an autochthonous mouse model of myd88- and bcl2-driven ......oct 31, 2020 · flümann et al. - 3 -...

Flümann et al.

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An autochthonous mouse model of Myd88- and BCL2-driven diffuse large B cell 1

lymphoma reveals actionable molecular vulnerabilities 2

3

Running Title: Vulnerabilities in a Myd88-/BCL2-driven DLBCL mouse model 4 5

Ruth Flümann1,2,3,4,*, Tim Rehkämper1,2,3,4,*, Pascal Nieper1,2,3,4,*, Pauline Pfeiffer1,2,3,4, 6 Alessandra Holzem1,2,3,4, Sebastian Klein5, Sanil Bhatia6, Moritz Kochanek1,2,3,4, Ilmars 7 Kisis1,2,3,4, Benedikt W. Pelzer1,2,3,4, Heinz Ahlert6, Julia Hauer7,8, Alexandra da Palma 8 Guerreiro1,2,4, Jeremy A. Ryan9, Maurice Reimann10, Arina Riabinska1,2,3,4, Janica 9 Wiederstein4, Marcus Krüger4, Martina Deckert2,16, Janine Altmüller11, Andreas R. 10 Klatt12, Lukas P. Frenzel1,2,4, Laura Pasqualucci13, Wendy Béguelin14, Ari M. Melnick14, 11 Sandrine Sander15, Manuel Montesinos-Rongen2,16, Anna Brunn2,16, Philipp Lohneis2,3,5, 12 Reinhard Büttner2,3,5, Hamid Kashkar3,4,17, Arndt Borkhardt6, Anthony Letai9, Thorsten 13 Persigehl2,18, Martin Peifer2,3,19, Clemens A. Schmitt10,20, Hans Christian Reinhardt21,‡,§, 14 Gero Knittel1,2,3,4,‡ 15 16 1 University of Cologne, Faculty of Medicine and University Hospital Cologne, Clinic I of Internal Medicine, Cologne, Germany. 17 2 Center for Integrated Oncology, University of Cologne, Cologne, Germany. 18 3 Center for Molecular Medicine, University of Cologne, Cologne, Germany. 19 4 Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases (CECAD), University of Cologne, 20

Cologne, Germany. 21 5 University of Cologne, Faculty of Medicine and University Hospital Cologne, Institute of Pathology, Cologne, Germany. 22 6 Heinrich Heine University Düsseldorf, Medical Faculty, Department of Pediatric Oncology, Hematology and Clinical Immunology, 23

Düsseldorf, Germany. 24 7 Department of Pediatrics, Pediatric Hematology and Oncology, University Hospital Carl Gustav Carus, Technische Universität 25

Dresden, Germany. 26 8 National Center for Tumor Diseases (NCT), Dresden, Germany 27 9 Dana-Farber Cancer Institute, Harvard Medical School, Boston, USA 28 10 Charité Universitätsmedizin Berlin, Medical Department of Hematology, Oncology and Tumor Immunology, and Molekulares 29

Krebsforschungszentrum - MKFZ, Virchow Campus, Berlin, Germany. 30 11 Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany. 31 12 University of Cologne, Faculty of Medicine and University Hospital Cologne, Institute of Clinical Chemistry, Cologne, Germany. 32 13 Department of Pathology and Cell Biology, Institute for Cancer Genetics and the Herbert Irving Comprehensive Cancer Center, 33

Columbia University, New York, USA 34 14 Division of Hematology/Oncology, Department of Medicine, Weill Cornell Medicine, Cornell University, New York, USA 35 15 Adaptive Immunity and Lymphoma Group, German Cancer Research Center/National Center for Tumor Diseases Heidelberg, 36

Heidelberg, Germany 37 16 University of Cologne, Faculty of Medicine and University Hospital Cologne, Institute of Neuropathology, Cologne, Germany. 38 17 University of Cologne, Faculty of Medicine and University Hospital Cologne, Institute for Medical Microbiology, Immunology and 39

Hygiene, Cologne, Germany. 40 18 University of Cologne, Faculty of Medicine and University Hospital Cologne, Department of Radiology and Interventional 41

Radiology, Cologne, Germany. 42 19 University of Cologne, Department of Translational Genomics, Cologne, Germany. 43 20 Kepler Universitätsklinikum, Medical Department of Hematology and Oncology, Johannes Kepler University, Linz, Austria 44 21 Department of Hematology and Stem Cell Transplantation, University Hospital Essen, University Duisburg-Essen, German 45

Cancer Consortium (DKTK partner site Essen), Essen, Germany 46 47 48 49 50 * These authors contributed equally to the manuscript 51 ‡ These authors contributed equally to the manuscript 52 § Corresponding authors: H. C. Reinhardt ([email protected]), Hufelandstraße 55, 45147 Essen, Germany, +49-53

(0)201-723-2413 54 55 56 57 58

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Declaration of Interests 1

H.C.R. received consulting and lecture fees from Abbvie, Astra-Zeneca, Vertex and 2

Merck. H.C.R. received research funding from Gilead Pharmaceuticals. The remaining 3

authors declare no competing financial interest. H.C.R. is a co-founder of CDL 4

Therapeutics GmbH. 5

6 7 Abstract 8

Based on gene expression profiles, diffuse large B cell lymphoma (DLBCL) is sub-9

divided into germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. 10

Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88, 11

as well as BCL2 copy number gains. Here, we employ immune phenotyping, RNA-Seq 12

and whole exome sequencing to characterize a Myd88 and Bcl2-driven mouse model of 13

ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We 14

further demonstrate an actionable dependence of our murine ABC-DLBCL model on 15

BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. 16

Moreover, human ABC-DLBCLs displayed increased PD-L1 expression, compared to 17

GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined 18

venetoclax and RMP1-14 significantly increased the overall survival of lymphoma 19

bearing animals, indicating that this combination may be a viable option for selected 20

human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations. 21

22

23

Statement of Significance 24

Oncogenic Myd88 and Bcl2 cooperate in murine DLBCL-lymphomagenesis. The 25

resulting lymphomas display morphologic and transcriptomic features reminiscent of 26

human ABC-DLBCL. Data derived from our Myd88/Bcl2-driven autochthonous model 27

demonstrate that combined BCL2 and PD1 blockade displays substantial preclinical 28

anti-lymphoma activity, providing preclinical proof-of-concept data, which pave the way 29

for clinical translation. 30

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Introduction 1

Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid neoplasm in 2

adults and accounts for approximately 35% of B cell Non-Hodgkin lymphomas (B-NHL) 3

(1). DLBCL is a morphologically, biologically and clinically heterogeneous disease that 4

has historically been subdivided into germinal center B cell-like (GCB) and activated B 5

cell-like (ABC) DLBCL, using gene expression profiling (1-3). This cell of origin (COO)-6

based classifier separates sub-entities with distinct biology, pathogenesis and clinical 7

response to frontline chemo-immune therapy (3-5). GCB-DLBCL has been proposed to 8

originate from light-zone GCBs (6), whereas ABC-DLBCL likely derives from post-9

germinal center plasmablasts (3, 6-8). To capture additional molecular heterogeneity in 10

DLBCL, two independent comprehensive genomic analyses of human DLBCL cases 11

were recently completed and led to the discovery of partially overlapping genetically-12

defined DLBCL categories (9, 10). One group classified ~50% of the primary cases in a 13

supervised approach to four genetically-defined DLBCL subtypes (10). These were 14

based on COO-associated alterations and identified tumors with co-occurring MYD88- 15

and CD79B mutations (MCD), BCL6 rearrangements and NOTCH2 mutations (BN2), 16

EZH2 mutations and BCL2 rearrangements (EZB), as well as NOTCH1 mutations (N1) 17

(10). An independent analysis first defined recurrent genetic drivers in DLBCL and used 18

a non-negative matrix factorization consensus clustering approach, allowing to classify 19

98% of cases into 5 clusters with specific coordinate genetic signatures (9). These 20

clusters were defined by 1) BCL6 structural variants in combination with NOTCH2 21

aberrations (C1 DLBCL), 2) Bi-allelic TP53 inactivation (TP53 mutations and 17p copy 22

number losses) in combination with haploinsufficiencies of 9p21.13/CDKN2A and 23

13q14.2/RB1 (C2 DLBCL), 3) BCL2 mutations with concordant BCL2 structural variants 24

in combination with EZH2-, CREBBP- and KMT2D mutations and additional activating 25

alterations of the PI3K pathway (C3 DLBCL), 4) mutations in linker and core histone 26

genes in combination with aberrations in immune evasion molecules, NFB and 27

RAS/JAK/STAT signaling molecules (C4 DLBCL) (9). An additional cluster was defined 28

by 18q gains in combination with MYD88- and CD79B mutations (C5 DLBCL) (9). 29

These large datasets, together with the recently published whole exome sequencing 30

results of 1,001 DLBCL cases have established a framework for the identification of 31



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potentially druggable genomic aberrations in human DLBCL (11). In this context it is 1

important to note that frontline chemo-immune therapy using R-CHOP, or R-CHOP-like 2

regimens, achieves cure rates of more than 60% (9, 12, 13). However, relapsed or 3

refractory disease represents a major clinical challenge, as these patients are often 4

difficult to salvage and even high-dose chemotherapy regimens with autologous stem 5

cell support frequently do not provide long-term disease control (14-17). Thus, there is a 6

pressing need for the development and preclinical validation of therapeutic strategies for 7

the treatment of relapsed/refractory disease, as well as strategies to treat elderly and 8

frail patients that do not qualify for intensive chemo-immune therapy. 9

A powerful tool to assess the biological effects of targeted therapeutic agents are 10

autochthonous mouse models, which are genetically engineered to carry genomic 11

aberrations that precisely match those observed in the corresponding human disease. 12

The advent of next generation sequencing technologies has enabled the fine-grained 13

cross validation of mouse models and human disease. Here, we report the detailed 14

molecular characterization and cross-species comparison of an autochthonous mouse 15

model of Myd88-driven ABC-DLBCL. 29% of human ABC-DLBCL harbor the p.L265P 16

mutation in the hydrophobic core of the MYD88 TIR domain (18, 19). In contrast, the 17

MYD88 p.L265P mutation is exceedingly rare in non-ABC-DLBCLs (18). To assess the 18

role of MYD88p.L265P in B cell lymphomagenesis, we recently generated a Myd88p.L252P 19

allele (Myd88c-p.L252P) that is expressed from the endogenous locus upon Cre-mediated 20

deletion of the endogenous exons 2-6, together with the entire 3' UTR (20). Of note, 21

murine Myd88p.L252P is at the orthologous position of human Myd88p.L265P (20). When the 22

Myd88c-p.L252P allele is crossed with an additional mutant strain that conditionally 23

overexpresses BCL2 from the Rosa26 locus upon Cre-mediated deletion of a STOP 24

cassette, the resulting Myd88c-p.L252P/wt;Rosa26LSL.BCL2.IRES.GFP/wt;Cd19Cre/wt (MBC) 25

animals develop an aggressive lymphoma (20). 26

Here, we demonstrate that this MBC model resembles human ABC-DLBCL with respect 27

to morphology, as well as on the transcriptomic and genomic level. In contrast, a 28

Kmt2d/Bcl2-driven lymphoma model displayed more similarity with human GCB-DLBCL. 29

Moreover, our analyses revealed a druggable dependence on BCL2 in murine and 30

human ABC-DLBCL cell lines and tumors, which was not detected in human GCB-31



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DLBCL cell lines. We further demonstrate that Myd88-driven lymphomas display a 1

single agent response to PD-1 blockade, which is synergistic with concurrent BCL2 2

inhibition, in vivo. Altogether, these data provide a detailed molecular and functional 3

description of our Myd88-driven ABC-DLBCL model and provide a biological rational for 4

the use of combined BCL2- and PD-1 inhibition for the treatment of ABC-DLBCL. 5

6

7

Results 8

Myd88p.L252 and BCL2 cooperate to induce splenomegaly and GC formation in 9

vivo 10

Two of the most common genomic aberrations in human ABC-DLBCL are oncogenic 11

MYD88 mutations and BCL2 copy number gains (7, 9-11). Moreover, copy number 12

gains of 18q21.33, where the BCL2 gene is localized, are significantly enriched in 13

MYD88-mutant DLBCL cases, compared to MYD88 wildtype (wt) cases (Fig. S1A (9)). 14

To assess the in vivo effects of these aberrations, we performed longitudinal monitoring 15

of wt, Myd88c-p.L252P/wt;Cd19Cre/wt (MC), Rosa26LSL.BCL2.IRES.GFP/wt;Cd19Cre/wt (BC) and 16

Myd88c-p.L252P/wt;Rosa26LSL.BCL2.IRES.GFP/wt;Cd19Cre/wt (MBC) animals using MRI scanning 17

to monitor splenomegaly (Fig. 1A-C). Amplifications involving 18q21.33, the 18

chromosomal location of BCL2, are associated with an overexpression of BCL2 (Fig. 19

