S31Poster Presentations / Experimental Hematology 42 (2014) S23–S68

P1033 - EVOLVING HETEROGENEITY IN ACUTE LYMPHOBLASTIC

LEUKEMIA

Stephanie Dobson2,1, Esm�e Waanders3, Ildiko Grandal2, Olga Gan1,

Jessica McLeod1, Mark Minden1,2, Cynthia Guidos2, Jayne Danska2,

Charles Mullighan3, and John Dick1,2

1Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario,

Canada; 2University of Toronto, Toronto, Ontario, Canada; 3St. Jude Children’s

Research Hospital, Memphis, Tennessee, USA

Despite high survival rates for children with acute lymphoblastic leukemia (ALL)

only 40% of adult patients will achieve long-term disease-free survival, and relapses

in both pediatric and adult ALL are often fatal. In 50% of patients the clones present

at relapse are not the dominant clone at diagnosis but have evolved from an ancestral

pre-leukemic clone. In order to investigate the functional consequences of clonal evo-

lution in disease progression and therapy resistance we performed in depth genomic

and functional analysis of 14 paired diagnosis/relapse samples from adult and pedi-

atric B-ALL patients with varying cytogenetics. Patient samples were subjected to

whole exome and/or genome sequencing and single nucleotide polymorphism micro-

array analysis for DNA copy number alterations. Limiting dilution analysis by trans-

plantation of CD19+ leukemic blasts into immune deficient mice (xenografts)

identified no significant trend in enrichment in leukemia-initiating cell (L-IC) fre-

quency between paired patient samples with a median frequency of 1 in 2691.

Only 5 pairs demonstrated a difference in L-IC frequency, with 2 higher at diagnosis

and 3 higher at relapse. Despite similar frequencies of L-IC, functional differences

within xenografts were noted among pairs including increased leukemic dissemina-

tion of relapse cells to the spleen and/or central nervous system in comparison to

the dissemination of diagnosis cells. Significant differences in engraftment were

also observed both between pairs as well as within the transplantation of single sam-

ples demonstrating the acquisition of functional differences between leukemic cells.

Future studies identifying the subclonal variation among xenografts and the func-

tional properties of each subclone will give insight into the role of diagnostic sub-

clones in the establishment of relapsed disease. Overall, this work will provide

further understanding of the functional and clonal heterogeneity identified in B-

ALL and the drivers of lymphoid leukemogenesis and disease relapse.

P1034 - NEWABELSON INTERACTOR-1(ABI-1)-DRIVEN MECHANISM

OF ACQUIRED DRUG RESISTANCE

Anna Chorzalska1, Ibrahem Salloum3, Diana Treaba1, Christoph Schorl2,

John Morgan3, Christine Bryke4, John Reagan1, Eric Winer1, Adam Olszewski5, and

Patrycja Dubielecka1

1Brown University/Rhode Island Hospital, Providence, Rhode Island, USA; 2Brown

University, Providence, Rhode Island, USA; 3Boston University/Roger Williams

Hospital, Providence, Rhode Island, USA; 4Quest Diagnostics Nichols Institute,

Chantilly, Virginia, USA; 5Brown University/Memorial Hospital of Rhode Island,

Pawtucket, Rhode Island, USA

In hematological malignancies, quiescent leukemic stem cells are responsible for

persistence of minimal residual disease and relapse. We have recently identified a

new signaling pathway that is significantly dysregulated in imatinib mesylate (IM)

resistant leukemic cells (Chorzalska et all, Leukemia in press). A key player in

this pathway is Abelson interactor-1 (Abi-1). Abi-1 was originally identified as

Abl kinase associating protein that was later confirmed to be one of the Bcr-Abl in-

teractors. Abi-1 was recently shown to interact directly with a4 integrin, which con-

trols lodging of hematopoietic and leukemic stem cells (HSCs/LSCs) in the bone

marrow microenvironment. We have recently obtained evidence that Abi-1 plays a

role in signaling cross-talk between Bcr-Abl and a4 integrin. We have found that

loss of Abi-1 leads to increased adhesion and quiescence, resulting in increased che-

moresistance of leukemic CD34+ progenitor cells. Comparison of Abi-1 (ABI-1) and

a4 integrin (ITGA4) gene expression in relapsing Bcr-Abl positive CD34+ progenitor

cells demonstrated a reduction in Abi-1 and an increase in a4 integrin mRNA in the

absence of Bcr-Abl mutations. This inverse correlation between Abi-1 and a4 integ-

rin expression, as well as linkage to elevated phospho-Akt and phospho-Erk

signaling, was confirmed in imatinib mesylate (IM) resistant leukemic cells. These

results indicate that the a4-Abi-1 signaling pathway may mediate acquisition of

the drug resistant phenotype of leukemic cells. Based on our findings, we hypothesize

that chemoresistance arises as a consequence of dysregulation of a pathway involving

a4 integrin and Abi-1, and is mediated through a previously unknown mechanism

that is independent of oncogene activity.

