S62 Poster Presentations/ Experimental Hematology 42 (2014) S23–S68

P1156 - CHARACTERIZATION OF IMMATURE MULTIPOTENT

PROGENITORS BY CELL TRANSPLANTATION IN FROG MODEL

Yuta Tanizaki1, Yoko Mochizuki2, Takato Otani2, and Takashi Kato1,2

1Department of Biology, School of Education, Waseda University, Tokyo, Japan;2Graduate School of Advanced Science and Engineering, Waseda University, Tokyo,

Japan

Hematopoiesis in animal models including non-mammalian zebrafish have demon-

strated that the system of hematopoiesis is largely conserved throughout vertebrate evo-

lution. We previously reported that hepatic cells of frog (X. laevis) differentiated into

megakaryocytic cells and thrombocytic cells in response to X. laevis thrombopoietin

(xlTPO). In this study, we identified the effects of xlTPO for immature hematopoietic

progenitors such as hematopoietic stem cells (HSCs). The hepatic cell suspension was

cultured in semi-solid culturewith xlTPO.After 24 days, multilineage colonies express-

ing various cell lineage markers appeared, and these colonies survived more than four

months. These cells differentiated tomature blood cells by hepatic cell-conditioned-me-

dia stimulation, demonstrating that the ability of these cells were multipotent in the cell

differentiation. Because inbred strains of X. laevis are not abundantly available, trans-

plantation studies in X. laevis are limited. We therefore developed the cell transplanta-

tionmodel to identify theHSCs inX. laevis. A left liver lobeofX. laeviswas resected and

cultured with xlTPO for 24 days. The cells were labeled by PKH26 and autologously

transplanted by intracardiac injection. After 30 days, PKH26+ cells detected in the sinu-

soid of the liver, and FACS analysis revealed that PKH26+ cells divided at least one time.

However it was hard to track the transplanted cells for long term by such fluorescent la-

beling, PKH26 labelled cellswere directly injected to the blastocele ofX. laevis embryos

at stage 8 which have no immune system. After 3 days of injection, PKH26+ cells were

detected all over the embryos, and part of transplanted cells flowed into blood vessels.

The cellular potency should be confirmed furthermore, but this unique transplantation

model developed here should have clues to resolve the functional evolution of TPO sys-

tem shared by hematopoietic progenitors.

P1157 - NUP98-PHF23 AND NUP98-HOXD13 ONCOGENES CONFER

ABERRANT SELF-RENEWAL POTENTIAL TO THYMOCYTE

PROGENITORS

Magalie Tardif, Bastien Gerby, and Trang Hoang

IRIC, Laval, Quebec, Canada

Background: In T-cell acute lymphoblastic leukemia (T-ALL), the homeobox (HOX)

A cluster is activated directly by chromosomal translocations or indirectly by two

fusion genes acting as upstream regulators, specifically NUP98-plant homeodomain

finger 23 (PHF23) and NUP98-HOXD13. Interestingly, HOXA genes control the self-

renewal activity of normal hematopoietic stem cells, suggesting a similar function in

cancer stem cells (CSCs). This hypothesis is reinforced by the recent observations

that two other oncogenic transcription factors in T-ALL, SCL and LMO1/2, confers

self-renewal potential to non-self-renewing thymocyte progenitors. Methods and

Results: Here we used two transgenic mouse models ectopically expressing

NUP98-PHF23 and NUP98-HOXD13 in hematopoietic stem/progenitor cells. Our re-

sults indicate that pre-leukemic thymocytes exhibit aberrant self-renewal potential as

assessed by their engraftment in the thymus after intravenous transplantation.

Furthermore, this activity is sustained over serial transplantations with 100-fold

expansion of the most primitive thymocyte populations, i.e. DN1 and DN2. At the

molecular level, engrafted thymocytes display a non-rearranged bTCR. Finally, puri-

fied early T-cell progenitors (ETP) and DN2 are the only cell types that can reconsti-

tute the thymus of transplanted mice, i.e. in cells in which bTcr genes are in a germ

line configuration. Conclusion and Relevance: We therefore conclude that NUP98-

PHF23 and NUP98-HOXD13 oncogenes reprogram ETP and DN2 thymocytes into

self-renewing progenitors. Our results open possibilities of targeted therapy towards

CSCs.

