Contributions
Abstract: S127
Type: Oral Presentation
Presentation during EHA22: On Friday, June 23, 2017 from 12:30 - 12:45
Location: Room N101
Background
ALCL is a high grade lymphoma characterized by anaplastic morphology, expression of CD30 (Ki-1) and T- or null cell phenotype. In 60% of systemic ALCL, the translocation t(2;5)(p23;q35) leads to expression of the oncogenic tyrosine kinase NPM-ALK. NIPA (Nuclear Interaction Partner of ALK) is an F-Box-Protein contributing to the timing of mitotic entry by defining an oscillating ubiquitin E3 ligase. NIPA deficient mice are viable but sterile due to impaired DNA double strand break repair. Co-expressed with NPM-ALK, NIPA is constitutively phosphorylated. However, the role of NIPA in NPM-ALK induced lymphomagenesis and the functional impact of this interaction remain unknown.
Aims
In this study, we aim to investigate the effect of Nipa deficiency on NPM-ALK driven cell proliferation and transformation in order to characterize the function of the protein in ALCL-induced lymphomagenesis.
Methods
Primary Nipa-/- MEFs infected with NPM-ALK were plated in softagar assays to evaluate their transformation ability. Moreover, NIPA was downregulated through targeted genetic approaches in Karpas299 and NPM-ALK infected Ba/F3 cells, which were analyzed regarding proliferation, signaling, and apoptosis. To assess the impact of Nipa deletion in vivo, we used a retroviral bone marrow transplantation model resembling human ALCL. Based on a Cre/loxP system under the LCK-Promotor, NPM-ALK expression and Nipa-deletion are restricted to early T cells. In wildtype background, mice die of systemic Thy1.2+ lymphoma with a latency of 4-6 months, developing neoplastic T-cell infiltration of bone marrow and lymphatic organs. Lymphomas were analyzed regarding immunphenotype and clinical presentation.
Results
Primary Nipa-/- MEFs plated in softagar showed significantly reduced colony formation potential upon NPM-ALK expression (38 vs. 79 CFUs; p<0.001). These results were substantiated in human and murine cell lines, where significantly reduced proliferation ability was observed in NIPA downregulating NPM-ALK expressing Ba/F3 cells (74% of ctrl; p<0.001) as well as in Karpas299 cells infected with NIPA miR (66% of wt growth; p<0.01). Moreover, treatment with the ALK inhibitor TAE-684 gave evidence of possible synergistic effects of ALK inhibition and NIPA knockdown. Mice transplanted with Lck-CreTG/wtNipaflox/flox MSNAIE infected bone marrow cells showed significantly prolonged disease development and progression (mean survival 143d vs. 121d in wt). Morphologically, mice presented with enlarged thymi, splenomegaly, lymphadenopathy, and bone marrow infiltration. Immunphenotyping showed a pure T-cell phenotype in Nipa-/- lymphomas, thus resembling wildtype. In a long-latency model of NPM-ALK expression in enriched HSCs, a significantly prolonged survival (110 vs. 80 days; p<0.01) and reduction of spleen colonies (10 vs. 28 colonies/spleen; p<0.001) in mice transplanted with MigNPM-ALKNipa-/- bone marrow compared to control animals were observed, thereby suggesting a crucial role of NIPA in NPM-ALK driven lymphomagenesis. To investigate the precise mechanism undelaying these results, we performed cell cycle analyses as well as cell viability assays. Indeed, we were able to detect significant differences in the cell viability in Nipa deficient NPM-ALK expressing cells, whereas cell cycle distribution seems not to be altered in knockout cells.
Conclusion
Session topic: 18. Non-Hodgkin & Hodgkin lymphoma - Biology
Keyword(s): NPM-ALK, Murine models, Lymphomagenesis, ALCL
Abstract: S127
Type: Oral Presentation
Presentation during EHA22: On Friday, June 23, 2017 from 12:30 - 12:45
Location: Room N101
Background
ALCL is a high grade lymphoma characterized by anaplastic morphology, expression of CD30 (Ki-1) and T- or null cell phenotype. In 60% of systemic ALCL, the translocation t(2;5)(p23;q35) leads to expression of the oncogenic tyrosine kinase NPM-ALK. NIPA (Nuclear Interaction Partner of ALK) is an F-Box-Protein contributing to the timing of mitotic entry by defining an oscillating ubiquitin E3 ligase. NIPA deficient mice are viable but sterile due to impaired DNA double strand break repair. Co-expressed with NPM-ALK, NIPA is constitutively phosphorylated. However, the role of NIPA in NPM-ALK induced lymphomagenesis and the functional impact of this interaction remain unknown.
Aims
In this study, we aim to investigate the effect of Nipa deficiency on NPM-ALK driven cell proliferation and transformation in order to characterize the function of the protein in ALCL-induced lymphomagenesis.
Methods
Primary Nipa-/- MEFs infected with NPM-ALK were plated in softagar assays to evaluate their transformation ability. Moreover, NIPA was downregulated through targeted genetic approaches in Karpas299 and NPM-ALK infected Ba/F3 cells, which were analyzed regarding proliferation, signaling, and apoptosis. To assess the impact of Nipa deletion in vivo, we used a retroviral bone marrow transplantation model resembling human ALCL. Based on a Cre/loxP system under the LCK-Promotor, NPM-ALK expression and Nipa-deletion are restricted to early T cells. In wildtype background, mice die of systemic Thy1.2+ lymphoma with a latency of 4-6 months, developing neoplastic T-cell infiltration of bone marrow and lymphatic organs. Lymphomas were analyzed regarding immunphenotype and clinical presentation.
Results
Primary Nipa-/- MEFs plated in softagar showed significantly reduced colony formation potential upon NPM-ALK expression (38 vs. 79 CFUs; p<0.001). These results were substantiated in human and murine cell lines, where significantly reduced proliferation ability was observed in NIPA downregulating NPM-ALK expressing Ba/F3 cells (74% of ctrl; p<0.001) as well as in Karpas299 cells infected with NIPA miR (66% of wt growth; p<0.01). Moreover, treatment with the ALK inhibitor TAE-684 gave evidence of possible synergistic effects of ALK inhibition and NIPA knockdown. Mice transplanted with Lck-CreTG/wtNipaflox/flox MSNAIE infected bone marrow cells showed significantly prolonged disease development and progression (mean survival 143d vs. 121d in wt). Morphologically, mice presented with enlarged thymi, splenomegaly, lymphadenopathy, and bone marrow infiltration. Immunphenotyping showed a pure T-cell phenotype in Nipa-/- lymphomas, thus resembling wildtype. In a long-latency model of NPM-ALK expression in enriched HSCs, a significantly prolonged survival (110 vs. 80 days; p<0.01) and reduction of spleen colonies (10 vs. 28 colonies/spleen; p<0.001) in mice transplanted with MigNPM-ALKNipa-/- bone marrow compared to control animals were observed, thereby suggesting a crucial role of NIPA in NPM-ALK driven lymphomagenesis. To investigate the precise mechanism undelaying these results, we performed cell cycle analyses as well as cell viability assays. Indeed, we were able to detect significant differences in the cell viability in Nipa deficient NPM-ALK expressing cells, whereas cell cycle distribution seems not to be altered in knockout cells.
Conclusion
Session topic: 18. Non-Hodgkin & Hodgkin lymphoma - Biology
Keyword(s): NPM-ALK, Murine models, Lymphomagenesis, ALCL