 and Y637 participate in numerous hydrogen bonds and salt-bridges with ARC1172 (e.g., Q625 contacts C29), and R632 is buried within a deep pocket of the aptamer. Botrocetin occupies a similar interface on VWF A1, which accounts for the ability of ARC1172 or ARC1779 to block botrocetininduced platelet aggregation. In addition, nucleotide T10 extends toward helix alpha3 (lime green) and obstructs residue K599, which is required for binding to platelet GPIbalpha. The results provide a structural explanation for many of the functional properties of aptamers ARC1172 and ARC1779.

Figure Figure
A Phase II Study of the Purine Nucleoside Phosphorylase (PNP) Inhibitor RO5092888 (BCX-4208) in Patients with Moderate to Severe Chronic Plaque Psoriasis: Safety, Tolerability and Lymphocyte Effects
Authors: Gomes Joseph P , Georgy Angela , Passe Sharon , Farid Adrienne , Bantia Shanta , Wolff Gerhard
Published: April 2020

Introduction: PNP is a purine-metabolizing enzyme that catalyzes the phosphorolysis of 2′-deoxyguanosine [dGuo] to guanine and deoxyribose-1-phosphate. In T-cells, PNP inhibition leads to accumulation of deoxyguanosine triphosphate (dGTP), triggering apoptotic cell death. Chronic-plaque type psoriasis (psoriasis) is an autoimmune disorder, in which T-cells contribute, at least in part, to the manifestations and maintenance of the disease. Therefore, targeting T-cells may be a beneficial treatment strategy. Since for chronic inflammatory diseases the safety of potential immunosuppressive drugs is a major consideration, we conducted this study to investigate safety and tolerability of oral RO5092888 (BCX-4208) in patients with moderate to severe chronic plaque psoriasis.

Methods: This was a randomized, double-blind, placebo-controlled, dose-ranging study. Sixty-six patients 18 to 70 years old were randomized into one of three groups: placebo, 20mg/d or 120mg/d. The study was conducted from August 2007 to June 2008. Patients received study drug over six weeks and were observed over an additional 4 weeks. Assessments for safety included tracking of adverse events (AEs) including infections; vital signs; ECGs; chemistry panel; LFTs; hematologic parameters including peripheral blood (PB) lymphocyte subsets CD3+, CD4+, CD8+, CD56+, CD20+; and urinalysis.

Results: 65 of the 66 enrolled patients were analyzed. One serious AE was observed in the 20mg/d group, a deep vein thrombosis (DVT) in a patient with a history of DVTs, and was considered unrelated to study drug. The percentage of patients experiencing at least one adverse event (AE) of any grade was placebo: 33% (7/21), 20mg: 41% (9/22), and 120mg: 59% (13/22). During treatment, two infections occurred in the placebo group (influenza and sinusitis), one in the 20mg group (nasopharyngitis), and 4 in the 120mg group (2-upper respiratory tract infections, 1 bronchitis, and 1 otitis externa). Reductions in PB lymphocytes and subsets were observed (Table 1). Nine patients showed decreased levels of CD4+ lymphocytes, below 350 cells/μL (20mg/d, 3; 120mg/d, 6).

Conclusion: Daily oral administration of 20 mg or 120 mg of RO5092888 for up to 6 weeks demonstrated adequate safety and tolerability. Reductions in PB T cells, T cell subsets and B cells were observed. Further investigation of RO5092888 is warranted in both T-cell and B-cell diseases.

Lymphocyte subpopulation Mean Nadir (% change from baseline) Placebo (n=21) 20 mg (n=22) 120 mg (n=22) CD3+ 24.0 30.6 47.5 CD4+ 23.6 28.9 44.9 CD8+ 26.1 31.8 53.9 CD20+ 37.5 42.6 64.2 CD56+ 40.1 47.7 72.6

Table 1
VWF-Cleaving Protease ADAMTS13 Reduces Brain Injury Following Ischemic Stroke in Mice: Essential Role for VWF in Stroke
Authors: Zhao Bing-Qiao , Chauhan Anil kumar , Patten Ian S. , Dockal Michael , Scheiflinger Friedrich , Wagner Denisa D.
Published: April 2020

