bluuebunny/crossref_metadata_2025 huggingface dataset:
The microRNA144/451 Locus Enhances Nuclear FOXO3a Activity to Protect Erythroid Cells against Oxidant Stress
Authors: dos Santos Camila O. , Yu Duonan , Amigo Julio , Kandros Eugene , Valentine Eric R , Shelat Suresh , Dore Louis C , Yao Yu , Souza Janine D’ , Ghaffari Saghi , Paw Barry H. , Orange Jordan S , Weiss Mitchell J.
Published: April 2020

MicroRNAs (miRNAs) regulate tissue development by destabilizing cellular mRNAs or blocking their translation. We previously described two conserved erythroid miRNAs, miR144 and miR451, which are encoded by a single gene locus that is regulated by the essential hematopoietic transcription factor GATA-1. Morpholino-induced inhibition of miR451 impairs erythropoiesis in zebrafish. To gain further insight into miR144/451, we deleted the locus in mice. Mutant animals are born at the normal Mendelian ratio and exhibit no obvious structural defects. At baseline, loss of miR 144/451 causes mild anemia and reticulocytosis with moderate abnormalities in erythrocyte morphology. However, compared to littermate controls, miR144/451 null animals exhibit 33% greater depletion of circulating erythrocytes after phenylhydrazine-induced oxidant stress. At baseline, the mutant erythrocytes exhibit increased levels of reactive oxidant species, exquisite sensitivity to hydrogen peroxide induced hemolysis, and 50% reduced catalase activity compared to controls. Catalase is a downstream effector in an erythroid anti-oxidant program regulated by the forkhead transcription factor Foxo3a. In the mutant erythroblasts, mRNAs encoding catalase and glutathione peroxidase 1, another Foxo3a-regulated anti-oxidant, were reduced 2 and 1.7 fold, respectively. Messenger RNAs encoding several additional Foxo3a target genes, including Cdkn1b and Btg1 were also significantly reduced. Quantitative confocal fluorescence microscopy demonstrated that although total cellular Foxo3a protein was similar in wt and miR144/451 null erythroblasts, nuclear Foxo3a was reduced by 40% in miR144/451 null erythroblasts with a corresponding relocation of the protein to the cytoplasm. To explain this, we showed that miR451 (but not miR144) directly blocks translation of Ywhaz mRNA encoding 14-3-3 zeta, a cytoplasmic protein that sequesters Foxo3a away from the nucleus. In agreement, 14-3-3 zeta protein is upregulated by approximately 2-fold higher levels in miR144/451 null erythroblasts. Together, our findings suggest a model in which miR451 represses 14-3-3 zeta expression, which releases Foxo3a for translocation to the nucleus, thereby activating an anti-oxidant program of gene expression. This illustrates a new miRNA-regulated pathway through which erythroid cells are protected against oxidant stress.
Bortezomib and Dexamethasone as Maintenance therapy in Relapse/Refractory multiple myeloma Patients
Authors: Benevolo Giulia , Larocca Alessandra , Pregno Patrizia , Gay Francesca , Botto Barbara , Orsucci Lorella , Evangelista Andrea , Palumbo Antonio , Vitolo Umberto , Boccadoro Mario
Published: April 2020

Background: Despite the advances in the treatment for multiple myeloma (MM) in the past decade, it still remains an incurable haematological disease and the majority of patients still experience relapse. Bortezomib is a novel proteasome inhibitor approved for the treatment of MM patients. Several studies have demonstrated its efficacy as front-line therapy and in relapsed, advanced MM but no data are available as maintenance therapy (MT) in refractory/relapsed MM.

Patients and methods: The aim of this study is to evaluate the safety and the efficacy of bortezomib/dexamethasone MT (1.3 mg/mq bortezomib on days 1 and 15; 20 mg dexamethasone on days 1–2 and 15–16 in a 28-day cycle for a total of 6 cycles) in patients with refractory/relapse MM who responded to salvage therapy.

Results: From October 2004 until April 2008, 40 MM patients have been enrolled. The characteristics of the patients were as follows: 20 males and 20 females, median age was 70 years (IQR:66–75). Median haemoglobin value was 10.85 (IQR:10.175–12.225) and 7 (18%) patients had a renal failure. Skeletal disease was present in 27 (68%) patients. The median number of prior therapies was 2 (1–4). The salvage therapy included bortezomib as single agent or in combination with steroids and/or thalidomide in 12 patients (30%). Median time from diagnosis to the first dose of MT was 41 months (IQR:26–59). The median number of bortezomib infusion was 8 (1–18). After a median follow up of 13 months (IQR:6–31), 10 patients died for PD and 4 patients for infections. The MT improved the quality of response and converted in 1 CR and 4 VGPR the PR after salvage therapy in 5 patients; moreover, we observed a remarkable decreased of M component in 11 patients. The median time to progression was 23 months (95%CI: 8-not reached) with a progression free-survival at 1 year of 69% (95%CI: 50–82). The overall survival at 1 years was 63% and the cumulative incidence of death due to PD adjusted for competitive risk event was 25% (95%IC:10–41). In a univariate analysis the response rate to MT was not significantly affected by age, sex, number or type of previous therapy and haemoglobin concentration. Non-dose-limiting toxicities included neuropathy grade 1 (12 pts), herpes zoster reactivation (2 pts) and gastrointestinal affections (1 pts).

