NPM1 Splice Variant R2 Reveals Biological and Clinical Consequences of Prognostic Value in Acute Myeloid Leukemia
Authors: Giannopoulos Krzysztof , Zajac Malgorzata , Dolnik Anna , Schunn Matthias , Correa Stephany , Glodkowska-Mrowka Eliza , Chakkarappan Sundaram Reddy , Jankowska-Lecka Olga , Schlenk Richard , Dohner Konstanze , Bullinger Lars 
Published: November 2019

Abstract
Background: The process of mRNA splicing has been reported to play an important role in human disease development and many cancer-related genes are regulated by alternative splicing. The genome-wide microarray analysis using Exon arrays discovered genes significantly spliced in AML. Recently we characterized nucleophosmin 1 (NPM1) splice variants expression in AML patients. We found that high expression of splice variant R2 may provide prognostic value for cytogenetically normal AML (CN-AML) patients. As the R2 splice variant represents a truncated form of NPM1 gene lacking of exons 11 and 12, this isoform might preferentially localize in the cytoplasm affecting signal pathways similarly as NPM1 mutation influencing thereby outcome or modulating treatment response. Methods: To evaluate the translation of the splice variant R2 to the protein level, Western Blot blot analyses in selected AML cell lines as well as patients samples were performed. To investigate whether R2 might disrupt localization of the NPM1 wild type protein, we also performed immunohistochemistry analysis for NPM1 in 23 AML bone marrow smears. Since we found prognostic significance of the expression level of R2 for the first cohort of patients, we validated findings in an independent cohort of AML cases. We consolidated 104 patients previously analyzed with 97 patients from the new cohort to a total of 201 cases and assessed the mRNA expression of R2 by qRT-PCR. Results: By Western blot analysis we confirmed that the R2 variant is also expressed at the protein level in AML patients samples as well as in 7 AML cell lines. By immunohistochemistry staining we were able to determine cytoplasmic localization of NPM1 in 3 samples showing high R2 expression levels even without concomitant NPM1 mutation. In contrast, exemplary samples without NPM1 mutation and with low level of R2 showed nuclear localization of NPM1. In a consolidated cohort of 201 AML patients, we found that the expression of R2 splice variant was significantly higher in all AML patients compared to normal controls with a median expression of 1.64 vs 0.33 (p= 0.009). High R2 splice variant expression was associated with longer overall survival (OS) when CN-AML patients were analyzed (880 vs 438 days, p= 0.028). Longer OS was observed in CN-AML patients with high R2 expression without concomitant FLT3-ITD mutations compared to the rest of groups. Of note, while in our cohort of CN-AML cases survival differences seen between the established ELN groups according to a NPM1/FLT3-ITD stratification were significant (p=0.03), differences between groups stratified according to R2 expression combined with FLT3-ITD mutational status were more pronounced (p=0.003). Conclusions: As the R2 splice variant represents a truncated form of the NPM1 gene lacking exons 11 and 12 (coding for the domain responsible for nucleolar localization of the protein), this isoform can localize to the cytoplasm, and thus might also have a biological impact in the malignant cells. In accordance, in cases with high R2 expression we were able to determine a cytoplasmic localization of NPM1 even in the absence of a concomitant NPM1 mutation. Therefore, we provide further evidence that the cytoplasmic localization of NPM1 might depend not only on its mutational status, but might also be influenced by the distribution of its splice variants. In line, R2 might interact with cellular proteins affecting signal pathways and thereby have an impact on the biology of the disease, which in turn is reflected in differences in treatment response and outcome. In summary, the expression of R2 might be of biological importance for CN-AML patients. Moreover, measuring of the R2 splice variant might provide prognostic value for CN-AML patients in addition to the NPM1 mutational status.

Disclosures
No relevant conflicts of interest to declare.