Ischemic stroke is the second leading cause of death and disability. The only approved therapy available is recombinant tissue plasminogen activator (tPA), but its use remains limited. Therefore, there is a need for an alternative drug. Platelets and their adhesion receptors play a crucial role in modulating infarct size during ischemic stroke. ADAMTS13 (A Disintegrin-like And Metalloprotease with Thrombospondin type I repeats-13) is a plasma metalloprotease that cleaves von Willebrand factor (VWF) an important adhesion molecule for platelets at sites of vascular injury. In patients, an increase in circulating levels of VWF and a decrease in ADAMTS13 activity are considered risk factors for ischemic stroke. By using genetically-modified mice we have previously shown that ADAMTS13 down regulates both thrombosis and inflammation and recombinant human ADAMTS13 down regulates platelet thrombi in injured arterioles. All these processes were dependent on VWF. We therefore hypothesize that ADAMTS13 has a protective role after ischemic stroke. In this study, we show that VWF deficiency or VWF heterozygosity in mice reduces infarct volume by two-fold after focal cerebral ischemia compared to wild-type (WT) in the middle cerebral artery occlusion (MCAO) stroke model. Furthermore, infusion of recombinant human VWF in WT mice not only accelerates thrombosis in the ferric-chloride injured artery model, but also increases infarct volume compared to vehicle-treated controls. These findings suggest an essential role of VWF in modulating infarction after stroke. We also show that ADAMTS13 deficiency in mice results in approximately 20% larger infarcts after cerebral ischemia compared to WT. The larger infarcts observed in ADAMTS13 deficient mice were due to VWF because mice deficient in both ADAMTS13 and VWF had infarct sizes similar to VWF deficient mice. Importantly, infusion of r-human ADAMTS13 immediately before reperfusion (two hour after occlusion) significantly reduced infarct volume (106.2 ± 9.7 mm3 vs 75.8 ± 6.9 mm3, P<0.05). Of note, we observed that ADAMTS13 protein was induced in the ischemic penumbra region of brain after ischemic stroke. Our findings reveal an important role for VWF in modulating infarct volume after ischemic stroke. In addition, recombinant-ADAMTS13 could become a new therapeutic agent for stroke therapy.
Confirmation of the Efficacy and Safety of Lenalidomide Oral Monotherapy in Patients with Relapsed or Refractory Mantle-Cell Lymphoma: Results of An International Study (NHL-003)
Authors: Zinzani Pier Luigi , Witzig Thomas E. , Vose Julie M. , Reeder Craig B. , Buckstein Rena , Haioun Corinne , Bouabdallah Reda , Polikoff Jonathan , Guo Pingshan , Ervin-Hayes Annette , Pietronigro Dennis , Zeldis Jerome B. , Czuczman Myron S.
Published: April 2020

Introduction: Mantle-cell lymphoma (MCL) is a type of B-cell non-Hodgkin lymphoma (NHL) that typically responds to initial chemo-immunotherapy, but with a relatively short duration of response (DR) and progression-free survival (PFS) when treated with conventional chemotherapy agents. Although the overall survival (OS) of MCL has improved, most patients are not cured and new agents are needed. A sub-analysis of a recent phase II trial (NHL-002) investigating the efficacy and safety of lenalidomide in patients with relapsed or refractory MCL, demonstrated a promising overall response rate (ORR) of 53% with a median DR of 11.9 months. A confirmatory international phase II trial (NHL-003) of single-agent lenalidomide was initiated for patients with relapsed/refractory aggressive NHL that had received at least one prior treatment and had measurable disease. In this report, we analyze the current results from the MCL patients enrolled in this trial.

Methods: Patients with relapsed or refractory MCL and measurable disease ≥2 cm after at least 1 prior treatment regimen were eligible. Patients received 25 mg of lenalidomide orally once daily on days 1–21 of every 28-day cycle. Patients continued therapy until disease progression or toxicity. The 1999 IWLRC methodology was used to assess response and progression.

Results: This report focuses on the 39 MCL patients that were enrolled and evaluable for response assessment. Median age was 66 (33–82) years and 29 (74%) patients were male. Median time from diagnosis to lenalidomide treatment was 3.4 (0.4–9) years. Patients had received a median of 3 (1–8) prior treatments, and 23% (9/39) of patients had received prior bortezomib treatment. The ORR with lenalidomide was 41% (16/39), including 13% (5/39) complete response (CR)/unconfirmed CR, and 28% (11/39) partial responses. Ten (26%) patients had stable disease. The most common grade 3 or 4 adverse events were neutropenia (51%) and thrombocytopenia (25%), anemia (13%), fatigue (10%) and febrile neutropenia (10%).

