bluuebunny/crossref_metadata_2025 huggingface dataset:
Leukocyte Adhesion and Activation in Atherothrombosis
Authors: Gawaz Meinrad
Published: October 2019

Platelets interact with a variety of cells, including leukocytes and progenitor cells. Upon activation, platelets release chemokines such as CXCL12/SDF-1 that modulate important functional aspects of vascular inflammation and atherogenesis. Platelet-derived SDF-1 and CyPA regulate chemotaxis, migration, and cell activation in the microenvironment of accumulating platelets. Previously we have shown that platelets are the major source of CXCL12/SDF-1. Platelet-derived SDF-1 provides a strong signal for recruitment of circulating progenitor cells and leukocytes towards vascular and myocardial injury. Patients with severe coronary artery disease have enhanced levels of plasma and platelet-associated SDF-1 that correlates with an enhanced number of circulating progenitor cells and the development of cardiovascular diseases. In patients with both acute myocardial infarction platelet-SDF-1 and an increase in circulating platelet/progenitor cell aggregates, the combination is associated with myocardial recovery and improved prognosis, indicating that platelet-SDF-1 is critical for repair and regeneration of vascular or myocardial injury. Administration of a stable recombinant SDF-1 fusion protein reduces infarct size and preserves myocardial infarction in mice. We showed that platelet-derived SDF-1 is an important survival factor both for platelets (in an autocrine manner) and for surrounding cells. SDF-1 released from activated platelets binds to its receptor CXCR4 and induces internalization of the SDF-1/CXCR4 complex. SDF-1-ligation of CXCR4 augments surface expression of CXCR7; a process coupled via ERK1/2 and cyclophilin A. SDF-1α caused downstream phosphorylation of Erk1/2 and cyclophilin A (CyPA). NIM-811 a CyPA-PPIase-activity inhibitor significantly abolished SDF-1α-driven CXCR7 surface exposure. Moreover, Cypa-/-murine platelets failed to show SDF-1α/CXCR4-mediated CXCR7 translocation. SDF-1α-induced ubiquitination of CXCR7 was essential for its surface translocation and was dependent on Erk1/2 and CyPA-PPIase activity, inhibited by U0126 and NIM-811, respectively. In contrast to wild-type, Cypa-/- murine platelets failed to exhibit a dynamic change in CXCR7 ubiquitination status upon SDF-1α exposure and thus failed in subsequent CXCR7 externalization. SDF-1/CXR4-driven CXCR7 translocation inhibits activation-induced apoptosis of platelets and prolongs platelet survival in vitro and in vivo. Similarly, platelet SDF-1 interacts with monocytes, reduces apoptosis and promotes differentiation of monocytes into macrophages. Inhibition of platelet release and adhesion to endothelial cells reduces monocyte migration and differentiation into macrophages in vitro and attenuates vascular inflammation and atheroprogression in apoE-deficient mice. In conclusion, platelets and platelet interaction with leukocytes are critical in regulation of vascular inflammation and atheroprogression and may offer promising targets for modulating inflammation in disease states.

Disclosures:

No relevant conflicts of interest to declare.
Ras Effector Pathways Differentially Support Proliferation and Survival of Acute Myeloid Leukemia
Authors: Eckfeldt Craig E , Pomeroy Emily J , Lee Robin DW , Hazen Katherine S , Moriarity Branden S , Largaespada David A
Published: October 2019

Mutations that lead to activation of Ras oncogenes and their effector pathways are common in acute myeloid leukemia (AML) making Ras signaling an attractive therapeutic target. To date, efforts to therapeutically target Ras directly have proven difficult and clinical outcomes have been disappointing. The rational development of strategies for targeting Ras effector pathways in AML have been hindered by a limited understanding of the how specific Ras pathways maintain AML proliferation and survival. To determine the roles of specific Ras effector pathways in AML we disrupted signaling through canonical Ras effector pathways (RAF-MEK-ERK, PI3K-AKT-mTOR, and RAL) in murine and human AML cells. Consistent with previously published data (Kim et al. Blood. 2009;113:1086), we found that suppression of NRAS(V12) expression in a tetracycline-repressible NRAS(V12) and Mll-AF9-driven murine AML model (tNM AML) led to AML remission in leukemic mice in vivo and almost completely suppressed leukemic-colony formation in vitro (20-fold reduction, p <0.01). In the tNM model suppression of the NRAS oncogene led to apoptosis of AML cells, but had little effect on cell cycle progression. Surprisingly, pharmacologic inhibition of the major oncogenic Ras effector pathways, RAF-MEK-ERK and PI3K-AKT-mTOR, alone or in combination suppressed AML cell growth by inducing G0/G1 cell cycle arrest in both tNM AML cells and human AML cell lines, but did not promote apoptosis in either. Conversely, pharmacologic inhibition of RALB signaling in both mouse and human AML cells induced apoptosis, but had little effect on cell cycle progression. Notably, these effects were seen in both NRAS mutant (tNM and THP1) and NRAS wild-type (MOLM13) AML cells. In preclinical studies, the RALB inhibitor Dinaciclib and the dual PI3K/mTOR inhibitor BEZ235 synergized to suppress AML growth in vitro, and had a potent anti-leukemic activity against tNM AML in vivo. We are currently evaluating the effects of a dominant negative mutant of RALB, RALB(N28), on AML growth and survival, and investigating whether RALB signaling supports AML survival through TBK1, NF-kB, and BCL2 family members as has been shown for KRAS-driven epithelial cancers (Barbie et al. Nature. 2009;462:108). By elucidating the distinct roles for RAS effector pathways in maintaining AML proliferation and survival, we have uncovered a critical role for RALB signaling in AML survival. This work suggests that combination therapy targeting both Ras effectors that promote proliferation (PI3K/mTOR) and survival (RALB) may provide a rational treatment approach for AML.

