Abstract 3245: Use of hypomethylating agents to reprogram the epigenome in large granular lymphocyte leukemia
Authors: Sabzevari Ariana , Pal Ipsita , Deddens Tess , Fox Todd , Ratan Aakrosh , Feith David J. , Loughran Thomas P. 
Published: March 2024

Large granular lymphocyte (LGL) leukemia is a rare heterogeneous blood cancer that is characterized by the clonal lymphoproliferation of CD8+ T cells (T-LGL leukemia) or, less frequently, natural killer cells (NK-LGL leukemia). These cells evade activation induced cell death, allowing them to accumulate as highly cytotoxic lymphocytes that contribute to autoimmune attacks, neutropenia, and anemia. Frontline treatments are limited to repurposed, nonspecific immunosuppressive agents with limited efficacy and incomplete response rates. As there are no FDA-approved antineoplastic agents for LGL leukemia, our research explores novel therapeutic avenues for this orphan disease. Somatic activating mutations in the transcription factor STAT3 are the most common molecular alteration found in &gt;50% of patients, and STAT3 signaling is dysregulated in almost all LGL leukemia patients. In the largest molecularly profiled LGL leukemia patient cohort to date, we found that STAT3 mutations frequently co-occur with mutations in chromatin and epigenetic modifying genes, suggesting crosstalk and cooperativity between STAT3 activation and epigenetic dysregulation. Moreover, TET2, which promotes DNA demethylation, is frequently inactivated in LGL leukemia via loss-of-function mutations and promoter hypermethylation. As DNA hypermethylation is an emerging hallmark of LGL leukemia, we evaluated the therapeutic potential of two epigenetic agents, azacitidine (AZA) and cladribine (CLAD), that directly and indirectly inhibit DNA hypermethylation, respectively. We hypothesize that AZA and CLAD will 1) exhibit cell intrinsic efficacy, 2) reprogram the epigenome of LGL leukemia through DNA hypomethylation to normalize aberrant gene expression, and 3) induce cytokine-mediated immune responses. AZA and CLAD cytotoxicity was assessed by the Cell Titer Glo viability assay in human LGL leukemia cell lines. Each exhibited potent anti-leukemic effects with IC50 values in the low micromolar and nanomolar ranges. Western blotting analysis demonstrated the decrease in protein expression of DNA methyltransferases and phosphorylated STAT3 levels with treatment in a time- and dose-dependent manner. We also explored the immunomodulatory effects of these hypomethylating agents since LGL leukemia is a pro-inflammatory malignancy with cytotoxic lymphocytes. Preliminary findings using the Isoplexis platform suggest that AZA and CLAD induce production of anti-inflammatory cytokines in LGL leukemia cells. Ongoing studies will identify differential methylation, chromatin accessibility, and gene expression using RRBS, ATAC-seq, and RNA-seq of AZA or CLAD treated samples to define the mechanisms and consequences of DNA demethylation. Uncovering the reprogramming capabilities of AZA and CLAD could allow for the potential repurposing of these inhibitors for treatment of LGL leukemia patients.

Citation Format: Ariana Sabzevari, Ipsita Pal, Tess Deddens, Todd Fox, Aakrosh Ratan, David J. Feith, Thomas P. Loughran. Use of hypomethylating agents to reprogram the epigenome in large granular lymphocyte leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3245.