bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dysregulation of C10orf55 Expression in Megakaryocytic Cell Lineage From Quebec Platelet Disorder Individuals
Authors: Bunimov Natalia , Blavignac Jessica , Tasneem Subia , Waye John S , Paterson Andrew D , Rivard Georges E , Hayward Catherine PM 
Published: October 2019

Abstract
Abstract 2274
Quebec platelet disorder (QPD) is a unique and serious bleeding disorder, associated with a gain-of-function defect in fibrinolysis that leads to a delayed onset bleeding after surgery or trauma in affected individuals. It has an autosomal dominant pattern of inheritance and affected individuals typically demonstrate an extensive personal and family history of bleeding. The disorder is caused by a tandem duplication mutation of a 78-kb genomic region located on chromosome 10 that contains PLAU (the gene that encodes urokinase plasminogen activator, uPA) in addition to the gene C10orf55, a gene of unknown function that encodes a transcript with partial complementary regions to PLAU mRNA. While QPD has minimal effects on uPA in urine, plasma and many other cell types, it elevates uPA levels >100 fold in QPD platelets compared to controls, due to overexpression of PLAU during megakaryopoiesis. As C10orf55 expression is postulated to have negative regulatory effects on uPA expression, we investigated if there is evidence of C10orf55 dysregulation in QPD. TaqMan assays were utilized for quantitative real-time RT-PCR analysis of C10orf55 transcripts in QPD and control CD34+ peripheral hematopoietic progenitors, platelets, lymphoblasts and saliva buccal cells (n=4–8 control and patient subjects for each cell type). QPD platelets contained elevated levels of C10orf55 mRNA that were >130 fold higher than in control platelets (p<0.001). Whereas in the other QPD cell types that were examined, the levels of C10orf55 mRNA were within the range expected for one additional copy of this gene, based on transcript levels measured in the control cells. These data demonstrate that the tandem duplication mutation of QPD leads to megakaryocyte specific increases in C10orf55 expression that parallel the pathological changes in PLAU expression during megakaryopoiesis. Although C10orf55 has been postulated to downregulate PLAU expression, due to complementarity of its transcripts, this seems unlikely given our findings. Our study indicates that the pathogenesis of QPD involves megakaryocyte-differentiation specific dysregulation of both genes contained in the 78-kb tandem duplicated locus on chromosome 10.

Disclosures:
No relevant conflicts of interest to declare.
Characterization of the Novel Function of Cyclin A1 to Influence the Stem Cell Niche and Microenvironment Signaling in Mouse Model
Authors: Miftakhova Regina R , Hedblom Andreas , Bredberg Anders , Wolgemuth Debra J , Persson Jenny L 
Published: October 2019

Abstract
Abstract 2338
The molecules and cellular mechanisms that regulate pool size of hematopoietic stem cells and its association with stem cell niches to protect HSC from cell cycle-dependent injury are unclear. The cell cycle regulatory factor, cyclin A1 is overexpressed in patients with hematopoietic malignancies. Further, targeted overexpression of cyclin A1 in myeloid progenitor cells initiated acute myeloid leukemia in transgenic mice.
In the present study, we investigated the role of cyclin A1 in controlling the HSC pool and its functional association with key molecules that regulate stem cell niches under steady-state conditions or following the cytokine stimulation or radiation exposure in vivo and in vitro.
We reported that cyclin A1 null bone marrow displayed a significant increase in the frequency of stem cells (P<0,01) and increased expression of P27kip and increased phosphorylation of Akt at ser-473 site in HSCs and hematopoietic progenitors.
We further showed that increased frequency and number of cyclin A1 null HSCs was associated with the increased expression BMP receptor type IA that is known as a key molecule controlling the HSC niche. In addition, cyclin A1 null HSCs exhibited increased ability to migrate as determined by in vitro migration assay, and bone marrow transplantation assay, and this correlated with the increased expression of MMP9, that is known for controlling the osteoblast cell expansion, and the accumulated nuclear localization of angiogenic and vascularization factor VEGFR2 in cyclin A1 null bone marrow cells. We also observed that IRSp53 that is a regulator for extracellular matrix signaling, was present in the nuclear compartments of cyclin A1 null bone marrow progenitor cells, but was absent in that of the wild-type controls. Further, flow cytometry and immunoblot analyses showed that cyclin A1 null HSCs and progenitor cells exhibited relatively resistant to TNF stimulation and the radiation exposure, and this was associated with the great increase in the expression of phosporylated of ser-473 Akt. Our findings suggest that the microenvironment may be altered in bone marrows from cyclin A1 null mice. Thus cyclin A1 may have important function in the decision of maintaining the HSC pools and protecting the HSCs and progenitors from exposure to the external agents by regulating the interaction between the HSCs/progenitor cells and bone marrow environment.

