scholarly journals Platelet (hem)ITAM Signaling Mediated Cancer-Associated Venous Thrombosis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2509-2509
Author(s):  
Xia Wang ◽  
Jingchang Wu ◽  
Haidi Gu ◽  
Jun Qian ◽  
Changgeng Ruan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
High Risk ◽  
Venous Thrombosis ◽  
Cell Lines ◽  
Platelet Activation ◽  
Tumor Type ◽  
Patients With Cancer ◽  
Increased Risk

Abstract Background-Patients with cancer are at a high risk of venous thromboembolism (VTE) and underlying mechanisms are unclear. Recent studies have shown that podoplanin (PDPN) expressing highly in primary brain tumors is associated with increasingly risk of VTE by activate platelets. The C-type lectin-like receptor 2(CLEC-2) is a type II transmembrane protein expressed on the surface of platelets and PDPN is the only known endogenous ligand for it. CLEC-2 mediates platelet activation through a hem-immunoreceptor tyrosine-based activation motif ((hem)ITAM). Objective: We tested the hypothesis that the platelet (hem)ITAM signaling is involved in the development of VTE in patients with malignant tumors, and targeted inhibitors may reduce thrombosis and improve prognosis in patients with cancer. Materials and Methods-We selected two human malignant cell lines (NCI-H226 and C8161) expressing high levels of PDPN as models to study the role of the platelet (hem)ITAM signaling in cancer-associated venous thrombosis in vitro and in vivo. Results and Conclusions-Our results showed that both types of cell lines expressing PDPN triggered platelet activation via CLEC-2 in vitro, which could be abrogated by an anti-PDPN antibody SZ-168 or syk inhibitor R406. Further, in vivo study showed that injection of NCI-H226 or C8161 in two mouse models of venous thrombosis activated platelets, increased platelet counts and enhanced thrombosis. These results suggested that platelet activation induced by PDPN was correlated with hypercoagulability, contributed to thrombosis in cancer patients. Importantly, PDPN enhanced thrombosis was reduced in mice treated with SZ168 or R406. Immunohistochemical staining using anti-PDPN antibody was also performed in pulmonary squamous cell carcinoma specimens of 166 patients. During up to 4-year-follow-up, 20 (12.05%) patients developed VTE. 105 tumor specimens stained positive for PDPN (27 high expression, 43 medium expression, 35 low expression). High PDPN expression was associated with an increased risk of VTE and poor prognosis, independently of age, sex and tumor type. In this study, we found that PDPN expression in tumors induced platelet aggregation and was associated with a high risk of VTE via platelet (hem)ITAM signaling. Our data also suggested that SZ168 inhibited PDPN-induced platelet aggregation in vitro and decreased the incidence of VTE in mice. In conclusion, our research demonstrated novel insights into the pathogenesis of cancer-associated venous thrombosis. Disclosures No relevant conflicts of interest to declare.

TAMI-73. GLIOBLASTOMA CELL POPULATIONS WITH DISTINCT ONCOGENIC PROGRAMS RELEASE PODOPLANIN AS PROCOAGULANT EXTRACELLULAR VESICLES

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi213-vi213
Author(s):  
Nadim Tawil ◽  
Rayhaan Bassawon ◽  
Brian Meehan ◽  
Laura Montermini ◽  
Ali Nehme ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Extracellular Vesicles ◽  
Glioblastoma Cell ◽  
Activation Markers ◽  
Increased Risk ◽  
Gradient Fractionation ◽  
D Dimer

