bluuebunny/crossref_metadata_2025 huggingface dataset:
Upregulation of MAGE-A1, MAGE-A3, NY-ESO-1 and MHC Molecules on Neuroblastoma Cells Following Exposure to Decitabine and Interferon-γ
Authors: Bao Lei , Dunham Kimberly , Lucas Kenneth
Published: October 2019

Abstract 5179

MAGE-A1, MAGE-A3, and NY-ESO-1 are cancer-testis (CT) antigens expressed on a number of malignant solid tumors, including neuroblastoma (NB), but many tumor cell lines down-regulate the expression of CT antigens as well as major histocompatibility (MHC) antigens, precluding recognition by antigen specific T cells. If expression of cancer antigens as well as MHC molecules could be enhanced pharmacologically, the efficacy of CT antigen specific immunotherapy could be improved. DNA methylation is an epigenetic mechanism used by cells to decrease the expression of several genes, including tumor suppressor genes. Decitabine (5-aza-2′-deoxycytidine, DAC) is a potent inhibitor of DNA methylation, and the doses associated with the demethylating action of this drug are much lower than those required for cytotoxicit. We have demonstrated that the expression of MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells following exposure to the DAC. When these cell lines are cultured in the presence of DAC at concentrations similar to levels achieved with clinical administration, we were able to achieve expression of NY-ESO-1, MAGE-A1, or MAGE-A3 in 11/11 neuroblastoma cell lines. Concentrations of 1uM DAC were associated with strongest expression of these antigens, which was optimal after 72 hours of drug exposure. Culture of NB cell lines with IFN-γ was also associated with an increased expression of either MHC Class I or II by cytofluorometry, and Class I upregulated in 10/11 of these cell lines, although to varying degrees. MAGE-A1, MAGE-A3, and NY-ESO-1 specific CTL were cultured from volunteer donors by stimulating peripheral blood mononuclear cells with dendritic cells pulsed with overlapping peptide mixes derived from full length proteins, and these CTL lysed HLA partially matched, DAC treated neuroblastoma as well as glioblastoma cell lines. These studies confirm that DAC and IFN-γ can be used to increase the expression of CT antigens and MHC molecules on neuroblastoma cells, and pre-treatment with these agents makes tumor cell lines more susceptible to CTL mediated killing.

Disclosures:

No relevant conflicts of interest to declare.
Frontline High Dose Chemotherapy Supported by Autologous PBSCT Might Improve Survival In Elderly Patients (≥60 Years) with High Risk Diffuse Large B-Cell Lymphoma (DLBCL)
Authors: Ivanov Vadim , Coso Diane , Rey Jerome , Aurran-Schleinitz Therese , Stoppa Anne-Marie , de Colella Jean-Marc Schiano , Blaise Didier , Bouabdallah Reda
Published: October 2019

Abstract 5190

Limited data is available concerning feasibility and efficacy of high dose therapy (HDT) supported by autologous PBSCT in elderly patients with non-Hodgkin lymphoma (NHL). In young patients with poor prognostic features intensification supported by PBSCT as a part of first-line treatment suggests survival benefit. It is not clear if the same strategy is applicable to the older patients.

The Institute Paoli-Calmettes database was reviewed for all DLBCL patients who received BEAM followed by PBSCT in patients >=60 years old between January 1998 and January 2010 (13 years). All patients were HIV-negative and received BEAM intensification as a part of front-line treatment. All of them were in a complete response after CHOP or R-CHOP induction prior to autograft. Thirty six auto-transplanted patients were identified (median age 63 y, range 60–68). This cohort was compared with closely matched group of 43 patients of same age range, who received first-line CHOP or R-CHOP regimen without intensification in the same 13-year interval. Only patients in a complete response after first line were included.

As frontline autoPBSCT was performed in high-risk patients, the group without HDT was naturally privileged in the terms of Ann-Arbor stage and aaIPI index. There was significant difference in the localised vs disseminated disease (stage I-II: 53,4% in no-HDT vs 14% in HDT group, p=0,00025) and aaIPI (0-1: 72% in no-HDT vs 28% in HDT, p=0,000086) between the two groups. Factors evaluated included treatment-related mortality (TRM), overall survival (OS) and event-free survival (EFS). There were no transplant-related deaths in the HDT group.

The estimated 5-year OS was 81,3% (95% CI 62,5-91,9 %) and 10-year OS was 65% (95%CI 34,2-86,9%) for HDT group compared to 91,5% (95% CI 77,5-97,1%) and 58,4% (95% CI 20–88,8%) in the no-HDT group (p=NS). There were 8 events (6 relapses and 2 secondary malignancy deaths) in the HDT group and 11 events (9 relapses and 2 secondary malignancy deaths) in no-HDT (5-year EFS 77,6% (95% CI 60,3-88,8%) vs 78,3% (95%CI 62,3-88,8%), p=NS)).

We conclude that front-line autologous PBSCT with BEAM conditioning can be safely performed in patients aged 60 years or above with DLBCL after CHOP of R-CHOP induction. There was no difference in OS and EFS between cohorts with and without intensification even if the auto-transplantation procedure was reserved for the high risk patients only. The first-line HDT with autologous PBSCT in older patients with high-risk IPI score might improve survival in this group and produce results similar to those in the low-risk group.

Disclosures:

No relevant conflicts of interest to declare.
Comorbidities Indexes In Patients Treated with 5-Azacitidine Are a An Useful and Easily Applicable Tool to Refine Prognostic Evaluation
Authors: Sanna Alessandro , Cannella Laura , Gozzini Antonella , Breccia Massimo , Sassolini Francesca , Bosi Alberto , Alimena Giuliana , Santini Valeria
Published: October 2019

Abstract 606

Myelodysplastic syndromes (MDS) are affecting mainly elderly patients, and age is considered per se a negative prognostic factor. Most of the elderly patients have comorbidity that presumably negatively impact the overall survival and quality of life and may influence response to therapy. A subanalysis of a recently concluded international trial demonstrated that MDS patients aged > 75 yrs treated with azacitidine have a significantly longer overall survival respect to best supportive care treated patients (Seymour, 2010).

