Inhibition of the nuclear export of p65 and IQCG in leukemogenesis by NUP98-IQCG

Abstract

NUP98 fuses with approximately 34 different partner genes via translocation in hematological malignancies. Transgenic or retrovirus-mediated bone marrow transplanted mouse models reveal the leukemogenesis of some NUP98-related fusion genes. We previously reported the fusion protein NUP98-IQ motif containing G (IQCG) in a myeloid/T lymphoid bi-phenoleukemia patient with t(3;11) and confirmed its leukemogenic ability. Herein, we demonstrated the association of NUP98-IQCG with CRM1, and found that NUP98-IQCG expression inhibits the CRM1-mediated nuclear export of p65 and enhances the transcriptional activity of nuclear factor-κB. Moreover, IQCG could be entrapped in the nucleus by NUP98-IQCG, and the fusion protein interacts with calmodulin via the IQ motif in a calcium-independent manner. Therefore, the inhibition of nuclear exports of p65 and IQCG might contribute to the leukemogenesis of NUP98-IQCG.

Keywords

NUP98-IQCG / nuclear export / NF-κB / CRM1

Introduction

The NUP98 gene encodes a 98-kDa nucleoporin (NUP98) that acts as a structural component of the nuclear pore complex and regulates the nucleocytoplasmic traffic of macromolecules across the nuclear envelope. A possible link between NUP98 and cancer has been proposed because of involvement of NUP98 in the chromosomal translocation of t(7;11) and its fusion with the HOXA9 gene in acute myeloid leukemia (AML) [ 1, 2]. Moreover, the NUP98 gene has been found in distinct chromosomal translocations with 34 different partner genes in hematological malignancies [ 3]. Most NUP98 translocations generate fusions between the phenylalanine–glycine (FG) repeats of NUP98 and the C-terminal domain of partner proteins. The FG repeats are speculated to function as docking sites with high affinity for nuclear transport receptors, such as importin b, CRM1, transportin, and the mRNA export receptor TAP [ 4– 7].

NUP98 fusion partner genes can be structurally and functionally divided into two categories: homeodomain (HD) genes and non-HD genes. HD genes can be subdivided into clustered “class I” HD genes (the HOX genes, such as HOXA9 [ 2], HOXC11 [ 8], and HOXD13 [ 9]) and non-clustered “class II” HD genes (such as HHEX [ 10] and PMX1 [ 11]). In fusion genes, the C-terminal DNA-binding HDs of HD genes are all retained, whereas the transactivation domains are replaced by the FG repeats of NUP98 [ 12]. The non-HD partners include a wide variety of genes, most of which perform unclear functions. Moreover, genes lacking known DNA binding domains may be involved in leukemogenesis. For example, the NUP98-DDX10 fusion contains an RNA helicase domain essential to sustain the transforming ability of the fusion gene in vitro [ 13]. Moreover, the NUP98-NSD1 fusion contains a SET2-family histone methyltransferase domain targeting H3K36, which is necessary to activate HOXA locus transcription and enforces myeloid progenitor self-renewal [ 14]. We have identified a novel fusion between NUP98 and IQCG from a de novo myeloid/T-lymphoid leukemia with t(3;11)(q29q13;p15)del(3)(q29), + 21 [ 15]. The FG repeats from NUP98 and the coiled-coil domain, along with the IQ motif from IQCG, were retained in the fusion protein. Although no homology among different non-HD partner genes for NUP98 has been observed, most of these unrelated molecules, including IQCG, possess a common coiled-coil domain believed to function in protein oligomerization [ 16].

Macrophage colony-stimulating factor (M-CSF) promotes mononuclear phagocyte survival and proliferation. The augmented proliferation of NUP98-IQCG-expressing BM cells upon M-CSF stimulation may partially result from the increased transcription of Csf1r [ 17]. The nuclear factor-kB (NF-kB) is an important transcription factor for expressions of cytokines and cell surface receptors in monocytes and its activation is crucial to M-CSF-induced monocyte survival [ 18, 19]. Five NF-kB family members have been identified in mammalian cells, namely, NF-kB1 (p105/p50), NF-kB2 (p100/p52), RELA (p65), cREL, and RELB, and most of which associate to form various hetero- and homodimeric dimers, which are sequestered in the cytoplasm by their inhibitor proteins, the IkBs. Many extracellular signals could activate NF-kB, that is, induce NF-kB nuclear translocation secondary to proteasome-mediated IkB degradation. Moreover, NUP98, as a mobile nucleoporin, is important in CRM1-mediated nuclear protein export [ 20]. Two other NUP98 fusions, namely, NUP98-HOXA9 and NUP98-DDX10, can entrap p65 in the nucleus and enhance NF-kB transcription in vitro [ 4]. We hypothesized that NUP98-IQCG may be associated to the NF-kB pathway via CRM1-mediated nuclear export regulation.

