Venetoclax resistance in preclinical KMT2A-rearranged acute lymphoblastic leukemia models is characterized by high inter- and intra-model heterogeneity.

The BCL-2 inhibitor venetoclax (VEN) has emerged as an important therapeutic backbone for hematological malignancies, but secondary resistance is a major challenge. In acute lymphoblastic leukemia (ALL), early clinical trials promise high efficacy as well. However, relapse is observed frequently, and so far, only a few resistance-inducing mechanisms have been discussed. We employed KMT2A-rearranged ALL cell lines and xenograft models to elucidate mechanisms of VEN resistance. Targeted DNA and mRNA sequencing, single-cell mRNAseq, as well as protein expression analyses were conducted. All models initially responded well but finally displayed secondary resistance. Novel as well as previously known pathogenic variants in tumor suppressor TP53 as well as pro-apoptotic molecule BAX, but not BCL2, were observed. Gene and protein expression studies demonstrated multifarious changes in resistant cells, with high inter- and intra-model heterogeneity. Finally, single-cell RNA sequencing revealed a likely contribution of the tumor microenvironment in the development of VEN resistance, as indicated by modulation of genes involved in cell-cell interaction and humoral signaling in resistance-specific clusters. Our data demonstrate and characterize the rise of VEN resistance in KMT2A-rearranged ALL models, suggesting that relapse must be expected in the clinical setting and that multifactorial processes are involved in this process.