Abstract

Abstract 120

MEIS1 is highly expressed in several leukemias, particularly those associated with MLL-translocations. Over-expression of MEIS1 accelerates leukemia development in experimental models of leukemia while shRNA mediated knock-down of MEIS1 inhibits leukemia cell growth, suggesting that MEIS1 may be a suitable target for therapy. These previous studies are however marred by potential off-target effects and were carried out on cultured cell lines. We thus studied the role of MEIS1 using a more robust inducible-deletion system. Mice engineered to carry loxp sites flanking the homeodomain of MEIS1 (MEIS1-flox) were crossed with Rosa26CreER mice. Tamoxifen treatment of the resulting MEIS1-flox/CreER mice resulted in efficient recombination and deletion of MEIS1. We transformed MEIS1-flox/CreER or CreER bone marrow cells with various oncogenes delivered via retroviral transduction and then evaluated the effect of MEIS1-deletion. Transformed cells were cultured in methylcellulose supplemented with 4-hydroxy-tamoxifen (4-OHT) or vehicle (ethanol) and colony numbers were counted after 7 days of culture. Loss of MEIS1 significantly reduced colony formation in MLL-AF9 and MLL-GAS7 transformed cells by 74.7 ± 3.2%, 43.9 ± 2.5%, respectively compared to controls. To validate the specificity of this effect, we studied the effect of MEIS1-deletion on cells transformed by the AML1-ETO fusion oncogene. These leukemias display normal or low levels of MEIS1, suggesting that MEIS1 is unlikely to play a role in transformation by AML1-ETO. As expected, MEIS1 deletion had no effect on colony formation by AML1-ETO transformed cells. To substantiate the role of MEIS1 in a more physiologic model of leukemia, we crossed the MEIS1-flox/CreER mice with MLL-AF9 knock-in mice. The knock-in mice carry a single copy of the MLL-AF9 fusion gene in every cell and develop AML with a medial latency of ∼6 months. In methylcellulose culture assays, MEIS1 deletion decreased colony formation of the MLL-AF9 knock-in cells by 81.5 ± 2.8% compared to controls (1.9 ± 0.1%). Further analysis of leukemia cells showed that MEIS1 deletion led to significant increase in apoptosis (as measured by Annexin V staining) to 24.6 ± 4.0% compared to that in controls (0.4 ± 0.33%). To assess the role of MEIS1 in leukemia in vivo, we transplanted MLL-AF9/MEIS1-flox/CreER cells into irradiated mice. Two weeks after transplantation, recipient mice were treated with either Tamoxifen or vehicle (corn oil). Deletion of MEIS1 was confirmed by PCR analysis on peripheral blood. Survival analysis showed that the two control groups - vehicle treated MLL-AF9/MEIS1-flox/CreER mice and Tamoxifen-treated MLL-AF9/CreER mice - succumbed to leukemia with a median time of 130 and 147 days, respectively. On the other hand, MEIS1-deletion led to a significantly prolonged survival with median time of 250 days (p < 0.003, long rank test). Re-expression of non-deletable MEIS1 by retrovirus rescued leukemia-development in MEIS1-deleted mice, confirming that the effect was MEIS1-mediated. On the other hand, re-expression of MEIS1 lacking the homeodomain, carboxyl-terminal domain or the PBX1-interacting domain did not rescue leukemia development, indicating that these domains and interactions of MEIS1 play important roles in the maintenance of leukemia. Finally, to determine the molecular pathways regulated by MEIS1 in leukemia, we performed gene expression profiling of control and MEIS1-deleted MLL-AF9 knock-in leukemia cells using RNA-sequencing. Preliminary analysis of the data revealed that MEIS1-deletion led to decreased expression of genes associated with the hypoxia-response pathway. Collectively, these results demonstrate that MEIS1 is plays an essential role in MLL-AF9 leukemia wherein it promotes survival. Thus, MEIS1-targeting could be a useful therapeutic strategy. Our data will also provide novel information on the downstream targets of MEIS1 in leukemia, revealing pathways that may be more readily amenable to suppression by available or novel inhibitory-agents.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.