We analyzed whether presence and number of comorbidities could have an impact on response and management of azacitidine treatment in elderly and very elderly patients treated in our Center, outside clinical trials. We evaluated outcome, survival and type of response, as well as adverse events in all patients.

We analyzed 59 elderly MDS patients (IPSS INT-1 15/59, INT-2 32/59 and High 12/59) treated with Azacitidine 75 mg/kg/day sc for 5 days every 28. Mean number of cycles was 9 (range:2-42). Mean age was 69 yrs (50-82); 30% of patients (18/59) were >= 75 yrs and 39% of the latter (7/18) >= 80 yrs.

We also evaluated Charlson comorbidity index (CCI) (10 patients scored =<1, 44 patients 2 or 3, and 5 patients >= 4), the Cumulative Illness Rating Scale (CIRS) (25 patients scored =<1, 25 patients 2 or 3 and 9 patients >= 4) and the Adult Comorbidity Evaluation-27 (ACE-27) (13 patients scored =<1, 43 patients 2,3 or 4 and 3 patients >=5).

Overall response rate (HI, CR and PR) according IWG criteria 2006 was 49.1%, stable disease was obtained in 37.1 % of MDS patients. We demonstrated by Fisher test that these IWG responses did not correlate with age.

We also evaluated CCI, CIRS, and the ACE-27 in relation to age and to hematological response and no correlation was showed. Hematological or non hematological adverse events were presented by 34% and 36% of patients, respectively. Adverse events were uniformly distributed independently from age. Median overall survival (OS) of our patient cohort was 21 months. Median OS in patients < 75yrs (20 monthts) and >= 75 yrs (16 months) was not significantly different (p value > 0.65).

OS in patients with higher CCI, CIRS, and the ACE-27 was respectly 10 months, 6,5 months and 6 months.

5 patients presented renal failure, but treatment with standard dose aza did not worsen creatinine clearance.

Very elderly patients with comorbidities may be treated with success with azacitidine, without any substantial increase in AE. Nevertheless comorbidities negatively influence overall survival. Evaluation of comorbidities with validated indexes is an useful and easily applicable tool to refine prognostic evaluation. ACE-27, although more complex, seems to give best prognostic evaluation.

Disclosures:

No relevant conflicts of interest to declare.
Targeting Myeloma Stem Cells through Simultaneous Inhibition of Wnt and Hedgehog (Hh) Signaling Pathways
Authors: Tricot Guido J. , Yang Ye , Xu Chujiao , Xu Hongwei , Zeng Ming , Zangari Maurizio , Zhan Fenghuang
Published: October 2019

Abstract 615

Cancer stem cells (CSC) have been identified in a variety of tumor types, including multiple myeloma (MM). Different signaling pathways, such as Wnt, Hedgehog (Hh), Notch and Bmi, are up-regulated in CSC. MM stem cells (MMSC) are enriched in the side population and have increased aldehyde dehydrogenase 1 A1 (ALDH1A1) activity. They are non-cycling and resistant to drugs typically used to treat MM. In this study, MMSC were isolated from 10 MM cell lines (CD138− fraction) and 4 primary MM patients by selecting the CD19+ CD138− cells from bone marrows heavily involved with MM. We confirmed that the CD19+/CD138− cells were part of the malignant clone by comparing expression levels of spiked genes, such as MAF-B and Cyclin D3, in MMSC and CD138+ MM cells. We also verified that CD138− from MM cell lines and the primary CD138−CD19+ had stem cell characteristics by clonogenic in vitro assays and tumor formation in NOD/SCID mice. GEP and real-time PCR analyses indicated that RARa2 levels were significantly higher levels in MM cell line-derived and primary MMSC than in CD138+ bulk MM cells. We also showed that over-expression of RARα2 in low-expressing MM cell lines resulted in increased Wnt and Hh activity, as evidenced by higher levels of nuclear β-catenin, Cyclin D1, TCF4, LEF1, Smo and GLI1. Expression levels of these proteins were also higher in MMSC than in CD138+ MM cells. We subsequently evaluated the efficacy of inhibition of Wnt (CAY10404, a COX-2 inhibitor) and Hh (cyclopamine and itraconazole) signaling on MMSC in vitro and also in vivo using the 5TGM1 mouse model. These drugs given separately induced potent cell death and growth inhibition in MMSC. Quantitative PCR and western blot revealed that CAY10404 decreased nuclear β-catenin and Gli1 expression, while cyclopamine and itraconazole inhibited nuclear GLI1 expression. The efficacy of CAY10404 and cyclopamine was further evaluated in the 5TGM1 mouse model. The CD138− fraction of this cell line has many of the same characteristics as human CD138− MMSC. After injection of 0.5 M cells into the C57BL/KaLwRij mice through the tail vein and allowing growth for 1 week, CAY10404 (20mg/kg, I.P.) and cyclopamine (20mg/kg, I.P) were given separately three times per week. Interestingly, we observed that the mice injected with untreated CD138+ cells survived longer than untreated mice receiving CD138− cells (P < 0.05). Treatment with CAY10404 and cyclopamine significantly reduced the tumor burden measured by idiotype IgG2 protein levels and dramatically prolonged survival (P < 0.05). We conclude that activation of Wnt and Hh pathway maintains the “stemness” features of MMSC. In MMSC, these pathways are activated by increased RARα2 expression, which we have shown to be related to drug resistance. MMSC can be eradicated by a combination of Wnt and Hh inhibitors.

