GYLATL1 promotes endocrine therapy resistance in luminal breast cancer and is regulated by estrogen-independent ERα signaling

Supplementary Information for study: GYLATL1 promotes endocrine therapy resistance in luminal breast cancer and is regulated by estrogen-independent ERα signaling Janina Müller1,2, Emre Sofyali1,2, Luisa Schwarzmüller1,2, Yael Aylon3, Eviatar Weizman4, Lisa Schlicker5, Katherine Kelly2,6, Simone Borgoni1,2, Simin Oz6, Birgitta E Michels1, Sara Burmester1, Angelika Wörner1, Sabine Karolus1, Daniela Heiss7, Rainer Will7, Veronica Rodrigues de Melo Costa1, Cole Stocker1,2, Pavlo Lutsik6, Dieter Weichenhan6, Ilse Hofmann8, Nishanth Belugali Nataraj9, Yosef Yarden9, Luca Magnani10, Christoph Plass6, Almut Schulze5, Cindy Körner1, Moshe Oren3, Stefan Wiemann1 Affiliations 1 Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany 2 Faculty of Biosciences, University of Heidelberg, Im Neuenheimer Feld 234, 69120 Heidelberg, Germany 3 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel 4 G-INCPM, Weizmann Institute of Science, Rehovot 76100, Israel 5 Division of Tumor Metabolism and Microenvironment, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg, Germany 6 Division of Cancer Epigenomics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany 7 Cellular Tools Core Facility, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany 8 Antibody Core Facility, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany 9 Department of Immunology and Regenerative Biology, Weizmann Institute of Science, Rehovot 76100, Israel 10 Department of Oncology and Hematology-Oncology, Università degli Studi di Milano, Via Festa del Perdono 7, 20122 Milan, Italy Experimental data Epigenetic-focused cytometry time-of-flight (EpiTOF) [1, 2] Experiment carried out by Janina Müller, in the lab of Yael Aylon and Moshe Oren. Data analyzed by Eviatar Weizmann and Luisa Schwarzmüller. MCF7 (RRID:CVCL_0031) single-cell suspensions were generated and washed with Maxpar PBS (#201058, Fluidigm, San Francisco, CA. USA) and subsequently stained with 1.25 µM Cisplatin for one minute to identify dead cells. The staining reaction was quenched using DMEM supplemented with 10% FBS. Cells were washed with Maxpar Cell Staining Buffer (#201068, Fluidigm, San Francisco, CA, USA), and about 3 × 106 cells per sample were fixed, permeabilized using the Maxpar Nuclear Antigen Staining Buffer Set (#201063, Fluidigm, San Francisco, CA, USA), and barcoded with the Cell-ID 20-Plex Pd Barcoding Kit (#201060, Fluidigm, San Francisco, CA, USA) according to the manufacturer's instructions. Following two washing steps with Maxpar Nuclear Antigen Staining Buffer Set permeabilization buffer, equal amounts of barcoded samples of the different MCF7 cell lines (i.e., parental, LTED, KO1, and KO2) were combined and incubated with an antibody cocktail (Supplementary Table 11) for 30 minutes at room temperature. Following antibody incubation, the cells were washed twice with Maxpar Cell Staining Buffer and fixed at 4 °C with fresh 4% formaldehyde (#28908, Thermo Fisher Scientific, Waltham, MA, USA), ensuring gentle rocking to prevent clumping. Following overnight incubation, 125 nM Cell-ID Intercalator-Ir (#201192A, Fluidigm, San Francisco, CA, USA) was added and incubated for 45 minutes at room temperature to label DNA. Afterward, cells were washed twice with Maxpar Cell Staining Buffer and once with Maxpar Water (#201069, Fluidigm, San Francisco, CA, USA). Cells were resuspended in a dilution of EQ Four Element Calibration Beads (#28908, Fluidigm, San Francisco, CA, USA) in Maxpar Water, achieving a concentration of approximately 250K cells/ml, and subsequently filtered through a 35 μm mesh. Data was acquired on a CyTOF Helios platform (Fluidigm, San Francisco, CA, USA). Normalization and data cleanup to isolate live single cells were conducted as described by Bagwell et al. [3]. Metal-conjugated antibodies were either procured from Fluidigm or conjugated utilizing appropriate Maxpar X8 Antibody Labeling Kits (Fluidigm, San Francisco, CA, USA). The mass cytometry antibody panel was designed to minimize signal spillover utilizing the Maxpar Panel Designer (Fluidigm, San Francisco, CA, USA). Analysis of EpiTOF data was executed through an R-based pipeline [4]. Data were imported into R (version 4.0.2) and transformed using arcsine transformation with a