Mutation in Retinoic X Receptor-γ is a possible mechanism of ATRA resistance in acute promyelocytic leukemia: Identifying genetic changes related to drug resistance using whole exome sequencing

Abstract

Background: Acute promyelocytic leukemia (APL) has the best prognosis among acute myeloid leukemia. However, a subset of APL patients are not cured with all-trans retinoic acid (ATRA) combined with anthracycline-based cytotoxic chemotherapy. Several mechanisms such as increased ATRA metabolism have been suggested to explain acquired resistance to ATRA. However, genetic mechanisms of ATRA resistance have not been characterized at all. Hence, in this study, I tried to reveal genetic alterations attributable to ATRA resistance. Methods: Whole exome sequencing (WES) was performed using DNA of three APL patients who showed resistance to ATRA based treatment. These include two patients who failed to achieve complete remission (CR) after induction chemotherapy, and one patient who experienced relapse after CR despite consolidation treatment. DNA extracted from bone marrow at the time of diagnosis was used for analysis, while DNA extracted from saliva at the time of CR was used as germline control. Calling of single nucleotide variants (SNVs) was performed using internal pipeline called Adiscan (http://www.syntekabio.com). Annotation was performed using Polyphen-2. SNVs found by WES were validated by direct Sanger sequencing. The frequency of those validated SNVs was defined in a separate APL cohort. Results: I identified 33 somatic non-synonymous SNVs in three patients. Polyphen-2 algorithm predicted 9 among 33 SNVs to damage protein function. Sanger sequencing validated 7 from 9 SNVs. These validated SNVs include RXRG M77R, N6AMT2 A78S, TXNDC15 D198E, B3GALTL A444T, RBBP8NL E182G, BHMT M185I and ADAMTS5 G85D. When these 7 SNVs were genotyped in a separate cohort, none of these SNVs were found in an APL cohort composed of 29 ATRA sensitive patients, suggesting these SNVs would be truly related to ATRA resistance in APL. Especially, when a simulation using amber molecular dynamics, I observed 1) increase in hydrogen bonding, 2) decreased helix folding structure and 3) decreased energy state in RXRG mutant case. Conclusions: WES identified seven SNVs which were unique in ATRA resistant cases. Among those mutations, RXRG mutation could be a promising genetic mechanism of ATRA resistance.