Nuclear Interaction Partner Of ALK Research Articles

NIPA (Nuclear Interaction Partner of ALK) Is Crucial for Effective NPM-ALK Mediated Lymphomagenesis.

The NPM-ALK fusion kinase is expressed in 60% of systemic anaplastic large-cell lymphomas (ALCL). A Nuclear Interaction Partner of ALK (NIPA) was identified as a binding partner of NPM-ALK. To identify the precise role of NIPA for NPM-ALK-driven lymphomagenesis, we investigated various NPM-ALK+ cell lines and mouse models. Nipa deletion in primary mouse embryonic fibroblasts resulted in reduced transformation ability and colony formation upon NPM-ALK expression. Downregulating NIPA in murine NPM-ALK+ Ba/F3 and human ALCL cells decreased their proliferation ability and demonstrated synergistic effects of ALK inhibition and NIPA knockdown. Comprehensive in vivo analyses using short- and long-latency transplantation mouse models with NPM-ALK+ bone marrow (BM) revealed that Nipa deletion inhibited NPM-ALK-induced tumorigenesis with prolonged survival and reduced spleen colonies. To avoid off-target effects, we combined Nipa deletion and NPM-ALK expression exclusively in T cells using a lineage-restricted murine ALCL-like model resembling human disease: control mice died from neoplastic T-cell infiltration, whereas mice transplanted with Lck-CreTG/wtNipaflox/flox NPM-ALK+ BM showed significantly prolonged survival. Immunophenotypic analyses indicated a characteristic ALCL-like phenotype in all recipients but revealed fewer “stem-cell-like” features of Nipa-deficient lymphomas compared to controls. Our results identify NIPA as a crucial player in effective NPM-ALK-driven ALCL-like disease in clinically relevant murine and cell-based models.

Loss of the Fanconi anemia-associated protein NIPA causes bone marrow failure.

Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of disorders characterized by defective hematopoiesis, impaired stem cell function, and cancer susceptibility. Diagnosis of IBMFS presents a major challenge due to the large variety of associated phenotypes, and novel, clinically relevant biomarkers are urgently needed. Our study identified nuclear interaction partner of ALK (NIPA) as an IBMFS gene, as it is significantly downregulated in a distinct subset of myelodysplastic syndrome-type (MDS-type) refractory cytopenia in children. Mechanistically, we showed that NIPA is major player in the Fanconi anemia (FA) pathway, which binds FANCD2 and regulates its nuclear abundance, making it essential for a functional DNA repair/FA/BRCA pathway. In a knockout mouse model, Nipa deficiency led to major cell-intrinsic defects, including a premature aging phenotype, with accumulation of DNA damage in hematopoietic stem cells (HSCs). Induction of replication stress triggered a reduction in and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the knockout mice with 100% penetrance. Taken together, the results of our study add NIPA to the short list of FA-associated proteins, thereby highlighting its potential as a diagnostic marker and/or possible target in diseases characterized by hematopoietic failure.

Proteomic Phosphosite Analysis Identified Crucial NPM-ALK-Mediated NIPA Serine and Threonine Residues.

Anaplastic large-cell lymphoma (ALCL) is an aggressive non-Hodgkin lymphoma that shows in 60% of cases a translocation t(2;5)(p23;q35), which leads to the expression of the oncogenic kinase NPM-ALK. The nuclear interaction partner of ALK (NIPA) defines an E3-SCF ligase that contributes to the timing of mitotic entry. It has been shown that co-expression of NIPA and NPM-ALK results in constitutive NIPA phosphorylation. By mass spectrometry-based proteomics we identified nine serine/threonine residues to be significantly upregulated in NIPA upon NPM-ALK expression. Generation of phospho-deficient mutants of the respective phospho-residues specified five serine/threonine residues (Ser-338, Ser-344, Ser-370, Ser-381 and Thr-387) as key phosphorylation sites involved in NPM-ALK-directed phosphorylation of NIPA. Analysis of the biological impact of NIPA phosphorylation by NPM-ALK demonstrated that the ALK-induced phosphorylation does not change the SCFNIPA-complex formation but may influence the localization of NIPA and NPM-ALK. Biochemical analyses with phospho-deficient mutants elucidated the importance of NIPA phosphorylation by NPM-ALK for the interaction of the two proteins and proliferation potential of respective cells: Silencing of the five crucial NIPA serine/threonine residues led to a highly enhanced NIPA-NPM-ALK binding capacity as well as a slightly reduced proliferation in Ba/F3 cells.

