Forkhead box M1 (FOXM1) is a transcription factor of the Forkhead family that induces the expression of genes involved in the execution of the mitotic program of normal cells, while it overexpressed in the majority of human cancer cells. In addition, FOXM1 regulatory network was a major predictor of adverse survival outcomes. We have previously shown that FOXM1 interacts with nucleophosmin (NPM) in cancer cells and NPM determines cellular localization of FOXM1. Mutations in NPM1 result in cytoplasmic relocalization of NPM (NPM1mut) and favorable outcome for the patients. We have also shown by immunofluorescence in the AML cell line OCI-AML3 and in AML primary samples with NPM1mut, FOXM1 and mutant NPM co-localize in the cytoplasm. Here we show the evidence that improved outcomes in the subset of NPM1mut AML may be partially explained by the cytoplasmic relocalization and consequent functional inactivation of FOXM1. First, we confirmed the colocalization of FOXM1 and NPMmut in cytoplasm of AML primary patient samples using Vectra imaging of AML diagnostic bone marrow biopsies. Strong cytoplasmic expression of FOXM1 was seen only in NPM1mut AML cells. We proceeded to test the role of FOXM1 in mediating chemoresistance in leukemia cell lines with nuclear FOXM1. Stable knockdown of FOXM1 in AML cell lines KG-1 with nuclear FOXM1/wild-type NPM resulted in increased sensitivity to the chemotherapeutic agent cytarabine. We overexpressed FOXM1 in the OCI-AML3 cell line with cytoplasmic localization of FOXM1 and confirmed overexpression of exogenous FOXM1 in the nuclear compartment by fractionation. Furthermore, we showed that nuclear overexpression of FOXM1 switches the sensitivity of these leukemia cell line to resistant phenotype. These data imply that suppressing of FOXM1 in AML could increase sensitivity to standard chemotherapy, while overexpression of FOXM1 increases chemoresistance of AML cells. Our data suggest that FOXM1 inhibitors may be useful for AML patients with FLT3 wild-type/NPM1 wild type with nuclear FOXM1. We used two compounds to target FOXM1 in AML: honokiol and ixazomib. Honokiol inhibits FOXM1-mediated transcription and FOXM1 protein expression and honokiol's inhibitory effect on FOXM1 is a result of direct binding of honokiol to FOXM1. We found that that honokiol suppresses FOXM1 in AML cell lines and sensitizes AML cells with nuclear FOXM1 to cytorabine. We also tested novel proteasome inhibitor ixazomib (Takeda) as a potential FOXM1 inhibitor in AML. We found that similarly to several other proteasome inhibitors ixazomib inhibits transcriptional activity of FOXM1, FOXM1 mRNA, and FOXM1 protein expression in primary AML cells and in AML cell line. In addition, it shows potential synergy with AML's most common drug cytarabine. Combination of cytarabine and ixazomib in AML KG1 cells led to strong apoptosis correlated with FOXM1 suppression. Since ixazomib is already approved for patients with multiple myeloma and it works as FOXM1 inhibitor in AML, we propose to test it as FOXM1 inhibitor in vivo. These results substantiate FOXM1 targeting to improve chemosensitivity for AML treatment. Our work is based on an intriguing premise that nuclear export of FOXM1 by mutant NPM1 in a subset of favorable risk AML is responsible for outcome. Furthermore, the idea that FOXM1 inactivation leads to favorable outcome gives rise to several strategies to develop FOXM1 inhibitors to increase the efficacy of chemotherapy in AML patients.

Citation Format: Irum Khan, Marianna Halasi, Andrei L. Gartel. The novel role of FOXM1 in AML [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 28.