SummaryThe frontline therapy R‐CHOP for patients with diffuse large B‐cell lymphoma (DLBCL) has remained unchanged for two decades despite numerous Phase III clinical trials investigating new alternatives. Multiple large studies have uncovered genetic subtypes of DLBCL enabling a targeted approach. To further pave the way for precision oncology, we perform genome‐wide CRISPR screening to uncover the cellular response to one of the components of R‐CHOP, vincristine, in the DLBCL cell line SU‐DHL‐5. We discover important pathways and subnetworks using gene‐set enrichment analysis and protein–protein interaction networks and identify genes related to mitotic spindle organization that are essential during vincristine treatment. The inhibition of KIF18A, a mediator of chromosome alignment, using the small molecule inhibitor BTB‐1 causes complete cell death in a synergistic manner when administered together with vincristine. We also identify the genes KIF18B and USP28 of which CRISPR/Cas9‐directed knockout induces vincristine resistance across two DLBCL cell lines. Mechanistic studies show that lack of KIF18B or USP28 counteracts a vincristine‐induced p53 response suggesting that resistance to vincristine has origin in the mitotic surveillance pathway (USP28‐53BP1‐p53). Collectively, our CRISPR screening data uncover potential drug targets and mechanisms behind vincristine resistance, which may support the development of future drug regimens.

The frontline therapy R-CHOP for patients with diffuse large B-cell lymphoma (DLBCL) has remained unchanged for two decades despite numerous phase III clinical trials investigating new alternatives. Multiple large studies have uncovered genetic subtypes of DLBCL enabling a targeted approach. To further pave the way for precision oncology, we perform genome-wide CRISPR screening to uncover the cellular response to one of the components of R-CHOP, vincristine, in the DLBCL cell line SU-DHL-5. We discover important pathways and subnetworks using gene-set enrichment analysis and protein-protein interaction networks and identify genes related to mitotic spindle organization that are essential during vincristine treatment. Inhibition of KIF18A, a mediator of chromosome alignment, using the small molecule inhibitor BTB-1 causes complete cell death in a synergistic manner when administered together with vincristine. We also identify the genes KIF18B and USP28 for which CRISPR/Cas9-directed knockout induces vincristine resistance across two DLBCL cell lines. Mechanistic studies show that lack of KIF18B or USP28 counteracts a vincristine-induced p53 response involving the mitotic surveillance pathway (USP28-53BP1-p53). Collectively, our CRISPR screening data uncover potential drug targets and mechanisms behind vincristine resistance, which may support the development of future drug regimens.