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Targeted nanopore sequencing for the identification of ABCB1 promoter translocations in cancer
Mark S Williams1, Naseer J Basma2, Fabio M R Amaral2
1Leukaemia Biology Laboratory, Cancer Research UK Manchester Institute, Oglesby Cancer Research Building, The University of Manchester, Manchester, M20 4GJ, UK. mark.williams-4@manchester.ac.uk.
Background:
Resistance to chemotherapy is the most common cause of treatment failure in acute myeloid leukemia (AML) and the drug efflux pump ABCB1 is a critical mediator. Recent studies have identified promoter translocations as common drivers of high ABCB1 expression in recurrent, chemotherapy-treated high-grade serous ovarian cancer (HGSC) and breast cancer. These fusions place ABCB1 under the control of a strong promoter while leaving its open reading frame intact. The mechanisms controlling high ABCB1 expression in AML are largely unknown. We therefore established an experimental system and analysis pipeline to determine whether promoter translocations account for high ABCB1 expression in cases of relapsed human AML.
Methods:
The human AML cell line THP-1 was used to create a model of chemotherapy resistance in which ABCB1 expression was driven by a promoter fusion. The THP-1 model was used to establish a targeted nanopore long-read sequencing approach that was then applied to cases of ABCB1high HGSC and AML. H3K27Ac ChIP sequencing was used to assess the activity of native promoters in cases of ABCB1high AML.
Results:
Prolonged in vitro daunorubicin exposure induced activating ABCB1 promoter translocations in human THP-1 AML cells, similar to those recently described in recurrent high-grade serous ovarian and breast cancers. Targeted nanopore sequencing proved an efficient method for identifying ABCB1 structural variants in THP-1 AML cells and HGSC; the promoter translocations identified in HGSC were both previously described and novel. In contrast, activating ABCB1 promoter translocations were not identified in ABCB1high AML; instead H3K27Ac ChIP sequencing demonstrated active native promoters in all cases studied.
Conclusions:
Despite frequent high level expression of ABCB1 in relapsed primary AML we found no evidence of ABCB1 translocations and instead confirmed high-level activity of native ABCB1 promoters, consistent with endogenous regulation.
Insights
Chemotherapy resistance in acute myeloid leukemia (AML) is often linked to ABCB1. Unlike other cancers, AML does not show ABCB1 promoter translocations; instead, its native promoters are highly active.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Chemotherapy resistance is a major challenge in acute myeloid leukemia (AML) treatment.
- The ABCB1 drug efflux pump is a key factor in this resistance.
- Promoter translocations driving high ABCB1 expression are known in ovarian and breast cancers, but mechanisms in AML are unclear.
Purpose of the Study:
- To investigate if promoter translocations cause high ABCB1 expression in relapsed AML.
- To establish and utilize an experimental system and analysis pipeline for this investigation.
Main Methods:
- Developed a chemotherapy-resistant THP-1 AML cell line model with ABCB1 promoter fusion.
- Employed targeted nanopore long-read sequencing for ABCB1 structural variant identification.
- Utilized H3K27Ac ChIP sequencing to assess native promoter activity in AML cases.
Main Results:
- Induced ABCB1 promoter translocations in THP-1 AML cells via daunorubicin exposure.
- Confirmed targeted nanopore sequencing's efficiency in detecting ABCB1 variants in AML and HGSC.
- Found no ABCB1 promoter translocations in high ABCB1 AML; instead, native promoters were highly active.
Conclusions:
- Relapsed AML frequently exhibits high ABCB1 expression.
- No evidence of ABCB1 translocations was found in relapsed AML.
- High-level activity of native ABCB1 promoters, indicating endogenous regulation, was confirmed in AML.
