Interference with the function of MYC – novel insights into the consequences of class I HDAC inhibition in Group 3 Medulloblastoma

High-risk medulloblastoma (MB) is a deadly disease with poor overall survival despite aggressive multi-modal treatment including surgery, irradiation and chemotherapy. Survivors suffer from important treatment-associated morbidity. Patients with Group 3 MB tumors with an amplification or high expression of the transcription factor MYC show particularly poor outcome. We have previously shown that MYC amplified Group 3 MB cell lines are highly susceptible to a pharmacological inhibition of class I histone deacetylases (HDACs) compared to MB cell lines without MYC amplification and low MYC protein levels. We here explore the potentially sensitizing effect of MYC to HDAC inhibition.

Three established MB cell lines, MYC amplified as well as single copy, were treated with pan- and class I-selective HDAC inhibitors. mRNA and protein expression of MYC and HDAC2 were analyzed, as well as the acetylation level of MYC protein and MYC protein stability. Gene expression profiling was determined in a MYC amplified cell lines. The direct interaction of HDAC2 and MYC protein was analyzed in co-immunoprecipitation and mass spectrometry studies. Chromatin immunoprecipitation with pulldown of MYC or HDAC2 and subsequent DNA-sequencing (ChIP-Seq) was performed in a MYC amplified cell line.

No significant alteration of MYC mRNA levels after class I HDAC inhibition or HDAC2 knockdown were observed. Treatment with class I HDAC inhibitors coincided with increased levels of acetylated MYC protein, acetylated MYC protein showed an increased half-life compared to non-acetylated MYC protein. Gene set enrichment analysis (GSEA) revealed a significant downregulation of MYC target genes 6 hours after treatment with the class I selective HDAC inhibitor MS-275. MYC and HDAC2 were found to be localized in a protein complex together with other potential drug targets. MYC and HDAC2 co-localize on the DNA with an overlap of DNA binding sites of 80%.

Our data suggests that inhibition of class I HDACs leads to a loss of transcriptional activity of MYC protein, mediated by MYC acetylation and subsequent inhibition of degradation. Inhibition of MYC protein turnover has previously been shown to be negatively correlated with its transcriptional activity. The direct interaction of MYC/HDAC2 in a multi-protein complex localized on the DNA represents a putative epigenetic target for novel combinatorial treatment approaches.