bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of Mcl-1 Inhibitors in Preclinical Models of Multiple Myeloma
Authors: Belmonte Matthew A , Adam Ammar , Borrelli Deanna , Bhavsar Deepa , Bebernitz Christina , Hird Alexander , Secrist Paul 
Published: October 2019

Abstract
Mcl-1, an anti-apoptotic Bcl-2 family member, is a known resistance factor to many cancer therapies, and a historically difficult target to drug. Recently however, we have made significant progress optimizing the potency and characterizing the mechanism of action of a novel class of selective Mcl-1 inhibitors. Here, we characterize the activity of one of our lead compounds and confirm on-target mechanistic activity in vitro and in vivo.
Using an Mcl-1 binding assay we demonstrate very potent activity (IC50 < 3nM), while maintaining selectivity over other anti-apoptotic Bcl-2 family proteins, Bcl-2 (IC50 > 33 µM) and Bcl-xL (IC50 > 33 µM). In an Mcl-1 dependent multiple myeloma cell line, MOLP-8, we demonstrate rapid Mcl-1/Bak complex disruption with 10 nM inhibitor by 15 min followed by caspase 9-dependent apoptosis by 1 hr (EC50= 50 nM), consistent with an Mcl-1 dependent mechanism of action. Importantly, by depleting Bak from cells using siRNA, we also confirm that the observed apoptosis is through a Bak-dependent mechanism. Moreover, our Mcl-1 inhibitor exhibits broad submicromolar activity across a panel of multiple myeloma cell lines.
In the absence of a validated selective Mcl-1 reference compound to benchmark in vivo activity, we engineered MOLP-8 cells to express the pro-apoptotic protein, Noxa, using a Tet-on expression system and grew these as subcutaneous tumors on the flanks of SCID mice. Within 24 hours of switching the mice to a diet containing doxycycline, we observe significant upregulation of Noxa, leading to an increase in cleaved caspase 3 (CC3) and cleaved PARP in tumor lysates, resulting in rapid tumor regression. In the same MOLP-8 xenograft model, we demonstrate that our Mcl-1 inhibitor induces rapid dissociation of Bak from Mcl-1 in tumors leading to accumulation of CC3 and cleaved PARP by 1 hr. Complete tumor regression was observed after a single 60 mg/kg or 100 mg/kg dose, while partial regression was observed after a single 30 mg/kg dose, and tumor growth inhibition was observed after a single 10 mg/kg dose. All doses were well tolerated with no significant body weight loss. Together, these data reinforce the potential utility of selective Mcl-1 inhibitors in hematological malignancies.

Disclosures
Belmonte: Astrazeneca: Employment, Equity Ownership. Adam:Astrazenenca: Employment, Equity Ownership. Borrelli:AstraZeneca: Employment, Equity Ownership. Bhavsar:AstraZeneca: Employment, Equity Ownership. Bebernitz:AstraZeneca: Employment, Equity Ownership. Hird:AstraZeneca: Employment, Equity Ownership. Secrist:AstraZeneca: Employment, Equity Ownership.
Relationship Between Metabolic Tumor Volume and Rituximab Pharmacokinetic Parameters in Non-Hodgkin’s Lymphoma
Authors: Monjanel Helene , Ternant David , Venel Yann , Prunier-Aesch Caroline , Tournamille Jean-François , Arbion Flavie , Colombat Philippe , Paintaud Gilles , Gyan Emmanuel 
Published: October 2019