Conclusion: The combination bortezomib/dexamethasone can be safely administered as a maintenance therapy in relapse/refractoy MM. These preliminary data suggest that bortezomib/dexametasone MT can improve remission duration and also quality of response with an acceptable toxicity.
The JAK2V617F Mutation Is Present in the Liver Endothelial Cells of Patients with Budd-Chiari Syndrome
Authors: Sozer Selcuk , Fiel Isabel M. , Schiano Thomas , Feller Faye , Mascarenhas John , Hoffman Ronald
Published: April 2020

Post-natal vasculogenesis has been reported to be derived from a hierarchy of circulating endothelial progenitor cells (EPC). Some of these EPC are of myeloid origin while others have a more robust proliferative potential and are solely of endothelial cell (EC) origin. A number of groups have hypothesized that EC dysfunction might contribute to the hypercoagulable state associated with polycythemia vera (PV) by orchestrating the recruitment of blood elements to sites of injury or by regulating vascular tone. The JAK2V617F mutation is present in >95% of patients with PV. In addition, some individuals with normal blood counts who develop splanchnic vein thrombosis, including Budd-Chiari syndrome (BCS) and portal vein thrombosis (PVT) have been reported to have JAK2V617F positive hematopoiesis (45% and 34%, respectively) indicating that this thrombotic tendency might precede the development of PV. We explored whether this activating mutation is present in EC in the vessels of patients with BCS. We tested this hypothesis by studying EC in venules of liver biopsy specimens of patients with BCS with (n=2) or without PV (n=1) and PVT (n=2) without PV using laser capture microdissection (LCM) followed by nested PCR or RT-PCR in order to determine if EC were JAK2V617F positive and were of hematopoietic or EC origin. EC from the hepatic venules of BCS and PVT patients were captured by LCM from hematoxylin and eosin stained sections of archival formalin-fixed paraffin-embedded liver biopsy tissue specimens. EC were identified by their fusiform nuclei and their location along the lining of the hepatic venules. Hepatocytes were identified by their morphology as having round centrally placed nuclei and abundant cytoplasm with a trabecular arrangement. At least 10 EC and 10 hepatocytes from each biopsy specimen were captured and DNA or RNA was extracted. The JAK2V617F mutation was detected using nested allele-specific PCR. The hepatocytes of each patient contained exclusively wild type JAK2 while the EC of the two BCS patients with PV were homozygous for the JAK2V617F. The EC of the other BCS patient and 2 PVT patients who did not have PV contained exclusively wild type JAK2. The EC identity of these cells in the PV patients was confirmed by the presence of the EC transcripts (VE-Cadherin and VEGF-R2) and the absence of hematopoietic cell transcripts (CD45 and CD14). These findings indicate that hepatic venule EC in BCS patients with PV are JAK2V617F positive. The presence of JAK2V617F in both endothelial cells and hematopoietic cells belonging to such patients suggests that PV originates in an adult hemangioblast like cell in such patients. Further studies are required to understand the consequence of JAK2 mutation in EC as related to their propensity to thrombosis in PV.
Impaired Function of Hematopoietic Stem Cells with Abrogated Hypoxia Induced Expression of Vascular Endothelial Growth Factor
Authors: Rehn Matilda , Reckzeh Kristian , Olsson André , Axelson Håkan , Cammenga Jörg
Published: April 2020

Hypoxia has been suggested to be a characteristic of the bone marrow (BM) microenvironment where hematopoietic stem cells (HSC) reside. The major molecular response to hypoxia is the activation of hypoxia inducible factors (HIF). These transcription factors activate gene expression by binding to hypoxia response elements (HRE) in the promoter region of target genes, such as vascular endothelial growth factor (VEGF). It has been demonstrated that VEGF is indispensable for HSC survival in the adult and that VEGF mediated HSC survival is cell intrinsic. We are therefore investigating the role of hypoxia-regulated VEGF expression in HSC survival. For our studies we use the transgenic mouse model VEGFδ/δ. In this model, the HRE in the VEGF promoter is mutated which prevents HIF binding. Thus, hypoxia-induced VEGF transcriptional activation is abrogated while basal VEGF expression remains unaffected. Steady state hematopoiesis was not affected by the mutation since distribution between the B-cell, T-cell and myeloid lineages in both peripheral blood and BM of mice homozygous for the mutation was comparable to wild type animals. Furthermore, we could not detect any differences in the frequency of lineage-, Sca1+, c-kit+, CD34- cells in the BM between VEGFδ/δ and wild type mice. However, when assessing HSC functional properties in transplantation assays, BM from mice homozygous for the mutation was demonstrated to have dramatically reduced HSC function. Only around 60 % of lethally irradiated recipients that were transplanted with VEGFδ/δ BM could be rescued while recipients receiving wild type BM survived as expected. In addition, during early hematopoietic reconstitution, a skewing towards myeloid differentiation at the expense of B-cell differentiation could be observed in peripheral blood of recipients reconstituted with VEGFδ/δ BM. Moreover, in a competitive repopulation assay, VEGFδ/δ BM was shown to have a severe competitive disadvantage compared to wild type BM. Our findings indicate an important role for hypoxia related VEGF expression in reconstituting HSCs and suggest that low oxygen levels might be an important characteristic of the HSC niche.
The Role of Akt3 in Platelet Activation
Authors: O’Brien Kelly A , Stojanovic Aleksandra , Hay Nissim , Du Xiaoping
Published: April 2020

