bluuebunny/crossref_metadata_2025 huggingface dataset:
FOG-1 Requires NuRD to Promote Hematopoiesis and Maintain Lineage Fidelity within the Megakaryocytic–Erythroid Compartment.
Authors: Gregory Gregory D , Miccio Annarita , Bersenev Alexey , Wang Yuhuan , Hong Wei , Zhang Zhe , Poncz Mortimer , Tong Wei , Blobel Gerd A
Published: October 2019

Abstract 702

Nuclear factors regulate the development of complex tissues by promoting the formation of one cell lineage over another. Transcription factors GATA-1 and GATA-2 co-operate with the cofactor FOG-1 to promote erythroid and megakaryocyte differentiation. In contrast, FOG-1 is inhibitory to the function of GATA factors in the context of mast cells. The mechanisms by which FOG-1 functions distinctly as co-activator and co-repressor are incompletely defined.

Purification of FOG-1-associated proteins led to the identification of the nucleosome remodeling and histone deacetylase NuRD complex (Hong, et al., EMBO J. 2005). To investigate the in vivo function of this interaction, we generated mice bearing point mutations in FOG-1 that disrupt NuRD binding. The phenotype of homozygous (Fog1ki/ki) mice revealed an essential role of NuRD for normal FOG-1 function in a broad range of cell types, including erythroid cells and megakaryocytes. Here we report that defects in hematopoietic development arise as early as the bipotent megakaryocyte-erythroid progenitor in adult mice. Strikingly, Fog1ki/ki megakaryocyte-erythroid progenitors (MEP) retained multi-lineage capacity, capable of generating mast cells and other myeloid lineages. We did not observe an increase in mast cell potential in E10.5 yolk sacs, as has been previously reported in Fog1null embryos, indicating a stage-specific role for FOG-1's interaction with NuRD.

Genome wide transcriptome analysis of purified Fog1ki/ki MEP revealed inappropriate expression of several mast cell-specific genes including Fcer1a, Fcer1b, Mcpt2, and Cpa3. Notably, aberrant mast cell gene expression persisted in committed Fog1ki/ki megakaryocyte and erythroid cells. Thus FOG-1 and NuRD are essential to constrain mast cell gene expression throughout erythroid and megakaryocyte ontogeny. A direct mechanism of repression is suggested as select mast cell genes are occupied by GATA-1 and FOG-1 in erythroid precursor cells. Furthermore, a mutant form of GATA-1 that is impaired for FOG-1 binding (GATA-1-V205M) failed to repress mast cell gene expression. Together, these observations suggest that a simple heritable epigenetic silencing mechanism is insufficient to permanently repress mast cell genes. Instead, the continuous presence of GATA-1, FOG-1 and NuRD is required to maintain lineage fidelity throughout megakaryocyte-erythroid ontogeny.

Disclosures:

No relevant conflicts of interest to declare.
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 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 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 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 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 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 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 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 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.
Preliminary Experience of the Spanish Compassionate Use Registry of Bendamustine in Patients with Relapsed and/or Refractory Multiple Myeloma
Authors: Bueno Beatriz Aguado , Andres Isabel VicuÑa , Entrena Laura , De Arriba Felipe , Krisnik Isabel , Navas BegoÑa , Callejas Marta , Teruel Anabel , Lopez Aurelio , Lopez Tomas Gonzalez , Castillo Inmaculada , De Oteyza Jaime Perez , De Miguel Dunia , Calvo Sara Rivas , Posada Raquel Rodriguez , Alegre Adrian
Published: October 2019

Abstract 4035

Introduction:

Bendamustine is a dual alkylating agent with demonstrated efficacy in de novo multiple myeloma (MM) and in relapsed/refractory (R/R MM). We present the preliminary results from the experience of Spanish compassionate use registry of this agent in different treatment schedules of R/R MM, promoted by the GEM/PETHEMA Group (Benda-MMRR-11). This study has been approved by local ethical committee, CEIC, HUP, Madrid, Spain.

