bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of PKB/Akt-Signaling and Oncogenic Ras in Multiple Myeloma.
Authors: Steinbrunn Torsten , Stühmer Thorsten , Chatterjee Manik , Gattenlöhner Stefan , Bargou Ralf C 
Published: October 2019

Abstract
Abstract 831

Introduction:
Dysregulated signaling pathways contribute to cell growth, survival and drug resistance in multiple myeloma (MM). In about 50 percent of primary MM samples the protein kinase B (PKB)/Akt pathway appears to be constitutively active and its blockade strongly impairs survival, thus defining Akt-dependent versus Akt-independent MM. Because illegitimate activation of survival pathways can be a consequence of oncogenic Ras mutations, we analyzed the role of Ras in Akt-dependent primary MM samples.

Methods:
Primary MM samples from different patients (n=35) were tested for cell death induction with the small molecule Akt-inhibitor Akti 1/2 and the sensitivity was correlated to the mutation status of N- or K-Ras in these samples, which was obtained by RT-PCR. Immunohistochemistry was used to demonstrate the presence of phosphorylated Akt in corresponding patient biopsies. Apoptosis was measured by flow cytometry with Annexin V-APC or Annexin V-FITC and propidium-iodide staining. We further analyzed the consequences of Ras depletion with regard to Akt activation in a Ras wildtype (AMO-1) versus a K-Ras mutated (MM.1s) and an N-Ras mutated (INA-6) myeloma cell line using siRNA knockdown and Western blotting. Isoform specificity of the siRNAs was confirmed through the effects on ectopically expressed HA-tagged Ras proteins.

Results:
Of the 35 primary MM samples tested 17 (48.6%) showed sensitivity to pharmacologic inhibition of Akt and thus constituted the Akt-dependent myeloma subgroup. Activating mutations of either K-Ras (n=7) or N-Ras (n=7) were found in 14 samples (40%). However, we did not observe any obvious correlation between the mutation status of Ras and sensitivity to the Akt inhibitor, suggesting that oncogenic Ras is not a prerequisite for the constitutive activation of Akt. We extended our analysis to MM cell lines and performed isoform-specific siRNA mediated Ras knockdown. Depletion of K-Ras induced apoptosis selectively in K-Ras mutated MM.1s cells, while N-Ras mutated INA-6 cells were most sensitive to N-Ras knockdown. Contrarily, knockdown of either of these isoforms had no effect on survival in the Ras wildtype cell line AMO-1. Ras knockdown did not lead to a notable decrease in Akt(Ser473) phosphorylation, again indicating that oncogenic Ras signaling may not be mediated through the PKB/Akt-pathway in MM.

Conclusion:
Whereas MM cell survival might well be promoted by activating Ras mutations, oncogenic Ras does not appear to be instrumental in the constitutive activation of the PKB/Akt pathway in primary MM. Targeting Ras isoforms themselves can, however, lead to apoptosis in Ras mutated MM cell lines. Thus, even though oncogenic mutated Ras is currently not a druggable target, these experiments underscore that it is nonetheless desirable to develop ways to specifically block its function in MM.

Disclosures:
No relevant conflicts of interest to declare.
Sickle Erythrocytes Have Increased Adducin Phosphorylation and Increased ROS Production Mediated by Signaling Pathways Involving Protein Kinase C and Rac GTPases.
Authors: Kalfa Theodosia A. , George Alex , Pushkaran Suvarnamala , An Xiuli , Joiner Clinton H. , Mohandas Narla , Zheng Yi 
Published: October 2019