Abstract BACKGROUND Vascular anomalies, including thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of dysregulated cancer cell genome and epigenome. Up-regulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk of venous thromboembolism in glioblastoma patients. Thus, regulation of this platelet activating protein by transforming events and release from cancer cells is of considerable interest. AIMS I. Investigate the pattern of PDPN expression and characterize PDPN-expressing cellular populations in GBM. II. Evaluate the contribution of oncogenic drivers to PDPN expression in GBM models. III. Investigate the potential involvement of extracellular vesicles (EVs) as a mechanism for systemic dissemination of PDPN and tissue factor (TF). IV. Examine the role of PDPN in intratumoral and systemic thrombosis. METHODS Bioinformatics (single-cell and bulk transcriptome data mining), GBM cell lines and stem cell lines, xenograft models in mice, ELISA assays for PDPN and TF, platelet (PF4) and clotting activation markers (D-dimer), EV electron microscopy, density gradient fractionation, and nano-flow cytometry. RESULTS PDPN is expressed by distinct glioblastoma cell subpopulations (mesenchymal) and downregulated by oncogenic mutations of EGFR and IDH1 genes, via changes in chromatin modifications (EZH2) and DNA methylation, respectively. GBM cells exteriorize PDPN and/or TF as cargo of exosome-like EVs shed both in vitro and in vivo. Injection of glioma PDPN-EVs activates platelets. Increase of platelet activation (PF4) or coagulation markers (D-dimer) occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Co-expression of PDPN and TF by GBM cells cooperatively increases tumor microthrombosis. CONCLUSION Distinct cellular subsets drive multiple facets of GBM-associated thrombosis and may represent targets for diagnosis and intervention. We suggest that the preponderance of PDPN expression as a risk factor in glioblastoma and the involvement of platelets may merit investigating anti-platelets for potential inclusion in thrombosis management in GBM.

scholarly journals Impact of extracellular matrix properties on neuroblastoma genomic heterogeneity

2020 ◽  
Author(s):  
Amparo López-Carrasco ◽  
Susana Martín-Vañó ◽  
Rebeca Burgos-Panadero ◽  
Ezequiel Monferrer ◽  
Ana P Berbegall ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
High Risk ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Chromosome 9 ◽  
Future Research ◽  
Knock Out ◽  
Specific Distribution

Abstract Background Increased tissue stiffness is a common feature of malignant solid tumors, often associated with metastasis and poor patient outcomes. Vitronectin, as an extracellular matrix anchorage glycoprotein related to a stiff matrix, is present in a particularly increased quantity and specific distribution in high-risk neuroblastoma. Furthermore, as cells can sense and transform the proprieties of the extracellular matrix into chemical signals through mechanotransduction, genotypic changes related to stiffness are possible. Methods We have applied high density SNPa and NGS techniques to in vivo and in vitro models (orthotropic xenograft vitronectin knock-out mice and 3D bioprinted hydrogels with different stiffness) using two representative neuroblastoma cell lines (the MYCN amplified SK-N-BE(2) and the ALK mutated SH-SY5Y), to discern how tumor genomics patterns and clonal heterogeneity of both cell lines are affected. Results We describe a remarkable subclonal selection of some genomic aberrations in SK-N-BE(2) cells grown in knock-out vitronectin xenograft mice that also emerged when cultured for long times in stiff hydrogels. Specially, we detected an enlarged subclonal cell population with chromosome 9 aberrations in both models. Similar abnormalities were found in human high-risk neuroblastoma with MYCN amplification. Genomics of the SH-SY5Y cell line remained stable when cultured in both models. Conclusions Focus on heterogeneous intratumor segmental chromosome aberrations and mutations, as a mirror image of tumor microenvironment, is a vital area of future research.

Improved Therapy of Multiple Myeloma by Combining Milatuzumab (Anti-CD74 Humanized mAb) and Bortezomib: In Vitro and In Vivo Results.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1176-1176
Author(s):  
Rhona Stein ◽  
David M. Goldenberg
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Leukemic Cells ◽  
Tumor Type ◽  
Term Survival ◽  
Significant Difference ◽  
Highly Effective ◽  
Ic50 Values

