bluuebunny/crossref_metadata_2025 huggingface dataset:
M-CSF Inhibition Selectively Targets Tumor Angiogenesis and Lymphangiogenesis
Authors: Kubota Yoshiaki , Suda Toshio
Published: April 2020

Anti-angiogenic therapy in cancer is desired to be selective for tumor angiogenesis. The best validated approaches for limiting tumor angiogenesis involve blockade of the VEGF pathway. However, recent studies show that VEGF blockade damages healthy vessels resulting in toxic side effects (Maharaj et al. J. Exp. Med. 2008) and that interrupting the VEGF blockade induces rapid vascular regrowth in tumors (Mancuso et al. J. Clin. Invest. 2006). Therefore, other targets are currently being explored. Macrophage-colony stimulating factor (M-CSF), a cytokine required for differentiation of monocyte-lineage cells including macrophages promotes formation of a high-density vessel network in tumors suggesting therapeutic potential of M-CSF inhibition (Lin et al. J. Exp. Med. 2001). Moreover, M-CSF is abundantly expressed in highly-metastatic cancers such as breast cancer and osteosarcoma, suggesting high dependency on macrophages. However, the physiological role of M-CSF in vascular and lymphatic development as well as the precise mechanisms underlying anti-angiogenic effects of M-CSF inhibition is not clarified. Here, utilizing osteopetrotic (op/op) mice, we show that M-CSF deficiency leads to lack of both LYVE-1+ and LYVE1− macrophages, resulting in defects not only in vascular but also lymphatic development. To clarify the anti-cancer effects of M-CSF inhibition, we utilized a novel mouse model of osteosarcoma, in which mice were transplanted with a newly established osteosarcoma cell line, AX, which were developed from c-Myc-overexpressing Ink4a/ARF−/− bone marrow-derived stromal cells. In this model, systemic treatment with small molecule c-fms inhibitors (Ki20227) effectively suppressed tumor angiogenesis and lymphangiogenesis disorganizing extracellular matrices. Consequently, c-fms inhibitors almost completely suppressed tumor metastasis, and protected mice from cancer death. Different from VEGF blockade, interruption of M-CSF inhibition did not promote rapid tumor regrowth. Continuous M-CSF inhibition did not affect healthy vascular and lymphatic systems outside the tumor. These results suggest M-CSF-targeted therapy is an ideal therapeutic strategy against cancer alternative to VEGF blockade.
The Second Signal in Antigen-Presenting Cells: Complementary JAK1/JAK2 and CD40 Signalling Mediate Pro-Inflammatory Activation.
Authors: Conzelmann Michael , Rodionova Elena , Hess Michael , Zota Annika , Giese Thomas , Wagner Andreas , Breit Stephen , Hecker Markus , Ho Anthony D. , Dreger Peter , Luft Thomas
Published: April 2020

CD40Ligand (CD40L) represents a strong endogenous danger signal associated with chronic inflammatory disease. CD40L induces pro-inflammatory activation of CD40 expressing antigen-presenting cells (APC) such as DCs, monocytes, B-cells and endothelial cells; however, CD40 activation alone is insufficient to induce interleukin (IL)-12p70 secretion. Using quantitative Western blotting and siRNA inhibition of protein expression we demonstrate that cytokine-induced Janus kinase (JAK)1 and/or JAK2 activation mediates a complementary second signal for the production of IL- 12p70, whereas tyrosine kinase (Tyk)2 activation has inhibitory effects. Furthermore, JAK1 activation reciprocally reduced IL-10 production. The mechanism of JAK modulation of CD40 signals in dendritic cells (DCs) involved transcriptional regulation. The Th1 and Th2 cytokines interferon (IFN)-γ and IL-4 both enhance IL-12p70 via activation of the same JAKs. However due to a weaker JAK activity, IL-4 signalling has to persist much longer than IFN-γ signalling in order to induce similar amounts of IL-12p70. This different dependence on signalling persistence may give rise to the distinct biological characteristics of Th1/Th2 cytokines and may explain the pleiotropic in vivo and in vitro effects of IL-4. Complementary CD40 and JAK activity is essential for IL-12p70 secretion by all human APC studied. This strict requirement of two complementary signals opens a new way of interfering with CD40L induced APC activation indirectly by modulating the “second signal” JAK.
Impact of Safety Concerns of Erythropoiesis-Stimulating Agents (ESAs) and Regulatory Changes on the Use of ESAs and Red Blood Cell (RBC) Transfusions at a Comprehensive Cancer Center
Authors: Vadhan-Raj Saroj , Hawkins Victoria E. , Zhou Xiao , Sizer Kurt , Lal Lincy S. , Roquemore Joyce , Arbuckle Rebecca B. , Benjamin Robert S. , Lichtiger Benjamin
Published: April 2020

Safety signals raised in the recent oncology clinical trials have led to various regulatory restrictions including FDA black-box warning, National Coverage Determination (NCD), and updated ASCO/ASH guidelines in 2007. The purpose of this study was to determine the impact of these changes on the utilization of ESAs and on transfusion (Tx) of RBCs in 2006 (prior to changes) and 2007. We identified the total number of unique patients that received any treatment including chemotherapy, radiation, transfusions, or any treatment in the out-patient and in-patient settings during this 2 year time period. All the data on the ESA doses dispensed by the hospital pharmacy and all the RBC transfusions dispensed by the Blood bank were also analyzed. The ESA units were calculated by converting 40,000 units of epoetin alfa or 100 mcg of darbepoetin alfa to one unit of ESA. When comparing 2007 to 2006, the number of patients that received ESAs decreased by 26% and the total ESA units decreased by 30%. The overall usage of ESAs decreased by 55%, from 2398 units in 1/2006 to 1080 units in 12/2007. However, the number of pts that received RBC transfusions increased only by 6% and the total number of RBC units transfused by 2% (from 38,218 units in 2006 to 38,948 units in 2007). The median Hgb on the day of transfusion was same for each year (Hgb 8.2 g/dL for both 2006 and 2007), suggesting that the lack of impact on RBC Tx may not be due to a change in Tx threshold. The total number of unique patients referred and treated at MDACC during 2007 (24,356) increased by 13% from 2006 (21,619), not accounting for a lack of impact on transfusions. We therefore examined Hgb at the initiation of ESAs in a subset of pts (n=212) that had not received ESA for at least 3 months. The median Hgb/HCT values at the initiation of ESAs were 9.5 g/dL/27.4. The most frequent utilization of ESAs and transfusions was in patients with hematological malignancies. Conclusion: These findings indicate that the recent ESA safety concerns and related regulatory changes have significantly affected the ESA utilization. The lack of significant impact of reduced ESA usage on RBC transfusions may be related to a lower Hgb threshold used at initiation of ESAs and/or the targeted patient population (less likely to respond) treated with ESAs. Further research is needed to establish the factors contributing to the lack of correlation and to optimize the use of ESAs.
Angiopoietin-Like 3 Deficient Bone Marrow Has Decreased Ability to Support Hematopoietic Stem Cells
Authors: Zheng Junke , Huynh HoangDinh , Zhang Chengcheng
Published: April 2020

