bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of Natural Killer Cell Interferon-Gamma Production By the Derivatives of the Natural Product Phyllanthusmins Via TLR-Mediated NF-Kb and STAT3 Signaling Pathways
Authors: Chen Luxi , Yi Long , Ren Yuning , Zhang Jianying , Kinghorn A. Douglas , Caligiuri Michael A. , Yu Jianhua
Published: October 2019

Natural killer (NK) cells are known to play a pivotal role in regulating immune defense against tumors and viruses. However, very few studies describe the effects of natural products or their derivatives on NK cell function. We previously showed that the natural product lignan phyllanthusmin C enhances human NK cell IFN-gamma production. In this study, we successfully synthesized dozens of phyllanthusmin derivatives and screened their activities to enhance IFN-gamma production by NK cells. We found that three of these derivatives possessed capacities to induce IFN-gamma production by NK cells. Among them, derivative No. 17 had the highest efficacy and was significantly superior (P < 0.05) to that of the original phyllanthusmin C. These three phyllanthusmin derivatives had no adverse effect on human NK cell survival or proliferation. All derivatives alone or in combination with IL-12 or IL-15 induced human NK cell interferon (IFN)-gamma in comparison with the corresponding vehicle control or cytokine alone (P < 0.05). These derivatives stimulated IFN-gamma production in both CD56bright and CD56dim human NK cell subsets (P < 0.01). Mechanistically, immunoblotting assays and chromatin immunoprecipitation (ChIP) analyses combined with promoter-reporter luciferase assays revealed that these phyllanthusmin derivatives induced the phosphorylation of NF-κB and STAT3, resulting in their increased binding on theIFN G promoter, which was dependent on the Toll-like receptor (TLR) 1 and the TLR3 signaling pathways, respectively. STAT3 knockdown with lentivirus shRNA and inhibition of NF-kB signaling with a specific inhibitor (TPCK) significantly attenuated IFN-gamma production induced by these phyllanthusmin derivatives in human NK cells (P < 0.05). Blockade of TLR1 or TLR3 with their corresponding neutralizing antibodies nearly abolished activation of NF-κB or STAT3 as well as IFN-γ induction by the phyllanthusmin derivatives (P < 0.05). In conclusion, we have successfully synthesized and screened phyllanthusmin derivatives and identified three compounds that induce IFN-gamma production in human NK cells, one of which had optimal potential. The induction of human NK cell IFN-gamma production by phyllanthusmin derivatives is mediated by TLR1-NF-κB and TLR3-STAT3 signaling pathways, rendering their potential for use as agents for cancer prevention or treatment.

Disclosures

No relevant conflicts of interest to declare.
Human Chorionic Villus Stem Cells May Contribute to Endogenous Factor VIII Production during Gestation
Authors: Miguelino Maricel G. , Kumar Priyadarsini , Powell Jerry S. , Wang Aijun
Published: October 2019

INTRODUCTION: The placenta is an organ that plays a vital role in providing nutrition, elimination of metabolic products and gas exchange for the growing fetus. Major organs that play these roles in an adult are undergoing development during gestation. The liver, a metabolic organ with a variety of endocrine and exocrine functions including the synthesis of coagulation proteins, is the major site of hematopoiesis at 11-15 weeks during gestation. Production of coagulation proteins during gestation remains unclear. We investigated the potential of very early gestation human chorionic villus mesenchymal stem cells (PMSCs) for Factor VIII (FVIII) mRNA and production of biologically active FVIII.

METHODOLOGY: PMSCs were isolated from human placenta at 9-13 weeks of gestational age by tissue explant culture and characterized via flow cytometry for mesenchymal stem cell (MSC) markers. Tri-lineage differentiation of PMSCs was investigated by inducing PMSCs in different culture conditions. Polymerase chain reaction (RT-PCR) was performed on cell lysates using FVIII primers. Chromogenic assay was performed on culture medium of PMSCs and HUVECs (negative control) after 72 hours in culture. Human plasma was used as positive control.

RESULTS: PMSCs isolated from preterm human placenta were plastic adherent, showed spindle-shaped morphology and demonstrated expression of MSC markers CD105, CD90, CD73, CD44, and CD29, and did not express CD184, HLA-DR or hematopoietic and endothelial markers CD45, CD34 and CD31. Tri-lineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages was observed under different culture conditions. These results show multi-potency and a surface marker profile analogous to bone marrow mesenchymal stem cells (BMSCs). PCR analysis revealed FVIII mRNA expression on PMSCs and was negative on HUVECs. Chromogenic assay showed ~0.4 U/ml on PMSCs and ~0.1 U/ml in HUVECs.

CONCLUSION: Here we report that early gestation PMSCs express and secrete biologically active FVIII. Further studies using different gestational ages of the placenta is needed to understand the potential contribution of chorionic villus to FVIII production during gestation.

Disclosures

Powell: CSL Behring: Employment.
Delayed Major Bleeding in Critically Ill Trauma Patients
Authors: Castellucci Lana A , Shaw Joseph , Le Gal Gregoire , Hawken Stephen , McIntyre Lauralyn , Carrier Marc
Published: October 2019

Introduction: Trauma is the leading cause of death in persons under the age of 40 years and hemorrhage is responsible for at least 40% of trauma related deaths within the first 24 hours following injury, with up to half of patients dying before arriving to hospital. During the last several decades, the focus of research in bleeding trauma patients has been targeted at the immediate post-injury period with little emphasis on the potential for delayed hemorrhage in survivors of the first 24 hours.

