bluuebunny/crossref_metadata_2025_split huggingface dataset:
Blockade of CD47-Sirpα Interactions Improved Myeloid Effector Cell Mediated Lymphoma Cell Killing By the HLA-DR Antibody Apolizumab
Authors: Petersen Judith , Rösner Thies , Kölling Max , Klausz Katja , Kellner Christian , Humpe Andreas , Weiner George J , Schewe Denis Martin , Peipp Matthias , Valerius Thomas 
Published: November 2019

Background:Apolizumab (APO) is a humanized IgG1 antibody targeting a HLA-DRβ chain specific epitope, which is expressed by approx. 50 % of B cell lymphomas. The antibody's clinical development was hampered by toxicity, potentially mediated by its strong CDC activity. However, previous studies demonstrated that APO efficiently triggered tumor cell killing by myeloid cells, especially by granulocytes. Myeloid effector cell engagement is strongly regulated by CD47-SIRPα interactions, and blockade of this axis has been demonstrated to improve the efficacy of therapeutic antibodies. Hence, our goal was to characterize the influence of CD47-SIRPα blockade on myeloid cell engagement by APO.
Methods: Expression of the APO epitope, CD20 and CD47 on different B lymphoma cell lines was measured by flow cytometry. The capacity of APO and Rituximab (RTX) to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) by mononuclear (MNC) or polymorphonuclear (PMN) effector cells was analysed by 51Cr release assays. Antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages was analysed by fluorescence microscopy. PMN-mediated ADCC and macrophage-mediated ADCP experiments were performed in the presence or absence of the CD47 blocking antibody 5F9-IgG2σ to analyse the impact of CD47-SIRPα blockade on APO-mediated lymphoma cell killing by myeloid cells.
Results: As anticipated we observed binding of APO on approx. 50% of the tested B lymphoma cell lines, including Raji (Burkitt's lymphoma), MINO (mantle cell lymphoma) and Carnaval (double hit DLBCL). Focusing on these cell lines, APO's CDC activity was more potent (Raji) or similar (Mino) compared to RTX. Furthermore, APO significantly induced tumor cell lysis by PMN or MNC in 51Cr release assays and achieved higher lysis rates in comparison to RTX. Blocking CD47-SIRPα interactions by 5F9-IgG2σ resulted in significantly increased APO-induced ADCC by PMN against all tested B cell lymphoma lines (Mino: 28.8 ± 10.2 % vs. 41.8 ± 13.3 %; Carnaval: 37.9 ± 17.9 % vs. 55.6 ± 17.6 %; Raji: 23.6 ± 11.7 % vs. 52.4 ± 19.4 % for APO alone vs. APO + 5F9-IgG2σ, respectively, p < 0.05 by ANOVA). However, no ADCC by PMN and RTX was observed - even in the presence of CD47-SIRPα blockade. Moreover, APO mediated similar or even stronger ADCP by macrophages compared to RTX, which could be further enhanced by 5F9-IgG2σ (Figure 1).
Conclusions: APO's potent myeloid cell activation could be further enhanced by disruption of CD47-SIRPα interactions. Our observations suggest to generate isotype variants of APO with lower CDC but preserved or enhanced ADCC activity. Subsequently, a combination of APO and CD47-SIRPα blocking strategies may improve B cell lymphoma immunotherapy.

Disclosures
No relevant conflicts of interest to declare.
Iron Catalyzed Hydrogen Sulfide and Its Prevalence during Oxidative Events in Sickle Cell Disease
Authors: Yang Jie , Bithi Nazmin , Link Christopher , Saunthararajah Yogenthiran , Hine Christopher 
Published: November 2019

