Nuclear Receptor-binding SET Domain Protein Research Articles

Identification of Novel NSD1 variations in four Pediatric cases with sotos Syndrome

ObjectiveSotos syndrome (SOTOS) is an uncommon genetic condition that manifests itself with the following distinctive features: prenatal overgrowth, facial abnormalities, and intellectual disability. This disorder is often associated with haploinsufficiency of the nuclear receptor-binding SET domain protein 1 (NSD1)gene. We investigated four pediatric cases characterized by early-onset overgrowth and developmental delay. The primary objective of this study was to achieve accurate genetic diagnoses.Design&MethodsA sequential analysis approach comprising chromosomal karyotyping, whole exome sequencing, and microarray analysis was conducted.ResultsAll four cases exhibited variations in the NSD1 gene, with the identification of four previously unreported de novo variants, each specific to one case.Specifically, Case 1 carried the NSD1 (NM_022455): c.2686 C > T(p.Q896X) variant, Case 2 had the NSD1 (NM_022455): c.2858_2859delCT(p.S953X) variant, Case 3 displayed a chromosomal aberration, chr5: 5q35.2q35.3(176,516,604–176,639,249)×1, which encompassed the 5′-untranslated region of NSD1, and Case 4 harbored the NSD1 (NM_022455): c.6397T > G(p.C2133G) variant.ConclusionThis study not only provided precise diagnoses for these cases but also supplied significant evidence to facilitate informed consultations. Furthermore, our findings expanded the spectrum of mutations associated with SOTOS.

NSD3 in Cancer: Unraveling Methyltransferase-Dependent and Isoform-Specific Functions.

NSD3 (nuclear receptor-binding SET domain protein 3) is a member of the NSD histone methyltransferase family of proteins. In recent years, it has been identified as a potential oncogene in certain types of cancer. The NSD3 gene encodes three isoforms, the long version (NSD3L), a short version (NSD3S) and the WHISTLE isoforms. Importantly, the NSD3S isoform corresponds to the N-terminal region of the full-length protein, lacking the methyltransferase domain. The chromosomal location of NSD3 is frequently amplified across cancer types, such as breast, lung, and colon, among others. Recently, this amplification has been correlated to a chromothripsis event, that could explain the different NSD3 alterations found in cancer. The fusion proteins containing NSD3 have also been reported in leukemia (NSD3-NUP98), and in NUT (nuclear protein of the testis) midline carcinoma (NSD3-NUT). Its role as an oncogene has been described by modulating different cancer pathways through its methyltransferase activity, or the short isoform of the protein, through protein interactions. Specifically, in this review we will focus on the functions that have been characterized as methyltransferase dependent, and those that have been correlated with the expression of the NSD3S isoform. There is evidence that both the NSD3L and NSD3S isoforms are relevant for cancer progression, establishing NSD3 as a therapeutic target. However, further functional studies are needed to differentiate NSD3 oncogenic activity as dependent or independent of the catalytic domain of the protein, as well as the contribution of each isoform and its clinical significance in cancer progression.

Chromatin regulation of transcriptional enhancers and cell fate by the Sotos syndrome gene NSD1

Nuclear receptor-binding SET-domain protein 1 (NSD1), a methyltransferase that catalyzes H3K36me2, is essential for mammalian development and is frequently dysregulated in diseases, including Sotos syndrome. Despite the impacts of H3K36me2 on H3K27me3 and DNA methylation, the direct role of NSD1 in transcriptional regulation remains largely unknown. Here, we show that NSD1 and H3K36me2 are enriched at cis-regulatory elements, particularly enhancers. NSD1 enhancer association is conferred by a tandem quadruple PHD (qPHD)-PWWP module, which recognizes p300-catalyzed H3K18ac. By combining acute NSD1 depletion with time-resolved epigenomic and nascent transcriptomic analyses, we demonstrate that NSD1 promotes enhancer-dependent gene transcription by facilitating RNA polymerase II (RNA Pol II) pause release. Notably, NSD1 can act as a transcriptional coactivator independent of its catalytic activity. Moreover, NSD1 enables the activation of developmental transcriptional programs associated with Sotos syndrome pathophysiology and controls embryonic stem cell (ESC) multilineage differentiation. Collectively, we have identified NSD1 as an enhancer-acting transcriptional coactivator that contributes to cell fate transition and Sotos syndrome development.

