Genetic manipulation of primary human natural killer cells to investigate the functional and oncogenic roles of PRDM1

Extra-nodal natural killer (NK)/T-cell lymphoma, nasal type (ENKTCL) is a highly aggressive lymphoma, in which the tumor suppressor gene PRDM1 is frequently lost or inactivated. We employed two different CRISPR/Cas9 approaches to generate PRDM1^-/primary NK cells to study the role of this gene in NK-cell homeostasis. PRDM1^-/- NK cells showed a marked increase in cloning efficiency, higher proliferation rate and less apoptosis compared with their wild-type counterparts. Gene expression profiling demonstrated a marked enrichment in pathways associated with proliferation, cell cycle, MYC, MYB and TCR/NK signaling in PRDM1^-/- NK cells, but pathways associated with normal cellular functions including cytotoxic functions were downregulated, suggesting that the loss of PRDM1 shifted NK cells toward proliferation and survival rather than the performance of their normal functions. We were also able to further modify a PRDM1-deleted clone to introduce heterozygous deletions of common tumor suppressor genes in ENKTCL such as TP53, DDX3X, and PTPN6. We established an in vitro model to elucidate the major pathways through which PRDM1 mediates its homeostatic control of NK cells. This approach can be applied to the study of other relevant genetic lesions and oncogenic collaborations in lymphoma pathogenesis.