S55Poster Presentations/ Experimental Hematology 42 (2014) S23–S68

P1128 - DPP4 TRUNCATION ALTERS THE SIGNALING AND

FUNCTIONAL ACTIVITY OF GM-CSF AND LL-3 IN NORMAL AND

LEUKEMIC HEMATOPOIETIC CELLS

Heather O’Leary, Charlie Mantel, Man Ryul Lee, Xianyin Lai, Scott Cooper,

Giao Hangoc, H. Scott Boswell, and Hal Broxmeyer

Microbiology and Immunology, Indiana University School of Medicine,

Indianapolis, Indiana, USA

Dipeptidylpeptidase 4 (DPP4) enzymatically cleaves select penultimate amino acids of

proteins, resulting in functional alterations. We published that the number of cytokines,

chemokines and growth factors that have putative DPP4 truncation sites were dramat-

ically underestimated. Functional andmechanistic roles of full length (FL) versusDPP4

truncated (T) factors, as well as the ability of DPP4 truncated proteins to induce

signaling that full length factors cannot, have not been investigated and may have yet

unappreciated clinical application. Utilizing the human, factor-dependent TF-1 cell

line, as well as primary samples from human cord blood (CB) and patients with Acute

Myeloid Leukemia (AML), we observed that DPP4 truncation of GM-CSF and IL-3 re-

sults in decreased colony stimulating factor (CSF) activity in normal and malignant he-

matopoietic progenitor cells (HPC). Receptor binding studies confirmed that both

DPP4 truncated GM-CSF (TGM) and IL-3 (T3) have enhanced receptor affinity and

can compete to blunt the receptor binding of both full length GM-CSF (FLGM) and

IL-3 (FL3). In vivo studies demonstrated that either exogenously added TGMorT3 sup-

pressed the effect of exogenously added FLGM or FL3 on progenitor cell numbers per

femur and diminished HPC cycling. Mechanistic investigation of miRNA, phosphory-

lation, and global protein analysis showed that both TGM and T3 were able to induce

unique signaling that the FLGM and FL3 did not induce, as well as signaling that over-

lappedwith their full length counterparts. Additionally, treatment with a 1:1 ratio of the

FL/T proteins resulted in both distinctive, and common, signaling compared to that de-

tected with treatment of only FL or T molecules, revealing the complexity of the

signaling interactions and heretofore unknown activities of DPP4 truncated proteins.

Further investigation into the roles and regulation of DPP4 in normal andmalignant he-

matopoiesis will allow for a better understanding of the significance, and potential clin-

ical utility, of DPP4 activity altering compounds as well as DPP4 truncated molecules.

P1129 - THROMBOPOIETIN-INDUCED DIFFERENTIATION AND

MATURATION OF SPLENIC AND HEPATIC THROMBOCYTES IN

XENOPUS LAEVIS

Takato Otani1, Yuta Tanizaki2, Yoko Mochizuki2, Ayaka Murase2, Shunji Sakai2, and

Takashi Kato1,2

1Graduate School of Advanced Science and Engineering, Waseda University, Tokyo,

Tokyo, Japan; 2Department of Biology, School of Education, Waseda University,

Tokyo, Tokyo, Japan

Non-mammalian vertebrates have nucleate thrombocytes instead of platelets. In adult

African clawed frog, Xenopus laevis (X. laevis), the process of thrombopoiesis occurs

predominantly in the liver and the spleen. We examined the deference and the degree

of the contribution of these organs in thrombopoiesis. The thrombocytic cells pre-

pared by cytocentrifugation were immune-stained with monoclonal antibody to X.

laevis thrombocytes (T12). The diameter of hepatic cells positive to T12 were ranged

from 20 to 30 while splenic cells and the peripheral thrombocytes were from 10 to 20.

The numbers of T12-positive cells recovered from the liver and spleen by collagenase

plus disperse tissue digestion were almost equal (1.5; 106 cells), despite the liver was

twenty-hold heavier than the spleen. Likewise the cellular morphology differed ac-

cording to the organs. Nuclear/cytoplasmic ratios of T12 positive cells were 58%

in the liver, 41% in the spleen and 42% in peripheral blood, respectively. We hypoth-

esized that T12 positive cells in the liver were more immature than in the spleen, and

we compared the DNA contents among respective T12 positive cells. By flowcytom-

etry in conjunction with Hoechst 33342, the fluorescence intensities of hepatic T12

positive cells displayed higher, compared to that of peripheral blood or the spleen.

