Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX, 77054, USA.
Targeted Therapeutic Drug Discovery and Development Program, Division of Chemical Biology & Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX, 78712, USA.
Cell Death Dis. 2024 Jan 22;15(1):80. doi: 10.1038/s41419-024-06423-0.
p38 mitogen-activated protein kinases (MAPKs) participate in autophagic signaling; and previous reports suggest that pyridinyl imidazole p38 MAPK inhibitors, including SB203580 and SB202190, induce cell death in some cancer cell-types through unrestrained autophagy. Subsequent studies, however, have suggested that the associated cytoplasmic vacuolation resulted from off-target inhibition of an unidentified enzyme. Herein, we report that SB203580-induced vacuolation is rapid, reversible, and relies on the class III phosphatidylinositol 3-kinase (PIK3C3) complex and the production of phosphatidylinositol 3-phosphate [PI(3)P] but not on autophagy per se. Rather, vacuolation resulted from the accumulation of Rab7 on late endosome and lysosome (LEL) membranes, combined with an osmotic imbalance that triggered severe swelling in these organelles. Inhibition of PIKfyve, the lipid kinase that converts PI(3)P to PI(3,5)P2 on LEL membranes, produced a similar phenotype in cells; therefore, we performed in vitro kinase assays and discovered that both SB203580 and SB202190 directly inhibited recombinant PIKfyve. Cancer cells treated with either drug likewise displayed significant reductions in the endogenous levels of PI(3,5)P2. Despite these results, SB203580-induced vacuolation was not entirely due to off-target inhibition of PIKfyve, as a drug-resistant p38α mutant suppressed vacuolation; and combined genetic deletion of both p38α and p38β dramatically sensitized cells to established PIKfyve inhibitors, including YM201636 and apilimod. The rate of vacuole dissolution (i.e., LEL fission), following the removal of apilimod, was also significantly reduced in cells treated with BIRB-796, a structurally unrelated p38 MAPK inhibitor. Thus, our studies indicate that pyridinyl imidazole p38 MAPK inhibitors induce cytoplasmic vacuolation through the combined inhibition of both PIKfyve and p38 MAPKs, and more generally, that p38 MAPKs act epistatically to PIKfyve, most likely to promote LEL fission.
p38 丝裂原活化蛋白激酶（MAPK）参与自噬信号转导；先前的报告表明，吡啶基咪唑 p38 MAPK 抑制剂，包括 SB203580 和 SB202190，通过不受控制的自噬诱导一些癌细胞死亡。然而，随后的研究表明，相关的细胞质空泡化是由于未鉴定的酶的非靶标抑制。在此，我们报告 SB203580 诱导的空泡化是快速、可逆的，依赖于 III 类磷脂酰肌醇 3-激酶（PI3K）复合物和磷脂酰肌醇 3-磷酸 [PI（3）P] 的产生，但本身并不依赖于自噬。相反，空泡化是由于 Rab7 在晚期内体和溶酶体（LEL）膜上的积累，以及由此引发这些细胞器严重肿胀的渗透失衡所导致的。PIKfyve 的抑制，即 PIKfyve 是将 LEL 膜上的 PI（3）P 转化为 PI（3，5）P2 的脂质激酶，在细胞中产生了类似的表型；因此，我们进行了体外激酶测定，并发现 SB203580 和 SB202190 均可直接抑制重组 PIKfyve。用两种药物处理的癌细胞也显示内源性 PI（3，5）P2 水平显著降低。尽管有这些结果，但 SB203580 诱导的空泡化并不完全归因于 PIKfyve 的非靶标抑制，因为一种耐药的 p38α 突变体抑制了空泡化；而 p38α 和 p38β 的联合基因缺失则显著增加了对包括 YM201636 和 apilimod 在内的已建立的 PIKfyve 抑制剂的敏感性。apilimod 去除后，空泡溶解（即 LEL 分裂）的速度也显著降低在 BIRB-796 处理的细胞中，BIRB-796 是一种结构上不相关的 p38 MAPK 抑制剂。因此，我们的研究表明，吡啶基咪唑 p38 MAPK 抑制剂通过 PIKfyve 和 p38 MAPK 的联合抑制诱导细胞质空泡化，更普遍地说，p38 MAPK 与 PIKfyve 具有上位性作用，很可能促进 LEL 分裂。