Evaluation of Cell Biology and Genetics using VIKOR Method

Cell biology and genetics are fields of heredity research. They enable experts to examine indigenous people's genetic data to identify their current state of health. By taking necessary precautions, one can protect their health using this technology. Cell biology has quickly expanded in the medical field and has emerged as the only treatment for issues with human reproduction. Genetics, or the science of genes and heredity, is all about the study of features that are passed down from one generation to the next. The subject of cell biology is the smallest units of life, cells, and their structures and functions. Understanding the structure and physiological functioning of single cells, and how cells interact and work together in great numbers to generate tissues and organisms, is a goal of cell biology. Cell biology is based on the notion that a cell is the basic unit of all life. Understanding the tissues and organisms that make up cells in great detail is made possible by concentrating on the cell. If you want to teach in a classroom, you can look into botany or zoology. If you're interested in biological or industrial sciences or medical research, you can choose from genetics, microbiology, or biotechnology. Cell biology encompasses both eukaryotic and prokaryotic cells and has a wide range of subtopics, including the research of cellular metabolism, cell communication, cell cycle, chemistry, and cell composition. Cells can be examined using a variety of microscopy techniques, cell culturing, and cell fractionation. There are two different cell types: prokaryotic and eukaryotic ones. Despite having differing morphologies (see Prokaryote, Eukaryote), eukaryotic organisms are remarkably comparable in terms of underlying molecular make-up and functions. Over the past 50 years, cell biology has seen remarkable expansion as a key area of basic science (1). Medicine makes use of information from basic science domains such as cell biology for the benefit of patients (2-4). A subfield of biology known as cell biology focuses on the cell, including its different types, structures, functions, and interactions with other cells. The subfields of cell biology include cell composition, cell cycle, cell communication, and cell metabolism. Research in several domains, including genetics, biochemistry, neuroscience, plant biology, molecular biology, microbiology, and immunology, is tied to those in cell biology. The VIKOR (VIšekriterijumsko Compromising Rangiranje) Optimal Replacement Select method is used in the evaluation of Epidermal Growth Factor Receptor, Fibroblast Growth Factor Receptors, Discoidin Domain Receptor, Mitogen-Activated Protein Kinase, and Nuclear Receptor-Binding SET Domain Protein alternatives for skin, lung, head/neck, and cervical cancer. Fibroblast Growth Factor Receptors have the highest rank, whereas Nuclear Receptor-Binding SET Domain Protein has the lowest rank.

Electrical Status Epilepticus during Sleep in a Male Filipino with Rare Nonsense Mutation Variant of Sotos Syndrome on Carbamazepine Monotherapy

Electrical status epilepticus during sleep (ESES) is an electrographic pattern associated with specific genetic disorders, brain malformations, and use of some antiseizure medications. This case report aims to present the management of ESES in Sotos syndrome (SoS) on carbamazepine.A nine-year-old Filipino male with clinical features suggestive of overgrowth syndrome presented with febrile seizure at one year old. Cranial imaging showed cavum septum pellucidum, corpus callosal dysgenesis, and ventriculomegaly. He was on carbamazepine monotherapy starting at three years old. A near continuous diffuse spike–wave discharges in slow wave sleep was recorded at nine years old hence shifted to valproic acid. Follow-up study showed focal epileptiform discharges during sleep with disappearance of ESES. Next generation sequencing tested positive for rare nonsense mutation of nuclear receptor binding set-domain protein 1 confirming the diagnosis of SoS.Advanced molecular genetics contributed to determination of ESES etiologies. To date, this is the first documented case of SoS developing ESES. Whether an inherent genetic predisposition or drug-induced, we recommend the avoidance of carbamazepine and use of valproic acid as first-line therapy.   &nbsp

