Nuclear Receptor-binding SET Domain Protein Research Articles

Frequency and Prognostic Impact of NUP98/NSD1 Translocations in Adult AML and MDS Patients

AbstractAbstract 1402Over 20 different fusion partner genes of NUP98 have been reported in hematological malignancies with the majority of NUP98 fusions occurring in acute myeloid leukemia (AML). Recently, a specific fusion of NUP98 with Nuclear receptor-binding SET domain protein 1 (NSD1) has been analyzed in a large cohort of pediatric and adult AML patients. In this report, 16.1% of pediatric AML samples and 2.3% of adult cytogentically normal (CN)-AML cases were found to be NUP98/NSD1-positive. NUP98/NSD1-positive patients had an adverse outcome. The aim of our study was to investigate the frequency, clinical features and the prognostic impact of NUP98/NSD1 in another large, uniformily treated adult AML cohort, and in patients with myelodysplastic syndromes, which frequently precede overt leukemia.We studied 504 younger AML patients (<60 years) and 193 MDS patients for the presence of NUP98/NSD1 translocations.Analysis in the AML cohort was also performed for mutations in FLT3-ITD, NPM1, DNMT3A, IDH1, IDH2. Additional mutation analyses were performed in the subgroup of CN-AML for CEBPA, MLL-PTD, WT1 and WT1 SNP rs16754, NRAS. The NUP98/NSD1 fusion was identified by a nested RT-PCR using patient-derived cDNA. cDNA from a patient with proven NUP98-NSD1 fusion was used as a positive control. NUP98/NSD1 fusions were identified in 7 out of 504 younger adult AML patients (1.4%) while the NUP98/NSD1 fusion was not found in any of the 193 MDS patients. In the AML cohort, NUP98/NSD1 positive patients showed a higher number of peripheral blood blasts (P=.002), while other clinical characteristics such as FAB-subtype, type of AML, hemoglobin levels, white cell count or platelet count did not significantly differ between patients with or without the NUP98/NSD1 fusion. The majority of NUP98/NSD1 positive patients (5 out of 7) showed a normal karyotype while one patient was found to have a del(9) and another patient an inv(3)(q21q26) with monosomy 7. We identified an association between FLT3-ITD and NUP98/NSD1 fusion in our cohort (P=.007, 5 patients with NUP98/NSD1 also had a FLT3-ITD). This finding confirms the data by Hollink et al. and suggests a possible functional link between FLT3-ITD and the NUP98/NSD1 fusion in leukemogenesis. NUP98/NSD1 fusions were not associated with other mutations like those suggested in epigenetic regulation (DNMT3A, IDH1 and IDH2). In CN-AML, we also looked for an association between BAALC, ERG, EVI1, MN1, MLL5 and WT1 expression but did not find a significant difference between the expression of these genes and NUP98/NSD1 fusion genes. Due to the low frequency of the aberration outcome analysis was performed for explorative purposes. Considering the whole AML cohort we did not detect a significant difference for OS and for RFS between NUP98/NSD1 positive and negative patients (OS, HR 1.6; 95%CI 0.66–3.88; P=.3; RFS, HR 2.33; 95%CI 0.74–7.30; P=.147). However, NUP98/NSD1 positive patients had significantly lower complete remission (CR) rates (43 vs. 77 percent, P=.037). When considering only patients with CN-AML the NUP98/NSD1 positive patients (n=5) had no significantly different OS and RFS (OS, HR 2.08; 95%CI 0.77–5.64; P=.15; RFS, HR 2.88; 95%CI 0.71–11.71; P=.14). Again, a significantly lower CR rate was observed in the NUP98/NSD1 positive patients compared to NUP98/NSD1 negative patients with CN-AML (40 vs. 80 percent, P=.03). Because of the association between FLT3-ITD and NUP98/NSD1 we also investigated the prognostic impact in the subgroup of CN-AML patients also positive for FLT3-ITD. Again, OS and RFS did not differ between NUP98/NSD1 positive and negative patients (OS, HR 1.31; 95%CI 0.47–3.64; P=.61; RFS, HR 2.04; 95%CI 0.49–8.57; P=.33). For this subgroup a trend towards a lower CR rate was observed for NUP98/NSD1 positive FLT3-ITD positive CN-AML patients (40 vs 75 percent, P=.08).In summary, our analysis confirms the presence but low incidence of the NUP98/NSD1 fusion gene in adult AML patients and the strong association with FLT3-ITD. The specific association of NUP98/NSD1 with FLT3-ITD mutations warrants clinical investigation with FLT3 inhibitors in these patients. Disclosures:No relevant conflicts of interest to declare.

