bluuebunny/crossref_metadata_2025_split huggingface dataset:
Direct Suppression of Fas-Mediated Apoptosis by PMLRARa through Forming An Apoptotic Inhibitory Complex with c-FLIP In Acute Promyelocytic Leukemia
Authors: Tao Rong-Hua , Berkova Zuzana , Wise Jillian F , Urszula Daniluk , Bai Li , Ao Xue , Samaniego Felipe 
Published: October 2019

Abstract
Abstract 3148
Fas plays a critical role in cell proliferation and in the selective killing of autoreactive lymphocytes and abnormal cells, including infected cells. To explain the common expression of Fas and the resistance to the Fas-induced apoptosis observed in some normal and cancer cells, we screened cells for potential regulators of the Fas death receptor. By using mass spectroscopy analysis of Fas-associated proteins, we identified peptides derived from promyelocytic leukemia (PML).
PML enhances pro-apoptotic signaling, while its dominant negative form, promyelocytic leukemia–retinoic acid receptor α (PMLRARα) fusion protein, activates pro-survival pathways. Given these opposing functions, we tested whether PMLRARα blocks Fas-mediated apoptosis.
Co-immunoprecipitation analysis demonstrated that PMLRARα interacts with Fas in acute promyelocytic leukemia (APL)-derived NB4 cells, U937/PR9 cells and APL primary cells isolated from patients. The PMLRARα-Fas binding was mapped to the PML B-box domain of PMLRARα and death domain of Fas. Flow cytometry analysis of propidium iodide- and Annexin V-stained cells challenged with Fas ligand (FasL) or agonistic anti-Fas antibody CH-11 indicated that PMLRARα blocks Fas-mediated apoptosis at early and late stages. In line with this finding, knockdown of PMLRARα with shRNA sensitized the NB4 cells to Fas-mediated apoptosis. Detailed analysis showed that expression of PMLRARα prevents procaspase-8 from binding to the Fas complex upon stimulation with the agonistic anti-Fas antibody (CH-11) and thus, also prevents cleavage/activation of procaspase-8. Further analysis indicated that PMLRARα recruits caspase-8 inhibitor c-FLIPL/S to Fas to suppress Fas signaling. A significantly higher number of mice transfected with PMLRARα-expressing plasmid than mice transfected with empty vector survived the treatment with the mouse agonistic anti-Fas antibody Jo2 (11 of 12 vs. 0 of 12; P < 0.001). Livers from PMLRARα-transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with vector-transfected mice.
These data suggest that PMLRARα is a cancer specific Fas-binding inhibitor of Fas-mediated apoptosis and thus, can contribute to cancer development and resistance to therapy. Our results may provide an explanation for the long-known role of PMLRARα and PML in the regulation of Fas signaling, which, as we have shown, can occur by regulation via direct interaction. The newly-discovered PMLRARα-Fas and PML-Fas complexes can be sites for modulation of apoptossis. By neutralizing the effect of death receptor inhibitors such as PMLRARα, we can improve responses to many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells.

Disclosures:
No relevant conflicts of interest to declare.
Glucocorticoids Inhibit Cell Death Induced by Oncogenic Stress After Imatinib Withdrawal In TKI Resistant p190Bcr/Abl Overexpressing Cells
Authors: Dengler Michael A. , Staiger Annette M. , Hofmann Ute , Gutekunst Matthias , van der Kuip Heiko , Aulitzky Walter E. 
Published: October 2019

