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Multicellular life requires altruistic cooperation between cells. The adaptive immune system is a notable exception, wherein germinal center B cells compete vigorously for limiting positive selection signals. Studying primary human lymphomas and developing new mouse models, we found that mutations affecting BTG1 disrupt a critical immune gatekeeper mechanism that strictly limits B cell fitness during antibody affinity maturation. This mechanism converted germinal center B cells into supercompetitors that rapidly outstrip their normal counterparts. This effect was conferred by a small shift in MYC protein induction kinetics but resulted in aggressive invasive lymphomas, which in humans are linked to dire clinical outcomes. Our findings reveal a delicate evolutionary trade-off between natural selection of B cells to provide immunity and potentially dangerous features that recall the more competitive nature of unicellular organisms.

BTG1 somatic mutations are exclusively found in mature B cell malignancies, ~12% diffuse large B cell lymphoma (DLBCL) and are particularly enriched in the MCD/cluster 5 subtype of ABC-DLBCL, characterized by extranodal dissemination and poor clinical outcome. However, the mechanism of action and clinical relevance of BTG1 mutations remain unknown. We find that BTG1 mutations score among the top mutations with DLBCL driver potential, using a rigorous genomic and epigenomic covariates analysis. Most notably, BTG1 mutant patients presented with inferior clinical outcome (p=0.0011) in ABC-DLBCL cases from 5 cohorts and BTG1 mutation was independently associated with lower overall survival in a multivariable Cox regression analysis (p=0.0190) DLBCL originates from mature B cells having experienced the germinal center (GC) reaction. We therefore generated a conditional knockin mouse model to express the most frequent Btg1 Q36H in B cells. Btg1 Q36H GC B cells showed a dramatic fitness advantage in in vivo competitive assays. This effect was specific to the GC compartment and was dependent on T cells. RNAseq showed that Btg1Q36H GC B cells were markedly enriched for genes normally transiently induced upon positive selection by T cells, including MYC targets. The same signatures were enriched in BTG1 mutant DLBCL patients and isogenic BTG1Q36H vs BTG1WT human DLBCL cell lines. We observed a higher proportion of MYC-expressing cells in Btg1Q36H GCs without an increase in maximal MYC levels per cell, also confirmed in human DLBCL lines and primary human tonsillar B cells, suggesting a lowered threshold for MYC induction in mutant cells. Mechanistically, ~800 transcripts associated with BTG1WT, but not BTG1Q36H. These belong to the same gene sets that characterize positively selected GC B cell, including MYC itself. BTG1Q36H therefore showed reduced association with the MYC mRNA. We found that this associated with facilitated MYC protein synthesis. Polysome profiling also showed higher translation capacity in BTG1Q36H DLBCL cells. Crossing our Btg1 Q36H mice to the VavP-Bcl2 model showed that VavP-Bcl2+Btg1Q36H mice displayed shorter survival, earlier onset of lymphoma and dysplastic B cell infiltration into non lymphoid organs. Immunoglobulin genes sequencing showed that VavP-Bcl2+Btg1Q36H lymphoma B cells were highly clonal, extensively mutated and selected. Importantly, the lack of BTG1 deletions suggested that BTG1 missense mutations do not cause a full loss-of-function of the protein. Indeed, we observed that shRNA-mediated knockdown of BTG1 resulted in apoptosis. Collectively, BTG1 mutations contribute to the formation of aggressive lymphomas through an entirely novel mechanism, by lowering the threshold to MYC induction in response to T cell selection signals, conferring dramatic fitness. This effect appears to correspond to a partial loss of function disrupting a novel GC context-specific check point, whereby BTG1 normally attenuates spurious MYC translation to tightly restrict fitness potential. Citation Format: Coraline Mlynarczyk, Matt Teater, Juhee Pae, Ling Wang, Jonatan Ersching, Antonin Papin, Darko Barisic, Ersilia Barin, Kenneth B. Hoehn, Zhengming Chen, Diu T. T. Nguyen, Chiara Evans, Ashley S. Doane, Michael G. Kharas, David W. Scott, Gabriel Victora, Ari Melnick. BTG1 mutations confer a fitness advantage and promote aggressive B cell lymphoma development by lowering the threshold for MYC induction [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A24.

Loss-of-function mutations in KMT2D are a striking feature of germinal center (GC) lymphomas, resulting in decreased histone 3 lysine 4 (H3K4) methylation and altered gene expression. We hypothesized that inhibition of the KDM5 family, which demethylates H3K4me3/me2, would reestablish H3K4 methylation and restore the expression of genes repressed on loss of KMT2D. KDM5 inhibition increased H3K4me3 levels and caused an antiproliferative response in vitro, which was markedly greater in both endogenous and gene-edited KMT2D mutant diffuse large B-cell lymphoma cell lines, whereas tumor growth was inhibited in KMT2D mutant xenografts in vivo. KDM5 inhibition reactivated both KMT2D-dependent and -independent genes, resulting in diminished B-cell signaling and altered expression of B-cell lymphoma 2 (BCL2) family members, including BCL2 itself. KDM5 inhibition may offer an effective therapeutic strategy for ameliorating KMT2D loss-of-function mutations in GC lymphomas.

Immunotherapies including the activation of endogenous immune cells have stimulated great interest for their anti-tumor functions. An initial focus has been on genetic lesions such as loss of MHC class I and class II molecules that facilitate tumor cell escape. More recently, epigenetic mechanisms such as the disruption of the SWI/SNF remodeling complex PBAF have been implicated in regulating tumor sensitivity to CD8 T cell-mediated killing. Here, I used an unbiased genetic screening approach to examine resistance and sensitivity of breast cancer cells to Natural Killer (NK) cells. Among expected hits including caspases, interferon receptors, and cell death executioners, the screen revealed a surprising role of alternate SWI/SNF complexes in controlling NK cell-mediated cytotoxicity that relate to interferon-γ and perforin/granzyme B, and may be pharmacologically reversible. Altogether, I will discuss a new epigenetic mechanism that affect NK cell-mediated cytotoxicity with a focus on SWI/SNF complexes. Citation Format: Joyce Pasion, Darko Barisic, Cem Meydan, Kong Y. Ng, Maria Tello Lafoz, Morgan Huse, Ari Melnick, Hans-Guido Wendel. Epigenetic control of tumor cell killing by natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3731.

Loss-of-function mutations in KMT2D are a striking feature of the germinal centre (GC) lymphomas, resulting in decreased H3K4 methylation and altered gene expression. We hypothesised that inhibition of the KDM5 family, which demethylates H3K4me3/me2, would re-establish H3K4 methylation and restore the expression of genes repressed upon loss of KMT2D. KDM5-inhibition increased H3K4me3 levels and caused an anti-proliferative response in vitro, which was markedly greater in both endogenous and CRISPR-edited KMT2D mutant DLBCL cell lines, whilst tumour growth was inhibited in KMT2D mutant xenografts in vivo. KDM5-inhibition reactivated both KMT2D-dependent and -independent genes, resulting in diminished B-cell receptor signalling and altered expression of BCL2 family members, including BCL2 itself, allowing it to synergise with agents targeting these pathways. KDM5-inhibition may offer an effective therapeutic strategy for ameliorating KMT2D loss-of-function mutations in GC-lymphomas.Statement of significanceWe detail a novel way of reverting the effects of loss-of-function mutations in the histone methyltransferase KMT2D by inhibiting the KDM5 demethylase family, increasing levels of H3K4me3 and restoring expression of KMT2D regulated genes.