bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clonal Features of Hematopoietic Stem and Progenitor Cells in Aging
Authors: Sun Jianlong , Camargo Fernando D. 
Published: October 2019

Abstract
It is traditionally thought that Hematopoietic Stem Cells (HSCs) maintain blood homeostasis through long-term self-renewal and multilineage differentiation. This concept, however, is challenged by two recent studies in which the fundamental features of unperturbed hematopoiesis are evaluated by different approaches of lineage tracing. Both the kinetic analysis of HSC output by the Rodewald group and our clonal analysis with transposon barcoding suggest a dominant role of non-transplantable short-term HSCs and progenitors, but not the long-term HSCs, in driving native blood cell production. In addition, our longitudinal analysis of peripheral blood demonstrates extensive clonal succession in granulocyte production. These findings collectively suggest a distinct mechanism of native hematopoiesis that differs significantly from what has been learned in transplantation experiments. At the same time, they bring to light new questions regarding the ultimate fate of the progenitor population and the exact contribution of HSCs under normal physiological conditions. To address these questions, we examined clonal features of HSCs and progenitors in aged mice. Our results show a progressive reduction in clonal complexity and a concurrent increase in clonal stability when blood granulocytes are analyzed up to a hundred ten weeks after transposon barcoding. As time elapses, clonal overlapping between granulocytes and B cells become much more extensive, suggesting an increased tendency toward multilineage differentiation during aging. Analysis of stem and progenitor cells in bone marrow of aged mice reveals prevalent lineage output by multipotent progenitors (MPPs), whereas a lower fraction of HSC clones are found to produce mature progeny. While this overall pattern of differentiation is reminiscent of what has been observed in young and middle-aged animals, a two-fold increase in HSC clonal output was observed in these old mice, indicating their increased contribution to blood cell production. A comparison of clonal compositions in blood and marrow cell populations demonstrates an MPP origin of stable peripheral blood clones, and a smaller fraction of these clones can even be traced back to HSCs. These observations hence suggest extensive self-renewal and asymmetric cell division of these two cell populations in aging. Taken together, our results indicate that the aged hematopoietic system is characterized by reduced clonal complexity, increased clonal persistence, and HSC activation. The higher propensity to self-renewal during aging may also explain the elevated risk of malignant transformation in the elderly population.

Disclosures
Camargo: Cell Signaling Technologies: Consultancy; Vital Therapies: Consultancy.
The Combination of ATRA and Dasatinib for Differentiation Therapy in Acute Myeloid Leukemias with IDH Mutations
Authors: Boutzen Helena , Saland Estelle , Cathebras Mathilde , Larrue Clément , Farge Thomas , Serhan Nizar , De Toni Fabienne , David Marion , Gales Lara , Castelli Florence , Junot Christophe , Delabesse Eric , Veronique Demas , Portais Jean-Charles , Ouerfelli Ouathek , Guangli Yang , Vergez Francois , Recher Christian , Sarry Jean-Emmanuel 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired differentiation programs due to recurrent mutations, amongst which IDH mutations occur in 15% of AML patients. Here, we show both in vitro as well as in a xenografted mouse model,
that clinically achievable doses of ATRA are sufficient to achieve a terminal granulocytic differentiation in primary AML samples and in AML cell lines harboring IDH1-R132H mutation. There is no effect at this concentration on the WT controls. This is associated with reduction of both proliferation and colony formation, and further leads to apoptosis, thereby improving overall survival of mutant xenografted mice.
We further showed, through transcriptomic and western blot analysis, that specific ATRA sensitivity is due to overexpression and activation of C/EBPα in the presence of IDH1-R132H mutation. This primes blasts into myeloid differentiation.
Moreover, IDH1 R132H mutation also reduces LYN activation, and thus, also sensitizes to clinically achievable doses of dasatinib, a LYN inhibitor. As ATRA induces a brief LYN activation, which transiently reduces ATRA activity, its combination with dasatinib synergistically increases differentiation. In vivo, the combination of ATRA and dasatinib reduces tumor growth of mutant xenografted mice.
The combination ATRA and dasatinib might also be considered for other IDH mutations that produce 2-hydroxyglutarate, since treatment with the mutant-specific oncometabolite (eg. 2-hydroxyglutarate) sensitizes AML cells to ATRA and dasatinib-induced differentiation.
Finally, ATRA also reduces BCL2 expression specifically in the presence of IDH1 R132H mutation. Since it has been shown that IDH mutations increase BCL2 dependence in leukemic cells, our results identified a subgroup of patients that is likely to respond to pharmacologic concentrations of ATRA.
To conclude, our data provide the preclinical rationale for investigating the use of the combination ATRA and dasatinib in a subgroup of patients who carry IDH1 R132H mutation, in clinical trials. The addition of a BCL2 inhibitor such as ABT-199 would also be considered.

