Targeting hells‑mediated chromatin accessibility to sensitize ALK‑negative anaplastic large cell lymphoma to JAK/STAT and DNA‑PK inhibition

Abstract Chromatin remodelers regulate DNA-nucleosome interactions, influencing chromatin structure, accessibility, and gene expression (Gourisankar, S., Nat Rev Genet, 2024). Their dysregulation is frequently observed in cancer and often represents a therapeutic vulnerability (Johnstone, S. E., Science, 2022). HELLS, a key chromatin remodeler, maintains genome stability and regulates gene expression in aggressive T-cell lymphomas, such as ALK-negative anaplastic large cell lymphoma (ALK- ALCL) (Fragliasso V, Leukemia 2020; Tameni A., Cell Death Dis. 2021). Previous studies have demonstrated that HELLS knockdown sensitizes ALCL cells to chemotherapy, supporting its potential as a therapeutic target (Tameni A., Nucleic Acids Res. 2024). This study aimed to define the transcriptional role of HELLS and evaluate its therapeutic relevance in ALCL. To achieve this, we analyzed formalin-fixed, paraffin-embedded biopsies from a cohort of 44 ALCL patients and performed an integrated in vitro multi-omics analysis (RNA-seq, ChIP-seq, ATAC-seq, and Connectivity Map), alongside functional assays. To understand how HELLS coordinates and supports transcription, we integrated HELLS Chromatin immunoprecipitation-sequencing (ChIP-seq) data from the TLBR-2 cell line with RNA-sequencing data from TLBR-2 HELLS-knockdown (KD) and control cells. Of the 729 genes significantly affected by HELLS KD, 467 (64%) were identified as direct HELLS targets based on ChIP-seq analysis. Gene-set enrichment analysis of these HELLS-direct genes (HDGs) revealed their association with diverse biological processes, including the JAK/STAT signaling pathway and chemokine- and cytokine-mediated inflammatory signaling. The clinical relevance of HDGs was confirmed by nCounter profiling in a cohort of 44 ALCL patients (15 ALK+, 29 ALK- ALCL). To assess the contribution of HELLS to HDG transcription, we investigated the distribution of RNA Polymerase II (RNAPII) via ChIP-seq in TLBR-2 HELLS-KD and control cells. RNAPII ChIP-seq data revealed RNAPII elongation defects in approximately 60% of HDGs and altered RNAPII occupancy in ~40% of HDGs following HELLS depletion. Gene ontology analysis of this latter group of HDGs highlighted an enrichment in immune-related pathways, including T-cell-mediated immunity, cytokine signaling, and JAK/STAT signaling. Furthermore, ATAC-seq and H3K4me3 profiling indicated that HELLS promotes chromatin accessibility and transcriptional activation at immune-related loci. To evaluate the therapeutic relevance of HELLS, we employed a drug repurposing approach based on the HDG signature. This led to the identification of 10 classes of synergistic pathways, with PI3K, JAK/STAT, and DNA-PK emerging as top-scoring synergistic targets. In several ALCL cell lines, HELLS depletion synergized with an IC20 dose of Ruxolitinib (a JAK inhibitor) or AZD7648 (a DNA-PK inhibitor), resulting in significant synthetic lethality compared to single-agent treatment or control cells. In conclusion, this study demonstrates that HELLS drives the expression of immune-related genes through chromatin remodeling in aggressive ALK- ALCL. Its inhibition uncovers combinatorial vulnerabilities, providing a rationale for dual-targeted therapies involving JAK/STAT or DNA-PK pathway inhibition.