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Overview of gene expression analysis. GSEA, gene signature enrichment analysis. Figure S2. DNA sequences of single nucleotide polymorphism at codon 762 of PARP1 gene in HT1080, SJSA-1, and SW684 cells. Figure S3. Distribution of trabectedin IC50 as single agent (TR alone) and in combination with veliparib (TR + VEL) or olaparib (TR + OL) among high-PARP1-expressing cells (red triangle) and low-PARP1-expressing cells (blue triangle). Figure S4. Dose- response curve obtained after 72-h treatment with trabectedin (2–0.125nM), olaparib (20–1.25 μM) as single agents and in constant combination. Figure S5. A, western blot analysis of PARylation and PARP1 expression in MES-SA and MES-SA-DX5 leiomyosarcoma cells; B, FISH analysis of PARP1 gene (red) and centromere of chromosome 1 (green) in MESSA and MESSA-DX5. Figure S6. Genomic status as obtained by aCGH analysis of TC-106, 402.91, DMR, SJSA-1, HT1080, SW684: gain (red) and loss (green) of chromosome regions. Figure S7. A, Western blot analysis of PARylation and PARP1 expression and B, quantitation of PAR in MSTO-H211, and PARP1-silenced MSTO-H211 untreated or treated with 10nM trabectedin, 20 μM cisplatin (Sandoz), 20 μM gemcitabine (Sandoz), 20 μM doxorubicin (Pfizer), 20 μM dacarbazine (Medac), 20 μM etoposide (Teva), 50 mM actinomycin-D (Thermo Fisher Scientific), β-actin was done as loading control. (DOCX 5982 kb)

Overview of gene expression analysis. GSEA, gene signature enrichment analysis. Figure S2. DNA sequences of single nucleotide polymorphism at codon 762 of PARP1 gene in HT1080, SJSA-1, and SW684 cells. Figure S3. Distribution of trabectedin IC50 as single agent (TR alone) and in combination with veliparib (TR + VEL) or olaparib (TR + OL) among high-PARP1-expressing cells (red triangle) and low-PARP1-expressing cells (blue triangle). Figure S4. Dose- response curve obtained after 72-h treatment with trabectedin (2–0.125nM), olaparib (20–1.25 μM) as single agents and in constant combination. Figure S5. A, western blot analysis of PARylation and PARP1 expression in MES-SA and MES-SA-DX5 leiomyosarcoma cells; B, FISH analysis of PARP1 gene (red) and centromere of chromosome 1 (green) in MESSA and MESSA-DX5. Figure S6. Genomic status as obtained by aCGH analysis of TC-106, 402.91, DMR, SJSA-1, HT1080, SW684: gain (red) and loss (green) of chromosome regions. Figure S7. A, Western blot analysis of PARylation and PARP1 expression and B, quantitation of PAR in MSTO-H211, and PARP1-silenced MSTO-H211 untreated or treated with 10nM trabectedin, 20 μM cisplatin (Sandoz), 20 μM gemcitabine (Sandoz), 20 μM doxorubicin (Pfizer), 20 μM dacarbazine (Medac), 20 μM etoposide (Teva), 50 mM actinomycin-D (Thermo Fisher Scientific), β-actin was done as loading control. (DOCX 5982 kb)

Analysis of differential genomic aberrations in HS-C and LS-C cells. (PDF 255 kb)

