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The Nup98::Nsd1 fusion gene induces CD123 expression in 32D cells

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Abstract

The NUP98::NSD1 fusion gene is associated with extremely poor prognosis in patients with acute myeloid leukemia (AML). NUP98::NSD1 induces self-renewal and blocks differentiation of hematopoietic stem cells, leading to development of leukemia. Despite its association with poor prognosis, targeted therapy for NUP98::NSD1-positive AML is lacking, as the details of NUP98::NSD1 function are unknown. Here, we generated 32D cells (a murine interleukin-3 (IL-3)-dependent myeloid progenitor cell line) expressing mouse Nup98::Nsd1 to explore the function of NUP98::NSD1 in AML, including comprehensive gene expression analysis. We identified two properties of Nup98::Nsd1 + 32D cells in vitro. First, Nup98::Nsd1 promoted blocking of AML cell differentiation, consistent with a previous report. Second, Nup98::Nsd1 increased dependence on IL-3 for cell proliferation, due to overexpression of the alpha subunit of the IL-3 receptor (IL3-RA, also known as CD123). Consistent with our in vitro data, IL3-RA was also upregulated in samples from patients with NUP98::NSD1-positive AML. These results highlight CD123 as a potential new therapeutic target in NUP98::NSD1-positive AML.

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Data availability

Original data presented in this manuscript are available upon request addressed to the corresponding author.

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Acknowledgements

This study was supported by grants-in-aid for scientific research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (17K10124 and 21K07759).

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Authors and Affiliations

  1. Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465, Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan

    Kenji Okamoto, Toshihiko Imamura, Seiji Tanaka, Takayo Urata, Hideki Yoshida & Tomoko Iehara

  2. Department of Pediatrics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan

    Norio Shiba

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  1. Kenji Okamoto
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  2. Toshihiko Imamura
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  3. Seiji Tanaka
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Correspondence to Toshihiko Imamura.

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12185_2023_3612_MOESM1_ESM.pdf

Supplementary file1 Fig. 1 Homology between human and murine NUP98::NSD1 amino acid sequences. Comparison between human and murine aa sequences; aa 518 of human NUP98 (Pro) is equivalent to aa 535 (Pro) in the murine Nup98, and aa 1166 of NSD1 (Ser) is equivalent to aa 1167 (Ser) in murine Nsd1. Human and murine NUP98 aa sequences fused to NSD1 exhibited approximately 98% homology, while human and murine NSD1 aa sequences fused to NUP98 had around 85% identity. Pro, Proline; Ser, Serine. Fig. 2 RT-PCR revealed expression of Nup98::Nsd1 mRNA in transduced 32D cells. Fig. 3 Western blotting did not show constitutive phosphorylation of Jak2 and Stat5 in Nup98::Nsd1+ 32D cells. Fig. 4 (a) qPCR revealed that the expression level of Meis1 was not upregulated in Nup98::Nsd1+ 32D cells. RQ, relative quantification. (b) RT-PCR did not reveal expression of Hoxa9 mRNA in either Nup98::Nsd1+ 32D or mock-transduced 32D cells. Fig. 5 Effects of Nup98::Nsd1 on colony formation. (a), (b) Neither C57BL/6 nor BALB/c Lin- bone marrow cells were immortalized by Nup98::Nsd1. KMT2A::Aff1 served as a positive control, which we previously confirmed has immortalization ability. (c), (d) RT-PCR revealed expression of Nup98::Nsd1 mRNA in murine Lin- BM cells (we used primers designed to straddle the fusion point; therefore, the size of the PCR product is 468 bp), indicating successful transduction of Nup98::Nsd1; however, Nup98::Nsd1 did not exhibit cell transformation activity. RT-PCR of Gapdh was performed using forward (5’-CATCACTGCCACCCAGAAGACTG-3’) and reverse (5’- ATGCCAGTGAGCTTCCCGTTCAG-3’) primers; therefore, the size of the PCR product is 153 bp. BM, bone marrow; PC, positive control; NTC, no template control. Fig. 6 The sensitivity of Nup98::Nsd1 to cytarabine and daunorubicin. Nup98::Nsd1+ and mock-transduced 32D cells were cultured in IL-3 containing medium at 1 × 105 cells per well with 1, 10, 100 and 1,000 nM cytarabine or daunorubicin. Viable cell numbers were measured at 24, 48, 72 and 96 hours. (a), (b) Cytarabine did not attenuate cell proliferation of Nup98::Nsd1+ 32D cells. (c), (d) Daunorubicin did not visibly attenuate cell proliferation of Nup98::Nsd1+ 32D cells. (e), (f) STK33 inhibitor had no effect to suppress the proliferation of Nup98::Nsd1+ 32D cells, because the vehicle control (indicated in green) was at the bottom. (PDF 691 KB)

12185_2023_3612_MOESM2_ESM.xlsx

Supplementary file2 Table 1 Lists of the top 10 up- and down-regulated genes in Nup98::Nsd1+ 32D cells, Table 2 Lists of the top 10 oncogenic signature gene sets (C6) enriched in Nup98::Nsd1+ 32D cells. (XLSX 15 KB)

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Okamoto, K., Imamura, T., Tanaka, S. et al. The Nup98::Nsd1 fusion gene induces CD123 expression in 32D cells. Int J Hematol 118, 277–287 (2023). https://doi.org/10.1007/s12185-023-03612-z

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  • DOI: https://doi.org/10.1007/s12185-023-03612-z

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