bluuebunny/crossref_metadata_2025_split huggingface dataset:
TIMP-1 Deficiency Subverts Cell Cycle Dynamics In HSCs.
Authors: Rossi Lara , Goodell Margaret A. 
Published: October 2019

Abstract
Abstract 1545
Although TIMP-1 was initially described as a mere inhibitor of Metalloproteinases (MMPs), recent findings have offered a different perspective on its biological role, contributing to unveil its multifaceted nature. In addition to inhibiting MMP activity, TIMP-1 has been proven to play MMP-independent, cytokine-like activities and to be involved in the regulation of numerous biological functions, including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of hematopoietic stem cells (HSCs), whose biological behavior is the synthesis of both microenvironmental and intrinsic cues. Bone marrow hematopoietic stem cells (HSCs) were isolated from TIMP-1-/- mice based on the phenotype Side Population c-Kit+Lin- Sca-1+ (SPKLS). In vitro cultural assays as well as in vivo transplantation assays were employed to investigate how TIMP-1 obliteration affects murine hematopoiesis. Cell-cycle dynamics in KO SPKLS HSCs were characterized by Pyronin Y/Hoechst staining, Ki-67 staining, as well as evaluation of RNA expression of cell cycle inhibitors, such as p53, p57, and p21.
We found that TIMP-1-/- mice have decreased HSC numbers and, consistent with this finding, TIMP-1-/- HSCs display reduced capability of long-term repopulation. Interestingly, the cell cycle distribution of TIMP-1-/- LT-HSCs is profoundly distorted, with a consistent proportion of the stem cell pool arrested in the G1 phase, suggesting that TIMP-1 is intrinsically involved in the regulation of the HSC proliferation dynamics. Of note, TIMP-1-/- HSCs present decreased levels of CD44 glycoprotein, whose expression has been proven to be controlled by p53, the master regulator of the G1/S transition. Interestingly, p53 RNA levels are indeed increased in TIMP-1-/- SPKLS HSCs compared to controls. Likewise, the expression level of other cell-cycle inhibitors, such as p57 and p21, were found to be higher in KO SPKLS HSCs, indicating a disregulation of cell-cycle dynamics.Our study highlights a novel biological role of TIMP-1 in the regulation of the HSC compartment and suggest a novel mechanism presiding over stem cell quiescence in the framework of the BM milieu.

Disclosures:
No relevant conflicts of interest to declare.
A MODEL to STUDY the Function of NPM-MLF1 IN MYELODYSPLASIA.
Authors: Lee Wen-hsin 
Published: October 2019

Abstract
Abstract 1571
Myelodysplastic syndromes (MDS) are bone marrow disorders characterized by ineffective haematopoiesis and peripheral cytopenia(s) with frequent evolution to acute myeloid leukemia (AML). Apoptosis is significantly deregulated in early MDS whereas advanced MDS is characterized by deregulation of DNA damage response. As MDS proceeds to AML, the ratio of apoptosis to proliferation decreases, resulting in clonal outgrowth of abnormal cells.
The t(3;5)(q25;q34) translocation, creating the NPM-MLF1 fusion, has been found as a sole cytogenetic abnormality in MDS. It is recurrent, with poor prognosis but the precise mechanism through which NPM-MLF1 induces malignant transformation remains unknown. We aimed to model this disease in vitro and in vivo by expressing NPM-MFL1 in mouse bone marrow hematopoietic progenitor cells (HPCs) and analyzing any changes in HPC self-renewal and response to DNA damage.
NPM-MLF1 did not impair haematopoiesis in vitro and in vivo. FLT3/ITD was frequently associated with NPM mutant in AML patients; however, NPM-MLF1 did not collaborate with FLT3/ITD in our system. To recapitulate NPM hemizygosity in t(3;5)-MDS patients, we have expressed NPM-MLF1 in HPCs derived from Npm+/− mice. A transient increase in the self-renewal of the NPM-MLF1-expressing Npm+/− HPCs was seen. These cells did not exhibit enhanced proliferation as confirmed by growth curve and analysis of DNA synthesis. Interestingly, unlike control cells, NPM-MLF1-expressing Npm+/− HPCs showed prolonged self-renewal ability in vitro, and an elevated expression of c-Myc, Hoxa9, Hoxa10 and Meis1 genes. In addition to altering HPC self-renewal, NPM-MLF1 was also found to modulate their DNA damage response. This study suggests that the ability of NPM-MLF1 to maintain HPC self-renewal and impaired DNA damage responses may favour the accumulation and outgrowth of the aberrant HPCs, contributing to the abnormal haematopoiesis.