Understanding the Roles of the NSD Protein Methyltransferases in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent non-skin cancer in the world. While immunotherapy has revolutionized the standard of care treatment in patients with recurrent/metastatic HNSCC, more than 70% of patients do not respond to this treatment, making the identification of novel therapeutic targets urgent. Recently, research endeavors have focused on how epigenetic modifications may affect tumor initiation and progression of HNSCC. The nuclear receptor binding SET domain (NSD) family of protein methyltransferases NSD1-NSD3 is of particular interest for HNSCC, with NSD1 and NSD3 being amongst the most commonly mutated or amplified genes respectively in HNSCC. Preclinical studies have identified both oncogenic and tumor-suppressing properties across NSD1, NSD2, and NSD3 within the context of HNSCC. The purpose of this review is to provide a better understanding of the contribution of the NSD family of protein methyltransferases to the pathogenesis of HNSCC, underscoring their promise as novel therapeutic targets in this devastating disease.

Identification of histone methyltransferase NSD2 as an important oncogenic gene in colorectal cancer

Colorectal cancer (CRC) is the second common cause of cancer-related human mortalities. Dysregulation of histone 3 (H3) methylation could lead to transcriptional activation of multiple oncogenes, which is closely associated with CRC tumorigenesis and progression. Nuclear receptor-binding SET Domain protein 2 (NSD2) is a key histone methyltransferase catalyzing histone H3 lysine 36 dimethylation (H3K36me2). Its expression, the potential functions, and molecular mechanisms in CRC are studied here. Gene Expression Profiling Interactive Analysis (GEPIA) bioinformatics results showed that the NSD2 mRNA expression is elevated in both colon cancers and rectal cancers. Furthermore, NSD2 mRNA and protein expression levels in local colon cancer tissues are significantly higher than those in matched surrounding normal tissues. In primary human colon cancer cells and established CRC cell lines, shRNA-induced silencing or CRISPR/Cas9-induced knockout of NSD2 inhibited cell viability, proliferation, cell cycle progression, migration, and invasion. Furthermore, NSD2 shRNA or knockout induced mitochondrial depolarization, DNA damage, and apoptosis in the primary and established CRC cells. Contrarily, ectopic NSD2 overexpression in primary colon cancer cells further enhanced cell proliferation, migration, and invasion. H3K36me2, expressions of multiple oncogenes (ADAM9, EGFR, Sox2, Bcl-2, SYK, and MET) and Akt activation were significantly decreased after NSD2 silencing or knockout in primary colon cancer cells. Their levels were however increased after ectopic NSD2 overexpression. A catalytic inactive NSD2 (Y1179A) also inhibited H3K36me2, multiple oncogenes expression, and Akt activation, as well as cell proliferation and migration in primary colon cancer cells. In vivo, intratumoral injection of adeno-associated virus (AAV)-packed NSD2 shRNA largely inhibited primary colon cancer cell xenograft growth in nude mice. Together, NSD2 exerted oncogenic functions in CRC and could be a promising therapeutic target.

5-Aminonaphthalene derivatives as selective nonnucleoside nuclear receptor binding SET domain-protein 2 (NSD2) inhibitors for the treatment of multiple myeloma.

