Reprogramming of MLL-AF9 Leukemic Cells Into Pluripotent Stem Cells in Mice

Abstract 205

Somatic cell reprogramming into a pluripotent state by the so called “iPS” technology not only holds great promise in regenerative medicine, but also provides a powerful tool to study pathological dedifferentiation processes such as tumorigenesis. In fact, the four “Yamanaka” reprogramming transcription factors used for iPS induction (Oct4, Sox2, and especially C-Myc and KLF4) are known for their direct or indirect oncogenic activities. In addition, the two most well-known tumor suppressor pathways, p53 and Rb, have been shown to also suppress iPS reprogramming. These suggest that tumorigenesis and somatic cell reprogramming may share some common mechanisms. Although it was previously reported that malignant cell lines or primary cancer cells could be reprogrammed by nuclear transfer or the iPS approach, it has not been definitively demonstrated whether primary transformed cells (not established tumor cell lines) can be reprogrammed into iPS cells with a full-term developmental potential in vivo.

To this end, we first established an acute myeloid leukemia (AML) mouse model by over-expressing the human MLL (mixed lineage leukemia)-AF9 fusion gene in hematopoietic cells harvested from transgenic mice that carry the Yamanaka reprogramming factors under the control of doxycycline (Dox) (Brambrink et al., 2008). We chose MLL leukemia (a group of aggressive forms of acute leukemia with poor prognosis) because the genome of MLL leukemic cells was shown to be relatively stable, thereby increasing the likelihood of successful reprogramming of the leukemic nuclei. The purified leukemic cells were then induced into iPS cells by an addition of Dox under mouse embryonic stem cell (ESC) culture conditions. The MLL-AF9 fusion gene was present in all the iPS colonies, but its expression was silent in the established iPS cell lines. The generated iPS cell lines were similar to normal ES cells lines, as shown by both genetic and epigenetic signatures. The MLL-AF9 iPS cell lines could give rise to teratomas consisting of three germ layers after injection into a SCID mouse. More importantly, some MLL-AF9 iPS cell lines were able to produce chimeras at a high rate through blastocyst injection. Noticeably however, most chimeras developed the same type of AML within 2 months, which was correlated with re-activation of the MLL-AF9 gene. Consistently, DNA methylation of a MLL-AF9 promoter differed significantly between original leukemic cells, derived iPS cells and the re-occurred leukemic cells from the chimera mice, for which the de novo DNA methtylase, DNA3b, rather than other methylating enzymes, seemed to be responsible. RNA-seq analysis is under way to further define the target genes involved in the differences of these interchangeable cell types.

In summary, this study demonstrates for the first time that primary leukemia cells can be fully reprogrammed into iPS cells with the potential of developing into three germ layers and contributing to chimeric mice. The interchangeable feature between leukemic cells and iPS cells offers a unique opportunity to define the distinct mechanisms between pluripotency and malignancy, thereby having implications for specific manipulations of iPS vs. cancer cells and particularly for selective targeting of the leukemic cells harboring a MLL fusion gene.