Integrative analyses reveal outcome-associated and targetable molecular partnerships between TP53, BRD4, TNFRSF10B, and CDKN1A in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a common, genomically heterogenous disease that presents a clinical challenge despite the success of frontline regimens and second-line chimeric antigen receptor T-cell (CAR-T) therapy. Recently, genomic alterations and tumor microenvironment features associated with poor CAR-T response have been identified, namely those to the TP53 tumor suppressor gene. This retrospective analysis aimed to integrate various data to identify genomic partnerships capable of providing further clarity and actionable treatment targets within this population. Publicly available data were analyzed for differential expression based on TP53 and 24-month event-free survival (EFS24) status, revealing enrichments of the BRD4 bromodomain oncogene (p < 0.0001, p = 0.001). High-BRD4 and TP53 alterations were significantly associated with lower CDKN1A (p21) and TNFRSF10B (TRAIL-R2), a key tumor suppressor and CAR-T modulator, respectively. Significant loss of CD8 T-cell presence within low-TNFRSF0B (p = 0.0042) and altered-TP53 (p = 0.0424) patients showcased relevant outcome-associated tumor microenvironment features. Furthermore, reduced expression of CDKN1A was associated with low TNFRSF10B (FDR < 0.0001) and increased BRD4 interactant genes (FDR < 0.0001). Promisingly, in vitro MDM2 inhibition with Idasnutlin and TP53 reactivation via Eprenetapopt was able to renew TNFRSF10B protein expression. Additionally, applying the BRD4-degrading PROTAC ARV-825 and the CDK4/6 inhibitor Abemaciclib as single-agents and in synergistic combination significantly reduced TP53-altered DLBCL cell line viability. Our analysis presents key associations within a genomic network of actionable targets capable of providing clarity within the evolving precision CAR-T treatment landscape.

Molecular classification and identification of an aggressive signature in low-grade B-cell lymphomas.

Non-follicular low-grade B-cell lymphomas (LGBCL) are biologically diverse entities that share clinical and histologic features that make definitive pathologic categorization challenging. While most patients with LGBCL have an indolent course, some experience aggressive disease, highlighting additional heterogeneity across these subtypes. To investigate the potential for shared biology across subtypes, we performed RNA sequencing and applied machine learning approaches that identified five clusters of patients that grouped independently of subtype. One cluster was characterized by inferior outcome, upregulation of cell cycle genes, and increased tumor immune cell content. Integration of whole exome sequencing identified novel LGBCL mutations and enrichment of TNFAIP3 and BCL2 alterations in the poor survival cluster. Building on this, we further refined a transcriptomic signature associated with early clinical failure in two independent cohorts. Taken together, this study identifies unique clusters of LGBCL defined by novel gene expression signatures and immune profiles associated with outcome across diagnostic subtypes.

Subtype-specific and co-occurring genetic alterations in B-cell non-Hodgkin lymphoma.

B-cell non-Hodgkin lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL, such as diffuse large B-cell lymphoma, have been comprehensively interrogated at the genomic level, but rarer subtypes, such as mantle cell lymphoma, remain less extensively characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth, including diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, and Burkitt lymphoma. We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumors from each subtype, including frequent genetic deregulation of the ubiquitin proteasome system. In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.

Crebbp loss cooperates with Bcl2 overexpression to promote lymphoma in mice.

CREBBP is targeted by inactivating mutations in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Here, we provide evidence from transgenic mouse models that Crebbp deletion results in deficits in B-cell development and can cooperate with Bcl2 overexpression to promote B-cell lymphoma. Through transcriptional and epigenetic profiling of these B cells, we found that Crebbp inactivation was associated with broad transcriptional alterations, but no changes in the patterns of histone acetylation at the proximal regulatory regions of these genes. However, B cells with Crebbp inactivation showed high expression of Myc and patterns of altered histone acetylation that were localized to intragenic regions, enriched for Myc DNA binding motifs, and showed Myc binding. Through the analysis of CREBBP mutations from a large cohort of primary human FL and DLBCL, we show a significant difference in the spectrum of CREBBP mutations in these 2 diseases, with higher frequencies of nonsense/frameshift mutations in DLBCL compared with FL. Together, our data therefore provide important links between Crebbp inactivation and Bcl2 dependence and show a role for Crebbp inactivation in the induction of Myc expression. We suggest this may parallel the role of CREBBP frameshift/nonsense mutations in DLBCL that result in loss of the protein, but may contrast the role of missense mutations in the lysine acetyltransferase domain that are more frequently observed in FL and yield an inactive protein.

FAM171B is a novel polyglutamine protein widely expressed in the mammalian brain.

Mutation in proteins containing polyglutamine (polyQ) tracts has been shown to underlie a number of severe human neurodegenerative disorders such as Huntington's Disease and Spinocerebellar Ataxia. In this study, we identify and describe FAM171B as a novel polyQ protein containing fourteen consecutive glutamine residues in its National Center for Biotechnology Information (NCBI) referenced sequence. Utilizing western blotting, in situ hybridization, and immunohistochemistry, we demonstrate that FAM171B is widely expressed in mouse brain with pronounced localization in the hippocampus, cerebellum, and cerebral cortex. Furthermore, immunofluorescence experiments reveal that FAM171B predominantly localizes to vesicle-like structures in the cytoplasm of neurons. Finally, bioinformatic analysis suggests that FAM171B is robustly expressed in human brain, and (similar to other polyQ disease genes) its polyQ tract is polymorphic within the general human population. Thus, as a polyQ protein that is expressed in brain, FAM171B should be considered a candidate gene for an as yet molecularly uncharacterized neurodegenerative disease.

