Targeted nanopore sequencing for the identification of ABCB1 promoter translocations in relapsed acute myeloid leukemia [ChIP-seq]

Resistance to chemotherapy is the most common cause of treatment failure in acute myeloid leukemia and the drug efflux pump ABCB1 is a critical mediator. Here we demonstrate that in vitro daunorubicin exposure can induce activating ABCB1 promoter translocations in human myeloid cells, similar to those recently described in recurrent high-grade serous ovarian and breast cancer. We then develop a targeted nanopore sequencing approach that enables efficient identification of ABCB1 structural variants in high-grade serous ovarian cancer. Finally, we confirm that ABCB1high cases of relapsed AML are not characterized by ABCB1 promoter translocations but instead show high-level activity of native promoters, consistent with endogenous regulation. Overall design: THP-1 cells were from DSMZ (Braunschweig, Germany) and cultured in RPMI 1640 medium (Sigma Aldrich) supplemented with 2mM L-Glutamine (Life Technologies, Carlsbad, CA) and 10% fetal bovine serum (Sigma Aldrich). Whilst under drug selection cells were counted and replated every third day. Cell lines were confirmed mycoplasma-free and authenticated by short tandem repeat DNA profiling. THP-1 were exposed to escalating doses of daunorubicin over 142 days, generating a resistant line (THP-1_R) which exhibited a 28.3-fold increase in daunorubicin IC50 compared with the sensitive parental cell line (THP-1_S). Primary human AML cells were obtained from the Manchester Cancer Research Centre Tissue Biobank (approved by the South Manchester Research Ethics Committee). The Biobank archive was searched for samples from patients with relapsed AML with high blast counts and sufficient viable cells from ChIP sequencing. H3K27 acetylation ChIP sequencing was performed using two replicates for both the sensitive (THP-1_S1 and THP-1_S2) and resistant (THP-1_R1 and THP-1_R2) lines and a single aliquot from each patient sample.