The RNA helicase DDX21 cooperates with ETS1 and FLI1 in cell cycle regulation and small nucleolar RNA processing to sustain the survival of DLBCL cells

Introduction: We have previously shown that the two ETS transcription factors ETS1 and FLI1, co-mapped in the 11q24.3 region, are recurrently gained in up to 25% of diffuse large B cell lymphomas (DLBCL), and largely co-regulate a series of genes involved in B cell signaling, differentiation and cell cycle (Bonetti et al., 2013; Priebe et al., 2020; Sartori et al., 2021). ABC-DLBCL is a more aggressive subtype of DLBCL compared to GCB-DLBCL, and it is associated with poor outcomes when treated with a standard therapy. As a result, there is an urgent need to elucidate new therapeutic venues for this dismal malignancy. While FLI1 is expressed at a higher level in DLBCL of the germinal center B-cell (GCB) type than in the activated B-cell-like (ABC) DLBCL, ETS1 is more expressed in the latter subgroup. We and others have reported preclinical anti-tumor activity in lymphomas and other tumors with small molecules blocking the binding of ETS factors and RNA helicases (Erkizan et al., 2009; Spriano et al., 2019). In this study, we identified additional therapeutic targets related to these transcription factors, by investigating the ETS1 interactome in ABC DLBCL. Methods: Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) was done on proteins obtained with pull down of strep-tagged ETS1. Proteins with a spectral count above 4 were considered candidate interactors, and validated by normal and reverse co-immunoprecipitation experiments. RNASeq and small RNASeq analysis was done after DDX21 siRNAs silencing in 2 ABC (HBL1 and U2932) and in 2 GCB (OCI-Ly1 and VAL) DLBCL cell lines. In addition, we performed ChIPSeq for DDX21 on the same 4 cell line models. Results: Proteins related to RNA processing, more specifically in spliceosome (NOP56 and ALYREF) and in ribosome biogenesis (SF3B1 and DDX21), were among the identified ETS1 interactors in the ABC DLBCL HBL-1 cell line. We focused on the novel ETS1 interactor DDX21, an RNA helicase also regulated by FLI1 (Sartori et al., 2021). DDX21 appeared more expressed in ABC than GCB DLBCL (P < 0.001) in 4 datasets (GSE98588, n = 117; phs001444.v2.p1, n = 432; GSE95013, n = 33; GSE10846, n = 350). When we silenced DDX21 with siRNAs, toxicity was seen in ABC (U2932) and not in GCB (OCI-Ly1) cell lines. Our results indicate DDX21 is involved in regulating proteins involved in cell cycle (FDR < 0.001), ribosomes (FDR < 0.001), spliceosome (FDR < 0.001) and small nucleolar RNAs (snRNAs). Conclusions: In ABC DLBCL, ETS1 interacts with proteins involved in spliceosome and in ribosome biogenesis, including DDX21. Highly expressed in ABC than GCB DLBCL, DDX21 sustains the survival of lymphoma cells by regulating cell cycle and RNA processing. Targeting the interaction between ETS1 and DDX21 represents a novel therapeutic modality against ABC DLBCL. The research was funded by: Swiss Cancer Research grant KLS-3580-02-2015 (to FB) and by Rotary Foundation grants GG1639200 and GG1756935 (to GS) Keywords: Aggressive B-cell non-Hodgkin lymphoma, Genomics, Epigenomics, and Other -Omics, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.