Disclosures:

No relevant conflicts of interest to declare.
Effect of Short-Term Simvastatin Treatment on Endothelial Adhesion Molecules in Sickle Cell Disease: A Pilot Study
Authors: Hoppe Carolyn , Kuypers Frans A , Larkin Sandra K , Hagar Robert , Vichinsky Elliott , Styles Lori A
Published: October 2019

Abstract 1070

Inflammation and abnormal adhesion of leukocytes and sickle red blood cells (RBC) to the vascular endothelium are postulated to play a central role in the pathogenesis of sickle cell disease (SCD). The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been shown to improve vascular function, independent of their lipid-lowering properties, by restoring nitric oxide (NO) bioavailability and suppressing the inflammatory response to endothelial injury. We previously documented an increase in plasma NO (nitrate) levels, as well as a reduction in plasma IL-6 and hs-CRP levels in SCD patients treated with simvastatin. In this study, we investigated the effect of simvastatin treatment on levels of circulating endothelial adhesion molecules, including vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecules (ICAM-1, ICAM-3), platelet endothelial cellular adhesion marker (PECAM), E- selectin, P-selectin as well as vascular endothelial growth factor (VEGF) in SCD patients. Twenty-eight subjects with SCD were treated with 20mg (n=16) or 40mg (n=12) of simvastatin daily for 21 days using a dose-escalating approach. Short-term simvastatin treatment resulted in decreased levels of plasma VCAM-1 (−22%, p=0.003), ICAM-1 (−7%, p=0.004), ICAM-3 (−24%, p=0.01), E-selectin (−9%, p=0.01) and VEGF (−35%, p=0.002) from baseline. The relative change in biomarker levels did not differ between the 20mg and 40mg treatment groups. Among HbSS subjects, ICAM-3 (p=0.02) and P-selectin (p=0.007) decreased to a greater extent in subjects who were not receiving hydroxyurea (HU) therapy compared to subjects who were on HU therapy. Simvastatin holds promise as a potential new treatment for patients with SCD.

Disclosures:

Off Label Use: Zocor (simvastatin) use in sickle cell disease.
ADAMTS13 Promotes Angiogenesis and Modulates VEGF-Mediated Angiogenic Activities
Authors: Lee Manfai , Baza Jonathan , Rodgers George M.
Published: October 2019

Abstract 1145

Severe plasma ADAMTS13 deficiency results in the clinical disorder thrombotic thrombocytopenic purpura. However, other potential pathophysiological roles of ADAMTS13 in endothelial cell biology remain unexplored. To assess the possible role of ADAMTS13 in angiogenesis, cell proliferation and migration of human umbilical vein endothelial cells (HUVEC) were studied in vitro. ADAMTS13 was found to be a highly potent chemoattractant, and additionally was capable of neutralizing VEGF activity in two angiogenesis assays-cell proliferation and cell migration. In the Boyden chamber cell migration assay, treatment of endothelial cells with exogenous recombinant ADAMTS13 promoted cell migration in a dose-dependent manner, with 1 ng/mL increasing cell migration across a gelatinized polycarbonate membrane by 14-fold. In the same model, 5 ng/mL VEGF165 (molar ratio of ADAMTS13:VEGF165 = 1/19) only increased cell migration by 7 fold. A steady decrease in endothelial cell migration was observed when the concentration of ADAMTS13 exceeded 1 ng/mL (Figure 1). Coincubation of 30 ng/mL ADAMTS13 with 6.16 ng/mL VEGF165 (molar ratio of ADAMTS13/VEGF165 = 1.3/1) inhibited endothelial cell migration by 45% compared to VEGF alone (Figure 2). A second model using an in vitro scratch-wound assay confirmed the Boyden chamber data.

Substitution of ADAMTS13 with ADAM17, an analog of ADAMTS13 without the thrombospondin domain reversed the inhibition of VEGF-mediated cell migration, suggesting that the thrombospondin domain of ADAMTS13 is responsible for the inhibitory interaction with VEGF165. This finding was in agreement with our previously published co-immunoprecipitation assay data (Blood 2010, 116, 4307). Similar patterns of inhibition were observed with VEGF121 and VEGF189, indicating that other isoforms of VEGF may interact with the TSP domain of ADAMTS13. Using a manual proliferation assay method, HUVEC treated with 30 ng/mL ADAMTS13 and 6.16 ng/mL VEGF165 proliferated 40% slower than the control treated with VEGF alone. Combined with our findings on the inhibition of endothelial cell-tube formation in a Matrigel assay with ADAMTS13 and VEGF165 previously reported, our cumulative data suggest that 1) ADAMTS13 promotes angiogenesis by increasing cell migration and 2) ADAMTS13 can modulate VEGF-mediated angiogenic activities.

Disclosures:

No relevant conflicts of interest to declare.
Live Cell Imaging Captures Immune Cell-Mediated Killing of Precursor-B Acute Lymphoblastic Leukemia Cells Targeted with Anti-CD19 (Medi-551) Antibodies: Support for Macrophage and NK Cell In Vivo Activity
Authors: Matlawska-Wasowska Ksenia , Cook Dennis , Stevens Samuel R. , Ward Elizabeth K. , Herbst Ronald , Winter Stuart S. , Wilson Bridget S.
Published: October 2019