In sickle cell disease (SCD), red blood cell (RBC) oxidation-reduction (redox) status is a critical determinant of hemolysis and RBC adhesion/vaso-occlusive crises (VOC). Iron, while essential for hemoglobin and RBC production, also is an important catalyst in processes that produce reactive oxygen species (ROS) which damage RBCs and ultimately other tissues. This has necessitated mechanisms for iron sequestration to control its activity, primarily achieved through binding of iron to heme, hemoglobin, and ferritin. In hemolytic disorders such as SCD, however, there is overload of protein-free, unbound iron, which by increasing ROS, significantly contributes to disease pathology. There is hence a need for novel druggable targets and treatments to attenuate this pathway of ROS production and pathophysiology. Previously, we determined a novel catalytic role of iron in which hydrogen sulfide (H2S) gas is produced both in vitro and in blood ex vivo by iron- and Vitamin B6- mediated catalysis of cysteine. We additionally determined that the bound versus unbound status of iron as well as the integrity of RBCs dictates this non-enzymatic capacity for H2S production. H2S serves as a potent regulatory redox factor throughout the body, and here we hypothesize that excess H2S in blood produced via iron catalysis protects against oxidative stress generated during SCD-related hemolytic crisis and VOC. In the current study, we further show that H2S produced in vitro from iron and Vitamin B6 catalysis of cysteine not only exists as a gas in the headspace, but itself reacts with iron in the solution. This was determined by the formation of acid labile iron sulfide, which formed a light yellow precipitate. The addition of a strong acid removed the yellow colored precipitate and resulted in further release of H2S in to the headspace. This indicates that H2S was interacting with free iron (Fe3+) and formed the iron sulfide product, which may have decreased capabilities for the formation of tissue- and cellular-damaging ROS. On-going work will examine the inhibition of H2S on iron-induced ROS generation in vitro and test the therapeutic potential of applying D-/L-cysteine and/or Vitamin B6- supplemented dietary interventions to enhance iron/B6 catalyzed H2S production in blood to lessen oxidative stress and VOC events in SCD mice in vivo. Free-iron in SCD is conventionally considered only to be a liability. However, here we exploit iron as a catalyst for beneficial H2S production in blood that can protect against oxidative stress and SCD-related pathologies. This recently discovered pathway has the potential to create novel opportunities and approaches to modulate the devastating pathophysiology of SCD.
Figure

Disclosures
Saunthararajah: Novo Nordisk: Consultancy; EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties.
Combination of Gatekeeper Mutations and Cysteine 481 Replacement Causes Super Resistance to the Irreversible BTK Inhibitors Ibrutinib, Acalabrutinib and Zanubrutinib
Authors: Estupiñan Velasquez Hernando Yesid , Shi Yuye , Mohammad Dara K. , Wang Qing , Vihinen Mauno , Zain Rula , Berglöf Anna , Smith Edvard CI 
Published: November 2019

Resistance to the irreversible Bruton´s tyrosine kinase (BTK) inhibitors is the main cause of disease progression in patients with Chronic Lymphocytic Leukemia (Quinquenel et. al. Blood 2019). Cysteine to serine substitution at the position 481 in BTK, is the most common resistance mutation. Other less frequent mutations like the constitutively active phospholipase C-gamma 2 (PLCG2) variant also occur (Woyach et. al. J. Clin. Oncology 2017). Different from many other tyrosine kinase inhibitors, BTK mutations less frequently affect the gatekeeper residue in the kinase domain (Maddocks et. al. JAMA Oncology 2015).
In this study, we have performed mutation scanning with substitutions replacing the gatekeeper residue. We have generated all the possible amino acid substitutions requiring a single nucleotide change in the gatekeeper and several variants requiring 2 or 3 nucleotide substitutions. Selected variants were also combined with substitutions at the C481, which is the binding site of irreversible BTK inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib.
Our results show unexpected, super-resistant variants and demonstrate that concomitant mutations, such as cysteine 481 to serine combined with threonine 474 to isoleucine or methionine, enhanced the resistance to irreversible BTK inhibitors. On the other hand, reversible BTK inhibitors displayed different inhibitory responses against the super-resistant mutants. Binding of the BTK inhibitors was subjected to molecular dynamics predictions, which correlated with the experimental binding data. Based on the available clinical and experimental results, the mechanisms underlying the spectrum of resistance mutations in BTK are presented.

