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Combination Treatment with GSK126 and Pomalidomide Induces B-Cell Differentiation in EZH2 Gain-of-Function Mutant Diffuse Large B-Cell Lymphoma.

ABSTRACT: Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), regulates genes involved in cell lineage and differentiation through methylating lysine 27 on histone H3 (H3K27me3). Recurrent gain-of-function mutations of EZH2 have been identified in various cancer types, in particular, diffuse large B-cell lymphoma (DLBCL), through large-scale genome-wide association studies and EZH2 depletion or pharmacological inhibition has been shown to exert an antiproliferative effect on cancer cells, both in vitro and in vivo. In the current study, a combination of pomalidomide and GSK126 synergistically inhibited the growth of EZH2 gain-of-function mutant Diffuse large B-cell lymphoma (DLBCL) cells. Furthermore, this synergistic effect appeared to be dependent on cereblon (CRBN), a cellular receptor of pomalidomide, but not degradation of IKAROS family zinc finger 1 (IKZF1) or IKAROS family zinc finger 3 (IKZF3). RNA sequencing analyses revealed that co-treatment with GSK126 and pomalidomide induced specific gene sets involved in B-cell differentiation and apoptosis. Synergistic growth inhibition and B-cell differentiation were further validated in xenograft mouse models. Our collective results provide a molecular basis for the mechanisms underlying the combined therapeutic effects of PRC2 inhibitors and pomalidomide on EZH2-mutated DLBCL.

SUBMITTER: Park S

PROVIDER: S-EPMC7565736 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Combination Treatment with GSK126 and Pomalidomide Induces B-Cell Differentiation in EZH2 Gain-of-Function Mutant Diffuse Large B-Cell Lymphoma.

Park Sungryul S Jo Seung-Hyun SH Kim Jong-Hwan JH Kim Seon-Young SY Ha Jae Du JD Hwang Jong Yeon JY Lee Myeong Youl MY Kang Jong Soon JS Han Tae-Su TS Park Sung Goo SG Kim Sunhong S Park Byoung Chul BC Kim Jeong-Hoon JH

Cancers 20200907 9

Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), regulates genes involved in cell lineage and differentiation through methylating lysine 27 on histone H3 (H3K27me3). Recurrent gain-of-function mutations of EZH2 have been identified in various cancer types, in particular, diffuse large B-cell lymphoma (DLBCL), through large-scale genome-wide association studies and EZH2 depletion or pharmacological inhib ...[more]

PMID: 32906688

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Project description:BackgroundDiffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma. Although durable remissions can be achieved in more than half of these patients, DLBCL remains a significant clinical challenge, with approximately 30% of patients not being cured. BCR-associated kinases (SYK, BTK, and PI3K) inhibitors have exhibited encouraging pre-clinical and clinical effects, as reported by many researchers. Early studies demonstrated that protein kinase C-β (PKCβ) inhibitors alter phosphorylation level the Bruton's tyrosine kinase (BTK), which leads to enhanced BTK signaling. Here, for the first time, we investigate whether the combination of PKCβ inhibitor enzastaurin and BTK inhibitor ibrutinib has synergistic anti-tumor effects in DLBCL.MethodsIn vitro cell proliferation was analyzed using Cell Titer-Glo Luminescent Cell Viability Assay. Induction of apoptosis and cell cycle arrest were measured by flow cytometry. Western Blotting analysis was used to detect the essential regulatory enzymes in related signaling pathways. RNA-seq was conducted to evaluate the whole transcriptome changes brought by co-treatment with low doses of enzastaurin and ibrutinib. The synergistic anti-tumor effects of enzastaurin and ibrutinib were also evaluated in vivo.ResultsCombination of enzastaurin and ibrutinib produced a lasting synergistic effect on the survival and proliferation of DLBCL cells, including reduction of proliferation, promoting apoptosis, inducting G1 phase arrest, preventing cell invasion and migration, and down-regulating activation of downstream signaling. More importantly, whole-transcriptome changes results showed that combination therapy worked synergistically to regulate whole-transcriptome expression compared with enzastaurin and ibrutinib alone. Co-treatment with low doses of enzastaurin and ibrutinib could effectively downregulate BCR, NF-κB, JAK and MAPK related signaling pathway. Furthermore, the mRNA expression analysis further indicated that co-treatment significantly decreased the mRNA levels of NOTCH1. The combination effect in inhibiting proliferation of DLBCL cells probably was realized through suppression of NOTCH1 expression. Finally, the anti-tumor activity of co-treatment also was demonstrated in vivo.ConclusionsCombination of enzastaurin and ibrutinib had synergistic anti-tumor effects in DLBCL, independent of molecular subtype. These results provided a sound foundation for an attractive therapeutic treatment, and the simultaneous suppression of BTK and PKCβ might be a new treatment strategy for DLBCL.