NIPA As a Novel Regulator of Aging and Stress Response of the Primitive HSC Pool

Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Although HSCs numerically expand with age, their functional activity declines over time and the protection mechanism from DNA damage accumulation remains to be elucidated.NIPA (Nuclear Interaction Partner of ALK) is highly expressed in hematopoietic stem and progenitor cells, especially in the most primitive long-term repopulating HSCs (CD34-Flt3-Lin-Sca1+cKit+). Loss of NIPA leads to a significant exhaustion of primitive hematopoietic cells, where Lin-Sca1+cKit+ (LSK) cells were reduced to 40% of wildtype (wt) littermates (p<0.001). All LSK-subgroups, LT-HSCs (p<0.001), ST-HSCs (CD34+Flt3-LSK; p<0.01) and MPPs (CD34+Flt3+LSK; p<0.05) of NIPA deficient animals are affected and failed to age-related increase, whereas the lineage differentiation of Nipako/ko progenitor cells showed no gross differences. Myeloid depression by 5-FU treatment led to severely reduced HSC self renewal in Nipako/ko mice independent of age (p<0.001). Moreover, weekly 5-FU activation showed reduced survival of Nipako/ko vs. wt animals (11 vs. 14.5 days).To further examine the role of NIPA in HSC maintenance and exhaustion, we performed in vivo repopulationexperiments, where Nipa deletion causes bone marrow failure in case of competition, as Nipako/ko cells contributed to less than 10% of transplanted BM cells 6 month after transplantation (TX). According to this, colony formation assays and limiting dilution transplantation showed a dramatic reduction of competitive repopulation units (p<0.0001) in Nipako/ko animals. Serial LSK transplantation assays revealed loss of Nipa-deficient LSKs shortly after TX, whereas long-term repopulation capacity seemed to be maintained, suggesting a role of NIPA in critical stress response.To further investigate the stress response in Nipa-deficient HSCs, we irradiated LSKs with 3 Gy and stained for DNA-Damage foci by pH2ax. Remarkably, loss of NIPA led to significant higher numbers of pH2ax foci in irradiated HSCs (46% > 6 foci vs. 17% > 6 foci in wt cells) and highly increased the rates of apoptotic cells especially in the primitive CD34-LSK population.Taken together our results highlight the importance of the DNA damage response at HSC level for lifelong hematopoiesis and establish NIPA as a novel regulator of aging and stress response of the primitive HSC pool. DisclosuresNo relevant conflicts of interest to declare.

The F-Box Protein NIPA Limits Hematopoietic Stem Cell Survival and Transplantation Efficiency

Hematopoietic stem cells (HSCs) are characterized by their ability to self-renewal and multilineage differentiation. Since mostly HSCs exist in a quiescent state re-entry into cell cycle is essential for their regeneration and differentiation. We previously characterized NIPA (Nuclear Interaction Partner of ALK) as a F-Box protein that defines an oscillating ubiquitin E3 ligase and contributes to the timing of mitotic entry. To examine the function of NIPA in vivo, we generated Nipa deficient animals, which are viable but sterile due to a defect in testis stem cell maintenance.To further characterize the role of NIPA in stem cell maintenance and self-renewal we investigated hematopoiesis in Nipa deficient animals. FACS analyses of spleen cells and bone marrow (BM) showed differences in Leucocyte subpopulations. Measuring the CD4 and CD8 positivity within all Thy1.2+ cells, the balance in NIPA-/- T-lymphocytes is destabilised in favour of CD4 positive cells. Besides CD43/CD19 positive as well as CD43/B220 positive cells within all leukocytes are increased in NIPA deficient spleen cells. Analysing more primitive cells, FACS data of bone marrow showed significantly decreased numbers of Lin-Sca1+cKit+ (LSK) cells in NIPA-/- mice (age > 20 month), where LSKs were reduced to 40% of wildtype (wt) littermates (p=0,0171). Additionally, in such older NIPA-/- mice, only half the number of multipotent myeloid progenitors were detected in comparison to wt mice. To examine efficient response of stem cells to myeloid depression, mice were treated with 5-FU four days before BM harvest. We found that in NIPA-/- mice, both the number of myeloid progenitors as well as the number of LSKs were severely reduced compared to those in wt levels after 5-FU treatment (p<0.001). Interestingly, the reduction of progenitors and LSK cells was not dependent on age of the NIPA ko mice, suggesting a role for NIPA in stem cell activation or regeneration. This statement was studied in vitro by methylcellulose assays with 10 000 BM cells seeded in methylcellulose with cytokines and replated for three times after 10 days. Nipa deficient hematopoietic progenitors showed a reduced ability to proliferate and differentiate into colonies compared to their controls with an increasing difference after each replating (p(third replating) < 0.0001). Dynamic cell cycle analysis of seeded BM cells with BRDU and PI uncovered delayed cell cycle progress and mitotic entry in NIPA-/- BM cells in contrast to wt BM cells. Using competitive BM transplantation assay we investigated the role of NIPA for hematopoietic reconstitution in vivo. These experiments showed that NIPA-/- BM cells were severely deficient in hematopoietic recovery as recipient mice of NIPA-/- BM cells showed only half the amount of donor-derived peripheral blood cells in contrast to recipient mice of wt BM cells after 4, 11, 17 and over 23 weeks after transplantation. Furthermore NIPA-/- cells contributed only 7% in BM of transplanted mice 6 month after transplantation compared to 33% in recipients transplanted with wt BM cells (p<0.005). To further explore this defect in hematopoietic repopulation capacity and apply to more primitive progenitors serial transplantation assays were conducted with LSK cells transplanted together with support BM cells.Taken together our results demonstrate a critical role of NIPA in regulating the primitive hematopoietic compartment as a regulator of self-renewal, cycle capacity and HSC expansion. Disclosures:No relevant conflicts of interest to declare.

