Abstract 4124: The Antioxidant heme oxygenase 1 promotes proliferation and survival of Flt3-ITD-positive AML.

Acute myelogenous leukemia (AML) afflicts ∼12,330 new patients per year in the United States. Regrettably, only 25% of patients will survive five years past diagnosis. The most common mutation in AML is internal tandem duplication (ITD) of the juxtamembrane domain of the fms-like tyrosine kinase receptor-3 (Flt3), which renders it constitutively active. Flt3-ITD regulates proliferation and survival, and also increases the production of reactive oxygen species (ROS), which act as secondary messengers for oncogenic signaling. ROS can cause the induction of a number of molecules, however, one protein, heme oxygenase 1 (HO-1), a well-known antioxidant, has been connected to both proliferation and drug resistance of various cancers. We hypothesized that Flt3-ITD-dependent signaling and ROS production increase constitutive expression of HO-1, leading to the activation of antioxidant and anti-apoptotic pathways, resulting in proliferation and drug resistance in AML. Western blotting revealed a two-fold increase of HO-1 protein in Flt3-ITD + cells as compared to Flt3-WT; a four-fold up-regulation of HO-1 mRNA was noted by quantitative real-time PCR suggesting transcriptional control. To determine if this up-regulation was due to the altered redox status of Flt3-ITD + cells, the flavonoid inhibitor diphenylene iodonium (DPI) was used. Consistent with published results implicating the flavonoid protein complex NADPH oxidase (NOX) as a primary source of ROS in these cells, DPI reduced ROS levels as early as two hours post treatment. This ROS reduction coincided with a decrease in HO-1 protein, suggesting that NOX may be involved in HO-1 up-regulation. Our previous results in chronic myeloid leukemia suggest that a Rac1-dependent isoform of NOX controls HO-1 expression, however, in Flt3-ITD + AML, dominant negative inhibition of Rac1 was insufficient to alter HO-1 expression suggesting that either a Rac1-independent NOX isoform is involved or that another flavonoid protein modulates HO-1 expression in this leukemia subtype. Interestingly, when HO-1 expression was knocked down using RNAi, there was a 50% reduction of proliferation and 40% reduction of viability as measured by trypan blue exclusion in Flt3-ITD + AML cells. These data suggest that the function of HO-1 up-regulation is to promote survival and proliferation of Flt3-ITD + AML. Additionally, we have created a model of acquired resistance to lestaurtinib, a Flt3 kinase inhibitor, by treating with increasing doses of the drug over time. Preliminary results suggest that HO-1 is further elevated in these resistant cells as compared to parental Flt3-ITD cells; thus, we will use this model to test the functional role of HO-1 in acquired resistance. Together, our data suggest that HO-1 is a growth and survival factor in Flt3-ITD + AML; therefore, targeting HO-1 or the mechanisms that control its expression may prove therapeutically valuable. Citation Format: Mary E. Irwin, Joya Chandra. The Antioxidant heme oxygenase 1 promotes proliferation and survival of Flt3-ITD-positive AML. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4124. doi:10.1158/1538-7445.AM2013-4124