Abstract Background: The humanized anti-CD74 monoclonal antibody, milatuzumab (hLL1, or IMMU-115; Immunomedics, Inc, Morris Plains, NJ), is in clinical evaluation for therapy of multiple myeloma (MM) after preclinical evidence of activity in this tumor type (Stein et al, Blood2004;104:3705). Here we examine the ability of milatuzumab to increase the efficacy of drugs in MM cell lines. Methods: MTT cytotoxicity assays were performed on a panel of MM cell lines, including CAG, KMS11, KMS12-PE, and MC/CAR, to examine the effects of bortezomib, doxorubicin (dox), and dexamethasone (dex) alone and combined with milatuzumab or milatuzumab + crosslinking 2nd Ab (goat anti-human IgG, GAH). In vivo studies used a CAG-SCID mouse model of disseminated disease. Results: Without drugs, crosslinked milatuzumab, but not milatuzumab alone, yielded significant anti-proliferative effects on the four MM cell lines. In combination studies, crosslinked milatuzumab produced significant reductions in the IC50 values of the anti-MM drugs. For example, in CAG, milatuzumab+GAH decreased the IC50 values 58%, 78%, and 98% for bortezomib, dox, and dex, respectively (P=0.0034, 0.0073, and 0.078, respectively). In vivo, milatuzumab at 100 μg/injection, 2x weekly for 4 weeks, starting 1 day after injection of CAG cells, more than doubled the median survival time (MST) from 42 days in untreated CAG-bearing SCID mice to 103 days. Combination therapy with milatuzumab and bortezomib or dox was compared to milatuzumab alone, with treatments initiated 5 days after injection of CAG cells. Bortezomib alone (1.0 mg/kg) increased MST from 33 to 44 days (P=0.0021 vs. untreated). Treatment with milatuzumab alone (100 μg/mouse) increased the MST to 73 days (P<0.0001 vs. untreated). When bortezomib and milatuzumab treatments were combined, the MST increased to 93 days (P=0.0441 vs. milatuzumab and P=0.0065 vs. bortezomib). Thus, the combination of milatuzumab and bortezomib increased survival significantly compared to either single treatment. Given alone, dox yielded little or no effect on survival compared with untreated animals, and there was no significant difference between milatuzumab monotherapy and milatuzumab plus doxorubicin in this model. In contrast, a milatuzumabdox immunoconjugate was found to be a highly effective therapeutic agent, with all mice achieving long-term survival. The inhibition of the NF-κB survival pathway of B-leukemic cells by milatuzumab supports its complementary effects when combined with drugs having different mechanisms of action, such as bortezomib. Conclusions: The therapeutic efficacies of bortezomib, dox, and dex are enhanced in vitro in MM cell lines when given in combination with milatuzumab. In vivo, milatuzumab alone or especially in combination with bortezomib is highly effective in MM. (Supported in part by USPHS grant P01CA103985 from the NCI, and grants from the Thomas and Agnes Carvel Foundation and the Walter and Louise Sutcliffe Foundation.)

Novel Small Molecule Inhibitors Of The Gas6/TAM Signaling Pathway Inhibit Platelet Aggregation In Vitro and Protect Mice From Arterial and Venous Thrombosis In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2296-2296
Author(s):  
Gilbert Acevedo ◽  
Brian R. Branchford ◽  
Christine Brzezinski ◽  
Susan Sather ◽  
Gary Brodsky ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Venous Thrombosis ◽  
Thrombus Formation ◽  
Patent Application ◽  
Mean Values ◽  
Inhibition Of Platelet Aggregation ◽  
Mean Time ◽  
Dose Dependent

