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. 2026 Feb 8;16:7792. doi: 10.1038/s41598-026-39353-2

A novel human acute myeloid leukemia cell line SDEY-AML1 with KMT2A: MLLT3, IKZF1: EVX1 fusions exhibits high tumorigenicity in NSG mice

Chenwei Yang 1,#, Weiliang Zhang 1,#, Yijun Wu 1,#, Xin Liu 1,#, Li Gao 1, Hui Zhang 1, Zhiheng Li 1, Shaoyan Hu 1,✉
PMCID: PMC12949064  PMID: 41656387

Abstract

Human leukemia cell lines play a crucial role in leukemia research, particularly in understanding the pathogenesis of acute leukemia and developing novel therapeutic strategies. We here describe the generation of a novel monocytic cell line SDEY-AML1, harboring alterations in KMT2A::MLLT3, IKZF1::EVX1, ETV6, and TP53 double mutations, and its biological characteristics. We established a cell line, SDEY-AML1, from the mononuclear cells isolated from the bone marrow of a patient with acute myeloid leukemia (AML-M5). Comprehensive morphological and cytogenetic characterization of SDEY-AML1 cells was performed using cytochemical staining, flow cytometry, and karyotype analysis. The molecular features of the cell line were identified by transcriptomic analysis and reverse transcription-polymerase chain reaction (PT-PCR). The proliferative capacity of the SDEY-AML1 cells was assessed using a clonogenic assay. And we conducted drug sensitivity experiments on SDEY-AML1 to tested its sensitivity to commonly used chemotherapy drugs and targeted drugs. Additionally, we created an in vivo tumor model of SDEY-AML1 cells by injecting 1×107 cells subcutaneously in NSG mice via the tail vein. The SDEY-AML1 cells were maintained in continuous culture for one year without the addition of exogenous cytokines. Cytochemical assays revealed that the cells were positive for α-naphthyl acetate esterase reactivity that was partially inhibited by sodium fluoride but negative for peroxidase and periodic acid-Schiff staining. Flow cytometry showed the expression of myeloid markers in SDEY-AML1 cells. Karyotypic analysis revealed a t(9;11) (p22;q23) translocation in SDEY-AML1 cells, mirroring the chromosomal aberrations identified in the primary leukemia cells of the patient. Genetic profiling confirmed the presence of alterations in KMT2A::MLLT3, IKZF1::EVX1, ETV6, and TP53 genes. SDEY-AML1 cultures were negative for EBV and mycoplasma. NSG mice showed tumor infiltration within 40-50 days of tail vein injection of SDEY-AML1 cells. The new AML cell line, SDEY-AML1, harbored KMT2A::MLLT3, IKZF1::EVX1, ETV6 gene alterations, and TP53 double mutations. These cells were highly tumorigenic in NSG mice. This cell line has applications in the research and development of new agents targeting KMT2A::MLLT3, IKZF1::EVX1-associated leukemia, making it a valuable tool in the study of leukemogenesis.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-39353-2.

Keywords: Leukemia cell line, TP53 double mutations, KMT2A:: MLLT3, IKZF1:: EVX1, NSG mice tumorigenicity

Subject terms: Haematological cancer, Paediatric cancer

Introduction

In the past two decades, the prognosis of acute myeloid leukemia (AML) has undergone a remarkable transformation, primarily due to the advent of innovative therapeutic agents and significant advancements in allogeneic hematopoietic stem cell transplantation (allo-HSCT)1–4. Notably, acute leukemia harboring rearrangements in the mixed lineage leukemia (MLL, also known as KMT2A) gene was historically characterized by alarmingly high relapse rates when managed with conventional chemotherapy regimens. KMT2A, located on chromosome 11q23, undergoes frequent rearrangements in both AML and acute lymphoblastic leukemia (ALL), posing a significant challenge to treatment5–8. Among cases traditionally regarded as monoallelic, patients with TP53 monoallelic mutations had better overall survival than those with a biallelic configuration of AML9,10. Current research suggests that mutated IKZF1 serves as an independent marker of adverse risk in AML11,12. At present, a novel case of EVX1 fusion has been discovered in the driver genome of immature T-cell acute lymphoblastic leukemia in children. In a case of a child undergoing transformation from refractory anemia with excessive primary cells to acute myeloid leukemia, the EVX1 gene has also been identified13,14. However, EVX1 has not been identified in AML and there is currently no specific research on the mechanism of its driving effect on leukemia.

