NUP98 rearrangements in adult AML patients: Evaluation of clinical implications and identification of novel fusion partners

NUP98 rearrangements in adult AML patients: Evaluation of clinical implications and identification of novel fusion partners | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article NUP98 rearrangements in adult AML patients: Evaluation of clinical implications and identification of novel fusion partners Yasuhito Nannya, Kazuhisa Chonabayashi, Makoto Iwasaki, Junya Kanda, and 28 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6926420/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Jan, 2026 Read the published version in Leukemia → Version 1 posted 9 You are reading this latest preprint version Abstract NUP98 rearrangements represent a distinct, high-risk subtype of acute myeloid leukemia (AML), particularly in pediatric patients. However, their prevalence, genetic features, and clinical implications in adult AML remain poorly characterized. Using targeted-capture sequencing, we identified 41 cases with NUP98 rearrangements among 1569 AML cases, representing the majority of 44 NUP98 -rearranged cases detected across 4,753 myeloid neoplasms. Fifteen distinct fusion partners were detected, with NUP98::NSD1 and NUP98::HOXA9 being the most frequent. Notably, two novel fusions— NUP98::MEOX2 and NUP98::HOXA6 —were identified. Co-mutations were relatively infrequent; FLT3 -ITD and WT1 mutations were the most common, while NPM1 mutations were exclusive. FLT3 -ITD was significantly enriched in NUP98::NSD1 cases, whereas TET2 mutations were more frequent in NUP98::HOXA9 cases. Clonal hierarchy analysis suggested that NUP98 rearrangements occur early in leukemogenesis. NUP98 -rearranged AML exhibited higher relapse rates and shorter event-free survival. Specifically, NUP98::NSD1 was associated with a poor induction response, whereas NUP98::HOXA9 and NUP98 fusions with other partners showed higher remission rates but frequent relapse. Allogeneic hematopoietic stem cell transplantation was associated with better survival, underscoring its significance. These findings reveal the genetic and clinical heterogeneity of NUP98 -rearranged AML in adults and support its classification as a distinct entity, highlighting the need for fusion partner-specific therapeutic strategies. Health sciences/Medical research/Genetics research Health sciences/Diseases/Haematological diseases/Haematological cancer/Leukaemia/Acute myeloid leukaemia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Nucleoporin 98 ( NUP98 ) fusion genes represent a distinct molecular subtype, particularly enriched in pediatric acute myeloid leukemia (AML), where they account for ~ 8% of cases and are associated with poor clinical outcomes [ 1 – 4 ]. In contrast, NUP98 fusions are rare in adult AML, and their clinical and prognostic implications remain poorly defined [ 5 – 8 ]. To date, more than 30 distinct NUP98 fusion partners have been identified across a range of hematologic malignancies, including de novo and therapy-related AML, myelodysplastic syndromes (MDS), chronic myeloid leukemia, T-cell acute lymphoblastic leukemia, and mixed-phenotype acute leukemia [ 9 ]. All NUP98 fusion proteins retain the N-terminal FG repeat domain of NUP98 fused to the C-terminal portion of the partner proteins, which often contains chromatin-modifying or transcriptional regulatory domains. Recent findings indicate that NUP98 fusion proteins exhibit a distinct subcellular distribution compared to wild-type NUP98 , forming aberrant biomolecular condensates that affect chromatin architecture and gene expression, ultimately leading to the development of AML [ 10 , 11 ]. Many NUP98 rearrangements, such as NUP98::NSD1 , are cryptic on conventional cytogenetic analysis and may be underrecognized without molecular diagnostics [ 12 ]. Recent advances in next-generation sequencing technologies have revealed their relatively high incidence, even among adult AML cases. NUP98 fusion partners can be classified into two groups: those with and without a homeodomain. The homeodomain-containing partners include nine clustered homeobox genes ( HOXA9 [ 13 , 14 ], HOXA11 [ 15 , 16 ], HOXA13 [ 16 ], HOXC11 [ 17 ], HOXC13 [ 18 ], HOXD8 [ 19 ], HOXD11 [ 20 ], HOXD12 [ 21 ], and HOXD13 [ 22 ]) and seven non-clustered homeobox genes ( HHEX [ 23 ], GSX2 [ 24 ], PRRX1 [ 25 ], PRRX2 [ 26 ], POU1F1 [ 27 ], POU6F2 [ 28 ], and EMX1 [ 29 ]). Among these, NUP98::HOXA9 is the most frequently observed and appears more prevalent in Asian populations than in Western cohorts [ 6 , 30 ]. Non-homeobox fusion partners are more commonly observed and typically contain coiled-coil domains that facilitate oligomerization. Additionally, several non-homeobox partners harbor conserved structural motifs, such as plant homeodomain (PHD) domains ( BPTF [ 31 ], JADE2 [ 32 ], KDM5A [ 33 ], MLLT10 [ 24 ], NSD1 [ 12 ], NSD3 [ 34 ], PHF23 [ 35 ], and ASH1L [ 36 ]) and SET domains ( KMT2A [ 37 ], NSD1 , and NSD3 ). Specific NUP98 fusion proteins show a preferential association with particular leukemia subtypes. For instance, NUP98::NSD1 is the most prevalent and is predominantly found in myelomonocytic leukemia, whereas NUP98::KDM5A is often associated with megakaryoblastic leukemia [ 3 ]. Co-mutations are relatively infrequent in NUP98 -rearranged leukemias. While FLT3 -ITD and WT1 mutations are observed across various NUP98 fusions and are associated with adverse prognosis, other co-occurring genetic events are limited to specific patient populations and remain poorly characterized. Recent updates in the World Health Organization classification and the International Consensus Classification (ICC) have defined AML with NUP98 rearrangements as a distinct disease entity, and the high specificity of this abnormality for AML allows it to be diagnosed even when the blast percentage is less than 20%. In pediatric patients with AML, NUP98 rearrangements with NSD1 and KDM5A have been most frequently observed and predict poor outcomes. In contrast, NUP98 translocations with other partners, including HOXA9 , are uncommon even in pediatric AML, and their prognostic significance remains unclear. Accumulation of clinical outcomes associated with gene alterations has enabled risk stratification of adult AML according to detailed mutational profiling, such as the 2022 European LeukemiaNet (ELN 2022) risk classification. Although NUP98 rearrangements were identified as AML with other rare recurring translocations and categorized as an intermediate-risk group in the ELN 2022 risk classification, their exact clinical significance in adult AML cohorts remains to be determined. To address these issues, we conducted a comprehensive characterization of NUP98 -rearranged AML in Japanese adults using targeted-capture sequencing. Materials and methods Patient samples We analyzed 4753 myeloid neoplasm cases, including 1569 AML cases (defined as having a blast count ≥ 20%), from 54 collaborating institutes in Japan, using targeted-capture sequencing in our laboratory between January 2001 and December 2022. From this cohort, we enrolled 44 patients harboring NUP98 rearrangements (41 AML and 3 MDS with excess blasts). We also enrolled 213 AML cases without NUP98 rearrangements for the control cohort. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committees of Kyoto University (G0608, G0697) and all participating institutions. All patients provided written informed consent. Detection of NUP98 fusions from patients with novel NUP98 rearrangements RNA was isolated using the miRNeasy Micro/Mini Kit (Qiagen), following the manufacturer’s protocol. cDNA was synthesized using SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme (Thermo Fisher Scientific). For reverse transcription polymerase chain reaction (RT-PCR), the KOD One PCR Master Mix (TOYOBO) was used following the manufacturer’s protocol. The primers used for this analysis are listed in Supplementary Table 1. PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen) and subjected to Sanger sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). Strand-specific RNA sequencing was performed by Novogene. RNA fastq files were mapped to the human genome (GRCh37) by hisat2 ( https://daehwankimlab.github.io/hisat2/ ) and manually inspected using Integrative Genomics Viewer (IGV). Molecular monitoring To evaluate molecular residual disease (MRD), quantification of NUP98::MEOX2 and NUP98::HOXA6 fusion mRNA transcripts was performed using real-time RT-PCR (qRT-PCR). A set of primers and a probe were specifically designed to amplify NUP98::MEOX2 mRNA detected in our patient (forward primer, 5’-CTCTTGGTACAGGAGCCTTTG-3’; reverse primer, 5’-TGGGTTTGCTGTTGACTTCT-3’; probe, 5’-FAM-ACTACGACA-ZEN-GCCACTTTGGGCTTT-IBFQ-3’) (Integrated DNA Technologies). Two sets of primers and probes were designed to amplify WT1 [ 38 ] and NUP98::HOXA6 mRNAs detected in our patient ( WT1 : forward primer, 5’-ACAGGGTACGAGAGCGATAACCA-3’; reverse primer, 5’-CACACGTCGCACATCCTGAAT-3’; probe, 5’-FAM-CAACGCCCATCCTCTGCGGAGCCCA-TAMRA-3’, NUP98::HOXA6 : forward primer, 5’-GATTTAATACTACGACAGCCACTTTGG-3’; reverse primer, 5’-ATGGCTCCCATACACAGCAC-3’; probe, 5’-Cy5-TTTGGAGCCCCCCAGGCCC-BHQ2-3’) (Thermo Fisher Scientific). In these experiments, ABL1 was used as the internal control [ 39 ]. NUP98::MEOX2 transcript levels were assessed by absolute quantification. The copy numbers of NUP98::MEOX2 and ABL1 transcripts were derived by extrapolating the data from the standard curve from which the normalized NUP98::MEOX2 / ABL1 copy number ratio was calculated. NUP98::HOXA6 and WT1 transcript levels were assessed by relative quantification (or ΔΔCq method) to determine the change in gene expression relative to a diagnostic sample. Results Clinical characteristics of patients with NUP98 rearrangements A total of 44 myeloid neoplasm patients with NUP98 rearrangements were identified by targeted-capture sequencing, including 26 males and 18 females, with a median age of 51 years (range, 18–87 years). All patients had ≥ 10% blasts in the bone marrow or peripheral blood, thus meeting the criteria for AML with recurrent genetic abnormalities according to the ICC 2022 classification. We analyzed these 44 NUP98 -rearranged AML patients (defined by ICC 2022) and compared them to 213 control AML patients without NUP98 rearrangements subjected to targeted sequencing during the same period (Table 1, Supplementary Fig. 1A). Based on the baseline characteristics, age was significantly lower in the NUP98 -rearranged AML group than in the control group. Fifteen NUP98 rearrangement partners were identified in our patient cohort. The most prevalent rearrangement was NUP98::NSD1 , which was cryptic in all cases, as previously reported [ 12 ]. This rearrangement was identified in 18 AML patients by targeted-capture sequencing (Supplementary Table 2). The second most frequent rearrangement was NUP98::HOXA9 , which was different from the pediatric cases where NUP98::KDM5A rearrangement ranks second, but is consistent with findings from other Asian AML cohorts of adults [ 7 ], suggesting a difference in translocation partner profile according to the age of the affected cases. NUP98::HOXA9 was identified in 12 AML patients, and cytogenetic analysis revealed the characteristic t(7;11)(p15;p15) translocation in nearly all NUP98::HOXA9 -positive patients, with the exception of one patient whose cytogenetic analysis result was unavailable (Supplementary Table 2). NUP98 rearrangements with partners other than NSD1 or HOXA9 , hereafter referred to as NUP98 ::Others, are rare. NUP98::HOXA11 was identified