We previously identified a group of angiopoietin-like proteins (Angptls) as new growth factors that stimulate ex vivo expansion of hematopoietic stem cells (HSCs). To investigate the physiological function of Angptl3 in bone marrow, we characterized the Angptl3 deficient mice, and identified several defects in the hematopoietic compartment. When we transplanted wild-type HSCs into lethally irradiated Angptl3 deficient mice, we found that the mutant bone marrow stroma have much lower ability to support in vivo expansion of HSCs. We sought to identify the Angptl3-producing cells in mouse bone marrow stroma, and showed that Angptl3 is highly expressed in CD45-SSEA4+ cells, which are mesenchymal stem cells (MSCs). Indeed, the co-culture of HSCs with CD45-SSEA4+ MSCs resulted in ex vivo expansion of HSCs. DNA microarray analysis, real-time RT-PCR, and flow cytometry were used to identify the intracellular factors that are responsible for Angptl3’s effects on HSCs. This investigation demonstrated that Angptl3-stimulated HSC expansion is contributed by its activities to support HSC self-renewal and inhibit hematopoietic differentiation. Our study will likely lead to the identification of a novel component of the niche for HSCs.
The Wnt Inhibitor sFRP2 Enhances Mesenchymal Stem Cell Engraftment, Granulation Tissue Formation and Myocardial Repair
Authors: Alfaro Maria Paula , Pagni Matthew , Vincent Alicia , Hill Michael F. , Lee Ethan , Young Pampee
Published: April 2020

Cell-based therapies using bone marrow-derived mesenchymal stem cells (MSCs) for organ regeneration are being pursued for cardiac disease, orthopedic injuries and biomaterial fabrication. The molecular pathways that regulate MSC-mediated regeneration or enhance their therapeutic efficacy are, however, poorly understood. In an attempt to elucidate a way to strengthen the regenerative potential of MSCs, preliminary studies in our lab were performed comparing MSCs isolated from wildtype and regenerative mouse strains. The MRL/MpJ mouse has been described as a “super healer” mouse that is able to repair soft tissue with minimal scaring. MSCs were isolated from the MRL/MpJ mouse (MRL-MSCs) and from C57/Bl6 mice (WT-MSCs) and their differing qualities assessed. Compared to WT-MSCs, MRL-MSCs demonstrated increased proliferation in vitro. We utilized a Poly-vinyl alcohol (PVA) sponge model of repair stimulation to assess their capacity to generate wound repair tissue. We observed that the MRL-MSCs demonstrated increased in vivo engraftment, experimental granulation tissue reconstitution, and tissue vascularity. The MRL-MSCs also reduced infarct size and improved cardiac function as compared to WT-MSCs in a murine acute myocardial infarct model. Genomic and functional analyses indicated a downregulation of the canonical Wnt pathway in MRL-MSCs characterized specifically by upregulation of secreted frizzled related proteins (sFRPs). In vitro proliferation studies confirmed that recombinant sFRP2 mediated enhanced proliferation of both mouse and human MSCs. Based on these observations, we hypothesized that sFRP2 served an important role in MSC-mediated repair and regeneration. We generated WT-MSCs overexpressing sFRP2 (sFRP2-MSCs) by retroviral transduction to test this hypothesis. sFRP2-MSCs maintained their ability for multilineage differentiation in vitro and proliferated faster than the vector only control MSCs (GFP-MSCs). When implanted in vivo in the PVA sponge model, the sFRP2-MSCs recapitulated the MRL phenotype by mediating greater, more vascularized granulation tissue. Moreover, periinfarct intramyocardial injection of sFRP2-MSCs resulted in reduced infarct size, favorable remodeling and better preserved left ventricular function following acute myocardial infarct in mice. These findings implicate sFRP2 as a key molecule for the biogenesis of a superior regenerative phenotype of MSCs.
Stabilin-1 and Stabilin-2 Are Expressed in Bone Marrow Sinusoidal Endothelial Cells and Mediate Scavenging and Cell Adhesive Functions
Authors: Qian Hong , Johansson Sophie , McCourt Peter , Smedsrød Bård , Ekblom Marja , Johansson Staffan
Published: April 2020

