bluuebunny/crossref_metadata_2025_split huggingface dataset:
STAT5b Gene Mutations and Their Association with a Unique Immunophenotype of CD4+CD56+TCRαβ Type LGL Leukemia
Authors: Tanahashi Takahiro , Matsuda Kazuyuki , Sekiguchi Nodoka , Nishina Sayaka , Senoo Noriko , Sakai Kaoko , Sakai Hitoshi , Ito Toshiro , Mitsui Takeki , Kwong Yok Lam , Ishida Fumihiro 
Published: October 2019

Abstract
Large granular lymphocyte leukemia (LGL L) includes T-cell LGL L and chronic lymphoproliferative disorders of NK cells (CLPD-NK) with indolent clinical course. STAT signaling system has been shown to play a crucial role in LGL L. We reported somatic mutations in the STAT3 gene in LGL L, which is associated with pure red cell aplasia among an Asian cohort. Since STAT5b mutations in LGL leukemia had also been discovered, we investigated mutations in STAT5b gene in Japanese patients with LGL L. Among 28 T-LGL L and 11 CLPD-NK, N642H and Y665F activating mutations in the SH2 domain of the STAT5b were found in 3 and 1 patient (pt)s, respectively, by direct sequencing, which was confirmed with the corresponding allele-specific (AS) PCR. The frequencies of STAT5b mutations in T-LGL L and CLPD-NK were 14.3% and 0%, respectively. Median age of the 4 pts was 76.5 years and mild neutropenia was recognized in 2pts, but no one was anemic. None of them showed apparent aggressive clinical courses, in contrast to previous reported cases (Rajala HLM et al, BLOOD 2013). Interestingly, STAT5b mutations were associated with a unique phenotype of LGL L with CD4+CD56+TCR αβ type (P = 0.0001). The mutations of STAT5b and STAT3 were mutually exclusive. In contrast to STAT3 mutation-positive patients, among whom significant part of the patients possessed STAT3-mutated subclones only detected with AS-PCR, no pt was identified to be positive for STAT5b mutation only with AS-PCR. We also investigated STAT5b mutations in 29 Chinese patients cohort with T cell LGL L by AS-PCR for N642H and Y665F, and found that no patient was positive for the mutations. Our results indicate that the STAT5b gene is mutated in T-LGL L, which would affect a specific immunophenotype of LGL. STAT5b and STAT3 mutations have significant but distinct contributions to the pathobiology of LGL L and STAT5b-mutated LGL L may consist a unique subtype of LGL L.

Disclosures
No relevant conflicts of interest to declare.
Diffuse Large B Cell Lymphoma (DLBCL) infiltrating T Cells Display an Activated and Exhausted Status and Are Inhibited By Ligands of Cosignaling Receptors Including PD-L1, PD-L2 and CD80 Expressed By Most DLBCL in Situ
Authors: Olive Daniel , Le Kieu Suong , Roussel Mikael , Broussais Florence , Azzaoui Imane , Rossille Delphine , Bouabdallah Reda , Fest Thierry , Xerri Luc , Lamy Thierry , Pangault Céline 
Published: October 2019