Abstract 1522

Precursor-B acute lymphoblastic leukemia (pre-B ALL) is the most common malignancy in children and can be cured in a majority of patients. However, cure remains elusive in approximately 20% of patients for reasons that are not well understood. Importantly, survivors commonly develop morbidities that result from dose-intensified treatment with cytotoxic drugs. Here, we investigate the tumoricidal effects of a novel humanized anti-CD19 monoclonal antibody (Medi-551). The a-fucosylated form of this antibody has increased affinity to human FcgammaRIII (CD16) receptor, present on the surface of NK cells and macrophages, mediating antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Medi-551/CD19 complexes internalize slowly and thus remain accessible for effector cells for prolonged periods. We evaluated in vitro ADCC and ADCP activities of primary human NK cells and macrophages (effector cells) against four pre-B ALL cell lines (697, Nalm 6, MHH-Call 3, RS 4;11), as well as freshly isolated patient blasts. We report results of live cell fluorescent imaging studies, characterizing the formation of immunological synapses between Medi 551-bound target leukemia cells and effector cells, as well as the kinetics of both NK-mediated killing and macrophage phagocytosis. The number of the CD19 receptors present on the cell surface is shown to be a factor in effector-mediated killing of Medi-551 targeted leukemia cells. Further, genetic polymorphisms in FcgammaRIII (158 F/V, V/V or F/F) affected in vitro ADCC and ADCP activities with FcgammaRIII 158 V homo- or heterozygotes showing the strongest activity. We also evaluated the efficacy of Medi-551 in a human pre-B ALL murine xenograft model. SCID mice were engrafted with 697 pre-B ALL cells and received either vehicle alone or Medi-551 (3 mg/kg; twice weekly for a total of 5 doses); treatment was started at day 5 after engraftment. Medi-551 treatment markedly lowered disease burden in blood, liver and bone marrow. The lack of cure is consistent with impaired roles for NK cells in this model, since murine NK cells lack FcgammaRIV. Experiments are in progress to improve the model through adoptive transfer of human NK cells. Taken together, the in vitro and in vivo data show that Medi-551 has strong activity against pre-B ALL and support a move forward to early phase trials in this disease.

Disclosures:

No relevant conflicts of interest to declare.
BMS-936564/MDX-1338: A Fully Human Anti-CXCR4 Antibody Induces Apoptosis In Vitro and Shows Anti Tumor Activity In Vivo
Authors: Kuhne Michelle R. , Mulvey Tanya , Chen Sharline , Pan Chin , Chong Colin , Niekro Wafa , Kempe Tom , Henning Karla A. , Cohen Lewis , Korman Alan , Cardarelli Pina M
Published: October 2019

Abstract 1543

BMS-936564/MDX-1338 is a fully human monoclonal antibody that specifically recognizes human CXCR4 and is currently in phase 1 studies in patients with relapsed/refractory acute myeloid leukemia (AML) and multiple myeloma (MM). CXCR4 has been identified as a prognostic indicator for AML and other malignancies, in which greater expression of CXCR4 correlates with disease severity. CXCR4 is a seven-transmembrane, G-protein-coupled receptor in the CXC chemokine receptor family. In response to stimulation by its ligand, the chemokine CXCL12, CXCR4 activates calcium flux, chemotaxis and mediates directional migration of hematopoietic cells. In healthy adults, the receptor is predominantly expressed on B and T cells, monocytes, macrophages, NK and dendritic cells, as well as lymphoid and myeloid precursor cells. Expression of CXCR4 is elevated in a variety of cancers and the interaction of CXCR4 on tumor cells with CXCL12 in the bone marrow promotes tumor cell survival and growth. An antagonist of this pathway is predicted to be efficacious in a variety of hematologic malignancies. In vitro studies demonstrate that BMS-936564/MDX-1338 binds to CXCR4expressing cells with low nanomolar affinity. The antibody blocks CXCL12 binding to CXCR4 expressing cells and inhibits CXCL12 induced migration and calcium flux with low nanomolar EC50 values. When given as monotherapy on established tumors, the antibody exhibits anti-tumor activity in multiple AML, NHL and MM xenograft models. BMS-936564/MDX-1338 is an IgG4 and thus does not elicit complement dependent cytotoxicity (CDC) or antibody dependent cell mediated cytotoxicity (ADCC). In vitro and in vivo studies suggest that BMS-936564/MDX-1338 induces apoptosis as one mechanism of tumor growth inhibition. Here we describe the in vitro and in vivo characterization and activities of BMS-936564/MDX-1338.

Disclosures:

Kuhne: Bristol-Myers Squibb: Employment. Mulvey:Bristol-Myers Squibb: Employment. Chen:Bristol-Myers Squibb: Employment. Pan:Bristol-Myers Squibb: Employment. Chong:Bristol-Myers Squibb: Employment. Niekro:Bristol-Myers Squibb: Employment. Kempe:Bristol-Myers Squibb: Employment. Henning:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Korman:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment.
A Restrospective Review of Peripheral T-Cell Lymphoma in a Single Institution Between 2000 and 2010
Authors: Broussais Florence , Coso Diane , Ivanov Vadim , Aurran Thérèse , Stoppa Anne-Marie , Bouabdallah Reda
Published: October 2019

Abstract 1623

Background:

Peripheral T-cell non-hodgkin lymphomas (NHL) comprise a heterogeneous group of malignancies, characterized by an aggressive disease course and a poor clinical outcome. T-cell lymphomas present in lymph nodes and they also frequently involve extra nodal sites. The first line treatment consists in CHOP-like regimen. Consolidation treatment in first line or in relapse is autologous stem cell transplantation (ASCT) or allogenic stem cell transplantation (SCT).

Patients and methods: We retrospectively analysed the data of 189 adults who had received chemotherapy in our institution between 2000 and 2010.