in two AML patients. NUP98::TNRC18 [ 21 ], NUP98::TOP1 [ 40 ], NUP98::KDM5A , NUP98::PRRX1 , NUP98::PRRX2 , NUP98::DDX10 [ 41 ], NUP98::HOXD8 , and NUP98::HOXC13 were all identified in only one AML patient. NUP98::TNRC18 , NUP98::KDM5A , and NUP98::PRRX2 were cryptic, as previously reported (Supplementary Table 2) [ 42 – 44 ]. Notably, three previously unreported NUP98 rearrangements were identified in a patient with AML: NUP98-HOXA6 with t(7;11)(p15;p15), NUP98-EVX2 with t(2;11)(q31;p15), and cryptic NUP98-MEOX2 . Additionally, in one AML patient, NUP98 was rearranged with an intergenic region of chromosome 4q12 (Table 2). It should also be noted that in de novo AML, the partner genes of NUP98 were NSD1 (18 cases), HOXA9 (10 cases), and Others (8 cases), whereas in therapy-related AML, only NUP98 ::Others (5 cases) was observed (Table 1). Identification of fusion transcripts in patients with novel NUP98 rearrangements RT-PCR analysis was performed on peripheral blood and bone marrow samples for the four novel rearrangements. RNA sequencing was conducted and compared with the expected conformational changes from targeted-capture sequencing. As expected, the fusion partners of the NUP98-MEOX2 and NUP98-HOXA6 rearrangements were confirmed to be MEOX2 and HOXA6 , respectively, by RNA sequencing. In the NUP98-EVX2 patient, no NUP98::EVX2 fusion was detected by RNA-sequencing, and alternatively, two previously reported fusions, NUP98::HOXD11 [ 20 ] and NUP98::HOXD13 [ 22 ], were detected by RT–PCR (Fig. 1 A-C). However, of the two NUP98 fusions, NUP98::HOXD11 was not detected by RNA sequencing, suggesting that NUP98::HOXD13 mainly contributed to the pathogenesis in this case. In a patient with the NUP98 rearrangement with 4q12, a single NUP98::GSX2 fusion was detected by RT-PCR, which was previously reported in only one case of AML [ 24 ]. For patients with two novel NUP98 fusions, NUP98::MEOX2 and NUP98::HOXA6 , specific qRT-PCR assays were designed to evaluate the efficacy of the treatment (Fig. 1 D, E). Coexisting variants in NUP98 -rearranged AML A comparison of coexisting variants in NUP98 -rearranged AML, along with their associations with partner genes of NUP98 rearrangements, was performed. As previously reported, among the 44 patients, the most common concomitant mutations were FLT3 -ITD (n = 17) and WT1 (n = 17), with both mutations occurring significantly more frequently in NUP98 -rearranged patients ( P < 0.05 and P < 0.01, respectively) [ 4 , 45 ]. NUP98 rearrangements are mutually exclusive with NPM1 mutations, consistent with previous reports [ 8 , 46 ]. Copy number alterations from targeted sequencing data revealed that del(17p) was not detected in NUP98 -rearranged patients. Although two NUP98 -rearranged patients showed monoallelic TP53 mutations, neither harbored del(17p) at least at initial onset, suggesting that NUP98 -rearranged AML is highly exclusive to biallelic TP53 alterations. In addition, no splicing factor mutations were detected in our NUP98 -rearranged patients, although statistical significance was not reached (Fig. 2 A, Supplementary Fig. 2A, B). We next examined the differences in coexisting mutations by translocation partners. As previously reported, FLT3 -ITD was more closely associated with NUP98::NSD1 than with NUP98::HOXA9 and NUP98 ::Others ( P < 0.001, 67%, 0%, and 36%, respectively) [ 6 ]. Conversely, TET2 mutations were only detected in patients with NUP98::HOXA9 and NUP98 ::Others in our NUP98 -rearranged cohort ( P < 0.01, 0%, 42%, and 7%, respectively) (Fig. 2 B). Next, using the Bradley-Terry model, the sequential order of mutation acquisition and the resulting clonal hierarchy of NUP98 -rearranged AML were reconstructed. NUP98 rearrangements preceded most other aberrations (such as FLT3 , NRAS , PTPN11 , and WT1 mutations), except for mutations in clonal hematopoiesis (CH)-related genes like DNMT3A and TET2 , indicating that NUP98 rearrangements are initiating and driving events in AML (Fig. 2 C, D, Supplementary Fig. 2C). In the case harboring the NUP98::HOXA6 fusion, the IDH1 mutation, which coexisted with the NUP98 rearrangement at the time of initial disease, disappeared after chemotherapy, while the IDH2 mutation was acquired. This suggests that NUP98 rearrangements precede IDH1 and IDH2 mutations. (Fig. 1 E) Clinical outcome and prognostic relevance Clinical outcome and prognostic relevance To investigate the clinical significance of NUP98 rearrangements, we first evaluated the response to initial induction therapy. The median follow-up duration among survivors was 3.0 years (interquartile range [IQR], 2.2–5.2) in the NUP98 -rearranged group and 4.2 years (IQR, 2.5–5.8) in the non- NUP98 -rearranged control group ( P = 0.683) (Table 1). The overall hematologic complete or partial remission (CR/PR) rate after one course of treatment was significantly lower in the NUP98 -rearranged cohort than in the control cohort (45.2% versus 67.6%, P = 0.008) (Fig. 3 A). This poor response of the NUP98 -rearranged cohort was largely explained by the markedly lower CR/PR rate in the NUP98::NSD1 subgroup (16.7%, P < 0.001), in contrast to the NUP98::HOXA9 (70.0%, P = 1) and NUP98 ::Others (64.3%, P = 1) subgroups, which showed response rates comparable to the control cohort (Fig. 3 B). Patients with NUP98 rearrangements exhibited a significantly higher risk of relapse and inferior event-free survival (EFS) in univariate analysis, although the overall survival (OS) did not differ significantly from the control group (median OS: 2.89 vs 2.74 years, P = 0.54; median EFS: 0.082 vs 1.06 years, P < 0.001; relapse: P < 0.001; non-relapse mortality [NRM]: P = 0.13) (Fig. 3 C-F, Supplementary Fig. 3A). We analyzed the prognostic impact of clinical variables within the NUP98 -rearranged cohort. We first examined the impact of age, sex, fusion partners, and co-mutations on OS and EFS using univariate analysis (Supplementary Table 3). As expected, age above the median (51 years) predicted a shorter OS (median OS: 0.86 vs not reached [NR] years, P = 0.019) but not affected EFS (median EFS: 0.082 vs. 0.082 years, P = 0.61). We assessed the prognostic impact of different fusion partners. Although not significant, NUP98::HOXA9 cases tended to have longer OS ( P = 0.17) and EFS ( P = 0.081) than NUP98::NSD1 and NUP98 ::Others subgroups (Supplementary Fig. 3B, C). Therefore, we combined the NUP98::NSD1 and NUP98 ::Others groups into a single NUP98 ::non- HOXA9 subgroup, which showed a trend toward shorter OS ( P = 0.062) and EFS ( P = 0.061) compared to NUP98::HOXA9 cases (Supplementary Fig. 3D, E). Next, we analyzed the impact of co-mutations on outcomes (Fig. 4 A-D, Supplementary Fig. 4A, B). Among the NUP98 -rearranged cohort, patients with FLT3 -ITD had significantly shorter OS (median: 0.767 vs. NA years, P < 0.001) and EFS (median: 0.082 vs. 0.090 years, P = 0.011), while patients with NRAS mutation had significantly longer OS (median: NR vs 1.59 years, P = 0.034) and EFS (median: 0.090 vs. 0.082 years, P = 0.025), compared to the FLT3 -ITD-negative or NRAS -unmutated group, respectively, in univariate analysis. Thus, age, fusion partner ( HOXA9 or not), and co-mutations with FLT3 -ITD or NRAS were subjected to multivariate analysis using the Cox regression model (Fig. 4 E). After correction, significantly inferior OS (hazard ratio [HR] = 11.48; 95% confidence interval [CI]: 3.15–41.75; P < 0.001) was observed only in FLT3 -ITD-positive patients compared to the reference group. Based on the multivariate model, we constructed a simple scoring model by assigning 1 point each to elderly age and FLT3 -ITD. The NUP98 -rearranged cohort was classified into three risk groups according to the total score (low risk: score = 0, intermediate risk: score = 1, high risk: score = 2), showing a significant difference in OS (median: NR vs. 2.047 vs. 0.516 years, P < 0.001, Fig. 4 F), but this risk stratification system was not effective in predicting EFS (median: 0.090 vs. 0.082 vs. 0.082 years, P = 0.31, Supplementary Fig. 4C). We then explored the impact of NUP98 rearrangements on outcomes in the context of ELN 2022 risk stratification (Fig. 5 A, B, Supplementary Fig. 5A-C). Compared to patients with favorable-risk ELN2022 AML without NUP98 rearrangements, those with NUP98 rearrangements had significantly lower OS (median: 2.89 vs NR years, P = 0.01) and EFS (median: 0.082 vs 2.13 years, P < 0.001). In contrast, patients with NUP98 rearrangements had comparable OS to both intermediate- and adverse-risk ELN2022 control groups (median:2.89 vs 4.07 years, P = 0.4; and 2.89 vs 1.60 years, P = 0.3, respectively), although significant differences were observed in EFS (median: 0.082 vs 1.06 years, P < 0.001; and 0.082 vs. 0.110 years, P = 0.03, respectively). Finally, we examined the prognostic impact of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the analysis including all patients, patients with NUP98 rearrangements who underwent allo-HSCT showed higher overall survival compared to those who did not, similar to patients without NUP98 rearrangement (median: 0.77 vs NR years, P = 0.005) (Fig. 5 C, D). When limited to patients aged 70 years or younger, OS showed a trend toward improvement, although the difference was not statistically significant in the NUP98 -rearranged cohort (median: 2.89 vs NR years, P = 0.14) (Supplementary Fig. 5D, E). Discussion NUP98 -rearranged AML has emerged as a distinct and heterogeneous disease entity, first identified in pediatric cohorts and more recently recognized in adult populations. Although several recent studies have provided deep insights into this fusion family in adult AML, a comprehensive study encompassing fusion partners, clinical phenotypes, coexisting mutations, and treatment outcomes remains lacking. In this study, we comprehensively analyzed the molecular and clinical features of NUP98 rearrangements in a large cohort of Japanese adults with AML. Among the 1569 cases of AML, we identified 41 cases (2.6%) harboring NUP98 rearrangements. Although less frequent than in pediatric AML, these fusions represent a distinct and non-negligible subset in adults. The most frequent fusion types identified were NUP98::NSD1 and NUP98::HOXA9 , which is consistent with prior reports, especially in Asian cohorts. Notably, we also detected novel fusions, such as NUP98::MEOX2 and NUP98::HOXA6 , highlighting the diversity and complexity of NUP98 rearrangements. To characterize these novel fusions, we developed fusion transcript-specific qRT-PCR assays, underscoring their diagnostic value and potential utility in MRD monitoring. Furthermore, in one case where a novel NUP98-EVX2 rearrangement was predicted at the DNA level, RNA analysis identified NUP98::HOXD13 as the actual fusion transcript, highlighting the importance of transcript-level validation for the accurate interpretation of atypical rearrangements. Co-mutation analysis revealed that FLT3 -ITD and WT1 mutations were significantly enriched in NUP98 -rearranged AML, supporting the notion of a synergistic leukemogenic effect. In contrast, mutations commonly observed in adult AML, such as NPM1 , splicing factor genes, and biallelic TP53 mutations, were notably absent, suggesting a distinct leukemogenic trajectory for NUP98 -rearranged AML. The underlying biology of NUP98 -rearranged AML appears to be influenced by specific fusion partners. We observed a significant overlap of cooperative WT1 mutations across different fusion partners, whereas FLT3 -ITD was markedly enriched in NUP98-NSD1 cases. While NUP98 rearrangements generally preceded most other aberrations, regardless