Abstract
Abstract 3150
Imatinib resistance is a major problem in treatment of Bcr-Abl positive leukemias, particularly in patients with ALL and advanced CML. One mechanism of this resistance is overexpression of Bcr-Abl. We investigated the effects of Imatinib deprivation in Bcr-Abl overexpressing Imatinib-resistant ALL cell lines. Removal of Imatinib from culture medium led to induction of a non-apoptotic cell death starting approximately 40 hours after Imatinib withdrawal. This cell death was preceded by a rapid and robust induction of Bcr-Abl autophosphorylation and concomitant overstimulation of PI3K-, MAPK-, and JAK/STAT signalling. Over-activation of Bcr-Abl downstream signalling was accompanied by a significant enhanced glucose and amino acid metabolism and an elevated intracellular protein and nucleic acid content. As a result of this enhanced metabolisms we observed a massive increase in cell size, multinucleation, cytoplasmic vacuolization, and reduced intracellular ATP level. The phase-lucent vacuoles did not incorporate the endosome/lysosome tracker, Lyso Tracker Red indicating that the vacuoles were formed by dilation of ER cisternae. To determine if Imatinib deprivation induces an ER stress response concurrent with vacuolization we investigated typical ER stress markers. Upon removal of Imatinib a massive induction of CHOP expression and eIF2 alpha phosphorylation, as well as alternative splicing of XPB could be detected. It has been demonstrated that severe ER stress promotes cell death either by induction of a BIM-dependent apoptitic process or by a TRAF2-RIP1 dependent pathway. Despite of a robust induction of BIM and posttranslational modification of RIP1, siRNA-mediated suppression of BIM and RIP1 had no effect on Imatinib deprivation-induced cell death whereas downmodulation of CHOP partially rescued those cells. Using different small molecule inhibitor libraries, we also identified inhibitors of glycogen synthase kinase-3 (GSK3) and p38-MAPK, as well as glucocorticoids as potent compounds which not only completely prevented metabolic stress and induction of cell death upon removal of Imatinib but also partially repressed induction of CHOP upon Imatinib withdrawal. Importantly, in the presence of glucocorticoids, we found a significant enhanced number of cell clones with Bcr-Abl overexpression in lympoid cell lines upon de novo transfection with Bcr-Abl.
In conclusion, acute over-activation of the oncogene Bcr-Abl led to metabolic stress and ER stress followed by a delayed induction of a necrosis-like cell death. These cellular responses to Bcr-Abl-mediated oncogenic stress could be completely blocked by glucocorticoids. Therefore, treatment of glucocorticoid resistant ALL may generate selective pressure towards Bcr-Abl overexpession.

Disclosures:
No relevant conflicts of interest to declare.
A Predictive Risk Score for Cancer-Associated Thrombosis: Role of Screening In A Prospective Study
Authors: Khorana Alok A. , Herman Kimberly , Rubens Deborah , Francis Charles W. 
Published: October 2019

Abstract
Abstract 3173

Background:
We evaluated the utility of screening for VTE using a previously developed clinical risk score (Khorana et al, Blood 2008) in a prospective cohort of cancer patients initiating outpatient chemotherapy but not receiving thromboprophylaxis.

Methods:
Cancer patients initiating a new chemotherapy regimen and deemed high-risk based on a predictive risk model (score ≥3) were enrolled on an ongoing prospective cohort study with informed consent. Patients were evaluated with baseline and Q4 (± 1) week serial ultrasonography for upto 16 weeks; additionally, computed tomography scans for restaging were also evaluated for VTE.

Results:
Of 30 patients enrolled on study, 8 (27%) developed a VTE. This included 5 patients with DVT alone (17%), 1 patient with PE alone (3%) and 2 (7%) with both. Twenty-seven patients underwent a baseline ultrasound. Of these, 3 asymptomatic DVTs were identified (11%). Subsequent ultrasounds were performed in 18 patients at week 4 (0 DVT), 17 patients at week 8 (0 DVT) and 15 patients at week 12 (1 DVT, 7%). An additional two patients developed symptomatic DVT between weeks 1 and 4. Restaging CT scans identified an asymptomatic PE in 1 patient at week 6 and asymptomatic PE in 1 patient at week 9 with subsequent symptomatic DVT at week 10.

Conclusions:
In a prospective observational study, 27% of cancer outpatients deemed high-risk using a clinical risk score developed VTE, a rate much higher than observed even in hospitalized acutely ill patients. Thus, this study confirms the validity of a previously described risk score. The role of thromboprophylaxis in this population is currently being tested. The value of screening ultrasonography should be considered in high-risk patients based on this risk score.