Patients and Methods:

72 patients, 42 males and 30 females, with advanced R/R MM after several lines of previous treatment received a schedule containing Bendamustine (Levact®, Mundipharma, UK). Median age was 66 (35–86), with a median of 4 previous salvage lines (1–11). Bendamustine dose used ranged between 60 and 120 mg/m2 iv on days 1 and 2 for each 28 days cycle. The median of cycles was 2 (1–9). The combinations used were: Bendamustine + Prednisone in 27 patients, Bendamustine + Thalidomide + Prednisone in 11 cases, Bendamustine + Dexamethasone in 14 patients, Bendamustine + Bortezomib + Dexamethasone in 7 patients, Bendamustine + Bortezomib + Prednisone in 3 patients, Bendamustine + Thalidomide + Dexamethasone in 2 cases, Bendamustine monotherapy in 5 patients, Bendamustine + Thalidomide in 1 case, Bendamustine with Caelyx in 1 case and Bendamustine with Rituximab in 1 case.

Results:

In the 69 evaluable patients, the response was: overall response rate (ORR) 30.24%%, with complete response (CR) 11.5%, partial response (PR) 13.04%. Minimal response (MR) was 5.7%, and stable disease (SD) was 17.39%. Progression was documented in 52.1% of patients. In general, treatment was well tolerated; the most common adverse effect was hematological toxicity with grade 3–4 neutropenia in 31.8%, grade 3–4 thrombocytopenia in 30.4% and grade 3–4 anemia in 17.3%. Other toxicities included grade 3–4 infections in 27% of patients and grade 3–4 asthenia in 11.5%. Clinical results will be updated with more details regarding TTP, OS and efficacy and toxicity with each schedule.

Comments:

Bendamustine is an effective salvage therapy in patients with advanced R/R MM. Our results are consistent with previous published data in larger series of patients. More experience is needed with this agent to define the best combination and to assess its grade of efficacy that will be probably superior in earlier stages of myeloma evolution.

Disclosures:

Off Label Use: compassionate use.
Phagocytosis Is A Mechanism of Action for Daratumumab
Authors: Overdijk Marije B. , Verploegen Sandra , Marijn Bögels , van Egmond Marjolein , Groen Richard W.J. , Martens Anton C.M. , van Bueren Jeroen Lammerts , Bleeker Wim , Parren Paul W.H.I.
Published: October 2019

Abstract 4054

Daratumumab (DARA) is a human IgG1 CD38 antibody with broad-spectrum killing activity. DARA induces killing of CD38-expressing tumor cells, including fresh cells from multiple myeloma (MM) patient samples, via diverse mechanisms. These prominently include the Fc-dependent effector mechanisms complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers et al. J. Immunol. 2011). In this study we show that DARA is also able to induce tumor cell killing via antibody-dependent cellular phagocytosis (ADCP) as an additional Fc-dependent effector mechanism.

In a first set of experiments, we studied ADCP with human macrophages as effector cells. Calcein-AM-labeled Daudi tumor cells, a Burkitt's lymphoma cell line, were mixed with human macrophages in the absence or presence of DARA. Specific DARA-induced phagocytosis was analyzed in flow cytometry by measuring the percentage of calcein-AM+/CD11b+ double-positive (DP) macrophages. Both classical GM-CSF activated and alternative M-CSF activated macrophages mediated DARA-specific ADCP of the Burkitt's lymphoma cells. To further explore the in vivo contribution of ADCP in the mechanism of action (MoA) of DARA, we studied DARA mediated phagocytosis with murine macrophages. In vitro ADCP with M-CSF-stimulated bone marrow-derived murine macrophages showed a dose-dependent DARA-specific effect on the Burkitt's lymphoma cell lines Ramos and Daudi, resulting in up to 24% and 43% DP macrophages and a 25% and 50% tumor cell reduction, respectively. Furthermore, dose-dependent DARA-specific phagocytosis was observed with patient-derived MM cell lines L363 and UM9, which were transduced with CD38 to obtain levels CD38 expression as they are generally observed in primary MM patient samples. With life-cell imaging we found that ADCP of Daudi and Ramos cells occurred very rapid and efficiently. Interestingly, our recordings document that single macrophages could engage multiple target cells and that they were able to engulf up to six tumor cells sequentially in a 30 min period. This suggests that ADCP might be a very potent MoA of DARA in vivo, which we are currently studying in a mouse xenograft model.

In conclusion, in addition to CDC and ADCC, we now show that DARA can also induce killing of CD38 expressing tumor cells via phagocytosis. This very fast and potent MoA might contribute to the treatment efficacy of DARA in hematological tumors, especially at sites where high numbers of macrophages reside, such as the bone marrow.