Disclosures
Off Label Use: ATRA and dasatinib for treatment of non APL AML. Recher:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Chugai: Research Funding.
Evidence for the Role of Biologically Active Exosomes in Acute Myeloid Leukemia Relapse
Authors: Boyiadzis Michael , Hong Chang Sook , Whiteside Theresa L 
Published: October 2019

Abstract
Introduction: Despite of the notable achievements of multi-modality treatments in patients with acute myeloid leukemia (AML), leukemic relapse remains a formidable clinical challenge. Studies in AML patients have shown that tumor-induced impairments of immune cells contribute to leukemia progression. One recently identified immunosuppressive mechanism operating in AML is the release by leukemia blasts of virus-size (30-100nm) membrane-bound vesicles called exosomes. We have reported that plasma of patients with newly-diagnosed AML prior to any therapy contains higher levels of exosomal proteins than that of normal donors. AML exosomes carry an immunosuppressive cargo, including membrane-associated TGF-β1, MICA/MICB and markers of myeloid blasts. We hypothesize that persistence of these exosomes may contribute to the high relapse rates seen in AML.
Methods: Venous blood (20-50 mL) was obtained from patients newly diagnosed with AML prior to any treatment, from AML patients who achieved CR following initial therapy and from age-matched healthy volunteers (NC). Exosome fractions were isolated from plasma by using mini-size exclusion chromatography with Sepharose 2A. Exosome protein levels, numbers and size of exosomes (qNano) and their morphology by transmission electron microscopy were determined. Exosomes were characterized by western blots for expression of exosome markers, Tsg101 and CD81, and myeloid cell-surface markers associated with AML, interleukin-3 receptor alpha chain (CD123) and C-type lectin-like molecule-1 (CLL-1), CD44 and CD96. Isolated normal human NK cells were co-incubated with AML exosomes and multiparameter flow cytometry was used to monitor changes in expression levels (mean fluorescence intensity) of NKG2D on NK cells.
Results: Exosome fractions isolated from AML patients' plasma at diagnosis (n=16) had higher mean protein content than fractions obtained from NC plasma (96 vs 37 µg protein/mL plasma, p=0.03). In exosome fractions isolated from plasma of AML patients in CR (n=13) protein levels remained elevated (mean value=120 µg protein/mL plasma) at the time when leukemic blasts were undetectable in the bone marrow by conventional hematopathological methods. AML exosomes in patients in CR carried blast markers, CD123, CLL-1, CD44, CD96 and were enriched in TGF-β1. Co-incubation of these exosomes with activated NK cells resulted in down-regulation of NKG2D expression with a concomitant reduction of NK-cell cytotoxicity. Ten/13 AML patients in CR whose plasma exosome levels remained elevated eventually relapsed.
Conclusion: The persistently elevated levels of biologically-active TGF-β1+ exosomes carrying leukemia blast markers in plasma of AML patients in CR impair anti-leukemia immune responses and might contribute to leukemia relapse.

Disclosures
No relevant conflicts of interest to declare.
Aberrant Expression of Homeobox Gene SIX1 in Hodgkin Lymphoma
Authors: Nagel Stefan , Meyer Corinna , Kaufmann Maren , MacLeod Roderick , Drexler Hans G. 
Published: October 2019

