Small molecule drug discovery has primarily focused on directly regulating protein activity, especially through inhibitors. Recently, targeted protein degradation (TPD) technologies have emerged utilizing the cell’s own degradation mechanisms using PROteolysis TArgeting Chimeras (PROTACs) [1], molecular glues, Lysosome-Targeting Chimeras [2], and Antibody-based PROTACs [3] to eliminate disease-associated proteins. These advances have not only broadened the scope of new target drug discovery but also enabled the pharmaceutical industry to tackle previously ‘undruggable’ targets, with significant clinical progress demonstrated in cancer therapies [4, 5].
Based on our previous research, we showed to demonstrate and compare the inhibitory effects of two drugs: the enhancer of zeste homolog 2 (EZH2) inhibitor Tazemetostat (iEZH2) and the novel PROTAC-based degrader MS1943 (dEZH2) [6]. Our results demonstrated that dEZH2 was significantly more effective at suppressing B cell lymphoma than the iEZH2 [7, 8]. Also, combination of dEZH2 with the Ibrutinib (Bruton’s tyrosine kinase inhibitor) led to a synergistic reduction in B cell lymphoma cells [7]. Additionally, dEZH2 showed effectiveness in inhibiting Burkitt’s lymphoma when used in combination with Lapatinib, an epidermal growth factor receptor/human epidermal growth factor receptor 2 targeted therapy [9]. These findings show that PROTAC-based drugs have significant therapeutic potential for targeting the same protein, offering promising new treatment strategies.
EZH2 is a methyltransferase, which contributes to tumor progression by silencing tumor suppressor genes, while methyltransferase 3 (METTL3) regulates mRNA modifications that promote oncogenic gene expression. Previous reports suggested that METTL3 modulated protein levels of EZH2 through m6A modification [10, 11]. By targeting both, we could more effectively disrupt the pathway that drive Burkitt’s lymphoma cell proliferation and potentially overcome resistance to single-drug therapy. METTL3 inhibitors provides the potential therapeutic targeting cancer therapy. The anticancer effects of METTL3 inhibitors have been demonstrated in diffuse large B-cell lymphoma [12, 13], acute myeloid leukemia (AML) [14, 15] and many different types of blood type cancer.
Burkitt’s lymphoma is a highly aggressive type of lymphoma that targets B-lymphocytes and constitutes about less 1% of all non-Hodgkin lymphomas [16, 17]. This study aims to demonstrate the efficacy of combining the PROTAC-based EZH2 degrader with a METTL3 inhibitor (iMETTL3) in a Burkitt’s lymphoma cell lines.
In this study, Burkitt’s lymphoma cell lines including, Daudi and Ramos cells were used. These cell lines were obtained from Korean Cell Line Bank. These cell lines were grown in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 2mM L-glutamine (Gibco), 1% antibiotics (10 U/mL penicillin and 10 g/mL streptomycin; Gibco). All cell lines were incubated at 37℃ and 5% CO2.
EZH2 degrader (MS1943) and METTL3 inhibitor (STM2457) were purchased from MedChemExpress. Both were dissolved in dimethyl sulfoxide (DMSO) as recommended by the manufacturer and stored at –80℃. All these drugs were diluted with the RPMI 1640 medium with 10% heat-inactivated FBS, 2 mM L-glutamine, and 1% antibiotics before being used for the treatment of cell lines.
Cell growth was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo) assay according to the manufacturer’s protocol. Daudi and Ramos cell lines were seeded at an initial cell density of 2×104 cells/100 μL culture medium in 96-well plates. These cells were treated with various doses of EZH2 degrader and METTL3 inhibitor drugs without any drugs, 2.5 μM, 5 μM, and 10 μM for each drug. The synergistic effects were assessed using 5 μM for both drugs. Cultures were maintained at 37℃ in a 5% CO2 atmosphere. CCK-8 solution was added in increments of 10 μL to each well after 24 hours, 48 hours, and 72 hours. The plates were incubated for 1∼4 hours in a CO2 incubator and the optical density was measured at 450 nm using a microplate reader.
One million Ramos or Daudi cells were lysed in 2× laemmli sample buffer (Bio-Rad) with β-mercaptoethanol and boiled at 95℃ for 10 minutes. After removal of the insoluble fraction by centrifugation at 10,000×g for 10 min, protein samples were separated by SDS gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were stained with cleaved poly (ADP-ribose) polymerase (PARP), PARP, cleaved caspase-3, tumor protein P53 (TP53) and p53 upregulated modulator of apoptosis (PUMA) antibodies (Cell Signaling Technology) at a dilution of 1:1,000 or GAPDH antibody (Cell Signaling Technology) at a dilution of 1:5,000 at 4℃ overnight. After overnight incubation at 4℃, HRP-conjugated secondary antibodies were added. After washing with Tris-buffered saline and Tween 20, the hybridized bands were detected using an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotech).
