Proteasome Addiction Defined in Ewing Sarcoma Is Effectively Targeted by a Novel Class of 19S Proteasome Inhibitors

Ewing sarcoma is the second most common bone malignancy in children, with a peak incidence in adolescence and is characterized by specific translocations leading to the fusion of EWSR1 to a gene of the ETS family of transcription factors (1, 2). Although localized disease is curable with highly intensive chemotherapy combined with surgery or radiation therapy (3, 4), patients with metastatic, recurrent, or refractory disease have dismal outcomes despite aggressive implementation of traditional chemotherapeutic agents (5).

To identify novel active agents against Ewing sarcoma, several high-throughput compound screening strategies have been employed. Stegmaier and colleagues characterized a gene expression profile signature that could act as a surrogate signal for inhibition of EWSR1–FLI1 (6). They performed a screen of 1,040 small molecules against Ewing sarcoma cell lines and identified cytarabine arabinoside as inducing a gene signature consistent with EWSR1–FLI1 inhibition. Cytarabine therapy demonstrated significant efficacy in preclinical models, but disappointingly, a subsequent study in a limited number of patients with relapsed/refractory Ewing sarcoma showed no objective responses (7). More recently, a chemical screen evaluating 50,000 compounds against Ewing sarcoma cell lines identified mithramycin as an agent that resulted in growth suppression as well as reduction of known targets of the EWSR1–FLI1 fusion protein (8). A trial assessing the safety and efficacy of mithramycin (Clinical Trial Identifier: NCT01610570) for children with relapsed Ewing sarcoma was recently completed, but the results are yet to be published.

We performed a broad, unbiased screen of more than 300,000 chemicals for growth-inhibitory activity against Ewing sarcoma using automated cell-based screening assays. The chemicals included synthetic compounds, as well as natural products from plants, micro-organisms, fungi, and deep sea algae. To broaden the biologic and therapeutic scope of the screen, we chose not to use EWSR1–FLI1 inhibition as the primary readout. Although the EWSR1–FLI1 fusion is widely recognized as the driving oncogenic alteration in Ewing sarcoma, an understanding of its complex role is still evolving, as highlighted by the recent demonstration of both activating and repressive transcriptional effects of this chimeric protein (9). Furthermore, effective disruption of critical EWSR1–FLI1 downstream targets may not lead to changes in EWSR1–FLI1 levels or function, and if used as a selection criterion for prioritization of compounds, could lead to dismissal of potentially relevant agents. In this report, we present the results of our broad chemical screen, which highlight a new class of inhibitors of the ubiquitin–proteasome system as having significant therapeutic potential in Ewing sarcoma. Proteasome inhibition was also defined as a specific vulnerability of Ewing sarcoma cells in a genome-wide shRNA screen.

A673, AK-PN-DW, SK-N-MC, and RD-ES were obtained from ATCC. CHP-100 and TC-71 were provided by Dr. Melinda Merchant (National Cancer Institute, Bethesda, MD). All cell lines were obtained in 2007 and reauthenticated within the past year by MSK-IMPACT sequencing, which includes 1,042 polymorphic SNPs (10). Antibodies to GAPDH and S6 were obtained from Cell Signaling Technology. Anti-UCHL5 antibody was purchased from Abcam. Anti-USP14 antibody was acquired from Bethyl Laboratories. Anti-ubiquitinylated proteins antibody (clone FK2) was purchased from EMD Millipore. Anti-rabbit secondary antibodies conjugated to horseradish peroxidase, enhanced chemiluminescence kit, AlamarBlue, and puromycin were obtained from Thermo Fisher Scientific. ApoOne caspase assay and HIV p21 ELISA kits were obtained from Promega. The 20S Proteasome Assay Kit was purchased from Cayman Chemicals. Lentiviral shRNA plasmids (The RNAi Consortium 1.0 library) were obtained from the MSK RNAi Core Facility. MG262 was purchased from Calbiochem. Bortezomib and all 19S proteasome inhibitors used in conformation and animal studies were synthesized by the MSK Organic Synthesis Core Facility (Supplementary Methods). VLX1570 was kindly provided by Hans Rosen at Vivolux Inc. Animal care was conducted in accordance with institutional guidelines.