Disclosures

Largaespada: NeoClone Biotechnology: Consultancy, Equity Ownership; Discovery Genomics, Inc.: Consultancy, Equity Ownership.
Flow Cytometric Leukemic Blasts Detection in Cerebrospinal Fluid of Children with Acute Lymphoblastic Leukemia
Authors: Levinsen Mette , Marquart Hanne Vibeke , Groth-Pedersen Line , Frandsen Thomas Leth , Albertsen Birgitte Klug , Pronk Cornelis J.H. , Heldrup Jesper , Ulvmoen Aina , Vaitkeviciene Goda , Wehner Peder Skov , Frost Britt-Marie , Abrahamsson Jonas , Harila-Saari Arja , Niinimäki Riitta , Lähteenmäki Päivi , Åsberg Ann , Lund Bendik , Gunnes Maria Winther , Norén-Nyström Ulrika , Behrendtz Mikael , Pesola Jouni , Taskinen Mervi , Schmiegelow Kjeld
Published: October 2019

Background: Central nervous system (CNS)-directed treatment has reduced risk of CNS relapse for childhood acute lymphoblastic leukemia (ALL), which accounts for 30-40% of initial relapses. Compared with CNS-negative patients, patients with CNS leukemia (>5 leukocytes/µL cerebrospinal fluid (CSF) and lymphoblasts) suffer from more CNS+ relapses. Conventional cytology (CC) is specific (>95%), but nonsensitive (<50%), since leukocytes in CSF decay within few hours after lumbar puncture.

Method: We prospectively assessed centralised multi-parameter flow cytometry (FCM) of fixated CSF versus local CC in Nordic/Baltic childhood ALL. Diagnostic samples from 172 children aged 0-18 years with de novo and eight children with relapsed ALL were investigated in 297 CSF samples from 180 patients. Kinetics of disappearance of leukemic cells in the CSF was evaluated until day 15. Antibody-combinations reflected the immunophenotype of leukemic blasts in bone marrow.

Result: Of 172 newly diagnosed patients, 51 (30%) had CSF involvement by FCM, while CC was positive in 16 patients (9%) (p<0.001). CSF involvement was detected by both FCM and CC in four of eight patients with relapse (50%). Among newly diagnosed patients, samples positive by FCM and CC had higher leukemic blast count compared to samples positive by FCM only (medians: 0.545 (range: 0.005-4.801) versus 0.016 (range: 0.003-1.38) leukemic blasts/µL; p<0.001). Among newly diagnosed patients with samples positive by FCM and CC, the CSF blast count was related to the CSF leukocyte count (rs=0.82; p=0.001). Compared to newly diagnosed patients who were FCM-negative, those with FCM-positivity had higher WBC (median: 37 versus 9 x 109/L; p<0.001), were younger (medians: 3 versus 5 years, p=0.04), and more often had T-cell ALL (12/51 (24%) versus (6/121 (5%), p<0.001). Five (16%) of 31 newly diagnosed patients with FCM detected blasts at diagnosis and available data at day 15 still had CSF leukemic blasts on day 15. So far the two patients who later developed CNS and/or bone marrow relapse were positive by flow (1.38 and 0.178 blasts/µL), but CNS negative by CC or had CSF leukocyte count <5/µL and lymphoblasts on CC, respectively.

Conclusion: Leukemic blasts are present in spinal fluid of one third of newly diagnosed ALL. CSF involvement is associated with other higher risk characteristics. The prognostic value of these findings awaits prospective evaluation.

Disclosures

No relevant conflicts of interest to declare.
Flamsa Reduced-Intensity Conditioning Regimen in AML Patients – Role of Re-Induction and Disease State Prior to Transplant
Authors: Holtick Udo , Shimabukuro-Vornhagen Alexander , Chakupurakal Geothy , Theurich Sebastian , Leitzke Silke , Wolf Dominik , Hallek Michael , von Bergwelt-Baildon Michael S. , Scheid Christof , Chemnitz Jens
Published: October 2019

Reduced intensity conditioning (RIC) regimens as compared to myeloablative protocols have demonstrated lower toxicity profiles at similar efficacy in the context of allogeneic haematopoietic stem cell transplantation (allo-SCT) for patients with acute myelogenous leukaemia (AML) in first or second remission. In addition, the FLAMSA RIC regimen, combining a cytoreductive part and a transplant-conditioning part, has been described to be efficacious in patients with refractory disease. Re-induction treatment after AML relapse or refractory disease is often accompanied by severe, in particular, infectious complications such as fungal pneumonia caused by long neutropenic phases, precluding a significant proportion of patients from completing allo-SCT. To have a closer look on the role of re-induction therapy prior to transplant, we retrospectively analysed clinical data of 118 consecutive patients with AML after allogeneic stem cell tranplantation following FLAMSA conditioning at our center. No prophylactic donor lymphocyte infusions were administered.

Median age of the cohort was 53 years (19-73 years). Donors were matched related, matched unrelated or mismatched unrelatred in 33, 49 and 18% of the transplants. Complete remission prior to transplant was detected in 29% and 21% of the patients after induction or re-induction, respectively. Twenty-five percent of the patients were transplanted with blast persistence, both in the group of patients after first induction and re-induction treatment. Median follow-up was 27 months.

The 4-yr overall survival of the whole cohort is 44% with a 4-yr relapse-free survival of 42%. Cumulative incidence of relapse was 26, 30 and 40% at one, two and four years, respectively. Cumulative incidence of non-relapse mortality (NRM) was 15, 18 and 18% at one, two and four years, respectively.

There were no significant differences regarding overall and relapse-free survival for patients transplanted in CR1, CR2 or blast persistence after induction treatment. Patients who failed remission after re-induction therapy showed significantly worse overall and relapse-free survival (p=0,049 and 0,003, repectively, log-rank test). NRM was similar in all cohorts.