Results:

Median age at time of presentation was 55 (range 17–89) years, 76% were <65 years. 50% had B symptoms and 50% serum elevated LDH. ECOG was 0–1 in 60% and 2–4 in 40%. According to the Ann Arbor classification, 15% were stage I-II and 85% were stage III-IV. 50% had low IPI (0-1-2) and 50% had elevated IPI (3-4-5). The histologic subtypes were 42% peripheral T-cell NHL unspecified (PTCL-U), anaplastic large cell lymphoma (ALCL), 8% had ALK+ and 10% had ALK-, 24% angioimmunoblastic lymphoma (AILT), 3% transformed mycosis fungoide, 2% instestinal T-cell lymphoma, 2% hepatosplenic γδ T-cell lymphoma and 1% adult T-cell lymphoma/leukemia. Primary extranodal lymphoma represented 17% and 8% were diagnosed with hemophagocytosis.

Five-year overall survival (OS) was 28.3% (21.8–36.8%), and five-year progression free survival (PFS) was 18.4% (13.1–25.7%). On multivariate analysis, ALCL-ALK+ (p=0.008), AILT (p<0.001), extranodal involvement (p=0.01), PS>1 (p<0.001), LDH>N (p=0.003) were independent adverse factors for OS. Moreover B symptoms (p<0.01) was a significant factor for PFS.

86% received CHOP-like induction treatment. The median number of chemotherapy was 2 (1–7). 59% experienced a complete response during the therapeutic procedure, while 22% were primary refractory. 44% had ASCT and 14% allogenic SCT. Only 28% of patients referred for allogenic SCT received this treatment.

Conclusions:

This 10-year review of patients treated in a single institution with initial conventional chemotherapy followed by more intensive treatments confirms the poor OS and PFS in the case of T-cell lymphoma. Despite a low incidence, T-cell lymphoma could be a priority area of research for new treatments with a potential to improve OS and PFS.

Disclosures:

No relevant conflicts of interest to declare.
Deletion or Inhibition of Fc Gamma Receptor IIb (CD32) Prevents the Memory B Cell Response to Factor VIII in a Hemophilia A Mouse Model
Authors: Werwitzke Sonja , von Hornung Marcus , Kalippke Katy , Trummer Arne , Ganser Arnold , Tiede Andreas
Published: October 2019

Abstract 204

The formation of inhibitory antibodies to factor VIII (FVIII) is the foremost complication of replacement therapy in hemophilia A. Patients with inhibitors are treated with very high doses of FVIII, over prolonged periods of time, to induce immune tolerance. Studies in a hemophilia A mouse model demonstrated that very high doses of FVIII can induce apoptosis in FVIII-specific memory B cells and prevent their differentiation into antibody-secreting cells. The Fc gamma receptor IIb (FcgRIIb) is expressed on B cells and mediates inhibitory signals after crosslinking with the B cell receptor. Here, we studied the potential role of this receptor in the regulation of memory B cell response to FVIII. FVIII knockout mice (B6;129S4-F8tm2Kaz/J) were crossed with FcgRIIb knockout mice (B6;129S4-Fcgr2btm1Ttk/J). Comparing F8−/− mice and F8−/−/FcgR2b−/− double knockout mice, the initial anti-FVIII antibody formation was similar after intravenous exposure to 4 weekly doses of 80 or 400 IU/kg. Similar numbers of FVIII-specific antibody-secreting cells were detected in the spleen and bone marrow by ELISPOT. As previously shown, in vitro re-stimulation of memory B cells from spleens of immunized F8−/− mice at doses of 1 to 200 ng/ml induced their differentiation into antibody-secreting cells. Higher doses of 400 to 800 ng/ml prevented differentiation. In F8−/−/FcgR2b−/− double knockout mice, however, formation of antibody-secreting cells was completely inhibited across all FVIII doses tested. Addition of B220-depleted splenocytes from F8−/− mice did not restore memory B cell function in F8−/−/FcgR2b−/− double knockout mice, indicating that the observed effect was not due to dysfunction of follicular dendritic cells or other antigen-presenting cells. Inhibition of FcgRIIb using a monoclonal antibody prevented the FVIII-specific memory B cell response in splenocytes from immunized F8−/− mice. Staining with propidium iodide, annexin V, or anti-caspase 3 indicated increased rates of apoptosis when FcgRIIb was blocked during re-stimulation. In summary, FcgRIIb plays a crucial role for the differentiation of FVIII-specific splenic memory B cells into antibody-secreting cells. Inhibition of FcgRIIb appears to sensitize B cells for apoptosis during re-stimulation with FVIII. This mechanism could potentially facilitate the eradication of FVIII-specific memory B cells during ITI.

Disclosures:

No relevant conflicts of interest to declare.
DISTINCT ROLES for Rap1b In PLATELET SECRETION and INTEGRIN aIIBb3 OUTSIDE-In SIGNALING
Authors: Zhang Guoying , Xiang Binggang , Shaojing Ye , Chrzanowska-Wodnicka Magdalena , Morris Andrew J. , Gartner T. Kent , Whiteheart Sidney W. , White Gilbert C. , Smyth Susan S. , Li Zhenyu
Published: October 2019

Abstract 2200

Rap1b is activated by platelet agonists, and plays a critical role in integrin aIIbb3 inside-out signaling and platelet aggregation. In this study, we identify two novel functions of Rap1b in platelets. We show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that aIIbb3 outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Defect in secretion of Rap1b deficient platelets was not due to reduced granule contents, because the amount of serotonin (5HT) and platelet factor 4 (PF4) in Rap1b deficient platelets is similar to that in wild type platelets. Data from transmission electron microscopy indicate that under resting conditions, wild type and Rap1b deficient platelets had normal discoid shapes with similar numbers of granules. When stimulated with thrombin, wild type platelets showed an irregular appearance with protruding filopodia and lack of granules. In contrast, thrombin-stimulated Rap1b−/– platelets showed that more Rap1b−/– platelets contained visible a granules than wild type platelets. Dense granules were also more obvious in thrombin-stimulated Rap1b−/– platelets than wild type platelets. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y12 or TP deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-BAPTA. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b deficient platelets compared with wild type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b deficient platelets were not rescued by addition of MnCl2, which elicits aIIbb3 outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin aIIbb3 outside-in signaling.