Disclosures:
No relevant conflicts of interest to declare.
Concurrent BCL2 and MYC Translocations In a Prospective Cohort of Diffuse Large B-Cell Lymphomas
Authors: Pedersen Mette Ølgod , Gang Anne Ortved , Poulsen Tim Svenstrup , Knudsen Helle , Lauritzen Anne F. , Talman MajLis M , Nielsen Signe Ledou , Norgaard Peter H 
Published: October 2019

Abstract
Abstract 319

Background:
Concurrent chromosomal translocations involving the BCL2 and MYC protooncogenes, so-called double-hit, is found both in diffuse large B-cell lymphoma (DLBCL) and in a newly defined B-cell lymphoma category with features overlapping between DLBCL and Burkitt lymphoma (BCLU, 2008 WHO classification). Few studies have been published on series of double-hit B-cell lymphomas, and to our knowledge only retrospective series, reporting an average frequency of around 5%. Therefore some authors suspected that the frequency of double-hit translocations was underestimated. In addition to unanimous reports of highly aggressive clinical behaviour; poor outcome and resistance to chemotherapy, double-hit lymphomas had GCB immunophenotype (Hans), varying morphology from classical Burkitt like to DLBCL-NOS and mostly high proliferation rates (Ki-67).

Purpose:
We conducted a prospective study of DLBCL to evaluate the frequency of double-hit BCL2/MYC translocations in DLBCL. Secondly, we wanted to analyse if any pathologic and/or clinical features correlated with the presence of a double-hit in order to evaluate the need for routine genetic analysis of large B-cell lymphomas in prognostic stratification.

Materials and methods:
All patients diagnosed with DLBCL or BCLU at the Haematopathology Section at Copenhagen University Hospital in Herlev were prospectively collected throughout 2009. Tumours were classified according to their morphology (2008 WHO classification), immunohistochemistry (CD10, BCL6, MUM1, BCL2, Ki-67) and FISH (BCL2, BCL6 and MYC translocations). Clinical data were collected from patient files.

Result:
Double-hit BCL2/MYC translocations were detected in 9 of 93 cases (10%); 7 DLBCL, 1 BCLU, 1 unclassified. All double-hit DLBCL were GCB immunophenotype and showed varying morphology. Ki-67 index ranged from 15–95%. Interestingly, 2 double-hit cases were late relapses (primary double-hit). Characteristic clinical parameters included a high International Prognostic Index (IPI) score, a high stage and an extranodal presentation.

Discussion and conclusion:
In this prospective cohort of DLBCL patients the incidence of BCL2/MYC double-hit was 10%. This suggests that double-hit is more frequent in DLBCL than previously estimated. Double-hit lymphomas had GCB-immunophenotype but could not be identified by morphology or proliferation rate. Therefore if these patients should be identified due to an inferior prognosis (which we could not significantly establish due to limited observation time) routine FISH analysis for double-hit translocations should be performed in all patients with BCLU or DLBCL of GCB-subtype.

Disclosures:
No relevant conflicts of interest to declare.
A novel P2Y12-Independent Signaling Pathway Mediating Akt Phosphorylation In Response to Thrombin Receptors
Authors: Xiang Binggang , Zhang Guoying , Liu Junling , Morris Andrew J. , Smyth Susan S. , Gartner T. Kent , Li Zhenyu 
Published: October 2019

