Summary of Study ST003410

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002111. The data can be accessed directly via it's Project DOI: 10.21228/M8RC0H This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003410
Study Title	Lipidomics Analysis of ER+ Breast Cancer Cells Treated with Giredestrant and Palbociclib
Study Summary	We observed in a previous experiment that giredestrant treatment had a profound impact on the lipid profile of MCF-7 cells, in particular through elevating PUFAs at the apparent expense of MUFAs. We hypothesize that this shift in PUFA/MUFA ratio underlies giredestrant-induced sensitivity to GPX4 inhibition. Emerging data from our lab and other suggests that palbociclib, a key combination partner for giredestrant in the clinic, also sensitizes to GPX4 inhibition, and that the combination of giredestrant and palbo may synergize to creating an even higher sensitivity to GPX4i. Here, we aim to directly compare the effects on the lipidome of giredestrant and palbociclib, in MCF-7 cells (the discovery cell line where we already have some lipidomics data), and also T-47D cells, in which giredestrant, palbociclib and their combination drive a very profound sensitization to GPX4i, that exceeds what was observed in MCF-7 cells. The cells were treated with DMSO, 1 nM giredestrant, 200 nM palbociclib, or 1nM giredestrant plus 200nM palbociclib in quadruplicates, and collected on Day 7 for lipid analysis. Despite their differences under basal conditions, drug treatments altered the lipid profiles of MCF7 and T47D cells in a similar manner, with 200nM palbociclib at the 7 day time point having a less pronounced effect than giredestrant, and with the greatest lipid changes occurring with the combination of palbociclib plus giredestrant. PUFA-ePLs were likewise elevated by giredestrant and/or palbociclib in both cell lines, with the combined action of both drugs generally driving greatest increases in individual PUFA-linked ePL species. Palbociclib combination with giredestrant enhance the accumulation of PUFAs-phospholipids compared to the single treatments.
Institute	Genentech Inc.
Last Name	Wong
First Name	Weng Ruh
Address	1 DNA Way, South San Francisco, CA 94080, USA
Email	wongw24@gene.com
Phone	4089048962
Submit Date	2024-08-15
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2024-09-08
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002111
Project DOI:	doi: 10.21228/M8RC0H
Project Title:	Inhibition of GPX4 enhances CDK4/6 inhibitor and endocrine therapy activity in breast cancer.
Project Type:	MS quantitative analysis
Project Summary:	CDK4/6 inhibition in combination with endocrine therapy is the standard of care for estrogen receptor (ER+) breast cancer, and although cytostasis is frequently observed, new treatment strategies that enhance efficacy are required. We performed two independent genome-wide CRISPR screens to identify genetic determinants of CDK4/6 and endocrine therapy sensitivity. Genes involved in oxidative stress and ferroptosis modulated sensitivity, with GPX4 the top sensitiser in both screens. Depletion or inhibition of GPX4 increased sensitivity to palbociclib and giredestrant, and their combination, in ER+ breast cancer models, with GPX4 null xenografts being highly sensitive to palbociclib. GPX4 perturbation additionally sensitised triple negative breast cancer models to palbociclib. Palbociclib and giredestrant induced oxidative stress and disordered lipid metabolism, leading to a ferroptosis-sensitive state. Lipid peroxidation was promoted by a peroxisome AGPAT3-dependent pathway in ER+ breast cancer models, rather than the classical ACSL4 pathway. Our data demonstrate that CDK4/6 and ER inhibition creates vulnerability to ferroptosis induction, that could be exploited through combination with GPX4 inhibitors, to enhance sensitivity to the current therapies in breast cancer.
Institute:	Genentech Inc.
Last Name:	Wong
First Name:	Weng Ruh
Address:	1 DNA Way, South San Francisco, CA 94080, USA
Email:	wongw24@gene.com
Phone:	4089048962
Contributors:	Herrera-Abrey MT, Guan J, Khalid U, Ning J, Costa MR, Chan J, Li Q, Fortin J-P, Perampalam P, Biton A, Sandoval W, Vijay J, Hafner M, Cutts R, Wilson G, Frankum J, Roumeliotis TI, Alexander J, Hickman O, Brough R, Haider S, Choudhary J, Lord CJ, Swain, A, Metcalfe C, Tuner NC

Subject:

Subject ID:	SU003536
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Genotype Strain:	MCF-7 and T-47D