Loss Of NIPA Leads To Defects In The Repair Of DNA Double Strand Breaks In Mice

Regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry.Using a conditional knockout strategy we inactivated the gene encoding Nipa. Nipa-deficient animals are viable, but show a lower birth rate and a reduced body weight. Furthermore, Nipa-deficient males were sterile due to a block of spermatogenesis during meiotic prophase. Virtually no spermatocytes progress beyond a late-zygotene to early-pachytene stage and reach an aberrant stage, with synaptonemal complex disassembly and incomplete synapsis. Nipa-/- females are sub-fertile with an early and severe meiotic defect during embryogenesis with extensive apoptosis in early prophase (E13.5-E14.5).Here we report, that Nipa-/- meiocytes exhibit persistent cytological markers for DNA double strand break repair proteins (like DMC1, RAD51) in meiotic prophase with more than twice as many DMC1 foci as control animals. Kinetic analysis of the first wave of spermatogenesis showed increased DMC1/RAD51 foci in Nipa-/- cells as soon as early-pachynema cells appear (13-14 days post partum). Moreover, we show that Nipa deficiency does not lead to a defect in meiotic sex chromosome inactivation despite epithelial stage IV apoptosis. Nipa-deficient spermatocytes exhibit numerous abnormalities in staining of chromosome axis associated proteins (like SYCP3 and STAG3) indicating that chromosome axis defects were associated with compromised chromosome axis integrity leading to overt chromosome fragmentation.Further in vitro analyses with bleomycin treated MEFs displayed high pH2AX levels in cells lacking NIPA. Repair of DNA DSB seemed to be abolished in these cells as the pH2AX-level were sustained and still visible after 90 min of timecourse, where wildtype cells already repaired sides of DNA Damage. Consistent with these findings NIPA-deficient spleen cells showed compromised DNA Damage repair measured in a comet assay with a significantly longer olive tail moment in NIPA knockout cells under repair conditions.Taken together, the phenotype of Nipa-knockout mice is a definitive proof of the meiotic significance of NIPA and our results show a new, unsuspected role of NIPA in chromosome stability and the repair of DNA double strand breaks. Disclosures:No relevant conflicts of interest to declare.

NIPA Deficiency Leads To Acceleration Of The Lymphoma Development In EµMyc Mice

Timely degradation of proteins that control cell proliferation and apoptosis is an essential mechanism in keeping normal growth from turning into runaway malignancy. We previously reported the cloning of NIPA (Nuclear-Interaction-Partner-of-ALK) and characterized it as a F-Box-protein that defines an oscillating E3-ubiquitin-ligase. Using a conditional-knockout strategy we inactivated Nipa and found Nipa-/- animals to be viable, but sterile due to a block of spermatogenesis. Our studies demonstrate that loss of Nipa has no substantive effect on physiological cell cycle progression of primary MEFs indicating that this cell cycle checkpoint is inactive under optimal proliferation conditions. Interestingly, Nipa checkpoint control can be unmasked by oncogenic c-Myc-transformation. Here we show significant differences in c-Myc-induced transformation: Focus formation ability of c-Myc-infected Nipa-/- MEFs was greatly reduced. Moreover, Nipa-deficiency leads to premature senescence in cultured primary MEFs. Ectopic reexpression of Nipa resulted vice versa in delayed senescence of knockout MEFs. Next, we sought to know, whether increased apoptosis in Nipa-/- c-Myc-transduced MEFs is dependent on a functional p53-Axis. Interestingly, the effect of Nipa deficiency on c-Myc-mediated transformation was totally abolished by p53-knockdown. We observed no differences in focus formation ability or growth behaviour in Nipa-/- MEFs with inactivated p53, suggesting the importance of p53 in Nipa-induced cell death. Looking in more detail on the c-myc-p53 axis we detected a substantial increase in Arf-p19 levels in Nipa-/- cells. Moreover, Nipa-knockdown in Zn-inducible-Arf-NIH/3T3 cells lead to stabilization of Arf p19. To test the impact of these findings in a relevant in-vivo model we intercrossed Nipa-/- animals with a transgene EµMyc-Strain. Nipa-/-EµMycTG/wt animals developed lymphomas within a significantly shorter latency than Nipa+/+EµMycTG/wt animals. Furthermore, lymphomas of knockout animals were more aggressive. FACS- and biochemical-analyses showed no gross differences between Nipa-/- and wt lymphomas except highly elevated Arf-p19 levels in Nipa-/- lymphomas, pointing to an important role of Nipa in Myc-p19-signalling. Taken together our results highlight the functional importance of the Nipa-p53-axis in cell cycle regulation and suggest that deregulation of the protein provides a substantial contribution during the process of tumorigenesis. Disclosures:No relevant conflicts of interest to declare.