Abstract Background Growth Arrest Specific gene 6 (Gas6) is a ligand for the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases found on the surface of platelets. Previous studies have shown that stimulation of these receptors results in amplification of platelet activation and thrombus stabilization via activation of phosphatidylinositol-3-kinase (PI3K) and Akt, leading to phosphorylation of the β3 integrin. Previous work (from our lab and others) demonstrated that inhibition of the Gas6/TAM pathway results in impaired platelet aggregation, reduced aggregate stability, and decreased platelet spreading. Additionally, knockout mice deficient in the receptor or ligand are protected from venous and arterial thrombosis, but retain normal tail bleeding times. Here, we describe development and characterization of novel Mer-selective small molecule inhibitors (SMIs) for thrombosis applications. Objectives To determine if Mer-selective SMIs can inhibit platelet aggregation and protect mice from thrombosis using in vitro and in vivo models Methods We used aggregometry and in vivo murine models of arterial and venous thrombosis to compare two Mer-selective SMIs (UNC Mer TKI1 and UNC Mer TKI2) and determine the most effective inhibitor of platelet aggregation and thrombus formation. The inhibitory effect of two doses (1µM and 5 µM) of the compounds were determined using standard light-transmission aggregometry after a 30 minute incubation with washed human platelets at 37 ¢ªC and compared to platelets treated with vehicle control or with a TKI control (UNC TKI Null), a SMI with similar structure but minimal anti-TAM activity. Both collagen/epinephrine-induced systemic venous thrombosis and FeCl3-induced carotid artery injury models were used to determine effects on thrombosis mediated by UNC TKIs. Wild type C57Bl/6 mice were treated with one of the two inhibitors and compared to mice treated with vehicle control. Mean values +/- SEM are shown and statistical significance (p<0.05) was determined using the student’s paired t-test. Results UNC Mer TKI1 exhibited more potent inhibition of platelet aggregation in vitro relative to UNC Mer TKI2, although both compounds mediated dose-dependent effects. At a concentration of 1uM, the maximum percent aggregation in UNC Mer TKI1-treated samples (n=7) was significantly greater than samples treated with UNC TKI Null (n=7), 20% DMSO vehicle (n=7), or UNC TKI2 (n=7), with mean values of 69 +/- 2.2%, 76.7 +/-1.8% (p<0.01), 76.9 +/- 2.1% (p=0.001), and 77 +/- 1.8% (p<0.001), respectively. At a concentration of 5 µM, UNC Mer TKI1-treated samples (n=7) exhibited a mean maximum percent aggregation of 23.7 +/- 2.4% compared to 50.4 +/- 4.8% for samples treated with UNC Mer TKI2 (n=7, p<0.001). UNC Mer TKIs also mediated protection from thrombus formation in mice. Following FeCl3 injury to the carotid artery, vehicle-treated mice (n=11) developed stable vessel occlusions with a mean time of 6.77 +/- 0.25 min. In contrast, stable occlusion occurred at a mean time of 46.6 +/- 7.72 min (n=9, p=0.001) for UNC Mer TKI1-treated mice. Survival times following venous injection of collagen and epinephrine were also significantly increased in mice treated with either UNC Mer TKI relative to the UNC TKI Null or vehicle controls. Mice pre-treated with UNC Mer TKI1 (n=9, p=0.04 compared to vehicle alone) or UNC Mer TKI2 (n=9, p=0.03 compared to vehicle alone) survived for 19.84 +/- 4.4 and 21.25 +/- 4.65 minutes, respectively. In contrast, mice given UNC TKI Null (n=3) or vehicle (n=21), only survived for 3.21 +/- 2.4 min and 3.09 +/- 0.22 minutes, respectively. Conclusion UNC Mer TKIs mediate dose-dependent inhibition of platelet aggregation and protect mice from arterial and venous thrombosis. Their pronounced activity compared to an inactive scaffold protein with minimal anti-TAM activity suggest that Gas6/TAM pathway inhibition is the mechanism of action for these novel compounds. UNC Mer TKI1 has more potent anti-thrombotic properties than UNC Mer TKI2. Disclosures: Branchford: University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Sather:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. DeRyckere:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Zhang:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Liu:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Earp:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Wang:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Frye:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Graham:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Di Paola:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties.

scholarly journals Novel Antibodies Against Glycoprotein Ibα Inhibit Pulmonary Metastasis By Affecting Vwf-Gpibα Interaction

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1133-1133
Author(s):  
Qi Yingxue ◽  
Wenchun Chen ◽  
Ke Xu ◽  
Fengying Wu ◽  
Xuemei Fan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Binding Sites ◽  
Tumor Metastasis ◽  
Cancer Metastasis ◽  
Platelet Glycoprotein ◽  
Induce Platelet Aggregation ◽  
Metastasis Model

