scholarly journals Circulating Extracellular Vesicles from Acute Myeloid Leukemia Patients Drive Distinct Metabolic Profile of Leukemic Cells and Reveal Crucial Lipidomic Biomarkers

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3471-3471
Author(s):  
Dorian Forte ◽  
Roberto Maria Pellegrino ◽  
Francesco Fabbri ◽  
Ivan Vannini ◽  
Samantha Bruno ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Mitochondrial Activity ◽  
Advisory Committees ◽  
Leukemic Cell Lines ◽  
Acute Myeloid

Abstract Background. Extracellular vesicles (EVs) are submicron vesicles released from various cell types including blood cells with pleiotropic effects on cell signalling and metabolism. EV cargos are enriched in nucleic acids, proteins, and lipids that can be delivered to target cells to influence surrounding microenvironment. Thus, EVs represent a powerful tool for liquid biopsy in hematological malignancies including acute myeloid leukemia (AML). AML is an aggressive disease with high relapse rate and less invasive tools are urgently needed to investigate disease (metabolic) dynamics. Accumulating evidence has reported a key role for EVs in shaping the AML bone marrow niche. However, at present, the metabolic function and the lipidomic signature driven by circulating EVs have yet to fully emerge. Methods. Peripheral blood (PB) and bone marrow (BM) were collected from AML patients at diagnosis (n=40) and PB from age/sex-matched healthy donors (HD, n=20). EVs were purified from platelet-poor plasma by size exclusion chromatography and quantified using the NanoSight technology. Immunomagnetically isolated CD34+ cells from umbilical cord blood (CB) or AML patients were characterized by analyzing the hematopoietic stem/progenitor cell (HSPC)-specific cluster of differentiation marker expression, redox metabolic profiling (using CellROX, glutathione detection reagent and MitoTracker) after 24 hours co-culture with EVs. Quantitative lipidomic profiling of circulating EVs was performed by Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC/HRMS). Seahorse extracellular flux analyses were performed in leukemia cell lines (including KG-1, KASUMI-1, MOLM-13, THP-1 and OCI-AML3). To functionally define the metabolic reprogramming of leukemic cellular components within their microenvironment, leukemic stem cell subsets were assessed by flow cytometry-based SCENITH (Single Cell ENergetic metabolism by profilIng Translation inHibition) method in both whole blood and BM samples (n=4). Results. In our work, plasma-derived EVs from AML patients showed a significant increase in the size and protein amounts compared to HD counterparts. To explore the metabolic perturbation triggered by EVs, we developed a co-culture system with circulating EVs from either HD or AML patients with CB or AML CD34+. We found a reduction in the frequency of AML CD34+ with high ROS levels in the presence of AML EVs without affecting the ROS levels in normal CB CD34+. In parallel, AML EVs increased the frequency of AML CD34+ with both high mitochondrial activity and glutathione, a key antioxidant molecule involved in many metabolic pathways. Similar metabolic profiles were also confirmed in human leukemic cell lines tested. Specifically, Seahorse flux analysis revealed that EVs induced a cell energy phenotype consistent with quiescent and chemoresistant state in human leukemic cell lines, showing a more glycolytic state in MOLM-13. Interestingly, both CD34+ and CD34+/CD38- leukemic fractions from whole blood and BM of the same AML patients were analyzed by SCENITH after co-cultures with HD/AML EVs. Remarkably, PB CD34+/CD38- leukemic fractions were more dependent on mitochondrial activity in the presence of AML EVs, suggesting a metabolic shift triggered by leukemic EV that apparently occur in the leukemic fractions out of the BM niche. In addition, to give insights into lipidomic signatures of EVs as disease biomarkers, we detected a total of 25 (out of 200) independent lipid species significantly different between AML-derived EVs and HD (n=20, respectively). We reported the abundance of both glycerolipid and fatty acids species in AML EVs. Also, through a multivariate statistical analysis of EV lipidomic profile, we revealed that AML EVs were depleted in sphingomyelin classes, a class of lipids that are interconnected to HSC metabolism. Finally, according to the 2017 ELN risk stratification system, we observed the depletion in important modulators of EV release and formation as ether-linked phosphatidylethanolamine and phosphatidylethanolamine species in adverse-risk AML patients. Conclusion. Overall, our study provides the basis for further investigations on the metabolic alterations trigger by EVs within the BM microenvironment and suggests prognostic biomarkers for leukemic patients that might reveal novel metabolic vulnerabilities in AML scenario. Disclosures Cavo: Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Accommodations, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Bristol-Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Curti: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.