FLAMSA is a highly effective conditioning regimen for AML patients even not in CR. Thus, the decision for re-induction therapy prior to allogeneic SCT has to be weighted against the potential toxicity of this approach.

Disclosures

No relevant conflicts of interest to declare.
Outcome and Prognostic Factors of 17p Deleted Multiple Myeloma Patients
Authors: Kull Miriam , Teleanu Veronica , Hayde Daniela , Wildbihler Katrin , Harsdorf Stephanie , Bunjes Donald , Liebisch Peter , Langer Christian
Published: October 2019

Introduction: During the last decade, the outcome of patients (pts) with symptomatic multiple myeloma (MM) has markedly improved. However, there is still a significant proportion of pts who do not achieve a longtime control of their disease. In particular, pts presenting with a deletion 17p (del17p) still have dismal prognosis. In order to better stratify this important group of MM pts we sought to investigate the prognostic impact of the following parameters in a larger cohort of del17p pts: del17p clone size, concomitant genetic abnormalities, treatment modalities and the incorporation of the novel agents lenalidomide and bortezomib.

Methods: We identified 54 MM pts diagnosed between 1998 – 2012 who had a del17p at diagnosis and were treated at the University Hospital of Ulm. The patients were screened for additional chromosomal aberrations by fluorescence in situ hybridization (FISH) performed on purified bone marrow plasma cells.

Results: The median age at MM diagnosis was 59 years and the proportion of male pts was 52%. At presentation the median del17p clone size was 83% (range: 28%-98%). In the vast majority of cases (83%) the presence of a del17p was associated with the presence of a del13q14. Other concomitant genetic abnormalities detected by FISH were t(4;14) in 17%, t(11;14) in 30% and gain at 1q21 (+1q21) in 31% of cases (figure 1). The median overall survival (OS) was poor (18.9 months) and did not change substantially over time (similar median survival in pts diagnosed before 2006 versus pts diagnosed thereafter). The del17p clone size had no impact on OS, neither the presence of a t(4;14) or a t(11;14). In patients with an additional +1q21 OS was significantly shorter (15.2 versus (v) 32.4 months; p=0,032). The incorporation of one of the novel agents into first-line treatment did not change the outcome significantly. In contrast, pts receiving at least one autologous transplantation showed a significantly longer OS (33.1 v 12.7 months; figure 2). On univariate analysis there was an improved median OS for pts undergoing an allogeneic transplantation (n=15; 32.4 v 14.4 months; p=0.025). In multivariate analysis ISS stage and the implementation of an autologous transplantation remained significant prognostic factors for OS.

Conclusions: The outcome of MM pts with a del17p remains poor, even after the introduction of lenalidomide and bortezomib into clinical practice. The development of novel therapeutic strategies therefore is urgently warranted.

Figure 1 Figure 1.

Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Elevated Adenosine Signaling Via Adenosine A2B Receptor Induces Normal and Sickle Erythrocyte Sphingosine Kinase 1 Activity
Authors: Sun Kaiqi , Zhang Yujin , Bogdanov Mikhail , Dowhan William , Idowu Modupe , Juneja Harinder S. , Blackburn Michael R. , Kellems Rodney E. , Xia Yang
Published: October 2019

Sickle Cell Disease (SCD) is one of the most devastating hemolytic genetic disorders affecting millions worldwide. Erythrocytes possess high sphingosine kinase 1 (Sphk1) activity and are considered to be the major cell type for supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is up-regulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte Sphk1 activity is regulated in SCD remains unknown. In an effort to identify specific factors and signaling pathways involved in regulation of erythrocyte SphK1 activities in SCD, we first chose to screen the effects of molecules known to induce SphK1 activities in other cell types and/or reported to be elevated in the circulation of SCD including tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), endothelin 1 (ET-1), vascular endothelial growth factor (VEGF), S1P and adenosine, on the activities of SphK1 in cultured primary mouse normal erythrocytes. Among all of those molecules tested, we found that adenosine is a previously unidentified hypoxia inducible molecule directly inducing SphK1 activity in vitro in a time and dosage-dependent manner. Next, using four adenosine receptor deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in cultured primary mouse normal and sickle erythrocytes. Subsequently, we provided in vivo genetic evidence that adenosine deaminase (ADA)-deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Mechanistically, we revealed that PKA functions downstream of ADORA2B mediating ERK activation and subsequently underlying adenosine-induced SphK1 activities in cultured mouse erythrocytes. Finally, we conducted human translational studies and reported that adenosine signaling via ADORA2B directly increases SphK1 activity in cultured primary human normal and sickle erythrocytes in a PKA/ERK-dependent manner. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms to control SphK1 activity in normal and sickle setting.

Disclosures

No relevant conflicts of interest to declare.
The Tissue Factor/Thrombin/Protease-Activated Receptor 1 Pathway Enhances Double-Strand RNA Induced Immune Responses in Macrophages
Authors: Antoniak Silvio , Tatsumi Kohei , Mackman Nigel
Published: October 2019

Introduction: Co-regulation of the immune response and the coagulation cascade after infection is thought to be an ancient response to limit pathogen spread. Recently, we showed that activation of the thrombin receptor, protease-activated receptor 1 (PAR1), on fibroblasts enhanced the innate immune responses to RNA virus infection. Here, we investigated whether PAR1 activation by the extrinsic coagulation pathway contributes to dsRNA-induced innate immune responses in macrophages.