Abstract Background: Platelet glycoprotein Ibα (GPIbα) extracellular domain, which is part of the receptor complex GPIb-IX-V, plays an important role in tumor metastasis. However, the mechanism through which GPIbα participates in the metastatic process remains unclear. In addition, potential bleeding complication remains an obstacle for the clinical use of anti-platelet agents in cancer therapy. Methods and Results: To generate antibodies that bind to mouse platelet GPIbα, washed mouse platelet lysate was used as the antigen for rat immunization. Obtained hybridoma clones were screened in ELISA for binding affinity to the GPIb-IX complex. Positive clones were further screened for their abilities to inhibit platelet-cancer cell adhesion. Finally, at static condition, two antibodies, 2B4 and 1D12, had virtually no effect on the activation of integrin αIIbβ3, which is used to indicate platelet activation. Then, we characterized the binding sites of 2B4 and 1D12 by 20 purified recombinant GPIbα fragments binding. Results showed that 2B4 and 1D12 shared the same binding sites with vWF. To determine whether 2B4 and 1D12 affect vWF binding, we tested the binding by flow cytometry using recombined mouse vWF, and then, we investigated platelet aggregation induced by several agonists, including vWF binding agonist ristocetin. Our data demonstrated clearly that 2B4 and 1D12 could inhibit vWF binding. To investigate whether the inhibition of vWF-GPIbα interaction was associated with tumor metastasis, we examined the effect of 2B4 and 1D12 in each of the following interactions in vitro: between activated platelets and tumor cells, platelets and endothelial cells. Meanwhile, We further investigated the inhibitory effect of these antibodies in vivo using the experimental metastasis model and the spontaneous metastasis model. Results showed that 2B4 and 1D12 could potently inhibit the adhesion of cancer cells in vitro, and metastasisin vivo. We next investigated whether 2B4 and 1D12 could affect platelet activation and/or induce thrombocytopenia in vivo. Results showed that the addition of 2B4 or 1D12 to PRP did not induce platelet aggregation and injection of 2B4 or 1D12 Fab at appropriate dose did not affect tail-bleeding time and platelet count. Based on the above findings, we obtained anti-human platelet GPIbα monoclonal antibody YQ3 using the same approach to explore the role of human GPIbα in cancer metastasis. As expected, YQ3 inhibited lung cancer adhesion and demonstrated similar value in metastasis. More importantly, for all three mAbs in our study, none of their Fabs induced thrombocytopenia. Conclusion: Our results therefore supported the hypothesis that GPIbα contributes to tumor metastasis, and suggested potential value of using anti-GPIbα mAb to suppress cancer metastasis. Disclosures Li: Neoletix: Consultancy, Equity Ownership.

Differential Effects of Reconstituted High-density Lipoprotein on Coagulation, Fibrinolysis and Platelet Activation during Human Endotoxemia

1997 ◽  
Vol 77 (02) ◽  
pp. 303-307 ◽  
Author(s):  
Dasja Pajkrt ◽  
Peter G Lerch ◽  
Tom van der Poll ◽  
Marcel Levi ◽  
Marlies Illi ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Plasma Levels ◽  
Density Lipoprotein ◽  
High Density ◽  
Tissue Type ◽  
High Density Lipoproteins ◽  
Double Blind

SummaryHigh-density lipoproteins (HDL) can bind and neutralize lipopoly- saccharides (LPS) in vitro and in vivo. HDL can also affect fibrinolytic activity and can directly influence platelet function by reducing platelet aggregation. In this study, the effects of reconstituted HDL (rHDL) on LPS-induced coagulation, fibrinolysis and platelet activation in humans were investigated. In a double-blind, randomized, placebo-controlled, cross-over study, eight healthy male volunteers were injected with LPS (4 ng/kg) on two occasions, once in conjunction with rHDL (40 mg/kg, given as a 4 h infusion starting 3.5 h prior to LPS injection), and once in conjunction with placebo. rHDL significantly reduced LPS-induced activation of coagulation (plasma levels of prothrombin fragment F1+2) and fibrinolysis (plasma levels of tissue type plasminogen activator antigen, t-PA). No effect was observed on LPS-induced inhibition of the fibrinolytic pathway (PAI-1) or on the transient thrombocytopenia elicited by LPS. Furthermore, rHDL treatment significantly enhanced the inhibition of collagen-stimulated inhibition of platelet aggregation during endotoxemia, but had no such effect on arachido- nate-stimulated platelet aggregation. rHDL treatment per se also reduced collagen-induced platelet aggregation. These results indicate that rHDL modifies the procoagulant state associated with endotoxemia.

Integration of mRNA and microRNA profiles as prognostic and predictive markers in lung adenocarcinoma

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 7522-7522
Author(s):  
J. H. Strickler ◽  
W. Mostertz ◽  
W. Kim ◽  
K. Walters ◽  
M. Stevenson ◽  
...  
Keyword(s):  
High Risk ◽  
Lung Adenocarcinoma ◽  
Cell Lines ◽  
Cisplatin Resistance ◽  
Early Stage ◽  
Gene Signature ◽  
Discovery Cohort ◽  
Risk Of Recurrence ◽  
Increased Risk

