In vivo CRISPR screening reveals cooperation of KMT2D and TP53 deficiencies in B-cell lymphomagenesis

Through bone marrow transplantation model, we performed in vivo CRISPR loss-of-function screening targeting 86 recurrently altered genes in lymphomas to examine oncogenicity of sgRNA-targeted genes, association between genotype and lineage, and cooperability among sgRNA-targeted genes and second-hit alterations in lymphoid malignancies. Although various murine hematologic malignancies develop, we observe strong enrichment of Pax5 and Kmt2d in B-lymphomas. sgPax5-targeted B-malignancies show various second-hit alterations including mutations, copy number amplifications, and fusions, mainly involving FLT3, JAK/STAT, and RAS pathways. sgKmt2d-targeted B-lymphomas show frequent second-hit mutations involving Trp53. Individual Kmt2d and Trp53 knockout confirms the development of B-lymphomas. KMT2D and TP53 mutations are the most frequent co-occurring combination in human DLBCL, particularly in relapsed/refractory DLBCL, and confer negative prognostic impact. Transcriptomic analysis identifies Yap1 overexpression in sgKmt2d-targeted and Trp53-altered B-lymphomas. Chromatic accessibility analysis demonstrates enrichment of TEAD1 motif in significantly more accessible regions and increased expression of their nearest genes. Importantly, pharmacological YAP1 inhibition suppressed cell proliferation of a human KMT2D- and TP53-mutated DLBCL cell line in vitro and prolongs the survival of mice transplanted with sgKmt2d-targeted and Trp53-altered B-lymphoma cells