of the partners, we found that NUP98::HOXA9 emerged on CH clones in a considerable number of patients in our study. In contrast, CH-related mutations were rare in patients with NUP98::NSD1 . Clinically, our study provides valuable data on treatment strategies for adult AML with NUP98 rearrangements. We observed that patients harboring NUP98 rearrangements had significantly higher relapse rates and lower EFS, although OS was not significantly different compared to those without NUP98 rearrangements. In a previous AML study, NUP98::NSD1 fusion was associated with poor outcomes in both pediatric and adult AML, with a 4-year OS as low as 11% in adult cases [ 46 ]. Similarly, another study on adult AML reported dismal outcomes for patients with the NUP98::HOXA9 fusion, with a median OS of 13.5 months [ 30 ]. Although cross-trial comparisons must be interpreted with caution, our cohort demonstrated relatively favorable survival outcomes. In our study, 22 out of 44 patients (50%) with NUP98 rearrangements underwent allo-HSCT, compared to only two out of 10 patients (20%) in the NUP98::NSD1 study and only four out of 11 patients (36%) in the NUP98::HOXA9 study. The relatively high allo-HSCT rate may have contributed to the improved outcomes observed in our cohort. A recent adult AML study reported 4-year OS rates of 65.2% in the NUP98::NSD1 group and 66.7% in the non- NUP98::NSD1 group (including NUP98::HOXA9 ), which appear higher than those observed in our cohort [ 6 ]. In that study, 25 of 51 patients (49%) underwent allo-HSCT, which is comparable to our cohort. Therefore, the superior OS observed in that cohort may be attributed to the greater proportion of patients receiving salvage regimens that included venetoclax or FLT3 inhibitors. We also noted differences in the efficacy of the therapeutic modalities between the subgroups. Adult patients with NUP98::NSD1 AML were at a particularly high risk for induction failure. Most patients failed to achieve hematological CR before allo-HSCT, emphasizing the need for further improvements in pre-transplant strategies for this subgroup. The high induction failure rate in the NUP98::NSD1 group aligns with previous reports [ 3 , 6 ]. NUP98::NSD1 -positive AML cells may be intrinsically resistant to topoisomerase II inhibitors, such as idarubicin, daunorubicin, or mitoxantrone [ 47 ], although the underlying mechanisms remain unclear. In contrast, patients with NUP98::HOXA9 and NUP98 ::Others achieved morphological CR rates similar to those of the non- NUP98 -rearranged control cohort. However, they showed a high relapse rate, and their OS did not differ significantly from that of NUP98::NSD1 patients, consistent with previous findings[ 3 , 6 ]. This high relapse rate has been attributed to persistently high MRD levels, even after achieving morphological CR [ 30 ]. Our findings emphasize that NUP98 -rearranged AML in adults represents not only a biologically distinct subtype but also a therapeutically challenging one. The relatively poor EFS and high relapse rates highlight the urgent need for novel treatment strategies, particularly for the NUP98::NSD1 subgroup, which appears to be associated with a unique set of adverse biological features, including FLT3 -ITD co-mutations and chemoresistance. Given the high prevalence of WT1 mutations and the distinct clonal hierarchies observed, future studies should aim to dissect the cooperative leukemogenic networks that define each fusion subtype. Our study further suggests that intensive upfront treatment approaches, including early consideration of allo-HSCT and incorporation of targeted agents, such as FLT3 inhibitors and venetoclax, may help mitigate some of the historically poor outcomes associated with these fusions. However, the persistently high MRD levels and relapse rates, particularly in the NUP98::HOXA9 and NUP98 ::Others subgroups despite initial responses, point to the need for MRD-guided post-remission strategies and maintenance therapies. The development of fusion-specific MRD monitoring tools and preclinical models will be essential for testing novel therapeutic approaches and understanding the mechanisms of resistance. The limitations of our study include its retrospective design, modest sample size, and absence of functional validation analyses. Future studies involving larger, multi-institutional cohorts and mechanistic investigations are needed to clarify the biological role of individual NUP98 fusion partners and identify actionable therapeutic targets. In conclusion, NUP98 -rearranged AML represents a high-risk, biologically distinct subset of adult AML cases. Our study underscores the need for risk-adapted, fusion-specific therapeutic strategies and highlights the urgency for prospective multicenter trials to validate these approaches and define optimal treatment guidelines. Declarations Competing Interests The authors declare no competing interests. Funding This work was supported in part by grants from the Japan Agency for Medical Research and Development (JP19cm0106235h0002, JP22bm0804004h0006 (K.C. and Y.Y.), JP15cm0106056h0005, JP19cm0106501h0004, JP16ck0106073h0003, JP19ck0106250h0003 (S.O.), JP19ck0106353h0003 (Y.Nannya.)), Core Research for Evolutional Science and Technology (JP19gm1110011) (S.O.), and JSPS KAKENHI (JP21K08414 (K.C.), JP25H01055 (M.S.-Y)), a grant from the Kobayashi Foundation for Cancer Research (K.C.), and a grant from the International Joint Usage/Research Center, IMSUT (24-2103 (K.C. and Y.Nannya). Author Contributions K.C., M.Iwasaki, J.K., and Y.Nannya conceived and designed the study, and wrote the manuscript. K.C., M.Iwasaki, and Y.Nannya collected and curated the data. S.O. and Y.Nannya performed targeted-capture sequencing. H.T., A.Y., S.O., and Y.Nannya analyzed and interpreted the sequencing data. K.C. performed most of the experiments. T.Kawata, S.M., and Y.Y. performed some experiments. M.S., T.Kondo, T.H., Y.U., A.G., M.W., S.K., Y.I., H.K., K.I., K.M., T.Kitano, Y.T., Y.Nakabou, N.S., N.K., T.F., M.Ichikawa, Y.M., S.F., M.S.-Y., and A.T.-K. collected patient samples and data. All authors reviewed and approved the final version of the manuscript. Acknowledgements The authors acknowledge the patients who participated in this study and their families. The authors thank the clinical research staff and caregivers at all participating sites. In preparing this work, ChatGPT was used to proofread the manuscript. Data Availability Statement The data generated in this study are available upon request from the corresponding author. References Bolouri H, Farrar JE, Triche T, Jr., Ries RE, Lim EL, Alonzo TA, et al. The molecular landscape of pediatric acute myeloid leukemia reveals recurrent structural alterations and age-specific mutational interactions. Nat Med. 2018;24:103–112. Struski S, Lagarde S, Bories P, Puiseux C, Prade N, Cuccuini W, et al. 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Acute myeloid leukemia with a cryptic NUP98/PRRX2 rearrangement developing after low-dose methotrexate therapy for rheumatoid arthritis. Ann Hematol. 2019;98:2841–2843. Kim JC, Zuzarte PC, Murphy T, Chan-Seng-Yue M, Brown AMK, Krzyzanowski PM, et al. Cryptic genomic lesions in adverse-risk acute myeloid leukemia identified by integrated whole genome and transcriptome sequencing. Leukemia. 2020;34:306–311. Ostronoff F, Othus M, Gerbing RB, Loken MR, Raimondi SC, Hirsch BA, et al. NUP98/NSD1 and FLT3/ITD coexpression is more prevalent in younger AML patients and leads to induction failure: a COG and SWOG report. Blood. 2014;124:2400–2407. Hollink IH, van den Heuvel-Eibrink MM, Arentsen-Peters ST, Pratcorona M, Abbas S, Kuipers JE, et al. NUP98/NSD1 characterizes a novel poor prognostic group in acute myeloid leukemia with a distinct HOX gene expression pattern. Blood. 2011;118:3645–3656. Kivioja JL, Thanasopoulou A, Kumar A, Kontro M, Yadav B, Majumder MM, et al. Dasatinib and navitoclax act synergistically to target NUP98-NSD1(+)/FLT3-ITD(+) acute myeloid leukemia. Leukemia. 2019;33:1360–1372. Tables Table 1 and 2 are available in the Supplementary Files section. Additional Declarations There is NO conflict of interest to disclose. 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University","correspondingAuthor":false,"prefix":"","firstName":"Seishi","middleName":"","lastName":"Ogawa","suffix":""}],"badges":[],"createdAt":"2025-06-19 01:05:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6926420/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6926420/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41375-025-02848-4","type":"published","date":"2026-01-07T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85646925,"identity":"51142524-4327-4b33-a4f9-181746eb80aa","added_by":"auto","created_at":"2025-06-30 08:45:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":537521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular characteristics of AML with the novel \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNUP98\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e rearrangements identified in this study.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Genomic breakpoint of \u003cem\u003eNUP98-MEOX2 \u003c/em\u003einvolving intron 12 of \u003cem\u003eNUP98\u003c/em\u003e and intron 1 of \u003cem\u003eMEOX2\u003c/em\u003e (upper panels) and in-frame fusion of the entire exon 12 of \u003cem\u003eNUP98\u003c/em\u003e to the entire exon 2 of \u003cem\u003eMEOX2\u003c/em\u003e(lower panels). The image was created using BioRender.com. \u003cstrong\u003eB\u003c/strong\u003eGenomic breakpoint of \u003cem\u003eNUP98-HOXA6\u003c/em\u003e involving intron 12 of \u003cem\u003eNUP98\u003c/em\u003eand intron 1 of \u003cem\u003eHOXA6\u003c/em\u003e (upper panels), and in-frame fusion of the entire exon 12 of \u003cem\u003eNUP98\u003c/em\u003e to the entire exon 2 of \u003cem\u003eHOXA6\u003c/em\u003e (lower panels). The image was created using BioRender.com. \u003cstrong\u003eC\u003c/strong\u003e Genomic breakpoint of \u003cem\u003eNUP98-EVX2\u003c/em\u003e involving intron 12 of \u003cem\u003eNUP98\u003c/em\u003e and intron 2 of \u003cem\u003eEVX2\u003c/em\u003e (upper panels) and in-frame fusion of the entire exon 12 of \u003cem\u003eNUP98\u003c/em\u003e to the entire exon 2 of \u003cem\u003eHOXD13\u003c/em\u003eor \u003cem\u003eHOXD11\u003c/em\u003e (lower panels). The image was created using BioRender.com. \u003cstrong\u003eD\u003c/strong\u003eFollow-up of the molecular disease through quantification of \u003cem\u003eNUP98::MEOX2\u003c/em\u003etranscript using real-time RT-PCR (qRT-PCR) in blood samples from case #28. \u003cstrong\u003eE\u003c/strong\u003e Changes in \u003cem\u003eNUP98::HOXA6\u003c/em\u003e and \u003cem\u003eWT1\u003c/em\u003etranscript levels measured by qRT-PCR, and variant allele frequencies (VAFs) of co-mutations in blood samples from case #2.\u003c/p\u003e","description":"","filename":"OnlineFig10603.png","url":"https://assets-eu.researchsquare.com/files/rs-6926420/v1/80beb49d98f87fc98d77e5a6.png"},{"id":85646918,"identity":"a5ce969d-e59b-4214-8dde-420c19811386","added_by":"auto","created_at":"2025-06-30 08:45:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":65678,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic characteristics of adult AML patients with and without \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNUP98\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e rearrangements.