DNMT3A encodes an enzyme that carries out de novo DNA methylation, which is essential for the acquisition of cellular identity and specialized functions during cellular differentiation. DNMT3A is the most frequently mutated gene in age-related clonal hematopoiesis. As such, mature immune cells harboring DNMT3A mutations can be readily detected in elderly persons. Most DNMT3A mutations associated with clonal hematopoiesis are heterozygous and predicted to cause loss of function, indicating that haploinsufficiency is the predominant pathogenic mechanism. Yet, the impact of DNMT3A haploinsufficiency on the function of mature immune cells is poorly understood. Here, we demonstrate that DNMT3A haploinsufficiency impairs the gain of DNA methylation at decommissioned enhancers, while simultaneously and unexpectedly impairing DNA demethylation of newly activated enhancers in mature human myeloid cells. The DNA methylation defects alter the activity of affected enhancers, leading to abnormal gene expression and impaired immune response. These findings provide insights into the mechanism of immune dysfunction associated with clonal hematopoiesis and acquired DNMT3A mutations.

Introduction: Enhancer of zeste homolog 2 (EZH2) catalyzes H3K27me3, an epigenetic modification linked to gene silencing, and its overexpression contributes to the progression of hematological malignancies. This study compares the efficacy of a conventional EZH2 inhibitor with a PROTAC-based EZH2 degrader in human lymphoma cell lines. Furthermore, we investigate the anti-tumor effects of combining EZH2 degrader with anti-PD-1, an immune checkpoint inhibitor, focusing on immune cell interactions and underlying mechanisms. Methods: The cytotoxic effects of the EZH2 degrader and EZH2 inhibitor were evaluated in Burkitt’s, B-cell, cutaneous T-cell, and Hodgkin’s lymphoma cell lines. Additionally, the combination therapy of the EZH2 degrader and anti-PD-1 was assessed both in vitro and in a hu-PBMC-CDX mouse model. Results: We evaluated the effects of an EZH2 degrader on seven lymphoma cell lines and observed significant reductions in cell viability compared to EZH2 inhibitor, particularly in Burkitt’s lymphoma cell lines. EZH2 degrader treatment reduced EZH2 and c-Myc expression, induced G2/M cell cycle arrest, and increased apoptosis markers, including cleaved caspase-3 and cleaved PARP. Furthermore, Burkitt’s lymphoma is a PD-L1 negative tumor; however, treatment with the EZH2 degrader resulted in a slight increase in PD-L1 expression. Combining EZH2 degrader with anti-PD-1 significantly enhanced anti-tumor effects compared to monotherapy. In vivo studies using a humanized lymphoma mouse model demonstrated a synergistic anti-tumor effect of EZH2 degrader and anti-PD-1, which was attributed to apoptosis-related pathways. Discussion: These findings aim to provide insights into the therapeutic potential of targeting EZH2 in combination with immune checkpoint inhibitors for improved treatment of lymphomas.

EZH2, a histone methyltransferase, contributes significantly to cancer cell survival and proliferation. Although various EZH2 inhibitors have demonstrated promise in treating lymphoma, they have not fully managed to curb lymphoma cell proliferation despite effective reduction of the H3K27me3 mark. We used MS1943, an EZH2 selective degrader, which successfully diminishes EZH2 levels in lymphoma cells. Additionally, lapatinib, a dual inhibitor of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases, targets a receptor protein that regulates cell growth and division. The overexpression of this protein is often observed in lymphoma cells. Our study aims to combine these two therapeutic targets to stimulate apoptosis pathways and potentially suppress Burkitt’s lymphoma cell survival and proliferation in a complementary and synergistic manner. We observed that a combination of MS1943 and lapatinib induced apoptosis in Daudi cells and caused cell cycle arrest at the S and G2/M phases in both Ramos and Daudi cells. This strategy, using a combination of MS1943 and lapatinib, presents a promising therapeutic approach for treating lymphoma and potentially Burkitt’s lymphoma.

Enhancer of zeste homolog 2 (EZH2) and Bruton’s tyrosine kinase (BTK) are both key factors involved in the development and progression of hematological malignancies. Clinical studies have demonstrated the potential of various EZH2 inhibitors, which target the methyltransferase activity of EZH2, for the treatment of lymphomas. However, despite their ability to effectively reduce the H3K27me3 levels, these inhibitors have shown limited efficacy in blocking the proliferation of lymphoma cells. To overcome this challenge, we employed a hydrophobic tagging approach utilizing MS1943, a selective EZH2 degrader. In this study, we investigated the inhibitory effects of two drugs, the FDA-approved EZH2 inhibitor Tazemetostat, currently undergoing clinical trials, and the novel drug MS1943, on Burkitt’s lymphoma. Furthermore, we assessed the potential synergistic effect of combining these drugs with the BTK inhibitor Ibrutinib. In this study, we evaluated the effects of combination therapy with MS1943 and Ibrutinib on the proliferation of three Burkitt’s lymphoma cell lines, namely RPMI1788, Ramos, and Daudi cells. Our results demonstrated that the combination of MS1943 and Ibrutinib significantly suppressed cell proliferation to a greater extent compared to the combination of Tazemetostat and Ibrutinib. Additionally, we investigated the underlying mechanisms of action and found that the combination therapy of MS1943 and Ibrutinib led to the upregulation of miR29B-mediated p53-upregulated modulator of apoptosis PUMA, BAX, cleaved PARP, and cleaved caspase-3 in Burkitt’s lymphoma cells. These findings highlight the potential of this innovative therapeutic strategy as an alternative to traditional EZH2 inhibitors, offering promising prospects for improving treatment outcomes in Burkitt’s lymphoma.

Chae-Eun YANG, Se Been KIM, Yurim JEONG, Jung-Yeon LIM. Korean J Clin Lab Sci. 2023;55:298-305. https://doi.org/10.15324/kjcls.2023.55.4.298

Minseo YU, Ra Eun KIM, Yurim JEONG, Hyewon JANG, Se Been KIM, Jung-Yeon LIM. Korean J Clin Lab Sci. 2024;56:198-206. https://doi.org/10.15324/kjcls.2024.56.3.198