Abstract
Abstract 2463
Acute myeloid leukemia is malignant disease, characterized by an accumulation of immature myeloid cells. Recent studies have demonstrated that myeloid leukemia appears to arise from a population of leukemia stem cells (LSCs). LSCs typically reside in a quiescent state and therefore do not respond to standard chemotherapeutic agents, which generally target more actively dividing cells. However, LSCs do display certain unique molecular properties that can be exploited to target this relatively rare population of cells that drive disease pathogenesis. Specifically, NF-kB, a pro-survival transcription factor, is constitutively active in LSCs but not in normal hematopoietic stem cells (HSCs). Targeting this pathway by pharmaceutical approaches has been suggested as a potential strategy in the treatment of leukemia; however, inhibiting this pathway alone is not sufficient to strongly induce AML-specific cell death. Further investigation of pathways, that are unique to AML, is a key in designing more effective pharmacologic agents that specifically target the LSC.
We have previously demonstrated that the naturally occurring compound parthenolide (PTL) induces apoptosis in primary AML cells, including the stem and progenitor cell. While the empirical anti-leukemic activity of PTL is clear, the underlying molecular mechanisms remain poorly understood. Here we investigate two properties associated with parthenolide-mediated cell death: i) activation of pro-apoptotic transcription factor p53, ii) inhibition of pro-survival transcription factor NF-kB.
In order to evaluate the role of p53 signaling, AML cells were challenged with PTL resulting in the phosphorylation of p53 at serine-15, indicating activation p53 in response to PTL. To further investigate the role of p53 in PTL mediated responses, we generated a lentiviral vector expressing shRNAs specifically targeting p53. Leukemia cells were infected with the lentiviral vector encoding p53 shRNA or scramble control and evaluated by qPCR and western blot analysis. The data showed a significant knockdown of p53 mRNA and protein levels, as well as strong inhibition of p21 expression, indicating the specificity of p53 knockdown. Exposure of cells to PTL in which p53 has been specifically disrupted results in partial rescue from PTL mediated cell death, implicating the role of p53 in this response.
Next, we performed a detailed analysis of the molecular mechanism by which PTL inhibits NF-kB pathway activity. Using a novel analog of PTL, we demonstrate that the compound directly binds to IKK-beta. Upon exposure to PTL, IKK-beta shows reduced kinase activity, indicating that binding of the drug directly impairs enzymatic function. Secondary to the inhibition of IKK-beta kinase activity, there is decreased phosphorylation of IkB-alpha at ser32/36, resulting in reduced proteosome mediated degradation. As expected, translocation of RelA/p65 to the nucleus was also impaired, resulting in decreased DNA binding activity as evidenced by electrophoretic mobility shift assay (EMSA). Interestingly, studies with a biotinylated analog also show that PTL appears to directly bind p65, we also observed a decreased phosphorylation of p65 at serine 536, an event mediating the transcriptional activity of DNA-bound p65. Inhibition of the NF-kB pathway by parthenolide also resulted in very significant inhibition of one of its well-known downstream target genes, ICAM-1 (CD54) at mRNA, protein and surface expression levels. Whether reduced ICAM-1 expression affects the biology of AML cells is as yet unknown. However, given the known role of ICAM-1 in integrin signaling, we propose that loss of ICAM-1 via NF-kB inhibition may impair the ability of AML cells to interact with their environment.
Taken together, this study further elucidates the mechanisms by which PTL mediates pro-apoptotic activity in primary AML cells. PTL induces activation of p53 pathway and therefore knockdown of p53 by shRNA results in partial rescue from PTL mediated cell death. PTL also inhibits the NF-kB pathway, which includes binding of PTL to both IKK-beta and RelA/p65, which leads to decreased phosphorylation of IkB-alpha and reduced DNA binding of p65. In addition, we have discovered the ICAM-1 expression in AML cells is regulated by NF-kB, and that loss of NF-kB DNA binding activity results in loss of ICAM-1 expression.
Disclosures:
No relevant conflicts of interest to declare.
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