CXCR4 Expression and Biological Activity Is Dependent on Oxygen Partial Pressure in Acute Myeloid Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 938-938
Author(s):  
Michael Fiegl ◽  
Ismael J. Samudio ◽  
Karen Clise Dwyer ◽  
Jared Burks ◽  
Herbert Fritsche ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Cxcr4 Expression ◽  
Leukemic Cell Lines ◽  
Acute Myeloid

Abstract CXCR4, the receptor for bone marrow stroma derived SDF-1, has recently been studied in normal hematopoiesis and hematologic malignancies. Increased expression of CXCR4 by leukemic blasts has been reported by us and others (Konoplev S. et al, Cancer 2007) to be associated with poor prognosis in acute myeloid leukemia (AML). However, all in-vitro studies are usually carried out under unphysiological, i.e. normoxic (21% O2) conditions. We hypothesized that the pO2 in vitro has major impact on the expression of CXCR4, a key receptor for cell migration and intracellular signalling. Thus, pO2 of bone marrow aspirates was measured using the i-STAT Portable Clinical Analyzer and a hypoxic workstation was used providing constant low oxygen content. Surface and total CXCR4 expression was examined in leukemic cell lines and patient samples by flow cytometry, confocal microscopy and Western blotting (WB). In 19 patients, the median pO2 of the bone marrow was determined as 46.1±12.8 mmHg (6.1±1.7%) with no significant difference between patients with AML (n=7, pO2 41.3±11.2 mmHg) and patients in CR (n=12, pO2 48.3±15.9 mmHg). This level of hypoxia significantly increases surface and total expression of CXCR4 in the leukemic cell lines U937 and OCI-AML3 as well as in samples from patients with AML, as compared to normoxic conditions (~2.8fold increase). This increase happened mainly within the first 2–8 hours of hypoxia and was unrelated to increased CXCR4 transcription, as shown by PCR. Re-oxygenation of leukemic cells resulted in a statistical significant degradation of CXCR4 (~3fold decrease) in all examined cell lines and patient samples (n=10). This loss of CXCR4 is very rapid (within 5 minutes of re-oxygenation) and was detected by flow cytometry, confocal microscopy and WB. This phenomenon was independent of proteasome activity and ATP. Detailed analysis of membraneous lipid rafts by sucrose density separation, cholesterol depletion and flow cytometry analysis for GM1 gangliosides showed structural (distinct re-distribution of Lck in lipid rafts) and quantitative changes (loss of cholesterol and CXCR4) during re-oxygenation. Moreover, part of the loss of CXCR4 can be attributed to sequestration of microparticles into the extracellular environment as shown by WB of supernatant of re-oxygenated cells and by a significant increase (~1.5fold) in the amount of microparticles released by cells (cell lines U937 and OCI-AML3 and additional patient samples) during the process of re-oxygenation, as measured by flow cytometry. In summary, this study determined the oxygen content of CR and leukemic bone marrow samples as 6.1±1.7%. This pO2 is associated with an increase in CXCR4 expression on AML cells, while re-oxygenation leads to a rapid decrease of CXCR4, perhaps in part by shedding of CXCR4- containing microparticles. These studies point to the importance of studying leukemic blasts under physiologic, i.e. hypoxic conditions.

scholarly journals Donor Cell Acute Myeloid Leukemia after Transplant for Follicular Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5697-5697
Author(s):  
Lacey S. Williams ◽  
Catherine E. Lai
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Follicular Lymphoma ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Donor Cell ◽  
Cell Leukemia ◽  
Advisory Committees ◽  
Donor Cell Leukemia ◽  
Acute Myeloid

Donor cell leukemia is postulated to account for up to 5% of all leukemia "relapses" after hematopoietic stem cell transplant (SCT), though in many cases this is the first leukemia diagnosis for the patient if their transplant was for non-leukemia primary diseases. The rarity of the condition and heterogeneity of disease create challenges in diagnosis and management. In the present case, donor cell leukemia (DCL) developed in a 68-year-old female after allogeneic SCT 18 years earlier for follicular lymphoma. Only one other case of DCL after transplantation for follicular lymphoma has been reported (Boulton-Jones et al., Bone Marrow Transplantation, 2005). Furthermore, this case is atypical in that the presentation occurred many years after transplantation, since very few cases of DCL occur more than 15 years after original transplant. Case In 1993, the patient was diagnosed with stage IIIA follicular lymphoma at age 50. She achieved a complete remission with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) for 4 years. She relapsed in 1998 and received treatment with fludarabine and mitoxantrone. In 1999, she enrolled in a toxitumomab clinical trial (NCT00268203) but discontinued therapy secondary to side effects. Due to persistent disease, she proceeded with SCT and received EPOCH-F (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and fludarabine) prior to allogeneic SCT from her brother in 2000 (6/6 HLA match), augmented with TH2 cells. She received graft versus host disease (GVHD) prophylaxis with cyclosporine, however her post transplant course was complicated by engraftment syndrome and gastrointestinal and skin GVHD. In 2019, she presented to hematology for evaluation of worsening chronic neutropenia and thrombocytopenia persistent for three years, noted during work-up for symptomatic cholelithiasis. Bone marrow biopsy revealed acute myeloid leukemia (AML) with a hypocellular marrow with 30% blasts and myelodysplasia related changes. Her cytogenetics showed 46XY, +1, der(1;7)(q10;p10)/47,sl,+8/46,XY. FISH analyses demonstrated deletion 7q31 D7S486 locus in 156/200 cells (78%). NGS panel showed IDH1 (VAF16%) and U2AF1 (VAF 26%) mutations. Based on cytogenetics and chimerism studies showing 100% donor, the patient was diagnosed with donor-derived AML secondary to allogeneic SCT from her brother. The brother currently has no known hematologic problems. The patient was treated with CPX-351 (liposomal cytarabine and daunorubicin) and achieved a complete remission, followed by consolidation with CPX-351. Given her complex cytogenetics and poor prognosis, the patient proceeded to non-myeloablative haploidentical peripheral blood SCT from her son, with post-transplant cyclophosphamide. She subsequently had complications of neutropenic fever and C. dificile colitis, with progressive colitis leading to her death on day 22 after SCT. Discussion Though cytogenetic and molecular studies along with functional status assist clinicians in treatment decisions for DCL patients, the benefits and risks of treatment remain difficult to balance for this unique subset of leukemia. Of patients that achieve remission for greater than 18 months, many undergo second allogeneic SCT, however a similar number of patients have remissions of at least 18 months treated with chemotherapy alone (Wiseman, Biology of Blood and Bone Marrow Transplantation, 2011). In 15 reported cases that went to SCT, approximately 50% lived longer than 12 months after their DCL diagnosis. Second allogeneic SCT is often favored after initial remission in patients with good performance status due to high risk for relapse. This case illustrates the challenge in management of donor cell leukemia, a rather rare entity with very few cases in the literature developing greater than 15 years after transplant. Limited robust evidence favoring a particular treatment supports the need for further prospective studies. Disclosures Lai: Agios: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Speakers Bureau; Astellas: Speakers Bureau.

