S17Oral Short Talk Presentations/ Experimental Hematology 42 (2014) S13–S21

O1017 - ROLE OF TP53 IN THE OXIDATIVE STRESS RESPONSE OF

ERYTHROID PRECURSORS

Michelle Carter, Rick Shimshock, Ashley Kramer, and Troy Lund

Pediatric Blood and Marrow Transplant, University of Minnesota, Minneapolis,

Minnesota, USA

Oxidative stress plays a key role in acute and chronic anemia. Erythroid precursors

and mature red cells have increased sensitivity to oxidative stress. As many of the

central genes in this pathway are necessary for life, it has been difficult to create an-

imal models to explore genetic components. Our zebrafish model permits pro-oxidant

exposure early in hematopoietic development, and gata1DsRed transgenic animals

allow clear identification of erythroid precursors, allowing us to examine the specific

effects of oxidative stress on these cells. After 72 hours of exposure to the strong pro-

oxidant naphthol, embryonic zebrafish up-regulated anti-oxidant genes including

hif1a, nrf2, fth1a, txn, and hmox1. Promoter analysis revealed tp53 binding sites

within 4 kb of the first exon in each gene. The tumor-suppressor protein tp53 is

thought to act primarily as a transcription factor. We showed by qRT-PCR that naph-

thol induced tp53 expression in zebrafish embryos 3-fold over baseline. The zebrafish

mutant tp53M214K contains a point mutation in the DNA binding region of tp53

eliminating this activity. Compared to wild-type, tp53M214K fish were highly sensi-

tive to pro-oxidants, showing a 3.2-fold increase in anemia and cardiac edema

(n5100/group, p!0.01); a 3-fold decrease in the number of hemoglobin-staining

cells (n530/group, p!0.01); and a doubling of generated ROS. The gata1DsRed1

zebrafish has labeled erythroid precursors, and we were able to specifically measure

a dose responsive induction of ROS in erythroid precursors after naphthol exposure.

Furthermore, gata1DsRed1 animals harboring tp53M214K showed a 20-fold increase

in ROS after naphthol exposure (versus tp53+/+ animals, n 5 10/group; p ! 0.01)

and displayed apoptosis as measured by flow cytometry. In conclusion, we show

that amongst the many functions of tp53, providing an anti-oxidant response is

also a mechanism though which erythroid precursors metabolize ROS after exposure

to pro-oxidants.. Understanding the mechanisms by which the anti-oxidant response

is regulated will assist in the discovery of more effective drug-able targets to treat

oxidative stress accompanying acute and chronic anemia.

O1018 - RNA EDITING IS THE PRIMARY IN VIVO FUNCTION OF ADAR1

AND IS ESSENTIAL FOR HEMATOPOIESIS

Brian Liddicoat1, Robert Piskol2, Jin Billy Li2, Alistair Chalk1, Peter Seeburg3,

Miyoko Higuchi3, Carl Walkley1, and Jochen Hartner4

1St Vincent’s Institute, Fitzroy, Victoria, Australia; 2Stanford University, Stanford,

California, USA; 3Max Planck Institute, Heidelberg, Germany; 4TaconicArtemis,

Cologne, Germany

The role of RNA and its regulation is becoming increasingly appreciated as a vital

component of hematopoietic development and disruptions to these pathways can

lead to blood diseases such as myelodysplastic syndromes and leukemia. RNA edit-

ing by ADAR1 is a form of post-transcriptional modification which converts adeno-

sine to inosine (A-to-I) in RNA. Germline deletion of murine ADAR1 resulted in

embryonic lethality at E12 from failed hematopoiesis and an upregulation of inter-

feron (IFN) stimulated genes (ISGs). Recently, non-editing roles for ADAR1 have

been implicated in transcription and miRNA processing. However, it is unclear

whether A-to-I RNA editing is the essential function of ADAR1. To determine the

role of A-to-I editing by ADAR1, we generated an editing dead knock-in allele of

ADAR1 (Adar1E861A). Mice homozygous for Adar1E861A allele died in utero at

E13.5. The fetal liver (FL) was small and had significantly lower cellularity than con-

trols. Analysis of FL hematopoiesis revealed a decrease in hematopoietic stem cells

(HSCs), all mature leukocytes and a severe loss of erythrocytes due to apoptosis.