S1B), which is modelled by the Rosa26LSL.BCL2.IRES.GFP allele employed in this study. As 20

shown in Fig. 1B-C, MBC animals displayed a significantly increased spleen volume, 21

compared to wt, BC and MC animals at 10, 20 and 30 weeks. Moreover, at the 20-, 30- 22

and 50-week time points, the spleen volume of BC animals was significantly larger than 23

that of wt and MC mice (Fig. 1C, S1C). We next performed a histological assessment of 24

spleen sections derived from wt, MC, BC and MBC animals (Fig. 1D, S1D). For that 25

purpose, 30-week old wt, MC, BC and MBC animals were sacrificed and spleens were 26

stained with antibodies detecting CD3 (labeling the splenic T cell zone) and B220 27

(labeling the splenic B cell zone), as well as PNA (labeling the germinal center), on 28

serial sections. Neither the number of germinal centers per spleen area, nor the 29

germinal center area per spleen area or the average size of the germinal centers 30

differed significantly between wt and MC animals (Fig. 1D, E, S1D). However, BC 31



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animals displayed significantly more and larger germinal centers, than wt and MC 1

animals (Fig. 1D, E). Similarly, MBC animals showed significantly more and larger 2

germinal centers, than MC mice and wt controls (Fig. 1D, E). Of note, we did not detect 3

clonal lymphoma infiltrates in MBC spleens at this timepoint. Furthermore, the global 4

splenic architecture was not disrupted in MBC mice. In a different set of experiments, 5

we employed a BCL6 antibody to stain splenic germinal centers (Fig. S1E). In these 6

experiments, BC animals displayed slightly more and significantly larger germinal 7

centers than wt animals (Fig. S1E). Similarly, MBC animals showed significantly more 8

and larger germinal centers than wt and MC controls (Fig. S1E). Altogether, these data 9

indicate that oncogenic Myd88 and BCL2 cooperate in enhancing reactive 10

splenomegaly and GC formation in vivo. 11

12

Myd88p.L252 and BCL2 cooperate to drive an expansion of CD138-positive cells in 13

vivo 14

To gain further insight into the cellular composition underlying the germinal center 15

hyperplasia that we observed in BC and MBC animals (Fig. 1D, E, S1D, E), we next 16

performed flow cytometry-based immune-phenotyping (Fig. 1F and S1F-H). We 17

particularly assessed the representation of total B cells (B220+ of CD45+), follicular B 18

cells (B220+/CD93-/CD21low/CD23+ of CD45+), marginal zone B cells (B220+/CD93-19

/CD21high/CD23- of CD45+), transitional B cells (B220+/CD93+ of CD45+), early 20

plasmablasts (CD138+/B220+/MHCII+ of CD45+) and late plasmablasts/plasma cells 21

(CD138+/B220-/MHCII- of CD45+) from spleens and bone marrow (BM) of 30-week old 22

wt, MC, BC and MBC animals (Fig. 1F, S1F), as well as 50-week old wt, MC and BC 23

mice (Fig. S1G, H). As shown in Fig. 1F, the relative percentage of splenic B cells was 24

significantly increased in BC and MBC mice, compared to wt animals. A further sub-25

classification of these B cells revealed that BC animals displayed significantly more 26

follicular B cells, compared to wt, MC and MBC mice (Fig. 1F). Marginal zone B cells 27

were significantly less prevalent in MC, BC and MBC mice, compared to wt animals 28

(Fig. 1F). MBC mice displayed a significantly increased percentage of transitional B 29

cells, compared to wt, MC and BC mice (Fig. 1F). The most striking differences were 30

observed when the percentages of CD138+ cells were analyzed (Fig. 1F). MC animals 31



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displayed significantly more CD138+ cells, compared to wt and BC mice, possibly 1

indicating that oncogenic Myd88 enhances GC transition (Fig. 1F). Moreover, 2

oncogenic Myd88 and BCL2 appear to cooperate in the accumulation of CD138+ early 3

plasmablasts (Fig. 1F). Similarly, MBC mice harbor a significantly higher percentage of 4

late plasmablasts/plasma cells in the spleen and bone marrow compared to wt, MC and 5

BC mice (Fig. 1F, S1F). The levels of CD138+ cells found in MBC animals at 30 weeks 6

of age were not reached even by 50-week old MC and BC mice (Fig. S1G, H). To 7

investigate whether these plasma cells passed through the germinal center reaction, or 8

whether the engineered mutations preferentially drive the development of extrafollicular 9

plasma cells, we performed full-length BCR sequencing on CD138+ cells isolated from 10

the spleens of 10 weeks old wt and MBC animals (n = 4 per genotype) (21). In short, 11

each individual Ig cDNA molecule is uniquely labeled with a barcode (unique molecular 12

identifier, UMI) during reverse transcription. This UMI allows the assignment of each 13

sequencing read to a cDNA molecule of origin. Reads with identical UMI are grouped 14

into a ‘molecular identifier group’ (MIG) and a nearly error-free sequence is derived for 15

each MIG by consensus assembly of the assigned reads. Analysis of the somatic 16

hypermutation frequency of the derived V(D)J region sequences revealed a minor, but 17

significant shift towards mutated Ighm sequences (51.6% and 39.4% of Ighm 18

sequences with more than one mutation for MBC and wt CD138+ cells, respectively,Fig. 19

S2A, B). No significant differences in the mutation rates of transcripts with Ighg1, 20

Ighg2c, Ighg3 and Igha constant regions were observed (Fig. S2A, B). Ig sequences 21

recovered from CD138+ MBC cells showed a significant reduction of Ighm transcripts 22

and a significant increase in Ighg2c and Ighg3 isotypes, compared to CD138+ wt cells 23

(Fig. S2C, D), in line with a role of Toll-like receptor (TLR) signaling in promoting class 24

switch recombination (22-24). Altogether, these data indicate that the CD138+ cells, 25

which accumulate in MBC mice, pass through the GC reaction with at least the same 26

frequency as CD138+ wt cells. These observations further suggest a role of oncogenic 27

Myd88 in promoting the transition through the GC reaction, which may be augmented 28

by the apoptosis-repressing effect of an increased BCL2 gene dosage, ultimately 29

leading to a substantial expansion of post-germinal center B cell stages in non-30

lymphoma-bearing animals. 31



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1

B cell-specific Myd88p.L252 and BCL2 expression disrupt self-tolerance in vivo 2

Given the robust expansion of post-germinal center B cell populations in MBC mice, we 3

next asked whether MBC animals displayed increased serum immunoglobulin levels, 4

compared to wt, MC and BC mice. To address this question, we initially performed 5

electrophoreses with serum samples isolated from 30-week-old animals (Fig. 1G). 6

Consistent with the massive expansion of early and late plasmablasts, as well as 7

plasma cells in MBC mice, we observed a substantially increased gamma-globulin 8

fraction in sera derived from MBC animals, compared to wt, MC and BC samples (Fig. 9

1G). The gamma-globulin levels of MC and BC animals were only mildly increased, 10

compared to wt mice (Fig. 1G). Of note, we detected a monoclonal gamma-globulin 11

band in one out of six MC animals, whereas no monoclonal band could be detected in 12

wt, BC and MBC mice at this 30-week time point (n = 6 each, Fig. 1G). To further 13

dissect this gammopathy, we next performed ELISA experiments to assess the 14

contribution of IgM and the IgG subclasses IgG1, IgG2b, IgG2c and IgG3 to the 15

gamma-globulin fraction. As shown in Fig. 1H, MBC animals displayed significantly 16

increased serum IgM, IgG1, IgG2b and IgG2c concentrations, compared to wt, MC and 17

BC animals, while there was no significant difference in IgG3 concentrations (Fig. 1H). 18

These data are consistent with the reduced percentage of splenic marginal zone cells, 19

which are a major source of IgG3 in response to T cell-independent antigens (25). 20

These results demonstrate that 30-week-old MBC animals display a marked polyclonal 21

gammopathy, consisting of class-switched and non-class-switched immunoglobulins. 22

Recent reports provided evidence indicating that chromatin/DNA-associated, as well as 23

RNA and RNA-associated antigens can potently activate autoreactive B cells through 24

sequential dual engagement of BCR and TLR/MYD88 signaling cascades (26-29). In 25

brief, AM14 RF+ B cells that bind autologous IgG2a with low affinity can be driven into 26

proliferation by in vitro exposure to affinity-purified IgG2aj monoclonal antibodies 27

specific for nucleosomes (30). Using this system, it was shown that activation of RF+ B 28

cells was driven by IgG2a:chromatin immune complexes, which was DNAse-sensitive 29

(26). Moreover, activation was dependent on the synergistic engagement of the BCR 30

and MYD88-dependent TLR signaling (26). The same BCR/TLR paradigm was shown 31



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to hold true for RNA-associated autoantigens, as well as CpG dsDNA antigens (27, 29). 1

It is important to note that full activation of these autoreactive B cells was abolished in 2

the absence of Myd88 (26, 27, 29). Moreover, the constitutive B cell-specific 3

overexpression of BCL2 was recently shown to impair tolerance induction in a series of 4

model systems and to induce a lupus-like serological phenotype with anti-nuclear 5

reactivity (31-37). Given the critical role of Myd88 in promoting the activation of 6

autoreactive B cells, as well as the impact of BCL2 on tolerance induction, we next 7

asked whether B cell-specific expression of the Myd88 p.L252P gain of function 8

mutation and/or BCL2 overexpression may promote the loss of self-tolerance. For that 9

purpose, we assessed the presence or absence of autoreactive serum antibodies, using 10

the HEp2 indirect immunofluorescence staining assay. As shown in Fig. 2A, wt and BC 11

sera produced only a dim anti-IgM fluorescence signal in the HEp2 assay, whereas MC 12

and MBC animals displayed significantly higher levels of autoreactive IgM antibodies. 13

The staining pattern obtained with MC and MBC sera was largely cytoplasmic, 14

indicating that these antibodies may not recognize DNA or DNA-associated antigens 15

(Fig. 2B). We next assessed the presence of IgG autoreactive antibodies. As shown in 16

Fig. 2C, we observed a robust staining with sera derived from MC, BC and MBC 17

animals, whereas wt serum only produced faint signal. Of note, BC serum primarily 18

reacted with nuclear antigens (Fig. 2D). Altogether, these data indicate that B cell-19

specific expression of Myd88p.L252P overcomes self-tolerance. Of note, Myd88p.L252P 20

expression primarily results in an increased presence of autoantibodies against 21

cytoplasmic structures, mainly of the IgM isotype, while BCL2 overexpression promotes 22

the generation of predominantly anti-nuclear IgG immunoglobulins. MBC animals 23

showed a mixed phenotype. 24

25

B cell-specific Myd88p.L252 and BCL2 expression lead to an enhanced B cell 26

reactivity in vivo 27

B cell responses are sub-divided into T cell-dependent (TD) and T cell-independent (TI) 28

responses (38). TD antigens are proteins that are processed and presented on MHC-II 29

surface molecules for detection by CD4+ T helper cells (38). Two types of TI antigens 30

exist (38). TI-I antigens, such as LPS, CpG and poly-IC mediate polyclonal B cell 31



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activation by engaging TLRs (38). TI-II antigens are typically polysaccharides that 1

induce antigen-specific B cell responses through BCR clustering (38). To ask whether B 2

cell-specific Myd88p.L252P expression and/or BCL2 overexpression may affect the 3

magnitude or persistence of the humoral immune response induced by TD or TI-II 4

antigens, we immunized wt, MC, BC and MBC mice with NP-CGG (TD) or NP-Ficoll (TI-5

II) and quantified the NP-specific IgM and IgG response 4, 7, 10, 21 and 40 days after 6

the vaccination. The maximal IgM response to NP-Ficoll was increased 7.5- and 6.6-fold 7

in MC and BC animals compared to wt, respectively (Fig. 2E). This effect was even 8

more pronounced in MBC animals, where we observed a 14.8-fold increase, compared 9

to wt (Fig. 2E). The NP-Ficoll induced IgG response was similarly enhanced in MC and 10

MBC animals (23.0- and 36.3-fold, respectively), compared to wt (Fig. 2E). However, in 11

contrast to the IgM response, the IgG response was only marginally (4.5-fold) increased 12

in BC, compared to wt animals, suggesting BCL2 overexpression may not suffice in 13

promoting class switch recombination following TI-II-mediated B cell activation (Fig. 14

2E). The humoral anti-NP response following exposure to the TD antigen NP-CGG 15

differed from that observed for NP-Ficoll. Both BC and MBC animals displayed a 16

massively enhanced maximal IgM (7.3- and 8.3-fold, respectively) and IgG response 17

