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


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.


Acute myeloid leukemia Gemtuzumab ozogamicin AZD2014 Lysosomal function Primary leukemia cells 



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)


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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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