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An mTORC1/2 dual inhibitor, AZD2014, acts as a lysosomal function activator and enhances gemtuzumab ozogamicin-induced apoptosis in primary human leukemia cells

  • Yu Mizutani
  • Aki Inase
  • Yimamu Maimaitili
  • Yoshiharu Miyata
  • Akihito Kitao
  • Hisayuki Matsumoto
  • Koji Kawaguchi
  • Ako Higashime
  • Hideaki Goto
  • Keiji Kurata
  • Kimikazu Yakushijin
  • Hironobu Minami
  • Hiroshi MatsuokaEmail author
Original Article
  • 36 Downloads

Abstract

Gemtuzumab ozogamicin (GO), an anti-CD33 antibody linked to calicheamicin via an acid-labile linker, is the first antibody–drug conjugate (ADC). The acidic environment inside lysosomes of target cells is an important intracellular determinant of the cytocidal action of GO, as the linker is hydrolyzed under acidic conditions. However, lysosomal activity in acute myeloid leukemia (AML) blasts in GO therapy has been insufficiently evaluated. It has been suggested that lysosome activity is suppressed in AML due to hyperactivation of the phosphoinositide 3-kinase/Akt pathway. We therefore hypothesized that agents which activate lysosomal function would potentiate the cytotoxicity of GO. Here, we found that a clinically useful mTORC1/2 dual inhibitor, AZD2014, reduced pH in the acidic organelles, including lysosomes, as shown by increased LysoTracker fluorescent intensity, and synergistically enhanced the cytotoxic effect of GO in primary leukemia cells. GO-induced cytotoxicity appeared to be enhanced with the increase in lysosomal activity by AZD2014. These results indicate that AZD2014 activated lysosomal function in primary leukemia cells, which in turn enhanced the cytotoxicity of GO. Enhancement of lysosomal activity may represent a new therapeutic strategy in the treatment of GO and other ADCs, particularly in cases with low lysosomal activity.

Keywords

Acute myeloid leukemia Gemtuzumab ozogamicin AZD2014 Lysosomal function Primary leukemia cells 

Notes

Acknowledgements

The authors sincerely thank the patients for their participation. We also thank Dr. Takashi Sonoki and Dr. Hideki Nakakuma for their valuable contribution and the many colleagues who read early drafts of the manuscript and provided critical comments and suggestions.

Compliance with ethical standards

Conflict of interest

This work was partly funded by Takeda Pharmaceutical Co., Ltd. and Novartis Pharma Co., Ltd. Neither Takeda Pharmaceutical Co., Ltd. nor Novartis Pharma Co., Ltd. had any involvement in the scientific content of this work.

Supplementary material

12185_2019_2701_MOESM1_ESM.docx (1.9 mb)
Supplement Fig. 1 AZD2014 inhibited both mTORC1 and mTORC2 activities without cytotoxicity in other AML cell lines. (A) Indicated cells were exposed to increasing concentrations of AZD2014 for 48 hours and specific apoptosis was determined in three individual experiments. (B) Indicated whole cell lysates obtained after AZD2014 treatment for 24 hours were analyzed by western blotting using the indicated antibodies. (AZD: AZD2014). Supplement Fig. 2 Cytotoxic effect of GO was also enhanced by 250 nM of AZD2014 treatment in AML cell lines. Indicated cell lines were treated with 2.5 μg/ml or 0.25 μg/ml (for NB4) of GO and 250 nM of AZD2014 either separately or in combination for 48 hours, and then specific apoptosis was determined in at least three individual experiments. Results are shown as the mean ± SD. The statistical significance of differences observed between GO and GO+AZD2014 was determined using two-tailed Student’s t test. *, ** and *** mean P < 0.05, P < 0.01 and P < 0.001, respectively. Supplement Fig. 3 ABCB1 expression was detected in KO52 cells but no other cell lines. Total RNA was extracted from the indicated AML cells and reverse transcription was performed. ABCB1 expression was then determined by PCR analysis. Expression of GAPDH was also determined as control. Supplement Fig. 4 AZD2014 activated lysosomal function in other cell lines. Left panel: Cells were exposed to the indicated reagents for 6 hours, followed by staining with LysoTracker Red DND-99 (300 nM) for 10 min, fixation with 4% paraformaldehyde and imaging. Right panel: LysoTracker fluorescent intensity of experimental cells compared with the control cells in the indicated cell lines. Results are shown as the mean ± standard error of the mean (SEM). Statistical significance of differences observed between GO and AZD2014 was determined using two-tailed Student’s t test. *, ** and *** mean P < 0.05, P < 0.01 and P < 0.001, respectively (AZD: AZD2014). Supplement Fig. 5 AZD2014 did not enhance GO-induced apoptosis in primary human leukemia cells with FLT3-ITD mutation. Primary leukemia cells were treated with 0.5 μg/ml of GO and 250 nM or 500 nM of AZD2014 either separately or in combination for 48 hours, and specific apoptosis was then determined. Supplement Fig. 6 Cases with FLT3-ITD mutation had high fluorescent intensity without AZD2014 treatment. Indicated primary leukemia cells were incubated without AZD2014 for 6 hours, followed by staining with LysoTracker Red DND-99 (300 nM) for 10 min, fixation with 4% paraformaldehyde and quantification of the amount of LysoTracker fluorescence. Results are shown as the mean ± SEM (DOCX 1914 kb)
12185_2019_2701_MOESM2_ESM.docx (15 kb)
Supplementary material 2 (DOCX 15 kb)