NUP98-NSD1 Related Gene Expression Signature Is Strongly Associated with a Poor Prognosis in Pediatric Acute Myeloid Leukemia

Abstract 1391 BackgroundAcute myeloid leukemia (AML) is a complex disease caused by mutations, epigenetic modifications, and deregulated expression of genes leading to increased proliferation and decreased differentiation of hematopoietic progenitor cells. Many important molecular markers have already been discovered in AML that have not only helped to better characterize patients, but also helped to improve risk stratification. However, in a subset of AML patients, no known prognosis-associated cytogenetic aberrations or mutations were detectable. In hematological malignancies, 11p15 translocations involving the nucleoporin 98-kDa (NUP98) protein gene are relatively rare, but more than 20 different chromosomal rearrangements have been identified. The cryptic t(5;11)(q35;p15.5), which is frequently accompanied by deletion of the long arm of chromosome 5, del(5q), creates a fusion gene between NUP98 and the nuclear receptor-binding SET-domain protein 1 (NSD1) gene. Recently, Hollink et al identified NUP98-NSD1 gene fusion by high-resolution genome-wide copy number analysis and reverse transcription (RT)-PCR in pediatric and adult AML patients, and reported that NUP98-NSD1 gene fusion was a predictor of poor prognosis. Thus, we explored the frequency, gene expression pattern, and clinical impact of NUP98-NSD1 in a large cohort of Japanese pediatric AML patients. MethodsFrom January 2000 to December 2002, 318 patients were newly diagnosed with de novo AML. Of these, samples from 124 patients whose gene expression profiling data were available were analyzed, including 10 patients with FAB-M3 and 6 patients with Down syndrome (DS) who were treated on different treatment protocols. For the detection of NUP98-NSD1 gene fusion, direct sequencing was performed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Gene expression profiling data, which were performed before, were available for these 124 patients. Hierarchical clustering analysis was performed using Cluster and Tree View software. ResultNUP98-NSD1 fusion transcript was detected in 6 (4.8%) of 124 pediatric AML patients and a characteristic gene expression pattern related to these patients was also detected. Further hierarchical clustering analysis using the 87 probe sets which were differentially expressed in 6 NUP98-NSD1-positive patients did not detect a simple cluster of 6 NUP98-NSD1-positive patients but a relatively large cluster including 18 NUP98-NSD1-negative patients. When compared with 100 NUP98-NSD1 signature-negative patients, the 24 NUP98-NSD1 signature-positive patients frequently had normal karyotype (62.5%), and del(9q) (8.3%), but did not have any favorable chromosomal translocations such as t(8;21), inv(16) and t(15;17). The frequencies of M4 and M5 subtypes (14/24, P < 0.001) were also higher than those in NUP98-NSD1 signature-negative patients. In addition, they frequently had FLT3-ITD (12/24, P < 0.001), MLL-PTD (7/24, P = 0.124), NPM1 (3/24, P = 0.007) and WT1 (5/24, P = 0.037) mutations. In total, NUP98-NSD1 related gene expression signature (NUP98-NSD1 signature) represented 19% (24/124) of total patients and 58% (15/26) of cytogenetically normal patients in our cohort. Their 4-year overall survival (OS) and event-free survival (EFS) were poor when compared to 100 NUP98-NSD1 signature-negative patients (37.5% vs. 86.0% and 37.5% vs. 72.0%). ConclusionOur results indicated that Patients with NUP98-NSD1 related gene expression signature had extremely poor clinical outcome, irrespective of the presence of NUP98-NSD1 gene fusion. Most of the patients displaying the NUP98-NSD1 signature have been classified into an intermediate risk group, but their unfavorable outcome suggests that a high risk group is a more suitable stratification. These results suggest the presence of a new poor prognostic subgroup which revealed the value of the NUP98-NSD1 signature as prognostic markers in pediatric AML. Although further investigation is necessary, we believe that our work contributes to improve the risk stratification in pediatric AML. Disclosures:No relevant conflicts of interest to declare.

Persistent falcine sinus and unilateral renal agenesis in a girl with Sotos syndrome

Sotos syndrome is an overgrowth syndrome characterized by distinctive facial features, developmental delay, and macrocephaly. Here, we present a 10-year-old girl with prenatal and postnatal overgrowth, prominent forehead, pointed chin, and advanced bone age. She also has a persistent falcine sinus in the posterior falx cerebri, patent ductus arteriosus, unilateral renal agenesis, and scoliosis. A pituitary macroadenoma was also found with external compression of the inferior aspect of the optic chiasm. We identified a de novo missense mutation of the NSD1 (nuclear receptor-binding SET domain protein 1) gene in this patient. Computational three-dimensional structural analysis revealed that the NSD1 mutation induced major alterations.

