FOXM1 Binds Nucleophosmin in AML and Confers Resistance to Chemotherapy

Background: The nucleo-cytoplasmic shuttle protein nucleophosmin (NPM1) is mutated in 50% of cytogenetically normal AML cases (CN-AML) resulting in the nuclear export of this protein and a favorable prognosis. The mechanism by which mutated NPM1 (NPM1^mut) confers this advantage in CN-AML is unclear.  We have previously demonstrated that FOXM1, an oncogenic transcription factor, co-localizes with NPM1 in cancer cells and NPM-binding is required for sustaining the level of FOXM1. In the NPM1^mut AML cell line OCI-AML3, as detected by immunofluorescence, NPM1 and FOXM1 co-localize in the cytoplasm where FOXM1 is transcriptionally inactive. We hypothesize that improved outcomes in the subset of NPM1^mut AML is partly attributable to the cytoplasmic relocalization and functional inactivation of FOXM1.

Methods: Patient samples were identified retrospectively using the University of Illinois Cancer Center Registry (IRB #: 2013-1245). Clinical, laboratory and molecular data were collected by chart review. Formalin fixed paraffin embedded bone marrow biopsies were retrieved to prepare slides. Patients with favorable or unfavorable karyotypes were excluded and only biopsies with >70% blasts were included. We performed a 2-target staining protocol with fluorescent antibodies to visualize NPM1 and FOXM1 in the same cells using the Vectra multispectral tissue imaging system. This system combines machine learning capability (inForm® software) with acquisition of spectral data from each pixel in the image, so that multiple targets can be quantified in the same image. Leukemia cell lines with stable knockdown of FOXM1 were generated using lentiviral transduction of HL60 cells (NPM1^wt )transduced with PLKO.1-shFOXM1 or PLKO.1-empty vector. In parallel, OCI-AML3 cells (NPM1^mut) were transduced with pBabe control or pBabe-FOXM1 expressing retrovirus to induce overexpression of FOXM1.

Results: FOXM1 is a well-characterized oncogenic transcription factor. In this study we examined its role in AML. In a series of AML cell lines, cellular fractionation followed by immunoblotting revealed strong nuclear expression of FOXM1 in HL-60 and KG1, both of which express the wild-type NPM1 gene. In comparison the OCI-AML3 cell line, carrying the mutated NPM1 gene, showed predominantly cytoplasmic expression of FOXM1 where it is inactive. To better understand the role of FOXM1 in mediating chemoresistance in AML, we inhibited FOXM1 expression in the HL60 leukemia cell line. Stable knockdown of FOXM1 in this cell line resulted in increased sensitivity to the chemotherapeutic agent cytarabine compared to empty vector transduced cells. This was demonstrated by increased caspase-3 cleavage as detected by immunoblotting and decreased cell viability by Trypan blue exclusion after 24 hour exposure to the drug. We then proceeded to validate our observations of the FOXM1-NPM interaction in primary AML samples. Using quantitative data generated from the Vectra imaging of 20 AML pre-treatment bone marrow biopsies we show a strong correlation between the nuclear:cytoplasmic ratios of FOXM1 and NPM, Pearson correlation co-efficient r=0.87, which is indicative of cellular co-localization. When the patients are stratified in 2 groups based on NPM1 mutation status, mean cytoplasmic expression of FOXM1 is 2-fold higher in NPM1^mutpatients (36% versus 18%).

Conclusion: We demonstrate that FOXM1, an oncogenic transcription factor is predominantly cytoplasmic and thereby inactive when NPM1 is mutated in AML. We show the functional significance of FOXM1 in mediating chemoresistance in this disease using AML cell lines. Using a novel quantitative imaging modality we establish for the first time the cellular co-localization of FOXM1 and NPM1 in AML primary blast cells. Studies are ongoing to manipulate FOXM1 expression in additional leukemia cell lines and xenograft models of AML to confirm our findings that FOXM1 inhibition in AML leads to increased sensitivity to chemotherapy.