Abstract
Abstract 901
Although sickle hemoglobin (HbSS) polymerization in hypoxic conditions is the trigger for the sickling deformation of red blood cells (RBC) containing HbSS, subsequent changes in the cytoskeleton and the mechanisms by which reversibly sickled cells become irreversibly malformed have not been fully elucidated. Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. The small Rho GTPases Rac1 and Rac2 regulate actin structures and mediate reactive oxygen species (ROS) production via NADPH oxidase in a variety of cells (Schwartz.M., J.Cell Science 2004). Deficiency of Rac1 and Rac2 GTPases in mice disrupts the normal hexagonal organization of the RBC cytoskeleton and reduces erythrocyte deformability. This is associated with increased phosphorylation of adducin (an F-actin capping protein) at Ser-726, a domain-target of protein kinase C (PKC) (Kalfa et al, Blood 2006).
We evaluated the cytoskeleton phosphorylation changes in RBCs from patients with HbSS and from control subjects with hemoglobin AA (HbAA) by immunoblotting with phospho-specific antibodies. We found that phosphorylation of adducin at Ser-726 and of band 4.1 at Ser-331 were significantly increased in the erythrocyte cytoskeleton of HbSS-RBCs. This was associated with elevated PKC activity in these cells, compared with normal RBCs. ROS concentration was also elevated in HbSS-RBCs by 150-250% compared to that in HbAA-RBC, as determined by flow cytometry using 5-(and 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, a peroxide-sensitive probe. Inhibition of PKC or Rac activity in HbSS-RBCs by small molecule-inhibitors, calphostin and NSC23766 respectively, resulted in decreased ROS production in a dose-dependent fashion, while PKC activation by phorbol 12-myristate 13-acetate (PMA) increased ROS production, implicating a PKC-Rac axis in erythrocyte ROS production. Additionally, Rac inhibition resulted in dose-dependent increase of adducin phosphorylation while PKC inhibition had the opposite effect. Preincubation of HbSS RBCs with calphostin appeared to decrease the subsequent susceptibility of these cells to sickling under hypoxic conditions, suggesting a role for PKC activity in the transition from normal to sickle morphology, likely through a combination of effects on volume regulation and membrane stability of the cells. Our results imply that aberrant cytoskeleton protein phosphorylation and ROS production in HbSS-RBCs, mediated by signaling pathways involving PKC and Rac GTPases, may contribute to membrane changes induced by cell sickling. Elucidation of these pathways may identify new therapeutic targets in sickle cell disease.

Disclosures:
No relevant conflicts of interest to declare.
Toll-Like Receptor and Autoimmunity.
Authors: Shlomchik Mark , Nickerson Kevin , Sweet Rebecca , Christensen Sean , Herlands Robin 
Published: October 2019

Abstract
Abstract SCI-24
While the paradigm that adaptive immunity to pathogens requires innate immune activation via pattern recognition receptors is well accepted, until recently how autoimmune responses are initiated and propagated has been less clear. In principle, it is less obvious how the requisite innate immune activation might occur. In 2002 landmark results demonstrated that autoreactive B cells could be activated in vitro by a self-Ags that contained both a BCR and a Toll-like receptor (TLR) ligand; the ability of endogenous chromatin antigens to engage TLR9, a DNA sensor, could explain how anti-DNA type antibodies were generated. We have extended these results in two ways. First, we have evaluated the roles of TLR9 and TLR7 (a ssRNA receptor) in vivo. We backcrossed TLR9 (DNA) and TLR7 (ssRNA) knockout alleles onto the MRL/lpr lupus-prone background. We found that TLR9 was required to generate the anti-chromatin response and TLR7 was required for anti-RNA associated responses. With respect to disease, TLR9 had an unexpected regulatory role: KO mice get more severe lupus, hypergammaglobulinemia, and die prematurely. Whereas, TLR7-deficient mice demonstrate ameliorated disease. This is surprising as TLR7 and TLR9 are highly homologous, are expressed in similar cells, and signal through the same pathway. To investigate the mechanism behind these differences, we have made TLR7 KO and TLR7/9 double KO MRL/lpr mice and I will discuss their phenotypes. In addition, we have used these animals to investigate B cell intrinsic roles for TLR9, and these data will be presented. These results suggest that innate immunity contributes to initiation and specificity of autoimmunity. In the second line of investigation, we have used a mouse that expresses an autoreactive BCR, specific for self-IgG (rheumatoid factor, RF) to investigate the roles of TLRs and T cells in the initial activation of these cells. Taken together, our results indicate that autoreactive B cells are activated in a TLR-dependent, T cell-independent fashion, but only by self molecules that provide a simultaneous BCR and TLR ligand. These cells then differentiate into autoantibody secreting plasmablasts and also are a vector for activating autoreactive T cells. Once this occurs, we propose that full-blown autoimmune disease is initiated and maintained by positive feedback between autoreactive B and T cells. The implications of this model for therapeutic approaches that target both B cells and TLRs will be discussed.

