bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Distinct Precursors of Hematopoietic Stem Cells and Endothelial Progenitors Characterized by the PCLP1 Expression.
Authors: Onitsuka Izumi , Takeuchi Masaki , Okabe Tomoya , Kamiya Yoshiko , Hirata Ayami , Yahara Ichiro , Miyajima Atsushi
Published: April 2020

Blood cells and endothelia are believed to arise from their common progenitor hemangioblast. However, it still remains unknown how these lineages develop. Here we report the existence of two distinct precursors for hematopoietic stem cells (HSCs) and endothelial progenitors in murine fetal liver (FL). Podocalyxin-like protein 1 (PCLP1) is a member of the sialomucin family and was shown to be expressed in hemangioblasts in the aorta-gonad-mesonephros region in murine embryo. To further analyze the fates of hematopoietic/endothelial cells, we focused on embryonic day 14.5 (E14.5) FL, since it is a major hematopoietic organ during embryonic period. Based on the PCLP1 expression levels, E14.5 FL cells could be fractionated into four distinct populations. In vitro colony-forming assay and in vivo transplantation analysis revealed that lineage-committed progenitors with colony-forming activities and long-term repopulating hematopoietic stem cells (LTR-HSCs) were in PCLP1neg cells. PCLP1dull cells contained erythroid lineage-committed cells. Interestingly, while PCLP1med cells lacked colony-forming activities, they showed LTR-HSC activity in vivo. To further characterize these cell populations, we cultured them with OP9 stromal cells, since OP9 cells have been used to induce hematopoietic and endothelial lineages from embryonic stem cells. In co-culture with OP9 cells, PCLP1neg cells immediately generated blood cells with colony-forming activity but lacking in vivo hematopoietic activity, indicating that OP9 cells failed to support hematopoietic progenitor/HSCs. However, PCLP1med generated colony-forming hematopoietic progenitors with LTR-HSC activities in the presence of OP9 cells. These results indicated that PCLP1med cells contained stromal cell-dependent immature precursors for HSCs. PCLP1high cells did not express the hematopoietic markers or endothelial cell markers such as PECAM1 and VE-cadherin. However, they formed endothelia-like cell colonies which were highly proliferative and serially transferable in OP9 co-culture. Interestingly, the addition of vascular endothelial growth factor (VEGF) to the culture strongly induced the expression of PECAM1 and VE-cadherin in these colonies. PCLP1high cells contributed to PECAM1+ endothelium in several organs in vivo when transplanted to conditioned neonatal liver. Therefore, PCLP1high cells contained immature precursors for endothelial progenitors. These results indicate that PCLP1 expression levels distinguish previously unrecognized early precursors for HSCs and endothelial progenitors, which are distinct from hemangioblasts.
Regulation of Hematopoiesis In Vitro and In Vivo by Invariant NKT Cells.
Authors: Kotsianidis Ioannis , Silk Jonathan D. , Patterson Scott , Almeida Antonio , Tsatalas Costas , Bourikas George , Cerundolo Vincenzo , Roberts Irene A.G. , Karadimitris Anastasios
Published: April 2020

Invariant NKT cells (iNKT cells) are a small subset of immunoregulatory T cells highly conserved in humans and mice. Upon activation by glycolipids presented by the MHC-like molecule CD1d, iNKT cells promptly secrete Th1/2 cytokines but also cytokines with hematopoietic potential such as IL-3 and GM-CSF. In mice, NKT cells activated by alpha-galactosylceramide (alphaGC), a potent glycolipid ligand, cause an in increase in extramedullary hematopoietic committed progenitor activity through secretion of these cytokines.

We tested the role of iNKT cells in regulating hematopoiesis under conditions of activation and in steady state hematopoiesis. We found that GM-CSF-secreting alphaGC -activated iNKT cells enhanced (by 64%, n=5, p<0.05) the myeloid clonogenic potential of human cord blood hematopoietic progenitors; conversely, in the absence of NKT cells short- and long-term progenitor activity is decreased: 48% reduction in GFU-GM frequency, n=6; p<0.05 and a reduction of 18–60% in LTC-IC frequency in 4 independent experiments. These findings suggest that NKT cells are implicated in the regulation of hematopoiesis both in the presence and absence of immune activation. In accordance with these findings, iNKT cell-deficient mice compared to wild type animals display impaired hematopoiesis characterized by peripheral blood cytopenia (43% and 32% reduction in leucocyte and platelet counts respectively, n=10, p<0.001), reduced marrow cellularity (reduced by 35%, n=13, p<0.001), lower frequency and absolute numbers per hind limb of hematopoietic stem cells (HSC) as assessed by the cKit+Lin-Sca-1+ phenotype (reduced by 40% n=8, p<0.05, and 61% n=8, p<0.001, respectively), and reduced early (50% and 68% reduction in frequency and absolute numbers of LTC-IC respectively, n=4, p<0.05) and late (40% and 57% reduction in frequency and total number of CFU-GM per hind limb respectively, n=8, p<0.05) hematopoietic progenitors. We also show that CD1d is expressed on human HSC and CD1d-expressing HSC display short- and long-term clonogenic potential and can present the glycolipid a-galactosylceramide to iNKT cells suggesting that the iNKT cell-mediated regulation of hematopoiesis might operate through cell contact of NKT cells with HSC and interaction of the invariant TCR with CD1d on HSC.

