TET2 deficiency in mesenchymal stem cells alters their ability to support hematopoietic stem cell proliferation and differentiation

Poster Presentations/ Experimental Hematology 53 (2017) S54-S136

them IM resistance. To investigate how HNRPDL acts, microarray data comparing HNRPDL silenced with control K562 cells were generated. The expression of preB-cell leukemia homeobox 1 (PBX1) was significantly decreased in HNRPDL silenced K562 and CML CD34+ cells (n53) compared with their control cells. Actinomycin D treatment impaired the stability of PBX1 upon HNRPDL silencing. The sequence analysis showed that PBX1 contained a HNRPDL consensus binding motif (5’- ACUAGC-3’) in its 3’-untranslated region (3’-UTR). A piece of 3’-UTR of PBX1 covering the consensus motif was sub-cloned in the 3’-end of a luciferase reporter vector, and it conferred elevated reporter activity in K562 cells than control vector (6-fold, P ! 0.05, n53). Importantly, PBX1 expression was higher in CML CD34+ cells (n510) compared with normal control cells (n57) significantly (4fold, P ! 0.05); and PBX1 silencing inhibited the growth of K562 cells, while PBX1 over-expression rescued the suppressed growth upon HNRPDL silencing. Taken together, we have revealed a novel HNRPDL-PBX1 pathway to promote the growth of CML cells.

3005 - ANALYSING THE IMPACT OF ERRORS IN SINGLECELL TRACKING EXPERIMENTS Thomas Zerjatke, Ingo Roeder, and Ingmar Glauche

3004 - INHIBITION OF THE PHOSPHATASE STS1 IN ACUTE MYELOID LEUKEMIA Jing Zhang, Olesya Vakhrusheva, and Christian Brandts

3006 - TET2 DEFICIENCY IN MESENCHYMAL STEM CELLS ALTERS THEIR ABILITY TO SUPPORT HEMATOPOIETIC STEM CELL PROLIFERATION AND DIFFERENTIATION Rong Li1, Zhigang Zhao2, Zizhen Chen1, Wen Xing1, Weiping Yuan1, Fengchun Yang3, Yuan Zhou1, and Mingjiang Xu3

Goethe University, Frankfurt am Main, Germany The class III receptor tyrosine kinases (RTKs) FLT3 and c-KIT play a central role in normal and malignant hematopoiesis. After binding to their natural ligands, these RTKs dimerize, become autophosphorylated and activate an intracellular signaling cascade. Negative RTK regulation by dephosphorylation, ubiquitination and degradation are equally important to prevent uncontrolled kinase activity and downstream signaling in normal and malignant hematopoiesis. Recently, we identified STS1/ STS2 as the phosphatases of FLT3 and c-KIT (Zhang J. et al, Stem Cell Reports, 2015). Loss of STS1/STS2 causes hyperphosphorylation of FLT3, enhanced AKT signaling and a strong proliferative advantage of hematopoietic stem and progenitors cells in STS1/ STS2 knockout mice. This indicates that STS1 and STS2 serve as negative regulators for wild-type FLT3 kinase activity and normal hematopoiesis. Internal tandem duplications of FLT3 (FLT3-ITD) are found in up to 30% of patients with acute myeloid leukemia (AML). Here, we investigated the function of STS1/ STS2 in FLT3-ITD induced malignant transformation. Surprisingly we found that loss of STS1 decreased the transforming capacity of the FLT3-ITD mutant, demonstrated by both reduced cell proliferation and colony forming ability. Importantly, the overexpression of STS1 enhanced FLT3-ITD induced cytokine independent cell growth in vitro in a phosphatase-dependent manner. Bone marrow transplantation results recapitulated the collaboration between FLT3-ITD and STS1/STS2, manifested by the increased engraftment of FLT3-ITD and STS1/STS2 co-expressing cells in vivo. Biological analyses showed prolonged half-life of FLT3-ITD, as well as increased downstream signaling in STS1 over-expressing cells. Mechanistically, the E3 ligase activity of CBL was negatively regulated by STS1. In summary, STS1/STS2 demonstrated two distinct roles in the context of FLT3wt and FLT3ITD. Our data suggests that the phosphatase inhibition of STS1/STS2 may both improve hematopoietic recovery of normal hematopoiesis and decrease the leukemic potential of FLT3-ITD expressing blasts. Inhibiting STS1 may provide a novel therapeutic in FLT3-ITD positive AML.

