Inhibition of FOXM1 By Ixazomib Confers Chemosensitivity in NPM1-Wild Type Acute Myeloid Leukemia

Background: FOXM1 represents an attractive therapeutic target owing to selective up-regulation in dividing cells. Proteasome inhibitors target FOXM1 by inhibiting FOXM1 through the stabilization of HSP70, which is the negative regulator of FOXM1 {Halasi. J Biol Chem. 2016}. Ixazomib has previously been shown to induce cell death in NPM1-mutant AML cells by oxidative stress {Garcia, Clin Cancer Research 2016} and is currently being tested in NPM1-mutant AML patients as a single agent. We have shown that FOXM1 is inactive in NPM1- mutant AML (Bhat UG. J Bio; Chem 2012) supporting an alternative mechanism of action such as reactive oxygen species induction.

In this abstract we propose a FOXM1 dependent mechanism of action in NPM1-wild type AML. We demonstrate that the novel proteasome inhibitor ixazomib is an inhibitor of FOXM1 and can enhance sensitivity to standard chemotherapy drug cytarabine in AML. With its favorable toxicity profile and oral route of administration, this is an attractive option to study as a chemosensitizing drug in leukemia.

Materials and Methods: Ixazomib was purchased from Selleck Chemicals. The doxycycline-inducible U2OS-derived C3-luc cell line expressing FOXM1-dependent firefly luciferase was developed in Dr. Gartel's lab (Radhakrishnan. Cancer Res. 2006). KG-1, MV4-11 and HL60 cell lines were purchased from ATCC. Patient samples were collected at the University of Illinois Cancer Center after informed consent using an IRB approved protocol.

Results: Using the C3-luc cell line, we studied the effect of ixazomib on FOXM1 transcriptional activity. Cells were treated with 1μg/ml doxycycline and ixazomib doses from 300-1000 nM overnight. The luciferase activity was determined by the Luciferase Assay System (Promega). We demonstrate significant dose-dependent inhibition of FOXM1 transcriptional activity at doses as low as 300nM.

Inhibition of FOXM1 protein expression was then confirmed in NPM1-wild type AML cells. We analyzed KG-1, MV4-11 and HL60 cell lines by immunoblotting for FOXM1 protein after 24 hour incubation with ixazomib at doses ranging from 10-1000 nM.

Given the well-studied role of FOXM1 in mediating chemoresistance, we studied the effect of ixazomib on increasing sensitivity of AML cells to the chemotherapeutic agent cytarabine. The MTS assay was used to calculate the IC50 of ixazomib in KG-1 and MV4-11 cells. The IC50 for KG1 cells was 38 nM (95% CI: 18-78nM) and for MV4-11 cells it was 27nM (95% CI: 16-45nM). We then used a range of doses of ixazomib (0-100nM) in combination with cytarabine at a fixed ratio in AML cell lines to assess cell proliferation using the MTS assay. A combination index (CI) was calculated using CompuSyn and the combination was synergistic in inhibiting proliferation in KG-1 and MV4-11 cells at all doses tested. Inhibition of FOXM1 expression with combination therapy was confirmed by immunoblotting and was accompanied by increased caspase-3 cleavage after 24 hour exposure to the drug.

Finally, to establish that this anti-leukemic effect is dependent on proteasome inhibition, we show a rescue effect with N-Acetyl L-Cysteine, which antagonizes the activity of proteasome inhibitors (Halasi. Biochemical J. 2013).

In primary NPM1-wild type AML mononuclear cells, treatment with ixazomib ex vivo for 24 hours in liquid culture resulted in dose dependent cell death measured by flow cytometry and caspase-3 cleavage. We are currently investigating FOXM1 expression and transcriptional activity in primary AML cells treated with ixazomib.

Conclusion:

Ixazomib demonstrates anti-neoplastic activity against NPM1-wild type AML and potentiates the effect of standard chemotherapy cytarabine. We present a novel mechanism of action whereby ixazomib, through its effect as a proteasome inhibitor, suppresses FOXM1 an oncogenic transcription factor that confers chemoresistance in AML. We are performing animal experiments to confirm the in vitro data and link the anti-leukemic activity of ixazomib with suppression of FOXM1. Our findings lay the groundwork for a target driven trial in AML. Using a novel, well-tolerated oral proteasome inhibitor to target FOXM1 we would expect increased efficacy with lower doses of the chemotherapy drug cytarabine in the treatment of NPM1-wild type AML.