A Novel Autochthonous Mouse Model Serves As a Preclinical Evaluation Platform and Explores Dual BTK and BCL2 Inhibition for Activated B Cell-like Diffuse Large B Cell Lymphoma
Authors: Flümann Ruth , Nieper Pascal , Lohmann Tim , Kisis Ilmars , Peifer Martin , Abedpour Nima , Thomas Roman , Kochanek Moritz , Pelzer Benedikt , Beleggia Filippo , Buettner Reinhard , Lohneis Philipp , Persigehl Thorsten , Hallek Michael , Calado Dinis Pedro , Pasqualucci Laura , von Tresckow Bastian , Chapuy Björn , Jachimowicz Ron , Reinhardt Christian , Knittel Gero 
Published: November 2021

Abstract
Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Both biologically and clinically, DLBCL represents a highly heterogeneous disease. DLBCL has been subdivided into germinal center B cell (GCB)-like and activated B cell (ABC)-like DLBCL, on the basis of gene expression profiling, which separates DLBCL according to the presumed cell of origin (COO). This COO-based classifier distinguishes sub-entities displaying distinct biological features, pathogenesis and clinical response to frontline therapy.
In addition to this classic transcriptome-based stratifier, recent genomic analyses of human DLBCL samples led to the discovery of partially overlapping genetically-defined DLBCL subsets. A study by Schmitz et al. employed a supervised clustering approach, allowing the classification of ~50% of the cases into four genetically-defined DLBCL subtypes, one of which is being characterized by co-occurring MYD88- and CD79B mutations as well as high expression of BCL2 (termed MCD). In a second approach by Chapuy et al., patient samples were clustered in an unsupervised manner. Also in this study, a cluster with recurrent mutations in MYD88 (specifically p.L265P) and CD79B, as well as gains of 18q (the location of BCL2) was identified (termed C5).
We previously reported the formation of B cell lymphoma in mice that were engineered to express Myd88 p.L252P in combination with overexpression of BCL2 (Myd88 p.L252P/wt;R26 LSL.BCL2/wt;Cd19 Cre/wt, abbr. MBC) in a B cell-specific manner. While the developing lesions display many features of human ABC DLBCL, their B220 -/CD138 + immunophenotype reflects plasmablastic characteristics. To refine this mouse model, we incorporated additional C5/MCD lesions by engineering a B cell-specific loss of Prmd1 or Spib overexpression generating Prdm1 fl/fl;Myd88 p.L252P/wt;R26 LSL.BCL2/wt;Cd19 Cre/wt (PPMBC) and Myd88 p.L252P/wt;R26 LSL.BCL2/LSL.Spib;Cd19 Cre/wt (SMBC) compound animals.
Both, the B cell-specific loss of Prdm1 and Spib overexpression on the MBC background resulted in a marked reduction of CD138 + cells in the spleens of 10 weeks old animals compared to control (Fig. 1A), accompanied by a decrease in serum immunoglobulins, indicative of a plasma cell differentiation block and in agreement with the reported function of PRMD1 and SPIB as transcription factors regulating plasma cell differentiation.
Both PPMBC and SMBC mice developed lymphoma significantly earlier than MBC animals. These tumors largely displayed a B220 +/CD138 - immunophenotype. As transcriptional profiling is the gold standard for differentiation between GCB and ABC DLBCL, we generated germinal center- and activated blood B cell gene sets from healthy donors. We then performed gene set enrichment analyses between SMBC/PPMBC tumors and either MBC or Kmt2d fl/fl;VavP-Bcl2;Cɣ1 Cre/wt (KBC) lymphomas, the latter being reminiscent of human GCB DLBCL. While both PPMBC and SMBC samples were enriched for GCB gene signatures when compared to MBC, they enriched for ABC gene sets in comparison to KBC, potentially suggesting a developmental stage between KBC and MBC lesions (Fig. 1B).
We next aimed to employ our PPMBC model of C5 DLBCL as a pre-clinical tool, in order to derive therapeutic approaches for this disease. In this regard, we note BCL2 has emerged as a potential therapeutic target in DLBCL. The BCL2 inhibitor venetoclax produces response rates of ~18% in relapsed/refractory DLBCL (Davids et al., 2017). Similarly, in a phase I/II clinical trial involving 80 patients with relapsed/refractory DLBCL, ibrutinib induced complete or partial remissions in 37% of ABC-DLBCL patients, but in only 5% GCB-DLBCL patients (Wilson et al., 2015). Building on these observations, we asked whether single agent or combined venetoclax and ibrutinib treatment might display pre-clinical activity in the PPMBC setting. Indeed, combination treatment with ibrutinib and venetoclax resulted in a significant survival benefit compared to single compound or untreated animals (Fig. 1C). Given this preclinical activity, we treated 6 relapsed/refractory (r/r) non-GCB DLBCL patients (determined by Hans algorithm) in an off-label setting and observed tumor shrinkage in 5 of 6 patients (Fig. 1D). Thus, our clinical data corroborate our preclinical observations and suggest that combined venetoclax and ibrutinib may display clinical activity in a subset of r/r non-GCB DLBCL.
Figure 1 Figure 1.

Disclosures
Hallek: Roche: Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Mundipharma: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Pharmacyclics: Honoraria, Speakers Bureau. Calado: Myricx Pharma: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties: Cancer Treatments. WO patent WO 2020/128475 A1 (2020). Pasqualucci: Sanofi: Research Funding; Astra Zeneca: Research Funding. von Tresckow: Amgen: Consultancy, Honoraria; AbbVie: Other: congress and travel support; Pentixafarm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS-Celgene: Consultancy, Honoraria, Other: congress and travel support; MSD: Consultancy, Honoraria, Other: congress and travel support, Research Funding; Novartis: Consultancy, Honoraria, Other: congress and travel support, Research Funding; AstraZeneca: Honoraria, Other: congress and travel support; Kite-Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Other, Research Funding. Chapuy: Gilead: Honoraria; BMS: Honoraria; Regeneron: Consultancy; Gilead Sciences: Research Funding; Astra Zeneca: Honoraria. Reinhardt: CDL Therapeutics: Current holder of individual stocks in a privately-held company; Gilead: Research Funding; Merck: Consultancy; Vertex: Consultancy; AstraZeneca: Consultancy; Abbvie: Consultancy.

OffLabel Disclosure:
Treatment of DLBCL patients with ibrutinib and venetoclax.