Chemical screens were conducted as described previously (11). In brief, chemicals were plated into clear-bottom, white 384-well tissue culture plates and then cells added at a density of 2,000 cells per well and incubated for 72 hours. During the last 24 hours with compounds, AlamarBlue proliferation dye was added at a final concentration of 10% (vol/vol) and fluorescence measured (Ex: 555 nm, Em: 585). A primary screen was conducted using 10 μmol/L compound in duplicates. Compounds that inhibited growth by ≥80% were considered “hits” and the growth inhibitory activity of these compounds was confirmed in secondary screens, using re-synthesized compounds. Dose–response studies were then carried out with candidate agents in a 12-point doubling dilution series. Data were processed and IC₅₀ determined using Sigma Plot graphing software.

For viability assays that were conducted outside of the chemical screen, cells were plated in clear-bottom, white 96-well plates at a density of 3,000 cells per well and incubated with compounds for 96 hours. To measure caspase-3/7 activity, cells were plated at density of 15,000 cells per well directly into inhibitors in white, clear-bottom 96-well plates, and the activity of caspase-3/7 was determined using ApoOne caspase-3/7 activity assay kit (Promega). Fluorescence was measured using a SpectraMax M2 plate reader (Ex: 485 nm, Em: 530).

20S proteasome activity was measured as per manufacturer instructions using a 20S proteasome assay kit (Cayman Chemicals). Briefly, the assay employs a substrate, SUC-LLVY-AMC, which upon cleavage by the 20S proteasome generates a highly fluorescent product (Ex: 380 nm, Em: 480 nm). A Jurkat cell lysate supernatant with high level 20S activity was used as a positive control. Cells were treated with known 20S inhibitors bortezomib and MG-262, as well as compounds ES-P and ES-W in 5 concentrations in triplicate.

CHP100 and TC-71 cell lines were treated with bortezomib (25 nmol/L), MG-262 (25 nmol/L), EWS-P (500 nmol/L), or EWS-W (25 nmol/L) for 6 hours. All experiments were performed in triplicate including DMSO-treated cells as a control. RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific) as per manufacturer instruction and analyzed on the Affymetrix Human Genome U133A 2.0 Array. The expression of probe sets was estimated with RMA (12). The genes whose expression was differentially up- or downregulated in treated versus control cells were determined with an empirical Bayes test using LIMMA (13). Significant genes were those with an adjusted P value (FDR) < 0.0001 and an absolute fold-change ≥ 2. Probe sets corresponding to significant genes were mapped to the U133A array annotation to ensure compatibility with, and query against, the Connective Map (CMAP) build 01 database.

We performed a genome-wide functional genomic screen using the Sigma-Aldrich shRNA Human Genome Library consisting of 63,093 shRNAs targeting 11,748 human genes with a median of 5 shRNA/gene [range, 1–58 shRNA/gene; 89.6% of genes with at least 5 shRNAs and 0.06% (7 of 11,748) of genes were targeted by a single shRNA]. shRNAs were introduced by lentivirus transduction using the pLKO.1 expression vector with puromycin selection. Specifically, lentivirus were generated using 293T cells. The titer of each preparation of virus was determined using a p21 ELISA assay (Promega). Cells were seeded at 350 cells per well in 35 μL medium and infected at 24 hours with multiplicity of infection (MOI) of 5 using 6 μg/mL polybrene. Antibiotic selection was initiated 6 days after infection with 1.0 μg/mL puromycin. Cells were grown for an additional 7 days in the presence of puromycin. On day 13, cells were fixed with 4% paraformaldehyde (PFA; vol/vol) in PBS, permeabilized with 0.05% Triton X-100, and nuclei stained with 10 μmol/L Hoechst. Nuclear counts were used as the readout for cell number.

For analysis of the shRNA screen data, outliers of the nuclei counts for puromycin-treated control were removed. shRNAs, for which the imaged nuclei count was less than or equal to the mean nuclei counts of the puromycin-treated controls, were considered positive. For each gene, the percentage of positive shRNA was computed and “hits” were defined as genes for which at least 80% of the shRNAs were positive. Pathway enrichment analysis of the “hits” for networks, biologic functions, and canonical pathways was performed using Ingenuity Pathway Analysis (IPA; http://www.ingenuity.com/products/ipa).