Disclosures:

No relevant conflicts of interest to declare.
Involvement of Lipid Rafts and Caveolae in gas6-Axl Signaling in Endothelial Cells
Authors: Laurance Sandrine , Lemarie Catherine A , Blostein Mark
Published: October 2019

Abstract 2211

Lipid rafts are localized regions of plasma membranes that contain primarily cholesterol and glycosphingolipids. Many receptor tyrosine kinases including the epidermal growth factor receptor, the platelet derived growth factor receptor, and the insulin receptor, localize specifically to these structures. Lipid rafts have been shown to play a role in intracellular signaling events. More specifically, diverse processes such as dimer formation, autophosphorylation, and intracellular adaptor protein recruitment/activation have been specifically involved in receptor tyrosine kinase/lipid raft interactions. Caveolae are defined as a subclass of lipid rafts and form well-shape invaginations on the cell surface. Caveolin-1 is the major constitutive protein of caveolae. Axl is a receptor tyrosine kinase whose association with lipid rafts and more specifically with caveolae has not been elucidated. Gas6, the ligand for Axl, is a vitamin K dependent protein that is post-translationally modified by an enzymatic process called g-carboxylation. We hypothesize that gas6/Axl mediated intracellular signaling is dependent on its localization to lipid rafts/caveolae in endothelial cells. First, we show that gas6-induced c-Src, Akt and ERK1/2 phosphorylation is mediated via Axl by using Axl siRNA in human umbilical vein endothelial cells (HUVECs). Then, lipid rafts were isolated from HUVECs by a detergent-free lysis method followed by a sucrose gradient centrifugation and western blot analysis. We found that Axl moved into the lipid raft-enriched fractions after 5 and 10 min of gas6 treatment as shown by its colocalization with caveolin-1. Interestingly, after gas6 treatment, c-Src, which is known to act as a transient docking platform for signaling molecules in lipid rafts, follow the same localization pattern to lipid raft-enriched fractions. The requirement of caveolae was then evaluated by transfecting HUVECs with caveolin-1 siRNA. Caveolin-1 knock down abolished completely gas6-induced phosphorylation of c-Src, Akt and ERK1/2 thus highlighting the role of caveolae in gas6-Axl signaling. Taken together these results demonstrate that activation of gas6-dependent signaling pathways involves Axl trafficking to lipid rafts/caveolae. These novel findings identify lipid rafts/caveolae as dynamic scaffolding systems for gas6/Axl interactions in endothelial cells.

Disclosures:

No relevant conflicts of interest to declare.
Age Dependent Alternations In Hematopoietic Stem Cell Niches
Authors: Li Geqiang , Arrieta Cesar Nombela , Wang Leo D. , Wagers Amy J. , Silberstein Leslie E
Published: October 2019

Abstract 2395

Age associated decline in hematopoietic system is a well-documented process which encompasses a substantial expansion of the hematopoietic stem cell (HSC) pool and a decline of their competitive repopulating ability on a per cell basis. Expansion occurs predominantly in the myeloid biased HSC subset, subsequently leading to a global skewing of lineage differentiation from lymphopoiesis toward myelopoiesis. These age-related effects are known to partially result from cell-intrinsic alterations of HSC, which include DNA damage accumulation, oxidative stress and activation of tumor suppressor pathways. HSCs exist in specialized microanatomical locations within the bone marrow (BM), where they receive critical regulatory cues that control HSC homeostasis. While the cell-autonomous effects of HSC aging have been extensively studied, little is known on how aging of the surrounding microenvironment may contribute to the age-associated alterations of HSC function, and thus the focus of our study.

Here, we have employed novel imaging approaches to analyze age-related changes of the BM microenvironment, and how these affect the global spatial distribution of HSPC-enriched subsets in the BM parenchyma. Our preliminary studies suggest during aging leads to a profound disruption of the BM microvascular compartment. By using imaging cytometry of two-dimensional (2D) sections, as well as confocal microscopy of 3D-BM slices, we demonstrate that the characteristic sinusoidal network found in young mice, degenerated with aging into a highly disorganized and expanded vascular bed, in which expression of the arterial marker Sca-1 was strongly upregulated. Flow cytometry analysis further revealed a 3–4 fold increase of CD45-Ter119-CD31+ Sca-1+cells in 22–24 month old mice. We next investigated how the changes in vasculature network impact HSC distribution. We employed imaging cytometry to track the presence in BM sections of a population of Lineage-Cd48-kit+ cells, 60% of which are long term HSC. Our preliminary results indicate that, in aged mice, HSPC-accumulation in endosteal zones (<100μm from endosteal surfaces) remains unchanged. Of note, consistent with the increase in vascularization, HSPCs display a significantly increased tendency to directly associate to BM microvessels in 22–24month old mice. We are currently investigating at the molecular level how altered vasculature influence HSC aging which would enable us to enhance the regenerative properties of the aged stem cells.