Abstract
The regulation of immune response through cosignaling receptors is the major mechanism that prevent normal responses against peripheral tissues but that is used by tumor to hijack the autologous immune responses. This therapy are currently among the most promising adjuvant therapy in cancers including lymphomas. In addition sPDL1 is an important marker of patient's prognosis (Rossille et al., Leukemia 2014)
Our objective was the study of the expression of ligands by activating receptors on diffuse large B cell lymphoma (DLBCL) in comparison to Follicular Lymphoma (FL), Hodgkin Lymphoma (HL) and hyperplasia and to investigate the status of the infiltrating immune system as well as the function of the sorted autologous interaction of T cells against autologous DLBCL
Methods: We have dissociated fresh tumor B cell samples ( FL= 13, HD= 15, DLBCL = 15) and analysed the immune microenvironment and DLBCL by flow cytometry using mAbs recognizing cosignaling molecules of 15 samples. In a second step we have sorted the T cells and autologous DLBCL and performed cocultures with the coincubation with antagonist PD1 (PD1.3.1) PDL1 (PDL1.3.1) PDL2 (PDL2.1), Tim3 and CD80 mAbs. T cell stimulation was performed for 5 days. We have previously described the proprietary antagonist PD1, PDL1, PDL2 and CD80 used (Serriari et al., J. Immunol., 2010; Ghiotto et al. , Int.Immunol., 2010)
Results: The immune cells including T cells present within the DLBCL microenvironment are characterized by a highly activated /exhausted status as evidenced by the expression of PD-1, PDL1, PDL2, CD80 and Tim3 Figure 1) . Importantly most of these receptors are involved in negative signals provided their ligand is expressed on their cognate target cell. PDL1, PDL2 and CD80 are both present on DLBCL with some degree of heterogeneity among tumors and surprisingly PD1. Autologous T cell and lymphoma cells were sorted and autologous responses such as proliferation and cytokine production were analysed after in vitro culture without or with antagonist mAbs directed against cosignaling receptors. We could evidence for the first time to our knowledge that blockade of PD1 and CD80 pathways induced /restored the autologous response of infiltrating T cells.
Conclusions: These data demonstrate that a subgroup of DLBCL patients might benefit from treatment with checkpoint blockade mAbs. A major objective is to understand the molecular basis of DLBCL heterogeneity that currently investigated.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.
Endogenous TGF-beta1 Mediated Osteogenic Impairment of Medullar Mesenchymal Stromal Cells in Primary Myelofibrosis
Authors: Martinaud Christophe , Desterke Christophe , Konopacki Johanna , Pieri Lisa , Golub Rachel , Schmutz Sandrine , Anginot Adrienne , Guerton Bernadette , Rochet Nathalie , Albanese Patricia , Henault Emilie , De Revel Thierry , Bourgeade Marie-Françoise , Alessandro Vanucchi , Lataillade Jean-Jacques , Le Bousse-Kerdilès Marie-Caroline 
Published: October 2019

Abstract
Primary myelofibrosis (PMF) is myeloproliferative neoplasm characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in bone marrow and spleen. Mesenchymal stromal cells (MSCs) are reported to play a pivotal role in fibrosis and stromal changes are considered as a reactive counterpart of the cytokine production by clonal hematopoietic cells. The present study shows that MSCs from patients demonstrate functional abnormalities that are unexpectedly maintained ex-vivo, in culture.
Material and Methods: we studied MSCs and bone marrow sections from PMF patients (n=12) as compared to healthy donors (HDs) (n=6). We tested their proliferation, immunophenotype, hematopoiesis supporting capacities, differentiation abilities, in-vivo osteogenic assays, and performed secretome and transcriptome analysis.
Results: We found that PMF-MSCs exhibit similar proliferative capacity and long-term hematopoiesis supporting abilities as compare to healthy donors. They overproduce interleukin 6, VEGF, RANTES, PDGF, BMP-2 and surprisingly TGF-beta1. MSCs from fibrotic PMF patients express high levels of glycosaminoglycans. Adipocytes and chondrocytes differentiation abilities were not different as compared to HDs but PMF-MSCs exhibit an increased in vitro potential. Implementation on scaffold in nude mice confirmed, in vivo, this increased osteogenic potential. We then looked into gene expression and discovered that PMF-MSCs show an original transcriptome signature related to osteogenic lineage and TGF-beta1. Indeed, osteogenic genes such as Runx2, Dlx5, Twist1, Noggin, Sclerostin, GDF5 and Serpine1 are deregulated and suggest a potential osteoprogenitor priming of PMF-MSCs. These molecular results also advocated for a TGF-beta1 impregnation that prompted us to study its impact on PMF-MSCs osteogenic differentiation. First, we then showed that Smad2 is intrinsically over-activated in PMF-MSC and that stimulation by TGF-beta1 is associated with an increase phospho-Smad2 level and an enhancement of bone master gene regulator Runx2 expression. Then, we inhibited TGF-beta1 pathway by by SB-431542 and evidenced a specific behavior of osteogenic MSCs differentiation in patients, suggesting involvement of TGF-beta1 in osteogenic impairment.
Conclusion: Altogether, our results identify a signature of PMF-MSCs and suggest that they participate in PMF osteogenic dysregulation independently from in vivo local stimulation by clonal hematopoietic cells

Disclosures
No relevant conflicts of interest to declare.
NEK2 Suppresses Cellular Senescence through Regulation of the β-Catenin/CCND2/RB Axis in Multiple Myeloma
Authors: Yang Ye , Tricot Guido J. , Jiliang Xia , Gu Zhimin , Xu Hongwei , Hao Mu , Zhan Fenghuang 
Published: October 2019

