PD-1/PD-L1 immune checkpoint and p53 loss facilitate tumor progression in activated B-cell diffuse large B-cell lymphomas

Abstract

Refractory or relapsed diffuse large B-cell lymphoma (DLBCL) often associates with the activated B-cell-like (ABC) subtype and genetic alterations that drive constitutive NF-κB activation and impair B-cell terminal differentiation. Here, we show that DNA damage response by p53 is a central mechanism suppressing the pathogenic cooperation of IKK2ca-enforced canonical NF-κB and impaired differentiation resulting from Blimp1 loss in ABC-DLBCL lymphomagenesis. We provide evidences that the interplay between these genetic alterations and the tumor microenvironment select for additional molecular addictions that promote lymphoma progression, including aberrant coexpression of FOXP1 and the B-cell mutagenic enzyme activation-induced deaminase, and immune evasion through major histocompatibility complex class II downregulation, PD-L1 upregulation, and T-cell exhaustion. Consistently, PD-1 blockade cooperated with anti-CD20-mediated B-cell cytotoxicity, promoting extended T-cell reactivation and antitumor specificity that improved long-term overall survival in mice. Our data support a pathogenic cooperation among NF-κB-driven prosurvival, genetic instability, and immune evasion mechanisms in DLBCL and provide preclinical proof of concept for including PD-1/PD-L1 blockade in combinatorial immunotherapy for ABC-DLBCL.

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

© 2019 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

Conditional deletion of p53 cooperates with constitutive canonical NF-κB and Blimp1 loss in ABC-DLBCL lymphomagenesis. (A) Schematic diagram of the mutant mice and targeted B-cell functions used in this study. YC controls, YFPstopF/+Cγ1Cre/+; BIC, Blimp1F/FIKK2castopF/stopFCγ1Cre/+; pBIC, p53F/FBIC. (B) Overall survival of control or multilesion mice. (C) Representative immunohistochemical staining of hematoxylin and eosin, B220 and ki67 to label proliferating B cells in normal splenic GCs and murine diffuse B-cell lymphomas. Scale bars, 200 or 20 μm, as indicated. (D) RNA-seq gene expression classifier distinguishes ABC-DLBCL subtype in the murine lymphomas, which is confirmed by qRT-PCR of FACS-sorted reporter-positive normal GCBs or PCs, and lymphoma B cells (n≥3 animals). Relative values are normalized to GCB expression levels. (E) Scatter plot of differentially expressed genes (N = 1387) as measured by RNA-seq from GFP+/YFP+ reporter splenic B cells, showing log2 fold-changes in lymphoma relative to normal GCBs (n ≥ 3 animals). Genes were stratified and colored according to whether they were found in both lymphoma models or differentially expressed in the more aggressive pBIC model. (F) Heat maps of gene expression levels (left) and gene ontology (GO) analysis (right) for the categories of differentially expressed genes stratified in panel E. (G) Comparative percentages of apoptotic cells within reporter-positive control or lymphoma cells. (H) Comparative percentages of reporter-positive B cells that are positive for γH2AX by intracellular FACS. Gray bars represent YFP/GFP-negative normal cells from the same tumors. (I) Bubble plot illustrating the enrichment of VDJ-IgH clonal groups within reporter-positive murine control or lymphoma cells that accumulate unique somatic mutations (y-axis) in intraclonally diverse V sequences (x-axis). Bubble sizes represent the abundance of clonal barcoded single-molecule, and therefore clon size, whereas colors indicate the dominant isotype.

Figure 2.