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Comparison of coexisting genomic alterations between patients with or without \u003cem\u003eNUP98\u003c/em\u003e rearrangements. Only genomic alterations with ≥3 events in single nucleotide variants (SNVs) or structural variants (SVs), and ≥5 events in copy number alterations across the entire cohort are shown. \u003cstrong\u003eB\u003c/strong\u003e Comparison of coexisting genomic alterations among \u003cem\u003eNUP98\u003c/em\u003e-rearranged patients with different partner genes. Only genomic alterations with ≥3 events are shown. \u003cstrong\u003eC\u003c/strong\u003eSize of coexisting mutations identified in the \u003cem\u003eNUP98\u003c/em\u003e-rearranged cohort. Only mutations with ≥2 events are shown. \u003cstrong\u003eD\u003c/strong\u003e Bradley-Terry plot based on pairwise precedences of gene mutations identified in the \u003cem\u003eNUP98\u003c/em\u003e-rearranged cohort. Only mutations with ≥2 events are shown. Genes are positioned along the x-axis according to their relative order of occurrence. The variant allele frequency was adjusted for sex and chromosomal aberrations.\u003c/p\u003e","description":"","filename":"OnlineFig20516.png","url":"https://assets-eu.researchsquare.com/files/rs-6926420/v1/34d4937bc6016300e37f3f4c.png"},{"id":85646922,"identity":"567d8f1f-50a3-402d-9cbe-4bb0cabb2929","added_by":"auto","created_at":"2025-06-30 08:45:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":29589,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResponse rate and survival of adult AML patients with and without \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNUP98\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003erearrangements. A-B\u003c/strong\u003e Response rates in (\u003cstrong\u003eA\u003c/strong\u003e) all AML patients with \u003cem\u003eNUP98\u003c/em\u003e rearrangements and (\u003cstrong\u003eB\u003c/strong\u003e) patients with different \u003cem\u003eNUP98\u003c/em\u003e fusion partners compared to the reference cohort without \u003cem\u003eNUP98\u003c/em\u003e rearrangements. \u003cstrong\u003eC-E\u003c/strong\u003eKaplan–Meier estimates of (\u003cstrong\u003eC\u003c/strong\u003e) overall survival (OS) and (\u003cstrong\u003eD\u003c/strong\u003e) event-free survival (EFS), and cumulative incidence curves of (\u003cstrong\u003eE\u003c/strong\u003e) relapse rate and non-relapse mortality (NRM) in adult AML patients with and without \u003cem\u003eNUP98\u003c/em\u003e rearrangements. Event rates were compared using the log-rank test for OS and EFS, and Fine and Gray regression for relapse and NRM.\u003c/p\u003e","description":"","filename":"OnlineFig30504.png","url":"https://assets-eu.researchsquare.com/files/rs-6926420/v1/ffaf058deafceb030be04753.png"},{"id":85646926,"identity":"c44d7e49-07d3-4431-bcbb-b4d4fb91e845","added_by":"auto","created_at":"2025-06-30 08:45:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of co-mutations on survival in adult patients with\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e NUP98\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-rearranged AML.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-D\u003c/strong\u003e Kaplan–Meier estimates of OS and EFS in patients with \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML according to (\u003cstrong\u003eA-B\u003c/strong\u003e) \u003cem\u003eFLT3\u003c/em\u003e-ITD and (\u003cstrong\u003eC-D\u003c/strong\u003e) \u003cem\u003eNRAS\u003c/em\u003e mutations. \u003cstrong\u003eE\u003c/strong\u003e Multivariable Cox regression analysis in the \u003cem\u003eNUP98\u003c/em\u003e-rearranged cohort. \u003cstrong\u003eF\u003c/strong\u003e Kaplan–Meier estimates of OS stratified by risk scores based on age and \u003cem\u003eFLT3\u003c/em\u003e-ITD status in the \u003cem\u003eNUP98\u003c/em\u003e-rearranged cohort. Event rates were compared using the log-rank test.\u003c/p\u003e","description":"","filename":"OnlineFig40504.png","url":"https://assets-eu.researchsquare.com/files/rs-6926420/v1/07e20d8d643f52753bde62f5.png"},{"id":85646929,"identity":"8d310e1c-b568-4744-9970-86680e47fd9c","added_by":"auto","created_at":"2025-06-30 08:45:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":57478,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNUP98\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e rearrangements and ELN 2022 risk classification on survival.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B\u003c/strong\u003e Kaplan–Meier estimates of (\u003cstrong\u003eA\u003c/strong\u003e) OS and (\u003cstrong\u003eB\u003c/strong\u003e) EFS in patients with \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML compared to the reference cohort according to ELN 2022 risk stratification. \u003cstrong\u003eC-D\u003c/strong\u003eKaplan–Meier estimates of OS showing the impact of allo-HSCT in AML patients (\u003cstrong\u003eC\u003c/strong\u003e) with or (\u003cstrong\u003eD\u003c/strong\u003e) without \u003cem\u003eNUP98\u003c/em\u003e rearrangements. Event rates were compared using the log-rank test.\u003c/p\u003e","description":"","filename":"OnlineFig50515.png","url":"https://assets-eu.researchsquare.com/files/rs-6926420/v1/d75fb846e1f7edf105e5ee72.png"},{"id":99766595,"identity":"00073683-95b3-476f-ae45-60e551ca0b94","added_by":"auto","created_at":"2026-01-08 08:12:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2165680,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6926420/v1/bfcbdb47-6a11-4b82-adad-70438d7abd62.pdf"},{"id":85648221,"identity":"1b7d0fca-7096-462a-99ba-33456fde3948","added_by":"auto","created_at":"2025-06-30 08:53:12","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28160,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"Table120250618.xls","url":"https://assets-eu.researchsquare.com/files/rs-6926420/v1/03274c2efe40f43e6cb3f608.xls"},{"id":85646919,"identity":"73a97705-17b0-4be5-85e2-134c6610e299","added_by":"auto","created_at":"2025-06-30 08:45:12","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":28160,"visible":true,"origin":"","legend":"Table 2","description":"","filename":"Table220250618.xls","url":"https://assets-eu.researchsquare.com/files/rs-6926420/v1/e650ae005e68e3f7d2c58cc6.xls"},{"id":85646934,"identity":"2f8ea4da-e197-4a05-a8f6-d1fb1950e9da","added_by":"auto","created_at":"2025-06-30 08:45:13","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2937829,"visible":true,"origin":"","legend":"Supplemental Material","description":"","filename":"SupplementaryInformation20250618.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6926420/v1/d7518ac9aee3e0020fac5605.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"NUP98 rearrangements in adult AML patients: Evaluation of clinical implications and identification of novel fusion partners","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eNucleoporin 98\u003c/em\u003e (\u003cem\u003eNUP98\u003c/em\u003e) fusion genes represent a distinct molecular subtype, particularly enriched in pediatric acute myeloid leukemia (AML), where they account for ~\u0026thinsp;8% of cases and are associated with poor clinical outcomes [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In contrast, \u003cem\u003eNUP98\u003c/em\u003e fusions are rare in adult AML, and their clinical and prognostic implications remain poorly defined [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. To date, more than 30 distinct \u003cem\u003eNUP98\u003c/em\u003e fusion partners have been identified across a range of hematologic malignancies, including de novo and therapy-related AML, myelodysplastic syndromes (MDS), chronic myeloid leukemia, T-cell acute lymphoblastic leukemia, and mixed-phenotype acute leukemia [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. All NUP98 fusion proteins retain the N-terminal FG repeat domain of NUP98 fused to the C-terminal portion of the partner proteins, which often contains chromatin-modifying or transcriptional regulatory domains. Recent findings indicate that \u003cem\u003eNUP98\u003c/em\u003e fusion proteins exhibit a distinct subcellular distribution compared to wild-type \u003cem\u003eNUP98\u003c/em\u003e, forming aberrant biomolecular condensates that affect chromatin architecture and gene expression, ultimately leading to the development of AML [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Many \u003cem\u003eNUP98\u003c/em\u003e rearrangements, such as \u003cem\u003eNUP98::NSD1\u003c/em\u003e, are cryptic on conventional cytogenetic analysis and may be underrecognized without molecular diagnostics [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Recent advances in next-generation sequencing technologies have revealed their relatively high incidence, even among adult AML cases.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNUP98\u003c/em\u003e fusion partners can be classified into two groups: those with and without a homeodomain. The homeodomain-containing partners include nine clustered homeobox genes (\u003cem\u003eHOXA9\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], \u003cem\u003eHOXA11\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], \u003cem\u003eHOXA13\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], \u003cem\u003eHOXC11\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], \u003cem\u003eHOXC13\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], \u003cem\u003eHOXD8\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], \u003cem\u003eHOXD11\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], \u003cem\u003eHOXD12\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and \u003cem\u003eHOXD13\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]) and seven non-clustered homeobox genes (\u003cem\u003eHHEX\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], \u003cem\u003eGSX2\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], \u003cem\u003ePRRX1\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], \u003cem\u003ePRRX2\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], \u003cem\u003ePOU1F1\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], \u003cem\u003ePOU6F2\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and \u003cem\u003eEMX1\u003c/em\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]). Among these, \u003cem\u003eNUP98::HOXA9\u003c/em\u003e is the most frequently observed and appears more prevalent in Asian populations than in Western cohorts [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Non-homeobox fusion partners are more commonly observed and typically contain coiled-coil domains that facilitate oligomerization. Additionally, several non-homeobox partners harbor conserved structural motifs, such as plant homeodomain (PHD) domains (\u003cem\u003eBPTF\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], \u003cem\u003eJADE2\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], \u003cem\u003eKDM5A\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], \u003cem\u003eMLLT10\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], \u003cem\u003eNSD1\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], \u003cem\u003eNSD3\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], \u003cem\u003ePHF23\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and \u003cem\u003eASH1L\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]) and SET domains (\u003cem\u003eKMT2A\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], \u003cem\u003eNSD1\u003c/em\u003e, and \u003cem\u003eNSD3\u003c/em\u003e). Specific \u003cem\u003eNUP98\u003c/em\u003e fusion proteins show a preferential association with particular leukemia subtypes. For instance, \u003cem\u003eNUP98::NSD1\u003c/em\u003e is the most prevalent and is predominantly found in myelomonocytic leukemia, whereas \u003cem\u003eNUP98::KDM5A\u003c/em\u003e is often associated with megakaryoblastic leukemia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCo-mutations are relatively infrequent in \u003cem\u003eNUP98\u003c/em\u003e-rearranged leukemias. While \u003cem\u003eFLT3\u003c/em\u003e-ITD and \u003cem\u003eWT1\u003c/em\u003e mutations are observed across various \u003cem\u003eNUP98\u003c/em\u003e fusions and are associated with adverse prognosis, other co-occurring genetic events are limited to specific patient populations and remain poorly characterized.