Disclosures:
No relevant conflicts of interest to declare.
Activation of Dormant Hematopoietic Stem Cells In Vivo by the Endotoxin LPS.
Authors: Essers Marieke , Lutz Raphael , Kern Andrea , Wurzer Stephan , Trumpp Andreas 
Published: October 2019

Abstract
Abstract 1613
Maintenance of the blood system is dependent on dormant haematopoietic stem cells (HSCs), which are characterized by pluripotency and life long self-renewal capacity. In order to both maintain a supply of mature blood cells and not exhaust HSCs throughout the lifespan of the organism, most adult HSCs remain deeply quiescent during homeostasis and only a limited number are cycling at any given time. The balance between self-renewal and differentiation of HSCs is controlled by external factors such as chemokines and cytokines, as well as interactions of HSCs with its niche environment.
We have recently shown that the cytokine IFNa very efficiently activates dormant HSCs in vivo. Within hours after treatment of mice with IFNa, HSCs exit G0 and enter an active cell cycle. In general, IFNa is produced in response to viral infections by cells of the immune system, and plays an important role in the antiviral host defense. We now questioned whether endogenous IFNa is also produced in response to other types of bone marrow stress and whether this affects the proliferation rate of HSCs. To monitor IFNa production in the bone marrow in vivo, we have generated MxCre; ROSA-R26-EYFP mice and found that treatment with both the chemotherapeutic agent 5-FU as well as the endotoxin LPS leads to the production of IFNa in the vicinity of HSCs and progenitors. In addition, LPS treatment in vivo induced a strong increase in HSC cycling. Surprisingly, since mice lacking the IFNa receptor (Ifnar-/-) still respond to LPS this effect is independent of IFNAR signaling. LPS binds and signals via the TLR4-CD14 receptor complex and HSCs of mice lacking TLR4 (TLR4-/-) are no longer activated by LPS. Strikingly, LPS induced HSC activation correlated with increased expression of Sca-1, similar to what occurs upon IFNa treatment. Moreover, as for IFNa, the upregulation of Sca-1 is required for LPS induced proliferation, since Sca-1-/- mice fail to respond to LPS stimulation.
In summary, these data suggest that not only virus inducible IFNa, but also infections by gram negative bacteria produced LPS induces cycling of progenitors and otherwise dormant HSCs. Finally, both IFNa and LPS induced activation of HSCs are dependent on the up-regulation of Sca-1, suggesting a more general role for Sca-1 in the activation of dormant stem cells in response to injury signals.

Disclosures:
No relevant conflicts of interest to declare.
Ribosomal RNA Expression In CD34+ Hematopoietic Progenitor Cells Inversely Correlates with Ribosomal DNA Methylation In Myelodysplastic Syndromes.
Authors: Raval Aparna , Pollyea Daniel A , Shridhar Kunju J , Patel Shripa , Greenberg Peter L. , Mitchell Beverly S. 
Published: October 2019

Abstract
Abstract 1682
Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis and, in the high risk setting, by progression to acute myelogenous leukemia (AML). The DNA hypomethylating agents, 5-azacytidine (5AC) and 5-aza-2’-deoxyazacytidine (DAC), are effective treatments for patients with MDS, increasing time to progression to AML and improving overall response rates and survival. Genome-wide DNA hypermethylation in MDS has been documented. However, promoter methylation signatures in MDS samples have not correlated with the clinical response to epigenetic drug therapies. Recently, attention has been drawn to the potential etiologic role of reduced expression of specific ribosomal proteins in del (5q) MDS, as well as in other bone marrow failure states. We therefore asked whether CD34+ cells from the bone marrow of MDS patients had reduced expression of ribosomal RNA (rRNA) and whether this correlates with hypermethylation of ribosomal DNA (rDNA). Using quantitative SYBR-Green RT-PCR, we found that rRNA expression was significantly reduced in CD34+ hematopoietic progenitor cells from 15/29 MDS patients as compared to normal controls (p value <0.01). We then studied quantitative rDNA methylation using pyrosequencing in DNA from CD34+ cells isolated from 5 normal controls and 6 MDS samples. Two of six MDS samples had reduced rRNA expression and increased DNA methylation, not only within the upstream core element but also in the intergenic rDNA sequences. For each of the CpGs evaluable within these highly CpG rich regions, the extent of DNA methylation in patients with reduced rRNA expression were increased by more than 2-fold compared to that in normal samples. This DNA hypermethylation was not observed in rDNA from 4/6 MDS samples where rRNA expression was not reduced in relation to controls. In addition, cells treated with DAC demonstrated a two-fold induction in rRNA gene expression and an up to four-fold reduction in rDNA promoter methylation as compared to controls, suggesting that promoter methylation may have a direct effect on rRNA expression. We are currently examining MDS patient samples before and after DAC treatment for levels of rDNA methylation and expression. It is possible that the methylation status at the rDNA locus may serve as a better predictor of response to hypomethylating agents than genome-wide methylation status. Regulation of rDNA gene expression is complex, but may be a fundamental underlying mechanism of bone marrow failure in a subgroup of MDS patients and perhaps in other bone marrow failure states.