Our previous study showed that the NUP98-IQCG fusion protein induced acute myelomonocytic leukemia in mice by dysregulating the Hox/Pbx3 pathway [ 17]. Moreover, Iqcg was essential in mouse spermatogenesis [ 21] and IQCG maintained definitive hematopoiesis in zebrafish by regulating calcium/CaM-dependent protein kinase IV (Camk4) [ 22], an important gene for maintenance of hematopoietic stem cells and proliferation of myeloid leukemia cells [ 23, 24]. In this report, NUP98-IQCG was associated with CRM1. Additionally, the expression of NUP98-IQCG caused p65 nuclear accumulation, which was correlated with enhanced transcriptional activity of NF-kB by luciferase reporter assay in vitro. Furthermore, NUP98-IQCG could entrap IQCG in the nucleus and interact with calmodulin (CaM) via IQ motif, which might be related to the dysregulated calcium signaling pathway.

Materials and methods

Plasmid construction

The NUP98-IQCG fusion gene was first cloned from the BM sample obtained from an acute leukemia patient exhibiting t(3;11). The full-length coding sequences of the human NUP98 and IQCG were obtained by reverse transcription PCR and then cloned into the MigR1, mCherry, pEGFP (green fluorescent protein), and pFlag-CMV4 vectors. Moreover, NUP98, IQCG, NUP98-IQCG without the IQCG portion (NUP98-IQCGΔIQCG), NUP98-IQCG without the NUP98 portion (NUP98-IQCGΔNUP98), NUP98-HOXA9 [ 1], and CRM1 were cloned into the pEGFP vector. pFlag-NUP98-IQCGΔIQ was constructed using the Mut Express® II Fast Mutagenesis Kit (Vazyme) with the following primers: forward: 5′-GGTAAAACAGGATCTCTTGGTTGATAGCAAGGATTCAAAAGGCAAAGG-3′; reverse: 5′-CAAGAGATCCTGTTTTACCTTCTTCTTGC-3′.

Quantitative RT-PCR, co-immunoprecipitation (Co-IP), and Western blot

For gene expression analysis, RNA was extracted from the BM cells of mice using TRIzol (Invitrogen). The RNA was reverse transcribed using M-MLV reverse transcriptase (Invitrogen) and quantified using SYBR (Takara) on an Applied Biosystems 7900 Real Time PCR machine (Applied Biosystems). b-actin was used as the internal control. All primers used are listed in Table S1 (supplementary file). Co-IP and Western blot analysis were performed as previously described [ 22].

Luciferase reporter assays

HEK293T cells (2e5) were transfected using Lipofectamine 2000 (Invitrogen) with 100 ng of NF-kB luciferase reporter and 500 ng of either empty pEGFP vector or vector expressing NUP98-HOXA9 or NUP98-IQCG. We included 5 ng of pRL-TK (Promega), which expressed Renilla luciferase as a control in determining transfection efficiency. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) 48 h post-transfection, and the results were normalized to Renilla luciferase.

Immunofluorescence

To determine the locations of NUP98-IQCG and CRM1, HEK293T cells were transfected with pEGFP vector or NUP98-IQCG-expressing vector, and seeded on coverslips 24 h post-transfection. After another 24 h, the cells were fixed in 4% PFA for 10 min and then treated with 0.1% Triton X-100 for 15 min at room temperature. Anti-CRM1 primary antibodies (C-20) (Santa Cruz) and Alexa Fluor 594-conjugated secondary antibody (Invitrogen) were used after fixation and permeabilization. Hoechst staining was used to render the nucleus visible. The slides were then mounted with an anti-fade fluorescent reagent and examined under a confocal microscope (Leica). Anti-p65 (Cell Signaling Technology), anti-p50 (Santa Cruz) or anti-IkBa (Cell Signaling Technology) primary antibodies, and Alexa Fluor 594-conjugated or 647-conjugated secondary antibodies (Invitrogen) were used to determine the locations of p65, p50, and IkBa. For the leptomycin B (LMB) group, cells were treated with 10 nmol/L LMB for 2 h before fixation. HEK293T cells were transfected with mCherry-NUP98-IQCG combined with a pEGFP vector expressing NUP98, IQCG, or NIΔIQCG through the calcium transfection method to test the subcellular localization relationships between these proteins. To further investigate the subcellular localization of IQCG, the HEK293T cells were transfected with pEGFP-IQCG with mCherry or NUP98-IQCG-expressing vectors.