Disclosures
No relevant conflicts of interest to declare.
Targeting Transcriptional Co-Activator PGC-1a for the Treatment of Sickle Cell Disease
Authors: Habara Alawi , LE Cuong , Chui David H.K. , Steinberg Martin H. , Cui Shuaiying 
Published: November 2019

Sickle cell disease (SCD) is the most common inherited human hematologic disease, which causes hemolytic anemia, pain, disability, progressive multi-organ damage and early mortality. Clinical studies have shown that increased synthesis of fetal hemoglobin (HbF) in sickled erythroid cells leads to diminished severity of many clinical features of SCD. Therefore, therapeutic agents that can increase HbF production will be of benefit to SCD patients. Hydroxyurea (HU) is a FDA-approved therapeutic for treatment of SCD, but not all patients respond favorably or adequately. Therefore, other methods of targeting HbF are highly desired, particularly those that act by different mechanisms that might be used in combination with HU or alone (for those who do not tolerate HU). Transcriptional co-activator PGC-1α has recently been showed to play a crucial role in globin gene regulation and erythropoiesis by our group. Loss of function in the PGC-1α knock out mice significantly reduced the expression of murine embryonic εy- and βh1-globin genes; both of them are homologues of the human ε- and γ-globin genes. Forced overexpression of PGC-1α in vitroby adenovirus infection in bone marrow cells from SCD mice resulted in significantly increased human γ- and murine εy- and βh1-globin genes. These results directly validate a role for PGC-1α in human γ-globin induction and provide significant support for the hypothesis that modulating PGC-1α activity, or the signaling pathways that it regulates, in SCD patients may be an effective approach that could therapeutically benefit individuals with SCD. Here we report the effects of PGC-1α in inducing HbF in human primary erythoid progenitor CD34+cells. We infected CD34+cells with lentivirus carrying PGC-1α and found that over-expression of PGC-1α resulted in significantly increased F-cells in the culture, from 1.6% up to 41.1%. We further discovered several PGC-1α agonists that are highly selective for PGC-1α. We found that one of the PGC-1α agonists, Compound SR-18292, induces PGC-1α mRNA expression in a dose dependent manner (2.6-fold at 1 μM, 5.7-fold at 10 μM) in cultured CD34+cells without effecting cell viability. Consistent with the increase of PGC-1α levels, the percentage of F-cells increased from 2.76% (vehicle control) to 32.1% when cells exposed to 1 µM SR-18292, which is comparable to that of 25 µM HU (32.2%). The mean fluorescence intensity of F-cells in SR-18292 treated cells was higher than in controls, suggesting that the treatment not only increased the number of F-cells but also increased the concentration of HbF in F-cells. The increased levels of PGC-1α reactivates the expression of fetal γ-globin genes resulting in significant HbF accumulation in adult erythrocytes, suggesting that the transcriptional co-activator PGC-1α comprises a new molecular target for possible therapeutic intervention in treating SCD.

Disclosures
No relevant conflicts of interest to declare.
Protein Phase Separation in Hematopoietic Stem Cell Aging
Authors: Maneix Laure , Iakova Polina , Moree Shannon , Sweeney Melanie , Moka Nagaishwarya , Yellapragada Sarvari Venkata , Catic Andre 
Published: November 2019

Imbalanced protein homeostasis (proteostasis) is a driver of aging and a vulnerability for long-lived cells such as hematopoietic stem cells. To determine critical proteostasis factors, we analyzed the proteome of hematopoietic stem and progenitor cells and found prolyl isomerases to be the dominant cytosolic chaperones. Genetic removal of prolyl isomerases led to accelerated aging in the stem cell compartment. We identified intrinsically disordered proteins as common substrates of these chaperones, including several key players that control phase transition. Phase separation allows the formation of supramolecular membrane-less organelles that regulate DNA and RNA biology as well as protein translation. Using microscopy and biochemistry, we show that prolyl isomerases promote phase separation and thereby increase cellular resistance to stress. Our research links a ubiquitously expressed chaperone family to phase transition and identifies macromolecular condensation dynamics as a driver of blood stem cell aging.