Project description:Ibrutinib, an FDA approved, orally administered BTK inhibitor, has demonstrated high response rates to diffuse large B‑cell lymphoma (DLBCL), however, complete responses are infrequent and acquired resistance to BTK inhibition can emerge. The present study investigated the role of the platelet‑derived growth factor D (PDGFD) gene and the ibrutinib resistance of DLBCL in relation to epidermal growth factor receptor (EGFR). Bioinformatics was used to screen and analyze differentially expressed genes (DEGs) in complete response (CR), partial response (PR) and stable disease (SD) in DLBCL treatment with ibrutinib, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to analyze enriched the signaling pathways increasing DEGs. The Search Tool for Interactions of Chemicals database was used to analyze the target genes of ibrutinib. An interaction network of DEGs, disease‑related genes and ibrutinib was constructed. The expression of PDGFD in tissues that were resistant or susceptible to DLBCL/ibrutinib was detected via immunohistochemistry (IHC), and the expression of PDGFD in DLBCL/ibrutinib‑resistant strains and their parental counterparts were examined via reverse transcription‑quantitative PCR and western blot analyses. Subsequently, a drug‑resistant cell model of DLBCL/ibrutinib in which PDGFD was silenced was constructed. The apoptosis of the DLBCL/ibrutinib‑resistant strains was examined using MTT and flow cytometry assays. EGFR gene expression was then assessed. At the same time, a PDGFD‑interfering plasmid and an EGFR overexpression plasmid were transfected into the DLBCL drug‑resistant cells (TMD8‑ibrutinib, HBL1‑ibrutinib) separately or together. MTT was used to measure cell proliferation and changes in the IC50 of ibrutinib. A total of 86 DEGs that increased in the CR, PR and SD tissues were screened, and then evaluated with GO and KEGG. The interaction network diagram showed that there was a regulatory relationship between PDGFD and disease‑related genes, and that PDGFD could indirectly target the ibrutinib target gene EGFR, indicating that PDGFD could regulate DLBCL via EGFR. IHC results showed high expression of PDGFD in diffuse large B‑cell lymphoma tissues with ibrutinib tolerance. PDGFD expression in ibrutinib‑resistant DLBCL cells was higher compared with in parental cells. Following interference with PDGFD expression in ibrutinib‑resistant DLBCL cells, the IC50 value of ibrutinib decreased, the rate of apoptosis increased and EGFR expression decreased. In brief, EGFR overexpression can reverse the resistance of DLBCL to ibrutinib via PDGFD interference, and PDGFD induces the resistance of DLBCL to ibrutinib via EGFR.

Project description:Primary bone diffuse large B-cell lymphoma (PB-DLBCL) is a rare extranodal lymphoma subtype. This retrospective study elucidates the currently unknown genetic background of a large clinically well-annotated cohort of DLBCL with osseous localizations (O-DLBCL), including PB-DLBCL. A total of 103 patients with O-DLBCL were included and compared with 63 (extra)nodal non-osseous (NO)-DLBCLs with germinal center B-cell phenotype (NO-DLBCL-GCB). Cell-of-origin was determined by immunohistochemistry and gene-expression profiling (GEP) using (extended)-NanoString/Lymph2Cx analysis. Mutational profiles were identified with targeted next-generation deep sequencing, including 52 B-cell lymphoma-relevant genes. O-DLBCLs, including 34 PB-DLBCLs, were predominantly classified as GCB phenotype based on immunohistochemistry (74%) and NanoString analysis (88%). Unsupervised hierarchical clustering of an extended-NanoString/Lymph2Cx revealed significantly different GEP clusters for PB-DLBCL as opposed to NO-DLBCL-GCB (P < .001). Expression levels of 23 genes of 2 different targeted GEP panels indicated a centrocyte-like phenotype for PB-DLBCL, whereas NO-DLBCL-GCB exhibited a centroblast-like constitution. PB-DLBCL had significantly more frequent mutations in four GCB-associated genes (ie, B2M, EZH2, IRF8, TNFRSF14) compared with NO-DLBCL-GCB (P = .031, P = .010, P = .047, and P = .003, respectively). PB-DLBCL, with its corresponding specific mutational profile, was significantly associated with a superior survival compared with equivalent Ann Arbor limited-stage I/II NO-DLBCL-GCB (P = .016). This study is the first to show that PB-DLBCL is characterized by a GCB phenotype, with a centrocyte-like GEP pattern and a GCB-associated mutational profile (both involved in immune surveillance) and a favorable prognosis. These novel biology-associated features provide evidence that PB-DLBCL represents a distinct extranodal DLBCL entity, and its specific mutational landscape offers potential for targeted therapies (eg, EZH2 inhibitors).

Dataset Information

Combination Treatment with GSK126 and Pomalidomide Induces B-Cell Differentiation in EZH2 Gain-of-Function Mutant Diffuse Large B-Cell Lymphoma.

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Combination Treatment with GSK126 and Pomalidomide Induces B-Cell Differentiation in <i>EZH2</i> Gain-of-Function Mutant Diffuse Large B-Cell Lymphoma.

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