Disclosures
Shlomchik: Coley Pharmaceuticals: Patents & Royalties.
Aberrant Expression of Hepatocyte Growth Factor Induces Autocrine MET Activation Providing a Novel Therapeutic Target In Acute Myeloid Leukemia.
Authors: Kentsis Alex , Sanda Takaomi , Ngo Vu , Rodig Scott J. , Kutok Jeffery , Tholouli Eleni , Byers Richard , Woude George Vande , Kung Andrew L. , Staudt Louis M. , Look A. Thomas 
Published: October 2019

Abstract
Abstract 1042
Despite improvements in the treatment of acute myeloid leukemia (AML), high risk disease such as complex aberrant karyotype AML remains largely refractory to current therapy, and is mostly fatal. Identification of effective therapeutic targets by using candidate gene approaches has been limited by the number and variety of genetic defects associated with AML. Thus, we carried out a genome-wide functional screen in complex karyotype AML using a retroviral library of short hairpin RNAs (shRNAs), and discovered that shRNA-mediated depletion of hepatocyte growth factor (HGF) specifically inhibits growth of AML cells but not a panel of lymphoid cancer cells. HGF was to found to be aberrantly expressed in about 15% of patients with AML, including most patients with complex karyotype disease. In contrast to normal CD34+ cells that express MET (but not HGF), 5 of 7 cell lines derived from patients with complex karyotype AML exhibited aberrant expression of HGF that was associated with autocrine activation of its receptor MET. Depletion of HGF or MET using multiple independent shRNAs profoundly reduced proliferation and induced cell death in AML cells lines that express HGF but not those that lack HGF expression. Inhibition of MET using the tyrosine kinase inhibitor (SU11274) or HGF using neutralizing anti-HGF antibody (R&D Systems) also inhibited growth and induced apoptosis in AML cell lines dependent on HGF/MET activation but not those that lack HGF expression. Thus, aberrant HGF expression causes autocrine MET activation and oncogene dependence in a subset of patients with AML, confers sensitivity to HGF/MET inhibition, and provides a novel therapeutic target for this otherwise lethal disease.

Disclosures:
No relevant conflicts of interest to declare.
The Multi-Kinase Inhibitor TG02 targets CD34+CD38-CD123+ AML Cells
Authors: Pallis Monica , Burrows Francis , Russell Nigel H. 
Published: October 2019

Abstract
Abstract 1823
In clinical trials, FLT3 inhibitors are reported to kill circulating AML blasts, but the bone marrow is protected. We have previously reported that niche-like conditions (fibronectin and a cytokine cocktail) significantly reduced the in vitro toxicity of the FLT3 inhibitor AG1296 to AML cells. Moreover, the toxicity of AG1296 to the chemoresistant leukaemic CD34+CD38-CD123+ subset was completely abolished under niche-like conditions. The novel multi-kinase inhibitor TG02 has selectivity against cell cycle and transcriptional CDKs and JAK2 as well as FLT3. TG02 has efficacy in in vivo models and induces apoptosis in primary AML cells. We have now evaluated its in vitro toxicity under niche-like conditions in bulk AML cells and in the CD34+CD38-CD123+ subset.
In a cohort of six FLT3-ITD and five FLT3-wildtype samples, 100nM TG02 induced decreases of 30% in bulk cells and 32% in CD34+CD38-CD123+ cells, whereas AG1296 (5μM) induced a median 21% decrease in bulk cells under niche-like conditions but a 0% decrease in CD34+CD38-CD123+ cells. Lestaurtinib, sorafenib and sunitinib were used as comparators (all at 100 nM) and induced, respectively, 13%, 4% and 13% decrease in bulk cells and 10%, 0% and 8% decrease in CD34+CD38-CD123+ cells. FLT3 wildtype as well as ITD samples were targeted. In order to establish the molecular pathways involved in niche-mediated chemoresistance and its reversal, we treated primary AML samples with TG02 or AG1296 for 3 hours in the presence and absence of niche proteins; we measured activating phosphorylations of STAT3 (tyr705), STAT5 (tyr694), ERK1/2 (thr202/tyr404) and AKT(ser473). Basal levels of activating phosphorylations were generally higher in the bulk cells than the CD34+CD38-CD123+ cells, possibly reflecting the increased quiescence of the latter subset. STAT3, STAT5 and ERK1/2 phosphorylation were reduced by TG02 to a slightly greater degree than by AG1296 in bulk cells. However, in CD34+CD38-CD123+ cells this contrast was enhanced, such that AG1296 was ineffective, whereas TG02 was at least as effective as in bulk cells. Niche-like conditions induced an increase in phosphorylation of STAT5, but not of the other proteins tested. TG02 reduced this to basal levels in both bulk cells and CD34+CD38-CD123+ cells. AG1296 partially blocked niche-induced STAT5 phosphorylation in bulk cells, but not in CD34+CD38-CD123+ cells. It had no effect on ERK signalling. AKT phosphorylation was not informative.
In conclusion, TGO2 is more cytotoxic than comparatively selective FLT3 inhibitors towards CD34+CD38-CD123+ AML cells as well as bulk cells under niche conditions and the toxicity is associated with downregulation of STAT3, STAT5 and ERK activation.