RNA-Seq

The total RNA was extracted from murine GFP⁺ c-Kit⁺ BM cells of the MigR1 empty vector control group or NUP98-IQCG-expressing group using an RNeasy Mini Kit (Qiagen). The RNA samples were prepared for sequencing using the TruSeq RNA Sample Preparation kit (Illumina). Libraries were multiplexed at two per lane and sequenced on HiSeq 2500 to obtain ~80 million paired end (2×101 bp) reads per sample.

Data analysis

Data were evaluated using Student’s t-test, and differences with a P<0.05 were considered statistically significant. The sequencing reads were aligned using TopHat [ 25] and read counts were extracted using HTSeq [ 26]. Differential expression analysis was performed using Edger R package [ 27]; hierarchical clustering was performed using the Ward method; and GO analysis was performed using the DAVID tool (http://david.abcc.ncifcrf.gov/) [ 28]. The statistical significance threshold for all gene ontology enrichment analyses was P<0.05 (Benjamini and Hochberg corrected for multiple comparisons). GSEA was performed using the GSEA Pre-ranked test in the GSEA software (version 2.1.0), with pathways defined by the KEGG PATHWAY Database (version 4.0) [ 29]. The significance of the gene set was empirically determined by 1000 gene set permutation tests.

Results

NUP98-IQCG dot formation is associated with CRM1

NUP98 is an important nucleoporin in CRM1-dependent nuclear protein export. Previous studies report that NUP98 and NUP98 fusion proteins, such as NUP98-HOXA9 and NUP98-DDX10, could colocalize and interact with CRM1 and, in turn, inhibit CRM1-mediated nuclear export [ 4, 20]. Moreover, NUP98-IQCG could form distinct dots in the nucleus similar to other NUP98 fusion proteins. Furthermore, CRM1 was mainly distributed in the nucleoplasm and the nuclear envelope in the HEK293T cells transfected with pEGFP empty vector; however, when we expressed NUP98-IQCG, the endogenous CRM1 colocalized with NUP98-IQCG in distinct dots in the nucleus. We then treated cells with LMB, a specific inhibitor of CRM1 cargo binding. After LMB for 2 h, the NUP98-IQCG nuclear dots were widely destroyed; CRM1 no longer colocalized with NUP98-IQCG and remained in the nucleoplasm and nuclear envelope, whereas the protein distribution of the cells transfected with empty vector was unchanged (Fig. 1). These results suggest that NUP98-IQCG dot formation, as well as the interaction between NUP98-IQCG and CRM1, depends on the cargo binding status of CRM1.

NUP98-IQCG causes significant nuclear retention of p65 but not of IkBa

Next, we hypothesized that NUP98-IQCG could change the localization of crucial transcription factors associated with CRM1-mediated nuclear export and dysregulate target genes involved in hematopoiesis regulation, which has a role in leukemogenesis. NUP98-IQCG-expressing BM cells showed augmented proliferation upon M-CSF stimulation, and NF-kB activation was crucial in M-CSF-induced monocyte survival [ 17– 19]. Therefore, we detected the location of p65 by transfecting either a pEGFP-empty or NUP98-IQCG-expressing vector into HEK293T cells (Fig. 2A). As shown in Fig. 2B, expression of NUP98-IQCG caused significant nuclear retention of p65 in human HEK293T cells, and the amount of the nuclear retention was related to the expression level of NUP98-IQCG. Moreover, NUP98 and NUP98-IQCG without the IQCG portion (NUP98-IQCGΔIQCG) were also able to entrap p65 in the nucleus to some extent. However, IQCG abolished the nuclear retention activity, as well as NUP98-IQCG without the NUP98 portion (NUP98-IQCGΔNUP98) (Fig. 2A and 2B). We counted the transfected cells with different subcellular localization of p65, and the relative proportions are shown in Fig. 2C.

