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A.K. Abdel-Aziz et al.
Mammalian target of rapamycin (mTOR) signaling is frequently hyperactive in AML.18 mTOR exists in two dis- tinct complexes; mTORC1 and mTORC2. mTORC1 pri- marily acts on substrates (as p70 S6 kinase) which controls glycolysis, protein synthesis and lipogenesis.19 mTORC2 regulates actin rearrangement, metabolism and survival (acting on substrates such as AKT).20 Inactivating mTORC1 significantly prolongs the survival of mice trans- planted with MLL-AF9 expressing AML cells.21 We and others have previously demonstrated that mTOR activa- tion acts as a fundamental adaptive response exploited by cancer cells to evade the cytotoxic stimuli triggered by several anticancer drugs including epigenetic therapies.22–24 Therefore, in this study, we investigated the potential implication of mTOR in mediating the sensitivity/resis- tance of AML cells to LSD1i.
Methods
Cell lines and cell culture
AML cell lines were obtained from either DSMZ or ATCC. KASUMI-1, NB4 and THP-1 cells were cultured in RPMI-1640 media supplemented with 2 mM L-glutamine, 10% FBS and 1% penicillin-streptomycin. SKNO-1 cells were cultured in RPMI- 1640 media supplemented with 10% FBS, 2 mM L-glutamine, 10 ng/mL GM-CSF and 1% penicillin-streptomycin. UF1 cells were cultured in RPMI-1640 media supplemented with 20% FBS and 2 mM L-glutamine. OCI-AML3 cells were cultured in a-MEM media supplemented with 20% FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin. PhoenixTM-Ampho cells were cultured in DMEM media supplemented with 2 mM L-glutamine, 10% FBS and 1% penicillin-streptomycin. Cells were maintained in a humidified tissue culture incubator at 37oC with 5% CO2.
Primary patient-derived AML blast and cord blood-derived CD34+ cells
A primary human AML sample (referred to as AML-IEO20; t(9;11);NPM WT;FLT3 WT) was obtained from the IEO Biobank according to the procedures approved by the Ethical Committee of the European Institute of Oncology. Mononuclear cells were isolated from the peripheral blood/bone marrow samples by Ficoll density centrifugation. For ex vivo studies, AML-IEO20 cells (pas- sage no. 3, ≥90% human leukemic blasts) were thawed and cul- tured in RPMI-1640 medium supplemented with 20% FBS, 1% S637 and 2mM L-glutamine. Primary human cord blood-derived CD34+ (non-transduced and hMLL-AF9 transduced) cells were cul- tured in HPGMTM Hematopoietic Growth Medium supplemented with 10% FBS, 2 mM L-glutamine, 100 ng/mL SCF, 100 ng/mL FLT3 and 100 ng/mL thrombopoietin. Before proceeding with in vitro experiments, cryopreserved cells were allowed to recover for at least three days.
In vivo studies
DDP38003 was dissolved in vehicle (40% PEG-400 in 5% glu-
cose solution). A stock solution of rapamycin (10 mg/mL) was pre- pared in 100% ethanol and stored at -20°C until use. Immediately before administration, rapamycin was diluted in vehicle com- posed of 5% PEG-400 and 5% Tween-80. AML-IEO20 cells (0.25x106 cells/mouse) were transplanted via tail vein injection of 8-10 weeks old NOD-SCID-IL2Rcγnull (NSG) mice. One week post-transplantation, mice were randomly assigned into four dif- ferent groups which were treated for five weeks. The first group served as vehicle treated group. The second group was adminis- tered DDP38003 (16.8 mg/kg, by oral gavage). The third group
received rapamycin (5 mg/kg, intraperitoneally). The fourth group received DDP38003 and rapamycin. The survival of the mice was analyzed and represented by a Kaplan-Meier survival plot. All ani- mal studies were conducted in compliance with the Italian Legislative Decree No.116 dated January 1992 and European Communities Council Directive No.86/609/EEC concerning the protection of animals used for experimental purposes and other scientific purposes according to the institutional policy regarding the care and use of laboratory animals. Mice were housed accord- ing to the guidelines set out in Commission Recommendation 2007/526/EC – June 18, 2007, guidelines of the accommodation and care of animals used for experimental and other scientific pur- poses. The study was approved by both the Ethical Committee of the European Institute of Oncology and Italian Ministry of Health (Project license number 199/2017).
For additional methods, please refer to the Online Supplementary Materials and Methods.
Results
Heterogeneous AML responses to LSD1i do not correlate with basal LSD1 level
To explore the anti-leukemic activity of inhibiting LSD1, we initially used DDP38003 (previously referred to as Compound 15),13 a potent selective and irreversible LSD1i, against a panel of AML cell lines belonging to different subtypes. As previously reported,12,17 AML cells demon- strated heterogeneous responses to LSD1 inhibition. DDP38003 dramatically diminished the proliferation and viability (cellular ATP level) of KASUMI-1, SKNO-1 and UF1 cells (Figure 1A and Online Supplementary Figure S1A). Indeed, DDP38003 induced apoptotic cell death in sensi- tive AML cells (Figure 1B and Online Supplementary Figure S1B). Conversely, the viability and proliferation of NB4, OCI-AML3 and THP-1 cells were not significantly affect- ed reflecting their resistance to DDP38003 (Figure 1A-B and Online Supplementary Figure S1A-B). Next, we investi- gated whether DDP38003 was efficiently inhibiting LSD1 in resistant AML. Indeed, genes reported to be directly repressed by LSD112,25 were upregulated post-DDP38003 treatment in both resistant THP-1 and sensitive KASUMI- 1 cells confirming efficient LSD1 inhibition (Online Supplementary Figure S1C). We then inquired whether such differential responsiveness of AML cells correlates with the basal level of LSD1. LSD1 levels in both sensitive and resistant AML were comparable ruling out this hypothesis (Figure 1C). Altogether, our findings indicate that assess- ing the changes in the transcript levels of direct target genes of LSD1 per se and/or basal LSD1 levels do not explain differential vulnerability/responsiveness of AML cells to LSD1i.
Activation of mTORC1 correlates with the resistance of AML cells to LSD1 inhibition
mTOR is constitutively activated in AML blasts26 and mediates chemoresistance.23 We therefore investigated the effect on mTOR signaling as a potential mechanism of resistance of AML cells to LSD1i. Indeed, DDP38003 trig- gered mTORC1 activation in resistant AML cells as shown by increased phosphorylation of its downstream targets: p70 S6 kinase (p70S6K), ribosomal S6 and 4 eukaryotic-binding protein 1 (4E-BP1) (Figure 1D). Conversely, treatment of sensitive AML with DDP38003 inactivated mTORC1 (Figure 1D). The activity of
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