Methods: A retrospective review of critically ill trauma patients admitted at a tertiary care center between September 2010 and December 2011 was performed to determine the incidence of delayed major bleeding. Delayed major bleeding was defined as bleeding that occurred more than 24 hours after presentation to the emergency department. The HEME bleeding assessment tool was used to identify delayed major bleeding events. Cox proportional hazards multivariate analysis was performed to assess for risk factors associated with delayed major bleeding events.

Results: One hundred and fifty critically ill trauma patients were included and the incidence of delayed major bleeding was 44%. Predictors that were statistically significantly associated with delayed major bleeding included: male gender (hazard ratio (HR): 2.22; 95% confidence interval (CI):1.15-4.17), pre-injury use of the antiplatelet agents aspirin and/or clopidogrel (HR: 2.11; 95%CI: 1.07-4.18), presence of intracranial bleeding (HR: 3.05; 95%CI: 1.70-5.47), higher injury severity scores (HR: 1.12; 95%CI: 1.03-1.22), requirement of massive transfusion (HR: 2.53; 95%CI: 1.05-6.08), and low pH values (HR: 1.41; 95%CI: 1.08-1.84). Use of anticoagulant prophylaxis was not associated with delayed major bleeding.

Conclusions: The rate of delayed major bleeding in this cohort of critically ill trauma patients is high. Prospective studies are needed to confirm the reported associations of gender, antecedent antiplatelet drugs, intracranial bleeding, injury severity scores, massive transfusion, and acidosis with delayed major bleeding.

Disclosures

Carrier: LEO Pharma: Consultancy, Research Funding; Pfizer: Consultancy; Bayer: Consultancy; BMS: Research Funding.
Id2 Is Required for the Self-Renewal and Proliferation of Hematopoietic Stem Cells
Authors: Sun Lei
Published: October 2019

The production of mammalian blood cells is sustained throughout life by the self-renewal and differentiation of hematopoietic stem cells (HSCs). Dysregulation in this system leads to different pathologies including anemia, bone marrow failure and hematopoietic malignancies. The Helix-Loop-Helix transcriptional regulator Id2 plays essential roles in regulating proliferation and cell fate of hematopoietic progenitors; however, its role in regulating HSC development remains largely unknown. To assess the function of Id2 in HSCs, we developed two mouse models, including an Id2 conditional knockout model and an Id2-EYFP model, in which EYFP expression is driven by endogenous Id2 promoter. When we examined HSC function by serial transplantation, we found that mice transplanted with Id2F/F Mx1-Cre+ conditionally deleted bone marrow cells became moribund more rapidly after primary and secondary transplantation, compared to those transplanted with Id2+/F Mx1-Cre+ bone marrow, suggesting that HSC self-renewal is impaired when Id2 is deleted. To further determine if self-renewal and maintenance of HSCs depends on the expression level of Id2, we purified HSCs with different levels of Id2 expression using Id2-EYFP mice to specifically address the role of Id2 in HSCs. First, we confirmed Id2 is highly expressed in HSCs in this model. Second, when HSCs with either low or high levels of Id2-EYFP were transplanted into irradiated mice, cells with high levels of Id2 reconstituted transplanted recipients faster than those with low levels of Id2 at 3 weeks and longer, suggesting that Id2 expression is associated with repopulation advantage. Furthermore, Ki-67 staining showed that HSCs with high levels of Id2 have 15-fold more cells in G2/M phase, and fewer cells in G0. BrdU staining also suggested that there are 5-fold more BrdU+ cells in HSCs with high levels of Id2, indicating that Id2 expression correlates with cell cycle progression in HSCs. In addition, p57 has been reported to be required for quiescence of HSCs. Our preliminary data showed that p57 is downregulated in HSCs with high levels of Id2, and p57 is correspondingly upregulated in Id2-null HSCs. Altogether, our data demonstrate that Id2 is required for the self-renewal and proliferation of HSCs, and suggest a link between Id2 and the transcriptional regulatory networks that regulate the functional hematopoietic system. Since Id2 is also expressed in other adult stem cells including muscle and neuronal stem cells, as well as cancer cells, we believe our results can improve our understanding of stem cell biology and cancer development, and contribute to the identification of novel molecules that may be targeted to eliminate cancer stem cells.

Disclosures

No relevant conflicts of interest to declare.
Regulation of Hematopoietic Stem Cell Function By the Histone H2A Deubiquitinase Usp16
Authors: Gu Yue , Yang Wei , Jones Amanda , Liu Shanrun , Dai Qian , Swindle C Scott , Ryan Thomas , Townes Tim M. , Klug Christopher , Wang Hengbin
Published: October 2019

Epigenetic mechanism plays important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of Polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it was unclear which H2A deubiquitinase pairs with PRC1 to control H2A ubiquitination (ubH2A) level in vivo and regulates hematopoiesis. Here we investigated the function of Usp16 in mouse hematopoiesis. Deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and mouse lethality. Usp16 deletion was associated with a dramatic reduction of progenitor cell populations and unchanged HSC number, revealing a critical role for Usp16 in HSC differentiation. The HSC differentiation defect was correlated with a great reduction of G1 phase cell population. RNA-seq and RT-qPCR studies revealed that Usp16 regulates the expression of many genes associated with HSC differentiation and self-renewal including cell cycle regulator p21. Significantly, knockdown of p21 largely rescued the undifferentiated phenotype of Usp16 deleted HSCs. Usp16 binds to regions flanking transcription start site and knockdown of PRC1 subunits, which reduced ubH2A levels, largely rescued the altered gene expression pattern associated with Usp16 deletion. Therefore, these studies identified Usp16 as the H2A deubiquitinase that pairs with PRC1 to regulate hematopoiesis in vivo and revealed that Usp16 regulates HSC function by affecting HSC cell cycle progression.

