Introduction.The ETS1 and FLI1 transcription factors are both located in the 11q24.3 region, which is commonly amplified in 25% of diffuse large B cell lymphomas (DLBCL) (Bonetti et al, 2013) and 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).

Methods.To explore interaction partners, protein precipitation was conducted utilizing streptavidin beads to capture strep-tagged ETS1 in the ABC DLBCL 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 and smallRNASeq after DDX21 siRNAs silencing in four DLBCL cell lines, two derived from ABC (HBL1 and U2932) and two from GCB (OCI-Ly1 and VAL). Direct targets were validated by DDX21 chromatin immunoprecipitation (ChIP).

Results. The ETS1 interactors included proteins associated with RNA processing. Among these, we focused on the novel interactor DDX21, an RNA helicase also regulated by pETS1 (Chung et al, 2023) and FLI1 (Sartori et al, 2021). DDX21 was found differentially expressed between GCB and ABC DLBCL, with higher expression in the latter (P<0.001). When we silenced DDX21 with siRNAs, toxicity was seen in ABC cell lines. To better characterize the role of DDX21 as a transcriptional factor in DLBCL we obtained RNASeq transcriptome after DDX21 silencing. Transcriptional profiling and functional annotation of transcripts regulated by DDX21 revealed genes coding proteins involved in cell cycle (FDR <0.001), ribosomes (FDR <0.001) and immune evasion (FDR <0.001). Based on the role of DDX21, smallRNAseq was also performed and miRNAs of some members of the let7 family (hsa-let-7d, hsa-let-7e and hsa-let-7f) were found downregulated by DDX21, together with miR-532. Among the upregulated miRNAs miR-222 and miR-155 were the most interesting. Different snoRNAs were also found deregulated, SNORA37 was further validated as a DDX21 upregulated direct target by RNA and ChIP qPCR.

Conclusions. In ABC DLBCL, 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 ABC lymphoma cells by regulating cell cycle, RNA processing and immune evasion. Several miRNAs and snoRNAs were found deregulated by DDX21. 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, Stefano Giuffrida, Andrea Rinaldi, Margot Thome Miazza, Francesco Bertoni. The RNA helicase DDX21 cooperates with ETS1 and FLI1 in cell cycle regulation, immune evasion and small nucleolar RNA processing to sustain the survival of DLBCL cells [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-015.