For patients with sarcoma, metastatic disease remains very difficult to cure, and outcomes remain less than optimal. Treatment options have not largely changed, although some promising gains have been made with single agents in specific subtypes with the use of targeted agents. Here, we developed a system to investigate synergy of combinations of targeted and cytotoxic agents in a panel of sarcoma cell lines. Agents were investigated alone and in combination with varying dose ratios. Dose-response curves were analyzed for synergy using methods derived from Chou and Talalay (1984). A promising combination, dasatinib and triciribine, was explored in a murine model using the A673 cell line, and tumors were evaluated by MRI and histology for therapy effect. We found that histone deacetylase inhibitors were synergistic with etoposide, dasatinib, and Akt inhibitors across cell lines. Sorafenib and topotecan demonstrated a mixed response. Our systematic drug screening method allowed us to screen a large number of combinations of sarcoma agents. This method can be easily modified to accommodate other cell line models, and confirmatory assays, such as animal experiments, can provide excellent preclinical data to inform clinical trials for these rare malignancies. 1. Introduction Sarcomas account for 10% of pediatric diagnoses, 8% of cancers in the adolescent/young adult population, and 1% of adult cancers [1]. This diverse group of malignancies is often lethal in surgically unresectable, recurrent, or metastatic settings. Different subtypes predominate in different age groups, with rhabdomyosarcoma, osteosarcoma, and Ewing sarcoma predominating in children and young adults and leiomyosarcoma, liposarcoma, and other soft tissue sarcomas predominating in older adults. Chemotherapy has demonstrated clinical benefit in patients with advanced disease; however, for patients with advanced metastatic soft tissue sarcoma, the prognosis remains poor, with disease-free survival of 5 years or less than 25%; therefore, novel therapeutic strategies are needed. Targeted therapy has shown promise in subtypes of sarcoma, with c-Kit mutant gastrointestinal stromal tumor demonstrating the most clinical efficacy to date [2]. Signaling pathways have long been known to be active in sarcomas, with Src being the first discovered oncogene. The success of single-agent targeted therapy in gastrointestinal stromal tumors has not been reproduced in other sarcomas, although investigations regarding targeted therapies with clinical benefit, particularly in combination, continue. Single agents
References
[1]
D. Reed and S. Altiok, “Metastatic soft tissue sarcoma chemotherapy: an opportunity for personalized medicine,” Cancer Control, vol. 18, no. 3, pp. 188–195, 2011.
[2]
C. D. Blanke, C. Rankin, G. D. Demetri et al., “Phase III randomized, intergroup trial assessing imatinib mesylate at two dose levels in patients with unresectable or metastatic gastrointestinal stromal tumors expressing the kit receptor tyrosine kinase: S0033,” Journal of Clinical Oncology, vol. 26, no. 4, pp. 626–632, 2008.
[3]
M. Van Glabbeke, J. Verweij, I. Judson, and O. S. Nielsen, “Progression-free rate as the principal end-point for phase II trials in soft-tissue sarcomas,” European Journal of Cancer, vol. 38, no. 4, pp. 543–549, 2002.
[4]
S. Tariq Mahmood, S. Agresta, C. E. Vigil et al., “Phase II study of sunitinib malate, a multitargeted tyrosine kinase inhibitor in patients with relapsed or refractory soft tissue sarcomas. Focus on three prevalent histologies: Leiomyosarcoma, liposarcoma and malignant fibrous histiocytoma,” International Journal of Cancer, vol. 129, no. 8, pp. 1963–1969, 2011.
[5]
T.-C. Chou and P. Talalay, “Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors,” Advances in Enzyme Regulation, vol. 22, pp. 27–55, 1984.
[6]
D. Siegel, M. Hussein, C. Belani et al., “Vorinostat in solid and hematologic malignancies,” Journal of Hematology and Oncology, vol. 2, article 31, 2009.
[7]
M. Fouladi, J. R. Park, C. F. Stewart et al., “Pediatric phase I trial and pharmacokinetic study of vorinostat: a children's oncology group phase I consortium report,” Journal of Clinical Oncology, vol. 28, no. 22, pp. 3623–3629, 2010.
[8]
S. M. Wilhelm, L. Adnane, P. Newell, A. Villanueva, J. M. Llovet, and M. Lynch, “Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling,” Molecular Cancer Therapeutics, vol. 7, no. 10, pp. 3129–3140, 2008.
[9]
L. J. Lombardo, F. Y. Lee, P. Chen et al., “Discovery of N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)- piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays,” Journal of Medicinal Chemistry, vol. 47, no. 27, pp. 6658–6661, 2004.
[10]
O. Hantschel, U. Rix, and G. Superti-Furga, “Target spectrum of the BCR-ABL inhibitors imatinib, nilotinib and dasatinib,” Leukemia and Lymphoma, vol. 49, no. 4, pp. 615–619, 2008.
[11]
A. Olivieri and L. Manzione, “Dasatinib: a new step in molecular target therapy,” Annals of Oncology, vol. 18, no. 6, pp. vi42–vi46, 2007.