Disclosures

No relevant conflicts of interest to declare.
B-Lymphoid Transcription Factors Restrict Glycolytic Energy Supply for Oncogenic Signaling
Authors: Chan Lai N , Chen Zhengshan , Braas Daniel , Geng Huimin , Hurtz Christian , Shojaee Seyedmehdi , Cazzaniga Valeria , Ng Carina , Ernst Thomas , Hochhaus Andreas , Kornblau Steven M , Cazzaniga Giovanni , Liu Grace , Milne Thomas , Koeffler H. Phillip , Armstrong Scott A. , Dickins Ross Alexander , Yamamoto Keith R , Graeber Thomas G , Muschen Markus
Published: October 2019

Oncogenic lesions in hematopoietic progenitor cells give rise to B-cell or myeloid malignancies. While often transformed by the same oncogenes, B-cell and myeloid leukemias markedly differ in biological and clinical characteristics. Our metabolic analyses revealed that B-cell-unlike myeloid-leukemia cells are massively restricted in their glycolytic capacity. Low glycolytic reserves in B cells resulted in a state of chronic energy depletion and engaged the energy sensor LKB1-AMPK. Myeloid cells strongly activated glucose transport through insulin receptor (INSR)-AKT signaling and lacked activity of LKB1-AMPK, reflecting energy abundance. Conversely, B-cells lacked INSR-AKT signaling and were critically dependent on LKB1-AMPK-mediated glucose uptake. Cre-mediated deletion of Lkb1 caused acute glycolytic exhaustion and cell death in B-lineage but increased glycolysis, energy levels and proliferation in myeloid leukemia. C/EBPa-mediated conversion of B-cell into myeloid identity reversed the detrimental effects of Lkb1-deletion and restored glycolysis, energy levels and survival of B→myeloid reprogrammed cells. In >80% of B-lineage leukemia cases, we found genetic lesions of transcription factors (e.g. deletion of PAX5, IKZF1, rearrangement of MLL) that caused a B→myeloid lineage shift. While previously of unknown functional significance, these lesions relieved B-cell-specific transcriptional repression of molecules that mediate glucose uptake and utilization (INSR, GLUT1, HK2, G6PD) and amplified glycolytic energy supply for transforming oncogenes. Likewise, glucocorticoid receptor (NR3C1)-mediated inhibition of glucose uptake and glycolysis was strictly dependent on a B-lymphoid transcriptional program. B→myeloid lineage conversion abolished NR3C1 expression and activity, which provides a mechanistic explanation for the empiric finding that glucocorticoids are highly active in the treatment of B-cell-but not myeloid malignancies. In conclusion, B-cell-specific restriction of glycolytic energy supply represents a previously unrecognized metabolic barrier against malignant transformation and reveals LKB1-AMPK as a novel target for the treatment of human B-lineage leukemia.

Disclosures

No relevant conflicts of interest to declare.
Incidence of Allergic Reactions to Pegaspargase (PEG) Administered Intramuscularly Versus Intravenously (IM vs. IV) in Children and Young Adults with High Risk B-Lymphoblastic Leukemia (HR B-ALL): Results of Children's Oncology Group (COG) Studies AALL0232/AALL1131
Authors: Salzer Wanda , Burke Michael J. , Larsen Eric C. , Chen Si , Gore Lia , Hilden Joanne M. , Loh Mignon L. , Raetz Elizabeth , Winick Naomi J. , Carroll William L. , Devidas Meenakshi , Hunger Stephen P
Published: October 2019

Introduction: Asparaginase is a vital component of multi-agent chemotherapy for patients with HR B-ALL, but rates of allergic reactions following administration of PEG range from 3-24%. Similar or increased rates of allergic reactions have been reported following IV vs. IM PEG when administered on varying chemotherapy backbones. The COG trials recently changed from IM to IV administration of PEG. We report the rate of allergic reactions/anaphylaxis (Grade 3-4) following IM vs. IV PEG on sequential trials for newly diagnosed patients with HR B-ALL.

Methods: Patients 1-30 years of age with newly diagnosed HR B-ALL were eligible for the sequential COG studies, AALL0232 and AALL1131. We limited this analysis to non-Down Syndrome patients who received highly similar chemotherapy regimens prior to maintenance therapy, including a 4-drug Induction with 14 days of dexamethasone or 28 days of prednisone, the COG augmented Berlin-Frankfurt-Munster (BFM) Consolidation, High Dose Methotrexate during Interim Maintenance, and Delayed Intensification (DI). Patients received a total of 5 doses of PEG (2,500 units/m2/dose) administered IM (AALL0232) or IV (AALL1131) during these phases of therapy: Induction (n=1), Consolidation (n=2), DI (n=2). Chi square analysis of the rates of Grade 3-4 allergic reactions/anaphylaxis (Common Terminology Criteria for Adverse Events v4.0) following IM vs. IV PEG was performed.

Results: Grade 3-4 allergic reactions/anaphylaxis rates in patients receiving IM vs. IV PEG (Table) were 0.54% vs. 0.22% during Induction (p=0.10), 14.4% vs. 12.64% during Consolidation (p=0.18), and 2.1% vs. 0.97% during DI (p=0.07).

Conclusion: Rates of Grade 3-4 allergic reactions/anaphylaxis were similar following IM vs. IV administration of PEG in patients with newly diagnosed HR B-ALL when treated on sequential COG studies, further establishing the safety of delivering IV PEG.

