Additional file 2 of The DNA repair pathway as a therapeutic target to synergize with trastuzumab deruxtecan in HER2-targeted antibody–drug conjugate–resistant HER2-overexpressing breast cancer
Creators: name: Lee, Jangsoon
name: Kida, Kumiko
name: Koh, Jiwon
name: Liu, Huey
name: Manyam, Ganiraju C.
name: Gi, Young Jin
name: Rampa, Dileep R.
name: Multani, Asha S.
name: Wang, Jing
name: Jayachandran, Gitanjali
name: Lee, Dae-Won
name: Reuben, James M.
name: Sahin, Aysegul
name: Huo, Lei
name: Tripathy, Debu
name: Im, Seock-Ah
name: Ueno, Naoto T.
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Supplementary Material 2: Supplementary Figure S1. Antiproliferation effect of T-DM1 and T-DXd in HER2-positive BC cell lines. A. HER2 gene copy variation in HER2+ BC cell lines. The TNBC cell line MDA-MB-231 was used as a negative control. Genomic DNA was used for the ddPCR assay. Each box shows mean with standard deviation. Data were collected from three biological replicates. B. T-DM1 and T-DXd inhibited the proliferation of HER2+ cell lines in a dose-dependent manner. Cells were treated with T-DM1 or T-DXd for 5 days, and viability was measured using SRB staining. The data shown are representative of 3 independent experiments with similar results. C. T-DM1 and T-DXd significantly reduced tumor volume in HER2+ BC cell xenograft models. SUM190 or HCC1954 cells were injected into the mammary fat pad of nude mice, and the treatment was started when tumors averaged 200 mm3. HER2-ADC (10 mg/kg) was administered one time on Day 0, and tumor size was monitored. An IHC assay was used to check the expression levels of HER2 and proliferation maker Ki-67 in tumor samples. The data shown represent three IHC staining experiments from each treatment group with similar results. 20× magnification. Scale bars, 200 μm. In vivo tumorigenicity data were compared using an analysis of the variance model. ** P &lt; 0.01, *** P&lt; 0.001.   Supplementary Figure S2. A. HER2-ADC cell lines did not show an increase in genomic instability. Thirty-five metaphases/anaphases were analyzed per cell line. B. Deletion of the amplified ERBB2 region was observed on chromosome 17 in the SUM190-TDXdR cell line. Karyotyping assay. C. The intrinsic T-DXd-resistant KPL4 cell line (KPL4-TDXdR) did not show reduced ERBB2, MIEN1, MIR4728, and PGAP3 gene copy numbers. Whole-genome sequencing analysis. Supplementary Figure S3. A. Overexpression of HER2 did not increase the antiproliferation effect of T-DXd in T-DXd resistant HER2+ BC cell lines. SUM190-TDXdR and HCC1954-TDXdR cells transfected with pcDNA3-HER2 plasmid (Addgene, Watertown, MA, USA) using the Neon transfection kit (ThermoFisher) and underwent an SRB proliferation assay with T-DXd. The remaining cells were used for immunoblotting to check overexpression of HER2. B. Cells were transfected with validated siRNA targeting EGR1 or SLC6A14 (Silencer select Hm genome siRNA library v4, ThermoFisher) using the Neon transfection kit and incubated for 48 hr; they then underwent an SRB proliferation assay with T-DXd. Supplementary Figure S2. A. HER2-ADC cell lines did not show an increase in genomic instability. Thirty-five metaphases/anaphases were analyzed per cell line. B. Deletion of the amplified ERBB2 region was observed on chromosome 17 in the SUM190-TDXdR cell line. Karyotyping assay. C. The intrinsic T-DXd-resistant KPL4 cell line (KPL4-TDXdR) did not show reduced ERBB2, MIEN1, MIR4728, and PGAP3 gene copy numbers. Whole-genome sequencing analysis. Supplementary Figure S3. A. Overexpression of HER2 did not increase the antiproliferation effect of T-DXd in T-DXd resistant HER2+ BC cell lines. SUM190-TDXdR and HCC1954-TDXdR cells transfected with pcDNA3-HER2 plasmid (Addgene, Watertown, MA, USA) using the Neon transfection kit (ThermoFisher) and underwent an SRB proliferation assay with T-DXd. The remaining cells were used for immunoblotting to check overexpression of HER2. B. Cells were transfected with validated siRNA targeting EGR1 or SLC6A14 (Silencer select Hm genome siRNA library v4, ThermoFisher) using the Neon transfection kit and incubated for 48 hr; they then underwent an SRB proliferation assay with T-DXd. Supplementary Figure S4. A. An Affymetrix Clariom D Human Transcriptome array data analysis identified targetable canonical pathways. TAC software was used to analyze and visualize global expression patterns of genes and pathways. The cut-off range was two-fold expression change (up and down) and P &lt; 0.001. Significance was calculated using a 2x2 contingency in a Fisher’s exact test (two-sided). After the P value was