(67.7- and 80.6-fold), compared to wt controls (Fig. 2F). In contrast MC animals 18

displayed only a marginally enhanced IgM and IgG response (2.8- and 27.6-fold), 19

compared to wt mice (Fig. 2F). These data, which are in line with previous reports on 20

the analysis of an Eµ:Bcl2 allele (35), indicate that BCL2 overexpression particularly 21

promotes the humoral response against TD antigens. The expression of Myd88p.L252P 22

promotes class switch in response to TI-II antigen, in agreement with previous studies 23

demonstrating the importance of TLR signaling for class switch to IgG, in response to 24

thymus-independent antigen (22, 39). 25

26

Expression of oncogenic Myd88 drives the formation of a protein super-complex 27

containing BCR- and Myddosome components 28

Recent data from human lymphoma cell lines indicate that oncogenic BCR signaling in 29

a subset of ABC-DLBCL cases is coordinated by a protein complex involving MYD88, 30

TLR9 and IgM (40). This so-called My-T-BCR complex co-localizes with mTOR on 31



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endolysosomes, where it promotes NFB-mediated pro-survival signaling (40). This 1

report prompted us to ask whether protein complexes involving BCR signaling 2

components, as well as MYD88 and Myddosome components, also assemble in non-3

transformed Myd88-mutant cells. To this end, we performed proximity ligation assays 4

(PLA), which label proteins that co-localize within a nanometer distance (41). We 5

isolated naïve B cells from wt and MC animals to explore the interactome of wt and 6

mutant MYD88. In these experiments, we detected significantly more complexes 7

involving MYD88 and IRAK1, as well as the Myddosome component IRAK4, in 8

unstimulated MC B cells, compared to wt controls (Fig. 3A, B). Moreover, naïve B cells 9

from MC animals also displayed significantly more protein complexes consisting of 10

MYD88 and the BCR signaling components IgM and BTK, compared to wt controls (Fig. 11

3A, B). To further substantiate the functional relevance of the increased MYD88-12

centered complex formation in MC-derived B cells, we next performed immunoblots 13

probing the Myddosome formation-induced autophosphorylation site Thr-345 in IRAK4, 14

the phospho-Tyr-223 residue in BTK and the IKK2-dependent phospho-Ser-536 site in 15

the NFB subunit p65, in unstimulated MC and wt B cells (Fig. 3C). In these 16

experiments (n=3 independent B cell isolations from distinct animals), we observed a 17

significantly increased p65 phosphorylation on Ser-536 in MC, compared to wt B cells 18

(Fig. 3C). While there was a trend towards increased IRAK4 autophosphorylation on 19

Thr-345 in MC B cells, this did not reach statistical significance (Fig. 3C). Similarly, 20

there was no differential BTK Tyr-223 phosphorylation in MC vs. wt B cells (Fig. 3C). 21

Altogether, these observations suggest that mutant MYD88 may constitutively nucleate 22

a signaling hub, which links BCR and TLR signaling components, even in non-23

transformed B cells. This constitutive protein complex formation is further associated 24

with increased p65 Ser-536 phosphorylation in MC B cells. 25

26

Myd88p.L252 and BCL2 cooperate in ABC-DLBCL lymphomagenesis in vivo 27

We next aimed to determine the effect of B cell-specific Myd88p.L252P and/or 28

Rosa26BCL2.IRES.GFP expression on overall survival and to determine the cause of death 29

in these mice. We previously reported that MC animals display a significantly reduced 30

overall survival, compared to wt mice and that MBC animals live significantly shorter 31



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than MC mice (20). However, it remained unclear whether BC animals display a 1

different overall survival than MC mice. Thus, we performed a direct head-to-head 2

comparison of MC, BC and MBC animals. As shown in Fig. 4A, BC mice show a 3

significantly reduced overall survival, compared to MC animals. Furthermore, and in line 4

with previous results, MBC mice died significantly earlier than MC and BC animals (Fig. 5

4A). We further determined the cause of death in MC, BC and MBC mice. While MBC 6

mice developed life-limiting lymphoma with 83 percent penetrance, MC and BC mice 7

developed lymphoma only in 20 and 50 percent, respectively (Fig. 4B). The malignant 8

lesions observed in MBC animals were largely located sub-diaphragmatically in 9

mesenteric lymph nodes, rarely in mediastinal and submandibular lymph nodes (Fig. 10

S3A, B). We did not detect any obvious central nervous system manifestations. While 11

spleens were frequently enlarged, we failed to detect evidence for infiltration by clonal 12

lymphoma. Instead, we observed the germinal center expansion detailed in Fig. 1D-E. 13

Moreover, spleen weights did not exceed 450mg, further indicating a reactive nature of 14

the observed splenomegaly (Fig. S3B). The average number of distinct lymph node 15

groups involved was 1.7 (Fig. S3C). Immuno-phenotyping revealed that BC- and MBC-16

derived lymphomas were almost exclusively B220- and largely CD138+ (Fig. 4B, C). We 17

note that the CD138 staining pattern displayed some inter-tumoral heterogeneity, which 18

is illustrated in Fig. S3D where 6 distinct lymph node manifestations derived from 6 19

distinct animals are displayed. The CD138 staining intensity varied between different 20

areas of the same lymphoma manifestation. Moreover, when analyzed at higher 21

magnification, we observed clearly distinct CD138 staining intensity in adjacent cells. It 22

is important to note that the same lymphoma areas stained uniformly positive for GFP, 23

which is only expressed in cells that have expressed the Cd19Cre allele during their 24

lifetime and therefore belong to the B cell lineage (Fig. S3D). Altogether, these data 25

suggest a post-germinal center plasmablastic differentiation pattern of MBC 26

lymphomas, whereas lymphomas developing in MC mice retained expression of the B 27

cell marker B220 and were CD138neg (Fig. 4B, C). The proliferation index in MBC 28

lymphomas was 42.7% (17.3%, n = 19). MC and BC lymphomas displayed similar 29

proliferation indices of 39.1 (14.5%, n = 4) and 35.9% (14.3%, n = 5), respectively 30

(Fig. 4C, S3E). Next to lymphomas, MC, BC and MBC mice displayed the indicated 31



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percentage of cases that had to be sacrificed, due to non-clonal B cell 1

lymphoproliferation or scratch wounds (Fig. 4B). Particularly the latter may represent 2

the manifestation of an exacerbated autoimmunity phenotype. To determine whether 3

lesions histologically classified as lymphoma truly represent clonal expansions, we next 4

performed RNA-based BCR sequencing (adapted from (42)). While the BCR repertoire 5

in the spleens of wt animals was highly poly-clonal, as expected, MBC lesions showed a 6

strongly reduced variation with the majority of cases being dominated by a single or only 7

a few parallel clones (Fig 4D, E). Perhaps not surprisingly, the BCR sequence of 8

isolated stable cell lines corresponded to a dominant clone found in the primary lesion 9

(Fig. 4D). 10

Cell lines derived from MBC lymphomas could be established in vitro and were 11

transplantable into Rag1-/- recipient animals. Upon transplantation, these MBC-derived 12

cell lines formed lymphomas that were indistinguishable from the original lymphomas 13

with respect to morphology, immune phenotype and proliferation index (Fig. 4F). 14

To further benchmark our mouse model against human ABC-DLBCL lymphomas, we 15

next used 3'-RNA-sequencing to assess the transcriptomes derived from MBC 16

lymphomas. Lymphomas derived from Kmt2dfl/fl;VavP-Bcl2;Ighg1Cre/wt (KBC) mice, 17

which develop follicular lymphoma and GCB-DLBCL-like disease, served as a reference 18

control (43). To specifically ask whether the transcriptome data derived from MBC and 19

KBC lymphomas co-cluster with transcriptome data derived from human GCB- or ABC-20

DLBCL, we performed a gene set enrichment analysis (GSEA) for gene signatures that 21

have previously been shown to effectively distinguish human GCB- from ABC-DLBCL 22

(11) and found a significant enrichment of the GCB- and ABC signatures in KBC and 23

MBC tumors, respectively (Fig. 4G). In addition to KBC lesions, we also benchmarked 24

our MBC tumors against the MYC/PI3K-driven R26LSL.Myc/LSL.P110*;Ighg1Cre/wt (MPC) 25

Burkitt lymphoma model (44), which constitutes an additional germinal center-derived 26

aggressive lymphoma model (Fig. S3F). We observed that MPC lymphomas displayed 27

a significant enrichment of GCB gene expression signatures, while MBC lymphomas 28

were enriched, albeit not significantly in this analysis, for ABC signatures (Fig. S3F). 29

Next to transcriptome profiling, we also performed whole exome sequencing of 17 MBC 30

cases, to ask whether the pattern of spontaneous mutations in these lymphomas also 31



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resembles the genomic aberrations detected in human DLBCL cases. As benchmarks, 1

we employed two recently published data sets reporting the mutational profiles of 878 2

human DLBCL cases (9, 10). We detected a number of mutations in our MBC 3

lymphomas, including aberrations in Pim1, Myc, Kmt2d, Nfkbia, Stat3, Pou2f2 and 4

Hist1h1e (Fig. 4H, Tab. S1), which were also frequently mutated in both human DLBCL 5

data sets (9, 11). 6

We also assessed the serum abundance of 32 distinct cytokines in lymphoma-bearing 7

MBC mice and age-matched wt controls, using multiplexed cytokine arrays. We 8

observed significantly higher levels of IFN- and TNF- in sera of lymphoma-bearing 9

MBC mice, compared to controls (Fig. 4I, Fig. S4A). In line with these observations, 10

increased TNF- and IL-10 plasma levels were recently shown to correlate with poor 11

prognosis in DLBCL (45). Moreover, the TNFA single nucleotide polymorphism 12

308G→A was shown to be associated with increased constitutive and inducible TNF- 13

expression and increased risk of DLBCL development (46-49). Furthermore, TNF- was 14

shown to induce an inhibitory gene expression signature in CD4+ T cells during chronic 15

viral infection, indicating that TNF- may impact on the ratio of CD4+:CD8+ T cells (50). 16

Conversely, IFN- was shown to act on CD8+ T cells to enhance their abundance, 17

mobility and cytotoxicity during viral infection and experimental graft rejection (51, 52). 18

Thus, our cytokine profiling data may indicate that lymphoma-bearing MBC mice display 19

signs of a pro-inflammatory environment favoring the activity of CD8+ T cells. 20

To further gauge whether cytokines were produced by the lymphoma cells themselves 21

or rather the non-malignant components of the lymphoma microenvironment, we 22

compared gene expression profiles of bulk primary MBC lymphoma samples and stable 23

cell lines derived from MBC lymphomas. The relative expression levels of Ccl11, Il1a, 24

Il6, Il12b, Cxcl1, Cxcl5, Ccl2, Csf1, Cxcl9, Ccl5 and Ifng were significantly lower in the 25

three analyzed cell lines, compared to the primary lymphoma material, suggesting that 26

these cytokines are, at least partially, synthesized by non-malignant cells within the 27

microenvironment. In contrast, we found that several cytokines, including Il2, Il10, Il15 28

(out of which particularly IL-2 and IL-15 were previously shown to promote B cell 29

proliferation (53, 54)), were expressed at high levels by the lymphoma cell lines. 30

Moreover, Tnf was expressed at high levels by two of the three cell lines (Fig. S4B). 31



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However, it is important to note that mRNA expression levels do not always correlate 1

with protein expression (55). Thus, the data presented here have to be interpreted with 2

care. 3

4

MBC-derived lymphomas display an actionable BCL2 inhibitor sensitivity 5

Our data indicate that the MBC model accurately mimics human ABC-DLBCL biology 6

with regard to morphology, as well as genomic and transcriptomic profiles. Thus, we 7

next aimed to employ this model as a preclinical tool. To this end, we initially performed 8

cellular viability assays to determine drug sensitivity. We specifically assessed the 9

sensitivity of 4 distinct MBC-derived lymphoma cell lines (M191, M190, M108, M552), 10

as well as 2 human ABC-DLBCL (U2932, RI1) and 2 human GCB-DLBCL cell lines 11

(SUDHL10, OCILY7) towards the IRAK4 inhibitor ND-2158, the IKK2 inhibitor LY-12

2409881 and the BCL2 inhibitor venetoclax. These experiments revealed that murine 13

MBC-derived cells, as well as the human ABC-DLBCL cell lines included in our screen 14

were sensitive against IKK2-, IRAK4- and BCL2 inhibition (Fig. 5A-C). Reminiscent of 15

previously published data (56, 57), we found the two GCB cell lines investigated here to 16

be resistant against BCL2 blockade (Fig. 5A). However, it is important to note that 17

several human GCB-DLBCL cell lines, such as HF, RC, McA and OCI-LY19 cells, have 18

been reported to be highly sensitive to BCL2 inhibition (58). Thus, our observations, 19

which are limited to two human GCB cell lines should not be generalized to all GCB-20

