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525 An In Vivo PiggyBac Insertional Mutagenesis Screen Reveals Oncogenic Lesions Cooperating with Myd88L265P

Program: Oral and Poster Abstracts
Type: Oral
Session: 621. Lymphomas: Translational – Molecular and Genetic: Signaling Pathways and MRD in Lymphoma
Hematology Disease Topics & Pathways:
Research, Translational Research, Lymphomas, non-Hodgkin lymphoma, B Cell lymphoma, Diseases, Lymphoid Malignancies, Study Population, Animal model
Sunday, December 10, 2023: 12:30 PM

Svenja Höfmann1*, Ruth Flümann2*, Julia Hansen2*, Sebastian Klein3*, Jörn Meinel4*, Pauline Pfeiffer2*, Hannah Goldfarb-Wittkopf2*, Anna Lütz2*, Jessica Wirtz2*, Michael Möllmann3*, Tanja Zhou3*, Areya Tabatabai3*, Tim Lohmann2*, Filippo Beleggia2*, Benedikt Pelzer, MD5*, Fabian Ullrich, MD3, Aastha Arora3*, Thorsten Persigehl6*, Reinhard Büttner4*, Bastian von Tresckow, MD3, Ron Jachimowicz2*, Gero Knittel, PhD3* and Hans Christian Reinhardt, MD3

1Department of Hematology and Stem Cell Transplantation, University Hospital Essen, Essen, Germany
2Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, University of Cologne, Cologne, NRW, Germany
3Department of Hematology and Stem Cell Transplantation, University Hospital Essen, Essen, NRW, Germany
4Faculty of Medicine and University Hospital Cologne, Institute of Pathology, University of Cologne, Cologne, NRW, Germany
5Division of Hematology/Oncology, Department of Medicine, Weill Cornell Medicine, Cornell University, New York, NY
6Faculty of Medicine and University Hospital Cologne, Department of Radiology and Interventional Radiology, University of Cologne, Cologne, NRW, Germany

Diffuse large B cell lymphoma (DLBCL) is the most common Non-Hodgkin lymphoma and originates from transformed germinal center-experienced B cells. Traditionally, DLBCL has been divided into two subtypes, depending on whether the transcriptional profile of the tumor relates to an activated B cell or a germinal center B cell (ABC and GCB DLBCL, Alizadeh et al., 2000). More recent efforts classified DLBCL cases based on their genetic aberrations and identified several clusters with distinct mutational profiles (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020). The MCD/C5 cluster is characterized by recurrent mutations in MYD88, PRDM1 and frequent amplifications of BCL2, amongst others. We recently showed that mice harboring a B cell-specific Myd88L252P mutation (orthologous position of the human p.L265P mutation) develop B cell proliferation and occasional transformation into DLBCL (Knittel et al., 2016). Lymphomagenesis is further increased when Myd88L252P is combined with BCL2 overexpression and a genetically engineered block in plasmacytic differentiation by loss of Prdm1 or overexpression of Spib (Flümann et al., 2021).

To ask which further genes and pathways cooperate with Myd88L252P in lymphomagenesis, we performed an in vivo piggyBac insertional mutagenesis screen. In this system, a conditionally expressed transposase mobilizes transposable elements. These elements can then reintegrate into the genome and either silence or drive the expression of genes, depending on the exact integration site and orientation of the transposon cassette (Rad et al., 2015). We crossed this piggyBac system onto a Myd88L252P background to identify genes that cooperate with Myd88 mutations in lymphomagenesis. Mice harboring both the Myd88 mutant allele and the piggyBac system lived significantly shorter than controls harboring only the Myd88 mutation or just the piggyBac system. Myd88/piggyBac animals developed B220+/CD138- lymphomas and DNA isolated from these lesions allowed the detection of common transposon insertion sites. Among the genes significantly enriched for integrations, we observed known genetic drivers of human MCD/C5 DLBCL, such as PIM1 and ETV6. We also identified TBL1XR1 and SPIB as common insertion sites, as well as BCL2, BIM1 and BCL-XL, further validating our approach of engineering a plasma cell differentiation block as well as anti-apoptotic BCL2 overexpression on a Myd88-mutant background to model MCD DLBCL. Of note, the hits identified in this screen were significantly distinct from hits identified to drive Myc-driven B cell lymphomagenesis (Weber et al., 2019). Additionally, we identified several candidate genes that are not reported to be frequently genetically altered in DLBCL, however associated with processes relevant to B cell biology, particularly to B cell receptor signaling.

As ‘B cell receptor signaling’ was a prominent term in our screen hits and CD79B ITAM mutations are an additional hallmark of MCD/C5 DLBCL (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020), we introduced a conditional Cd79b p.Y195H allele to our Cd19Cre/wt;Myd88cond.p.L252P/wt;Rosa26LSL.BCL2-IRES-GFP/wt;Prdm1fl/fl MCD/C5 DLBCL model. While we did not observe significant differences in the immunohistochemial and transcriptional phenotype of Cd79b WT and mutant lymphomas, Cd79b p.Y195H tumors showed increased B cell receptor (BCR) signaling activity, indicated by increased levels of phosphorylated SYK and PLCg2. Cd79b mutant lymphomas also showed an increased formation of cytoplasmic signaling complexes comprised of MYD88 and several components of the BCR pathway, including MALT1 and BTK. The formation of these complexes depended on active BTK, as treatment with the BTK inhibitor ibrutinib reduced complex formation to levels found in Cd79b WT lymphomas. Consequently, we investigated the effects of ibrutinib treatment in Cd79b-mutant and wildtype MCD DLBCL mouse models and found Cd79b-mutant lymphomas to be significantly more sensitive to ibrutinib treatment than their Cd79b WT counterparts.

Disclosures: von Tresckow: Pfizer: Consultancy; Takeda: Consultancy, Honoraria, Other: Travel Support, Research Funding; Noscendo: Consultancy; Lilly: Consultancy, Honoraria, Other: Travel Support; AstraZeneca: Honoraria, Other: Travel Support; IQVIA: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel Support, Research Funding; BMS/Celgene: Consultancy, Honoraria; Cerus: Consultancy; Gilead Kite: Consultancy, Other: Travel Support; AbbVie: Other: Travel Support; MSD: Consultancy, Honoraria, Other: Travel Support, Research Funding; Roche: Consultancy, Honoraria, Other: Travel Support; Miltenyi: Consultancy; Incyte: Consultancy, Honoraria; Allogene: Consultancy; Pentixapharm: Consultancy; Amgen: Consultancy; Pierre Fabre: Other: Travel support. Reinhardt: Gilead: Research Funding; Merck: Consultancy; Vertex: Consultancy; AstraZeneca: Research Funding; AbbVie: Consultancy; BMS: Consultancy; Novartis: Consultancy; Roche: Consultancy; CDL Therapeutics GmbH: Consultancy.

*signifies non-member of ASH