Methods: Activation of the type-I interferon (IFN) pathway in the murine macrophage cell line RAW264.7 and bone-marrow derived macrophages (BMDM) from WT and PAR1-/- was analyzed after dsRNA (poly I:C) and/or PAR-1 stimulation. In addition, innate immune responses in the spleen were analyzed in vivo 4 hours after poly I:C (8mg/kg) injection in mice with reduced tissue factor expression (LowTF) or global PAR1 deletion (PAR1-/-) as well as in WT mice with a thrombin inhibitor (dabigatran etexilate, 10g/kg chow) or PAR-1 inhibitor (SCH79797, 25μg/kg). Lastly, we investigated the innate immune response in the spleen of WT and PAR1-/-mice after infection with the single-stranded RNA virus coxsackievirus B3.

Results: RAW264.7 and BMDM exhibited a toll-like receptor 3 dependent induction of IFNβ and CXCL10 after poly I:C stimulation. Activation of PAR-1 with either thrombin or agonist peptide enhanced poly I:C induction of IFNβ and CXCL10. A deficiency of tissue factor levels, thrombin inhibition, PAR-1 inhibition or PAR1 deficiency resulted in reduced expression levels of type-I IFNs and IFN-response genes such as CXCL10 in the spleen and plasma in mice given poly I:C. Last, PAR1-/-mice exhibited impaired IFNβ immune response 4 days after coxsackievirus B3 infection compared to WT mice.

Conclusion: Our study indicates that the coagulation dependent activation of PAR1 on macrophages is important for anti-viral responses to dsRNA. We speculate that PAR1 inhibition may interfere with anti-viral responses in humans.

Disclosures

No relevant conflicts of interest to declare.
Very High Efficiency of ICE (Ifosfamide-Carboplatin-Etoposide) in Relapse/Refractory (R/R) Primary Central Nervous System (PCNSL) and Vitreo-Retinal (VRL) Non Hodgkin Lymphoma. a LOC Network Multicenter Retrospective Study on 58 Cases
Authors: Choquet Sylvain , Grenier Adrien , Houillier Caroline , Soussain Carole , Moles Marie Pierre , Gastinne Thomas , Cassoux Nathalie , merle Beral Helene , roos Weil Damien , Leblond Veronique , hoang Xuan Khe
Published: October 2019

Background: R/R PCNSL and VRL carry a very poor prognosis (i.e. 4,5 months, Jahnke, J neuro-oncol 2006)). Autologous stem-cell transplantation (ASCT) is effective in this setting but essentially if the disease is in good response (CR and PR). Since recent recommendations for first line treatment recommend high dose Mtx and AraC, new salvage chemotherapies must use other drugs. ICE regimen contain three drugs known to pass the blood-brain barrier and is validated in R/R systemic lymphoma but not in PCNSL/VRL.

Methods: From June 2010 to May 2015, all relapse/refractory PCNSL and VRL already treated by Mtx and AraC in 5 departments of haematology or neuro-oncology in France, where treated by ICE: ifosfamide (5g/m² at day 2), carboplatine (AUC 5 at day 2) and etoposide (100mg/m²/d days 1 to 3) every 3 to 4 weeks. ASCT with thiotepa-busulfan-cyclophosphamide was proposed when possible.

Results: Fifty-eight patients have been treated, 25 females and 33 males, median age was 64 [28-84]. Eight received rituximab at day 1 of each cycle. Twelve where refractory, 46 in relapse, with a mean progression free survival (PFS) of 159 days [0-1763] before ICE. Thirty patients (51%) were in second line, 12 (21%) in third, 4 in fourth. Localizations where CNS in 32 cases, CNS + spinal fluid (SF) in 11, CNS + VR in 2, VRL in 9, SF in 3 and CNS+VR+SF in 1. Patients received a median of 3 cycles and 25 needed a dose reduction. Grade 3/4 WHO toxicities where: 29% neutropenic fever, 50% anemia, 69% neutropenia, 78% thrombocytopenia and 3 neurological reversible complications. ASCT have been made in 19 patients (33%): 13 in CR, 4 in PR, 2 in progression. Response rate is 70% (48% CR and 22% PR). With a mean follow-up of 670 days, 25/41 patients in response relapsed (only 7/19 after ASCT), median PFS is 133 days (NR for ASCT), 35 patients died (30 by progression, 4 by sepsis and one by sudden heart arrest in PR). Median Overall survival (OS) was 238 days for all patients but was not reached in case of ASCT, mean follow up for surviving ASCT is 856 days [278-1628]).

Conclusion: ICE regimen is very effective in relapse/refractory PCNSL and LVR heavily treated by high dose Mtx and AraC, with a manageable toxicity. If ASCT is performed, OS is very high and relapses rare. ICE + ASCT can represent a new standard in this setting.

Disclosures

Leblond: Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Mundipharma: Honoraria.
USP47 Is a New Target in Chronic Myelogenous Leukemia
Authors: Lei Hu , Ma Chunmin , Liu Zhen , Gao Shenmeng , Zhou Li , Wang Weiwei , Xu Hanzhang , Gao Fenghou , Wu Yingli , Liu Han
Published: October 2019

Despite of the great success of tyrosine kinase inhibitor in the therapy of chronic myelogenous leukemia (CML), CML is still not curable. Further investigation on the molecular mechanism of CML pathogenesis and identification of novel therapeutic target are required. In this study, we found that overexpression of CML characterized Bcr/Abl oncoprotein could significantly upregulate the expression of ubiquitin-specific protease Usp47. Also, increased expression of Usp47 was observed in primary CML cells and its expression was associated with the treatment response of imatinib, the first generation of tyrosine kinase inhibitor. Furthermore, Usp47 knockdown could inhibit the proliferation of K562 cells in vitro and in vivo. P22077, a Usp47 inhibitor, could inhibit the proliferation of CML cell lines, which comprise BCR/ABL mutation, including the T315I mutation. P22077 inhibits the proliferation of primary CML cells sensitive or resistant to imatinib. The colony forming activity of CD34+ from CML primary cells but not from normal cord blood cells were also inhibited by P22077. We further demonstrated that inactivation of STAT5 significantly reduce the expression of Usp47, indicating that BCR/ABL-induced upregulation of Usp47 is mediated by STAT5 activition. Interestingly, Usp47 could interact with Sirt1, another STAT5 target gene. Inactivation of Usp47 reduces the expression of Sirt1 in CML cell lines and primary CML cells. Taken together, we demonstrated that Usp47 plays an critical role in CML, revealing a novel BCR/ABL/STAT5/Usp47/Sirt1 signaling pathway in CML, providing a potential target to overcome tyrosine kinase inhibitor resistance.