Abstract
Background: Multiple myeloma (MM) is a B-cell malignancy characterized by complex cytogenetic abnormalities and drug-resistance. We have previously shown that increased NEK2 enhances MM cell growth, survival and drug-resistance in vitro and in vivo. In this study, we explore whether high NEK2 is involved in overcoming cellular senescence, a state of irreversible proliferation arrest.
Materials and Methods: MM cell lines (ARP1, OPM2, OCI-MY5, and KMS28PE) with either over-expressed or knocked-down (shRNA) NEK2 (NEK2 OE or KD) by a lentiviral delivery system were used in this study. Cell senescence status was evaluated by staining SA β-galactosidase (SA β-gal). Flow cytometry was performed to examine cell cycle state in NEK2-KD or NEK2-OE MM cells treated with doxorubicin (30nM) for 48 hrs. Mouse embronic fibroblasts (MEFs) derived from NEK2-transgenic mice and wild type control mice were also used to verify the role of NEK2 in suppressing senescence and to elucidate signaling pathways related to NEK2 anti-senescence. Real-time PCR, immunofluorescence, co-immunoprecipitation (Co-IP) assay and western blot were employed for NEK2 mechanistic studies.
Results: NEK2-KD induced MM cellular senescence and cell cycle arrest, while NEK2 OE decreased doxorubicin-induced senescence and cell cycle arrest. Also, MEFs derived from NEK2 transgenic mice showed suppressed cell senescence. Mechanistic studies showed that NEK2 stabilized β-catenin and prevented degradation by the ubiquitin/proteasome pathway in MM cells. Intriguingly, immunofluorescence revealed that NEK2 co-located with β-catenin in the cell nucleus in NEK2 transgenic MEFs. Importantly, increased nuclear β-catenin up-regulated CCND2 expression, (but not CCND1 and CCND3) and phosphorylated RB in NEK2 over-expressed MM cells or NEK2 transgenic MEFs. Furthermore, LiCl, an inhibitor of GSK3β, was used to restore the activity of β-catenin in NEK2-KD cells. LiCl significantly increased CCND2 expression and blocked NEK2-shRNA induced cellular senescence in MM cells.
Conclusion: NEK2 prevented MM cell senescence. This may play an important role in NEK2 induced MM cell proliferation, survival and drug resistance. Prevention of senescence by NEK2 depended on its interaction with β-catenin. Increased β-catenin activity, caused by high NEK2 resulted in up-regulation of CCND2 and hyper-phosphorylation of RB.

Disclosures
No relevant conflicts of interest to declare.
Re-Educating Myeloma Associated Macrophages with Lenalidomide
Authors: Bruns Heiko , Gehlen Hanna , Nolting Jens , Pasemann Shirin , Brossart Peter , Mackensen Andreas , Gerbitz Armin 
Published: October 2019

Abstract
Introduction: The bone marrow niche plays a critical role in determining the fate of malignant plasma cells in multiple myeloma (MM). Macrophages are an abundant component of the stromal cell compartment and are believed to support proliferation, survival, and drug resistance of MM cells. Conversely, macrophages can directly kill tumor cells and participate in antitumor immune responses as effector cells. Moreover, macrophages are key immune effector cells for the therapeutic effect of monoclonal antibodies. Lenalidomide, an immunomodulatory drug (IMiD®) is used for the treatment of MM, also in the combination with therapeutic antibodies. Lenalidomide is thought to target the stromal support, but its precise influence on the phenotype or the effector functions of macrophages is still unclear.
Methods: To investigate the effect of lenalidomide on the interaction between macrophages and malignant plasma cells in vitro, we coincubated lenalidomide pretreated macrophages with several MM cell lines, and analysed the viability, proliferation and phenotype. For in vivo studies we utilized 5TMM mice, a suitable animal model for MM. Animals were treated with lenalidomide (50 mg/kg 5days/week) for 3 weeks, and the effector functions and phenotype of isolated bone marrow macrophages were analyzed. In addition, macrophages in the bone marrow of MM patients treated with lenalidomide were characterized by immunohistochemistry and flow cytometry.
Results: We showed, that infiltrating macrophages in the bone marrow of MM patients display an anti-inflammatory M2-like phenotype characterized by the expression of surface marker CD163, CD206, PD-L1 and cytokine/chemokine secretion (e.g. IL10, CXCL10, APRIL, BAFF and RANKL). Incubation of macrophages with lenalidomide in vitro, substantially changed their transcriptional program (e.g. downregulation of IRF4 and upregulation of IRF5) and their phenotype (e.g. downregulation of the surfaces marker CD163, CD206, and upregulation of CD16, CD64, CD40 and CD86). Furthermore, we show that lenalidomide treatment decreases the expression of RANKL, BAFF and APRIL, while tumoricidal effector molecules (e.g. TRAIL, cathelicidine, Granzyme B) were increased. When lenalidomide treated macrophages were cocultured with MM cells significant cytotoxicity was detected, for all MM cell lines tested. In contrast, untreated macrophages promote tumor growth and viability of MM cells.
Conclusion: Lenalidomide in vitro influences macrophages by reverting an anti-inflammatory M2 like profile to a more immunogenic phenotype. In addition it impacts on the support function by decreasing the secretion of important growth factors for B-cells. Similar results were observed in first in vivo studies. Taken together our results imply that lenalidomide interrupts an important stromal cell function thereby influencing survival of MM cells.

