Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

MOLECULAR TARGETS FOR THERAPY

Kinase-independent role of mTOR and on-/off-target effects of an mTOR kinase inhibitor

Leukemia volume 37, pages 2073–2081 (2023)Cite this article

Subjects

Abstract

mTOR, as a serine/threonine kinase, is a widely pursued anticancer target. Multiple clinical trials of mTOR kinase inhibitors are ongoing, but their specificity and safety features remain lacking. Here, we have employed an inducible kinase-inactive D2338A mTOR knock-in mouse model (mTOR−/KI) together with a mTOR conditional knockout model (mTOR−/−) to assess the kinase-dependent/-independent function of mTOR in hematopoiesis and the on-/off-target effects of mTOR kinase inhibitor AZD2014. Despite exhibiting many similar phenotypes to mTOR−/− mice in hematopoiesis, the mTOR−/KI mice survived longer and showed differences in hematopoietic progenitor cells compared to mTOR−/− mice, suggesting a kinase-independent function of mTOR in hematopoiesis. Gene expression signatures in hematopoietic stem cells (HSCs) further revealed both kinase-dependent and independent effects of mTOR. AZD2014, a lead mTOR kinase inhibitor, appeared to work mostly on-target in suppressing mTOR kinase activity, mimicking that of mTOR−/KI HSCs in transcriptome analysis, but it also induced a small set of off-target responses in mTOR−/KI HSCs. In murine and human myeloid leukemia, besides kinase-inhibitory on-target effects, AZD2014 displayed similar off-target and growth-inhibitory cytostatic effects. These studies provide new insights into kinase-dependent/-independent effects of mTOR in hematopoiesis and present a genetic means for precisely assessing the specificity of mTOR kinase inhibitors.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: mTOR KI mice show similar phenotypes to mTOR KO mice in hematopoiesis.
Fig. 2: mTOR has kinase-independent function in HSPC regulation.
Fig. 3: mTOR KI mouse model is useful for precisely assessing on-/off-target effects of mTOR kinase inhibitor AZD2014.
Fig. 4: AZD2014 induces on-/off-target and exerts cytostatic effects in BCR-ABL-driven leukemia mice.
Fig. 5: AZD2014 induces similar on-/off-target and cytostatic effects in R/R AML PDX mice.

Similar content being viewed by others

Data availability

All the RNA-seq and ATAC-seq data reported in this paper have been deposited in the NCBI Gene Expression Omnibus (GEO) database under accession number GSE172250, which is composed of three subseries GSE172247, GSE172248, and GSE235097.

References

  1. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149:274–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. Cell. 2017;168:960–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kalaitzidis D, Sykes SM, Wang Z, Punt N, Tang Y, Ragu C, et al. mTOR complex 1 plays critical roles in hematopoiesis and Pten-loss-evoked leukemogenesis. Cell Stem Cell. 2012;11:429–39.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ghosh J, Kapur R. Role of mTORC1-S6K1 signaling pathway in regulation of hematopoietic stem cell and acute myeloid leukemia. Exp Hematol. 2017;50:13–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Fernandes H, Moura J, Carvalho E. mTOR signaling as a regulator of hematopoietic stem cell fate. Stem Cell Rev Rep. 2021;17:1312–22.

    Article  CAS  PubMed  Google Scholar 

  6. Guo F, Zhang S, Grogg M, Cancelas JA, Varney ME, Starczynowski DT, et al. Mouse gene targeting reveals an essential role of mTOR in hematopoietic stem cell engraftment and hematopoiesis. Haematologica. 2013;98:1353–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Fan C, Zhao C, Zhang F, Kesarwani M, Tu Z, Cai X, et al. Adaptive responses to mTOR gene targeting in hematopoietic stem cells reveal a proliferative mechanism evasive to mTOR inhibition. Proc Natl Acad Sci USA. 2021;118:e2020102118.

    Article  CAS  PubMed  Google Scholar 

  8. Hua H, Kong Q, Zhang H, Wang J, Luo T, Jiang Y. Targeting mTOR for cancer therapy. J Hematol Oncol. 2019;12:71.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Calimeri T, Ferreri AJM. m-TOR inhibitors and their potential role in haematological malignancies. Br J Haematol. 2017;177:684–702.

    Article  CAS  PubMed  Google Scholar 

  10. Popova NV, Jucker M. The role of mTOR signaling as a therapeutic target in cancer. Int J Mol Sci. 2021;22:1743.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Martelli AM, Buontempo F, McCubrey JA. Drug discovery targeting the mTOR pathway. Clin Sci. 2018;132:543–68.

