Introduction. The two ETS transcription factors ETS1 and FLI1 are co-mapped in the 11q24.3 region recurrently gained in up to 25% of diffuse large B cell lymphomas (DLBCL) (Bonetti et al, 2013). The two proteins largely co-regulate a series of genes involved in B-cell signaling, differentiation and cell cycle (Priebe et al, 2020; Sartori et al, 2021). 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), and encouraging clinical data are coming from the first trials enrolling Ewing Sarcoma patients (Ludwig et al, 2020). Here, proteomic experiments were performed to investigate ETS1 interactome in DLBCL. Methods. To investigate interaction partners, protein precipitation was performed using streptavidin beads to pull down strep-tagged ETS1 in DLBCL ABC cell line, HBL-1, followed by Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS). Shotgun proteomics analysis identified biologically relevant ETS1 protein interactors, with a spectral count above four, that were validated by normal and reverse co-immunoprecipitation experiments. Transcriptome analysis was done via RNASeq after DDX21 siRNAs silencing in four DLBCL cell lines, two derived from ABC (HBL1 and U2932) and two from GCB (OCI-Ly1 and VAL). Results. Identified ETS1 interactors in the ABC DLBCL HBL-1 included proteins related to RNA processing, more specifically in spliceosome (NOP56 and ALYREF) and in ribosome biogenesis (SF3B1 and DDX21). Among these, we focused on the novel interactor DDX21, an RNA helicase also regulated by FLI1 (Sartori et al, 2021). DDX21 was found differentially expressed between GCB and ABC DLBCL, with higher expression in the latter (P<0.001) (GSE98588: n= 117 cases, phs001444.v2.p1: n= 432 cases, GSE95013: n= 33 cases, and GSE10846, n= 350 cases). When we silenced DDX21 with siRNAs, toxicity was seen in both ABC and GCB cell lines. To better characterize the role of DDX21 as a transcriptional factor in DLBCL and to further investigate its link with ETS1 and FLI1 we obtained RNASeq transcriptome after DDX21 silencing. Transcriptional profiling and functional annotation of transcripts regulated by DDX21 identified genes coding proteins involved in cell cycle (FDR <0.001), ribosomes (FDR <0.001) and spliceosome (FDR <0.001).Conclusions. ETS1 interacts with proteins involved in spliceosome and in ribosome biogenesis, including DDX21. More expressed in ABC than GCB DLBCL, DDX21 appears essential for the survival of lymphoma cells by regulating cell cycle and RNA processing. The interaction between ETS1 and DDX21 offers an additional therapeutic opportunity against DLBCL cells.

Citation Format: Giulio Sartori, Valdemar Priebe, Luciano Cascione, Elaine Y.L. Chung, Mélanie Favre-Juilland, Sara Napoli, Alberto Arribas, Andrea Rinaldi, Margot Thome Miazza, Francesco Bertoni. The RNA helicase DDX21 cooperates with ETS1 and FLI1 in cell cycle and RNA processing [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A14.