Stabilin-1 and stabilin-2 have been identified as scavenging receptors in sinusoidal capillaries in some tissues, including liver and spleen. They function as endocytic receptors for SPARC and hyaluronan, respectively, and for several other ligands. In the present study, we show by real-time PCR, Western blot and immunohistochemistry that bone marrow (BM) sinusoidal endothelial cells (SECs) ubiquitously express stabilin-1 and stabilin-2. To test the ability of BM SECs to function as a scavenging endothelium, we analyzed the uptake of specific scavenger receptor ligands after intravenous injection. Accumulation of TRITC labelled formaldehyde-treated serum albumin (FSA) was observed one hour after the injection in the BM SECs. Injection of unlabelled FSA together with TRITC-FSA reduced the fluorescence in the SECs, indicating that the uptake was due to specific recognition of FSA. Likewise, FITC-conjugated advanced glycation end products (AGEs)-modified BSA accumulated in BM SECs. These two ligands are primarily cleared by the stabilins. These results suggested that bone marrow SECs have a scavenging function. Stabilin-2 has been identified as a major receptor for hyaluronan. Hyaluronan is synthesized by primitive hematopoietic cells and has been shown to influence stem and progenitor (HSPC) functions, including mobilization and homing into BM (Nilsson et al., Blood.2003;101:856). Consequently, it is possible that adhesion of hyaluronan on HSPCs to stabilin-2 is a recognition mechanism in BM SECs, directing circulating HSPCs into BM. To investigate this, we studied adhesion of mouse BM lin-Sca-1+Kit+ (LSK) HSPCs to stabilin-1 or stabilin-2 transfected human embryonic kidney cells (HEK 293 cells). We found increased adhesion of LSK cells to stabilin-2 expressing cells, as compared to stabilin-1 expressing or non-transfected cells. Notably, hyaluronidase treatment abolished the increased adhesion of the LSK cell to stabilin-2 transfected cells. These findings indicate that stabilin-2 mediates adhesion of HSPCs to bone marrow SECs. In conclusion, this study shows a novel function for BM SECs as a scavenging endothelium expressing stabilin-1 and stabilin-2. The specific function of stabilins in recognition and endocytosis of extracellular matrix molecules, including hyaluronan and SPARC (Kzhyshkowska J et al., J Cell Mol Med.2006;10:635), suggests that these receptors are involved in tissue remodelling and cell trafficking in BM. Importantly, hyaluronan-mediated binding of HSPCs to SEC stabilin-2 may be a specific recognition mechanism for HSPCs in BM sinusoids. Similarly, binding of tumour cell-associated hyaluronan by stabilin-2 might cause tumour cells in blood to halt in bone marrow, thereby increasing the likelihood for metastasis at this site.
GATA-2 Controls Proliferation of Megakaryocyte Progenitors and Its Expression Contributes to Human DS-AMKL.
Authors: Huang Zan , Crispino John
Published: April 2020

GATA2 is an essential transcription factor that regulates multiple aspects of hematopoiesis. Mutations in GATA2 are associated with blast crisis phase of chronic myelogenous leukemia (CML) while overexpression of GATA2 is a hallmark of Down syndrome acute megakaryoblastic leukemia (DS-AMKL), a malignancy that is defined by the combination of trisomic chromosome 21 and a GATA1 mutation. Here, we show thatGATA2 is essential for megakaryocyte development from both wild-type and GATA-1mutant hematopoietic progenitors. Furthermore, we reveal that GATA2 is indispensable for cell cycle progression and that its expression cooperates with activated JAK3 to promote TPO-independent expansion of megakaryocytes. Genome-wide microarray analysis revealed that GATA2 regulates a wide set of genes including cell cycle regulators and lineage-specific genes that are essential for terminal differentiation of megakaryocytes. Of note, a subset of genes within the GATA2 signature is contained within human DS-AMKL patient specimens. These observations implicate overexpression of GATA2 in the aberrant gene expression and proliferation of AMKL. Taken together, our data demonstrate thatGATA2 is a critical regulator of normal and malignant megakaryopoiesis.
Hedgehog Signaling Is Dispensable for Adult Murine Hematopoiesis and Leukemogenesis.
Authors: Zhang Inga Hofmann , Stover Elizabeth H. , Cullen Dana E. , Mao Junhao , Morgan Kelly J. , Lee Benjamin H. , Kharas Michael G. , Miller Peter G. , Okabe Rachel , Armstrong Scott A. , Ghilardi Nico , Gould Stephen , deSauvage Fred J. , McMahon Andrew P. , Gilliland D. Gary
Published: April 2020

Hedgehog (Hh) pathway proteins are a highly conserved family of intracellular signaling molecules that are critical for the development of multiple organs and tissues, and play a role in cell fate determination of self-renewing tissues in the adult. Mutations that impair Hh signaling have been associated with developmental abnormalities, and recent studies indicate that Hh plays an important role in hemangioblast formation and in adult hematopoiesis, as well as in the differentiation and proliferation of hematopoietic stem cells (HSC) and progenitor cells. We used a genetic and pharmacologic approach to define the role of the Hh pathway in adult hematopoiesis and leukemogenesis. We report the unexpected finding that loss of Hh signaling through conditional deletion of Smoothened (Smo) in the adult hematopoietic compartment has no effect on adult hematopoiesis, including peripheral blood count, number or cell cycle status of stem and progenitor cells, hematopoietic colony forming potential, long-term repopulating activity in competitive repopulation assays, or stress-response to serial 5-fluorouracil treatment. In support of these observations based on genetic inactivation of the pathway, we observed that pharmacologic inhibition of Hh signaling with a potent and highly selective small molecule antagonist of Smo has no apparent effect on hematopoiesis in the mouse in vivo. In addition, we observed that Hh signaling is not required for the development of MLL-AF9 mediated leukemia. Taken together, these data indicate that Hh signaling is dispensable for normal hematopoietic development and leukemogenesis, and that pharmacologic inhibition of Hh signaling, as a therapeutic strategy in treatment of solid tumors with constitutive Hh pathway activation is not likely to be associated with unmanageable hematopoietic toxicity.
The Ski oncoprotein regulates Hematopoietic Stem Cell Fitness and Functions as a Corepressor for Gfi1.
Authors: Velu Chinavenmeni S. , Berk Michael , Xu Haiming , Bourdeau Tristan , Kazanjian Avedis , Williams David A. , Colmenares Clemencia , Grimes H. Leighton
Published: April 2020

