Epiproteomic Landscape and Histone Code of Cutaneous T-Cell Lymphoma/Sézary Syndrome

Background: The genetic basis and the genome-wide abnormalities underlying most forms of cancer are being comprehensively annotated in many human malignancies. However, the contribution of epigenetic aberrations, particularly the post-translational modifications (PTM) of histone tails has not been investigated. This is largely due to lack of approaches to comprehensively interrogate the status of the numerous PTMs that define histone marks which control gene expression and complex biologic processes such as cancer. Sézary Syndrome (SS) is an aggressive form of cutaneous T-cell lymphoma (CTCL) characterized by poor outcomes and complex genetic alterations frequently targeting epigenetic regulators and chromatin remodelers. CTCLs represent the first FDA-approved disease for treatment using histone deacetylase inhibitors (HDACi) such as romidepsin, but the direct consequences of this treatment on histone PTMs remain unknown. Here we define the histone PTM signatures of CTCL/SS by using a novel unbiased strategy leveraging tandem mass spectrometry (MS/MS)-based quantitative proteomics to study the comprehensive combinatorial histone PTM code in a cohort of primary SS samples, and 4 CTCL/SS cell lines in comparison with normal reactive CD4⁺ T-lymphocytes from healthy individuals.

Methods: CD4⁺ T-cells were isolated using CD4 immunomagnetic beads from PBMC's of healthy volunteers (n=8) or patients with diagnosed SS (n=20). CD4⁺ cells were frozen and subsequently analyzed in batches. The CTCL/SS-derived cell lines (HH and MJ, HuT78 and H9) were also analyzed each with three biological replicates. Histones were acid extracted from isolated nuclei. Total bulk histones were propionylated and trypsinized prior to MS analysis. Nano-scale liquid chromatography followed by -tandem mass spectrometry (nano LC-MS/MS) data acquisition was performed in an Orbitrap Fusion^TM Tribrid^TM MS in technical triplicates for each sample with a data-independent acquisition (DIA)-method using a 50 m/z quadrupole-isolation windows that steps across the 200-1500 m/z ranges. Data analysis was performed using EpiProfile for single and combinatorial histone PTM analysis and quantification. Orthogonal validation for selected modifications was performed using western blotting and single-cell mass cytometry by time of flight (CyTOF) analysis.

Results: Our quantitative epiproteomic strategy interrogated the relative ratios of a total of 228 histone PTM combinations, and 20 histone variants in a well-characterized cohort of SS patient samples, four CTCL cell lines and a cohort of normal primary CD4⁺ T-lymphocytes isolated from healthy individuals. With this approach, we were able to identify and quantify 23 unique histone peptides on histone H4, 105 on histone H3, 80 on histone H2A, 12 on histone H2B and 28 on histone H1 across every sample. Pearson correlation and principal component analysis of overall histone PTMs profiles across all samples provided accurate discrimination. We found a distinct pattern of histone PTMs in both SS patient samples and CTCL cell lines that were strikingly different from CD4⁺ T-cells obtained from healthy individuals. Notably, differences between cell lines and primary patient samples are more marked than those within individual SS primary patient samples, implying that cell lines may poorly recapitulate some aspects of in vivo biology. Unsupervised hierarchical clustering of the primary patient samples, cell lines and the normal CD4⁺ T-cells performed based on abundances of the identified histone PTMs revealed disease-specific ubiquitous marks as well as disease-specific unique marks for SS. Among the analyzed histone PTMs, H3K27ac, H3.3K27ac, H4K8ac, H4K20ac, H3K4me3, H3K18me1, H3K79me3 and H4K20me3 were distinct between healthy and CTCL CD4⁺ T-cells. Selected differential marks such as H4K20me3 were orthogonally corroborated by Western blotting and CyTOF mass cytometry analysis.

Conclusions: For the first time, we have defined the histone code of CTCL/SS using global mass spectrometry based quantitative proteomics with high specificity and sensitivity. The results of our MS-based epiproteomic profiling revealed disease-specific histone PTM signatures and opportunities to exploit tractable changes induced by HDACi treatment as clinically actionable molecular biomarkers and therapeutic targets.