To examine the ability to undergo differentiation into megakaryocytes/thrombocytes,

cells recovered from the liver and spleen were cultured in semi-solid media under the

stimulation of recombinant X. laevis thrombopoietin (xlTPO). The number of col-

onies derived from the liver cells was much higher than those from the spleen, indi-

cating that thrombocytic progenitors at earlier stages resided in the liver. We discuss

about distinct properties of the microenvironment, including the vascular niche, and

the origins of megakaryocytes/thrombocytes.

P1130 - SUSTAINED P16INK4A EXPRESSION IS REQUIRED TO PROTECT

AGAINST IR-INDUCED LYMPHOMA IN MICE

Lina Palacio1, Krishna Vimal1, Oahn Le1, Norman Sharpless2, and

Christian Beausejour1

1CHU Ste-Justine, l’Universit�e de Montr�eal, Montr�eal, Quebec, Canada;2Departments of Medicine and Genetics, University of North Carolina School of

Medicine, Chapel Hill, North Carolina, USA

We recently showed that mice and human tissues exposed to ionizing radiation (IR)

have an increased expression of p16INK4a, a tumor suppressor gene and proven

senescence marker. Expression of p16INK4a prevents adequate tissue renewal and

is hypothesized to be an underlying mechanism by which most childhood cancer sur-

vivors develop IR treatment-related health conditions. Intriguingly, increased

p16INK4a expression is delayed several weeks post exposure to IR. Hence, it is un-

clear if delayed expression occurs as an orchestred tumor suppressive pathway or

simply as a bystandard effect to IR-induced loss of tissue homeostasis. Using a mouse

model where the p16INK4a gene can be conditionally deleted upon treatment with

tamoxifen, we evaluated if inactivation of p16INK4a post exposure to IR could pro-

mote tissue renewal without increasing the risk of cancer. Following exposure to a

single dose of IR (2.5 G) we found that p16INK4a expression was increased in a de-

layed manner in mice tissues and that such an expression was abolished upon tamox-

ifen injection (0.05). As expected, inactivation of p16INK4a post-IR was shown to

favour the incorporation of BrdU in cells in vitro and in various tissues in vivo

(0.05). However, we observed that inactivation of p16INK4a 8 weeks post IR signif-

icantly increased the incidence of lymphoma in treated mice compared to non-in-

jected mice, reducing their life expectancy by an average of 20 weeks (0.001).

Overall our results suggests that delayed p16INK4a expression post IR interferes

with tissue renewal but that its sustained expression is required to protect mice

against IR-induced tumorigenesis.

P1131 - DIRECT CONVERSION FROM MOUSE FIBROBLASTS INFORMS

THE IDENTIFICATION OF HEMOGENIC PRECURSOR CELLS IN VIVO

Carlos-Filipe Pereira1, Betty Chang1, Xiaohong Niu1, Andreia Gomes1,

Gemma Swiers2, Emanuele Azzoni2, Christoph Schaniel1, Marella de Brujin2,

Ihor Lemishka1, and Kateri Moore1

1Icahn School of Medicine at Mount Sinai, New York, New York, USA; 2Weatherall

Institute of Molecular Medicine, Oxford, United Kingdom

Definitive hematopoiesis emerges via an endothelial-to-hematopoietic transition in

the aorta-gonad-mesonephros (AGM) region and placenta. We have recently de-

monstrated the induction of hematopoietic stem/progenitors (HSPCs) from mouse

fibroblasts with a combination of transcription factors progressing through endothe-

lial-like precursors. Here, guided by our in vitro programming experiments we

analyzed mouse placentas for the presence of the precursor phenotype. We identified

a small population of CD34+ Sca1+Prom1+ (34PS) cells in mid-gestation placentas

that do not express the pan-hematopoietic marker CD45. After isolation and culture

34PS cells acquire CD45 and generate large hematopoietic as well as cobblestone

colonies. Prom1+ cells localize to the placental vascular labyrinth where HSPCs

emerge. 34PS cells express markers associated with the hemogenic endothelium

(CD31, Tie2, VE-Cadherin, Sox17, Runx1, Scl) and also markers identified by direct

induction (Itga6/CD49f). This population is heterogeneous for the early hematopoi-

etic marker CD41 and expresses the programming transcription factors. Remarkably,

global gene expression profiles of placental 34PS cells correlate with AGM-derived

hemogenic endothelium and fibroblast-derived precursors. Finally, when co-cultured

with stroma placental 34PS cells give rise to B/T lymphoid cells as well as mixed

colonies containing erythroid, myeloid and megakaryocytic cell lineages. In sum-

mary, we show that direct in vitro conversion provided a cell surface phenotype

for the isolation of hemogenic precursors in vivo. Our findings provide insights

into the specification of definitive hemogenesis in the placenta, in depth characteriza-

tion of hemogenic precursor populations and the first evidence that direct in vitro con-

version approaches can be used as a valuable tool to address basic developmental

questions in vivo.