Disclosures

No relevant conflicts of interest to declare.
Carfilzomib Enhances Natural Killer Cell-Mediated Lysis of Myeloma Linked with Decreasing Expression of HLA Class I
Authors: Shi Jumei , Yang Guang , Kong Yuanyuan , Gao Minjie , Tao Yi , Zhang Yiwen , Wu Xiaosong
Published: October 2019

Multiple myeloma (MM) is a malignant disorder characterized by uncontrolled monoclonal plasma cell proliferation. It accounts for 10% of all hematological malignancies and causes 15-20% of deaths from hematological malignancies. Although new therapies were introduced and overall survival of MM was improved in the last 10 years, MM still remains an incurable disease due to drug resistance. Natural killer (NK) cell-based treatments are promising therapies for multiple myeloma (MM). Carfilzomib (CFZ), a second-generation proteasome inhibitor, is used to treat patients with MM who are refractory or intolerant to both bortezomib and lenalidomide (or thalidomide). In this study, we determined that CFZ treatment enhanced the sensitivity of MM cells to NK cell-mediated lysis. Here, we report that CFZ decreased the expression of human leukocyte antigen (HLA) class I on MM cell lines and primary MM cells, the mean reduction was 47.7 ± 9.4% and 42.8 ± 12.4%, respectively. The down-regulation caused by CFZ occurred in a dose- and time- dependent manner. We compared the cell surface levels of HLA class I on MM cells in the presence or absence of CFZ after acid treatment. CFZ also down-regulated the expression of newly formed HLA class I on MM cells. CD107a expression levels were used to measure NK-cell degranulation. When NK cells were incubated with MM cells with CFZ treatment, the percentage of NK cells expressing CD107a on the surface greatly increased (mean ± SD: 33.6 ± 2.1%, for treated cells vs 16.7 ± 2.3%, for control cells, P < 0.05). We also showed that CFZ augmented NK-cell cytotoxity by a perforin/granzyme-mediated mechanism, because such enhancement was abolished when CMA, but not anti-TRAIL or anti-Fas-L antibodies, was added. Treatment of MM with CFZ significantly sensitized patients' MM cells to NK cell-mediated lysis (mean ± SD: 43.1 ± 6.4%, for treated cells vs 16.1 ± 4.0%, for control cells at effector/target (E/T) ratio of 10:1, n = 9, P < 0.01). Furthermore, the exogenous HLA-C binding peptides, used in the CFZ treated group rescued the down-regulation of HLA-C and reduced NK cell-mediated lysis to a similar level as in the untreated group. Blocking NKG2D, NCRs and TRAIL did not have a significant impact on NK cell lysis of myeloma cells. These implied the enhancement of NK cell-mediated lysis was mainly linked with the decreased expression of HLA class I. Our findings show a novel activity of CFZ as an immunomodulating agent and suggest a possible approach to therapeutically augment NK cell function in MM patients.

Disclosures

No relevant conflicts of interest to declare.
Efficacy and Safety of Recombinant Fusion Protein Linking Coagulation Factor IX with Albumin (rIX-FP) in Previously Treated Patients with Hemophilia B Undergoing Major Orthopedic Surgeries
Authors: Negrier Claude , Lepatan Lynda Mae , Karim Faralzah , Lienhart Anne , Wolko Denise , Voigt Christine , Jacobs Iris C. , Santagostino Elena
Published: October 2019

A fusion protein genetically linking recombinant human coagulation FIX with recombinant human albumin (rIX-FP) has been developed with an improved PK profile, improving hemophilia B treatment by allowing less frequent dosing and FIX consumption for perioperative management. A surgical sub-study was included in 3 Phase III studies, including an on-going Phase IIIb extension study in subjects with hemophilia B, as part of the global PROLONG-9FP clinical program. This report will focus on the use of rIX-FP for perioperative prophylaxis.

Efficacy was evaluated by Investigator assessment (4-point scale), predicted and estimated actual blood loss, and consumption of rIX-FP during the 14-day perioperative period. Safety, including inhibitors to FIX, was assessed. The pre-operative dose of rIX-FP was based on individual incremental recovery, targeting 80-100% FIX activity. FIX activity was monitored at local laboratories to determine repeat doses during the surgical period.

Five major orthopedic surgeries have been assessed to date. Hemostatic response was rated by investigators as excellent or good for all surgeries. A single dose of rIX-FP prior to surgery was sufficient to maintain hemostasis during surgery. Estimated actual blood loss was consistent with predicted blood loss in patients without hemophilia B. The total number of rIX-FP injections ranged from 6-7 over the 14 day post-operative period. The median rIX-FP consumption prior to surgery, Days 1-3, Days 1-7 and Days 8-14 post surgery were 84, 33, 81 and 107 IU/kg, respectively. There were no inhibitors to FIX or antibodies to rIX-FP during the study. The extension study is still ongoing, and additional orthopedic surgeries data may be presented.

These results support the efficacy and safety of rIX-FP for perioperative management in patients with hemophilia B, with prolonged dosing intervals and lower FIX consumption than conventional therapies.