Table 1. Allergic Reactions Following IM vs IV PEG Allergic reaction/Anaphylaxis (Grade 3-4) AALL0232(IM PEG) AALL1131(IV PEG) p-value(Chi square test) Induction 15/2771 (0.54%) 4/1797 (0.22%) 0.10 Consolidation 342/2369 (14.4%) 114/902 (12.64%) 0.18 DI 22/1060 (2.1%) 7/715 (0.97%) 0.07

Disclosures

Hunger: Spectrum Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Sigma Tau: Consultancy; Merck: Equity Ownership.
Whole Exome Sequencing of FLT3-ITD Sorafenib-Resistant Acute Myeloid Leukaemia
Authors: Lam Stephen Sze Yuen , Cher Chae Yin , Ng Nelson K. L. , Man Cheuk Him , Leung Anskar Y.H.
Published: October 2019

Acute myeloid leukaemia (AML) is a heterogeneous disease characterised by an abnormal increase in myeloblasts. Current treatment protocol employs the standard "7+3" chemotherapy regimen complemented with haematopoietic stem cell transplantation. However overall cure rate remains at 30-40%. Targeted therapy, especially the use of tyrosine kinase inhibitors, has been the main focus in the pursuit of an alternative solution. Internal tandem duplication at the juxatmembrane domain of fms-like tyrosine kinase 3 (FLT3-ITD) was found in around 30% of AML cases, conferring unfavourable treatment outcome and prognosis. Sorafenib, and other FLT3 inhibitors, successfully induced remission or partial remission in most of the relapsed/refractory FLT3-ITD AML patients (CR/CRi/nCR = 82.4%, n=34 in our sorafenib monotherapy cohort). Although consolidation with combination of sorafenib and chemotherapy or hypomethylating agents was reported to significantly extend the remission period, all patients relapsed with drug resistance. Mutations at the tyrosine kinase domain (TKD) were reported to alter the binding affinity of the drugs to the receptor and hence generate resistance. However, they were found only in around 25% of the patients while other non-mutational mechanisms of resistance have also been reported. Their relative importance in patients remains uncertain. We hypothesised that there is an emergence of drug-resistant clones acquiring new mutations or pathways to develop the resistance.

We have performed whole exome sequencing (HiSeq pair-end sequencing of 101 bp) for sorafenib-sensitive and sorafenib-resistant samples obtained from 8 FLT3-ITD relapsed/refractory AML patients before sorafenib treatment and at relapse with drug resistance. Genomic content of the bulk population was observed to change drastically among the 8 patients, with disappearance of single nucleotide polymorphisms (SNPs) and emergence of new SNPs, suggesting the emergence of drug-resistant clones. Emergence of TKD D835Y mutation was observed in 3 patients with variant allele frequencies (VAF) at 11%, 44% and 50% that were undetectable in the sorafenib-sensitive samples. Mutations at other reported sites of TKD were not found. Filtering of the generated list of single nucleotide changes and insertion/deletions (indels) to sort out emerging non-synonymous mutations with high VAF at resistant samples but undetectable in the sensitive samples was done. They include SLC15A1, CDC27, NADKD1, DGAT2, UBXN11, TFR2 and TTBK1, potentially implicated in sorafenib resistance. They are involved in different pathways, including membrane transporter, cell cycle regulation and energy metabolism. Further functional validation of their role in sorafenib resistance is required to understand the mechanisms involved.

Disclosures

No relevant conflicts of interest to declare.
Chemoresistant Leukemia-Initiating Cell Expansion Is Inhibited By Targeting Oncogenic Self-Renewal
Authors: Perry John M. , Roy Anuradha , Lu Xiuling , He Xi C. , Tao Fang , Zhao Meng , Qian Pengxu , Li Linheng
Published: October 2019

Leukemia is often maintained by a relatively rare subset of tumor-initiating cells, which may be resistant to traditional chemotherapy. Targeting these leukemic stem cells is expected to eliminate or reduce acquired resistance to chemotherapy and disease recurrence, but this goal has been elusive. It's also not clear whether eliminating the 'root' of cancer is sufficient or whether targeting both 'root' and 'branch' (i.e. LSCs and their bulk progeny) is necessary. To test this, we established and characterized a mouse model with clearly defined populations of rare leukemic stem cells (LSCs), abundant leukemic blast cell progeny, and normal hematopoietic stem/progenitor cells (HSPCs). This was achieved by simultaneously activating the Wnt/β-catenin and PI3K/Akt signaling pathways in HSPCs to drive stem cell self-renewal-resulting in successive expansion of HSPCs, LSCs, and leukemic blasts. Functional analysis showed a 1,300-fold increase in leukemia-initiating activity of sorted LSCs compared to bulk blast cells or sorted HSPCs. Since Akt activates β-catenin by phosphorylation at serine 552 and thus represents a molecular link mediating the synergistic, self-renewal promoting activity of these pathways, we sought to specifically target pS552-β-catenin. Unexpectedly, high-throughput screening (HTS) and subsequent validation assays found that doxorubicin (DXR) inhibits pS552-β-catenin with minimal effects on total β-catenin.

DXR exhibits the broadest spectrum of anti-cancer activity known and has been employed as a standard chemotherapeutic agent for decades, but severe side-effects limit its use. We found that while standard chemotherapeutic treatment reduced the bulk leukemic blast cells as expected, it also induced pS552-β-catenin uniquely in LSCs and stimulated LSC expansion. Using long-circulating nanoparticles (NanoDXR) to deliver very low, sustained doses of DXR, we repurposed DXR as a targeted therapy for pS552-β-catenin inhibition rather than a broadly cytotoxic agent. In vivo, this treatment reduced pS552-β-catenin levels in LSCs, prevented LSC expansion, essentially eliminated LSC tumorigenic activity in transplant recipients, and was accompanied by recovery of HSPCs and substantially increased median survival from 44.5 days to 139 days.