[12]
N. Berndt, H. Yang, B. Trinczek et al., “The Akt activation inhibitor TCN-P inhibits Akt phosphorylation by binding to the PH domain of Akt and blocking its recruitment to the plasma membrane,” Cell Death and Differentiation, vol. 17, no. 11, pp. 1795–1804, 2010.
[13]
C. R. Garrett, D. Coppola, R. M. Wenham et al., “Phase I pharmacokinetic and pharmacodynamic study of triciribine phosphate monohydrate, a small-molecule inhibitor of AKT phosphorylation, in adult subjects with solid tumors containing activated AKT,” Investigational new drugs, vol. 29, no. 6, pp. 1381–1389, 2011.
[14]
T. A. Yap, L. Yan, A. Patnaik et al., “First-in-man clinical trial of the oral pan-AKT inhibitor MK-206 in patients with advanced solid tumors,” Journal of Clinical Oncology, vol. 29, no. 35, pp. 4688–4695, 2011.
[15]
G. Neale, X. Su, C. L. Morton et al., “Molecular characterization of the pediatric preclinical testing panel,” Clinical Cancer Research, vol. 14, no. 14, pp. 4572–4583, 2008.
[16]
P. J. Houghton, C. L. Morton, C. Tucker et al., “The pediatric preclinical testing program: description of models and early testing results,” Pediatric Blood & Cancer, vol. 49, no. 7, pp. 928–940, 2007.
[17]
R. Gorlick, J. M. Maris, P. J. Houghton, et al., “Testing of the Akt/PKB inhibitor MK-2206 by the pediatric preclinical testing program,” Pediatric Blood & Cancer, vol. 59, no. 3, pp. 518–524, 2012.
[18]
S. T. Keir, J. M. Maris, R. Lock et al., “Initial testing (stage 1) of the multi-targeted kinase inhibitor sorafenib by the pediatric preclinical testing program,” Pediatric Blood & Cancer, vol. 55, no. 6, pp. 1126–1133, 2010.
[19]
H. Carol, P. J. Houghton, C. L. Morton et al., “Initial testing of topotecan by the pediatric preclinical testing program,” Pediatric Blood & Cancer, vol. 54, no. 5, pp. 707–715, 2010.
[20]
N. Keshelava, P. J. Houghton, C. L. Morton et al., “Initial testing (stage 1) of vorinostat (SAHA) by the pediatric preclinical testing program,” Pediatric Blood & Cancer, vol. 53, no. 3, pp. 505–508, 2009.
[21]
E. A. Kolb, R. Gorlick, P. J. Houghton et al., “Initial testing of dasatinib by the pediatric preclinical testing program,” Pediatric Blood & Cancer, vol. 50, no. 6, pp. 1198–1206, 2008.
[22]
P. J. Houghton, C. L. Morton, R. Gorlick et al., “Stage 2 combination testing of rapamycin with cytotoxic agents by the pediatric preclinical testing program,” Molecular Cancer Therapeutics, vol. 9, no. 1, pp. 101–112, 2010.
[23]
R. Aplenc and C. Ahern, “Study ADVL0516 Progress Report,” Study Committee Progress Report—Children's Oncology Group, August 2009.
[24]
M. Fouladi, W. L. Furman, T. Chin et al., “Phase I study of depsipeptide in pediatric patients with refractory solid tumors: a children's oncology group report,” Journal of Clinical Oncology, vol. 24, no. 22, pp. 3678–3685, 2006.
[25]
J. Knipstein and L. Gore, “Entinostat for treatment of solid tumors and hematologic malignancies,” Expert Opinion on Investigational Drugs, vol. 20, no. 10, pp. 1455–1467, 2011.
[26]
J. Cortes, P. Rousselot, D.-W. Kim et al., “Dasatinib induces complete hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in blast crisis,” Blood, vol. 109, no. 8, pp. 3207–3213, 2007.
[27]
R. G. Maki, J. K. Wathen, S. R. Patel et al., “Randomized phase II study of gemcitabine and docetaxel compared with gemcitabine alone in patients with metastatic soft tissue sarcomas,” Journal of Clinical Oncology, vol. 25, no. 19, pp. 2755–2763, 2007.
[28]
R. Aplenc, S. M. Blaney, L. C. Strauss et al., “Pediatric phase I trial and pharmacokinetic study of dasatinib: a report from the children's oncology group phase I consortium,” Journal of Clinical Oncology, vol. 29, no. 7, pp. 839–844, 2011.
[29]
E. Choy, F. Hornicek, L. Macconaill et al., “High-throughput genotyping in osteosarcoma identifies multiple mutations in phosphoinositide-3-kinase and other oncogenes,” Cancer, vol. 118, no. 11, pp. 2905–2914, 2011.
[30]
E. Hernando, E. Charytonowicz, M. E. Dudas et al., “The AKT-mTOR pathway plays a critical role in the development of leiomyosarcomas,” Nature Medicine, vol. 13, no. 6, pp. 748–753, 2007.