Disclosures
No relevant conflicts of interest to declare.
Lenalidomide Immunomodulation with an Allogeneic Myeloma GVAX in a Near Complete Remission Induces Durable Clinical Remissions
Authors: Noonan Kimberly , Huff Carol Ann , Sidorski Amy , Ferguson Anna , Rudraraju Lakshmi , Casildo Andrea , Borrello Ivan 
Published: October 2019

Abstract
Background: Increasing evidence suggests that the depth of response in myeloma (MM) correlates with delayed time to progression1.We have previously shown that lenalidomide (Len) can augment vaccine efficacy to the pneumococcal conjugate vaccine (PCV), Prevnar2.We extend those observations to examine whether vaccinating patients on Len in a near complete remission (nCR) (negative M-spike, IFE positive) could further deepen the clinical response and generate measurable myeloma specific immunity in a small Phase II study.
Design:Patients needed to be on a Len-containing regimen and needed to be in one of the following categories for enrollment: 1) in a stable nCR for at least 4 months; 2) converting from IFE negative to IFE positive; or 3) showed signs of early relapse from a nCR to an M-spike <0.3g/dL. Patients would be continued on single agent Len and receive 4 vaccinations consisting of two allogeneic MM lines, H929, U266 admixed with K562 transduced to express GM-CSF as well as PCV. Patients received 3 monthly vaccines and a boost at 6 months.
Results: To date 32 patients have been screened: 12 had relapsed and 5 entered into an IFE negative CR during the 4 month observation period; 11 have been enrolled. Median follow-up for the study is 13.9 months. Of the vaccinated patients, category 1: 4 patients: 3 are in a true CR (IFE-),1 lost the original clone and is still in a nCR. Category 2: 2 patients both achieved a true CR. Category 3: 5 patients: 2 stable disease, 3 progressive disease. The overall true CR rate is 54%.of the patients that achieved a CR with a median follow-up of 17.1 months. Laboratory analysis showed that the patients achieving a CR had more central memory CD8 T cells at baseline in the BM and blood, a greater activation phenotype in CD8 cells in the BM and greater tumor specific IFNg production. Interestingly, LAG-3, TIM-3 and PD-1 on CD8 T cells were more expressed in the BM of CR patients.
Conclusions: Vaccination in combination with Len appears to durably deepen the clinical response in patients in a nCR whereas patients with early relapse appear less likely to respond. The deepening the clinical response correlates with the development of myeloma-specific immunity. Vaccination of MM patients in nCR in combination with Len shows promising early clinical activity that warrants further investigation as an approach to maintaining durable clinical remissions.
1.Chanan-Khan, A.A., and S. Giralt. 2010. Importance of achieving a complete response in multiplemyeloma, and the impact of novel agents. J Clin Oncol 28:2612-2624.
2. Noonan, K., L. Rudraraju, A. Ferguson, A. Emerling, M.F. Pasetti, C.A. Huff, and I. Borrello. 2012. Lenalidomide-induced immunomodulation in multiple myeloma: impact on vaccines and antitumor responses. Clin Cancer Res 18:1426-1434.

Disclosures
Noonan: Celgene: Speakers Bureau. Sidorski:Celgene: Speakers Bureau.
Maintenance of Respiration Capacity Is an Early Predictor of Drug Resistance in Cancer Cells
Authors: Brendel Cornelia A , Henkenius Katharina , Maerken Melanie , Kaiser Tom , Greif Larissa , Metzelder Stephan , Rehberger Miriam , Neubauer Andreas 
Published: October 2019

