Loss of nuclear SMARCB1 (INI1/hSNF5/BAF47) protein expression due to biallelic mutations of the SMARCB1 tumor suppressor gene is a hallmark of atypical teratoid/rhabdoid tumors (ATRT), but the presence of cytoplasmic SMARCB1 protein in these tumors has not yet been described. In a series of 102 primary ATRT, distinct cytoplasmic SMARCB1 staining on immunohistochemistry was encountered in 19 cases (19%) and was highly over-represented in cases showing pathogenic sequence variants leading to truncation or mutation of the C-terminal part of SMARCB1 (15/19 vs. 4/83; Chi-square: 56.04, p = 1.0E−10) and, related to this, in tumors of the molecular subgroup ATRT-TYR (16/36 vs. 3/66; Chi-square: 24.47, p = 7.6E−7). Previous reports have indicated that while SMARCB1 lacks a bona fide nuclear localization signal, it harbors a masked nuclear export signal (NES) and that truncation of the C-terminal region results in unmasking of this NES leading to cytoplasmic localization. To determine if cytoplasmic localization found in ATRT is due to unmasking of NES, we generated GFP fusions of one of the SMARCB1 truncating mutations (p.Q318X) found in the tumors along with a p.L266A mutation, which was shown to disrupt the interaction of SMARCB1-NES with exportin-1. We found that while the GFP-SMARCB1(Q318X) mutant localized to the cytoplasm, the double mutant GFP-SMARCB1(Q318X;L266A) localized to the nucleus, confirming NES requirement for cytoplasmic localization. Furthermore, cytoplasmic SMARCB1(Q318X) was unable to cause senescence as determined by morphological observations and by senescence-associated β-galactosidase assay, while nuclear SMARCB1(Q318X;L266A) mutant regained this function. Selinexor, a selective exportin-1 inhibitor, was effective in inhibiting the nuclear export of SMARCB1(Q318X) and caused rapid cell death in rhabdoid tumor cells. In conclusion, inhibition of nuclear export restores nuclear localization and residual tumor suppressor function of truncated SMARCB1. Therapies aimed at preventing nuclear export of mutant SMARCB1 protein may represent a promising targeted therapy in ATRT harboring truncating C-terminal SMARCB1 mutations.

由于SMARCB1抑癌基因的双等位基因突变导致核 SMARCB1 (INI1/hSNF5/BAF47) 蛋白表达缺失是非典型畸胎样/横纹肌样肿瘤 (ATRT) 的标志，但这些肿瘤中细胞质 SMARCB1 蛋白的存在尚未被发现描述。在一系列 102 例原发性 ATRT 中，19 例（19%）在免疫组织化学上发现了不同的细胞质 SMARCB1 染色，并且在显示导致 SMARCB1 C 末端部分截断或突变的致病序列变异的病例中高度过度表达（15 /19 对 4/83；卡方：56.04，p  = 1.0E-10），并且与此相关，在分子亚组 ATRT-TYR 的肿瘤中（16/36 对 3/66；卡方： 24.47, p = 7.6E-7)。先前的报道表明，虽然 SMARCB1 缺乏真正的核定位信号，但它具有掩蔽的核输出信号 (NES)，并且 C 末端区域的截断导致该 NES 的暴露导致细胞质定位。为了确定在 ATRT 中发现的细胞质定位是否是由于 NES 的暴露，我们生成了在肿瘤中发现的 SMARCB1 截短突变之一（p.Q318X）的 GFP 融合物以及 p.L266A 突变，这被证明会破坏相互作用带有 exportin-1 的 SMARCB1-NES。我们发现，虽然 GFP-SMARCB1(Q318X) 突变体定位于细胞质，但双突变体 GFP-SMARCB1(Q318X;L266A) 定位于细胞核，证实了 NES 对细胞质定位的要求。此外，细胞质 SMARCB1(Q318X) 通过形态学观察和衰老相关 β-半乳糖苷酶试验确定不能引起衰老，而核 SMARCB1(Q318X;L266A) 突变体恢复了这一功能。Selinexor 是一种选择性的 exportin-1 抑制剂，可有效抑制 SMARCB1(Q318X) 的核输出，并导致横纹肌样肿瘤细胞快速死亡。总之，核输出的抑制恢复了截短的 SMARCB1 的核定位和残留的肿瘤抑制功能. 旨在防止突变 SMARCB1 蛋白核输出的疗法可能代表一种有前途的靶向疗法，用于治疗含有截断 C 末端SMARCB1突变的 ATRT。