Differential Role of Myeloid Transcription Factors, C/EBPα and PU.1 In Leukemogenesis by MLL-Fusion Oncogenes.

Abstract 1568

MLL translocations found in acute leukemia possess unique clinical characteristics. They have over 50 different fusion partners and show poor prognosis. These MLL fusion proteins lost H3K4 methyltransferase activity of wild-type MLL, but gained the ability to induce aberrant expression of HoxA cluster genes. Moreover, these proteins are able to transform hematopoietic stem/progenitor cells into leukemic stem cells (LSCs).

Previous studies have shown that C/EBPα and PU.1, well-known myeloid specific transcription factors, were common molecular targets of myeloid malignancies. We and others have recently shown that C/EBPα and PU.1 are negative regulators of hematopoietic stem cells, suggesting that these transcription factors may play a role in the generation of LSCs. Because we have little knowledge on the role of C/EBPα and PU.1 in MLL-leukemia, we asked whether these key myeloid transcription factors were involved in the leukemogenesis by MLL-fusion proteins, especially in the stages of leukemia initiation and/or progression.

First, we investigated the role of C/EBPα and PU.1 by in vitro self-renewal capacity and in vivo leukemia formation by MLL-fusion oncogenes. Bone marrow (BM) cells were harvested from C57BL/6J mice treated with 5-FU (150 mg/kg), and pre-stimulated with recombinant mouse (rm) SCF, rmIL-6, rhFL, rhTPO (50 ng/ml each). Cells were then transduced with pMYs-IG-MLL-ENL or pMXs-IG-MLL-Septin6, serially replated in methylcellulose, and transferred to rmIL-3 (10 ng/ml) containing liquid culture (immortalized cells), or were transplanted into lethally irradiated recipients (primary leukemic cells). MLL-ENL (or MLL-Septin6) immortalized cells or MLL-ENL primary leukemic cells were transduced with pMXs-IRES-DsRed-C/EBPα-ER or pMXs-IRES-DsRed-PU.1-ER. GFP⁺DsRed⁺ cells were sorted and serially replated in methylcellulose with or without 4-hydroxytamoxifen (4-HT) (1 mM), or were treated with or without 4-HT (1 mM) for 5 days followed by transplantion into sublethally irradiated secondary recipients. The results showed that overexpression of PU.1, but not C/EBPα, completely suppressed the serial replating capacity of MLL-ENL- and MLL-Septin6-immortalized cells. Moreover, activation of PU.1 suppressed propagation of MLL-ENL leukemic cells in the secondary recipients. In contrast, activation of C/EBPα did not eradicate leukemic cells in the same settings.

To elucidate the role of PU.1 in the initiation of leukemia by MLL-ENL, we took PU.1^+/− BM cells, or E14.5 fetal liver (FL) cells from PU.1^-/- or ^+/− mice, and examined their capability to initiate leukemia when they were transduced with MLL-ENL. The result showed that leukemia did not develop in the absence of PU.1, and PU.1 haploinsufficiency prolonged survival of the recipients. A role of PU.1 in leukemia progression/maintenance by MLL-ENL was also tested using PU.1 conditional knockout mice. BM cells from PU.1^flox/flox or ^flox/- mice were transduced with pMYs-IG-MLL-ENL, and transplanted into lethally irradiated recipients. PU.1-flox allele was conditionally deleted in primary leukemia cells by induction of Cre recombinase, whose effect was assessed by transplanting Cre-treated cells into secondary recipients. The result showed that conditional inactivation of PU.1 perturbed propagation of MLL-ENL leukemic cells, indicating that PU.1 is absolutely required not only for initiation, but also for maintenance of MLL-leukemia.

Taken together, these results suggest that the dosage of PU.1 activity has profound impact on the self-renewal of LSCs and in vivo leukemia formation induced by MLL-fusion oncogenes. Therefore, PU.1 may serve as a potential therapeutic target for MLL-leukemia.