In conclusion, iNKT cells when activated enhance the clonogenic capacity of myeloid progenitors and can thus modulate innate immune responses. Additionally, they are required for maintenance of normal hematopoiesis in the absence of immune activation. Our findings offer further evidence for a concerted regulation of the immune and hematopoietic systems and the potential for new therapeutic approaches for the manipulation of hematopoiesis.
FLT3 Mutant Activation Is Still Dependent on FLT3 Ligand.
Authors: Zheng Rui , Piloto Obdulio , Small Donald
Published: April 2020

FLT3 is a member of the class III receptor tyrosine kinase family and is primarily expressed on hematopoietic stem/progenitor cells. FLT3 ligand (FL) interacts with wild-type FLT3 (wtFLT3) receptors leading to the activation of FLT3 signaling. Constitutive activation of wt FLT3 through autocrine activation by FL is also observed in a number of cell lines and primary AML samples. Incubation of leukemic blasts expressing FLT3 with FL results in enhanced proliferation and decreased spontaneous apoptosis in many cases of acute leukemia. Somatic mutations of FLT3 involving internal tandem duplication (ITD) of the juxtamembrane domain or point mutations in the tyrosine kinase domain (TKD) have been identified in approximately 17–34% and 7–9% of acute myeloid leukemia (AML) patients, respectively. The ITD and TKD mutations appear to activate the tyrosine kinase domain of FLT3 in a FL-independent manner. However, these data were all obtained in human and murine cell lines that express FL. Thus, it is not clear whether FL also plays a role in the activation of FLT3 mutants. In order to determine whether or not FLT3 mutants are completely or somewhat dependent on FL, a FL deficient murine embryo fibroblast cell line (MEF) was derived from FL deficient (FL−/−) mice by SV40 large T antigen transformation. This eliminates the possibility of autocrine stimulation of transfected FLT3 receptor by FL. Expression of FLT3/ITD and FLT3/TKD mutations in FL−/−MEF cells resulted in some constitutive phosphorylation of FLT3 receptor. However, a more than 4 fold increase of FLT3 activation was induced by addition of FL. Retroviral introduction of FL in FL−/− MEF cells expressing FLT3 mutants led to more than 3 fold and 2 fold increase of tyrosine phosphorylation of FLT3 and AKT, respectively. In addition, FL expression resulted in a 3 fold increase of phosphorylation of STAT5 in FL−/−MEF cells expressing FLT3/ITD. In these FL expressing cells, addition of exogenous FL showed no further stimulation of FLT3, AKT and STAT5. These data strongly suggest that the level of FLT3 activation mediated by FLT3 mutants is still largely dependent on FL.
Over-Expression of Stromal Cell-Derived Factor-1 in Non-Specialized Endothelium Enhances the Endothelium-Supported Transmigration, Maintenance, and Proliferation of Hematopoietic Progenitor Cells.
Authors: Hwang Jin-Hee , Kim Seong-Woo , Yun Hwan-Jung , Lee Younghee , Kim Samyong , Jo Deog-Yeon
Published: April 2020

Stromal cell-derived factor-1 (SDF-1) plays a central role in the homing of hematopoietic stem cells and progenitor cells (HPCs) to bone marrow (BM). Tissue damage, most notably ischemia, induces the recruitment of bone marrow (BM)-derived stem/progenitor cells, which can lead to tissue repair. Moreover, damaged tissues and endothelium express and produce SDF-1. However, the effects of aberrant expression and production of SDF-1 in human endothelium on the biology of BM-derived stem/progenitor cells are not fully understood. To clarify this, we over-expressed the SDF-1 gene in human umbilical vein endothelial cells using an adenoviral vector (HUVEC/AdeSDF-1) and examined the transmigration, maintenance, and proliferation of HPCs supported by the endothelium. HUVEC/AdeSDF-1 monolayers induced the migration of MO7e cells and mobilized peripheral blood (mPB) CD34+ cells underneath the endothelium within a few hours, while HUVEC monolayers transfected with adenoviral vectors expressing LacZ (HUVEC/AdeLacZ) did not. A transmigration assay revealed that HUVEC/AdeSDF-1 monolayers supported the spontaneous transmigration of CD34+ cells to a greater degree than did HUVEC/LacZ monolayers (15 ± 4% vs. 5 ± 3%) and that transmigration was blocked with pretreatment of the input cells with pertussis toxin (PTX). The co-culturing of mPB CD34+ cells with HUVEC/AdeSDF-1 for up to 2 weeks led to a greater expansion of CD45+ cells and colony-forming cells, and reduced cell apoptosis. Furthermore, the co-culturing of mPB CD34+ cells with HUVEC/AdeSDF-1 led to the formation of numerous cobblestone areas, while mPB CD34+ cells plus HUVEC/AdeLacZ supported the formation of only a few cobblestone areas (253 ± 28 vs. 10 ± 3 per 2 x 104 mPB CD34+ cells at week-2), which were further enhanced by adding a combination of early-acting hematopoietic growth factors at low concentrations. To further elucidate whether SDF-1 is directly involved in the formation of cobblestone areas, mPB CD34+ cells were co-cultured with murine BM MS-5 stromal cells. Weekly PTX treatment, beginning 24 hours after start of co-culturing, markedly decreased the numbers of cobblestone areas by weeks 2 and 5, which indicates that SDF-1 is involved not only in the localization of primitive HPCs underneath the endothelium or BM stroma but also in the maintenance and proliferation of the cells. These results indicate that aberrant SDF-1 over-expression in any non-specialized endothelium induces BM-derived progenitor cell trafficking into extravasculr sites and promotes the maintenance and proliferation of the migrated cells.
Immunohistologic Identification and Quantification of Human Bone Marrow Hematopoietic Stem Cells.
Authors: Quaini Federico , Lazzaretti Mirca , Carranza Lorena , Ferraro Francesca , Bonomini Sabrina , Urbanek Konrad , Mancini Cristina , Giuliani Nicola , Anversa Piero , Rizzoli Vittorio
Published: April 2020