Akt is a family of serine/threonine kinases that are activated downstream of Phosphoinositide 3 Kinases (PI3K). There are three known Akt isoforms: Akt1, Akt2, and Akt3. Both Akt1 and Akt2 have been shown to be expressed in platelets and play important roles in PI3K-dependent platelet activation signaling. Previous studies have concluded that Akt3 is not expressed in platelets; however, we show that Akt3 is in fact a major Akt isoform present in platelets, and demonstrate that Akt3 plays an important role in platelet activation. In order to determine if Akt3 mRNA is present in platelets, RNA was isolated from platelets and RT-PCR experiments were performed using Akt3 specific primers. Akt3 mRNA was detected in both human and mouse platelets, and the possibility that Akt3 mRNA came from leukocyte contamination was excluded because Akt3 was not detected with RT-PCR using RNA purified from the same number of leukocytes as that in platelet preparation. To assess if Akt3 protein is present in platelets, western blot analysis was performed using an Akt3 specific antibody. Results revealed that Akt3 is highly expressed in human and mouse platelets. In order to assess the relative amount of Akt3 present in platelets, total Akt levels and phosphorylated Akt levels were measured in Akt3 knockout mouse platelets in comparison with wild type mouse platelets. Akt3 knockout mouse platelets showed a significant decrease in total Akt, and also a significant reduction in phosphorylated Akt, as indicated by Western blot analysis of phospho-Thr308-Akt and phospho-Ser473-Akt antibodies. Thus, our data indicates Akt3 is a major Akt isoform expressed in platelets. To determine the role of Akt3 in platelet activation, platelets from wild type mice and Akt3 knockout mice were stimulated with low concentrations of platelet agonists. Akt3 knockout mouse platelets exhibited impaired aggregation and ATP secretion in response to low dose thrombin and collagen compared with wild type controls, indicating that Akt3 plays an important role in promoting platelet activation. In order to assess the signaling molecules downstream from Akt3, Akt3 knockout and wild type mouse platelets were stimulated with low dose thrombin and phosphorylation of GSK-3β, an enzyme recently reported to negatively regulate platelet function downstream from Akt1 or Akt2 in platelets, was assessed. Western blot for phospho-Ser9 of GSK-3β in Akt3 knockout mouse platelets stimulated with thrombin showed less phosphorylation compared to Akt1 or Akt2 knockout mouse platelets, indicating that Akt3 is the major Akt isoform responsible for GSK-3β phosphorylation. Thus, our data shows that Akt3 is a major Akt isoform in platelets and plays an important role in platelet activation. Since phosphorylation of GSK-3β at Ser9 has been reported to reverse the inhibitory effect of GSK-3β on platelet function, our data also suggest that the stimulatory role of Akt3 in platelet activation may be mediated by negative regulation of GSK-3β function.
Negative Regulation of Gq-Mediated Pathways in Platelets by G12/13 Pathways through Fyn Kinase
Authors: Kim Soochong , Kunapuli Satya P.
Published: April 2020

Platelets contain high levels of Src family kinases (SFKs) suggesting an important role for these enzymes in platelet function. In this study, we have investigated the regulation of platelet function by SFKs downstream of G12/13 pathways. Calcium-independent platelet shape change induced by selective G12/13 stimulation with YFLLRNP was potentiated with a small mobilization of intracellular calcium in the presence of SFK inhibitors PP1 or PP2, which was abolished by the chelation of intracellular calcium, suggesting that SFKs downstream of G12/13 negatively regulate calcium mobilization in platelets. In addition, PP1 or PP2 caused a leftward shift of human platelet aggregation, secretion, and calcium response induced by low concentrations of agonists that activate platelets through G12/13 signaling such as PAR1-activating peptide SFLLRN and PAR4-activating peptide AYPGKF. However, 2-MeSADP-induced calcium response and platelet aggregation were not affected by the presence of PP1 or PP2, suggesting that SFKs downstream of G12/13, but not Gq/Gi, pathways are involved in this platelet response. Moreover, platelet aggregation and secretion caused by combined stimulation of G12/13 and Gi/Gz (YFLLRNP + 2-MeSADP with P2Y1 antagonist/epinephrine) were also potentiated in the presence of PP1 or PP2 confirming the contribution of SFKs downstream of G12/13 as a negative regulator to platelet activation. Potentiation of platelet aggregation in the presence of SFK inhibitors was not affected in the presence of GF109203X, while PP2 failed to potentiate platelet aggregation in the presence of 5,5′-dimethyl-BAPTA indicating that potentiation of cytosolic calcium may have an important role in this enhanced platelet responses by SFK inhibition. Moreover, PP1 or PP2 failed to potentiate platelet responses in the presence of Gq selective inhibitor YM-254890 or in Gq-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of Gq signaling pathways. Importantly, AYPGKF-induced platelet aggregation, secretion, and calcium response were potentiated in Fyn-, but not in Lyn-, deficient platelets compared to the wild-type mouse platelets, whereas 2-MeSADP-induced platelet response was not affected in these platelets. We conclude that SFKs activated downstream of G12/13, but not Gq/Gi, pathways negatively regulate platelet responses by inhibiting intracellular calcium mobilization in platelets through Gq signaling pathways. Specifically, we define that Fyn plays a major role in this negatively regulatory pathway.
Role of Sialic Acid for Platelet Lifespan and Function
Authors: Sørensen Anne Louise , Rumjantseva Viktoria , Nayeb-Hashemi Sara , Patel-Hett Sunita , Richardson Jennifer , Italiano Joseph , Clausen Henrik , Hartwig John H. , Wandall Hans H. , Hoffmeister Karin M.
Published: April 2020

