One form of modification in cancer lines is alteration in the epigenetic status of the cells. Cancer lineages are heterogeneous with many diverse sublineages arising over time ( Heppner, 1984 Vogelstein et al., 2013), and these are subjected to intensive selection pressure as the tumor evolves ( Fortunato et al., 2017). A high level of genomic instability in cancer cells allows for novel forms of effector molecules to be produced, but also imposes a genetic load of potentially detrimental mutations. The development of a tumorigenic lineage from healthy cells is usually associated with a wide range of genetic changes, including point mutations, small and large deletions and insertions (indels) and chromosomal rearrangements ( Vogelstein et al., 2013). 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. Similar selection pressures occur during the evolution of a cancer in vivo, and our results can be seen as a case study in leukemic cell adaptation. 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. 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. Other network clusters showed effects prior to or after differentiation in the homozygous clones. However, three homozygotes failed to fully induce USP3 during differentiation. In contrast, a number of genes were up-regulated in the homozygous cells compared to wild type at baseline, including other deubiquitinases ( USP12, BAP1, and MYSM1). There was also apparent loss of interferon signaling. Prior to PMA treatment, the homozygous clones had lower levels than wild type of genes relating to metabolism and mitochondria, including SRPRB, encoding an interaction partner of USP16. 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. Three clones showed sustained expression of the progenitor cell marker MYB, indicating that differentiation had not completely blocked proliferation in these clones. One line was highly proliferative prior to PMA treatment and shut down proliferation upon differentiation, like wild type. All were able to proliferate and to differentiate in response to phorbol ester (PMA) treatment.
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We mutated the USP16 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. USP16 is a histone deubiquitinase which facilitates G2/M transition during the cell cycle, regulates DNA damage repair and contributes to inducible gene expression.
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Hume 2‡, Andreas Lengeling 1†‡ and Kim M. Iveta Gažová 1†‡, Lucas Lefevre 1†‡, Stephen J.