We then speculated whether the nuclear retention of p65 induced by NUP98-IQCG expression was common. Therefore, we investigated the subcellular localization of p65 in Cos-7 cell line. As expected, the p65 nuclear accumulation was found in Cos-7 cells transfected with NUP98-IQCG (Fig. 2D). We have established a bone marrow transplantation (BMT) mouse model expressing NUP98-IQCG using a retroviral transduction system and confirmed the leukemogenic activity of this fusion gene in our previous study [ 17]. Immunofluorescence experiments were performed on the bone marrow cells from control (empty vector) and NUP98-IQCG mice to determine the impact of NUP98-IQCG on nuclear export of p65. In line with the mentioned cell lines, significant nuclear retention was seen in NUP98-IQCG mice through immunofluorescence staining (Fig. 2E).

It is well known that p65/p50 is the most predominant heterodimer that can translocate to the nucleus and bind to kB consensus sequences in many gene enhancers, which regulate diverse cellular functions, such as immune response, cell growth, and development. The IkB family can function as cytoplasmic inhibitor of NF-kB dimers in resting cells. Therefore, detecting the subcellular localizations of p50 and IkBs in NUP98-IQCG expressing cells is necessary. As shown in Fig. 3A, the fluorescence of p50 is dense in the nucleus, though some exist in cytoplasm. HEK293T cells were similar whether transfected with pEGFP-empty or NUP98-IQCG-expressing vector. Moreover, the nucleocytoplasmic traffic of IkBa, an important component of IkBs in the cytoplasm, was mediated by CRM1, and IkBa could be sequestered in the nucleus after LMB treatment [ 30]. Our results showed significant IkBa nuclear accumulation after LMB treatment, whereas no effect on the subcellular IkBa localization was observed in NUP98-IQCG-expressing cells (Fig. 3B). The same results were found in Cos-7 cells transduced with NUP98-IQCG (Fig. 3C and 3D). Thus, we infer that the p65 nuclear accumulation may correlate with CRM1 inhibition, and the retained p65 in nucleus can form complexes with nuclear p50 to activate NF-kB pathway.

NUP98-IQCG-induced p65 nuclear retention and NF-kB activation are associated with the inhibition of CRM1-mediated nuclear export

To determine the former notion, we cloned CRM1 and investigated its role in LMB and NUP98-IQCG-induced nuclear retention of p65 in HEK293T cell line. As shown in Fig. 4A and 4B, overexpressed CRM1 could relieve the nuclear retention of p65 induced by LMB treatment and NUP98-IQCG expression. Moreover, the NUP98-IQCG nuclear dots were destroyed in the presence of LMB, which was in accordance with the results in Fig. 1. The overexpressed CRM1 could protect the distinct dots of NUP98-IQCG from LMB-induced destruction and remain colocalized with the fusion protein (Fig. 4B). These results suggest that the nuclear retention of p65 is associated with the CRM1 inhibition. Afterwards, we detected the protein levels of p65, p50, and IkBa in HEK293T cells transfected with pEGFP or NUP98-IQCG-expressing vector, and found no significant protein level alteration induced by NUP98-IQCG (Fig. 4C). Finally, we used a NF-kB luciferase reporter plasmid harboring several NF-kB binding sites to investigate whether NUP98-IQCG expression could increase NF-kB-dependent transcriptional activities. Similar to the positive control for NUP98-HOXA9, NUP98-IQCG could significantly augment the transcriptional level of NF-kB (Fig. 4D). Therefore, our results indicate that NUP98-IQCG could induce the nuclear retention of p65 by inhibiting CRM1-mediated nuclear export, which might contribute to the activation of NF-kB.

NUP98-IQCG causes nuclear retention of IQCG

In contrast to most reported NUP98 fusion partners that localize in the nucleus, IQCG is a cytoplasmic protein. As shown in Fig. 5B, NUP98-IQCG formed distinct dots in the nucleus, both NUP98-IQCG without the IQCG portion and wild-type NUP98 colocalized with the fusion protein, indicating the occurrence of interactions among these proteins, and consistent with the results of a previous report [ 15]. Moreover, pEGFP-tagged IQCG could partially locate in the nucleus when co-transfected with mCherry-tagged NUP98-IQCG (Fig. 5B). To clarify this further, we tested and counted the subcellular localizations of pEGFP-IQCG in HEK293T cells when co-transfected with empty mCherry vector with or without LMB treatment (Fig. 5C). The statistical analysis further supported the conclusion that NUP98-IQCG expression caused nuclear retention of IQCG (Fig. 5D).