Although sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet half-life is not fully clarified. We and others have previously provided evidence that thrombocytopenia in mice deficient in the ST3Gal-IV sialyltransferase gene (ST3Gal-IV−/− mice) is caused by rapid clearance of the platelets due to recognition of surface galactose by asialoglycoprotein receptor-expressing scavenger cells. Here we report new insight into clearance mechanisms, activation and production of sialic acid deficient platelets. Immunofluorescence staining of organ specimens harvested shortly after platelet transfusion demonstrated the predominant clearance of ST3Gal-IV−/− platelets by liver macrophages and, as previously reported, hepatocytes. The differential cellular clearance pathways were further explored following macrophage depletion in mice using clodronate encapsulated liposomes, a procedure that restored ST3Gal-IV−/− platelet circulation to approximately 40% of normal values, confirming that macrophages play a major role in the clearance of sialic acid deficient platelets and that other clearance pathways are equally important. Ingestion of ST3Gal-IV−/− platelets by hepatocytes was confirmed by in vitro HepG2 phagocytosis assays. We next investigated the function of desialylated platelets. Platelet binding of von Willebrand factor (vWf) upon botrocetin stimulation was 3 fold higher for ST3Gal-IV−/− platelet rich plasma compared to ST3Gal- IV+/+ platelet rich plasma. The circulation time of wild-type platelets transfused into ST3Gal-IV−/− mice was 20% reduced compared to control platelets, indicating that platelet removal could be accelerated due to binding of desialylated vWf to platelets. Loss of sialic acid did not affect platelet production in vitro and in vivo as cultivated megakaryocytes was found to produce proplatelets normally and measurements of reticulated platelets by flow cytometry showed a 3-fold increased thrombocytopoietic activity in the ST3Gal- IV−/− mice compared to wild-type mice. Interestingly, thiazole orange staining revealed a significant negative correlation between platelet size and platelet age for both genotypes, In conclusion, depletion of the ST3Gal-IV gene promotes binding of vWf to platelets, exposes platelet surface galactose residues to the lectin domain of asialoglycoprotein receptors on both hepatocytes and liver macrophages, resulting in rapid platelet clearance from the circulation and supporting previous findings that platelets decrease in size while aging in circulation.
CIB1 Is Required for FAK Activation during Outside-in Signaling through Platelet Integrin αIIbβ3
Authors: Naik Ulhas Pandurang , Naik Meghna Ulhas
Published: April 2020

Platelets play an important role in the processes of hemostasis and thrombosis. Platelet integrin αIIbβ3 mediates bi-directional signaling during these processes. Agonist-dependent activation of integrin αIIbβ3 through inside-out signaling results in high-affinity binding of soluble ligands, such as fibrinogen. Fibrinogen binding induces a cascade of signaling through the integrin, termed outside-in signaling that results in platelet aggregation and clot retraction. Previously, we have characterized CIB1, a calcium- and integrin-binding protein that specifically interacts with the cytoplasmic domain of αIIb. Previous reports using in vitro and ex vivo studies implicated that CIB1 is involved in

maintaining αIIbβ3 in its resting state, agonist-induced activation of the integrin, and outside-in signaling resulting in platelet spreading.

Here, we show that platelet filopodia formation induced by fibrinogen binding to integrin αIIbβ3 needs Ca2+, but is independent of the Ca2+-dependent interaction of CIB1 with αIIb. Additionally, dynamic rearrangement of the cytoskeleton is required for the recruitment of FAK to the CIB1-αIIb complex at the filopodia and FAK activation. Moreover, disruption of the association of CIB1 and αIIb by incorporation of αIIb peptide or CIB1 antibody inhibited FAK activation. Furthermore, Cib1 null platelets acquired a spiky morphology and failed to fully spread on immobilized fibrinogen. Interestingly, FAK activation was significantly reduced in Cib1 null platelets exposed to immobilized fibrinogen. Our results suggest that during outside-in signaling, a rise in the intracellular Ca2+ level and filopodia formation occurs prior to the interaction of CIB1 with αIIb. Additionally, Ca2+ bound CIB1 recruits FAK to the αIIbβ3 complex at the filopodia, where FAK is activated, resulting in platelet spreading. Thus, our results have provided a mechanism through which CIB1 regulates outside-in signaling through integrin αIIbβ3.
Actinomycin D Blocks Transcription of Surfactant B mRNA in Marrow Cells Co-Cultured with Lung
Authors: Aliotta Jason M , de Paz Nicole , Pereira Mandy , Quesenberry Peter J.
Published: April 2020

We have previously described that RNA-filled microvesicles are shed from lung cells and are capable of entering whole bone marrow (WBM) cells in culture (Aliotta et al, Stem Cells, 2007;25(9):2245–56). WBM cells cultured with these lung-derived microvesicles appear to express lung cell-specific genes after 7 days in culture. However, it is not known if the lung cell-specific mRNA detected in these cells represents de novo transcription or previously transcribed mRNA carried into the cell via microvesicles. To address this issue, we co-cultured WBM cells extracted from C57BL/6 mice and co-cultured them opposite lung from C57BL/6 mice exposed to 500 centigrey (cGy) of total body irradiation (TBI) or no radiation. The two cell types were separated by a cell-impermeable membrane (pore size, 0.4μm). After 7 days of co-culture, WBM cells were removed, extensively washed and cultured alone for an additional 36 hours, with or without the transcriptional blocking agent Actinomycin-D (Act-D, 10 ug/ml). Control WBM cells were co-cultured without lung and not exposed to Act-D (control). WBM cells were then analyzed for the presence of a variety of hematopoietic and lung-specific markers by Real Time RT-PCR and fold difference in target gene expression was determined (relative to control cells). WBM cells co-cultured with or without lung expressed high levels of hematopoietic markers including Sca-1, c-kit and CD45. This expression was nearly or completely eliminated after exposure to Act-D in culture. Expression of the surfactant protein B (Sp-B) gene was also significantly decreased in WBM co-cultured with lung (irradiated or non-irradiated) after exposure to Act-D (mean ± SE, from 16±4.9 to 1.1±0.4-fold increase vs. control, p=0.012). Interestingly, exposure to Act-D resulted in an increase in the expression of other lung-specific genes, including surfactant protein C (Sp-C) and clara cell specific protein (CCSP), in WBM co-cultured with lung (from 165±103 to 3,700±1,625 and 2±0.9 to 93±38-fold increases vs. control, p<0.015). In separate experiments, WBM cells co-cultured with cell-free lung conditioned media (CM) that was pre-treated with RNase had nearly 70% lower Sp-B expression compared with WBM cells co-cultured with untreated cell-free CM. These data suggest that lung cells release a transcriptional factor that induces marrow cell expression of the Sp-B gene in culture. This transcriptional factor is RNase-sensitive and could potentially represent microRNA transferred to marrow cells via lung-derived microvesicles. The increase in expression of other lung-specific genes after Act-D exposure suggests alternative transcriptional control mechanisms for lung-specific genes other than Sp-B.
High-Dose RHAMM-R3 Peptide Peptide Vaccination for Patients with Vaccination for Patients with Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS), Multiple Myeloma (MM) and Chronic Lymphocytic Leukemia (CLL)
Authors: Greiner Jochen , Schmitt Anita , Giannopoulos Krzysztof , Funk Isabel , Heyduk Marta , Rojewski Markus , Chen Jinfei , Goetz Marlies , Bommer Martin , Ritter Gerd , Guillaume Phillipe , Dmoszynska Anna , Döhner Hartmut , Schmitt Michael
Published: April 2020