Conclusion: These results confirm that lenalidomide oral monotherapy is effective in the treatment of patients with relapsed or refractory MCL, with manageable side effects.
Inhibitors of Heat Shock Protein 72 Induction Enhance Apoptosis Induced by Hsp90 Inhibitors
Authors: Davenport Emma , Zeisig Athanasia , Wiseglass Lauren , Moore Hannah E. , Hockley Sarah L. , Gonzalez David , Smith Emma , Sharp Swee , Workman Paul , Morgan Gareth J , Davies Faith
Published: April 2020

Exposure of myeloma cells to Hsp90 inhibitors (eg 17-AAG) or proteasome inhibitors (eg Bortezomib) results in a time-dependent induction of heat shock protein 72 (Hsp72), protecting cells from drug-induced apoptosis. Hsp72 is a member of the heat shock protein 70 (Hsp70) family and enables cells to cope with harmful aggregations of proteins during and after stress, protecting cells from apoptosis in addition to acting as a chaperone for proteins such as immunoglobulin. We show that inhibition of Hsp72 upregulation induced by these anti-myeloma drugs, by either inhibitors of Hsp70 induction or siRNA, increases susceptibility of myeloma cells to drug-induced apoptosis. This provides a rationale for using such inhibitors in combination with anti-myeloma drugs. A number of commercially available inhibitors of Hsp70 induction, including KNK437 and triptolide, were used to inhibit stress-induced upregulation of Hsp72. Combining Hsp90 inhibitors (Hsp90i) with these compounds increased cell death to 73% from 33% when Hsp90i were used alone. Cell cycle analysis indicated that combining these compounds with Hsp90 inhibitors overcame the G2 growth arrest induced by Hsp90 inhibitors and increased the proportion of cells in sub G1, indicative of apoptosis. Analysis of their anti-proliferative effects demonstrated that, in H929 cells, Hsp90 inhibitor/triptolide combinations had additive effects whilst in U266 cells their effects were synergistic. To determine the underlying mechanisms responsible for the effects of the combination, key proteins involved in endoplasmic reticulum (ER) stress, Unfolded Protein Response and caspase-dependent and independent signalling pathways were analysed. We show that ER stress signalling through IRE1 and PERK pathways, measured by cleavage of XBP1 and induction of CHOP, were initiated earlier and sustained longer, when cells were treated with the combination. Similarly, analysis of caspase-dependent and independent signalling demonstrated that the combination induced early cleavage of caspases 9, 8 and 3 which was sustained. Nuclear translocation of AIF, the caspase-independent cell death signal, was strongly induced suggestive of strong activation of this pathway. We demonstrate that death induced by triptolide or its combination with 17-AAG is p53-independent using of a panel of myeloma cell lines. To confirm our findings, we used lentiviral siRNA constructs targeting the inducible forms of Hsp70 and then analysed response to Hsp90 inhibitors. These experiments confirm the results generated using the chemical inhibitors of Hsp70 expression. In conclusion, inhibiting drug-induced Hsp72 expression appears to overcome cellular resistance to Hsp90 inhibitors, supporting the clinical development of Hsp70 inhibitors and their combination with Hsp90 and proteasome inhibitors.
NK Cell Anti-Tumor Immune Responses Are Impaired by Sorafenib but Not by Sunitinib
Authors: Krusch Matthias , Schlicke Manuela , Salih Julia , Baessler Tina , Kanz Lothar , Salih Helmut
Published: April 2020