Aberrant coexpression of FOXP1 and AID characterizes NF-κB-driven murine and human ABC-DLBCL. (A) Representative immunohistochemical staining of FOXP1 (nuclear) and AID (cytoplasmic) in normal splenic GCs and lymphomas. Scale bars, 200 μm or 10 μm at insets. (B) Expression analysis by qRT-PCR of FACS-sorted reporter-positive normal resting or GCBs, and lymphoma B cells (n5 animals). Relative values were normalized to resting or GCB expression levels for mFoxp1 or mAID, respectively. (C) Expression analysis by qRT-PCR of paired NBC or GCBs magnetically sorted from human tonsils or FACS sorted from normal murine spleens (n4). Relative values are normalized to endogenous levels h/mGAPDH. (D) Representative images of inverse correlation of FOXP1 and AID expression in reactive human tonsil or murine spleen examined by IHC, using combined labeling of anti-FOXP1 (brown) and anti-AID (red). Scale bars, 200 μm; magnification, ×20. (E) ChIP-seq data from OCI-Ly1 cells showing enrichment of FOXP1 on the hAID locus. Distribution of FOXP1 occupancy is overlapped with chromatin-state models predicted by ENCODE ChromHMM from GM12878 (human B lymphoblastoid) or CH12 (mouse B-cell lymphoma) cells, and with the predicted conserved mAID gene locus according to Genome Browser comparison of hg19 and mm9 sequence data. Black boxes on top indicate genomic regions (R1-4) associated with transcriptional regulation of AID expression. (F) Comparison of hFOXP1 ChIP-seq peaks at intronic region 2 of the hAID locus observed here in OCI-Ly1 cells or previously in other GCB-DLBCL or ABC-DLBCL cell lines (GSE69009). (G) Validation of FOXP1 occupancy at AID intronic peaks and measured by ChIP-qPCR in human OCI-Ly1 DLBCL cells, as well as in resting or activated murine cells (ie, CH12 lymphoma cells and primary magnetically sorted CD19+ splenic cells). CIT, combination of anti-CD40 plus IL-4 and TNF-β; LPS, lipopolysaccharides. (H) Scatter plot of gene expression array data from OCI-Ly1 cells showing average log2 fold-changes (n = 3 replicates) in the expression of FOXP1-bound genes (according to ChIP-seq data from OCI-LY1) relative to scramble control after FOXP1 silencing with 2 different siRNAs. (I) Heat map of average fold-changes (n = 3 replicates) in the expression of FOXP1 and AID relative to scramble control after siRNA-mediated silencing of FOXP1 in DLBCL cell lines and in CIT-activated CH12 cells. (J) Forest graph plot of Pearson r coefficients measuring the correlation of hFOXP1 and hAID expression in previously published GSE series. (K) Scatter plot of FOXP1 and AID gene expression array data from R-CHOP-treated patients with DLBCL (n = 233, GSE10846). Median expression levels for FOXP1 (223287_s_at) and AID (219841_at) are indicated and were used as cutoff values for patient stratification. (L) Overall survival of R-CHOP-treated patients with DLBCL stratified by FOXP1/AID expression levels in panel K. (M) Distribution of COO-based subtypes in the FOXP1/AID HiHi and LoLo expression DLBCL subgroups stratified in panel K. COO subtypes were defined by expression signatures and were available in metadata from GSE10846. (N) Scatter plot of FOXP1 and AID protein expression data as measured by IHC scoring from CHOP-treated patients with DLBCL (n = 112). Median IHC scores are indicated and were used as cutoff values for patient stratification. (O) Overall survival of CHOP-treated patients with DLBCL stratified by FOXP1/AID IHC scores in panel N. (P) Distribution of COO-based subtypes in the FOXP1/AID HiHi and LoLo expression DLBCL subgroups stratified in panel N. COO subtypes were defined by the Hans IHC algorithm. HiHi, FOXP1highAIDhigh; LoLo, FOXP1lowAIDlow; NBC, naive B cells; UNC, unclassified.

Figure 2.

