Gadd45a Loss Enhances MLL Leukemogenesis By Promoting Beta-Catenin Mitochondrial Translocation and Inhibiting Apoptosis

Gadd45a is a mediator of growth arrest and its expression is suppressed in primary human MLL-rearranged AML (predominantly MLL-AF9) and AML1-ETO AML with increased FLT3 expression (Valk et al., N Engl J Med 2004; Perugini et al., Leukemia 2009). While Gadd45a is frequently silenced in several human cancers, including leukemia, little is know about how Gadd45a loss contributes to leukemogenesis. Our studies have shown that Gadd45a loss promotes MLL-AF9 mediated development in AML in mice and protects leukemic cells from apoptosis via novel β-catenin/mitochondria pathway.

To explore the functional role of Gadd45a loss in MLL-rearranged AML, we retrovirally introduced MLL-AF9 into c-Kit^high progenitors isolated from Gadd45a knockout mouse bone marrow cells. Our results showed that Gadd45a knockout markedly reduced the colony forming capacity of pre-leukemic cells and significantly shortened the latency for MLL leukemia development in mice (p= 0.037, N = 6 mice). As Gadd45a could induce cell death through the mitochondria pathway (Tong et al., Mol Cell Biol 2005), we next examined the expression of proteins involved in the mitochondrial apoptosis in Gadd45a knockout pre-leukemic cells. Our western blot analysis showed that Gadd45a knockout dramatically decreased expression of pro-death proteins Txnip, Bim and Bax but interestingly, significantly increased active β-catenin expression in mitochondria. Given that Gadd45a has been reported to regulate β-catenin distribution (Ji et al., Oncogene 2007), we postulate that Gadd45a loss facilitates the translocation of β-catenin into mitochondria. We have previously demonstrated that β-catenin is highly expressed in MLL-AF9 transformed pre-leukemic cells and is lower in Hoxa9/Meis1 transformed pre-leukemic cells, while increased β-catenin expression is correlated with a poor survival rate in mice. Here, we retrovirally introduced a constitutively active form of β-catenin into Hoxa9/Meis1 pre-leukemic cells. As expected, activation of β-catenin reduced endogenous expression of Txnip and Bim in mitochondria, exhibiting an expression pattern similar to that regulated by Gadd45a knockout. Consistently, both Gadd45a knockout and β-catenin activation not only reduced the basal cellular reactive oxygen species (ROS) levels but also inhibited Antimycin A (an ROS generator)-induced mitochondrial ROS production.

Taken together, our study discovers a previously unrecognized Gadd45a loss/β-catenin/mitochondria pathway that prevents mitochondrial ROS accumulation and protects leukemic cells from cell death in MLL leukemogenesis.