We have demonstrated immunological responses and positive clinical effects of a peptide vaccination for patients with AML, MDS, MM and CLL with a limited tumor load or a minimal residual disease over-expressing RHAMM using 300 μg RHAMM-R3 peptide (Schmitt et al., Blood 2008; Giannopoulos et al., abstract submitted). To date, 26 patients were enrolled in this clinical peptide vaccination trial. Here, we report on the second cohort of nine patients with AML, MDS and MM vaccinated with a higher peptide dose (1000 μg RHAMM-R3 peptide). The vaccine was given four times at a biweekly interval and GM-CSF was added for five days each vaccination. Similar to the patients vaccinated with 300 μg peptide only mild drug-related adverse events were observed such as erythema and induration of the skin. Immunomonitoring was performed using ELISpot assays for Interferon gamma and Granzyme B, tetramer-based flow cytometry and chromium release assays. Moreover, the frequency of regulatory T cells was quantified at different time points of vaccination. In this second cohort of patients treated with 1,000 μg peptide we detected specific immune responses in a lower frequency (4/9 patients) in contrast to patients in the 300 μg cohort (7/10 patients). In these patients with immune responses we found an increase of CD8+/HLA-A2/RHAMM-R3 tetramer+/CD45RA+/CCR7−/CD27−/CD28− effector T cells in flow cytometry in accordance with an increase of R3-specific CD8+ T cells in ELISpot assays. Two patients with positive immune responses showed a significant decrease of regulatory T cells. One patient without positive immune and clinical effects showed an increase of the frequency of regulatory T cells (5.03% to 15.9%). Three out of nine patients treated with 1,000 μg showed positive clinical effects: One patient with MDS RAEB-2 showed a reduction of leukemic blasts in the bone morrow to lower than 5%, one MDS patient achieved a normalization of the peripheral blood counts and one patient with multiple myeloma experienced a reduction of light chain in serum. The patients in the 300 μg cohort showed also a higher frequency of positive clinical effects (5 out of 10 patients).

Taken together, RHAMM-R3 peptide vaccination induced both immunological and clinical responses. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematological malignancies. However, higher doses of peptide do not improve the frequency and intensity of immune responses in this clinical trial.
The Myeloid Cell Surface Receptor IREM-1: A Novel Monoclonal Antibody Target for Acute Myeloid Leukemia (AML)
Authors: Zhao Xiaoxian , Korver Wouter , Singh Shweta , Pardoux Cecile , Hsi Eric D. , Abo Arie
Published: April 2020

Introduction: Immune Receptor Expressed by Myeloid Cells (IREM-1), a member of the CD300L gene family, is an inhibitory receptor expressed by myeloid cells. We reported the characterization of IREM-1 expression in AML blasts and demonstrated the therapeutic potential of IREM-1 specific mouse monoclonal antibodies (mAb) against AML blasts through complement-dependent cytotoxicity assays. Here we present our latest expression and in vivo studies to further evaluate the potential of IREM-1 for antibody-mediated AML therapy.

Methods: High affinity mAbs to IREM-1 were generated with Kd values in low nM range. For in vivo models, the IREM-1 chimeric (huIgG1) monoclonal antibody D12 was generated. In these models, growth of established tumors in an HL-60 xenograft model in nude mice was monitored upon dosing with anti-IREM1 mAb. Immunohistochemistry (IHC, Discovery, Ventana Medical Systems) or flow cytometry (FC, FACSCalibur, BD Bioscience) were performed using MAbs (Alexa-488 conjugated for FC).

Results: We compared the expression of IREM-1 and CD33 in normal human peripheral blood (n=10) by flow cytometry. Both molecules are expressed in monocytes, dendritic cells and granulocytes, while no expression in lymphocytes was observed. The expression pattern of CD33 was similar to the IREM-1 pattern. IHC staining also demonstrated the expression of IREM-1 in AML and normal bone marrow. Flow cytometry indicated IREM-1 was expressed in 37/52 (71.2%) of AML cases with a range of positive blasts from 20–99% (mean 72.1%). The IREM-1 chimeric monoclonal antibody D12 showed antibody-dependent cell-mediated cytotoxicity activity against IREM-1 transfected human embryonic kidney 293 cells with EC50 at 65 ng/ml. Antibody-mediated IREM-1 internalization was observed in both transfected cells as well as AML cell lines. In an HL-60 AML xenograft model, anti-tumor activity of D12 antibody delayed tumor growth by 40%.