\u003c/p\u003e \u003cp\u003eRecent updates in the World Health Organization classification and the International Consensus Classification (ICC) have defined AML with \u003cem\u003eNUP98\u003c/em\u003e rearrangements as a distinct disease entity, and the high specificity of this abnormality for AML allows it to be diagnosed even when the blast percentage is less than 20%. In pediatric patients with AML, \u003cem\u003eNUP98\u003c/em\u003e rearrangements with \u003cem\u003eNSD1\u003c/em\u003e and \u003cem\u003eKDM5A\u003c/em\u003e have been most frequently observed and predict poor outcomes. In contrast, \u003cem\u003eNUP98\u003c/em\u003e translocations with other partners, including \u003cem\u003eHOXA9\u003c/em\u003e, are uncommon even in pediatric AML, and their prognostic significance remains unclear. Accumulation of clinical outcomes associated with gene alterations has enabled risk stratification of adult AML according to detailed mutational profiling, such as the 2022 European LeukemiaNet (ELN 2022) risk classification. Although \u003cem\u003eNUP98\u003c/em\u003e rearrangements were identified as AML with other rare recurring translocations and categorized as an intermediate-risk group in the ELN 2022 risk classification, their exact clinical significance in adult AML cohorts remains to be determined.\u003c/p\u003e \u003cp\u003eTo address these issues, we conducted a comprehensive characterization of \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML in Japanese adults using targeted-capture sequencing.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient samples\u003c/h2\u003e \u003cp\u003eWe analyzed 4753 myeloid neoplasm cases, including 1569 AML cases (defined as having a blast count\u0026thinsp;\u0026ge;\u0026thinsp;20%), from 54 collaborating institutes in Japan, using targeted-capture sequencing in our laboratory between January 2001 and December 2022. From this cohort, we enrolled 44 patients harboring \u003cem\u003eNUP98\u003c/em\u003e rearrangements (41 AML and 3 MDS with excess blasts). We also enrolled 213 AML cases without \u003cem\u003eNUP98\u003c/em\u003e rearrangements for the control cohort. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committees of Kyoto University (G0608, G0697) and all participating institutions. All patients provided written informed consent.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of\u003c/b\u003e \u003cb\u003eNUP98\u003c/b\u003e \u003cb\u003efusions from patients with novel\u003c/b\u003e \u003cb\u003eNUP98\u003c/b\u003e \u003cb\u003erearrangements\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRNA was isolated using the miRNeasy Micro/Mini Kit (Qiagen), following the manufacturer\u0026rsquo;s protocol. cDNA was synthesized using SuperScript\u0026trade; IV VILO\u0026trade; Master Mix with ezDNase\u0026trade; Enzyme (Thermo Fisher Scientific). For reverse transcription polymerase chain reaction (RT-PCR), the KOD One PCR Master Mix (TOYOBO) was used following the manufacturer\u0026rsquo;s protocol. The primers used for this analysis are listed in Supplementary Table\u0026nbsp;1. PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen) and subjected to Sanger sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). Strand-specific RNA sequencing was performed by Novogene. RNA fastq files were mapped to the human genome (GRCh37) by hisat2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://daehwankimlab.github.io/hisat2/\u003c/span\u003e\u003cspan address=\"https://daehwankimlab.github.io/hisat2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and manually inspected using Integrative Genomics Viewer (IGV).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular monitoring\u003c/h3\u003e\n\u003cp\u003eTo evaluate molecular residual disease (MRD), quantification of \u003cem\u003eNUP98::MEOX2\u003c/em\u003e and \u003cem\u003eNUP98::HOXA6\u003c/em\u003e fusion mRNA transcripts was performed using real-time RT-PCR (qRT-PCR). A set of primers and a probe were specifically designed to amplify \u003cem\u003eNUP98::MEOX2\u003c/em\u003e mRNA detected in our patient (forward primer, 5\u0026rsquo;-CTCTTGGTACAGGAGCCTTTG-3\u0026rsquo;; reverse primer, 5\u0026rsquo;-TGGGTTTGCTGTTGACTTCT-3\u0026rsquo;; probe, 5\u0026rsquo;-FAM-ACTACGACA-ZEN-GCCACTTTGGGCTTT-IBFQ-3\u0026rsquo;) (Integrated DNA Technologies). Two sets of primers and probes were designed to amplify \u003cem\u003eWT1\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and \u003cem\u003eNUP98::HOXA6\u003c/em\u003e mRNAs detected in our patient (\u003cem\u003eWT1\u003c/em\u003e: forward primer, 5\u0026rsquo;-ACAGGGTACGAGAGCGATAACCA-3\u0026rsquo;; reverse primer, 5\u0026rsquo;-CACACGTCGCACATCCTGAAT-3\u0026rsquo;; probe, 5\u0026rsquo;-FAM-CAACGCCCATCCTCTGCGGAGCCCA-TAMRA-3\u0026rsquo;, \u003cem\u003eNUP98::HOXA6\u003c/em\u003e: forward primer, 5\u0026rsquo;-GATTTAATACTACGACAGCCACTTTGG-3\u0026rsquo;; reverse primer, 5\u0026rsquo;-ATGGCTCCCATACACAGCAC-3\u0026rsquo;; probe, 5\u0026rsquo;-Cy5-TTTGGAGCCCCCCAGGCCC-BHQ2-3\u0026rsquo;) (Thermo Fisher Scientific). In these experiments, \u003cem\u003eABL1\u003c/em\u003e was used as the internal control [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. \u003cem\u003eNUP98::MEOX2\u003c/em\u003e transcript levels were assessed by absolute quantification. The copy numbers of \u003cem\u003eNUP98::MEOX2\u003c/em\u003e and \u003cem\u003eABL1\u003c/em\u003e transcripts were derived by extrapolating the data from the standard curve from which the normalized \u003cem\u003eNUP98::MEOX2\u003c/em\u003e/\u003cem\u003eABL1\u003c/em\u003e copy number ratio was calculated. \u003cem\u003eNUP98::HOXA6\u003c/em\u003e and \u003cem\u003eWT1\u003c/em\u003e transcript levels were assessed by relative quantification (or ΔΔCq method) to determine the change in gene expression relative to a diagnostic sample.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eClinical characteristics of patients with\u003c/b\u003e \u003cb\u003eNUP98\u003c/b\u003e \u003cb\u003erearrangements\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 44 myeloid neoplasm patients with \u003cem\u003eNUP98\u003c/em\u003e rearrangements were identified by targeted-capture sequencing, including 26 males and 18 females, with a median age of 51 years (range, 18\u0026ndash;87 years). All patients had\u0026thinsp;\u0026ge;\u0026thinsp;10% blasts in the bone marrow or peripheral blood, thus meeting the criteria for AML with recurrent genetic abnormalities according to the ICC 2022 classification. We analyzed these 44 \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML patients (defined by ICC 2022) and compared them to 213 control AML patients without \u003cem\u003eNUP98\u003c/em\u003e rearrangements subjected to targeted sequencing during the same period (Table\u0026nbsp;1, Supplementary Fig.\u0026nbsp;1A). Based on the baseline characteristics, age was significantly lower in the \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML group than in the control group. Fifteen \u003cem\u003eNUP98\u003c/em\u003e rearrangement partners were identified in our patient cohort. The most prevalent rearrangement was \u003cem\u003eNUP98::NSD1\u003c/em\u003e, which was cryptic in all cases, as previously reported [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This rearrangement was identified in 18 AML patients by targeted-capture sequencing (Supplementary Table\u0026nbsp;2). The second most frequent rearrangement was \u003cem\u003eNUP98::HOXA9\u003c/em\u003e, which was different from the pediatric cases where \u003cem\u003eNUP98::KDM5A\u003c/em\u003e rearrangement ranks second, but is consistent with findings from other Asian AML cohorts of adults [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], suggesting a difference in translocation partner profile according to the age of the affected cases. \u003cem\u003eNUP98::HOXA9\u003c/em\u003e was identified in 12 AML patients, and cytogenetic analysis revealed the characteristic t(7;11)(p15;p15) translocation in nearly all \u003cem\u003eNUP98::HOXA9\u003c/em\u003e-positive patients, with the exception of one patient whose cytogenetic analysis result was unavailable (Supplementary Table\u0026nbsp;2). \u003cem\u003eNUP98\u003c/em\u003e rearrangements with partners other than \u003cem\u003eNSD1\u003c/em\u003e or \u003cem\u003eHOXA9\u003c/em\u003e, hereafter referred to as \u003cem\u003eNUP98\u003c/em\u003e::Others, are rare. \u003cem\u003eNUP98::HOXA11\u003c/em\u003e was identified in two AML patients. \u003cem\u003eNUP98::TNRC18\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], \u003cem\u003eNUP98::TOP1\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], \u003cem\u003eNUP98::KDM5A\u003c/em\u003e, \u003cem\u003eNUP98::PRRX1\u003c/em\u003e, \u003cem\u003eNUP98::PRRX2\u003c/em\u003e, \u003cem\u003eNUP98::DDX10\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], \u003cem\u003eNUP98::HOXD8\u003c/em\u003e, and \u003cem\u003eNUP98::HOXC13\u003c/em\u003e were all identified in only one AML patient. \u003cem\u003eNUP98::TNRC18\u003c/em\u003e, \u003cem\u003eNUP98::KDM5A\u003c/em\u003e, and \u003cem\u003eNUP98::PRRX2\u003c/em\u003e were cryptic, as previously reported (Supplementary Table\u0026nbsp;2) [\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Notably, three previously unreported \u003cem\u003eNUP98\u003c/em\u003e rearrangements were identified in a patient with AML: \u003cem\u003eNUP98-HOXA6\u003c/em\u003e with t(7;11)(p15;p15), \u003cem\u003eNUP98-EVX2\u003c/em\u003e with t(2;11)(q31;p15), and cryptic \u003cem\u003eNUP98-MEOX2\u003c/em\u003e. Additionally, in one AML patient, \u003cem\u003eNUP98\u003c/em\u003e was rearranged with an intergenic region of chromosome 4q12 (Table\u0026nbsp;2). It should also be noted that in \u003cem\u003ede novo\u003c/em\u003e AML, the partner genes of \u003cem\u003eNUP98\u003c/em\u003e were \u003cem\u003eNSD1\u003c/em\u003e (18 cases), \u003cem\u003eHOXA9\u003c/em\u003e (10 cases), and Others (8 cases), whereas in therapy-related AML, only \u003cem\u003eNUP98\u003c/em\u003e::Others (5 cases) was observed (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of fusion transcripts in patients with novel\u003c/b\u003e \u003cb\u003eNUP98\u003c/b\u003e \u003cb\u003erearrangements\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRT-PCR analysis was performed on peripheral blood and bone marrow samples for the four novel rearrangements. RNA sequencing was conducted and compared with the expected conformational changes from targeted-capture sequencing. As expected, the fusion partners of the \u003cem\u003eNUP98-MEOX2\u003c/em\u003e and \u003cem\u003eNUP98-HOXA6\u003c/em\u003e rearrangements were confirmed to be \u003cem\u003eMEOX2\u003c/em\u003e and \u003cem\u003eHOXA6\u003c/em\u003e, respectively, by RNA sequencing. In the \u003cem\u003eNUP98-EVX2\u003c/em\u003e patient, no \u003cem\u003eNUP98::EVX2\u003c/em\u003e fusion was detected by RNA-sequencing, and alternatively, two previously reported fusions, \u003cem\u003eNUP98::HOXD11\u003c/em\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and \u003cem\u003eNUP98::HOXD13\u003c/em\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], were