Mutation in Retinoic X Receptor-γ Is a Possible Mechanism of All-Trans Retinoic Acid Resistance in Acute Promyelocytic Leukemia(APL): Identifying Genetic Changes Related to Drug Resistance in APL Using Whole Exome Sequencing

Background

Acute promyelocytic leukemia (APL) has the best prognosis among acute myeloid leukemia (AML). However, a subset of APL patients is not cured with all-trans retinoic acid (ATRA) combined with anthracycline-based cytotoxic chemotherapy. Some mechanisms such as increased ATRA metabolism have been suggested to acquired resistance to ATRA. However genetic mechanism of ATRA resistance has not been characterized at all. Hence, in this study, we tried to reveal genetic alterations attributable to ATRA resistance.

Methods

First, we performed whole exome sequencing (WES) using DNA of three APL patients who showed resistance to ATRA based treatment. These included two patients who failed to achieve complete remission (CR) after induction chemotherapy, and one patient who experienced relapse after CR despite of 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 (SNV) was performed using internal pipeline called Adiscan (http://www.syntekabio.com). Annotation was performed using Polyphen-2. SNV’s found by WES were validated by direct Sanger sequencing. The frequency of those validated SNV’s was defined in a separate APL cohort.

Results

We identified 34 somatic non-synonymous SNV’s in three patients. Polyphen-2 algorithm predicted 9 among 34 SNV’s to damage protein function. Sanger sequencing revealed 8 over 9 SNV’s to be validated. These validated SNV’s include RXRG M77R, N6AMT2 A78S, TXNDC15 D198E, B3GALTL A444T, RBBP8NL E182G, TNPO3 L173W, BHMT M185I and ADAMTS5 G85D. When these 8 SNV’s were genotyped in a separate cohort, none of these SNV’s was found in the APL cohort composed of 30 ATRA sensitive patients, suggesting these SNV’s would be truly related to ATRA resistance in APL. Especially, when a simulation using amber molecular dynamics was performed, we observed 1) Increase in hydrogen bonding, 2) Decreased helix folding structure, 3) Decreased energy state in RXRG mutant case.

Conclusions

WES identified eight SNV’s which were unique in ATRA resistant cases. Among those mutations, RXRG could be a promising nonsynonymous mutation that explains the genetic mechanism of ATRA resistance.