scholarly journals EZH2 Mutations and Impact on Clinical Outcome Analyzed in 1604 Patients with Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1528-1528
Author(s):  
Sebastian Stasik ◽  
Jan Moritz Middeke ◽  
Michael Kramer ◽  
Christoph Rollig ◽  
Alwin Krämer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Employment Equity ◽  
Advisory Committees ◽  
Mutational Status ◽  
Equity Ownership ◽  
Acute Myeloid

Abstract Purpose: The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and key epigenetic regulator involved in transcriptional repression and embryonic development. Loss of EZH2 activity by inactivating mutations is associated with poor prognosis in myeloid malignancies such as MDS. More recently, EZH2 inactivation was shown to induce chemoresistance in acute myeloid leukemia (AML) (Göllner et al., 2017). Data on the frequency and prognostic role of EZH2-mutations in AML are rare and mostly confined to smaller cohorts. To investigate the prevalence and prognostic impact of this alteration in more detail, we analyzed a large cohort of AML patients (n = 1604) for EZH2 mutations. Patients and Methods: All patients analyzed had newly diagnosed AML, were registered in clinical protocols of the Study Alliance Leukemia (SAL) (AML96, AML2003 or AML60+, SORAML) and had available material at diagnosis. Screening for EZH2 mutations and associated alterations was done using Next-Generation Sequencing (NGS) (TruSight Myeloid Sequencing Panel, Illumina) on an Illumina MiSeq-system using bone marrow or peripheral blood. Detection was conducted with a defined cut-off of 5% variant allele frequency (VAF). All samples below the predefined threshold were classified as EZH2 wild type (wt). Patient clinical characteristics and co-mutations were analyzed according to the mutational status. Furthermore, multivariate analysis was used to identify the impact of EZH2 mutations on outcome. Results: EZH2-mutations were found in 63 of 1604 (4%) patients, with a median VAF of 44% (range 6-97%; median coverage 3077x). Mutations were detected within several exons (2-6; 8-12; 14-20) with highest frequencies in exons 17 and 18 (29%). The majority of detected mutations (71% missense and 29% nonsense/frameshift) were single nucleotide variants (SNVs) (87%), followed by small indel mutations. Descriptive statistics of clinical parameters and associated co-mutations revealed significant differences between EZH2-mut and -wt patients. At diagnosis, patients with EZH2 mutations were significantly older (median age 59 yrs) than EZH2-wt patients (median 56 yrs; p=0.044). In addition, significantly fewer EZH2-mut patients (71%) were diagnosed with de novo AML compared to EZH2-wt patients (84%; p=0.036). Accordingly, EZH2-mut patients had a higher rate of secondary acute myeloid leukemia (sAML) (21%), evolving from prior MDS or after prior chemotherapy (tAML) (8%; p=0.036). Also, bone marrow (and blood) blast counts differed between the two groups (EZH2-mut patients had significantly lower BM and PB blast counts; p=0.013). In contrast, no differences were observed for WBC counts, karyotype, ECOG performance status and ELN-2017 risk category compared to EZH2-wt patients. Based on cytogenetics according to the 2017 ELN criteria, 35% of EZH2-mut patients were categorized with favorable risk, 28% had intermediate and 37% adverse risk. No association was seen with -7/7q-. In the group of EZH2-mut AML patients, significantly higher rates of co-mutations were detected in RUNX1 (25%), ASXL1 (22%) and NRAS (25%) compared to EZH2-wt patients (with 10%; 8% and 15%, respectively). Vice versa, concomitant mutations in NPM1 were (non-significantly) more common in EZH2-wt patients (33%) vs EZH2-mut patients (21%). For other frequently mutated genes in AML there was no major difference between EZH2-mut and -wt patients, e.g. FLT3ITD (13%), FLT3TKD (10%) and CEBPA (24%), as well as genes encoding epigenetic modifiers, namely, DNMT3A (21%), IDH1/2 (11/14%), and TET2 (21%). The correlation of EZH2 mutational status with clinical outcomes showed no effect of EZH2 mutations on the rate of complete remission (CR), relapse free survival (RFS) and overall survival (OS) (with a median OS of 18.4 and 17.1 months for EZH2-mut and -wt patients, respectively) in the univariate analyses. Likewise, the multivariate analysis with clinical variable such as age, cytogenetics and WBC using Cox proportional hazard regression, revealed that EZH2 mutations were not an independent risk factor for OS or RFS. Conclusion EZH mutations are recurrent alterations in patients with AML. The association with certain clinical factors and typical mutations such as RUNX1 and ASXL1 points to the fact that these mutations are associated with secondary AML. Our data do not indicate that EZH2 mutations represent an independent prognostic factor. Disclosures Middeke: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Rollig:Bayer: Research Funding; Janssen: Research Funding. Scholl:Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Abbivie: Other: Travel support; Alexion: Other: Travel support; MDS: Other: Travel support; Novartis: Other: Travel support; Deutsche Krebshilfe: Research Funding; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Hochhaus:Pfizer: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Research Funding. Brümmendorf:Janssen: Consultancy; Takeda: Consultancy; Novartis: Consultancy, Research Funding; Merck: Consultancy; Pfizer: Consultancy, Research Funding. Burchert:AOP Orphan: Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Research Funding. Krause:Novartis: Research Funding. Hänel:Amgen: Honoraria; Roche: Honoraria; Takeda: Honoraria; Novartis: Honoraria. Platzbecker:Celgene: Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding; Roche: Research Funding; Johnson & Johnson: Research Funding; Affimed: Research Funding. Serve:Bayer: Research Funding. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