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Treatment	Time
SA376182	MCF7_DMSO_2	MCF7 breast cancer cells	Control	Day_7
SA376183	MCF7_DMSO_4	MCF7 breast cancer cells	Control	Day_7
SA376184	MCF7_DMSO_1	MCF7 breast cancer cells	Control	Day_7
SA376185	MCF7_DMSO_3	MCF7 breast cancer cells	Control	Day_7
SA376190	MCF7_9plusP_4	MCF7 breast cancer cells	Giredestrant_1nM_plus_Palbociclib_200nM	Day_7
SA376191	MCF7_9plusP_1	MCF7 breast cancer cells	Giredestrant_1nM_plus_Palbociclib_200nM	Day_7
SA376192	MCF7_9plusP_2	MCF7 breast cancer cells	Giredestrant_1nM_plus_Palbociclib_200nM	Day_7
SA376193	MCF7_9plusP_3	MCF7 breast cancer cells	Giredestrant_1nM_plus_Palbociclib_200nM	Day_7
SA376186	MCF7_9545_1	MCF7 breast cancer cells	Giredestrant_1nM	Day_7
SA376187	MCF7_9545_2	MCF7 breast cancer cells	Giredestrant_1nM	Day_7
SA376188	MCF7_9545_4	MCF7 breast cancer cells	Giredestrant_1nM	Day_7
SA376189	MCF7_9545_3	MCF7 breast cancer cells	Giredestrant_1nM	Day_7
SA376194	MCF7_Palbo_4	MCF7 breast cancer cells	Palbociclib_200nM	Day_7
SA376195	MCF7_Palbo_1	MCF7 breast cancer cells	Palbociclib_200nM	Day_7
SA376196	MCF7_Palbo_3	MCF7 breast cancer cells	Palbociclib_200nM	Day_7
SA376197	MCF7_Palbo_2	MCF7 breast cancer cells	Palbociclib_200nM	Day_7
SA376198	T47D_DMSO_1	T47D breast cancer cells	Control	Day_7
SA376199	T47D_DMSO_4	T47D breast cancer cells	Control	Day_7
SA376200	T47D_DMSO_2	T47D breast cancer cells	Control	Day_7
SA376201	T47D_DMSO_3	T47D breast cancer cells	Control	Day_7
SA376206	T47D_9plusP_3	T47D breast cancer cells	Giredestrant_1nM_plus_Palbociclib_200nM	Day_7
SA376207	T47D_9plusP_1	T47D breast cancer cells	Giredestrant_1nM_plus_Palbociclib_200nM	Day_7
SA376208	T47D_9plusP_4	T47D breast cancer cells	Giredestrant_1nM_plus_Palbociclib_200nM	Day_7
SA376209	T47D_9plusP_2	T47D breast cancer cells	Giredestrant_1nM_plus_Palbociclib_200nM	Day_7
SA376202	T47D_9545_3	T47D breast cancer cells	Giredestrant_1nM	Day_7
SA376203	T47D_9545_2	T47D breast cancer cells	Giredestrant_1nM	Day_7
SA376204	T47D_9545_1	T47D breast cancer cells	Giredestrant_1nM	Day_7
SA376205	T47D_9545_4	T47D breast cancer cells	Giredestrant_1nM	Day_7
SA376210	T47D_Palbo_3	T47D breast cancer cells	Palbociclib_200nM	Day_7
SA376211	T47D_Palbo_2	T47D breast cancer cells	Palbociclib_200nM	Day_7
SA376212	T47D_Palbo_1	T47D breast cancer cells	Palbociclib_200nM	Day_7
SA376213	T47D_Palbo_4	T47D breast cancer cells	Palbociclib_200nM	Day_7

Showing results 1 to 32 of 32

Showing results 1 to 32 of 32

Collection:

Collection ID:	CO003529
Collection Summary:	All cell lines were obtained from American Type Culture Collection (ATCC) or Asterand and maintained according to the supplier's instructions. MCF-7 and T47D cells were seeded at same density, 4x106 cells in RPMI medium in T175 flask (16 flasks each for each cell line) overnight. On day 0, the cells were treated with DMSO, 1 nM giredestrant, 200 nM palbociclib, or 1nM giredestrant plus 200nM palbociclib in quadruplicates. On day 4, after the cells were passaged, and 4x106 cells from each flask were reseeded into another T175, and continued with the same treatment for 3 more days. On day 7, the cells were trypsinized and counted, and 1.5-2 x106 cells from each flask were collected and frozen for lipid analysis.
Sample Type:	Breast cancer cells

Treatment:

Treatment ID:	TR003545
Treatment Summary:	Time-points (n = 4; 2x10^6 cells for pelleting and submission) - Time: 7 days - Treatment: DMSO; 1 nM Giredestrant (GDC-9545); 200nM Palbociclib; Giredestrant (GDC-9545) + Palbo Total samples: 32

Sample Preparation:

Sampleprep ID:	SP003543
Sampleprep Summary:	Cells were homogenized in dichloromethane (DCM):methanol (1:1, v:v). After centrifuging, homogenate containing the same amount of proteins was transferred into a v-bottom glass tube. 0.5 ml water, 0.45 ml DCM and 1.0 ml methanol were added to the supernatant to form a single phase. After 30 minutes, isotope labeled internal standards were added to the mixture, followed by 0.45 ml DCM and 0.5 ml water. The mixture was centrifuged at 1000x g for 20 minutes. Phase separation was achieved after centrifuge. The bottom layer was then collected into a clean glass tube, and the upper layer was re-extracted by adding 1.8 ml of DCM. The bottom layer was combined and dried under a gentle stream of nitrogen. The residue was reconstituted in 300 μl of DCM:Methanol (1:1), 10 mM ammonium acetate for LipidyzerTM Platform direct infusion analysis [ref:Zhijun Cao, Thomas C. Schmitt, Vijayalakshmi Varma et al. Evaluation of the Performance of Lipidyzer Platform and Its Application in the Lipidomics Analysis in Mouse Heart and Liver; J. Proteome Res. 2020, 19, 7, 2742–2749] on AB Sciex 6500+ LC-MS/MS. Flow rate is set at 7 μl/min. The injection volume is 50 μl. The autosampler temperature was kept at 15 °C. Buffer A and B are the same as the reconstitution buffer [DCM:Methanol (1:1), 10 mM ammonium acetate]. Lipids concentrations were calculated by the LipidyzerTM platform based on the known concentrations of spiked internal standards. Heatmap was generated using R.

Sampleprep ID:

SP003543

Sampleprep Summary:

Cells were homogenized in dichloromethane (DCM):methanol (1:1, v:v). After centrifuging, homogenate containing the same amount of proteins was transferred into a v-bottom glass tube. 0.5 ml water, 0.45 ml DCM and 1.0 ml methanol were added to the supernatant to form a single phase. After 30 minutes, isotope labeled internal standards were added to the mixture, followed by 0.45 ml DCM and 0.5 ml water. The mixture was centrifuged at 1000x g for 20 minutes. Phase separation was achieved after centrifuge. The bottom layer was then collected into a clean glass tube, and the upper layer was re-extracted by adding 1.8 ml of DCM. The bottom layer was combined and dried under a gentle stream of nitrogen. The residue was reconstituted in 300 μl of DCM:Methanol (1:1), 10 mM ammonium acetate for LipidyzerTM Platform direct infusion analysis [ref:Zhijun Cao, Thomas C. Schmitt, Vijayalakshmi Varma et al. Evaluation of the Performance of Lipidyzer Platform and Its Application in the Lipidomics Analysis in Mouse Heart and Liver; J. Proteome Res. 2020, 19, 7, 2742–2749] on AB Sciex 6500+ LC-MS/MS. Flow rate is set at 7 μl/min. The injection volume is 50 μl. The autosampler temperature was kept at 15 °C. Buffer A and B are the same as the reconstitution buffer [DCM:Methanol (1:1), 10 mM ammonium acetate]. Lipids concentrations were calculated by the LipidyzerTM platform based on the known concentrations of spiked internal standards. Heatmap was generated using R.

Combined analysis:

Analysis ID	AN005599	AN005600	AN005601
Analysis type	MS	MS	MS
Chromatography type	None (Direct infusion)	None (Direct infusion)	None (Direct infusion)
Chromatography system	Sciex QTRAP 6500+ with SelexION	Sciex QTRAP 6500+ with SelexION	Sciex QTRAP 6500+
Column	none	none	none
MS Type	ESI	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole	Triple quadrupole
MS instrument name	ABI Sciex 6500+ QTrap	ABI Sciex 6500+ QTrap	ABI Sciex 6500+ QTrap
Ion Mode	NEGATIVE	POSITIVE	POSITIVE
Units	nmol/250 ug protein	nmol/250 ug protein	nmol/250 ug protein

Chromatography:

Chromatography ID:	CH004255
Chromatography Summary:	DMS on with switching pos/neg polarity
Chromatography Comments:	Reference: Ubhi, B.K. (2018). Direct Infusion-Tandem Mass Spectrometry (DI-MS/MS) Analysis of Complex Lipids in Human Plasma and Serum Using the Lipidyzer™ Platform. In: Giera, M. (eds) Clinical Metabolomics. Methods in Molecular Biology, vol 1730. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7592-1_15
Instrument Name:	Sciex QTRAP 6500+ with SelexION
Column Name:	none
Column Temperature:	none
Flow Gradient:	none
Flow Rate:	none
Sample Injection:	50 ul
Solvent A:	none
Solvent B:	none
Chromatography Type:	None (Direct infusion)