NUP98-IQCG interacts with CaM via IQ motif and the calcium signaling pathway is dysregulated in NUP98-IQCG mice

IQCG could interact with CaM, which is a primary receptor for calcium, in a calcium-independent manner [ 22]. Therefore, we investigated whether NUP98-IQCG could retain the ability to interact with CaM. In the co-immunoprecipitation (Co-IP) assays, NUP98-IQCG precipitated CaM when calcium was chelated by EGTA. Deletion of the IQ motif abolished the ability of NUP98-IQCG to precipitate CaM under the same condition (Fig. 6A). Thus, NUP98-IQCG retains the ability to interact with CaM via IQ motif in a calcium-independent manner. In addition to the nuclear localization of NUP98-IQCG, we speculated that the redistribution of IQ motif of IQCG would influence calcium homeostasis. As expected, GO analysis of dysregulated genes (FC>2; FDR<0.05) revealed the altered cytosolic calcium ion homeostasis in NUP98-IQCG-expressing c-Kit⁺ cells (Fig. 6B). GSEA analysis further showed the calcium signaling pathway was dysregulated (Fig. 6C). Quantitative RT-PCR confirmed that some genes that belong to CaM kinase cascades, such as Camk4, Camk2, and Camk kinase (Camkk), were downregulated in NUP98-IQCG mice (Fig. 6D).

Discussion

NUP98-IQCG was identified in a bi-phenotypic acute myeloid/T-lymphoid leukemia case exhibiting a complex karyotype containing a chromosomal translocation t(3;11)(q29q13;p15)del(3)(q29) and trisomy 21 [ 15]. We have established a murine BMT model with NUP98-IQCG showing an acute myelomonocytic leukemia phenotype that was similar to the myeloid involvement of the initial clinical case, suggesting that NUP98-IQCG could act as a driver of leukemogenesis. Mechanistically, we found that the Hox/Pbx3 pathway may be the dominant mechanism underlying the leukemogenesis of NUP98-IQCG [ 17].

In this report, careful analysis of the subcellular localization of NUP98-IQCG and CRM1 with or without LMB treatment indicated the association between NUP98-IQCG and CRM1. We then hypothesized that the CRM1-mediated nuclear export of some important transcription factors may be affected by NUP98-IQCG, which ultimately contributed to the leukemogenic effects of the fusion protein. Moreover, p65 was entrapped in nucleus by NUP98-IQCG, whereas p50 and IkBa were not affected in cell lines. The p65 nuclear accumulation was found in the bone marrow cells of NUP98-IQCG mice. We sought to reveal the mechanism underlying the abnormal localization of p65 induced by NUP98-IQCG fusion protein and found that the nuclear retention of p65 was associated with CRM1 inhibition. Luciferase reporter assay revealed that the expression of NUP98-IQCG enhanced the transcriptional activity of NF-kB, which was important in M-CSF-induced monocyte survival [ 19]. Our data suggest that nuclear retention of p65 might induce NF-kB activation. Because NUP98-IQCG-induced AML exhibits a relatively long latency, the fusion gene might require additional genetic abnormalities to induce full-blown leukemia. Therefore, we speculated that the dysregulated NF-kB pathway might, at least partially, contribute to the leukemogenic effects of NUP98-IQCG, and there remain other transcription factors that might be abnormally accumulated in the nucleus.

Current evidence indicates that many of the NUP98 fusion proteins act as aberrant transcription factors, and the coiled-coil domain-mediated oligomerization of a transcription factor (such as PML and ETO) could alter the interactions of this factor with transcriptional coregulators and promote oncogenic transformation [ 31]. Given that NUP98-IQCG lacks a known DNA binding domain, we suspect that the effect of NUP98-IQCG on the trans-regulation of genes may be mediated through protein oligomerization by the coiled-coil domain of the IQCG portion, including NUP98-IQCG and retained IQCG in nucleus.