Our data reveals a dynamic relationship between rare LSCs and bulk leukemic blast cells in their response to cytotoxic chemotherapy and show how oncogenic self-renewal in chemoresistant LSCs can be targeted while sparing normal HSPCs. In distinguishing the unique properties of LSCs and their progeny, we find that both populations must be differentially targeted at both the 'root' and 'branch' of leukemia and can be effectively reduced or eliminated while allowing for recovery of normal HSPCs.

Disclosures

No relevant conflicts of interest to declare.
Antithymocyte Globulin Treatment at the Time of Transplantation Impairs Donor Hematopoietic Stem Cell Engraftment
Authors: Hu Zheng , Jin Feng , He Jin , Jin Chunhui , Fan Wei , Shan Yanhong , Zhang Zefeng , Sun Liguang , Yang Yong-Guang
Published: October 2019

Antithymocyte globulin (ATG) is often included in the conditioning regimen to prevent graft-vs.-host disease in allogeneic hematopoietic stem cell (HSC) transplantation. However, because ATG contains antibodies targeting a wide range of antigens on human cells, its potential off-target effects remain a concern. Here, we explored this question in humanized mice that were made by transplantation of human fetal thymic grafts (under the renal capsule) and CD34+ human fetal liver cells (i.v.). We showed that ATG binds to almost all lineages of human hematopoietic cells including HSCs, and accordingly, it is capable of depleting almost all human hematopoietic cells. Interestingly, the efficacy of ATG was highly variable depending on the tissue of residence, with human cells in bone marrow significantly less susceptible than those in the blood and spleen. Recovery of multilineage human lymphohematopoietic reconstitution in humanized mice that received ATG 3 weeks after HSC transplantation indicates that ATG had a minimal effect on human HSCs that have settled in bone marrow niches. However, efficient human HSC depletion and engraftment failure were seen in mice receiving ATG immediately following transplantation. Our data indicate that the efficacy of ATG is tissue-dependent, and suggest a risk of impairing donor hematopoietic engraftment when ATG is used in preparative conditioning regimens in HSC transplantation.

Disclosures

No relevant conflicts of interest to declare.
Extramedullary Disease at Diagnosis of Acute Myeloid Leukemia Does Not Influence Outcome of Patients Undergoing Allogeneic Hematopoietic Cell Transplant in First Complete Remission
Authors: Michelis Fotios V. , Messner Hans A. , Alam Naheed , Gupta Vikas , Kim Dennis Dong Hwan , Kuruvilla John , Lambie Anna , Viswabandya Auro , Lipton Jeffrey H.
Published: October 2019

Occurrence of extramedullary (EM) disease at diagnosis of acute myeloid leukemia (AML) has been associated with increased risk of relapse and worse outcomes post-chemotherapy. There is minimal data in the literature concerning the association with outcomes following allogeneic hematopoietic cell transplantation (HCT). The purpose of this single-centre study was to retrospectively investigate the impact of EM disease at diagnosis on the outcome of 303 patients with AML in first complete remission (CR1) that underwent HCT during the time period 2000-2013.

Median age at HCT was 51 years (range 18-71), 151 (50%) patients were female. Myeloablative conditioning (MAC) was used in 202 (67%) patients, reduced-intensity (RIC) in 101 (33%) patients. Donors were related for 194 (64%) patients, unrelated for 109 (36%) patients. Grafts were peripheral blood stem cells (PBSC) in 253 (83%) patients and bone marrow in 50 (17%) patients. Median follow-up of patients alive was 63 months (range 12-168). Cytogenetics at diagnosis were available for 263 (87%) of patients, of which 16 (5%) were favorable, 185 (61%) were intermediate and 62 (20%) were unfavorable risk (MRC classification). Primary induction failure prior to achievement of CR was seen in 67 (22%) patients. In vivo T-cell depletion was performed in 71 (23%) patients. A total of 124 patients (41%) underwent HCT during the years 2000-2006 and 179 patients (59%) during the years 2007-2013. EM disease at diagnosis was seen in 39 patients (13%), of whom 11 patients had CSF disease, 7 patients had gingival infiltration and 5 patients had leukemia cutis.

Univariate analysis for overall survival (OS) demonstrated that EM disease at diagnosis had no influence (HR=0.96 for EM, 95%CI=0.60-1.51, p=0.85, Figure 1). Multivariable analysis for OS including the previously described variables verified this observation. EM disease did not influence cumulative incidence of relapse (CIR) on univariate analysis (HR=0.94 for EM, 95%CI=0.45-1.96, p=0.86, Figure 2), and this also was confirmed on multivariable analysis. Moreover, EM disease did not influence cumulative incidence of non-relapse mortality on both univariate (HR=0.94 for EM, 95%CI=0.53-1.66, p=0.83) and multivariable analysis.

In conclusion, EM disease at diagnosis of AML in patients achieving CR1, does not seem to influence outcomes post allogeneic HCT. This is significant in the consideration of allogeneic HCT for the treatment of this unfavorable subtype of AML. We are unable to comment on whether a similar percentage of patients with EM disease versus without EM disease, achieve CR1.

Figure 1. Figure 1. Figure 2. Figure 2.

Disclosures

Kim: Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.
Prevalence of Extramedullary Hematopoiesis, Vertebral, Hepatic and Splenic Hemangioma and Renal Cyst Among Thalassaemic Patients: A Retrospective Study from the Myocardial Iron in Thalassemia (MIOT) Network
Authors: Pepe Alessia , Meloni Antonella , Briatico Vangosa Alessandra , Terrazzino Fabrizia , Neri Maria Giovanna , Maddaloni Domenico , Roccamo Gaetano , Armari Sabrina , Maggio Aurelio , Salvatori Cristina , Positano Vincenzo , Ricchi Paolo
Published: October 2019

BACKGROUND: The prevalence and the clinical relevance of several extracardiac findings (EF) at Magnetic Resonance Imaging (MRI) have been recently estimated in cohorts of non thalassaemic patients.