Leukemia cell lines carrying KMT2A have emerged as invaluable tools for unraveling the intricate pathogenesis of the disease and screening potential therapeutic agents. Notably, over 100 distinct chaperone genes fused with KMT2A have been identified, highlighting the diversity of genetic rearrangements in this context. In 2017, approximately 40 leukemia cell lines harboring the 11q23 translocation were established, with seven featuring the KMT2A::MLLT3 fusion. Each of these seven cell lines exhibits unique breakpoints and fusion sites, rendering them all essential instruments in the KMT2A-associated leukemia research landscape15. However, there are currently very few cell lines established from bone marrow mononuclear cells of AML patients that harbor both TP53 double mutations and the IKZF1::EVX1 fusion gene.

Recently, we developed a novel human cell line, SDEY-AML1, derived from a patient with acute monocytic leukemia. SDEY-AML1 cells harbor a characteristic translocation, t(9;11) (p22;q23), leading to the rearrangement of the MLL gene, along with a KMT2A::MLLT3, IKZF1::EVX1, TP53 double mutation, and ETV6 gene alterations. These cells demonstrated a high degree of tumorigenicity when transplanted into NSG mice. This study aims to contribute to the scientific community’s understanding of this disease and potentially facilitate the development of targeted therapeutic approaches.

Methods

Case report

A 14-year-old boy was diagnosed with AML in July 2022 at the Children’s Hospital of Soochow University. Notable physical findings included bilateral inguinal lymphadenopathy and hepatosplenomegaly. Laboratory results revealed anemia (hemoglobin, 86 g/L), thrombocytopenia (platelet count 116X109/L), and leukocytosis (white blood cell count 259X109/L) with an abnormal differential showing a high percentage of lymphocytes (80.3%) and a low absolute neutrophil count (2.1X109/L). Additionally, elevated CRP level (33.92 mg/L) and recurrent fever were observed.

A bone marrow biopsy confirmed the diagnosis of AML-M5. The patient was categorized into a high-risk group with a newly diagnosed white blood cell count greater than 100 × 109 and no low-risk genes. He was treated with DAE [(cytarabine (100mg/m2, q12h, 20 doses), daunorubicin (50mg/m2/day, 3 doses), and etoposide (100mg/m2/day, five doses]) from August 18, 2022. On September 22, 2022, bone marrow evaluation showed 69% proto-monocytes and 72.54% MRD, indicating no complete remission (CR) after the first induction therapy. The patient was transferred to low-dose chemotherapy with decitabine [idarubicin (5mg/m2/day, three doses), cytarabine (10mg/m2, q12h, 20 doses), and decitabine (20mg/m2/day, five doses)] but he still did not achieve CR. A third course of chemotherapy, including decitabine (10mg/m2/day, five doses), homoharringtonine (1mg/m2/day, seven doses), and cytarabine (10mg/m2, q12h, 20 doses). Based on the results of a drug sensitivity test performed on April 14, 2023, the patient was treated with cytarabine (10mg/m2, q12h, 20 doses), venetoclax (400 mg/day, 20 doses), and cedarbene (the trade name of the drug Azacitidine) (30 mg/day, twice weekly for 2 weeks) with no relief. The patient died of pulmonary mucormycosis and disseminated intravascular coagulation in May 2023 due to the primary disease.

This study was approved by the Ethics Committee of the Children’s Hospital of Soochow University (No. 2017047-3) and was in accordance with Declaration of Helsinki. All research was performed in accordance with relevant guidelines, Consent was obtained from all subjects and/or their legal guardian(s).

