ONC201/TIC10 enhances durability of mTOR inhibitor everolimus in metastatic ER+ breast cancer

  1. Elena Farmaki
  2. Aritro Nath
  3. Rena Emond
  4. Kimya L Karimi
  5. Vince K Grolmusz
  6. Patrick A Cosgrove
  7. Andrea H Bild  Is a corresponding author
  1. Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, United States
7 figures, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
ONC201/TIC10 inhibits the proliferation of everolimus sensitive and resistant cells in 2D.

(A) Dose–response curves of CAMA-1, MCF7, and T47D everolimus sensitive and resistant cells under everolimus treatment. Cells were treated with increasing concentration of everolimus for 4 days and …

Figure 1—figure supplement 1
ONC201/TIC10 inhibits the proliferation of everolimus sensitive and resistant cells in 2D.

Dose–response curves of CAMA-1, MCF7, and T47D everolimus sensitive and resistant cells under ONC201/TIC10 treatment. Cells were treated with increasing concentration of ONC201/TIC10 for 4 days and …

Combination of ONC201/TIC10 and everolimus inhibits spheroid growth in 3D.

(A) Representative images of spheroid growth of sensitive (Venus, green) or resistant (mCherry, red) cells cultured in everolimus, ONC201/TIC10, or combination treated media at the indicated …

Figure 3 with 1 supplement
Combination therapy of ONC201/TIC10 and everolimus inhibits the growth of primary patient-derived cell spheroids.

(A) 3D cultures of primary patient-derived ER+ BC cells from ascites or pleural effusion treated with everolimus, ONC201/TIC10, or combination at the indicated concentrations for 4 days. Cell …

Figure 3—figure supplement 1
Combination therapy of ONC201/TIC10 and everolimus inhibits the growth of primary patient-derived cell spheroids.

Representative images of spheroid growth of primary patient-derived ER+ BC cells collected during everolimus treatment or post-everolimus and therapy resistant, cultured and treated with everolimus, …

Figure 4 with 1 supplement
ONC201/TIC10 causes loss of mitochondrial proteins and activation of stress response in everolimus sensitive and resistant cells.

(A) CAMA-1, MCF7, T47D everolimus sensitive and resistant cells were treated for 24 hr with ONC201/TIC10 at the indicated concentrations, and cell lysates were immunoblotted for TUFM, TFAM, BiP, …

Figure 4—figure supplement 1
ONC201/TIC10 mechanism in everolimus sensitive and resistant cells is TRAIL independent.

CAMA-1, MCF7, T47D everolimus sensitive and resistant cells were treated for 24 hr with everolimus, ONC201/TIC10, and combination at the indicated concentrations. Cell lysates were immunoblotted for …

Change in pathway activity over time in response to ONC201/TIC10.

Scatter plots displaying the enrichment scores (Y-axis) of (A) REACTOME cell cycle signature, (B) REACTOME ATF4 activated genes in response to endoplasmic reticulum stress signature, (C) REACTOME …

Figure 6 with 2 supplements
ONC201/TIC10 inhibits mitochondrial respiration in everolimus sensitive and resistant cells.

Cells were treated for 18 hr with indicated concentrations of everolimus, ONC201/TIC10, and combination. Mitochondrial respiration was measured using Seahorse XF Cell Mito Stress assay and oxygen …

Figure 6—figure supplement 1
Mitochondrial respiration in everolimus sensitive and resistant cells.

Cells were cultured for 18 hr. Mitochondrial respiration was measured using Seahorse XF Cell Mito Stress assay and oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) graphs …

Figure 6—figure supplement 2
ONC201/TIC10 inhibits mitochondrial respiration in everolimus sensitive and resistant cells.

Cells were treated for 18 hr with indicated concentrations of ONC201/TIC10 and everolimus. Mitochondrial respiration was measured using Seahorse XF Cell Mito Stress assay and oxygen consumption …

Cell cycle and oxidative phosphorylation pathway activity and neoadjuvant everolimus response.

Box plots comparing cell cycle pathway activity indicated by (A) REACTOME cell cycle signature enrichment scores and (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) oxidative phosphorylation …

