Abstract
BackgroundThe small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy.ResultsNutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intracellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells.ConclusionsAltogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML.
Highlights
The small-molecule Mouse double minute 2 (MDM2) antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML)
Nutlin-3 enhances the acetylation of histone Histone H2B (H2B) and heat shock proteins Hsp27 and Hsp90 Based on the critical role of acetylation in nutlininduced p53 activation, we wanted to examine if nutlin3 could enhance the acetylation of other proteins than p53
Intracellular levels of heat shock proteins and sensitivity to nutlin-3 in primary AML cells To investigate if levels of different heat shock proteins could affect sensitivity to nutlin-3, intracellular protein levels of Hsp27, Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were quantified in Stable isotope labeling with amino acids in cell culture (SILAC)
Summary
The small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). Small-molecule MDM2 inhibitors like nutlin-3 have emerged as a potent and promising treatment option for cancers harboring wild type TP53, including AML [3,4,5], and the oral formulation of nutlin-3, RG7112, has completed the first early phase clinical trials for both solid cancers and hematological malignancies [6,7,8] These small-molecule p53 activators have demonstrated selective toxicity for cancer cells versus normal cells [4,9], and may induce reversible cell cycle arrest of normal cells to protect them from adverse effects of conventional chemotherapy [10]. The direct effect of nutlin-3 on regulation of histones and heat shock proteins has not been determined
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