Targeted inactivation of nuclear interaction partner of ALK disrupts meiotic prophase

NIPA (nuclear interaction partner of ALK) is an F-box-like protein that monitors the timing of mitotic entry. Constitutively active NIPA delays mitotic entry by preventing accumulation of nuclear cyclin B1. Here, we have investigated the consequences of Nipa inactivation by using a conditional knockout strategy. Nipa-deficient animals are viable but show a lower birth rate and reduced body weight. Furthermore, Nipa-deficient males are sterile owing to a block of spermatogenesis during meiotic prophase. Whereas Nipa-/- mouse embryonic fibroblasts show no severe phenotype, Nipa-/- spermatocytes arrest during stage IV of the epithelial cycle with subsequent TUNEL-positive apoptosis resulting from improper synapsis, defects in the repair of DNA double-stranded breaks and synaptonemal complex formation. Moreover, we show nuclear accumulation of cyclin B1 with a subsequent premature increase in G2/M kinase activity in Nipa-/- spermatocytes. Together, these results reveal a novel role for NIPA in meiosis.

The F-Box Protein NIPA Regulates the Hematopoietic Stem Cell Pool

Abstract 2330Hematopoietic stem cells (HSCs) are characterized by their ability to self-renewal and multilineage differentiation. Since mostly HSCs exist in a quiescent state re-entry into cell cycle is essential for their regeneration and differentiation and the expression of numerous cell cycle regulators must be tightly controlled. We previously characterized NIPA (Nuclear Interaction Partner of ALK) as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. To examine the function of NIPA on vivo, we generated NIPA deficient animals, which are viable but sterile due to a defect in recombination and testis stem cell maintenance.To further characterize the role of NIPA in stem cell maintenance and self-renewal we investigated hematopoiesis in NIPA deficient animals. Peripheral blood counts taken at different ages revealed no apparent difference between NIPA knockout and wild type mice in numbers and differentiation. In contrast, looking at the hematopoietic stem cell pool, FACS analyses of bone marrow showed significantly decreased numbers of Lin-Sca1+cKit+ (LSK) cells in NIPA deficient animals, where LSKs were reduced to 40% of wild type littermates (p=0,0171). This effect was only apparent in older animals, where physiologically higher LSK numbers have to compensate for the exhaustion of the stem cell pool. Additionally, older NIPA deficient mice have only half the amount of multi myeloid progenitors (MMPs) in contrast to wild type animals. To examine efficient activation of stem cells to self-renew in response to myeloid depression, we treated young and old mice with the cytotoxic drug (5-FU) four days before bone marrow harvest. As expected, 5-FU activated hematopoietic progenitors in wild type animals, whereas NIPA deficient progenitors failed to compensate to 5-FU depression, e.g. LSKs of NIPA knockout mice were reduced to 50% of wild type levels (p<0.001), CD150+CD34+ Nipa deficient cells to 20% of wild type levels (p<0.0001). Interestingly, these effects were seen in all NIPA deficient animals independent of age, allowing us to trigger the self-renewal phenotype by activating the hematopoietic stem cell pool. Using competitive bone marrow transplantation assays, CD45.2 positive NIPA deficient or NIPA wild type bone marrow cells were mixed with CD45.1 positive wild type bone marrow cells and transplanted into lethally irradiated CD45.2 positive recipient mice. Thirty days after transplantation, FACS analysis of peripheral blood and bone marrow showed reduced numbers of NIPA knockout cells in comparison to NIPA wild type bone marrow recipient mice. This result was even more severe with aging of transplanted mice, where NIPA deficient cells were reduced to less than 10% of the level of wild type cells in bone marrow of sacrificed mice 6 months after transplantation, pointing to a profound defect in repopulation capacity of NIPA deficient HSCs.Taken together our results demonstrate a unique and critical role of NIPA in regulating the primitive hematopoietic compartment as a regulator of self-renewal, cycle capacity and HSC expansion. Disclosures:No relevant conflicts of interest to declare.