Disclosures:
No relevant conflicts of interest to declare.
Clinical, Biological Profile and Outcome of Biphenotypic Acute Leukemia: a Case Series.
Authors: Zhang Yanming , Depei Wu , Sun Aining , Qiu Huiying , He Guangsheng , Jin Zhengming , Tang Xiaowen , Miao Miao , Fu Chengcheng , Han Yue , Zhu Mingqing , Xue Yongquan , Chen Zixing , Ruan Changgeng 
Published: October 2019

Abstract
Abstract 1683
Biphenotypic acute leukemia (BAL) is a very rare type of acute leukemia, which presents a high heterogeneity and poor prognosis. We identified 51 cases (3.0%) BAL from 1693 newly diagnosed acute leukemia patients according to the EGIL scoring system between January 2003 and July 2009. The biological features, treatment and outcome of 39 evaluable BAL patients were analyzed retrospectively. There were 23 (59.0%) cases of myeloid and B-lymphoid (M/B) phenotype, 14 (35.9%) cases of myeloid and T-lymphoid (M/T) phenotype, one case (2.6%) of trilineage phenotype or B-lymphoid and T-lymphoid phenotype respectively. The high expressions of CD34 (84.6%) and HLA-DR (54.5%) on the blast cells of BAL support the notion that BAL probably arises from hemopoietic stem/progenitor cells. It seemed that CD7-positive patients had poorer median survivals comparing with those CD7-negative patients, however, there was no statistical difference (P=0.076). Abnormal karyotypes were detected in 75.7% of 37 BAL patients with valid analysis and displayed a high heterogeneity, which were associated with structural rearrangement and numerical abnormalities including t(8;21) (16.2%), t(9;22) (13.5%), structural rearrangement of 11 chromosome (16.2%), 11q23 (5.4%), complex karyotype (21.6%) and other abnormal karyotypes (10.8%). Combined regimens for both AML and ALL, ALL-type regimens appeared a better complete remission (CR) rate than AML-type regimens (71.4% vs. 63.6% vs. 33.3%). The median survival for overall survival (OS) and disease-free survival (DFS) in our series was 14 and 12 months, the probability of OS and DFS at 2 years was 26.0% and 18.5%, respectively. No statistical differences were observed in CR rate, OS and DFS between M/B and M/T cases. It showed that BAL patients with complex karyotype or rearrangement of 11 chromosome had a significantly worse survival in contrast to normal karyotype, t(8;21) and t(9;22) group (P=0.001). Although BAL with t(8;21) seemed to be appeared a better survival than normal karyotype and t(9;22) group, there were no statistical significance (P=0.436). Our data indicate that combined-type regimens or ALL-based protocols are more effective and complex karyotype, rearrangement of 11 chromosome have the unfavorable prognosis for BAL.

Disclosures:
No relevant conflicts of interest to declare.
Is There a Role for Autologous Stem Cell Transplantation (Auto SCT) for Patients with Acute Myelogenous Leukaemia? A Retrospective Analysis.
Authors: Novitzky Nicolas , Thomas Valda , Toit Cecile du , McDonald Andrew 
Published: October 2019

Abstract
Abstract 1689

Introduction:
To better counsel our patients on the role of auto SCT for patients with AML we studied consecutive individuals in CR 1 who had tissue compatible siblings and underwent allogeneic (allo) SCT with subjects who had no HLA donor and actually underwent auto SCT.

Methods:
Patients were in CR 1 following induction combinations containing 7 days of cyatarabine and etoposide with 3 days daunorubicin followed by similar consolidation therapy. The choice for the type of graft was based on availability of HLA identical siblings. Allogeneic donors underwent PBPC mobilisation with filgrastim and for GvHD prophylaxis grafts were exposed ex vivo to alemtuzumab 1mg/1010 mononuclear cells. Patients were prescribed cyclosporin until day 90 post transplant. Individuals lacking a donor underwent PBPC mobilization with etoposide 2 gr/m2 and harvested PBPC were cryopreserved. Patients received similar myeloablative conditioning followed by infusion of the grafts. Patients were stratified by clinical and laboratory factors as well as cytogenetic risk. The end points were TRM, DFS and OS.