One million A673 or TC-71 cells were mixed with Matrigel and injected subcutaneously into a single flank of female NOD scid gamma (NSG) or athymic mice (Harlan Labs). NSG mice were transduced with lentivirus-expressing GFP-luciferase. NSG mice bearing A673 xenografts were treated with DMSO, bortezomib, or b-AP15 after they reached 80 to 100 mm³ and weekly tumor size measurements were taken. Athymic mice bearing A673 and TC-71 xenografts were employed for VLX1570 experiments. When tumors reached 80 to 100 mm³, mice were randomized into 3 groups, 5 mice/group. Mice were treated with vehicle only, b-AP15 (25 mg/kg), or VLX1570 (4.4 mg/kg) daily. Drugs were administered via intraperitoneal injection. Tumors were measured twice weekly, and tumor volume was calculated using the formula: length × width² × 0.52. Body weight was also assessed twice weekly.

Ewing sarcoma cells were treated with b-AP15 for 3 hours followed by lysis [50 mmol/L HEPES, 50 mmol/L NaCl, 5 mmol/L MgCl₂, 0.25% Triton X-100, 2 mmol/L ATP, 1 mmol/L dithiothreitol (DTT), 250 mmol/L sucrose]. Twenty-five micrograms of protein was labeled with 1 μmol/L UbVS for 10 minutes, followed by quenching in loading buffer, fractionation on SDS-PAGE gels and immunoblotting with indicated antibodies.

The cellular thermostability assay (CETSA) was performed as described (14). In brief cells were collected, subject to freeze thaw, soluble fraction was extracted, and lysates were exposed to DMSO or drug for 30 minutes at room temperature. Lysates were heated at 53°C, fractionated by SDS-PAGE. Thermostabilization was observed by immunoblotting with USP14 or UCHL5 antibodies.

Recombinant UCHL1, UCHL3, and purified 26S proteasomes were obtained from Boston Biochem. 26S proteasomes (5 nmol/L) in reaction buffer (50 mmol/L HEPES, 50 mmol/L NaCl, 5 mmol/L MgCl₂, 2 mmol/L ATP, 1 mmol/L DTT, 250 mmol/L sucrose) were treated with b-AP15 for 5 minutes prior to the addition of 500 nmol/L Ub-AMC. Cleavage of AMC was monitored at 380 nm excitation and 460 nm emission wavelengths using Tecan plate reader.

We performed a screen of a chemical library of 309,989 compounds to find agents with antiproliferative activity in Ewing sarcoma cell lines. The primary screen was conducted against the TC-71 cell line, which contains the type 1 EWSR1–FLI1 fusion using 10 μmol/L of each compound at the Memorial Sloan Kettering Cancer Center High-Throughput Drug Screening Facility (MSKCC HTSCF; New York, NY). Compounds that inhibited growth by ≥80% were moved forward for further analysis; 209 compounds met this criterion (Fig. 1). To identify highly active compounds against multiple Ewing sarcoma cell lines, we tested the 209 compounds against 5 Ewing sarcoma cell lines with the EWSR1–FLI1 fusion (TC-71, A673, RD-ES, SK-N-MC, SK-PN-DW). Dose–response studies were conducted, and chemicals for which the IC₅₀ for inhibition of growth was ≤1 μmol/L were selected as potential growth inhibitors of Ewing sarcoma cell lines. This identified 23 compounds as inhibitors of the growth of Ewing sarcoma cells, based on IC₅₀ concentrations of ≤1 μmol/L in all 5 Ewing sarcoma cell lines.

Reasoning that pan‐active compounds with low specificity for Ewing sarcoma would less likely be clinically novel or relevant and would be less helpful in providing insight into the biology of Ewing sarcoma, we sought to identify which of these 23 compounds had relatively selective activity against Ewing sarcoma compared with other non–Ewing sarcoma cancer cell lines. We assembled a panel of 11 non‐-Ewing sarcoma cell lines from 10 different cancer types to perform a lateral screen with the 23 compounds identified to be active against the 5 Ewing sarcoma cell lines (Supplementary Table S1). Cell lines for which the IC₅₀ for inhibition of growth by any of the 23 compounds was <1 μmol/L were designated as sensitive. From the group of 23 compounds highly active against Ewing sarcoma cell lines, we identified 8 compounds to which at least 5 of 11 non‐-Ewing sarcoma cell lines were resistant in our comparative screening panel, suggesting selective or preferential activity against Ewing sarcoma. Two of the 8 compounds are analogues of daunorubicin and ellipticine (NCT608747, NCT176327), both of which are from chemotherapy families currently used as effective frontline chemotherapy for Ewing sarcoma (anthracyclines and topoisomerase-II inhibitors). Two of the remaining 6 compounds (NCT666038, NCT669441) were analogues that share a benzyl‐4‐piperidone scaffold, which prompted us to further explore this novel class of compounds. These compounds were designated EWS-P (NCT666038) and EWS-W (NCT669441).