On the other hand, both NUP98-IQCG and nuclear-retained IQCG can interact with CaM via the IQ motif in a calcium-independent manner. Because cells have a limited CaM pool, high levels of intra-nuclear NUP98-IQCG could influence CaM availability and, therefore, broadly affect the network of CaM targets in the nucleus [ 32]. GO and GSEA analyses of the gene expression profiles of the NUP98-IQCG leukemia BM cells provided evidence supporting the calcium signaling pathway dysregulation. Calcium signaling could be critical in cytokine synthesis and lymphocyte activation, development, and maturation [ 33]. Remodeling of calcium ion homeostasis is responsible for some of the aberrant behaviors of cancer cells, including their growth advantage, abnormal migration, invasion, and metastasis [ 34]. Therefore, IQCG nuclear export inhibition may contribute to the leukemogenic effects of NUP98-IQCG; the mechanism of this phenomenon, however, requires further investigation.

In summary, the present study elucidates that NUP98-IQCG can inhibit the CRM1-mediated nuclear export, and might result in p65 and IQCG nuclear accumulations, as well as NF-kB activation, thereby contributing to leukemogenesis.

References

[1] Nakamura T, Largaespada DA, Lee MP, Johnson LA, Ohyashiki K, Toyama K, Chen SJ, Willman CL, Chen IM, Feinberg AP, Jenkins NA, Copeland NG, Shaughnessy JD Jr. Fusion of the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia. Nat Genet 1996; 12(2): 154–158

[2] Borrow J, Shearman AM, Stanton VP Jr, Becher R, Collins T, Williams AJ, Dubé I, Katz F, Kwong YL, Morris C, Ohyashiki K, Toyama K, Rowley J, Housman DE. The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9. Nat Genet 1996; 12(2): 159–167

[3] Franks TM, Hetzer MW. The role of Nup98 in transcription regulation in healthy and diseased cells. Trends Cell Biol 2013; 23(3): 112–117

[4] Takeda A, Sarma NJ, Abdul-Nabi AM, Yaseen NR. Inhibition of CRM1-mediated nuclear export of transcription factors by leukemogenic NUP98 fusion proteins. J Biol Chem 2010; 285(21): 16248–16257

[5] Fontoura BM, Blobel G, Yaseen NR. The nucleoporin Nup98 is a site for GDP/GTP exchange on ran and termination of karyopherin beta 2-mediated nuclear import. J Biol Chem 2000; 275(40): 31289–31296

[6] Radu A, Moore MS, Blobel G. The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex. Cell 1995; 81(2): 215–222

[7] Blevins MB, Smith AM, Phillips EM, Powers MA. Complex formation among the RNA export proteins Nup98, Rae1/Gle2, and TAP. J Biol Chem 2003; 278(23): 20979–20988

[8] Taketani T, Taki T, Shibuya N, Kikuchi A, Hanada R, Hayashi Y. Novel NUP98-HOXC11 fusion gene resulted from a chromosomal break within exon 1 of HOXC11 in acute myeloid leukemia with t(11;12)(p15;q13). Cancer Res 2002; 62(16): 4571–4574

[9] Raza-Egilmez SZ, Jani-Sait SN, Grossi M, Higgins MJ, Shows TB, Aplan PD. NUP98-HOXD13 gene fusion in therapy-related acute myelogenous leukemia. Cancer Res 1998; 58(19): 4269–4273

[10] Jankovic D, Gorello P, Liu T, Ehret S, La Starza R, Desjobert C, Baty F, Brutsche M, Jayaraman PS, Santoro A, Mecucci C, Schwaller J. Leukemogenic mechanisms and targets of a NUP98/HHEX fusion in acute myeloid leukemia. Blood 2008; 111(12): 5672–5682

[11] Nakamura T, Yamazaki Y, Hatano Y, Miura I. NUP98 is fused to PMX1 homeobox gene in human acute myelogenous leukemia with chromosome translocation t(1;11)(q23;p15). Blood 1999; 94(2): 741–747

[12] Gough SM, Slape CI, Aplan PD. NUP98 gene fusions and hematopoietic malignancies: common themes and new biologic insights. Blood 2011; 118(24): 6247–6257

[13] Yassin ER, Abdul-Nabi AM, Takeda A, Yaseen NR. Effects of the NUP98-DDX10 oncogene on primary human CD34⁺ cells: role of a conserved helicase motif. Leukemia 2010; 24(5): 1001–1011

[14] Wang GG, Cai L, Pasillas MP, Kamps MP. NUP98-NSD1 links H3K36 methylation to Hox-A gene activation and leukaemogenesis. Nat Cell Biol 2007; 9(7): 804–812