Disclosures:
No relevant conflicts of interest to declare.
Enhanced Cytokine Responsiveness Counteracts Age-Induced Decline in Hematopoietic Stem Cell Function
Authors: Norddahl Gudmundur L , Wahlestedt Martin , Gisler Santiago , Sigvardsson Mikael , Bryder David 
Published: October 2019

Abstract
Abstract 2342
In recent years, it has become increasingly clear that physiologic aging has several profound cell intrinsic effects on hematopoietic stem cell (HSC) aging. This includes an altered output of mature progeny and an expansion of the HSC pool, although the latter is accompanied with several functional shortcomings. Because of the hierarchical structure of hematopoiesis, improving HSC function should present a promising avenue to restore aberrant hematopoietic function.
The signal adaptor protein Lnk, a relay of cytokine signaling, has been shown to negatively regulate hematopoiesis at several cellular stages. As a consequence of Lnk deficiency, mice display increased B lymphopoiesis and increased HSC numbers. The increase in HSC numbers has been suggested to arise by an increased ability of HSCs to self-renew. However, it has remained unclear how the enhanced sensitivity to cytokine signaling would affect hematopoiesis upon physiological age. In the present study, we investigated the effects of physiological aging on hematopoiesis in the Lnk−/− mice. Aged wild-type mice showed a number of age-related alterations in the hematopoietic system including; increased HSC numbers, decreased lymphopoiesis and decreased reconstitution potential upon competitive transplantation. Most of these parameters were dramatically altered as a consequence of Lnk deficiency. When competitively transplanted, aged Lnk−/− HSCs displayed a dramatically increased overall reconstitution potential compared to WT mice. Increases in reconstitution potential were observed in both primary and serially reconstituted recipient animals. Upon physiologic age, the lineage distribution of transplanted aged wild-type HSCs is skewed towards the myeloid lineage. By contrast, transplanted aged Lnk−/− HSCs gave rise to a hematopoietic system with a balanced lineage distribution resembling that generated by young wild-type HSCs. The enhanced HSC potential of aged Lnk−/− mice was uncoupled from effects on telomere length maintenance but displayed a distinct molecular profile in whole genome expression analysis. In summary, we demonstrate that deficiency of the signal adaptor molecule Lnk appears to functionally protect HSCs from many of the consequences of physiologic age, which at a broader level perhaps suggest a potential of cytokines to counteract age-related HSC decline.

Disclosures:
No relevant conflicts of interest to declare.
Latent Cardiogenic Potential in Endocardium and Hemogenic Endothelium Revealed in the Absence of Scl/tal1
Authors: Montel-Hagen Amelie , Handel Ben Van , Ferrari Roberto , Sasidharan Rajkumar , Org Tonis , Boogerd Kees , Nakano Haruko , Zhou Jian , Li Xinmin , Pellegrini Matteo , Orkin Stuart H. , Nakano Austin , Kurdistani Siavash , Evans Sylvia , Mikkola Hanna 
Published: October 2019