Disclosures:

Overdijk: Genmab BV: Employment. Verploegen:Genmab BV: Employment. Groen:Genmab BV: Research Funding. Martens:Genmab BV: Research Funding. Lammerts van Bueren:Genmab BV: Employment. Bleeker:Genmab BV: Employment. Parren:Genmab BV: Employment.
The Novel Anti-Myeloma Agent Chaetocin Enhances Tumor-Specific Dendritic Cell Response Via the Induction of Heat Shock Protein and Cancer Testis Antigens On Myeloma Cells
Authors: Nguyen-Pham Thanh-Nhan , Lee Hyun Ju , Tran Huong Thi Thanh , Vo Manh-cuong , Hoang My-Dung , Choi Nuri , Kim Hyeoung-Joon , Lee Je-Jung
Published: October 2019

Abstract 4066

Background:

Dendritic cell (DC) based immunotherapy is emerging as a useful tool to treat multiple myeloma (MM). Although several tumor target antigens have been used, the clinical effectiveness of the vaccination is still limited. Chaetocin is a small molecule thiodioxopiperazine natural-product produced by Chaetomium species fungi currently in development as a candidate antimyeloma therapeutic, and has potent in vitro and in vivo activity conveyed by its ability to impose increased levels of cellular oxidative stress. Chaetocin has also been found to be used as histone methyl-transferase inhibitor with interest in the compound sufficient to kill various cancer cells. We investigated whether chaetocin could be used to induce apoptotic bodies for loading into DCs to enhance myeloma specific antitumor responses.

Methods:

Chaetocin was used to induce the apoptotic U266 myeloma cells, which were used as the sources of tumor antigens to loading onto DCs to generate myeloma-specific cytotoxic T lymphocytes (CTLs).

Results:

Chaetocin showed to induce apoptotic cells mostly from tumor cells and led to increase the expression of heat shock protein 90 (HSP90) at a level comparable to bortezomib. The transfer of activating signal from chaetocin-induced tumor cells to induce DCs maturation is mediated by the exposure of HSP90 on the surface of apoptotic cells. The HSP90 inhibitor geldanamycin can block this immunogenicity. Chaetocin up-regulate the expression of cancer testis antigens MAGEA3 and MAGEC1/CT7, which are commonly expressed in MM and the potent targets for active immunotherapies. The cytokines production during uptake tumor antigens and maturation was comparable in DCs loaded with either irradiated MM cells or chaetocin-induced MM cells. However, DCs loaded with chaetocin-induced MM cells can reduce the production of an inhibitory cytokine (IL-10) after stimulation with CD40 ligand. CTLs stimulated by chaetocin-induced MM cells-loaded DCs showed the increasing of CD8+T cells and displayed a greater number of IFN-g-secreting cells than did those stimulated by other DCs loaded with MM cell made by other conditions.

Conclusion:

These results indicate that anti-myeloma drug-induced apoptotic cells can be used as the source of myeloma antigens to loading onto DCs that could elicit potent anti-myeloma activity of CTLs due to the expression of heat shock proteins and cancer testis antigens as a mechanism of immunogenic death of human MM cells.

Disclosures:

No relevant conflicts of interest to declare.
Alcam Mediated Interaction Regulates Normal Hematopoietic Stem Cell Function and Cbfβ-SMMHC Induced Leukemogenesis
Authors: Jeannet Robin , Cai Qi , Liu Hongjun , Vu Hieu , Kuo Ya-Huei
Published: October 2019

