S66 Poster Presentations/ Experimental Hematology 42 (2014) S23–S68

P1172 - REGULATION OF HEMATOPOIETIC STEM CELL FATE BY

SPECIAL AT-RICH SEQUENCE BINDING PROTEIN 1

Britta Will1, Tihomira Todorova1, Amit Verma1, Ulrich Steidl1, and Ari Melnick2

1Albert Einstein College of Medicine, Bronx, New York, USA; 2Weill Cornell

Medical College, New York, New York, USA

Hematopoietic stem cells (HSCs) harbor extensive self-renewal capacity and the abil-

ity to commit to multilineage differentiation, thereby providing a life-long supply of

mature peripheral blood cells. Cell fate decisions of self-renewal or differentiation-

commitment are tightly regulated by the interplay of transcription factors instructing

HSCs to function in a highly plastic manner during tissue maintenance, regeneration

and repair. We have identified Special AT-rich sequence-binding protein 1 (Satb1) as

a novel regulator integrating different HSC functions by promoting quiescence and

repressing differentiation commitment through promoting symmetric self-renewal

divisions of HSCs. In a Satb1-deficient murine model system we determined

Satb1-dependent HSC function and molecular regulation utilizing congenic stem

cell transplantation, cell cycle and division analyses, in vivo tracing of HSC commit-

ment, asymmetric division assays, and clonogenic differentiation cultures. HSCs

lacking Satb1 display defective self-renewal, were significantly less quiescent, and

showed significantly increased and accelerated differentiation commitment caused

by reduced symmetric self-renewal and increased symmetric differentiation divi-

sions. Mechanistically, we found that Satb1 represses the expression of c-Myc and

Numb, two factors known to be involved in HSC activation. Global DNA cytosine

methylation analysis uncovered that Satb1 KO HSCs harbor premature differentia-

tion-specific DNA methylation changes which closely resemble the pattern found

in differentiation-committed multipotent progenitors. Our results also suggest that

Satb1 can be retained at certain loci during mitosis, ensuring their appropriate tran-

scriptional activity immediately upon cell division. Our findings uncover Satb1 as a

novel transcriptional regulator in HSCs which promotes quiescence and represses dif-

ferentiation commitment and thereby ensures the sustained self-renewal of HSCs.

P1173 - PROBING IKAROS FUNCTIONS IN B-ALL USING NOVEL MOUSE

MODELS

Matthew Witkowski1, Yifang Hu1, Priyanka Sathe, Dr.1, Mark McKenzie1,

Luisa Cimmino, Dr.2, Grace Liu1, Andrew Keniry, Dr.1, Gordon K. Smyth1,

Nicholas Huntington, Dr.1, and Ross Dickins1

1Walter and Eliza Hall Institute, Parkville, Victoria, Australia; 2NYU Department of

Pathology, New York, New York, USA

Loss-of-function mutations in the transcription factor IKAROS correlate with poor

prognosis in human precursor B cell acute lymphoblastic leukemia (B-ALL) and

are prevalent in the high-risk BCR-ABL+ and Ph-like B-ALL subtypes. Recent

studies using mouse models of Ikaros-deficient B-ALL have uncovered a multitude

of Ikaros-regulated genes, however the mechanisms underlying therapeutic resistance

in IKAROS-mutant B-ALL remain unclear. We have generated a novel, transgenic

mouse model that allows inducible, shRNA-mediated Ikaros knockdown or restora-

tion in normal lymphocytes and leukemias in vivo. Retroviral expression of a

BCR-ABL fusion oncogene co-operates with regulatable (tet-off) Ikaros knockdown

to significantly accelerate B-ALL development in mice, recapitulating a common ge-

netic interaction in high-risk pediatric B-ALL. Following leukemia transplant into

multiple recipient mice, we used RNA-seq to assess the dynamic transcriptional re-

sponses to acute, Dox-induced Ikaros restoration in B-ALL in vivo. Combining this

data with Ikaros ChIP-seq from normal B cell progenitors identified direct Ikaros-

regulated target genes in murine B-ALL. In complementary studies aimed at identi-

fying IKAROS target genes in human B-ALL, we have virally transduced primary

human CD34+ cord blood hematopoietic stem cells with vectors encoding BCR-

ABL and the leukemia-associated dominant negative IKAROS isoform IK6. These

cells display augmented proliferation in vitro and readily engraft a humanized mouse

model. These mice are currently being monitored for disease progression and will be

used to investigate IKAROS target genes based on transcriptional differences in B-

ALL resulting from BCR-ABL and IK6 co-expression relative to BCR-ABL expres-

sion alone. We anticipate that this humanized system will provide new insights into

the in vivo contribution of IKAROS deletion/mutation to human B-ALL genesis and

therapeutic resistance.