Disclosures:
Pallis: Tragara Pharmaceuticals: Research Funding. Burrows:Tragara Pharmaceuticals: Employment.
A Phase 1/2, Open-Label, Prospective, Multicenter Study to Evaluate the Efficacy and Safety of the Lower Dose of Bortezomib Plus Busulfan and Melphalan As a Conditioning Regimen in Patients with Multiple Myeloma Undergoing Autologous Peripheral Blood Stem Cell Transplantation: Result of Phase 1 Trial
Authors: Kim Jin Seok , Min Chang Ki , Yoon Sung-Soo , Lee Jae Hoon 
Published: October 2019

Abstract
Abstract 1937

Background:
Despite the advantages of autologous stem cell transplantation (ASCT) over conventional chemotherapy, the results of ASCT in multiple myeloma (MM) are still unsatisfactory. Intravenous (iv) melphalan at a dose of 200 mg/m2 is the most commonly used conditioning regimen for ASCT. However, better conditioning regimens are still needed, and more intensive conditioning regimens have been investigated. We evaluated the role of bortezomib-containing regimen (escalated dose of bortezomib + iv busulfan and iv melphalan) in the MM patients who had a sensitive response to bortezomib during the induction chemotherapy. In the phase 1 trial, we assessed the MTD (maximum tolerated dose) of bortezomib. The trial is registered on National Cancer Institute website, number NCT01255527.

Methods:
Nine MM patients who were candidate for ASCT enrolled in this study. M:F=5:4 and median age were 59 (34–62). They were treated with escalating doses of bortezomib as a conditioning regimen (N=3/group; 0.7, 1.0, and 1.3 mg/m2 on D-6, D-3 & D+1) and a fixed dose of busulfan (3.2 mg/kg from D-5 to D-3) and melphalan (140 mg/m2 on D-2). Dose limiting toxicities (DLT) were defined as any toxicity of grade 3 or greater, with the following exceptions: vomiting, fatigue, alopecia, libido, amenorrhea, nausea, febrile neutropenia, infection, anorexia, depression, anxiety, and cytopenias. Grade 3/4 hematological toxicity was acceptable. Grade 4 of mucositis, °Ã Grade 3 diarrhea and °Ã Grade 3 cardiac toxicity were regarded as DLT. Only one patients had grade 3 oral mucositis and there were no other non hematologic toxicities of grade 3 or greater. DLT refers only to toxic events (symptomatic grade °Ã 3 toxicity) that occur until day +28 after ASCT and are attributed as possibly, probably, or definitely due to treatment.

Results:
Four patients had grade 3 oral mucositis (2 in 0.7 mg/m2 group, 1 in 1.0 mg/m2 group and 1 in 1.3 mg/m2 group), but no grade 4 (considered as DLT) oral mucositis. No other non hematologic toxicities of grade 3 or greater. Therefore, there were no dose limiting toxicities in this phase 1 trial and MTD of bortezomib was 1.3 mg/m2. Median times to ANC > 0.5 · 106/L and platelet > 20 · 106/L were 12 and 10 days, respectively, with no graft failures. All patients achieved at least very good partial response. Six patients (67%) achieved complete remission(CR) after ASCT.

Conclusion:
In the phase 1 trial, MTD of bortezomib was 1.3 mg/m2. Addition of bortezomib to busulfan and melphalan conditioning regimen shows to be safe, well-tolerated and worthy of phase 2 trial.