Concentrations inhibiting 50% of cell viability (IC50) after 72- h treatment with serial dilutions of trabectedin (2–0.125 nM), olaparib (20–1.25 μM), and veliparib (80–5 μM) as single agents (a) or in constant combination (b, c), and their 95% confidence intervals (95% CI). Population doubling time. Combination index ± estimated standard deviation (est st dev) calculated at IC50 by the Chou-Talalay method for trabectedin and olaparib in combination and trabectedin and veliparib in combination in cell lines of different histotypes: leiomyosarcoma (LMS), undifferentiated pleomorphic sarcoma (UPS), myxoid liposarcoma (LMS), dedifferentiated liposarcoma (DLS), fibrosarcoma (FSA), synovial sarcoma (SS), Ewing’s sarcoma (ES), and osteosarcoma (OS). Supplier and culture conditions are reported for each cell line. Table S2. Direct correlation between combination index (calculated at IC50) and PARP1 protein intensity expressed as Pearson score (r Pearson). IC50 and 95% confidence intervals (95% CI) were calculated after 72-h treatment with serial dilutions of trabectedin (2–0.125 nM), olaparib (20–1.25μM), and their constant combination. Cell line characteristics, population doubling time, purchasers and culture conditions were included. Table S3. Gene expression (ΔCT) of DNA-damage response and repair key components and drug synergism expressed by combination index (CI). The correlation between each gene expression and the CI was evaluated by Pearson score (r); t distribution and their relative P value were shown. Yellow cells highlight significant direct correlation. Table S4. PARP1 gene (chromosome 1 q42.12d) copy number obtained by FISH. Table S5. PARP1, BRCA1, RAD51 gene copy number obtained by real- time PCR on genomic DNA. The gene copy number of PARP1, RAD51, and BRCA1 did not correlate with the Combination index (CI) as shown by Pearson score. Table S6. Genomic status of selected genes analyzed by MLPA and DHPLC /Sequencing. Red cells indicate increased copy number, while blue cells indicate reduced copy number as obtained by DHPLC analysis. Table S7. Immunohistochemistry score of intensity for PARP1, BRCA1, and RAD51 protein expression in formalin-fixed paraffin-embedded sarcoma samples. Table S8. 2 × 2 contingency tables of immunohistochemistry (IHC) expression of PARP1, BRCA1, and RAD51 in patient-derived soft tissue and bone sarcoma specimens (a, b, c) and related concordance rates (d). (DOCX 2086 kb)

Concentrations inhibiting 50% of cell viability (IC50) after 72- h treatment with serial dilutions of trabectedin (2–0.125 nM), olaparib (20–1.25 μM), and veliparib (80–5 μM) as single agents (a) or in constant combination (b, c), and their 95% confidence intervals (95% CI). Population doubling time. Combination index ± estimated standard deviation (est st dev) calculated at IC50 by the Chou-Talalay method for trabectedin and olaparib in combination and trabectedin and veliparib in combination in cell lines of different histotypes: leiomyosarcoma (LMS), undifferentiated pleomorphic sarcoma (UPS), myxoid liposarcoma (LMS), dedifferentiated liposarcoma (DLS), fibrosarcoma (FSA), synovial sarcoma (SS), Ewing’s sarcoma (ES), and osteosarcoma (OS). Supplier and culture conditions are reported for each cell line. Table S2. Direct correlation between combination index (calculated at IC50) and PARP1 protein intensity expressed as Pearson score (r Pearson). IC50 and 95% confidence intervals (95% CI) were calculated after 72-h treatment with serial dilutions of trabectedin (2–0.125 nM), olaparib (20–1.25μM), and their constant combination. Cell line characteristics, population doubling time, purchasers and culture conditions were included. Table S3. Gene expression (ΔCT) of DNA-damage response and repair key components and drug synergism expressed by combination index (CI). The correlation between each gene expression and the CI was evaluated by Pearson score (r); t distribution and their relative P value were shown. Yellow cells highlight significant direct correlation. Table S4. PARP1 gene (chromosome 1 q42.12d) copy number obtained by FISH. Table S5. PARP1, BRCA1, RAD51 gene copy number obtained by real- time PCR on genomic DNA. The gene copy number of PARP1, RAD51, and BRCA1 did not correlate with the Combination index (CI) as shown by Pearson score. Table S6. Genomic status of selected genes analyzed by MLPA and DHPLC /Sequencing. Red cells indicate increased copy number, while blue cells indicate reduced copy number as obtained by DHPLC analysis. Table S7. Immunohistochemistry score of intensity for PARP1, BRCA1, and RAD51 protein expression in formalin-fixed paraffin-embedded sarcoma samples. Table S8. 2 × 2 contingency tables of immunohistochemistry (IHC) expression of PARP1, BRCA1, and RAD51 in patient-derived soft tissue and bone sarcoma specimens (a, b, c) and related concordance rates (d). (DOCX 2086 kb)