Disclosures

Negrier: CSL Behring: Speakers Bureau. Wolko:CSL Behring: Employment. Voigt:CSL Behring: Employment. Jacobs:CSL Behring: Employment. Santagostino:CSL Behring: Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Baxter/Baxalta: Speakers Bureau; Biotest: Speakers Bureau; Kedrion: Speakers Bureau; Roche: Speakers Bureau; Octapharma: Speakers Bureau; Biogen/Sobi: Speakers Bureau; Novo Nordisk: Speakers Bureau; Bayer: Speakers Bureau.
A Comparison of Von Willebrand Factor Indexes in Heartmate II Compared to Heartware Left Ventricular Assist Devices
Authors: Gupta Bhanu P , Leoni Juan , Patel Parag , Chen Dong , Blackshear Joseph
Published: October 2019

Background: Von Willebrand factor (VWF) is a multimeric glycoprotein synthesized in endothelium and megakaryocytes. Elevated total VWF antigen has been associated with arterial thrombosis, while in patients with left ventricular assist devices (LVAD), loss of high molecular weight multimers is associated with spontaneous bleeding. We hypothesized that the lower rotational speed of the Heartware LVAD would be associated with less severe disruption of VWF.

Methods: A total of 22 patients were included. We compared samples from a) 17 patients with Heartmate II LVAD (HM II), 76% male, median age 67 (54-73) years, 24% non-white, 45 (33-59) months after implant, with b) 5 patients with Heartware (HW) LVAD, all male, age 64 (59-65) years, 20% non-white, 27 (14-29) months after implant. All patients received warfarin. All HW patients took aspirin, while 82% of HM II patients took aspirin. Samples were analyzed for VWF antigen, activity, platelet function analyzer-100 collagen adenosine diphosphate closure time (PFA) and VWF multimers by gel electrophoresis. Multimers were assessed qualitatively and quantitatively by dividing mers > 15 by mers 2-15 (multimer ratio) and with a normalized multimer ratio (multimer ratio of patient / normal plasma control from the same gel).

Results: Although flow rates did not differ significantly between HM II and HW, 5.2 (4.6-6.5) versus 4.3 (4.2-5.4) liters per minute respectively, rotational speeds were very different, mean 9472 versus 2680 rpms, p<0.0001). There were no differences in hemoglobin, hematocrit, or platelet count at the time of sampling. Qualitative VWF multimers were abnormal in all patients, and PFA was non-clotting in 88% of HM II patients and 80% of HW patients. VWF activity to antigen ratio was not different: HM II 0.68 (0.63-0.74) versus HW 0.70 (0.63-0.71). However, VWF antigen was significantly higher in HM II patients, 199 (174-227) compared to HW, 139 (122-160), p=0.039 and the normalized VWF multimer ratio was significantly lower, suggesting greater loss of high molecular weight multimers in HM II, 0.43 (0.41-0.48) compared to HW, 0.53 (0.48-0.56), p=0.0025 (figure).

Conclusion: The lower rotational speed associated with the Heartware LVAD appears to produce less elevation of VWF antigen, and slightly less loss of VWF high molecular weight multimers compared to the Heartmate II LVAD. Additional studies are needed to describe whether or not lower pump thrombosis rates and rates of spontaneous bleeding are associated with these differences.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Comparison of Rethrombosis-Free Survival in Children Versus Adults Suffering from Antithrombin-, Protein C- and Protein S-Deficiency: An Observational Multicenter Cohort Study
Authors: Limperger Verena , Kenet Gili , Heller Christine , Knoefler Ralf , Kurnik Karin , Klostermeier Ulrich C , Mesters Rolf , Goldenberg Neil A , Young Guy , Nowak-Gottl Ulrike
Published: October 2019

Background: Venous thromboembolism [TE] is a multifactorial disease and antithrombin [ATD]-, protein C [PCD]- or protein S [PSD]-deficiency constitutes a major risk factor. Since screening for thrombophilia is controversial, individuals at high risk for recurrence who benefit from screening need to be identified. Primary study objective was to determinethe individual recurrence risk in children compared to adults with TE with respect to their thrombophilia status.

Methods: In 142 consecutively enrolled TE patients (children n=85; adults n=57) with ATD, PCD or PSD after exclusion of six children with purpura fulminans due to homozygous PCD or PSD we calculated i) the cumulative recurrence rates (CRR) at 1, 5 and 10 year following the first TE onset and, ii) in addition, the absolute recurrence risk (ARR) per 100 patient years (%).

Results: At first TE onset in univariate analysis a higher rate of unprovoked TE was found in children, whereas adults presented with a higher rate of a positive TE family history and a higher rate of recurrence: 40 out of 136 patients showed a second TE after withdrawal of anticoagulation (AC) or insufficient AC [n=6]. Two events in children were fatal [pulmonary embolism]. The overall CRR at 1, 5 and 10 years was 10.9%, 20.4% and 29.2% with total ARR [95% CIs] of 5.3 [3.4-7.8] in adults compared to 2.1 [1.0-5.3] in children [p=0.004]. Whereas the ARR was no different between adults and children in ATD patients [5.1 versus 4.7; p=0.85] and in symptomatic PCD subjects [3.9 versus 1.6; p=0.17], adults with PSD showed a higher ARR compared to children [6.3 versus 0.1; p=0.001]. Positive family TE history did not predict recurrence.

Conclusion: Given the high ARR of 5.3% in adults and 2.1% in children we suggest screening for ATD, PCD and PSD in adult and pediatric TE patients. The high rate of ARR should be taken into account when initiating future therapeutic trials. Duration and intensity of AC should be carefully reevaluated in patients with ATD, PCD or PSD.

