bluuebunny/crossref_metadata_2025_split huggingface dataset:
Maintenance of Hematopoietic Stem Cells Through Regulation of Wnt and mTOR Pathways.
Authors: Huang Jian , Klein Peter S. 
Published: October 2019

Abstract
Abstract 2309
Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our previous study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a b-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by b-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. In the current study, we found that suppression of the mammalian target of rapamycin (mTOR) pathway, an established nutrient sensor, combined with activation of canonical Wnt/ß-catenin signaling, allows the ex vivo maintenance of human and mouse long-term HSCs under cytokine-free conditions. We also show that combining two clinically approved medications that activate Wnt/ß-catenin signaling and inhibit mTOR increases the number of long-term HSCs in vivo.

Disclosures:
No relevant conflicts of interest to declare.
Autologous HSCT for Ph-Positive Adult Acute Lymphoblastic Leukemia: A Curative Option in the Era of Tyrosine Kinase Inhibitors? an Analysis From the Acute Leukemia Working Party of the EBMT
Authors: Giebel Sebastian , Labopin Myriam , Gorin Norbert Claude , Caillot Denis , Leguay Thibaut , Schaap Nicolaas , Michallet Mauricette , Socie Gerard , Mohty Mohamad 
Published: October 2019

Abstract
Abstract 233
Outcome of patients with Ph-positive acute lymphoblastic leukemia (Ph+ ALL) improved markedly with the introduction of tyrosine kinase inhibitors (TKIs) used in combination with chemotherapy. However, despite very high rate of complete remissions the possibility of cure with conventional-dose treatment remains questionable. Hence, allogeneic hematopoietic stem cell transplantation (HSCT) is still considered a standard of care. Patients lacking appropriate donor are usually treated with TKI-based maintenance. Autologous HSCT could be an alternative approach, however, due to “negative” results of a series of prospective studies run in XX. century its use has been limited. The goal of the current analysis was to retrospectively analyze if results of autoHSCT for Ph+ ALL changed over time.
Results of 171 autologous transplantations performed in the first complete remission between 1996–2010 and reported to the EBMT registry have been analyzed. Median patient age was 48.3 (19–65) years. Conditioning regimen was based on either TBI (63%) or chemotherapy (37%). Peripheral blood was used as a source of stem cells in 84% cases.
With the median follow-up of 2 years, in the whole study group, the probability of the overall survival (OS) at 2 years was 45% (+/−4%) and leukemia-free survival (LFS) was 32% (+/−4%). The cumulative incidence of relapse (RI) and non-relapse mortality (NRM) was 54% (+/−4%) and 13% (+/−3%), respectively. LFS rates were comparable for TBI and chemotherapy-based conditioning (34% vs. 29%, p=0.53). As well, the source of stem cells had no impact on LFS (32% for PB vs. 33% for BM, p=0.91). The 2 year probability of LFS increased from 22% for transplants performed between 1996–2000 (n=70) to 32% between 2001–2006 (n=61) and 54% between 2007–2010 (n=40), p<0.001 (Fig). In respective periods the RI decreased from 65% to 47% and 46% (p=0.01), while NRM was 11%, 21% and 0% (p=0.03). In a multivariate analysis the year of transplantation (<2007 vs. 2007–2010) was the only factor independently influencing the risk of treatment failure (HR=2.98, 95%CI, 1.54–5.79; p=0.001).
We conclude that results of autologous HSCT for Ph+ ALL improved markedly in recent period with more than half of patients being alive and leukemia-free at 2 years. Therefore, it appears than in the era of TKIs autologous HSCT may be considered potentially curative option. The advantage is probably associated with more profound responses achieved with TKIs, which, however requires confirmation in a separate analysis. As well, the role of post-transplant maintenance remains to be determined.

Disclosures:
No relevant conflicts of interest to declare.
TGFβ Restores Hematopoietic Homeostasis After Chemotherapy.
Authors: Brenet Fabienne A. , Kermani Pouneh , Rafii Shahin , Scandura Joseph M. 
Published: October 2019

Abstract
Abstract 2344

Background:
Hematopoietic stem cells (HSCs) are required for lifelong blood cell production and, to prevent exhaustion, the majority of HSCs are deeply quiescent during steady-state hematopoiesis. Paracrine factors produced by specialized bone marrow niche cells maintain HSC quiescence. Yet, evolution demands a rapid hematopoietic response to stressors such as infection, bleeding or toxin exposure. These triggers set off a remarkable adaptation in hematopoiesis that sacrifices HSPC quiescence, and the protection it affords, to meet an urgent need for new blood cell production. A great deal is known about how the HSPCs are mobilized during these periods of stress but mechanisms driving the return to steady-state hematopoiesis after stress have not been identified.