References

  1. 1.
    Fernandez HF, Sun Z, Yao X, Litzow MR, Luger SM, Paietta EM, et al. Anthracycline dose intensification in acute myeloid leukemia. N Engl J Med. 2009;361:1249–59.CrossRefGoogle Scholar
  2. 2.
    Robak T, Wierzbowska A. Current and emerging therapies for acute myeloid leukemia. Clin Ther. 2009;31(Pt 2):2349–70.CrossRefGoogle Scholar
  3. 3.
    Rollig C, Bornhauser M, Thiede C, Taube F, Kramer M, Mohr B, et al. Long-term prognosis of acute myeloid leukemia according to the new genetic risk classification of the European LeukemiaNet recommendations: evaluation of the proposed reporting system. J Clin Oncol. 2011;29:2758–65.CrossRefGoogle Scholar
  4. 4.
    Schaich M, Rollig C, Soucek S, Kramer M, Thiede C, Mohr B, et al. Cytarabine dose of 36 g/m(2) compared with 12 g/m(2) within first consolidation in acute myeloid leukemia: results of patients enrolled onto the prospective randomized AML96 study. J Clin Oncol. 2011;29:2696–702.CrossRefGoogle Scholar
  5. 5.
    Hamann PR, Hinman LM, Hollander I, Beyer CF, Lindh D, Holcomb R, et al. Gemtuzumab ozogamicin, a potent and selective anti-CD33 antibody-calicheamicin conjugate for treatment of acute myeloid leukemia. Bioconjugate Chem. 2002;13:47–58.CrossRefGoogle Scholar
  6. 6.
    Godwin CD, Gale RP, Walter RB. Gemtuzumab ozogamicin in acute myeloid leukemia. Leukemia. 2017;31:1855–68.CrossRefGoogle Scholar
  7. 7.
    Walter RB, Appelbaum FR, Estey EH, Bernstein ID. Acute myeloid leukemia stem cells and CD33-targeted immunotherapy. Blood. 2012;119:6198–208.CrossRefGoogle Scholar
  8. 8.
    Larson RA, Sievers EL, Stadtmauer EA, Lowenberg B, Estey EH, Dombret H, et al. Final report of the efficacy and safety of gemtuzumab ozogamicin (Mylotarg) in patients with CD33-positive acute myeloid leukemia in first recurrence. Cancer. 2005;104:1442–52.CrossRefGoogle Scholar
  9. 9.
    Giles F, Estey E, O’Brien S. Gemtuzumab ozogamicin in the treatment of acute myeloid leukemia. Cancer. 2003;98:2095–104.CrossRefGoogle Scholar
  10. 10.
    Petersdorf SH, Kopecky KJ, Slovak M, Willman C, Nevill T, Brandwein J, et al. A phase 3 study of gemtuzumab ozogamicin during induction and postconsolidation therapy in younger patients with acute myeloid leukemia. Blood. 2013;121:4854–60.CrossRefGoogle Scholar
  11. 11.
    Ricart AD. Antibody–drug conjugates of calicheamicin derivative: gemtuzumab ozogamicin and inotuzumab ozogamicin. Clin Cancer Res. 2011;17:6417–27.CrossRefGoogle Scholar
  12. 12.
    Chalouni C, Doll S. Fate of antibody–drug conjugates in cancer cells. J Exp Clin Cancer Res. 2018;37:20.CrossRefGoogle Scholar
  13. 13.
    LaMarr WA, Yu L, Nicolaou KC, Dedon PC. Supercoiling affects the accessibility of glutathione to DNA-bound molecules: positive supercoiling inhibits calicheamicin-induced DNA damage. Proc Natl Acad Sci USA. 1998;95:102–7.CrossRefGoogle Scholar
  14. 14.
    Laszlo GS, Estey EH, Walter RB. The past and future of CD33 as therapeutic target in acute myeloid leukemia. Blood Rev. 2014;28:143–53.CrossRefGoogle Scholar
  15. 15.
    Saftig P, Klumperman J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat Rev Mol Cell Biol. 2009;10:623–35.CrossRefGoogle Scholar
  16. 16.
    Settembre C, Zoncu R, Medina DL, Vetrini F, Erdin S, Erdin S, et al. A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. EMBO J. 2012;31:1095–108.CrossRefGoogle Scholar