The 5×105 cells were treated with the drugs described. After incubating for 72 hours in CO2 incubator, the cells were harvested and centrifuged 2,000 rpm 5 minutes with DPBS (Gibco). These cells were performed twice for wash and discarded the supernatant. For cell cycle analysis, the Annexin V-propidium iodide (PI) Apoptosis Kit (BioVision) was used following the manufacturer’s protocol. The cell cycle was detected using the Novocyte Advanteon (Agilent).
All data were statistically evaluated by Student’s t-test using GraphPad Prism 8 (GraphPad Software Inc.). All experiments were conducted at least thrice. The values are depicted using the mean and SEM. Differences between the means of the groups were considered to be statistically significant if P<0.05, P<0.01, and P<0.001.
We examined the effects of dEZH2 and iMETTL3 on the proliferation of Daudi and Ramos cells using the CCK-8 assay. Both cell lines were treated with various concentrations of the EZH2 degrader and METTL3 inhibitor (ranging from 2.5 to 10 μM) at different time points: 24 hours (Figure 1A), 48 hours (Figure 1B), and 72 hours (Figure 1C). And we showed dEZH2, iMETTL3 and comb cell viability (%) in same concentration by time period in Daudi (Figure 1D) and Ramos (Figure 1E). Our findings clearly demonstrated that these treatments significantly inhibited the proliferation of Burkitt’s lymphoma cells.
To evaluate the inhibitory effects of dEZH2 and iMETTL3 on the proliferation of Burkitt’s lymphoma cells in vitro, we performed cell proliferation assays on cultured Daudi and Ramos cells, with or without these drugs. Our results demonstrated that the combination of the dEZH2 and iMETTL3 significantly reduced cell growth compared to individual treatments at the 24 hours (Figure 2A), 48 hours (Figure 2B), and 72 hours (Figure 2C). We also performed the toxicity assays in HEK293T cells showed no significant difference with dEZH2 and iMETTL3 (data not shown). These findings suggest that the combined therapy effectively suppresses the survival and proliferation of Burkitt’s lymphoma cells, indicating a synergistic effect.
To verify that dEZH2 degrader and iMETTL3 induce cell death, we conducted annexin V/PI staining analysis. The data indicated a pronounced combined effect of the drugs in the Q2 (late apoptosis) and Q3 (early apoptosis) quadrants. The combination treatment promoted both early and late apoptosis in Daudi (Figure 3A, 3B) and Ramos cells (Figure 3C, 3D).
To investigate the relationship between cell proliferation inhibition and cell cycle regulation, we performed flow cytometric analysis to assess cell distribution across different cell cycle phases. Treatment with a combination of dEZH2 and iMETTL3 resulted in a significant increase in Burkitt’s lymphoma cells in the G2/M phase after 72 hours. Concurrently, there was a decrease in the number of cells in the G0/G1 phase in both Daudi (Figure 4A) and Ramos cells (Figure 4B).
After confirming apoptosis induction with the dual inhibition of EZH2 and METTL3, we examined the involvement of specific apoptosis-related pathways using western blot analysis. PARP, a key protein in DNA repair, becomes cleaved upon activation by Caspase-3, leading to DNA damage and apoptosis [18, 19]. Compared to the control group, Daudi cells showed significantly increased levels of cleaved PARP and cleaved caspase-3 (Figure 5A). We further investigated the role of the pro-apoptotic gene TP53, which induces cell cycle arrest and activates PUMA, promoting cell death in cancer cells [20, 21]. The combination therapy increasd the expression of TP53 and PUMA in Daudi cells (Figure 5B). In summary, our study demonstrates that the combination therapy enhances p53-dependent apoptosis, as indicated by the upregulation of cleaved PARP, cleaved caspase-3, TP53, and PUMA.
PROTAC-based drugs offer several distinct advantages over traditional small molecule inhibitors. First, PROTAC-based drugs induce TPD, leading to the complete removal of disease-causing proteins, which ensures a sustained therapeutic effect since the target protein needs to be resynthesized to regain functionality [5, 22]. Second, this approach can overcome drug resistance, as PROTAC-based drugs can degrade mutated proteins that are not effectively inhibited by conventional small molecules [23]. Third, PROTAC-based drugs can target a broader range of protein conformations and mutations, enhancing their therapeutic versatility. Additionally, PROTACs often require lower dosages than traditional inhibitors, potentially reducing side effects [24]. These advantages collectively underscore the transformative potential of PROTACs in developing more effective and durable cancer treatments [25, 26].