AIM: In this study we sought to determine the prevalence of EF potentially related to anaemia and ipoxia in age- and sex- matched populations with thalassaemia major (TM) and Thalassaemia Intermedia (TI).

METHODS: We retrospectively considered 159 TM patients regularly transfused (73 F; 34±9.20 years) and 159 TI patients (73 F; 34±9.18 years) consecutively enrolled in the Myocardial Iron in Thalassemia (MIOT) Network to quantify cardiac and liver iron by T2* technique. Patients with TI were further subdivided in non transfusion dependent (NTD-TI) and transfusion dependent (TD-TI). All MRI images were used to assess the presence or absence of Extramedullary Hematopoiesis (EMH), kidney, splenic and liver cysts, and vertebral hemangiomas.

RESULTS: Overall, EF were detected in 33% and 26% of patients with TI and TM, respectively. Table 1 shows the comparison of MRI data between TM and TI patients. Both groups of patients had elevated but comparable prevalence of kidney, splenic and liver cysts and vertebral haemangiomas. TI patients were found to have significant higher EMH as compared to TM. The prevalence of total EF increased with advancing age and renal cysts were found in the 28.6% of the cohort aged 45-55 years. Patients with renal cysts had comparable serum creatinin level with respect to those without. Within the population of TI-TD it was observed the highest prevalence of patients with renal cysts, splenic cyst and vertebral haemangioma (26%, 3% and 3%, respectively).

Thalassemic patients had a was significant higher prevalence of renal cysts than the general population (19.2 vs 7.8%; P<0.0001).

CONCLUSIONS: EF are well-recognized features in patients undergoing MRI, being hepatic cysts or hemangiomas and renal cyst the most frequently observed. Our data on hemangiomas and renal cysts, particularly among patients with TI-TD, indicate a significant higher prevalence of EF compared to the general population. These data seem to suggest the role of the inappropriate activation of the Hypoxia-Inducible Factor (HIF) system linked to the chronic hypoxia as observed in the Chuvash polycythemia or in the the von Hippel-Lindau (VHL) syndrome. Furthermore, HIF in general population has been directly involved in the development and/or progression of clear cell renal cancer also described among thalassemic patients.

Table 1. Table 1.

Disclosures

Pepe: Chiesi: Speakers Bureau; ApoPharma Inc: Speakers Bureau; Novartis: Speakers Bureau.
Neutrophil Infiltration Is Partially Inhibited By EC-18 in the LPS-Induced Acute Lung Injury
Authors: Shin Su-Hyun , Lee Ha-Reum , Yoo Nina , Yoon Sun Young , Kim Myung-Hwan , Suh Cheolwon , Sohn Ki-Young , Kim Heung-Jae , Han Yong-Hae , Chong Saeho , Kim Jae Wha
Published: October 2019

Acute lung injury (ALI) is an acute respiratory failure and linked closely to neutrophil accumulation. It can lead to acute respiratory distress syndrome (ARDS). Mouse model of ALI was established by lipopolysaccharide (LPS) administration. LPS, an outer membrane of gram negative bacteria, is considered as immune stimulator via recognition as pathogen-associated molecular patterns (PAMP). ALI and neutrophil infiltration were readily induced by intranasal injection of LPS. In this study, we investigated whether EC-18 treatment attenuates LPS-induced ALI. EC-18 (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) is an immune modulator in the allergic asthma response through modulation of the balance between Th1 and Th2. To analyze the role of EC-18 in LPS-induced ALI mice, Balb/c mice were divided into three separate groups: control, LPS treated, and EC-18/LPS co-treated (n=5 per group). 25 mg/kg of LPS was administered by an intranasal route, and 250 mg/kg of EC-18 was orally administered. Mice were sacrificed after 15hrs, and various samples were collected. Bone marrow cells, whole blood cells, cells in lung and bronchoalveolar lavage fluid (BALF) were analyzed using complete blood count (CBC) assay. As results, the number of neutrophil in the bone marrow was decreased in the LPS treated group, and the circulating neutrophil, neutrophil in the lung and BALF were significantly increased in the LPS treated group. Neutrophils in the lung and BALF were dramatically decreased in the EC-18/LPS co-treated group. Also, Evans Blue staining of the lung indicated that capillary permeability was enhanced in the LPS injected mice, and this permeability was lessened in the EC-18/LPS co-treated group as much as that of control. These findings suggested that EC-18 could effectively block neutrophil transmigration into the lung and vesicular leakage. Consequently, EC-18 could be utilized as a potential therapeutic agent for acute and chronic inflammation related disease like ALI.