Cell culture

The Children’s Hospital of Soochow University strictly abides by ethical principles and ensures the formal signing of informed consent forms. Bone marrow samples (5 ml) were collected on April 9, 2023, during the diagnostic procedure, with informed consent obtained. Mononuclear cells were isolated from these samples using red blood cell lysis solution. The cells were cultured in T25 flasks at a density of 1×10^6 cells per milliliter in RPMI 1640 medium supplemented with 20% heat-inactivated fetal calf serum (FCS) (Zhejiang Meisen Biology Co., Ltd.). The FCS contained a cocktail of cytokines, including 10 ng/ml interleukin-3 (IL-3), 100 ng/ml stem cell factor (SCF), and 100 ng/ml Fms-related tyrosine kinase 3 ligand (Flt-3L) (Pepro Tech). The cells were maintained in a humidified incubator at 37°C with 80-95% humidity and 5% CO2, ensuring optimal growth conditions. The culture medium was replaced every 4-5 days, depending on the observed rate of cellular proliferation. The SDEY-AML1 cells were cryopreserved in a freezing medium consisting of 90% fetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO). Following storage in liquid nitrogen, the cells could be successfully thawed with a viability rate of 85% or higher and further expanded in culture.

Morphological and cytochemical studies

Preparations for Wright and Giemsa staining were meticulously centrifuged and examined under a light microscope for detailed cellular morphology. Additionally, the samples were stained for peroxidase staining, periodic acid-schiff staining reactivity, and non-specific esterase activity staining using established protocols. Furthermore, ultrastructural analysis was performed and validated to gain insights into the subcellular architecture.

Cell surface marker analysis

Comprehensive surface immunotyping of both primary leukemia cells and the SDEY-AML1 cells was performed by employing a diverse panel of monoclonal antibodies sourced from BD Biosciences and BioLegends. Flow cytometry (Beckman Gallios™ flow cytometer) was used to measure the fluorescence intensity of cells labeled with either fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies.

Cytogenetic analysis

Cytogenetic analysis of chromosomes from both primary leukemia cells and SDEY-AML1 cells was performed. The chromosomes were prepared using a standardized methodology and subsequently banded using the R-banding technique. The identified karyotypic abnormalities were classified based on the guidelines outlined in the International System for Human Cytogenetic Nomenclature (ISCN, 1995), ensuring accuracy and consistency in their classification.

Fluorescence in situ hybridization (FISH) assay

The FISH assay was performed according to the manufacturer’s instructions. The bone marrow sample was preprocessed for the MLL rearrangement probe targeting 11q23. Cells were isolated, fixed, and denatured to ensure optimal conditions for the probe to bind specifically to the target DNA. A fluorescent-labeled probe was precisely aligned with the DNA within the sample. This process leveraged fluorescence signals as indicators, revealing the presence or absence of translocations and rearrangements within the MLL genes. Using a fluorescence microscope, we captured images of the hybridized samples and analyzed the precise location and intensity of the emitted fluorescence signals. Based on the signal patterns observed, a definitive conclusion was drawn regarding the status of MLL gene translocation and rearrangement, including the specific types of rearrangement.

Detection of Epstein-Barr virus (EBV) and mycoplasma

The presence of EBV genomic DNA was detected by real-time PCR (Shanghai Huzhen Industrial Co., Ltd.), as per the manufacturer’s instructions. DNA was extracted from the cells using the DNA extraction kit DP304 for blood/cell/tissue genome following the kit instructions., and amplified by PCR. The PCR reaction system included the reagent Hhv-4 reaction solution (19.5 μl) and enzyme solution (0.5 μl). The reagent was added into a centrifuge tube, mixed thoroughly, and packed into eight rows of PCR reaction tubes (20 ul per tube). The DNA and the control (positive and negative) samples (5ul each) were added to the corresponding reaction tube, covered, mixed, and centrifuged briefly (Supplementary Figure 1A-B). The reaction tube was then placed in a fluorescence-quantitative PCR reactor. FAM was selected as the detection channel. PCR reaction included 45 cycles at 95 ° C for 3 min, 95 ° C for 15 sec and 55 ° C for 30 sec. The data for each reaction were analyzed using ABI7500 software for each.

