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.

Abstract: Diffuse large B cell lymphoma (DLBCL) is a clinically and genetically very heterogeneous disease. Several [GK1] [RW2] recent studies attempted to give structure to the genetic heterogeneity by clustering cases based on their mutational profiles. Clusters MCD by Schmitz et al. and C5 by Chapuy et al. are amongst the clusters enriched for cases with particularly inferior prognosis. Both clusters are defined by mutations in MYD88 and CD79B as well as high levels of BCL2 expression and recurring mutations in transcriptional regulators of the plasmacytic differentiation process (PRDM1, SPIB). We have generated genetically-engineered mouse models that mimic key alterations of MCD/C5 DLBCL: Hallmark mutations in MYD88 and CD79B, loss of PRDM1 and overexpression of BCL2/SPIB. We could show that those mice develop clonal lesions, which morphologically resemble DLBCL. In the presence of a lesion that prevents plasmacytic differentiation (overexpression of Spib, loss of Prdm1), the forming lesions display signs of an earlier stage of B cell development, specifically histologic and transcriptomic similarities to germinal center cells. In contrast, allelic combinations without differentiation block lead to lymphomas that express CD138 and lack B220 expression, suggesting a more plasmacytic/plasmablastic differentiation status. The presence or absence of a Cd79b p.Y195H mutation has no notable effect on the immunophenotype of the lymphoma. However, its presence correlates with enrichment of BCR signaling gene set expression in lymphoma tissue and significantly increased sensitivity to inhibition of Bruton’s Tyrosine Kinase (BTK) by ibrutinib in our model systems. Taken together, we have established a set of murine models which mimic different genetic features of C5/MCD DLBCL. We have extensively characterized these models and show their usability as preclinical tools for therapeutic studies. Citation Format: Ruth Fluemann, Julia Hansen, Benedikt Pelzer, Tim Lohmann, Ilmars Kisis, Reinhard Büttner, Thorsten Persighel, Nima Abedpour, Martin Peifer, Ron Daniel Jachimowicz, Hans Christian Reinhardt, Gero Knittel. Novel autochthonous mouse models as preclinical tools in the study of MCD/C5 DLBCL [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A21.

Abstract: Genomic profiling revealed the identity of at least 5 subtypes of diffuse large B-cell lymphoma (DLBCL), including the MCD/C5 cluster characterized by aberrations in MYD88, BCL2, PRDM1, and/or SPIB. We generated mouse models harboring B cell–specific Prdm1 or Spib aberrations on the background of oncogenic Myd88 and Bcl2 lesions. We deployed whole-exome sequencing, transcriptome, flow-cytometry, and mass cytometry analyses to demonstrate that Prdm1- or Spib-altered lymphomas display molecular features consistent with prememory B cells and light-zone B cells, whereas lymphomas lacking these alterations were enriched for late light-zone and plasmablast-associated gene sets. Consistent with the phenotypic evidence for increased B cell receptor signaling activity in Prdm1-altered lymphomas, we demonstrate that combined BTK/BCL2 inhibition displays therapeutic activity in mice and in five of six relapsed/refractory DLBCL patients. Moreover, Prdm1-altered lymphomas were immunogenic upon transplantation into immuno-competent hosts, displayed an actionable PD-L1 surface expression, and were sensitive to antimurine-CD19-CAR-T cell therapy, in vivo. Significance: Relapsed/refractory DLBCL remains a major medical challenge, and most of these patients succumb to their disease. Here, we generated mouse models, faithfully recapitulating the biology of MYD88-driven human DLBCL. These models revealed robust preclinical activity of combined BTK/BCL2 inhibition. We confirmed activity of this regimen in pretreated non–GCB-DLBCL patients. See related commentary by Leveille et al., p. 8. This article is highlighted in the In This Issue feature, p. 1

Abstract: Based on gene expression profiles, diffuse large B-cell lymphoma (DLBCL) is subdivided into germinal center B-cell–like (GCB) and activated B-cell–like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88 as well as BCL2 copy-number gains. Here, we employ immune phenotyping, RNA sequencing, and whole-exome sequencing to characterize a Myd88- and BCL2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression compared with GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and PD-1 blockade significantly increased the overall survival of lymphoma-bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations. Significance: Oncogenic Myd88 and BCL2 cooperate in murine DLBCL lymphomagenesis. The resulting lymphomas display morphologic and transcriptomic features reminiscent of human ABC-DLBCL. Data derived from our Myd88/BCL2-driven autochthonous model demonstrate that combined BCL2 and PD-1 blockade displays substantial preclinical antilymphoma activity, providing preclinical proof-of-concept data, which pave the way for clinical translation. This article is highlighted in the In This Issue feature, p. 1

Abstract: The transport efficiency (TE) describes the performance of a transport protein for a specific substrate. To compare the TE of different transporters, the number of active transporters in the plasma membrane must be monitored, as it may vary for each transporter and experiment. Available methods, like LC–MS quantification of tryptic peptides, fail to discriminate inactive intracellular transporters or, like cell-surface biotinylation followed by affinity chromatography and Western blotting, are imprecise and very laborious. We wanted to normalize active transporters by the activity of a second transporter. A transporter tandem, generated by joining two transporter cDNAs into a single open reading frame, should guarantee a 1 : 1 stoichiometry. Here we created a series of tandems with different linkers between the human ergothioneine (ET) transporter ETT (gene symbol SLC22A4) and organic cation transporter OCT2 (SLC22A2). The linker sequence strongly affected the expression strength. The stoichiometry was validated by absolute peptide quantification and untargeted peptide analysis. Compared with wild-type ETT, the normalized ET clearance of the natural variant L503F was higher (f = 1.34); G462E was completely inactive. The general usefulness of the tandem strategy was demonstrated by linking several transporters with ETT; every construct was active in both parts. Transporter tandems can be used - without membrane isolation or protein quantification — as precise tools for transporter number normalization, to identify, for example, relevant transporters for a drug. It is necessary, however, to find suitable linkers, to check the order of transporters, and to verify the absence of functional interference by saturation kinetics.