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Malignant peripheral nerve sheath tumor (MPNST) is a type of soft-tissue sarcoma strongly associated with dysfunction in neurofibromin; an inhibitor of the RAS pathway. We performed high-throughput screening of an array of FDA approved and promising agents in clinical development both alone and in combination at physiologically achievable concentrations against a panel of established MPNST cell line models. We found that drugs targeting a variety of factors in the RAS pathway can effectively lead to cell death in vitro with considerable drug combination synergy in regimens that target MEK or mTOR. We observed that the degree of relative sensitivity to chemotherapeutic agents was associated with the status of neurofibromin in these cell line models. Using a combination of agents that target MEK and mTORC1/2, we effectively silenced RAS/PI3K/MEK/mTOR signaling in vitro. Moreover, we employed RNAi against NF1 to establish that MPNST drug sensitivity is directly proportional to relative level of intracellular neurofibromin. Thus, two-drug combinations that target MEK and mTORC1/2 are most effective in halting the RAS signaling cascade, and the relative success of this and related small molecule interventions in MPNSTs may be predicated upon the molecular status of neurofibromin.

Purpose Systemic therapy has improved rhabdomyosarcoma event-free and overall survival; however, approximately 40 % of patients will have progressive or recurrent disease which is difficult to cure and remains a considerable challenge. Minimal progress has been made in improving outcomes for metastatic or relapsed RMS due to a lack of effective therapeutic agents. Targeted therapies are likely to be incorporated into regimens which rely on conventional cytotoxic chemotherapy. A system to evaluate novel combinations of interest is needed. Methods In this study, we explored 8 agents, 5 that are routinely used or similar to agents used in the clinical management of RMS and 3 biologically targeted agents with novel mechanisms of action, the Wee1 inhibitor AZD1775, the tyrosine kinase inhibitor cabozantinib, and the proteasome inhibitor bortezomib. All were tested individually at clinically achievable concentrations for activity in 4 RMS cell lines and then for potential synergy in two-drug combinations. Results We found single-agent activity in five of the agents (or their active metabolites) that constitute the standard of care in RMS and for AZD1775 with mean IC50 values of 207 ng/ml, well below clinically achievable levels. In addition, the combination of individual cytotoxic chemotherapeutics currently used for RMS demonstrated largely synergistic activity with higher, but clinically achievable concentrations of AZD1775 in our assays. Conclusions Prioritization of chemotherapeutics in RMS is possible using an in vitro system that can define novel drug combinations worthy of future investigation. AZD1775 exhibits single-agent activity, as well as synergy with conventional cytotoxic chemotherapy, and is a novel targeted agent that warrants further study in RMS. Electronic supplementary material The online version of this article (doi:10.1007/s00280-016-3077-8) contains supplementary material, which is available to authorized users.

Malignant peripheral nerve sheath tumor (MPNST) is an aggressive soft tissue sarcoma. To more fully characterize the genomic landscape of this tumor type, we performed next generation sequencing studies for mutational and copy number analysis. We analyzed whole exome sequencing data from 12 MPNST and SNP arrays for a subset of these. We additionally conducted a literature review of prior next generation sequencing studies in this disease and compared to the current study. We report recurrent mutations in NF1, SUZ12, EED, TP53 and CDKN2A in our study cohort. Combined with prior studies, we calculate the disease specific incidence of mutation in these genes to be: NF1 (56/64 = 87.5%). SUZ12 (69/123 = 56.1%), EED (40/123 = 32.5%), TP53 (29/72 = 40.3%), and CDKN2A (54/72 = 75.0%). Notably, we also identified frequent Ras pathway activating somatic mutations outside of these previously reported recurrently mutated genes. Five of the 12 MPNST in our cohort (42%) contained such a mutation. In conclusion, our study adds to the growing understanding of the genomic complexity of MPNST. We report a previously underappreciated frequency and variety of secondary or tertiary Ras pathway activating mutations, though not highly recurrent in a single gene.