Disclosures
Yellapragada: Novartis: Employment, Other: Spouse Employment ; Celgene: Research Funding; BMS: Research Funding; Takeda: Research Funding.
Mesenchymal COX2-Derived PGD2 Activates an ILC2-Treg Axis to Promote Proliferation of Normal and Malignant HSPCs
Authors: Wu Limei , Lin Qiqi , Ma Zhilin , Chowdhury Fabliha , Mazumder Md Habibul , Du Wei 
Published: November 2019

Cyclooxygenase (COX)-dependent production of prostaglandins (PGs) is known to play important roles in tumorigenesis. PGD2 has recently emerged as a key regulator of tumor- and inflammation-associated functions. We previously reported that mesenchymal stromal cells (MSCs) from patients with acute myeloid leukemia (AML) overexpressed COX-2 and secreted high levels of PGs including PGD2. Since little is known about the role of PGD2 in normal and malignant hematopoiesis, we prioritized this mesenchymal source of PG for further investigation. We observed that AML MSCs or normal MSCs overexpressing COX-2 promotes proliferation of co-cultured hematopoietic stem and progenitor cells (HSPCs), which can be prevented by treatment with COX-2 knockdown or TM30089, a specific antagonist of the PGD2 receptor CRTH2. Mechanistically, we demonstrate that PGD2-CRTH2 signaling acts directly on type 2 innate lymphoid cells (ILC2s), potentiating their expansion and driving them to produce Interleukin-5 (IL-5) and IL-13. We further show that IL-5 but not IL-13 expands CD25+Foxp3+ IL5Ra+ T regulatory cells (Tregs) and promotes HSCP proliferation. Disruption of the PGD2-activated ILC2-Treg axis by specifically blocking the PGD2 receptor CRTH2 or IL-5 impedes proliferation of normal and malignant HSPCs. Conversely, co-transfer of Lin-CD127+CRTH2+ ILC2s and CD4+CD25+IL5Ra+ Tregs promotes malignant HSCP proliferation and accelerates leukemia development in xenotransplanted mice. Collectively, these results indicate that the mesenchymal source of PGD2 promotes proliferation of normal and malignant HSPCs through activation of the ILC2-Treg axis. These findings also suggest that this PGD2-activated ILC2-Treg axis may be a valuable therapeutic target for cancer and inflammation-associated diseases.

Disclosures
No relevant conflicts of interest to declare.
3D Telomeric Fingerprint of Advanced Cutaneous T-Cell Lymphoma
Authors: Bienz Marc Nicolas , Taylor-Kashton Cheryl , Benlimame Naciba , Petrogiannis-Haliotis Tina , Pehr Kevin , Mai Sabine , Knecht Hans 
Published: November 2019