DLBCL cases. Of note, BH3 profiling (59) confirmed that human ABC-DLBCL cell lines 21

and murine MBC-derived cell lines were BCL2-dependent, whereas the limited number 22

of human GCB-DLBCL cell lines displayed a more prominent sensitivity against MCL1 23

inhibition (Fig. 5D). 24

As cell viability assays do not distinguish between cell death and growth arrest, we next 25

performed flow-cytometry-based apoptosis measurements, using Annexin V/propidium 26

iodide (PI) co-staining. Venetoclax and LY-2409881 effectively induced apoptosis in the 27

MBC lines (Fig. 5E, F). Moreover, venetoclax induced massive apoptosis in both human 28

ABC-DLBCL cell lines, whereas LY-2409881-induced apoptosis was prominent in RI1 29

and relatively mild in U2932 ABC-DLBCL cells (Fig. 5E, F). ND-2158 treatment did not 30

lead to a significant induction of apoptosis in any of the investigated cell lines. 31



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Altogether, these data indicate that venetoclax, ND-2158 and LY-2409881 display 1

therapeutic efficacy in the human and murine ABC-DLBCL cell line models investigated 2

in this study. However, only venetoclax and LY-2409881 appear to induce apoptosis, 3

whereas ND-2158 likely reduces proliferation (Fig. 5G, Fig. S5A). 4

Of note, in an extension of this focused candidate approach to discover potentially 5

actionable aberrations in GCB and ABC cell lines, we also conducted a larger discovery 6

screen on human GCB (OCI-LY7, SUDHL10) and ABC cell lines (RI-1, U2932), as well 7

as three MBC-derived murine lymphoma cell lines. We specifically determined the IC50 8

values of 167 additional distinct drug compounds (Fig. S5B, Tab. S2). Fitting with our 9

transcriptome data, RI-1 and U2932 cells co-clustered with the MBC cell lines. 10

To ask whether the therapeutic efficacy of venetoclax, ND-2158 and LY-2409881 was 11

reproducible in vivo, we next performed preclinical drug sensitivity studies in a 12

transplant model. To this end, we transplanted 107 murine M108 cells intraperitoneally 13

into Rag1-/- recipient animals. Treatment was initiated 14 days after transplantation. Of 14

note, in a series of preparatory experiments, we had verified that 4/4 transplanted 15

animals developed necropsy-verified clonal lymphoma 14 days following 16

transplantation. Animals were treated with venetoclax (200mg/kg, q.d., orally), ND-2158 17

(150 mg/kg, q.d., intraperitoneally), LY-2409881 (100 mg/kg, q.d., intraperitoneally), or 18

left untreated. As shown in Kaplan-Meier format in Fig. 5H, venetoclax induced a 19

significantly increased overall survival (50.0 4.5 days after transplantation following 20

completion of a 3-week treatment course), compared to vehicle control (32.5 5.9 21

days). Somewhat surprisingly, ND-2158 did not produce a significant survival gain in 22

these experiments. All ND-2158-treated animals succumbed to lymphoma, indicating a 23

lack of in vivo activity of this compound at the chosen dose in our model. Similarly, LY-24

2409881 did not lead to significant survival gains in this model. We note, however, that 25

LY-2409881-treated animals uniformly reached pre-defined termination criteria (weight 26

loss >10%, hunched posture, ragged fur, apathy). Importantly, we did not detect 27

lymphoma in any of the LY-2409881-treated animals at the time of sacrifice, indicating 28

that LY-2409881 may display preclinical activity, which is masked by life-limiting toxicity. 29

This pro-inflammatory adverse effect of LY-2409881 is in line with previously reported 30

toxicities of IKK inhibitors (60). Collectively, our preclinical data indicate that venetoclax 31



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may display activity against a subset of BCL2-altered ABC-DLBCL lymphomas, 1

whereas our data suggest that ND-2158 and LY-2409881 may not be candidates for 2

further development in this entity, due to lack of preclinical efficacy or toxicity. 3

4

Human ABC-DLBCL and murine MBC-derived lymphomas display an actionable 5

PD-L1 expression 6

Immune checkpoint blockade is emerging as a potential route for therapeutic 7

intervention in relapsed/refractory DLBCL (61). For instance, the anti-PD-1 monoclonal 8

antibody nivolumab achieved an overall response rate of 36% in DLBCL patients in a 9

phase I, open-label, dose-escalation, cohort-expansion study (62). Moreover, NFB has 10

been shown to be a potent inducer of PD-L1 expression (63). As increased NFB 11

activity is a hallmark of ABC-DLBCL, we next assessed the PD-L1 mRNA expression 12

levels in a publicly available data set comprised of 1,001 human DLBCL cases (11). 13

This analysis revealed that PD-L1 and PD-1 were significantly overexpressed in ABC-, 14

compared to GCB-DLBCL, whereas CD86 was expressed at significantly higher levels 15

in GCB-, compared to ABC-DLBCL (Fig. 6A). No entity-specific differences were 16

observed in the expression levels of PD-L2 and CTLA4 (Fig. 6A). These data are in line 17

with recently reported analyses, which demonstrate that cytogenetic aberrations 18

affecting the CD273/CD274 locus were more frequently observed in the non-GCB 19

subtype of DLBCL (64). To verify that human ABC-DLBCL displays increased PD-L1 20

protein expression compared to GCB-DLBCL, we next performed 21

immunohistochemistry on 38 human DLBCL cases. As shown in Fig. 6B, we observed 22

a significantly higher fraction of PD-L1-positive cases among the ABC-DLBCL samples, 23

compared to GCB-DLBCL. Moreover, in a series of primary central nervous system 24

lymphomas, we found PD-L1 expression in 22 out of 23 cases (Fig. 6C). Intriguingly, 25

the single PD-L1-negative case harbored a non-canonical MYD88p.P267L. Among the 26

PD-L1-expressing cases, we found 16 harboring a MYD88p.L265P mutation, whereas the 27

remaining 5 cases were MYD88 wt. These data suggest that ABC-DLBCL cases may 28

be sensitive to immune checkpoint blockade by disrupting the PD-1/PD-L1 axis. 29

In addition to the PD-1/PD-L1 axis, BCL2 has emerged as a therapeutic target in 30

DLBCL during the last years. The BCL2 inhibitor venetoclax produces response rates of 31



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approx. 18% in relapsed/refractory DLBCL (65). To ask whether there might be a 1

biological rational for combining these agents, we reassessed the available data set 2

consisting of 1,001 human DLBCL cases (11). This analysis revealed that 22% of 3

DLBCL cases display a higher than average combined expression of CD274 and BCL2 4

and that of these cases, 71% are of the ABC subtype (Fig. S6A). In contrast, only 29% 5

of cases with a lower than average expression of both CD274 and BCL2 were ABC 6

tumors. Building on the observation that human ABC DLBCL cases frequently co-7

expressed CD274 and BCL2, and given that our MBC model phenocopies the clinical 8

scenario of ABC-DLBCL, we next asked whether combined BCL2- and PD-1 inhibition 9

may display synergistic cytotoxic activity in this model. For that purpose, we generated 10

a cohort of MBC animals, in which lymphoma development was surveilled by 11

longitudinal MRI scans. Upon lymphoma detection, defined by a lesion larger than 75µl 12

with detectable volume increase in two consecutive scans, animals were randomized in 13

a 1:1:1:1 fashion to receive either vehicle solution (control), venetoclax (200mg/kg, p.o., 14

q.d., day 1-21), the anti-PD-1 antibody RMP1-14 (10mg/kg, i.p., twice weekly, until 15

death), or combined venetoclax plus RMP1-14. Therapy response was longitudinally 16

assessed through weekly MRI scans, which enabled us to gauge depth of remission 17

and duration of response (Fig. 6D, E, S6B). While both single agent RMP1-14 and 18

venetoclax produced objective responses in the MBC model (Fig. 6E), these responses 19

were not durable (Fig. S6B). The median survival of RMP1-14- or venetoclax-treated 20

mice was 11.5 and 10.1 weeks, respectively, compared to 5.1 weeks in untreated 21

animals. While the survival difference between untreated and venetoclax-treated mice 22

failed to reach statistical significance, RMP1-14 induced a significant survival gain, 23

compared to untreated controls (Fig. 6F). 24

The significant single agent activity of RMP1-14 prompted us to further explore the 25

effects of PD1 blockade on the composition and activation status of the cells within the 26

lymphoma microenvironment. For that purpose, we employed mass cytometry from 27

lymphoma tissue derived from RMP1-14-treated (250 µg/dose, day 1, 4 and 7, i.p.) or 28

untreated animals (Fig. 6G-K). Our analysis revealed that PD1 blockade in MBC 29

lymphoma-bearing animals induced distinct phenotypic changes in lymphoma-infiltrating 30

CD4+ and CD8+ T cells, which are in line with reversal of an exhausted phenotype of 31



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lymphoma-infiltrating T cells upon treatment (Fig. 6H-I). While the CD4+ and CD8+ T cell 1

population sizes did not show significant changes (Fig. 6J), we particularly detected a 2

significant reduction in IRF4 and TIM3 expression in both, CD4+ and CD8+ T cells, upon 3

RMP1-14 exposure (Fig. 6I, K). Moreover, CD8+ T cells displayed significantly reduced 4

4-1BB and LAG3 expression, following PD1 blockade. Further, CD4+ T cells displayed 5

reduced CD69 expression, upon RMP1-14 exposure (Fig. 6I, K). These data are in line 6

with a reinstated anti-lymphoma immune response following PD1 blockade, as LAG3, 7

TIM3 and CD69 are well-established exhaustion markers in CD4+ and CD8+ T cells (66-8

70). Similarly, IRF4 was shown to induce exhaustion of CD8+ T cells, during chronic 9

stimulation (71). In addition, 4-1BB is an established marker of exhausted CD8+ T cells 10

(72, 73). Lastly, the p53 target gene Cdkn1a (p21) is a potent CDK inhibitor involved in 11

p53-mediated cell cycle arrest (74). Reduced expression of Cdkn1a in CD4+ T cells 12

following PD1 blockade thus might be in line with restored proliferation potential. 13

Altogether, these data suggest that PD1 blockade in lymphoma-bearing MBC mice 14

promotes a phenotypic switch away from an exhausted CD4+ and CD8+ T cell state in 15

treatment-naïve lymphomas. We note that a parallel assessment of serum cytokine 16

levels (n=32 distinct cytokines) in RMP1-14- and vehicle-treated animals did not reveal 17

any significant differences (Fig. S7). 18

In addition to our assessment of single agent activities, we also analyzed the effect of 19

combined venetoclax plus RMP1-14, which resulted in a significant overall survival gain 20

(median overall survival 20 weeks), compared to untreated animals or mice exposed to 21

the single agents (Fig. 6D-F, S6B). Altogether, these functional in vivo experiments 22

indicate that combined BCL2 and PD-1 blockade may represent a viable treatment 23

strategy for a molecularly-defined subset of ABC-DLBCL cases. 24

25

26

Discussion 27

Here, we characterized a mouse model of Myd88 and BCL2-driven DLBCL. In essence, 28

we show that Myd88 p.L252P and BCL2 cooperate in DLBCL lymphomagenesis. The 29

resulting lymphomas display gene expression profiles that are strikingly similar to 30

human ABC-DLBCL (Fig. 4G, S3F). Moreover, in addition to the engineered aberrations 31



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in Myd88 and BCL2, these lymphomas also spontaneously acquire single nucleotide 1

variants that are also detectable in human DLBCL, including mutations in Pim1, Myc, 2

Pou2f2, Nfkbia and Kmt2d (Fig. 4H, Tab. S1). 3

We also assessed the effects of Myd88 p.L252P expression in non-transformed B cells. 4

We specifically analyzed spontaneous, MYD88-centered protein complex formation in 5

naïve B cells and found significantly more complexes involving MYD88 together with 6

IRAK1, IRAK4, IgM and BTK, compared to wt controls (Fig. 3). These ex vivo 7

experiments suggest that MYD88p.L252P constitutively nucleates a signaling complex, 8

physically linking BCR and TLR signaling molecules in non-transformed B cells. These 9

data are in line with the recently reported presence of the so-called My-T-BCR complex 10

in ABC-DLBCL lymphoma cell lines (40). Moreover, these data are supported by a 11

recent report, indicating that BTK localizes in a protein complex with MYD88 in 12

p.L265P-expressing OCI-Ly3 DLBCL cells (75). 13

Further investigation into the impact of Myd88 p.L252P expression in non-transformed B 14

cells revealed the presence of auto-reactive antibodies in MC, BC and MBC animals 15

