Gažová Iveta, Lefevre Lucas, Bush Stephen J, Rojo Rocio, Hume David A, Lengeling Andreas, Summers Kim M
The Roslin Institute, University of Edinburgh, Easter Bush, United Kingdom.
Mater Research Institute - University of Queensland, Translational Research Institute, Woolloongabba, QLD, Australia.
Front Cell Dev Biol. 2021 May 31;9:679544. doi: 10.3389/fcell.2021.679544. eCollection 2021.
USP16 is a histone deubiquitinase which facilitates G2/M transition during the cell cycle, regulates DNA damage repair and contributes to inducible gene expression. We mutated the gene in a high differentiation clone of the acute monocytic leukemia cell line THP-1 using the CRISPR-Cas9 system and generated four homozygous knockout clones. All were able to proliferate and to differentiate in response to phorbol ester (PMA) treatment. One line was highly proliferative prior to PMA treatment and shut down proliferation upon differentiation, like wild type. Three clones showed sustained expression of the progenitor cell marker , indicating that differentiation had not completely blocked proliferation in these clones. Network analysis of transcriptomic differences among wild type, heterozygotes and homozygotes showed clusters of genes that were up- or down-regulated after differentiation in all cell lines. Prior to PMA treatment, the homozygous clones had lower levels than wild type of genes relating to metabolism and mitochondria, including , encoding an interaction partner of USP16. There was also apparent loss of interferon signaling. In contrast, a number of genes were up-regulated in the homozygous cells compared to wild type at baseline, including other deubiquitinases (, and ). However, three homozygotes failed to fully induce during differentiation. Other network clusters showed effects prior to or after differentiation in the homozygous clones. Thus the removal of USP16 affected the transcriptome of the cells, although all these lines were able to survive, which suggests that the functions attributed to USP16 may be redundant. Our analysis indicates that the leukemic line can adapt to the extreme selection pressure applied by the loss of USP16, and the harsh conditions of the gene editing and selection protocol, through different compensatory pathways. Similar selection pressures occur during the evolution of a cancer , and our results can be seen as a case study in leukemic cell adaptation. USP16 has been considered a target for cancer chemotherapy, but our results suggest that treatment would select for escape mutants that are resistant to USP16 inhibitors.
USP16是一种组蛋白去泛素化酶，它在细胞周期中促进G2/M期转换，调节DNA损伤修复，并有助于诱导基因表达。我们使用CRISPR-Cas9系统在急性单核细胞白血病细胞系THP-1的一个高分化克隆中对该基因进行突变，生成了四个纯合敲除克隆。所有克隆都能够增殖，并在佛波酯（PMA）处理后发生分化。其中一个细胞系在PMA处理前具有高增殖能力，并且在分化时停止增殖，与野生型相似。三个克隆显示祖细胞标志物持续表达，表明这些克隆中的分化并未完全阻断增殖。对野生型、杂合子和纯合子之间的转录组差异进行网络分析，结果显示在所有细胞系分化后上调或下调的基因簇。在PMA处理前，纯合克隆中与代谢和线粒体相关的基因水平低于野生型，包括编码USP16相互作用伴侣的基因。同时还明显存在干扰素信号传导缺失。相比之下，与野生型相比，一些基因在纯合细胞的基线水平上上调，包括其他去泛素化酶（、和）。然而，三个纯合子在分化过程中未能完全诱导。其他网络簇在纯合克隆的分化之前或之后显示出影响。因此，尽管所有这些细胞系都能够存活，但USP16的缺失影响了细胞的转录组，这表明归因于USP16的功能可能是冗余的。我们的分析表明，白血病细胞系可以通过不同的补偿途径适应USP16缺失所施加的极端选择压力以及基因编辑和选择方案的苛刻条件。在癌症进化过程中会出现类似的选择压力，我们的结果可被视为白血病细胞适应性的一个案例研究。USP16一直被认为是癌症化疗的靶点，但我们的结果表明，治疗会选择出对USP16抑制剂耐药的逃逸突变体。