Abstract 4091

The inv(16) acute myeloid leukemia (AML)-associated CBFβ-SMMHC fusion protein impairs hematopoietic differentiation and predisposes to leukemic transformation. Alcam, which encodes the activated leukocyte cell adhesion molecule (CD166), is a cell surface immunoglobulin superfamily member mediating homophilic adhesion as well as heterotypic interactions with CD6. We found that Alcam expression marks long-term repopulating HSCs (LT-HSC), multipotent progenitors (MPP), a subset of granulocyte-macrophage progenitors (GMP), and that Alcam expression is lost or reduced in subsets of pre-leukemic and leukemic progenitors expressing the Cbfβ-SMMHC fusion protein. We characterized the role of Alcam in HSC differentiation and self-renewal using an Alcam-null (Alcam−/−) mouse model (Weiner et al. 2004 Mol Cell Neurosci 27:1, 59–69). We show that Alcam is highly expressed in LT-HSCs where its level progressively increases with age. Young adult Alcam−/− mice had normal homeostatic hematopoiesis, and normal numbers of phenotypic HSCs. However, Alcam−/− HSCs had reduced long-term replating capacity in vitro and reduced long-term engraftment potential upon transplantation. We show that Alcam−/− BM contain a markedly lower frequency of long-term repopulating cells than wild type (WT). Further, the long-term repopulating potential and engraftment efficiency of Alcam−/− LT-HSCs was greatly compromised despite a progressive increase in phenotypic LT-HSC numbers during long-term serial transplantation. In addition, an age-associated increase in phenotypic LT-HSC cellularity was observed in Alcam−/− mice. This increase was predominately within the CD150hi fraction, and was accompanied by significantly reduced leukocyte output. Moreover, Alcam−/− LT-HSCs display premature elevation of Selp expression, a hallmark of HSC aging. To understand the role of Alcam in leukemic transformation, we generated conditional Cbfb-MYH11 knock-in (Cbfb56M/+/Mx1-Cre), Alcam-deficient (Alcam−/−) mice. Interestingly, we found that loss of Alcam drastically delayed or reduced leukemia incidence. Transplantation of Alcam−/−/Cbfb56M/+/Mx1-Cre pre-leukemic bone marrow cells into WT recipients also led to delayed and reduced incidence of leukemia development. These results suggest that Alcam contributes to leukemia transformation in a cell-intrinsic manner. Collectively, our study reveals that Alcam regulates the functional integrity and self-renewal of LT-HSCs, and contributes to leukemia initiation induced by CBFβ-SMMHC.

Disclosures:

No relevant conflicts of interest to declare.
Prophylactic Use of Enoxaparin in Morbidly Obese Adolescents During Bariatric Surgery
Authors: Mushtaq Alvina , Vaughns Janelle D , Verdun Nicole , Williams Elaine , Ziesenitz Victoria , Nadler Evan P , van den Anker John
Published: October 2019

Abstract 4367

Introduction:

Obesity has increased exponentially in recent years and currently a substantial number of children and adolescents are morbidly obese. This development has resulted in the need for bariatric surgery in adolescents. Therefore, a bariatric surgery program has been initiated at Children's National Medical Center (CNMC) and enoxaparin has been used as prophylaxis for venous thromboembolism (VTE). Enoxaparin is the drug of choice for prevention and treatment of VTE in children and adults. The standard of care at CNMC is to dose enoxaparin 40 mg SC q 12 hrs in patients with a BMI < 50 and 60 mg SC q 12 hrs for patients with a BMI > 50 with no routine use of anti-FXa monitoring. Nationally and internationally there are no well defined guidelines for prophylactic doses of enoxaparin and the approach varies among different institutions. This study was therefore designed and conducted to investigate if the current dosing regimens are indeed appropriate.

Patients and Methods:

Four morbidly obese adolescents aged 16 to 18 years and with a body weight ranging between 118 and 174 kg were studied. Anti-FXa assay concentrations were collected before (predose) and at 1, 2, 4, 6, and 12 hrs after the administration of the first dose of enoxaparin. In addition, after an extensive literature search, two pharmacokinetic (PK) studies were found that investigated comparable doses of enoxaparin in adult lean healthy volunteers with a mean age of 24 years.

Results:

The results are shown in Figure 1 and Table 1. The peak anti-FXa level (Cmax) ranged from 0.2–0.23 IU/ml in the 4 studied adolescents and these values are significantly lower as compared to the range of concentrations (0.38–0.5 IU/ml) detected in the two control groups from the literature. The time to reach Cmax (T max) was delayed, along with a lower area under the time vs concentration curve and the mean residence time.

Conclusions:

We conclude, based on our results, that the use of 40–60 mg enoxaparin as the current standard of care for VTE prophylaxis in morbidly obese adolescents results in different and probably insufficient anti-FXa levels as compared to those found in lean patients of comparable age. The clinical significance of this finding still needs to be determined.