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TU Dresden, Dresden, Germany Time-lapse video microscopy is an increasingly popular method to study the temporal and spatial behaviour of single cells. It is used for a broad range of applications, e.g. analysing cell migration, proliferation properties, or clonal composition, or reconstructing the complete divisional history of cells that is represented by cellular genealogies. A large number of automated methods have been developed for segmenting and tracking single cells. Although these methods are increasingly sophisticated to cope with a broad spectrum of situations they inevitably produce errors in the reconstruction of cellular tracks. Using post-processing tools for the manual correction of automatically created tracks can reduce the number of errors. However, ambiguous situations can still occur that lead to different subjective decisions of individual raters in the assignment of cellular objects. The number of these ambiguous situations and hence the number of differences in the reconstructed cellular tracks depends on cell type specific properties like migration speed or proliferation rate, as well as on specific properties of the experimental setting like the spatial and temporal resolution of the image sequence or the density of seeded cells. To study the interrater variability in single-cell tracking we exemplarily use time-lapse movie experiments of in vitro cultured haematopoietic stem and progenitor cells. We analyse the impact of the observed variability on the reconstruction of cellular genealogies and consequently the reliability of statistical measures defined on these data structures. This analysis is complemented by computer simulations of cell cultures that allow us to comprehensively mimic a broad range of cell type specific and experimental properties. Specifically, we aim to quantify maximum error rates that are admissible to reliably measure particular statistical outcomes. These maximum admissible error rates can then be accounted for in the design of the experimental set- up and the choice of the cell tracking procedure.

1 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China, Tianjin, China (People’s Republic); 2Department of Hematology and Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, China, Tianjin, China (People’s Republic); 3Sylvester Comprehensive Cancer Center, Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, United States

TET2 is a member of TET family which can demethylate 5mC primarily to 5hmC as well as 5fC and 5caC. TET2 mutations frequently occur in myeloid malignancies. Although the role of TET2 in HSCs and ESCs are well demonstrated, it remains to be explored whether TET2 has important function in stem cells of mesenchymal origin. As a vital component of hematopoietic microenvironment, MSCs and their cellular progenies osteoblasts are essential for HSC maintenance and regulation of blood production. To detect the role of TET2 in MSCs, TET2 was knocked down in healthy individual bone marrow derived MSCs (shTET2 MSCs), and empty vector infected MSCs were used as control. We found that shTET2 MSCs obtained significant decrease 5hmC level. Importantly, shTET2 MSCs displayed enhanced selfrenewal, proliferation and osteoblast differentiation potential. The hematopoietic supportive ability of shTET2 MSCs was significantly higher than that of control MSCs. LTC-IC assay revealed that shTET2 MSCs resulted in a skewed differentiation of HSCs towards myeloid cells. Consistently, conditional deletion of Tet2 in MSCs using Prx1–Cre mice demonstrated enhanced proliferation ability, skewed lineage commitment towards osteoblast and enhanced hematopoietic supportive activity. Furthermore, whole genomic 5-hmC profiling and RNA-sequencing analysis showed that Tet2-/-MSCs exhibited dramatically decreased hydromethylation signatures and dysregulated gene expression. We confirmed by qPCR 11 dysregulated genes (5 upregulated genes Trp63, Fbn2, Sfrp2, Adamts12, Eya1 and 6 down-regulated genes

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Poster Presentations/ Experimental Hematology 53 (2017) S54-S136

Hes1, Wnt9a, Syk, Comp, IL-7, Nox4) which could lead to enhanced osteoblast differentiation potential and 5 down-regulated genes (Ddit4, Pmaip, Ifit3, Clmn and Egln) which could contribute to the increased proliferation capacity. Briefly, these results indicate that TET2 loss in MSCs enhances self-renewal, proliferation and osteoblast differentiation potential, which may in turn alters their ability to support HSC proliferation and differentiation. These studies provide further insights for the identification of additional potential therapeutic targets for patients with TET2 mutated myeloid malignancies.

3008 - TRANSCRIPTIONAL MECHANISMS THAT CONFER SELF-RENEWING POTENTIAL IN MLL-REARRANGED LEUKEMIA Akihiko Yokoyama1, Hiroshi Okuda1, and Satoshi Takahashi2 1

National Cancer Center, Tsuruoka Metabolomics Laboratory, Tsuruoka, Japan; Kyoto University Graduate School of Medicine, Tsuruoka, Japan