scholarly journals Genetic Dissection of p53 Driven Senescence of Bone Marrow Mesenchymal Cells in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2625-2625
Author(s):  
Rasoul Pourebrahim ◽  
Peter P. Ruvolo ◽  
Steven M. Kornblau ◽  
Carlos E. Bueso-Ramos ◽  
Michael Andreeff
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Bone Marrow Mscs ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Bone Marrow Mesenchymal Cells ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy characterized by bone marrow infiltration of abnormally proliferating leukemic blasts which results in fatal anemia, bleeding and infectious complications due to compromised normal hematopoiesis. Patients with complete remission (CR) but incomplete blood cell count recovery (CRi) have significantly shorter survival compared to CR patients. Although there is a correlation between CRi and minimal residual disease (MRD), the two variables were shown to be independent risk factors for relapse development (1). The mechanism by which AML induces bone marrow failure in patients is largely unknown. Here, we demonstrate that AML derived MSCs highly express p53 and p21 proteins and are more senescent compared to their normal age-matched controls as demonstrated by high β-galactosidase staining (figure 1. A, B&C). Emerging evidence indicates that the aging of endosteal niche cells results in lower reconstitution potential of hematopoietic stem cells (2). To functionally evaluate the effects of AML on bone marrow MSCs, we utilized a murine leukemia model of the AML microenvironment. We transplanted Osx-Cre;mTmG mice with AML cells and compared the senescence of MSCs in normal bone marrow (Figure 1.D) with AML (Figure 1.E). Consistent with our initial findings in human, AML strongly induced senescence of osteoblasts. This suggests that AML suppresses normal hematopoiesis by inducing senescence in the hematopoietic niche. To address the role of p53 signaling in senescence of MSCs we generated a traceable conditional p53 gain/loss model specifically in bone marrow MSCs using Osx-Cre;mTmG; Mdm2fl/+ and Osx-Cre;mTmG;p53fl/fl mice respectively (Figure 1.F). Deletion of p53 in bone marrow MSCs resulted in an increased population of osteoblasts (GFP+) in Osx-Cre;mTmG;p53fl/fl mice in comparison to Osx-Cre;mTmG mice suggesting that p53 loss in osteoblasts inhibits senescence of osteoblasts. In order to evaluate p53 activity after recombination of p53fl alleles in the osteoblasts, we isolated MSCs from bone marrows and analyzed the expression of p21.P21 was significantly down regulated in osteoblasts (GFP+) derived from Osx-Cre;mTmG;p53fl/fl mice whereas its expression in the hematopoietic cells from same tissue (tdTomato+) remained comparable to p53 wild type suggesting that p21 as the master regulator of senescence is regulated by p53 in bone marrow mesenchymal cells. To evaluate the effect of p53 loss in osteoblasts and its impact on hematopoietic cells, we isolated the GFP+ cells (osteoblasts) and RFP + cells (hematopoietic) by FACS. Senescent cells, non-cell autonomously, modulate the bone marrow microenvironment through the senescence-associated secretory phenotype (SASP). We analyzed the expression of fifteen SASP cytokines by QPCR. Deletion of p53 in bone marrow mesenchymal cells strongly abrogated the expression of several SASP cytokines. Interestingly several Notch target genes such as Hey1 and Hey2 were highly induced in MSCs following p53 deletion suggesting a role for Notch signaling in hematopoietic failure following AML induced MSCs senescence. Our data suggest that AML induces senescence of endosteal niche resulting in hematopoietic failure. These findings contribute to our understanding of the role of p53 in leukemia MSCs and could have broad translational significance for the treatment of hematopoietic failure in patients with AML.Chen X, et al. (2015) Relation of clinical response and minimal residual disease and their prognostic impact on outcome in acute myeloid leukemia. J Clin Oncol 33(11):1258-1264.Li J, et al. (2018) Murine hematopoietic stem cell reconstitution potential is maintained by osteopontin during aging. Sci Rep 8(1):2833. Disclosures Andreeff: Astra Zeneca: Research Funding; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Celgene: Consultancy; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SentiBio: Equity Ownership; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Jazz Pharma: Consultancy; Reata: Equity Ownership.

