Hells (LSH) Controls Gene Expression in T-Cell Lymphomas By Regulating RNA Polymerase II with a Bimodal Function

Chromatin modifiers are emerging as determinants of many types of cancer, including anaplastic large cell lymphoma (ALCL), a family of highly heterogeneous T-cell lymphomas for which therapeutic options are still limited. We recently identified the DNA helicase HELLS - a member of the SWI/SNF2 family- as a genetic vulnerability of ALK^- ALCLs. HELLS is a multifunctional chromatin remodeler that affects genomic instability. Although its transcriptional function has been suggested, no clues on how HELLS controls transcription are currently available.

To explore its transcriptional function in ALCLs, we integrated HELLS Chromatin immunoprecipitation‐sequencing (ChIP‐seq) analysis in the TLBR-2 cell line with RNA-sequencing data TLBR-2 HELLS^KD and control cells. Out of 729 genes significantly affected by HELLS KD, 467 genes (64% of the total) resulted directly bound by HELLS. We termed these genes HELLS-direct genes (HDGs). Analysis of HELLS binding profile at HDGs site showed that 67% of HELLS binding sites at HDGs has intragenic localization being associated with promoters, introns, and exons. Gene-set enrichment analysis of HDGs revealed several pathogenic processes associated with deregulated genes including JAK/STAT signaling pathway, inflammation mediated by chemokines, and cytokines signaling pathway. To assess the contribution of HELLS to the transcription of its HDGs, the distribution of RNA‐PolymeraseII (RNAPII) was investigated by ChIP‐seq in TLBR-2 HELLS^KD and control cells. We observed that HELLS depletion caused a bimodal alteration of RNAPII. On genes belonging to biological processes involved in the dysregulation of circuitries that controls T-cells dependent host interactions (n=195, 40% of HDG promoters), the depletion of HELLS caused a significant impairment of RNAPII recruitment suggesting that HELLS partakes in transcription regulation of these pathways acting as a co-transcriptional factor. Whereas, on the remaining 60% of HDGs (n=272), which are enriched for selective biological functions including cytoskeleton regulation and DNA metabolic processes, we observed a selective loss of RNAPII signal in the regions immediately downstream of the TSS coherent with a potential proximal promoter pausing of RNAPII. Target analysis of phospho-CTD modification ser2P of RNAPII confirmed the RNAPII pausing after HELLS depletion. Stacked RNAPII during elongation drives DNA-RNA hybrids (R-loops) formation and it is a primary source of DNA break accumulation. We hypothesized that HELLS could promote RNAPII elongation by relieving R-loops, thereby decreasing potential obstacles to the transcription machinery. We investigated the effect of HELLS depletion on R-loops accumulation by performing immunofluorescences using the S9.6 antibody to detect R-loops in control and HELLS^KD cells. We observed a significant increase in the nuclear intensity of R-loops in several models of ALCL HELLS^KD cells. The increased S9.6 signal was accompanied by a different pattern of nuclear Ser2P-RNAPII with clusters adjacent to R-loop structures suggesting that R-loops are associated with stalled transcription. After HELLS KD, we also observed an overall increase in the intensity of the nuclear γH2AX signal – used as a readout of DNA damage accumulation- and a significant increase in the formation of γH2AX foci in all cell lines tested. The increase in γH2AX foci was found in S9.6 positive cells and was associated with a distinctive pattern of ser2P-RNAPII. By using reporter-GFP-based assays, we investigated which pathway mediates HELLS ability to repair DNA damage discovering that the loss of HELLS results in decreased NHEJ and MMEJ efficiency (37% and 36% respectively over time).

To validate the clinical impact of HELLS in vivo, we interrogated a cohort of 44 ALCL patients using the NCounter platform. Gene expression analysis showed that HELLS was significantly associated with its HDGs showing a positive correlation with 68% of genes and a negative correlation with 32% of genes in ALK^-ALCL samples.

Our data indicate that i) HELLS works with a bi-modal function in controlling gene expression by regulating both the initiation and the elongation of RNAPII and ii) the different work modalities underline different biological consequences. At the crossroads between transcription and DNA repair, HELLS is as an attractive target for developing novel treatments in hematological malignancies