Targeting of inflammatory pathways with R2CHOP in high-risk DLBCL.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma, and front line therapies have not improved overall outcomes since the advent of immunochemotherapy. By pairing DNA and gene expression data with clinical response data, we identified a high-risk subset of non-GCB DLBCL patients characterized by genomic alterations and expression signatures capable of sustaining an inflammatory environment. These mutational alterations (PIM1, SPEN, and MYD88 [L265P]) and expression signatures (NF-κB, IRF4, and JAK-STAT engagement) were associated with proliferative signaling, and were found to be enriched in patients treated with RCHOP that experienced unfavorable outcomes. However, patients with these high-risk mutations had more favorable outcomes when the immunomodulatory agent lenalidomide was added to RCHOP (R2CHOP). We are the first to report the genomic validation of a high-risk phenotype with a preferential response towards R2CHOP therapy in non-GCB DLBCL patients. These conclusions could be translated to a clinical setting to identify the ~38% of non-GCB patients that could be considered high-risk, and would benefit from alternative therapies to standard RCHOP based on personalized genomic data.

Somatic copy number gains in MYC, BCL2, and BCL6 identifies a subset of aggressive alternative-DH/TH DLBCL patients.

Double/triple hit lymphoma (DH/TH), known as high-grade B-cell lymphoma (HGBL), is an aggressive diffuse large B cell lymphoma (DLBCL), defined as having concurrent MYC, BCL2, and/or BCL6 gene rearrangements. While gene rearrangements represent significant genetic events in cancer, copy number alterations (CNAs) also play an important role, and their contributions to rearrangements have yet to be fully elucidated. Using FISH and high-resolution CNA data, we defined the landscape of concurrent gene rearrangements and copy gains in MYC, BCL2, and BCL6, in a cohort of 479 newly diagnosed DLBCL. We also show that concurrent translocations and copy number alterations, in combinations similar to DH/TH, identify a unique subset of DLBCL, alternative DH/TH, that have survival outcomes similar to DH/TH DLBCL patients.

Amplification of 9p24.1 in diffuse large B-cell lymphoma identifies a unique subset of cases that resemble primary mediastinal large B-cell lymphoma.

Copy number alterations (CNAs) of 9p24.1 occur frequently in Hodgkin lymphoma, primary mediastinal large B-cell lymphoma (PMBCL), primary central nervous system lymphoma, and primary testicular lymphoma, resulting in overexpression of PD-L1 and sensitivity to PD-1 blockade-based immunotherapy. While 9p24.1 CNA was also reported in diffuse large B-cell lymphoma (DLBCL), little is known about its molecular or clinical significance. In this study, we analyzed the prevalence of 9p24.1 CNA in newly diagnosed DLBCL and examined its association with PD-L1, PD-L2, and JAK2 expression, clinical characteristics, and outcome. We found that 10% of DLBCL cases had CNA of 9p24.1, with 6.5% gains, and 3.5% amplifications. Only the cases with a 9p24.1 amplification had high levels of PD-L1, PD-L2, and JAK2 expression. Gains or amplifications of 9p24.1 were associated with a younger age and the ABC/non-GCB subtype. Compared with DLBCL cases without 9p24.1 CNA, the cases with a 9p24.1 amplification had a trend of better event-free survival. Furthermore, the amplification cases had a gene expression and mutation profile similar to those of PMBCL. Our data suggest that amplification of 9p24.1 identifies a unique subset of DLBCL with clinical and molecular features resembling PMBCL that may be amenable to PD-1 blockade-based immunotherapy.

Targetable genetic alterations of TCF4 (E2-2) drive immunoglobulin expression in diffuse large B cell lymphoma.

The activated B cell (ABC-like) subtype of diffuse large B cell lymphoma (DLBCL) is characterized by chronic activation of signaling initiated by immunoglobulin µ (IgM). By analyzing the DNA copy number profiles of 1000 DLBCL tumors, we identified gains of 18q21.2 as the most frequent genetic alteration in ABC-like DLBCL. Using integrative analysis of matched gene expression profiling data, we found that the TCF4 (E2-2) transcription factor gene was the target of these alterations. Overexpression of TCF4 in ABC-like DLBCL cell lines led to its occupancy on immunoglobulin (IGHM) and MYC gene enhancers and increased expression of these genes at the transcript and protein levels. Inhibition of TCF4 activity with dominant-negative constructs was synthetically lethal to ABC-like DLBCL cell lines harboring TCF4 DNA copy gains, highlighting these gains as an attractive potential therapeutic target. Furthermore, the TCF4 gene was one of the top BRD4-regulated genes in DLBCL cell lines. BET proteolysis-targeting chimera (PROTAC) ARV771 extinguished TCF4, MYC, and IgM expression and killed ABC-like DLBCL cells in vitro. In DLBCL xenograft models, ARV771 treatment reduced tumor growth and prolonged survival. This work highlights a genetic mechanism for promoting immunoglobulin signaling in ABC-like DLBCL and provides a functional rationale for the use of BET inhibitors in this disease.