Disclosures

No relevant conflicts of interest to declare.
T Regulatory Cells Exhibit Surface Expression of FoxP3 Derived Peptides Presented within Class I MHC
Authors: Schultz Liora M. , Olsson Niclas , Khodadoust Michael , Narayan Rupa , Sagiv-Barfi Idit , Elias Joshua , Levy Ronald
Published: October 2019

Background:

FOXP3 is a transcription factor of central importance to the inhibitory function of CD4+, CD25+ FOXP3+ T regulatory cells (Tregs). Tregs function as critical inhibitory immunoregulatory cells and have been reported to be increased in tumor-bearing individuals, inhibiting optimal T effector mediated anti-tumor cytotoxicity. Treg depletion targeting CD25, a surface marker of Tregs as well as activated T cells, has been validated in pre-clinical models as a promising adjunctive therapeutic approach to improving T cell mediated therapy and is currently being explored in clinical trials. However, because CD25 expression is not limited to Tregs, CD25-directed therapies may also result in the undesired depletion of effector T cells. Although FOXP3 expression is more restricted to Tregs as compared to CD25, its intranuclear localization has made it challenging to target therapeutically. Vaccination of mice against FOXP3 using FOXP3 mRNA-transfected dendritic cells has been explored and can elicit FOXP3 specific CTL mediated cytotoxicity. This work implies that FOXP3 mRNA-transfected dendritic cells process FOXP3 and present FOXP3 derived peptides on the cell surface. Direct expression of FOXP3 derived peptides on the surface of Tregs has however, not been previously demonstrated.

Methods:

We utilized mass spectrometry to investigate if FOXP3 derived peptides presented within MHC class I complex are expressed on the surface of Tregs. CD4+, CD25+ T cells were isolated from healthy donors using magnetic bead selection to enrich for Tregs. Surface class I MHC was immunoprecipitated using a pan-MHC class I anti- HLA-A, HLA-B and HLA-C antibody and associated peptides were eluted from their MHC complex using acid elution. Peptide and fragment masses were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the spectra of resulting epitopes were matched to known protein sequences by SEQUEST.

Results:

Four unique FOXP3 derived peptides were identified from the primary donor samples enriched for FOXP3+ cells as described. Predictive HLA binding algorithms demonstrate that the peptides identified are predicted binders to the specific HLA alleles of the original donors. No FOXP3 derived peptides were identified from the CD4+, CD25- T cell fraction analyzed to date.

Conclusions:

This work directly demonstrates for the first time to our knowledge that, although FOXP3 is an intra-nuclear transcription factor, FOXP3 derived peptides are presented within class I MHC on the surface of Tregs, representing a targetable Treg associated surface protein complex.

Disclosures

Levy: Bullet Biotechnology, Inc.: Consultancy.
Parstatin Mediates Platelet Activation That Is Unaffected By PAR Antagonism
Authors: Dunphy Paige Selvy , Kotha Jayaprakash , Dixon Mason L. A. , Jalenak Jay M. , Jennings Lisa K.
Published: October 2019

Thrombin cleaves PAR-1 at amino acids arg41-ser42 to yield the canonical tethered ligand as well as a soluble peptide, parstatin, which has been reported to have divergent physiological functions. In animal models, this peptide demonstrates anti-angiogenic, anti-inflammatory, and cardioprotective properties. In ex vivo studies, parstatin was also demonstrated to affect platelet aggregation and enhance GPIIb/IIIa-mediated adhesion, suggesting this cleaved peptide may participate in pathological thrombosis. With recent approval of a first-in-class PAR-1 antagonist as an antiplatelet agent, it is clinically imperative to fully appreciate the physiologic mechanisms through which thrombin-activation of PAR-1 contributes to platelet aggregation and thrombosis. As such, the effects of PAR antagonism in modulating parstatin-mediated platelet activation requires evaluation.

In this study, we characterized parstatin-mediated effects on platelets and investigated the potential involvement of platelet thrombin receptor (PAR-1, PAR-4)-associated signaling in this phenomenon. Light-transmission aggregometry was used to measure aggregation response in washed platelet preparations, and flow cytometry was used to assess expression of protein markers indicative of platelet activation. Consistent with previous reports, we demonstrated parstatin induces P-Selectin surface expression (degranulation), GPIIb/IIIa activation (PAC-1 binding), and aggregation independent of thrombin receptor cleavage (n=10, healthy donors). Interestingly, platelet shape change was not observed following parstatin treatment, even in the presence of PAR-1 activating peptide (PAR-1-AP, SFLLRN), suggesting parstatin-mediated activation does not signal through G12/13-dependent mechanisms, and may override canonical G12/13-associated PAR-1 signaling. Pretreatment with Gq-selective PAR-1 antagonist, ML161 (3 µM), or PAR-4-selective antagonist, ML354 (500 nM) did not inhibit parstatin-mediated platelet activation. These findings are consistent with previous reports suggesting this peptide may signal through a Gi-dependent mechanism. Platelet PAR receptors couple to Gαq and Gα12/13, but direct coupling to Gαi is controversial; therefore, parstatin-mediated activation may occur through a signaling cascade unrelated to canonical PAR-associated mechanisms.

Disclosures

No relevant conflicts of interest to declare.
Use of Clofarabine in Pediatric Multisystem Langerhans Cell Hystiocytosis
Authors: Pita Valentina , Betancourt Neiva , Ramirez Bertha , Bello Adriana C.
Published: October 2019

Langerhans cell hystiocytosis has been defined as an inflammatory and/or neoplasm of the hematopoietic system, mainly of myeloid precursors. There is recent evidence of mutations in the BRAFF V600 progenitor cells, which underline its neoplastic nature -myeloprolipherative condition.

The diagnosis of LCH relies on a positive biopsy identifying characteristic LCH cells by morphology and immunohystochemistry (CD1a and Langherin). Genetic tests have been added to the diagnostic armamentarium, with prognostic value.

Children younger than 2 years, and with risk organ involvement, have an unpredictable, often bad prognosis.

There is no current international consensus on LCH treatment, and guidelines are based on the best currently known treatment approaches.

