PRDM16::SKI is a predictor of aberrant expression of the short variant of PRDM16 in pediatric acute myeloid leukemia

PRDM16::SKI is a predictor of aberrant expression of the short variant of PRDM16 in pediatric acute myeloid leukemia | 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 PRDM16::SKI is a predictor of aberrant expression of the short variant of PRDM16 in pediatric acute myeloid leukemia Norio Shiba, Masahiro Yoshitomi, Tomoya Komori, Junji Ikeda, Kenichi Yoshida, and 27 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6187243/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The RNA-sequencing data from the Japanese Children’s Cancer Group (JCCG)’s AML-05 study was re-analyzed to clarify the mechanisms related to high PRDM16 expressions, which is independently associated with adverse outcomes. Results showed that 19 of 139 patients presented with out-of-frame PRDM16::SKI fusions. Thus, the gene expression levels of PRDM16::SKI in 369 and 329 patients from the AML-05 and AML-12 studies, respectively, were measured. In total, 119 (32%) of 369 patients in the AML-05 study and 58 (18%) of 329 patients in the AML-12 study presented with an aberrant expression of PRDM16::SKI . This fusion was a 48-base-pair product that immediately formed a stop codon on the SKI side. The introduction of this product in mice did not cause AML. Intriguingly, none of the patients presented with SKI::PRDM16 , which is reciprocal. Moreover, partner fusion genes were not detected in front of truncated PRDM16 , indicating that a short form of PRDM16 , which lacked exon 1, existed by itself. Patients with high PRDM16::SKI expression had significantly worse overall survival and event-free survival than those with a low PRDM16 expression. The cleavage between exons 1 and 2 of PRDM16 induces aberrant PRDM16 expression, and a strong associations was observed between PRDM16::SKI and PRDM16 expression. Biological sciences/Cancer/Cancer genomics Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia PRDM16::SKI NUP98-rearrangements KMT2A-PTD UBTF-TD nucleoporin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The pathogenesis of acute myeloid leukemia (AML) is heterogeneous. AML is caused by various chromosomal aberrations, gene mutations/epigenetic modifications, and downregulated/upregulated gene expression, leading to increased proliferation and decreased hematopoietic progenitor cell differentiation. 1 – 5 Various genetic analyses are performed on patients with pediatric AML, and many genetic abnormalities have been identified and used for prognostic stratification. In particular, RUNX1::RUNX1T1 , CBFB::MYH11 , FUS::ERG , CBFA2T3::GLIS2 , NUP98 -rearrangements, and KMT2A rearrangements and bZIP CEBPA , NPM1 , and FLT3 -internal tandem duplication (ITD) mutations are frequently observed and considered useful for prognostic prediction. 6 – 14 A distinctive NUP98::NSD1 fusion gene-related signature, forming a unique entity, was identified in pediatric patients with AML. 15 This signature is characterized by elevated expression of PR domain containing 16 ( PRDM16 ; also known as MEL1 ). 15 , 16 This signature is closely associated with KMT2A -PTD, FLT3 -ITD, NPM1 , and UBTF -TD mutations, 15 – 17 and is significantly related to poor prognosis other than NPM1 mutations. 18 The translocation t(1;3)(p36;q21)/ PRDM16::MECOM occurs in a subset of myelodysplastic syndrome (MDS) and AML. 19 PRDM16 is located near the 1p36.3 breakpoint and is specifically expressed in t(1:3)(p36,q21)-positive MDS/AML. The protein encoded by this gene is a zinc finger transcription factor, and it contains an N-terminal PR domain. The translocation results in the overexpression of a truncated version of this protein (sPRDM16), which lacks the PR domain and may play an important role in the pathogenesis of MDS and AML. 20 , 21 However, the cause of the high PRDM16 expression has not yet been elucidated in most patients. In this study, we re-analyzed RNA-sequencing data of 139 patients enrolled in the AML-05 study, conducted by the Japanese Children’s Cancer Group (JCCG), to elucidate the cause of this high expression. We identified an out-of-frame PRDM16::SKI fusion gene in 19 patients. This fusion gene has been reported in previous case reports and was associated with high PRDM16 expression. 22 This fusion gene was only the product of 48-base-pair product due to the immediate appearance of a stop codon on the SKI side. In addition, the reciprocal SKI::PRDM16 was not detected, and no other genes were observed upstream of truncated PRDM16 . Hence, the short form of PRDM16 (s PRDM16 -∆exon1) plays an important on causing leukemia. s PRDM16 is associated with leukemia progression in mouse experiments. 23 Therefore, PRDM16::SKI is a useful predictor of the presence of truncated PRDM16 . This fusion gene is strongly correlated with a high PRDM16 expression. The current study aimed to examine the clinical and functional significance of this fusion gene and its impact on patient prognosis. Methods Patients The current study enrolled 369 patients with de novo AML who were registered in the JCCG’s AML-05 study between November 2006 and December 2010. 24 , 25 In addition, among the 387 patients with AML who were registered in the JCCG AML-12 study, 329 with known mutation status who had available gene expression data were included in the validation cohort. 26 Patients diagnosed with acute promyelocytic leukemia or Down syndrome-associated AML were excluded from this analysis. This study was conducted in accordance with the Declaration of Helsinki and approved by the institutional review board of Yokohama City University Hospital and the ethical review board of the JCCG. A written informed consent from all patients or their parents/guardians was obtained. Treatments The protocol treatment regimen for the AML-05 and AML-12 studies have already reported. 26 , 27 RNA sequencing We performed RNA-seq for 139 out of the 369 patients with pediatric AML in order to obtain a complete registry of gene rearrangements, other genetic lesions, and gene expressions in pediatric AML. The RNA-seq data were available at the European Genome-Phenome Archive (EGAD00001005078). The cytogenetic characteristics of the analyzed patients are shown in supplementary Table S1 . The study population mainly included patients with a normal karyotype (60/70 patients), FLT3- ITD (33/47 patients), KMT2A -PTD (12/13 patients), and high PRDM16 expression (65/84 patients). Among 369 patients, all 137 patients with core binding factor AML (CBF-AML) and many patients with KMT2A rearrangements were excluded from RNA-seq, because the main purpose of this study was to identify unknown fusion genes and to investigate the gene expression patterns of cases with poor prognoses. The quality of the extracted RNA was assessed using TapeStation system (Agilent Technologies, Santa Clara, CA). Sequencing libraries were prepared using a NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA), and prepared libraries were run on a HiSeq 2500 high-throughput sequencing system. Sequencing reads were aligned using bowtie and blat, and fusion genes were analyzed using Genomon-fusion. 28 Candidate gene fusions were validated by RT-PCR. Obtained reads were also analyzed using an in-house pipeline, GenomonExpression ( https://github.com/Genomon-Project/GenomonExpression ), to obtain fragments per kilobase million (FPKM) values. Gene expression analysis using reverse transcription-polymerase chain reaction All leukemia samples were obtained from bone marrow or peripheral blood at the time of diagnosis; total RNA was prepared using an RNeasy Mini Kit (Qiagen, Hilden, Germany). Quantitative RT-PCR analysis was performed using a CFX 96 Deep Well Real-Time System (Bio-Rad, Foster City, CA). cDNA was pre pared using 0.8–1.0 µg and 0.2µg of total RNA in AML-05 and AML-12 studies, respectively, and Ready-To-Go RT-PCR Beads (GE Healthcare, Buckinghamshire, UK); 1/200 and 1/50 of the prepared cDNA in AML-05 and AML-12 studies, respectively, was used as a template for each PCR reaction. Supplementary Table S2 depicts the primer and probe information used for the PRDM16::SKI expression measurement (IDT, Coralville, IA, USA). Quantitative reverse transcription-polymerase chain reaction for PRDM16 and ABL1 was performed using with TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assay (Thermo Fisher Scientific Inc., Waltham, MA). ABL1 was used as the control gene. 16 , 18 A previous study used TaqMan gene expression assays to analyze PRDM16 and ABL1 . 16 , 18 The expression level of PRDM16 in exon 17 was measured. 