Results:
The median presentation age for both transplant groups was 35 (14-60) years. Of the 112 consecutive patients achieving remission 37 had HLA identical siblings, but 3 relapsed and donors became unavailable in 2. Thus, autologous or allogeneic grafts were actually transplanted to 43 and 32 patients respectively. There was no significant difference in the presentation clinical features, laboratory parameters, marrow morphology or proportion of low and intermediate cytogenetic risk for both transplant options. Treatment mortality as well as relapse rate was similar (14 and 15%; 39 and 27%, respectively). At a median of 1609 and 1819 post transplant days, 56% and 63% in each group survive. In univariate analysis performance status, cytogenetic risk, morphological features of dysplasia, blast count and LDH were significant factors for survival. While for the entire group there was no difference in survival between both modalities, all patients with unfavourable cytogenetics receiving an autologous graft died of disease recurrence (3 year survival 35% vs 0%; p= 0.05).

Conclusions:
We conclude that patients with AML who have low or intermediate cytogenetic risk undergoing myeloablative conditioning followed by autologous or allogeneic T-cell depleted stem cell transplantation appeared to have similar outcome. However, those with unfavourable karyotype are unlikely to be cured with autologous grafts and are candidates for experimental modalities.

Disclosures:
No relevant conflicts of interest to declare.
A Proteomic Platform for EBV and KSHV Serological Screening
Authors: Zheng Dasheng , Wang Leyao , Chiou C.-J. , Liao Gangling , Zhu Heng , Hayward Gary S , Ambinder Richard F. , Hayward S.D. 
Published: October 2019

Abstract
Abstract 1747
Individuals with HIV infection have an increased susceptibility to EBV- and KSHV-associated malignancies. Evaluating a possible role for specific humoral responses to individual viral proteins in the control of viral load and prevention of EBV or KSHV related disease development in this population has been hampered by the limited number of viral antigens currently used for screening serum samples. We have developed a protein array platform that can be utilized for EBV and KSHV serological screening. The array consists of EBV and KSHV proteins printed onto glass slides. All eighty four EBV open reading frames and eighty six KSHV open reading frames were cloned into bacterial vectors and the inserts validated by DNA sequencing in both directions. Authenticated inserts were transferred to a yeast vector that expresses proteins as N-terminal GST-fusions. Seventy nine EBV proteins and eighty one KSHV proteins were successfully purified from yeast. First generation EBV/KSHV protein arrays have been printed with these proteins plus a variety of control proteins that include GST, and human IgG, IgM and IgA. Initial serological assays compared IgG antibody responses in HIV positive individuals, cancer patients and healthy adults. The HIV positive group (i) had a broader EBV antigenic response than the cancer patients or healthy normal's and (ii) recognized a wider range of EBV antigens compared to KSHV. This may reflect more extensive exposure to replicating EBV versus KSHV in these patients. In addition to the previously described dominant antigenic responses to EBV and KSHV, the assays identified multiple additional immunogenic viral proteins. The ability to compare serological responses to the complete repertoire of individual EBV and KSHV encoded proteins should provide new insight into B cell mediated immune regulation of these viruses.

Disclosures:
No relevant conflicts of interest to declare.
Induction of Autophagic Cell Death Circumvents Azacitidine-Resistance In Myelodysplastic Syndrome-Derived Cell Lines
Authors: Cluzeau Thomas , Robert Guillaume , Karsenti Jean-Michel , Luciano Frederic , Puissant Alexandre , Raynaud S. Dominique , Mannone Lionel , Cassuto Jill-Patrice , Mounier Nicolas , Auberger Patrick 
Published: October 2019