Our previous research demonstrated the superior efficacy of the PROTAC-based EZH2 degrader, MS1943, compared to the clinical EZH2 inhibitor, Tazemetostat in B cell lymphoma cells [27, 28]. In this study, we extended these findings by exploring the synergistic effects of combining EZH2 degrader with the METTL3 inhibitor on Burkitt’s lymphoma cells. The combination therapy was strikingly reduced in Ramos and Daudi cell proliferation and significantly increased levels of apoptosis related protein, highlighting the therapeutic potential of PROTAC-based strategies.
The results of our study provide compelling evidence that the combination of dEZH2 and iMETTL3 can effectively suppress the growth of Burkitt’s lymphoma cells and induce apoptosis through a p53-dependent pathway. This results are significant given the challenges associated with targeting EZH2 in clinical settings. And, we showed that treatment with a combination of dEZH2 and iMETTL3 resulted in a significant increase in the G2/M phase. We also observed an increase in the S phase of the cell cycle following combination treatment. These results suggest that inhibition of METTL3 increases PLK1 expression, leading to accumulation in the S phase and potentially causing arrest in the G2/M phase.
The clinical implications of these findings are important in Burkitt’s lymphoma therapy. The enhanced efficacy of the combination of dEZH2 and iMETTL3 offers a promising new approach for Burkitt’s lymphoma therapy. The ability of this combination to target and degrade disease-associated proteins more effectively than conventional inhibitors underscores the potential of TPD technologies in oncology.
Moreover, the successful use of this combination therapy need to perform the preclinical models for potential clinical trials. The broader application of PROTAC-based strategies need in various cancer types such as breast cancer, lung cancer, and prostate where EZH2 is overexpressed, particularly those that have been challenging to treat with traditional small molecule inhibitor [29, 30].
In conclusion, our study demonstrates that the combination dEZH2 degrader and iMETTL3 significantly suppresses Burkitt’s lymphoma cell growth and induces p53-dependent apoptosis pathway. These findings show a strong rationale for the continued development and clinical evaluation of PROTAC-based therapies, providing new hope for improved treatment outcomes in Burkitt’s lymphoma patients and potentially other hematologic malignancy.
PROteolysis TArgeting Chimeras (PROTACs)은 기존의 저분자(small moleule)로는 타겟팅이 어려웠던 단백질을 제거하기 위한 새로운 치료 전략을 제공한다. EZH2는 림프종 치료의 중요한 타겟인 메틸트랜스퍼라제(methyltransferase)이지만, 임상에서 널리 사용되지 않는다. 이전 연구에서 우리는 임상에서 사용되는 EZH2 억제제와 PROTAC 기반의 EZH2 분해제를 비교했으며, PROTAC 기반 분해제가 훨씬 더 효과적임을 확인했다. 이를 바탕으로, 우리는 PROTAC 기반의 EZH2 분해제와 기존 림프종 치료제인 METTL3 억제제를 병용하여 버킷림프종 세포의 증식과 세포 사멸에 미치는 영향을 조사했다. CCK-8 분석을 통해 두 약물이 단독 및 병용 처리 시 농도 의존적으로 Daudi 및 Ramos 세포 성장을 유의미하게 억제하는 것을 확인했다. 병용 치료는 세포 증식을 현저히 억제하고, annexin V/PI 염색을 통해 세포 사멸을 유도하는 것으로 확인되었다. 유세포 분석에서는 G0/G1 단계의 감소와 함께 G2/M 단계에서의 세포 주기 정지를 보였다. 웨스턴 블럿(western blot) 분석에서는 cleaved PARP, cleaved caspase-3, TP53 및 PUMA의 증가된 수준을 나타내어 p53 의존적인 세포 사멸이 강화됨을 증명했다. 우리는 이 연구 결과가 이 병용 요법이 버킷림프종 치료에 유망한 접근법임을 제안한다.
None
This work was supported by grant from Inje University, 2023 (No. 20230017).
None
Yu M, Graduate student; Kim RE, Graduate student; Jeong Y, Master graduate student; Jang H, Graduate student; Kim SB, Graduate student; Lim JY, Professor.
-Conceptualization: Yu M, Kim RE, Jeong Y.
-Data curation: Yu M, Kim RE, Jeong Y.
-Formal analysis: Yu M, Kim RE.
-Methodology: Jeong Y, Kim SB.
-Validation: Lim JY.
-Writing - original draft: Lim JY, Yu M, Kim RE.
-Writing - review & editing: Yu M, Kim RE, Jang H, Jeong Y, Lim JY.
This article does not require IRB/IACUC approval because there are no human and animal participants.