Inhibitor of DNA binding 2 (ID2) is epigenetically regulated during lymphomagenesis and constitutes a novel therapeutic target in mature T-cell lymphoma Free

Mature T-cell lymphomas (TCL) are a heterogeneous group of non-Hodgkin lymphomas arising from the malignant transformation of post-thymic T and NK cells. These lymphomas are characterized by poor response to chemotherapy, high relapse rates, and dismal survival outcomes. Genomic analyses of TCL have revealed recurrent alterations affecting intracellular signaling pathways and epigenetic regulators. The frequent involvement of epigenetic modifiers suggests that epigenetic dysregulation is a critical step in TCL pathogenesis.

To identify the epigenetic changes driving TCL transformation, we analyzed chromatin modifications and gene expression profiles in murine TCL models that closely recapitulate human disease. Our data revealed that Id2, a repressor of basic helix-loop-helix proteins, is consistently overexpressed at both RNA and protein levels across multiple murine lymphoma models. Supporting this, analysis of the Cancer Cell Encyclopedia identified TCL as one of the tumor types with the highest ID2 expression, which we confirmed by Western blot in human TCL-derived cell lines. Furthermore, analysis of single-nuclei RNAseq data from a cohort of primary angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma not otherwise specified (PTCL, NOS) patient samples generated in our laboratory, demonstrated significant ID2 overexpression in a subset of cases (12/27; 44%) compared to tonsil and lymph node controls. Interestingly, all relapsed TCL cases in our cohort showed significant overexpression of ID2. Based on our data, we hypothesize that ID2 is epigenetically regulated during transformation and contributes to T-cell lymphomagenesis.

To define the mechanisms leading to ID2 overexpression in TCL, we performed H3K27ac and H3K4me1 ChIP-seq, ATAC-seq, reverse-ChIP coupled with mass spectrometry, and dual-luciferase reporter assays. These analyses identified a conserved region near the ID2 locus enriched for H3K27ac and H3K4me1 marks that co-localizes with ATAC-seq peaks and is ranked as a super-enhancer by the ROSE algorithm. This region is specifically activated in mature TCL cells compared to control CD4⁺ T cells. Reporter assays using the most conserved segment within the enhancer region showed more than twofold increased luciferase activity relative to a minimal promoter, confirming its enhancer function. Interestingly, reverse-ChIP identified CTCF and ASCL1 as candidate regulators of enhancer looping and transcriptional activation, respectively. To characterize the proteins mediating the epigenetic function of ID2 in TCL, we employed a biotin ligase proximity labeling assay (BioID) approach coupled with mass spectrometry. Our results identified known ID2 partners, including the E-proteins TCF3, TCF4 and TCF12, key regulators of T-cell differentiation, as well as novel partners including elements of the SWI/SNF chromatin-remodeling complex, suggesting that ID2 overexpression indirectly regulates transcriptional programs critical for lymphomagenesis.

To determine the functional role of ID2 in TCL, we characterized the effects of both genetic and pharmacological inhibition of ID2, using shRNA and CRISPR-Cas9 mediated knockout and novel pan-ID inhibitors AGX51 and AGXA, respectively. Loss of ID2 leads to decreased cell viability and increased apoptosis in in vitro TCL models. Interestingly, RNA-seq analysis revealed enrichment in signatures associated with glucose metabolism, suggesting a novel role for ID2 in tumor metabolic regulation. Finally, in vivo treatment with the pan-ID inhibitor AGX51 in a murine lymphoma model induced a modest but consistent reduction in tumor burden, spleen weight, and activated CD69⁺ T cells population in AGX51-treated mice compared to vehicle-treated controls.

In summary, we have identified a novel TCL-specific super-enhancer that drives ID2 overexpression in TCL and demonstrated an essential role for ID2 in supporting lymphoma proliferation and survival. More importantly, pharmacological targeting of ID2 reduces tumor burden in vitro and in vivo, highlighting its potential as a therapeutic target in mature T-cell lymphomas.