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.

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.

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

No abstract

The human CMG helicase (Cdc45-MCM-GINS) is a novel target for anti-cancer therapy due to tumor-specific weaknesses in CMG function induced by oncogenic changes and the need for CMG function during recovery from replicative stresses such as chemotherapy. Here, we developed an orthogonal biochemical screening approach and identified selective CMG inhibitors (CMGi) that inhibit ATPase and helicase activities in an ATP-competitive manner at low micromolar concentrations. Structure-activity information and in silico docking indicate that CMGi occupy ATP binding sites and channels within MCM subunits leading to the ATP clefts, which are likely used for ATP/ADP ingress or egress. CMGi inhibit cell growth and DNA replication using multiple molecular mechanisms. CMGi block helicase assembly steps that require ATP binding/hydrolysis by the MCM complex, specifically MCM ring assembly on DNA and GINS recruitment to DNA-loaded MCM hexamers. During S-phase, inhibition of MCM ATP binding/hydrolysis by CMGi causes a ‘reverse allosteric’ dissociation of Cdc45/GINS from the CMG that destabilizes the replisome and disrupts interactions with Ctf4, Mcm10, and DNA polymerase-α, -δ, -ε, resulting in DNA damage. These novel CMGi are selectively toxic toward tumor cells and define a new class of CMG helicase-targeted anti-cancer compounds with distinct mechanisms of action.

BACKGROUND: Osteosarcoma is the most common bone sarcoma in children, adolescents, and young adults. Osteosarcoma primarily arises through tumor suppressor inactivation, most typically due to TP53 attenuation mechanisms. Increasingly additional oncogenic and tumor suppressor pathways are being recognized in subsets of osteosarcoma. We hypothesized that novel insights into mechanisms of sensitivity and resistance for emerging targeted therapies could be elucidated by combining activity and genomic information across cell line models of osteosarcoma. METHODS: In this study we evaluated 6 established osteosarcoma cell lines (U2-OS, MG-63, OS252, SAOS-2, 143B, MNNG) representing several TP53 and other tumor suppressor inactivation mechanisms with human osteoblasts as a control. We have obtained RNA-seq profiles detailing genomic variants, gene fusions, and expression of both protein-coding and miRNA genes for these cell lines. In addition to genomic changes, we evaluated 140 chemical probes and candidate drug molecules across these cell lines. RESULTS: RNAseq identified TP53 changes in 5 of 6 cell lines with OS252, SAOS2 and MG-63 having intron 1 translocations and 143B and MNNG sharing a TP53 gain-of-function mutation. Variant calling from the whole exome sequencing data revealed additional putatively oncogenic mutations. Additionally, we observed differential gene expression patterns compared to control samples in genes associated with genomic stability, epigenetic regulation, and transcriptional activity. Differential sensitivities to several classes of small molecules were seen across the cell lines and were associated with genomic changes that warrant additional investigation such as: RAD51 pathway genomic findings associated with sensitivity to RS-1, disulfuram activity varying according to aldehyde dehydrogenase activity, and variable response to inhibitors of ATR and specific CDKs that are associated with the expression of their respective targets. Additional pathways of interest that demonstrate differential response to small molecules include histone/protein deacetylation, telomerase activity, protein translation, and others. Unique vulnerabilities associated with tumor suppressor or oncogenic changes specific to an individual cell line will be presented. CONCLUSIONS: By characterizing response to small molecules in the context of gene expression, underlying pathway vulnerabilities may be determined for further testing suggesting potential biomarkers for basket trials. Other agents with shared sensitivities to agents across cell lines may be translated in a different manner. Citation Format: Elliot J. Kahen, Darcy Welch, Jamie Teer, Andrew S. Brohl, Sean J. Yoder, Yonghong Zhang, Ling Cen, Damon Reed. Osteosarcoma cell lines display both shared and unique vulnerabilities to 140 targeted small molecules with RNA-seq revealing putative mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3010.