Case #1:

4 year old girl with MS-LCH with hematologic, bone and lung involvement. She presented early at 18 months of age with pancytopenia, became transfusion dependant of red blood cells and occasional platelets. She was started on LCH-III after a lymph node biopsy showed extramedullary hemopoiesis and positive for LCH (Langherin, Cd1a). She received 12 weeks of initial therapy with vinblastine and prednisolone, with no improvement. Disease progressed with additional bilateral pleural effusion and fever. She was given Clofarabine 25mg/m2/d x5 during 2 courses, with improvement of counts and symptoms. A PET FDG scan was pending to assess disease status.

Case #2:

18 month old girl with systemic manifestations of MS-LCH since age 3 months, with fever, splenomegaly and anemia. Multiple cranial lesions were found, and skull biopsy at age 13 months was positive for LCH (langherin, Cd1a). BRAFF V600 mutation by PCR of biopsy was negative. cerebral MRI was normal. She was started on LCH-III (vinlastine plus steroids) during 6 weeks. Due to disease progression, worsening splenomegaly, anemia, fever, she was given clofarabine 25mg/m2/d x5. Fever disappeared post 1st course of treatment. Rest of manifestations were gone after 2nd cycle. Treatment was administered as inpatient, with no significant side effects.

Clofarabine is a second generation nucleoside analog with activity in refractory AML. It has been used with success as salvage therapy in LCH patients.

There is growing optimism with the use of nucleoside analogs as effective treatment of LCH, with dose-dependent effects on therapeutic efficacy, as well as toxicity.

Disclosures

Off Label Use: Clofarabine. Second generation nucleoside analog.
Lymphocyte Activation Gene 3-a Novel Therapeutic Target in Chronic Lymphocytic Leukemia
Authors: Shapiro Mika , Katz Ben-Zion , Perry Chava , Sun Clare , Dezorella Nili , Kay Sigi , Avivi Irit , Wiestner Adrian , Yair Herishanu
Published: October 2019

A novel therapeutic approach in cancer attempting to stimulate host anti-tumor immunity involves blocking of immune checkpoints. Lymphocyte-activation gene 3 (LAG3) is an immune checkpoint receptor expressed on activated/exhausted T-cells. When engaged by MHC Class II (MHCII) molecules, LAG3 negatively regulates T-cell function thereby contributing to tumor escape. Intriguingly, a soluble LAG3 variant was reported to activate both immune and malignant MHCII-presenting cells. In this study, we examined the role of LAG3 in the pathogenesis of CLL and show that CLL cells express and secrete LAG3. High levels of surface and soluble LAG3 were associated with the unmutated immunoglobulin variable heavy chain CLL and a more aggressive disease. It was evident that recombinant soluble LAG3-Ig fusion protein activated CLL cells, induced increased levels of Bcl-2 and Mcl-1 and protected the cells from spontaneous apoptosis, utilizing a mechanism mediated through MHCII engagement. Our data suggest that autocrine secretion of soluble LAG3 from CLL cells and its subsequent interaction with MHCII on their surface, promotes CLL cell activation and anti-apoptotic effects. Moreover, CLL cells may impose immune exhaustion on their microenvironment by expressing MHCII, thus affecting the surrounding, LAG3-presenting immune cells. Hence, blocking LAG3-MHCII interactions is a potential therapeutic target in CLL.

Disclosures

Avivi: Tel Aviv Sourasky Medical center: Consultancy, Other: consultancy to :BMS Roche. Wiestner:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding.
Inhibition of Casein Kinase 2 Impairs Wnt Signaling and Cell Survival in Chronic Lymphocytic Leukemia
Authors: Wu Christina , Lao Fitzgerald S , Nan Emily , Li Hongying , Choi Michael Y. , Messer Karen , Carson Dennis A.
Published: October 2019

The oncogenic Wnt pathway is aberrantly activated in most CLL clones, and hence is an attractive target for therapy. The casein kinase 2 (CK2) enzyme is an established positive regulator of Wnt signaling. The inhibitor Silmitasertib, also known as CX-4945, is a nanomolar inhibitor of CK2. It has been reported that CK2 is overexpressed in CLL. Here we have investigated the effects of CX-4945 on WNT signaling in primary CLL cells.

We confirmed that CX-4945 displayed in vitro cytotoxic activity toward CLL cells at very low µM concentration, as previously reported by others. However, at least 2-3 fold higher concentration of CX-4945 was required to achieve a similar toxicity against normal PBMC. Previously, our laboratory has successfully utilized a short-term CLL "parking" model in immunodeficient RAG/gamma chain knock out (RG-KO) mice to evaluate the in vivo efficacy and potential toxicity of anti-CLL agents. CX-4945 at dosages of 0.3-10 mg/kg was administered by oral gavage daily for 6 days to mice injected i.p. with 10 million CLL cells. These dosages of drug were well tolerated, and potently inhibited CLL persistence in the xenotransplanted mice.

In a reporter gene assay, CX-4945 dose-dependently inhibited Wnt target gene expression. Furthermore, inhibition of dishevelled-2 (Dvl-2) protein expression was observed in primary CLL patient samples treated with 3-10 µM CX-4945 for 4-16 hours. Similar reduction in p-GSK3b(S9) protein was also observed. Quantitative RT-PCR also confirmed down regulation of b-catenin gene expression in primary CLL patient samples treated with 10 µM CX-4945 for 4h. Further molecular analyses of predictive or correlative biomarkers is ongoing using Nanostring PanCancer multipathway gene analysis. In a preliminary study, we found that CX-4945 perturbed the expression of multiple genes implicated in CLL development and survival.

In summary, the CK2 inhibitor CX-4945 inhibited Wnt signaling and CLL survival, and displayed oral activity in mice. CK2 inhibitors are thus potential therapeutic agents for CLL.

Disclosures

No relevant conflicts of interest to declare.