Method/Results:
Because TGFβ is one of few negative regulators of hematopoiesis, we measured the levels of active TGFβ and assessed the effects of its downstream signaling during hematopoietic recovery after chemotherapy. During late hematopoietic regeneration, as hematopoietic homeostasis is restored, the levels of active TGFβ spike in whole bone marrow and downstream signaling (as reported by Smad2 phosphorylation) increases in hematopoietic stem and progenitor cells (HSPCs). To assess how TGFβ signaling alters hematopoietic regeneration, we administered myelosuppressive chemotherapy to mice and then, during recovery, treated cohorts with either an antibody that specifically neutralizes active TGFβ (1D11), an isotype control antibody (13C4) or nothing at all. We found that TGFβ blockade accelerated blood count and bone marrow regeneration, and promoted HSCP self-renewal/proliferation by delaying the return of HSPCs to quiescence (G0). In contrast, TGFβ blockade during homeostasis elevates neither blood counts nor bone marrow cellularity and fails to induce HSPCs to emerge from quiescence.

Conclusions:
The de facto paradigm is that homeostasis is passively reestablished as stress mediators normalize. Our data strongly suggest that this paradigm is incorrect. Rather than being a passive process, it appears that steady-state hematopoiesis is actively reimposed. TGFβ pathway activation marks regenerating HSPCs returning to quiescence and this context-depending signaling helps reestablish homeostasis during recovery from chemotherapy. Together, our data demonstrate that myelosuppression does not drive hematopoiesis using only a cytokine-fueled gas pedal but also taps an active braking mechanism once sufficient recovery has been attained. More directly, our data strongly suggests that TGFb pathway inhibitors could be used to promote multi-lineage hematopoietic reconstitution. This finding significantly extends prior work in the field by opening the door to new, potentially superior, ways to modulate the hematopoietic adaptation to stress.

Disclosures:
No relevant conflicts of interest to declare.
AML1-ETO Collaborates with the TALE Homeobox Genes MEIS1 and MEIS2 in Inducing AML.
Authors: Vegi Naidu M , Klappacher Josef , Oswald Franz , Claus Rainer , Mulaw Medhanie M , Thiel Verena , Rawat Vijay P.S , Fend Leticia Quintanilla , Plass Christoph , Döhner Konstanze , Döhner Hartmut , Hiddemann Wolfgang , Buske Michaela Feuring , Buske Christian 
Published: October 2019

Abstract
Abstract 2387
AML1-ETO (AE) is the most frequent fusion gene in human AML. Previously, we and others have demonstrated that the fusion is not able to cause leukemia on its own in experimental murine models, but that it needs collaborative partners. Following the ‘two-hit model’ hypothesis that class I and class II mutations have to collaborate to induce leukemia, we could previously demonstrate that AE (class II) collaborates with FLT3-length mutation (class I) to generate AML in the murine bone marrow transplantation model (BMT model). We now demonstrate that AE can collaborate with mutations of its own class in inducing AML. We focussed our analyses on the TALE homeobox gene family, namely Meis1 and Meis2, as already known in the case of Meis1 to be a potent co-factor for Hox gene associated leukemias and being classified as other homeobox genes as a class II mutation.
First, real-time RT - PCR confirmed that MEIS1 is expressed at high levels in a subgroup of AE positive AML patients. Furthermore, MEIS2 was highly and aberrantly expressed virtually in all AE patients (n=70) compared to normal bone marrow. Expression patterns of both MEIS genes correlated with their promoter methylation in the t(8;21) patients and cell lines. To test the functional relevance of MEIS expression in human AE patients, we analysed collaboration of AE with Meis genes in the BMT model: Meis1 and Meis2 were retrovirally expressed alone or in combination with AE in 5-FU treated primary murine bone marrow and injected into lethally irradiated recipients (AML1-ETO alone, n=10; EGFP control, n=7; AE+Meis1, n=14; AE+Meis2, n=4). None of the mice in the AE as well as in the control group developed disease. In contrast, mice transplanted with BM co-expressing AE+Meis1 and AE+Meis2 developed lethal disease after a median latency of 102 and 255 days respectively. AE+Meis1 induced MPS in three, AML in seven and ALL in three cases, whereas AE+Meis2 induced AML in all cases. Functional relevance of MEIS expression was further confirmed in human AML cell lines as shRNA mediated depletion of MEIS1 as well as MEIS2 impaired cell growth in AE positive AML cell lines and so far one primary AE AML sample. To understand the mechanisms of AE/MEIS collaboration co-immunoprecipitation assays were performed documenting weak binding of Meis1, but strong interaction of Meis2 with AE. ChIP-Seq is currently being performed to test whether DNA binding of MEIS genes alter in collaboration with AE. Taken together, we could demonstrate that AE collaborates with Meis genes in AML. It furthermore shows that a class II mutation can be converted into an overt oncogene by a mutation of its own class.