Aberrant coexpression of FOXP1 and AID characterizes NF-κB-driven murine and human ABC-DLBCL. (A) Representative immunohistochemical staining of FOXP1 (nuclear) and AID (cytoplasmic) in normal splenic GCs and lymphomas. Scale bars, 200 μm or 10 μm at insets. (B) Expression analysis by qRT-PCR of FACS-sorted reporter-positive normal resting or GCBs, and lymphoma B cells (n5 animals). Relative values were normalized to resting or GCB expression levels for mFoxp1 or mAID, respectively. (C) Expression analysis by qRT-PCR of paired NBC or GCBs magnetically sorted from human tonsils or FACS sorted from normal murine spleens (n4). Relative values are normalized to endogenous levels h/mGAPDH. (D) Representative images of inverse correlation of FOXP1 and AID expression in reactive human tonsil or murine spleen examined by IHC, using combined labeling of anti-FOXP1 (brown) and anti-AID (red). Scale bars, 200 μm; magnification, ×20. (E) ChIP-seq data from OCI-Ly1 cells showing enrichment of FOXP1 on the hAID locus. Distribution of FOXP1 occupancy is overlapped with chromatin-state models predicted by ENCODE ChromHMM from GM12878 (human B lymphoblastoid) or CH12 (mouse B-cell lymphoma) cells, and with the predicted conserved mAID gene locus according to Genome Browser comparison of hg19 and mm9 sequence data. Black boxes on top indicate genomic regions (R1-4) associated with transcriptional regulation of AID expression. (F) Comparison of hFOXP1 ChIP-seq peaks at intronic region 2 of the hAID locus observed here in OCI-Ly1 cells or previously in other GCB-DLBCL or ABC-DLBCL cell lines (GSE69009). (G) Validation of FOXP1 occupancy at AID intronic peaks and measured by ChIP-qPCR in human OCI-Ly1 DLBCL cells, as well as in resting or activated murine cells (ie, CH12 lymphoma cells and primary magnetically sorted CD19+ splenic cells). CIT, combination of anti-CD40 plus IL-4 and TNF-β; LPS, lipopolysaccharides. (H) Scatter plot of gene expression array data from OCI-Ly1 cells showing average log2 fold-changes (n = 3 replicates) in the expression of FOXP1-bound genes (according to ChIP-seq data from OCI-LY1) relative to scramble control after FOXP1 silencing with 2 different siRNAs. (I) Heat map of average fold-changes (n = 3 replicates) in the expression of FOXP1 and AID relative to scramble control after siRNA-mediated silencing of FOXP1 in DLBCL cell lines and in CIT-activated CH12 cells. (J) Forest graph plot of Pearson r coefficients measuring the correlation of hFOXP1 and hAID expression in previously published GSE series. (K) Scatter plot of FOXP1 and AID gene expression array data from R-CHOP-treated patients with DLBCL (n = 233, GSE10846). Median expression levels for FOXP1 (223287_s_at) and AID (219841_at) are indicated and were used as cutoff values for patient stratification. (L) Overall survival of R-CHOP-treated patients with DLBCL stratified by FOXP1/AID expression levels in panel K. (M) Distribution of COO-based subtypes in the FOXP1/AID HiHi and LoLo expression DLBCL subgroups stratified in panel K. COO subtypes were defined by expression signatures and were available in metadata from GSE10846. (N) Scatter plot of FOXP1 and AID protein expression data as measured by IHC scoring from CHOP-treated patients with DLBCL (n = 112). Median IHC scores are indicated and were used as cutoff values for patient stratification. (O) Overall survival of CHOP-treated patients with DLBCL stratified by FOXP1/AID IHC scores in panel N. (P) Distribution of COO-based subtypes in the FOXP1/AID HiHi and LoLo expression DLBCL subgroups stratified in panel N. COO subtypes were defined by the Hans IHC algorithm. HiHi, FOXP1highAIDhigh; LoLo, FOXP1lowAIDlow; NBC, naive B cells; UNC, unclassified.

Figure 3.