Abstract
The pharmacokinetics (PK) of the monoclonal antibody rituximab in B-cell lymphomas presents a high interindividual variability. In the pivotal study, inverse correlation between rituximab concentration and tumor burden estimated by the measurement of tumor bulk at baseline has been observed (Berinstein and al., Ann. Oncol. 1998). Using a pharmacokinetic/pharmacodynamic (PK/PD) model, it has been demonstrated in a murine model of lymphoma-expressing CD20 that tumor burden influences rituximab concentration (Dayde and al., Blood 2009). The aim of the present study was to evaluate the relationship between rituximab pharmacokinetic (PK) parameters and metabolic tumor volume (MTV) in patients with a previously untreated CD20-positive non-Hodgkin's lymphoma.
Twenty-five patients (12 follicular and 13 diffuse large B-cell lymphoma) received a combination of rituximab (dose of 375 mg/m2) with 6 to 8 cycles of CHOP. All patients provided written informed consent for the study. Rituximab serum levels were measured using a validated enzyme-linked immunosorbent assay (ELISA). Tumor volume delineations were performed on 18F-FDG PET-scan at baseline (Planet Onco, Dosisoft, France). Rituximab PK was assessed using population compartmental modelling. A relationship between MTV and survival was described.
Estimations of typical (interindividual standard deviation) systemic clearance (CLc) and volume of distribution (Vc) were 0.00972 L.h-1 and 3.12 L, respectively. Median MTV was 600 mL (range, 7 - 6217 mL). The central volume of distribution and the systemic clearance increased with MTV (p<0.05) (Table). A high MTV (>600 mL) was significantly associated with decreased event-free survival (p<0.05).
This pilot study is the first to describe a correlation between initial imaging-calculated metabolic tumor volume as a continuous variable and rituximab PK parameters in patients treated with R-chemotherapy for B-cell lymphomas. An increase in MTV leaded to a decrease in exposure to rituximab. Further prospective investigations on rituximab PK and dose adjustments in relationship with tumor burden are needed. Increased rituximab dose should be considered in patients with high tumor burden.
Table. Estimated pharmacokinetic parameters Parameter (units) Estimate Precision (%) p Vc (L) 3,12 10 b Vc (MTV) (L) 0,00012 36 0,0059 Cl (L.h-1) 0,00972 14 b Cl (MTV) (L.h-1) 0,000146 46 0,03

Disclosures
Paintaud: LFB and Pierre-Fabre Laboratoire: Consultancy; Roche Pharma, Chugai, Pfizer, Novartis, Janssen: Research Funding. Gyan:Roche: Research Funding.
Sensory Neuropeptides Prime the Splenic Marginal Zone for Optimal Humoral Immunity
Authors: Tsui Hubert , Ghazarian Magar , Winer Shawn , Adissu Hibret , Dosch Michael , Reis Marciano D. , Winer Daniel , Keating Armand 
Published: October 2019

Abstract
Effective spleen-derived humoral responses are essential for protecting hosts from blood-borne pathogens. Increased susceptibility to infection is therefore a risk post-splenectomy but is also associated with smoking, chemotherapy and aging. In fact, decreased vaccine efficacy in the elderly may be due to splenic hypofunction. While investigating the role of sensory nerves in hematopoiesis, we discovered a lymphocytosis phenotype involving the splenic marginal zone (MZ). Dividing red and white pulp in the spleen, the MZ is a specialized micro-anatomic structure facilitating innate and adaptive crosstalk to incoming antigen. Sensory nerves were found to synapse at this site and in mice deficient in the major sensory neurotransmitter substance P (Tac-1-/-), MZs were expanded histologically and confirmed by flow cytometry as an increase involving the IgMhiIgDloCD21hiCD23lo MZ B cell population. Tac-1-/- mice also exhibited splenomegaly with overall Ki-67 proliferation indices of 40% vs. 5% in controls. In the peripheral blood, Tac-1-/- mice had persistent polyclonal pan-B cell lymphocytosis with an absolute increase of 200% relative to wildtype. Aberrant B cell homeostasis had functional consequences as demonstrated by significantly decreased total basal plasma IgG (P<0.001) but not IgM, suggesting a transitional defect from the low-affinity T-independent (TI) polyspecific response to T-dependent (TD) mature germinal center derived antibodies. Mechanistically, MZ B cell migration to follicles is required for optimal TD responses where they shuttle antigen-laden IgM immune complexes. Since MZ B cell activation can occur independently of the B cell receptor, relying on Toll-like receptors or other pattern recognition receptor ligation – it is reasonable to include neuropeptide release as an additional stimulus coordinating appropriate MZ B cell mobilization. A working hypothesis therefore is inflammatory stimuli conveyed by the sensory nervous system, which may or may not involve perceptible pain sensation, is rapidly conveyed to the splenic MZ via release of substance P. This may provide an integrated communication network facilitating optimal antibody formation, more efficient than relying on diffusion of conventional danger signals or chemokines alone. TI and TD antigen challenge, cell tracing and electrode stimulation experiments are underway to confirm this theory. Splenic sensory nerve manipulation or pharmacological modulation of the substance P axis may have therapeutic applications for boosting humoral immunity in a variety of immune compromised states.