7522 Background: Lung adenocarcinoma (ADC) is a distinct biologic entity with unique gene amplifications (Weir B, Nature 2008). Yet, comprehensive transcriptomic analysis, including microRNAs, specific to lung ADC are lacking. Methods: Using mRNA expression data from a discovery cohort of 154 patients with histologically proven early stage (I and II) lung ADC, signatures of oncogenic pathway and tumor microenvironment status were applied and further organized by hierarchical clustering to develop a metagene model. Further, using in vitro assays in a large cohort of lung ADC cell lines (n = 42) with corresponding mRNA and microRNA data, novel microRNAs associated with a poor prognosis and their relationship to cisplatin resistance was elucidated. Results: In the discovery cohort of 154 patients with early stage disease, activation of oncogenic pathways associated with wound healing (angiogenesis), chromosomal instability, and STAT signaling were associated with an increased risk of recurrence (p<0.001). Utilizing the extremes of survival to identify cohorts of patients as high and low risk phenotypes, using bayesian regression, a 100 gene signature (‘metagene') that captured the diversity of signaling pathways unique to patients at increased risk of recurrence was identified and validated in an independent cohort (n = 364) of lung ADC samples with 78.3% accuracy. Kaplan Meier survival analysis and multivariate analysis further confirmed the independent prognostic value of the 100 gene signature (p= 0.007). Using in vitro cell proliferation assays, predicted high risk lung ADC cell lines were identified as being more resistant to cisplatin therapy than those predicted to be low risk (p=0.001). In a novel manner, we also identified several microRNAs (miR-215, miR-98, miR- 643, let-7b, miR-665, miR-629) associated with a high risk of recurrence and more importantly cisplatin resistance. Conclusions: mRNA and microRNA profiles reflect unique aspects of individual tumors and may characterize histology-specific tumor heterogeneity in lung ADC, providing an opportunity to better characterize the oncogenic process and refine therapeutic options. No significant financial relationships to disclose.

Abstract 456: Antiphospholipid Antibodies Promote Platelet Activation and Thrombosis by Inhibition of Platelet eNOS

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Craig Morrell ◽  
AnneMarie Swaim ◽  
Tanika Martin ◽  
Guillermina Girardi ◽  
Jane E Salmon ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Antiphospholipid Antibodies ◽  
Thrombus Formation ◽  
Ldl Receptor ◽  
Wild Type ◽  
Human Igg ◽  
Increased Risk ◽  
Receptor Family

The antiphospholipid syndrome (APS) is an autoimmune systemic disorder characterized by the persistent presence of antiphospholipid antibodies (aPL Ab) and increased risk of thrombosis, coronary artery disease and myocardial infarction. Although platelets are known direct targets of aPL Ab action, the molecular basis of aPL Ab actions on platelets remains unclear. Platelet endothelial NO synthase (eNOS) is a key regulator of platelet function, with NO causing blunted activation. We therefore determined whether aPL Ab modulate platelet eNOS. Normal human IgG (NH IgG) and human IgG containing polyclonal aPL Ab were obtained from healthy individuals and APS patients, respectively, and purified using protein G-Sepharose chromatography. Using both human and mouse platelets, we found that aPL Ab increased agonist-induced platelet activation whereas NH IgG did not. In contrast to the enhanced activation by aPL Ab in platelets from wild-type mice, aPL Ab had no effect on platelets isolated from eNOS null mice. Pre-treatment of platelets with aPL Ab also inhibited insulin-mediated eNOS stimulation as evidenced by diminished cGMP production and DAF2 fluorescence. Receptor associated protein (RAP), an antagonist of ligand binding to members of the LDL receptor family, blocked aPL Ab-induced increases in platelet activation. RAP also prevented aPL Ab-mediated antagonism of platelet eNOS, indicating that aPL Ab signal through the platelet ApoER2â ϵ™ (LRP8) to attenuate eNOS activity. Furthermore, using intravital microscopy of the mouse mesenteric circulation, we demonstrated that platelets from wild-type mice treated with aPL Ab have increased rolling on a stimulated endothelium and a decreased time to thrombus formation in vivo versus platelets treated with NH IgG. In contrast, aPL Ab did not alter the in vivo function of platelets from eNOS null mice. These cumulative in vitro and in vivo findings demonstrate that aPL Ab antagonism of platelet eNOS through LDL receptor family member binding underlies aPL Ab-mediated thrombosis.

