THE ROLE OF THE NUCELOPHOSMIN-1 SPLICE VARIANT R2 IN ACUTE MYELOID LEUKEMIA DEVELOPMENT
(Abstract release date: 05/19/16)
EHA Library. Zajac M. 06/09/16; 132457; E908
Mrs. Malgorzata Zajac
Contributions
Contributions
Abstract
Abstract: E908
Type: Eposter Presentation
Background
A close cytogenetic and molecular analogy between de novo MDS and AML of elderly people indicates a common pathogenic mechanism for these conditions. The frequent mutations of the splicing machinery in MDS suggest that alternative splicing of certain molecules of the crucial signaling pathways might drive progression to AML. Recently, we found that high expression of the NPM1 splice variant R2 may provide prognostic value for CN-AML patients.
Aims
Assuming the common origin of MDS and AML we aimed to characterize the NPM1 R2 splice variant expression in groups with MDS, sAML and AML patients. Moreover, NPM1 stabilizes the ARF and interacts with the tumor suppressor p53, regulates the increase in stability and transcriptional activation of p53, thus contributing to modulating growth-suppressive pathways. Therefore, we characterized expression pattern of ARF, MDM2, TP53 genes with additional downstream molecules (p21, miR-34a) in AML, s-AML and MDS patients.
Methods
For 128 samples (58 AML, 62 MDS and 8 samples with sAML) expression levels of NPM1 R2, ARF, MDM2, TP53 and p21 were assessed by qRT-PCR. We also measured expression levels of miR-34a, miR-34b and miR-34c in CD33+ cells from 20 AML patients samples. To investigate whether R2 might disrupt localization of the NPM1 wild type protein, we used constructs with GFP-tagged NPM1-R2 and NPM1-wt under cytomegalovirus promotor to transfect WI-38 fibroblasts and performed immunohistochemistry analysis for NPM1 in 23 AML bone marrow smears.
Results
The expression of NPM1 R2 was significantly higher in AML, s-AML 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). NPM1 R2 positively correlated 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). No differences were found in miR34a, miR-34b and miR-34c expression between groups with high or low R2 expression. Transfections analyses showed that GFP-tagged NPM1-R2 was detected in nucleoplasm, whereas the GFP-NPMwt was detected in the nucleoli. 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. Therefore, we provide further evidence that the cytoplasmic localization of NPM1 might depend not only on its mutational status, but might be influenced by the distribution of its splice variants.
Conclusion
In our study we found that the expression levels of R2 were elevated in AML, sAML and MDS groups compared to HVs suggesting that R2 might play a role in the process of the tumorigenesis not only in AML cases but also in early stages of development of this disease. As the NPM1 R2 splice variant represents a truncated form of NPM1 gene, our transfections analyses confirmed that this isoform mostly localizes in the nucleoplasm, and thus might also have a biological impact in the malignant cells by interaction with other proteins. 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 sAML and MDS patients.This work was supported by National Centre for Science Grant HARMONIA (UMO-2013/10/M/NZ5/00313).
Session topic: E-poster
Keyword(s): AML, Expression, MDS
Type: Eposter Presentation
Background
A close cytogenetic and molecular analogy between de novo MDS and AML of elderly people indicates a common pathogenic mechanism for these conditions. The frequent mutations of the splicing machinery in MDS suggest that alternative splicing of certain molecules of the crucial signaling pathways might drive progression to AML. Recently, we found that high expression of the NPM1 splice variant R2 may provide prognostic value for CN-AML patients.
Aims
Assuming the common origin of MDS and AML we aimed to characterize the NPM1 R2 splice variant expression in groups with MDS, sAML and AML patients. Moreover, NPM1 stabilizes the ARF and interacts with the tumor suppressor p53, regulates the increase in stability and transcriptional activation of p53, thus contributing to modulating growth-suppressive pathways. Therefore, we characterized expression pattern of ARF, MDM2, TP53 genes with additional downstream molecules (p21, miR-34a) in AML, s-AML and MDS patients.
Methods
For 128 samples (58 AML, 62 MDS and 8 samples with sAML) expression levels of NPM1 R2, ARF, MDM2, TP53 and p21 were assessed by qRT-PCR. We also measured expression levels of miR-34a, miR-34b and miR-34c in CD33+ cells from 20 AML patients samples. To investigate whether R2 might disrupt localization of the NPM1 wild type protein, we used constructs with GFP-tagged NPM1-R2 and NPM1-wt under cytomegalovirus promotor to transfect WI-38 fibroblasts and performed immunohistochemistry analysis for NPM1 in 23 AML bone marrow smears.
Results
The expression of NPM1 R2 was significantly higher in AML, s-AML 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). NPM1 R2 positively correlated 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). No differences were found in miR34a, miR-34b and miR-34c expression between groups with high or low R2 expression. Transfections analyses showed that GFP-tagged NPM1-R2 was detected in nucleoplasm, whereas the GFP-NPMwt was detected in the nucleoli. 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. Therefore, we provide further evidence that the cytoplasmic localization of NPM1 might depend not only on its mutational status, but might be influenced by the distribution of its splice variants.
Conclusion
In our study we found that the expression levels of R2 were elevated in AML, sAML and MDS groups compared to HVs suggesting that R2 might play a role in the process of the tumorigenesis not only in AML cases but also in early stages of development of this disease. As the NPM1 R2 splice variant represents a truncated form of NPM1 gene, our transfections analyses confirmed that this isoform mostly localizes in the nucleoplasm, and thus might also have a biological impact in the malignant cells by interaction with other proteins. 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 sAML and MDS patients.This work was supported by National Centre for Science Grant HARMONIA (UMO-2013/10/M/NZ5/00313).