Abstract
Abstract 1817
Azacitidine (AZA) is the first line treatment for IPSS (index prognostic scoring system) high-risk myelodysplastic syndrome (HR-MDS). To date, only Khan et al. (Exp Hematol. 2008) and Hollenbach et al. (PLoS One. 2009) have reported apoptosis as a mechanism of AZA effect on MDS cell lines. Nevertheless, approximately 40% of patients treated with AZA are refractory to this molecule. To investigate the possible mechanisms of AZA resistance in MDS cells, we developped AZA-resistant cell clones (AZA-R) from the well-characterized MDS cell line SKM1. The bulk resistant SKM1 cell line (AZA-R) was obtained following long time exposure of cells to iterative and increasing doses of AZA ranging from 0.1 to 8mM. We first showed that AZA triggered loss of cell metabolism in SKM1 parental cells but not in their AZA-resistant counterpart at a maximally effective dose of 1mM. AZA-mediated loss of cell metabolism accounted mainly for induction of apoptosis as judged by both an increase in caspase 9 and 3 activities triggered by this compound and by a significant protection in the presence of the pan-caspase inhibitor Z-VAD-fmk in SKM1 parental cells. Conversely, no or very few activation of caspases and apoptosis were detected in AZA-R cells strongly suggesting that apoptosis is impaired in AZA-R SKM1 cells. Finally, unlike in SKM1 cells, AZA failed to induce mitochondrial membrane permeabilization in AZA-R SKM1 cells. Importantly, basal autophagy was increased in AZA-R versus AZA-S cells as shown by LC3-I cleavage into LC3-II, p62/SQSTM1 protein expression, cathepsin B activation, mTOR and S6 ribosomal protein dephosphorylation and finally electronic microscopy experiments. In addition, Acadesine, an adenosine derivative and AMPK agonist which targets autophagy was capable to circumvent AZA resistance in both AZA-R SKM1 cells and in medullary cells from five MDS patients resistant to AZA after 6 cycles of Azacitidine. In conclusion, targeting autophagy appears as an attractive therapeutical strategy to circumvent AZA resistance in both established MDS cell lines and cells from MDS patients. Therefore, drugs capable of inducing autophagy or autophagic cell death, such as Acadesine (Robert et al., PLoS One. 2009) which is currently in phase II clinical trials for the treatment of Chronic Lymphoblastic Leukemia could be also beneficial for HR-MDS patients resistant to AZA.

Disclosures:
Cluzeau: Celgene: Consultancy. Raynaud:Celgene: Consultancy.
Hedgehog Pathway Inhibition Is a Novel Therapeutic Approach in Lymphomas and Its Efficacy is Inversely Related to PI3 Kinase Inhibition
Authors: Walsh Katherine J. , Fan Siyao , Patel Amee , Jacobs Cassandra L , Smith Jason , Liu Qingquan , Dave Sandeep 
Published: October 2019

Abstract
Abstract 1848
Background:
The hedgehog signaling pathway has been shown to be important in the development of malignancies, however, the role of this pathway in lymphomas is uncertain. GDC-0449 is a novel small molecule inhibitor that has been shown to block hedgehog activity by inhibiting SMO. The purpose of this study was to determine a potential role for hedgehog inhibition in the treatment strategies for lymphomas. We further sought to understand the determinants of sensitivity and potential resistance to therapeutic inhibition of this pathway.
Methods and Results:
We tested the small molecule inhibitor GDC-0449 in cell lines that span the spectrum of lymphoma including Burkitt lymphoma (N=4), ABC DLBCL (N=3), GCB DLBCL (N=7), mantle cell lymphoma (N=2), multiple myeloma (N=2), lymphoid leukemia (N=2), and T cell leukemia (N=2). Using the MTT assay, we identified the IC50 of drug response in these cell lines. We performed gene expression profiling in these cell lines and applied the Cox proportional hazards model using IC50 as the survival variable to identify genes that were associated with either sensitivity or resistance to the drug.
We found that the drug was lethal in these cell lines at physiologically achievable concentrations with IC50s ranging from 5 to 22μM. We applied hierarchical clustering to the genes associated with sensitivity and resistance and found that the method distinguished two groups of samples that differed in their average IC50s by over 2-fold (8μM vs 17μM). There was a suggestion of association with lymphoma cell type with Burkitt lymphoma lines being the dominant cell type in the resistant group and DLBCL being the dominant cell type in the sensitive group.
Resistance to hedgehog inhibition was found to be highly associated with the phosphatidylinositol 3′-kinase (PI3K) signaling pathway, suggesting that the hedgehog pathway and PI3K pathways act through independent, complementary mechanisms to promote tumor proliferation. In order to test the hypothesis that PI3K signaling is independent of hedgehog signaling we tested the lethality of PI3K inhibition using a dual PI3K/mTOR inhibitor (BEZ235) in the same cell lines.
We found an inverse relationship between the hedgehog inhibitor and PI3K inhibitor, i.e. the cell lines that were relatively sensitive to hedgehog inhibition (mean IC50 of 8μm) were resistant to PI3K inhibition(mean IC50 of 0.69μm) and the cell lines that were relatively resistant to hedgehog inhibition (17μm) were relatively sensitive to PI3K inhibition (0.32μm).
Conclusion:
Hedgehog pathway inhibition is a promising therapeutic approach in a carefully defined subset of patients with lymphoma. Hedgehog and PI3K inhibitors appear to act by distinct mechanisms and may be complementary in the treatment of lymphomas.

Disclosures:
No relevant conflicts of interest to declare.