Disclosures
No relevant conflicts of interest to declare.
BCL6 Orchestrates Tfh Differentiation Via Multiple Distinct Mechanisms
Authors: Hatzi Katerina , Nance J. Philip , Kroenke Mark , Haddad Elias K. , Melnick Ari M. , Crotty Shane 
Published: October 2019

Abstract
Follicular Helper CD4 T Cells (Tfh) cells are required for T cell help to B cells and are essential for germinal centers and the development of most high affinity antibody responses that are a hallmark of protective immunity. BCL6 is the defining transcription factor of Tfh cells. However, the functions of Bcl6 in Tfh have largely remained unclear. Intriguingly, Bcl6 is essential in both Tfh cells and germinal center B cells, two cells with very different functions that exist in the same place at the same time and interact extensively. How Bcl6 accomplishes different tasks in these two different cell types has remained unknown. We defined the BCL6 cistrome in primary human germinal center Tfh cells to assess mechanisms of BCL6 regulation of CD4 T cells, comparing and contrasting BCL6 function in T and B cells. Bcl6 binds over 3,000 genes in GC Tfh cells and predominantly occupies promoters, but also enhancers. In these cells BCL6 primarily acts as a repressor and BCL6 binding was associated with control of Tfh cell migration, TCR signaling, and repression of alternative cell fates. Interestingly, although some BCL6 bound genes possessed BCL6 DNA binding motifs, many BCL6-bound loci were instead characterized by the presence of DNA motifs for AP1 or STAT. AP1 complexes are key positive downstream mediators of TCR signaling and external stimuli. We show that BCL6 can directly bind AP1, and AP1 and BCL6 co-occupied BCL6 binding sites with AP1 motifs, suggesting that BCL6 subverts AP1 activity. These findings reveal that BCL6 has broad and multifaceted effects on Tfh biology, and provide insight into how this master regulator mediates distinct cell-context dependent phenotypes.

Disclosures
Melnick: Genentech: Speakers Bureau; Janssen: Research Funding; Calgene: Consultancy; Bioreference: Scientific Advisory Board, Scientific Advisory Board Other.
Mitochondrial Uncoupling Protein 2 Is Present in Human Platelets and Regulates Platelet Activation
Authors: Shiva Sruti , Novelli Enrico M , Bullock Grant C , Kenny Elizabeth , Hill Gabrielle , Corey Catherine G 
Published: October 2019

Abstract
It has long been recognized that platelets contain functional mitochondria and accumulating data suggest that several aspects of mitochondrial function, including reactive oxygen species production, modulate platelet thrombotic function. We have recently shown that platelet mitochondria isolated from patients with Sickle Cell Disease generate significantly greater concentrations of reactive oxygen species than healthy African American subjects and that this mitochondrial oxidant generation contributes to higher levels of basal platelet activation in these patients (Blood. 2014 May 1;123(18):2864-72). Based on these data, we have now investigated the factors that regulate platelet mitochondrial superoxide generation in healthy and Sickle Cell Disease platelets. Here we demonstrate that human platelets express mitochondrial uncoupling protein-2 (UCP2), a protein that is known to decrease the efficiency of oxidative phosphorylation and oxidant generation in other cell types but has previously not been identified in platelets. In this study we show that UCP2 protein is expressed in healthy human platelets and is fully functional as it facilitates proton leak across the inner mitochondrial membrane, leading to decreased mitochondrial membrane potential. Further, we demonstrate that the expression of this protein attenuates platelet mitochondrial superoxide generation, as treatment of platelets with Genipin (2-10µM), a pharmacological inhibitor of UCPs, concentration-dependently increases mitochondrial membrane potential and reactive oxygen species production. Further, an approximately 70% inhibition of UCP activity results in platelet activation demonstrated by increased membrane p-selectin expression (65±7% versus 6±3% in untreated controls) and augmented glycoprotein IIb/IIIa activation (57±9% versus 9±4% in untreated controls). The use of the mitochondrial-targeted antioxidant mitoTEMPO (10µM) decreases genipin-induced superoxide generation and significantly attenuates platelet activation. Notably, preliminary data presented here also suggest that UCP expression is decreased in platelets isolated from Sickle Cell Disease patients (3-fold) compared to healthy African American subjects. Additionally, ongoing studies are investigating platelet function in mice deficient in UCP2. Taken together, these data demonstrate a novel mechanism of regulation of platelet thrombotic function whose physiological relevance is apparent in the context of Sickle Cell Disease. More broadly this study advances the understanding of the role of the mitochondrion in platelet biology and thrombotic disease.