Natural killer (NK) cells play an important role in the surveillance of tumors by exerting cellular cytotoxicity and also due to their ability to secrete immunoregulatory cytokines. A variety of intracellular kinases like PI3K, MAPKs or STAT molecules are involved in the regulation of NK cell functions. Therefore, treatment of tumor patients with small molecule inhibitors targeting various protein kinases may also largely affect NK cell anti-tumor reactivity. The multikinase inhibitors Sunitinib (SUTENT®) and Sorafenib (NEXAVAR®) have been approved for the treatment of patients with metastatic Renal Cell Carcinoma (RCC), and are now also being evaluated in various other tumor entities including AML. Both compounds target, among others, PDGF receptors, VEGF receptors, Flt3 and c-kit. Sorafenib additionally inhibits the MAPK pathway members B-RAF and c-RAF. Here we report that Sorafenib but not Sunitinib substantially reduces NK cell cytotoxicity as revealed by analysis of cell surface CD107a expression as a surrogate marker for mobilization of cytolytic granules on NK cells from ten healthy donors. In line, pharmacological levels of Sorafenib but not Sunitinib largely reduced lysis of several solid tumor and leukemia cell lines by primary NK cells, and this inhibitory effect of Sorafenib occurred concentration dependently. Furthermore, Sorafenib but not Sunitinib concentration dependently impaired NK cell IFN-γ production in coculture experiments with different tumor cell lines. Since Sorafenib, in contrast to Sunitinib, also potently inhibits RAF which is involved in MAPK activation we determined the influence of the two compounds on ERK phosphorylation which is important for the activation of NK cell anti-tumor reactivity. While Sunitinib had no effect, Sorafenib, alike the specific MEK inhibitor PD98059, markedly reduced ERK phosphorylation in NK cells. Taken together, out results indicate that NK cell-mediated anti-tumor immune responses are impaired by Sorafenib but not by Sunitinib via inhibition of signaling pathways crucial for NK cell effector functions. Thus, Sunitinib is exquisitely suitable in diseases and for therapeutic approaches which rely on a sufficient anti-tumor immune response like e.g. allogenic stem cell transplantation in AML or immunotherapeutic approaches in RCC patients.
Efficient Binding, but Moderate Modulation, of Human Dendritic Cell Functions by Milatuzumab, a Humanized Anti-CD74 Monoclonal Antibody
Authors: Chen Xiaochuan , Chang Chien-Hsing , Goldenberg David
Published: April 2020

Milatuzumab (hLL1, Immunomedics, Inc.), a humanized anti-CD74 immunoglobulin-G monoclonal antibody (MAb), has been shown to have therapeutic activity against CD74-expressing B-cell malignancies in vitro and in xenografts models, and is in clinical evaluation as a therapeutic MAb for non-Hodgkin lymphoma, chronic lymphocytic leukemia, and multiple myeloma. Since it is unclear whether this MAb has any effects on human antigen-presenting cells that express CD74, we investigated the binding efficiency, viability, and functional modulation of human dendritic cells (DC), the professional and most potent antigen-presenting cells, exposed to milatuzumab. We found that milatuzumab bound efficiently with B cells, monocytes, and different subsets of blood DCs including myeloid DC1 (BDCA-1+), myeloid DC2 (BDCA-3+) and plasmacytoid DC (BDCA-2+) in human PBMC, as well as with monocyte-derived immature DCs, but not LPS-matured DCs. The side-by-side comparative cytotoxicity assay showed that milatuzumab, in the presence of a second antibody for cross-linking (GAH, the F(ab′)2 of goat anti-human IgG Fcgamma-specific), dramatically reduced the cell viability of Daudi B-lymphoma cells, but did not influence the cell viability or induce apoptosis in monocyte-derived DCs, even at high concentations up to 50 μg/ml. At the concentrations ranging from 0.05 to 5 μg/ml, milatuzumab upregulated the expression of the antigen-presenting molecule, HLA-DR, and costimulatory molecules, CD54 and CD86, in human monocyte-derived DCs in a moderate, but dose-dependent manner, suggesting that milatuzumab could enhance DC constitutive maturation. Although this effect was not reflected by an enhanced T-cell expansion, as shown by unaltered CFSE-low, -medium, and –high peaks in total and CD4+ and CD4− T cells, milatuzumab-treated DCs could moderately promote the differentiation of CD4+ naïve T cells toward more Th1 effector cells, suggesting that milatuzumab can modulate DC functions, inducing the polarization and differentiation of functional Th cells. These data highlight the prospects of milatuzumab as a novel immunotherapeutic agent that possesses not only direct anti-proliferative effects against CD74+ hematological malignancies, but also some regulatory effects on DC-mediated immune functions, and no cytotoxic effect on DCs.
New Prognostic Data on Rare Cytogenetic Abnormalities in MDS: A Collaborative Study of the International Working Group on MDS Cytogenetics
Authors: Schanz Julie , Slovak Marilyn L. , Ohyashiki Kazuma , Solé Francesc , Mallo Maria del Mar , Steidl Christian , Pfeilstoecker Michael , Noesslinger Thomas , Hildebrandt Barbara , Luebbert Michael , Andersen Mette K , Giagounidis Aristoteles , Valent Peter , Fonatsch Christa , Germing Ulrich , Haase Detlef
Published: April 2020