16 , 18 The high or low PRDM16::SKI expression was determined based on the PRDM16::SKI /ABL1 ratio of each patient, which was calculated using the receiver operating characteristic curve (data not shown). Targeted sequencing to detect PRDM16::SKI gene fusion in DNA level In order to identify the cleavage points of PRDM16 and SKI at the DNA level, we designed primers for the entire PRDM16 intron 1–2 and the entire SKI intron 1–2 regions (primer data sets are not shown), and performed target sequencing using a NovaSeq 6000 high-throughput sequencing system. Leukemogenicity of the PRDM16::SKI gene transfer to the AML cell line Retroviral bone marrow transduction assays : To generate pMSCV-PRDM16-SKI-IRES-GFP plasmids, PRDM16::SKI was cloned into the XhoI/EcoRI sites of the pMSCV-IRES-GFP (pMIG) vector. Next, 293T cells were transiently transfected with pMIG vectors and the pCL-Eco plasmid using polyethylenimine. Lineage low Sca-1 − c-kit + (LSK) cells were harvested and incubated in X-Vivo 15 (Lonza, Allendale, NJ) supplemented with 50 ng/mL of stem cell factor (SCF), 50 ng/mL of thrombopoietin, 10 ng/mL of interleukin (IL)-3, and 10 ng/mL of IL-6 (Peprotech, Rocky Hill, NJ) for 24 h. After incubation, the cells were spin-infected with a retroviral supernatant supplemented with polybrene (8 µg/mL) in retroNectin (Clontech, Mountain View, CA)-coated plates at 490 g for 45 min at 20°C. Colony-forming assay and histology Using colony assays, 10,000 GFP-positive cells were plated in MethoCult M3234 (StemCell Technologies, Vancouver, Canada) medium supplemented with 20 ng/mL of SCF, 10 ng/mL of granulocyte-macrophage colony-stimulating factor, 10 ng/mL of IL-3, and 10 ng/mL of IL-6, according to the manufacturer’s instructions. Statistical analysis All analyses were performed using EZR (version 1.68; Saitama Medical Centre, Jichi Medical University, Saitama, Japan) 29 , which is a graphical user interface for R (version 4.3.1. The R Foundation for Statistical Computing, Vienna, Austria). Survival distributions were assessed using the Kaplan-Meier method and the differences were compared using the log-rank test. EFS and OS were defined as the times from diagnosis to event (relapse or death of any cause) and from diagnosis to death from any cause, respectively. Continuous variables are presented as means standard deviations (SD) and/or medians with ranges. Categorical variables are represented by frequencies and percentages. For all analyses, the P values were 2-tailed and a P value < 0.05 was considered statistically significant. Results Detection of PRDM16::SKI in pediatric AML we re-analyzed RNA-sequencing data of 139 patients enrolled in the AML-05 study, and identified an out-of-frame PRDM16::SKI fusion gene in 19 patients (Supplementary Table S3). This fusion gene was only the product of 48-base-pair product due to the immediate appearance of a stop codon on the SKI side. This gene fusion was confirmed via Sanger sequencing in these 19 patients (Fig. 1 A). On the other hand, a reciprocal SKI::PRDM16 was not detected, and no fused sequences were identified upstream of PRDM16 _exon 2 based on the RNA-sequencing data on Integral Genome Viewer. Hence, PRDM16 did not fuse with any other genes after cleavage, and it existed as the s PRDM16 (Fig. 1 B). Since s PRDM16 lacking exon 1 initiates transcription from ATG within exon 2 and is not expected to undergo a frameshift compared to the full form, s PRDM16 expression is expected to be maintained, indicating PRDM16 functionality (Fig. 1 C). Although we performed targeted sequencing in order to identify the cleavage points of PRDM16 and SKI at the DNA level, including intron regions, we were unable to identify the cleavage points. Thus, it was thought that this fusion gene occurs at the RNA level as the result of trans-splicing. Prognostic impact of PRDM16::SKI on pediatric AML To determine the frequency of positive PRDM16::SKI expression, the expression level of PRDM16::SKI was measured using real-time PCR in 369 and 329 patients in the AML-05 and AML-12 studies, respectively. Approximately 32% (119/369) and 18% (58/329) of the patients in the AML-05 and AML-12 studies, respectively, presented with a high PRDM16::SKI expression (Fig. S1 ). Patients with a high PRDM16::SKI expression had significantly worse OS and event-free survival (EFS) than those with a low PRDM16::SKI expression (3-year OS: 66% vs. 88%, log-rank P < 0.001 and EFS: 50% vs. 75%, log-rank P < 0.001 in the AML-05 study; 3-year OS: 66% vs. 88%, log-rank P < 0.001 and EFS: 50% vs. 75%, log-rank P < 0.001 in the AML-12 study) (Fig. 2 ). Association between PRDM16::SKI and PRDM16 expression Next, the association between PRDM16::SKI and PRDM16 expression was examined. PRDM16 expression at exon17 was measured. The mean gene expression levels of PRDM16 were 13.45 in PRDM16::SKI -positive patients and 0.93 in PRDM16::SKI -negative patients. Thus, PRDM16 was significantly highly expressed in PRDM16::SKI- positive patients in the AML-05 study (Fig. 3 ). Of the 250 PRDM16::SKI -negative patients in the AML-05 study, 9 had a high PRDM16 expression. Supplementary Table 4 shows the characteristics of these nine patients. RUNX1::PRDM16 , but not PRDM16::SKI , was detected in the patient with the highest PRDM16 expression. Hence, cleavage of PRDM16 at exons 1 and 2 was important in increasing PRDM16 expression. This simultaneously indicated that PRDM16::SKI was not a technical artifact of the sequence detected in RNA-sequencing and not depending on high PRDM16 expression. The remaining patients presented with a high PRDM16 expression that was attributed to other factors such as different cleavage points, but this notion could not be validated in this analysis. The patients were classified into four groups according to PRDM16::SKI and PRDM16 expression (Fig. S2). Neither PRDM16 nor PRDM16::SKI was expressed in 241 of 369 patients in the AML-05 study. Sixty-six patients had a high expression of PRDM16::SKI alone. Further, nine patients had a high expression of PRDM16 only, thereby indicating that the presence of PRDM16::SKI is likely induced by the high PRDM16 expression. Both were highly expressed in 53 patients in the AML-05 study (Fig. S2). The survival rates of these four groups indicated that the high PRDM16 expression group had a worse prognosis than the low PRDM16 expression group, regardless of PRDM16::SKI gene expression. By contrast, patients with a high PRDM16::SKI expression were more likely to have a worse prognosis than those with a low PRDM16::SKI expression in the low PRDM16 expression group (Fig. 4 ). These results were confirmed using data from the AML-12 study. Association between typical genetic abnormalities and PRDM16::SKI expression levels The association between representative genetic abnormalities and the expression level of PRDM16::SKI was examined. The PRDM16::SKI expression was high in KMT2A -PTD-, UBTF -TD-, NUP98::NSD1- , NUP98::KDM5A- , FLT3 -ITD-, and NPM1 -positive patients. Meanwhile, the PRDM16::SKI expression was low in RUNX1::RUNX1T1- , CBFB::MYH11- , bZIP CEBPA- , and CBFA2T3::GLIS2- positive patients (Fig. 5 A and 5 B). Figure 5 C and 5 D present each genetic abnormality in PRDM16::SKI expression-positive patients. Transfection of PRDM16::SKI into the Lin(-)Sca-1(+)c-Kit(+) (LSK) cells and investigation of its association with leukemia development To examine the role of PRDM16::SKI in the development of myeloid leukemia, the fusion gene was introduced into the LSK cells and its colony-forming ability was evaluated. The LSK cells transfected with PRDM16::SKI and the control LSK cells did not significantly differ in terms of colony-forming ability, and the colonies disappeared in the fourth plating assay (Fig. 6 ). Discussion PRDM16 is highly expressed in patients with AML with a poor prognosis. 15 – 18 , 30 , 31 This gene is essential for transcriptional regulation as a repressor by DNA binding, maintenance of stemness, granulocyte differentiation regulation, and TGF-beta signaling. However, the mechanism by which PRDM16 is upregulated in AML has been unknown. In this study, we recurrently detected PRDM16::SKI by re-analyzing RNA-sequencing data and determining the gene expression of all patients. PRDM16 expression increased with the formation of the PRDM16::SKI fusion gene, and the significant association was observed between a high PRDM16 expression and this fusion gene. This can be attributed to gene truncation between exons 1 and 2 of PRDM16 , where the exon 2 side of PRDM16 does not fuse with other partner genes but exists as a short form, causing excessive PRDM16 expression. By contrast, exon 1 of the truncated PRDM16 forms a fusion gene with SKI. Nevertheless, our analysis indicates that this fusion gene is not leukemogenic. Interestingly, our previous DNA methylation analysis using the Infinium Methylation EPIC BeadChip (Illumina,San Diego, CA) showed that the DNA methylation pattern was drastically changed between exon 1 and after exon 2 of the PRDM16 gene in AMLs with high PRDM16 expression. 32 This may be related to the fact that PRDM16 is truncated between exons 1 and 2. However, this could not be revealed the details in this analysis. High PRDM16 and PRDM16::SKI expression was frequently observed in FLT3 -ITD, NUP98::NSD1 , UBTF -TD, KMT2A -PTD, FUS::ERG , and NPM1 , thereby indicating that these gene alterations may induce a high PRDM16 expression. Intriguingly, many of these gene alterations were associated with MENIN-dependent transcription profiles. Recently, in AML cells expressing NUP98::HOXA9 fusion oncoprotein, phase-separated droplets/condensates are formed by the intrinsically disordered region derived from NUP98. 33 , 34 Phase separation forms membrane-less bodies, which regulate gene expression and genome organization. 35 From the perspective of phase separation, other genetic alternations in patients with AML, such as FUS and KMT2A , also possess intrinsically disordered regions that enable phase separation in vivo . 36 Moreover, UBTF and NPM1 are the components of the nucleolus known as a multiphase condensate. 