scholarly journals Dissecting Signaling Network Responses to PAK4 Allosteric Modulators (PAMs) in Cell Subsets within Primary Human Acute Myeloid Leukemia Samples

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3686-3686
Author(s):  
Paul Brent Ferrell ◽  
William Senapedis ◽  
Alexander Cook ◽  
Erkan Baloglu ◽  
Yosef Landesman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Leukemia Stem Cells ◽  
Allosteric Modulators ◽  
Advisory Committees ◽  
Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults and has a poor outcome with limited treatment options in patients with relapsed or resistant disease. Therapy resistance in AML is likely related to the inadequacy of therapy within leukemia cell subsets, including leukemia stem cells (LSCs). The p21-activated kinase (PAK) family of proteins was shown to be overexpressed in cancer cells and to play a key role in proliferation, survival, and maintenance of cellular structure. The series of orally bioavailable PAK4 allosteric modulators (PAM) have previously been shown to have activity in hematological cancer cell lines, including those derived from acute myeloid leukemia (AML) (Senapedis et al. Blood124, 2208-2208). Understanding how therapies target cellular subsets within primary patient samples could aid drug development by revealing any subset specific drug effects. In this project, we studied the effects of p21-activated kinase 4 (PAK4) modulation in AML samples. PAK4 modulation has been shown to have significant effects on many intracellular signaling pathways, including PI3K/AKT, MAPK/ERK and WNT/β-catenin pathways (Senapedis et al. Blood124, 2208-2208). It is unknown whether PAMs will have similar activity in primary leukemia cells. Likewise, it is currently unclear to what extent PAMs will differentially impact primary cell subsets including leukemia stem cells and non-malignant cell subsets that may be critical to recovery of bone marrow functions. We have previously shown that the single cell biology platform of flow cytometry is well-suited for dissecting clinically relevant signaling network mechanisms in primary human AML (Irish et al. Cell, 118(2):217-28). Methods: Flow cytometry was used to dissect the impact of an orally bioavailable PAM in AML cell lines and primary patient tissue. Cell lines chosen for this study included NRAS mutant KG-1 and Kasumi-1, which carry t(8;21) and express the AML1:ETO fusion protein. Primary AML biopsies were acquired from bone marrow or blood prior to any treatment and patients were identified and consented for this study according to a local Institutional Review Board-approved protocol. AML tissue samples were viably cryopreserved and then assayed ex vivo. Established protocols were used for phospho-specific flow cytometry, fluorescent cell barcoding, and data analysis in Cytobank (Irish et al. Cell, 118(2):217-28, Doxie and Irish, Curr Top Microbiol Immunol. 377:1-21). Results: Differential effects of PAK4 inhibition were observed between cell lines and among cell subsets from AML patient bone marrow. In leukemia cell lines and patient samples, p-ERK and p-S6 showed marked inhibition via PAM, though degree of inhibition varied. In AML patient samples, PAMs blocked signaling responses in p-ERK specifically in AML blasts, but spared normal CD45hi mononuclear cells (0.88 vs. 0.29-fold reduction (arcsinh scale) in p-ERK at 10 nM). Within the AML blast population, CD34+ CD38- and CD34+ CD38+ AML subsets showed similar PAM dose response via p-ERK. Conclusions: Single cell analysis effectively distinguishes effects of PAK4 inhibition via a series of allosteric modulators of PAK4 (PAMs) on leukemia and non-leukemia subsets in the same sample. PAM reduced immediate p-ERK and p-S6 levels in primary leukemia and cell lines. Notably, inhibition in various subsets within human AML was successfully measured by phospho-flow cytometry. Signaling changes in p-ERK were minimal within non-leukemic mature CD45+ mononuclear cells found in primary patient biopsies. Analysis of CD34+ CD38- cells indicates that PAMs could have activity within leukemia stem cells, and, at least, effect the AML progenitors. These findings support further investigation into the mechanism of action and treatment potential of PAMs in AML. Disclosures Senapedis: Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Irish:Novartis: Honoraria; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; InCyte: Research Funding. Savona:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Polo-like kinase 1 is overexpressed in acute myeloid leukemia and its inhibition preferentially targets the proliferation of leukemic cells

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 659-662 ◽  
Author(s):  
Annelies G. Renner ◽  
Cédric Dos Santos ◽  
Christian Recher ◽  
Christian Bailly ◽  
Laurent Créancier ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Leukemic Cell Lines ◽  
Clonogenic Potential ◽  
Established Cell ◽  
Interfering Rna ◽  
Acute Myeloid