P1174 - DELINEATING KEY MEIS1 CIS-REGULATORY ELEMENTS

THROUGH CRISPR/CAS9 MEDIATED TARGETING

Ping Xiang, Patricia Rosten, Wei Wei, Pamela Hoodless, and R. Keith Humphries

BCCRC, Vancouver, British Columbia, Canada

MEIS1 is a critical regulator of normal hematopoiesis and its overexpression is impli-

cated in a wide range of leukemias. As demonstrated in our previous study (Xiang et

al., Leukemia, 2014, 28(2):433-6), in addition to the MEIS1 promoter, multiple cis-

regulatory elements reside within the 140 kb MEIS1 genomic locus and are potential

key components contributing to increased MEIS1 expression seen in leukemia. To

further validate these elements, we are exploiting the CRISPR/Cas9 system as a

powerful gene editing tool. As a first step we have targeted the Meis1 translational

start site using CRISPR/Cas9 system to insert a GFP-P2A-HA tag 5’ of the

MEIS1 ATG through homologous recombination. This design simplifies and enables

the measurement of endogenous MEIS1 expression level by using GFP level as a re-

porter as well as allowing MEIS1 protein detection with an anti-HA antibody. Using

P19 embryonic carcinoma cells as a model cell line, we obtained a 10% targeting ef-

ficiency. In wild type P19, Meis1 mRNA is up-regulated O 500 fold after 3 days of 1

micro M retinoic acid (RA) treatment. Similarly Clones bearing Meis1 targeted al-

leles showed dramatic upregulation of the GFP marker following RA induction

with O95% of cells positive by FACS analysis and high level expression of HA-

tagged Meis1 detected by Western blot. Using these Meis1-GFP reporter lines we

then used CRISPR/Cas9 editing to introduce deletions of the E1 enhancer region

located 2kb upstream of the Meis1 promoter. Clones bearing sequence verified dele-

tions of O50 bp of the E1 enhancer sequence had a significantly blunted response to

retinoic induction of Meis1 expression as evidenced decreased induction of GFP

expression and reduced Meis1 mRNA. These data provide important new functional

evidence of the regulatory role of the E1 enhancer region for Meis1 expression and

demonstrate the power of CRISPR/Cas9 mediated editing to introduce reporter genes

to transcription factors and dissect their regulation. Preliminary data indicate that this

system is also highly efficient for genome editing of human leukemic cell lines and

experiments are now in progress to extend analysis of key MEIS1 regulatory domains

to primary hematopoietic cells.

P1175 - TRANSPLANTABLE B-1 PROGENITOR CELLS ARE PRESENT IN

THE FETAL LIVER OF HEMATOPOIETIC STEM CELL DEFICIENT

EMBRYOS

Michihiro Kobayashi1, Williams Shelly1, Wooseok Seo2, Sasidhar Vemula1, Yan Liu1,

Ichiro Taniuchi2, and Momoko Yoshimoto1

1Indiana University School of Medicine, Indianapolis, Indiana, USA; 2RIKEN Center

for Integrative Medical Sciences, IMS-RCAI, Yokohama, Japan

The fetal liver is a major site containing progenitor cells to give rise to B-1 cells,

especially CD5+ B-1a cells. We previously reported that hemogenic endothelial cells

in the yolk sac (YS) and para-aortic splanchnopleure (P-Sp) (prior to hematopoietic

stem cell [HSC] emergence) produced B-1 progenitors in vitro that engraft in immu-

nodeficient neonates. Since the fetal liver is not a de novo site of hematopoietic cell

emergence and must be seeded by various hematopoietic progenitor/stem cells

derived from other hematopoietic tissues, B-1 progenitors in the fetal liver may be

a mixture of YS/P-Sp derived progenies, and not only derivatives of HSCs that

also seed the fetal liver at E11. Until now, it has been impossible to determine

whether HSC independent B-1 progenitor cells exist in the fetal liver. We have uti-

lized embryos genetically engineered to be devoid of HSC (CBFb-/-) but carrying a

transgene expressing CBFb under the TEK promoter (CBFb-/-:TEK-CBFb/GFP

mice). We have found the presence of transplantable B-1 and marginal zone B pro-

genitors in the fetal liver despite the absence of HSCs. YS/P-Sp cells from

CBFb-/-:TEK-CBFb/GFP embryos could produce B-1 progenitors in vitro. While

TEK is expressed in hemogenic endothelial cells, it is not expressed in B-1 progen-

itors, and transplanted CBFb-/-:TEK-CBFb/GFP fetal liver cells failed to expand/

maintain B-1 cells in the host peritoneum, unless CBFb expression was rescued by

retrovirus transduction.

Our data provide solid evidence for the presence of the first wave of HSC indepen-

dent B-1 progenitors in the fetal liver as postulated 20 years ago in the immune

layered theory of B lymphopoiesis.