The SDEY-AML1 cells were tested for mycoplasma using the Myco-Lumi™ Luminescent Mycoplasma Detection Kit for High Sensitivity Instrument.

Clonogenic assay

The SDEY-AML1 cells were inoculated into a soft agar medium sourced from Sigma and cultivated for 14–21 days. The colonies formed were stained and counted using Giemsa solution.

Whole transcriptome sequencing

Total RNA was extracted from SDEY-AML1 cells using the TRIzol method, and mRNA was subsequently isolated by magnetic bead capture. The mRNA was reverse transcribed into cDNA, which served as a template for constructing a sequencing library. The Illumina NovoSeq sequencing system was used to ensure a total data output of no less than 16G. The quality of all data was rigorously analyzed using bioinformatics, adhering to a Q30 threshold of > 85%. To analyze the fusion genes, we leveraged databases such as Fusion Cancer, My Cancer Genome, Atlas of Genetics, and Cytogenetics in Oncology and Hematology. For gene annotation, dbSNP, 1000G, EXAC, COSMIC, HGMD, GnomAD, ClinVar, and other similar comprehensive resources. For bioinformatic analysis of fusion genes and genetic mutations, STAR-fusion and other advanced software were employed, while genetic mutations were aligned against the hg19 reference genome. The Integrative Genomics Viewer (IGV) software was used for sequence alignment and visualization of Bam files.

Establishment of luciferase-expressing SDEY-AML1 stable cell line and its high tumorigenicity in NSG mice

Target cells in good condition were seeded in 24-well cell culture plates by adding 500 µl of cell suspension (5-10 ×105 cells/ml) per well. The plated wells had a cell density of 30%-40%. The cells were transduced with lentivirus at MOIs of 0,10,20,40,80,160,320. The blank group received only polybrene (5 µg/ml). The amount of virus added per well (µl) was equal to the number of cells/titer (TU/mL) ×103 for any MOI. After 12-16 hours of lentivirus infection, the medium was removed and replaced with fresh, complete medium. The optimal MOI was determined by preliminary observation, and the transduction was repeated at the optimal MOI. After 72 h of culture, the cells were treated with the luciferase protein every three days. Luciferase expression was measured in a small animal imager to confirm the generation of luciferase-overexpressing SDEY-AML1 stable cells.

Five- to six-week-old NSG mice were purchased from Shanghai Model Organisms Center, Inc. To assess the tumorigenicity of SDEY-AML1 cells, NSG mice (n=14) were injected with luciferase-expressing SDEY-AML1 cells (1×107) via the tail vein.

All animal experiments were conducted in compliance with the animal care guidelines approved by the Institutional Laboratory Animal Care and Use Committee of Soochow University (SUDA20230802A05) and were in accordance with the ARRIVE guidelines. Mice were anesthetized with isoflurane and euthanized by carbon dioxide (CO₂) inhalation.

Drug sensitivity test

Chidamide, LBH589, S63845, Olverembatinib, Cladribine, Afatinib were purchased by MedChemExpress. Powder was dissolved in DMSO in 10–50 mM stock solutions and stored at −20°C.

Results

Establishment of the SDEY-AML1 cell line

One month after initiating the culture (April 2024), the cells exhibited a gradual and consistent proliferation pattern. After three months, the cells transitioned into a rapid proliferation phase. Remarkably, these cells were maintained in continuous culture for one year without the necessity of cytokines, thereby qualifying them as a stable and continuous cell line, designated as SDEY-AML1.

SDEY-AML1 cells exhibited a gradual and consistent proliferation pattern when co-cultured with cytokines over the initial three months (from April 2023 to July post-isolation). Notably, after these three months, a marked acceleration was noted in the proliferation rate of these cells, which became capable of proliferating independent of cytokines. This significant increase in proliferation was statistically validated (Figure 1A). Microscopic images of SDEY-AML1 and primary cells are shown in Figure 1B. It was preserved in October 2023 at the General Microbiology Centre of the China Commission (CGMCC No. 45734).