Tables

Table 1
Treatment history of patients included in the study.
Patient diagnosisSampleTherapy linesDrugsTherapy lines post-everolimus
P1Metastatic ER+/HER2−Ascites6122
P2Metastatic ER+/PR+/HER2−Ascites792
P3Metastatic ER+/PR+/HER2−Pleural effusion791
P4Metastatic ER+/PR+/HER2−Ascites10145
P5Metastatic ER+/PR+/HER2−Ascites561
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Homo-sapiens) femaleCAMA-1 Breast; Mammary gland: adenocarcinomaATCCHTB-21
Cell line (Homo-sapiens) femaleMCF7 Breast; Mammary gland: adenocarcinoma epithelial cellATCCHTB-22
Cell line (Homo-sapiens) femaleT47D Breast; Mammary gland: carcinoma ductal epithelial cellATCCHTB-133
Cell line (Homo-sapiens) femalePrimary, P1, P2, P3, P4, P5This paper. City of Hope IRB #07047 and #17334P1, P2, P3, P4, P5Pleural effusion or ascites samples from patients
Transfected construct (mammalian)LeGO-V2 (Venus)Weber et al., 2008 Addgene #27340Addgene #27340; RRID:Addgene_27340Lentiviral construct to
transfect and express Venus fluorescent protein
Transfected construct (mammalian)LeGO-C2 (mCherry)Weber et al., 2008 Addgene #27339Addgene #27339; RRID:Addgene_27339Lentiviral construct to transfect and express mCherry fluorescent protein
Antibodyanti-BiP (C50B12) (rabbit monoclonal)Cell Signaling TechnologyCat#3177;
RRID:AB_2119845
WB (1:1000)
Antibodyanti-pFoxO3a (Ser294) (rabbit polyclonal)Cell Signaling TechnologyCat#5538; RRID:AB_10696878WB (1:1000)
Antibodyanti-FoxO3a (D19A7) (rabbit monoclonal)Cell Signaling TechnologyCat#12829; RRID:AB_2636990WB (1:1000)
Antibodyanti-pERK1/2 Thr202/Tyr204 (197G2) (rabbit monoclonal)Cell Signaling TechnologyCat#4377; RRID:AB_331775WB (1:1000)
Antibodyanti-ERK1/2 (137F5) (rabbit monoclonal)Cell Signaling TechnologyCat#4695; RRID:AB_390779WB (1:1000)
Antibodyanti-pAKT Ser473 (D9E) (rabbit monoclonal)Cell Signaling TechnologyCat#4060; RRID:AB_2315049WB (1:1000)
Antibodyanti-AKT (C67E7) (rabbit monoclonal)Cell Signaling TechnologyCat#4691; RRID:AB_915783WB (1:1000)
Antibodyanti-pS6 Ser240/244 Ribosomal Protein (D68F8) (rabbit monoclonal)Cell Signaling TechnologyCat#5364; RRID:AB_10694233WB (1:1000)
Antibodyanti-S6 Ribosomal Protein (54D2) (mouse monoclonal)Cell Signaling TechnologyCat#2317; RRID:AB_2238583WB (1:1000)
Antibodyanti-β-actin (8H10D10) (mouse monoclonal) (HRP Conjugate)Cell Signaling TechnologyCat#12262; RRID:AB_2566811WB (1:2000)
Antibodyanti-β-actin (13E5) (rabbit monoclonal) (HRP Conjugate)Cell Signaling TechnologyCat#5125; RRID:AB_1903890WB (1:2000)
Antibodyanti-rabbit IgG, HRP-linked (goat polyclonal)Cell Signaling TechnologyCat#7074; RRID:AB_2099233WB (1:2000)
Antibodyanti-mouse IgG, HRP-linked (horse polyclonal)Cell Signaling TechnologyCat#7076; RRID:AB_330924WB (1:2000)
Antibodyanti-CREB-2/ATF-4 (B-3) (mouse monoclonal)Santa Cruz BiotechnologyCat#390063; RRID:AB_2810998WB (1:300)
Antibodyanti-mtTFA (TFAM) (C-9) (mouse monoclonal)Santa Cruz BiotechnologyCat#376672; RRID:AB_11150497WB (1:200)
Antibodyanti-TRAIL (55B709.3) (mouse monoclonal)Thermo Fisher ScientificCat# MA1-41027; RRID:AB_1087999WB (1.5 μg/ml)
Antibodyanti-CHOP (rabbit polyclonal)ProteintechCat#15204-1-AP; RRID:AB_2292610WB (1:1000)
Antibodyanti-TUFM (rabbit polyclonal)Thermo Fisher ScientificCat#PA5-27511; RRID:AB_2544987WB (1:500)
Antibodyanti-Total OXPHOS (mouse monoclonal)AbcamCat#ab110411; RRID:AB_2756818WB (1:1000)
Chemical compound, drugEverolimus (RAD001)SelleckchemCat#S1120Dissolved in DMSO
Chemical compound, drugONC201/TIC10SelleckchemCat#S7963Dissolved in DMSO
Commercial assay or kitCellTiter-Glo Luminescent Cell Viability AssayPromegaCat#G7573
Commercial assay or kitCellTiter-Glo 3D Cell Viability AssayPromegaCat#G9682
Commercial assay or kitEasySep CD45 Depletion Kit IIStemCell TechnologiesCat#17898
Commercial assay or kitEasySep Dead Cell Removal (Annexin V) KitStemCell TechnologiesCat#17899
Commercial assay or kitAgilent Seahorse XF Cell Mito Stress Test KitAgilent TechnologiesCat#103015-100
SoftwareGraphPad Prism softwareGraphPad (https://graphpad.com)RRID:SCR_002798Version 9.3.1
SoftwareGen5 softwareBiotek Instruments (https://www.agilent.com/)Version 3.05
SoftwareSeahorse Wave Desktop Software 2.6Agilent Technologies (https://www.agilent.com/)RRID:SCR_014526Version 2.6
SoftwareImageJ softwareImageJ (http://imagej.nih.gov/ij/)RRID:SCR_003070

Additional files

Download links