Disclosures

Young: Baxter, Grifols: Consultancy, Honoraria. Nowak-Gottl:Bayer, LFB: Membership on an entity's Board of Directors or advisory committees.
Bloodspot: A Web Resource Facilitating the Analysis of Transcriptional Programs in Normal and Malignant Hematopoiesis
Authors: Bagger Frederik Otzen , Sasivarevic Damir , Sohi Sina Hadi , Laursen Linea Gøricke , Pundhir Sachin , Winther Ole , Rapin Nicolas , Porse Bo T
Published: October 2019

Decades of studies on developing cells in human and murine haematopoiesis have resulted in a large number of gene-expression datasets that may answer questions regarding normal and aberrant blood formation. To researchers and clinicians with limited bioinformatics experience, these data remain available, yet largely inaccessible. Current resources provide information about gene-expression patterns but disregard key aspects such as genetic co-regulation of genes, and the effects on patient survival.

Here, we present a new web-based resourced termed, BloodSpot, which provides a) a comprehensive representation of gene-expression throughout haematopoiesis, b) a single gene-based Kaplan-Meier analysis and c) a novel, simpler, yet more informative, type of expression plot. Significantly, users can compare their own expression profiles to normal haematopietic populations within the statistical framework of Bloodspot. We illustrate the potential of key features in BloodSpot to identify new putative C/EBPa targets.

Accessible at http://servers.binf.ku.dk/bloodspot/

Disclosures

No relevant conflicts of interest to declare.
ER71/ETV2 Is Essential for Hematopoietic Regeneration after Injury
Authors: Xu Canxin , Choi Kyunghee , Lee Tae-Jin , Sakurai Nagisa
Published: October 2019

Hematopoietic stem and progenitor cell (HSPC) expansion and regeneration is requisite for effective hematopoietic transplantation and recovery from anticancer chemotherapy drug induced bone marrow injury. In chemotherapy-mediated hematopoietic injury model, we found that key developmental genes that are critical for HSPC generation were upregulated in HSPCs when treated with 5-FU. ETS transcription factor Er71/ETV2 is essential for the development of endothelial and blood cells during mammalian embryogenesis. Here, we generated different tissue specific Er71 conditional knockout (CKO) mice as well as transgenic mice that coexpress Er71, GATA2 and Scl (EGS) in a doxycycline inducible manner to investigate the Er71 role in adult hematopoiesis. While Vav-Cre; Er71 CKO and Tie2-Cre; Er71 CKO mice did not display any hematopoietic defects in homeostatic conditions, Vav-Cre; Er71 CKO bone marrow was significantly compromised in hematopoietic reconstitution potential compared to controls. In addition, Vav-Cre; Er71 CKO and Tie2-Cre; Er71 CKO but not VECadherin-Cre; Er71 CKO mice showed reduced HSPC proliferation and deficient HSPC and myeloid cell recovery after 5-FU injury. On the other hand, temporal Er71, GATA2 and Scl (EGS) coexpression promoted HSPC proliferation, hematopoietic reconstitution potential and regeneration after 5-FU injury. These results demonstrate that hematopoietic Er71 is critical for HSPC expansion and regeneration. Future studies warrant Er71 application for in vitro hematopoietic stem cell expansion and bone marrow regeneration in response to hematopoietic injury.

Disclosures

No relevant conflicts of interest to declare.
PRMT1-Mediated Methylation of DUSP4 Determines Megakaryocyte-Erythroid Lineage Choice By Regulating p38 Signaling
Authors: Su Hairui , Tran Ngoc-Tung , Guo Han , Luo Minkui , Zhao Xinyang
Published: October 2019

Protein arginine methyltransferase 1 (PRMT1) expression level is correlated with short survival rate of acute myeloid leukemia patients. PRMT1 contributes to leukemogenesis via direct interaction with leukemic fusion proteins such as AML1-ETO and MLL-EEN. Our previous studies have shown that PRMT1 blocks megakaryocyte (MK) terminal differentiation. Inhibition of PRMT1 enzymatic activity promotes apoptosis and differentiation of acute megakaryocytic leukemia (AMKL) cells. To understand the molecular mechanisms of PRMT1-mediated leukemogenesis, we apply bioorthognal profiling of protein methylation technology to identify PRMT1 substrates. A dual specificity protein phosphatase, DUSP4 was found a PRMT1 substrate. In literature, high expression of DUPS4 is linked to idiopathic CD4 lymphopenia and silencing of DUSP4 expression causes chemo-resistance in solid tumors and abnormal innate immune response to parasitic infections. To understand the role of DUSP4 in megakaryocytic differentiation, we ectopically expressed DUSP4 and found that it enhances megakaryocyte development. In liquid culture system, DUSP4 knockdown in human CD34+ cells crippled MK differentiation induced by TPO. In TPO+EPO culture system, DUSP4 knockdown in human CD34+ cells gave rise to more erythroid cells (CD71+) at the expense of MK cells (CD41+). Therefore, DUSP4 is essential for MK differentiation. To understand how PRMT1-mediated methylation of DUSP4 blocks MK differentiation, we mapped the DUSP4 methylation site. We found that the DUSP4 methylation mutant has a longer half-life compared to DUSP4 wild type. Further studies confirmed that DUSP4 protein stability is controlled by methylation-induced ubiquitylation.

Although DUSP4 has been shown to dephosphorylate ERK, JNK and p38 kinases in overexpression systems, we found that during megakaryocytic differentiation DUSP4 specifically targets p38. These results are consistent with the findings in literature that p38 is required for erythroid differentiation, while ERK is required for megakaryocytic differentiation. Therefore, DUSP4 is responsible for turning off p38 signaling pathway, which pushes cells to erythroid lineage. Furthermore, we found that DUSP4 is associated with chromatin and dephosphorylates histone H3S10; while the methylation mutant DUSP4 locates in cytoplasm, therefore can still dephosphorylate p38 but not H3S10. Given that H3S10 phosphorylation may play a critical role in endomitosis, we also studied whether overexpression of DUSP4 antagonize aurora kinase activity to promote megakaryocytic differentiation in AMKL cell lines, containing OTT-Mal translocation or trisomy 21/down syndrome. We have tested an array of PRMT1 inhibitors to boost DUSP4 activity for potential curative therapy using AMKL leukemia mouse models. Our data support the notion that targeting PRMT1-DUSP4 pathway is a valid therapeutic approach for AMKL.

