SUMMARY The EZH2 histone methyltransferase mediates the humoral immune response and drives lymphomagenesis through formation of bivalent chromatin domains at critical germinal center (GC) B cell promoters. Herein we show that the actions of EZH2 in driving GC formation and lymphoma precursor lesions require site-specific binding by the BCL6 transcriptional repressor and the presence of a non-canonical PRC1-BCOR-CBX8 complex. The chromodomain protein CBX8 is induced in GC B cells, binds to H3K27me3 at bivalent promoters, and is required for stable association of the complex and the resulting histone modifications. Moreover, oncogenic BCL6 and EZH2 cooperate to accelerate diffuse large B cell lymphoma (DLBCL) development and combinatorial targeting of these repressors results in enhanced anti-lymphoma activity in DLBCLs.

The EZH2 histone methyltransferase is required for B cells to form germinal centers (GC). Here we show that EZH2 mediates GC formation through repression of cyclin-dependent kinase inhibitor CDKN1A (p21Cip1). Deletion of Cdkn1a rescues the GC reaction in Ezh2 −/− mice. Using a 3D B cell follicular organoid system that mimics the GC reaction, we show that depletion of EZH2 suppresses G1 to S phase transition of GC B cells in a Cdkn1a-dependent manner. GC B cells of Cdkn1a −/− Ezh2 −/− mice have high levels of phospho-Rb, indicating that loss of Cdkn1a enables progression of cell cycle. Moreover, the transcription factor E2F1 induces EZH2 during the GC reaction. E2f1 −/− mice manifest impaired GC responses, which is rescued by restoring EZH2 expression, thus defining a positive feedback loop in which EZH2 controls GC B cell proliferation by suppressing CDKN1A, enabling cell cycle progression with a concomitant phosphorylation of Rb and release of E2F1.

TET2 somatic mutations occur in ~10% of DLBCLs but are of unknown significance. Herein we show that TET2 is required for the humoral immune response and is a DLBCL tumor suppressor. TET2 loss of function disrupts transit of B-cells through germinal centers (GC), causing GC hyperplasia, impaired class switch recombination, blockade of plasma cell differentiation and a pre-neoplastic phenotype. TET2 loss was linked to focal loss of enhancer hydroxymethylation and transcriptional repression of genes that mediate GC exit such as PRDM1. Notably, these enhancers and genes are also repressed in CREBBP-mutant DLBCLs. Accordingly, TET2 mutation in patients yields a CREBBP-mutant gene expression signature, CREBBP and TET2 mutations are generally mutually exclusive, and hydroxymethylation loss caused by TET2 deficiency impairs enhancer H3K27 acetylation. Hence TET2 plays a critical role in the GC reaction and its loss of function results in lymphomagenesis through failure to activate genes linked to GC exit signals.

KRAS is the most frequently mutated oncogene in cancer.Tumor sequencing has revealed a complex spectrum of KRAS mutations across different cancer types, yet there is little understanding how specific KRAS alterations impact tumor in initiation, progression, or therapy response. Using highfidelity CRISPR-based engineering, we created an allelic series of new LSL-Kras mutant mice, reflecting codon 12 and 13 mutations that are highly prevalent in lung (KRAS G12C ), pancreas (KRAS G12R ) and colon (KRAS G13D ) cancers. Induction of each mutation in the developing mouse pancreas reveal striking quantitative and qualitative differences in the degree of ductal transformation and pre-malignant progression. Further, using organoid models we show that KRAS G13D mutants respond to EGFR inhibition, while the anti-proliferative effect of KRAS G12C -selective inhibitors can be overcome by upstream EGFR signaling. Together, these new mouse strains provide an ideal for investigating KRAS biology in vivo, and for developing pre-clinical precision oncology models of KRAS-mutant pancreas (G12R), colon (G13D), and lung (G12C) cancers.KRAS mutations in tumor development. However, these models alone do not recapitulate the spectrum of KRAS alterations in human cancer. Here we describe an efficient pipeline for engineering allelic series of conditional alleles that significantly expands repertoire of pre-clinical KRASdriven cancer models. Using high-fidelity CRISPR targeting in embryonic stem cell (ESC)-based mouse models (GEMM-ESCs) 11-14 , we engineered six new LSL-Kras mutant alleles (G12V, G12C, G13D, G12R, G12A, G12S) that represent the most frequent mutations at the G12/G13 hotspot, after G12D. Guided by clinical data, we generated conditional mice representing three tissue-selective alterations observed in colorectal (G13D), pancreatic (G12R), and lung cancer (G12C) and show that, even subtle mutational changes in the Kras oncogene, have a profound impact on tumor initiation in the pancreas.Conditional animal models, such as Lox-Stop-Lox (LSL)-Kras G12D and LSL-Kras G12Vgeo mice developed almost 20 years ago 9,10 , have been critical tools to dissect the role of . CC-BY-NC-ND 4.

During the germinal center (GC) reaction, B cells undergo extensive redistribution of cohesin complex and 3D reorganization of their genomes. Yet, the significance of cohesin and architectural programming in the humoral immune response is unknown. Herein we report that homozygous deletion of Smc3 encoding the cohesin ATPase subunit abrogated GC formation, yet in marked contrast Smc3 haploinsufficiency induced GC hyperplasia, skewing of GC polarity and impaired plasma cell differentiation. Genome-wide chromosomal conformation and transcriptional profiling revealed defects in GC B cell terminal differentiation programs controlled by lymphoma epigenetic tumor suppressors Tet2 and Kmt2d , and failure of Smc3 wt/– GC B cells to switch from B cell to plasma cell defining transcription factors. Smc3 haploinsufficiency preferentially impaired connectivity of enhancer elements controlling various lymphoma tumor suppressor genes, and accordingly Smc3 haploinsufficiency accelerated lymphomagenesis in mice with constitutive Bcl6 expression. Collectively, our data indicate a dose-dependent function for cohesin in humoral immunity to facilitate the B cell to plasma cell phenotypic switch, while restricting their malignant transformation.