Fig. 1.

Fig. 1

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Analyses of SDEY-AML1 cells. (A) CCK8 assay was used to assess the proliferation of SDEY-AML1 cells in culture with and without the addition of SCF (100 ng/ml), Flt3 (100 ng/ml), and IL-3 (10 ng/ml). (B) Microscopic visualization of SDEY-AML1 cells and primary leukemia cells (scale bar: 200 μm). (C) peroxidase staining and non-specific esterase activity staining of SDEY-AML1 cells with positive control and negative control (scale bar: 40 ×). (D) Wright’s, Giemsa’s and periodic acid-schiff staining of SDEY-AML1 cells (scale bar: 40 ×). Data are presented as the mean ± SD (n=3). Comparisons were evaluated using the two-tailed Student’s t-test, and multiple groups were analyzed using a one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < .0001, NS, not significant.

Morphology of SDEY-AML1 cells

SDEY-AML1 cells exhibited a characteristic morphology featuring a regular cell shape with a distinctively folded nucleus containing prominent nucleoli. The chromatin within these nuclei appeared to be finely reticular, contributing to their distinct appearance. Furthermore, a portion of the cells displayed a blue-gray cytoplasm dotted with fine azurophilic granules. In terms of enzymatic activity, SDEY-AML1 cells were positive for non-specific esterase activity staining, which was further confirmed using the sodium fluoride (NaF) inhibition test. Conversely, 95% of the cells tested negative for peroxidase staining and periodic acid-schiff staining, which provided valuable insights into their biochemical profiles (Figure 1C-D).

Immunophenotypic profiles

The primary leukemic cells and the derived SDEY-AML1 cells showed remarkably similar expression of immune surface markers. Both cells consistently expressed a characteristic array of myeloid surface antigens, including CD13, CD15, CD33, and CD64 (Figure 2A), underscoring their common myeloid lineage and phenotypic congruence.

Fig. 2.

Fig. 2

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Analyses of SDEY-AML1 cells. (A) Flow cytometric immunophenotyping of the patient’s primary cells and SDEY-AML1 cells for AML (B) FISH (fluorescence in situ hybridization) analysis of SDEY-AML1 cells.

FISH studies

The FISH analysis indicated a positive MLL rearrangement in 99% of the cells, with 1% of them displaying 2Y signals and 99% exhibiting 1Y1R1G signals. In Figure 2B, the R, G, and Y represent the red, green, and yellow fluorescent signals, respectively. The FISH analysis indicated 11q23/MLL rearrangement abnormality. According to the WHO 2022 classification, if a patient is diagnosed with a myeloid neoplasm, the presence of an MLL rearrangement, regardless of the percentage of blast cells, is sufficient for the diagnosis of AML. In the context of AML, t(9;11)/MLL-AF9 is classified as an intermediate-risk group with a moderate prognosis, whereas other 11q23/MLL abnormalities are categorized as high-risk with a poor prognosis.

Detection of Epstein-Barr virus (EBV) and mycoplasma

Following cryopreservation, SDEY-AML1 cells maintained a stable proliferation rate, demonstrating resilience and purity, and were free from mycoplasma and EBV contaminations (Supplementary Figure 1C). Rigorous short tandem repeat (STR) profiling was performed by Suzhou Jinda Biotechnology Co., Ltd. to ensure the authenticity and genetic integrity of the cell line (Supplementary Figure 2).

Whole transcriptome sequencing

Genomic analysis identified two translocation events involving critical genes implicated in leukemia pathogenesis. Specifically, we identified a fusion between the 5’ end of the KMT2A gene located at CHR11:118353210 (positive strand) and the 3’ end of the MLLT3 gene positioned at CHR9:20365742 (negative strand), resulting in a translocation fusion type. Another fusion type involving the 5’ end of the IKZF1 gene at CHR7:50367353 (positive strand) and the 3’ end of the EVX1 gene at CHR7:27284667 (negative strand) was also identified.