NIPA Checkpoint Control In Oncogenic Transformation Is Dependent on p53

AbstractAbstract 4178The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. Using a conditional knockout strategy we inactivated the gene encoding NIPA. NIPA-deficient animals are viable, but sterile due to a block of spermatogenesis. Moreover, our studies demonstrate that loss of NIPA has no substantive effect on the physiological cell cycle progression of primary MEFs indicating that this cell cycle checkpoint is inactive under optimal proliferation conditions. Interestingly, NIPA checkpoint control can be unmasked by oncogenic transformation by c-Myc. Here we show that transformed focus formation assays revealed highly significant differences in c-Myc-induced transformation in NIPA-deficient and wild-type MEFs. c-Myc transduction caused a pronounced upregulation of cyclin-B in NIPA-null MEFs, which was completely reversible by ectopic NIPA expression. The increased cyclin-B1 expression after c-Myc transduction in the absence of NIPA has considerable functional consequences for the cells: Focus formation ability of c-Myc-infected Nipa-/- MEFs was greatly reduced in comparison to wild-type MEFs (24.6% vs. 100%). Moreover, c-Myc expression caused 12.8% apoptotic subG1 cells in wild-type MEFs, whereas Nipa-/- MEFs were more affected by c-Myc-induced apoptosis (22.45%). Next, we sought to know, whether increased apoptosis in Nipa-deficient c-Myc transduced MEFs is dependent on a functional p53-Axis. Therefore, Nipa-wildtype and knockout MEFs were first infected with a retroviral Supernatant encoding for a p53Mir- and thereafter with the oncogene c-Myc. Interestingly the effect of Nipa knockout on c-Myc-mediated oncogenic transformation was totally abolished by the knock-down of p53. We observed no differences in focus formation ability or growth behaviour in Nipa-/- MEFs with inactivated p53 in comparison to wildtype cells, suggesting the importance of functional p53 in Nipa-induced cell death. Taken together, our data demonstrate that NIPA is required for efficient c-Myc transformation in a p53-dependent manner. Moreover, our results highlight the functional importance of the NIPA-p53 axis in cell cycle regulation and suggest that deregulation of the protein provides a substantial contribution during the process of tumorigenesis. Disclosures:No relevant conflicts of interest to declare.

NIPA Phosphorylation and Inactivation at G2/M Is Mediated by ERK2.

Abstract 2513Poster Board II-490Regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome. We previously reported cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, contributing to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3-ligase. The SCF-NIPA complex targets nuclear cyclin B1 for ubiquitination in interphase while phosphorylation of NIPA in late G2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1, a critical event for proper G2/M transition. Thus, SCF-NIPA executes an important G2/M checkpoint control. We recently specified three serine residues Ser 354, 359 and 395 implicated in NIPA phosphorylation at G2/M. These data suggest a sequential NIPA phosphorylation, where initial Ser 354 and 359 phosphorylation is most crucial for SCF-NIPA inactivation by dissociating the SCF-NIPA complex. Here we aimed to find the kinase responsible in this initial most important phosphorylation step. Using in vitro kinase assays we identified both ERK1 and ERK2 to phosphorylate NIPA with high efficiency. Mutation of either Ser 354 or Ser 359 abolished ERK-dependent NIPA phosphorylation. Inhibition of ERK1/2 activity in cell lines by specific inhibitors resulted in decreased NIPA phosphorylation at G2/M. To differentiate between phosphorylation by ERK1 and ERK2, we combined cell cycle analysis with stable expression of microRNA's targeting both isoforms. To this end NIH/3T3 cells were retrovirally transduced with microRNAs targeting ERK1 and 2 and cell cycle progression was analysed by BRDU/PI labeling. Using this approach, we are able to show that ERK2 but not ERK1 mediates NIPA phosphorylation at G2/M. Furthermore, ERK2 silencing leads to a distinct phenotype in cell cycle progression with a delay of ERK2 knockdown cells at the G2/M transition. Thus, our data indicate, that the recently described divergent functions of ERK1 and ERK2 in cell cycle regulation could be in part due to the differential ability of these kinases to phosphorylate and inactivate NIPA at G2/M. Since checkpoint proteins such as NIPA are constitutively inactivated in tumor cells ERK2 might represent an interesting target to reconstitute important cell cycle checkpoint controls in malignant cells. Disclosures:No relevant conflicts of interest to declare.

