To better understand the role of histone variants and their post-translational modifications on the development, progression and treatment of chronic lymphocytic leukemia (CLL), we intend to develop and apply high mass accuracy mass spectrometry based assays to profile CLL-specific patterns of histone isoforms and conduct detailed molecular characterization to identify the specific histone isoforms present in different genetic subtypes of CLL and their impact on patient treatment and outcome. We will carry out these goals by profiling the state of histone modifications and characterize the changes in histone modifications that are induced by treatment with chromatin remodeling reagents and changes that are associated with specific genetic mutations. We will focus on processes that correlate with induction of apoptosis and differentiation in vitro and in vivo in CLL cells. The rationale is to define the changes in chromatin induced by these therapies and determine chromatin's potential to predict risk and outcome for the therapy. The techniques developed and validated in this proposal will allow basic scientists to detect and understand specific changes observed in histone isoforms and clinical-translational scientists to effectively predict outcome and apply therapies that modify histones in patients with CLL.
The aims of this application are:
Aim 1. To combine multidimensional liquid chromatography with high-mass accuracy mass spectrometry to profile histone proteins and characterize histone protein isoforms in primary malignant hematopoietic tumor cells. These techniques will be applied to profile malignant cells from genetic subtypes of CLL. We hypothesize these techniques will facilitate the characterization of histone variants and post-translational modifications and permit the targeting of specific CLL-specific sites not identifiable with currently available immunological techniques;
Aim 2. Obtain the histone profiles of CLL patients and determine the clinical relevance of these profiles relative to other biomarkers used to predict early progression of this disease. We hypothesize that the application of LC-MS based histone profiling will lead to new markers that will facilitate the stratification of CLL patients according to risk;
and Aim 3. To identify, map and characterize sites of histone post-translational modifications in malignant cells from CLL subtypes by high- mass accuracy liquid chromatography tandem mass spectrometry. We will determine the histone H1 phosphorylation isoforms present in nuclear and cytosolic extracts due to H1 release induced by flavopiridol therapy in CLL patients. We hypothesize that proteomics will allow the identification of select histone modifications that will serve as markers for mechanistic endpoints and patient outcome in flavopiridol therapy of CLL patients.
Core histones play a vital role in the regulation of cellular processes that involve access to chromosomal DNA and subsequent gene transcription. These processes are misregulated in a large proportion of cancers including chronic lymphocytic leukemia (CLL). To better understand the role of histone variants and their post- translational modifications on the development, progression and treatment of CLL, we intend to develop and apply high mass accuracy mass spectrometry based assays to profile CLL-specific patterns of histone isoforms and conduct detailed molecular characterization to identify the specific histone isoforms present in different genetic subtypes of CLL and their impact on patient treatment and outcome.
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