Disclosures:
No relevant conflicts of interest to declare.
FLT3-ITD Interacts with and Phosphorylates IL-3β, and JAK1/2 Dependent IL-3β Activation Bypasses FLT3-ITD in FLT3 Kinase Independent Inhibitor Resistance in Vitro: Evidence for the Significance of IL-3β for FLT3-ITD Dependent Oncogeneic Signaling in AML.
Authors: Rummelt Christoph , Gorantla Sivahari P. , Sigl Michael , Saenger Jana , Götze Katharina , Peschel Christian , Duyster Justus , von Bubnoff Nikolas 
Published: October 2019

Abstract
Abstract 2423
Activating FLT3 mutations are found in 30% of AML patients. Internal tandem duplication (ITD) mutations are most common, and are associated with poor prognosis. FLT3 tyrosine kinase inhibitors (TKI) display limited activity in FLT3 mutant AML. Most patients experience primary or secondary TKI resistance. Only single cases of FLT3 kinase-dependent TKI resistance by secondary FLT3-ITD kinase domain mutations were reported. We therefore examined the mechanism of FLT3-ITD kinase-independent TKI resistance using an in vitro model. FLT3-ITD cell lines resistant to PKC412 or Sorafenib in 50% of the cases did not harbor secondary FLT3-ITD kinase domain mutations. However, 30% of these lines displayed a persistent phosphorylation of JAK2, IL-3βc and STAT5, while FLT3 phosphorylation was suppressed, indicating that these cell lines bypassed FLT3-ITD by activation of IL-3β by JAK2. Strikingly, TKI sensitive FLT3-ITD cells also displayed IL-3β phosphorylation, which in contrast to TKI resistant cells was JAK2 independent and was suppressed upon FLT3 TKI treatment. Expression of FLT3-ITD in IL-3β and JAK2 deficient g2A cells was sufficient for IL-3β phosphorylation. Co-IP experiments indicated a direct interaction of IL-3β and FLT3-ITD in TKI-sensitive and -resistant cells, which did not require interaction of IL-3β with IL-3a. This indicates that FLT3-ITD “physiologically” uses IL-3β as signaling intermediate. Mapping experiments showed that FLT3-ITD binding occurs in the membrane proximal region of IL-3β, independent of 8 tyrosines present in the cytoplasmic part. In search of the kinase bypassing FLT3-ITD in TKI-resistant cell lines, we identified an activating JAK1 mutation V658F in all TKI resistant FLT3-ITD cell lines displaying phosphorylation of JAK2, IL-3β and STAT5. JAK1 V658F is homologous to JAK2 V617F present in PV, phosphorylates IL-3β and STAT5, and confers TKI resistance in FLT3-ITD positive cells, which could be overcome by the JAK1/2 inhibitor Ruxolitinib. In summary, these data indicate that IL-3β not only mediates “physiological” FLT3-ITD dependent STAT5 activation, but also serves as a signaling module mediating JAK1/2-dependent TKI resistance. Thus, JAK inhibitors might represent an attractive therapeutic option in addition to FLT3 inhibitors in FLT3-ITD positive AML.

