Regen BioPharma Inc (RGBP)

[Kryo]

Christine Ichims Poster:

http://stemcellmeetingonthemesa.com/scientific-symposium/poster-session/

15. Silencing of the Novel Leukemia Target NR2F6 Using DiffronC siRNA Technology Induces Terminal Differentiation of Murine Hematopoietic Stem & Progenitor Cells

Presented by: Christine Ichim, Regen BioPharma
Authors: Ichim, Christine (Regen BioPharma); Dervovic, Dzana (University of Toronto); Chesney, Alden (Sunnybrook Health Sciences Centre); Reis, Marciano (Sunnybrook Health Sciences Centre); Ichim, Thomas (Regen BioPharma); Koos, David (Regen BioPharma); Wells, Richard (Regen BioPharma)

Abstract:
To identify therapeutic targets, we previously adopted a novel single-cell approach that used microarray analysis of clonal siblings to identify the molecular signature of leukemia stem cells. Our work led to the discovery of the orphan nuclear receptor NR2F6 (EAR-2) as a potential leukemia stem cell therapeutic target. Previously we showed that NR2F6 is expressed greater in individual leukemia cells with clonogenic capacity than in leukemia cells that do not divide. In vitro, overexpression of NR2F6 inhibits differentiation of cell lines and primary bone marrow cells. In vivo, overexpression of NR2F6 leads to a rapidly fatal leukemia that is preceded by expansion of the stem cell compartment, c-kit+, sca-1+, lineage- hematopoietic cells (KSL cells) in mice. The discovery of the role of NR2F6 in leukemogenesis and the induction of differentiation of leukemia cell lines following silencing of NR2F6 expression suggests that this is a logical therapeutic target for differentiation therapy using gene silencing technology. Hence, we wished to show proof-of-concept that silencing of NR2F6 using the DiffronC siRNA gene silencing technology in primary mouse hematopoietic stem and progenitor cells promoted terminal differentiation. Herein, we show that while silencing of NR2F6 did not significantly reduce the number of colony forming units, it did significantly increase colony size. We then showed that silencing of NR2F6 reduced the clonal longevity of bone marrow cells: In replating experiments we observed a significant decrease in secondary colonies in cells treated with NR2F6 shRNA. Furthermore, silencing of NR2F6 resulted in a drastic reduction in KSL cells after six days of ex vivo culture, and caused a dramatic decline in lineage negative cells, and a concurrent increase in cells expressing the myeloid markers CD11b and Gr-1, suggesting that they had differentiated into cells of the neutrophil lineage. This was confirmed by examination of the cytomorphology of the bone marrow cultures. Taken together, these results establish that NR2F6 is a negative regulator of terminal differentiation of the hematopoietic lineage and lays the foundation for use of gene silencing technology against NR2F6 as differentiation therapy for diseases with blocked differentiation such as leukemia and myelodysplastic syndromes.

Key Words: cancer stem cell differentiation nuclear receptor gene silencing hematopoietic leukemia progenitor