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Background: Ewing Sarcoma (ES) is the second most common primary bone cancer affecting children and young adults. Despite advances in treatment that have led to survival rates of approximately 73% for localized disease, outcomes for patients with metastatic or recurrent ES remain poor. A distinguishing feature of ES is the presence of the EWS/FLI1 fusion in 85% of cases. The fusion has been shown to alter expression of a number of oncogenic genes. Mechanistic studies have demonstrated that the NuRD co-repressor complex interacts with EWS/FLI1. The associated protein LSD-1 contributes to the repressive function by histone modifications. While reversible LSD1 inhibitors demonstrate single agent activity, in preclinical models, a system to evaluate combinations may be needed for optimizing effect in clinical trials. Methods: Here, we seek to confirm promising single drug activity and evaluate combination therapies using active chemotherapies currently utilized in ES care (4-HC, etoposide, SN-38, vincristine and doxorubicin) along with the LSD1 Inhibitors SP2509 and SP2577 and romidepsin, an HDAC inhibitor. We evaluated these combinations in high-throughput screening platforms and well-established cell line models for ES (A-673, TC-32, RD-ES, TC-71). Taking into consideration past lessons learned from in vitro experiments, we designed stringent screening conditions that assess the candidate compounds and combinations at clinically-relevant concentrations and exposure times that mimic the in vivo pharmacokinetics in an effort to maximize the translational potential of these results to the clinical setting. All combinations of agents were studied in two-drug combinations to evaluate for synergy in addition to efficacy. Results: IC50 for SP2509 was found to be in the submicromolar range across cell lines with SP2577 being more potent. A-673 and TC-71 were 5-10 fold less sensitive than RD-ES and TC-32. Agents currently utilized in clinic were universally active at clinically achievable concentrations and exposure times. Combinations showed additivity frequently and demonstrated promising activity that can be used to inform further decision making once LSD1 inhibition toxicities are better known. These findings suggest potentially promising opportunities for developing combination clinical trials to maximize development of LSD1 inhibitors. Citation Format: Darcy Welch, Elliot Kahen, Christopher L. Cubitt, Damon R. Reed. LSD1 inhibition alone and in combination with chemotherapy in Ewing sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2034. doi:10.1158/1538-7445.AM2017-2034

Background: Malignant Peripheral Nerve Sheath Tumor (MPNST) is a malignant sarcoma that derives from a peripheral nerve or plexiform neurofibroma. Neurofibromatosis type 1 (NF-1) patients are particularly susceptible, with a higher risk, earlier onset, and worse prognosis. The major factor associated with MPNST and NF-1 is Neurofibromin 1, coded by the NF1 gene. NF1 mutation results in RAS hyperactivation. Chemotherapy for MPNST is currently limited, with poor prognosis for metastatic or unresectable tumors. Thus, the development of promising treatment solutions for MPNST to translate to clinical trials is required. Methods: Here, we seek to identify efficacious chemotherapeutic treatments for MPNST with a combination of drug screening and biological pathway analysis. We used our previously established preclinical system to test FDA approved or promising developmental agents against five cell line models for MPNST. We screened sixty agents with diverse mechanisms of action below published maximum plasma concentrations, and measured effects with a CellTiter-Glo viability assay. Promising agents were then tested in two-drug combinations, allowing for determination of synergism. We then examined the molecular effects of the top candidates with use of antibody arrays that permit detection of a series of phosphorylated proteins. Results: The group of most efficacious drugs was enriched with agents that target factors downstream of RAS, including MEK, mTOR, and PI3K inhibitors, with microtubule inhibitors, genotoxics, and HDAC inhibitors also demonstrating good results. Strong synergism was observed across our cell line models particularly in combinations containing the dual mTORC1/2 inhibitor INK128. Interestingly, drug sensitivity varied greatly between cell lines, correlating with relative NF1 protein and RAS-GTP levels. We analyzed the activation of the RAS pathway in response to drug treatment with antibody arrays and found that, following treatment, relative phosphorylation signal was more decreased compared to controls in cell lines with lower relative NF1 protein levels. Doxorubicin was able to reduce phosphorylation signal compared to controls to a level near comparable to targeted inhibitors, which could contribute to doxorubicin’s current usefulness against MPNSTs. Importantly, we identified combination treatments that were able to greatly reduce the relative phosphorylation signal of RAS pathway members versus control. Combinations containing INK128 resulted in the most pathway shutdown. These findings suggest that MPNSTs may be susceptible to combination treatments targeting RAS pathway members. Moreover, it may be possible to use pathway analysis as a diagnostic tool to predict drug tolerance. Citation Format: Elliot Kahen, Darcy Welch, Diana Yu, Christopher Cubitt, Jae Lee, Andrew Brohl, Damon R. Reed. RAS pathway activation and sensitivity to therapeutic agents is correlated with NF1 residual activity in malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1340. doi:10.1158/1538-7445.AM2017-1340

Background: Alveolar Soft Part Sarcoma (ASPS) is a rare soft tissue sarcoma subtype occurring mainly in children and young adults. Malignant tumors develop in the connective tissues, typically in the lower extremities of the legs and thighs, but can also be found in the head and neck regions. ASPS is slow growing, highly angiogenic, and may have unique metabolic properties. ASPS is characterized by an unbalanced translocation between the ASPSCR1 gene (whose product tethers GLUT4 transporters) and the TFE3 gene (encoding a transcription factor). The resulting ASPSCR1-TFE3 fusion gene is responsible for an aberrant transcription factor that is thought to be involved in the pathogenesis of the disease. Methods to assess a number of different agents in combination are needed for practical use in clinical trials. Methods: Here, we seek to examine the therapeutic activity of single agent and combinations of epigenetic, anti-angiogenic and a diverse mix of other mechanisms of action agents using high-throughput screening platforms and well-established cell line models for ASPS (ASPS-1 and ASPS-KY). We designed stringent screening conditions that assess the compounds at clinically-relevant concentrations and time of exposures that mimic the in vivo pharmacokinetics in an effort to maximize the translational potential of these results to the clinical setting. 54 agents were screened across the 2 cell lines with attention to existing clinical data and angiogenesis inhibitors. The cell titer glo assay was used to determine cell viability at three clinically achievable concentrations for all agents. The most promising 8 agents were then studied in all two-drug combinations to evaluate for synergy. Results: The results of our screening showed that several agents significantly reduced cell viability at clinically relevant concentrations and exposure times with belinostat (an HDAC inhibitor) and dinaciclib (a CDK 1, 2, 5 and 9 inhibitor) in particular having good activity. Several combinations with doxorubicin showed good efficacy and synergy. Belinostat and doxorubicin affected both ASPS-KY and ASPS-1 cell lines similarly, with an average fraction affected (FA) of 0.87. The combination index (CI) indicated strong synergy in the ASPS-KY (CI = 0.09) and some synergy in ASPS-1 (CI = 0.74). Similarly, dinaciclib and doxorubicin produced a FA of 0.85 with good synergy (CI of 0.51-0.66). In addition, the combination of belinostat and tivantinib showed additive effects in both cell lines (CI of 0.88-1.2) with a FA of ∼0.8. These findings suggest potentially promising opportunities for developing combinational approach to treating childhood soft tissue sarcomas. Citation Format: Justine Clark, Elliot Kahen, Diana X. Yu, Christopher L. Cubitt, Daniel M. Sullivan, Damon R. Reed. Assessing combinations of chemotherapy in alveolar soft part sarcoma cell lines. [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 274.