Introduction: Several recent publications have advanced our knowledge of the prognostic significance of clonal cytogenetic abnormalities in MDS, yet the genetic risk assessment of the rare karyotypic aberrations in MDS patients (pts) remains unknown. Using the German-Austrian (G-A) Cytogenetics Database, we previously defined 24 cytogenetic prognostic subgroups; however, 12 subgroups characterized by non-complex (isolated or one additional abnormality only) karyotypes with del(9q), del(15q), t(15q), del(12p), −X, t(1q), t(7q), t(17q), −21, t(11q23), +19, t(5q) were observed infrequently (<10 pts) and considered too few for an informative risk assessment. To increase the number of informative pts and expand/validate the statistical robustness of these rare cytogenetic subgroups, an international collaboration was initiated and promoted under the auspices of the MDS Foundation.

Patients and Methods: A total of 90 new MDS pts with rare recurring abnormalities was collected from 12 MDS Foundation Centers of Excellence, of which 66 pts fulfilled the non-complex requirement. Survival was estimated by Kaplan-Meier analysis and restricted to pts treated by supportive care only. The final analysis included 108 pts: del(9q)(n=10); del(12p)=17; −X=10; t(1q)=13; t(7q)=14; −21=12; t(11q23)=12; +19=10; t(5q)=10. The frequency of non-complex del(15q), t(15q) and t(17q) remained below ten pts and deemed ineligible for further analysis at this time.

Results: The pooled international data showed an excellent correlation with the G-A data set in 6 of 10 cytogenetic subgroups: t(7q): 34.7 months (mo.) median survival (G-A) vs. 34.7 mo. (new data), del(9q): not reached vs. 63.1 mo., t(11q23): 20.0 vs. 28.0 mo, del(12p): 108.0 mo. vs. not reached, +19: 19.8 vs. 21.7 mo., −21: 32.0 vs. 35.0 mo. A moderate correlation was found for t(1q): 34.7 mo. vs. not reached and for del(15q): not reached vs. 26.7 mo. Discordant median survival was observed for t(5q): 4.4 mo. vs. not reached and −X: 56.4 mo. vs. 15.7 mo.

Conclusions: Through an international collaboration, we were able to define the prognostic impact of nine distinct yet infrequent, recurring cytogenetic aberrations observed in MDS pts. The discordant median survival differences observed in two subgroups may be attributed to various translocation partners and/or secondary abnormalities. Even though data collection remains ongoing, the current results expand and refine the cytogenetic prognostic classification system for pts suffering from MDS and underscore the need for standardized cytogenetic testing in MDS for further international collaborations.
Prevalence and Cause of Death in Patients with Monoclonal Gammopathy in Atomic Bomb Survivors in Hiroshima
Authors: Fujimura Kingo , Kasagi Fumiyoshi , Sasaki Hideo , Ito Chikako
Published: April 2020

Purpose: To examine the effect of Atomic bomb (A-bomb) radiation exposure condition on the development M-proteinemia in A-bomb survivors in Hiroshima.

Back ground: The influence of radiation exposure on the development of M-proteinemia remained unclear. The screening test for monoclonal gammopathy in A- bomb survivors in Hiroshima was started from Sep. 1988 at the Hiroshima Atomic Bomb Casualty Council Health management center to challenge this problem with the free access policy.

Materials: The peak year of the examined population was 1989, which was examined 42915 survivors. This population was designed as study population. This population consisted of five groups of irradiated conditions as follows, 60% was exposed directly in all over the Hiroshima city at A- bombing, 27% entered to center of Hiroshima city within 14 days after A-bombing, 9% helped the victims of A- bombing at the suburbs and 1% was exposed in prenatal state.

Examination study: Each participant was examined one test yearly. Routine laboratory tests, such as complete blood cell counts, serum biochemical examination and serum protein electrophoresis on cellulose acetate were performed for the first step screening. M-protein or low level of gammaglobulin was subsequently assessed in a detailed analysis by immunoelectrophoresis of serum and urine, and measurement of serum concentration of immunoglobulin andβ2-Microglobulin.

