ABSTRACT

Introduction

B-cell acute lymphoblastic leukemia (B-ALL) is the most prevalent malignant tumor affecting children. While the majority of B-ALL patients (90%) experience successful recovery, early relapse cases of B-ALL continue to exhibit high mortality rates. MZ1, a novel inhibitor of Bromodomains and extra-terminal (BET) proteins, has demonstrated potent antitumor activity against hematological malignancies. The objective of this study was to examine the role and therapeutic potential of MZ1 in the treatment of B-ALL.

Methods

In order to ascertain the fundamental mechanism of MZ1, a sequence of in vitro assays was conducted on B-ALL cell lines, encompassing Cell Counting Kit 8 (CCK8) assay, Propidium iodide (PI) staining, and Annexin V/PI staining. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to examine protein and mRNA expression levels. Transcriptomic RNA sequencing (RNA-seq) was utilized to screen the target genes of MZ1, and lentiviral transfection was employed to establish stably-expressing/knockdown cell lines.

Results

MZ1 has been observed to induce the degradation of Bromodomain Containing 4 (BRD4), Bromodomain Containing 3 (BRD3), and Bromodomain Containing 2 (BRD2) in B-ALL cell strains, leading to inhibited cell growth and induction of cell apoptosis and cycle arrest in vitro. These findings suggest that MZ1 exhibits cytotoxic effects on two distinct molecular subtypes of B-ALL, namely 697 (TCF3/PBX1) and RS4;11 (MLL-AF4) B-ALL cell lines. Additionally, RNA-sequencing analysis revealed that MZ1 significantly downregulated the expression of Cyclin D3 (CCND3) gene in B-ALL cell lines, which in turn promoted cell apoptosis, blocked cell cycle, and caused cell proliferation inhibition.

Conclusion

Our results suggest that MZ1 has potential anti-B-ALL effects and might be a novel therapeutic target.

中文翻译：

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BRD4 PROTAC 降解剂 MZ1 通过靶向 CCND3 表现出抗 B 细胞急性淋巴细胞白血病作用

摘要

介绍

B 细胞急性淋巴细胞白血病 (B-ALL) 是影响儿童的最常见的恶性肿瘤。虽然大多数 B-ALL 患者 (90%) 成功康复，但 B-ALL 早期复发病例仍然表现出高死亡率。MZ1 是一种新型溴结构域和末端外 (BET) 蛋白抑制剂，已证明对血液恶性肿瘤具有有效的抗肿瘤活性。本研究的目的是探讨 MZ1 在治疗 B-ALL 中的作用和治疗潜力。

方法

为了确定 MZ1 的基本机制，对 B-ALL 细胞系进行了一系列体外测定，包括细胞计数试剂盒 8 (CCK8) 测定、碘化丙啶 (PI) 染色和膜联蛋白 V/PI 染色。采用蛋白质印迹和定量实时聚合酶链反应（qRT-PCR）来检查蛋白质和 mRNA 表达水平。利用转录组RNA测序（RNA-seq）筛选MZ1的靶基因，并利用慢病毒转染建立稳定表达/敲低的细胞系。

结果

据观察，MZ1 可诱导 B-ALL 细胞株中的 Bromodomain Containing 4 (BRD4)、Bromodomain Containing 3 (BRD3) 和 Bromodomain Containing 2 (BRD2) 降解，从而抑制细胞生长并诱导细胞凋亡和周期停滞体外。这些发现表明，MZ1 对 B-ALL 的两种不同分子亚型，即 697 (TCF3/PBX1) 和 RS4;11 (MLL-AF4) B-ALL 细胞系表现出细胞毒性作用。此外，RNA测序分析显示，MZ1显着下调B-ALL细胞系中Cyclin D3（CCND3）基因的表达，进而促进细胞凋亡、阻断细胞周期并导致细胞增殖抑制。

结论

我们的结果表明 MZ1 具有潜在的抗 B-ALL 作用，可能是一个新的治疗靶点。