Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the malignant expansion of dysfunctional myeloid progenitors and the suppression of normal hematopoiesis. Despite of the intensive efforts in developing novel therapeutic strategies in the past decades, the standard chemotherapy, i.e., the "5+3" regimen, is still the main approach for treating AML, and resistance to the standard chemotherapy remains the major cause of relapse and therapeutic failure. Therefore, it is crucial to understand the mechanisms underlying AML pathogenesis and chemotherapy resistance, and thus identify new therapeutic targets.
The arachidonate5-lipoxygenase gene (Alox5) is known to be involved in various physiological and pathological processes, including oxidative stress response, inflammation and cancer. Previous reports show that loss of Alox5 impairs the function of leukemic stem cells in BCR-ABL-induced chronic myelogenous leukemia (CML), as well as the long-term self-renewal of hematopoietic cells of AML with RUNX1-ETO9a, MLL-AF9 and PML-RARA. To our surprise, our Affymetrix microarray-based, genome-wide gene expression profiling of primary AML patient samples reveals that Alox5 is expressed at a particularly lower level in certain AML subtypes (e.g., MLL-rearranged AML) that are associated with poor prognosis than in AML subtypes (e.g., t(8;21) and inv(16) AMLs) that are associated with favorable prognosis. The aim of this study is to identify the influence and mechanism of Alox5 overexpression in AML pathogenesis and therapeutic response.
To assess the potential effect of Aloxon leukemic cell viability and drug response/resistance, AML cell lines with stable ectopic expression of Alox5 or control plasmids induced by lentivirus were treated with DMSO control, doxorubicin (DOX) or cytarabine (Ara-C). Cell viability and proliferation assays, i.e., MTT assays, were performed. To determine the influence of Alox5 on the transformation capacity, viability and drug response of mouse BM progenitor cells bearing MLL-AF9, colony-forming/replating assay (CFA) and MTT assay were conducted. To evaluate the effect of Alox5 on AML progression and chemotherapeutic response in vivo, we first carried out primary bone marrow transplantation (BMT) by use of hematopoietic progenitor cells retrovirally co-infected with MLL-AF9 and Alox5 or empty vector as primary donors, and then applied the leukemic blast cells isolated from the primary leukemic recipients as donor cells in a secondary BMT followed by "5+3" chemotherapy.Gene Set Enrichment Analysis (GSEA) was used to analyze the signal pathway enrichments in the BMT samples based on RNA sequencing results.
In AML cell lines, lentivirus induced overexpression of Alox5 resulted in a moderate repression of cell viability, while significantly sensitizing the cells to DOX and Ara-C treatment. In mouse BM progenitor cells with MLL-AF9, forced expression of Alox5 remarkably enhanced the inhibitory effect of DOX and Ara-C on the colony forming capacity and cell viability.
Secondary BMT assay results showed that overexpression of Alox5 inhibited MLL-AF9-induced AML progression (MLL-AF9+Alox5+Ctrl, with medium survival of 44.5 days; MLL-AF9+Ctrl, with medium survival of 39.5 days; P=0.001) in vivo. The "5+3" chemotherapy alone indeed improved survival as compared with MLL-AF9+Ctrl (MLL-AF9+DOX/Ara-C, with medium survival of 50 days; P<0.001). The therapeutic effect of DOX+Ara-C treatment was remarkably enhanced by Alox5 overexpression, and the medium survival was prolonged to >200 days (MLL-AF9+Alox5+DOX/Ara-C) (P<0.001) (Fig. 1A).
RNA-seqand GSEA suggested that overexpressing Alox5 activates the E2F signaling and oxidative stress response, while repressing the K-ras and Stat pathways (Fig. 1B).
Our results suggest that while forced expression of Alox5 shows a moderate tumor suppressor role in the progression of MLL-AF9-mediated AML, it significantly enhanced the effect of the standard "5+3" chemotherapy both in vitro and in vivo. The potential mechanism is through targeting E2F, oxidative stress response, K-ras and Stat signaling pathways, which were all known to be closely associated with tumorigenesis and drug response/resistance. Our study indicates the restoration of Alox5 in combination with the classic chemotherapy as a potential novel therapeutic strategy in treating AML.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.