Introduction
Cutaneous T-cell lymphomas (CTCLs) are characterized by dermal and epidermal infiltration of skin homing clonal CD4+ memory T-cells. Little is known about the oncogenic events driving either the progression of skin-limited disease such as Mycosis Fungoides (MF) to a leukemic form or Sézary Syndrome (SS), and there are no histologic means to predict evolution. Genetic instability is a hallmark of malignancy progression and telomere remodeling has been shown to play a role in the progression of hematological malignancies. Thus, the aim of this study is to characterize the three-dimensional (3D) telomeric organization in early and advanced CTCLs.
Methods
We performed 3D telomeric quantitative fluorescent in situ hybridization (3D Telo-Q-FISH) of 5mm skin tissue slides of 10 patients with MF and SS and of CD4+ lymphocytes of 3 healthy controls (Figure 1). Using the program TeloView (Vermolen et al., 2005), the proportion of telomeres of low intensity (TLI) (&lt;5000 u), the nuclear volume and the total number of telomeric signals per cell were calculated. Patients were stratified based on CD30 expression (CD30 high, n=3 versus CD30 low, n=7) and clinical stage (early stages I-IIA, n=6 versus advanced stages IIB-IV, n=4).
Results
TLI represented 27% of telomeres in CTCL cells compared to 16% in control lymphocytes (p&lt;0.0001). The highest proportion of TLIs was found in tumors CD30 high (34%), compared to 22% in tumors CD30 low (p&lt;0.0001). Similar findings were observed when stratifying for advanced and early clinical stages (30% vs 24%, p&lt;0.0001). Nuclear volume and total number of telomeric signals increased in CTCL cells expressing CD30 (both p&lt;0.0001), and in CTCL cells associated with advanced clinical stages (p=0.0021 and p=0.001, respectively). However, as expected, nuclear volume and total number of telomeric signals were higher in control cells compared to CTCL cells (both p&lt;0.0001).
Conclusion
We report clear evidence that CTCLs with either CD30 expression or advanced clinical stage are associated with loss of telomeres and telomeric signal intensity. These very small telomeres, termed 't-stumps', are a hallmark feature in many tumor cells. Analysis of the nuclear volume and total number of telomeric signals suggest that CTCL cells undergo in a first step telomere shortening and loss compared to healthy controls. In a second step further telomeric shortening associated with chromosomal rearrangements and bridge-breakage-fusion cycles may be involved in the progression of CTCL.

Disclosures
Pehr: Merck: Consultancy, Other: Clinical research on Alzheimers Drug; Eli Lilly: Consultancy, Other: Clinical research on Alzheimers Drug; galderma: Consultancy, Other: tactupump Forte; Actelion: Consultancy; valeant: Consultancy; CeraVe: Consultancy; Janssen-Cilag: Other: Train the trainer.
Superior Graft-Versus-Leukemia Effect in Haploidentical Transplantation Compared with HLA-Matched Sibling Transplantation for High-Risk Acute Myeloid Leukemia in First Complete Remission: A Prospective Multicentre Cohort Study
Authors: Yu Sijian , Liu Qifa 
Published: November 2019

This study aimed to investigate graft-versus-leukemia (GVL) of haploidentical donor (HID) compared with HLA-matched sibling donor (MSD) for high-risk acute myeloid leukemia (H-AML) in first complete remission (CR1). One hundred and eighty-nine patients with H-AML in CR1 were enrolled in this multicentre prospective cohort study. Patients were assigned to groups transplanted with HID (n=83) or MSD (n=106) based on donor availability (biological randomization). The primary endpoint was the incidence of MRD positivity post-transplantation (post-MRD+). All post-MRD+ patients received preemptive interventions. The cumulative incidences of post-MRD+ were 18% and 42% in HID and MSD groups, respectively, (p<.001). Fifty-two patients received preemptive DLI, including 13 (16%) in HID and 39 cases (37%) in MSD groups (p=.001). Among HID and MSD groups, the 3-year cumulative incidence of relapse were 14% and 24% (p=0.101); the 3-year cumulative incidence of treatment-related mortality were 15% and 10% (p=0.368); the 3-year overall survival rates were 72% and 68% (p=0.687); the 3-year disease-free-survival were 71% and 66% (p=0.579); the 3-year graft-versus-host disease and relapse free survival were 63% and 43% (p=0.035), respectively. HID has a stronger GVL than MSD in H-AML patients. HID transplantation as post-remission therapy should be recommended as one of the optimal choices for H-AML in CR1.