37 – 39 These fusion and mutated proteins without a three-dimensional structure can form the aberrant condensates in the nucleoplasm, likely facilitating a drastic alteration in the high-order genome structure, the fusion of PRDM16 with SKI , and the subsequently high expression of PRDM16 . By contrast, patients with RUNX1::RUNX1T1 , CBFB::MYH11 , CBFA2T3::GLIS2 , monosomy 7, bZIP CEBPA mutation, or KMT2A arrangement had a lower expression of PRDM16::SKI . Therefore, these patients may have fundamentally different AML pathological mechanisms. The components of the NUP98::NSD1 -signature specified for the high PRDM16 expression were NUP98 rearrangements, DEK::NUP214 , NPM1 , UBTF -TD, and KMT2A -PTD. This finding aligns with that of other reports. 16 – 18 , 40 In this study, there was a difference in the frequency of high expression of PRDM16::SKI between the AML-05 and AML-12 studies. One reason for this was that in the AML-12 study, it was not possible to obtain sufficient amounts of RNA compared to AML-05 study, so that the amount of RNA used to create cDNA was different. The prognosis of patients with high PRDM16 expression in the AML-12 study improved compared with that of patients with high PRDM16 expression in the AML-05 study. This finding may be attributed to the use of FLT3 inhibitors in patients who dropped out from the AML-12 study due to non-remission or relapse and those patients may have been rescued. Indeed, FLT3 -ITD-positive patients had a significantly higher PRDM16::SKI expression (Fig. 7 ), thereby contributing to the improved prognosis of patients with high PRDM16::SKI and PRDM16 expression. In conclusion, the PRDM16::SKI gene fusion, which contributes to a high PRDM16 expression in pediatric AML, has a high frequency. Further, it is a major contributor to the development of AML, along with core binding factor-AML, and MECOM ( EVI1 ) high expression. The patients in this study commonly had a poor prognosis. Hence, appropriate treatments for this patient group must be urgently developed, and reducing the expression levels of PRDM16 can be a therapeutic target. Declarations Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research (KAKEN, grant number JP19K08350 to N.S. and JP21K15870 to S.T.) and Exploratory Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Practical Research for Innovative Cancer Control from Japan Agency for Medical Research and Development (AMED, grant number JP20ck0106634 to N.S. and JP23ck0106853 to D.T.); Kawano Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics, a research grant from the Japanese Society of Hematology to N.S.; and a research grant from the Takeda Science Foundation to N.S. The authors also thank Enago for English language review. Authorship Contributions Contribution: M.Y., T. Komori, and N.S. wrote the paper; M.Y., T. Komori, J.I., S.T., Y.H., G.Y., K.Y. and N.S. performed the experiments; M.Y, T. Komori, J.I., S.T., Y.H., G.Y., K.Y., and N.S. analyzed the data the clinical data and genome samples were provided from JCCG; N.S. designed the study and supervised the work; and all authors critically reviewed and revised the manuscript. Conflict-of-interest disclosure The authors declare no competing financial interests. References Frohling S, Scholl C, Gilliland DG, Levine RL. Genetics of myeloid malignancies: pathogenetic and clinical implications. J Clin Oncol. 2005;23:6285–95. Marcucci G, Haferlach T, Dohner H. Molecular genetics of adult acute myeloid leukemia: prognostic and therapeutic implications. J Clin Oncol. 2011;29:475–86. Pui CH, Carroll WL, Meshinchi S, Arceci RJ. Biology, risk stratification, and therapy of pediatric acute leukemias: an update. J Clin Oncol. 2011;29:551–65. Patel JP, Gonen M, Figueroa ME, et al. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med. 2012;366:1079–89. 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;24:103–12. Kong XT, Ida K, Ichikawa H, et al. Consistent detection of TLS/FUS-ERG chimeric transcripts in acute myeloid leukemia with t(16;21)(p11;q22) and identification of a novel transcript. Blood. 1997;90:1192–9. de Rooij JD, Hollink IH, Arentsen-Peters ST, et al. NUP98/JARID1A is a novel recurrent abnormality in pediatric acute megakaryoblastic leukemia with a distinct HOX gene expression pattern. Leukemia. 2013;27:2280–8. Hollink IH, van den Heuvel-Eibrink MM, Arentsen-Peters ST, et al. NUP98/NSD1 characterizes a novel poor prognostic group in acute myeloid leukemia with a distinct HOX gene expression pattern. Blood. 2011;118:3645–56. Matsuo H, Kajihara M, Tomizawa D, et al. EVI1 overexpression is a poor prognostic factor in pediatric patients with mixed lineage leukemia-AF9 rearranged acute myeloid leukemia. Haematologica. 2014;99:e225–7. Preudhomme C, Sagot C, Boissel N, et al. ALFA Group. Favorable prognostic significance of CEBPA mutations in patients with de novo acute myeloid leukemia: a study from the Acute Leukemia French Association (ALFA). Blood. 2002;100:2717–23. Hollink IH, Zwaan CM, Zimmermann M, et al. Favorable prognostic impact of NPM1 gene mutations in childhood acute myeloid leukemia, with emphasis on cytogenetically normal AML. Leukemia. 2009;23:262–70. Shimada A, Taki T, Tabuchi K, et al. Tandem duplications of MLL and FLT3 are correlated with poor prognoses in pediatric acute myeloid leukemia: a study of the Japanese childhood AML Cooperative Study Group. Pediatr Blood Cancer. 2008;50:264–9. Taketani T, Taki T, Sugita K, et al. FLT3 mutations in the activation loop of tyrosine kinase domain are frequently found in infant ALL with MLL rearrangements and pediatric ALL with hyperdiploidy. Blood. 2004;103:1085–8. Pratcorona M, Brunet S, Nomded´eu J, et al. Grupo Cooperativo Para el Estudio y Tratamiento de las Leucemias Agudas Mielobl ´ asticas. Favorable outcome of patients with acute myeloid leukemia harboring a low-allelic burden FLT3-ITD mutation and concomitant NPM1 mutation: relevance to postremission therapy. Blood. 2013;121:2734–8. Shiba N, Ichikawa H, Taki T, Park MJ, Jo A, Mitani S, et al. NUP98-NSD1 gene fusion and its related gene expression signature are strongly associated with a poor prognosis in pediatric acute myeloid leukemia. Genes Chromosomes Cancer. 2013;52:683–93. Jo A, Mitani S, Shiba N, et al. High expression of EVI1 and MEL1 is a compelling poor prognostic marker of pediatric AML. Leukemia. 2015;29:1076–83. Umeda M, Ma J, Huang BJ, Hagiwara K, Westover T, Abdelhamed S, et al. Integrated Genomic Analysis Identifies UBTF Tandem Duplications as a Recurrent Lesion in Pediatric Acute Myeloid Leukemia. Blood Cancer Discov. 2022;3:194–207. Shiba N, Ohki K, Kobayashi T, et al. High PRDM16 expression identifies a prognostic subgroup of pediatric acute myeloid leukaemia correlated to FLT3-ITD, KMT2A-PTD, and NUP98-NSD1: the results of the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-05 trial. Br J Haematol. 2016; 172:581–91. Mochizuki N, Shimizu S, Nagasawa T, Tanaka H, Taniwaki M, Yokota J, et al. A novel gene, MEL1, mapped to 1p36.3 is highly homologous to the MDS1/EVI1 gene and is transcriptionally activated in t(1;3)(p36;q21)-positive leukemia cells. Blood. 2000;96:3209–14. Shing DC, Trubia M, Marchesi F, Radaelli E, Belloni E, Tapinassi C, et al. Overexpression of sPRDM16 coupled with loss of p53 induces myeloid leukemias in mice. J Clin Invest. 2007;117:3696–707. Dong S, Chen J. SUMOylation of sPRDM16 promotes the progression of acute myeloid leukemia. BMC Cancer. 2015;15:893. Masetti R, Togni M, Astolfi A, Pigazzi M, Indio V, Rivalta B, et al. Whole transcriptome sequencing of a paediatric case of de novo acute myeloid leukaemia with del(5q) reveals RUNX1-USP42 and PRDM16-SKI fusion transcripts. Br J Haematol. 2014;166:449–52. Hu T, Morita K, Hill MC, Jiang Y, Kitano A, Saito Y, et al. PRDM16s transforms megakaryocyte-erythroid progenitors into myeloid leukemia-initiating cells. Blood. 2019;134:614–25. Kudo K, Kojima S, Tabuchi K, Yabe H, Tawa A, Imaizumi M, et al. 2007. Prospective study of a pirarubicin, intermediate-dose cytarabine, and etoposide regimen in children with Down syndrome and acute myeloid leukemia: The Japanese Childhood AML Cooperative Study Group. J Clin Oncol. 25:5442–7. Tsukimoto I, Tawa A, Horibe K, Tabuchi K, Kigasawa H, Tsuchida M, et al. Risk-stratified therapy and the intensive use of cytarabine improves the outcome in childhood acute myeloid leukemia: the AML99 trial from the Japanese Childhood AML Cooperative Study Group. J Clin Oncol. 2009;27:4007–13. Tomizawa D, Matsubayashi J, Iwamoto S, Hiramatsu H, Hasegawa D, Moritake H, et al. High-dose cytarabine induction therapy and flow cytometric measurable residual disease monitoring for children with acute myeloid leukemia. Leukemia. 2024;38:202–6. Tomizawa D, Tawa A, Watanabe T, et al. Excess treatment reduction including anthracyclines results in higher incidence of relapse in core binding factor acute myeloid leukemia in children. Leukemia. 2013;27:2413–6. Shiraishi Y, Fujimoto A, Furuta M, et al. Integrated analysis of whole genome and transcriptome sequencing reveals diverse transcriptomic aberrations driven by somatic genomic changes in liver cancers. PLoS One. 2014;9:e114263. Kanda, Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplantation. 2013;48:452–8. Yamato G, Yamaguchi H, Handa H, et al. Clinical features and prognostic impact of PRDM16 expression in adult acute myeloid leukemia. Genes Chromosomes Cancer. 2017;56:800–9. Dao FT, Chen WM, Long LY, et al. High PRDM16 expression predicts poor outcomes in adult acute myeloid leukemia patients with intermediate cytogenetic risk: a comprehensive cohort study from a single Chinese center. Leuk Lymphoma. 