Disclosures:

No relevant conflicts of interest to declare.
Galactorrhea and Acute Lymphoblastic T-Cell Leukemia: A Case Presentation and Evaluation of the Role of Prolactin
Authors: Grimes Allison C
Published: October 2019

Abstract 4828

Acute lymphoblastic T-cell leukemia (T-ALL) accounts for nearly 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia (ALL). Historically, T-ALL has conferred a worse prognosis than other forms of ALL. Although multi-agent pulse chemotherapy has improved EFS over the past several decades, the lack of clinical or laboratory based prognosticators as well as high rate of relapse have remained barriers in management. Described is a case of acute lymphoblastic T-cell leukemia in a previously healthy sixteen-year-old girl initially presenting with galactorrhea and hyperprolactinemia without evidence of CNS infiltrate. Although galactorrhea has been described in a few adults as an initial presentation of acute myeloginous leukemia (AML) and a single adult case of T-cell lymphoma, to date there has been no association with childhood cancers or T-ALL in particular. Hypothesized in AML cases as a rare paraneoplastic phenomenon, this case explores this theory and employs direct prolactin staining of tumor cell cytoplasm, serial prolactin monitoring throughout treatment, and investigation into underlying causes for hyperprolactinemia in the context of T-ALL. The possibilities for prolactin as a novel tumor marker are explored.

Disclosures:

No relevant conflicts of interest to declare.
Pilot Study of Sorafenib for Myelodysplastic Syndrome
Authors: Rao Arati , Rizzieri David A. , Vanegas Eduardo , Moore Joseph O. , Gockerman Jon P. , Diehl Louis F. , Adams Donna , Warzecho Julie , Decastro Carlos
Published: October 2019

Abstract 4948

Background:

MDS (Myelodysplastic syndrome) is a hematologic malignancy in which one or multiple conditions affects the fabrication (or dysplasia) of the myeloid class of blood cells. While treatable, therapies provide short term benefit and are not considered curable, short of an allogeneic stem cell transplant. The RAS pathway has been noted to be abnormally regulated in many malignancies, including MDS and AML. Thus, we performed a phase 2 efficacy study using sorafenib, a RAS inhibitor, for patients with MDS by WHO criterion.

Methods:

Adults with primary or secondary MDS, estimated CrCl >30 ml/min, and liver function tests 2 2. 5 × ULN were eligible. Patients of all IPSS risk categories were included. Patients received Sorafenib 400mg po bid daily for 28 days for a 28 day cycle (continuous therapy) and continued till progression or withdrawal for toxicity. Patients were evaluated after the first 2 cycles for response and then every 3 cycles thereafter until an endpoint was reached (up to five years).

Results:

Patient and disease characteristics are noted in the table below. Sixteen evaluable patients were treated on study: median 71 (range 56–88 years), median time from diagnosis to starting this study was 34 months (range 9–103 months), median number of prior therapies was 2 (range 0–6), median number of cycles on study was 1. 5 (range 1–7). Responses lasted for 4 months in 4 and a year in 1.

The agent was tolerated in this population though some toxicities were notable: 2 (12%) had grade 2 rash (though many with grade 1), 5 with grade 2 (31%) and 1 (6%) grade 3 nausea, vomiting or diarrhea, 2 (12% with grade 2 and 1 (6%) with grade 3 liver function abnormalities, and 1 (6%) grade 4 myositis likely related to the agent. Unfortunately, many of the toxicities occurred shortly after initiating therapy, leading to early withdrawal from therapy before a full cycle and evaluation for response could be performed.

Conclusions:

Sorafenib was tolerated and provided some responses in this difficult to treat group of unselected patients. Due to many early withdrawals, the study was terminated early. Utility would be enhanced in future studies by devising methods to apriori select this targeted therapy for those most likely to respond.

Disclosures:

No relevant conflicts of interest to declare.
Drp1 Activation Overcomes Diffuse Large B-Cell Lymphoma Cells Radioresistance
Authors: Pang Qingsong , Liu Ningbo , Liu Fengting , Agrawal Samir , Wang Ping
Published: October 2019

Abstract 5121

Mitochondria undergo fusion and fission in response to physiological or pathological changes. Mitochondrial fusion is regulated by mitofusin-1 and 2 (MFN-1/2) and optic atrophy 1(OPA-1), whereas, mitochondrial fission (or fragmentation) is controlled by a Dynamin-related protein 1 (Drp1). Drp1 activation is triggered by dephosphorylation of Drp1 on its serine 637 site. Recent studies demonstrated that Drp1 activation plays an important role in mitochondrial fragmentation-induced cell death. The phosphorylation of Drp1 by serine kinase inhibits its GTPase activity and prevents mitochondrial fragmentation. Inhibition of Drp1 activation or loss of Drp1 function leads to slow down apoptosis and necrosis.