[15] Pan Q, Zhu YJ, Gu BW, Cai X, Bai XT, Yun HY, Zhu J, Chen B, Weng L, Chen Z, Xue YQ, Chen SJ. A new fusion gene NUP98-IQCG identified in an acute T-lymphoid/myeloid leukemia with a t(3;11)(q29q13;p15)del(3)(q29) translocation. Oncogene 2008; 27(24): 3414–3423

[16] Hussey DJ, Dobrovic A. Recurrent coiled-coil motifs in NUP98 fusion partners provide a clue to leukemogenesis. Blood 2002; 99(3): 1097–1098

[17] Pan MM, Zhang QY, Wang YY, Liu P, Ren RB, Huang JY, Chen LT, Xi XD, Chen Z, Chen SJ. Human NUP98-IQCG fusion protein induces acute myelomonocytic leukemia in mice by dysregulating the Hox/Pbx3 pathway. Leukemia 2016; 30(7): 1590–1593 doi:10.1038/leu.2015.347

[18] Baldwin AS Jr. The NF-kB and IkB proteins: new discoveries and insights. Annu Rev Immunol 1996; 14(1): 649–681

[19] Wang Y, Mo X, Piper MG, Wang H, Parinandi NL, Guttridge D, Marsh CB. M-CSF induces monocyte survival by activating NF-kB p65 phosphorylation at Ser276 via protein kinase C. PLoS ONE 2011; 6(12): e28081

[20] Oka M, Asally M, Yasuda Y, Ogawa Y, Tachibana T, Yoneda Y. The mobile FG nucleoporin Nup98 is a cofactor for Crm1-dependent protein export. Mol Biol Cell 2010; 21(11): 1885–1896

[21] Li RK, Tan JL, Chen LT, Feng JS, Liang WX, Guo XJ, Liu P, Chen Z, Sha JH, Wang YF, Chen SJ. Iqcg is essential for sperm flagellum formation in mice. PLoS ONE 2014; 9(5): e98053

[22] Chen LT, Liang WX, Chen S, Li RK, Tan JL, Xu PF, Luo LF, Wang L, Yu SH, Meng G, Li KK, Liu TX, Chen Z, Chen SJ. Functional and molecular features of the calmodulin-interacting protein IQCG required for haematopoiesis in zebrafish. Nat Commun 2014; 5(5): 3811

[23] Kitsos CM, Sankar U, Illario M, Colomer-Font JM, Duncan AW, Ribar TJ, Reya T, Means AR. Calmodulin-dependent protein kinase IV regulates hematopoietic stem cell maintenance. J Biol Chem 2005; 280(39): 33101–33108

[24] Monaco S, Rusciano MR, Maione AS, Soprano M, Gomathinayagam R, Todd LR, Campiglia P, Salzano S, Pastore L, Leggiero E, Wilkerson DC, Rocco M, Selleri C, Iaccarino G, Sankar U, Illario M. A novel crosstalk between calcium/calmodulin kinases II and IV regulates cell proliferation in myeloid leukemia cells. Cell Signal 2015; 27(2): 204–214

[25] Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 2013; 14(4): R36

[26] Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 2015; 31(2): 166–169

[27] Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010; 26(1): 139–140

[28] Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009; 4(1): 44–57

[29] Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 2005; 102(43): 15545–15550

[30] Huang TT, Kudo N, Yoshida M, Miyamoto S. A nuclear export signal in the N-terminal regulatory domain of IkBα controls cytoplasmic localization of inactive NF-kB/IkBα complexes. Proc Natl Acad Sci USA 2000; 97(3): 1014–1019

[31] Minucci S, Maccarana M, Cioce M, De Luca P, Gelmetti V, Segalla S, Di Croce L, Giavara S, Matteucci C, Gobbi A, Bianchini A, Colombo E, Schiavoni I, Badaracco G, Hu X, Lazar MA, Landsberger N, Nervi C, Pelicci PG. Oligomerization of RAR and AML1 transcription factors as a novel mechanism of oncogenic activation. Mol Cell 2000; 5(5): 811–820

[32] Tran QK, Black DJ, Persechini A. Intracellular coupling via limiting calmodulin. J Biol Chem 2003; 278(27): 24247–24250

[33] Colomer J, Means AR. Physiological roles of the Ca²⁺/CaM-dependent protein kinase cascade in health and disease. Subcell Biochem 2007; 45: 169–214

[34] Stewart TA, Yapa KT, Monteith GR. Altered calcium signaling in cancer cells. Biochim Biophys Acta 2015; 1848(10 Pt B): 2502–2511

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg