Foxos Suppress Lipid Peroxidation to Promote AML Progression and Chemotherapy Resistance
Authors: Aghayev Turan , Zhou Chun , Klein Alyssa , Martinez Esteban , Scholl Claudia , Fröhling Stefan , Sykes Stephen Matthew 
Published: October 2019

Abstract
Over 30% of acute myeloid leukemia (AML) patients do not respond to first-line chemotherapy, and a significant portion of patients that do initially respond subsequently relapse with resistant disease. These unsatisfactory outcomes indicate that AML cells either rapidly evolve or inherently possess mechanisms for evading standard chemotherapeutic approaches. Several studies have suggested that AML cells utilize pathways that regulate intracellular redox biology to promote chemotherapy resistance; however, the precise pathways governing resistance and redox biology in AML have yet to be fully determined.
We recently discovered that the FOXO family of transcription factors, which have been traditionally considered tumor suppressor genes, actually support AML cell survival and the differentiation blockade. Specifically, we observed that the expression of FOXO1 and FOXO3 are significantly increased in approximately 40% of primary human AML samples (p<0.0001) and that short hairpin RNA (shRNA)-mediated inhibition of FOXO3 caused human AML cells to acquire characteristics of myeloid differentiation such as increased CD11b expression, cytoplasmic volume, size and granularity. Moreover, we also found that compound ablation of the FoxO family members FoxO1, FoxO3 and FoxO4 significantly extends disease latency and improves survival (p=0.0009) in a murine model of AML driven by the leukemogenic allele MLL-AF9.
Previous studies have shown that, in variety of cell types, FOXOs influence the intracellular redox environment by suppressing the production of reactive oxygen species (ROS). Therefore, to determine the molecular role of FOXOs in AML, we initially focused on the impact of FOXO inhibition on AML cell redox biology. Using fluorogenic probes that detect either total intracellular ROS content (CellRox) or superoxide production (MitoSox), we found that shRNA-mediated inhibition of FOXO3 did not affect total levels of intracellular ROS or superoxides. However, using a lipid peroxidation sensor (BODIPY¨ 581/591 C11), we did observe that two distinct FOXO3-targeting shRNAs increased both homeostatic and stress-induced levels of lipid peroxides in AML cells (shFOXO3-1, p=0.0004; shFOXO3-2, p=0.0023). Consistent with this, we also found that AML cells treated with a chemical inhibitor of FOXOs (AS1842856) display increased steady-state levels of intracellular lipid peroxides (p=0.0076) as well as increased signs of differentiation (CD11b and morphological changes) and death (Annexin V staining).
To elucidate the importance of lipid peroxidation in AML, we evaluated how two chemical anti-oxidants, N-acetyl-L-cysteine (NAC) and butylated hydroxyanisole (BHA), impact the anti-leukemia effects and increased lipid peroxidation mediated by FOXO inhibition. From these analyses, we have observed that BHA treatment suppresses lipid peroxide production and partly blocks AML cell death induced by shRNA-mediated FOXO3 inhibition (shFOXO3-1, p=0.0001; shFOXO3-2, p<0.0001). Interestingly, NAC treatment, which does protect healthy hematopoietic stem and progenitor cells from FOXO inhibition, is unable to reverse the anti-leukemia effects or lipid peroxidation induced by FOXO inhibition, suggesting that FOXOs may differentially regulate redox biology between normal and malignant hematopoietic progenitors.
Both basic and clinical studies have shown that anthracyclines such as daunorubicin (DNR) induce lipid peroxidation; however, the role of lipid peroxidation in chemotherapy effectiveness is largely unknown. We have discovered that co-treatment of AML cells with DNR and BHA (but not NAC) completely blocks the cytotoxic effects of DNR (p<0.0001), suggesting that suppression of lipid peroxides could promote chemotherapy resistance. Consistent with this idea, we have observed that shRNA-mediated inhibition of FOXO3 enhance DNR-mediated AML cell death (p<0.0001), whereas enforced expression of FOXO3 protects human AML cells from DNR cytotoxicity (p=0.003).
Collectively, these results suggest that FOXOs are critical mediators of AML progression and chemotherapy resistance by directly regulating intracellular lipid peroxide levels.

Disclosures
No relevant conflicts of interest to declare.