Dynamic Regulation of Histone Acetylation By Nuclear Proteolysis Controls Cell Cycle Gene Expression and Chromosome Integrity in Multiple Myeloma

Transcription factors are generally short-lived proteins that undergo active turnover. The dynamic interaction of transcription factors and co-regulators with promoters and enhancers allows cells to continuously adjust gene expression. Whereas the composition and binding of transcription factors at genomic sites is the focus of a widespread research effort, relatively little is known about how these complexes are being removed by the ubiquitin-proteasome system (UPS).

Multiple myeloma (MM), the second most common hematopoietic malignancy, has become a model disease for drugs that interfere with the UPS through either blocking or facilitating protein elimination. The proteasome inhibitor Bortezomib, for instance, is used as first-line treatment in myeloma; yet, the process by which myeloma cells are killed by this drug is ill-defined. Since transcription factors are prime targets of proteasomal degradation, our research is focused on defining how proteolysis regulates transcriptional dynamics in this disease and determining the therapeutic relevance for treatment with proteasome inhibitors.

Following proteasome inhibition in multiple myeloma cell lines, we performed chromatin-immunoprecipitation for histone H3 acetylation (K27) and multiple histone deacetylases and used next generation sequencing (ChIP-seq) to identify unique gene clusters that are actively regulated by the proteasome and quantify epigenetic changes in dependence of protein turnover. Our findings reveal that cell cycle genes, particularly subsets of genes involved in centromere formation and sister chromatid segregation during mitosis, are associated with nuclear protein turnover and are transcriptionally repressed by proteasome inhibition. Notably, proteasome inhibition increased the recruitment of specific histone deacetylases (HDACs) to the promoters of genes involved in centromere formation. We are investigating the mechanisms for UPS regulation of HDAC abundance at specific genomic locations. Moreover, data analysis of a panel of multiple myeloma patients shows that the expression levels of HDACs correlate with patient survival, specifically when treated with proteasome inhibitors. We are currently exploring how histone modifications and proteasome activity crosstalk in a therapeutically relevant manner in multiple myeloma.

Finally, to determine whether epigenetic regulation of centromere stability might play a role in sensitivity or the development of resistance to proteasome inhibitors, we established and optimized long-term treatment of multiple myeloma cell lines with low doses of proteasome inhibitors. Remarkably, we observed reduced immunofluorescence staining for centromeric protein A (CENP-A) in multiple myeloma cells receiving drugs over an extended period, suggesting a loss of centromere integrity.

This research project will contribute to our understanding of epigenetic and transcriptional dynamics in MM. With our focus on the continuously changing abundance of transcription factors and co-regulators at promoters of cell cycle genes, we seek to unlock new pathways for molecular therapy, as well as identify more specific targets for treatment compared to blunt proteasome inhibition. Elucidating the mechanisms of action and resistance to proteasome inhibition will help advance future MM therapy.