Regarding clinically significant variants, three notable alterations were detected:

In ETV6, located on chromosome 12p13.2, a nucleotide substitution from C to A at position c.392 within exon 4 of the Nm transcript was observed. This mutation results in a premature stop codon (p.SER131TER), suggesting truncation of the protein. A variant allele was detected at a frequency of 45.7%. A C to T substitution at nucleotide position c.817 within exon 8 of TP53, located on chromosome 17p13.1 (transcript NM-000546.5), results in an amino acid change from arginine to cysteine (p.ARG273CYS). This mutation is highly prevalent, with a variant allele frequency of 96.6%. A second mutation in TP53, a G-to-T substitution at nucleotide position c.1015 within exon 10, leads to a premature stop codon (p.GLU339TER). Although this variant allele occurred at a low frequency (3.4 %), it represents a significant alteration with potential clinical implications.

These findings highlight the presence of complex genomic rearrangements, including oncogene fusions KMT2A::MLLT3(Figure 3A) and IKZF1::EVX1 (Figure 3B) and clinically relevant mutations in key tumor suppressor genes(TP53 (double mutation, Figure 4A) and ETV6 (Figure 4B) that potentially contribute to the pathogenesis and progression of the underlying hematological malignancy. Specific alterations in ETV6 and dual mutations in TP53 further underscore the importance of comprehensive genomic profiling for guiding precision medicine approaches and developing targeted therapeutic strategies.

Fig. 3.

Fig. 3

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Whole transcriptome sequencing of SDEY-AML1. Whole transcriptome sequencing of SDEY-AML1 detected gene fusions involving (A) KMT2A::MLLT3 and (B) IKZF1::EVX1 genes.

Fig. 4.

Fig. 4

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Whole transcriptome sequencing of SDEY-AML1. Whole transcriptome sequencing of SDEY-AML1 detected (A) double mutations in the TP53 and (B) alterations in the ETV6 genes.

Cytogenetics

Karyotype analysis of SDEY-AML1 and primary cells was performed during the logarithmic growth phase. The preparation protocol employed was C24 + D, whereas the detection method involved the analysis of chromosome R banding (RHG). Notably, In the karyotype images, we consistently and clearly observed a significantly shortened long arm of one chromosome 11 (11q–), which strongly suggests a break and rearrangement at the 11q23 region. The appearance of an elongated short arm of chromosome 9 (9p+) was atypical or inconsistent across cells, possibly due to banding quality issues. Therefore, we cautiously denoted it as "?9p+" in the karyotype description to reflect the limitations of G-banding. Based on the integrated results of FISH and whole-transcriptome sequencing, we have defined this abnormality as a t(9;11) translocation. Furthermore, a subset of SDEY-AML1 cells showed multiple aberrant and complex karyotypes (Figure 5A-B).

Fig. 5.

Fig. 5

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Karyotypic analysis. (A-B) Karyotypic analysis of SDEY-AML1 cells.

Clonogenic assay

After incubation for 14 and 21 days, colonies were counted under an inverted microscope (Figure 6A). The clone formation assay suggests that SDEY-AML1 has a strong proliferative capacity.

Fig. 6.

Fig. 6

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Luciferase-expressing SDEY-AML1 stable cell line and its tumorigenicity in NSG mice. (A) The tumorigenicity and proliferation of SDEY-AML1 cells was tested in a colony formation assay. (B) Luciferase expression was confirmed in the SDEY-AML1 cell line. (C) Cancer ascites extracted from mouse tumor xenograft models was centrifuged and cells were taken for Giemsa staining. (D) Tumor infiltration in NSG mice injected with SDEY-AML1 cells via the tail vein. (E) Flow cytometry analysis of CD45 stained bone marrow-derived mononuclear cells in a mouse xenograft model. Data are presented as the mean ± SD (n=3). Comparisons were evaluated using the two-tailed Student’s t-test, and multiple groups were analyzed using a one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < .0001, NS, not significant.