Disclosures
No relevant conflicts of interest to declare.
Integrin αIIbβ3-Mediated Outside-in Signaling: Brake or Amplifier of Platelet Activation?
Authors: Dai Baiyun , Wu Peng , Xue Feng , Yang Renchi , Yu Ziqiang , Dai Kesheng , Ruan Changgeng , Zhu Jieqing , Newman Peter J. , Gao Cunji 
Published: October 2019

Abstract
αIIbβ3 is the most prominent integrin in platelets, and binding to its ligands, in addition to supporting platelet aggregation, also results in the transmission of so-called αIIbβ3-mediated outside-in signals into the cell interior. While it is well accepted that integrin-mediated outside-in signaling functions as an amplifier of platelet activation, accumulating evidence suggests that outside-in signaling can, under certain conditions, function as an inhibitor of platelet activation. In this regard, previous studies have shown that ligand binding and platelet aggregation activate the inositol phosphatase SHIP-1, a negative regulator of the PI3K/Akt signaling pathway, to shift activated integrins back to their resting state, leading to dissociation of platelet aggregates. Because the PI3K/Akt signaling pathway is also involved in platelet granule secretion, we examined whether ligand binding to αIIbβ3 might transmit inhibitory signals that suppress platelet granule secretion. Interestingly, we found that antagonists of integrin αIIbβ3 promote both platelet dense- and α-granule secretion stimulated by low dose agonists. In support of this finding, both mouse and human platelets lacking expression of αIIbβ3 exhibited increased granule secretion compared to their wild-type counterparts. Conversely, Mn++-induced fibrinogen binding to αIIbβ3 inhibited low-dose agonist-induced platelet granule secretion. Biochemical analysis revealed that blocking ligand binding to, or absence of, αIIbβ3, enhanced agonist-induced Akt phosphorylation, while at the same time prevented the activation of the inhibitory enzyme, SHIP-1. To further investigate the role of SHIP-1 in inhibitory signaling, we examined the effect on platelet secretion of 3AC, a specific inhibitor of SHIP-1. We found that 3AC not only restores ADP-induced platelet granule secretion, but also increases CRP- or TRAP-induced platelet granule secretion. Taken together, these data demonstrate that integrin αIIbβ3-mediated outside-in signaling act as a brake to restrict unnecessary platelet activation that occurs in the presence of low-dose agonist stimulation.

Disclosures
No relevant conflicts of interest to declare.
Novel Mouse Hemostasis Model for Real-Time Determination of Bleeding Time and Hemostatic Plug Composition
Authors: Bergmeier Wolfgang , Piatt Raymond , Petrich Brian G. , Monroe Dougald , Mackman Nigel , Getz Todd M 
Published: October 2019

