Skipping of ATP-Binding Cassette Transporter A3 Exon 19 in AML Cells Is an Independent Prognostic Factor in Patients with Normal Cytogenetics

ATP binding cassette (ABC) transporters are a superfamily of highly conserved membrane proteins that transport a wide variety of substrates across cell membranes and confer drug resistance against a wide range of chemotherapeutic agents. We recently found that WT1, which is regularly overexpressed in AML and interact with the splicing machinery, modifies the splicing of ABC transporters A2, A3, A5, and C2. For ABCA3, WT1 knock-down in three AML cell line coupled with Affymetrix HTA2 exon arrays analysis confirmed by exon-specific PCR revealed that WT1 influences the skipping of exon 19. ABCA3 belongs in the ABC subclass and induces a significant reduction in cytotoxicity observed following exposure to DNR, mitoxantrone, etoposide, Ara-C and vincristine. The ABCA3 domain encoded by exon 19 (amino acid 805-847) is localized at the junction of the first nucleotide-binding domain and the second transmembrane domain, and is involved in ATP hydrolysis. In silico, skipping of exon 19 deletes a sequence of 32 amino acids rich in positively charged residues and is thereby assumed to increase drug efflux through increased ATP hydrolysis. The effects of the skipping of exon 19 on chemoresistance and DNR efflux are currently investigated while for the present study, we hypothesized that skipping of exon 19 of ABCA3 might negatively influence outcome in AML patients.

Analyzing 132 bone marrow AML samples harvested at diagnostic confirmed the statistically significant correlation between WT1 expression and ABCA3 splicing in vivo (p<10-4, Spearman Rank Correlation). This correlation was specific because it was not observed in 37 control samples deriving from bone marrow donors or in the AML cases with 2 TET2 alternative exon usages found WT1-independent ex vivo. In the 106 patients treated with intensive chemotherapy (IC), skipping of ABCA3 exon 19 was associated with a significantly lower response rate: 39/55 (70.9%) vs 44/51 (86.3%, p = 0.045, Pearson’s chi-squared test). In complete remitters, skipping of ABCA3 exon 19 was associated with a significantly higher relapse rate: 77.1% vs 52.6% (p=0.026, Pearson’s chi-squared test). Median follow-up was 25 months. The 25 allografted patients were censored at the time of allograft. By univariate analysis ABC-A3 exon skipping of exon 19 significantly affected OS (HR=4.50 (95% confidence interval: 2.10-9.63), p<10-4), DFS (HR=3.76 (95% confidence interval: 1.87-7.42), p<10-4), and EFS (HR 3.73 (95% confidence interval: 1.38-5.13), p=0.004); higher the level of exon 19 skipping, poorer the outcome. In multivariate analysis, age, cytogenetic, and ABC A3 exon 19 skipping were identified to be independent prognostic factors for OS, EFS and DFS. Age and ABC-A3 exon exclusion were identified to be independent prognostic factor for OS in the 49 patients with normal karyotype.

In order to confirm these results we investigated the prognostic impact of ABC A3 exon 19 skipping in a validation cohort of 108 additional AML case with normal cytogenetics. FLT3 internal tandem duplication (FLT3-ITD) and nucleophosmin (NPM1) exon-12 gene mutation were identified in 37 (34.3%) and 66 cases (61.1%). In the 86 patients treated with IC, the CR rate was 89,5% without significant difference between patients with or without exon 19 skipping. The relapse rate was higher in cases with exon 19 skipping (47,1% vs 23.5%) but the difference was not statistically significant. The 29 allografted patients were censored at the time of allograft. Median follow-up was 12 months. By univariate analysis ABC-A3 exon skipping of exon 19 significantly affected DFS (HR=2.03 (95% confidence interval: 1.22-3.39), p=0.007, and EFS (HR 1.62 (95% confidence interval: 1.06-2.48), p=0.027); higher the level of exon 19 skipping, poorer the outcome. In multivariate analysis, age, FLT3-ITD, ABC A3 exon 19 skipping but not NPM1 mutation were identified to be independent prognostic factors for EFS while FLT3-ITD and ABC A3 exon 19 skipping were independent prognostic factors for DFS.

In conclusion ABC-A3 missplicing possesses a strong prognostic impact in AML indicating that besides whole gene transcription, quantitative analysis of alternative splicing might represent a promising tool for assessing AML aggressiveness at the time of diagnostic in patients with normal or abnormal karyotype.