NIPA Checkpoint Control Is Essential for Efficient Oncogenic Transformation by C-Myc

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. Using a conditional knockout strategy we inactivated the gene encoding NIPA. NIPA-deficient animals are viable, but sterile due to a block of spermatogenesis. Moreover, our studies demonstrate that loss of NIPA has no substantive effect on the physiological cell cycle progression of primary MEFs indicating that this cell cycle checkpoint is inactive under optimal proliferation conditions. Interestingly, NIPA checkpoint control can be unmasked by oncogenic transformation by c-Myc. Here we show that transformed focus formation assays revealed higly significant differences in c-Myc-induced transformation in NIPA-null and wild-type MEFs. c-Myc transduction caused a pronounced upregulation of cyclin-B in NIPA-null MEFs, which was completely reversible by ectopic NIPA expression. This increased cyclin-B1 expression after c-Myc transduction in the absence of NIPA has considerable functional consequences for the cells: Focus formation ability of c-Myc-infected Nipa−/− MEFs was greatly reduced in comparison to wild-type MEFs (24.6% vs. 100%). Moreover, c-Myc expression caused 12.8% apoptotic subG1 cells in wild-type MEFs, whereas Nipa−/− MEFs were more affected by c-Myc-induced apoptosis (22.45%). By contrast, transduction with other oncogenes like k-Ras in p53 knockdown Nipa−/− and Nipa+/+ MEFs showed no differences in various transformation and apoptosis assays pointing out the exclusive role of the G2/M checkpoint NIPA in c-Myc induced transformation. Furthermore, we investigated the impact of these findings for the pathogenesis of c-Myc induced tumorigenesis in vivo. Recipient mice transplanted with c-Myc transduced wild-type bone marrow rapidly developed an AML-like disease (median survival 33 days) characterized by bone marrow infiltration and expression of the myeloid lineage markers CD11b and Gr1. In contrast, animals transplanted with c-myc transduced NIPA knockout BM showed a substantially delayed onset of leukemia and survived significantly longer compared to the control group (median survival 52 days, p<0.01). Taken together, our data demonstrate that NIPA is required for efficient c-Myc transformation in vitro and and in vivo in a murine bone marrow transplantation model. Moreover, our results highlight the functional importance of NIPA in cell cycle regulation and suggest that deregulation of the protein provides a substantial contribution during the process of tumorigenesis.

Multisite Phosphorylation of NIPA at G2/M.

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein (FBP) that defines an oscillating ubiquitin E3 ligase. The SCFNIPA complex targets nuclear cyclin B1 for ubiquitination in interphase while phosphorylation of NIPA in late G2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1. Here, we identify the region of NIPA that mediates binding to its substrate cyclin B1. In addition to the recently described serine residue 354, we specify 2 new residues, Ser-359 and Ser-395, implicated in the phosphorylation process at G2M within this region. Moreover, we found cyclin B1/Cdk1 to phosphorylate NIPA at Ser-395 in mitosis. Mutation of both Ser-359 and Ser-395 impaired effective inactivation of the SCFNIPA complex, resulting in reduced levels of mitotic cyclin B1. Furthermore, we aimed to identify the kinases involved in the initial phosphorylation of Ser-345. Therefore, we tested a panel of different kinases active at the G2M transition such as GSK3?, Casein kinase 2, PLK-1 and Erk1. Effective in vitro phosphorylation of NIPA could only be demonstrated with Erk-1. Moreover, we demonstrate an interaction of Erk-1 and NIPA at G2M but not in interphase cells. Binding of Erk-1 and NIPA led to phosphorylation at Ser-354 in vivo and could be blocked by the MEK-1/MEK-2 inhibitor PD98059. Together these data suggest a process of sequential phosphorylation where NIPA is initially phosphorylated by Erk-1 leading to the dissociation of NIPA from the SCF core complex. Once Ser-354 is phosphorylated, cyclin B1/CDK1 amplifies phosphorylation of NIPA, thus contributing to the regulation of its own abundance in early mitosis. In ALK positive lymphomas enhanced phosphorylation of NIPA at Ser 354 can be observed. We demonstrate that NPM-ALK leads to the activation of Erk-1, thereby phosphorylating and inactivating the SCFNIPA E3 ligase. Inactivation of SCFNIPA may have an important impact on the cell cycle turnover of lymphoma cells and thus for the pathogenesis of NPM-ALK induced lymphomas.

Multisite Phosphorylation of Nuclear Interaction Partner of ALK (NIPA) at G2/M Involves Cyclin B1/Cdk1

Nuclear interaction partner of ALK (NIPA) is an F-box-containing protein that defines a nuclear skp1 cullin F-box (SCF)-type ubiquitin E3 ligase (SCFNIPA) implicated in the regulation of mitotic entry. The SCFNIPA complex targets nuclear cyclin B1 for ubiquitination in interphase, whereas phosphorylation of NIPA in late G2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1. Here, we identify the region of NIPA that mediates binding to its substrate cyclin B1. In addition to the recently described serine residue 354, we specify 2 new residues, Ser-359 and Ser-395, implicated in the phosphorylation process at G2/M within this region. Moreover, we found cyclin B1/Cdk1 to phosphorylate NIPA at Ser-395 in mitosis. Mutation of both Ser-359 and Ser-395 impaired effective inactivation of the SCFNIPA complex, resulting in reduced levels of mitotic cyclin B1. These data are compatible with a process of sequential NIPA phosphorylation where cyclin B1/Cdk1 amplifies phosphorylation of NIPA once an initial phosphorylation event has dissociated the SCFNIPA complex. Thus, cyclin B1/Cdk1 may contribute to the regulation of its own abundance in early mitosis.