P1158 - WILMS’ TUMOR 1 MUTATION DRIVES DNA

HYPERMETHYLATION IN AML AND RESPONDS TO EZH2-INHIBITOR

Daniel Thomas1, Subarna Sinha3, Andrew Gentles1, Namyoung Jung2,

Andrew Feinberg2, David Dill3, and Ravindra Majeti1

1Department of Medicine, Stanford University, Stanford, California, USA; 2Johns

Hopkins School of Medicine, Baltimore, Maryland, USA; 3Department of Computer

Science, Stanford University, Stanford, California, USA

Acute myeloid leukemia (AML) is associated with widespread deregulation of DNA

methylation but the upstream signaling events responsible in the majority of patients

is poorly understood. Sequencing of purified cell populations in our laboratory and

others has revealed mutations in enzymes that regulate cytosine methylation (such

as TET2 and IDH), not only in bulk leukemia, but also in residual non-leukemic

stem cells at the time of diagnosis, suggesting that deregulation of DNA methylation

is an early step in the evolution of AML (Jan et al, 2012, Sci Transl Med 4:149). In

order to find additional mutations that may drive DNA hypermethylation we explored

the TCGA AML dataset using a novel application of Boolean implications (if-then

rules). Boolean implications identify strict L-shaped relationships (subset and mutual

exclusion) between the presence of a somatic mutation and CpG methylation after

discretization of beta values. We found that mutation in the Wilms’ Tumor 1

(WT1mut) gene (10% of normal karyotype AML) strongly linked to CpG hyperme-

thylation, similar to mutation in IDH2, but acting upon different genes. Expression of

mutant WT1 protein in AML cells induced differentiation block and DNA hyperme-

thylation as measured by 450K bead-chip arrays, validating WT1 mutation to be an

active driver of DNA hypermethylation. Strikingly, the pattern of methylation in both

WT1mut patient samples and induced cells was enriched for polycomb repressor

complex (PRC) 2 target genes (1.6E-87), implicating this chromatin-remodeling

complex in WTmut leukemogenesis. WT1mut AMLs showed marked repression of

PRC2 targets and treatment of purified WT1mut progenitors with PRC2 EZH2 inhib-

itor GSK-126 promoted differentiation. Our results reveal WT1mut as a novel driver

of DNA hypermethylation in AML that is mutually exclusive to IDH2 mutation.

Exploring mutation-specific methylation patterns by Boolean implications has utility

in finding upstream signaling pathways that are perturbed and druggable in leukemia.

P1159 - A NOVEL TUMOR SUPPRESSOR SH2B3 IN ACUTE

LYMPHOBLASTIC LEUKEMIA

Ying Cheng1, Kudakwashe Chikwavak1, Chao Wu1, and Wei Tong1,2

1Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; 2Perelman

School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania,

USA

B-precursor acute lymphoblastic leukemia (B-ALL) is the leading cause of cancer-

related death in young people and commonly has poor outcomes in adults. Dysregu-

lation of cytokine signaling frequently occur in high-risk ALL and correlates with

poor prognosis. Molecular insight into activation of oncogenic cytokine signaling

comes from the recent discovery of loss-of-function mutations in the adaptor protein

LNK (or SH2B3) and constitutive active JAK2 mutations in Ph-like pediatric B-ALL,

the subtype that lacks Philadelphia (Ph) chromosome translocation, yet displays a

similar activated kinase signature to Ph+ ALL. While clearly established at the ge-

netic level, mechanisms by which LNK mutation impacts ALL initiation or progres-

sion have not been established. Here we show that LNK synergizes with TP53 in

suppressing B-ALL development in mice. Lnk-/-Tp53-/- B-ALLs are highly aggressive

with 100% penetrance. More importantly, Lnk-/-Tp53-/- B blasts specifically exhibit

gene signatures of human Ph-like ALL. Mechanistically, we demonstrate that B pro-

genitors from preleukemic Lnk-/-Tp53-/- mice are hypersensitive to IL-7 and display

marked self-renewal ability in vitro and in vivo. Using a novel phosphoflow strategy

that enables interrogation of early transformative signaling events in B progenitors at

different stages, we find that Lnk deficiency confers a heightened sensitivity to IL-7

in activating pStat5 in pro B progenitors. Furthermore, Lnk-/-Tp53-/- B blasts show

constitutive activation of pAkt and pS6, but still depend on IL-7 for activation of

Stat5. Moreover, Lnk-/-Tp53-/- B blasts are less sensitive to JAK inhibitors than

normal B progenitors, consistent with the inability of JAK inhibition to eradicate

B-ALL in humans. High-risk B-ALLs that show genetic mutations in cytokine depen-

dent pathways and elevated cytokine signaling have poor survival rates. Our research

focuses on a novel B-ALL mouse model to decipher mechanisms underlying high-

risk ALL, and will enhance our understanding of this incurable disease and provide

novel insights into therapeutic strategies.