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Additional file 1: Figure S1. Characterisation of LMS cell lines: A. Heat map of copy number variations between STS39 tumor, STS39 cell lines passages 4, 9, 14, 24, 34, SKLMS1 and HUVEC (Human umbilical vein endothelial cells) as a control, showing genomic stability of cell lines over time. The scale represents the percentage of genetic differences, where white represents minimal to no genetic change and dark purple represents maximum genetic change. For example, the genetic difference between HUVEC and STS39 is 23%. B. Immunocytochemistry of both cell lines using DAPI, Desmin, Smooth Muscle Actin (SMA) and mouse IgG (msIgG) as an isotype control. SKLMS1 demonstrates focally positive SMA staining, while STS39 shows focal positivity for Desmin and SMA (n=3). C. Immunoblot analysis showing protein stability of PI3K pathway proteins with serial passaging of STS39 and SKLMS1 cells. Increased phosphorylation of p85, a subunit of the PI3K receptor, was seen in SKLMS1 cells. In addition, elevated levels of RICTOR, a binding partner of mTOR, were observed. Both of these modifications can potentially lead to increased pathway activation. HeLa and Jurkat cell lines were used as controls for protein expression. siRNA against 4EBP1 was used to create a negative control for 4EBP1 and p-4EBP1 antibodies. Figure S2. Treatment with BEZ235 and/or Dox induces cell death via apoptosis. A. Cells were treated with BEZ235 (500nM), Dox (500nM) and BEZ/Dox for 72h and then analysed for apoptosis by flow cytometry for Annexin V and 7-ADD staining. Combination of BEZ235 and Dox significantly induced apoptosis in SKLMS1 and STS-39 cells (data not shown). B. Quantification of apoptotic cells at 72h post-treatment of SKLMS1 cells (n=3) and STS39 cells (n=3). Table S1. Sequencing primers (5â ˛-3â ˛) used to determine the presence of mutations in mTOR and in exon 9 and 20 of the kinase domain of PI3K. Table S2. Combination Index (CI) tables with BEZ235, BKM120 and/or Dox at 3 dosing schedules. Viability was determined using an ATPlite assay and analysed using CalcuSyn software. Treatment with BEZ235 (15-240nM) and Dox (125-2000nM) showed synergy in all 3 schedules (CI

11560 Background: Leiomyosarcomas (LMS) are genetically heterogeneous tumors that arise from smooth muscle. Currently, the mainstay of systemic treatment for patients with advanced/metastatic disease is doxorubicin (Dox) based CTx. Several genomic analyses of LMS reveal defects in homologous recombination (HR) DNA repair pathway in about half of patients, consistent with a druggable “BRCAness” phenotype. Thus, we sought to determine which combinations of standard CTx and PARPi might be synergistic promising therapeutic strategies for LMS. Methods: Dox, Docetaxel (Doc), Temozolomide (Tmz) were evaluated in combination with PARPi (Olaparib [Ola], Niraparib [Nira] and Talazoparib [Tala]) at 12 different drug concentrations. Four LMS cell lines of different origins (gynecological - GY, abdominal - A, extremity - E) were tested in a high throughput manner. All drug concentrations were chosen according to EC50. Cells were incubated with each combination for 7 days. Viability was assessed by ATPlite Luminescence Assay System.Evaluation of drug combination effect was performed using a Bliss synergy score. This system quantifies the degree of synergy as multiplicative effect of single drugs as if they acted independently. With a synergy score of -5 to 5, the interaction between two drugs is considered as additive; 5 synergistic, and therefore a promising combination. Results: Anticancer activity, ranging from additive to synergistic was seen with all combinations. Results were consistent among all cell lines, independent of site of cell line origin (Table) Most synergistic combination in the majority of LMS cell lines were Dox or Tmz when combined with Tala, reaching up to 15 % and 27% above Bliss respectively. In contrast, Doc showed only additive effect with all analyzed PARPi. Conclusions: The data suggest that the combination of Dox or Tmz with PARPi may represent promising treatment options for LMS patients. Recent clinical studies support this notion in uterine LMS. Importantly these results suggest that such approach may be extended to all sites of LMS. Pre-clinical studies are underway to identify the most promising combinations for future clinical trial design. [Table: see text]

Additional file 1: Figure S1. Characterisation of LMS cell lines: A. Heat map of copy number variations between STS39 tumor, STS39 cell lines passages 4, 9, 14, 24, 34, SKLMS1 and HUVEC (Human umbilical vein endothelial cells) as a control, showing genomic stability of cell lines over time. The scale represents the percentage of genetic differences, where white represents minimal to no genetic change and dark purple represents maximum genetic change. For example, the genetic difference between HUVEC and STS39 is 23%. B. Immunocytochemistry of both cell lines using DAPI, Desmin, Smooth Muscle Actin (SMA) and mouse IgG (msIgG) as an isotype control. SKLMS1 demonstrates focally positive SMA staining, while STS39 shows focal positivity for Desmin and SMA (n=3). C. Immunoblot analysis showing protein stability of PI3K pathway proteins with serial passaging of STS39 and SKLMS1 cells. Increased phosphorylation of p85, a subunit of the PI3K receptor, was seen in SKLMS1 cells. In addition, elevated levels of RICTOR, a binding partner of mTOR, were observed. Both of these modifications can potentially lead to increased pathway activation. HeLa and Jurkat cell lines were used as controls for protein expression. siRNA against 4EBP1 was used to create a negative control for 4EBP1 and p-4EBP1 antibodies. Figure S2. Treatment with BEZ235 and/or Dox induces cell death via apoptosis. A. Cells were treated with BEZ235 (500nM), Dox (500nM) and BEZ/Dox for 72h and then analysed for apoptosis by flow cytometry for Annexin V and 7-ADD staining. Combination of BEZ235 and Dox significantly induced apoptosis in SKLMS1 and STS-39 cells (data not shown). B. Quantification of apoptotic cells at 72h post-treatment of SKLMS1 cells (n=3) and STS39 cells (n=3). Table S1. Sequencing primers (5â ˛-3â ˛) used to determine the presence of mutations in mTOR and in exon 9 and 20 of the kinase domain of PI3K. Table S2. Combination Index (CI) tables with BEZ235, BKM120 and/or Dox at 3 dosing schedules. Viability was determined using an ATPlite assay and analysed using CalcuSyn software. Treatment with BEZ235 (15-240nM) and Dox (125-2000nM) showed synergy in all 3 schedules (CI