Clinical and Biological Role of Accumulation of the NPM-1 Splice Variant R2 in AML, MDS and sAML

Background Acute myelogenous leukemia (AML) represents a heterogeneous group of myeloid malignancies harboring different chromosomal abnormalities, gene mutations, and epigenetic modifications. Recent clinical and biological studies indicate that myelodysplastic syndromes (MDS) and AML could be considered as part of the same continuous disease spectrum rather than as distinct disorders. NPM1 is a multifunctional protein involved in both biological and pathological processes controlling development, cell proliferation, ribosome biogenesis, transformation and genomic stability. It interacts with many cellular proteins, including ARF and the tumor suppressor p53. Recently, we found that high expression of the NPM1 splice variant R2, which encodes a truncated form of NPM1, may provide prognostic value for CN-AML patients. Aims Therefore, our aim was evaluation of NPM1 R2 splice variant significance for MDS and sAML cases, as well as assignment if different expression levels of R2 might have influence on the expression pattern of each of the components of the ARF-MDM2-p53-p21 signaling pathway and additional downstream molecules (miR-34a, miR-34b and miR-34c). In order to determine the impact of NPM1 R2 on NPM1 localization and to compare it with the NPM1mut effect, transfection analyses and IHC stainings were performed. Methods NPM1 R2, CDKN2A (encoding ARF), MDM2, TP53 and CDKN1A(encoding p21) genes expression levels were assessed for 128 samples (58 AML, 62 MDS and 8 sAML) using qRT-PCR. Additionally, expression level of miR-34a (n=29), miR-34b (n=20) and miR-34c (n=20) was measured in CD33+ cells derived from AML patient samples. WI-38 fibroblasts and HEK-293 cells were transfected with constructs containing eGFP-tagged NPM1-R2, NPM1-mut and NPM1-wt under a cytomegalovirus promoter, stained and visualized with confocal microscope. Immunochemistry analysis was performed for NPM1 in 23 AML bone marrow smears. Results NPM1 R2 expression levels differed between AML, sAML, MDS and healthy volunteers (HV) groups and were significantly higher in AML, sAML and MDS groups compared to HVs (median 0.023 vs 0.005, p<0.001, 0.025 vs 0.005, p<0.001 and 0.017 vs 0.005, p<0.001, respectively). CDKN2A, MDM2, TP53 and CDKN1A expression analysis in these sample groups showed also significant differences. Expression of TP53 was elevated in groups with high R2 expression in comparison to groups with low R2 expression in AML and MDS patients (median 0.01 vs 0.005, p<0.001 and 0.007 vs 0.004, p<0.001, respectively). Moreover, we found strong positive correlation of R2 expression with TP53 expression in AML (r=0.77, p<0.001) and MDS (r=0.68, p<0.001). We observed elevated expression of miR-34c in HVs group compared to AML (0.11 vs 0.07, p<0,001) and trend to decreased expression of miR-34a in AML in comparison with HVs. No differences were found in miR-34a, miR-34b and miR-34c expression between groups with high or low R2 expression. Transfection analyses showed various localization of each eGFP-tagged NPM1 forms. NPM1-wt localized mainly in nucleoli, NPM1-R2 was detected in the nucleoplasm and nucleoli, whereas eGFP-NPM1-mut displayed cytoplasmic localization. However, the IHC stainings for AML samples revealed that in cases with high R2 expression we were able to determine a cytoplasmic localization of NPM1 even in the absence of its concomitant mutation. Conclusions The elevated level of NPM1 R2 splice variant in AML, sAML and MDS groups versus HVs suggests that R2 might play some role in neoplasia process also in early stages of this hematological malignancy. Transfection analyses established that NPM1 R2 mostly localizes in the nucleoplasm, where it might interact with other proteins e.g. ARF and p53. Nucleolar localization of this NPM1 form might be determined both by lack of nucleolar localization signal present in the wt form of NPM1 and nuclear export signal occurring in mutated NPM1. Moreover, strong positive correlation between R2 and TP53 expression was found in AML and MDS groups suggesting biological link between these transcripts. In summary, the expression of NPM1 R2 might be of biological importance for AML as well as for transformation of MDS into sAML. This work was supported by National Centre for Science Grant HARMONIA (UMO-2013/10/M/NZ5/00313). Disclosures Grzasko: Celgene: Honoraria; Munipharma: Honoraria; Janssen: Honoraria.