2021;62:185–93. Yamato G, Kawai T, Shiba N, Ikeda J, Hara Y, Ohki K, et al. Genome-wide DNA methylation analysis in pediatric acute myeloid leukemia. Blood Adv. 2022;6:3207–19. Ahn JH, Davis ES, Daugird TA, Zhao S, Quiroga IY, Uryu H, et al. Phase separation drives aberrant chromatin looping and cancer development. Nature. 2021;595:591–5. Chandra B, Michmerhuizen NL, Shirnekhi HK, Tripathi S, Pioso BJ, Baggett DW, et al. Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation. Cancer Discov. 2022;12:1152–69. Hyman AA, Weber CA, Jülicher F. Liquid-liquid phase separation in biology. Annu Rev Cell Dev Biol. 2014;30:39–58. Patel A, Lee HO, Jawerth L, et al. A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell. 2015;162:1066–77. Mitrea DM, Cika JA, Guy CS, Ban D, Banerjee PR, Stanley CB, et al. Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA. Elife. 2016;5:e13571. Lafontaine DLJ, Riback JA, Bascetin R, Brangwynne CP. The nucleolus as a multiphase liquid condensate. Nat Rev Mol Cell Biol. 2021;22:165–82. Ide S, Imai R, Ochi H, Maeshima K. Transcriptional suppression of ribosomal DNA with phase separation. Sci Adv. 2020;6:eabb5953. Kaburagi T, Shiba N, Yamato G, et al. UBTF-internal tandem duplication as a novel poor prognostic factor in pediatric acute myeloid leukemia. Genes Chromosomes Cancer. 2023;62:202–9. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files Supplementary250309F.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6187243","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":427115895,"identity":"1a8bc2de-aaa1-4c2a-8100-a66e88a85f75","order_by":0,"name":"Norio 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high or low \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRDM16::SKI \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression in pediatric AML.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Overall survival in the AML-05 study. \u003cstrong\u003e(B)\u003c/strong\u003e Event-free survival in the AML-05 study.\u003cstrong\u003e (C) \u003c/strong\u003eOverall survival in the AML-12 study.\u003cstrong\u003e (D)\u003c/strong\u003e Event-free survival in the AML-12 study.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6187243/v1/10c35fcb3c50f1055dedf03b.png"},{"id":78644836,"identity":"34674fb9-1bed-4db4-a89a-8279328dcdf7","added_by":"auto","created_at":"2025-03-17 07:30:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":898515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssociations between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRDM16::SKI \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRDM16 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eAML-05 study. \u003cstrong\u003e(B) \u003c/strong\u003eAML-12 study.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6187243/v1/2b38e8152a30f2101e7a6c63.png"},{"id":78644137,"identity":"e9fc8850-ca48-4fcb-88ce-45ed795d631f","added_by":"auto","created_at":"2025-03-17 07:22:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":590123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaplan–Meier analysis based on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRDM16\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRDM16;::SKI\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Overall survival in the AML-05 study. \u003cstrong\u003e(B)\u003c/strong\u003eEvent-free survival in the AML-05 study.\u003cstrong\u003e (C) \u003c/strong\u003eOverall survival in the AML-12 study.\u003cstrong\u003e (D)\u003c/strong\u003e Event-free survival in the AML-12 study.\u003c/p\u003e","description":"","filename":"OnlineFigure4300dpi.png","url":"https://assets-eu.researchsquare.com/files/rs-6187243/v1/afc488a16d13f2d628b482a5.png"},{"id":78644134,"identity":"00d69e4f-ad4b-473f-b89a-5328eae6ee23","added_by":"auto","created_at":"2025-03-17 07:22:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":204233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssociations between 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(A and B) \u003c/strong\u003e\u003cem\u003ePRDM16::SKI \u003c/em\u003eexpression value in each cytogenetic alterations in AML-05 and AML-12 studies, respectively.\u003cstrong\u003e (C and D)\u003c/strong\u003e Frequencies of positivity with high \u003cem\u003ePRDM16::SKI \u003c/em\u003eexpression in various cytogenetic alterations in AML-05 and AML-12 studies.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6187243/v1/6f21e912438c02d9be7e906e.png"},{"id":78644135,"identity":"d3a618f9-c79d-45a9-9f4f-679edd5fee36","added_by":"auto","created_at":"2025-03-17 07:22:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":21491,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe colony numbers for each round of replating are indicated on the graph.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ePRDM16::SKI\u003c/em\u003efusion gene was introduced into the LSK cells, and the colony-forming ability was evaluated. The LSK cells transfected with \u003cem\u003ePRDM16::SKI \u003c/em\u003eand the control\u003cem\u003e \u003c/em\u003epMIG cells did not significantly differ in terms of colony-forming ability, and the colonies disappeared in the fourth plating assay.\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6187243/v1/96a8d10ffda6de1968cd1e69.png"},{"id":78643816,"identity":"debbaa6b-e082-421e-8678-9ebffad8abd6","added_by":"auto","created_at":"2025-03-17 07:14:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":18046,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRDM16::SKI \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression level with or without \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFLT3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-ITD.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6187243/v1/dcf1f232502b3c45afcdde3e.png"},{"id":79827521,"identity":"d54861cd-fd24-4c05-9c48-cf0cf222853a","added_by":"auto","created_at":"2025-04-03 09:49:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4867650,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6187243/v1/226b54fe-894e-4b35-9a4a-14b04777853e.pdf"},{"id":78643817,"identity":"927c8ad5-211a-4545-8bfa-15408f603286","added_by":"auto","created_at":"2025-03-17 07:14:16","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":633856,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary250309F.doc","url":"https://assets-eu.researchsquare.com/files/rs-6187243/v1/e20111d228b27b22a64e0055.doc"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"PRDM16::SKI is a predictor of aberrant expression of the short variant of PRDM16 in pediatric acute myeloid leukemia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe pathogenesis of acute myeloid leukemia (AML) is heterogeneous. AML is caused by various chromosomal aberrations, gene mutations/epigenetic modifications, and downregulated/upregulated gene expression, leading to increased proliferation and decreased hematopoietic progenitor cell differentiation.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Various genetic analyses are performed on patients with pediatric AML, and many genetic abnormalities have been identified and used for prognostic stratification. In particular, \u003cem\u003eRUNX1::RUNX1T1\u003c/em\u003e, \u003cem\u003eCBFB::MYH11\u003c/em\u003e, \u003cem\u003eFUS::ERG\u003c/em\u003e, \u003cem\u003eCBFA2T3::GLIS2\u003c/em\u003e, \u003cem\u003eNUP98\u003c/em\u003e-rearrangements, and \u003cem\u003eKMT2A\u003c/em\u003e rearrangements and bZIP \u003cem\u003eCEBPA\u003c/em\u003e, \u003cem\u003eNPM1\u003c/em\u003e, and \u003cem\u003eFLT3\u003c/em\u003e-internal tandem duplication (ITD) mutations are frequently observed and considered useful for prognostic prediction.\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12 CR13\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e A distinctive \u003cem\u003eNUP98::NSD1\u003c/em\u003e fusion gene-related signature, forming a unique entity, was identified in pediatric patients with AML.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e This signature is characterized by elevated expression of PR domain containing 16 (\u003cem\u003ePRDM16\u003c/em\u003e; also known as \u003cem\u003eMEL1\u003c/em\u003e).\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e This signature is closely associated with \u003cem\u003eKMT2A\u003c/em\u003e-PTD, \u003cem\u003eFLT3\u003c/em\u003e-ITD, \u003cem\u003eNPM1\u003c/em\u003e, and \u003cem\u003eUBTF\u003c/em\u003e-TD mutations,\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and is significantly related to poor prognosis other than \u003cem\u003eNPM1\u003c/em\u003e mutations.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe translocation t(1;3)(p36;q21)/\u003cem\u003ePRDM16::MECOM\u003c/em\u003e occurs in a subset of myelodysplastic syndrome (MDS) and AML.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e \u003cem\u003ePRDM16\u003c/em\u003e is located near the 1p36.3 breakpoint and is specifically expressed in t(1:3)(p36,q21)-positive MDS/AML. The protein encoded by this gene is a zinc finger transcription factor, and it contains an N-terminal PR domain. The translocation results in the overexpression of a truncated version of this protein (sPRDM16), which lacks the PR domain and may play an important role in the pathogenesis of MDS and AML.