Disclosures:
Duyster: Novartis: Honoraria, Speakers Bureau. von Bubnoff:Novartis: Honoraria, Research Funding, Speakers Bureau.
A Genome-Wide Analysis of Variants Influencing Methotrexate Clearance Replicates SLCO1B1.
Authors: Ramsey Laura B. , Panetta John C , Smith Colton , Yang Wenjian , Fan Yiping , Winick Naomi , Martin Paul L. , Cheng Cheng , Devidas Meenakshi , Pui Ching-Hon , Evans William E. , Hunger Stephen P. , Loh Mignon L. , Relling Mary V. 
Published: October 2019

Abstract
Abstract 2466
High-dose methotrexate (HDMTX) is an important element of chemotherapy for acute lymphoblastic leukemia (ALL) and other malignancies. Methotrexate clearance influences cure and toxicity in children with acute lymphoblastic leukemia (ALL). HDMTX schedules and doses vary widely among treatment protocols. The Children's Oncology Group (COG) tested the efficacy of 6 courses of 2 g/m2 over 4 hours versus 1 g/m2 over 24 hours (P9904 and P9905 protocols). Patients were assigned to one of four arms for consolidation: A, 24-hour methotrexate infusion (1 g/m2) and no delayed intensification (DI); B, 4-hour methotrexate infusion (2 g/m2) with no DI; C, 24-hour methotrexate infusion with DI; D, 4-hour methotrexate infusion with DI. We estimated methotrexate clearance for 1279 patients treated on these protocols, with two plasma MTX concentrations per course, using a Bayesian pharmacokinetic modeling approach. Germline genetic variation was assessed using the Affymetrix 6.0 array, and other single nucleotide polymorphisms (SNPs) were imputed based on 1000 Genomes reference data, yielding 5.2 million SNP genotypes evaluable per patient.
Average MTX clearance was highly variable, with a median (range) of 164 (65–355) and 109 (49–290) ml/min/m2 for the 24-hour and 4-hour infusions, respectively. Methotrexate clearance was lower in older children (p = 7 × 10−7), girls (p = 2.7 × 10−4), and patients who received a delayed intensification phase during consolidation (p = 0.0022). Adjusting for age, gender, race, and treatment arm, a genome-wide analysis showed that methotrexate clearance was associated with polymorphisms in SLCO1B1(p = 2.1 × 10−11), a gene that encodes for an organic anion transporter that is known to transport methotrexate. This replicates our previous findings (Trevino et al, J Clin Oncol. 2009;27(35):5972-8) that polymorphisms in SLCO1B1 influence methotrexate clearance in ALL patients treated on St. Jude protocols with three different HDMTX schedules. In a combined meta-analysis including the 1279 COG patients and 699 St. Jude patients, and adjusting for age, gender, race, and treatment arm, the association of methotrexate clearance with SLCO1B1 SNP rs4149056 yields a p-value of 3.1 × 10−19 (Figure). Even after adjustment for the rs4149056 SNP, other polymorphisms in SLCO1B1 remained significantly related to methotrexate clearance, indicating that there are multiple variants in SLCO1B1 that can influence methotrexate clearance. Validation of the association of this gene with five different treatment regimens of methotrexate solidifies the robustness of this pharmacogenomic determinant of methotrexate clearance.

Disclosures:
No relevant conflicts of interest to declare.
Whole Exome Sequencing Analysis in AML with Normal Karyotype Not Harboring FLT3/ITD Mutation Reveals Novel Genetic Alterations.
Authors: Lee Il-Kwon , Kim Namshin , Kim Yeo-Kyeoung , Kim Dennis Dong Hwan , Trinh Quang , Minden Mark D , Jung Chul Won , Jang Jun-Ho , Kim Hee-je , Moon Joon Ho , Sohn Sang Kyun , Won Jong-Ho , Kim Sung-Hyun , Huh Jungwon , Kim Nan Young , Yu Li , Lee Yun Gyeong , Chung Sung-Hoon , Ahn Jae-Sook , Yang Deok-Hwan , Lee Je-Jung , Kim Hyeoung-Joon 
Published: October 2019

Abstract
Abstract 2593

Background:
NK-AML represents genetically heterogeneous group of disease. However genetic lesions affecting treatment outcome in patients with NK-AML are relatively unknown.