Abstract
Abstract 3191
The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. It has been previously shown that Akt phosphorylation in response to thrombin receptors is dependent on Gi signaling activated primarily by secreted ADP through its receptor P2Y12. By using P2Y12 knockout mice combined with recombinant CHO models, we demonstrate a Gi-independent pathway for Akt phosphorylation in response to thrombin. Although Akt phosphorylation in response to low dose thrombin or the PAR4 thrombin receptor peptide AYPGKF was abolished in P2Y12 deficient platelets, at high concentrations, they stimulated substantial phosphorylation of both Akt residues Thr308 and Ser473 in P2Y12 deficient platelets, demonstrating that there are P2Y12-dependent and -independent pathways contributing to Akt phosphorylation in response to thrombin receptors. Thrombin or AYPGKF repressed forskolin-induced cAMP production in the wild type mouse platelets but not in the P2Y12 deficient platelets, suggesting that Gi activation by thrombin or AYPGKF is dependent on ADP receptor P2Y12. Therefore, thrombin- or AYPGKF-induced Akt phosphorylation in P2Y12 deficient platelets is Gi independent. AYPGKF-induced Akt phosphorylation was enhanced by expression of recombinant PAR4 cDNA in CHO cells, demonstrating that stimulation of thrombin receptor PAR4 is able to elicit Akt phosphorylation in the absence of platelet secretion. It is unlikely that PAR4-induced Akt phosphorylation in CHO cells involves the P2Y12 pathway, because CHO cells apparently do not express functional P2Y12. This conclusion is supported by the observation that ADP failed to stimulate Akt phosphorylation and inhibit forskolin-induced cAMP production in CHO cells. Unlike thrombin, U46619-induced Akt phosphorylation was dramatically decreased by P2Y12 deficiency, suggesting that TXA2-induced Akt phosphorylation is largely P2Y12 dependent. In addition, P2Y12-independent Akt phosphorylation was not inhibited by the integrin inhibitor peptide RGDS or integrin β3 deficiency, demonstrating that integrin αIIbβ3 outside-in signaling is not required for thrombin-induced, P2Y12-independent, Akt phosphorylation. Furthermore, Akt phosphorylation in response to thrombin or AYPGKF in P2Y12 deficient platelets was inhibited by the calcium chelator dimethyl-BAPTA, the Src family kinase inhibitor PP2, or PI3K inhibitors, but was not affected by PKC inhibitors. Thus, our results reveal a novel P2Y12-independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors.

Disclosures:
No relevant conflicts of interest to declare.
Platelet 12-Lipoxygenase Is Required for Dense Granule Secretion and Platelet Aggregation: Role of 12-hLO In Platelet Hemostasis and Thrombosis
Authors: Apopa Patrick , Patel Megha , Boutaud Olivier , Holinstat Michael 
Published: October 2019

Abstract
Abstract 3203
Platelet activation plays a central role in regulating hemostasis. Uncontrolled activation of circulating platelets can result in the formation of occlusive thrombi and stroke. Following activation, metabolism of arachidonic acid by 12-lipoxygenase (12-hLO) may play a significant role in regulating the degree and stability of platelet reactivity. Using specific inhibitors for 12-hLO which do not interact with other lipoxygenases or enzymes in the COX-1 pathway, we were able for the first time to asses the involvement of 12-hLO in platelet reactivity. In order to assess the role of 12-hLO in platelet activation and thrombosis, dense granule secretion, platelet aggregation, alpha granule secretion, and platelet adhesion and clot formation under flow were measured. Inhibiting 12-hLO results in a complete inhibition of dense granule secretion with only a partial attenuation of alpha granule secretion indicating a novel regulatory scheme for modulating positive autocrine reinforcement of platelet reactivity and clot formation. Addition of the 12-hLO metabolite, 12-HETE (as low as 250 nM), resulted in a significant (25%) increase in PAR1-mediated dense granule secretion compare to agonist alone indicating that 12-HETE may be the crucial metabolite formed by 12-hLO metabolism of arachidonic acid. Importantly, platelet aggregation and adhesion are also significantly attenuated in the absence of 12-hLO. In fact, collagen-mediated platelet aggregation was shifted over 25 fold to the right in the absence of 12-hLO. These studies support the role of 12-hLO in hemostasis and may be a good target for anti-platelet therapy.

Disclosures:
No relevant conflicts of interest to declare.
Diabetic Platelets Are Not Resistant to Aspirin Inhibition
Authors: Schwartz Kenneth A. , Schwartz Dianne , Gossain Ved , Myneni Anjana , Nelson Peggy , Defranco Anthony 
Published: October 2019

Abstract
Abstract 3324
A daily aspirin in patients with myocardial infarctions (MI) effectively reduces the risk of occlusive vascular disease by 25%. This protective effect is reduced or absent in diabetics possibly because aspirin induced inhibition of platelet function is diminished. We tested the hypothesis that the amount of aspirin induced inhibition of platelets in diabetics is decreased.