Abstract
Abstract 2362
The endothelium in embryonic and extraembryonic hematopoietic tissues has the capacity to generate hematopoietic stem and progenitor cells (HS/PC). However, it is unknown how this unique endothelium is specified. Microarray analysis of endothelial cells from hematopoietic tissues of embryos deficient for the bHLH transcription factor Scl/tal1 revealed that Scl establishes a robust hematopoietic transcriptional program in the endothelium. Surprisingly, lack of Scl also induced an unexpected fate switching of the prospective hemogenic endothelium to the cardiac lineage. Scl deficient embryos displayed a dramatic upregulation of cardiac transcription factors and structural proteins within the yolk sac vasculature, resulting in the generation of spontaneously beating cardiomyocytes. Ectopic cardiac potential in Scl deficient embryos arose from endothelial-derived CD31+Pdgfrα+ cardiogenic progenitor cells (CPCs), which were present in all sites of HS/PC generation. Analysis of Runx1-deficient embryos revealed, that although Runx1 acts downstream of Scl during the emergence of definitive HS/PCs, it is not required for the suppression of the cardiac fate in the endothelium. The only wild type tissue that contained CD31+Pdgfrα+ putative CPCs was the heart, and this population was greatly expanded in Scl deficient embryos. Strikingly, endocardium in Scl−/− hearts also activated a robust cardiomyogenic transcriptional program and generated Troponin T+ cardiomyocytes both in vivo and in vitro. Although CD31+Pdgfrα+ CPCs from wild type hearts did not generate readily beating cells in culture, they produced cells expressing endothelial, smooth muscle and cardiomyocyte specific genes, implying multipotentiality of this novel CPC population. Furthermore, CD31+Pdgfrα+ CPCs were greatly reduced in Isl1−/− hearts, which fail to generate functional, multipotential CPCs. Lineage tracing using VE-cadherin Cre Rosa-YFP mouse strain demonstrated that, in addition to generating HS/PCs in hematopoietic tissues, endothelial cells are also the cell of origin for CD31+Pdgfrα+ CPCs in the heart. Together, these data suggest a broader role for embryonic endothelium as a potential source of tissue-specific stem and progenitor cells and implicate Scl/tal1 as an important regulator of endothelial fate choice.

Disclosures:
No relevant conflicts of interest to declare.
Evaluation of Role for flt3L in Human Early B and T/NK Lymphopoiesis in a Novel Stromal Cell Culture System
Authors: Nakamori Yoshiki , Ohishi Kohshi , Liu Bing , Masuya Masahiro , Hamada Hirofumi , Katayama Naoyuki 
Published: October 2019

Abstract
Abstract 2385
The regulatory mechanism of human early lymphopoiesis remains less defined. A major limitation of conventional in vitro assay is that B and T lymphopoiesis cannot be assessed simultaneously. We exploded a novel culture assay and found that the hTERT-transduced telomerized human stromal cells support the generation of CD19+CD34lo/-CD10+cyCD79a+CD20+/−VpreB− pro B- and CD7+CD34+CD45RA+CD56−cyCD3− early T-cell precursors from human hematopoietic progenitors without cytokines, which was enhanced by flt3L (2010, ASH). We further characterized the generated lymphoid precursors, and verified that lineage-specific transcription factors for B (Pax-5 and EBF) and T/NK-cell precursors (GATA-3, HEB, Id2) were expressed by the CD19+ and CD7+CD56− cells, respectively. The CD7+CD56− cells showed the differentiation potential to T- and NK-lineage cells, by replating with OP9-Delta1 in the presence of flt3L+IL-7, and with the telomerized-stromal cells in the presence of flt3L+IL-7+IL-15, respectively. In serum-free culture condition, B-cell differentiation was minimally supported by the stromal cells, while low number of CD7+ cells was observed. Nonetheless, a significant number of CD7+, CD19-cyCD79a+, and CD19+ cells developed in the presence of flt3L, suggesting an important role for flt3L in the generation of early T/NK- and, particularly, B-lineage cell precursors on the stromal cells. The cellular interaction between Notch and Notch ligand Delta-1 or -4 in the presence of appropriate cytokines is considered to be crucial in T-lineage cell development in mice. We therefore examined whether Notch pathway is involved in the T/NK-cell development under this culture condition. While the gene expression of Delta-1 or -4 was detected in the telomerized stromal cells, the protein expression of these ligands was not detected with Western-blotting analysis. After co-culture of hematopoietic progenitor cells with the telomerized stromal cells, the gene expression of Notch target gene, HES1, was not increased. These data suggest that Notch signaling is not involved in the generation of early T/NK cell precursors on the stromal cells. This novel in vitro culture system suggests that the development of early B- and T/NK- cell precursors from hematopoietic precursors take places on the stromal cells in a Notch-independent manner and that flt3L plays a principal role in the stimulation of the early B and T/NK lymphopoiesis.