  17. 17.
    Evangelisti C, Evangelisti C, Chiarini F, Lonetti A, Buontempo F, Neri LM, et al. Autophagy in acute leukemias: a double-edged sword with important therapeutic implications. Biochim Biophys Acta. 2015;1853:14–26.CrossRefGoogle Scholar
  18. 18.
    Nyakern M, Tazzari PL, Finelli C, Bosi C, Follo MY, Grafone T, et al. Frequent elevation of Akt kinase phosphorylation in blood marrow and peripheral blood mononuclear cells from high-risk myelodysplastic syndrome patients. Leukemia. 2006;20:230–8.CrossRefGoogle Scholar
  19. 19.
    Follo MY, Mongiorgi S, Bosi C, Cappellini A, Finelli C, Chiarini F, et al. The Akt/mammalian target of rapamycin signal transduction pathway is activated in high-risk myelodysplastic syndromes and influences cell survival and proliferation. Cancer Res. 2007;67:4287–94.CrossRefGoogle Scholar
  20. 20.
    Tamburini J, Elie C, Bardet V, Chapuis N, Park S, Broet P, et al. Constitutive phosphoinositide 3-kinase/Akt activation represents a favorable prognostic factor in de novo acute myelogenous leukemia patients. Blood. 2007;110:1025–8.CrossRefGoogle Scholar
  21. 21.
    Zhang H. Targeting autophagy in lymphomas: a double-edged sword? Int J Hematol. 2018;107:502–12.CrossRefGoogle Scholar
  22. 22.
    Watson AS, Riffelmacher T, Stranks A, Williams O, De Boer J, Cain K, et al. Autophagy limits proliferation and glycolytic metabolism in acute myeloid leukemia. Cell Death Discov. 2015;1:15008.CrossRefGoogle Scholar
  23. 23.
    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.Google Scholar
  24. 24.
    Basu B, Dean E, Puglisi M, Greystoke A, Ong M, Burke W, et al. First-in-human pharmacokinetic and pharmacodynamic study of the dual m-TORC 1/2 inhibitor AZD2014. Clin Cancer Res. 2015;21:3412–9.CrossRefGoogle Scholar
  25. 25.
    Maimaitili Y, Inase A, Miyata Y, Kitao A, Mizutani Y, Kakiuchi S, et al. An mTORC1/2 kinase inhibitor enhances the cytotoxicity of gemtuzumab ozogamicin by activation of lysosomal function. Leuk Res. 2018;74:68–74.CrossRefGoogle Scholar
  26. 26.
    Klco JM, Spencer DH, Lamprecht TL, Sarkaria SM, Wylie T, Magrini V, et al. Genomic impact of transient low-dose decitabine treatment on primary AML cells. Blood. 2013;121:1633–43.CrossRefGoogle Scholar
  27. 27.
    Weir MC, Hellwig S, Tan L, Liu Y, Gray NS, Smithgall TE. Dual inhibition of Fes and Flt3 tyrosine kinases potently inhibits Flt3-ITD+ AML cell growth. PLoS One. 2017;12:e0181178.CrossRefGoogle Scholar
  28. 28.
    Wu Y, Giaisi M, Kohler R, Chen WM, Krammer PH, Li-Weber M. Rocaglamide breaks TRAIL-resistance in human multiple myeloma and acute T-cell leukemia in vivo in a mouse xenograft model. Cancer Lett. 2017;389:70–7.CrossRefGoogle Scholar
  29. 29.
    Foucquier J, Guedj M. Analysis of drug combinations: current methodological landscape. Pharmacol Res Perspect. 2015;3:e00149.CrossRefGoogle Scholar
  30. 30.
    Kim SK, Im GJ, An YS, Lee SH, Jung HH, Park SY. The effects of the antioxidant alpha-tocopherol succinate on cisplatin-induced ototoxicity in HEI-OC1 auditory cells. Int J Pediatr Otorhinolaryngol. 2016;86:9–14.CrossRefGoogle Scholar
  31. 31.
    Scott RC, Schuldiner O, Neufeld TP. Role and regulation of starvation-induced autophagy in the Drosophila fat body. Dev Cell. 2004;7:167–78.CrossRefGoogle Scholar
  32. 32.