Recent reports showed that patients who received consolidative radiotherapy (RT) combine R-CHOP had significant better 5-year event-free and overall survival rates than patients with diffuse large B-cell lymphoma who did not receive RT combination therapy. Although RT combination therapy significant increased survival of DLBCL, the mechanisms of RT induced cell death are reminded unclear.

In this study, we investigated the roles of Drp1 activation in the sensitivity of DLBCL cells to radiotherapy. Radiation causes damage in both nuclear and mitochondrial DNAs and generation of reactive oxygen species (ROS) followed by caspase activation. In the radio-resistant DLBCL cells, radiation induced activation of Drp1, and led to mitochondrial fragmentation, caspase-3 activation and cell death. However, the sensitive DLBCL cells underwent cytochrome c release, caspase-3 activation and PARP cleavage but not the activation of Drip1. To confirm the role of Drp1 activation in radiation-induced cell death on radio-resistant cells, Drp1 was dephosphorylated by a serine kinase inhibitor STS. Co-treatment of radio-resistant DLBCL cells with STS and radiation greatly enhanced mitochondrial fragmentation and significantly increased the sensitivity of radio-resistant DLBCL cells to radiation-induced caspase-3 activation, PARP cleavage and apoptotic cell death. However, co-treatment with STS did not further increase the sensitivity of radio-sensitive cells to caspsase-3 activation and cell death. Our data indicate that radiation-induced cell death in radio-resistant DLBCL cells is regulated by Drp1. We therefore propose that Drp1 could be a potential target for overcoming radio-resistance.

Disclosures:

No relevant conflicts of interest to declare.
SF3B1 Mutations in CLL Are Equivalent to p53/ATM Dysfunction and Cause Defective Puma Upregulation in Response to Chemotherapy
Authors: te Raa Doreen , Derks Ingrid AM , Luijks Dieuwertje M , van Laar Jacoline , Monsuur Hanneke , Oldreive Ceri , Jethwa Alexander , Hüllein Jennifer , Stankovic Tatjana , Zenz Thorsten , Van Oers Marinus H.J. , Kater Arnon P. , Eldering Eric
Published: October 2019

Abstract 711

Mutations or deletions of the tumor suppressor p53 or its upstream kinase ATM are well-known determinants of poor prognosis in Chronic Lymphocytic Leukemia (CLL). In recent years, genome wide sequencing has uncovered novel gene mutations that correspond with poor prognosis. Specifically, recurrent mutations in the splicing factor SF3B1 and the Notch and NRAS/KRAS oncogenes have been found. These mutations were (in part) mutually exclusive with p53 and/or ATM mutations, which suggested overlap in biological function.

Here, we report results of a comparative analysis of p53 target genes and in vitro drug responses in CLL samples with either p53 (n=9), ATM (n=10), SF3B1 (n=11), Notch (n=6), or NRAS/KRAS (n=4) gene deletions/mutations. We found that upon irradiation, mRNA induction of all tested p53 targets genes (p21, Puma, CD95, Bax, PCNA, FXDR) was clearly decreased in all SF3B1 mutated CLL samples (overall p<0.001). SF3B1 samples resembled ATM mutated/11q− CLL in displaying a defective but not absent p53 response. In contrast, Notch and KRAS/NRAS mutations did not affect RNA induction of apoptosis inducers Puma and Bax. At the protein level, Puma and p21 induction were defective or absent in SF3B1 mutated CLL. This corresponded with decreased apoptosis after in vitro treatment with fludarabine. Treatment with nutlin, either alone or in combination with fludarabine, restored cell death induction, again indicating an overlap with ATM dysfunction. To establish possible causality between SF3B1 mutation and ATM dysfunction, more genetic and functional studies are ongoing and will be reported.

In conclusion, the recently described mutations in a splicing factor in CLL can be linked at the functional level to defective ATM and/or p53 target gene responses, providing an explanation for the poor clinical prognosis of CLL patients with SF3B1 mutations.

Disclosures:

No relevant conflicts of interest to declare.