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHowever, the cause of the high \u003cem\u003ePRDM16\u003c/em\u003e expression has not yet been elucidated in most patients. In this study, we re-analyzed RNA-sequencing data of 139 patients enrolled in the AML-05 study, conducted by the Japanese Children\u0026rsquo;s Cancer Group (JCCG), to elucidate the cause of this high expression. We identified an out-of-frame \u003cem\u003ePRDM16::SKI\u003c/em\u003e fusion gene in 19 patients. This fusion gene has been reported in previous case reports and was associated with high \u003cem\u003ePRDM16\u003c/em\u003e expression.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e This fusion gene was only the product of 48-base-pair product due to the immediate appearance of a stop codon on the \u003cem\u003eSKI\u003c/em\u003e side.\u003c/p\u003e \u003cp\u003eIn addition, the reciprocal \u003cem\u003eSKI::PRDM16\u003c/em\u003e was not detected, and no other genes were observed upstream of truncated \u003cem\u003ePRDM16\u003c/em\u003e. Hence, the short form of \u003cem\u003ePRDM16\u003c/em\u003e (s\u003cem\u003ePRDM16\u003c/em\u003e-∆exon1) plays an important on causing leukemia. s\u003cem\u003ePRDM16\u003c/em\u003e is associated with leukemia progression in mouse experiments.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Therefore, \u003cem\u003ePRDM16::SKI\u003c/em\u003e is a useful predictor of the presence of truncated \u003cem\u003ePRDM16\u003c/em\u003e. This fusion gene is strongly correlated with a high \u003cem\u003ePRDM16\u003c/em\u003e expression. The current study aimed to examine the clinical and functional significance of this fusion gene and its impact on patient prognosis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eThe current study enrolled 369 patients with de novo AML who were registered in the JCCG\u0026rsquo;s AML-05 study between November 2006 and December 2010.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e In addition, among the 387 patients with AML who were registered in the JCCG AML-12 study, 329 with known mutation status who had available gene expression data were included in the validation cohort.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003ePatients diagnosed with acute promyelocytic leukemia or Down syndrome-associated AML were excluded from this analysis. This study was conducted in accordance with the Declaration of Helsinki and approved by the institutional review board of Yokohama City University Hospital and the ethical review board of the JCCG. A written informed consent from all patients or their parents/guardians was obtained.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTreatments\u003c/h3\u003e\n\u003cp\u003eThe protocol treatment regimen for the AML-05 and AML-12 studies have already reported.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eRNA sequencing\u003c/h3\u003e\n\u003cp\u003eWe performed RNA-seq for 139 out of the 369 patients with pediatric AML in order to obtain a complete registry of gene rearrangements, other genetic lesions, and gene expressions in pediatric AML. The RNA-seq data were available at the European Genome-Phenome Archive (EGAD00001005078). The cytogenetic characteristics of the analyzed patients are shown in supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The study population mainly included patients with a normal karyotype (60/70 patients), \u003cem\u003eFLT3-\u003c/em\u003eITD (33/47 patients), \u003cem\u003eKMT2A\u003c/em\u003e-PTD (12/13 patients), and high \u003cem\u003ePRDM16\u003c/em\u003e expression (65/84 patients). Among 369 patients, all 137 patients with core binding factor AML (CBF-AML) and many patients with \u003cem\u003eKMT2A\u003c/em\u003e rearrangements were excluded from RNA-seq, because the main purpose of this study was to identify unknown fusion genes and to investigate the gene expression patterns of cases with poor prognoses.\u003c/p\u003e \u003cp\u003eThe quality of the extracted RNA was assessed using TapeStation system (Agilent Technologies, Santa Clara, CA). Sequencing libraries were prepared using a NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA), and prepared libraries were run on a HiSeq 2500 high-throughput sequencing system. Sequencing reads were aligned using bowtie and blat, and fusion genes were analyzed using Genomon-fusion.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Candidate gene fusions were validated by RT-PCR. Obtained reads were also analyzed using an in-house pipeline, GenomonExpression (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/Genomon-Project/GenomonExpression\u003c/span\u003e\u003cspan address=\"https://github.com/Genomon-Project/GenomonExpression\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), to obtain fragments per kilobase million (FPKM) values.\u003c/p\u003e\n\u003ch3\u003eGene expression analysis using reverse transcription-polymerase chain reaction\u003c/h3\u003e\n\u003cp\u003eAll leukemia samples were obtained from bone marrow or peripheral blood at the time of diagnosis; total RNA was prepared using an RNeasy Mini Kit (Qiagen, Hilden, Germany). Quantitative RT-PCR analysis was performed using a CFX 96 Deep Well Real-Time System (Bio-Rad, Foster City, CA). cDNA was pre pared using 0.8\u0026ndash;1.0 \u0026micro;g and 0.2\u0026micro;g of total RNA in AML-05 and AML-12 studies, respectively, and Ready-To-Go RT-PCR Beads (GE Healthcare, Buckinghamshire, UK); 1/200 and 1/50 of the prepared cDNA in AML-05 and AML-12 studies, respectively, was used as a template for each PCR reaction. Supplementary Table S2 depicts the primer and probe information used for the \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression measurement (IDT, Coralville, IA, USA). Quantitative reverse transcription-polymerase chain reaction for \u003cem\u003ePRDM16\u003c/em\u003e and \u003cem\u003eABL1\u003c/em\u003e was performed using with TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assay (Thermo Fisher Scientific Inc., Waltham, MA). \u003cem\u003eABL1\u003c/em\u003e was used as the control gene.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e A previous study used TaqMan gene expression assays to analyze \u003cem\u003ePRDM16\u003c/em\u003e and \u003cem\u003eABL1\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e The expression level of \u003cem\u003ePRDM16\u003c/em\u003e in exon 17 was measured.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e The high or low \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression was determined based on the \u003cem\u003ePRDM16::SKI /ABL1\u003c/em\u003e ratio of each patient, which was calculated using the receiver operating characteristic curve (data not shown).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTargeted sequencing to detect\u003c/b\u003e \u003cb\u003ePRDM16::SKI\u003c/b\u003e \u003cb\u003egene fusion in DNA level\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to identify the cleavage points of \u003cem\u003ePRDM16\u003c/em\u003e and \u003cem\u003eSKI\u003c/em\u003e at the DNA level, we designed primers for the entire \u003cem\u003ePRDM16\u003c/em\u003e intron 1\u0026ndash;2 and the entire \u003cem\u003eSKI\u003c/em\u003e intron 1\u0026ndash;2 regions (primer data sets are not shown), and performed target sequencing using a NovaSeq 6000 high-throughput sequencing system.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLeukemogenicity of the\u003c/b\u003e \u003cb\u003ePRDM16::SKI\u003c/b\u003e \u003cb\u003egene transfer to the AML cell line\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eRetroviral bone marrow transduction assays\u003c/b\u003e: To generate pMSCV-PRDM16-SKI-IRES-GFP plasmids, \u003cem\u003ePRDM16::SKI\u003c/em\u003e was cloned into the XhoI/EcoRI sites of the pMSCV-IRES-GFP (pMIG) vector. Next, 293T cells were transiently transfected with pMIG vectors and the pCL-Eco plasmid using polyethylenimine. Lineage\u003csup\u003elow\u003c/sup\u003eSca-1\u003csup\u003e\u0026minus;\u003c/sup\u003ec-kit\u003csup\u003e+\u003c/sup\u003e (LSK) cells were harvested and incubated in X-Vivo 15 (Lonza, Allendale, NJ) supplemented with 50 ng/mL of stem cell factor (SCF), 50 ng/mL of thrombopoietin, 10 ng/mL of interleukin (IL)-3, and 10 ng/mL of IL-6 (Peprotech, Rocky Hill, NJ) for 24 h. After incubation, the cells were spin-infected with a retroviral supernatant supplemented with polybrene (8 \u0026micro;g/mL) in retroNectin (Clontech, Mountain View, CA)-coated plates at 490 g for 45 min at 20\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eColony-forming assay and histology\u003c/strong\u003e \u003cp\u003eUsing colony assays, 10,000 GFP-positive cells were plated in MethoCult M3234 (StemCell Technologies, Vancouver, Canada) medium supplemented with 20 ng/mL of SCF, 10 ng/mL of granulocyte-macrophage colony-stimulating factor, 10 ng/mL of IL-3, and 10 ng/mL of IL-6, according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll analyses were performed using EZR (version 1.68; Saitama Medical Centre, Jichi Medical University, Saitama, Japan)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, which is a graphical user interface for R (version 4.3.1. The R Foundation for Statistical Computing, Vienna, Austria). Survival distributions were assessed using the Kaplan-Meier method and the differences were compared using the log-rank test. EFS and OS were defined as the times from diagnosis to event (relapse or death of any cause) and from diagnosis to death from any cause, respectively. Continuous variables are presented as means standard deviations (SD) and/or medians with ranges. Categorical variables are represented by frequencies and percentages. For all analyses, the P values were 2-tailed and a