Leukemic stem cells have several dysregulated pathways and the Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) pathway are prominent among them. High STAT3 expression in leukemia has been associated with an increased risk for relapse and decreased overall survival. In addition, phosphorylated STAT3 can be found following prolonged activation of the anaplastic lymphoma kinase (ALK) ligand binding site, but the level of phospho-STAT3 engendered by ligand-independent activation of ALK was exponentially higher. In ALK positive tumors, ALK-STAT3 activation promotes tumor cell outgrowth and directly prevents apoptosis. The role of ALK in several types of ALK-expressing solid tumors as well as the therapeutic use of ALK inhibitors in treating these cancers also have been reviewed in a number of recent papers.However, clinical resistance to ALK inhibitors invariably occurs. Amplified ALK or mutated ALK was identified in ~14% of neuroblastomas (NB), the most common and aggressive childhood malignancy, and phase I trial of ALK inhibitor such as crizotinib showed a lack of response in patients harboring certain ALK mutations. In this study, we implemented a high throughput chemical screen in 4 NB-derived cell lines to identify compounds with the potential of inhibiting oncogenic activity of ALK in NB, using a curated library of ~450 compounds. Brefeldin A (BFA),an inhibitor of the transport of proteins from the endoplasmic reticulum (ER) to the Golgi, was identified as a suitable candidate. It was previously reported that BFA inhibited phosphorylation of ALK and its downstream molecule, STAT3. Since an increasing number of studies have found that STAT3 abnormal expression and activation are accompanied by leukemia development, which indicates the potential role of STAT3 in the pathogenesis of leukemia, we analyzed the cytotoxicity of BFA against 12 T-ALL and 6 AML cell lines. Assessment of BFA antitumor effect using an MTT assay revealed BFA to possess potent cytotoxic activity across a broad spectrum of hematopoietic malignancies, with 10 T-ALL and 5 AML being especially responsive. For example, T-ALL cell lines, as well as AML lines, were potently inhibited by BFA ( IC50 values: ICH-TALL-UK, 168.1 nM; MOLT-4, 23.2nM; Jurkat, 66.6 nM; MOLT14, 97.3 nM; ICH-TALL-SM, 54.9 nM). In an expanded panel of 20 NB cell lines, those with or without MYCN-amplification or 11q loss of heterozygousity which have been identified as two major oncogenic events in NB pathogenesis, especially in the high-risk group were the most sensitive to low nanomolar concentrations of BFA. In NB cell lines harboring F1174L or R1275Q-mutated ALK,BFAshowed cytotoxicity.BFA reduced the phosphorylation of ALK in NIH3T3 cells stably expressed F1174L ALK or TGW, cell line harboring R1275Q-mutated ALK. Of the molecules that are phosphorylated downstream of ALK that have been reported to be important for ALCL cells, the phosphorylation of STAT3, AKT and ERK was inhibited by BFA. These results suggested that BFA can be used to target STAT3 signaling pathways for leukemia treatment. Furthermore these results indicated that the effect of BFA on ALK mutated NB cell lines is accompanied by inhibition of the phosphorylation of ALK and its downstream key effector molecule STAT3.These findings provide the significance of ALK and STAT3 as they relate to NB or leukemia, explores the significance of STAT3, AKT and ERK downstream of ALK, and touches on the potential for new chemotherapeutics targeting ALK and STAT3 to improve high risk NB and leukemia patient prognosis.
Disclosures
No relevant conflicts of interest to declare.
Comprehensive Analysis and Validation Reveal Potential MYCN Regulatory Biomarkers Associated with Prognosis of Neuroblastoma