Abstract
Introduction: Hemostasis is a rapid response by the body to stop bleeding at sites of vessel injury. Both platelets and fibrin are important for the formation of a hemostatic plug. Mice have been used to uncover the molecular mechanisms that regulate the activation of platelets and coagulation under physiological conditions. However, measuring hemostasis in mice is quite variable and current methods do not quantify platelet adhesion or fibrin formation at the site of injury.
Methods: We describe a novel hemostasis model that uses intravital fluorescence microscopy to quantify platelet adhesion, fibrin formation, and time to hemostatic plug formation in real-time. Repeated vessel injuries of ~50-100µm in diameter were induced using laser ablation technology in the saphenous vein of mice.
Results: Hemostasis in this model was strongly impaired in mice deficient in glycoprotein (GP)Ibα (IL4R/GPIb-tg) or talin-1 (talin1f/f PF4-Cre), important regulators of platelet adhesiveness. In contrast, the time to hemostatic plug formation was only minimally affected in mice defective in the extrinsic (tissue factor (TF)low) or the intrinsic (FIX-/-) coagulation pathways, even though platelet adhesion was significantly reduced. Interestingly, fibrin accumulation was markedly increased in lesions of talin1f/f PF4-Cre, IL4R/GPIb-tg and FIX-/- mice, while it was decreased in TFlow mice. These findings suggest that prolonged plasma exposure to TF leads to increased thrombin and fibrin generation in the surrounding tissue. A partial reduction of platelet adhesiveness using clopidogrel led to instability within the hemostatic plug, especially when combined with impaired coagulation in TFlow mice.
Conclusions: In summary, we present a novel, highly sensitive method to quantify hemostatic plug formation in mice. This new model has several defining characteristics, including the use of intravital fluorescence microscopy to monitor the hemostatic process, the novel use of laser ablation technology to generate vascular lesions with a defined diameter, the ability to repeatedly disrupt the hemostatic process at the same site of injury, and the possibility to perform multiple injuries along the exposed saphenous vein. Based on its sensitivity towards platelet adhesion defects and its real-time imaging capability, we propose this model as an ideal tool to study the efficacy and safety of antiplatelet agents.

Disclosures
No relevant conflicts of interest to declare.
A Lesson from the Use of Optional Inferior Vena Cava Filters of Type Optease in Trauma Patients- a Single Type of Filter in a Single Medical Center
Authors: Salomon Ophira , Lavan Orly , Rimon Uri , Simon Dani , Khaitovich Boris , Segal Batia , Grossman Ehud , Kleinbaum Yeroham , Steinberg David M 
Published: October 2019

Abstract
Venous thromboembolism (VTE) is a frequent complication of patients who experienced major trauma and is accountable for almost 11% of deaths in those who survive at least 24 hours. Prevention of VTE is usually feasible through thromboprophylaxis and/or mechanical devices, but some trauma patients have injuries that make thromboprophylaxis impossible. Using inferior vena cava filter (IVC) is optional but it is not devoid of complications. Therefore the ASH in 2013 choosing Wisely® recommended avoiding routine usage of IVC filter. In this work our goal was to assess complications related to a single type of filter used prophylactically in trauma patients in a tertiary center and to search for risk factors associated with failure to remove the filter. The study included 136 patients aged 16 through 60 who experienced a major trauma and underwent prophylactic IVC filter insertion of type Optease at Sheba Medical Center during the years 2007-2013. The mean age of the patients was 36.7 with 79.6% men and 20.4% women. The predominant cause of injury was motor vehicle accidents (52%) and the mean Glasgow coma scale of patients when first seen by a traumatologist was 7.5. The median time from trauma to filter insertion was 2 days. Anticoagulation in prophylactic dose was started in 92.1% patients (117/127, no information for 9) once filter was inserted. The filter was removed in 68 (50%) of patients. In 13 patients the attempted filter removal was not successful for mechanical reasons. Nine patients developed IVC thrombosis and one of them died because of development of bilateral phelgmasia cerulea dolens leading to ischemic foot. Another 8 patients developed lower extremities DVT and therefore the filter was left in place. Among patients with a successful removal, the median dwelling time was 30 days, with a minimum of 8 days and a maximum of 207 days. As seen in the table, no relationship was found between age, sex, BMI, or GCS to successful filter removal.
Table 1. Successful Unsuccessful Mean age 35.3 (sd = 12.8) 38.1 (sd = 12.7) p = 0.20 Sex 81% Male 80% Male p = 0.93 BMI 25.3(sd = 4.7)kg/m2 26.9(sd = 7.3) kg/m2 p = 0.17 GCS 7.4(sd = 6.1) 7.8(sd = 5.8) p = 0.70
Of concern are the persistent complications related to the filter such as IVC thrombosis even with the prophylactic use of anticoagulants. Further, the long-term effects of radiation needed for insertion and extraction of the filter are worrisome, especially when it involves young patients. Thus the routine use of IVC filter in trauma patients may not be desirable. Careful assessment of the risks and the benefits is needed especially for young patients.

Disclosures
No relevant conflicts of interest to declare.