A recent experimental and clinical observation on the use of bone marrow-derived stem/progenitor cells (BM-SC) to treat non-hematologic degenerative diseases has renovated the long-standing knowledge of their biology and function. However, the paradoxical lack of histological studies limits the identification of SC within the human BM, constraining their purification and further obscuring the sites where SC fate is decided. In an attempt to identify human hematopoietic BM-SC and their relationships with the tissue environment, bone marrow biopsies from 31 subjects without BM involvement by hemo-lymphoproliferative diseases were analysed with stringent morphometric criteria for the quantification of cells expressing CD117 (c-kit) and CD34, as candidate SC markers, and/or CD45, CD3, CD20, myeloperoxidase (MPOX), CD68, Glycophorin A (GlyA) and Factor VIII (vWF), as lineage commitment. Immunofluorescence combined with confocal examination was also performed to allow the simultaneous detection of multiple surface/cytoplasmic antigens together with transcription factors. Hematopoietic cells constituted 54% of the tissue among which MPOX, GlyA and vWF positive cells represented 43.8%, 13.5% and 0.06% of marrow cells, respectively. The frequency of CD34pos cells was 93.5±43.6 each 105 total nucleated cells; c-kit involved 2.7±2.6/105 cells and c-kit-CD34 co-expression was present in 1.14±0.98/105cells. When the incidence of CD45 was evaluated among these two cell populations only 15% of c-kitpos and 49% of CD34pos cells showed hematopoietic lineage commitment, indicating the presence in the human bone marrow of undifferentiated SC. The analysis of the expression of Terminal Deoxynucleotidyl Transferase (TdT) in hematopoietic cells demonstrated that 10% of c-kitpos, 20% of CD34pos and 0.62% of CD45pos cells possessed the nuclear transcription factor. The distribution of BM-SC was predominantly scattered throughout the tissue although interstitial and paratrabecular clusters were detected and are currently under intense scrutiny in search of BM-SC sites of activation. In a subset of patients, the histological analysis was complemented by the cytofluorimetric estimation of the same BM cells populations. The results of this study may contribute to the improvement in the isolation and characterization of BM-SC aimed to the identification of their niches and to an appropriate clinical application in the field of regenerative medicine.
Abnormal Pulmonary Function in Adults and Children with Sickle Cell Disease.
Authors: Morris Claudia R. , Gardner Jennifer , Wen Andrew , Robertson Shanda , Hagar Ward , Vichinsky Elliott
Published: April 2020

Introduction: Chronic lung disease is a fatal complication in sickle cell disease (SCD) often undiagnosed until late stages. Early detection and treatment of risk factors may improve survival. Since asthma, pulmonary hypertension (PHT), and acute chest syndrome (ACS) are potential risk factors, in 1998, we began screening patients with history of ACS or wheezing with pulmonary function tests (PFT) and echocardiogram (echo).

Methods: A chart review was completed on the 362 adults and children evaluating history of asthma, lung function (PFT) and PHT (echo, tricuspid regurgitant jet velocity ≥ 2.5).

Results:

PFT Findings All Ages Adults Children PHT (All Ages) Study performed 34% (124/362) 41% (95/232) 22% (29/130) Echo: 90% (111/124) Abnormal 85% (106/124) 87% (83/95) 79% (23/29) 51% (54/106) Obstructive (OBS) only 31% (33/106) 31% (26/83) 30% (7/23) 42% (14/33) Restrictive (REST) only 30% (32/106) 28% (23/83) 39%(9/23) 38% (12/32) Both OBS + REST pattern 27% (29/106) 29% (24/83) 22% (5/23) 69% (20/29) “Abnormal,” not specified 11% (12/106) 12% (10/83) 9% (2/23) 67% (8/12) DLCO < 65% predicted 48% (45/94) 53% (42/80) 21% (3/14) 54% (26/48) Diagnosis & Treatment Clinical asthma diagnosis 13% (47/362) 14% (33/232) 11% (14/130) 43% (20/47) Receiving asthma treatment 51% (24/47) 45% (15/33) 64% (9/14) 50% (12/24) Bronchodilator only 23% (11/47) 24% (8/33) 21% (3/14) 45% (5/11) Inhaled steroid 23% (11/47) 18% (6/33) 36% (5/14) 55% (6/11) Singulair 4% (2/47) 3% (1/33) 7% (1/14) 50% (1/2) PFT suggests asthma 50% (62/124) 53% (50/95) 41% (12/29) 53% (33/62) +PFT, + asthma treatment 16% (10/62) 14% (7/50) 25% (3/12) 80% (8/10)

One hundred twenty-four patients underwent PFTs, of which 111 (85%) were abnormal. Obstructive and/or restrictive disease with abnormal diffusion capacity were widespread. Of patients with an obstructive pattern, only 16% were receiving any asthma treatment. Forty-seven patients (33 adults and 14 children) were diagnosed with asthma. However, only half were receiving any treatment: 23% bronchodilators, and 23% inhaled steroids. Echo was performed on 90% of patients with PFT data. Half of all patients with abnormal PFTs, and 69% of those with obstructive/restrictive patterns, had PHT.

Conclusion: While a more severe population may have been tested, this data suggests abnormal lung function is prevalent in SCD and is associated with PHT. Recent data suggests abnormal NO metabolism may link asthma and PHT in SCD. In conclusion, our data suggests patient morbidity will be decreased by regular screening with PFT and echo followed by early treatment for asthma and PHT.
Lactadherin Enhances the Adhesion of Sickle Cells to Vascular Endothelial Cells.
Authors: Guchhait Prasenjit , Dasgupta Swapan K. , Lopez Jose A. , Thiagarajan Perumal
Published: April 2020

Adherence of red blood cells to the endothelium initiates vaso-occlusion in sickle cell anemia. The increased adhesiveness of sickle erythrocytes is accompanied by several changes in the lipids of the erythrocyte membrane, including increased expression of phosphatidylserine (PS). One important PS-binding protein is lactadherin (also known as milk fat globule-EGF factor 8), a 45-kDa glycoprotein containing an Arg-Gly-Asp (RGD) sequence. Lactadherin is secreted by many cell types, including macrophages, and is known to have a role in the clearance of apoptotic lymphocytes by binding PS on the cell surface and thus presenting a binding site for macrophage integrins, including αvβ3. We recently showed that lactadherin, added exogenously to both normal and sickle erythrocytes, enhances phagocytosis of these cells by macrophages. Here, we investigated the effect of bovine lactadherin on the adhesion of sickle erythrocytes to the endothelium in flowing blood. Lactadherin (100 nM) promoted the adhesion of sickle erythrocytes to histamine-stimulated human umbilical vein endothelial cells (HUVECs) at shear stresses from 2.5 to 10 dyne/cm2. Adhesion was inhibited significantly by abciximab (10μ g/ml), an antibody against αvβ3 (P<0.05). Normal erythrocytes were induced to adhere to stimulated HUVEC in a lactadherin-dependent manner by treatment with 10 mM N-ethylmaleamide and 4 μ M calcium ionophore A23187—treatment that exposes phosphatidylserine on the erythrocyte surface. These results indicate that lactadherin mediates sickle cell adhesion to the endothelium by bridging PS on erythrocytes to αvβ3 integrin on the endothelium. We propose that sickle erythrocytes acquire lactadherin as they traverse macrophage-rich zones such as spleen, liver, and lymph nodes. Those that are not ingested immediately will become more adhesive for endothelium. Thus, lactadherin appears to be involved in both hemolysis and vaso-occlusion in sickle cell anemia.
Parallel Analyses Disclose Novel Genomic Imbalances in Acute Myeloid Leukemia with Complex Karyotypes.
Authors: Rucker Frank G. , Bullinger Lars , Kestler Hans A. , Lichter Peter , Dohner Konstanze , Dohner Hartmut
Published: April 2020

