ENHANCER RNAS (ERNAS) PLAY A ROLE IN THE RESPONSE TO SMALL MOLECULES AND IN THE DEVELOPMENT OF ACQUIRED RESISTANCE IN MARGINAL ZONE LYMPHOMA (MZL)

Abstract

Introduction: The therapeutic options for MZL patients with disseminated disease include chemotherapy, rituximab alone or rituximab with chemotherapy but several small molecules are also available. Resistance development is always an issue. Stable non-genetic resistance can be acquired by transcriptional adaptation through alternative pattern of enhancers sustaining the expression of survival genes. Most active enhancers are also transcribed in long noncoding RNAs (lncRNAs) called eRNAs. They play a regulatory role helping the correct chromatin conformation required to transactivate promoters. eRNAs can occasionally be stabilized and regulate distal loci in trans. We studied the contribution of eRNAs, but also other lincRNAs, expressed in MZL in the response to agents as inhibitors of PI3K (copanlisib, umbralisib), BTK (ibrutinib), BCL2 (venetoclax) or anti CD20 (rituximab). In particular, for ibrutinib we took advantage of a resistant cell line in vitro stabilized by prolonged exposure to the drug. Methods: To identify eRNAs expressed in MZL, we applied de novo reconstruction to total RNA-Seq profiling of VL51 parental, ibrutinib-Res and idelalisib-Res derivatives (Arribas et al., Haematologica 2022). We looked for novel and known reconstructed transcripts associated to the superenhancers (SEs), identified by ROSE algorithm. We associated to these eRNAs a TSS derived from publicly available CAGE data sets. We designed 5825 paired gRNAs against 659 targets (312 eRNAs, 255 lincRNAs) and, as controls, 90 essential and 30 not expressed genes. The pgRNAs were included in a custom lentiviral library to infect VL51 expressing dCas9KRAB-ZIM3 mCherry. Cells were kept for 14 days under DMSO, ibrutinib, rituximab, copanlisib, umbralisib or copanlisib/venetoclax and gRNAs dropout was measured. Results: Unsupervised clustering of eRNAs differentially enriched at day 14 ver sus day0 discriminated parental and ibrutinib-Res cells, indicating the dependency of the cells on different transcriptome program. A series of eRNAs and lincRNA were significantly enriched after DMSO (12 and 19, respectively) ibrutinib (27 and 28), rituximab (12 and 18), copanlisib (8 and 17), umbralisib (12 and 13) or copanlisib/venetoclax (14 and 15). The inhibition of transcription of those molecules in some cases represented a proliferative advantage under exposure to the drug, in other cases a disadvantage. For further investigation, we considered eRNAs essential under treatment since they may regulate pathways involved in therapeutic escape, as the calcium-calcineurin pathway, or G-protein coupled receptors or molecules already known to interact with epigenetic regulators as EZH2. Conclusions: Individual eRNAs revealed to be essential in drug response, in a treatment specific manner and to play a role in resistance development, as regulator of relevant pathways. Encore Abstract—previously submitted to regional or national meetings (up to <1000 attendees) The research was funded by: Work supported from the Swiss National Science Foundation (SNSF Sparks CRSK-3_190808 and SNSF 310030_197466). Keywords: genomics, epigenomics, and other-omics, molecular targeted therapies, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.