Ski is a corepressor protein originally identified as a retrovirally transduced oncoprotein. Genetic deletion of Ski has revealed essential roles in multiple developmental processes. Suggestion that Ski may play a role in hematopoiesis first came from expression of v-Ski and c-Kit, which induced the continuous in vitro growth of primary avian multipotent progenitors. However, the hematopoietic phenotype of Ski−/− mice has not been described. Here, we show that Ski loss of function results in loss of hematopoietic stem cell (HSC) fitness and abnormal regulation of myeloid progenitor numbers. Fetal liver Ski−/− HSC engraft well in ablated recipients, but are not competitive in engraftment. Moreover, Ski null embryonic stem cells generate many tissues in chimeras, but infrequently participate in hematopoiesis. Thus, Ski null HSC are not competitive in both transplant and chimera settings, indicating a defect in stem cell fitness. Engrafted Ski−/− fetal liver cells generate fewer myeloid lineage cells than wild type littermates, and accumulate granulocytemonocyte progenitors. Growth factor independent -1 (Gfi1) is a transcriptional repressor that controls HSC maintenance and myeloid progenitor differentiation. Gfi1−/− and Ski−/− hematopoietic stem and myeloid progenitor phenotypes are strikingly similar. We find that Ski functions as a corepressor for Gfi1. Both endogenous and synthetic Gfi1 and Ski physically interact in vitro and upon Gfi1 target genes. Knockdown of Gfi1 or Ski results in derepression of these targets. Thus, our results provide a molecular link between the similar HSC and myeloid progenitor phenotypes engendered by Gfi1 or Ski deletion.
GATA-2 Regulates Granulocyte-Macrophage Progenitor Cell Function.
Authors: Rodrigues Neil P , Boyd Ashleigh S , Fugazza Cristina , May Gillian E , Guo YanPing , Tipping Alex J , Scadden David T. , Vyas Paresh , Enver Tariq
Published: April 2020

The zinc finger transcription factor GATA-2 has been implicated in the regulation of hematopoietic stem cells. Herein we explored the role of GATA-2 as a candidate regulator of the hematopoietic progenitor cell compartment. We showed that bone marrow from GATA-2 heterozygote (GATA-2+/-) mice displayed attenuated granulocyte-macrophage progenitor function in colony-forming cell (CFC) and serial replating CFC assays. This defect was mapped to the Lin−CD117+Sca-1−CD34+CD16/32high granulocyte-macrophage progenitor (GMP) compartment of GATA-2+/− marrow, which was reduced in size and functionally impaired in CFC assays and competitive transplantation. Similar functional impairments were obtained using a RNA interference approach to stably knockdown GATA-2 in wild-type GMP. While apoptosis and cell cycle distribution remained unperturbed in GATA-2+/− GMP, quiescent cells from GATA-2+/− GMP exhibited altered functionality. Gene expression analysis revealed attenuated expression of HES-1 mRNA in GATA-2 deficient GMPs. Binding of GATA-2 to the HES-1 locus was detected in the myeloid progenitor cell line 32Dcl3 and enforced expression of HES-1 expression in GATA-2+/− GMP rectified the functional defect, suggesting that GATA-2 regulates myeloid progenitor function through HES-1. These data collectively point to GATA-2 as novel, pivotal determinant of GMP cell fate.
Prospective Evaluation of the International Prognostic Score (IPS) in All Stages of Hodgkin’s Lymphoma Treated with ABVD Plus Involved-Field Radiotherapy (IFRT).
Authors: Pavlovsky Santiago , Corrado Claudia , Pavlovsky Miguel A , Prates Virginia , Zoppegno Lucia , Giunta Mario , Cerutti Ider , Palomino Elsa , Avila Graciela , Lastiri Francisco
Published: April 2020

Background: The prognostic score for Hodgkin’s lymphoma was defined as the number of adverse prognostic factors presented at diagnosis. Seven factors had similar independent prognostic effects. This model was validated retrospectively in advanced disease using different therapeutic approaches (D Hasenclever et al N Eng J Med339:1506–14, 1998).

Methods: From December 1996 up to October 2005, the GATLA completed a risk-adapted therapy with ABVD and IFRT. Patients with stages I-IIIA without bulky disease, who achieved complete remission (CR) after three cycles of ABVD, favorable group (FG) received only IFRT 25 GY to areas of >2 cm at diagnosis. Patients with FG not in CR after three cycles of ABVD, slow responders (FGSR), all stages IIIB-IV and all bulky disease, unfavorable group (UG) received six cycles of ABVD and IFRT 30 GY at remaining areas after 3 cycles of ABVD. A total of 584 patients, completed therapy; of them 513 were evaluated with the IPS. Patients were divided in three groups according to the number of adverse prognostic factors 0–1, 2–3, and ≥ 4.

Results: The number of patients, complete remission (CR) rate, event-free survival (EFS) and overall survival (OSV) at 5 years according to prognostic factors in the 513 patients were as follows:

IPS # patients (%) # CR (%) % EFS % OSV 0–1 224 (44) 217 (97) 86 95 2–3 241 (47) 213 (88) 73 90 ≥4 48 (9) 40 (83) 65 72 P< 0.020 0.001 0.001

A total of 200 patients with FG had a 5 years EFS and OSV of 89% and 98% while 53 patients with FGSR had an EFS and OSV of 66% and 88% respectively (P<0.001). The IPS in FG and FGSR was 0–1 of 61% versus 49%, 2–3 of 38.5% versus 43% and ≥4 of 0.5% versus 8% respectively (p=0.003). In UG with an EFS and OSV of 72% and 87%, the incidence of IPS 0–1 was 29%, 2–3 was 54% and ≥4 was 17%.

