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Leukemias induced by altered TRK-signaling are sensitive to mTOR inhibitors in preclinical models

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Abstract

Rapamycin is a potent allosteric mTORC1 inhibitor with clinical applications as an anticancer agent. However, only a fraction of cancer patients responds to the drug, and no biomarkers are available to predict tumor sensitivity. Recently, we and others have obtained evidence for potential involvement of tropomyosin-related kinase (TRK) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) in leukemia. In the present study, we tested the therapeutic effect of Rapamycin and its analog RAD001 on altered TRK-induced leukemia in a murine model. Daily treatment with Rapamycin (2 mg/kg) or RAD001 (1 mg/kg) significantly prolonged the survival of treated animals (n = 40) compared with the placebo group. Consistently, both mTOR and S6 proteins were strongly dephosphorylated in vitro and in vivo after treatment with Rapamycin or RAD001. However, Rapamycin did not completely inhibit mTORC1-dependent phosphorylation of 4E-BP1. With exception of one mouse showing slight reactivation of Akt after treatment, no reactivation of MAPK or Akt pathways was observed in other resistant tumors. Interestingly, leukemic cells isolated from a Rapamycin-resistant mouse were still highly sensitive to Rapamycin in vitro. Our findings suggest that altered TRK signaling may be a good predictor of tumor sensitivity to mTOR inhibition and that pathways other than MAPK and Akt exist that may trigger resistance of leukemic cells to Rapamycin in vivo.

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Acknowledgements

This study was supported by the Deutsche Krebshilfe (grants: 10-2090-Li I and 108245) and DFG excellence cluster REBIRTH. AS is a student of the PhD program for Molecular Medicine in Hannover Medical School. We are very grateful to Rena-Mareike Struß, Jessica Wenzl, and Thomas Neumann for technical assistance; Michael Morgan for critical reading of this paper; and Hans Grundtke, Jörg Frühauf, and Martin Werner (Radiotherapy, Hannover Medical School) for irradiation of animals.

Conflict of interest

Authors declare no competing financial interests.

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Authors and Affiliations

  1. Department of Experimental Hematology OE6960, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany

    Mathias Rhein, Adrian Schwarzer, Min Yang, Martijn Brugman, Johann Meyer, Christopher Baum & Zhixiong Li

  2. Pharmacology, Hannover Medical School, 30625, Hannover, Germany

    Volkhard Kaever

  3. Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, 30625, Hannover, Germany

    Arnold Ganser

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  1. Mathias Rhein
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Correspondence to Zhixiong Li.

Additional information

Mathias Rhein and Adrian Schwarzer contributed equally to this study.

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Fig. S1

Phenotyping of #483 cells by FACS analysis. Panels show representative stainings of bone marrow cells (upper row) and thymus cells against markers of lymphoid (CD4, CD8, and CD19) and myeloid lineages (CD11b, Gr1). Additionally, thymus cells were further characterized, showing expression of CD25, CD44 and CD3 (all lymphoid lineage), but not Ter119 (data not shown). The #483 cell line was established from thymus cells of a mouse transplanted with a retroviral vector expressing ∆TrkA and eGFP [1] (PDF 248 kb)

Fig. S2

Establishment of a murine model for in vivo application of Rapamycin. a Kaplan–Meier plot for the titration of #483 cells. After sublethal irradiation with 7.5 Gy, 4 C57Bl/6J mice per group were transplanted with 107 (dashed line, mean survival 21 days), 106 (dotted line, mean survival 35 days), or 105 (solid line, survived observation time) cells per mouse. All animals with 107 cells died on day 21 after transplantation, and 50% of the animals from the 106 group died around day 35, while none of the 105 group developed leukemia after observation for 8 months. Thus, we chose to transplant animals with 106 cells in further experiments. b Rapamycin levels in whole blood. Blood samples of daily treated healthy mice (2 mg/kg Rapamycin) via intraperitoneal injection (i.p.) were taken at the respective time points. Rapamycin levels are comparable with levels achieved in other murine models (15–72 ng/ml) [2] and patients (70 ng/ml max). c Leukocytes (WBC) in peripheral blood of animals treated with Rapamycin. d Pharmacokinetic study of RAD001 in mice: steady state RAD001 levels after i.p. injection of three different doses. e Treatment of RAD001 induced slight leukopenia of healthy mice treated with RAD001. (PDF 1324 kb)

Fig. S3

Kaplan–Meier plot of animals treated with Rapamycin. Animals were transplanted with #481 cells [1]. The progression of leukemia was significantly delayed in the cohort treated with apamycin (p < 0.05) (PDF 162 kb)

Fig. S4

Monitoring of leukemia cells in peripheral blood of animals. In placebo group (a), the population of GFP positive cells went up in two animals 1 week after treatment with carrier (the rest of the group died around 1 week after treatment start). In RAD001 treated animals (b), the leukemic population went down initially, but eventually rose up (PDF 133 kb)

Fig. S5

a Western blots of Rapamycin-treated cells. #483 cells were incubated with inhibitor for 24 h and lysed using standard procedures. There is a strong reduction of phosphorylated protein in #483 cells for S6 protein, a downstream substrate of mTOR. There was no reactivation of ERK. b PhosFow analysis showed no reactivation of Akt or ERK in #483 cells treated with Rapamycin in vitro. c Reduced phosphorylation of 4E-BP1 (Thr-37/46) in #483 cells after treatment. However, phosphorylation of Ser-65 was not significantly changed. Gel shifts can be observed in samples treated with Rapamycin. The α-β-γ isoforms represent the phosphorylation status of 4E-BP1 with α being hypophosphorylated and γ being hyperphosphorylated [3]. d No reactivation of Akt in Rapamycin-treated animal (#1011) in comparison with a control animal (#1009). e Slightly high activation of Akt in mouse #958, but not in #840 (both Rapamycin-treated). #953 is from the placebo group. Note the higher activation of the parent cells #483 in vitro (PDF 560 kb)

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Rhein, M., Schwarzer, A., Yang, M. et al. Leukemias induced by altered TRK-signaling are sensitive to mTOR inhibitors in preclinical models. Ann Hematol 90, 283–292 (2011). https://doi.org/10.1007/s00277-010-1065-3

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