CRISPR Cas9 Screens in Mice to Identify Tumor Suppressors of Aggressive Human Lymphoma

Diffuse Large B Cell Lymphoma (DLBCL), the most common form of aggressive Non-Hodgkin's Lymphoma, has been subdivided into genetic subtypes that respond differentially to therapy. The MCD subtype, characterized by MYD88L265P and CD79B mutations, is a subtype of DLBCL with some of the worst clinical outcomes. Patients have the lowest response rates to the standard of care - R-CHOP immunochemotherapy. To study MCD biology, we have developed a mouse model harboring four MCD-associated genetic alterations. With age, these mice develop DLBCL that arises in the spleen following an accumulation of spontaneous germinal center B Cells (GC Bs) suggesting they play a role in the transformation to malignancy. These MCD mice develop DLBCL with age which suggests additional genetic events are necessary to accelerate tumor development. The MCD genetic subclass of DLBCL is enriched for mutations in many genes that could potentially function as tumor suppressors. Thus, we conditionally expressed Cas9 in the MCD murine model enabling loss-of-function assays to study putative tumor suppressors in a premalignant setting. We screened candidate tumor suppressor genes using CIRSPR/Cas9 mediated knockout, which revealed that loss of Setd1b, a histone methyltransferase, and Tbl1xr1, a subunit of a transcriptional repressor complex, caused the largest increase in premalignant splenic GC Bs, suggesting they play a key role in driving MCD pathogenesis. Furthermore, the combined loss of these genes causes an even larger increase in this premalignant subpopulation, indicating a functional relationship between these two genes. Single cell RNA sequencing data showed that loss of Setd1b and Tbl1xr1 alters the phenotype of splenic GC B, resulting in a neomorphic cell state with features of both GC Bs and memory B cells. The combinatorial loss of these genes also induced gene expression signatures characteristic of the oncogenic signaling pathways that human MCD tumors depend upon. These data point towards a cooperative functional relationship between these genes, a discovery that is further supported by analysis of human MCD tumors, in which SETD1B and TBL1XR1 are genetically inactivated in the same tumors significantly more often than expected by chance. Thus, we aged MCD-Cas9 mice transduced with either guide-RNAs against both Setd1b and Tbl1xr1 or a non-targeting control and found the double knockout mice had accelerated tumorigenesis. As we continue to investigate the role of these now validated tumor suppressors in the development of MCD-DLBCL, we hope to shed valuable light on their contribution to lymphomagenesis, which may reveal new therapeutic vulnerabilities in this aggressive cancer.