Session: 603. Lymphoid Oncogenesis: Basic: Mechanisms in Lymphomagenesis
Hematology Disease Topics & Pathways:
Research, Fundamental Science, immune mechanism, Biological Processes, molecular biology
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.
Disclosures: Melnick: Astra Zeneca: Consultancy, Research Funding; Canaan Management, LLC: Consultancy; Celgene Corporation: Consultancy; Constellation Pharmaceuticals: Consultancy, Other: Scientific Advisory Board; Daiichi Sankyo: Consultancy, Research Funding; Epizyme: Consultancy, Other: Scientific Advisory Board, Research Funding; Exo Therapeutics: Other: Scientific Advisory Board; Janssen: Research Funding; Janssen Global Advisory: Other: Scientific Advisory Board; KDAC Therapeutics: Current holder of stock options in a privately-held company; Treeline Biosciences: Consultancy; CASMA Therapeuctics: Consultancy. Vega: Geron Corporation: Research Funding; Allogene: Research Funding; Crisp Therapeutics: Research Funding. Nastoupil: ADC Therapeutics, BMS, Caribou Biosciences, Epizyme, Genentech/Roche, Gilead/Kite, Genmab, Janssen, MEI, Morphosys, Novartis, Takeda: Honoraria; Genentech/Roche, MEI, Takeda: Other: DSMC; BMS, Caribou Biosciences, Epizyme, Genentech, Gilead/Kite, Genmab, Janssen, IGM Biosciences, Novartis, Takeda: Research Funding. Green: KDAc Therapeutics: Current holder of stock options in a privately-held company; Allogene: Research Funding; Tessa Therapeutics: Honoraria; Sanofi: Research Funding; Abbvie: Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Monte Rosa Therapeutics: Honoraria; Kite/Gilead: Research Funding.
See more of: Oral and Poster Abstracts