[31]
I. Vivanco and C. L. Sawyers, “The phosphatidylinositol 3-kinase-AKT pathway in human cancer,” Nature Reviews Cancer, vol. 2, no. 7, pp. 489–501, 2002.
[32]
Y. Tomita, T. Morooka, Y. Hoshida et al., “Reassessment of the 1993 Osaka grading system for localized soft tissue sarcoma in Japan,” Anticancer Research, vol. 26, no. 6C, pp. 4665–4669, 2006.
[33]
R. Nitschke, J. Parkhurst, J. Sullivan, M. B. Harris, M. Bernstein, and C. Pratt, “Topotecan in pediatric patients with recurrent and progressive solid tumors: a pediatric oncology group phase II study,” Journal of Pediatric Hematology/Oncology, vol. 20, no. 4, pp. 315–318, 1998.
[34]
N. Hijiya, C. F. Stewart, Y. Zhou et al., “Phase II study of topotecan in combination with dexamethasone, asparaginase, and vincristine in pediatric patients with acute lymphoblastic leukemia in first relapse,” Cancer, vol. 112, no. 9, pp. 1983–1991, 2008.
[35]
M. L. Metzger, C. F. Stewart, B. B. Freeman III et al., “Topotecan is active against Wilms' tumor: results of a multi-institutional phase II study,” Journal of Clinical Oncology, vol. 25, no. 21, pp. 3130–3136, 2007.
[36]
J. Shamash, T. Powles, K. Mutsvangwa et al., “A phase II study using a topoisomerase I-based approach in patients with multiply relapsed germ-cell tumours,” Annals of Oncology, vol. 18, no. 5, pp. 925–930, 2007.
[37]
N. C. Daw, V. M. Santana, L. C. Iacono et al., “Phase I and pharmacokinetic study of topotecan administered orally once daily for 5 days for 2 consecutive weeks to pediatric patients with refractory solid tumors,” Journal of Clinical Oncology, vol. 22, no. 5, pp. 829–837, 2004.
[38]
D. O. Walterhouse, E. R. Lyden, P. P. Breitfeld, S. J. Qualman, M. D. Wharam, and W. H. Meyer, “Efficacy of topotecan and cyclophosphamide given in a phase II window trial in children with newly diagnosed metastatic rhabdomyosarcoma: a children's oncology group study,” Journal of Clinical Oncology, vol. 22, no. 8, pp. 1398–1403, 2004.
[39]
A. Garaventa, R. Luksch, S. Biasotti et al., “A phase II study of topotecan with vincristine and doxorubicin in children with recurrent/refractory neuroblastoma,” Cancer, vol. 98, no. 11, pp. 2488–2494, 2003.
[40]
K. Kramer, B. H. Kushner, and N.-K. V. Cheung, “Oral topotecan for refractory and relapsed neuroblastoma: a retrospective analysis,” Journal of Pediatric Hematology/Oncology, vol. 25, no. 8, pp. 601–605, 2003.
[41]
W. L. Furman, C. F. Stewart, M. Kirstein et al., “Protracted intermittent schedule of topotecan in children with refractory acute leukemia: a Pediatric Oncology Group study,” Journal of Clinical Oncology, vol. 20, no. 6, pp. 1617–1624, 2002.
[42]
H. Rubie, B. Geoerger, D. Frappaz et al., “Phase I study of topotecan in combination with temozolomide (TOTEM) in relapsed or refractory paediatric solid tumours,” European Journal of Cancer, vol. 46, no. 15, pp. 2763–2770, 2010.
[43]
C. Rodriguez-Galindo, K. R. Crews, C. F. Stewart et al., “Phase I study of the combination of topotecan and irinotecan in children with refractory solid tumors,” Cancer Chemotherapy and Pharmacology, vol. 57, no. 1, pp. 15–24, 2006.
[44]
W. T. van der Graaf, J. Y. Blay, S. P. Chawla, et al., “Pazopanib for metastatic soft-tissue sarcoma (PALETTE): a randomised, double-blind, placebo-controlled phase 3 trial,” The Lancet, vol. 379, no. 9829, pp. 1879–1886, 2012.
[45]
S. Kumar, R. B. Mokhtari, R. Sheikh et al., “Metronomic oral topotecan with pazopanib is an active antiangiogenic regimen in mouse models of aggressive pediatric solid tumor,” Clinical Cancer Research, vol. 17, no. 17, pp. 5656–5667, 2011.
[46]
M. Talpaz, N. P. Shah, H. Kantarjian et al., “Dasatinib in imatinib-resistant Philadelphia chromosome-positive leukemias,” The New England Journal of Medicine, vol. 354, no. 24, pp. 2531–2541, 2006.
[47]
M. A. Smith and P. Houghton, “A proposal regarding reporting of in vitro testing results,” Clinical Cancer Research, vol. 19, no. 11, pp. 2828–2833, 2013.
[48]
M. Gerlinger, A. J. Rowan, S. Horswell et al., “Intratumor heterogeneity and branched evolution revealed by multiregion sequencing,” The New England Journal of Medicine, vol. 366, no. 10, pp. 883–892, 2012.