Conclusion: Our results further confirm IREM-1 as a cell surface antigen expressed in majority of AML cases and IREM-1 has a similar expression pattern as CD33 among normal blood specimen. Our high affinity chimeric monoclonal antibody against IREM-1 showed anti-tumor activity both in vitro and in vivo. IREM-1 internalization upon antibody engagement also identifies IREM-1 as a suitable target antigen for toxin-conjugated antibody therapy. Taken together, this study supports the rationale for further development of anti-IREM-1 D12 as an immunotherapeutic agent in AML.
Energy Metabolism of Leukemia Cells: Glycolysis Vs Oxidative Phosphorylation
Authors: Miwa Hiroshi , Suganuma Kazuto , Shikami Masato , Imai Norikazu , Sakai Mayuko , Hiramatsu Akihito , Yamamoto Hidesuke , Wakabayashi Motohiro , Watarai Masaya , Hanamura Ichiro , Imamura Akira , Mihara Hidetsugu , Nitta Masakazu
Published: April 2020

Cancer cells are more dependent on glycolysis than oxidative phosphorylation in the mitochondria for generation of ATP as energy source. By using 2-deoxy-D-glucose (2-DG: glycolysis inhibitor) and oligomycin (inhibitor of oxidative phosphorylation), we examined the energy metabolism of various leukemia cell lines. The growth of the cell lines was measured by MTS assay, which detects viable cells in proliferation. 2-DG suppressed the growth of all leukemia cell lines examined in dose-dependent manners. The IC50 of each cell line was as follows: Kasumi-1 0.5±0.1mM, KG-1a 1.8±0.6mM, HL-60 3.3±0.1mM, NB4 3.8±0.4mM, and THP-1 23.1±3.8mM. The concentration of lactic acid (the final product of glycolytic pathway) in the culture supernatant was greatly reduced by the treatment with 0.2mM 2-DG for 24 hours in Kasumi-1 (54.5% of the control), compared with THP-1 (92.2%). It is suggested that the growth of Kasumi-1 was strongly suppressed by 2-DG through inhibition of glycolysis, which is supposed to be a main metabolic pathway in this cell line. On the other hand, treatment with oligomycin (1μg/ml) for 48 hours potently suppressed the growth of THP-1 (44.7%), then Kasumi-1 (72.1%). The growth of NB4, KG-1a and HL-60 was minimally suppressed (more than 90%) by oligomycin. Cell cycle was analyzed after 24 hours treatment with 2-DG or oligomycin. Sub-G1 fraction (apoptosis) was greatly increased by 2-DG (5mM) in Kasumi-1 (56.5%) and NB4 (30.6%), compared with THP-1 (7.6%). The apoptosis inducing effect was confirmed by annexinV staining. Oligomycin treatment (1μg/ml) increased apoptosis (subG1) in THP-1 (35.8%), then Kasumi-1 (16.6%) and NB4 (12.2%). Oligomycin treatment also increased G1 population (G1 arrest) in THP-1 (35.9% to 69.4%). AMP-activated protein kinase (AMPK) is activated by an elevated AMP/ATP ratio, which means the energy-deprived status of the cell. Western blot analysis using phospho-AMPK α (Thr172) antibody revealed that treatment with 2-DG or oligomycin induced prompt (30 min) phosphorylation of AMPK in leukemia cell lines. The extent of AMPK phosphorylation was almost proportional to the suppression of the growth. Collectively, it is suggested that leukemia cells are dependent almost exclusively on either glycolysis or oxidative phosphorylation in the mitochondria for energy production. Then, inhibition of glycolysis by 2-DG or oxidative phosphorylation by oligomycin results in growth suppression by inducing apoptosis and/or cell cycle arrest through activation of AMPK. Our data clarified the characteristics of the energy metabolism of each leukemia cell, and showed the key to produce novel therapeutic approach targeting metabolic pathway.
Low Expression of p14ARF in De Novo AML with Normal Karyotype Is Associated with Short Survival but Increased in Vitro Sensitivity to p53 Modulating Drugs
Authors: Paul Esbjörn , Uggla Bertil , Wiman Kals G , Paul Christer , Lehmann Soren , Nahi Hareth
Published: April 2020

Background: The p53 protein is strongly regulated by the E3 ligase HDM-2, which specifically binds to p53 and causes proteosomal degradation. The p14ARF protein activates p53 by binding to and inhibiting HDM-2.

Design and methods: To further study the prognostic impact of p14ARF in AML, leukemic cells from 57 adult patients with normal karyotype de novo AML were analysed for p14ARF mRNA expression level using real-time PCR. We also measured the cytotoxicity against conventional cytostatics and PRIMA-1, a novel small molecule shown to activate mutated p53 (Nature Medicine2002; 8: 282–288) using an ATP-assay. Intracellular p53 protein was measured by FACS after incubation with PRIMA-1 in combination with the HDM-2 blocking agent RITA (Nature Medicine2004; 10: 1321–1328)

Results: Patients whose cells expressed more p14ARF mRNA than the 75th percentile (0.26) showed significantly better survival compared with those with lower levels, 61% vs. 30% 3-year survival (p=0.033). The difference remained significant also when NPM1/FLT3 status was considered. The mean effects of all tested conventional antileukemic drugs were greater in leukemic cell samples expressing p14ARF mRNA ≥0.26, but the differences did not reach statistical significance. In contrast, PRIMA-1 had a significantly greater effect on leukemic cell samples with low levels of p14ARF mRNA and PRIMA-1 and RITA significantly increased intracellular p53 levels.