Potent Efficacy of BCL2 Inhibition with ABT-199 in High-Risk Aggressive B-Lymphoma Models When Combined with Knockdown of MCL1

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 506-506
Author(s):  
Lingxiao Li ◽  
Praechompoo Pongtornpipat ◽  
Timothy Tiutan ◽  
Samantha L. Kendrick ◽  
Soyoung Park ◽  
...  
Keyword(s):  
High Risk ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Double Hit Lymphoma ◽  
Apoptotic Protein ◽  
Resistant Lines ◽  
Double Hit ◽  
Factor C

Abstract Avoiding apoptosis is a hallmark of cancer. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma and carries a poor prognosis in cases at high-risk of failing up-front R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). Frequent expression of the anti-apoptotic protein BCL2 is well-described in DLBCL in numerous studies and is clear a negative prognostic marker when co-expressed with the oncogenic transcription factor c-MYC. Expression of the anti-apoptotic protein MCL1 also is found in about half of cases. BCL2 and MCL1 have redundant function in protecting cells from apoptosis. Direct inhibitors of MCL1 are not clinically available, but its short half-life permits knock-down through inhibition of cyclin-dependent kinase 9 (CDK9), which regulates transcriptional elongation. Older multi-CDK inhibitors have anti-tumor activity from loss of MCL1 but are not approved clinically due to off-target toxicities. Dinaciclib is a more potent and specific multi-CDK inhibitor with activity against CDK9. We tested dinaciclib against a panel of >20 DLBCL cell lines and found high potency, with IC50 < 20 nM in most lines. Both in vitro and in vivo, dinaciclib results in rapid loss of MCL1 protein and corresponding induction of apoptosis. Interestingly, both sensitive and resistant lines show loss of MCL1 in response to the compound. Thus, we hypothesized BCL2 activity compensates for loss of MCL1 in resistant lines. Correspondingly, over-expression of BCL2 in sensitive cells renders them completely insensitive to dinaciclib without effecting MCL1 knockdown. In 59 DLBCL cases with known BCL2 status, we assessed MCL1 protein by immunohistochemistry and found no significant difference in MCL1 expression between BCL2 positive (66%, 10/15) and negative (57%, 25/44) cases (p=0.5576). Expression of MCL1, BCL2, or both in DLBCL and the proteins’ redundant function led to the hypothesis that knockdown of MCL1 combined with direct BCL2 inhibition would synergize in the killing of high-risk DLBCL tumors. ABT-199 is a third-generation BH3 mimetic direct inhibitor of BCL2, which has shown remarkable clinical activity in chronic lymphocytic leukemia but less activity in DLBCL and other more aggressive lymphomas. We found ABT-199 combines potently and synergistically with dinaciclib in DLBCL cell lines with none of 23 lines resistant to the combination. We confirmed this in vivo using the line U2932, which is resistant in vitro to both drugs as single agents. U2932 xenografts showed dramatic reduction of tumor burden in response to the combination, a response far superior to either drug alone. We next evaluated a genetically defined immunocompetent mouse model of MYC-BCL2 double-hit lymphoma, based on MYC expression in the VavP-Bcl2 transgenic model, replicating the genetics, pathology, and aggressive clinical behavior of the human disease. Tumors from this model in vitro, interestingly, show little response to single-agent ABT-199, but the combination with dinaciclib is again synergistic. Treatment of tumor-bearing mice in vivo showed animals treated with either drug alone had no significant survival difference from vehicle-treated controls, while those treated with the combination had dramatically improved survival by Kaplan-Meier analysis (p<0.0001). Finally, we assessed the effect of combining ABT-199 with standard lymphoma chemotherapy drugs that are thought to affect MCL1 protein levels due to global effects on transcription. Doxorubicin, etoposide, and cytarabine all result in loss of MCL1 at peak in vivo attainable concentrations and synergize with ABT-199 to kill DLBCL cells otherwise resistant to the single agents. In sum, we propose therapeutic strategies combining direct inhibition of BCL2 with knockdown of MCL1 expression will be effective and tolerable for poor-prognosis lymphomas such as high-risk DLBCL and double-hit lymphoma. Disclosures No relevant conflicts of interest to declare.