Conclusion: The IPS is an excellent tool to predict outcome. Patients with stages I-IIIA without bulky tumour who did not achieve CR after three cycles of ABVD (FGSR) had poorer IPS than FG. In spite of receiving six cycles of ABVD, those with FGSR instead of three of those with FG had statistically a poor outcome. In the PET-TC era, patients who remain positive after three cycles of ABVD will need an intensified therapy with the purpose of improving the bad prognosis.
A High Throughput Experimental Approach to Identify miRNA Target Genes in Hodgkin Lymphoma.
Authors: Tan Lu Ping , Seinen Erwin , Duns Gerben , Jong Debora , Schakel Rikst Nynke , Harms Geert , Sibon Ody , Kok Klaas , Poppema Sibrand , Kroesen Bart-Jan , van den Berg Anke
Published: April 2020

MiRNAs are small regulatory RNAs which control gene expression at the post-transcriptional level. Tumor cells of Hodgkin lymphoma (HL), the so-called Hodgkin Reed-Sternberg (HRS) cells, originate from defective germinal center B cells. HRS cells are characterized by their loss of B cell phenotype, large multi-nucleated cell size, a disturbed cell cycle and resistance to undergo apoptosis. The aim of our study is to investigate the role of deregulated miRNA expression to the pathogenesis of HL. To identify genes which are regulated by miRNAs in a high throughput manner, an approach called Ribonucleoprotein ImmunoPrecipitation – microarray (RIP-CHIP) was applied. As a first step, the AGO2 protein, which is part of the RISC complex, is immunoprecipitated (IP) with an AGO2 specific antibody. RNA isolation of the IP fraction followed by microarray analysis thus leads to the identification of miRNA targets from the whole transcriptome. With this approach, 1255 genes were found to be commonly regulated by miRNAs in both L1236 and L428 HL cell lines. Genes known to be absent or down regulated in HRS cells, like CDKN1A, CDKN1B, and PRDM1, were included in this gene list. Gene ontology analysis of these candidate miRNA target genes using Database for Annotation, Visualization and Integrated Discovery (DAVID) revealed a significant enrichment in KEGG pathways termed p53 signaling pathway and cell cycle. TargetScan prediction and seed sequence search at the 3′UTRs of these genes both indicated that about half of these genes were targets of the highly abundant miRNAs found in Hodgkin lymphoma cell lines. 10 randomly selected target genes were analyzed using luciferase reporter assays. For all genes, targeting by the predicted miRNA was confirmed using specific antisense Locked Nucleic Acids (LNA) inhibitors. In conclusion, we demonstrated an approach for large scale identification of endogenous miRNA targets without prior manipulation of the cells. HL specific target genes that may contribute to the characteristic phenotype of HRS cells and to the malignant transformation of germinal center B cells were identified.
Targeting of alpha4beta1 Integrin and CD4/CD8 on Human T Cells, in Addition to CD3 and CD28, Induces Maximal Activation, as Assessed by Single-Cell Analysis of MAP Kinase Pathway Activation and Cytokine Production.
Authors: Kim Tae Kon , Billard Matthew J. , Wieder Eric D. , McIntyre Bradley W. , Komanduri Krishna V.
Published: April 2020

Optimal T cell activation requires the formation of immunological synapses between T cells and antigen-presenting cells (APC). Immunological synapse formation involves the binding of ligands on APC to co-stimulatory receptors on T cells, resulting in maximal activation. Most prior in vitro studies examining the process of T cell activation have utilized immobilized mAbs or ligands to assess the roles of co-stimulatory molecules in T cell activation. Micro-domain formation in such models may be inflexible, potentially yielding misleading results about the relative importance of individual costimulatory pathways. To more physiologically mimic immunologic synapse formation, we stimulated T cells with soluble primary mAbs that were then cross-linked by secondary antibodies. Specifically, we induced cross-linking of surface proteins on purified human T cells using a combination of mAbs recognizing four potential co-stimulatory receptors (CD3, CD4 or CD8, CD28 and VLA4/a4b1 integrin), and assessed phospho-Erk1/2 and cytokine production at a single cell level using flow cytometry. Consistent with the predictions of the Bretscher-Cohn two-signal model, CD28 induced a modest increase in Erk1/2 phosphorylation (in CD4) and IL-2 production (in CD4+ and CD8+ T cells) and IFNg (in CD8+ T cells), while low dose (0.1mcg/ml) anti-CD3 mAb alone failed to induce significant activation (P<0.05). However, CD28 stimulation consistently augmented activation in both CD4+ and CD8+ T cells in the presence of anti-CD4 and anti-CD8 mAbs (P<0.05). More interestingly, stimulation by CD4 or CD8 mAbs induced an additive effect on Erk1/2 phosphorylation and IL-2 and IFNg production regardless of CD28 stimulation (P<0.05). In addition, stimulation via a4b1/VLA4 increased the level of T cell activation in the presence of three distinct signals in both CD4+ T cells and also in CD8+ T cells (P<0.05). We then examined the dependence of T cell activation on all four types of stimuli (i.e., via CD3, CD28, CD4 or CD8, and VLA4). Following stimulation with low-dose anti-CD3 mAb (0.1mcg/ml), the extent of Erk1/2 phosphorylation and IL-2/IFNg production was found to be dependent on the numbers of stimulatory signals received through all four pathways in a statistically meaningful manner (see Figure). In conclusion, optimal T cell activation requires not only CD3 and CD28, but also signaling via CD4/CD8 and VLA4, supporting a “four-signal” model of T cell activation. These data suggest that

additional co-stimulations through a4b1/VLA4 and CD4 or CD8 may be necessary to induce optimal T cell signaling, targeting of a4b1 and CD4/CD8 by specific agonists or antagonists may have therapeutic benefit in immunomodulation (e.g., augmentation of vaccine responses and/or abrogation of pathogenic T cell activation).

Figure Figure
Telomere Elongation in B-Cells Is Independent of Class Switching.
Authors: Roeth Alexander , de Beer Dirk , Seifert Marc , Mueller Astrid , Duehrsen Ulrich , Kueppers Ralf , Baerlocher Gabriela M.
Published: April 2020

BACKGROUND: In contrast to human granulocytes and T-cells with a quite homogenous decrease in telomere lengths over age, telomere lengths of peripheral blood B-cell populations are highly heterogeneous with average telomere lengths of B-cells remaining relatively constant from middle age onward (Baerlocher et al, J. Leuk. Biol. 2003). So far, telomere elongation has been described in highly proliferating germinal center B-cells during the development from naive B-cells to memory B-cells (Norrback et al, Eur J Haematol 2001). During this same time period the process of class-switch recombination (CSR) occurs. Our aims were to analyze the telomere lengths in different B-cell subsets in order to elucidate telomere length dynamics in relationship to CSR.

