Missense mutations in PML-RARA are critical for the lack of responsiveness to arsenic trioxide treatment

Abstract

Arsenic trioxide (As 2 O 3 ) is a highly effective treatment for patients with refractory/ relapsed acute promyelocytic leukemia (APL), but resistance to As 2 O 3 has recently been seen. In the present study, we report the findings that 2 of 15 patients with refractory/relapsed APL treated with IntroductionAcute promyelocytic leukemia (APL) is characterized by the reciprocal chromosomal translocation t(15;17)(q22;q21), leading to fusion of the promyelocytic leukemia gene (PML) on chromosome 15 and the retinoic acid receptor-␣ gene (RARA) on chromosome 17. 1 PML-RARA fusions are detectable in Ͼ 95% of patients with APL. In 1985, all-trans retinoic acid (ATRA) was introduced for the treatment of APL as a differentiation therapy, and a dramatic improvement in the overall survival of patients with APL has been obtained. [2][3][4] However, approximately 10%-30% of patients eventually relapse after treatment with combination chemotherapies with ATRA. [5][6][7] Arsenic trioxide (As 2 O 3 ) is a critical drug for the treatment of APL and is clinically effective even in ATRA-resistant patients. 8 As 2 O 3 is a natural substance that has been used medically for over 2400 years. In the 1970s, a group in China identified As 2 O 3 as a component of an anticancer reagent. 9 Over the last 18 years, clinical trials conducted worldwide have demonstrated the efficacy of As 2 O 3 for the treatment of relapsed patients with APL. 10,11 Recently, it was also reported that As 2 O 3 improves event-free survival and overall survival of adult APL when As 2 O 3 is used as a consolidation treatment after obtaining the first remission. 12 Currently, the role of As 2 O 3 in frontline therapy is under investigation. 10,13 Rapid degradation of PML-RARA via targeting of PML has been reported as a molecular mechanism for the effectiveness of As 2 O 3 . 14 Furthermore, As 2 O 3 induces posttranslational modifications of PML-RARA with small ubiquitin-related modifier (SUMO) and ubiquitin, resulting in the transfer of PML-RARA from the soluble fraction to the insoluble nuclear matrix 14 and the degradation of both PML and PML-RARA. [14][15][16][17] In addition to the significant clinical effectiveness of As 2 O 3 for patients with APL, acquired resistance to As 2 O 3 therapy has been recognized in clinical practice. 18 Several studies have indicated that arsenicresistant NB4 cells in vitro show higher glutathione levels than in parental cells. [19][20][21] However, the detailed molecular mechanisms of resistance to As 2 O 3 remain unclear.Very recently, 2 studies reported that As 2 O 3 binds directly to cysteine residues in zinc fingers located within the RBCC motif that contains 3 cysteine-rich zinc-binding domains, a RING-finger (R), 2 B-box motifs (B1 and B2), and a coiled-coil (CC) domain, 22,23 in PML-RARA and PML. 24,25 An intriguing hypothesis is that impairment of As 2 O 3 binding to PML-RARA due to conformational changes may result from genetic mutations and/or abnormal posttranslational modifications. These events may be related to resis...

How to manage acute promyelocytic leukemia

“…Recently, a Japanese working group demonstrated from a study of two cases of relapsed patients that the mechanism for ATO-resistance may be missense mutations in PML --RARa coding sequence, resulting in amino-acid substitutions of A216V and L218P in the PML B2 motif of the RBCC domain. 101 On the other hand, we recently found DNMT3A mutations in a relapsed patient. 102 These results have prompted us to further identify the mechanisms of drug resistance in future work, so that the subset of patients who may develop resistance could be screened out earlier in the treatment, and specific therapeutic strategies can be given from the very beginning, to finally alter the prognosis of these patients.…”

The arsenic-based cure of acute promyelocytic leukemia promotes cytoplasmic sequestration of PML and PML/RARA through inhibition of PML body recycling

“…[26][27][28] Thirdly, the ability of PML to support CyPN formation is strictly dependent on cysteine residues within the BB2 motif of the PML protein, the same amino acids that recently have been shown to be important for ATO-PML interactions as well as the ATO-mediated cure of APL in mouse. 6 Finally, single amino acid substitutions within the PML protein, which recently were identified in 2 APL patients with clinical resistance to ATO, 35 severely reduced the ability of PML and PML/RARA to support CyPN formation both in the presence and in the absence of ATO. Because these mutations are also expected to cause reduced PML/RARA SUMOylation, 6 they may have a double effect in causing arsenic resistance through both inhibiting RNF4-mediated degradation as well as postmitotic PML body recycling.…”

“…6 Recently, 2 APL patients with clinical resistance to ATOmediated therapy were found to harbor single amino acid substitutions that clustered proximal to the zinc-binding cysteines within the BB2 motif of PML/RARA. 35 To assess whether these mutations affected CyPN assembly, we introduced them into the EYFP-PML1-expressing lentivirus-based plasmid construct and analyzed the localization of the expressed proteins in the PML-depleted HaCaT/ shPML cell line. Similar to EYFP-PML1.⌬BB2, both EYFP-PML1.A216V and EYFP-PML1.L218P (which expressed EYFP-PML1 with amino acid substitutions derived from ATO-resistant patient 1 and 2, respectively) supported formation of seemingly normal PML bodies, but were severely impaired in their ability to support CyPN formation ( Figure 4D-E).…”

Acute Promyelocytic Leukemia: A Model Disease for Targeted Cancer Therapy

“…Interestingly, one study identified mutations within the intact PML locus of APL patients with ATRA-resistance and poor prognosis (Gurrieri et al, 2004). Recently, PML/RARA mutations have also been discovered in two APL cases with poor response to ATO (Goto et al, 2011). In both cases, the mutations were located within the second B-box motif of the PML protein.…”