Approximately 20% of multiple myeloma (MM) are caused by a chromosomal translocation t (4; 14) that leads to the overexpression of the nuclear receptor binding SET domain-protein 2 (NSD2) histone methyltransferase. NSD2 catalyzes the methylation of lysine 36 on histone H3 (H3K36me2) and is associated with transcriptionally active regions. Using high-throughput screening (HTS) with biological analyses, a series of 5-aminonaphthalene derivatives were designed and synthesized as novel NSD2 inhibitors. Among all the prepared compounds, 9c displayed a good NSD2 inhibitory activity (IC50=2.7μM) and selectivity against both SET-domain-containing and non-SET-domain-containing methyltransferases. Preliminary research indicates the inhibition mechanism of compound 9c by significantly suppressed the methylation of H3K36me2. Compound 9c specifically inhibits the proliferation of the human B cell precursor leukemia cell line RS4:11 and the human myeloma cell line KMS11 by inducing cell cycle arrest and apoptosis with little cytotoxicity. It has been reported that the anti-cancer effect of compound 9c is partly achieved by completely suppressing the transcriptional activation of NSD2-targeted genes. When administered intraperitoneally at 25mg/kg, compound 9c suppressed the tumor growth of RS4:11 xenografts invivo and no body weight loss was detected in the tested SCID mice.

High WHSC1L1 Expression Reduces Survival Rates in Operated Breast Cancer Patients with Decreased CD8+ T Cells: Machine Learning Approach.

Nuclear receptor-binding SET domain protein (NSD), a histone methyltransferase, is known to play an important role in cancer pathogenesis. The WHSC1L1 (Wolf-Hirschhorn syndrome candidate 1-like 1) gene, encoding NSD3, is highly expressed in breast cancer, but its role in the development of breast cancer is still unknown. The purpose of this study was to analyze the survival rates and immune responses of breast cancer patients with high WHSC1L1 expression and to validate the results using gradient boosting machine (GBM) in breast cancer. We investigated the clinicopathologic parameters, proportions of immune cells, pathway networks and in vitro drug responses according to WHSC1L1 expression in 456, 1500 and 776 breast cancer patients from the Hanyang University Guri Hospital, METABRIC and TCGA, respectively. High WHSC1L1 expression was associated with poor prognosis, decreased CD8+ T cells and high CD274 expression (encoding PD-L1). In the pathway networks, WHSC1L1 was indirectly linked to the regulation of the lymphocyte apoptotic process. The GBM model with WHSC1L1 showed improved prognostic performance compared with the model without WHSC1L1. We found that VX-11e, CZC24832, LY2109761, oxaliplatin and erlotinib were effective in inhibiting breast cancer cell lines with high WHSC1L1 expression. High WHSC1L1 expression could play potential roles in the progression of breast cancer and targeting WHSC1L1 could be a potential strategy for the treatment of breast cancer.

Mutation and expression alterations of histone methylation-related NSD2, KDM2B and SETMAR genes in colon cancers

Epigenetic dysregulation is a hallmark of cancers, and examples of its cancer-associated expression and mutation alterations are rapidly growing. Histone methylation, a process by which methyl groups are transferred to amino acids of histone proteins, is crucial for the epigenetic gene regulation. NSD2 (nuclear receptor-binding SET domain protein 2) and SETMAR are epigenetic regulators for histone methylation. KDM2B, also known as FBXL10, is a histone demethylase that targets histone methylation processes. They are known to be altered in many cancers, but somatic frameshift mutation and expression of these genes remain undetermined in many other subsets of cancers, including high microsatellite instability (MSI-H) colon cancer (CC). In this study, we analyzed mononucleotide repeats in coding sequences of NSD2, KDM2B and SETMAR genes, and found frameshift mutations in 10 %, 2 % and 1 % of CCs with MSI-H, respectively. Of note, there was no frameshift mutation of these genes in microsatellite stable (MSS) CCs. In addition, we discovered that 2 and 2 of 16 CRCs (12.5 % and 12.5 %) harbored intratumoral heterogeneity (ITH) of the NSD2 and KDM2B frameshift mutations, respectively. In the immunohistochemistry for NSD2, intensity of NSD2 immunostaining in MSI-H CC is decreased compared to that in MSS. These results suggest that NSD2 might be altered at multiple levels (frameshift mutation, mutational ITH and expression) in MSI-H CCs, and could be related to MSI-H cancer pathogenesis.

