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Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem–like cells (sCSC) that are likely responsible for relapses and drug resistance. In this study, we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS, including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs, autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4, a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients, we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas, including putative sCSCs, supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease. Cancer Res; 74(1); 119–29. ©2013 AACR.

Purpose of study. An attractive strategy to improve antitumor treatments is to inflict cytotoxic DNA damage with chemotherapy, and then impede DNA repair by molecular targeting. TR is a new drug characterized by a peculiar mechanism of action: TR traps DNA repair machinery leading to DNA damage, particularly in BRCA1/2-deficient tumors. We speculated that TR might activate PARP1, a key player in DNA-repair, and that subsequent PARP1 inhibition perpetuates TR-induced DNA damage leading to cell death. Experimental procedures and results. We developed a preclinical platform of 31 cell lines from different histotypes to explore the potential synergy between TR and the PARP1-Is olaparib (OL) and veliparib. We demonstrated that, regardless of BRCA1/2 status, PARP1-Is significantly increased TR activity, but a 15-fold range of sensitivity to the combination was observed. OL was proven the best PARP-I to combine with TR, probably due to its PARP1 trapping activity. In selected experiments, whole-genome expression profiling and GSEA analysis comparing cells displaying high vs. low synergism of the combination (HS-C vs. LS-C) revealed that DDR, G2/M cell cycle checkpoint, and DNA repair pathways were mechanistically involved in TR+OL synergy. TR induced PARP1 activation in 3/6 cell lines and PARP1-Is completely blocked both basal and TR-induced PARP1 activation. OL enhanced DNA damage response in 6/6 cell lines, but unrepairable DNA fragmentation was obtained in cells with high PARP1expr only. In two independent cell panels TR+OL synergism was directly related to PARP1expr both at mRNA (Pearson score r: 0.70, p = 0.00079) and protein level (r: 0.71, p = 0.015). Silencing and overexpression experiments validated the functional role of PARP1expr in determining TR+OL synergism: in HS-C the downmodulation of PARP1expr reduced sensitivity to TR+OL while the overexpression of PARP1 in LS-C rose TR+OL activity to levels observed in HS-C. Subcutaneous, intravenous and orthotopic xenografts of one HS-C (DMR) and one LS-C (SJSA-1) in NOD/SCID mice revealed OL significantly increased antitumor and antimetastatic activity of TR in cells with high PARP1expr only. Finally, we demonstrated that basal PARP1expr PARP1 activation by other cytotoxics with a stronger PARP1 activation observed in cells with high vs. low PARP1expr, regardless of the considered drug. Conclusions. OL enhances and potentially broaden TR cytotoxicity. TR+OL combination is particularly attractive in tumors harboring high PARP1expr and specific DDR-R gene signatures that might become predictive biomarkers of response. Future clinical validation of TR+OL combination may extend the use of PARP1-Is beyond BRCA1/2 defective tumors. Indeed, the crucial role of PARP1expr is confirmed regardless of tumor histotype and BRCA1/2 status. Further studies of combination between PARP1-Is and other cytotoxics should consider basal PARP1expr and activation after drug exposure. Citation Format: Ymera Pignochino, Federica Capozzi, Lorenzo D’ambrosio, Carmine Dell’aglio, Marco Basiricò, Paola Boccone, Erica Palesandro, Loretta Gammaitoni, Dario Sangiolo, Maria Serena Benassi, Massimo Aglietta, Giovanni Grignani. PARP1 expression (PARP1expr) drives synergy between PARP1 inhibitors (PARP1-Is) and trabectedin (TR). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3709.

Background. TR is a DNA-binding alkaloid approved in Europe for the treatment STS as second/third line therapy. TR interferes with gene transcription and nucleotide excision repair, inducing DNA double strand breaks (DSBs). There is a strong clinical interest to increase TR activity by combination with other anti-cancer drugs. PARP-1 inhibitors (PARPi) which disable DNA base-excision repair mechanism causing the accumulation of DSBs, look like a worth to explore TR therapeutic partners. Our preclinical studies were focused on the effects of the combination of TR with the PARPi (OL and veliparib). Methods: We explored DNA damage by comet assay and P-H2AX determination, PARP-1 activity by polyADPribosylation assay in a panel of 18 sarcoma cell lines, evaluating cell viability after 72h treatment with escalating doses of TR, PARPi, and their constant combination. Colony growth, cell cycle, apoptosis and DNA damage were evaluated. Antitumoral activity was checked after 4 weeks of treatment with 3 endovenous injection of TR once a week and OL intraperitoneally 5 days/week, and compared to control and single agents in bone (SJSA-1) and soft tissue sarcoma (DMR) xenografted NOD/SCID mice. TUNEL assay, P-H2AX, PCNA and CD31 immunohistochemistry was done on explanted xenografts. Results: We showed TR-induced DNA damage and PARP-1 activation in sarcoma sensitive cell line. The addition of PARPi completely blocked basal and TR-induced PARP-1 activation. DNA damage response and checkpoints (ATM, ATR, BRCA1, CHK1, CHK2, p53) were activated after single agents and combination treatment inducing cell cycle arrest in S/G2 phase. This block was released after 48h in single agent-treated cells but not in combination-treated cells leading to apoptotic cell death. Colony growth assay and viability tests revealed a strong synergism of the two drugs (Combination Index < 1, based on Chou-Talalay method). Different sarcoma histotypes displayed differences in sensitivity to both drugs and combination (Ewing's sarcoma the most sensitive> myxoid liposarcoma > leiomyosarcoma > osteosarcoma ≥ undifferentiated pleomorphic > fibrosarcoma). The antitumor effect of combined TR and OL was shown after in vivo treatment of SJSA-1 and DMR xenografted mice. A significant tumor volume reduction was observed if compared to both control and single agent and was attributed to increased DNA damage, apoptosis and reduced proliferation, but not to anti-angiogenic potentiation. In fact, a direct effect on tumor vasculature was shown in TR and combination treated group vs control. Conclusions: Our results validate the biological rationale to combine TR and PARPi in sarcoma and suggest exploring this pharmacological approach in the clinical setting. Citation Format: Ymera Pignochino, Federica Capozzi, Carmine Dell'aglio, Marco Basiricò, Lorenzo D'ambrosio, Danilo Galizia, Erica Palesandro, Maria Serena Benassi, Massimo Aglietta, Giovanni Grignani. The antitumor effect of trabectedin (TR) is potentiated by olaparib (OL) in preclinical models of bone and soft tissue sarcomas (STS). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-213. doi:10.1158/1538-7445.AM2013-LB-213

OS is the most frequent primary bone tumor in children and young adults.The mammalian Target of Rapamycin (mTOR) and the Extracellular Regulated Kinases (ERK)1/2 pathways have been shown to cooperate in survival advantage of OS. Inhibition of this signaling can be a rational pharmacological strategy to impinge on OS progression. This study pursued the dual blockage of ERK1/2 and mTOR pathways by combining the multikinase inhibitor SOR with the rapamycin analog EV. The synergistic anti-profilerative effect was found against 5 out 7 OS cell lines (combination index Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-366. doi:1538-7445.AM2012-LB-366