Disclosures:
No relevant conflicts of interest to declare.
Flt3 Is Required for MLL-ENL-Induced Leukemogenesis In Vivo, but Not for LSC Function in Vitro
Authors: Kamezaki Kenjiro , Snoeck Hans-Willem 
Published: October 2019

Abstract
Abstract 2450
Flt3 is a type III tyrosine kinase receptor expressed on hematopoietic multipotential progenitors and more downstream progenitor cells in the mouse bone marrow (BM).Flt3 is one of the most frequently mutated genes in acute myeloblastic leukemia (AML) patients, leading to constitutive activation. Several Flt3 kinase inhibitors are in clinical development in AML with Flt3 mutations, although disappointing results suggest that Flt3-inhibitors as monotherapy are unlikely to become reality. Wild type (wt) Flt3, which differs in its signaling characteristics and downstream targets from mutated Flt3, is highly expressed in particular in AML and ALL (acute lymphoblastic leukemia) with MLL (Mixed Lineage Leukemia) rearrangements. These observations suggest a role for wt Flt3 in pathogenesis as well, although little experimental or clinical evidence has been published yet supporting this notion. To our knowledge, there is only one report suggesting that wt Flt3 is required for the development of leukemia in a murine model of c-cbl mutation-induced leukemogenesis (Rathinam C, et al. Cancer Cell 2010). For this reason, we focused on the role of wt Flt3 in leukemogenesis and using leukemia mouse models established by retroviral transduction of MLL-ENL, MLL-AF9 and AML1-ETO9a. Mice transplanted with MLL-ENL-transduced Flt3−/− BM cells showed a higher survival rate (wt 61.3%, n=34 vs Flt3−/− 96.8%, n=31, median follow-up period; 255 days, p=0.038). No difference in survival was observed in the MLL-AF9 and AML1-ETO9a models however. Surprisingly, MLL-ENL-transduced Flt3−/− BM cells showed no significant difference in serial replating capacity of BM cells in methylcellulose colony forming assays in vitro compared to wt BM cells, and putative lin-kit+ leukemic stem cells (LSCs) did not express Flt3. In vivo, however, putative MLL-ENL LSCs, but not MLL-AF9 LSCs expressed Flt3. Furthermore, in non- and pre-leukemic MLL-ENL-transduced mice BM cells the frequency of lin-kit+ putative LSCs among MLL-ENL positive populations was decreased in Flt3−/− group (wt 1.468% vs Flt3−/− 0.466%, p=0.031). Flt3+ lin-kit+ populations were increased in MLL-ENL transduced leukemic wt mice compared to non-leukemic mice (leukemic mice 26.7% vs non-leukemic mice 9.9%, p=0.028). These results suggest that Flt3 signaling is required for MLL-ENL-induced leukemogenesis and maintenance of LSCs in vivo, but not in vitro. Importantly, the data suggest that the in vitro replatable leukemia stem cell (LSC) paradigm might not reflect LSC function or phenotype in vivo.

Disclosures:
No relevant conflicts of interest to declare.
Clofarabine Combinations in Adults with Refractory/Relapsed Acute Lymphoblastic Leukemia (ALL): A GRAALL Report
Authors: Pigneux Arnaud , Sauvezie Mathieu , Vey Norbert , Raffoux Emmanuel , Marcais Ambroise , Huguet Françoise , Rousselot Philippe , Marolleau Jean Pierre , Leprêtre Stéphane , Turlure Pascal , Chevallier Patrice , Hunault Mathilde , Bonmati Caroline , Cluzeau Thomas , Leguay Thibaut , Ifrah Norbert , Dombret Herve 
Published: October 2019

Abstract
Abstract 2586
Even when intensively treated within pediatric-inspired protocols, about 25 to 30% of adults with ALL eventually relapse. With standard 4-drug or Hyper-CVAD salvages, post-relapse outcome remains very poor. Clofarabine is one of the new agents approved in US and EU to treat relapsing B-cell precursor (BCP) and T-cell ALL in children and young adults (up to 21 at initial diagnosis). In these patients, clofarabine is associated with a salvage rate around 30% when used as single agent. Combining clofarabine with conventional drugs in an intensive schedule may provide a chance to improve results in this difficult to treat population.

Methods:
Fifty-five patients were treated between March 2008 and February 2011. Thirty-seven patients received the VANDEVOL chemotherapy combining dexamethasone 10 mg/m2/12h day 1–5, mitoxantrone 8 mg/m2/d day 3–4, etoposide 150 mg/m2/d day 3–5, Peg-asparaginase 2.500 UI/m2 day 7, and Clofarabine 30 mg/m2/day day 1–5 and eighteen patients received the ENDEVOL chemotherapy combining cyclophosphamide 300 mg/m2/d day 1–3 and clofarabine 30 mg/m2/d day 1–5. Median age was 34 (19–67) and 53 (18–78) years in the VANDEVOL and ENDEVOL cohort, respectively. The proportion of first relapsing patients was 68% in the VANDEVOL cohort and 39% in the ENDEVOL cohort. Fifty patients had BCP-ALL (including 8 Ph+ ALL) and 5 patient had T-ALL.