Long non-coding RNAs MACC1-AS1 and FOXD2-AS1 mediate NSD2-induced cisplatin resistance in esophageal squamous cell carcinoma

The nuclear receptor-binding SET domain (NSD) protein family encoding histone lysine methyltransferases is involved in cancer progression. However, the role of NSDs in esophageal squamous cell carcinoma (ESCC) remains unclear. Here we examined the expression of NSDs in cisplatin-resistant and parental ESCC cells and revealed the upregulation of NSD2 in cisplatin-resistant cells. Ectopic expression of NSD2 increased cisplatin resistance and attenuated cisplatin-induced apoptosis. Colony formation assay indicated that NSD2 overexpression enhanced long-term survival of ESCC cells after treatment with cisplatin. In contrast, knockdown of NSD2 inhibited ESCC cell proliferation and sensitized ESCC cells to cisplatin. Depletion of NSD2 augmented the cytotoxic effect of cisplatin on EC109 xenograft tumors. NSD2 stimulated long non-coding RNA MACC1-AS1 in ESCC cells. Knockdown of MACC1-AS1 impaired NSD2-induced cisplatin resistance. Moreover, MACC1-AS1 overexpression promoted ESCC cell proliferation and cisplatin resistance. Clinically, MACC1-AS1 was upregulated in ESCC relative to adjacent noncancerous tissues. High MACC1-AS1 levels were significantly associated with reduced overall survival of ESCC patients. There was a positive correlation between MACC1-AS1 and NSD2 expression in ESCC specimens. Taken together, MACC1-AS1 induced by NSD2 mediates resistance to cisplatin in ESCC and may represent a novel target to improve cisplatin-based chemotherapy.

High-throughput screening with nucleosome substrate identifies small-molecule inhibitors of the human histone lysine methyltransferase NSD2

The histone lysine methyltransferase nuclear receptor-binding SET domain protein 2 (NSD2, also known as WHSC1/MMSET) is an epigenetic modifier and is thought to play a driving role in oncogenesis. Both NSD2 overexpression and point mutations that increase its catalytic activity are associated with several human cancers. Although NSD2 is an attractive therapeutic target, no potent, selective, and bioactive small molecule inhibitors of NSD2 have been reported to date, possibly due to the challenges of developing high-throughput assays for NSD2. Here, to establish a platform for the discovery and development of selective NSD2 inhibitors, we optimized and implemented multiple assays. We performed quantitative high-throughput screening with full-length WT NSD2 and a nucleosome substrate against a diverse collection of bioactive small molecules comprising 16,251 compounds. We further interrogated 174 inhibitory compounds identified in the primary screen with orthogonal and counter assays and with activity assays based on the clinically relevant NSD2 variants E1099K and T1150A. We selected five confirmed inhibitors for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. We found that all five NSD2 inhibitors bind the catalytic SET domain and one exhibited apparent activity in cells, validating the workflow and providing a template for identifying selective NSD2 inhibitors. In summary, we have established a robust discovery pipeline for identifying potent NSD2 inhibitors from small-molecule libraries.

Incidentalome in Neurogenetics: Pathogenic Variant of NSD1 in a Patient With Spinocerebellar Ataxia (SCA).

Background: Genetic studies of late-onset sporadic ataxias (>40 years of age) are not routinely indicated. For unresolved cases, next-generation sequencing (NGS) tools, such as whole-exome sequencing (WES), are available for a definitive diagnosis.Case presentation: Our patient is a woman with a usual facial phenotype and anthropometry, who developed ataxia at 45 years of age, with no relevant family history and an initial clinical approach that ruled out common aetiologies. WES was performed when the patient was 54 years old. The results identified the heterozygous pathogenic variant c.248delA (p.N83MfsX4) in the nuclear receptor-binding SET domain protein 1 (NSD1; MIM 606681) gene (related to Sotos syndrome), which was not associated with ataxia and is not related to the patient's phenotype. Sanger sequencing of NSD1 in two different laboratories confirmed the variant.Conclusions: NGS findings generally offer valuable information that can be used for clinical decision-making. However, an incidental finding that leads to defining new clinical and bioethical actions is also possible. Consequently, the biological importance of this type of genetic “incidentalome” must be determined.

