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MPL overexpression induces a high level of mutant-CALR/MPL complex: a novel mechanism of ruxolitinib resistance in myeloproliferative neoplasms with CALR mutations

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Abstract

Ruxolitinib (RUX), a JAK1/2-inhibitor, is effective for myeloproliferative neoplasm (MPN) with both JAK2V617 F and calreticulin (CALR) mutations. However, many MPN patients develop resistance to RUX. Although mechanisms of RUX-resistance in cells with JAK2V617 F have already been characterized, those in cells with CALR mutations remain to be elucidated. In this study, we established RUX-resistant human cell lines with CALR mutations and characterized mechanisms of RUX-resistance. Here, we found that RUX-resistant cells had high levels of MPL transcripts, overexpression of both MPL and JAK2, and increased phosphorylation of JAK2 and STAT5. We also found that mature MPL proteins were more stable in RUX-resistant cells. Knockdown of MPL in RUX-resistant cells by shRNAs decreased JAK/STAT signaling. Immunoprecipitation assays showed that binding of mutant CALR to MPL was increased in RUX-resistant cells. Reduction of mutated CALR decreased proliferation of the resistant cells. When resistant cells were cultured in the absence of RUX, the RUX-resistance was reversed, with reduction of the mutant-CALR/MPL complex. In conclusion, MPL overexpression induces higher levels of a mutant-CALR/MPL complex, which may cause RUX-resistance in cells with CALR mutations. This mechanism may be a new therapeutic target to overcome RUX-resistance.

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Acknowledgements

We thank Ms. Okada and Ms. Liu Meiou for technical supports. We thank Dr. Nami Masubuchi for her supports for qPCR experiments. We thank Dr. Toshiaki Ohteki and Dr. Masashi Kanayama in Department of Biodefence Research, Medical Research Institute at TMDU for their supports for animal experiments. We also thank Ms. Inoue’s help for IHC staining. This research was supported by Nagao-Takeshi Research grant (Yoshihiro Umezawa) and TMDU Young Investigator Research grant (Yoshihiro Umezawa).

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Authors and Affiliations

  1. Department of Immunotherapy for Hematopoietic Disorders, Tokyo Medical and Dental University, TMDU, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan

    Shunichiro Yasuda, Satoru Aoyama, Huixin Li, Daisuke Watanabe & Norihiko Kawamata

  2. Department of Hematology, TMDU, Tokyo, Japan

    Shunichiro Yasuda, Satoru Aoyama, Ryoto Yoshimoto, Daisuke Watanabe, Hiroki Akiyama & Osamu Miura

  3. Department of Comprehensive Pathology, TMDU, Tokyo, Japan

    Kouhei Yamamoto

  4. Department of Global Health Promotion, TMDU, Tokyo, Japan

    Takeo Fujiwara

  5. Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan

    Yuho Najima & Noriko Doki

  6. Department of Hematology, Chiba University, Chiba, Japan

    Emiko Sakaida

  7. Department of Hematology, Juntendo University Graduate School of Medicine, Tokyo, Japan

    Yoko Edahiro, Misa Imai & Norio Komatsu

  8. Leading center for the development and Research of Cancer Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan

    Misa Imai

  9. Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University Graduate School of Medicine, Tokyo, Japan

    Marito Araki

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  1. Shunichiro Yasuda
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Contributions

SY, SA, DW, MA, OM and NKa designed the experiments. SY, RY, HL, HA, MI and MA performed the experiments. SY, YE, ES, NKo, YN, ND and NKa contributed to collection of clinical samples. SY, RY, HL and KY examined histopathological samples. SY, SA, MA, ES, YN, ND and NKa analyzed the data. SY, TF and NKa performed statistical analysis. SY, SA, MA, NKo, OM and NKa wrote the paper. All the authors have approved the manuscript.

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Correspondence to Norihiko Kawamata.

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Yasuda, S., Aoyama, S., Yoshimoto, R. et al. MPL overexpression induces a high level of mutant-CALR/MPL complex: a novel mechanism of ruxolitinib resistance in myeloproliferative neoplasms with CALR mutations. Int J Hematol 114, 424–440 (2021). https://doi.org/10.1007/s12185-021-03180-0

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