Abstract Polo-like kinase 1 (Plk1) is a major mitotic regulator overexpressed in many solid tumors. Its role in hematopoietic malignancies is still poorly characterized. In this study, we demonstrate that Plk1 is highly expressed in leukemic cell lines, and overexpressed in a majority of samples from patients with acute myeloid leukemia compared with normal progenitors. A pharmacologic inhibitor, BI2536, blocks proliferation in established cell lines, and dramatically inhibits the clonogenic potential of leukemic cells from patients. Plk1 knockdown by small interfering RNA also blocked proliferation of leukemic cell lines and the clonogenic potential of primary cells from patients. Interestingly, normal primary hematopoietic progenitors are less sensitive to Plk1 inhibition than leukemic cells, whose proliferation is dramatically decreased by the inhibitor. These results highlight Plk1 as a potentially interesting therapeutic target for the treatment of acute myeloid leukemia.

scholarly journals Validation of NMT1 and NMT2 As Novel Drug Targets in Adult Acute Myeloid Leukemia: Rationale for N-Myristoyltransferase Inhibition with Pclx-001 for Clinical Trials

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4344-4344
Author(s):  
Jay Gamma ◽  
Aishwarya Iyer ◽  
Megan Yap ◽  
Zoulika Zak ◽  
Krista Vincent ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Advisory Committees ◽  
Acute Myeloid ◽  
Privately Held

Abstract Background: N-terminal myristoylation is the transfer of the saturated fourteen carbon fatty acid myristate to an N-terminal glycine residue. This co- or post-translational protein modification promotes protein-protein and protein-lipid interactions and is essential for proper membrane localization and/or activity of up to 600 human intracellular proteins. N-myristoyltransferases (NMTs) are the enzymes responsible and two isoforms are found in humans. NMT1 (ubiquitous and essential for cell survival) and NMT2 (more variably expressed) differ in activity level and substrate specificities. NMT expression levels vary in some cancers, and with myristoylation being essential for activity of certain oncogenes including Src Family Kinases (SFKs). NMTs have therefore been proposed as an anti-cancer target. Dysregulation and oncogenic activity of SFKs occurs frequently in acute myeloid leukemia (AML), suggesting NMT inhibition could provide therapeutic benefit. PCLX-001 is a low nanomolar small molecule pan-NMT inhibitor with high oral bioavailability in clinical trials as once daily oral therapy for lymphoma and solid tumors. Methods and Results: Data from the TCGA Transcriptome database showed high NMT1 and low NMT2 were associated with reduced overall and event-free survival in adult AML, and high NMT1 - but not NMT2 - expression is associated with proliferative gene sets in AML cell lines. AML cell lines treated with PCLX-001 showed a significant reduction in total protein myristoylation, as well as reduced levels of SFK proteins and SFK phosphorylation. PCLX-001 induced apoptosis in AML cell lines and patient blasts at concentrations which spared a large proportion of peripheral blood lymphocytes and monocytes from healthy individuals. AML cell lines showed significant increase in BIP protein and ER stress in response to PCLX-001, along with caspase 3 cleavage. In an AML cell line derived xenograft (CDX) and two AML patient derived xenograft (PDX) series (n=1 DX for MV-4-11 and n=2 PDX), PCLX-001 monotherapy had dose-dependent anticancer activity and resulted in complete remissions in subcutaneous AML cell deposits. In tail-vein injection PDX models, PCLX-001 treatment resulted in up to 95% reduction of human CD45+ cells in peripheral blood and bone marrow. Conclusions: These findings validate NMT inhibition as a novel therapeutic strategy for AML. PCLX-001 preferentially targeted AML cells that rely on oncogenic activity of myristoylated proteins, inducing apoptosis and reducing leukemic burden. PCLX-001 warrants evaluation in clinical trials for adult AML. Disclosures Gamma: Pacylex Pharmaceuticals: Current holder of individual stocks in a privately-held company. Yap: Pacylex Pharmaceuticals: Current holder of individual stocks in a privately-held company, Patents & Royalties. Beauchamp: Pacylex Pharmaceuticals: Current Employment, Current holder of individual stocks in a privately-held company, Patents & Royalties. Mackey: Pacylex Pharmaceuticals, Inc.: Current holder of individual stocks in a privately-held company. Pemmaraju: Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Aptitude Health: Consultancy; Sager Strong Foundation: Other; Celgene Corporation: Consultancy; LFB Biotechnologies: Consultancy; Plexxicon: Other, Research Funding; MustangBio: Consultancy, Other; Roche Diagnostics: Consultancy; Daiichi Sankyo, Inc.: Other, Research Funding; DAVA Oncology: Consultancy; Cellectis S.A. ADR: Other, Research Funding; Springer Science + Business Media: Other; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; Samus: Other, Research Funding; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; CareDx, Inc.: Consultancy; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; Incyte: Consultancy; Affymetrix: Consultancy, Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Clearview Healthcare Partners: Consultancy; Blueprint Medicines: Consultancy; Bristol-Myers Squibb Co.: Consultancy; ImmunoGen, Inc: Consultancy; Pacylex Pharmaceuticals: Consultancy. Borthakur: Astex: Research Funding; Ryvu: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; ArgenX: Membership on an entity's Board of Directors or advisory committees; University of Texas MD Anderson Cancer Center: Current Employment; Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy. Brandwein: AbbVie: Honoraria; Jazz: Honoraria; Taiho: Honoraria; Astellas: Honoraria; Bristol Myers Squibb: Honoraria; Roche: Honoraria; Pfizer: Honoraria; Amgen: Honoraria. Berthiaume: Pacylex Pharmaceuticals, Inc.: Current holder of individual stocks in a privately-held company.