Approximately 10 to 15 % of acute myeloid leukemia (AML) cases exhibit complex karyotypes, i.e., three or more chromosome abnormalities without presence of a specific fusion transcript. To identify novel genomic regions of interest in AML with complex karyotypes we applied comparative genomic hybridization to microarrays (matrix-CGH) allowing high-resolution genome-wide screening of genomic imbalances. We designed a microarray consisting of 2799 different BAC- or PAC-vectors. A set of 1500 of these clones covers the whole human genome with a physical distance of approximately 1.5 Mb. The remaining 1299 clones either contiguously span genomic regions known to be frequently involved in hematologic malignancies (e.g., 1p, 2p, 3q, 7q, 9p, 11q, 12q, 13q, 17p, 18q) (n=600) or contain oncogenes or tumor suppressor genes (n=699).

Using this microarray platform, 83 AML cases with complex karyotypes were analyzed. Genomic losses were found more frequently than gains; the most frequent losses were deletions of 5q (71%), 17p (53%), 7q (48%); followed by deletions of 18q (30%), 16q (28%), 3p and 12q (20% each), 12p (18%), 20q (17%), and 11q (12%). The most frequent genomic gains were trisomies of 11q (39%) and 8q (31%); followed by trisomies of 1p (22%), 21q (20%), 9p (14%), 22q (13%), 13q (12%), and 6p (10%). In part, some critical segments were delineated to genomic fragments of 0.8 to a few megabase pairs in size. In lost/gained regions parallel analysis of gene expression using microarray technology detected a gene dosage effect with significant lower/higher average gene expression levels across the genes located in the respective regions. Furthermore, 47 high-level DNA amplifications in 19 different regions were identified; amplifications occurring in at least two cases mapped to (candidate genes in the amplicon) 11q23.3-q24.1 (n=10; ETS, FLI1); 11q23.3 (n=8; MLL, DDX6); 21q22 (n=5; ERG, ETS2); 13q12 (n=3; CDX2, FLT1, FLT3, PAN3); 8q24 (n=3; C8FW, MYC); 9p24 (n=2; JAK2); 12p13 (n=2; FGF6, CCND2); and 20q11 (n=2; ID1, BCL2L1). Parallel analysis displayed overexpressed candidate genes in critical amplified region, e.g., C8FW and MYC in 8q24.

In conclusion, using high-resolution genome-wide screening tools such as matrix-CGH allows to unravel the enormous genetic diversity of AML cases with complex karyotypes, and correlation with global gene expression studies facilitates the delineation of disease-related candidate genes located in the critical regions.
SCL Oncogene Perturbs HEB/E2A Function on Thymocyte Differentiation.
Authors: Tremblay Mathieu , Herblot Sabine , Desrosiers Marianne , Todorova Margarita , Paquet Eric , Major Francois , Hoang Trang
Published: April 2020

The SCL and LMO oncogenes are frequently activated in childhood T cell acute leukemia (T-ALL). SCL is a transcription factor of the basic helix-loop-helix (bHLH) family that forms heterodimers with other members of the family, specifically HEB and E2A. SCL can activate or repress transcription but the mechanism through which SCL functions as an oncogene remains to be clarified. Ectopic expression of SCL and LMO in the thymus of transgenic mice causes thymocyte differentiation arrest during the preleukemic phase with aberrant differentiation at the DN3-DN4 stage, prior to the acquisition of CD4 and CD8. We therefore took several approaches to define the mechanism underlying differentiation arrest in these cells. We first analyzed global gene expression of pre-leukemic DN3 thymocytes from SCLtg/LMOtg mice against their wild type littermates. We found that in this context, these oncogenes act as global transcriptional repressor as 90% of the genes with more than two fold differences are repressed when compared to wild type controls. Furthermore, we identify the HEB/E2A pathway as being targeted by SCL/LMO and used different approaches to show that the HEB/E2A activity is repressed by these oncogenes. First, using real-time quantitative PCR, we confirmed the repression of known HEB/E2A target genes (pTa and Rag1/2). In addition, we identified new HEB/E2A target genes that were confirmed as direct target through chromatin immunoprecipitation of primary thymocytes. This array also reveals that SCL associates with HEB and/or E2A on DNA to repress their function. Furthermore, we took a genetic approach to show that SCL/LMO collaborates with HEB haploinsufficiency in inducing leukemia. Our observations therefore reveal that the repression of the HEB pathway is crucial for T cell transformation. The importance of this repression is underscored by the fact that the E2A/HEB target genes that we identify here are expressed at low levels in primary leukemic cells from T-ALL patients when compared to B-ALL or AML samples. Together, these results show that SCL/LMO repress HEB/E2A activity to block T cell differentiation, an important step for T cell leukemia.
Immunotherapy of Primary Ovarian Cancer Using Autologous Cytokine Induced Killer Cells Retargeted with Bispecific Antibodies: A Preclinical Study.
Authors: Chan John K. , Hamilton Chad A. , Cheung Michael K. , Karimi Mobin , Baker Jeanette , Gall Jonathan M. , Lum Lawrence G. , Schulz Stephan , Thorne Steve H. , Teng Nelson N. , Contag Christopher H. , Negrin Robert S.
Published: April 2020

Cytokine induced killer (CIK) cells are ex-vivo activated and expanded CD8+ natural killer T cells that have been shown to have anti-tumor activity. This is the first study exploring cell killing of primary ovarian carcinoma with and without bispecific antibodies (BSAbs). Primary cancer cells and autologous CIK cells were collected from women with epithelial ovarian cancer. BSAbs against CA125 (BSAbxCA125) and Her2 (BSAbxHer2) were developed using chemical heteroconjugation. On FACS analysis, the expansion and stimulation of CIK cells resulted in a significant increase of CD3+CD8+ and CD3+CD56+ T cells. With enhancement by BSAbs, the mean percent lysis in a 51Cr release assay of fresh ovarian cancer cells exposed to autologous CIK cells increased from 22% (±0.3) to 89% (±2.1) at an effector to target ratio (E:T) of 100:1. Anti-NKG2D antibodies significantly attenuated the CIK activity by 57% on primary cells. In a xenograft SCID mouse model, real-time tumor regression and progression was visualized using a non-invasive in vivo bioluminescence imaging system. Four hours after CIK cell injection, we were able to visualize CD8+NKG2D+ CIK cells infiltrating Her2-expressing cancer cells on fluorescence microscopy. Mice that underwent adoptive transfer of CIK cells redirected with BSAbxCA125 (p=0.0002) and BSAbxHer2 (p=0.0002) had a significant reduction in tumor burden and improvement in survival versus those treated with CIK cells alone (p=0.03). BSAbs significantly enhanced the cytotoxicity of CIK cells in primary ovarian cancer cells and in our in vivo mouse model. The mechanism of cytolysis appears to be mediated in part by the NKG2D receptor.
Pyrimethamine Does Not Diminish Lymphoproliferation in MRL/lpr−/− Mice and Patients with Autoimmune Lymphoproliferative Syndrome (ALPS).
Authors: Rao V. Koneti , Dowdell Kennichi C. , Dugan Faith , Dale Janet K. , Pesnicak Lesley , Bi Lilia , Penzak Scott , Avila Nilo A. , Fleisher Thomas A. , Puck Jennifer M. , Straus Stephen E.
Published: April 2020

ALPS is an inherited disorder of lymphocyte apoptosis leading to childhood onset of chronic lymphadenopathy, hepatosplenomegaly, autoimmunity, and an increased risk of lymphoma in a subset of patients with mutations in the intracellular domain of Fas (ALPS Type Ia). Similarly, MRL/lpr −/− mice are homozygous for Fas mutations and develop massive lymphadenopathy and splenomegaly associated with hypergammaglobulinemia, glomerulonephritis, and expansion of TCR αβ+, CD4−/CD8−double-negative (DN) T cells that are pathognomonic of ALPS. There remain no proven therapies for the lymphoproliferation underlying ALPS itself, although investigators in Europe reported that some children with ALPS showed reductions in spleen and lymph node sizes while on a weekly regimen of pyrimethamine/sulfadoxine (Fansidar@) (Br J Haematol.2002; 117:176). However, in vitro studies using stimulated human PBMCs, revealed that lymphocyte apoptosis is induced by the antifolate agent, pyrimethamine (Pyr) at an EC50 of 2.5ug/mL, and not sulfadoxine. This suggested that Pyr alone might prove clinically beneficial, thus avoiding risks of allergy and hypersensitivity associated with sulfa drugs. Hence, studies were conducted in the MRL/lpr−/− murine model of ALPS with Pyr alone given 2mg/kg twice weekly by gavage for 8 weeks, and in combination with sulfadoxine. Neither Pyr nor Fansidar yielded significant shrinkage of lymphoid mass in mice. A pilot study of the safety and efficacy of Pyr was conducted under an FDA IND in 6 children with ALPS Type 1a and one with ALPS Type III (with no identifiable mutation). Aged 6 to 17 yrs (median; 13 y), these 6 males and 1 female patient were enrolled following informed consent to be treated with oral Pyr at escalating doses (25–50mg) given twice weekly for 12 weeks. Computerized tomography was done before and after treatment to determine whether these patients would show 40 and 50% reductions in the sizes of their lymphadenopathy and/or splenomegaly, respectively. Lymph node sizes were assessed as sums of the bi-dimensional products of 3 to 5 nodes. Spleen sizes were measured as products of their craniocaudal and anterioposterior dimensions at the superior mesenteric artery level, as well as spleen volumes computed by digital image processing. The effects of treatment on other well-defined laboratory features of ALPS were also assessed. Pyr was discontinued after 11 days in one patient due to skin rash. Dose escalation was interrupted transiently in 3 others because of neutropenia, aphthous ulcer, or leucopenia. There were no significant changes in study outcome measures. The aggregate experience with Pyr and Fansidar in mice, as well as with Pyr alone in ALPS patients (Table) lead us to conclude that these agents do not manifest sufficient effectiveness to warrant larger trials.
The Novel, Orally Active Proteasome Inhibitor, NPI-0052, Induces Apoptosis in Leukemia Lymphoma Cell Lines and Patient Specimens.
Authors: Miller Claudia P. , Ban Kechen , Ruiz Stacey L. , Neuteboom Saskia , Palladino Michael , McConkey David , Chandra Joya
Published: April 2020