P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDetection of\u003c/b\u003e \u003cb\u003ePRDM16::SKI\u003c/b\u003e \u003cb\u003ein pediatric AML\u003c/b\u003e\u003c/p\u003e \u003cp\u003ewe re-analyzed RNA-sequencing data of 139 patients enrolled in the AML-05 study, and identified an out-of-frame \u003cem\u003ePRDM16::SKI\u003c/em\u003e fusion gene in 19 patients (Supplementary Table S3). This fusion gene was only the product of 48-base-pair product due to the immediate appearance of a stop codon on the \u003cem\u003eSKI\u003c/em\u003e side. This gene fusion was confirmed via Sanger sequencing in these 19 patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). On the other hand, a reciprocal \u003cem\u003eSKI::PRDM16\u003c/em\u003e was not detected, and no fused sequences were identified upstream of \u003cem\u003ePRDM16\u003c/em\u003e_exon 2 based on the RNA-sequencing data on Integral Genome Viewer. Hence, \u003cem\u003ePRDM16\u003c/em\u003e did not fuse with any other genes after cleavage, and it existed as the s\u003cem\u003ePRDM16\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Since s\u003cem\u003ePRDM16\u003c/em\u003e lacking exon 1 initiates transcription from ATG within exon 2 and is not expected to undergo a frameshift compared to the full form, s\u003cem\u003ePRDM16\u003c/em\u003e expression is expected to be maintained, indicating \u003cem\u003ePRDM16\u003c/em\u003e functionality (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Although we performed targeted sequencing in order to identify the cleavage points of \u003cem\u003ePRDM16\u003c/em\u003e and \u003cem\u003eSKI\u003c/em\u003e at the DNA level, including intron regions, we were unable to identify the cleavage points. Thus, it was thought that this fusion gene occurs at the RNA level as the result of trans-splicing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePrognostic impact of\u003c/b\u003e \u003cb\u003ePRDM16::SKI\u003c/b\u003e \u003cb\u003eon pediatric AML\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the frequency of positive \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression, the expression level of \u003cem\u003ePRDM16::SKI\u003c/em\u003e was measured using real-time PCR in 369 and 329 patients in the AML-05 and AML-12 studies, respectively. Approximately 32% (119/369) and 18% (58/329) of the patients in the AML-05 and AML-12 studies, respectively, presented with a high \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Patients with a high \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression had significantly worse OS and event-free survival (EFS) than those with a low \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression (3-year OS: 66% vs. 88%, log-rank P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and EFS: 50% vs. 75%, log-rank P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 in the AML-05 study; 3-year OS: 66% vs. 88%, log-rank P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and EFS: 50% vs. 75%, log-rank P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 in the AML-12 study) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAssociation between\u003c/b\u003e \u003cb\u003ePRDM16::SKI\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ePRDM16\u003c/b\u003e \u003cb\u003eexpression\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNext, the association between \u003cem\u003ePRDM16::SKI\u003c/em\u003e and \u003cem\u003ePRDM16\u003c/em\u003e expression was examined. \u003cem\u003ePRDM16\u003c/em\u003e expression at exon17 was measured. The mean gene expression levels of \u003cem\u003ePRDM16\u003c/em\u003e were 13.45 in \u003cem\u003ePRDM16::SKI\u003c/em\u003e-positive patients and 0.93 in \u003cem\u003ePRDM16::SKI\u003c/em\u003e-negative patients. Thus, \u003cem\u003ePRDM16\u003c/em\u003e was significantly highly expressed in \u003cem\u003ePRDM16::SKI-\u003c/em\u003epositive patients in the AML-05 study (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Of the 250 \u003cem\u003ePRDM16::SKI\u003c/em\u003e-negative patients in the AML-05 study, 9 had a high \u003cem\u003ePRDM16\u003c/em\u003e expression. Supplementary Table\u0026nbsp;4 shows the characteristics of these nine patients. \u003cem\u003eRUNX1::PRDM16\u003c/em\u003e, but not \u003cem\u003ePRDM16::SKI\u003c/em\u003e, was detected in the patient with the highest \u003cem\u003ePRDM16\u003c/em\u003e expression. Hence, cleavage of \u003cem\u003ePRDM16\u003c/em\u003e at exons 1 and 2 was important in increasing \u003cem\u003ePRDM16\u003c/em\u003e expression. This simultaneously indicated that \u003cem\u003ePRDM16::SKI\u003c/em\u003e was not a technical artifact of the sequence detected in RNA-sequencing and not depending on high \u003cem\u003ePRDM16\u003c/em\u003e expression. The remaining patients presented with a high \u003cem\u003ePRDM16\u003c/em\u003e expression that was attributed to other factors such as different cleavage points, but this notion could not be validated in this analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe patients were classified into four groups according to \u003cem\u003ePRDM16::SKI\u003c/em\u003e and \u003cem\u003ePRDM16\u003c/em\u003e expression (Fig. S2). Neither \u003cem\u003ePRDM16\u003c/em\u003e nor \u003cem\u003ePRDM16::SKI\u003c/em\u003e was expressed in 241 of 369 patients in the AML-05 study. Sixty-six patients had a high expression of \u003cem\u003ePRDM16::SKI\u003c/em\u003e alone. Further, nine patients had a high expression of \u003cem\u003ePRDM16\u003c/em\u003e only, thereby indicating that the presence of \u003cem\u003ePRDM16::SKI\u003c/em\u003e is likely induced by the high \u003cem\u003ePRDM16\u003c/em\u003e expression. Both were highly expressed in 53 patients in the AML-05 study (Fig. S2). The survival rates of these four groups indicated that the high \u003cem\u003ePRDM16\u003c/em\u003e expression group had a worse prognosis than the low \u003cem\u003ePRDM16\u003c/em\u003e expression group, regardless of \u003cem\u003ePRDM16::SKI\u003c/em\u003e gene expression. By contrast, patients with a high \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression were more likely to have a worse prognosis than those with a low \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression in the low \u003cem\u003ePRDM16\u003c/em\u003e expression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results were confirmed using data from the AML-12 study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAssociation between typical genetic abnormalities and\u003c/b\u003e \u003cb\u003ePRDM16::SKI\u003c/b\u003e \u003cb\u003eexpression levels\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe association between representative genetic abnormalities and the expression level of \u003cem\u003ePRDM16::SKI\u003c/em\u003e was examined. The \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression was high in \u003cem\u003eKMT2A\u003c/em\u003e-PTD-, \u003cem\u003eUBTF\u003c/em\u003e-TD-, \u003cem\u003eNUP98::NSD1-\u003c/em\u003e, \u003cem\u003eNUP98::KDM5A-\u003c/em\u003e, \u003cem\u003eFLT3\u003c/em\u003e-ITD-, and \u003cem\u003eNPM1\u003c/em\u003e-positive patients. Meanwhile, the \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression was low in \u003cem\u003eRUNX1::RUNX1T1-\u003c/em\u003e, \u003cem\u003eCBFB::MYH11-\u003c/em\u003e, bZIP \u003cem\u003eCEBPA-\u003c/em\u003e, and \u003cem\u003eCBFA2T3::GLIS2-\u003c/em\u003epositive patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD present each genetic abnormality in \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression-positive patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTransfection of\u003c/b\u003e \u003cb\u003ePRDM16::SKI\u003c/b\u003e \u003cb\u003einto the Lin(-)Sca-1(+)c-Kit(+) (LSK) cells and investigation of its association with leukemia development\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo examine the role of \u003cem\u003ePRDM16::SKI\u003c/em\u003e in the development of myeloid leukemia, the fusion gene was introduced into the LSK cells and its colony-forming ability was evaluated. The LSK cells transfected with \u003cem\u003ePRDM16::SKI\u003c/em\u003e and the control LSK cells did not significantly differ in terms of colony-forming ability, and the colonies disappeared in the fourth plating assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003ePRDM16\u003c/em\u003e is highly expressed in patients with AML with a poor prognosis.\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e This gene is essential for transcriptional regulation as a repressor by DNA binding, maintenance of stemness, granulocyte differentiation regulation, and TGF-beta signaling. However, the mechanism by which \u003cem\u003ePRDM16\u003c/em\u003e is upregulated in AML has been unknown. In this study, we recurrently detected \u003cem\u003ePRDM16::SKI\u003c/em\u003e by re-analyzing RNA-sequencing data and determining the gene expression of all patients. \u003cem\u003ePRDM16\u003c/em\u003e expression increased with the formation of the \u003cem\u003ePRDM16::SKI\u003c/em\u003e fusion gene, and the significant association was observed between a high \u003cem\u003ePRDM16\u003c/em\u003e expression and this fusion gene. This can be attributed to gene truncation between exons 1 and 2 of \u003cem\u003ePRDM16\u003c/em\u003e, where the exon 2 side of \u003cem\u003ePRDM16\u003c/em\u003e does not fuse with other partner genes but exists as a short form, causing excessive \u003cem\u003ePRDM16\u003c/em\u003e expression. By contrast, exon 1 of the truncated \u003cem\u003ePRDM16\u003c/em\u003e forms a fusion gene with \u003cem\u003eSKI.