Control of Meiotic and Mitoic Progression by the F-Box Protein NIPA In Vivo.

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein (FBP) that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry.Using a conditional knockout strategy we inactivated the gene encoding the FBP NIPA to determined the consequences of NIPA deletion in vivo. The targeting construct was designed to flank exon 1 and 2 of the NIPA gene with loxP-sites. Deletion of this region was obtained by crossing the floxed mice to a cre-transgenic mouse strain expressing cre ubiquitously. NIPA deficiency did not affect the viability of NIPA −/− animals. Mating of heterozygotes yielded NIPA +/+, NIPA +/− and NIPA −/− offspring approximately at the expected Mendelian ratio. Although copulatory behavior was normal and vaginal plugs were produced, NIPA-deficient animals have a fertility defect. 100% of the tested NIPA −/− males and 60% of NIPA −/− females never produced progeny with young fertile wild-type mice. Interestingly, histological evaluation showed progressive testis atrophy in NIPA −/− males. Further analyses indicate a block in germ cell differentiation at the stage of meiotic prophase, no spermatides or spermatozoa were observed in NIPA-deficient animals. High levels of nuclear cyclin B1, a previously reported NIPA substrate, were present in NIPA-deficient germ cells. Furthermore, inactivation of NIPA leads to premature mitotic entry and subsequent mitotic catastrophe and TUNEL positive apoptosis in germ cells. Long-term studies of NIPA deficient mice may display a potential role of NIPA in tumor development.Since we found the most striking phenotype in high proliferating germ cells our results strongly confirm the cell cycle regulatory function of NIPA.

Inactivation of NIPA Results in Premature Mitotic Entry and Subsequent Mitotic Catastrophe.

Ubiquitin-mediated destruction of regulatory proteins marks a vital means of controlling cell cycle progresssion. The E3 ubiquitin-ligases are prominent in this process as they determine specificity of the ubiquitination process and thus regulate proteasomal degradation of target proteins. Recently, a class of E3 ligases referred to as SCF complexes has been identified. The substrate binding specifity within this class of E3 ligases is mediated by a class of molecules termed F-box proteins. We previously reported the cloning of NIPA (nuclear interaction partner of ALK) in complex with constitutively-active oncogenic fusions of ALK, which contribute to the development of certain lymphomas and sarcomas. Subsequently we characterized NIPA as a human F-box protein that defines an oscillating ubiquitin ligase (SCF-NIPA) which targets nuclear cyclin B1 in interphase. We have now determined the consequence of inactivating NIPA with regard to cell cycle regulation using an RNAi approach. Kinetic analysis of cell cycle phase transition times revealed a premature onset of Cdk1/cyclin B1 kinase activity and early mitotic entry in cells treated with NIPA siRNA. Cyclin B1 was shown to accumulate within the nucleus in these cells correlative to a reduced ubiquitination activity of the SCF-NIPA complex. Subsequent to premature mitotic entry, NIPA inactivated cells arrested in prometaphase and mitotic catastrophe was observed thereafter. We searched for relevant proteins involved in this process and found Survivin, a member of the inhibitor of apoptosis proteins (IAP) to be strongly downregulated in NIPA siRNA treated cells. Survivin has been shown to function as a mitotic checkpoint molecule that induces mitotic catastrophe subsequent to aberant mitosis when inactivated or downregulated. While the precise functional relationship between Survivin and NIPA is still under investigation, the current data distinguishes NIPA as a central molecule in timing mitotic entry. Given this function, NIPA directly influences the fidelity of DNA replication and segregation. Interference with NIPA function may therefore be an oncogenic principle that favours genomic instability in tumor cells.

NIPA Defines an SCF-Type Mammalian E3 Ligase that Regulates Mitotic Entry

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-box subunit of the SCF specifically recruiting a given substrate to the SCF core. Here we identify NIPA (nuclear interaction partner of ALK) as a human F-box-containing protein that defines an SCF-type E3 ligase (SCF(NIPA)) controlling mitotic entry. Assembly of this SCF complex is regulated by cell-cycle-dependent phosphorylation of NIPA, which restricts substrate ubiquitination activity to interphase. We show nuclear cyclin B1 to be a substrate of SCF(NIPA). Inactivation of NIPA by RNAi results in nuclear accumulation of cyclin B1 in interphase, activation of cyclin B1-Cdk1 kinase activity, and premature mitotic entry. Thus, SCF(NIPA)-based ubiquitination may regulate S-phase completion and mitotic entry in the mammalian cell cycle.