NPI-0052 is the second proteasome inhibitor with potential for clinical use since the FDA approval of bortezomib. NPI-0052 is a novel, orally active, non-peptide small molecule inhibitor discovered by Nereus Pharmaceuticals during the fermentation of a new marine Gram-positive actinomycete, Salinospora sp. In human erythrocyte derived 20S proteasomes, with EC50 values in the picomolar and nanomolar range, NPI-0052 inhibits all three proteolytic activities: the chymotrypsin-like, the trypsin-like and caspase-like activities. In the present study, exposure of 1uM NPI-0052 for 1 h inhibited the chymotryptic and caspase-like activities by greater than 90% in Jurkat and ML-1 cells. The trypsin-like activity was also inhibited to a lesser extent. NPI-0052 demonstrated varying degrees of apoptosis in cell lines representative of AML (ML-1), ALL (Jurkat), and Burkitt lymphoma (BL-41) and in mononuclear cells isolated from CLL and ALL patients, as measured by propidium iodide staining and subsequent FACS analysis. In addition, treatment of Jurkat cells with NPI-0052 resulted in activation of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP). Further experiments revealed that caspase activity might be initiated differently in myeloid versus lymphoid leukemia cell lines. NPI-0052 caused cleavage of caspase-8 as demonstrated by SDS-PAGE analysis and when combined with an inhibitor specific for caspase-8 (IETD-fmk), Jurkat cells (of lymphoid origin) were protected against NPI-0052 induced apoptosis whereas ML-1 (of myeloid origin) were not. The cleaved product of Bid was detected by immunoblotting in NPI-0052 treated Jurkat cells, suggesting amplification of caspase-8 activity through mitochondria. NPI-0052 induced loss of mitochondrial membrane potential and release of cytochrome c in Jurkat cells. Cell lines of lymphocytic origin exposed to 4h of NPI-0052 resulted in increased levels of peroxide and superoxide prior to cell death. Furthermore, the antioxidant, N-acetyl cysteine (NAC), conferred protection in Jurkat cells against NPI-0052 induced apoptosis. CLL and Ph+ ALL patient material confirmed that lymphocytes from these patients are protected from NPI-0052 induced apoptosis by antioxidants. In summary, NPI-0052 inhibits all three major proteolytic activities of 20S proteasome in leukemia cells and induces apoptosis in leukemic cells and patient samples. The cytotoxic effects of NPI-0052 in leukemia and lymphoma cells warrant further testing to determine if this compound is clinically effective.
Inhibition of AKT/mTOR Signaling Induces Cell Cycle Arrest and Apoptosis in Mantle Cell Lymphoma.
Authors: Peponi Evangelia , Drakos Elias , Reyes Guadalupe , Leventaki Vassiliki , Medeiros L. Jeffrey , Rassidakis George Z.
Published: April 2020

Mantle cell lymphoma (MCL) is a distinct type of B-cell lymphoma associated with aggressive clinical course. The PI3K/AKT pathway is activated in a variety of human malignancies including hematopoeitic tumors. Activated (serine 473-phosphorylated) AKT (pAKT) is believed to mediate its effects, at least in part, via phosphorylation/activation of the mammalian target of rapamycin (mTOR). However, the biologic significance of AKT/mTOR signaling in MCL is unknown. We hypothesized that the PI3K/AKT signaling pathway is activated in MCL and contributes to tumor cell survival through activation of mTOR and its downstream effectors 4EBP1, p70S6K, ribosomal protein (rp) S6 (rpS6) and eIF-4E. We used 3 mantle cell lymphoma cell lines (Mino, Jeko and Z-138), a PI3K inhibitor (LY294002), and small interfering RNA (siRNA) specific for mTOR gene to investigate the biologic effects after inhibition of AKT/mTOR signaling in MCL. Treatment of MCL cells with LY294002 resulted in apoptotic cell death in a dose-dependent manner that was associated with downregultion of the anti-apoptotic proteins cFLIP and Mcl-1. Inhibition of PI3K also resulted in decreased cyclin D1 levels and cell cycle arrest as shown by decreased S-phase fraction assessed by BrdU incorporation. Changes in cell cycle and apoptosis were associated with decreased phosphorylation (activation) of mTOR and its downstream targets 4EBP1, p70S6K, and rpS6. Silencing of mTOR expression, using mTOR-specific siRNA also decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis in MCL cell lines. These biologic effects were associated with downregulation of cyclin D1 and modulation of apoptosis regulators. In addition, expression of activated AKT, and effectors downstream of AKT/mTOR pathway (p-p70S6K, p-rpS6, 4EBP1, eIF-4E) was assessed in MCL tumors. Using a 10% cutoff to define positivity, 10/20 (33%) expressed Ser473pAKT, 22/22 (100%) p-p70S6K, 5/20 (25%)p- rpS6, 13/14 (93%) 4E-BP1, and 16/29 (55%) eIF-4E. p-p70S6K phosphoprotein was detected predominantly in the nucleus of tumor cells. All other phosphoproteins were expressed with a cytoplasmic pattern. We conclude that activation of PI3K/AKT may contribute to cell cycle progression and tumor cell survival in MCL cells through activation of the mTOR signaling pathway. Our data suggest that inhibition of mTOR with rapamycin analogues currently used in clinical trials may be a novel therapeutic strategy for patients with MCL.
Activation of mTOR Signaling Pathway Contributes to Tumoral Cell Survival in ALK-Positive Anaplastic Large Cell Lymphoma.
Authors: Vega Francisco , Medeiros L. Jeffrey , Atwell Coralyn , Cho Jeong Hee , Tian Ling , Claret Francois-Xavier , Rassidakis George Z.
Published: April 2020