Abstract
Inherent or acquired drug resistance is the major obstacle in cancer therapy. An individual prediction of response to cancer therapy would be highly appreciated. It has been reported that the mitochondrial function of cancer cells correlates with the treatment response in different cancer entities (Chonghaile et al., Science 2011 and Vo et al., Cell 2012).
However, as the method of BCL-2 peptide priming measurement (“BH3 profiling”) seemed to be rather complicated and error-prone in our hand we sought to determine mitochondrial function by analyzing cellular respiration. We established three different drug resistant human cancer cell lines (MV4-11, HL-60 and NCl-H82) by continuous exposure to Sorafenib, Cytarabine (Ara-C) or Etoposid (VP-16) respectively. Cellular respiration was measured as oxygen consumption rate (OCR) 24h after drug exposure employing the XF96 extracellular Flux Analyzer. Viability, reduction equivalents NADH/NADPH and mitochondrial membrane potential of the cells were concomitantly determined by flow cytometry via 4’, 6-diamidino-2-phenylindole (DAPI) exclusion, (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Tetramethylrhodamine ethyl ester (TMRE) staining with flow cytometric analysis, respectively.
All resistant cancer cells maintained their respiration capacity upon exposure to tyrosine kinase inhibitors, chemotherapeutics or topoisomerase inhibitors, whereas the respiration of drug sensitive cancer cells decreased significantly. Moreover, resistant MV4-11 and HL-60 cells exhibited a metabolic shift towards glycolysis after drug treatment. In contrast sensitive cancer cells showed a decline in respiration and glycolysis in a dose dependent manner. Decrease of respiration was evident at 24h post treatment, while the onset of apoptosis was measurable 48-120h later. Reduction equivalents NADH/NADPH but not mitochondrial membrane potential were diminished in sensitive cells after 24h drug exposure, but dose dependency was more distinct with OCR analysis. The increase of glycolysis in resistant cell lines was also not detectable with the MTT assay.
Our data indicate cellular metabolism and in particular respiration as an early, sensitive and reliable surrogate parameter of drug sensitivity in viable cells that might therefore be applicable in order to determine drug responsiveness in primary cancer cells in a prospective manner.

Disclosures
No relevant conflicts of interest to declare.
Targeting C-Myc Promoter: Helquats As Novel G-Quadruplex Stabilizing Ligands
Authors: Kuzmova Erika , Kozak Jaroslav , Komarkova Veronika , Pytlik Robert , Teply Filip , Miroslav Hajek 
Published: October 2019

Abstract
Influencing stability of G-quadruplexes that can be formed in specific guanine-rich sequences has emerged as a promising route to anticancer drug design in recent years. These sequences are highly prevalent in human genome and occur in the telomeres as well as in promoters of certain genes upregulated in cancer (e.g. c-Myc, K-ras, VEGF, bcl-2, c-kit, or HIF-1α). Functional role of these G-quadruplex structures has been tested using G-quadruplex-stabilizing ligands in cell-based assays, resulting in suppression of transcription of oncogenes. As c-Myc deregulation is found in many types of human leukemia and lymphoma and its overexpression is often associated with disease progression, we chose it as our primary target.
We designed a library of helical structures with cationic aromatic moieties called helquats to find a novel class of G-quadruplex specific ligands. Their non-planar structure minimizes potential of intercalation into DNA duplex. To screen a library of about 1500 compounds synthesized in-house and find ligands that stabilize c-Myc G-quadruplex at low micromolar concentrations, we used G-quadruplex forming sequence NHE III1 of c-Myc promoter in various biophysical and biochemical experiments such as FRET (Förster resonance energy transfer) melting experiments, CD spectroscopy, and DNA polymerase stop assay. We tested promising candidates for reducing c-Myc mRNA and protein expression in several leukemic cell lines and in reporter assay using an experimental vector with firefly luciferase under control of c-Myc promoter region containing either wild-type or mutated G-quadruplex NHE III1 sequence. The most potent helquats showed more than 50 % inhibition of c-Myc mRNA and protein expression (at 25 μM, after 24 hours) in HL-60, Ramos, and U937 cell lines overexpressing c-Myc. On the other hand, no such an effect of helquats was observed in CA46 Burkitt lymphoma cells in which NHE III1 element has been lost during the translocation. Similar results as with cell lines were obtained in primary cells with or without c-Myc translocation.
Our results provide the foundation for the development of new helquat derivatives as effective inhibitors of c-Myc expression and as potential anticancer agents in hematooncology.
This study was supported by IOCB, AS CR, v.v.i. (RVO: 61388963), Project InterBioMed LO1302 from Ministry of Education of the Czech Republic and PRVOUK-27/LF1/1

Disclosures
No relevant conflicts of interest to declare.