Disclosures
No relevant conflicts of interest to declare.
A Rare Case of Kimura Disease in the Wrist
Authors: Tizro Parastou , Hashemi Zonouz Taraneh , Almutairi Fawaz , Karcher Donald 
Published: November 2019

Kimura disease is a rare benign chronic inflammatory disorder of unknown etiology that typically involves lymph nodes and soft tissues. This disease is most common in middle-aged Asian men. Although the disorder most frequently involves the head and neck region, it has also been reported to involve the extremities. Lesions typically show follicular lymphocytic hyperplasia with prominent interfollicular venules and a mixed inflammatory infiltrate, including numerous eosinophils. We describe a case of Kimura disease in a 29-year-old female presenting with a slowly enlarging, painless soft tissue mass in her left wrist for five months. The lesion was non-tender, mobile, and compressible. Lab results were significant for peripheral blood eosinophilia and elevated IgE. Microscopically, the mass was circumscribed, with apparent fibrous capsule or pseudocapsule, prominent high-endothelial venules, and inflammatory cells consisting of lymphocytes and eosinophils. There were prominent secondary B-lymphocytic follicles with germinal centers containing many penetrating small blood vessels, and some follicles exhibiting follicle lysis and a small amount of eosinophilic extracellular material. Polykaryocytes were noted in many germinal centers. Angiolymphoid hyperplasia with eosinophilia, a main differential diagnosis of Kimura disease, was considered; however, the combined clinical and morphologic findings appeared to be more consistent with Kimura disease, particularly in the context of eosinophilia and elevated IgE level. Kimura disease may present as a rare cause of a soft tissue mass and a diagnostic challenge for clinicians and pathologists, especially in less common sites such as the wrist.

Disclosures
No relevant conflicts of interest to declare.
Phase Ib of Copanlisib in Combination with Ibrutinib in Recurrent/Refractory Primary CNS Lymphoma (PCNSL)
Authors: Grommes Christian , Gavrilovic Igor , Miller Alexandra M , Stone Jacqueline B , Kaley Thomas , Madzsar Juli T , Schumpp Alyssa , Butler Brittany T , Schaff Lauren , Mellinghoff Ingo K 
Published: November 2019

BACKGROUND: PCNSL is an aggressive primary brain tumor with median progression free survival (PFS) after upfront methotrexate-based chemotherapy of 2-3 years. Outcome and treatment options are poor for recurrent/refractory (r/r) disease. This trial combines the pan-PI3K inhibitor copanlisib with Ibrutinib in patients with r/r PCNSL.
METHODS: Eligible patients had r/r PCNSL, age≥18, ECOG≤2, normal end-organ function, and an unrestricted number of CNS directed prior therapies. Ibrutinib was given orally daily; copanlisib intravenously on days 1, 8, and 15 of each 28-day cycle.
RESULTS: Six patients have been enrolled so far and received copanlisib at 60 mg and ibrutinib at 560 mg. Median age was 68 (range 50-77); 3 were women. Median ECOG was 1 (0: 2, 1: 3, 2: 1). All had recurrent parenchymal disease. Three had additional cerebrospinal fluid (CSF) involvement. Two had received prior single-agent ibrutinib. Initially, no prophylactic antimicrobial treatment was required. PCP prophylaxis was made mandatory after one patient developed PCP pneumonia leading to a grade 5 lung infection. Four patients are still on trial and one withdrew due to personal choice. Two grade 4 adverse events were observed in 2 patients (LFT elevation, lymphopenia); four grade 3 events in 3 patients (rash, lymphopenia, LFT elevation). The most common toxicities at any grade were transient, infusion-related hyperglycemia and hypertension. No Aspergillus infections have been observed. Enrollment into the next dose level (copanlisib 60 mg, ibrutinib 840 mg) is ongoing. After a median follow-up of 180 days (range 46-249), all patients were evaluated for response with an overall response rate of 67% with 1 CR, 3 PR, 1 SD and 1 PD as best response.
CONCLUSION: Treatment with copanlisib and ibrutinib in patients with PCNSL has manageable adverse events after initiation of mandatory PCP prophylaxis. Clinical response was seen in 67% of patients.

Disclosures
Grommes: Squipps: Speakers Bureau; Kite: Consultancy; BTG: Consultancy.

OffLabel Disclosure:
The combination of the BTK inhibitor ibrutinib and the pan PI3K inhibitor copanlisib in recurrent PCNSL will be discussed