detected by RT\u0026ndash;PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). However, of the two \u003cem\u003eNUP98\u003c/em\u003e fusions, \u003cem\u003eNUP98::HOXD11\u003c/em\u003e was not detected by RNA sequencing, suggesting that \u003cem\u003eNUP98::HOXD13\u003c/em\u003e mainly contributed to the pathogenesis in this case. In a patient with the \u003cem\u003eNUP98\u003c/em\u003e rearrangement with 4q12, a single \u003cem\u003eNUP98::GSX2\u003c/em\u003e fusion was detected by RT-PCR, which was previously reported in only one case of AML [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For patients with two novel \u003cem\u003eNUP98\u003c/em\u003e fusions, \u003cem\u003eNUP98::MEOX2\u003c/em\u003e and \u003cem\u003eNUP98::HOXA6\u003c/em\u003e, specific qRT-PCR assays were designed to evaluate the efficacy of the treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCoexisting variants in\u003c/b\u003e \u003cb\u003eNUP98\u003c/b\u003e\u003cb\u003e-rearranged AML\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA comparison of coexisting variants in \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML, along with their associations with partner genes of \u003cem\u003eNUP98\u003c/em\u003e rearrangements, was performed. As previously reported, among the 44 patients, the most common concomitant mutations were \u003cem\u003eFLT3\u003c/em\u003e-ITD (n\u0026thinsp;=\u0026thinsp;17) and \u003cem\u003eWT1\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;17), with both mutations occurring significantly more frequently in \u003cem\u003eNUP98\u003c/em\u003e-rearranged patients (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. \u003cem\u003eNUP98\u003c/em\u003e rearrangements are mutually exclusive with \u003cem\u003eNPM1\u003c/em\u003e mutations, consistent with previous reports [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Copy number alterations from targeted sequencing data revealed that del(17p) was not detected in \u003cem\u003eNUP98\u003c/em\u003e-rearranged patients. Although two \u003cem\u003eNUP98\u003c/em\u003e-rearranged patients showed monoallelic \u003cem\u003eTP53\u003c/em\u003e mutations, neither harbored del(17p) at least at initial onset, suggesting that \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML is highly exclusive to biallelic \u003cem\u003eTP53\u003c/em\u003e alterations. In addition, no splicing factor mutations were detected in our \u003cem\u003eNUP98\u003c/em\u003e-rearranged patients, although statistical significance was not reached (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;2A, B). We next examined the differences in coexisting mutations by translocation partners. As previously reported, \u003cem\u003eFLT3\u003c/em\u003e-ITD was more closely associated with \u003cem\u003eNUP98::NSD1\u003c/em\u003e than with \u003cem\u003eNUP98::HOXA9\u003c/em\u003e and \u003cem\u003eNUP98\u003c/em\u003e::Others (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, 67%, 0%, and 36%, respectively) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Conversely, \u003cem\u003eTET2\u003c/em\u003e mutations were only detected in patients with \u003cem\u003eNUP98::HOXA9\u003c/em\u003e and \u003cem\u003eNUP98\u003c/em\u003e::Others in our \u003cem\u003eNUP98\u003c/em\u003e-rearranged cohort (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, 0%, 42%, and 7%, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, using the Bradley-Terry model, the sequential order of mutation acquisition and the resulting clonal hierarchy of \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML were reconstructed. \u003cem\u003eNUP98\u003c/em\u003e rearrangements preceded most other aberrations (such as \u003cem\u003eFLT3\u003c/em\u003e, \u003cem\u003eNRAS\u003c/em\u003e, \u003cem\u003ePTPN11\u003c/em\u003e, and \u003cem\u003eWT1\u003c/em\u003e mutations), except for mutations in clonal hematopoiesis (CH)-related genes like \u003cem\u003eDNMT3A\u003c/em\u003e and \u003cem\u003eTET2\u003c/em\u003e, indicating that \u003cem\u003eNUP98\u003c/em\u003e rearrangements are initiating and driving events in AML (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D, Supplementary Fig.\u0026nbsp;2C). In the case harboring the \u003cem\u003eNUP98::HOXA6\u003c/em\u003e fusion, the \u003cem\u003eIDH1\u003c/em\u003e mutation, which coexisted with the \u003cem\u003eNUP98\u003c/em\u003e rearrangement at the time of initial disease, disappeared after chemotherapy, while the \u003cem\u003eIDH2\u003c/em\u003e mutation was acquired. This suggests that \u003cem\u003eNUP98\u003c/em\u003e rearrangements precede \u003cem\u003eIDH1\u003c/em\u003e and \u003cem\u003eIDH2\u003c/em\u003e mutations. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE)\u003c/p\u003e\n\u003ch3\u003eClinical outcome and prognostic relevance\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eClinical outcome and prognostic relevance\u003c/div\u003e \u003cp\u003eTo investigate the clinical significance of \u003cem\u003eNUP98\u003c/em\u003e rearrangements, we first evaluated the response to initial induction therapy. The median follow-up duration among survivors was 3.0 years (interquartile range [IQR], 2.2\u0026ndash;5.2) in the \u003cem\u003eNUP98\u003c/em\u003e-rearranged group and 4.2 years (IQR, 2.5\u0026ndash;5.8) in the non-\u003cem\u003eNUP98\u003c/em\u003e-rearranged control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.683) (Table\u0026nbsp;1). The overall hematologic complete or partial remission (CR/PR) rate after one course of treatment was significantly lower in the \u003cem\u003eNUP98\u003c/em\u003e-rearranged cohort than in the control cohort (45.2% versus 67.6%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This poor response of the \u003cem\u003eNUP98\u003c/em\u003e-rearranged cohort was largely explained by the markedly lower CR/PR rate in the \u003cem\u003eNUP98::NSD1\u003c/em\u003e subgroup (16.7%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), in contrast to the \u003cem\u003eNUP98::HOXA9\u003c/em\u003e (70.0%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1) and \u003cem\u003eNUP98\u003c/em\u003e::Others (64.3%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1) subgroups, which showed response rates comparable to the control cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Patients with \u003cem\u003eNUP98\u003c/em\u003e rearrangements exhibited a significantly higher risk of relapse and inferior event-free survival (EFS) in univariate analysis, although the overall survival (OS) did not differ significantly from the control group (median OS: 2.89 vs 2.74 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.54; median EFS: 0.082 vs 1.06 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; relapse: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; non-relapse mortality [NRM]: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.13) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F, Supplementary Fig.\u0026nbsp;3A).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe analyzed the prognostic impact of clinical variables within the \u003cem\u003eNUP98\u003c/em\u003e-rearranged cohort. We first examined the impact of age, sex, fusion partners, and co-mutations on OS and EFS using univariate analysis (Supplementary Table\u0026nbsp;3). As expected, age above the median (51 years) predicted a shorter OS (median OS: 0.86 vs not reached [NR] years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019) but not affected EFS (median EFS: 0.082 vs. 0.082 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.61). We assessed the prognostic impact of different fusion partners. Although not significant, \u003cem\u003eNUP98::HOXA9\u003c/em\u003e cases tended to have longer OS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.17) and EFS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.081) than \u003cem\u003eNUP98::NSD1\u003c/em\u003e and \u003cem\u003eNUP98\u003c/em\u003e::Others subgroups (Supplementary Fig.\u0026nbsp;3B, C). Therefore, we combined the \u003cem\u003eNUP98::NSD1\u003c/em\u003e and \u003cem\u003eNUP98\u003c/em\u003e::Others groups into a single \u003cem\u003eNUP98\u003c/em\u003e::non-\u003cem\u003eHOXA9\u003c/em\u003e subgroup, which showed a trend toward shorter OS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.062) and EFS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.061) compared to \u003cem\u003eNUP98::HOXA9\u003c/em\u003e cases (Supplementary Fig.\u0026nbsp;3D, E).\u003c/p\u003e \u003cp\u003eNext, we analyzed the impact of co-mutations on outcomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D, Supplementary Fig.\u0026nbsp;4A, B). Among the \u003cem\u003eNUP98\u003c/em\u003e-rearranged cohort, patients with \u003cem\u003eFLT3\u003c/em\u003e-ITD had significantly shorter OS (median: 0.767 vs. NA years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and EFS (median: 0.082 vs. 0.090 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011), while patients with \u003cem\u003eNRAS\u003c/em\u003e mutation had significantly longer OS (median: NR vs 1.59 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.034) and EFS (median: 0.090 vs. 0.082 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025), compared to the \u003cem\u003eFLT3\u003c/em\u003e-ITD-negative or \u003cem\u003eNRAS\u003c/em\u003e-unmutated group, respectively, in univariate analysis. Thus, age, fusion partner (\u003cem\u003eHOXA9\u003c/em\u003e or not), and co-mutations with \u003cem\u003eFLT3\u003c/em\u003e-ITD or \u003cem\u003eNRAS\u003c/em\u003e were subjected to multivariate analysis using the Cox regression model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). After correction, significantly inferior OS (hazard ratio [HR]\u0026thinsp;=\u0026thinsp;11.48; 95% confidence interval [CI]: 3.15\u0026ndash;41.75; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed only in \u003cem\u003eFLT3\u003c/em\u003e-ITD-positive patients compared to the reference group. Based on the multivariate model, we constructed a simple scoring model by assigning 1 point each to elderly age and \u003cem\u003eFLT3\u003c/em\u003e-ITD. The \u003cem\u003eNUP98\u003c/em\u003e-rearranged cohort was classified into three risk groups according to the total score (low risk: score\u0026thinsp;=\u0026thinsp;0, intermediate risk: score\u0026thinsp;=\u0026thinsp;1, high risk: score\u0026thinsp;=\u0026thinsp;2), showing a significant difference in OS (median: NR vs. 2.047 vs. 0.516 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), but this risk stratification system was not effective in predicting EFS (median: 0.090 vs. 0.082 vs. 0.082 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.31, Supplementary Fig.\u0026nbsp;4C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then explored the impact of \u003cem\u003eNUP98\u003c/em\u003e rearrangements on outcomes in the context of ELN 2022 risk stratification (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, Supplementary Fig.