Generation of Natural Killer Cell Lymphoma Models In Vitro by Gene Editing
Authors: Dong Gehong , Li Yuping , Jiang Bei , Yee Jiing-Kuan , Lee Dean A. , McKeithan Timothy W. , Chan Wing Chung 
Published: October 2019

Abstract
Introduction: Natural killer cell lymphoma (NKCL) constitutes a rare and aggressive form of non-Hodgkin lymphoma. We and others have identified PRDM1 as an important tumor suppressor gene (TSG) that is inactivated in 60%-70% of NKCL. Other frequent genetic and epigenetic abnormalities have been reported recently. However, the functional consequences of these alterations and how they cooperate in generating a lymphoma have not been defined. Recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas9) system has been developed to provide highly efficient targeted gene editing. Here we report our experience using the CRISPR/Cas9 technology for targeted gene modifications of normal NK-cells in vitroto generate human model systems to study NKCL initiation and progression.
Materials and Methods: Primary NK cells were isolated from peripheral blood lymphocytes of healthy donors and cocultured with an engineered feeder cell line, K562-Cl9-mb21, in RPMI 1640 containing 10% FBS and IL2. The purity of NK cells was 96%, as determined by CD56 and CD3 staining by flow cytometry. Four pairs of small guide RNA (sgRNA) targeting regions within exon 4 of PRDM1 were designed using an online CRISPR design tool (http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design) and cloned into the pSpCas9 (BB)-2A-GFP vector. The cleavage efficiency of sgRNAs was assessed using HEK 293T cells. The best performing PRDM1 sgRNA construct was delivered into primary NK cells by electroporation. After 48 hours, NK cells expressing GFP were seeded by FACS as single cells into 96-well plates containing feeder cells. PCR and Sanger sequencing were used to genotype the resulting clones. A similar approach was used to derive mutants of TP53, DDX3X, and PTPN6. We used unmodified NK cells for comparative analysis.
Results: The PRDM1-modified NK cell population (GFP+ cells) could be cloned with a frequency of 52% (198/384) versus 11% (45/384) for normal primary NK cells. 61 of 92 clones were identified with PRDM1 frameshift deletions at the targeted site within exon 4, including two heterozygous deletions (numbering from Refseq NM_001198: △C [420]; △29bp [409-437]), one homozygous deletion (△29bp [409-437]), and one clone with a 79-bp deletion in one allele (comprising 35 bp from exon 4 [411-445] and 44 bp from the adjacent intron 3) and a 11-bp deletion in the other allele (413-424). This last clone, named PRDM1 C5 was used in subsequent studies, and it has been maintained for up to 54 million-fold expansion for 2 months. Using propidium iodide staining, we observed an increase in the percentage of cells in G2/M phase in PRDM1 C5 (22.75% vs 8.08% in normal NK cells). An MTS assay demonstrated that PRDM1 C5 grew more rapidly than normal NK cell controls with or without feeder cells. Next we introduced BCOR, TP53, DDX3X, and PTPN6 heterozygous deletions into PRDM1 C5 using the CRISPR/Cas9 technology. Three PRDM1-/-/TP53+/, three PRDM1-/-/DDX3X+/-, and two PRDM1-/-/PTPN6-/- clones were confirmed by Sanger sequencing. To accelerate this process, we have put up to three different sgRNA into a single plasmid to simultaneously disrupt multiple TSGs. We are in the process of assessing the biology of the double deletion clones and producing multiple triple deletion mutants to assess cooperativity of these TSGs.
Conclusion: CRISPR/Cas9 technology can be applied to genetically alter primary NK cells and has been used successfully to target PRDM1 and other TSGs in NKCL in different combinations. These deletion mutants had a higher cloning efficiency than normal primary NK cells and can be expanded for prolonged periods in vitro. This approach is promising in building human in vitro model systems to study NKCL development and to identify the functional consequences of alterations of TSGs individually and in combination with other TSGs and oncogenes. We will determine which modified cells will be able to establish a NK cell lymphoproliferative disorder in immunodeficient mice and identify pathway alterations in the various genetically modified cell models.

Disclosures
No relevant conflicts of interest to declare.