PATIENTS and METHODS: Buffy coats from 14 healthy donors were enriched for CD19+ B cells by magnetic cell separation. Naive B cells (IgM+/IgD+/CD27−), IgM-only B cells (IgM+/IgD−/low/CD27+), double-positive B cells (IgM+/IgD+/CD27+) and class-switched memory B cells (IgM−/IgD−/CD27+) were further separated by cell sorting. Telomere length of sorted B-cell subpopulations was measured by automated multicolour flow-FISH.

RESULTS: Naive B-cells presented the shortest telomere length values (average telomere length, n=14: 7.2 kb ± 0.7 kb) compared to the other B-cell subsets. In comparison to the naive B-cell subset, the IgM only B-cell subset had 13% longer telomeres (average telomere length, n=14: 8.1 kb ± 1.3 kb), the double-positive B-cell subset had 10% longer telomeres (average telomere length, n=14: 7.9 kb ± 0.8 kb) and the memory B-cell subset had 22% longer telomeres (average telomere length, n=14: 8.7 kb ± 1.1 kb) (p < 0.0001).

CONCLUSIONS: We are able to confirm longer telomeres in memory B cells than in naive B cells. For the first time, however, we can demonstrate that longer telomeres are also found in non-class switched B-cells. Based on these results telomere elongation does not coincide with the process of CSR. Additional studies are needed to assess whether telomere elongation can only take place in the germinal center or whether certain B-cell subsets are able to elongate their telomeres independent from the germinal center.
Characterization of MDX-1401, a Human Anti-CD30 Antibody with Enhanced Effector Function, for Therapy of Malignant Lymphoma.
Authors: Cardarelli Pina M. , Loomis Cristina , Preston Ben , Black Amelia , Passmore David , Chen Tseng-Hui , Chen Sharline , Liu Jie , Kuhne Michelle R , Srinivasan Mohan , Assad Albert , Witte Alison , Coccia Marco , Graziano Robert F. , King David J.
Published: April 2020

Therapeutic monoclonal antibodies kill target cells by multiple mechanisms including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), complement-dependent cytotoxicity (CDC), and direct induction of apoptosis. Antibody binding to FcgRIIIA (CD16), which is expressed on NK cells, macrophages, and CD8+ cells, is essential for ADCC. MDX-1401 is a fully human monoclonal antibody of IgG1 (kappa) isotype that binds to human CD30. This second generation anti-CD30 antibody was derived from parental antibody MDX-060 but differs in that the oligosaccharides of the IgG1 lack fucose, a property that conveys increased antibody affinity for Fc receptor CD16 (FcgRIIIa) and thus stimulates both more potent and efficacious ADCC than a fucosylated IgG1. The KD of the parental antibody for CD30 expressed on L540 Hodgkin’s lymphoma cells is approximately 1.4 nM while the KD of MDX-1401 is 1.9 nM, demonstrating that binding affinity for antigen are nearly identical. However, the EC50 for MDX-1401 binding to CD16-transfected cells is approximately 3 nM compared to barely detectable binding by parental antibody, confirming that the absence of fucose conveys increased affinity for FcgRIIIa. Data is presented that demonstrates MDX-1401 has significantly improved potency and efficacy in an ADCC assay as evidenced by a decrease in EC50 as well as an increase in maximum specific lysis. Importantly, the enhanced ADCC activity is observed among a large panel of cell lines with varying numbers of CD30 receptors per cell, including one cell line with low antigen expression in which parental antibody failed to induce ADCC. MDX-1401 enhances ADCC activity with all polymorphic variants including lower affinity FcgRIIIa Phe/Phe158 and FcgRIIIa Phe/Val158 effector cells. Furthermore, MDX-1401 is efficacious in inhibiting growth of CD30+ tumor xenografts in mice. The low doses of antibody required for ADCC activity irrespective of donor genotype, the capacity to mediate ADCC of target cells expressing low levels of CD30, and increased in vivo efficacy support the development of MDX-1401 for treatment of malignant lymphoma. Phase 1 studies in subjects with Hodgkin’s lymphoma are currently underway.
Bortezomib-Doxorubicin-Dexamethasone as Induction Prior to Reduced Intensity Autologous Transplantation Followed by Lenalidomide as Consolidation/Maintenance in Elderly Untreated Myeloma Patients
Authors: Palumbo Antonio , Falco Patrizia , Gay Francesca , Montefusco Vittorio , Crippa Claudia , Patriarca Francesca , Gilestro Milena , Liberati Anna M. , Rossini Fausto , Caravita Tommaso , Falcone Antonietta , Cavallo Federica , Corradini Paolo , Rossi Giuseppe , Boccadoro Mario
Published: April 2020

Background: New agents have been introduced as induction prior to autologous stem cell transplant (ASCT) and as consolidation/maintenance thereafter to improve complete response (CR) rates. In this trial we evaluate Bortezomib plus Pegylated-lyposomal-doxorubicin and Dexamethasone (PAD) as induction therapy prior to reduced intensity ASCT, followed by consolidation with Lenalidomide and Prednisone (LP) and maintenance with Lenalidomide alone (L).

Methods: Newly diagnosed multiple myeloma (MM) patients aged 65–75 years were eligible. Induction regimen consisted of 4 21-day PAD cycles (Bortezomib 1.3 mg/m2 days 1, 4, 8, 11, Pegylated-lyposomal-doxorubicin 30 mg/m2 day 4 and Dexamethasone 40 mg days 1–4, 8–11, 15–18). Two cycles of Cyclophosphamide 3 g/m2 plus Granulocyte-Colony Stimulating Factor were used to harvest stem cells. Patients were conditioned with tandem Melphalan 100 mg/m2 (MEL100) followed by stem cell support. After ASCT patients received consolidation with 4 28-day LP cycles (Lenalidomide 25 mg days 1–21 plus, Prednisone 50 mg every other day) followed by Lenalidomide alone maintenance (10 mg days 1–21 every 28 day). Primary objectives were safety (grade 3 non-hematologic toxicity < 30%) and efficacy (near CR rate > 35%).