\u0026nbsp;5A-C). Compared to patients with favorable-risk ELN2022 AML without \u003cem\u003eNUP98\u003c/em\u003e rearrangements, those with \u003cem\u003eNUP98\u003c/em\u003e rearrangements had significantly lower OS (median: 2.89 vs NR years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) and EFS (median: 0.082 vs 2.13 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, patients with \u003cem\u003eNUP98\u003c/em\u003e rearrangements had comparable OS to both intermediate- and adverse-risk ELN2022 control groups (median:2.89 vs 4.07 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.4; and 2.89 vs 1.60 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.3, respectively), although significant differences were observed in EFS (median: 0.082 vs 1.06 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; and 0.082 vs. 0.110 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, we examined the prognostic impact of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the analysis including all patients, patients with \u003cem\u003eNUP98\u003c/em\u003e rearrangements who underwent allo-HSCT showed higher overall survival compared to those who did not, similar to patients without \u003cem\u003eNUP98\u003c/em\u003e rearrangement (median: 0.77 vs NR years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). When limited to patients aged 70 years or younger, OS showed a trend toward improvement, although the difference was not statistically significant in the \u003cem\u003eNUP98\u003c/em\u003e-rearranged cohort (median: 2.89 vs NR years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.14) (Supplementary Fig.\u0026nbsp;5D, E).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML has emerged as a distinct and heterogeneous disease entity, first identified in pediatric cohorts and more recently recognized in adult populations. Although several recent studies have provided deep insights into this fusion family in adult AML, a comprehensive study encompassing fusion partners, clinical phenotypes, coexisting mutations, and treatment outcomes remains lacking.\u003c/p\u003e \u003cp\u003eIn this study, we comprehensively analyzed the molecular and clinical features of \u003cem\u003eNUP98\u003c/em\u003e rearrangements in a large cohort of Japanese adults with AML. Among the 1569 cases of AML, we identified 41 cases (2.6%) harboring \u003cem\u003eNUP98\u003c/em\u003e rearrangements. Although less frequent than in pediatric AML, these fusions represent a distinct and non-negligible subset in adults.\u003c/p\u003e \u003cp\u003eThe most frequent fusion types identified were \u003cem\u003eNUP98::NSD1\u003c/em\u003e and \u003cem\u003eNUP98::HOXA9\u003c/em\u003e, which is consistent with prior reports, especially in Asian cohorts. Notably, we also detected novel fusions, such as \u003cem\u003eNUP98::MEOX2\u003c/em\u003e and \u003cem\u003eNUP98::HOXA6\u003c/em\u003e, highlighting the diversity and complexity of \u003cem\u003eNUP98\u003c/em\u003e rearrangements. To characterize these novel fusions, we developed fusion transcript-specific qRT-PCR assays, underscoring their diagnostic value and potential utility in MRD monitoring. Furthermore, in one case where a novel \u003cem\u003eNUP98-EVX2\u003c/em\u003e rearrangement was predicted at the DNA level, RNA analysis identified \u003cem\u003eNUP98::HOXD13\u003c/em\u003e as the actual fusion transcript, highlighting the importance of transcript-level validation for the accurate interpretation of atypical rearrangements.\u003c/p\u003e \u003cp\u003eCo-mutation analysis revealed that \u003cem\u003eFLT3\u003c/em\u003e-ITD and \u003cem\u003eWT1\u003c/em\u003e mutations were significantly enriched in \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML, supporting the notion of a synergistic leukemogenic effect. In contrast, mutations commonly observed in adult AML, such as \u003cem\u003eNPM1\u003c/em\u003e, splicing factor genes, and biallelic \u003cem\u003eTP53\u003c/em\u003e mutations, were notably absent, suggesting a distinct leukemogenic trajectory for \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML. The underlying biology of \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML appears to be influenced by specific fusion partners. We observed a significant overlap of cooperative \u003cem\u003eWT1\u003c/em\u003e mutations across different fusion partners, whereas \u003cem\u003eFLT3\u003c/em\u003e-ITD was markedly enriched in \u003cem\u003eNUP98-NSD1\u003c/em\u003e cases. While \u003cem\u003eNUP98\u003c/em\u003e rearrangements generally preceded most other aberrations, regardless of the partners, we found that \u003cem\u003eNUP98::HOXA9\u003c/em\u003e emerged on CH clones in a considerable number of patients in our study. In contrast, CH-related mutations were rare in patients with \u003cem\u003eNUP98::NSD1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eClinically, our study provides valuable data on treatment strategies for adult AML with \u003cem\u003eNUP98\u003c/em\u003e rearrangements. We observed that patients harboring \u003cem\u003eNUP98\u003c/em\u003e rearrangements had significantly higher relapse rates and lower EFS, although OS was not significantly different compared to those without \u003cem\u003eNUP98\u003c/em\u003e rearrangements. In a previous AML study, \u003cem\u003eNUP98::NSD1\u003c/em\u003e fusion was associated with poor outcomes in both pediatric and adult AML, with a 4-year OS as low as 11% in adult cases [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Similarly, another study on adult AML reported dismal outcomes for patients with the \u003cem\u003eNUP98::HOXA9\u003c/em\u003e fusion, with a median OS of 13.5 months [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Although cross-trial comparisons must be interpreted with caution, our cohort demonstrated relatively favorable survival outcomes. In our study, 22 out of 44 patients (50%) with \u003cem\u003eNUP98\u003c/em\u003e rearrangements underwent allo-HSCT, compared to only two out of 10 patients (20%) in the \u003cem\u003eNUP98::NSD1\u003c/em\u003e study and only four out of 11 patients (36%) in the \u003cem\u003eNUP98::HOXA9\u003c/em\u003e study. The relatively high allo-HSCT rate may have contributed to the improved outcomes observed in our cohort. A recent adult AML study reported 4-year OS rates of 65.2% in the \u003cem\u003eNUP98::NSD1\u003c/em\u003e group and 66.7% in the non-\u003cem\u003eNUP98::NSD1\u003c/em\u003e group (including \u003cem\u003eNUP98::HOXA9\u003c/em\u003e), which appear higher than those observed in our cohort [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In that study, 25 of 51 patients (49%) underwent allo-HSCT, which is comparable to our cohort. Therefore, the superior OS observed in that cohort may be attributed to the greater proportion of patients receiving salvage regimens that included venetoclax or FLT3 inhibitors.\u003c/p\u003e \u003cp\u003eWe also noted differences in the efficacy of the therapeutic modalities between the subgroups. Adult patients with \u003cem\u003eNUP98::NSD1\u003c/em\u003e AML were at a particularly high risk for induction failure. Most patients failed to achieve hematological CR before allo-HSCT, emphasizing the need for further improvements in pre-transplant strategies for this subgroup. The high induction failure rate in the \u003cem\u003eNUP98::NSD1\u003c/em\u003e group aligns with previous reports [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eNUP98::NSD1\u003c/em\u003e-positive AML cells may be intrinsically resistant to topoisomerase II inhibitors, such as idarubicin, daunorubicin, or mitoxantrone [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], although the underlying mechanisms remain unclear. In contrast, patients with \u003cem\u003eNUP98::HOXA9\u003c/em\u003e and \u003cem\u003eNUP98\u003c/em\u003e::Others achieved morphological CR rates similar to those of the non-\u003cem\u003eNUP98\u003c/em\u003e-rearranged control cohort. However, they showed a high relapse rate, and their OS did not differ significantly from that of \u003cem\u003eNUP98::NSD1\u003c/em\u003e patients, consistent with previous findings[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This high relapse rate has been attributed to persistently high MRD levels, even after achieving morphological CR [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur findings emphasize that \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML in adults represents not only a biologically distinct subtype but also a therapeutically challenging one. The relatively poor EFS and high relapse rates highlight the urgent need for novel treatment strategies, particularly for the \u003cem\u003eNUP98::NSD1\u003c/em\u003e subgroup, which appears to be associated with a unique set of adverse biological features, including \u003cem\u003eFLT3\u003c/em\u003e-ITD co-mutations and chemoresistance. Given the high prevalence of \u003cem\u003eWT1\u003c/em\u003e mutations and the distinct clonal hierarchies observed, future studies should aim to dissect the cooperative leukemogenic networks that define each fusion subtype. Our study further suggests that intensive upfront treatment approaches, including early consideration of allo-HSCT and incorporation of targeted agents, such as FLT3 inhibitors and venetoclax, may help mitigate some of the historically poor outcomes associated with these fusions. However, the persistently high MRD levels and relapse rates, particularly in the \u003cem\u003eNUP98::HOXA9\u003c/em\u003e and \u003cem\u003eNUP98\u003c/em\u003e::Others subgroups despite initial responses, point to the need for MRD-guided post-remission strategies and maintenance therapies. The development of fusion-specific MRD monitoring tools and preclinical models will be essential for testing novel therapeutic approaches and understanding the mechanisms of resistance. The limitations of our study include its retrospective design, modest sample size, and absence of functional validation analyses. Future studies involving larger, multi-institutional cohorts and mechanistic investigations are needed to clarify the biological role of individual \u003cem\u003eNUP98\u003c/em\u003e fusion partners and identify actionable therapeutic targets.\u003c/p\u003e \u003cp\u003eIn conclusion, \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML represents a high-risk, biologically distinct subset of adult AML cases. Our study underscores the need for risk-adapted, fusion-specific therapeutic strategies and highlights the urgency for prospective multicenter trials to validate these approaches and define optimal treatment guidelines.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported in part by grants from the Japan Agency for Medical Research and Development (JP19cm0106235h0002, JP22bm0804004h0006 (K.C. and Y.Y.), JP15cm0106056h0005, JP19cm0106501h0004, JP16ck0106073h0003, JP19ck0106250h0003 (S.O.), JP19ck0106353h0003 (Y.Nannya.)), Core Research for Evolutional Science and Technology (JP19gm1110011) (S.O.), and JSPS KAKENHI (JP21K08414 (K.C.), JP25H01055 (M.S.-Y)), a grant from the Kobayashi Foundation for Cancer Research (K.C.), and a grant from the International Joint Usage/Research Center, IMSUT (24-2103 (K.C. and Y.Nannya).\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eK.C., M.Iwasaki, J.K., and Y.Nannya conceived and designed the study, and wrote the manuscript. K.C., M.Iwasaki, and Y.Nannya collected and curated the data. S.O. and Y.Nannya performed targeted-capture sequencing. H.T., A.Y., S.O., and Y.Nannya analyzed and interpreted the sequencing data. K.C. performed most of the experiments. T.Kawata, S.M., and Y.Y. performed some experiments. M.S., T.Kondo, T.H., Y.U., A.G., M.W., S.K., Y.I., H.K., K.I., K.M., T.Kitano, Y.T., Y.Nakabou, N.S., N.K., T.F., M.Ichikawa, Y.M., S.F., M.S.-Y., and A.T.-K. collected patient samples and data. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThe authors acknowledge the patients who participated in this study and their families. The authors thank the clinical research staff and caregivers at all participating sites. In preparing this work, ChatGPT was used to proofread the manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability Statement\u003c/h2\u003e\n\u003cp\u003eThe data generated in this study are available upon request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBolouri H, Farrar JE, Triche T, Jr., Ries RE, Lim EL, Alonzo TA, et al. The molecular landscape of pediatric acute myeloid leukemia reveals recurrent structural alterations and age-specific mutational interactions. Nat Med. 2018;24:103\u0026ndash;112.