Disclosures

No relevant conflicts of interest to declare.
Replication Associated Stress in the Fetal Stem Cell Pool of FA Mice Can be Rescued By Pharmacologic Inhibition of Constitutively Activated P38 MAPK
Authors: Yoon Youngme , Kamimae-Lanning Ashley N. , Storm Kelsie , Goloviznina Natalya A , Kurre Peter
Published: October 2019

Fanconi Anemia (FA) is a rare, recessively heritable disorder with prominent failure of hematopoiesis. The physiologic role of FA proteins has not been fully resolved to date. While several existing model systems delineate its role in DNA damage response caused by alkylating agents, aldehydes, and inflammatory cytokines, all rely on experimental induction. We previously demonstrated the in utero onset of hematopoietic failure in mice with genetic disruption of Fancc. Herein, we found significant deficits in the fetal liver (FL) hematopoietic stem and progenitor cell (HSPC) pool in Fancd2 mice. Both AA4.1+ Sca-1+ Lin- expressing progenitors (ASL) and CD48- CD150+ Lin- Sca-1+ (SLAM) cells were decreased in frequency in Fancd2-/- versus WT FL. Similarly, we observed a significant decrease in progenitor colony formation and deficits in primary and secondary transplantation among Fancd2-/- FL compared to WT. Fancd2-/- FL cells were characteristically sensitive to mitomycin C and had significantly fewer SLAM cells in the G0 phase of cell cycle and elevated p21 expression, indicating canonical P53 activation. Consistent with prior reports by other groups on embryonic stem cells and our own Fancc-/- FL studies, we found neither exaggerated frequency of apoptotic cells, nor transcriptional induction of Puma or Noxa. We hypothesized that the observed deficits in developmental HSPC pool expansion reflect replication-associated stress. At the transcriptional level, we found activation of the DNA damage response via Rad51 and Prkdc, corroborated by immunofluorescent imaging of Rad51 foci as well as comet assays in FL cells. Next, we tested P38 MAPK as a stress response previously found to confer repopulation deficits in postnatal BM failure among Fancc and Fanca mice; here, our experiments revealed baseline (unprovoked) activation of phospho-p38 and rescue of Fancd2-/- progenitor colony formation using a pharmacological inhibitor, SB203580. Results were further strengthened by transplantation, revealing increased Fancd2-/- donor chimerism after in vivo administration of SB203580. The gains in donor chimerism persisted even after cessation of drug administration. These results suggest that replication-associated stress in the rapidly cycling fetal Fancd2-/- HSPC pool evokes a cellular stress response that constrains physiological expansion. Our work emphasizes the prenatal onset of hematopoietic failure and reveals pharmacological rescue by inhibition of constitutively active P38 MAPK. Furthermore, FA fetal hematopoiesis is an original model of unprovoked hematopoietic failure that allows the study of physiologic role of FA proteins in HSPC.

Disclosures

No relevant conflicts of interest to declare.
FOXR1 Activation in B-Cell Lymphoma
Authors: Quentmeier Hilmar , Pommerenke Claudia , Nagel Stefan , Hauer Vivien , Zaborski Margarete , Amini Rose Marie , Berglund Mattias , Geffers Robert , Drexler Hans G
Published: October 2019

Several recurrent chromosomal aberrations targeting 11q23 have been described in diffuse large B-cell lymphoma (DLBCL). Here, we describe a novel fusion between RPS25 and FOXR1, two neighboring genes on 11q23 in the DLBCL cell line U-2932. RNAseq identified the fusion mRNA and genomic cloning localized the breakpoint to intron 2/3 of RPS25 and to the promoter region of FOXR1 (bp -3532). The cell line consists of two genetically distinct clones that represent subclones of the patients tumor.1 We confirmed RPS25/FOXR1 fusion in the patients DNA and in one of the two cell line subclones suggesting that the fusion had occurred at some later stages of tumor development. Physiological FOXR1 expression is restricted to the early stages of embryogenesis. Ectopic expression as result of 11q23 intrachromosomal deletion-fusion had so far only been described in neuroblastoma.2 In-frame fusions with the 5´ genes MLL and PAFAH1B2 led to the overexpression of FOXR1.2 In accordance with the notion that also in DLBCL a constitutively expressed 5´ gene (RPS25) might be responsible for the ectopic expression of FOXR1, FOXR1 levels were 1000x higher in the fusion-positive than in the fusion-negative U-2932 subclone. Expression array analyses showed that the RPS25/FOXR1 positive U-2932 subclone had the highest FOXR1 expression level of 55 B-lymphoma cell lines tested, three log-scales higher than the average expression level. Santo et al.2 reported that FOXR1 acts as negative regulator of fork-head box factor-mediated transcription and suggested a possible role in tumorigenesis. We describe for the first time that FOXR1 fusions also occur in lymphoma. In-silico analyses show that the aberrant expression of FOXR1 is rare, but recurrent in various forms of lymphoma. Given the potential oncogenic role of FOXR1, cell line U-2932 with one FOXR1-positive and one FOXR1-negative subclone appear to be a promising model for functional analysis of FOXR1-mediated cellular events.

1 Quentmeier H, Amini RM, Berglund M, et al. U-2932: two clones in one cell line, a tool for the study of clonal evolution. Leukemia. 2013;27(5):1155-1164.

2 Santo EE, Ebus ME, Koster J, et al. Oncogenic activation of FOXR1 by 11q23 intrachromosomal deletion-fusions in neuroblastoma. Oncogene 2012;31(12):1571-1581.

Disclosures

No relevant conflicts of interest to declare.