Abstract
Abstract 772
Chromosomal inversion inv(16)(p13.1q22) is found in approximately 12% of acute myeloid leukemia (AML) patients, and leads to the fusion of the transcription factor gene CBFb and the MYH11 gene, and encodes a fusion protein CBFβ-SMMHC. Previous studies revealed that CBFβ-SMMHC is a dominant inhibitor of core-binding factor (CBF) function, and impairs hematopoietic differentiation. Expression of CBFβ-SMMHC predisposes for leukemia transformation, however, the molecular mechanism underlying the leukemogenic function of CBFβ-SMMHC remains elusive. The tumor suppressor p53 is considered the master genomic guardian that is frequently mutated in a wide variety of tumors but is rarely mutated in inv(16) AML. Thus, we examined whether CBFβ-SMMHC fusion protein might impair p53 function. We found that p53 acetylation (Ac-p53) level was reduced in the presence of CBFβ-SMMHC fusion protein in the myeloid progenitor 32D cell line as well as in primary pre-leukemic bone marrow progenitor cells isolated from our conditional Cbfb-MYH11 knock-in (Cbfb56M/+/Mx1-Cre) mice (Kuo et al, Cancer Cell 2006, 9:1,57-68). We assessed the effect of CBFβ-SMMHC on p53 transcriptional activity by quantitative RT-PCR analysis of p53 target genes including TP53 and p21 Cdkn1a, Mdm2, Bid, Bax, Stag1, LincRNA-p21, Gadd45b in 32D cells. The result showed that expression of these p53 target genes are reduced in the presence of CBFβ-SMMHC fusion protein, consistent with the impaired Ac-p53 by CBFβ-SMMHC. To understand how CBFβ-SMMHC impairs p53 function, we tested whether CBFβ-SMMHC fusion protein might interact with the p53 protein by co-immunoprecipitation (co-IP) assays. We found that CBFβ-SMMHC fusion protein interacts with p53 both in 32D cells and primary bone marrow cells. Although CBFβ-SMMHC fusion protein is detected both in the nucleus and the cytoplasm, the complex with p53 is present exclusively in the nucleus. It has been reported that CBFβ-SMMHC interacts with histone deacetylase 8 (HDAC8) through the C-terminal SMMHC region. Therefore, we assessed the interaction between CBFβ-SMMHC, p53 and HDAC8 in 32D cell line by co-IP and sequential co-IP. We were able to detect a multimeric protein complex containing CBFβ-SMMHC, p53, and Hdac8. To access whether HDAC8 contributes to the deacetylation of p53, we used two independent small-hairpin (sh)-RNA to knock-down Hdac8 in 32D-CBFβ-SMMHC cells. Hdac8 knock-down led to robust increase in Ac-p53 levels while total p53 levels were modestly stabilized. To test whether this effect is dependent on the deacetylase function of HDAC8, we used HDAC8 selective pharmacological inhibitors (HDAC8i including PCI-34051 and PCI-48012) directed against its catalytic sites (Balasubramanian et al Leukemia 2008, 22:5,1026-34). Treatment with HDAC8i remarkably increased Ac-p53 in both control and CBFβ-SMMHC cells. Since p53 protein levels were also increased upon HDAC8i treatment, we included Mdm2 inhibitor Nutlin-3 to stabilize p53. HDAC8i treatment alone or in combination with Nutlin-3 was able to enhance Ac-p53 compared to Nutlin-3 treatment, confirming its effect in restoring p53 acetylation. Collectively, our study shows that the CBFβ-SMMHC fusion protein forms an aberrant complex with p53 and HDAC8, leading to the aberrant deacetylation and impaired activity of p53. In addition, this deacetylation of p53 conferred by CBFβ-SMMHC is mediated by HDAC8. Our study reveals a novel leukemogenic mechanism in which CBFβ-SMMHC disrupts p53 activation through aberrant protein-protein interaction and recruitment of HDAC8.
Disclosures:
No relevant conflicts of interest to declare.
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