(Fig. 2A-D). Particularly the robust detection of auto-reactive antibodies in MC animals 16

was surprising, as it suggests that B cell-specific expression of Myd88 p.L252P is 17

tolerated in vivo. This observation is in contrast to the results of a recently reported 18

transplantation experiment, where mature B cells were first transduced with MYD88 19

p.L265P and subsequently transplanted into Rag1-/- recipients (76). In these 20

experiments, MYD88 p.L265P was sufficient to initiate a spontaneous proliferation burst 21

in mature B cells in vitro and in vivo (76). Nevertheless, the MYD88 p.L265P-induced 22

aberrant clonal growth was rapidly limited in a Bim-dependent manner (76). However, it 23

is important to note that an overexpression system was used in those experiments, 24

while we employ Myd88 expression from its endogenous locus in vivo (Fig. 1A). 25

Moreover, as we use Cd19Cre to mediate recombination, the entire B cell pool in our 26

experimental system carries the Myd88 p.L252P mutation (Fig. 1A). Thus, B cell 27

competition effects are very limited in our mouse model. 28

The observation of an increased presence of autoreactive IgM and IgG antibodies in 29

lymphoma-prone MBC mice (Fig. 2A-D) is intriguing, as this might suggest a role for 30

autoantigens in promoting DLBCL lymphomagenesis. A role for BCR stimulation in 31



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lymphomagenesis has long been postulated (77). For instance, there is ample evidence 1

indicating that chronic infections, such as hepatitis C (HCV) or Helicobacter pylori are 2

associated with the development of splenic marginal zone lymphoma (SMZL) and 3

Mucosa Associated Lymphoid Tissue (MALT) lymphoma, respectively (78). Moreover, 4

in HCV‐associated SMZL, a single BCR specific to a glycoprotein in the viral envelope 5

has been identified, strongly suggesting that HCV itself contributes to driving SMZL 6

BCR signaling (78). Next to infection-driven BCR signaling, our detection of autoreactive 7

antibodies in MBC animals is in line with the hypothesis that the constant engagement 8

of the BCR by a self‐antigen might account for the sustained nature of chronic active 9

BCR signaling (79). In fact, BCR stimulation by self‐antigens has been correlated with 10

lymphomagenesis. Epidemiological analyses revealed associations between B cell-11

activating autoimmune diseases, such as Sjögren's syndrome (SS) and systemic lupus 12

erythematosus (SLE), with increased DLBCL risk after controlling for all other risk 13

factors (80). Furthermore, analyses of VH gene segment in DLBCL revealed that 14

segment VH4‐34 is utilized in approximately 30% of ABC-DLBCL cases (79). 15

Experiments in cell line models of ABC-DLBCL with chronic active BCR signaling 16

provided further evidence for autoreactivity of the VH4‐34 segment. The viability of the 17

VH4-34-positive ABC cell line HBL1 depended on the V region-mediated ability of its 18

BCR to bind to self-glycoproteins on its own cell surface (79). Moreover, chronic active 19

BCR signaling in the ABC‐DLBCL cell line OCI‐Ly10 is driven by BCR recognition of an 20

antigen in the debris of apoptotic cells (79). OCI‐Ly10 was shown to depended on BCR 21

specificity mediated by charged amino acids within CDR2 and CDR3 of the VH3‐7 22

region (79). Lastly, the survival of the ABC‐DLBCL cell line TMD8 was shown to depend 23

on homotypic interactions with its own FR2 domain, which sustained chronic active BCR 24

signaling (79). These data indicate that a diverse array of self-antigens is responsible 25

for maintaining the survival of ABC-DLBCL cells. Building on these data, it is intriguing 26

to speculate that in our MBC model, the enlarged pool of B cells that are activated by 27

self-antigen and in which anergy is repressed by MYD88-driven TLR signaling might 28

increase the pool of cells that are prone for malignant transformation. 29

We also employed our MBC model as a preclinical tool, which mimics central features 30

of ABC-DLBCL (surface marker profile, expression profile, driver mutations and 31



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spontaneously developing mutational profile). Particularly the analysis of murine and 1

human tissue specimens and transcription profiles revealed that ABC-DLBCL cases 2

display higher CD274 (encoding PD-L1) levels, than GCB-DLBCL cases. These 3

observations are in line with a previous report, in which the authors demonstrate an 4

increased Cd274 expression in the murine C1Cre/wt;Ikk2CAGFPLSL/LSL;Prdm1fl/fl- and 5

C1Cre/wt;Tp53fl/fl;Ikk2CAGFPLSL/LSL;Prdm1fl/fl-derived murine lymphoma cells, compared 6

to C1Cre/wt;YFPLSL/wt germinal center B cells (81). There is further substantial evidence 7

that mechanistically supports the high PD-L1 expression levels in our MBC model: IFN- 8

and Myd88-dependent TLR signaling was recently shown to drive PD-L1 expression in 9

multiple myeloma cells (82). Intriguingly, we found significantly higher IFN- levels in the 10

serum of MBC mice, compared to wt serum (Fig. 4I). Moreover, an in silico analysis 11

revealed that a sizeable fraction of human DLBCL cases (22%) harbors high BCL2 and 12

CD274 expression levels. To particularly target this population, we assessed the 13

preclinical efficacy of combined BCL2- and PD1 blockade. While both single agents 14

displayed only mild activity in the MBC model, combined venetoclax and RMP1-14 led 15

to significantly increased tumor volume control and overall survival benefit, compared to 16

the single agents or vehicle. These results are in line with previous reports, 17

demonstrating that the anti-PD1 antibody RPM1-14 as single agent did not lead to a 18

significant overall survival benefit in C1Cre/wt;Tp53fl/fl;Ikk2CAGFPLSL/LSL;Prdm1fl/fl 19

lymphoma-bearing mice, compared to untreated controls, whereas combined anti-20

PD1/anti-CD20 blockade synergistically increased the overall survival in these animals 21

(81). 22

Our observations provide further evidence for the clinical development of BCL2 and PD-23

1/PD-L1 inhibitors in the clinical arena. In this context, it is important to reiterate that 24

single agent venetoclax was recently shown to achieve an ORR of 18% in 25

relapsed/refractory DLBCL (65). Similarly, single agent nivolumab achieved an ORR of 26

36% in DLBCL (62). Our data now indicate that DLBCL patients displaying high-level 27

PD-L1 and BCL2 exist and that these patients may be particularly well-suited to receive 28

combined BCL2- and PD-1 blockade. This strategy may be particularly useful in 29

relapsed/refractory ABC-DLBCL, or those patients that are not eligible for intensive 30

consolidation regimens, involving autologous stem cell support. In our experiments, 31



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venetoclax was used at 200mg/kg q.d., which is a dose that is typically used for in vivo 1

experiments in mice (83, 84). While venetoclax at 200mg/kg q.d. did not induce any 2

obvious toxicities in our experiments, it is important to note that this dose exceeds the 3

doses that are clinically applied to human patients (typically 5-20mg/kg, depending on 4

venetoclax dosing regimen and body weight). Altogether, we provide a detailed 5

molecular analysis of the MBC model, including the comparison with Kmt2d/BCL2-6

driven lymphomas and a large series of human DLBCL cases. These experiments 7

indicate that the MBC model reflects key aspects of human ABC-DLBCL. Moreover, we 8

employ the MBC model to derive a combination strategy involving PD-1-and BCL2 9

blockade for the treatment of MYD88- and BCL2-altered aggressive lymphomas. 10

11

12

Materials and Methods 13

14

Experimental mice 15

The generation of the Cd19Cre, Myd88cond.p.L252P and Rosa26LSL.BCL2-IRES-GFP alleles has 16

been described previously (20, 85). For survival analyses, animals that succumbed to 17

disease or had to be killed due to satisfied termination criteria were recorded as events. 18

Animals that died from genotype-unrelated criteria in rare cases (appendicitis, abnormal 19

teeth, injuries inflicted by cage mates) were censored. For transplantation experiments, 20

107 cells were transplanted intraperitoneally into Rag1-/- recipients. In autochthonous 21

treatment studies, onset of lymphoma was defined by a lesion larger than 75µl 22

detectable by MRI, with a detectable volume increase in two consecutive scans. In 23

transplantation studies, onset of clonal lymphoma was determined in a preceding 24

experiment. ABT-199 was administered as a suspension in 60% Phosal 50PG, 30% 25

polyethylene glycol 400, 10% ethanol by oral gavage at 200 mg/kg daily. ND-2158 was 26

dissolved in 10% -cyclodextrin at 15 mg/ml and administered intraperitoneally at 150 27

mg/kg. The anti-PD1 antibody RMP1-14 (BioXCell) was administered i.p. on three days 28

per week (250 µg/administration). 29

All animals were housed in a specific-pathogen-free facility and animal breedings and 30

experiments were approved by the local animal care committee and the relevant 31



Flümann et al.

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authorities (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, 1

AZ: 84-02.04.2014.A146, 84-02.04.2017.A131, 81-02.04.2019.A009). 2

3

MR imaging 4

MR imaging was performed as described previously(20). In brief, mice were 5

anesthetized with 2.5% isoflurane and scanned on a 3.0T MRI system (Igenia, Philips) 6

with a small rodent solenoid coil (diameter 40 mm, Philips Research Europe). Axial T2-7

weighted images of the abdomen were acquired (TSE factor: 10, TR: 2674 ms, TE: 65 8

ms, slice thickness: 1.0 mm. Images were exported in DICOM format and spleen and 9

tumor volumes were measured by segmentation using the Horos software. 10

11

Immunohistochemistry 12

Formalin-fixed and paraffin-embedded tissue from mice were cut into 4 µm sections and 13

stained for BCL6 (Santa Cruz, clone C-19), B220 (BD, clone RA3-6B2), CD138 (BD, 14

Cat. No. 553712), Ki67 (Cell Marque), CD3 (Thermo Fisher, clone RM-9107), CD4 15

(abcam, Cat. No. ab183685), CD8 (abcam, Cat. No. ab203035,), biotinylated PNA 16

(Vector Laboratories, B-1075-5). Germinal centers were quantified from BCL6 and PNA 17

stainings using the software ImageJ. Ki67 and CD3 positivity was quantified using the 18

ImmunoRatio plugin for ImageJ (86). 19

Human samples were cut into 4 µm sections and stained for CD10 (NCL-L-CD10-279, 20

Novocastra), IRF4 (Dako, Cat. No. M7259), BCL6 (Dako, Cat. No. M7211), BCL2 ( 21

Dako, Cat. No. M0877), and PD-L1 (Dako, clone 28-8), CD3 (Biorad, clone 145-2C11), 22

CD4 (abcam, clone EPR19514), CD8 (abcam, Cat. No. ab203035). Human PD-L1 23

stainings were graded according to the Cologne Score (87). 24

25

Flow cytometry 26

For immunotypisation, splenocytes were stained with fluorophore-coupled primary 27

antibodies against CD23 PE, (Ebioscience, clone B3B4), CD93 (PE-Cy7, Biolegend, 28

clone AA4.1), CD21/35 (APC, BD, clone 7G6), CD45 (APC-Cy7, BD, clone 30-F11), 29

B220 (Pacific-Blue, BD, clone RA3-6B2), CD138 (PE-Cy7, Biolegend, Cat. No. 142514), 30

MHCII (AF700, Biolegend, clone M5-1142) and measured on a Gallios flow cytometer 31



Flümann et al.

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(Beckman Coulter). Data was analyzed with the software Kaluza (Beckman Coulter). 1

The gating strategy is depicted in Fig. S8. 2

For annexin V/PI measurements, cells were cultured at an initial density of 106 cells/ml 3

and treated with the indicated compounds and doses for 48 hours. Cells were then 4

washed and stained with a FITC-coupled antibody against annexin V (diluted 1:600, BD, 5

Cat. No. 550474) and propidium iodide (0.5 mg/ml). Cells were analyzed on a flow 6

cytometer (Gallios, Beckman Coulter) after 15 minutes of incubation and the double-7

positive population was measured using the software Kaluza (Beckman Coulter). 8

9

Serum gel electrophoresis 10

Serum protein electrophoresis was performed with HYDRAGEL IF 2/4 agarose gels 11

(Sebia, Fulda, Germany) on the HYDRASIS electrophoresis system (Sebia, Fulda, 12

Germany). Therefore, 4 µl serum was diluted 8 µl HYDRAGEL IF diluent (Sebia, Fulda, 13

Germany), electrophoretically separated and stained with acid violet according to the 14

manufacturer’s instructions. 15

16

HEp2 assay 17

Serum was diluted 1:40 in PBS and 30 µl per sample were added on a Kallestad HEp-2 18

slide (Biorad, Cat. No. 26101), incubated for 20 min in a wet chamber and washed with 19

PBS for 10 min. The samples were then stained with Alexa Fluor 488-coupled 20

secondary antibodies against either murine IgM (Thermo Fisher, Cat. No. A-21042) or 21

IgG (Thermo Fisher, Cat. No. A-11001) for 20 minutes, washed and covered. 22

Fluorescence intensities were quantified from stainings produced in the same run by 23

analyzing images generated with identical exposure times by ImageJ. 24

25

Immunization experiments 26

Animals were injected intraperitoneally with 50 µg of either NP-ficoll (Biocat, Cat. No. F-27

1420-10-BS) dissolved in 200 µl PBS or NP-CGG (Biocat, Cat. No. N-5055E-1-BS) 28

dissolved in 100 µl PBS + 100 µl Imject Alum (Thermo Scientific, Cat. No. 77161). 30 µl 29

of blood were drawn from the tail vein at the indicated days. Antibody levels against NP 30

in the collected sera were measured by ELISA. High-protein-binding plates were coated 31



Flümann et al.