Results: One thousand and hundred eighty two cases of M-proteinemia were detected for 18 years. The over all prevalence rate was 2.75%, and 4.1% in males and 2.1% in females. Nine hundred twenty nigh cases of MGUS(78.6%), 147 cases of MM(12.4%), 80 cases of IgM Macroglobulinemia(6.8%) and 26 cases of two clone M-proteinemia(2.2%) were detected. Of these M-proteinemia, 669cases(2.6%) were developed in directly exposed population, 368cases(3.1%) were in entered population and 133 cases(3.4%) were in helper population. The prevalence rate of these M-proteinemia was increased depend on aging. Forty three cases (4.6%) of MGUS moved to overt MM for 18 years. The mean duration time from MGUS to overt MM was 8.9 years (2~18 years). Four hundred sixteen cases (35.2%) were died with MM or lymphoproliferative diseases (99cases,23.8%), cancer (93cases,22.3%), infection (50cases,12%), cardio or cerebral vascular diseases (89 cases,21.4%) and others (85cases,21%). The 101 cases (13.2%) of 766 living cases combined some cancer throughout the follow up. No significant differences in the prevalence rate of M-proteinemia and death from MM or combination with cancer were observed among any A-bomb irradiated conditions.

Conclusions: There were no clear correlations between the development or disease state of M-proteinemia and the irradiated conditions. We are now starting to analyze the individual radiation dose effects longitudinally on the development of M-proteinemia.
Osteoclast Gene Expression Profiling in Multiple Myeloma
Authors: Moreaux Jerome , Hose Dirk , Rème Thierry , Moine Philippe , Mahtouk Karène , Moos Marion , Requirand Guilhem , De Vos John , Rossi Jean François , Goldschmidt Hartmut , Klein Bernard
Published: April 2020

Multiple myeloma (MM) is a fatal hematologic malignancy associated with clonal expansion of malignant plasma cells within the bone marrow and the development of a destructive osteolytic bone disease. The principal cellular mechanisms involved in the development of myeloma bone disease are an increase in osteoclastic bone resorption, and a reduction in bone formation. Myeloma cells (MMC) are found in close association with sites of active bone resorption, and the interactions between myeloma cells and other cells within the specialized bone marrow microenvironment are essential, both for tumor growth and the development of myeloma bone disease. In order to investigate the gene expression profile (GEP) of osteoclastic cells, we compare GEP of osteoclastic cells (7 samples) with normal B cells (7 samples), normal bone marrow plasma cells (7 samples), bone marrow stromal cells (5 samples), bone marrow CD3 cells (5 samples), CD14 cells (7 samples), CD15 cells (7 samples), CD34 cells (7 samples) and primary MMC (123 samples). Using SAM analysis, a set of 552 genes was overexpressed in osteoclasts compared to others cell subpopulations with a FDR ≤ 1% and a ratio ≥ 2. Osteoclasts specifically overexpressed genes coding for chemokines (CCL2, CCL7, CCL8, CCL13, CCL18, CXCL5 and CCL23) and MMC growth factors (IGF-1, APRIL and IL-10). Anti- IGF-1 receptor and TACI-Fc inhibit MMC growth induced by osteoclasts. Among the chemokines overexpressed by osteoclasts, the majority of them have a common receptor: CCR2 expressed by MMC. Anti-CCR2 MoAb inhibits migration of the CCR2+ HMCL in response to osteoclasts. Expression data of purified MMC were analyzed by supervised clustering of group with higher (CCR2high) versus lower (CCR2low) CCR2 expression level. Patients in the CCR2high group are characterized by a higher bone disease. A set of 176 genes was differentially expressed between CCR2high and CCR2low MMC. CCR2high displayed a gene signature linked to the dependency of MMC on the interactions with the BM osteoclastic subpopulation and the osteoclastic bone resorption.

Taken together, our findings suggest addition of chemokine antagonists to current treatment regimens for MM should result in better therapeutic responses because of the loss of both the protective effect of the bone marrow environment on the MMC and the osteoclastic cells activity.
OH-2, a Hyperdiploid Myeloma Cell Line without An IGH Translocation, Has a Complex Translocation Juxtaposing MYC near MAFB and the IGK Locus
Authors: Våtsveen Thea Kristin , Tian Erming , Kresse Stine H , Meza-zepeda Leonardo A , Gabrea Ana , Dai Hong Yan , Kuehl Michael , Sundan Anders , Borset Magne
Published: April 2020