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStruski S, Lagarde S, Bories P, Puiseux C, Prade N, Cuccuini W, et al. NUP98 is rearranged in 3.8% of pediatric AML forming a clinical and molecular homogenous group with a poor prognosis. 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Cancer Res. 1998;58:4269\u0026ndash;4273.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJankovic D, Gorello P, Liu T, Ehret S, La Starza R, Desjobert C, et al. Leukemogenic mechanisms and targets of a NUP98/HHEX fusion in acute myeloid leukemia. Blood. 2008;111:5672\u0026ndash;5682.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoler G, Kaltenbach S, Dobbelstein S, Broccardo C, Radford I, Mozziconacci MJ, et al. Identification of GSX2 and AF10 as NUP98 partner genes in myeloid malignancies. Blood Cancer J. 2013;3:e124.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura 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;94:741\u0026ndash;747.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGervais C, Mauvieux L, Perrusson N, Helias C, Struski S, Leymarie V, et al. A new translocation t(9;11)(q34;p15) fuses NUP98 to a novel homeobox partner gene, PRRX2, in a therapy-related acute myeloid leukemia. Leukemia. 2005;19:145\u0026ndash;148.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLisboa S, Cerveira N, Bizarro S, Correia C, Vieira J, Torres L, et al. POU1F1 is a novel fusion partner of NUP98 in acute myeloid leukemia with t(3;11)(p11;p15). Mol Cancer. 2013;12:5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi YJ, Lee S, Kim Y, Shin S, Lee KA. POU6F2, a novel fusion partner of NUP98 in acute myeloid leukaemia: A case report. Br J Haematol. 2024;205:1632\u0026ndash;1635.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeald JS, Lopez AM, Pato ML, Ruiz-Xiville N, Cabezon M, Zamora L, et al. Identification of novel NUP98 fusion partners and comutations in acute myeloid leukemia: an adult cohort study. Blood Adv. 2024;8:2691\u0026ndash;2694.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChou WC, Chen CY, Hou HA, Lin LI, Tang JL, Yao M, et al. Acute myeloid leukemia bearing t(7;11)(p15;p15) is a distinct cytogenetic entity with poor outcome and a distinct mutation profile: comparative analysis of 493 adult patients. Leukemia. 2009;23:1303\u0026ndash;1310.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoussy M, Bilodeau M, Jouan L, Tibout P, Laramee L, Lemyre E, et al. NUP98-BPTF gene fusion identified in primary refractory acute megakaryoblastic leukemia of infancy. Genes Chromosomes Cancer. 2018;57:311\u0026ndash;319.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwartz JR, Ma J, Lamprecht T, Walsh M, Wang S, Bryant V, et al. The genomic landscape of pediatric myelodysplastic syndromes. Nat Commun. 2017;8:1557.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Zutven LJ, Onen E, Velthuizen SC, van Drunen E, von Bergh AR, van den Heuvel-Eibrink MM, et al. Identification of NUP98 abnormalities in acute leukemia: JARID1A (12p13) as a new partner gene. Genes Chromosomes Cancer. 2006;45:437\u0026ndash;446.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosati R, La Starza R, Veronese A, Aventin A, Schwienbacher C, Vallespi T, et al. NUP98 is fused to the NSD3 gene in acute myeloid leukemia associated with t(8;11)(p11.2;p15). Blood. 2002;99:3857\u0026ndash;3860.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReader JC, Meekins JS, Gojo I, Ning Y. A novel NUP98-PHF23 fusion resulting from a cryptic translocation t(11;17)(p15;p13) in acute myeloid leukemia. Leukemia. 2007;21:842\u0026ndash;844.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTembrink M, Gerding WM, Wieczorek S, Mika T, Schroers R, Nguyen HP, et al. Novel NUP98::ASH1L Gene Fusion in Acute Myeloid Leukemia Detected by Optical Genome Mapping. Cancers (Basel). 2023;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaltenbach S, Soler G, Barin C, Gervais C, Bernard OA, Penard-Lacronique V, et al. NUP98-MLL fusion in human acute myeloblastic leukemia. Blood. 2010;116:2332\u0026ndash;2335.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWillasch AM, Gruhn B, Coliva T, Kalinova M, Schneider G, Kreyenberg H, et al. Standardization of WT1 mRNA quantitation for minimal residual disease monitoring in childhood AML and implications of WT1 gene mutations: a European multicenter study. Leukemia. 2009;23:1472\u0026ndash;1479.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeillard E, Pallisgaard N, van der Velden VH, Bi W, Dee R, van der Schoot E, et al. Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using 'real-time' quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) - a Europe against cancer program. Leukemia. 2003;17:2474\u0026ndash;2486.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhuja HG, Felix CA, Aplan PD. The t(11;20)(p15;q11) chromosomal translocation associated with therapy-related myelodysplastic syndrome results in an NUP98-TOP1 fusion. Blood. 1999;94:3258\u0026ndash;3261.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArai Y, Hosoda F, Kobayashi H, Arai K, Hayashi Y, Kamada N, et al. The inv(11)(p15q22) chromosome translocation of de novo and therapy-related myeloid malignancies results in fusion of the nucleoporin gene, NUP98, with the putative RNA helicase gene, DDX10. Blood. 1997;89:3936\u0026ndash;3944.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao L, Zhang F, Wen L, Wang Z, Ruan C, Chen S. Novel NUP98:TNRC18 fusion transcript in acute myeloid leukemia: a case report and literature review. Blood Sci. 2025;7:e00232.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChonabayashi K, Yoshida Y, Kitawaki T, Nannya Y, Nakamura M, Oshima S, et al. Acute myeloid leukemia with a cryptic NUP98/PRRX2 rearrangement developing after low-dose methotrexate therapy for rheumatoid arthritis. Ann Hematol. 2019;98:2841\u0026ndash;2843.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JC, Zuzarte PC, Murphy T, Chan-Seng-Yue M, Brown AMK, Krzyzanowski PM, et al. Cryptic genomic lesions in adverse-risk acute myeloid leukemia identified by integrated whole genome and transcriptome sequencing. Leukemia. 2020;34:306\u0026ndash;311.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOstronoff F, Othus M, Gerbing RB, Loken MR, Raimondi SC, Hirsch BA, et al. NUP98/NSD1 and FLT3/ITD coexpression is more prevalent in younger AML patients and leads to induction failure: a COG and SWOG report. Blood. 2014;124:2400\u0026ndash;2407.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHollink IH, van den Heuvel-Eibrink MM, Arentsen-Peters ST, Pratcorona M, Abbas S, Kuipers JE, et al. NUP98/NSD1 characterizes a novel poor prognostic group in acute myeloid leukemia with a distinct HOX gene expression pattern. Blood. 2011;118:3645\u0026ndash;3656.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKivioja JL, Thanasopoulou A, Kumar A, Kontro M, Yadav B, Majumder MM, et al. Dasatinib and navitoclax act synergistically to target NUP98-NSD1(+)/FLT3-ITD(+) acute myeloid leukemia. Leukemia. 2019;33:1360\u0026ndash;1372.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6926420/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6926420/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eNUP98\u003c/em\u003e rearrangements represent a distinct, high-risk subtype of acute myeloid leukemia (AML), particularly in pediatric patients. However, their prevalence, genetic features, and clinical implications in adult AML remain poorly characterized. Using targeted-capture sequencing, we identified 41 cases with \u003cem\u003eNUP98\u003c/em\u003e rearrangements among 1569 AML cases, representing the majority of 44 \u003cem\u003eNUP98\u003c/em\u003e-rearranged cases detected across 4,753 myeloid neoplasms. Fifteen distinct fusion partners were detected, with \u003cem\u003eNUP98::NSD1\u003c/em\u003e and \u003cem\u003eNUP98::HOXA9\u003c/em\u003e being the most frequent. Notably, two novel fusions\u0026mdash;\u003cem\u003eNUP98::MEOX2\u003c/em\u003e and \u003cem\u003eNUP98::HOXA6\u003c/em\u003e\u0026mdash;were identified. Co-mutations were relatively infrequent; \u003cem\u003eFLT3\u003c/em\u003e-ITD and \u003cem\u003eWT1\u003c/em\u003e mutations were the most common, while \u003cem\u003eNPM1\u003c/em\u003e mutations were exclusive. \u003cem\u003eFLT3\u003c/em\u003e-ITD was significantly enriched in \u003cem\u003eNUP98::NSD1\u003c/em\u003e cases, whereas \u003cem\u003eTET2\u003c/em\u003e mutations were more frequent in \u003cem\u003eNUP98::HOXA9\u003c/em\u003e cases. Clonal hierarchy analysis suggested that \u003cem\u003eNUP98\u003c/em\u003e rearrangements occur early in leukemogenesis. \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML exhibited higher relapse rates and shorter event-free survival. Specifically, \u003cem\u003eNUP98::NSD1\u003c/em\u003e was associated with a poor induction response, whereas \u003cem\u003eNUP98::HOXA9\u003c/em\u003e and \u003cem\u003eNUP98\u003c/em\u003e fusions with other partners showed higher remission rates but frequent relapse. Allogeneic hematopoietic stem cell transplantation was associated with better survival, underscoring its significance. These findings reveal the genetic and clinical heterogeneity of \u003cem\u003eNUP98\u003c/em\u003e-rearranged AML in adults and support its classification as a distinct entity, highlighting the need for fusion partner-specific therapeutic strategies.\u003c/p\u003e","manuscriptTitle":"NUP98 rearrangements in adult AML patients: Evaluation of clinical implications and identification of novel fusion partners","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-30 08:45:07","doi":"10.21203/rs.3.rs-6926420/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-07-28T10:56:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-07-20T22:59:17+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-07-13T14:38:38+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-07-06T07:08:43+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-06-27T12:35:45+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-06-24T09:32:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-19T09:56:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-19T09:55:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Leukemia","date":"2025-06-19T01:04:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ba009645-574f-44c2-90fb-26d438fa8b28","owner":[],"postedDate":"June 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50508099,"name":"Health sciences/Medical research/Genetics research"},{"id":50508100,"name":"Health sciences/Diseases/Haematological diseases/Haematological cancer/Leukaemia/Acute myeloid leukaemia"}],"tags":[],"updatedAt":"2026-01-08T08:12:41+00:00","versionOfRecord":{"articleIdentity":"rs-6926420","link":"https://doi.org/10.1038/s41375-025-02848-4","journal":{"identity":"leukemia","isVorOnly":false,"title":"Leukemia"},"publishedOn":"2026-01-07 05:00:00","publishedOnDateReadable":"January 7th, 2026"},"versionCreatedAt":"2025-06-30 08:45:07","video":"","vorDoi":"10.1038/s41375-025-02848-4","vorDoiUrl":"https://doi.org/10.1038/s41375-025-02848-4","workflowStages":[]},"version":"v1","identity":"rs-6926420","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6926420","identity":"rs-6926420","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}