Disclosures:
No relevant conflicts of interest to declare.
Is Recovery of Gonadal function possible in Very Long-Term Adult Male Survivors of Hodgkin's Lymphoma?
Authors: van Dorp Wendy , van der Geest Ivana , Laven Joop , Hop Wim C.J. , Neggers Sebastian J.C.M.M. , de Vries Andrica , Pieters Rob , van den Heuvel Marry M. 
Published: October 2019

Abstract
Abstract 2065

Background:
Significant improvements in childhood cancer survival rates over recent decades have increased the importance of long-term treatment effects. Gonadal toxicity is a major complication in survivors of childhood cancer, which can especially occur in Hodgkin Lymphoma survivors, since they have been treated with alkylating agents. Inhibin B levels reflect gonadal function in men, and therefore this marker can be used to identify subgroups of childhood cancer survivors at risk for impaired gonadal function. Hitherto in male childhood cancer survivors, follow-up studies of gonadal function are lacking.

Objective:
To evaluate possible recovery of gonadal dysfunction over time in adult male survivors.

Methods:
In this retrospective study, adult male long-term childhood cancer survivors (n=201) of whom 24 (12%) were survivors of Hodgkin lymphoma were included. Serum inhibin B levels were used as a surrogate marker for gonadal function.

Results:
Median age at diagnosis was 6.0 years (range 0.0–17.5) and discontinuation of treatment was reached at a median age of 8.3 years (range 0.0–20.8). Inhibin B levels were first measured after a median follow-up time of 15.7 years (range 3.0–37.0). Median interval between the first (T1) and second measurement (T2) was 3.3 years (range 0.7–11.3). Median inhibin B level was 127 ng/L (range 5–366) at T1 and 156 ng/L (range 10–507) at T2. Survivors with an inhibin B level at first assessment≥105 ng/L have 50% chance to reach normal inhibin B levels, while this probability of recovery is negligible when the first inhibin B level is below 60 ng/L. The latter group consists of survivors of Hodgkin lymphoma treated with alkylating agents and survivors with an AAD score≥3.

Conclusions:
Our results suggest that recovery of gonadal function is possible even long after discontinuation of treatment. However, this recovery does not seem to occur in survivors who already reached critically low inhibin B levels after discontinuation of treatment, such as in survivors of a Hodgkin lymphoma treated with alkylating agents.

Disclosures:
No relevant conflicts of interest to declare.
Clearance of Desialylated Platelets by the Hepatic Asialoglycoprotein Receptor Regulates TPO Homeostasis in Vivo.
Authors: Grozovsky Renata , Begonja Antonija Jurak , Hartwig John H. , Hoffmeister Karin M. 
Published: October 2019

Abstract
Abstract 2170
The human body produces and removes 1011 platelets daily to maintain a normal steady-state platelet count, and the level of production can be greatly increased under conditions of platelet destruction. Here, we provide the experimental evidence that platelets with impaired Siaa2–3Galb1–4GlcNAc (LacNAc) structures are removed by the hepatic asialoglycoprotein receptor Asgr1/2, indicating that survival of platelets is intimately tied to surface glycans. Mice lacking Asgr2 subunit that is necessary to assemble a functional receptor have increased platelet survival (t1/2 = 49.5 ± 2h) compared to wild type mice (t1/2 = 31 ± 4h). Surprisingly, platelets from Asgr2-null mice have diminished surface sialic acid, as evidenced by lectins that bind exposed Gal moieties. Hence, desialylated platelets circulate in Asgr2-null mice. Besides “terminating” platelet circulation, the liver is the main source of thrombopoietin (TPO), the major hormone regulating platelet production. We hypothesized that desialylated platelet uptake by hepatic Asgr1/2 would affects TPO mRNA synthesis and have found that liver tissue from Asgr2-null mice has a 40% decrease in TPO mRNA levels compared to liver tissue from WT mice. In contrast, ST3Gal4-null mice, which have high rates of platelet turnover and increased desialylated platelet uptake by the Asgr2, have a 30% increase in TPO mRNA content in their livers. Both plasma TPO levels and platelet TPO contents are similarly altered in both mutant mice. In contrast, and in agreement with published data, antibody-mediated platelet clearance did not affect hepatic TPO mRNA levels. Taken together, these data show that the clearance of desialylated platelets by the hepatic Asgr1/2 regulates TPO homeostasis in vivo.

Disclosures:
No relevant conflicts of interest to declare.