The DNA Helicase Hells Is a New Unconventional Player in ALK- Anaplastic Large Cell Lymphoma Biology
Authors: Fragliasso Valentina , Tameni Annalisa , Verma Akanksha , Bareja Rohan , Heavican Tayla , Iqbal Javeed , Manzotti Gloria , Fiore Danilo , Mularoni Valentina , Chan Wing C. , Merli Francesco , Elemento Olivier , Ciarrocchi Alessia , Inghirami Giorgio 
Published: November 2019

Anaplastic Large Cell Lymphoma (ALCL) is an aggressive subtype of T-cell lymphoma. ALCLs are stratified based on the presence of anaplastic lymphoma kinase (ALK) translocations. ALK negative (ALK-ALCLs) are heterogeneous subtypes characterized by higher aggressiveness and poorer outcome than ALK+ALCL. The molecular and genetic asset of ALK-ALCLs has recently begun to emerge (i.e. JAK/STAT3 activating mutations, DUSP22, TP63, TP53 and IRF4 rearrangements), but an exhaustive picture of the molecular drivers leading to ALK-ALCLs transformation, progression, and immune evasion is still lacking.
Long non coding RNAs (LncRNAs) are transcripts longer than 200 nucleotides with different regulatory functions ranging from transcriptional regulation to structural functions, which are emerging as relevant players in many cellular processes including cancer.
Using deep RNA-sequencing profiling combined with de novo transcriptome assembly, we explored and validated the potential contribution of non-coding RNAs in a large series of ALCLs. 24 lncRNAs were found specifically enriched in ALCL samples. Among these, a 70Kb chromatin associated lncRNA (BlackMamba) was identified as preferentially associated with the ALK-ALCLs subtypes and was shown to be a target of the JAK/STAT3 signaling. BlackMamba was overexpressed in ALK-ALCL patient samples as well as in patient-derived tumor xenograft (PDTX) models. Its shRNA mediated knockdown (KD) in ALK-ALCL cells reduces cell proliferation and clonogenicity. BlackMamba KD cells also showed a remarkable increase in the number of multinucleated cells (without ploidy alteration) providing evidence that this lncRNA may be required for correct cytokinesis. To better characterize the role of BlackMamba we performed RNA-sequencing profiling in BlackMamba KD ALK-ALCL cells showing that this lncRNA affects primarily the expression of genes involved in cytoskeleton organization and remodeling.
Noticeably, the DNA-helicase HELLS emerged among the most relevant BlackMamba target gene in ALK-ALCL cells and patients. Loss of BlackMamba causes profound reduction of HELLS concomitant with a reorganization of chromatin markers (reduction of K4me3 and increase of K27me3) in the HELLS locus.
To test whether HELLS enforces BlackMamba-mediated transcription, we silenced HELLS by shRNA approach in ALK-ALCLs. Noticeably, loss of HELLS led to a reduction in cell growth, a delay in the duplication rate and a dramatic drop of clonogenicity potential of ALK-ALCL cells. This phenotype was also associated with an increased number of multinucleated cells, phenocopying the BlackMamba KD cells. We also showed that, HELLS KD causes expression changes in a subset of cytoskeleton-related genes previously identified as BlackMamba targets confirming that HELLS is a crucial mediator of BlackMamba function. Indeed, ectopic over-expression of HELLS in BlackMamba KD cells rescued the cell growth defects induced by the loss of lncRNA mitigated the polynucleation phenotype and restored the baseline expression of BlackMamba target genes.
Being established that lncRNAs affect gene expression by recruiting chromatin remodeling complexes to target promoters or enhancers we also investigated whether BlackMamba may associates with HELLS and dictates its chromatin positioning in ALK-ALCL cells.
By RNA-immunoprecipitation (RIP) we showed that HELLS binds to BlackMamba at in two distinct regions the 3'-end of the lncRNA. Next, by Chromatin Immunoprecipitation (ChIP) we demonstrated that HELLS is associated to BlackMamba target gene promoters exclusively in ALK-ALCLs in which BlackMamba is expressed.
Collectively, these data demonstrate the existence of a new lncRNA-dependent mechanism controlling the recruitment of HELLS on chromatin sites and its expression in lymphomas. The axis BlackMamba-HELLS sustains the neoplastic phenotype of ALK-ALCL representing a potential vulnerability of ALCL cells.

Disclosures
Merli: Mundipharma: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Research Funding; Sandoz: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Janssen: Honoraria; Takeda: Honoraria, Other: Travel Expenses; Gilead: Honoraria.