Tumorigenicity of SDEY-AML1-Luciferase cells in NSG mice

Fourteen NSG mice were intravenously injected with 1×107 luciferase-expressing SDEY-AML1 cells (Figure 6B) via the tail vein. After 40-50 days, most of the mice(n=8) developed abdominal distension, as shown in supplementary figure 3A. Abdominal paracentesis was performed using disposable insulin needles, and hemorrhagic ascites was aspirated, as shown in supplementary figure 3B. The ascitic fluid was centrifuged, washed with PBS, and stained with Giemsa stain (Figure 6C). The presence of deeply stained blue-purple cells suggested the likely occurrence of cancerous ascites in the abdominal cavity of the mice. Tumor infiltration was observed in the remaining 6 mice using a small animal fluorescence imaging system (Figure 6D). Subsequently, these remaining mice also developed ascites. Bone marrow samples from healthy mice and mouse xenograft models were stained with CD45 antibody and analyzed by flow cytometry. The results indicated the presence of human CD45-positive cells in the bone marrow of the xenograft models(Figure 6E). This finding underscores the aggressive and tumorigenic potential of SDEY-AML1 cells and highlights their suitability for use in preclinical studies on leukemia and other hematological malignancies.

Drug sensitivity test

We have conducted drug sensitivity tests on this cell line. We found that the following drugs Chidamide, LBH589, S63845, Olverembatinib, Cladribine and Afatinib are sensitive to this cell line, which has guiding significance for the treatment of this leukemia (Figure 7A).

Fig. 7.

Fig. 7

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Drug sensitivity experiment of SDEY-AML1 cell. (A)Testing the sensitivity of Chidamide, LBH589, S63845, Olverembatinib, Cladribine, Afatinib to SDEY-AML1.

Discussion

In this study, we generated a novel cell line, SDEY-AML1, derived from a patient diagnosed with AML M5. Morphological, cytochemical, and immunological analyses corroborated the diagnosis. SDEY-AML1 cells expressed myeloid and monocytic lineage-specific cell surface antigens, including CD13, CD15, CD33, and CD64. FISH analysis revealed the presence of KMT2A rearrangements, while transcriptome sequencing further identified a novel fusion gene IKZF1::EVX1 and TP53 double mutations, providing valuable insights into genetic alterations. And tested its sensitivity to commonly used chemotherapy drugs and targeted drugs. The in vivo tumorigenicity of cell lines is of significant importance in tumor research and provides a crucial model for the evaluation of novel anticancer drugs and drug combination regimens. However, not all AML cell lines exhibit robust in vivo tumorigenic potential, thereby limiting their application in research. We successfully established a mouse xenograft model NSG mice following subcutaneous injection of SDEY-AML1 cells (1×107), highlighting potential applications for this AML cell line.

Human leukemia-lymphoma (LL) cell lines serve as valuable tools for investigating both fundamental and applied aspects of cell biology. Their unparalleled advantages include abundant and continuous supply, ensuring global accessibility to identical cellular materials. Furthermore, these cell lines can be conveniently cryopreserved for long-term storage. Notably, they are characterized by their monoclonal origin, which guarantees genetic uniformity, as well as their arrested differentiation state and sustained proliferation in vitro while preserving the majority of cellular characteristics and harboring specific genetic alterations. Collectively, these features contribute to their exceptional utility in advancing cancer research16,17.

KMT2A rearrangements at 11q23 have varied profiles and outcomes based on the fusion partners. Incorporating five adverse fusions refined the risk stratification of pediatric AML, revising 1q21/KMT2A::MLLT11 to intermediate risk. Future studies should validate the links between ACA outcomes and explore their pathogenesis. A distinct phosphoproteomic signature in KMT2A::MLLT3 AML identifies two disparate groups, justifying targeted therapies for MLLGA-signature patients18–20. Therefore, it is essential to construct a cell line harboring the KMT2A::MLLT3 fusion gene.