Session topic: E-poster
Keyword(s): AML, Expression, MDS
Abstract: E908
Type: Eposter Presentation
Background
A close cytogenetic and molecular analogy between de novo MDS and AML of elderly people indicates a common pathogenic mechanism for these conditions. The frequent mutations of the splicing machinery in MDS suggest that alternative splicing of certain molecules of the crucial signaling pathways might drive progression to AML. Recently, we found that high expression of the NPM1 splice variant R2 may provide prognostic value for CN-AML patients.
Aims
Assuming the common origin of MDS and AML we aimed to characterize the NPM1 R2 splice variant expression in groups with MDS, sAML and AML patients. Moreover, NPM1 stabilizes the ARF and interacts with the tumor suppressor p53, regulates the increase in stability and transcriptional activation of p53, thus contributing to modulating growth-suppressive pathways. Therefore, we characterized expression pattern of ARF, MDM2, TP53 genes with additional downstream molecules (p21, miR-34a) in AML, s-AML and MDS patients.
Methods
For 128 samples (58 AML, 62 MDS and 8 samples with sAML) expression levels of NPM1 R2, ARF, MDM2, TP53 and p21 were assessed by qRT-PCR. We also measured expression levels of miR-34a, miR-34b and miR-34c in CD33+ cells from 20 AML patients samples. To investigate whether R2 might disrupt localization of the NPM1 wild type protein, we used constructs with GFP-tagged NPM1-R2 and NPM1-wt under cytomegalovirus promotor to transfect WI-38 fibroblasts and performed immunohistochemistry analysis for NPM1 in 23 AML bone marrow smears.
Results
The expression of NPM1 R2 was significantly higher in AML, s-AML 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). NPM1 R2 positively correlated 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). No differences were found in miR34a, miR-34b and miR-34c expression between groups with high or low R2 expression. Transfections analyses showed that GFP-tagged NPM1-R2 was detected in nucleoplasm, whereas the GFP-NPMwt was detected in the nucleoli. 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. Therefore, we provide further evidence that the cytoplasmic localization of NPM1 might depend not only on its mutational status, but might be influenced by the distribution of its splice variants.
Conclusion
In our study we found that the expression levels of R2 were elevated in AML, sAML and MDS groups compared to HVs suggesting that R2 might play a role in the process of the tumorigenesis not only in AML cases but also in early stages of development of this disease. As the NPM1 R2 splice variant represents a truncated form of NPM1 gene, our transfections analyses confirmed that this isoform mostly localizes in the nucleoplasm, and thus might also have a biological impact in the malignant cells by interaction with other proteins. 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 sAML and MDS patients.This work was supported by National Centre for Science Grant HARMONIA (UMO-2013/10/M/NZ5/00313).
Session topic: E-poster
Keyword(s): AML, Expression, MDS
Type: Eposter Presentation
Background
A close cytogenetic and molecular analogy between de novo MDS and AML of elderly people indicates a common pathogenic mechanism for these conditions. The frequent mutations of the splicing machinery in MDS suggest that alternative splicing of certain molecules of the crucial signaling pathways might drive progression to AML. Recently, we found that high expression of the NPM1 splice variant R2 may provide prognostic value for CN-AML patients.
Aims
Assuming the common origin of MDS and AML we aimed to characterize the NPM1 R2 splice variant expression in groups with MDS, sAML and AML patients. Moreover, NPM1 stabilizes the ARF and interacts with the tumor suppressor p53, regulates the increase in stability and transcriptional activation of p53, thus contributing to modulating growth-suppressive pathways. Therefore, we characterized expression pattern of ARF, MDM2, TP53 genes with additional downstream molecules (p21, miR-34a) in AML, s-AML and MDS patients.
Methods
For 128 samples (58 AML, 62 MDS and 8 samples with sAML) expression levels of NPM1 R2, ARF, MDM2, TP53 and p21 were assessed by qRT-PCR. We also measured expression levels of miR-34a, miR-34b and miR-34c in CD33+ cells from 20 AML patients samples. To investigate whether R2 might disrupt localization of the NPM1 wild type protein, we used constructs with GFP-tagged NPM1-R2 and NPM1-wt under cytomegalovirus promotor to transfect WI-38 fibroblasts and performed immunohistochemistry analysis for NPM1 in 23 AML bone marrow smears.
Results
The expression of NPM1 R2 was significantly higher in AML, s-AML 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). NPM1 R2 positively correlated 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). No differences were found in miR34a, miR-34b and miR-34c expression between groups with high or low R2 expression. Transfections analyses showed that GFP-tagged NPM1-R2 was detected in nucleoplasm, whereas the GFP-NPMwt was detected in the nucleoli. 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. Therefore, we provide further evidence that the cytoplasmic localization of NPM1 might depend not only on its mutational status, but might be influenced by the distribution of its splice variants.
Conclusion
In our study we found that the expression levels of R2 were elevated in AML, sAML and MDS groups compared to HVs suggesting that R2 might play a role in the process of the tumorigenesis not only in AML cases but also in early stages of development of this disease. As the NPM1 R2 splice variant represents a truncated form of NPM1 gene, our transfections analyses confirmed that this isoform mostly localizes in the nucleoplasm, and thus might also have a biological impact in the malignant cells by interaction with other proteins. 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 sAML and MDS patients.This work was supported by National Centre for Science Grant HARMONIA (UMO-2013/10/M/NZ5/00313).
Session topic: E-poster
Keyword(s): AML, Expression, MDS
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