To evaluate the mechanism of action of the EWS-P and EWS-W compounds, we studied the change in gene expression profiles of two Ewing sarcoma cell lines, CHP100 and TC-71, treated with these two agents compared with DMSO-treated controls (see Materials and Methods). Statistically significantly up‐ and downregulated genes were queried against the Connectivity Map (CMAP) platform (http://www.broadinstitute.org/cmap/), a database of gene expression profiles generated from human cells treated with a wide range of bioactive small molecules (15). To validate this approach, we also generated expression profiles of Ewing sarcoma cell lines treated with two compounds identified from the screen that were derivatives of camptothecin (EWS-A) and the topoisomerase II inhibitor ellipticine (EWS-S). The genes differentially expressed upon treatment with these compounds were highly similar to those expression profiles inferred from analogous compounds in CMAP (data not shown). The gene signatures inferred from both Ewing sarcoma cell lines treated with compounds EWS-P and EWS-W were highly congruent with expression signatures obtained for the proteasome inhibitor MG‐262 (Supplementary Fig. S1). Pairwise expression profile comparisons of Ewing sarcoma cell lines treated with MG-262 and its analogue, bortezomib, demonstrated a statistically significant overlap with the expression profiles generated by compounds EWS-P and EWS-W. Furthermore, we identified 13 core genes that were upregulated in both CHP100 and TC-71 following treatment with all 4 agents; bortezomib, MG-262, EWS-P, and EWS-W (Supplementary Fig. S1A). The majority of these core genes are well-established in the literature as being upregulated following inhibition of the proteasome (Supplementary Fig. S1B; refs. 16–19).

Activity of the proteasome is dependent upon the polyubiquitination of peptides destined for degradation, and therefore, inhibition of proteasomal activity leads to intracellular accumulation of polyubiquitinated proteins (20). To better define the effect of EWS-P and EWS-W on the proteasome, we treated TC-71 and CHP100 cell lines with these compounds and then looked at global protein ubiquitination by Western blot analysis. Treatment with compounds EWS-P and EWS-W resulted in significant accumulation of polyubiquitinated proteins providing further evidence that these agents are inhibitors of the proteasome (Supplementary Fig. S1C). The human 26S proteasome consists of a central, barrel-shaped 20S subunit and two outer structures designated as the 19S proteasome subunit. The 19S proteasome subunit is responsible for recognition of polyubiquitinated proteins, unfolding of the peptide structure, and passage of the peptide into the 20S subunit, the primary site of peptide degradation (21–23). Agents such as bortezomib and MG‐262 bind to the active site of the 20S proteasome, leading to inhibition (24). To assess activity against the 20S proteasome, we performed an assay that employs a specific 20S substrate, which upon cleavage by the active proteasome generates a highly fluorescent product. As predicted, inhibition with bortezomib and MG‐262 resulted in a marked decrease in fluorescence. Treatment with compounds EWS-P and EWS-W, however, did not result in a statistically significant reduction in fluorescence, indicating that these compounds inhibit proteasomal activity through a mechanism not dependent on the 20S subunit.

We generated analogues of EWS-P and EWS-W (MSK-EWS-4 and MSK-EWS-5) and tested their ability to inhibit growth of four Ewing sarcoma cell lines. Three of the benzyl-4-piperidone compounds inhibited the growth of all Ewing sarcoma lines tested, whereas one compound (MSK-EWS-4) was inactive (Supplementary Table S2). Compound MSK-EWS-5, which was previously published under the name b-AP15 (25), was the most potent analogue (Fig. 2A and Supplementary Table S2). b-AP15 was previously shown to inhibit the deubiquitinating enzymes (DUB) USP14 and UCHL5 (25). These enzymes associate with the 19S proteasome subunit and function to deubiquitinate proteins as they enter the 20S proteolytic core subunit (26–28). These results are consistent with our finding that these compounds, as a class, inhibit proteasomal activity through a 20S independent mechanism.