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of nucleophosmin (NPM)-ALK. Previously, NPM-ALK has been shown to activate phosphatidylinositol 3-kinase (PI3K) and its downstream effector, the serine/threonine kinase AKT. Recently, we have shown that mTOR signaling proteins are activated in ALK-positive ALCL tumors and that mTOR activation depends, at least in part, on activation of AKT (Lab Invest2005; 85: 255A). In this study, we investigate the biological effects of inhibition of mTOR on two ALK-positive ALCL cell lines, Karpas 299 and SU-DHL1. For this purpose, we used rapamycin to inhibit mTOR-raptor complex and mTOR-specific small interfering RNA (siRNA) to silence the endogenous mtor gene. Treatment with rapamycin, resulted in a marked concentration-dependent decrease of phosphorylated (p)-mTOR, and its downstream targets, p-p70S6K, p-S6K, p-4E-BP1 and total eIF4E. Similarly, silencing the expression of mtor resulted in a decrease in the activation/phosphorylation level of these proteins as well as in the level of p-AKT. Both treatments induced apoptosis and cell cycle arrest in both ALK-positive ALCL cell lines as demonstrated by trypan blue exclusion, annexin V staining, BrdU incorporation, and cell cycle studies. There was a concentration-dependent decrease in the anti-apoptotic proteins BCL-2, BCL-XL, MCL-1 and c-FLIP (L and S) with increasing concentrations of rapamycin or after mTOR siRNA treatment. The cyclin dependent kinase inhibitors p21waf1 and p27kip1 and underphosphorylated (Un-p)-RB protein were upregulated, after treatment with rapamycin or after mTOR siRNA treatment. In conclusion, we provide evidence that inhibition of mTOR induces cell cycle arrest and apoptosis in ALK-positive ALCL cells. The decrease of p-AKT by silencing mtor suggests that mTOR is necessary to activate AKT in ALK-positive ALCL, and thus, mTOR can function as a feedback signal activity of its own pathway.
Initial Safety and Efficacy Results of a Second-Generation Humanized Anti-CD20 Antibody, IMMU-106 (hA20), in Non-Hodgkin’s Lymphoma.
Authors: Morschhauser Franck , Leonard John P. , Coiffier Bertrand , Petillon Marie-Odile , Coleman Morton , Bahkti Amina , Sapra Puja , Teoh Nick , Wegener William A. , Horak Ivan D. , Goldenberg David M.
Published: April 2020

Background. Infusion reactions, prolonged infusion times and immunogenicity of the chimeric anti-CD20 antibody, rituximab, may in part be related to its murine component. The humanized anti-CD20 antibody, IMMU-106 (hA20), may offer potential advantages because only the CDRs are of murine origin and the remaining >90% human framework regions are identical to humanized anti-CD22 IgG1 antibody epratuzumab, whose safety and short infusion times have been reported.

Methods. An open-label, multicenter, phase I/II, dose escalation study conducted in patients with recurrent B cell lymphoma to establish the safety, tolerance, pharmacokinetics, and immunogenicity of hA20 administered once weekly for 4 doses.

Results. Twenty patients (11 M/9 F, 40–81 y.o) with grade 1–2 follicular (n=16) or other CD20+ B-cell lymphomas (mantle cell, small lymphocytic, marginal zone x 2) were enrolled in the dose escalation portion of this study, receiving 120 mg/m2 (n=7), 200 mg/m2 (n=6), 375 mg/m2 (n=4) or 750 mg/m2 (n=3) weekly x 4. These patients had previously received 1-7 prior therapies (median, 2.5), including treatments with one (n=12) or 2–4 (n=6) rituximab-containing regimens (without progression within 6 months). Five patients (25%) had grade 1–2 infusion reactions predominantly with first hA20 infusion. Median infusion times for 375 mg/m2 of hA20 were 3.3 hours for first infusion, 2.0 hours for subsequent infusions, and were generally shorter at lower doses. Standard laboratory values obtained at each infusion were generally observed to be within normal limits, and human anti-human antibody (HAHA) serum evaluations are negative to date. Peripheral blood B-cell depletion occurred after first infusion and persisted for samples obtained up to 12 weeks following fourth infusion with analysis ongoing. Pharmacokinetic results after first and fourth infusions demonstrate a mean antibody serum half-life of 5.3 ± 5.3 and 12.0 ± 8.7 days, respectively. To date, of 15 pts with at least one post-treatment assessments now completed, there are at least 8 objective responses (53%) by Cheson criteria, including 6 patients (40%) with complete responses [CR, CRu] at a median follow-up of 12 weeks. Complete responses have been observed at all dose levels, including 3/7 patients receiving 120 mg/m2/wk.

Conclusion. This study demonstrates the safety, tolerability, and preliminary efficacy of this humanized anti-CD20 antibody administered weekly for 4 doses, mostly to patients with follicular lymphomas who relapsed after rituximab-containing regimens. The demonstration of CRs, tolerability, and relatively short infusion times is encouraging. This study is continuing and expanding to assess the durability of responses and the optimal dose for subsequent studies.
Long-Term Follow-Up of Patients Treated in a Phase 2 Trial with MyVax® Personalized Immunotherapy (Recombinant Id-KLH with GM-CSF) after Chemotherapy as Initial Treatment for Follicular Non-Hodgkin’s Lymphoma (NHL).
Authors: Timmerman John M. , Vose Julie , Levy Ronald , Mayo Martha , Denney Dan
Published: April 2020

Background: The tumor-specific variable regions of the clonal immunoglobulin (idiotype or Id) expressed by malignant B cell NHL can be used as a target for active immunotherapy. MyVax® Personalized Immunotherapy is a patient-specific, recombinant Id protein conjugated to Keyhole Limpet Hemocyanin (Id-KLH) and administered with Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF). Several Phase 2 clinical trials have been conducted to determine the immunogenicity of MyVax® Personalized Immunotherapy. Patients and

Methods: The patients all had previously untreated follicular lymphoma and achieved at least a partial response to either CVP chemotherapy (16 patients) or CVP + CHOP chemotherapy (5 patients) to be eligible to receive 5 series of immunizations with MyVax® Personalized Immunotherapy. Each immunization series consisted of MyVax® Personalized Immunotherapy administered SQ on Day 1 and GM-CSF alone on Days 2–4. The immunizations were administered over 24 weeks. The first 4 immunization series were administered every 4 weeks. The 5th immunization series was administered 12 weeks after the 4th immunization.

Results: There were a total of 21 evaluable patients in the study. Both cellular and humoral immune anti-idiotype responses (IRs) were elicited using this treatment schedule. Thirteen of the 21 patients (62%) mounted an anti-idiotype immune response. Ten of the 21 patients mounted a humoral anti-idiotype response while 7 of 16 patients evaluated mounted a cellular anti-idiotype response. Immunizations with MyVax® Personalized Immunotherapy appear to be safe and well tolerated. Adverse events reported in this trial were mostly mild to moderate and transient in nature. Follicular Lymphoma International Prognostic Index (FLIPI) scores for these patients indicate that most of the patients were in the intermediate or high-risk categories. The median follow-up is over 5.5 years in this trial. The median time to disease progression was 37.7 months measured from the end of chemotherapy. Ten of the 21 patients in this trial remain progression free between 47 months and 71 months post chemotherapy as of their last follow-up. Four of the patients that remain in remission are in the high risk category by FLIPI score.