    Rodriguez-Enriquez S, Kim I, Currin RT, Lemasters JJ. Tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes. Autophagy. 2006;2:39–46.CrossRefGoogle Scholar
  33. 33.
    Kurimoto M, Matsuoka H, Hanaoka N, Uneda S, Murayama T, Sonoki T, et al. Pretreatment of leukemic cells with low-dose decitabine markedly enhances the cytotoxicity of gemtuzumab ozogamicin. Leukemia. 2013;27:233–5.CrossRefGoogle Scholar
  34. 34.
    Amico D, Barbui AM, Erba E, Rambaldi A, Introna M, Golay J. Differential response of human acute myeloid leukemia cells to gemtuzumab ozogamicin in vitro: role of Chk1 and Chk2 phosphorylation and caspase 3. Blood. 2003;101:4589–97.CrossRefGoogle Scholar
  35. 35.
    Zhou J, Tan SH, Nicolas V, Bauvy C, Yang ND, Zhang J, et al. Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion. Cell Res. 2013;23:508–23.CrossRefGoogle Scholar
  36. 36.
    Selvarajah J, Elia A, Carroll VA, Moumen A. DNA damage-induced S and G2/M cell cycle arrest requires mTORC2-dependent regulation of Chk1. Oncotarget. 2015;6:427–40.CrossRefGoogle Scholar
  37. 37.
    Musa F, Alard A, David-West G, Curtin JP, Blank SV, Schneider RJ. Dual mTORC1/2 inhibition as a novel strategy for the resensitization and treatment of platinum-resistant ovarian cancer. Mol Cancer Ther. 2016;15:1557–67.CrossRefGoogle Scholar
  38. 38.
    Saeki K, Okuma E, Yuo A. Recurrent growth factor starvation promotes drug resistance in human leukaemic cells. Br J Cancer. 2002;86:292–300.CrossRefGoogle Scholar
  39. 39.
    Takeshita A. Efficacy and resistance of gemtuzumab ozogamicin for acute myeloid leukemia. Int J Hematol. 2013;97:703–16.CrossRefGoogle Scholar
  40. 40.
    Folkerts H, Hilgendorf S, Wierenga ATJ, Jaques J, Mulder AB, Coffer PJ, et al. Inhibition of autophagy as a treatment strategy for p53 wild-type acute myeloid leukemia. Cell Death Dis. 2017;8:e2927.CrossRefGoogle Scholar
  41. 41.
    Heydt Q, Larrue C, Saland E, Bertoli S, Sarry JE, Besson A, et al. Oncogenic FLT3-ITD supports autophagy via ATF4 in acute myeloid leukemia. Oncogene. 2018;37:787–97.CrossRefGoogle Scholar
  42. 42.
    Jawad M, Seedhouse C, Mony U, Grundy M, Russell NH, Pallis M. Analysis of factors that affect in vitro chemosensitivity of leukaemic stem and progenitor cells to gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukaemia. Leukemia. 2010;24:74–80.CrossRefGoogle Scholar
  43. 43.
    Kantarjian HM, DeAngelo DJ, Stelljes M, Martinelli G, Liedtke M, Stock W, et al. Inotuzumab ozogamicin versus standard therapy for acute lymphoblastic leukemia. N Engl J Med. 2016;375:740–53.CrossRefGoogle Scholar

Copyright information

© Japanese Society of Hematology 2019

Authors and Affiliations

  • Yu Mizutani
    • 1
  • Aki Inase
    • 1
  • Yimamu Maimaitili
    • 1
  • Yoshiharu Miyata
    • 1
  • Akihito Kitao
    • 1
  • Hisayuki Matsumoto
    • 2
  • Koji Kawaguchi
    • 1
  • Ako Higashime
    • 1
  • Hideaki Goto
    • 1
  • Keiji Kurata
    • 1
  • Kimikazu Yakushijin
    • 1
  • Hironobu Minami
    • 1
    • 3
  • Hiroshi Matsuoka
    • 1
    Email author
  1. 1.Division of Medical Oncology/Hematology, Department of MedicineKobe University Graduate School of MedicineKobeJapan
  2. 2.Department of Clinical LaboratoryKobe University HospitalKobeJapan
  3. 3.Cancer CenterKobe University HospitalKobeJapan

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