    Article  CAS  Google Scholar 

  12. Magaway C, Kim E, Jacinto E. Targeting mTOR and metabolism in cancer: lessons and innovations. Cells. 2019;8:1584.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Nepstad I, Hatfield KJ, Gronningsaeter IS, Reikvam H. The PI3K-Akt-mTOR signaling pathway in human acute myeloid leukemia (AML) cells. Int J Mol Sci. 2020;21:2907.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tabe Y, Tafuri A, Sekihara K, Yang H, Konopleva M. Inhibition of mTOR kinase as a therapeutic target for acute myeloid leukemia. Expert Opin Ther Targets. 2017;21:705–14.

    Article  CAS  PubMed  Google Scholar 

  15. Zhong L, Li Y, Xiong L, Wang W, Wu M, Yuan T, et al. Small molecules in targeted cancer therapy: advances, challenges, and future perspectives. Signal Transduct Target Ther. 2021;6:201.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Benjamin D, Colombi M, Moroni C, Hall MN. Rapamycin passes the torch: a new generation of mTOR inhibitors. Nat Rev Drug Discov. 2011;10:868–80.

    Article  CAS  PubMed  Google Scholar 

  17. Tian T, Li X, Zhang J. mTOR signaling in cancer and mTOR inhibitors in solid tumor targeting therapy. Int J Mol Sci. 2019;20:755.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kalim KW, Zhang S, Chen X, Li Y, Yang JQ, Zheng Y, et al. mTOR has a developmental stage-specific role in mitochondrial fitness independent of conventional mTORC1 and mTORC2 and the kinase activity. PLoS One. 2017;12:e0183266.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Shor B, Cavender D, Harris C. A kinase-dead knock-in mutation in mTOR leads to early embryonic lethality and is dispensable for the immune system in heterozygous mice. BMC Immunol. 2009;10:28.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Harada M, Benito J, Yamamoto S, Kaur S, Arslan D, Ramirez S, et al. The novel combination of dual mTOR inhibitor AZD2014 and pan-PIM inhibitor AZD1208 inhibits growth in acute myeloid leukemia via HSF pathway suppression. Oncotarget. 2015;6:37930–47.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Ly C, Arechiga AF, Melo JV, Walsh CM, Ong ST. Bcr-Abl kinase modulates the translation regulators ribosomal protein S6 and 4E-BP1 in chronic myelogenous leukemia cells via the mammalian target of rapamycin. Cancer Res. 2003;63:5716–22.

    CAS  PubMed  Google Scholar 

  22. Kharas MG, Janes MR, Scarfone VM, Lilly MB, Knight ZA, Shokat KM, et al. Ablation of PI3K blocks BCR-ABL leukemogenesis in mice, and a dual PI3K/mTOR inhibitor prevents expansion of human BCR-ABL+ leukemia cells. J Clin Invest. 2008;118:3038–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Arrigoni E, Del ReM, Galimberti S, Restante G, Rofi E, Crucitta S, et al. Concise review: chronic myeloid leukemia: stem cell niche and response to pharmacologic treatment. Stem Cells Transl Med. 2018;7:305–14.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Mojtahedi H, Yazdanpanah N, Rezaei N. Chronic myeloid leukemia stem cells: targeting therapeutic implications. Stem Cell Res Ther. 2021;12:603.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Burchert A, Wang Y, Cai D, von Bubnoff N, Paschka P, Muller-Brusselbach S, et al. Compensatory PI3-kinase/Akt/mTor activation regulates imatinib resistance development. Leukemia. 2005;19:1774–82.

    Article  CAS  PubMed  Google Scholar 

  26. Kesarwani M, Kincaid Z, Gomaa A, Huber E, Rohrabaugh S, Siddiqui Z, et al. Targeting c-FOS and DUSP1 abrogates intrinsic resistance to tyrosine-kinase inhibitor therapy in BCR-ABL-induced leukemia. Nat Med. 2017;23:472–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wunderlich M, Brooks RA, Panchal R, Rhyasen GW, Danet-Desnoyers G, Mulloy JC. OKT3 prevents xenogeneic GVHD and allows reliable xenograft initiation from unfractionated human hematopoietic tissues. Blood. 2014;123:e134–44.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Yang H, Rudge DG, Koos JD, Vaidialingam B, Yang HJ, Pavletich NP. mTOR kinase structure, mechanism and regulation. Nature. 2013;497:217–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Harwood FC, Klein Geltink RI, O’Hara BP, Cardone M, Janke L, Finkelstein D, et al. ETV7 is an essential component of a rapamycin-insensitive mTOR complex in cancer. Sci Adv. 2018;4:eaar3938.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Rodrik-Outmezguine VS, Okaniwa M, Yao Z, Novotny CJ, McWhirter C, Banaji A, et al. Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor. Nature. 2016;534:272–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Fan Q, Aksoy O, Wong RA, Ilkhanizadeh S, Novotny CJ, Gustafson WC, et al. A kinase inhibitor targeted to mTORC1 drives regression in glioblastoma. Cancer Cell. 2017;31:424–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kuroshima K, Yoshino H, Okamura S, Tsuruda M, Osako Y, Sakaguchi T, et al. Potential new therapy of Rapalink-1, a new generation mammalian target of rapamycin inhibitor, against sunitinib-resistant renal cell carcinoma. Cancer Sci. 2020;111:1607–18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. La Manna F, De Menna M, Patel N, Karkampouna S, De Filippo MR, Klima I, et al. Dual-mTOR Inhibitor Rapalink-1 reduces prostate cancer patient-derived xenograft growth and alters tumor heterogeneity. Front Oncol. 2020;10:1012.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Zheng B, Mao JH, Qian L, Zhu H, Gu DH, Pan XD, et al. Pre-clinical evaluation of AZD-2014, a novel mTORC1/2 dual inhibitor, against renal cell carcinoma. Cancer Lett. 2015;357:468–75.