Results: One-hundred and two patients have been enrolled. After PAD cycles at least partial response (PR) rate was 94%, at least very good partial response (VGPR) was 59% including 13% CR. After tandem MEL100, 88% of patients achieved at least VGPR and 41% CR. After LP consolidation all patients obtained PR, 88% at least VGPR and 53% immunofixation negative CR. After a median follow-up of 14 months, 1-year progression free survival (PFS) was 92%, 1 year time to progression was 97% and 1 year overall survival was 92%. PFS was not significantly affected β2-microglobulin levels (p=0.10), presence of chromosome 13 deletion (p=0.5) or t(4;14) (p=0.61).

During PAD, grade 3–4 adverse events included thrombocitopenia (13%), neutropenia (11%), infections (18%), gastrointestinal toxicities (12%), peripheral neuropathy (11%) and deep vein thrombosis (6%). During LP consolidation, grade 3–4 toxicities included neutropenia (18%), thrombocytopenia (6%), infections (6%) and deep vein thrombosis (6%). The other grade 3–4 toxicities occurred in less than 5% of patients.

Conclusions: Bortezomib as induction regimen prior to reduced intensity ASCT, followed by Lenalidomide as consolidation maintenance is a highly effective regimen in elderly patients. Updated results will be presented at the meeting.
Molecular Mechanism by Which Rituximab Sensitizes B-NHL Cells to TRAIL Apoptosis: Induction of RKIP and DR5 Expression Via Inhibition of NF-Kb, Snail, and YY1.
Authors: Vega Mario I. , Martinez-Paniagua Melisa , Huerta-Yepez Sara , Kam Yeung , Baritaki Stavroula , Bonavida Benjamin
Published: April 2020

There have been significant advances in the treatment of patients with B-NHL using combination of rituximab and CHOP. However, a subset of patients does not initially respond or develop resistance to further treatments; hence, the need for alternative therapies to overcome resistance. TRAIL and agonist DR4/DR5 monoclonal antibodies have been examined clinically against a variety of tumors in Phase I/II. However, the majority of B-NHL derived from patients and cell lines are resistant to TRAIL-induced apoptosis. Recent findings demonstrated that treatment of TRAIL-resistant-B-NHL with rituximab sensitizes the tumor cells to TRAIL apoptosis. The underlying mechanism of rituximab-induced sensitization to TRAIL, however, is not clear. We have recently reported that treatment of tumor cells with sensitizing agents (example CDDP, proteasome inhibitors) resulted in the reversal of resistance to TRAIL via induction of Raf-1 kinase inhibitor protein (RKIP) and demonstrated the pivotal role of RKIP in the regulation of tumor cell sensitivity to TRAIL. Hence, since rituximab induces the expression of RKIP in B-NHL, we determined the role of RKIP induction by rituximab in the sensitization of B-NHL to TRAIL apoptosis. Various B-NHL cell lines were used as models for study. Treatment of B-NHL cells with rituximab (20 ng/ml) and TRAIL (5–10 ng/ml) resulted in significant potentiation of apoptosis and synergy was achieved. Rituximab induced the expression of RKIP as determined by RT-PCR and western concomitantly with inhibition of NF-kB. The inhibition of NF-kB resulted in upregulation of RKIP expression and was mediated, in large part, by inhibition of the transcription repressor Snail (downstream of NF-kB). Further, RKIP-induced inhibition of NF-kB by rituximab resulted in downstream inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) and concomitantly with the upregulation of DR5 expression. The role of RKIP induction by rituximab in the upregulation of DR5 and sensitization to TRAIL apoptosis was corroborated by the use of cells over expressing RKIP which were sensitive to TRAIL apoptosis in the absence of rituximab. Our findings reveal a novel mechanism of rituximab-induced sensitization of B-NHL to TRAIL apoptosis via inhibition of NF-kB and Snail and upregulation of RKIP and DR-5. The combination of rituximab and TRAIL may be effective in the treatment of B-NHL. Further, our studies suggest that agents other than rituximab that can induce RKIP can reverse resistance to TRAIL in B-NHL that are unresponsive to rituximab treatment.
DNA Repair Gene, XRCC1 Polymorphisms Are Associated with Chemotherapy Induced Toxicity in Patients with Diffuse Large B Cell Lymphoma Who Received Frontline R-CHOP Chemotherapy.
Authors: Kim In-Suk , Lee Gyeong-Won , Kim Hoon-Gu , Kong Sun-Young , Eom HyeonSeok
Published: April 2020

BACKGROUND. X-ray repair cross-complementing group I (XRCC1) is a DNA repair gene. Polymorphisms in DNA repair genes may alter protein function and therefore the efficacy of DNA damaging chemotherapy. We retrospectively evaluated the association of three polymorphisms in XRCC1 codon 194 (Arg to Trp), 280 (Arg to His), and 399 (Arg to Gln), with grade 3 or 4 toxicities of primary rituximab plus cyclophosphamide/doxorubicin/vincristine/prednisone (R-CHOP) therapy in 145 patients with diffuse large B cell lymphoma (DLBCL).

METHODS. A total of 145 patients who received R-CHOP chemotherapy as a frontline regimen DLBCL were included in this retrospective study from 3 hospitals in Korea. The genotypes of XRCC1 at the Arg194Trp, Arg280His, and Arg399Gln were determined by direct sequencing methods. The clinical characteristics, treatment outcomes and grade 3 and 4 toxicities of the patients were compared using Chi-square, Fisher exact, Mann-Whitney U tests, or logistic regression according to the XRCC1 polymorphisms.