Ewing Sarcoma (ES) is characterized by recurrent translocations between EWSR1 and members of the ETS family of transcription factors. The transcriptional activity of the fusion oncoprotein is dependent on interaction with the nucleosome remodeling and deactylase (NuRD) co-repressor complex. While inhibitors of both histone deacetylase (HDAC) and lysine-specific demethylase-1 (LSD1) subunits of the NuRD complex demonstrate single agent activity in preclinical models, combination strategies have not been investigated. We selected 7 clinically utilized chemotherapy agents, or active metabolites thereof, for experimentation: doxorubicin, cyclophosphamide, vincristine, etoposide and irinotecan as well as the HDAC inhibitor romidepsin and the reversible LSD1 inhibitor SP2509. All agents were tested at clinically achievable concentrations in 4 ES cell lines. All possible 2 drug combinations were then tested for potential synergy. Order of addition of second-line conventional combination therapy agents was tested with the addition of SP2509. In two drug experiments, synergy was observed with several combinations, including when SP2509 was paired with topoisomerase inhibitors or romidepsin. Addition of SP2509 after treatment with second-line combination therapy agents enhanced treatment effect. Our findings suggest promising combination treatment strategies that utilize epigenetic agents in ES.

Box H/ACA small nucleolar and Cajal body ribonucleoprotein particles comprise the most complex pseudouridine synthases and are essential for ribosome and spliceosome maturation. The multistep and multicomponent-mediated enzyme mechanism remains only partially understood. Here we report a crystal structure at 2.35 A of a substrate-bound functional archaeal enzyme containing three of the four proteins, Cbf5, Nop10 and L7Ae, and a box H/ACA RNA that reveals detailed information about the protein-only active site. The substrate RNA, containing 5-fluorouridine at the modification position, is fully docked and catalytically rearranged by the enzyme in a manner similar to that seen in two stand-alone pseudouridine synthases. Structural analysis provides a mechanism for plasticity in the diversity of guide RNA sequences used and identifies a substrate-anchoring loop of Cbf5 that also interacts with Gar1 in unliganded structures. Activity analyses of mutated proteins and RNAs support the structural findings and further suggest a role of the Cbf5 loop in regulation of enzyme activity.

Systemic therapy has improved osteosarcoma event-free and overall survival, but 30–50% of patients originally diagnosed will have progressive or recurrent disease, which is difficult to cure. Osteosarcoma has a complex karyotype, with loss of p53 in the vast majority of cases and an absence of recurrent, targetable pathways. In this study, we explored 54 agents that are clinically approved for other oncologic indications, agents in active clinical development and others with promising preclinical data in osteosarcoma at clinically achievable concentrations in 5 osteosarcoma cell lines. We found significant single-agent activity of multiple agents and tested 10 drugs in all permutations of two-drug combinations to define synergistic combinations by Chou and Talalay analysis. We then evaluated order of addition to choose the combinations that may be best to translate to the clinic. We conclude that the repurposing of chemotherapeutics in osteosarcoma by using an in vitro system may define novel drug combinations with significant in vivo activity. In particular, combinations of proteasome inhibitors with histone deacetylase inhibitors and ixabepilone and MK1775 demonstrated excellent activity in our assays.

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.

The structural basis for accurate placement of substrate RNA by H/ACA proteins is studied using a nonintrusive fluorescence assay. A model substrate RNA containing 2-aminopurine immediately 3′ of the uridine targeted for modification produces distinct fluorescence signals that report the substrate's docking status within the enzyme active site. We combined substrate RNA with complete and subcomplexes of H/ACA ribonucleoprotein particles and monitored changes in the substrate conformation. Our results show that each of the three accessory proteins, as well as an active site residue, have distinct effects on substrate conformations, presumably as docking occurs. Interestingly, in some cases these effects are exerted far from the active site. Application of our data to an available structural model of the holoenzyme, enables the functional role of each accessory protein in substrate placement to come into view.