scholarly journals A Novel Proteomic Profiling of the Bone Marrow Microenvironment Reveals Elevated Levels of the Chemokine CCL23 Isoforms in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2709-2709
Author(s):  
Haydar Celik ◽  
Katherine E. Lindblad ◽  
Bogdan Popescu ◽  
Giovanna Fantoni ◽  
Gege Gui ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Serum Samples ◽  
Advisory Committees ◽  
Extracellular Compartment ◽  
Acute Myeloid

The bone marrow (BM) microenvironment is increasingly recognized as an important contributor to acute myeloid leukemia (AML) pathogenesis. However, despite growing interest in characterizing different components and cellular architecture of the BM niche and their biological significance in leukemogenesis, the proteomic constitution of the BM extracellular compartment that distinguishes a leukemic niche from its normal counterpart has not yet been fully described. We therefore performed a quantitative, large-scale proteomic analysis of 1,305 human proteins of the non-cellular compartment of BM (plasma) samples from ten relapsed or refractory AML patients and from ten age- and sex-matched healthy donors (HDs) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). This screen identified a total of 168 differentially abundant proteins, of which 91 were significantly more and 77 proteins significantly less abundant in leukemic BM compared with healthy marrow (FC ≥ 1.5, FDR ≤ 0.05). Comparative analysis of BM plasma and peripheral blood (PB) serum samples from the same AML patients and HDs revealed 65 similarly regulated proteins (37 up-regulated vs. 28 down-regulated) and 1 differently regulated protein between the two compartments. Out of the total 168 proteins, 102 proteins were specifically dysregulated only in the BM compartment. TruSeq Stranded Total RNA-sequencing (Illumina) was also performed using paired-end 75bp sequencing on a HiSeq 3000. RNA was isolated from PAXgene BM RNA tubes (Qiagen) collected in parallel with samples for proteomic analysis. Results of analysis of differentially expressed transcripts only partially overlapped with those candidates identified from our validated proteomic approach, indicating that sequencing of RNA derived from cellular sources of BM may be a suboptimal screening strategy to determine the true proteomic composition of the extracellular compartment of the AML marrow microenvironment. In addition to several previously reported proteins, our proteomics screen discovered numerous aberrantly expressed proteins in leukemic marrow whose role in AML pathogenesis is currently unknown. Using pathway analysis, we identified sets of proteins enriched for specific biological pathways including RAS, ephrin, PDGF, PI3K/AKT, MAPK, Notch, TLR, JAK-STAT, NFκB, Rap1, and Tie2 signaling pathways. A systems biology analysis approach revealed the highly connected network of cytokines and chemokines as the most striking AML-associated proteomic alteration in the BM. We identified IL-8 as a differentially expressed and key central molecule of this network in AML, consistent with recent reports. Importantly, we also identified significantly elevated levels of CKβ8 and CKβ8-1, alternatively spliced isoforms of the myelosuppressive chemokine CCL23 also known as myeloid progenitor inhibitory factor 1 (MPIF-1) or CKβ8, in both leukemic marrow and PB serum samples (Figure 1). Given the critical importance of cytopenias, often disproportional to the degree of leukemic marrow involvement, in the morbidity and mortality of patients with myelodysplastic syndrome (MDS) and AML, we subsequently confirmed this striking finding by performing orthogonal validation in a larger cohort of MDS and AML patients using an ELISA-based immunoassay. This novel finding suggests the possibility that CCL23 may play a role in suppression of normal hematopoiesis in MDS and AML. In support of this hypothesis, we demonstrated in vitro myelosuppressive effects of CCL23 isoforms on colony formation by human CD34+ hematopoietic stem and progenitor cells (HSPCs) in an in vitro colony forming unit assay, resulting in an approximately 2.5-fold decrease in CFU-GM and an evident decrease in CFU-GEMM counts. In summary, our broad and quantitative proteomic dataset of extracellular factors present in leukemic and normal aging bone marrow has already provided novel mechanistic insights into AML pathogenesis and should serve, together with paired RNA-sequencing information, as a useful public resource for the research community. Disclosures Lai: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Speakers Bureau; Astellas: Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Hourigan:SELLAS Life Sciences Group AG: Research Funding; Merck, Sharpe & Dohme: Research Funding.

scholarly journals RNF5 Defines Acute Myeloid Leukemia Growth and Susceptibility to Histone Deacetylase Inhibitors

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-32
Author(s):  
Ali Khateb ◽  
Anagha Deshpande ◽  
Yongmei Feng ◽  
Ikrame Lazar ◽  
Joo Sang Lee ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Histone Deacetylase Inhibitors ◽  
Hdac Inhibitors ◽  
Therapeutic Modality ◽  
Advisory Committees ◽  
Acute Myeloid