\u003c/em\u003e Nevertheless, our analysis indicates that this fusion gene is not leukemogenic. Interestingly, our previous DNA methylation analysis using the Infinium Methylation EPIC BeadChip (Illumina,San Diego, CA) showed that the DNA methylation pattern was drastically changed between exon 1 and after exon 2 of the \u003cem\u003ePRDM16\u003c/em\u003e gene in AMLs with high \u003cem\u003ePRDM16\u003c/em\u003e expression.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e This may be related to the fact that \u003cem\u003ePRDM16\u003c/em\u003e is truncated between exons 1 and 2. However, this could not be revealed the details in this analysis.\u003c/p\u003e \u003cp\u003eHigh \u003cem\u003ePRDM16\u003c/em\u003e and \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression was frequently observed in \u003cem\u003eFLT3\u003c/em\u003e-ITD, \u003cem\u003eNUP98::NSD1\u003c/em\u003e, \u003cem\u003eUBTF\u003c/em\u003e-TD, \u003cem\u003eKMT2A\u003c/em\u003e-PTD, \u003cem\u003eFUS::ERG\u003c/em\u003e, and \u003cem\u003eNPM1\u003c/em\u003e, thereby indicating that these gene alterations may induce a high \u003cem\u003ePRDM16\u003c/em\u003e expression. Intriguingly, many of these gene alterations were associated with MENIN-dependent transcription profiles. Recently, in AML cells expressing \u003cem\u003eNUP98::HOXA9\u003c/em\u003e fusion oncoprotein, phase-separated droplets/condensates are formed by the intrinsically disordered region derived from NUP98.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Phase separation forms membrane-less bodies, which regulate gene expression and genome organization.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e From the perspective of phase separation, other genetic alternations in patients with AML, such as \u003cem\u003eFUS\u003c/em\u003e and \u003cem\u003eKMT2A\u003c/em\u003e, also possess intrinsically disordered regions that enable phase separation \u003cem\u003ein vivo\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Moreover, \u003cem\u003eUBTF\u003c/em\u003e and \u003cem\u003eNPM1\u003c/em\u003e are the components of the nucleolus known as a multiphase condensate.\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e These fusion and mutated proteins without a three-dimensional structure can form the aberrant condensates in the nucleoplasm, likely facilitating a drastic alteration in the high-order genome structure, the fusion of \u003cem\u003ePRDM16\u003c/em\u003e with \u003cem\u003eSKI\u003c/em\u003e, and the subsequently high expression of \u003cem\u003ePRDM16\u003c/em\u003e. By contrast, patients with \u003cem\u003eRUNX1::RUNX1T1\u003c/em\u003e, \u003cem\u003eCBFB::MYH11\u003c/em\u003e, \u003cem\u003eCBFA2T3::GLIS2\u003c/em\u003e, monosomy 7, bZIP \u003cem\u003eCEBPA\u003c/em\u003e mutation, or \u003cem\u003eKMT2A\u003c/em\u003e arrangement had a lower expression of \u003cem\u003ePRDM16::SKI\u003c/em\u003e. Therefore, these patients may have fundamentally different AML pathological mechanisms. The components of the \u003cem\u003eNUP98::NSD1\u003c/em\u003e-signature specified for the high \u003cem\u003ePRDM16\u003c/em\u003e expression were \u003cem\u003eNUP98\u003c/em\u003e rearrangements, \u003cem\u003eDEK::NUP214\u003c/em\u003e, \u003cem\u003eNPM1\u003c/em\u003e, \u003cem\u003eUBTF\u003c/em\u003e-TD, and \u003cem\u003eKMT2A\u003c/em\u003e-PTD. This finding aligns with that of other reports.\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, there was a difference in the frequency of high expression of \u003cem\u003ePRDM16::SKI\u003c/em\u003e between the AML-05 and AML-12 studies. One reason for this was that in the AML-12 study, it was not possible to obtain sufficient amounts of RNA compared to AML-05 study, so that the amount of RNA used to create cDNA was different. The prognosis of patients with high \u003cem\u003ePRDM16\u003c/em\u003e expression in the AML-12 study improved compared with that of patients with high \u003cem\u003ePRDM16\u003c/em\u003e expression in the AML-05 study. This finding may be attributed to the use of FLT3 inhibitors in patients who dropped out from the AML-12 study due to non-remission or relapse and those patients may have been rescued. Indeed, \u003cem\u003eFLT3\u003c/em\u003e-ITD-positive patients had a significantly higher \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), thereby contributing to the improved prognosis of patients with high \u003cem\u003ePRDM16::SKI\u003c/em\u003e and \u003cem\u003ePRDM16\u003c/em\u003e expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, the \u003cem\u003ePRDM16::SKI\u003c/em\u003e gene fusion, which contributes to a high \u003cem\u003ePRDM16\u003c/em\u003e expression in pediatric AML, has a high frequency. Further, it is a major contributor to the development of AML, along with core binding factor-AML, and \u003cem\u003eMECOM\u003c/em\u003e (\u003cem\u003eEVI1\u003c/em\u003e) high expression. The patients in this study commonly had a poor prognosis. Hence, appropriate treatments for this patient group must be urgently developed, and reducing the expression levels of \u003cem\u003ePRDM16\u003c/em\u003e can be a therapeutic target.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a Grant-in-Aid for Scientific Research (KAKEN, grant number JP19K08350 to N.S. and JP21K15870 to S.T.) and Exploratory Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Practical Research for Innovative Cancer Control from Japan Agency for Medical Research and Development (AMED, grant number JP20ck0106634 to N.S. and JP23ck0106853 to D.T.); Kawano Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics, a research grant from the Japanese Society of Hematology to N.S.; and a research grant from the Takeda Science Foundation to N.S. The authors also thank Enago for English language review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eContribution: M.Y., T. Komori, and N.S. wrote the paper; M.Y., T. Komori, J.I., S.T., Y.H., G.Y., K.Y. and N.S. performed the experiments; M.Y, T. Komori, J.I., S.T., Y.H., G.Y., K.Y., and N.S. analyzed the data the clinical data and genome samples were provided from JCCG; N.S. designed the study and supervised the work; and all authors critically reviewed and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict-of-interest disclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFrohling S, Scholl C, Gilliland DG, Levine RL. Genetics of myeloid malignancies: pathogenetic and clinical implications. J Clin Oncol. 2005;23:6285\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarcucci G, Haferlach T, Dohner H. Molecular genetics of adult acute myeloid leukemia: prognostic and therapeutic implications. J Clin Oncol. 2011;29:475\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePui CH, Carroll WL, Meshinchi S, Arceci RJ. Biology, risk stratification, and therapy of pediatric acute leukemias: an update. J Clin Oncol. 2011;29:551\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel JP, Gonen M, Figueroa ME, et al. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med. 2012;366:1079\u0026ndash;89.\u003c/span\u003e\u003c/li\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;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong XT, Ida K, Ichikawa H, et al. Consistent detection of TLS/FUS-ERG chimeric transcripts in acute myeloid leukemia with t(16;21)(p11;q22) and identification of a novel transcript. Blood. 1997;90:1192\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Rooij JD, Hollink IH, Arentsen-Peters ST, et al. NUP98/JARID1A is a novel recurrent abnormality in pediatric acute megakaryoblastic leukemia with a distinct HOX gene expression pattern. Leukemia. 2013;27:2280\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHollink IH, van den Heuvel-Eibrink MM, Arentsen-Peters ST, 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;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsuo H, Kajihara M, Tomizawa D, et al. EVI1 overexpression is a poor prognostic factor in pediatric patients with mixed lineage leukemia-AF9 rearranged acute myeloid leukemia. Haematologica. 2014;99:e225\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePreudhomme C, Sagot C, Boissel N, et al. ALFA Group. Favorable prognostic significance of CEBPA mutations in patients with de novo acute myeloid leukemia: a study from the Acute Leukemia French Association (ALFA). Blood. 2002;100:2717\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHollink IH, Zwaan CM, Zimmermann M, et al. Favorable prognostic impact of NPM1 gene mutations in childhood acute myeloid leukemia, with emphasis on cytogenetically normal AML. Leukemia. 2009;23:262\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimada A, Taki T, Tabuchi K, et al. Tandem duplications of MLL and FLT3 are correlated with poor prognoses in pediatric acute myeloid leukemia: a study of the Japanese childhood AML Cooperative Study Group. Pediatr Blood Cancer. 2008;50:264\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaketani T, Taki T, Sugita K, et al. FLT3 mutations in the activation loop of tyrosine kinase domain are frequently found in infant ALL with MLL rearrangements and pediatric ALL with hyperdiploidy. Blood. 2004;103:1085\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePratcorona M, Brunet S, Nomded\u0026acute;eu J, et al. Grupo Cooperativo Para el Estudio y Tratamiento de las Leucemias Agudas Mielobl \u0026acute; asticas. Favorable outcome of patients with acute myeloid leukemia harboring a low-allelic burden FLT3-ITD mutation and concomitant NPM1 mutation: relevance to postremission therapy. Blood. 