Crispr/cas9 screening reveals sos1 participate in asparaginase resistance in nk/t cell lymphoma

Introduction: Extranodal nature killer/T cell lymphoma (NKTCL) accounts for about 6% of all kinds of lymphoma. Asparaginase based chemotherapy regime greatly improved the outcome of NKTCL. However, asparaginase resistance still exists and patients who failed first line asparaginase treatment appeared a poor prognosis. At present, the mechanism of asparaginase resistance in NKTCL was few elucidated. CRISPR/Cas9 genome-wide screening has been widely used to explore disease mechanisms and novel drug targets. In this study, we explored asparaginase-resistant mechanism in NKTCL by CRISPR/Cas9 whole-genome screening, and sequencing after inducing resistant cells from sensitive cells, with a view to improve the clinical efficacy of asparaginase. Methods: Exploring the sensitivity to asparaginase of NKTCL cell lines in our center, and selecting relative drug-resistant cell line for subsequent screening process. CRISPR/Cas9 library was transfected and whole genome knockout was performed. The transfected cells were selected by puromycin, and the transfection efficiency was controlled at about 30%. After treated with PEG-asparaginase or PBS as control group, DNA was extracted and sent for sequencing to explore drug-resistant genes. Long-term drug treatment was performed in sensitive cell lines to induce drug-resistant cell lines, and then RNA-Sequencing was conducted in parental and induced cells. Drug resistant targets were selected and in vitro validation was performed. Results: CRISPR/Cas9 whole genome knockout library (TKvO3) was first transfected into KHYG-1 cells to screen the asparaginase resistant genes. The transfected cells were treated with asparaginase or vehicle for 5 days, the DNA of transfected cells was extracted for amplification and sequencing. Compared with the control group, SOS1 was one of the genes most significantly depleted in asparaginase-treated cells. Next the SOS1 targeting sgRNA were transfected into KHYG-1 cell line and improved the sensitivity of KHYG-1 towards asparaginase treatment. And then the SOS1 over-expression plasmid induced KHYG-1 cells more resistant to asparaginase treatment. Besides CRISPR/Cas9 whole genome screening, we also developed the asparaginase resistant cell lines by a long time of low-dose asparaginase treatment in NKYS and YT cells. The gene expression profile analysis indicated that SOS1 associated KRAS signal pathway was also significantly enriched in these related resistant cell lines. Keywords: Extranodal non-Hodgkin lymphoma, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.