The Drosophila histone methyltransferase NSD is positively regulated by the DRE/DREF system.

The Drosophila nuclear receptor-binding SET domain protein (NSD) gene encodes the Drosophila ortholog of mammalian NSD family members that are important in many aspects of development and disease in humans. In this study, we observed that overexpression of Drosophila NSD in imaginal discs induces organ atrophy. Thus, to gain an understanding of the transcriptional regulation of the gene, we analyzed the NSD promoter region. First, we identified the presence of three putative DNA replication-related element (DRE) sequences in its promoter region, where DRE-binding factor (DREF) could bind for transcriptional activation. In the experiments with the fly GAL4-UAS system, we demonstrated that overexpressed DREF increased the endogenous NSD transcription. To confirm the role of DREF as a transcriptional activator on the NSD expression, we generated a series of luciferase reporter gene constructs containing deleted portions of the 5'-flanking regions as well as point mutations in the putative DRE sites. When transiently transfected into S2 cells, the deletion construct containing no DRE sites showed dramatic decrease in the NSD promoter activity, but only two sites near the transcriptional start site were important. Furthermore, we verified the direct interaction of DREF with the two positively cis-acting sequences on the NSD promoter by chromatin immunoprecipitation assay. Taken together, these results demonstrated that NSD is one of the downstream targets of the DRE/DREF pathway that is associated with various cellular processes in Drosophila, indicating that our findings may contribute to the understanding of molecular mechanisms in complex disorders associated with NSD family members in humans.

Overexpression of histone methyltransferase NSD in Drosophila induces apoptotic cell death via the Jun-N-terminal kinase pathway

The nuclear receptor-binding SET domain protein gene (NSD) family encodes a group of highly conserved SET domain-containing histone lysine methyltransferases that are important in multiple aspects of development in various organisms. The association of NSD1 duplications has been reported with growth retardation diseases in humans. In this study, to gain insight into the molecular mechanisms by which the overexpression of NSD1 influences the disease progression, we analyzed the gain-of-function mutant phenotypes of the Drosophila NSD using the GAL4/UAS system. Ubiquitous overexpression of NSD in the fly caused developmental delay and reduced body size at the larval stage, resulting in pupal lethality. Moreover, targeted overexpression in various developing tissues led to significant phenotype alterations, and the gain-of-function phenotypes were rescued by NSD RNAi knockdown. We also demonstrated that NSD overexpression not only enhanced the transcription of pro-apoptotic genes but also activated caspase. The atrophied phenotype of NSD-overexpressing wing was strongly suppressed by a loss-of-function mutation in hemipterous, which encodes a Drosophila Jun N-terminal kinase. Taken together, our findings suggest that NSD induces apoptosis via the activation of JNK, and thus contributes to the understanding of the molecular mechanisms involved in NSD1-related diseases in humans.

Nucleosome Binding Alters the Substrate Bonding Environment of Histone H3 Lysine 36 Methyltransferase NSD2.

Nuclear receptor-binding SET domain protein 2 (NSD2) is a histone H3 lysine 36 (H3K36)-specific methyltransferase enzyme that is overexpressed in a number of cancers, including multiple myeloma. NSD2 binds to S-adenosyl-l-methionine (SAM) and nucleosome substrates to catalyze the transfer of a methyl group from SAM to the ε-amino group of histone H3K36. Equilibrium binding isotope effects and density functional theory calculations indicate that the SAM methyl group is sterically constrained in complex with NSD2, and that this steric constraint is released upon nucleosome binding. Together, these results show that nucleosome binding to NSD2 induces a significant change in the chemical environment of enzyme-bound SAM.

