BTG1 Mutation Promotes Aggressive Lymphoma Development By Lowering the Threshold to MYC Activation and Generating "Super-Competitor" B Cells

Somatic missense mutations of BTG1 are exclusive to germinal center (GC)-derived B cell lymphomas (~12% of DLBCLs) and are most prevalent in ABC-DLBCL (p=0.0184 vs GCB-DLBCL), particularly in the MCD/cluster 5 subtype, which features extranodal dissemination and unfavorable outcome. However, the relevance, mechanism of action and biological contribution of BTG1 mutations have not been studied. Using a rigorous genomic covariate analysis, we identified BTG1 mutations as a top genetic driver in DLBCL. Furthermore, molecular dynamics simulations indicated that BTG1 recurrent mutations, including the most frequent Q36H, disrupted the protein structure, with likely deleterious functional consequences.

To investigate the effect of BTG1 mutation in GC B cells, we generated a conditional Btg1^Q36H knock in mouse crossed to the B cell specific Cd19^Cre line. Surprisingly, there was no apparent phenotype in GC B cells or other B cell populations. However, placing Btg1 ^Q36H and WT GC B cells in competition within the same mouse through adoptive transfer revealed a dramatic competitive advantage of Btg1 ^Q36H cells, virtually taking over the GC reaction.

To gain further insight into this striking fitness advantage, we performed RNAseq in Btg1 ^Q36H GCs, which showed marked enrichment for genes induced in positively selected GC B cells, including MYC targets and biosynthetic pathways. The same genes were also enriched in BTG1 mutant DLBCL patients in 2 independent cohorts. Furthermore, Btg1 ^Q36H GC B cells displayed greater RNA content and cell size, reflecting increased fitness. Positive selection normally triggers a brief Myc pulse in GC B cells. We therefore crossed our Btg1^Q36H mice to the Myc^GFPprotein fusion reporter and observed higher proportion of Myc ^GFP+ cells in Btg1 ^Q36H GCs.

For mechanistic studies, we generated isogenic BTG1 ^Q36H or BTG1 ^WT human DLBCL cell lines. BTG1 ^Q36H cells exhibited enrichment for the same positively selected GC B and MYC target genes, as well as greater RNA content and cell size. BTG1 family members were suggested to interact with RNA. Performing RNA-immunoprecipitation, we discovered that ~800 transcripts associated with BTG1 ^WT, but not BTG1 ^Q36H. Notably, these corresponded to the same positively selected GC B and MYC target genes, including MYC itself. BTG1 was shown to regulate mRNA stability in other cell types. However, BTG1 ^Q36H did not alter MYC mRNA stability and instead facilitated MYC protein synthesis, thus disrupting a novel GC context-specific checkpoint mechanism, whereby BTG1 normally attenuates spurious MYC translation to tightly restrict fitness potential.

In GC B cells, Myc induction coincides with S phase entry, but G2/M progression requires re-entry into the proliferative dark zone. To characterize GC dynamics in vivo, we performed targeted single cell RNAseq in competing Btg1 ^Q36H and WT GC B cells and noted earlier and higher proportion of positively selected Btg1 ^Q36H GC B cells having committed to G2/M and the proliferative program. We confirmed faster S phase completion in competing Btg1 ^Q36H GC B cells by in vivo EdU/BrdU labelling and greater re-entry into the proliferative dark zone by in vivo antigen delivery to synchronize GC B cells at the time of positive selection.

Given that MCD-DLBCLs express high levels of BCL2, we crossed our Btg1^Q36H mice to the VavP-Bcl2 model. As compared to VavP-Bcl2, VavP-Bcl2+Btg1 ^Q36H mice displayed shorter survival (p=0.0005), earlier onset of lymphoma, dysplastic B cell infiltration into non lymphoid organs and they contained highly mutated, selected and clonal tumor B cells. Moribund VavP-Bcl2+Btg1 ^Q36H mice uniquely featured sheets of large, immunoblastic lymphoma cells, characteristic of ABC-DLBCLs. Most notably, examining ABC-DLBCLs from 5 independent cohorts showed inferior clinical outcome for BTG1 mutant patients (p=0.0011) and independent association of BTG1 mutation with inferior overall survival by multivariable Cox regression (p=0.0190).

Collectively, we find that BTG1 mutations mediate lymphomagenesis through an entirely novel mechanism of action that recapitulates the embryonic MYC-dependent "super-competitive" phenotype originally described in Drosophila imaginal disc cells. In the GC, "super-competition" is provided by BTG1 mutation via a subtle acceleration of MYC induction and GC dynamics, conferring dramatic fitness and the potential to transform into aggressive lymphomas.