AN IN VIVO PIGGYBAC INSERTIONAL MUTAGENESIS SCREEN TO SEARCH FOR MODIFIERS OF Myd88L265P-DRIVEN DLBCL LYMPHOMAGENESIS

Introduction: Based on genetic features, human DLBCL cases can be subdivided into several distinct clusters. Recurrent mutations in MYD88, CD79B, PRDM1 and frequent BCL2 copy number gains are characteristic of the MCD/C5 cluster. Mice with B cell-specific expression of Myd88^L252P (orthologous to human p.L265P) develop a lymphoproliferative phenotype at old age and occasional lymphoma. We aimed to identify genes cooperating with Myd88^p.L252P in DLBCL lymphomagenesis by performing an in vivo piggyBac (PB) insertional mutagenesis screen.

Methods: We crossed the PB transposon system with the Myd88^cond_p.L252P allele. Both the PB system and expression of Myd88 p.L252P were activated B cell-specifically with Cd19^Cre. Animals were aged and samples were collected when mice became moribund. The isolated tumors were characterized immunohistochemically and transcriptionally. The malignant nature was verified by B cell receptor clonality analysis. Lastly, common transposon integrations were identified by QiSeq and subsequent bioinformatic analysis.

One of the most prominent hits from this screen was Etv6, which is commonly affected by deleterious mutations in MCD/C5 DLBCL. To investigate the role of ETV6 in B cell biology and lymphomagenesis, we utilized an Etv6-flox allele in combination with Cd19^Cre/wt. Changes in the B cell compartment were determined by flow cytometry in steady state and after immunization. Cohorts were aged and developing lesions were characterized histologically and transcriptionally.

Results: The presence of the PB system on the Myd88^L252P background reduced overall survival. Myd88/PB mice formed clonal B220⁺ lymphomas. We were able to identify ∼1000 genes with significantly enriched integrations. Reminiscent of MCD DLBCL, hits were strongly enriched for the KEGG gene set ‘B cell receptor signaling’. Furthermore, we observed a distinct overlap between the PB hits and genes mutated in MCD DLBCL, including Tbl1xr1, Pim1 and Etv6. To investigate the effects of a B cell-specific loss of Etv6, we crossed a conditional knockout-allele to Cd19^Cre. Germinal center (GC) B cells were increased in 30wks old unimmunized Etv6-KO animals compared to Cd19^Cre controls. In contrast, post-GC species were reduced in Etv6-KO. Unexpectedly, the number of NP-specific GCB cells was lower in the Etv6-KO cohort ten days after NP-OVA immunization. To assess the lymphomagenic potential of Etv6 loss in a Myd88-mutant setting, we generated Myd88^p.L252P animals with or without Etv6-KO. Indeed, Etv6-KO resulted in a drastically increased development of lymphoma.

Conclusion: The PB transposon system identified several genes cooperating with Myd88^L265P in lymphomagenesis. Known genetic drivers of MCD DLBCL were observed, including Etv6. We show that Etv6 loss accelerates lymphomagenesis on a Myd88 p.L252P background. Lastly, the described GC phenotype leads us to speculate that Etv6 regulates both early steps in GC formation as well as GC exit/resolution.

Keywords: aggressive B-cell non-Hodgkin lymphoma; other basic and translational science

Potential sources of conflict of interest:

B. v. Tresckow

Employment or leadership position: Regeneron (Inst) and Takeda

Consultant or advisory role: Allogene, Amgen, BMS/Celgene, Cerus, Gilead Kite, Incyte, IQVIA, Janssen-Cilag, Lilly, Merck Sharp & Dohme, Miltenyi, Novartis, Noscendo, Pentixapharm, Pfizer, Pierre Fabre, Qualworld, Regeneron, Roche, Sobi and Takeda

Honoraria: AbbVie, AstraZeneca, BMS/Celgene, Gilead Kite, Incyte, Janssen-Cilag, Lilly, Merck Sharp & Dohme, Novartis, Roche and Takeda

Educational grants: AbbVie, AstraZeneca, Gilead Kite, Janssen-Cilag, Lilly, Merck Sharp & Dohme, Pierre Fabre, Roche, Takeda, and Novartis

Other remuneration: Research funding: Esteve (Inst), Merck Sharp & Dohme (Inst), Novartis (Inst), and Takeda (Inst)

H. C. Reinhardt

Employment or leadership position: CDU Therapeutics GmbH

Consultant or advisory role: Merck, Vertex, Novartis, Janssen-Cilag, Roche, AstraZeneca, Abbvie

Honoraria: Merck, Vertex, Novartis, Janssen-Cilag, Roche, AstraZeneca, Abbvie