P1035 - ROLE OF THE POLARITY PROTEIN, SCRIBBLE, IN

HEMATOPOIESIS AND LEUKEMIA

Sarah Ellis2,1, Naomi Campanale2,1, Judy Borg2,1, Matthew Reardon2,1,

Leah Adolph2,1, Samantha Williams2,1, Patrick Humbert2,1, Carl Walkley3,4,

Louise Purton3,4, and Sarah Russell2,1,5

1Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville,

Victoria, Australia; 2Peter MacCallum Cancer Centre, East Melbourne, Victoria,

Australia; 3St Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia;4Department of Medicine, University of Melbourne, Parkville, Victoria, Australia;5Swinburne University of Technology, Hawthorn, Victoria, Australia

The polarity protein, Scribble, is a member of a group of proteins responsible for the

apical basal polarity in epithelia. Scribble is highly conserved from flies to humans

and is deregulated in a number of human epithelial cancers including cervical, colon,

breast and prostate but its role in blood cancers has not been explored. As Scribble

knockout mice are embryonic lethal, dying from a severe neural tube closure defect,

we have developed conditional knockout mice using the Mx1-Cre model to explore

the role of Scribble in hematopoiesis in both steady state and leukemia. Expression of

Scribble in hematopoietic organs and specific lineages was confirmed using multiple

approaches. We established that loss of PTEN results in myeloproliferative disease

with progression to T-ALL or AML/T-ALL. Pten/Scribble double knockouts have

similar symptoms of disease as Pten single knockouts: splenomegaly, hepatomegaly,

enlarged lymph nodes and thymus, terminal deoxynucleotidyl transferase (Tdt)-pos-

itive cells in the thymus and/or an abundance of blasts in the spleen. Both Pten single

knockouts and Pten/Scribble double knockouts have a block in the Pre-Pro B stage of

B cell development in the bone marrow and in the DN1 stage of T cell differentiation

in the thymus. Preliminary examination of disease burden between Pten single

knockouts and Pten/Scribble double knockouts suggests a shift from T-ALL to

AML in Pten/Scribble double knockouts. We are currently investigating this and

other differences between double and single knockout mice and the underlying mech-

anisms for these differences with the ultimate aim of generating novel chemothera-

peutic targets for treatment of T-ALL and AML

P1036 - DISTINCTION AMONG LONG-TERM HSCS, SHORT-TERM HSCS,

AND REPOPULATING CMPS BY A NOVEL IN VITRO SINGLE-CELL

ASSAY

Hideo Ema1,2, Aled O’Neill2,3, and Toshio Suda2

1Institute of Hematology and Blood Hospital, Tianjin, China; 2Keio University

School of Medicine, Tokyo, Japan; 3Institute of Medical Science, University of

Tokyo, Tokyo, Japan

Long-term (LT 12 months) and short-term (ST 6 months) hematopoietic stem cells

(HSCs), and repopulating common myeloid progenitors (rCMPs) have recently

been identified. These cells have previously been characterized by in vivo single-

cell transplantation. In this study, we attempted to characterize them using a novel

in vitro single-cell assay composed of cell division analysis and flow cytometery-

based colony analysis. Here we report that LT-HSCs, ST-HSCs, and rCMPs can be

distinguished from one another by single-cell culture. c-Kit+Sca1+Lineage- cells

were further separated into 8 populations (3 HSC and 5 HPC populations) on which

single-cell cultures were performed with stem cell factor, thrombopoietin, inter-

leukin-3, and erythropoietin. Cell numbers were counted at 12 hour-intervals during

the first 4 days of culture to calculate the mean length of one cell cycle (cycle length)

and the length of the initial quiescent phase (latency). Colonies were individually

stained with antibodies and analyzed by flow cytometry on days 7, 10, and 14 of cul-

ture. The number of cells per colony was measured by adding fluorescent beads to

cells upon flow cytometric analysis. Colonies were classified based on the presence

of myeloid lineage markers. We found that the colony frequency and sizes were

similar between LT-HSCs and rCMPs, but the colony frequency and sizes of LT-

HSCs were significantly greater than those of ST-HSCs. Also of note was that LT-

HSCs and rCMPs gave rise to a large proportion of colonies containing all myeloid

lineages while a majority of ST-HSCs colonies were found to contain only neutro-

phil/macrophage lineages. We also found that both cycle length and latency of LT-

HSCs were significantly greater than those of rCMPs. Unlike single-cell transplanta-

tion, a large number of single cells can be analyzed in a short time frame by single-

cell culture. Single-cell culture and single-cell transplantation work complementarily

for characterization of HSCs and their immediate progeny.