Methods:
The discovery cohort consists of 67 NK-AML patients in complete remission (median age: 49.2, ranges: 19–70) without FLT-3 mutations. Genomic DNA was extracted from enriched AML cells at diagnosis or control specimens obtained after complete remission. Whole exomes were captured using Agilent SureSelect and sequencing were performed by HiSeq2000 with 41∼89× coverage. Bioinformatics analysis and identification of somatic mutation has been done by series of software such as BWA, Picard, GATK, VarScan 2, and custom-made scripts. All the data has been re-checked by manual inspection. Validation has been done independent set of cohort (358 NK-AML patients, median age: 51, ranges: 15–85) with Sanger sequencing on highly mutated target sites.

Results:
Filtering against dbSNP and COSMIC database generated 485 genes with somatic and structural variations. Among them, 41 genes were detected in more than two patients. In addition to well-known 28 mutations, 13 novel mutations with different frequencies were identified including genes responsible for structural maintenance of chromosome (SMC1A, 6.0%) and tumor suppressor function (FAT1, 6.0%). Most common type of mutation was missense mutation (70.8%), and substantial fraction of mutation was splicing site mutations (3.8%). The hematological system development and hematologic function were most highly enriched by the Ingenuity Pathway Analysis (IPA) as expected. CIRCOS plot analysis showed similar co-occurring pattern of recurrent mutations with previous reports. Hierarchical clustering analysis divided into four different groups according to the number of harboring mutations. In network analysis four distinct subgroups were observed ranging 21 to 3 gene network.

Conclusion:
Using whole exome sequencing approach, a catalog of recurrent mutations was successfully defined in the patients with NK-AML without FLT3/ITD mutation. This candidate list of novel mutations should be tested further for therapeutic target and prognostic marker in the patients with NK-AML.

Disclosures:
No relevant conflicts of interest to declare.
Single Cell Proteomics Reveals Novel Cytokine-Producing Function of Hematopoietic Stem and Progenitor Cells
Authors: Zhao Jimmy L. , Ma Chao , O'Connell Ryan , Rao Dinesh S. , Heath James , Baltimore David 
Published: October 2019

Abstract
Abstract 26
During infection, hematopoietic stem and progenitor cells (HSPCs) are called upon to proliferate and differentiate to produce more innate and adaptive immune cells to combat infection. Traditionally, HSPCs are thought to respond to depletion of downstream hematopoietic cells during infection. More recent evidence suggests that HSPCs may respond directly to infection and pro-inflammatory cytokines. However, little is known about the direct immune response of HSPCs and the molecular signaling regulating this response upon sensing an infection. In this study, we have combined transgenic and genetic knockout mouse models with a novel single cell barcode proteomics microchip technology to tackle these questions. We show that although long-term hematopoietic stem cells (HSCs) (defined by Lineage-cKit+Sca1+CD150+CD48-) do not secrete cytokines upon toll-like receptor (TLR) stimulation, short-term HSCs and multipotent progenitor cells (MPPs) (defined by Lineage-cKit+Sca1+, referred to as LKS thereafter) can produce copious amounts of cytokines upon direct TLR-4 and TLR-2 stimulation, indicating that LKS cells can directly participate in an immune response by producing a myriad of cytokines, upon a bacterial infection. Within the population of LKS cells we detect multiple functional subsets of cells, specialized in producing myeloid-like, lymphoid-like or both types of cytokines. Moreover, we show that the cytokine production by LKS cells is regulated by the NF-κB activity, as p50-deficient LKS cells show reduced cytokine production while microRNA-146a (miR-146a)-deficient LKS cells show significantly increased cytokine production.
As long-term HSCs differentiate, they start to gain effector immune function much earlier than we had originally anticipated. In light of this finding, we should start to view the stepwise differentiation scheme of HSCs, and perhaps all other stem cells, as a strategy to sequentially gain functional capacity, instead of simply losing stemness and self-renewal ability. The remarkable ability of LKS cells to produce copious amounts of cytokines in response to bacteria may provide some protective immunity during severe neutropenia and lymphopenia or in the early stage of HSC transplantation. This study further extends the functions of NF-κB to include the regulation of primitive hematopoietic stem and progenitor cells and provides direct evidence of the bacteria-responding ability of HSPCs through the TLR/NF-κB axis. The single cell barcode proteomics technology can be widely applied to study proteomics of other rare cells or heterogeneous cell population at a single cell level.

Disclosures:
No relevant conflicts of interest to declare.