Disclosures:

Wagers: iPierian, Inc.: Membership on an entity's Board of Directors or advisory committees.
Alternative Methods of Activation of the Stem Cell Niche Reveal a Spatial Localization of Primitive Cells in the Bone Marrow
Authors: Rashidi Narges M , Spindler Tassja J , Barsky Lora W , Adams Gregor B.
Published: October 2019

Abstract 2396

Osteoblasts are key constituents of the murine hematopoietic stem cell (HSC) endosteal niche, evidenced by the fact that increasing their activity leads to an increase in the number of HSCs. However, recent studies have also suggested a role of the bone resorbing osteoclast in the HSC niche. In these studies we wished to examine whether activation of the osteoblast or the osteoclast had differing effects on the primitive hematopoietic cell populations. To achieve this we pharmacologically activated osteoclasts and osteoblasts using receptor activator of nuclear factor-κB ligand (RANK-L) and parathyroid hormone (PTH), respectively. Our in vitro results demonstrated that both PTH and RANK-L treatment of bone marrow stromal cell populations activated osteoblasts and osteoclasts. Yet, RANK-L treatment, which is able to expand the osteoclastic population to a much higher degree, demonstrated enhanced support of primitive hematopoietic cells in cobblestone area forming cell assays. We next examined the effects of in vivo treatment with RANK-L and PTH by performing histological analyses to study the effect of these treatments on bone composition, and examining the effects on different HSC sub-populations. These data suggested that the effects of our treatment regimens were different according to the region of the bone, with RANK-L treatment having no effect or leading to a reduction in trabecular bone in the metaphysis, but an increase in bone remodeling activity at the endosteal surface of the cortical bone in the diaphysis, while PTH treatment increases bone formation and remodeling activity both in the diaphysis and the trabecular bone of the metaphysis. Correlating with this was our observations that LT-HSC frequency was increased following RANK-L and PTH treatment, yet an increased ST-HSC frequency was only observed following PTH treatment. Direct examination of HSCs in specific regions of the bone indicated that RANK-L treatment preferentially increased the LT-HSCs in the diaphysis region of the bone marrow, while PTH treatment specifically increased the ST-HSC population in the metaphysis. These data not only reveal a supportive role for osteoclasts in the HSC niche suggesting that osteoclast interaction with osteoblasts is an essential component for maintaining HSC population in the bone marrow niche, they also suggest a possible structural organization for localization of LT-HSC vs. ST-HSCs in the long bone with LT-HSCs being more localized in the endosteal surface of the diaphysis area of the long bones as oppose to the ST-HSCs having a preference for the metaphysis of these bones.

Disclosures:

No relevant conflicts of interest to declare.
The TLE1 Tumor Suppressor Regulates Myc Induced Leukemogenesis
Authors: Sweetser David , Ramasamy Selvi , Blackburn Jessica S , Langenau David M.
Published: October 2019

Abstract 2473

The Groucho/TLE family of corepressors has been described as master regulatory genes during development, affecting multiple cell fate decisions. These proteins bind to a variety of transcription factors and recruit inhibitory proteins to repress transcription. We previously identified TLE1 as a novel tumor suppressor gene that is deleted or methylated in subgroups of acute myeloid leukemia (AML) and other hematological malignancies. We find the loss of Tle1 alone is insufficient to induce leukemia in mice and apparently requires cooperation with additional oncogenes. Our studies, and those from other groups, have shown that over-expression of TLE1 in leukemia cells slows cell cycle progression, colony formation and tumor growth in xenografts, while silencing results in increased cell proliferation. The pathways by which TLE1 affects oncogenesis is unclear, but this gene family is capable of interacting with effectors of Myc, Wnt, Notch, TGFB signaling–prominent pathways dysregulated in malignancies.

Myc is important for hematopoietic stem cell proliferation, survival and differentiation and is over-expressed in most AML samples. The TLE homologue Groucho binds and represses Drosophila Myc expression of target genes, thus we postulated that TLE1 could be an important regulator of Myc activity in leukemia. Using hematopoietic progenitor cells from Tle1 knockout and wild-type fetal livers we found that the loss of Tle1 dramatically increased proliferation and serial replating efficiency. Expression of N-Myc by itself in wild type fetal liver cells triggered significant cell death and apoptosis. However, when N-Myc expression was combined with the additional loss of Tle1, not only was N-Myc induced apoptosis inhibited, but a dramatic cell proliferation, well in excess of that seen with Tle1 loss by itself, was seen. Furthermore, mice transplanted with N-Myc transduced hematopoietic cells from Tle1 knockout mice fetal liver developed a more aggressive leukemia, compared to N-Myc transfected wild type mice fetal liver hematopoietic cells, with increased proliferation of leukemic cells as demonstrated by in vitro colony assays and higher secondary transplantability.

We extended these studies to a zebrafish model of Rag2-Myc mediated T-ALL. Using these zebrafish we demonstrated over-expression of the TLE homologue, Groucho, completely blocked the initiation and progression of Myc induced leukemia development. Expression of a truncated version of Groucho reduced the initiation of T-ALL and prolonged the survival of fish developing leukemia. These studies demonstrate TLE1 can inhibit the oncogenicity of Myc, and suggests modulation of expression of this gene family may be of importance for a variety of malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Studying Evolution of Multiple Myeloma with CanEvolve
Authors: Shah Parantu K , Samur Mehmet Kemal , Li Cheng , Munshi Nikhil C.
Published: October 2019

Abstract 2886

Multiple Myeloma (MM) evolves from monoclonal gammopathy of undetermined significance (MGUS), its pre-malignant stage at the rate of 1% per year. Moreover all MM patients eventually develop drug resistance. Currently, very little is known about this process of MM evolution, specifically about the changes in regulatory networks and signaling pathways responsible for it. One major difficulty in studying this is the lack of suitable functional genomics profiles covering all the stages of MM evolution from the same patients.

We have now modeled this process using the knowledge stored in the canEvolve portal for cancer integrative genomics and evolution (available at http://www.canevolve.org). The current version of the portal stores gene and miRNA expression and copy number alterations information derived from microarray and next generation sequencing platforms for more than 10 cancer types. It also stores hundreds of thousands of instances of protein-protein interactions, and metabolic and signaling pathway information for the Human proteome. The primary analysis capabilities include identification of differential gene expression, survival analysis and reconstructed gene regulatory networks and their visualization. Finally, canEvolve stores integrative analysis carried out using Gene set enrichment (GSEA) and GemiNI.