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with NP-BSA (5 µg/ml) over night. Plates were washed with 0.05% PBS-T and blocked 1

with 1% BSA. The samples were prediluted with 1% BSA in PBS at 1:10,000 or higher 2

and incubated on the plate for two hours at room temperature. Plates were washed with 3

PBS-T and secondary antibody against murine IgG (Antibodies-online, Cat. No. 4

ABIN376241) or IgM (Novus, Cat. No. NB7497). After 20 min, the reaction was stopped 5

by 1 M phosphoric acid and the plates were read on a plate reader (Tecan). 6

7

Immunoblotting 8

Single cell suspensions were generated by pressing spleens through a cell strainer (70 9

µm) and naïve B cells were purified using a CD43-depletion kit (Miltenyi, 130-049-801). 10

Cells were lysed in 4% SDS containing phosphatase and protease inhibitors (Merck, 11

4906845001 and 05892970001). Protein concentration was measured by BCA assay 12

(Thermo Fisher, 23225) and concentration was adjusted to 800 µg/ml before the 13

addition of Laemmli buffer. 20 µl per sample were loaded onto 10% polyacrylamide gels 14

and blotted onto a PVDF membrane. Membranes were blocked (5% BSA in TBST) and 15

stained overnight with primary antibody (pp65, Cell Signaling, 3033; pIRAK4, Abnova, 16

MAB2538; pBTK, Cell Signaling, 5082, GAPDH, Cell Signaling, 5174). Membranes 17

were washed and incubated with HRP-coupled secondary antibody for one hour at 18

room temperature. After washing, membranes were incubated with ECL solution 19

(Amersham) and imaged on a ChemiDoc (Bio-Rad). Densitometric analysis was 20

performed using ImageJ. 21

22

Clonality analysis 23

RNA was isolated from cryo-preserved tumor tissue using a commercial kit (Qiagen, 24

Cat. No. 74104) and clonality analysis was performed using an adaption of a published 25

BCR clonality analysis approach (42). The cDNA was then synthesized using a reverse 26

transcriptase generating poly-dC overlaps (SMARTScribe, Takara, Cat. No. 639537), 27

allowing for template switching and adapter ligation, which contains a unique barcode 28

(unique molecular identifier, UMI). For first-strand synthesis, primers specific for the 29

constant regions of Ighg, Ighm and Igha were used. Two rounds of nested PCR were 30

performed, resulting in an amplification product containing the V(D)J junctions, which 31



Flümann et al.

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was then sequenced. Purified PCR-amplicons were end-repaired, A-tailed and adapter 1

ligated with unique dual indices using the Illumina TruSeq nano kit and protocol but 2

without further PCR amplification. After validation (2200 TapeStation; Agilent 3

Technologies) and quantification (Qubit System; Invitrogen, Waltham, USA) amplicon 4

libraries were individually quantified using the KAPA Library Quantification kit (Peqlab, 5

Erlangen, Germany) and the 7900HT Sequence Detection System (Applied Biosystems, 6

Waltham, USA) and subsequently spiked-in in larger pools of libraries. The pools were 7

sequenced on an Illumina NovaSeq6000 sequencing instrument using a paired-end 8

2×100 bp protocol. The samples were then error-corrected using the published MIGEC 9

algorithm (42). UMIs present with less than three reads were discarded. It was then 10

quantified with how many UMIs one specific BCR sequence was present, defining the 11

size of a clone. Clones differing by one base were connected to clusters, and the size of 12

these clusters was quantified as the sum of the individual clone sizes. BCR repertoires 13

were visualized using the software Gephi (https://gephi.org/). Primer sequences and 14

barcode-sample mapping are given in Supplementary Table 3. 15

16

Determination of SHM frequency in CD138+ cells 17

CD138-positive cells were selected using a magnetic antibody-based cell separation kit 18

(Miltenyi, 130-098-257). RNA was isolated from these cells using a commercial kit 19

(Qiagen). Full-length B cell receptor sequencing was performed as published by 20

Turchaninova et al. (21). Primer sequences and barcode-sample mapping are given in 21

Supplementary Table 3. 600 ng of RNA were used for cDNA synthesis, and a cDNA 22

equivalent of 500 plasma cells was used for further PCR amplification steps. Amplicon 23

sequencing was then performed on a MiSeq (asymmetric 400+100-nt paired-end 24

sequencing), with 8 samples on a full lane. Processing of the sequencing data was 25

performed according to the protocol using the MIGEC software (21, 42). Mapping of the 26

identified BCR sequences was done using the MiXCR (for isotype information) and 27

MiGMAP (for determination of differing bases from germline sequence) (88). 28

29

3'-RNA-sequencing 30


https://gephi.org/


Flümann et al.

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RNA was isolated from cryo-preserved tumor tissue using a commercial kit (Qiagen, 1

Cat. No. 74104). 3´mRNA libraries were generated from total RNA using the Lexogen 2

QuantSeq kit according to the standard protocol. After validation (2200 TapeStation; 3

Agilent Technologies) and quantification (Qubit System; Invitrogen, Waltham, USA) 4

pools of cDNA libraries were generated. The pools were quantified using the KAPA 5

Library Quantification kit (Peqlab, Erlangen, Germany) and the 7900HT Sequence 6

Detection System (Applied Biosystems, Waltham, USA) and subsequently sequenced 7

on an Illumina HiSeq4000 sequencer using a 1x50 bp protocol. Reads were mapped to 8

the murine genome (mm10) and quantified using Salmon. Data was normalized and 9

statistics were calculated using DESeq2 (89). To perform gene set enrichment analysis 10

for published human gene sets, the murine genes were mapped to their human 11

orthologues using the biomaRt package for R (90). Gene set enrichment analysis was 12

then performed using the FGSEA package (91). To analyze the published human RNA 13

sequencing dataset (11), reads were mapped to the human transcriptome (GRCh38) 14

and quantified using Salmon (92). 15

16

CyTOF analysis 17

Single cell suspensions were generated from untreated and anti-PD1-treated tumors 18

(RMP1-14-treated, 250 µg on days 1, 4 and 7 after tumor detection by MRI, i.p., 19

collected on day 8) by pressing the tissue through a 70 µm cell strainer. Cell 20

suspensions were frozen in FCS/10% DMSO at -80˚C for collective staining and 21

measurement. Samples were barcoded, using a commercial kit following the 22

manufacturer’s instructions (Fluidigm, 201060). Cells were then pooled and stained with 23

a set of surface and intracellular markers following standard protocol. Antibodies (clone 24

or manufacturer and order number in case of polyclonal): IRF4 (IRF4.3E4), QA1b 25

(6A8.6F10.1A6), p16 (2D9A12), TIM-3 (RMT3-23), CD179 (MFL3), LAG-3 (631501), 26

p21 (WA-1), CTLA-4 (UC10-4B9), CCR6 (29-2L17), CD45R (RA3-6B2), CD4 (RM4-5), 27

CD19 (6D5), CD8a (53-6.7), CD279 (R&D, AF1021), CD95 (R&D, AF435), BCL6 28

(K112-91), CD3e (145-2C11), 4-1BB (158332), NK1.1 (PK136), CD69 (H1.2F3), FOXP3 29

(FJK-16s), ICOS (7E.17G9), CD45 (30-F11). Cell acquisition was done on a Helios 30

mass cytometer (Fluidigm). Debarcoding, normalization and compensation was 31



Flümann et al.

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performed with the R package CATALYST (93). CD3+/CD8+ and CD3+/CD4+ cells were 1

gated and mean signal intensities between the two groups were compared for each 2

marker, significance was calculated by unpaired two-sided Welch’s t-test and adjusted 3

for multiple hypothesis testing (Benjamini & Hochberg). 4

5

Whole exome sequencing 6

DNA was isolated from MBC tumors with high clonal fractions. Mouse exomes were 7

individually prepared using 200 ng of DNA, the standard protocol SureSelectXT 8

Automated Target Enrichment for Illumina Paired-End Multiplexed Sequencing, and the 9

Agilent Bravo automated liquid handling platform. After validation (2200 TapeStation; 10

Agilent Technologies) and quantification (Qubit System; Invitrogen, Waltham, USA) 11

pools of libraries were generated. The pools were quantified using the KAPA Library 12

Quantification kit (Peqlab, Erlangen, Germany) and the 7900HT Sequence Detection 13

System (Applied Biosystems, Waltham, USA) and subsequently sequenced on an 14

Illumina NovaSeq6000 sequencing instrument using a paired-end 2×100 bp protocol. 15

We aligned raw sequencing reads to the mouse reference genome (mm10) by using the 16

BWA mem aligner (version 0.7.13-r1126). Concordant read pairs that represent 17

possible PCR duplicates were masked out after alignment. Furthermore, all overlapping 18

regions between the read pairs are considered only once in the analysis. Due to a lack 19

of matched normals for all tumor specimens, we generated a representative non-tumor 20

sample by combining normals matching to two tumor samples. This combined normal is 21

used for mutation calling with the latest version of our in-house cancer genome analysis 22

pipeline (94). To correct for genotypes that are not captured by representative normal, 23

we filtered out called mutations that were exactly the same in two or more tumor 24

samples. 25

26

Multiplex cytokine assay 27

Mouse serum was collected by retro-orbital or tail-vein bleeding. Serum levels of 28

cytokines and chemokines were determined using the Mouse 31-Plex 29

Cytokine/Chemokine Array (Eve Technologies, Calgary, Canada). 30

31



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Cell lines 1

Human cell lines were purchased from DSMZ. Cell line identity was verified by STR 2

analysis. Cell lines were cultivated in RPMI with 20% FCS and 1% 3

penicillin/streptomycin. Murine cell lines were established from primary tumors and 4

cultivated in DMSO, containing 4.5g/l glucose, with the following supplements: 10% 5

FCS, 2 mM l-glutamine, 1mM sodium pyruvate, 10 mM HEPES, non-essential amino 6

acids, 1% penicillin/streptomycin, 50 µM beta-mercaptoethanol. No mycoplasm-testing 7

was performed. 8

9

Cell viability assays 10

5000 cells per well were treated with the indicated drugs and doses (dissolved in 11

DMSO, DMSO concentrations were adjusted). After 96 hours of incubation, viability was 12

measured by CellTiter-Glo assay (Promega, Cat. No. G7572) diluted 1:6 in PBS and the 13

luminescence was measured (Infinite M1000 pro, Tecan). The values were normalized 14

to untreated controls on each plate. 15

16

High-throughput cell line screening 17

High throughput screening (HTS) was performed on the mouse and human lymphomas 18

cells, as described earlier with minor modifications (95-97). Briefly the DMSO dissolved 19

compound library (MedChemExpress, NJ, USA) was dispensed with increasing 20

concentrations of the inhibitors in 6 dilution steps (0.008 - 25 µM) on a white 1536-well 21

plate (Corning, NY, USA) using digital dispenser (D300e, Tecan, Männedorf, 22

Switzerland), which ensures precise and robotic compound application in randomized 23

fashion. The compound selection involves majority of FDA/EMA approved routinely 24

used chemotherapeutic and targeting drugs, inhibitors in the early to late clinical trial 25

phase and several investigational compounds (see supplements for the detailed list). 26

The printed plates were sealed with parafilm, followed by packing in vacuum sealed 27

bags to avoid evaporation during storage at -80oC. The cells (>90% viability) were 28

seeded on the thawed pre-dispensed inhibitor plates using an automated Multidrop 29

Combi Reagent Dispenser (Thermo Fisher Scientific, Waltham, USA). Differential 30

responses were monitored with ATP-dependent CellTiter-Glo Luminescent cell viability 31



Flümann et al.