Restricted expression of ADAR1E861A in adult HSCs also resulted in failed hema-

topoiesis. To understand the mechanism through which ADAR1 mediated A-to-I ed-

iting regulates hematopoiesis, RNA-seq was performed. Gene expression profiles

showed that a loss of ADAR1 mediated A-to-I editing resulted in an upregulation

of ISGs, as observed in ADAR1 null mice. Analysis of A-to-I mismatches in

RNA-seq data revealed 2,050 ADAR1-specific editing sites. Whilst no editing events

could directly explain the hematopoietic defect, we identified a cluster of 118 A-to-I

mismatches in the 3’UTR of Klf1, which may be important for the erythroid require-

ment of ADAR1 mediated A-to-I editing. These results demonstrate that A-to-I edit-

ing by ADAR1 is the primary in vivo function of ADAR1 and is essential for the

maintenance of hematopoiesis. Furthermore, ADAR1 mediated A-to-I RNA editing

is required for suppressing the IFN response in hematopoietic cells.

O1021 - SUPPRESSION OF NOTCH1-DEPENDENT T-CELL LEUKEMIA BY

b-CATENIN INHIBITION

Christos Gekas, Teresa d’Altri, Lluis Espinosa, and Anna Bigas

IMIM, Barcelona, Spain

T-cell acute lymphoblastic leukemia (T-ALL) is frequently associated with activating

mutations of Notch1, which however are not sufficient to recapitulate the disease.

Here, we investigated the role of b-catenin as a co-operating factor for Notch1 in

T-ALL, prompted by the observations that b-catenin and Notch1 share the regulation

of functions associated with cancer and stemness. Utilizing a small molecule

(PKF115-584), which inhibits the transcriptional activity of b-catenin, we show

that Notch and Wnt pathways are synergistically required for T-ALL cell growth.

We assessed the effect of b-catenin in T-ALL in vivo by using two independent ge-

netic models dependent on Notch1 activation; 1) retrovirally induced intracellular

domain of Notch1 (N1IC) in bone marrow cells from b-cateninfl/fl:VavCre+ mice,

or 2) triple-transgenic N1ICLSL:b-cateninfl/fl:VavCre+ fetal liver (FL) cells. In the

presence of b-catenin, N1IC rapidly induced T-cell leukemia, whereas deletion of

b-catenin significantly abrogated T-ALL in both systems. In vivo treatment of

mice receiving N1IC+b-catenin+/+VavCre+ FL cells with PKF115-584 showed a

marked reduction in T-ALL growth and improved disease-free survival without

affecting normal hematopoiesis. Notably, Notch activation caused an expansion of

Lin-sca1+ckit+ (LSK) CD48+CD150- hematopoietic stem cells in the FL indepen-

dently of b-catenin, suggesting both b-catenin dependent and independent functions

of Notch1 in stem cells and leukemia. Gene expression analyses of FL LSK cells sup-

port this notion, revealing categories of genes that in the absence of b-catenin fail to

be differentially expressed by Notch, as well as b-catenin-independent genes down-

stream of Notch1. Interestingly, most classical Notch1 targets (e.g. Hes1, Dtx1, Hey1,

Hey5, Dll1) fall into the latter category, whereas those in the former likely constitute

the oncogenic program of Notch1:b-catenin. Together, these results uncover impor-

tant heretofore unrecognized roles of b-catenin in Notch1-driven T-ALL and could

constitute a novel avenue of treatment.

O1022 - CDKN1A (P21) IS REQUIRED FOR QUIESCENCE, THERAPEUTIC

RESISTANCE AND CLONAL EVOLUTION OF PRE-LEUKEMIC STEM

CELLS

Cedric Tremblay, Jesslyn Saw, and David Curtis

Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria,

Australia

T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous malig-

nancy, with 20% of patients dying from resistant or relapsed disease. Recent studies

support the concept that cells responsible for relapse frequently develop from a small

population of ancestral or pre-leukemic stem cells (pre-LSCs) that give rise to clonal

heterogeneity. Using the Lmo2 transgenic mouse model, we have shown previously

that these pre-LSCs have long-term self-renewal potential and resistance to high dose

radiation. To determine if quiescence is an important property of pre-LSCs, we first

used the doxycycline inducible H2B-GFP transgenic mouse model to show the exis-

tence of rare (!1%) pre-LSCs that cycle less than once a month in Lmo2 transgenic

mice. We then used mice lacking the cyclin-dependent kinase inhibitor Cdkn1a (p21)

to address the importance of quiescence in therapeutic resistance. Absence of p21 had

no effect on the formation of pre-LSCs but reduced the proportion of quiescent pre-

LSCs in vivo. Importantly, pre-LSCs lacking p21 were sensitive to killing by radia-

tion therapy. Finally, we aged cohorts of mice to determine the role of p21 in the

clonal evolution of pre-LSCs. Remarkably, absence of p21 completely abrogated

the development of T-ALL from pre-LSCs. These results provide the most

convincing in vivo evidence that p21 is required for the quiescence, therapeutic resis-

tance and clonal evolution of pre-LSCs. Understanding how p21 controls the fate of

pre-LSCs may provide new therapeutic avenues for improving cure rates in T-ALL.