Current research suggests that C-FOS serves as a cofactor with IKZF1 in determining lenalidomide sensitivity and mediating drug resistance in multiple myeloma cells, thereby presenting itself as a promising novel therapeutic target for enhancing the prognosis of MM patients21. Overcoming resistance to immunomodulatory drugs (IMiDs) in T-cell lymphomas can be achieved through the potent degradation of key factors, such as ZFP91 and IKZF1, offering a novel therapeutic approach to combat this challenging condition22. IKZF1 deletions are closely linked to treatment resistance and poorer clinical outcomes23. The relationship between IKZF1 and drug resistance, as well as relapsed AML, remains unclear. A cell line derived from the bone marrow of a patient with recalcitrant AML is a valuable resource for elucidating the intricate connection between IKZF1 and drug resistance in this malignancy. Furthermore, while EVX1 is sometimes described as a potential competitor gene for IKZF1, our groundbreaking work established, for the first time, a cell line harboring the IKZF1::EVX1 fusion gene, offering fresh insights into this unexplored area.

TP53 disruption (deletion and/or mutation) is the strongest prognostic factor for refractoriness to chemotherapy24. Consequently, resistance to therapy and unfavorable clinical outcomes often accompany p53-related abnormalities in chronic lymphocytic leukemia (CLL)25. Currently, the relationship between TP53 double mutations and AML resistance has not been explored. We constructed a TP53 double-mutated AML cell line for the first time with the aim of using it as a tool to study the role of TP53 double mutations in AML.

In summary, SDEY-AML1 represents a novel and valuable cell line derived from AML, characterized by KMT2A::MLLT3 and IKZF1::EVX1 fusions, TP53 double mutation, ETV6 gene alterations, and high tumorigenicity in NSG mice. With its well-defined biological underpinnings, SDEY-AML1 serves as a pivotal tool for advancing leukemia research and facilitating deeper insights into the mechanisms of the disease.

Supplementary Information

Supplementary Information. (1.7MB, ppt)

Author contributions

C.Y. conceived the study and analyzed data; C.Y.,W.Z.,Y.W and X.L. performed animal studies; S.H., L.G. and H.Z. provided clinical AML samples and collected patient information; C.Y.,W.Z.,Y.W and X.L. conceived the cell experiments, S.H., Z.L.and Y.W. helped organize the paper and conceived the experiments, C.Y. and S.H. designed the study and wrote the manuscript.

Funding

This study was supported by the following grants: The National Key Research and Development Program of China (no. 2022YFC2502700), Jiangsu Key project, BE2021654 to Shaoyan Hu, the National Natural Science Foundation of China (NSFC 82200177 to Li Gao, 82170218, and 81970163 to Shaoyan Hu), and Suzhou Projects GSWS2020039, 2020ZKPB02, and the Suzhou Municipal Key Laboratory (SZS201615 to Shaoyan Hu).

Data availability

Our sequencing and processed data files were submitted to the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) repository GSE269021. Other relevant data are available from the corresponding authors upon reasonable request.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethics

Patient: This study was approved by the Ethics Committee of the Children’s Hospital of Soochow University (No. 2017047-3) and was in accordance with Declaration of Helsinki. All research was performed in accordance with relevant guidelines, Informed Consent was obtained from all subjects and/or their legal guardian(s). Animal: All animal experiments were conducted in compliance with the animal care guidelines approved by the Institutional Laboratory Animal Care and Use Committee of Soochow University (SUDA20230802A05) and were in accordance with the ARRIVE guidelines.

Consent for publication

Consent for publication was obtained from the participants.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Chenwei Yang, Weiliang Zhang, Yijun Wu, Xin Liu are authors contributed equally to this work.

Reference

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information. (1.7MB, ppt)

Data Availability Statement

Our sequencing and processed data files were submitted to the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) repository GSE269021. Other relevant data are available from the corresponding authors upon reasonable request.

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