    Article  CAS  PubMed  Google Scholar 

  35. Guichard SM, Curwen J, Bihani T, D'Cruz CM, Yates JW, Grondine M, et al. AZD2014, an inhibitor of mTORC1 and mTORC2, is highly effective in ER+ breast cancer when administered using intermittent or continuous schedules. Mol Cancer Ther. 2015;14:2508–18.

    Article  CAS  PubMed  Google Scholar 

  36. Liao H, Huang Y, Guo B, Liang B, Liu X, Ou H, et al. Dramatic antitumor effects of the dual mTORC1 and mTORC2 inhibitor AZD2014 in hepatocellular carcinoma. Am J Cancer Res. 2015;5:125–39.

    PubMed  Google Scholar 

  37. Pi R, Yang Y, Hu X, Li H, Shi H, Liu Y, et al. Dual mTORC1/2 inhibitor AZD2014 diminishes myeloid-derived suppressor cells accumulation in ovarian cancer and delays tumor growth. Cancer Lett. 2021;523:72–81.

    Article  CAS  PubMed  Google Scholar 

  38. Li S, Sheng J, Liu Z, Fan Y, Zhang C, Lv T, et al. Potent antitumour of the mTORC1/2 dual inhibitor AZD2014 in docetaxel-sensitive and docetaxel-resistant castration-resistant prostate cancer cells. J Cell Mol Med. 2021;25:2436–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Mizutani Y, Inase A, Maimaitili Y, Miyata Y, Kitao A, Matsumoto H, et al. An mTORC1/2 dual inhibitor, AZD2014, acts as a lysosomal function activator and enhances gemtuzumab ozogamicin-induced apoptosis in primary human leukemia cells. Int J Hematol. 2019;110:490–9.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank James Johnson for mouse technical assistance, Meenu Kesarwani, and Zachary Kincaid for help with BCR-ABL-driven leukemia model, and Aditi Paranjpe for bioinformatics support. We are also thankful to the Comprehensive Mouse and Cancer Core and the Research Flow Cytometry Core at Cincinnati Children’s Hospital Medical Center for technical support. This work was supported by NIH grants R01 CA204895 and U54 DK126108, and by a Cancer Free Kids grant 308269.

Author information

Authors and Affiliations

  1. Division of Experimental Hematology and Cancer Biology, Cancer and Blood Diseases Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 45229, USA

    Cuiqing Fan, Mark Wunderlich, Xiongwei Cai, Feng Zhang, Ashley Kuenzi Davis, Lingli Xu, Fukun Guo, Q. Richard Lu, Mohammad Azam & Yi Zheng

  2. State Key Laboratory of Genetic Engineering, Department of Biostatistics and Computational Biology, School of Life Sciences, Fudan University, Shanghai, 200438, China

    Zijun Yan & Weidong Tian

  3. Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    Fukun Guo, Q. Richard Lu, Mohammad Azam & Yi Zheng

Authors
  1. Cuiqing Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mark Wunderlich
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiongwei Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zijun Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Feng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ashley Kuenzi Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lingli Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Fukun Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Q. Richard Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Mohammad Azam
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Weidong Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Yi Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

CF designed and performed research, analyzed data, and wrote the paper; MW performed research and analyzed data; XC performed research; ZY, FZ, AKD, and LX analyzed the data; FG, QRL, MA, and WT contributed new reagents or analytic tools; YZ supervised the study, designed the research, analyzed data, and wrote the paper.

Corresponding author

Correspondence to Yi Zheng.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fan, C., Wunderlich, M., Cai, X. et al. Kinase-independent role of mTOR and on-/off-target effects of an mTOR kinase inhibitor. Leukemia 37, 2073–2081 (2023). https://doi.org/10.1038/s41375-023-01987-w

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41375-023-01987-w

Search

Advanced search

Quick links