Conclusions: Low level of p14ARF mRNA in leukemic cells from patients with normal karyotype AML is a strong marker for poor prognosis and decreased sensitivity to conventional cytostatics. Treatment with drugs targeting p53 can be a future possibility to improve the outcome for these patients.
GFI1 Is a Tumor Suppressor in Myeloid Progenitors
Authors: Chaubey Aditya , Hormon Shane , Velu Chinavenmeni S. , Bourdeau Tristan , Zhu Jinfang , Paul William , Jegga Anil , Gebelein Brian , Grimes H. Leighton
Published: April 2020

In severe congenital neutropenia (SCN) patients and mice with Growth factor independent-1 (Gfi1) loss of function, arrested progenitors are suspended in a hyperproliferative state while terminal granulpoiesis is blocked. SCN patients are at increased risk for the development of acute myeloid leukemia. We demonstrate that Gfi1 directly targets HoxA9, Pbx1 and Meis1 during normal myelopoiesis. Gfi1−/− progenitors exhibit elevated levels of HoxA9, Pbx1 and Meis1, exaggerated HoxA9-Pbx1-Meis1 activity, and increased persistence in vivo and in vitro. Limiting HoxA9 alleles corrects, in a dose dependent manner, in vivo and in vitro phenotypes observed with loss of Gfi1. Moreover, in a manner conserved in Drosophila anterior/posterior patterning, we demonstrate that these factors can compete for occupancy of DNA sequences encoding composite Gfi1-HoxA9-Pbx1-Meis1 binding sites. Finally, the expression of Gfi1 and HoxA9 are inverse and stratify human myeloid leukemias, suggesting a role for HoxA9- Gfi1 antagonism in human AML. In agreement with this, a myeloproliferative disorder progresses into a rapid, lethal and transplantable myeloid leukemia in a Gfi1−/− setting. We conclude that the lifespan and oncogenic transformation of hematopoietic progenitor cells is regulated through a conserved competition between Gfi1 and HoxA9-Pbx1-Meis1.
Low Dose Ara-C Versus Low Dose Ara-C and Tipifarnib: Result of the UK NCRI AML16 “Pick a Winner” Comparison
Authors: Burnett Alan K. , Hills Robert , Milligan Donald , Kell William J. , Wheatley Keith , Virchis Andres E. , Russell Nigel H.
Published: April 2020

Novel treatments are needed for older patients with AML who are not thought likely to benefit from standard chemotherapy. A recent randomised trial demonstrated that Low Dose Ara-C (LD Ara-C) was superior to best supportive care (BSC) (Burnett et al Cancer2007: 109: 1114–1124). The Farnesyl Transferase inhibitor, Tipifarnib, has produced encouraging responses in this patient group (Lancet 109; 1387–1394) although in a subsequent randomised comparison vs BSC it was not superior (Haroussea et al Blood1997:110,135a)

The UK NCRI AML16 trial aims to test novel agents or combinations in untreated older patients with AML or high risk MDS (marrow blasts >10%) following a “pick a winner” design. The intention is to randomise up to 50 patients per arm with the expectation that a “winner” will achieve a remission rate in excess of 30%, compared with 15–20% with LD Ara-C. If that is achieved randomisation will continue with OS and DFS as endpoints. We report our experience of LD Ara-C (20mg bd days 1–10 for 4 courses versus LD Ara-C combined with Tipifarnib (300mg bd days 1–21) for 4 courses at 6–8 week intervals.

Patient Details: Sixty-five patients were randomised between December 2006 and October 2007. The median age was 74.4 years with 82% of patients over 70 years. There were no differences between the treatment arms with respect to age distribution, gender, performance status, de novo/secondary AML, high risk MDS presenting WBC or cytogenetic risk group.

Because of concerns about excess deaths in the Tipifarnib arm and therefore the low probability that LD Ara-C + Tipifarnib would be superior to LD Ara-C alone, the DMEC recommended premature closure of the Tipifarnib arm.

Conclusions: While other agents combined with Tipifarnib may continue to show promise, the combination with LD-Ara-C in this patient population was not beneficial.
Front-Line Therapy with Arsenic Trioxide/All-Trans-Retinoic Acid Combination and Chemotherapy Improves Cure Rates in Newly Diagnosed Patients with Acute Promyelocytic Leukemia
Authors: Hu Jiong , Liu Yuan-fang , Wu Chuan-feng , Zhou Guang-biao , Shen Zhi-Xiang , Li Jun-min , Wang Zhen-Yi , Chen Sai-Juan , Chen Zhu
Published: April 2020

BACKGROUND: In this study, we analyzed the clinical benefit and safety of upfront use of all-trans-retinoic acid (ATRA), arsenic trioxide (ATO) and chemotherapy in patients with newly-diagnosed acute promyelocytic leukemia (APL) during Apr 2001 and Dec 2005.

METHODS: A total of 85 patients were treated with ATRA and ATO as induction therapy, followed by consolidation/maintenance therapy composed of ATRA, ATO and chemotherapy. All patients were followed-up to evaluate the long-term efficacy and safety.

RESULTS: A total of 81 (95.3%) patients entered complete remission (CR) with a median of 27 days. Among these 81 patients, 4 patients relapsed and 2 patients died from the disease with a median follow-up of 70 months (15–87). The 5-year leukemia-free survival (5-yr-LFS) and overall survival (5-yr-OS) for all patients were 89.2±3.4% and 91.7±3.0% while for patients who achieved CR (n=81), the 5-yr-LFS and 5-yr-OS were 96.2±2.1% and 96.2±2.1% respectively. With careful monitoring of in vivo arsenic levels in 33 evaluable long-term survivors, we demonstrated that the serum and urine arsenic concentrations were within safety limits, although a slight but significantly increase in arsenic levels was observed as compared to healthy donors. Overall, no obvious arsenic associated long-term toxicity was documented in these patients.

