Identifying the mechanistic differences between hypomethylating agents for the treatment of peripheral T cell lymphoma

Abstract

Introduction: Peripheral T-cell lymphomas (PTCL) are a rare and heterogeneous group of malignancies that share a unifying feature of epigenetic dysregulation. Recent evidence suggests that PTCL, unlike other forms of cancer or even lymphoma, exhibits a marked vulnerability to hypomethylating agents (HMAs) alone and especially in combination with HDAC inhibitors. HMAs like azacitidine (AZA) and decitabine (DAC) have been shown to reverse transcriptional repression secondary to hypermethylation. Cladribine (CLAD) is FDA-approved for the treatment of hairy cell leukemia and is postulated to inhibit both DNA and histone methylation. Here, we sought to define the mechanistic differences between different DNA HMAs in order to inform a rationale on the optimal combinations that might exploit the epigenetic vulnerabilities of PTCL. Methods: Cell viability, caspase activity assay, and western blotting (WB) were performed on a panel of T-cell lymphoma cell lines to identify the effects of AZA/DAC/CLAD on cell survival, apoptosis, and DNA methyltransferase (DNMT) levels. Liquid chromatography-based mass spectrometry (LC-MS) was done to quantify 5-methyl cytosine (5-mC) levels. Results: Cell viability analysis confirmed that the dose and time-dependent sensitivity of the six PTCL cell lines differed following exposure to AZA/DAC/CLAD, where CLAD showed the lowest IC50 value (0.1–1 µM) across all cell lines, followed by AZA (IC50: 1–10 µM), and DAC had the least effect on cell viability (IC50: >100 µM, except T-ALL). A differential threshold of caspase activation was observed across the PTCL cell lines where CLAD showed the most superior caspase activity leading to the highest apoptotic potential. However, WB analysis showed that after 24 hours of treatment, DNMT1 and DNMT3A protein levels were significantly depleted at low concentrations of DAC (<0.01 µM) compared to AZA whereas CLAD did not affect the DNMT1 and DNMT3A levels. LC-MS-based quantification of 5-mC showed a similar relationship in methylation, with initial changes observed at doses >0.01 μM of DAC and >0.1 μM of AZA/CLAD. Taking the short half-life of AZA/DAC into account, cell viability assays with daily addition of AZA/DAC showed increased cell cytotoxicity for AZA/DAC treated samples, which was comparable to CLAD. Ongoing analyses of gene expression and metabolism of the universal methyl donor S-adenosylmethionine (SAM) will help to understand the mechanistic difference between these HMAs. Conclusions: These data suggest that the HMAs have a distinct mechanism of action for hypomethylation. While AZA/DAC induces hypomethylation by depletion of DNMTs, CLAD acts in a DNMT depletion-independent pathway. A mechanistic understanding of AZA/DAC/CLAD will inform how best to use them in clinic as well as assist in the strategic development of biological correlates to further support their therapeutic application. The research was funded by: Translational Orphan Blood Cancer Research Initiative Fund and RO1 # FD-R-006814-01. Keywords: Genomics, Epigenomics, and Other -Omics, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.