DTX1 Controls Germinal Center B-Cell Development and Lymphomagenesis

DTX1 is highly expressed in germinal center B (GCB)-cells and is targeted by coding mutations in 17% of DLBCL, including 50% of the BN2 genetic subtype that is enriched for the ABC-like cell of origin subtype (Wright et al., Cancer Cell 2020). DTX1 has also been recognized as a prominent target of AID-mediated mutagenesis, with a high rate of non-coding regulatory element (RE) mutations being observed across DLBCL subtypes. The DTX1 gene encodes a ring finger ubiquitin ligase that regulates NOTCH1 recycling. However, the role of DTX1 in GC B-cell development and lymphomagenesis has not been thoroughly investigated.

We characterized mutations within the regulatory element of DTX1 using whole genome sequencing data from ICGC and TCGA, together with H3K27Ac ChIP-sequencing data from tonsillar GCB-cells to define active regulatory elements (REs). We observed non-coding mutations of DTX1-proximal REs in 59% of DLBCL tumors. We characterized these putative REs by knocking GFP into the start codon of the endogenous DTX1 gene and performing CRISPR/Cas9 tiling, using reductions of GFP expression as a surrogate for reduced DTX1 RE activity. We observed a significant loss of GFP expression with DTX1 RE-targeted gRNAs compared control gRNAs, confirming these recurrently mutated regions as active REs.

We therefore generated a novel floxed Dtx1 genetically engineered mouse model and crossed with Aid-cre for GCB-specific conditional knock-out (cKO). Immunized mice were euthanized at the peak of the GC reaction and analyzed by flow cytometry and multispectral immunofluorescence (mIF) imaging. Mice with monoalleleic (Dtx1^+/-) or bialleleic (Dtx1^-/-) cKO of Dtx1 had a significant increase in GCB cell frequency compared to wild-type (Dtx1^+/+) littermates, with a significant skew of polarization towards the light zone. This was confirmed by mIF analysis, which showed significantly larger and more abundant GCs, and a significantly higher fraction of Ki67+ cells within the GCs, in Dtx1^+/- compared to Dtx1^+/+ mice. We therefore investigated whether Dtx1 loss may confer a competitive advantage within the GC reaction by generating Dtx1^+/+ / Dtx1^+/- mixed chimera models in Rag-deficient recipients. Dtx1^+/- cells were significantly over-represented within the GCB compartment and showed a significant light-zone skew, and significantly higher frequencies of proliferating (Edu+) GCB cells within the light-zone relative to Dtx1^+/+ cells. We therefore investigated changes in GC development using scRNA-sequencing PNA-enriched GCB cells from SRBC-immunized Dtx1^+/+ (n=4) and Dtx1^+/- littermates (n=4). This confirmed a significant light-zone polarization within GCB cells, and highlighted a dramatic reduction in pre-memory cells within Dtx1^+/- mice. Furthermore, differential gene expression analysis identified significant alterations in light-zone gene expression programs within Dtx1^+/- GCB cells. To test whether Dtx1 loss may participate in the early stages of lymphomagenesis, we performed serial re-challenge with SRBCs monthly for 3 months and 10 days later euthanized mice. Histological analysis of major organs revealed a significant increase in the frequency of lymphoid aggregates within the lungs, liver and kidneys, indicating extranodal dissemination.

In conclusion, these data demonstrate for the first time that highly-recurrent mutations in DLBCL target an active RE for DTX1, resulting in inhibition of DTX1 expression. Dtx1 haploinsufficiency confers a selective advantage to GCB cells that promotes GC hyperplasia with light-zone polarization and alters GCB cell differentiation trajectories, and can result in extranodal dissemination of Dtx1^+/- GCB cells following chronic antigen stimulation.