Despite heterogeneity of phenotypes, multiple myeloma (MM) can be classified into two major groups: hyperdiploid (HRD) tumors with 48–74 chromosomes, which typically have extra copies of at least four of the eight odd chromosomes 3, 5, 7, 9, 11, 15, 19, and 21; and non-hyperdiploid (NHRD) tumors, which usually have immunoglobulin heavy chain (IGH) translocations and <46 or >74 chromosomes. Human myeloma cell lines (HMCL) with a typical HRD genotype are lacking. The OH-2 HMCL was derived from extramedullary myeloma, and retains the same HRD phenotype as the primary tumor, with extra copies of chromosomes 3, 7, 15, 19, and 21, as demonstrated by array comparative genomic hybridization. This provides a unique example of an HMCL and the corresponding primary tumor that share the same HRD phenotype. Also Spectral Karyotyping showed the same HRD phenotype. By fluorescence in situ hybridization it was shown that OH-2 does not have an IGH or Ig lambda light chain translocation. Instead, both the HMCL and the primary tumor have complex translocations involving chromosomes 2, 8, and 20. The translocation juxtaposes MAFB near MYC and the Ig kappa (IGK) enhancer. Both MYC and MAFB are highly expressed, as shown by microarray analysis, and are thought to be dysregulated by the IGK enhancer. Both OH-2 HMCL and the primary tumor cells express high levels of Cyclin D2, an expected consequence of the enhanced MAFB level. OH-2 has a phenotype similar to the 20% of HRD MM tumors that express increased levels of Cyclin D2 but not Cyclin D1 and could be a good in vitro model for studies of this subgroup of MM. OH-2 is dependent on medium containing human serum and growth-promoting cytokines like IL-6 or IL-21, reflecting the critical role of the microenvironment for growth of MM cells.
Promoting Osteoblastogenesis Using a Novel Dkk-1 Neutralizing Antibody in the Treatment of Multiple Myeloma Related Bone Disease
Authors: Pozzi Samantha , Yan Hua , Vallet Sonia , Vaghela Nileshwari , Fulciniti Mariateresa , Cirstea Diana , Santo Loredana , Mukherjee Siddhartha , Hideshima Teru , Schirtzinger Linda , Kuhstoss Stuart , Munshi Nikhil C. , Anderson Kenneth C , Scadden David , Raje Noopur
Published: April 2020

Treatment of bone disease in multiple myeloma (MM) has largely focussed on the osteoclast axis while the osteoblast axis has been underexploited. Dkk1, an inhibitor of the wingless int (wnt) pathway, is important in osteoblastogenesis. Increased expression of Dkk1 in a subset of MM patients and its association with lytic bone disease opens up the potential of targeting the osteoblast axis. The aim of this study was to test the effect of a Dkk-1 neutralizing chimeric antibody (Mab B3) on osteoblasts (OB), osteoclasts (OC) and MM cells in the context of the bone microenvironment. First, we tested the expression of Dkk1 in plasma and bone marrow of 16 MM patients and 10 MM cell lines. Dkk1 levels were >18 ng/mL in 2 out of 16 patients; levels were comparable in blood and bone marrow plasma. In contrast, very little Dkk1 (2–9 ng/ml) was produced by bone marrow stromal cells (BMSC). One out of 10 MM cell lines (INA-6) expressed low concentrations of Dkk1 in the supernatant. Next, we tested the effect of Mab B3 on MM cell lines, in the presence or absence of BMSC, and on OB and OC from MM patient derived bone marrow. The effects on OC were evaluated by TRAP staining and pit formation. Effects on OB were assayed by alkaline phosphatase staining and alizarin red assays for calcium deposition. Mab B3 treatment did not demonstrate direct cytotoxic effects on MM cell lines negative for Dkk1. Mab B3, however, enhanced OB differentiation and calcium deposition in a dose dependent manner and inhibited OC differentiation and function, as evidenced by a decrease in number of multinucleated TRAP+ cells and a decrease in pit formation. Ongoing studies are addressing the effect of Mab B3 on MM cells in the context of OC and OB. Mab B3 is also undergoing in vivo testing in a SCID-hu model bearing INA-6 MM cells. These studies and the underlying mechanism of action of Mab B3 will be presented. Our preliminary data suggests that Mab B3 has anabolic bone effects; a corresponding human monoclonal antibody may be useful for the treatment of MM related bone disease. Future studies will evaluate Mab B3 in combination with catabolic agents such as bisphosphonates with the goal of restoring normal bone homeostatsis.