CONCLUSIONS: Use of up-front ATRA/ATO/chemotherapy combination treatment in newly-diagnosed APL has proven relatively safe and has lead to a significant improvement in long-term LFS/OS.
Mito-FLAG with Ara-C as Bolus Vs. Continuous Infusion in Recurrent AML – Results of a Prospective Randomized Intergroup Study of the East German Study Group Hematology/Oncology (OSHO) and the Study Alliance Leukemias (SAL)
Authors: Hanel Mathias , Friedrichsen Kai , Kroschinsky Frank , Poenisch Wolfram , Schaefer-Eckart Kerstin , Aulitzky Walter , Peter Norma , Neugebauer Sascha , Herbst Regina , Hanel Annette , Wilhelm Martin , Niederwieser Dietger , Ehninger Gerhard , Fiedler Friedrich
Published: April 2020

We started a prospective randomized multicenter trial to examine the importance of the infusion schedule of Ara-C as part of the Mito-FLAG regimen in patients (pts) with relapsed or primary refractory acute myeloid leukemia (AML). The treatment consisted of mitoxantrone (7 mg/m2, days 1/3/5), fludarabine (15 mg/m2, q-12h, days 1–5), Ara-C as bolus (B) (1000 mg/m2 over 1 h, q-12h, days 1–5) or continuous infusion (CI) (125 mg/m2 over 24 h, days 1–5) and G-CSF (5 μg/kg/day, day 0 until a neutrophile count of 0.5 × 109/μl). Since 11/1999, 253 pts from 36 centers in Germany entered the study (131 men, 122 women, aged 19–75 years, median 59 years). 131 pts (52%) showed a primary refractory AML or had relapsed within 6 months after 1st complete remission (CR), whereas 120 pts had suffered their 1st relapse after 6–18 months (79 pts/31%) or later (41 pts/16%). Only 2 pts with a 2nd relapse were enrolled. The diagnoses according to FAB criteria were as follows: M0 - 17 pts, M1 - 55 pts, M2 - 71 pts, M3 - 2 pts, M4 - 44 pts, M5 - 27 pts, M6 - 9 pts, M7 - 2 pts, 26 pts were not classified. 62 pts (25%) had a poor risk karyotype (defined as aberrations on the chromosomes 5, 7, 8 or multiple). In 50 pts (20%) an elevated leukocyte count (> 20 × 109/μl) was observed. 22 pts (9%) had relapsed after prior hematopoietic stem cell transplantation (HSCT). Currently 249 pts are evaluable for response and 231 pts for toxicity and survival. Following Mito-FLAG, 120 pts (48%) achieved CR and 43 pts (17%) partial remission (PR), for an overall response rate of 65%. 206 pts experienced at least one episode of febrile neutropenia with a median duration of 8 (1–53) days. 19 pts (8%) suffered an early death because of toxicity (n=11) or progressive AML (n=8). Out of 163 responders, 8 pts were consolidated by high-dose therapy with autologous HSCT and 48 pts underwent allogeneic HSCT after dose-reduced conditioning. With a median follow-up of 40.2 months (0–93), the probabilities of event-free survival (EFS) and overall survival (OS) after 3 years were 13% and 19%, respectively. In responding pts the median duration of EFS and OS were 8.4 and 10 months, the 3-year-rates of EFS and OS were 28% and 33%, respectively. In conclusion, Mito-FLAG is an effective and well tolerated regimen in the salvage therapy of pts with AML.
Decitabine and Gemtuzumab Ozogamicin in Acute Myelogenous Leukemia and High-Risk Myelodysplastic Syndrome
Authors: Borthakur Gautam , Estrov Zeev , Garcia-Manero Guillermo , Williams Betsy , Wathen Jay K. , Konopleva Marina , Burger Jan , Ferrajoli Alessandra , George Solly , Kantarjian Hagop
Published: April 2020

Background: Hypomethylating agents are approved for the treatment of myelodysplastic syndrome (MDS) and have activity in acute myelogenous leukemia (AML) (Issa, JP et al. Blood. 2004;103:1635–40)(Silverman LR et al. Journal of Clinical Oncology2006; 24: 3895–3903). Exposure of AML cells to hypomethylating agents increase their CD33 expression (Balain and Ball. Leukemia. 2006; 20:2093–2101). This establishes a rationale for the combination of hypomethylating agent with CD33 antibody for treatment of high-risk MDS and AML. A preliminary report of the combination of 5-azacitidine (Aza) and gemtuzumab ozogamicin (GO) in elderly patients with previously untreated AML and MDS showed promise (Nand, S. et al. Blood2006;108: Abstract 1981).

Patients and Method: We have started enrolling patients in a phase 2 study of Decitabine (DAC) with GO in patients with AML and high-risk myelodysplastic syndrome (MDS). Treatment includes DAC 20 mg/m2/day intravenously (IV) × 5 days and GO 3 mg/m2 IV on day 5. GO is repeated on day 15±2 of first cycle if peripheral blood blast count is > 109/L. Six such cycles are planned every 4–8 weeks. Patients achieving clinically relevant response e.g. complete remission (CR), CR with incomplete platelet recovery (CRp), marrow clearance/reduction of blasts etc. can continue on DAC alone approximately every 4–8 weeks for up to 24 cycles of total therapy duration. Based on historical expectations patients are divided into four groups according to study design:

relapsed AML 1st CR duration <= 1 year or beyond first relapse, relapsed AML 1st CR duration > 1 year, untreated AML (not eligible for standard induction)/MDS, previously treated MDS, secondary MDS or AML.

Results: Thirty-seven patients have been treated till date; Group 1=19, group 2=1, Group 3=12 and group 4=5. Median age is 63.5 years (range, 26–82), performance status =1 (range, 0–3), prior therapy =1 (range, 0–7). Eighteen (49%) patients has adverse cytogenetics and 14 (38%) has prior malignancies. Two patients received day 15±2 administration of GO. Median number of treatment cycles is 2 (range, 1–5). Eight patients died while on study. Except for cytopenias, infections, infusion related reactions etc. no significant grade 3/4 toxicities have been encountered. Three patients have achieved CR/CRp and 5 patients achieved other clinically relevant responses with an overall response rate of 22%. Responses according to patient groups are; Group 1=2/19 (4 ongoing), group 2=1/1, group 3=2/12 (3 ongoing), group 4=3/5. Seven additional patients are continuing on study with stable disease. Accrual to the study is ongoing.

Conclusion: The combination of DAC and GO appears promising particularly in less heavily pre-treated patients with high-risk MDS and AML.