Abstract
In Hodgkin lymphoma (HL) we recently identified deregulated expression of homeobox gene MSX1 which physiologically controls development of the embryonal neural plate border region. This region forms the neural crest and placodes which in turn give rise to fundamental structures and functions of the head including jaw and sensory organs. Here, we examined in HL another homeobox gene, SIX1, an additional regulator of this embryonal region mediating differentiation of placodal precursors. In silico data (GSE12453) show aberrantly activated SIX1 in 12 % of HL patient samples, indicating a pathological role in a subset of this malignancy. In addition, SIX1 expression was detected in HL cell lines which then were used as models to reveal regulators and target genes of this basic developmental factor. Cytogenetics and quantitative genomic PCR revealed copy number gains of the SIX1 locus at chromosomal band 14q23 correlating with enhanced expression while chromosomal translocations were absent. Moreover, comparative expression profiling data and pertinent gene modulation experiments indicated that the WNT-signalling pathway activates and transcription factor MEF2C suppresses SIX1 expression. MAPK7/ERK5 and HDAC9 phosphorylate and deacetylate MEF2C, respectively, boosting or constraining its inhibitory capacity. MEF2C, MAPK7 and HDAC9 show aberrant expression levels in both HL cell lines and patient samples, collectively restricting the inhibitory activity of MEF2C. Genes encoding the transcription factors GATA2, GATA3, MSX1 and SPIB - all basic lymphoid regulators - were identified as targets of SIX1 in HL, contributing to deregulated B-cell differentiation in this malignancy. In addition, cofactors EYA1 and TLE4 contrastingly mediated activation and suppression of SIX1 target gene expression, respectively. Thus, the protein domain interfaces may represent therapeutic targets in SIX1-positive HL subsets to prevent cofactor interactions and subsequent target gene regulation. Collectively, our data reveal a gene regulatory network where SIX1 centrally deregulates lymphoid differentiation. Additionally, our data strengthen the emerging concordance of lymphopoiesis/lymphomagenesis and the neural plate border region ontogeny.

Disclosures
No relevant conflicts of interest to declare.
CD30/CD16A Tandab AFM13-Induced Target Cell Lysis By NK-Cells Is Enhanced By CD137 Co-Stimulation and Blocking PD-1
Authors: Zhao Xing , Rajasekaran Narendiran , Reusch Uwe , Marschner Jens-Peter , Treder Martin , Kohrt Holbrook 
Published: October 2019

Abstract
Introduction: AFM13 is a CD30/CD16A bispecific tetravalent TandAb antibody that recruits and activates NK-cells by specific binding to CD16A for targeted lysis of CD30+ tumor cells. Given promising clinical activity and safety profile of AFM13 and proof-of-mechanism demonstrating dependence on the immune response, potential synergy of AFM13 and checkpoint modulators was evaluated.
Methods: Efficacy of AFM13 alone or in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies was assessed by in vitro cytotoxicity assays with human PBMCs or enriched NK-cells and CD30+ target cells as well as patient-derived xenograft in vivo models with autologous PBMC.
To evaluate NK-cell-mediated lysis of CD30+ lymphoma cell lines, 4 hour cytotoxicity assays were performed with PBMCs or enriched NK-cells as effector cells in the presence of suboptimal concentrations of AFM13 alone, and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies. For the in vivo model tumor fragments derived from surgical specimens of newly diagnosed patients with CD30+ Hodgkin Lymphoma were xenografted (PDX) in immuno-deficient mice. After 28 days mice were reconstituted with autologous patient-derived PBMC and treated with AFM13 alone and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies weekly for a total of three weeks. Tumor size, tumor-infiltrating human lymphocytes and intra-tumoral cytokines were evaluated on day 58.
Results: AFM13 as a single agent at suboptimal concentrations induced effector-to-target cell-dependent lysis of CD30+ lymphoma cells up to 40% using enriched NK-cells as effector cells in a 4 hour in vitro assay. Immune-modulating antibodies alone mediated substantially lower lysis (<25%). However, the addition of anti-PD-1 or anti-CD137 to AFM13 strongly enhanced specific lysis up to 70%, whereas the addition of anti-CTLA-4 to AFM13 showed no beneficial effect. The most impressive increase of efficacy was observed when AFM13 was applied together with a combination of anti-PD-1 and anti-CD137. In vivo, reduction of tumor growth was observed when AFM13 and anti-PD-1 were used as single agents or when AFM13 was combined with anti-CD137. Synergy was most impressive in these PDX models for the combination of AFM13 and anti-PD-1 which led to a very strong reduction of tumor size. Of note, reduction of tumor growth was strongly correlated with infiltrating NK- and T-cells and intra-tumoral cytokines.
Conclusions:
The combination trials performed with companion intra-tumoral assessment of lymphocytes and cytokines may enhance the efficacy of AFM13 in patients. This may be explained by a potential cross-talk between NK-cells and T-cell which was enhanced when AFM13 was used in combination with checkpoint modulators.