Chromatography ID:	CH004256
Chromatography Summary:	DMS off with switching pos/neg polarity
Chromatography Comments:	Reference: Ubhi, B.K. (2018). Direct Infusion-Tandem Mass Spectrometry (DI-MS/MS) Analysis of Complex Lipids in Human Plasma and Serum Using the Lipidyzer™ Platform. In: Giera, M. (eds) Clinical Metabolomics. Methods in Molecular Biology, vol 1730. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7592-1_15
Instrument Name:	Sciex QTRAP 6500+
Column Name:	none
Column Temperature:	none
Flow Gradient:	none
Flow Rate:	none
Sample Injection:	50 ul
Solvent A:	none
Solvent B:	none
Chromatography Type:	None (Direct infusion)

MS:

MS ID:	MS005324
Analysis ID:	AN005599
Instrument Name:	ABI Sciex 6500+ QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	DI-MS/MS Analysis 1. A QTRAP® system with SelexION Technology (SCIEX) is used for targeted profiling (SCIEX, MA, USA). 2. This method is using a flow injection analysis (FIA): one injection with the SelexION voltages turned ON 3. The lipid molecular species are measured using multiple reaction monitoring (MRM) and positive/negative switching. The Negative ion mode detected the following lipid classes: LPE/LPC/PC/PE 4. A flow injection analysis (FIA) setup is employed by using the LC to flow at an isocratic rate of 7 μL/min with a ramp up to 30 μL/min for the last 2 min of the experiment to allow for washing. 5. Data acquisition is around 20 min per sample, and 50 μL of the reconstituted sample is infused and the area under the flat infusion line reported and corrected to the appropriate internal standard . 6. Samples are quantified using the LWM software which reports all the detected lipids Refence: Ubhi, B.K. (2018). Direct Infusion-Tandem Mass Spectrometry (DI-MS/MS) Analysis of Complex Lipids in Human Plasma and Serum Using the Lipidyzer™ Platform. In: Giera, M. (eds) Clinical Metabolomics. Methods in Molecular Biology, vol 1730. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7592-1_15
Ion Mode:	NEGATIVE

MS ID:	MS005325
Analysis ID:	AN005600
Instrument Name:	ABI Sciex 6500+ QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	DI-MS/MS Analysis 1. A QTRAP® system with SelexION Technology (SCIEX) is used for targeted profiling (SCIEX, MA, USA). 2. This method is using a flow injection analysis (FIA): one injection with the SelexION voltages turned ON 3. The lipid molecular species are measured using multiple reaction monitoring (MRM) and positive/negative switching. The Positive ion mode detected the following lipid classes: SM 4. A flow injection analysis (FIA) setup is employed by using the LC to flow at an isocratic rate of 7 μL/min with a ramp up to 30 μL/min for the last 2 min of the experiment to allow for washing. 5. Data acquisition is around 20 min per sample, and 50 μL of the reconstituted sample is infused and the area under the flat infusion line reported and corrected to the appropriate internal standard . 6. Samples are quantified using the LWM software which reports all the detected lipids. Refence: Ubhi, B.K. (2018). Direct Infusion-Tandem Mass Spectrometry (DI-MS/MS) Analysis of Complex Lipids in Human Plasma and Serum Using the Lipidyzer™ Platform. In: Giera, M. (eds) Clinical Metabolomics. Methods in Molecular Biology, vol 1730. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7592-1_15
Ion Mode:	POSITIVE

MS ID:	MS005326
Analysis ID:	AN005601
Instrument Name:	ABI Sciex 6500+ QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	DI-MS/MS Analysis 1. A QTRAP® system with SelexION Technology (SCIEX) is used for targeted profiling (SCIEX, MA, USA). 2. This second method is also using a flow injection analysis (FIA): a separate injection with the SelexION voltages turned OFF. 3. The lipid molecular species are measured using multiple reaction monitoring (MRM) and positive/negative switching. Positive ion mode detected the following lipid classes: DAG/CE/CER/LCER/HCER/TAG. Negative ion mode detected the following lipid classes: FFA (data not used in this study) 4. A flow injection analysis (FIA) setup is employed by using the LC to flow at an isocratic rate of 7 μL/min with a ramp up to 30 μL/min for the last 2 min of the experiment to allow for washing. 5. Data acquisition is around 20 min per sample, and 50 μL of the reconstituted sample is infused and the area under the flat infusion line reported and corrected to the appropriate internal standard . 6. Samples are quantified using the LWM software which reports all the detected lipids. Refence: Ubhi, B.K. (2018). Direct Infusion-Tandem Mass Spectrometry (DI-MS/MS) Analysis of Complex Lipids in Human Plasma and Serum Using the Lipidyzer™ Platform. In: Giera, M. (eds) Clinical Metabolomics. Methods in Molecular Biology, vol 1730. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7592-1_15
Ion Mode:	POSITIVE