ABSTRACTBackgroundVenetoclax, a B‐cell lymphoma 2 (BCL‐2) inhibitor, is a promising treatment for blastic plasmacytoid dendritic cell neoplasm (BPDCN) and is currently under evaluation in clinical trials. However, several case reports have reported relapses after prolonged treatment, and development of resistance. To date, no study has investigated resistance to venetoclax in BPDCN. Therefore, we sought to investigate the mechanisms that trigger resistance during treatment.MethodsWe developed an in vitro model of chronic exposure of a BPDCN cell line to venetoclax that leads to resistance to the drug. We then characterized the resistant cell line using genomic and proteomic analyses and evaluated drug combinations to overcome the resistance.ResultsWe found an upregulation of BCL‐xL, both at genomic and protein levels in the resistant BPDCN cell line. By combining venetoclax with a hypomethylating agent, we were able to overcome venetoclax resistance and induce resistant cell‐line death. However, association with a proteasome inhibitor, known to be efficient against BPDCN, failed to restore sensitivity to venetoclax, probably due to modification of the NF‐κB pathway observed by bulk‐RNAseq analysis.ConclusionsCollectively, these data suggest that acquired venetoclax resistance in BPDCN is mediated by a modification of the balance of BCL‐2 family proteins. Also, the mechanisms involved in resistance may also lead to resistance to other molecules among the drugs that might otherwise have been considered as alternative therapies.Clinical Trial Registration (Including Trial Number)The authors have confirmed clinical trial registration is not needed for this submission.