Mutation in the Unrearranged PML Allele Confers Resistance to Arsenic Trioxide in Acute Promyelocytic Leukemia

Abstract

Arsenic trioxide (ATO) is able to selectively target and degrade the disease-causing PML::RARα (P/R) oncoprotein in acute promyelocytic leukemia (APL) for curing the disease. However, some relapsed patients develop resistance to ATO due to mutations in the promyelocytic leukemia (PML) part of the PML::RARα fusion gene. A relapsed APL patient had shown resistance to ATO and chemotherapy and was identified to harbor a point mutation (A216V) in the unrearranged PML allele rather than the PML::RARα fusion gene. Here, we report that mutations in the unrearranged PML allele impede the ATO-induced destabilization and degradation of the wild-type P/R oncoprotein. Deletion of the coiled-coil domain in a PML mutant completely reversed wild-type P/R protein resistance to ATO by abolishing the interaction between PML and P/R proteins. Collectively, our findings reveal that a point mutation in the unrearranged PML allele can confer ATO resistance through a protein-protein interaction. Therefore, the unrearranged PML allele should also be screened for drug-resistant mutations in relapsed APL patients.

Fig. 1.

Promyelocytic leukemia (PML) mutants prevent arsenic trioxide (ATO)-induced destabilization, PML nuclear body (PML-NB) re-formation, and degradation of the wild-type (WT) PML::RARα (P/R)fusion protein. (A) The treatment history of a 47-year-old ATO-resistant acute promyelocytic leukemia (APL) patient. Initially, this patient was diagnosed as typical APL harboring the PML::RARα fusion gene and received ATO plus all-trans-retinoic acid (ATRA) treatment, achieving complete remission (CR) in January 2017. For consolidation, an ATO plus idarubicin (IDA) regimen was applied, and PML::RARα detection in the bone marrow (BM) turned negative. After 30 months, the patient experienced extramedullary relapse in the skin (R1). Consequently, after treatment with ATO plus ATRA and IDA as well as resection of the skin lesion, this patient achieved CR in January 2020. Two months later, the patient relapsed (R2) again and showed no response to ATO plus ATRA and IDA. Notably, there was a rapid increase in the level of P/R fusion within the bone marrow from 0.81% to 81%. Unfortunately, the patient died due to rapid disease progression. (B) APL is characterized by the reciprocal translocation t(15;17)(q24.1;q21.2), which fuses one PML gene on chromosome 15 and one RARα gene on chromosome 17 to generate the PML::RARα fusion gene while the PML allele is still present. (C) Domain structure of the unrearranged PML protein and P/R fusion proteins. Note the same RBCC domain (e.g., RING finger, B-box, and coiled-coil) in the N-terminus but different in the C-terminus. (D) Genetic analysis of the unrearranged PML allele and the PML::RARα fusion gene in this ATO-resistant patient. (E and F) Primary APL blast cells were obtained from the 47-year-old ATO-resistant patient (blast 1) and an ATO-sensitive patient (blast 2). Subsequently, primary blast cells were exposed to ATO (1 μM) for 3 h, and the P/R fusion protein degradation was analyzed by western blotting. NB4 cells were used as positive control. (G and H) Green fluorescent protein (GFP)-labeled WT P/R plasmid was transiently cotransfected with Flag-tagged WT PML, A216V-PML mutant, and L218P-PML mutant in PML^−/− HeLa for 24 h and then exposed to ATO (1 μM) for 3 and 6 h. Solubility changes in the P/R fusion protein and PML proteins were detected by western blotting. (I) Changes in PML-NBs were determined by confocal microscopy. (J) PML^−/− HeLa cells coexpressing WT P/R with WT PML, A216V-PML mutant, and L218P-PML mutant were exposed to ATO (1 μM) for 3, 6, 12, and 24 h to determine protein degradation. Total proteins were extracted from cells by a urea buffer as described in Supplementary Materials and Methods. Green fluorescence indicates the PML protein, red fluorescence indicates the P/R fusion protein, and blue fluorescence (4′,6-diamidino-2-phenylindole [DAPI]) indicates the nucleus. S indicates supernatant; P indicates insoluble pellet. The scale bar is 5 μm. RARα, retinoic acid receptor alpha.

Fig. 2.

Unrearranged PML mutants alter the sensitivity of the WT P/R fusion protein to ATO through protein–protein interaction mediated by the coiled-coil (CC) domain. (A) A GFP-labeled WT P/R plasmid was transiently cotransfected with Flag-tagged WT PML, A216V-PML mutant, and L218P-PML mutant into PML^−/−HeLa cells for 24 h and then exposed to 1 μM ATO for 3 and 6 h. SUMOylation of PML proteins was determined by western blotting with PML antibody. (B) Colocalization of PML-NBs with small ubiquitin like modifier 1 (SUMO-1) in cells was detected by confocal microscopy. (C) Interactions between speckled protein 100 (SP100) and Flag-P/R in the presence of each WT PML, A216V-PML mutant, and L218P-PML mutant were determined by co-immunoprecipitation (co-IP) in PML^−/− HeLa cells after treatment with ATO (1 μM) for 6 h. (D) PML^−/− HeLa cells coexpressing GFP-P/R with each Flag-PML (i.e., WT, A216V, and L218P) were exposed to ATO at 1 μM for 6 h. Interaction of PML proteins with the P/R fusion protein was determined by co-IP. (E) Schematic diagram of the RING (ΔR), B-box1 (ΔB1), B-box2 (ΔB2), and coiled-coil (ΔCC) domain truncated PMLs. (F) Changes in PML multimerization in truncated PML proteins were determined by native gel electrophoresis. M indicates monomer, D indicates dimer, and P indicates polymer. (G) Interactions between GFP-P/R and each of ΔR, ΔB1, and ΔB2 as well as ΔCC-A216V-PML mutants in PML^−/− HeLa were analyzed by co-IP after treatment with ATO (1 μM) for 6 h. Solubility changes of the P/R fusion protein and PML protein were determined by western blotting. (H) PML^−/− HeLa cells expressing GFP-P/R and CC truncated Flag-PML-A216V were exposed to ATO (1 μM) for 3, 6, 12, and 24 h, and then the changes in the total protein levels of the GFP-P/R fusion protein and ΔCC-A216V-PML protein were determined. Green fluorescence indicates PML protein, red fluorescence indicates P/R fusion protein, purple fluorescence indicates SUMO-1, and blue fluorescence (DAPI) indicates the nucleus. S indicates supernatant, P indicates insoluble pellet, and T indicates total protein. The scale bar is 5 μm.