Acute myeloid leukemia (AML) remains an incurable blood cancer largely due to rapid emergence of resistance to conventional treatments. Thus, new therapeutic modalities are greatly needed to halt AML development. Here, using genetic and xenograft mouse models, we reveal that inhibition of the ubiquitin ligase RNF5 in human AML cell lines and in MLL-AF9-driven AML severely decreased the leukemogenic potential of those cells and prolonged survival of model leukemic mice. These findings suggest the possibility that targeting a single gene, namely RNF5, could effectively inhibit different AML subtypes. We initially focused on RNF5 as its expression is upregulated in AML patient cohorts as well as in AML-derived cell lines compared with normal hematopoietic cells. Furthermore, high RNF5 expression in AML patient specimens correlated with poor prognosis, relapse and short overall patient survival. By contrast, specimens from AML patients who responded to therapy exhibited low RNF5 levels. In vitro, RNF5 loss impaired the clonogenic potential of MLL-AF9-transduced bone marrow cells and markedly attenuated growth and survival of AML but not CML or T-ALL cell lines, in which RNF5 is also highly expressed. High-throughput screen and bioinformatics analysis identified RNF5 and ER-associated degradation (ERAD) components, as augmenting AML cell sensitivity to histone deacetylase (HDAC) inhibition. Indeed, inhibition of RNF5 sensitized AML cells to HDAC inhibitors. Correspondingly, a favorable prognosis was observed in AML patients exhibiting low expression of RNF5 and HDAC. Collectivity, our studies identify a potential new therapeutic modality based on targeting RNF5 to inhibit AML and suggest that RNF5 expression could serve as a prognostic marker and means to stratify patients for treatment with HDAC inhibitors. Disclosures Ofran: AbbVie: Membership on an entity's Board of Directors or advisory committees. Vuori:Bionano Genomics: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Multinational Study on the Clinical and Genetic Features of the ERCC6L2-Disease

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 864-864
Author(s):  
Marja Hakkarainen ◽  
Suvi P. M. Douglas ◽  
Tom Vulliamy ◽  
Inderjeet Dokal ◽  
Jean Soulier ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Germ Line ◽  
Bone Marrow Failure ◽  
Intensive Therapy ◽  
Advisory Committees ◽  
Genetic Characteristics ◽  
Acute Myeloid

Abstract Biallelic mutations in ERCC6L2 were first reported to cause bone marrow failure (BMF). Additionally, we recently described a strong predisposition to erythroid lineage-restricted acute myeloid leukemia (AML-M6). Today, 31 ERCC6L2-mutated cases have been reported. This study aims to further explore the clinical and molecular features, as well as outcomes, of the ERCC6L2 patients. By June 2021, we have gathered clinical and genetic characteristics of 46 subjects in 31 families with biallelic germ line ERCC6L2 mutations from Finland (n = 21), France (n = 8), Israel (n = 1), Sweden (n = 4), and referred to a center in the United Kingdom (n = 12). Extension of the data collection from additional countries is ongoing. According to our data, ERCC6L2-disease often presents first as mild and fluctuating cytopenias with underlying bone marrow (BM) hypoplasia. With increasing age, patients develop clonal hematopoiesis with somatic mutations in TP53, and ultimately myelodysplastic syndrome (MDS) leading to a very high-risk acute myeloid leukemia. The median age of the patients at first referral to a hematologist was 18 years (range 6-65 years old). Characteristic changes in complete blood count (CBC) were mild thrombocytopenia, leukopenia, and sometimes macrocytosis. In individual pediatric or adolescent cases, more pronounced pancytopenia has also been noted. Severe BM hypoplasia was detected in many patients despite only mild changes in the CBC indicating that examining the histology of the BM biopsy, in addition to BM aspirate and CBC, is crucial. Also spontaneous, possibly transient, recovery of CBC in a few patients has occurred. All but one patient above 10 years of age, and with data from somatic mutation analysis (n = 17), carried one to four somatic TP53 mutations in their bone marrow. Among the 46 subjects, nine have been diagnosed with MDS and nine with AML (six with The French-American-British subtype M6, erythroleukemia; three with non-specific subtypes). Interestingly, increased reticulin fibrosis in the BM has been identified in at least three out of nine patients with MDS. In the nine patients diagnosed with AML, the median age of the appearance of leukemia was 37 years (range 20-65). Characteristic of TP53-mutated AML, all leukemia patients had complex hematologic karyotype and have deceased, despite intensive therapy, within one year of diagnosis. Approximately half of the patients with ERCC6L2-disease have been identified in Finland. The great majority of the patients (20/21) carry the same biallelic ERCC6L2 mutation (NM_020207.7) c.1424delT (p.Ile475ThrfsTer36). None of the Finnish families (n = 14) are consanguineous, but according to them, the ancestries reside in North-Eastern Finland indicating a founder effect. A more detailed genealogical analysis is ongoing and we suggest ERCC6L2-disease to be added to the Finnish Disease Heritage as the first cancer predisposition syndrome. Like many of the conditions identified in genetic isolates, ERCC6L2-disease is not restricted to Finland. Thus the current global effort to define the phenotype, as well as further molecular studies, will bring guidance to clinicians for tailoring follow-up and therapies for patients with ERCC6L2-disease. As a novel entity in the field of inherited bone marrow syndromes, we want to increase the awareness of ERCC6L2 -disease and encourage clinics to integrate ERCC6L2 into their germ line testing. Disclosures Siitonen: celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees; Brystol Myers Squibb: Consultancy; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; amgen: Honoraria.

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