NF-κB is a potential therapeutic target for histone deacetylase inhibitor-resistant cutaneous T-cell lymphoma

Abstract Background: Cutaneous T-cell lymphoma (CTCL) is a malignant T-cell lymphoma that proliferates and progresses in the skin. Advanced-stage CTCL has a poor prognosis, with no established curative treatment, except for allogeneic transplantation. Despite the development of novel agents, the effective treatment of advanced-stage CTCL remains a considerable unmet medical need. Histone deacetylase (HDAC) inhibitors, including vorinostat and romidepsin, are used clinically to restore the expression of tumor suppressor genes known to be epigenetically suppressed in CTCL. Although HDAC inhibitors initially demonstrate efficacy, resistance eventually emerges, which presents a substantial therapeutic challenge. Detailed mechanisms through which CTCL acquires resistance to HDAC inhibitors remain elusive. Accordingly, new strategies need to be considered to develop effective treatments for HDAC-inhibitor-resistant CTCL. Methods: To elucidate the underlying mechanisms and identify potential countermeasures, we established HDAC inhibitor-resistant CTCL cell lines via prolonged exposure to vorinostat. Specifically, the parental MyLa and MJ CTCL cell lines were subjected to continuous and progressively increasing exposure to vorinostat over an extended period to develop resistance. Subsequently, we comprehensively profiled gene expression in these cell lines using microarrays and in silico analytical approaches, such as ChIP-Atlas and Enrichr. Results: We confirmed that resistant cell lines (designated as MyLa-resistant and MJ-resistant) exhibited significantly increased IC50 values for vorinostat compared with their parental counterparts, indicating markedly reduced sensitivity to the drug. Additionally, both resistant cell lines demonstrated cross-resistance to romidepsin, a clinically relevant HDAC inhibitor. To elucidate the potential mechanisms underlying acquired HDAC inhibitor resistance, we conducted a comprehensive gene expression analysis using microarrays. In total, 83 genes were significantly upregulated in both MyLa-resistant and MJ-resistant cell lines compared with their respective parental cell lines. Next, enrichment analyses were performed using the ChIP-Atlas and Enrichr databases. ChIP-Atlas analysis indicated the potential regulation of these upregulated genes by several transcription factors, including RELA (also known as p65), HIC1, MED12, GATA3, and EP300. Similarly, enrichment analysis further highlighted RELA, along with BCL3, STAT3, SOX2, and EGR1, as candidate regulators. Among these, RELA (p65), a key subunit of NF-κB, exhibited the highest composite enrichment score, strongly suggesting its involvement in driving alterations in gene expression associated with acquired HDAC inhibitor resistance. Although reverse transcription-quantitative PCR analysis revealed only modest changes in the expression of RELA mRNA, western blot analysis demonstrated a marked increase in NF-κB p65 protein levels in both resistant cell lines relative to parental controls. This discrepancy between mRNA and protein expression suggests the involvement of posttranslational regulatory mechanisms. Given that acetylation of NF-κB p65 has been reported to stabilize the protein by inhibiting its ubiquitination and subsequent degradation, and that members of the HDAC family, namely HDAC1, HDAC2, and HDAC3, deacetylate p65, we next evaluated the acetylation status of p65. Western blotting analysis revealed elevated levels of acetylated p65 in resistant cell lines compared with those in parental cells, suggesting that the mechanism involved acetylation-mediated inhibition of p65 ubiquitination, which resulted in protein stabilization and enhanced transcriptional activity. Finally, we confirmed that HDAC inhibitor-resistant cell lines displayed heightened sensitivity to inhibition of the NF-κB pathway by bortezomib, a proteasome inhibitor that prevents IκB degradation and thereby blocks NF-κB activation, and dimethyl fumarate, an immunomodulatory and anti-inflammatory drug that suppresses NF-κB signaling by reducing the nuclear translocation and phosphorylation of p65. Conclusion: These findings suggest that aberrant NF-κB activation is a central driver of HDAC inhibitor resistance in CTCL. Our results provide a strong rationale for exploring NF-κB inhibition as a therapeutic strategy to restore or enhance the efficacy of HDAC-inhibitor-based therapies, overcome HDAC inhibitor resistance, and improve the outcomes of patients with CTCL.