Chemotherapy resistance in CRISPR/Cas9-mediated WEE1 knockout cells
Author
Issa, Issa Ismail
Term
4. term
Publication year
2019
Submitted on
2019-05-31
Pages
72
Abstract
Diffuse large B‑cell lymphoma (DLBCL) is an aggressive and highly heterogeneous cancer, and despite standard immunochemotherapy (R‑CHOP), about 40% of patients die from relapsed disease. The mechanisms that govern treatment response remain incompletely understood. WEE1 is a kinase that controls the G2/M checkpoint, and its inhibition can enhance the effects of antimitotic drugs such as vincristine. This study examined whether WEE1 contributes to chemotherapy resistance and whether its loss increases vincristine sensitivity. Four WEE1‑targeting sgRNAs (plus a non‑targeting control) were designed, cloned into CRISPR/Cas9 plasmids, validated by PCR and Sanger sequencing, and transfected into HEK293T cells to induce WEE1 knockout. Editing efficiency was assessed using the ICE tool, and bulk‑edited cells were characterized by proliferation assays, flow cytometric cell‑cycle analysis, and droplet digital PCR quantification of WEE1 mRNA. Vincristine dose–response screens were also performed. The sgRNA constructs produced effective insertions/deletions in WEE1. Edited cells showed reduced proliferation without a detectable change in cell‑cycle distribution, while ddPCR indicated increased WEE1 mRNA levels. Despite heterogeneous populations, significant growth inhibition after vincristine was observed in some WEE1‑knockout populations, correlating with editing/knockout efficiency. Overall, the findings suggest that WEE1 knockout can sensitize cells to vincristine and highlight WEE1 as a promising candidate target to overcome chemoresistance in DLBCL.
Diffuse large B‑cell lymphoma (DLBCL) er en aggressiv og meget heterogen kræftform, hvor trods standard immunkemoterapi (R‑CHOP) omkring 40 % af patienterne dør af recidiv. Mekanismerne bag behandlingsrespons er ikke fuldt klarlagt. WEE1 er en kinase, der regulerer G2/M‑overgangen i cellecyklus, og hæmning af WEE1 kan forstærke effekten af antimitotiske lægemidler som vincristin. Dette studie undersøgte, om WEE1 bidrager til kemoresistens, og om tab af WEE1 øger følsomheden over for vincristin. Fire sgRNA’er rettet mod WEE1 (samt en ikke‑målrettet kontrol) blev designet, klonet i CRISPR/Cas9‑plasmider, valideret med PCR og Sanger‑sekventering og transficeret i HEK293T‑celler for at inducere knockout. Redigeringseffektivitet blev vurderet med ICE, og de bulk‑redigerede celler blev karakteriseret ved proliferationsmåling, flowcytometrisk cellecyklusanalyse og kvantificering af WEE1‑mRNA med droplet digital PCR. Derudover blev vincristin‑dose‑respons‑skærme udført. Transfektion med sgRNA‑konstrukter gav effektive insertioner/deletioner i WEE1. De redigerede celler viste nedsat proliferation, men ingen tydelig ændring i cellecyklusfordeling; samtidig indikerede ddPCR en øget WEE1‑mRNA‑mængde. På trods af heterogene cellepopulationer blev der observeret signifikant væksthæmning efter vincristin i nogle WEE1‑knockout‑populationer, som korrelerede med redigerings/knockout‑effektiviteten. Samlet peger resultaterne på, at knockout af WEE1 kan sensibilisere celler over for vincristin og dermed kan være et lovende spor til at overvinde kemoresistens i DLBCL.
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