FOXO1 Is Involved in the Regulation of B-Cell Precursor Acute Lymphoblastic Leukemia Survival and Serves As a Novel Target for Directed Therapy

Acute lymphoblastic leukemia (ALL) is the most common pediatric and adolescent malignancy. Although current treatment provides five-year event-free survival, in up to 20% conventional chemotherapy fails resulting in relapse with inferior prognosis.

FOXO1 is a member of the forkhead family of transcription factors, which is preferably expressed in B-cells with high expression at the early B-cell stage. FOXOs are involved in several cellular processes including cell death and proliferation, anti-cancer drug resistance and protection from oxidative stress. Since FOXO1 can enhance tumor growth and potentiate metastasis, we aimed to investigate the effects of FOXO1 inactivation on B-cell precursor (BCP)-ALL, including preclinical in vivo evaluation.

FOXO1 expression levels were compared among 497 cancer samples using the Genevestigator online software. Expression of FOXO1 in BCP-ALL was significantly higher than in any of the other cancer types. Next, we investigated FOXO1 expression and subcellular localization in 3 BCP-ALL cell lines by cellular fractionation and fluorescent microscopy. Both methods showed localization of FOXO1 in the nucleus, indicating transcriptionally active FOXO1 in BCP-ALL.

In order to study the potential anti-tumor effect of FOXO1 repression, we investigated genetically modified, FOXO1 deficient BCP-ALL cell lines (n=5) and observed no cell death induction in control transduced cells, in contrast to a clear reduction of cell viability of up to 80% upon FOXO1 knock-down, clearly indicating dependency of BCP-ALL cells on FOXO1. Moreover, lentiviral mediated FOXO1 knockdown did not induce cell death in the Hodgkin's lymphoma cell line cHL, suggesting a BCP-ALL specific importance for FOXO1.

Based on these results indicating the importance of FOXO1 expression for BCP-ALL maintenance, we investigated the feasibility of pharmacological interference with FOXO1. Exposure of 7 BCP-ALL, 4 T-ALL, 3 B-cell NHL, 2 DLBCL and 3 cHL cell lines to the small molecular weight FOXO1 inhibitor AS1842856 showed effectivity in BCP-ALL lines, reflected by significantly higher half maximal inhibitory concentrations (IC50) by MTT test. The most sensitive cell line was the BCP-ALL line RS4;11, while the cHL cell line SUP-HD1 showed insensitivity for FOXO1 inhibition (IC50: 3 nM and 26 µM), again indicating that BCP-ALL is particularly dependent on FOXO1 activity. Caspase 3 cleavage detected upon exposure to AS1842856 showed induction of apoptosis as mechanism of cell death.

Furthermore, we evaluated the sensitivity of primary BCP-ALL primograft samples (n=9) exposing the ALL cells to increasing pharmacologically relevant concentrations of AS1842856. The inhibitor increased cell death as measured by flow cytometry (FSC/SSC criteria) in all of the samples tested in a time and dose dependent manner. Importantly, FOXO1 inhibition also showed activity on high risk leukemias including MLL-rearranged and early or second-relapse cases.

Moreover, we investigated the in vivo effectivity of AS1842856. Two different patient derived leukemias were transplanted onto NOD/SCID mice and upon leukemia manifestation vehicle or AS1842856 was administered for a time of 11 days. At the end of the experiment, all mice were sacrificed and tumor loads were quantified in spleen, bone marrow and central nervous system (CNS). Importantly, tumor loads of all compartments and spleen sizes were significantly reduced in AS1842856 treated animals (p=0.028, U-test). Moreover, in an early-relapse sample leukemia-free survival upon AS1842856 treatment was evaluated. Mice were treated by vehicle or AS1842856 (n=10/group) during 11 days. Leukemia-free survival was significantly prolonged in mice which received AS1842856 (p=0.003, Log-rank test).

Taken together, we show that the active form of FOXO1 is highly expressed in BCP-ALL cells as compared to other cancers, and that viability of BCP-ALL cells is regulated by FOXO1 activity. Importantly, silencing or pharmacological inhibition of FOXO1 induces cell death in BCP-ALL primogafts including high risk cases, both ex vivo and preclinically in vivo. Thus, targeting FOXO1 provides a promising novel strategy for therapeutic intervention in these high-risk subtypes of BCP-ALL.