RESULTS. Carrying at least one variant XRCC1 Arg194Trp alleles (194Arg/Trp or 194Trp/Trp) and XRCC1 Arg399Gln variant alleles (399Arg/Gln or 399Gln/Gln) was associated with a significantly increased risk of grade 3 or 4 gastrointestinal toxicity (Arg194Trp alleles; odds ratio, 3.489; 95% confidence interval, 0.126–1.568; P=0.001, Arg399Gln alleles; odds ratio, 2.577; 95% confidence interval, 0.144–1.096; P=0.011, respectively). The carriers of XRCC1 Arg280His variant alleles (280Arg/His or 280His/His) was associated with grade 3 or 4 neutropenia (odds ratio, 2.047; 95% confidence interval, 0.008–0.450; P=0.043). No differences in the patient characteristics, disease characteristics, response, and survival in patients with DLBCL who received frontline R-CHOP chemotherapy were observed according to three XRCC1 polymorphims.

CONCLUSIONS. This study demonstrates that the patients carrying at least one variant XRCC1 Arg194Trp or XRCC1 Arg399Gln alleles have a 2.5-to 3.5-fold increased risk of grade 3 or 4 gastrointestinal toxicity and the patients carrying at least one variant XRCC1 Arg280His allele have a 2.0-fold increased risk of grade 3 or 4 neutropenia when treated with frontline R-CHOP chemotherapy and this has implications for optimizing treatment with such agents.
Bortezomib Overcomes Cell Adhesion-Mediated Drug Resistance Via Down-Regulation of VLA-4 Expression in Multiple Myeloma.
Authors: Hatano Kaoru , Kikuchi Jiro , Takatoku Masaaki , Shimizu Rumi , Wada Taeko , Ueda Masuzu , Nobuyoshi Masaharu , Oh Iekuni , Sato Kazuya , Suzuki Takahiro , Ozaki Katsutoshi , Mori Masaki , Nagai Tadashi , Muroi Kazuo , Kano Yasuhiko , Furukawa Yusuke , Ozawa Keiya
Published: April 2020

Multiple myeloma (MM) is incurable, mainly because of cell adhesion-mediated drug resistance (CAM-DR). In this study, we performed functional screening using shRNA to define the molecule(s) responsible for CAM-DR of MM. Using 4 bona fide myeloma cell lines (KHM-1B, KMS12-BM, RPMI8226, and U266) and primary myeloma cells, we identified CD29 (b1-integrin), CD44, CD49d (a4-integrin, a subunit of VLA-4), CD54 (ICAM-1), CD138 (syndecan-1) and CD184 (CXCR4) as major adhesion molecules expressed on MM. Short hairpin RNA-mediated knockdown of CD49d but not CD44, CD54, CD138, and CD184 significantly reversed CAM-DR of myeloma cells to bortezomib, vincristine, doxorubicin, and dexamethasone. Experiments using blocking antibodies yielded almost identical results. Bortezomib was relatively resistant to CAM-DR because of its ability to specifically down-regulate CD49d expression. This property was unique to bortezomib and was not observed in other anti-myeloma drugs. Pretreatment with bortezomib was able to ameliorate CAM-DR of myeloma cells to vincristine and dexamethasone. These results suggest that VLA-4 plays a critical role in CAM-DR of MM cells. The combination of bortezomib with conventional anti-myeloma drugs may be effective in overcoming CAM-DR of MM.
Obesity Alters the Pharmacokinetics of Vincristine in Mice.
Authors: Behan James W , Avramis Vassilios I , Yun Jason P , Butturini Anna , Mittelman Steven D
Published: April 2020

Obesity has been associated with increased risk of development and poor outcome from a variety of hematological malignancies. There is a lack of consensus as to how to dose chemotherapies in obese patients, and therefore inadequate dosing represents a potential factor contributing to poor outcome. We have previously shown that diet-induced obese mice transplanted with p190BCR/ABL ALL cells and then treated with vincristine (VCR) proportional to body weight (15 mg/kg/wk × 4 wks) have a poorer outcome than non-obese mice (event free survival: Obese = 0.42, Control = 0.75, p=0.07, log rank). The present study was designed to quantify the pharmacokinetic (PK) parameters of vincristine in control and obese mice and determine whether differences in these parameters could explain the poorer outcome in the obese mice. Twenty obese and 20 control mice received tail-vein injections of tritiated vincristine (specific activity 75 mCi/mg; dose 0.5 mg/kg). Blood samples were taken at 5 minutes, and the mice sacrificed at various time-points between 15 minutes and 24 hours post-injection. Whole blood and tissue specimens were solubilized, decolorized, and read on a scintillation counter. Blood vincristine concentrations were fit to a 3-compartment model using the two-stage PK in 3 subsets of VCR concentrations vs. time method. Non-compartmental modeling was used to confirm the results. Due to body weight differences, obese mice received ~ 28% more vincristine per injection than controls. Blood vincristine concentrations in both groups of animals followed a triexponential decay, as has been previously described. Blood levels tended to be higher (by 20±15%) in the obese mice at all time-points tested. While the t1/2α was longer in the obese mice (10.6 vs. 6.0 minutes), the t1/2b and t1/2γ were both shorter (22.7 vs. 27.1 minutes and 114.4 vs. 345.8 hours). Therefore the AUC0 → 24 was higher in the obese mice (3,279 vs. 2,776 ng/ml*hr), while the AUC0 → ∞ was lower in the obese mice (21,705 vs. 53,581 ng/ml*hr). Vincristine concentrations in spleen and bone marrow were similar at all time-points in the obese and control groups. In summary, obesity caused alterations in vincristine pharmacokinetics, characterized by longer initial half-life but shorter terminal half-lives. Thus, it is possible that the decreased AUC0 → ∞ contributed to the poorer outcome in this model. Despite this, blood and tissue concentrations of vincristine were equal or higher in the obese mice during the first 24 hours after injection of the drug when it was dosed proportional to body weight. These data suggest that dosing vincristine in obese patients based on body surface may lead to lower levels in blood and tissue, particularly if the dose is capped. As the prevalence of obesity continues to increase, pharmacokinetics studies should be performed in obese and lean subjects to further optimize chemotherapy dosing regimens in obese patients.