Decreased MHC-II gene expression and immune checkpoint deregulation cooperate with ABC-DLBCL genetic hallmarks to promote immune evasion. (A) Comparative gene expression levels of the MHC-II transactivator CIITA in GFP+/YFP+ reporter murine B cells from control or tumoral spleens (left) and human DLBCL (right, GSE10846). (B) Heat map of gene expression levels for MHC-II genes (KEGG pathway mmu04612) in GFP+/YFP+ reporter murine B cells from control or tumoral spleens. (C) Representative FACS histograms and comparative levels of surface PD-L1 in GFP+/YFP+ reporter murine B cells (left), and PD-L1 gene expression levels in human DLBCL (right, GSE10846). (D) Pie charts showing average percentages (n = 4 animals) of different immune cells in the control or tumoral spleens. (E) Comparative percentages of different T-cell populations in the control or tumoral spleens of indicated mice. (F-G) Multidimensional depiction by t-SNE of surface marker levels in aggregated total CD8+ T cells (dots) from control or tumoral spleens (n = 4 animals). Expression by FACS of coinhibitory and exhaustion markers (F) or activation and differentiation markers (G) were overlaid onto the overall t-SNE maps. Interpretation of the overall phenotype for each CD8+ T cell is color coded and annotated as nonexhausted (nEx; PD-1negLAG-3neg2B4neg), exhausted (Ex; PD-1hi), hyperexhausted cells that coexpress multiple inhibitory receptors besides PD-1 (hEx), naive (N; CD44lowCD62L+), central memory (CM; CD44highCD62L+), and effector or effector memory (E/EM; CD44highCD62Lneg). Percentages indicate average abundance of CD8+ T cells with the different exhaustion/differentiation phenotypes in YC control spleens or BIC/pBIC lymphomas.

Figure 4.

Immunotherapy with anti-PD-1 enhances anti-CD20 efficacy in the aggressive immunocompetent ABC-DLBCL mouse model. (A) Overall survival of murine ABC-DLBCL pBIC mice treated with different immunotherapy combinations. (B) Relative changes in splenic transversal areas measured by ventral ultrasound of pBIC mice (>180 days, with evidence of splenomegaly, n = 3) at 4 sequential times during immunotherapy treatment, as indicated in the scheme at the top. Representative ultrasound sections of the spleen are shown on the right. Scale bars, 1 mm. (C) Representative immunohistochemical staining of GFP, CD4, and CD8 to label T-cell infiltration in murine pBIC lymphomas (>180 days) that had received 4 weeks of immunotherapy. Scale bars, 200 μm. (D) Comparative percentages of splenic B220+GFP+ lymphoma cells from pBIC lymphomas (>180 days) in response to different 4-week immunotherapy regimens (n3). (E) Pie charts showing percentages of immune cells in the spleen pBIC mice (>180 days) after 4-week immunotherapy (n = 3). (F) Comparative fractions of PD-1-positive T cells in the TME of pBIC mice (>180 days) after 4-week immunotherapy (n = 3). (G) Heat maps of mean expression intensity of coinhibitory/exhaustion (top) and activation/differentiation (bottom) surface markers within lymphocyte subsets detected by RPhenograph clustering (n = 3 mice × 4 groups). Distribution of these cell populations in response to 4-week immunotherapy (n = 3) is represented in t-SNE maps and colored. Percentages indicate average abundance of each cell population in the corresponding treatment group. (H) Comparative fractions of PD-L1-positive or PD-L1-negative cells within the compartment of lymphoma cells (CD19+GFP+) or neighbor normal B cells (CD19+GFP−) from pBIC mice (>180 days) after 4-week immunotherapy (n = 3). (I) FACS analysis to assess the specific depletion of lymphoma or normal B cells in the spleen of pBIC mice (>180 days) after 4-week immunotherapy (n = 3). (J) Percentages of reappearing splenic lymphoma cells (CD20+GFP+) or neighbor normal B cells (CD20+GFP−) during a resting period of 8 weeks after anti-CD20–based immunotherapy that can efficiently deplete the B-cell compartment in pBIC mice (>180 days, n3). MS, median survival; UNT, untreated.