Development of a Novel Phthalimide Derivative, Preclinical Effects on High-Risk Myeloma Cells and Osteoclasts

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5718-5718
Author(s):  
Yutaka Hattori ◽  
Maiko Matsushita ◽  
Noriko Tabata ◽  
Hirokazu Shiheido ◽  
Hiroshi Yanagawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Cell Lines ◽  
Pharmacokinetic Study ◽  
Bone Disease ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Phthalimide Derivative

Abstract BACKGROUND: Despite recent advances in the use of newly developed drugs including immune-modulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide and proteasome inhibitors such as bortezomib, carfilzomib, and MLN9708, MM is still an incurable disease. In particular, MM patients harboring 17p deletion, t(14;16), t(14;20), or t(4;14) are classified as a high-risk group and have shown significantly shorter survival. With the goal of helping prolong the survival of these high-risk MM patients, we screened 29 synthetic phthalimide derivatives and found a novel compound, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione (TC11), which induced the apoptosis of KMS34 cells with t(4;14) and del17p13. PURPOSE:The purpose of this project is to clarify preclinical effects of the synthetic phthalimide derivative, TC11, on high-risk MM cell lines and osteoclasts. Namely, anti-myeloma and anti-osteoclastogenic activities and pharmacokinetic study in mice were shown. We also try to isolate directly binding molecules. Safety issues including hematological toxicities and teratogenicity were also discussed. METHODS AND RESULTS: TC11 significantly inhibited growth of MM cell lines (IC50 4-8μM) including KMS34 and KMS11 cells which have high-risk chromosomal abnormalities. TC11 also suppressed the proliferation of all of the bone marrow cells obtained from the MM patients, in a dose-dependent manner. TC11 increased annexin V-positive fraction and induced apoptosis. TC11 was injected intraperitonealy into myeloma (KMS34 and KMS11 cells)-bearing lcr/SCID mice, and anti-myeloma activity was evaluated in vivo. Twenty mg/kg of TC11 significantly inhibited growth of KMS34 or KMS11-derived plasmacytomas. Apoptosis of MM cells was observed by histopathological examination. In order to evaluate hematological toxicity of TC11, growth of colony-forming cells was examined. In the presence of 5μM of TC11, formation of CFCs was not significantly suppressed, suggesting low hematopoietic toxicity. In the pharmacokinetic analyses using lcr mice, the plasma concentrations of TC11 was examined; Cmaxwas 18.1μM at 1.5hr (Tmax), and T1/2 was 2.5hr, when 100mg/kg of TC11 was injected. If 20mg/kg was injected, Cmaxwas 2.1μM at 1.0hr (Tmax), and T1/2 was 1.2hr. Oral administration of TC11 to Icr mice was safely carried out, and results of pharmacokinetic study will be shown. Aiming at the therapeutic use of TC11 to bone disease, anti-osteoclastogenic activity was examined. Mouse bone marrow mononuclear cells were incubated in the presence of M-CSF and RANK-ligand. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in number in the presence of 1μM of TC11. It was also found that 1μM of TC11 inhibited bone resorption by pit assay. We have identified nucleophosmin 1 (NPM1) and α-tubulin as TC11-binding molecules using our unique in vitro selection system using mRNA display, in vitro virus (IVV) method. However, cereblon (CRBN) was not detected as a TC11-binding protein by this method. The immunofluorescent analysis showed that TC11-treated cells exhibited elevated levels of α-tubulin fragmentation. Together with our previous observation of induction of centrosomal disruption of HeLa cells by NPM1-knock down, TC11 may cause anti-myeloma effects via mitotic catastrophe. CONCLUSION: We have demonstrated that TC11, a novel phthalimide derivative, has anti-tumor activity against MM cells with high-risk genetic abnormality including del 17p and t(4;14), in vitro and in vivo. This novel compound also down-regulates the differentiation and function of osteoclasts. Our data provide a strong preclinical rationale for TC11 as a safe and effective drug for the treatment of high-risk MM patients with bone disease. The actions of this drug relating to α-tubulin and NPM1 remain to be further investigated. TC11 exerts its anti-myeloma effect via molecular interactions which do not involve CRBN. In addition, TC11 does not form racemate and is expected to lack teratogenicity. The results of our present study suggest that new phthalimide derivatives other than thalidomide, lenalidomide and pomalidomide could be developed by drug designing for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

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