Results:
Complete remission was achieved in 15/37 (41%) VANDEVOL patients and in 9/18 (50%) ENDEVOL patients. Early death rate was 5/37 (14%) in patients treated with VANDEVOL and 1/18 (6%) in patients treated by ENDEVOL. Grade 3–4 infectious, neurological, GI, and liver toxicities were observed in 22, 5, 5, and 11 VANDEVOL patients and in 10, 0, 0, and 2 ENDEVOL patients, respectively. Thirteen patients in the VANDEVOL group and three in the ENDEVOL group received subsequent allogeneic stem cell transplantation (overall transplant rate, 29%). After a median follow-up of 6 months, the median OS was 6.5 months.

Conclusion:
Combination of clofarabine with standard chemotherapy seems to be a promising and relatively safe approach to treat adult patients with refractory/relapsing ALL. This approach may be considered as a bridge to transplant in patients eligible for subsequent allogeneic stem cell transplantation. Updated data will be presented. The GRAALL will soon initiate a large multicenter prospective Phase II study using the VANDEVOL regimen.

Disclosures:
No relevant conflicts of interest to declare.
Mitotic Spindle Assembly Inhibition Through Aurora A (MLN8237) Plus Vincristine Is Synthetic Lethal and Synergizes with Rituximab As a Curative Therapy in Aggressive B-Cell Non-Hodgkin Lymphoma
Authors: Mahadevan Daruka , Cooke Laurence , Stejskal Amy , Manziello Ann , Morales Carla , Persky Daniel O. , Fisher Richard I , Miller Thomas P. , Qi Wenqing 
Published: October 2019

Abstract
Abstract 2721
Most aggressive B-cell non-Hodgkin lymphomas (B-NHL) are not curable with current chemo-immunotherapy combinations. Synthetic lethal interactions with novel agents may yield effective combination therapies with minimal toxicity for aggressive B-NHL. Auroras (A and B) are a family of mitotic oncogenic serine/threonine kinases intimately involved in high fidelity regulation of cell division. Aberrant over-expression of Auroras leads to genetic instability, polyploidy, tumor initiation and progression. Over-expression of Auroras in aggressive B-NHL promotes resistance to microtubule targeted agents (MTA, taxanes and vinca alkaloids). Si-RNA knockdown or pharmacologic inhibition of Aurora with MLN8237 [M], an ATP-site small molecule inhibitor, leads to enhanced sensitivity of B-NHL cells to MTAs. We hypothesized that promotion of microtubule de-polymerization with vincristine [V] would be more effective in synergizing with M than a microtubule polymerizing agent docetaxel [D]. We demonstrated that M plus V is synergistic while M plus D is additive in B-NHL cell culture models. Further, the addition of rituximab [R] enhanced apoptosis of B-NHL cells treated with MV or MD therapy. Mouse xenograft models of mantle cell lymphoma show modest single agent activity for M, R, D and V with tumor growth inhibition (TGI) of ∼10–15% (p=0.01). Of the doublets, MV caused tumor regression while MD and MR caused TGI (∼55–60%) and (∼20–25% (p=0.001) respectively. Although MV caused tumor regression, mice relapsed after 2 weeks of stopping therapy. In contrast, MVR had no relapses 120 days after stopping therapy, while MDR led to TGI of ∼85% (p=0.001). Kaplan-Meier analysis of overall survival showed that the mice treated with MV and MVR had a statistically significant improvement in overall survival when compared with the control (p<0.0001) or MR (p=0.0043). Gene expression profiling (human HG-U133A) and confirmatory Western blotting of harvested tumors at the end of treatment (3 weeks) confirmed reactivation of cell cycle regulatory genes including PCNA, Aurora B, cyclin B1 and cyclin D1, in MV versus MVR. Moreover, MVR therapy continues to inhibit Aurora B by repressing genes regulating mitotic sister chromatid segregation by repressed gene expression analyzed by Gene Ontology. Thus, addition of R to MV represents a novel therapeutic strategy that warrants clinical trial evaluation in aggressive B-NHL [Funded by the Lymphoma SPORE 1 P50 CA 130805 01A1].

Disclosures:
No relevant conflicts of interest to declare.