Disclosures
No relevant conflicts of interest to declare.
Pathway-Directed High Throughput Drug Screen Identifies PI3K Inhibitors That Synergistically Potentiate Anti-Tumor Activity of HDAC Inhibitors in Mycosis Fungoides and Sezary Syndrome
Authors: Ai Weiyun , Yang Chen-Yen , Faraj Razan , Rakhshandhroo Taha , Afghani Shervin , Pincus Laura , Bandyopadhyay Sourav , McCormick Frank 
Published: October 2019

Abstract
Introduction and Objectives: Mycosis fungoides and Sezary syndrome (MF/SS) represent a group of heterogeneous diseases. Recent studies demonstrated dysregulation of several signaling pathways in MF/SS, including PI3K/AKT, JAK/STAT, RAS and NFkB pathways. We performed a high throughput drug screen to determine the potential of novel agents targeting these pathways for the treatment of MF/SS.
Materials and Methods: We compiled a libraryof 94 compounds targeting pathways known to be relevant in cancer biology. These included kinases involved in growth factor receptor signaling, HDACs, proteasome, DNA repair and regulators of apoptosis. The compounds were screened for anti-proliferative activity against four MF/SS cell lines in high throughput proliferation assays. Selected hits were further studied in xenograft models of MF/SS and in primary T cell lymphomas. Promising candidates from different classes were also tested in combination therapy assays using a matrix block method across dose gradients of each compound designed to detect synergistic activities.
Results: From the high throughput screen, we identified 14 compounds with anti-proliferative activity in MF/SS, including multiple inhibitors of the PI3K pathway. PI3K inhibitors emerged as preliminary hits in this screen and secondary validation assays confirmed the class effect of PI3K inhibitors. From this class, the PI3K inhibitor BKM120 was selected for in vivo studies. In a xenograft model of MF, BKM120 exhibited striking anti-tumor activity measured by a marked suppression of tumor growth and prolonged survival of tumor-bearing mice compared with vehicle control. In a search for even more effective combination therapies, we identified that BKM120 and the HDAC inhibitor class of compounds exhibit synergistic anti-proliferative effects in MF/SS tumor cells. Each of three HDAC inhibitors including LBH, Romidepsin and Vorinosat showed synergistic activity with BKM120, most evident at the GI50 concentrations of each drug, and apparent in both growth inhibition and apoptotic assays.
Conclusion: BKM120 is highly active in preclinical models of MF/SS. Furthermore, it synergistically potentiates the effect of HDAC inhibitors against MF/SS tumor cells. These are highly promising approaches for the treatment of MF/SS and warrant clinical investigation.

Disclosures
No relevant conflicts of interest to declare.
Increased Expression of Circulating Mir-22 in the Peripheral Blood of Patients with Myelodysplastic Syndromes Was Associated with Poorer Prognosis
Authors: Meng Yuesheng , Hua Fanli , Li Ying , Gao Song 
Published: October 2019

Abstract
The molecular pathogenesis of myelodysplastic syndromes (MDS) has not been completely elucidated. Deregulation of expression of some microRNA has been implicated in hematological disorders including MDS. An oncogenic role for miR-22 was recently suggested in MDS in which the expression of miR-22 was increased. However, there were corroborations showing that it could be a tumor suppressor in acute myeloid leukemia. In this study, we examined the expression levels of circulating miR-22 in the plasma of patients with MDS and evaluated its significance in clinical context. The diagnosis of MDS was made according to the WHO classifications and subtypes included 8 cases of RA, 14 cases of RCMD and 6 cases of RARS/RCMD-RS, 10 cases RAEB1 and 7 cases RAEB2. Twenty cases of healthy donors were enrolled as normal control. Written informed consent for sample collection was obtained from all subjects enrolled. EDTA-anticoagulated peripheral blood samples were collected and centrifuged. Circulating microRNAs were purified from the plasma with miRNeasy serum kit (Qiagen). Expression of miR-22 was reverse- transcripted and measured with stem-loop real time quantitative polymerase chain reaction assay. First of all, we proved that the expression of miR-22 was detectable in the plasma of patients with MDS as well as of healthy donors. Our result showed that the expression levels of circulating miR-22 in MDS patients were higher than that in healthy donors (medians 1.360 vs 1.000, P<0.05), among which 13 patients (37.1%) showed marked increase of miR-22 expression (>2 times). Due to the high heterogeneity of MDS and relatively smaller sample size, the subtypes were grouped into two major categories based on IPSS: the low-risk group (including RA, RCMD and RARS/RCMD-RS, totally 18 cases) and the high-risk group (RAEB1 and RAEB2, 17 cases in total). Relative levels of circulating miR-22 were higher in the high-risk group than in the low-risk group (medians= 1.081 vs 2.317, P<0.05). A follow-up study revealed an association of the expression levels of miR-22 with prognosis. Patients with increased expression of miR-22 had poorer clinical response (Chi-square analysis, P<0.05) and lower overall survival rate (Kaplan-Meier analysis, P<0.05). We compared the levels of miR-22 in the plasma with that of mononuclear cells of bone marrow in some MDS patients and found that the relative levels of miR-22 in the plasma and bone marrow cells were not directly co-related (P>0.05), indicating it could be an independent prognostic factor. In summary, this study confirmed an oncogenic presence of miR-22 in MDS. Given the fact that there is a lack of blastic cells in the bone marrow of patients with low-risk MDS but their plasma or serum is easy to obtain, the detection of circulating miR-22 may be of value as a molecular marker in the prognosis of MDS.