Conclusions and Discussion: These results suggest that patients with a poorer prognosis according to their FLIPI score can achieve significant remissions following treatment with MyVax® Personalized Immunotherapy. MyVax® Personalized Immunotherapy is currently in a late-stage Phase 3 trial.
Atiprimod Blocks STAT3 and STAT5 Phosphorylation, Inhibits Proliferation, and Induces Apoptosis in Acute Myeloid Leukemia (AML) but Not Normal Cells.
Authors: Faderl Stefan , Ferrajoli Alessandra , Harris David , Van Quin , Kantarjian Hagop M. , Estrov Zeev
Published: April 2020

Cytokines and growth factors stimulate AML cell proliferation by activating various signaling pathways and high levels of cytokines have been associated with poor prognosis. Blocking signal transduction of cytokine-stimulated pathways may therefore inhibit AML growth and proliferation. Atiprimod is a cationic amphiphilic azaspirane. Although their mechanisms of action are not completely understood, azaspiranes were demonstrated among others to downregulate various cytokine receptors such as for interleukin (IL)-1, IL-2, IL-6, interferon-γ, and tumor necrosis factor-α. Owing to this spectrum of activities, we hypothesized that atiprimod inhibits activation of intracellular signaling pathways in AML cells resulting in apoptosis and growth inhibition. We first studied the antiproliferative effect of atiprimod on 5 AML cell lines (K562, HL-60, KG-1, OCIM2, OCI/AML3) using the MTT assay. Cells were incubated for 72 hours without and with increasing concentrations of atiprimod (1, 2, 3, and 4 μM), then harvested and their metabolic activity and viability determined as optical density measurements. To corroborate the results, we also studied the effect of atiprimod on OCIM2 cells using a clonogenic cell line assay. Next we determined expression of Stat3 and Stat5, as well as Phospho-stat3 and phospho-stat5 in the K562 cells by Western Immunoblotting where cells were incubated in the absence and presence of increasing atiprimod concentrations (0.5, 1, 2, 3, 4 μM). We further evaluated induction of apoptosis of OCIM2 and K562 cells following incubation with atiprimod at 1 and 4 μM using the annexin V-FITC assay and finally analyzed caspase 3 and PARP cleavage in K562 cells at atiprimod concentrations of 0.5, 1, 2, 3, and 4 μM using Western Immunoblotting. To demonstrate the effect of atiprimod on marrow cells from AML patients and healthy volunteers we incubated marrow cells with atprimod at increasing concentrations and used the blast colony assay to measure inhibition of proliferation. Our results demonstrate that: 1) atiprimod inhibits proliferation of AML cell lines and AML blast proliferation from patient samples, but not significantly normal hematopoietic progenitors from samples of healthy controls; 2) atiprimod inhibits phosphorylation of Stat 3 and 5; and 3) atiprimod induces apoptosis in OCIM2 and K562 cells by cleavage of caspase 3 and PARP. In summary, our data suggest that atiprimod inhibits phopshorylation of Stat 3 and 5, induces caspase-dependent apoptosis, and blocks AML cell proliferation. Further evaluation of atiprimod in clinical trials of AML and MDS should be considered.
RNA Silencing of Aurora Kinase Genes Sensitizes Human Leukemia Cells to the Ara-C-Induced Cell Death Via Caspase-Independent Pathway.
Authors: Min Yoo-Hong , Lee Jin Koo , Eom Ju In , Park In Hae , Park Sun Young , Kim Yuri , Kim Jin Seok , Cheong June-Won
Published: April 2020

Aurora kinases play critical roles in chromosome segregation and cell division. Furthermore, Aurora A and B kinases are both frequently amplified and overexpressed in a various kinds of human cancers including leukemia disorders. To address its possibility as a therapeutic target for leukemia, we employed the RNA interference technique to knockdown Aurora kinase gene expression and analyzed its effect on the cell cycle distribution and the chemotherapy-induced cell death in leukemia cells. The expression of Aurora kinase A (AURKA) and Aurora kinase B (AURKB) was observed in all the leukemia cell lines evaluated and 15 out of 18 cultured leukemic cell specimens obtained from untreated patients with acute myeloid leukemia (AML). An arrest in the G(2)/M phase 24 h after specific knockdown of AURKA was shown in U937 leukemia cells. Following the silencing of AURKA, we observed a striking increase in the proportion of cells in the S phase, tetraploid state, and showing multi-nucleated morphologic changes. Cell death was observed in 17.0 ± 0.9% and 20.1 ± 2.5% of U937 cells after AURKA and AURKB silencing, respectively. The proportion of cells in the subG(0)/G(1) phase was 23% and 11% with AURKA and AURKB silencing, respectively. Although the disruption of mitochondrial membrane potential was observed after AURKA or AURKB silencing in U937 cells, the cleavage of caspase-3, -8, -9, and PARP was not observed in the apoptotic cells. Pretreatment of U937 cells with the caspase-3 inhibitor, DEVD-CHO, did not abrogate the AURKA or AURKB silencing-induced cell death. The U937 leukemia cells are resistant to Ara-C-induced cell death. However, we observed a striking synergistic enhancement of the cytotoxicity of Ara-C (10 μM), an important antimetabolic chemotherapeutic agent used for acute leukemia, by the RNA interference-mediated knockdown of AURKA and AURKB through a caspase-independent manner in U937 cells. Collectively, we demonstrate that the specific inhibition of either AURKA or AURKB can potentially regulate the cell cycle distribution of leukemia cells and induce synergistic interaction with Ara-C for inducing the cell death through the mechanism of mitotic catastrophe. These results indicate that Aurora kinase inhibition provides a novel approach for the treatment of human acute leukemia.