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kit (Promega, Madison, USA) after 72 h of inhibitor exposure using Microplate reader 1

(Spark® 10M, Tecan). The outer three wells of the plate were excluded from analysis to 2

circumvent the evaporation effect on the plate edges. Dose response curves (n=3) for 3

the inhibitors were determined by plotting raw data (normalized to controls) with non-4

linear regression (log(inhibitor) vs. normalized response) variable slope function 5

(GraphPad Prism Inc., San Diego, CA). Average drug response values (n=3) 6

normalized to the mean IC50 for each individual compound over all lines were plotted 7

for the Heatmap visualization followed by unsupervised hierarchical clustering (R 8

package gplots). 9

10

Proximity ligation assay 11

B cells were isolated from spleens of 15 – 20-week old wt and MC mice by MACS 12

sorting according to protocol (CD43 (Ly-48) Micro beads, Miltenyi Biotec, Cat. No. 130-13

049-801). PLA was performed with the Duolink-In-Situ-Orange kit (Sigma, Cat. No. 14

DUO92007), as previously published (98). In brief, cells were plated on a 10 well 15

polylysine coated microscope slide (Thermo Scientific, 3 wells per genotype, condition 16

and experiment), allowed to adhere for 30 minutes at 37°C and subsequently fixed with 17

1% PFA at room temperature for 20 minutes. Cells were then blocked and 18

permeabilized with 0,5% Saponin in Duolink Blocking buffer (Sigma). For PLA-probes 19

against specific targets the following primary antibodies were used: anti-Myd88 20

(Abcam), anti-BTK (NovusBiologicals, Cat. No. NBP1-78295SS), anti-IRAK1 (Novus 21

Biologicals, Cat. No. NBP1-77068SS), anti-IRAK4 (Invitrogen, Cat. No. 700026), anti-22

IgM (Jackson Laboratories, Cat. No. 115-007-020). Primary antibodies were labeled 23

with Duolink In-Situ Probemaker Plus (Sigma, Cat. No. DUO92009) or Minus (Sigma, 24

Cat. No. Cat. No. DUO92010) according to protocol. Fixed cells were incubated with 25

Plus and Minus probes over night at 4 °C. Ligation, rolling circle amplification and 26

mounting of cells were performed according to protocol. 3 Images per well, each 27

capturing at least 120 cells, were acquired on a Leica SP8 confocal microscope using 28

the Leica LAS X software. Nuclei and PLA signals were counted using BlobFinder 29

version 3.0.0 (99). 30

31



Flümann et al.

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BH3 profiling 1

BH3 Profiling was performed using the iBH3 plate-based method as previously 2

published (56). In brief, cell lines were seeded at a density of 1 x 106 cells/mL 24 hours 3

before profiling. Four million cells of each cell line were pelleted at 300 x g for 5 minutes 4

and resuspended in 2mL MEB-P25 (150mM Mannitol, 10mM HEPES-KOH pH 7.5, 5

150mM KCl, 1mM EGTA, 1mM EDTA, 0.1% BSA, 5 mM Succinate, 0.25% Polaxamer 6

188[Fisher, MT61161RM]). Cells were permeabilized with digitonin (Sigma, D5628) 7

exposed to BH3 peptides for 60 minutes at 25°C and mitochondrial Cytochrome C 8

release was measured by flow cytometry using a FITC-conjugated antibody 9

(BioLegend, 983502). BH3 peptides were synthesised by New England Peptide using 10

published sequences (100). The Bcl-XL-selective inhibitor A-1331852 (Selleckchem, 11

S7801), the Bcl-2-selective inihibtor ABT-199 (Selleckchem, S8048) and the MCL-12

inhibitor AZD5991 (Selleckchem, S8643) were used at a concentration of 10 µM. 13

Results were only deemed valid where cell cytochrome C release in presence of DMSO 14

control was <10% and cytochrome C release in presence of 50ug/mL(25uM) 15

Alamethicin (Enzo, BML-A150-0005) was >90%. Figure represents mean of three 16

independent experiments. 17

18

Data accessibility 19

Murine exome and 3’ RNA sequencing data are available at the Sequence Read 20

Archive (SRA) under the accession number PRJNA668334. The BCR repertoire 21

sequencing data is available under PRJNA672930. A dataset consisting of DNA and 22

RNA sequencing data generated by the lab of Sandeep S. Dave (11) was reanalyzed 23

for this work and is accessible over the European genome-phenome archive (accession 24

number EGAD00001003600). Some analyses conducted in this work were based on 25

previously published supplementary data by the labs of Margaret A. Shipp and Louis M. 26

Staudt (9, 10). 27

28

29

Acknowledgements 30



Flümann et al.

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We are indebted to our patients, who provided primary material. We thank Alexandra 1

Florin, Marion Müller and Ursula Rommerscheidt-Fuß from the Institute of Pathology, 2

University Hospital Cologne, for their outstanding technical support. We acknowledge 3

the Institute of Forensic Medicine, University of Cologne for help with short tandem 4

repeat (STR)-based cell line authentication. We thank the CECAD Imaging Facility and 5

Christian Jüngst for their support in microscopy. This work was funded through the 6

German-Israeli Foundation for Research and Development (I-65-412.20-2016 to 7

H.C.R.), the Deutsche Forschungsgemeinschaft (KFO-286-RP2 and RE 2246/13-1 to 8

H.C.R.), the Deutsche Jose Carreras Leukämie Stiftung (R12/08 to H.C.R., 9

Promotionsstipendium to P.N.), the Else Kröner-Fresenius Stiftung (EKFS-2014-A06 to 10

H.C.R., 2016_Kolleg.19 to H.C.R., S.K.), the Deutsche Krebshilfe (1117240 and 11

70113041 to H.C.R.) and the German Ministry of Education and Research (BMBF 12

e:Med 01ZX1303A to H.C.R.). J.H. and A.B. have been supported by the Deutsche 13

Krebshilfe (Translational Oncology Program 70112951) and Deutsches Konsortium für 14

Translationale Krebsforschung (DKTK), Joint funding (Targeting MYC L*10). L.P. has 15

been supported by the NIH/NCI (2R01CA172492) and the Leukemia and Lymphoma 16

Society (TRP Grant #6575-19). 17

18

19

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29



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Figure Captions 1

2

Figure 1 - Germinal center hyperplasia and increased plasma cell pools in MBC 3

animals. A) Schematic illustrations of the employed alleles. Exons 2-6 of the 4

endogenous Myd88 locus were flanked by loxP-sites (triangles). Downstream of the 5

second loxP-site, a second set of the exons 2-6 was inserted, harboring the L252P point 6

mutation (asterisk). Read-through is prevented by a strong polyadenylation signal (‘pA’). 7

Human BCL2 cDNA expression is controlled by a CAGGs promoter and prevented by a 8

lox-stop-lox cassette. GFP expression is coupled to BCL2 expression by an internal 9

ribosomal entry site (IRES). The construct is a knock-in into the Rosa26 locus. Both 10

alleles have been previously published (20). The Cd19Cre allele is a knock-in into the 11

Cd19 locus and has been previously published (85). B) Exemplary axial MR images of 12

30 weeks old animals. Spleens are outlined. C) Spleen volumes of wt (n = 5), MC (n = 13

5), BC (n = 7) and MBC (n 7) mice were quantified from MR images. D) 14

Immunohistochemical stainings for B220, PNA and CD3 of splenic sections of 30 weeks 15

old wt, MC, BC and MBC animals. E) The germinal center (GC) structures stained by 16

PNA in splenic sections of 30 weeks old WT (n = 8), MC (n = 4), BC (n = 5) and MBC 17

animals (n = 5) were quantified. F) Splenocytes of 30 weeks old wt (n 7), MC (n = 8), 18

BC (n = 8) and MBC (n 7) were analyzed by flow cytometry and the relative amounts 19

of different B cell developmental stages were quantified. G) Serum protein 20

electrophoresis was performed with serum of 30 weeks old wt, MC, BC and MBC 21

animals (n = 6 per genotype). H) Serum immunoglobulin levels of 30 weeks old wt, MC, 22

BC and MBC animals (n 4 per genotype) were measured by ELISA. *, p 0.05; **, p 23

0.01; ***, p 0.001; Welch’s unpaired two-tailed t-test. 24

25

26

Figure 2 - MBC animals show exaggerated immune responses to self and foreign 27

antigen. A) Self-reactive antibodies of the IgM isotype were visualized by a Kallestad 28

HEp-2 assay adapted to the murine system and mean fluorescence intensities (MFI) 29

were quantified (n = 6 per genotype, two exemplary cases per genotype are shown). B) 30

Quantification of the observed staining patterns. C) Autoreactive IgG immunoglobulins 31



Flümann et al.

- 43 -

were visualized by an adapted Kallestad HEp-2 assay and MFI values were quantified 1

(wt, n = 7; MC, n = 8; BC, n = 8; MBC, n = 9. Two exemplary cases per genotype are 2

shown). D) Quantification of the observed staining patterns. E) and F) wt, MC, BC and 3

MBC animals (n = 3) were immunized intraperitoneally with either NP-Ficoll (50 µg) or 4

NP-CGG (50 µg) at day 0 and the NP-specific IgM and IgG levels in the sera of animals 5

were measured at days 0, 4, 7, 10, 21 and 40 after immunization by ELISA. Envelopes 6

represent SEM.*, p 0.05; **, p 0.01; ***, p 0.001; Welch’s unpaired two-tailed t-test. 7

8

9

Figure 3 - Myd88 p.L252P mutation enhances the formation of the My-T-BCR 10

supercomplex. A) Exemplary images of PLA assays for MYD88 proximity with IRAK4, 11

IRAK1, BTK and IgM, respectively. Blue, DAPI; red, PLA signal. Scale bars represent 12

10 µm. B). Quantification of the data shown in A). Each dot represents the average 13

number of PLA signals per cell and experimental well. The mean and SEM of at least 14

three independent experiments are depicted. Significant differences between samples 15

was calculated by Welch’s unpaired t-test. C) Lysates were generated from CD43-16

depleted splenocytes from 10 weeks old MC and WT. Immunoblots were generated for 17

the indicated targets. Blots were quantified using ImageJ. 18

19

Figure 4 - MBC animals develop ABC DLBCL-like tumors. A) Survival curves of wt 20

(n = 10), MC (n = 104, median 101.9 weeks), BC (n = 74, median 68.7 weeks) and MBC 21

animals (n = 107, median 42.3 weeks). B) Quantification of the terminal phenotype of 22

MC (n =15), BC (n = 12) and MBC animals (n = 30). C) Exemplary illustration of H/E, 23

B220, CD138 and Ki67 stainings of MC, BC and MBC tumors. D) BCR sequencing-24

based clonality analysis of wt spleens and two MBC primary tumors (‘M552 tumor’ and 25

‘M108 tumor’) and derived cell lines. Each circle represents a unique BCR sequence 26

with the circle area representing the clone size. Clones differing in one base are 27

connected by lines to clusters. Clusters consisting of 10% of reads are highlighted by 28

color and the exact percentages are given. E) Summary of the clonalities observed in 29

MC (n = 3), BC (n = 5) and MBC lesions (n = 25) compared to the polyclonal scenario 30

observed in wt spleens (n = 3). F) Comparison of the immunohistochemical phenotype 31



Flümann et al.

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of two primary tumors and transplanted tumors. For transplantation, stable cell lines 1

derived from the primary tumors M108 and M552 were injected intraperitoneally into 2

Rag1-/- recipients. G) Gene set enrichment analysis for ABC and GCB DLBCL 3

signatures (11) on MBC (n = 4) and KBC (Kmt2dfl/fl;VavP-Bcl2;Cγ1-Cre (43), n = 6) 4

tumors. H) WES was performed on 17 MBC tumors. Identified mutated genes (see also 5

Tab. S1) were plotted for the mutation frequencies of the orthologous human genes in 6

two published DLBCL WES data sets (9, 11). I) Cytokine levels in the sera of 7

lymphoma-bearing MBC animals (n = 15) were measured and compared to wt levels (n 8

= 7). Solid lines represent the mean and envelopes the standard deviation. Cytokines 9

with significant differences between MBC and wt are highlighted in red. *, p 0.05; **, p 10

0.01; ***, p 0.001; A) log-rank test. G, I) Welch’s unpaired two-tailed t-test adjusted 11

for multiple comparisons. Scale bars represent 50 µm. 12

13

Figure 5 - MBC tumors are responsive to BCL2-inhibition by venetoclax. A-C) 14

Murine MBC and MYC cell lines as well as human ABC and GCB DLBCL cell lines were 15

treated with increasing doses of ABT-199 (0 – 300 nM), LY-2409881 (0 – 15 µM) or ND-16

2158 (0 – 5 µM) and cell viability was measured after 96 hours by CTG. The mean of a 17

minimum of three independent experiments is shown, each experiment consisting of 18

three technical replicates. Error bars represent the SEM. D) BH3-Profiling (56) of MBC, 19

ABC, MYC and GCB DLBCL cell lines. Cells were exposed to either BH3 peptides or 20

small molecule inhibitors for one hour. The coloring indicates the fraction of Cytochrome 21

C releasing (i.e. apoptotic) cells after exposure measured by flow cytometry. E-G) 22

Apoptosis was measured by flowcytometric analysis of the Annexin V/PI double-positive 23

population 48 hours after treatment of the cell lines M-191, M-108, M-552, MYC-14, 24

RYS-202, U2932, RI1, SUDHL10, OCILY7 with 300 nM ABT-199, 5 µM ND-2158 or 5 25

µM LY-2409881. H) 107 M-108 cells were injected intraperitoneally into RAG1-/- 26

recipients. Two weeks after transplantation, animals were treated with ABT-199 (200 27

mg/kg daily, oral gavage), ND-2158 (150 mg/kg, i.p., daily), LY-2409881 (100 mg/kg, 28

i.p., q.a.d.) or left untreated and survival after transplantation was recorded. *, p 0.05; 29

**, p 0.01; ***, p 0.001. E-G) Welch’s unpaired two-tailed t-test adjusted for multiple 30

comparisons. H) Log-rank test. 31



Flümann et al.