A Role for NIPA in DNA Damage Response.

The nuclear interaction partner of ALK (NIPA) is a nuclear protein identified by our group in a screen for NPM-ALK interaction partners. We recently reported that NIPA is an F-box protein that assembles with SKP1, Cul1 and Roc1 to establish a novel SCF-type E3 ubiquitin ligase. The formation of the SCFNIPA complex is regulated by cell cycle-dependent phosphorylation of NIPA that restricts SCFNIPA assembly from G1- to late S-phase, thus allowing its substrates to be active from late S-phase throughout mitosis.Proteins involved in cell cycle regulation frequently play a role in DNA damage checkpoints. We therefore sought to determine whether NIPA has a function in the cellular response to genotoxic stress. For this reason we treated NIH/3T3 cells with various DNA-damaging agents. Surprisingly, we observed phosphorylation of NIPA in response to some of these agents, including UV radiation. This phosphorylation was cell cycle phase independent and thus independent of the physiological cell cycle dependent phosphorylation of NIPA. The relevant phosphorylation site is identical to the respective site in the course of cell cycle-dependent phosphorylation of NIPA. Thus, phosphorylation of NIPA upon genotoxic stress would inactivate the SCFNIPA complex in a cell cycle independent manner. Interestingly, this phosphorylation site lies within a consensus site of the Chk1/Chk2 checkpoint kinases. These kinases are central to DNA damage checkpoint signaling. Chk1 is activated by ATR in response to blocked replication forks as they occur after treatment with UV. We performed experiments using the ATM/ATR inhibitor caffeine and the Chk1 inhibitor SB218078 to investigate a potential role of Chk1 in NIPA phosphorylation. Indeed, we found both inhibitors to prevent UV-induced phosphorylation of NIPA. Current experiments applying Chk1 knock-out cells will unravel the role of Chk1 in NIPA phosphorylation. Additional experiments were performed to investigate a function for NIPA in DNA-damage induced apoptosis. In this regard, we observed overexpression of NIPA WT to induce apoptosis in response to UV, whereas no proapoptotic effect was seen with the phosphorylation deficient NIPA mutant. Therefore, the phosphorylated form of NIPA may be involved in apoptotic signaling pathways.In summary, we present data suggesting a cell cycle independent function for NIPA. This activity is involved in DNA damage response and may be involved in regulating apoptosis upon genotoxic stress.

NIPA Targets Nuclear Cyclin B1 in a Cell-Cycle Dependent Manner.

Ubiquitin-mediated destruction of regulatory proteins marks the vital means of controlling cell cycle progresssion. The E3 ubiquitin-ligases are prominent in this process, as they allow the transfer of ubiquitin to the target protein and mediate substrate binding specificity. Recently, a new class of E3 ligases referred to as SCF complexes has been identified that consists of four subunits:SKP1, Cul1, Roc1 and an F-box protein, the latter of which determines substrate specifity.We previously reported the cloning of NIPA (nuclear interaction partner of ALK) in complex with constitutively-active oncogenic fusions of ALK, which contribute to the development of certain lymphomas and sarcomas. Subsequently we characterized NIPA as a human F-box protein that determines a novel SCF complex (SCFNIPA) whose cell cycle regulated activity is restricted to interphase to allow for substrate expression at G2/M and mitosis. Phosphorylation of NIPA in late S-phase was found to be the underlying mechanism of SCFNIPA inactivation.We have now identified the key mitotic regulator cyclin B1 to serve as the relevant substrate of the SCFNIPA complex. This targeting process is restricted to interphase and directed towards the nuclear pool of cyclin B1. Inactivation of NIPA by siRNAs results in nuclear accumulation of cyclin B1 in interphase and an elevation of cells in S-phase and mitosis. In contrast, expression of a phosphorylation deficient NIPA mutant that retains constitutive SCFNIPA activity throughout the cell cycle arrests cells at early prophase thus delaying mitotic entry. Both effects are likely attributable to either cyclin B1 accumulation in the case of NIPA inactivation by siRNA or untimely cyclin B degradation at G2/M upon expression of the constitutively active SCFNIPA complex. Cyclin B1 is physiologically kept cytoplasmic during interphase and premature nuclear accumulation has been associated with untimely mitotic entry, loss of checkpoint control and genomic instability. Our data provides a mechanism to inhibit premature nuclear accumulation of cyclin B1 in the mammalian cell cycle. NIPAs association with NPM-ALK of ALCL has been shown to be associated with NIPA phosphorylation and thus to the inactivation of the SCFNIPA complex. The mechanism described above may therefore provide a framework for understanding how this oncogene interferes with the physiologic regulation of cyclin B - a potential mechanism by which NPM-ALK transforms hematopoietic cells.