Castration-resistant prostate cancer (CRPC) is an incurable form of prostate cancer (PCa), with DNMT1 and G9a being reported as overexpressed, rendering them highly attractive targets for precision medicine. CM-272 is a dual inhibitor of both methyltransferases’ activity. Herein, we assessed the response of different PCa cell lines to CM-272, in both 2D and 3D models, and explored the molecular mechanisms underlying CM-272 inhibitory effects.

CRPC tissues displayed significantly higher DNMT1, G9a and H3K9me2 expression than localized PCa. In vitro, CM-272 caused a significant decrease in PCa cell viability and proliferation alongside with increased apoptotic levels. We disclose that, under the evaluated dose, CM-272 led to G9a activity inhibition, while not significantly affecting DNMT1 activity. Upon G9a knockdown, DU145 and PC3 showed decreased cell viability. Remarkably, DU145 cells treated with CM-272 or with G9a knockdown displayed no differences in viability, suggesting a SET-dependent mechanism. Contrarily, PC3 cell viability impact was higher in G9a knockdown, compared with CM-272 treatment, suggesting an additional G9a function. Moreover, DU145 cells overexpressing catalytically functional G9a disclosed higher resistance to CM-272 treatment, reinforcing that the drug mechanism of action is dependent on G9a catalytic function.

Importantly, we successfully assembled spheroids from several prostate cell lines. Our results showed that CM-272 retained its anti-tumoral effects in 3D PCa models, leading to a clear reduction in cancer cell survival.

We concluded that inhibition of G9a methyltransferase activity by CM-272 has anti-tumor effect in PCa cells, holding therapeutic potential against CRPC.

去势抵抗性前列腺癌 (CRPC) 是一种无法治愈的前列腺癌 (PCa)，据报道 DNMT1 和 G9a 过度表达，这使得它们成为精准医学极具吸引力的目标。CM-272 是两种甲基转移酶活性的双重抑制剂。在此，我们在 2D 和 3D 模型中评估了不同 PCa 细胞系对 CM-272 的反应，并探索了 CM-272 抑制作用的分子机制。

CRPC 组织的 DNMT1、G9a 和 H3K9me2 表达明显高于局部 PCa。体外, CM-272 导致 PCa 细胞活力和增殖显着降低，同时凋亡水平增加。我们披露，在评估的剂量下，CM-272 导致 G9a 活性抑制，而不显着影响 DNMT1 活性。在 G9a 敲低后，DU145 和 PC3 显示细胞活力降低。值得注意的是，用 CM-272 或 G9a 敲低处理的 DU145 细胞在活力方面没有差异，表明存在 SET 依赖性机制。相反，与 CM-272 处理相比，G9a 敲低对 PC3 细胞活力的影响更高，这表明 G9a 具有额外的功能。此外，过表达具有催化功能的 G9a 的 DU145 细胞对 CM-272 治疗具有更高的抗性，这表明药物的作用机制依赖于 G9a 催化功能。

重要的是，我们成功地从几个前列腺细胞系中组装了球体。我们的研究结果表明，CM-272 在 3D PCa 模型中保留了其抗肿瘤作用，导致癌细胞存活率明显降低。

我们得出结论，CM-272 对 G9a 甲基转移酶活性的抑制在 PCa 细胞中具有抗肿瘤作用，具有抗 CRPC 的治疗潜力。