Relapse Mechanism of FLT3-ITD Positive AML and Cell Differentiation Blockage

Introduction: Activating mutation of FLT3 by internal tandem duplications (ITD) is the most common molecular aberration found in AML. FLT3-ITD mutation induces a proliferation signal and is associated with leukocytosis and poor prognosis. The co-existence of FLT3-ITD with translocation t(5:11) that yields fusion transcript of Nucleoporin98 and the nuclear receptor binding SET-domain protein 1 (NUP98/NSD1) was recently reported to be associated with chemo-resistance. Relapse risk of the majority of FLT3-ITD positive AML patients who are negative to NUP98/NSD1 is increased compared to other AML with normal karyotype, despite similar remission rates. The mechanism of FLT3-ITD contribution to relapse development in patients who achieved remission is unknown.Aims: To explore potential mechanisms that allow relapse in FLT3-ITD positive AML in the presence or absence of NUP98/NSD1 fusion gene. In addition, time sequence of the emergence of both mutations is suggested from a case with relapse.Methods: Leukemic blasts derived from newly diagnosed or relapsed FLT3-ITD positive AML patients were enriched for FLT3-ITD mutated sub-clones by sorting according to CD34 expression. Genomic DNA was extracted from each sorted fraction and FLT3-ITD mutation allele load was quantitatively determined by GeneScane. Total RNA was extracted from FLT3-ITD positive patients and RT-PCR was performed to detect NUP98/NSD1 fusion mRNA. All patients received induction with intensive chemotherapy combination of Daunorubicin and Cytarabine and their outcome was recorded. Chemo-sensitivity assays using Ara-C were conducted on different leukemia cell lines sub-populations divided by their CD34 expression.Results: NUP98/NSD1 was identified in 4 of 19 (21%) FLT3- ITD positive adult AML patients with normal karyotype who had a compatible donor and were considered transplant eligible. Patients harboring both NUP98/NSD1 and FLT3-ITD (75%) had higher rate of induction failure than FLT3-ITD patients without NUP98/NSD1 (40%). To explore the correlation between FLT3-ITD and differentiation capacity, primary AML FLT3-ITD positive blasts were sorted into two distinct sub-populations according to CD34 expression (example is shown in fig. 1). In 14/19 patients DNA from CD34+ and CD34- leukemic blasts was successfully extracted and FLT3- ITD allele load was tested and recorded as the ratio between FLT3 normal and mutated alleles. Of these 14 patients, 3 experienced induction failure and 8 (58%) achieved complete remission but unfortunately eventually relapsed. FLT3-ITD allelic ratio (AR) was equally measured in both CD34+ and CD34- sub-populations in 7 patients (50%) while in 5 patients (35.7%) the mutated allele was restricted to the CD34 positive cells and 4/5 (80%) patients experienced relapse. In two patients (14%) who also expressed NUP98/NSD1, the FLT3-ITD mutated allele was restricted to CD34 negative sub-population. In one patient, NUP98/NSD1 was detected in relapse but not in diagnostic specimen. Cytotoxic assay confirmed that differentiation stage as determined by CD34 expression is fundamental for chemo-sensitivity of leukemic cells regardless of their genetic profile. CD34 positive cell lines as well as CD34+ sub-population of Kasumi-1 cell line were resistant to the Ara-C compare to CD34 negative cell lines or the matched Kasumi-1 CD34- sub-population (fig. 2).Conclusions: FLT3-IDT mutation is a late event during leukemogenesis. We observed NUP98/NSD1 that emerged as a new mutation on relapse in a FLT3-IDT positive patient, assuming this may also be a later event. Our strategy to sort blasts according to their CD34 expression enable us to describe the accumulation of FLT3-ITD sub-clones at the primitive stage that express CD34, therefore, these leukemic sub-clones may be enriched with early leukemic precursors. Such differentiating blockage results a higher portion of cells which survive chemotherapy among FLT3-ITD and hence increases relapse risk as an indirect effect. Inducing differentiation in FLT3-ITD positive AML should be further studied as a therapeutic strategy. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.