Philadelphia Chromosome positive acute lymphoblastic leukemia (ALL) is associated with particularly poor clinical outcome. Fusion of the ABL1 oncogene to a breakpoint cluster region (BCR) results in constitutively activated BCR-ABL1 tyrosine kinase activity that can be specifically targeted by small molecule tyrosine kinase inhibitors (TKI) such as Imatinib. After initially good clinical response to TKI, BCR-ABL1+ ALL patients invariably relapse into a more aggressive, TKI resistant disease. FOXM1 belongs to the forkhead box transcription factor family and is a key regulator of cell growth by promoting cell cycle progression. While a functional role of the transcription factor FOXM1 in terms of disease progression and drug resistance is well established for solid tumors, it remains elusive whether it has a similar function in ALL.
We have identified high FOXM1 protein expression levels in patient-derived BCR-ABL1+ ALL samples compared to healthy B cells and B cell precursors. Consistent with this finding, the FOXM1 promoter region was de-methylated in 83 BCR-ABL1+ ALL patient samples compared to normal pre-B cells and Lymphoma samples. We used a mouse-model for BCR-ABL1+ ALL to characterize the role of BCR-ABL1 in the induction of FOXM1 expression: we cultured murine bone marrow B cell precursors in the presence of IL7 and induced transformation with a retroviral BCR-ABL1 expression vector. Consistent with findings for other oncogenes, i.e. activating mutation of Ras, BCR-ABL1 expression increased levels of FOXM1 compared to the normal IL7-dependent pre-B cells. In order to evaluate a potential clinical relevance of FOXM1 expression levels in ALL disease progression, we correlated FOXM1 mRNA levels with clinical outcome. High FOXM1 expression levels correlate with poor clinical outcome of ALL. In addition, the group of Sanders et al. (Genome Biol., 2013) has recently identified a set of 38 genes that are directly regulated by FOXM1 and associated with poor prognosis in breast cancer. 34 of the described 38 FOXM1 target genes were associated with high risk in ALL based on a data set collected by the German ALL-REZ BFM 2002 of the Berlin-Frankfurt-Münster study group (n=60). To study the functional role of FOXM1 in BCR-ABL1+ ALL, we performed experimental genetic deletion experiments using bone-marrow cells derived from Foxm1fl/fl mice. Deletion of Foxm1 decreases cell viability, colony formation, and proliferative capacity in vitro as well as leukemia formation in vivo of BCR-ABL1-driven leukemia. FOXM1-deleted ALL cells revealed a strikingly higher sensitivity towards TKI-treatment compared to the control cells in Imatinib dose-response curves (IC50EV: 420 nM vs IC50 CreERT2: 160 nM) as well as annexin V staining. As FOXM1 is a critical regulator of oxidative responses, we analyzed the intracellular reactive oxygen species (ROS) formation in the presence and absence of FOXM1 in BCR-ABL1 transduced ALL cells and observed consistently higher ROS levels after FOXM1 deletion. As a functional mediator of this effect, we analyzed the expression of the described FOXM1 target and ROS scavenger Catalase and found reduced levels after FOXM1 deletion. We determined Catalase expression in ALL cells after Imatinib treatment and found significant transcriptional upregulation. To evaluate whether this effect is FOXM1-dependent, we treated ALL cells with Imatinib and evaluated Catalase expression in the presence and absence of FOXM1. Only the FOXM1 expressing ALL cells were capable of upregulating Catalase after Imatinib treatment. Single locus chromatin immunoprecipitation (ChIP) analysis of a described binding site of FoxM1 in intron 1 of the catalase gene showed specific binding of FOXM1, albeit low enrichment compared to the positive control cyclin B1. As potential therapeutic agents to target FOXM1, we evaluated the effects of a previously described ARF peptide and the natural occurring antibiotic Thiostrepton. Both bind FOXM1 and inhibit its function and induced apoptosis in Ph+ ALL. In line with our observation of the genetic deletion, we observed induction of ROS with the treatment of either substance along with a decrease in Catalase expression.
Taken together, our data identify FOXM1 as a valid therapeutic target for the treatment of TKI sensitive and resistant BCR-ABL1+ ALL – either together with TKI or as a single agent.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.
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