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1

Figure 6 - -PD-1 treatment is an effective strategy for MBC tumors. A) Expression 2

levels of the indicated genes were compared between ABC (n = 310) and GCB (n = 3

328) DLBCL cases in a previously published RNA sequencing dataset (11). B) 38 4

human DLBCL samples were categorized into the ABC and GCB subtypes employing 5

the Hans algorithm (101) and PD-L1 was stained immunohistochemically. Grades 0 and 6

1 were classified as negative/low expression and 2 to 5 as medium/high expression. C) 7

Expression of PD-L1 in PCNSL samples was analyzed by immunohistochemistry. MBC 8

animals were monitored for lymphoma development by MRI and upon tumor detection 9

treatment was initiated with either ABT-199 (200 mg/kg daily by oral gavage for 3 10

weeks), -PD-1 antibody (250 µg twice weekly for 8 weeks) or a combination of both. 11

Exemplary MRI scans three weeks after treatment initiation are illustrated in D), E) 12

shows best tumor volume change within 8 weeks of untreated (n = 4), ABT-199 treated 13

(n = 6), -PD-1 treated (n = 5) or combination treated (n = 7) MBC. F) Survival after 14

tumor detection of untreated (n = 4), ABT-199 treated (n = 6), -PD-1 treated (n = 5) or 15

combination treated (n = 7) MBC animals. G) Timescale of sample collection from -16

PD-1 treated MBC tumors. H) Mass cytometric analysis of untreated and -PD-1 treated 17

tumors. Cells were gated for CD45+DNA+ (not illustrated) and CD3+CD4+ as well as 18

CD3+CD8+ events were selected for further analysis. The adjusted p value and log2-fold 19

change between -PD-1 (n = 4) and untreated samples (n = 14) for each marker 20

(individually for the CD4+ and CD8+ populations) is depicted in I)and significant markers 21

are highlighted. J) CD3+/CD4+ and CD3+/CD8+ population sizes are given as 22

percentages of the DNA+/CD45+ population. K) Differentially expressed markers in the 23

CD3+/CD4+ and CD3+/CD8+ populations. *, p 0.05; **, p 0.01; ***, p 0.001; ****, p 24

0.0001. A, I, K) Welch’s unpaired two-tailed t-test, B) Fisher’s exact test; F), log-rank 25

test. Scale bars represent 50 µm. 26



AFlümann et al., Figure 1

B

C

D

1 2 3 4 5 pA 2 3 4 5*

Myd88 conditional knock-in allele

6 6

1 2CAGGs Neo WSS BCL2 IRES GFP

Rosa26 conditional BCL2 knock-in allele

*

B cells Follicular Marginal Zone Transitional

MCWT

BC MBC

WT MC BC MBC

*******

***

*

****

***

***

**

*******

*

***

****

10 20 300

100

200

300

400

500

Sple

en v

olum

e (µ

l)

Age (weeks)

020406080

100***

0

20

40

60*

2468

10

0

****

*

0.00.51.01.52.02.5 *

0

1

2

3

4

Early PB

**

**

**

***

0

5

10

15

Late PB/PC

****

******

*

WT MC BC MBC

G

F

BC MBC

MCWT

% o

f CD4

5+

H

IgM IgA IgG1 IgG2b IgG2c IgG3100

102

104

106

108

1010

**

********

*****

*****

Ig ti

ter (

µg/m

l)

Cd19 constitutive Cre knock-in allele1 2 cre neoR 2 3 4

WT

MC

BC

MB

C

B220 PNA CD3

E

200µm

0

2

4

6

GCs

per

mm

2 spl

een

10

20

30

GC

area

(%)

0

2

4

6

8

Mea

n G

C si

ze (x

104 µm

2 )

*******

*****

****

*



IgG

MFI

(a.u

.)

IgM

C MC BC MBC

* ****

*****

Flümann et al., Figure 2A

E

C

0

50

100

% o

f cas

es

nuclearcytoplasmic

nuc. + cyt.

none

0

50

100

% o

f cas

es

D

0 4 7 10 21 40

Abs

585n

m (a

.u.)

IgG NP-CGG

0 4 7 10 21 40

IgG NP-Ficoll

Abs

585n

m (a

.u.)

0 4 7 10 21 40

Abs

585n

m (a

.u.)

IgM NP-CGG

0 4 7 10 21 40

Abs

585n

m (a

.u.)

0

.2

.4

.6

.8

1IgM NP-Ficoll

0

.2

.4

.6

.8

1

0

.2

.4

.6

.8

1

0

.2

.4

.6

.8

1

WT MC BC MBC

time after immunization (days) time after immunization (days)

time after immunization (days) time after immunization (days)

CM

CB

CM

BC

0

10

20

30

40

50

CM

CB

CM

BC

B

0

5

10

15

20

MFI

(a.u

.)

C

MC

BC

MB

C

CM

CB

CM

BC

F



WT

MC

IRAK4 IRAK1 BTK IgMA

BMYD88:BTK

punc

ta/c

ell

CIRAK4 pT345

p65 pS536

WT MC

GAPDHC MC

0

2

4

6

8

p65

pS53

6 (n

orm

.)

0

1

2

3

IRAK

4 pT

345

(nor

m.)

C MC

0.008 0.1

MYD88:IRAK4 MYD88:IRAK1 MYD88:IgM

WT MC WT MC WT MC WT MC

<0.0001<0.0001<0.0001 <0.0001

0.0

0.4

0.8

0.0

0.6

1.2

0.00

0.35

0.70

0.0

0.2

0.4

BTK pY223

0

1

2

BTK

pY22

3 (n

orm

.)

C MC

0.3

Flümann et al., Figure 3



Flümann et al., Figure 4A

B

H/E CD138 Ki67B220

BC

MC

reactiveScratch wounds

Lymphoma

B220+/CD138-B220-/CD138+

Non-malignant

MC BC MBC

WT 1spleen

WT 2spleen

C4: 12.0%

C2: 34.0%C3: 13.1%

C1: 20.2%M552 tumor M552 cell line

C1: 99.4% C1: 89.1%M108 tumor

C1: 99.9%M108 cell line

D

E GF

0 50 1000

50

100

Perc

ent s

urvi

val

MBCBCMCWT * ***** ****

C

CCL1

1CS

F3CS

F2IF

Nγ*

IL-1α

IL-1ß

IL-2

IL-3

IL-4

IL-5IL-6

IL-7IL-9IL-10

IL-12 p40

IL-12 p70

IL-1

3

IL-1

5

IL-1

7

CXCL1

0

CXCL1LIFLIX

CCL2CSF1

CXCL9

CCL3

CCL4

CXCL2

CCL5TN

Fα*

VEGF

I

MBC WT

MB

C

WT

MC

BC

MB

C

0

20

40

60

80

100

Larg

est c

lone

(% o

f rea

ds)

Pim1

Kmt2d

Hist1h1eStat3Pou2f2 Myc

Nfkbia

H

1 10 1000

1

10

100

0Schm

itz e

t al.

(% m

utat

ed)

Chapuy et al. (% mutated)

n=12n=15 n=30

C5: 11.1%

HE CD138 B220 Ki67

.tua255

M801

M .psnart.tua

.psnarttime (weeks)

0.0

0.2

0.4

0.6

0.8

0 10000 20000 30000rank

enric

hmen

t sco

re

Reddy ABC Up

−0.8

−0.6

−0.4

−0.2

0.0

0 10000 20000 30000rank

enric

hmen

t sco

re

Reddy GCB Up

KBCMBC

KBCMBC

p = 0.001

p = 0.003



cell v

iab

ilit

y

A

B

An

nexin

V+/P

I+ (

%) ABC

**

**

**

***

****

C

D

E

F

G

0 2 4 6 8 10 12

0

50

100

time (weeks)

Perc

en

t su

rviv

al Untreated

ND-2158

LY-2409881 **

venetoclax

ND-2158

- + - + - + - + - + - + - + - +

ND-2158

LY-2409881

- + - + - + - + - + - + - + - +

LY-2409881

***

*

*

***

0

0.2

0.4

0.6

0.8

1.0MBC ABC GCB

BIM

PUMA

BAD

MS1

FS1

HRK

A1331852

AZD5991

ALM

H


M191

M108

M552

U2932

RI1

OC

ILY

7

SU

DH

L10

fractio

n c

yto

ch

rom

e c

- neg

ativ

e

venetoclax

venetoclax

venetoclax

0.0

0.5

1.0

1.5MBC ABC GCB

murine human

M191 M190 M108 M552 U2932 RI1 SUDHL10 OCILY7

0.0

0.5

1.0

1.5

MBC ABC GCB

murine human


MBC ABC GCB

murine human


cell v

iab

ilit

y

0.0

0.5

1.0

1.5

cell v

iab

ilit

y

0

50

100

GCB

murine human

MBC

- + - + - + - + - + - + - + - +

0

50

100

ABC GCB

murine human

MBC

An

nexin

V+/P

I+ (

%)

0

50

100

An

nexin

V+/P

I+ (

%)

ABC GCB

murine human

MBC



1.51.61.71.81.9

IRF4

groupMBC

MBC_aPD1

PD-L

1

0 3+ 5+B

GCB ABC

PD-L1 neg/low PD-L1 med/high

p = 0.02

179

12

FD

A

5

156

C01+2+

PD-L1

0

2

4

6

8

10

12lo

g 2(TPM

)

PD-L

1

PD-1

PD-L

2

CTL

A4

CD

86

*****

****

GCBABC

0 3+ 5+

PD-L

1

4+5+

3+

untreated

PD-1 + venetoclax

PD-1venetoclax

**** *

-150-100-50

050

100150

untreatedαPD-1

venetoclaxvenetoclax + αPD-1

best

cha

nge

(% fr

om b

asel

ine)


untreated Evenetoclax+αPD-1

base

line

3 w

eeks

% s

urvi

val

time after tumor detection (weeks)

G

�le2ebc3f1ea133

2 4 6 8

2

4

6

8

CD3E CD3E

CD4

CD4

2000

4000

6000

count

arcsinh(CD3e)

arcs

inh(

CD

4)

�le2ebc3f1ea133

2.5 5.0 7.5

2

4

6

8

CD3E CD3E

CD8A

CD

8A

1000

2000

30004000

count

arcsinh(CD3e)

arcs

inh(

CD

8a)

CD3+/CD4+ CD3+/CD8+

tumordetection αPD-1 αPD-1 αPD-1

Samplecollection

-1 0 3 6 7time (days)

H

-0.4 -0.2 0.0 0.2 0.4

1

2

3

4

log2(αPD-1/untreated)

IRF4CCR5

p21QA1b TIM3CD69

IRF4

LAG341BB

CD3+/CD8+

CD3+/CD4+

I

1.4

1.6

1.8

IRF4

groupMBC

MBC_aPD1

1.1

1.2

1.3

1.4

1.5

TIM

3 groupMBC

MBC_aPD1

CD3+/CD8+CD3+/CD4+

** * *** *

K

TIM3

J

1.71.81.92.02.12.2

p21 group

MBC

MBC_aPD1

1.2

1.3

1.4

1.5

1.6

CD

69

* *

untreated αPD-1

1.4

1.6

1.8

2.0

41BB

groupMBC

MBC_aPD1

3.25

3.50

3.75

4.00

4.25

LAG

3 groupMBC

MBC_aPD1

1.3

1.4

1.5

1.6

TIM

3

*

untreated αPD-1

0

5

10

15

CD3+ /C

D4+ (

%)

010203040

CD3+ /C

D8+ (

%)

0 20 400

50

100



Published OnlineFirst November 2, 2020.Blood Cancer Discov Ruth Flümann, Tim Rehkämper, Pascal Nieper, et al. vulnerabilitiesdiffuse large B cell lymphoma reveals actionable molecular An autochthonous mouse model of Myd88- and BCL2-driven

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