Disclosures
No relevant conflicts of interest to declare.
Brca1 Deficiency Causes Bone Marrow Failure and Spontaneous Hematologic Malignancies in Mice
Authors: Vasanthakumar Aparna , Arnovitz Stephen , Marquez Rafael , Lepore Janet , Rafidi George , Davis Elizabeth M. , Asom Anase , Weatherly Madison , Neistadt Barbara , Duszynski Robert , Vardiman James W. , Le Beau Michelle M. , Godley Lucy A. , Churpek Jane E. 
Published: October 2019

Abstract
Background: Fanconi anemia (FA) is caused by mutations in one of seventeen genes that make up the FA DNA double strand break (DSB) repair pathway. Recently, two individuals with biallelic germline BRCA1 mutations, each consisting of one null and one hypomorphic mutation, were identified and noted to have features consistent with FA, including congenital anomalies and increased chromosomal breakage of lymphocytes on exposure to diepoxybutane (Domchek et al. Cancer Discov. 2013 Apr; (4):399-405; and Sawyer et al. Cancer Discov. Epub. 2014 Dec 3.), adding BRCA1 as the newest FA gene. However, neither patient developed bone marrow failure (BMF), making the bone marrow effects of BRCA1 deficiency still unclear.
Methods: To test the hypothesis that Brca1 is also essential in hematopoiesis, we developed a conditional mouse model with Mx-1 Cre-mediated Brca1 deletion and examined the effects of Brca1 deficiency on hematopoiesis in this model.
Results: At baseline, Brca1-/- mice have macrocytic anemia and leukopenia. Further, by 6 months of age, 30% and 50% of the Brca1-/- mice develop spontaneous BMF or hematologic malignancies (HM), respectively. Brca1-/- mice develop a diverse range of HM, including T-cell lymphomas and acute myeloid leukemias, suggesting a defect in an early hematopoietic progenitor population. Methylcellulose colony forming assays also demonstrate a defect in progenitor cell function with Brca1-/- bone marrow cells forming fewer colonies (44.4±31.9) than Brca1+/+ cells (200.3±30.5, p=0.004) at baseline, and show FA-like hypersensitivity to the DNA cross-linking agent, Mitomycin C (MMC) (mean colony survival % at 10 nM MMC 40% versus 82% and at 50 nM 1% vs 56%). Spectral karyotyping of bone marrow cells from mice that developed BMF demonstrated chromatid exchanges and breaks. Similarly, multiple chromosomal translocations were seen in the myeloid leukemia cells, implicating genomic instability in the pathogenesis of these disorders.
Conclusions: Taken together, our results show that loss of Brca1 in murine bone marrow causes hematopoietic defects and MMC sensitivity similar to that seen in humans with FA, providing strong evidence that Brca1 is critical for normal hematopoiesis and that Brca1 is a bona fide FA gene. This novel mouse model provides the opportunity to gain functional insight into the key stage(s) of hematopoiesis that require Brca1 and the effects of Brca1 haploinsufficiency, as seen in humans, on hematopoiesis. Further, as nearly all of the single gene FA mouse models to-date have failed to recapitulate the bone marrow phenotype of human FA, this model will be critical for deepening our understanding of the pathogenesis of FA.

Disclosures
No relevant conflicts of interest to declare.