2

Cancer stem cells need to self-renew to propagate indefinitely. However, the transcriptional machinery that confers self-renewing potential is poorly understood. The eleven-nineteen leukemia (ENL) protein family, composed of ENL and AF9, is a common component of three transcriptional modulators: AF4/ENL/P-TEFb complex (AEP), DOT1L/AF10/ENL complex, and Polycomb repressive complex 1 (PRC1). Each ENL-containing complex associates with chromatin via distinct mechanisms, conferring different transcriptional properties including activation, maintenance, and repression. The mixed-lineage leukemia (MLL) gene often fuses with ENL and AF10 family genes in leukemia. These MLL fusion proteins directly associate with some of these ENL-containing complexes to induce leukemia. Therefore, it is thought that ENL-containing complexes are engaged in transcriptional regulation to maintain the stemness of cancer stem cell. However, the functional interrelationship among these various ENL-containing complexes in leukemic transformation remains largely elusive. Here, we show that MLL-ENL and MLL-AF10 constitutively activate transcription by aberrantly inducing both AEP-dependent transcriptional activation and DOT1L-dependent transcriptional maintenance, mostly in the absence of PRC1, to confer stemness to leukemia initiating cells. These results reveal a significant role of cooperative transcriptional activation mechanism of AEP and DOT1L in self-renewal and suggest a molecular rationale for the simultaneous inhibition of the MLL fusion/AF4 complex and DOT1L for better treatment efficacy of MLL-rearranged leukemia.

3007 - HSC-INDEPENDENT AND DEPENDENT PATHWAYS FOR GENERATING PERITONEAL INNATE B-1A CELLS Momoko Yoshimoto1, Michihiro Kobayashi1, and Mervin Yoder2

3009 - THE INFLUENCE TO HSC EMERGING BY PTPR INACTIVATION IN MOUSE EMBRYO Tomoko Yamada-Inagawa and Yuko Sekine

1

Chiba University, Chiba, Japan

University of Texas Health Science Center at Houston, Houston, United States; Indiana University, Indianapolis, United States

2

Innate immune cells such as tissue resident macrophages have been reported to develop independently from hematopoietic stem cells (HSCs) and persist into postnatal life. Similarly, the peritoneal innate B-1a cells have been postulated to be of fetal origin because they are not produced from adult bone marrow (BM) HSCs efficiently. Recent reports argue for B-1a cell potential in fetal liver (FL) HSCs by transplantation assay and barcoding system, and these data contradict other papers reporting that B-1a cells emerge in an HSC independent fashion. We have recently shown that the peritoneal innate B-1 progenitor cells are derived from hemogenic endothelial cells at a pre-HSC stage and develop in the fetal liver in the absence of HSCs. However, this evidence supports only the presence of HSC-independentB-1 progenitor cells. Therefore, we now ask 1) Are B-1a cells produced from fetal liver HSCs and 2) What is the main ontogenic origin of B-1a cells. We transplanted 4 populations among FL KSL population based on CD150 and CD48 expression into NSG neonates and adult BoyJ mice and confirmed the recent report that FL LT-HSCs do not give rise to B-1a cells efficiently; the CD150-CD48+ population repopulated B-1a cells most efficiently. In order to examine if the first HSC in the AGM region possesses B-1a cell potential, we transplanted 1 or 10 CD45+VEcad+c-kit+EPCR+ cells into the irradiated adult and neonatal NSG mice, and found that this population contained B-1 cell specific progenitor cells in addition to LT-HSCs that possess both B-1a and B-2 lineage potential. Taken together, B-1a cells seem to be derived from both HSC-independent and dependent pathways, although B-1a cell production from LT-HSCs is detectable in a very limited developmental time window.

In mouse, adult hematopoietic stem cells (HSCs) emerge in the AGM region at the middle ontogeny. These cells are found in clusters closely associated with the endothelial layer of the aorta. From the complete gene expression profiling of these HSCs and their precursors (*1), it was found that receptor protein tyrosine phosphatase beta/zeta (RPTPb/z) expressed in hemogenic endothelial cells during the endothelial to hematopoietic cell transition (EHT) process. During EHT the shape of endothelial cell changes from flat to round with migrating toward the lumen of the aorta. The EHT process is important for emerging of HSC during ontogeny. RPTPb/z is known of myelination of neuroaxis, and it is known to be involved in the regulation of selfrenewal and homing of adult HSC and differentiation to blood cells. However, the function of RPTPb/z is unclear in the developmental process of HSC emerging in embryo. To elucidate the function of RPTPb/z in the process of EHT, in vitro AGM tissue of embryo at E10.5 and E 11 was cultivated with ligand of RPTPb/z which disturbs its phosphatase activity. As results in this experiment, the number of each ckit+/CD31+ cells and CD31+ cells were decreased, and CD31-/ckit- cell population was increased. Because RPTPb/z is involved in migration and differentiation of cells, it is analysed whether RPTPb/z might transform the morphology of cell for EHT with regulating the expression of identical marker for endothelial cells or HSCs. *1 J.Exp.Med 212, 93-106, 2015