Role for the nuclear receptor-binding SET domain protein 1 (NSD1) methyltransferase in coordinating lysine 36 methylation at histone 3 with RNA polymerase II function

The NSD (nuclear receptor-binding SET domain protein) family encodes methyltransferases that are important in multiple aspects of development and disease. Perturbations in NSD family members can lead to Sotos syndrome and Wolf-Hirschhorn syndrome as well as cancers such as acute myeloid leukemia. Previous studies have implicated NSD1 (KMT3B) in transcription and methylation of histone H3 at lysine 36 (H3-K36), but its molecular mechanism in these processes remains largely unknown. Here we describe an NSD1 regulatory network in human cells. We show that NSD1 binds near various promoter elements and regulates multiple genes that appear to have a concerted role in various processes, such as cell growth/cancer, keratin biology, and bone morphogenesis. In particular, we show that NSD1 binding is concentrated upstream of gene targets such as the bone morphogenetic protein 4 (BMP4) and zinc finger protein 36 C3H type-like 1 (ZFP36L1/TPP). NSD1 regulates the levels of the various forms of methylation at H3-K36 primarily, but not exclusively, within the promoter proximal region occupied by NSD1. At BMP4 we find that this reduces the levels of RNAP II recruited to the promoter, suggesting a role for NSD1-dependent methylation in initiation. Interestingly, we also observe that the RNAP II molecules that lie within BMP4 have inappropriate persistence of serine-5 phosphorylation and reduced levels of serine-2 phosphorylation within the C-terminal domain (CTD) of the large subunit of RNAP II. Our findings indicate that NSD1 regulates RNAP II recruitment to BMP4, and failure to do so leads to reduced gene expression and abrogated levels of H3K36Me and CTD phosphorylation.

Abstract 4863: The histone lysine-methyltransferase NSD1 and its functional role in non-small cell lung cancer (NSCLC)

Abstract NSD-1 (Nuclear receptor-binding SET domain protein 1) belongs to the epigenetic family of histone methyltransferases (HMTs), which are frequently found to be mutated in overgrowth syndromes. In cancer, NSD-1 is found as part of fusion proteins (e.g., NUP98-NSD1), which play a major role in the etiology of AML. In contrast to data from hematological tumors, little is known regarding the role of NSD-1 in cancers from solid origin, except that high level expression of NSD-1 has been correlated with poor survival in breast cancer patients (Miller, 2005). NSD-1 over-expression as well as copy number gain for the NSD-1 gene has been described in a variety of tumor cell lines. We studied the role of NSD-1 in the NSCLC line NCI-H460 using an inducible knockdown system (pSLIK). We found that knockdown of NSD-1 (&amp;gt;70% reduction in target mRNA expression) led to a significant decrease in cell proliferation as well as nearly 100% inhibition of anchorage-independent growth, whereas an empty vector transfected stable cell line did not. NSD-1 knockdown induced apoptosis in a time-dependent manner as measured by flow cytometry-based Annexin V staining. Cell death was found to follow a PARP-dependent mechanism suggesting that apoptosis is a contributor to growth inhibition/cell death. By conducting immuno-blot experiments against a panel of histone modifications, we examined putative in vivo histone substrates of NSD1. Our data suggest that NSD1 knockdown in NCI-H460 cells leads to a decrease in histone H4K20 trimethylation (64% inhibition after 24h), but did not affect H3K36 trimethylation as previously reported for NSD1 fusion proteins in AML. Further studies of changes in additional histone modifications are ongoing. In summary, we describe for the first time a functional role for NSD-1 in a NSCLC cell line with implications for this HMT in solid tumor growth/survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4863.

NUP98–NSD1 links H3K36 methylation to Hox-A gene activation and leukaemogenesis

Nuclear receptor-binding SET domain protein 1 (NSD1) prototype is a family of mammalian histone methyltransferases (NSD1, NSD2/MMSET/WHSC1, NSD3/WHSC1L1) that are essential in development and are mutated in human acute myeloid leukemia (AML), overgrowth syndromes, multiple myeloma and lung cancers. In AML, the recurring t(5;11)(q35;p15.5) translocation fuses NSD1 to nucleoporin-98 (NUP98). Here, we present the first characterization of the transforming properties and molecular mechanisms of NUP98-NSD1. We demonstrate that NUP98-NSD1 induces AML in vivo, sustains self-renewal of myeloid stem cells in vitro, and enforces expression of the HoxA7, HoxA9, HoxA10 and Meis1 proto-oncogenes. Mechanistically, NUP98-NSD1 binds genomic elements adjacent to HoxA7 and HoxA9, maintains histone H3 Lys 36 (H3K36) methylation and histone acetylation, and prevents EZH2-mediated transcriptional repression of the Hox-A locus during differentiation. Deletion of the NUP98 FG-repeat domain, or mutations in NSD1 that inactivate the H3K36 methyltransferase activity or that prevent binding of NUP98-NSD1 to the Hox-A locus precluded both Hox-A gene activation and myeloid progenitor immortalization. We propose that NUP98-NSD1 prevents EZH2-mediated repression of Hox-A locus genes by colocalizing H3K36 methylation and histone acetylation at regulatory DNA elements. This report is the first to link deregulated H3K36 methylation to tumorigenesis and to link NSD1 to transcriptional regulation of the Hox-A locus.