ARID1A Mutations Promote Immune Evasion of Follicular Lymphoma Through Reduced FAS-Mediated Apoptosis by Hindering RUNX3-ETS1 Cooperativity.

The cell death receptor FAS and its ligand (FASLG) play a crucial role in the negative selection of B cells during the germinal center (GC) reaction. Failure to eliminate potentially harmful B cells can lead to the development B cell malignancies. Follicular lymphoma (FL) is a prototypic GC-derived B cell malignancy. Translocation and overexpression of antiapoptotic BCL2 is a hallmark of FL. Additional alterations have been shown to be clinically relevant, including mutations in ARID1A. ARID1A is part of the SWI/SNF nucleosome remodeling complex that regulates DNA accessibility (“openness”). However, the contribution of ARID1A mutations to the development and the biology of FL remains unclear. Based on a functional screen, we investigated the link between ARID1A mutations and FAS/FASLG-induced apoptosis in FL. We analyzed 151 diagnostic FL biopsies and found that ARID1A mutations were recurrent and mainly disruptive, with an overall frequency of 18%. Additionally, we observed that ARID1A mutant FL showed significantly lower FAS expression in the FL tumor cell population. Functional experiments in t(14;18)+ B lymphoma cells demonstrated that ARID1A is directly involved in the regulation of FAS, and ARID1A loss leads to decreased FAS protein and gene expression. However, ARID1A loss did not affect FAS promoter openness. Instead, we identified and experimentally validated a previously unknown co-transcriptional complex consisting of RUNX3 and ETS1 that regulates FAS expression, and ARID1A loss leads to reduced RUNX3 promotor openness and gene expression. The reduced FAS levels induced by ARID1A loss rendered lymphoma cells resistant to both soluble and T cell membrane-anchored FASLG-induced apoptosis. Furthermore, we observed an additional accumulation of ARID1A mutations in relapsed/refractory FL biopsies, indicating that we have identified a functionally and clinically relevant mechanism how FL cells can escape T cell mediated killing, which may also profoundly impact the efficacy of subsequent therapies, including CAR T cells. A total of fifteen single cell-derived clones was assayed in technical duplicates in OCI-Ly1 and OCI-Ly8. For each cell line three control clones (ctrl#1,ctrl#7, and ctrl#9 in OCI-Ly1, ctrl#3, ctrl#6, and ctrl#11, in OCI-Ly8) transduced with the empty pSpCas9(BB)-2A-GFP backbone, were analysed. In OCI-Ly1, two single cell clones with heterozygous ARID1A mutation (#F9 and #F10), and three ARID1A knock-out clones (#E8, #G1, and #H3), were sequenced. In OCI-Ly8, three single cell clones with heterozygous ARID1A mutation (#A9, #G5 and #G10), and one ARID1A knock-out clone (#G6), were sequenced.