Identification of FoxM1 As Therapeutic Target in TKI-Resistant Ph+ ALL
Authors: Buchner Maike , Klemm Lars , Zhengshan Chen , Geng Huimin , Muschen Markus 
Published: October 2019

Abstract
Abstract 874

Background:
Despite initial responsiveness of primary Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) to tyrosine kinase inhibition (TKI), the majority of patients will relapse and develop TKI-resistant disease. Foxm1 belongs to the forkhead box transcription factor family and is a key regulator of malignant growth by promoting cell cycle and survival through increased DNA damage repair. Foxm1 has been implicated in the progression and chemoresistance in a wide range of solid tumors, including hepatocellular carcinoma and breast cancer. Foxm1 is expressed in dividing cells and regulates the expression of critical regulators for G2/M entry of the cell cycle including Cdc25B, cyclin-B1, Plk-1 and Aurora B kinase. In addition it decreases protein stability of p27kip and p21cip and regulates the expression of antioxidant defense machinery of the cell, e.g. by superoxide dismutase expression.

Results:
We compared Foxm1 expression levels in Ph+ ALL patient samples and CD19+ B cell precursors from healthy donors and found 12-fold higher levels in the leukemic cells (p=0.011). More importantly, Foxm1 levels at the time of diagnosis in a clinical trial for patients with high risk acute lymphoblastic leukemia (ALL) were predictive of poor outcome (COG P9906; n=207). Comparative analysis of microarray data from matched sample pairs at diagnosis and relapse revealed a significant upregulation of Foxm1 in the relapse samples (n=42; p=0.0025). To further study the role of Foxm1 in Ph+ ALL, we developed a genetic model for inducible inactivation of Foxm1 in Ph+ ALL. To this end, B cell precursors of Foxm1fl/fl mice were transformed with BCR-ABL1 and transduced with a tamoxifen (4-OHT)-inducible Cre. Interestingly, 4-OHT-mediated deletion of Foxm1 resulted in reduced cell viability and an arrest in G0/G1 with a significant decrease of the S-phase of the cell cycle following deletion of Foxm1. The ability to form colonies in vitro was significantly decreased by deletion of Foxm1. In addition, Foxm1−/− ALL cells revealed a strikingly higher sensitivity towards TKI-treatment (Imatinib dose-response curve) compared to the control cells. As a potential therapeutic agent to pharmacologically inhibit Foxm1 function, we evaluated the effects of a previously described ARF peptide that binds and inhibits Foxm1 function. We treated TKI-resistant (BCR-ABL1T315I) and TKI-sensitive patient-derived xenograft Ph+ ALL cells with various ARF peptide concentrations and found significant growth inhibition after 72h (IC50 16.8±4.3μM, n=4), regardless of TKI responsiveness. In addition, treatment of ARF peptide in combination with TKI reduced the viability from 65.7%±1.7 after TKI treatment alone (10μM) to 19%±0.8 after 48h (TKI 10μM ARF peptide 12μM). Ph+ ALL cells treated with similar concentrations of a mutated ARF control peptide revealed 77%±0.9 viable cells and ARF peptide treatment alone decreased the viability to 29.6%±0.4. Hence treatment with the ARF peptide alone induces apoptosis in patient-derived Ph+ ALL cells and enhances the effect of TKI, which confirms the findings of the ALL mouse model for human Ph+ ALL xenografts. In a complementary approach, we used the natural antibiotic Thiostrepton, which functions via Foxm1 blockade. To test the ability of Thiostrepton as a potential anti-leukemia agent, we studied patient-derived TKI-resistant (BCR-ABL1T315I) Ph+ ALL cells. Treatment of these patient derived Ph+ALL cells induced cytotoxicity in nanomolar concentrations of Thiostrepton along with a significant downregulation of Foxm1 protein levels. By contrast, Non-BCR-ABL1 tumor cells including lymphoma cells were not responsive to Thiostrepton treatment at similar concentrations.

Conclusion:
Our analyses reveal that Foxm1 is a valid therapeutic target for the treatment of TKI sensitive and resistant Ph+ ALL, including BCR-ABL1T315I. We show that Foxm1 has a crucial function in Ph+ ALL and impacts a) leukemia proliferation, b) colony formation, and c) TKI-resistance. These findings identify Foxm1 a rational target for combination therapy with TKI or as a single agent for TKI-resistant Ph+ ALL.

Disclosures:
No relevant conflicts of interest to declare.