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

• In vivo CRISPR screening allows efficient evaluation of DLBCL drivers, showing cooperation of Kmt2d and Trp53 losses in B-lymphomagenesis.

• Combined Kmt2d and Trp53 inactivation induces Yap1 overexpression in B-lymphomas and Yap1 inhibition suppresses their tumorigenesis.

Although recent genetic studies have identified numerous genetic alterations in diffuse large B-cell lymphoma (DLBCL), their biological relevance remains elusive. Here we performed in vivo CRISPR loss-of-function screening targeting 86 genes recurrently altered in DLBCL to examine oncogenicity of sgRNA-targeted genes, association between genotype and lineage, occurrence of second-hit alterations, and cooperability among sgRNA-targeted genes and second-hit alterations. Transplantation of the CRISPR library-transduced hematopoietic stem/progenitor cells induces various hematologic malignancies, including B-lymphomas in mice. Enrichment analysis of sgRNA-targeted genes demonstrates significant overrepresentation of Kmt2d, Pax5, and Trp53 in B-lymphomas. Whole-exome sequencing identifies recurrent second-hit driver alterations, showing significant enrichment of Trp53 alterations in sgKmt2d-targeted B-lymphomas. Importantly, KMT2D and TP53 mutations are found to be the most prevalent co-occurring combination in human DLBCL, which is more prominent in relapsed/refractory DLBCL. Moreover, this combination confers significantly worse prognosis independent of clinical factors. Transcriptomic sequencing identifies overexpression of Yap1, the Hippo pathway component, in double sgKmt2d-targeted/Trp53-altered B-lymphomas. Furthermore, chromatin accessibility analysis demonstrates enrichment of TEAD1 binding motifs in regions that gained accessibility and increased expression of their nearest genes in these B-lymphomas. Most importantly, genetic and pharmacological inhibition of YAP1 suppresses in vitro cell proliferation and in vivo tumor growth of a human KMT2D/TP53-altered DLBCL cell line and prolongs survival of mice transplanted with double sgKmt2d-targeted/Trp53-altered B-lymphoma cells. Our findings demonstrate the utility of in vivo CRISPR screening to integrate human cancer genomics with mouse modeling, and highlight the functional interplay between KMT2D and TP53 aberrations, providing insights into therapeutic strategies in DLBCL.