2013;121:2734\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShiba N, Ichikawa H, Taki T, Park MJ, Jo A, Mitani S, et al. NUP98-NSD1 gene fusion and its related gene expression signature are strongly associated with a poor prognosis in pediatric acute myeloid leukemia. Genes Chromosomes Cancer. 2013;52:683\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJo A, Mitani S, Shiba N, et al. High expression of EVI1 and MEL1 is a compelling poor prognostic marker of pediatric AML. Leukemia. 2015;29:1076\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUmeda M, Ma J, Huang BJ, Hagiwara K, Westover T, Abdelhamed S, et al. Integrated Genomic Analysis Identifies UBTF Tandem Duplications as a Recurrent Lesion in Pediatric Acute Myeloid Leukemia. Blood Cancer Discov. 2022;3:194\u0026ndash;207.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShiba N, Ohki K, Kobayashi T, et al. High PRDM16 expression identifies a prognostic subgroup of pediatric acute myeloid leukaemia correlated to FLT3-ITD, KMT2A-PTD, and NUP98-NSD1: the results of the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-05 trial. Br J Haematol. 2016; 172:581\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMochizuki N, Shimizu S, Nagasawa T, Tanaka H, Taniwaki M, Yokota J, et al. A novel gene, MEL1, mapped to 1p36.3 is highly homologous to the MDS1/EVI1 gene and is transcriptionally activated in t(1;3)(p36;q21)-positive leukemia cells. Blood. 2000;96:3209\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShing DC, Trubia M, Marchesi F, Radaelli E, Belloni E, Tapinassi C, et al. Overexpression of sPRDM16 coupled with loss of p53 induces myeloid leukemias in mice. J Clin Invest. 2007;117:3696\u0026ndash;707.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong S, Chen J. SUMOylation of sPRDM16 promotes the progression of acute myeloid leukemia. BMC Cancer. 2015;15:893.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasetti R, Togni M, Astolfi A, Pigazzi M, Indio V, Rivalta B, et al. Whole transcriptome sequencing of a paediatric case of de novo acute myeloid leukaemia with del(5q) reveals RUNX1-USP42 and PRDM16-SKI fusion transcripts. Br J Haematol. 2014;166:449\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu T, Morita K, Hill MC, Jiang Y, Kitano A, Saito Y, et al. PRDM16s transforms megakaryocyte-erythroid progenitors into myeloid leukemia-initiating cells. Blood. 2019;134:614\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKudo K, Kojima S, Tabuchi K, Yabe H, Tawa A, Imaizumi M, et al. 2007. Prospective study of a pirarubicin, intermediate-dose cytarabine, and etoposide regimen in children with Down syndrome and acute myeloid leukemia: The Japanese Childhood AML Cooperative Study Group. J Clin Oncol. 25:5442\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsukimoto I, Tawa A, Horibe K, Tabuchi K, Kigasawa H, Tsuchida M, et al. Risk-stratified therapy and the intensive use of cytarabine improves the outcome in childhood acute myeloid leukemia: the AML99 trial from the Japanese Childhood AML Cooperative Study Group. J Clin Oncol. 2009;27:4007\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomizawa D, Matsubayashi J, Iwamoto S, Hiramatsu H, Hasegawa D, Moritake H, et al. High-dose cytarabine induction therapy and flow cytometric measurable residual disease monitoring for children with acute myeloid leukemia. Leukemia. 2024;38:202\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomizawa D, Tawa A, Watanabe T, et al. Excess treatment reduction including anthracyclines results in higher incidence of relapse in core binding factor acute myeloid leukemia in children. Leukemia. 2013;27:2413\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShiraishi Y, Fujimoto A, Furuta M, et al. Integrated analysis of whole genome and transcriptome sequencing reveals diverse transcriptomic aberrations driven by somatic genomic changes in liver cancers. PLoS One. 2014;9:e114263.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanda, Y. Investigation of the freely available easy-to-use software \u0026lsquo;EZR\u0026rsquo; for medical statistics. Bone Marrow Transplantation. 2013;48:452\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamato G, Yamaguchi H, Handa H, et al. Clinical features and prognostic impact of PRDM16 expression in adult acute myeloid leukemia. Genes Chromosomes Cancer. 2017;56:800\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDao FT, Chen WM, Long LY, et al. High PRDM16 expression predicts poor outcomes in adult acute myeloid leukemia patients with intermediate cytogenetic risk: a comprehensive cohort study from a single Chinese center. Leuk Lymphoma. 2021;62:185\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamato G, Kawai T, Shiba N, Ikeda J, Hara Y, Ohki K, et al. Genome-wide DNA methylation analysis in pediatric acute myeloid leukemia. Blood Adv. 2022;6:3207\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhn JH, Davis ES, Daugird TA, Zhao S, Quiroga IY, Uryu H, et al. Phase separation drives aberrant chromatin looping and cancer development. Nature. 2021;595:591\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChandra B, Michmerhuizen NL, Shirnekhi HK, Tripathi S, Pioso BJ, Baggett DW, et al. Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation. Cancer Discov. 2022;12:1152\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHyman AA, Weber CA, J\u0026uuml;licher F. Liquid-liquid phase separation in biology. Annu Rev Cell Dev Biol. 2014;30:39\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel A, Lee HO, Jawerth L, et al. A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell. 2015;162:1066\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitrea DM, Cika JA, Guy CS, Ban D, Banerjee PR, Stanley CB, et al. Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA. Elife. 2016;5:e13571.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLafontaine DLJ, Riback JA, Bascetin R, Brangwynne CP. The nucleolus as a multiphase liquid condensate. Nat Rev Mol Cell Biol. 2021;22:165\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIde S, Imai R, Ochi H, Maeshima K. Transcriptional suppression of ribosomal DNA with phase separation. Sci Adv. 2020;6:eabb5953.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaburagi T, Shiba N, Yamato G, et al. UBTF-internal tandem duplication as a novel poor prognostic factor in pediatric acute myeloid leukemia. Genes Chromosomes Cancer. 2023;62:202\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PRDM16::SKI, NUP98-rearrangements, KMT2A-PTD, UBTF-TD, nucleoporin","lastPublishedDoi":"10.21203/rs.3.rs-6187243/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6187243/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe RNA-sequencing data from the Japanese Children\u0026rsquo;s Cancer Group (JCCG)\u0026rsquo;s AML-05 study was re-analyzed to clarify the mechanisms related to high \u003cem\u003ePRDM16\u003c/em\u003e expressions, which is independently associated with adverse outcomes. Results showed that 19 of 139 patients presented with out-of-frame \u003cem\u003ePRDM16::SKI\u003c/em\u003e fusions. Thus, the gene expression levels of \u003cem\u003ePRDM16::SKI\u003c/em\u003e in 369 and 329 patients from the AML-05 and AML-12 studies, respectively, were measured. In total, 119 (32%) of 369 patients in the AML-05 study and 58 (18%) of 329 patients in the AML-12 study presented with an aberrant expression of \u003cem\u003ePRDM16::SKI\u003c/em\u003e. This fusion was a 48-base-pair product that immediately formed a stop codon on the \u003cem\u003eSKI\u003c/em\u003e side. The introduction of this product in mice did not cause AML. Intriguingly, none of the patients presented with \u003cem\u003eSKI::PRDM16\u003c/em\u003e, which is reciprocal. Moreover, partner fusion genes were not detected in front of truncated \u003cem\u003ePRDM16\u003c/em\u003e, indicating that a short form of \u003cem\u003ePRDM16\u003c/em\u003e, which lacked exon 1, existed by itself. Patients with high \u003cem\u003ePRDM16::SKI\u003c/em\u003e expression had significantly worse overall survival and event-free survival than those with a low \u003cem\u003ePRDM16\u003c/em\u003e expression. The cleavage between exons 1 and 2 of \u003cem\u003ePRDM16\u003c/em\u003e induces aberrant \u003cem\u003ePRDM16\u003c/em\u003e expression, and a strong associations was observed between \u003cem\u003ePRDM16::SKI\u003c/em\u003e and \u003cem\u003ePRDM16\u003c/em\u003e expression.\u003c/p\u003e","manuscriptTitle":"PRDM16::SKI is a predictor of aberrant expression of the short variant of PRDM16 in pediatric acute myeloid leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-17 07:14:11","doi":"10.21203/rs.3.rs-6187243/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b9ecad5e-1d58-4062-96a1-fdfc524b3558","owner":[],"postedDate":"March 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45510999,"name":"Biological sciences/Cancer/Cancer genomics"},{"id":45511000,"name":"Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia"}],"tags":[],"updatedAt":"2025-04-03T09:41:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-17 07:14:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6187243","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6187243","identity":"rs-6187243","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}