established using Fisher’s exact test, it was converted to -log10. B-E. DNA repair pathway network analysis of microarray data from HER2-ADC-resistant cell lines. The cut-off range is the two-fold expression change (up and down) and P &lt; 0.001. Significance was calculated using a 2x2 contingency in a Fisher’s Exact Test (two-sided). SUM190-TDM1R (B), HCC1954-TDM1R (C), SUM190-TDXdR (D), and HCC-1954TDXdR (E). Data were collected from three biological replicates. E-G. DNA repair pathway related proteins were elevated in HER2-ADC-resistant cell lines compared to is of microarray data from HER2-ADC-resistant cell lines. ATR, pATR, Chk1, Chk2, ATM, Rad50, and Rad51 were elevated in HER2-ADC-resistant cell lines. Reverse-phase protein array data (F). Hierarchical Clustering of reverse-phase protein array data using Morpheus software (https://software.broadinstitute.org/morpheus) Median expression values used for analysis (G). STRING interactome analysis of ATR, pATR, Chk1, Chk2, ATM, Rad50, and Rad51 (H). Supplementary Figure S5. DNA repair pathway–targeting drug enhances the efficacy of T-DXd in HER2-ADC-resistant HER2+ BC cell lines. A. Knockdown of ATR significantly reduces viability of HCC1954-TDXdR cell line. Two ATR RNAi were individually transfected, and SRB proliferation assay conducted for 7 days. B. ATR RNAi significantly enhances the efficacy of T-DXd in HER2+ BC. Cell lysates were collected at 72 hr after transfection for Western blotting analysis. The data shown are representative of three independent experiments with similar results. C and D. Clonogenic assay. Cells were treated with T-DXd and/or selected kinase inhibitor for 14 days, and cell viability was measured by SRB staining. SUM190-TDM1R and SUM190-TDXdR cell lines (C). HCC1954-TDM1R and HCC1954-TDXdR cell lines (D). Data are presented as mean ± standard deviation. Two-tailed unpaired Student’s t-test; *, P &lt; 0.05, **, P &lt; 0.01, ***, P &lt; 0.001, ****, P &lt; 0.0001, n.s. not significant. Experiments were repeated in triplicate. The data shown are representative of three independent experiments with similar results. E - H. Bliss independence dose-response assay. Cells were treated with T-DXd and selected inhibitors for 7 days, and viability was measured using SRB staining. The cell images were captured using GelCounter , the table indicates viability, and the Bliss synergy score was evaluated and visualized using the Synergyfinderplus software (right, www.synergyfinderplus.org). The color indicates a synergist (red) or antagonist (green) effect in two-drug combinations. Supplementary Figure S6. A. T-DXd and elimusertib increased DNA damage stress and apoptosis. Western blotting assay. Cells were treated with T-DXd (1 µg/ml) and/or elimusertib (100 nM) for 48 h, and whole-cell lysates were collected for immunoblotting. Protein expression was normalized with actin level in control cells from each TDM1R and TDXdR cell line using ImageJ software. B. T-DM1 or T-DXd treatment did not induce the HER2 downstream molecules, pAKT and pMAPK. Reverse-phase protein array data. Cells  C and D. Western blotting. Basal levels of HER, pHER2, pAKT, and pERK in parent and HER2-ADC-resistant cell lines (C). T-DXd treatment does not induces pAKT and pERK expression (D). The ImageJ program was used to measure intensity. Supplementary Figure S7. T-DXd and elimusertib did not show toxicity in xenograft models. T-DXd (10 mg/kg) was administered one time on Day 0 via tail-vein injection. Elimusertib (10 mg/kg) was administered via oral gavage twice a day (6-hr interval) for 3 consecutively days per week. Data are presented as mean ± standard deviation. Supplementary Figure S8. Reduced HER2 expression level is retained in HER2-ADC-resistant cell lines without T-DM1 or T-DMd treatment. To match xenograft assay conditions, HER2-ADC-resistant cell lines were maintained without the drug for 2 months. A. Western blotting. TDM1R and TDXdR cell lines retained reduced HER2 expression compared to the parent cell line. The ImageJ program was used for measuring intensity. B. FACS analysis. TDM1R and TDXdR cell lines showed reduced cell-surface HER2 expression. C. The sections (5-μm thick) were used for IHC staining, as described in the Methods section. The slides were then incubated with anti-HER2. Immunostained slides were scanned using an Aperio AT2 slide scanner and captured at 20× magnification using Aperio ImageScope software (Leica Biosystems). Scale bars = 200 µm. Supplementary Figure S9. Genes mutation profiling of the DNA repair pathway in HER2-ADC-resistant cell lines. Whole-genome sequencing data.