We carried out meta analysis of gene expression profiles of MGUS, smoldering Myeloma, MM and relapsed patients publicly available from multiple Gene expression omnibus (GEO) datasets. We carried out geneset enrichment analysis of normal-MGUS, normal-MM, normal-relapsed, MGUS-SMM, MGUS-MM and MM-relapsed pairs with different gene sets available from MSigDB. We identified transcription factors, miRNA, metabolic and signaling pathways whose targets/members significantly change expression. For example, targets of mir-17 and NF-Kappa B significantly change as MGUS turns to MM. Similarly, targets of miR-484 and CREL significantly changes as MM patients relapse. The CanEvolve portal thus provides us the ability to serially monitor and analyze various genomic datasets to provide targets that may play a crucial role in myelomagenesis as well as propgression of the disease. We are now validating some of the identified targets.

Disclosures:

No relevant conflicts of interest to declare.
Successful Treatment with Bortezomib in Combination with Bendamustine and Prednisone of Patients with Newly Diagnosed/Untreated Multiple Myeloma and Light Chain Induced Renal Failure
Authors: Pönisch Wolfram , Andrea Marc , Wagner Ina , Hammerschmidt Doreen , Kreibich Ute , Schwarzer Andreas , Zehrfeld Thomas , Schwarz Maik , Winkelmann Cornelia , Petros Sirak , Bachmann Anette , Lindner Tom , Niederwieser Dietger
Published: October 2019

Abstract 2938

Introduction:

Renal impairment is one of the most severe complications of Multiple Myeloma (MM) at diagnosis. These patients are at increased risk for infections and have a significantly worse prognosis. Small phase I/II studies suggest that treatment with chemotherapy and/or new substances results in recovery of renal function in up to 25%. The window of opportunity to reverse renal impairment is rather small, making an immediate and highly active treatment strategy mandatory. Bortezomib as well as Bendamustine have turned out to be effective, rapid action drugs in the treatment of MM. Bendamustine is a bifunctional alkylating agent with low toxicity that produces both single- and double-strand breaks of DNA, and shows only partial cross resistance with other alkylating drugs.

Methods:

Between June 2006 and May 2011, 18 patients (median age 69; range 43 – 86 years) with newly diagnosed/untreated MM and renal insufficiency (creatinine clearance < 35 ml/min) were treated with Bendamustine 60 mg/qm day 1 and 2, Prednisone 100 mg on day 1, 2, 4, 8 and 11, and Bortezomib 1.3 mg/qm on day 1, 4, 8 and 11 (BPV). Cycles were repeated every 21 days up to the stage of maximum response or disease progression. MM response was assessed using IMWG criteria modified to include near complete response (nCR) and minimal response (MR). Eight patients were on dialysis at the time of diagnosis.

Results:

Fifteen patients (83%) responded after at least one cycle of chemotherapy with three sCR, five nCR, five VGPR, and two PR. With a median follow up of 17 months, PFS at 12 months was 57 % and OS was 61 %. The median number of the BPV-treatment cycles was 2 (1–5) cycles. The myeloma protein decreased rapidly, reaching the best response after the first cycle in 4 patients and after the second cycle in a further 7. Six patients showed a complete remission of the kidney function (creatinine clearance > 60 ml/min) and in seven patients a partial remission (creatinine clearance > 30 ml/min) was attained. Transient grade 3 – 4 neutropenia was reported in one patient, and grade 3 – 4 thrombocytopenia occurred in 6 patients. One patient experienced a new grade 3 polyneuropathy.

These results indicate that the combination of Bortezomib, Bendamustine and Prednisone is effective and tolerated in patients with newly diagnosed/untreated MM and renal failure.

Disclosures:

Pönisch: Mundipharma: Honoraria, Research Funding. Niederwieser:Mundipharma: Research Funding.
Rapid and Efficient Mobilization of Murine Hematopoietic Stem and Progenitor Cells with Nox-A12, a New Spiegelmers®-Based CXCR4/SDF-1(CXCL12) Antagonist
Authors: Scheller Marina , Schwoebel Frank , Vossmeyer Doerte , Leutz Achim
Published: October 2019

Abstract 2995

Mobilization of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) is important in many hematological therapies. However, up to 30% of the patients respond poorly to standard granulocyte colony-stimulating factor (G-CSF) treatment, highlighting the need for more effective mobilizing strategies. The CXCR4/stromalcell-derived factor 1 (SDF-1) axis plays a crucial role in the interaction between HSCs and the marrow niche and is involved in HSC mobilization. NOX-A12 is a structured mirror-image RNA oligonucleotide, a so-called Spiegelmer®, that was identified to bind SDF-1 thereby inhibiting its activity with subnanomolar IC50. HSC/HPC mobilization by NOX-A12 was examined in the mouse. Single NOX-A12 administration induced reversible mobilization of HSC/HPC populations within a few hours. NOX-A12 synergized with G-CSF to strongly enhance HSC/HPC mobilization. In particular, the progenitor compartment mobilized by single NOX-A12 administration contained more differentiated short-term HSCs (ST-HSCs), and combined administration of NOX-A12 and G-CSF mobilized a significantly higher proportion of primitive and more potent murine long-term repopulating cells that successfully engrafted primary and secondary lethally-irradiated recipients. These results characterize NOX-A12 as a potent HSCs/HPCs mobilizing therapeutic in mammals and suggest its clinical potential.

Disclosures:

No relevant conflicts of interest to declare.