cofactor of 5 and regressed to H3 and H3.3 expression. For statistical comparison, the median arcsine-transformed intensities were used (n = 2). Using a two-sided, unpaired T-test, LTED was compared against the parental cells and cells from both GLYATL1 KO clones simultaneously. The tables contain raw data (signal intensities for every identified cell and for every tested mark) from two replicate experiments/batches. WT: parental MCF7 cell line (ATCC); LTED: long-time estrogen deprived MCF7 cell line (proxy for resistance to aromatase inhibition) [5]; KO1 and KO2: LTED cell line with CRISPR/Cas9 knockout of GLYATL1 gene, clones LTED GLYATL1 KO1 and LTED GLYATL1 KO2 [this study]. References: 1. Aylon Y, Furth N, Mallel G, Friedlander G, Nataraj NB, Dong M, Hassin O, Zoabi R, Cohen B, Drendel V, Salame TM, Mukherjee S, Harpaz N, Johnson R, Aulitzky WE, Yarden Y, Shema E & Oren M (2022) Breast cancer plasticity is restricted by a LATS1-NCOR1 repressive axis. Nat Commun 13, 7199, doi: 10.1038/s41467-022-34863-9. 2. Harpaz N, Mittelman T, Beresh O, Griess O, Furth N, Salame TM, Oren R, Fellus-Alyagor L, Harmelin A, Alexandrescu S, Marques JG, Filbin MG, Ron G & Shema E (2022) Single-cell epigenetic analysis reveals principles of chromatin states in H3.3-K27M gliomas. Mol Cell 82, 2696-2713 e2699, doi: 10.1016/j.molcel.2022.05.023. 3. Bagwell CB, Inokuma M, Hunsberger B, Herbert D, Bray C, Hill B, Stelzer G, Li S, Kollipara A, Ornatsky O & Baranov V (2020) Automated Data Cleanup for Mass Cytometry. Cytometry A 97, 184-198, doi: 10.1002/cyto.a.23926. 4. Nowicka M, Krieg C, Crowell HL, Weber LM, Hartmann FJ, Guglietta S, Becher B, Levesque MP & Robinson MD (2017) CyTOF workflow: differential discovery in high-throughput high-dimensional cytometry datasets. F1000Res 6, 748, doi: 10.12688/f1000research.11622.3. 5. Nguyen VT, Barozzi I, Faronato M, Lombardo Y, Steel JH, Patel N, Darbre P, Castellano L, Gyorffy B, Woodley L, Meira A, Patten DK, Vircillo V, Periyasamy M, Ali S, Frige G, Minucci S, Coombes RC & Magnani L (2015) Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion. Nat Commun 6, 10044, doi: 10.1038/ncomms10044. List of antibodies used in the experiment: Name target metal isotope provider cat# Histone H3 Antibody (D1H2) - 115In H3 In 115 IonPath 711501 Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade H3.3 Gd 155 Abcam ab208690 Histone H4 (D2X4V) Rabbit mAb #13919 H4 Tb 159 Cell Signaling 13919 Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb H3K4me3 Nd 145 Cell Signaling 9751 Di-Methyl-Histone H3 (Lys36) (C75H12) Rabbit mAb H3K36me2 Sm 149 Cell Signaling 2901 Acetyl-Histone H4 (Lys16) (E2B8W) Rabbit mAb #13534 H4K16AC Sm 152 Cell Signaling 13534 Mono-Methyl-Histone H3 (Lys4) (D1A9) XP® Rabbit mAb (BSA and Azide Free) H3K4me1 Sm 154 Cell Signaling 53138 Anti-Histone H3 (acetyl K64) antibody [EPR20713] - BSA and Azide free H3K64ac Gd 156 Abcam ab251549 Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb H3K27ac Gd 160 Cell Signaling 8173P Histone H3K27me3 antibody (mAb) - 168Er H3K27me3 Er 168 Active Motif 61017 Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb #13969 H3K9me3 Er 170 Cell Signaling 13969 Anti-Histone H3 (di methyl K27) antibody - ChIP Grade H3K27me2 ND 142 Abcam ab24684 Anti-Histone H3 (di methyl K9) antibody [Y49] - BSA and Azide free (ab173325) H3K9me2 Eu 151 Abcam ab173325 Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (BSA and Azide Free) #45772 H2Aub Eu 153 Cell Signaling 45772 Anti-Histone H4 (tri methyl K20) antibody [EPR17001(2)] - BSA and Azide free H4K20me3 Dy 161 Abcam ab239410 Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb #4909 H3K36me3 Ho 165 Cell Signaling 4909 Histone H3K9ac antibody (mAb) H3K9ac Tm 169 Active Motif 61663 Anti-Human CD326/EpCAM (9C4)-141Pr EpCAM Pr 141 FLUIDIGM 3141006B Anti-Human CD24 (ML5)-166Er CD24 Er 166 FLUIDIGM 3166007B Anti-Human/Mouse CD49F (GoH3)-164Dy CD49f Dy 164 FLUIDIGM 3164006B Anti-Human/Mouse CD44 (IM7)-171Yb CD44 Yb 171 FLUIDIGM 3171003 Anti-Cytokeratin 5 antibody [SP27] - BSA and Azide free K5 Ce 140 Abcam ab236216 Anti-Estrogen Receptor alpha antibody [E115] - Low endotoxin, Azide free ERa Dy 163 Abcam ab167611 (3174014A) Anti-Human Keratin 8/18 (C51)-174Yb K8/18 Yb 174 FLUIDIGM 3174014A Anti-pHistone H3 [S28] (HTA28)-175Lu pH3 Lu 175 FLUIDIGM 3175012A (3162012B) Anti-Ki-67 (B56)-162Dy Ki-67 Yb 172 FLUIDIGM 3162012B