Methods:
Each subject was evaluated at 2 time points: off-aspirin and 2-hours after observed ingestion of a 325mg aspirin tablet. Platelets were stimulated in a light aggregometer with platelet prostaglandin agonist (PPA). The slope of the PPA aggregation curve decreases with aspirin inhibition and is a sensitive measure of the amount of aspirin induced platelet inhibition (Schwartz J. Lab Clin Med 2002;139:227). Light aggregometry with arachidonic acid was used to verify that the patients had or had not taken aspirin. Maximal aspirin induced decrease in platelet function was defined as the difference between the PPA aggregation curve slopes at the 2 time points, off-aspirin and 2-hours post aspirin ingestion. 187 adult subjects (62% male 38% female) divided into four groups were studied, MI patients without diabetes n=98, diabetics with an MI n=41, diabetics without an MI n=9, and normal subjects n=39.

Results:
Comparisons of the PPA slopes of the 4 groups showed that diabetics had higher slopes than non-diabetics. Platelets from diabetics with and without an MI had significantly (p<0.05) increased off aspirin platelet reactivity (PPA slope) compared to normals. At the 2 hr time point patients with diabetes and an MI had a significantly higher PPA slope than the MI without diabetes group. Maximum aspirin induced decrease in platelet function was negatively correlated with age (r=-0.32, p=0.002). When controlled for age, there was no difference in the maximum decrease in platelet function among the 4 groups.

Conclusions:
1. Platelets from diabetic patients with and without MI have increased platelet reactivity to PPA. 2. The maximum decrease in platelet function observed in diabetic patients with and without an MI was similar to that observed in MI patients without diabetes and in normals. This suggests that the decreased protection from future vascular events observed with aspirin in diabetic patients is not because of an intrinsic resistance to aspirin inhibition of platelets.

Disclosures:
No relevant conflicts of interest to declare.
Identification and Characterization of Recurrent Venous Thrombosis In Cancer Patients
Authors: Weber Catherine , Adel Nelly G. , Riedel Elyn , Soff Gerald A. 
Published: October 2019

Abstract
Abstract 3328

Background:
Venous thromboembolism (VTE) is a common cause of morbidity and mortality in cancer patients. Standard of care for treatment is Low Molecular Weight Heparin, but recurrence of VTE remains a concern. We performed a retrospective analysis of our institutional experience, to characterize the patients who had a recurrence of VTE while on therapeutic doses of Dalteparin.

Objectives:
1. To determine the VTE recurrence rate for cancer patients on therapeutic Dalteparin. 2. To elucidate potential risk factors for recurrence. 3. To determine the impact of recurrent VTE on overall survival.

Methods:
Patients beginning treatment for VTE with dalteparin between 1/1/2008 and 12/10/ 2009 were retrospectively identified through the hospital's electronic medical records system and cases of recurrent VTE were characterized. Overall survival was estimated using the Kaplan-Meier method and the influence of VTE recurrence on overall survival was analyzed as a time-dependent covariate using a Cox proportional hazards model.

Results:
1,392 patients, treated for VTE with dalteparin were included in this study. 34 recurrent VTE episodes were identified. The overall incidence of recurrent thrombosis by six months was 2.3% (95% CI: 1.7%-3.3%). Older age was significantly associated with recurrence (p=0.04). Lung cancer patients had a significantly elevated risk of recurrence (5.6%, p=0.03). No other cancer types were associated with a significant trend to increased recurrent VTE rates. The incidence of recurrent VTE was higher among females compared to males (3.0% vs. 1.6%), although this trend was not statistically significant (P = 0.08). After adjusting for gender, sex and cancer diagnosis, developing a recurrent VTE was associated with a 3.0-fold hazard ratio of death (<0.0001).

Conclusions:
The rate of recurrent VTE in cancer patients at MSKCC is low in comparison with previously published reports. However, we identified both older age and lung cancer diagnosis as statistically significant risk factors for recurrent VTE. Females also experienced a higher rate of recurrent thrombosis when compared to males, although this result was not statistically significant. The hazard ratio for death was three times that for a patient with recurrent thrombosis when compared to one without subsequent VTE, suggesting recurrence of VTE remains an important influence on cancer-associated mortality.

Disclosures:
No relevant conflicts of interest to declare.