To confirm DUB inhibition as a mechanism of action in Ewing sarcoma, we performed activity labeling using the suicide substrate Ub-vinylsulphone (UbVS) on cells following treatment with b-AP15 (1 μmol/L) for 3 hours. Immunoblotting following UbVS labeling showed the inhibition of USP14 and UCHL5 in all Ewing sarcoma cell lines tested (Fig. 2A). As previously reported (25, 29), USP14 appeared to be more effectively inhibited by b-AP15 compared with UCHL5. Building upon this, we also examined the binding of b-AP15 to USP14 in Ewing sarcoma cells using CETSA (14). We found stabilization of USP14 after Ewing sarcoma cell exposure to 1 μmol/L b-AP15 (Fig. 2B). Thermostabilization of UCHL5 was not observed at this drug concentration, further supporting the idea that USP14 is the more sensitive DUB target for these compounds. Furthermore, b-AP15 did not demonstrate inhibition of other DUBs, including recombinant UCHL1 and UCHL3 (Supplementary Fig. S2).

VLX1570 is an analogue of b-AP15 with minor structural modifications improving its chemical properties for clinical use (29). Cell viability assays in 4 Ewing sarcoma cell lines demonstrate that VLX1570 is slightly more potent that b-AP15 at inhibiting growth in the 4 Ewing sarcoma cell lines tested (Fig. 3A and Supplementary Table S2). To determine whether the reduction in growth of Ewing sarcoma cells by these benzyl-4-piperidone compounds is due in part to induction of apoptosis, we measured the enzymatic activity of caspase-3/7 following drug treatment. Four Ewing sarcoma cell lines were treated with b-AP15 or VLX1570 for 48 hours followed by fluorescence-based measurement of caspase-3/7 activity. We observed a dose-dependent increase in caspase-3/7 activity, approximately 2- to 7-fold above DMSO-treated controls, in cells following treatment with b-AP15 or VLX1570, indicating activation of apoptosis (Fig. 3B).

To evaluate the in vivo activity of benzyl-4-piperidone compounds, we first treated a GFP-luciferase expressing A673 xenograft model with b-AP15 following injection of cells to study the effect on tumor formation. Treatment with b-AP15 resulted in undetectable tumors by imaging at 4 weeks (Supplementary Fig. S3A). Next, we compared bortezomib and b-AP15 in an A673 xenograft model and demonstrated superior growth inhibition and improved survival with the former (Supplementary Fig. S3B and S3C). We then tested the ability of b-AP15 and VLX1570 to inhibit the growth of 2 Ewing sarcoma cell line xenografts, A673 and TC-71. Athymic mice were treated with b-AP15 (25 mg/kg), VLX1570 (4.4 mg/kg), or vehicle daily via intraperitoneal administration. We initiated treatment when tumors reached a volume of approximately 100 mm³. Growth of A673 and TC-71 xenograft tumors was significantly reduced by both compounds with VLX1570 being more potent (Fig. 3C). There was no weight loss in any treatment group (Supplementary Fig. S3D).

To provide orthogonal confirmation of Ewing sarcoma sensitivity to proteasome inhibition, we analyzed data from a functional genomic screen in Ewing sarcoma cells. Specifically, the CHP100 cell line was screened with the Sigma‐Aldrich shRNA Human Genome Library by lentivirus transduction. The screen queried 11,748 genes with ≥5 shRNAs targeting 89.6% of the genes. Effects on proliferation and viability were measured by nuclear counts (Hoechst stain). The screen identified 627 genes with ≥80% shRNA scoring positive (Fig. 4A). Pathway analysis of these 627 genes revealed an enrichment of genes involved in the protein ubiquitination pathway (P = 0.0004, Supplementary Table S3), including PSMB2/3/5/6/8, PSMC3, PSMD1/3/7, UBC, and USP1/14/26. Furthermore, among these 627 “hits,” 26S proteasome component genes and associated DUBs were significantly overrepresented (P < 0.0001, Fisher exact test; Tables 1 and 2). Overall screen data and IPA of the hits are provided in Supplementary Tables S3 and S4.

To further evaluate USP14 and UCHL5 as potential targets in Ewing sarcoma, we performed shRNA-mediated knockdown (2 shRNAs per gene) in A673 and TC-71 cells. RNAi of these genes caused a significant reduction in expression level of the encoded proteins and a concomitant decrease in cell numbers (Fig. 4B).

Given the current outcomes for patients with metastatic and recurrent Ewing sarcoma, there is a critical need for novel therapeutic agents. Proteasome inhibitors such as bortezomib have been evaluated in both preclinical and clinical studies of Ewing sarcoma. Bortezomib is a dipeptide boronic acid analogue that inhibits the chemotryptic activity of the 20S proteasome (30). It is currently FDA-approved for the treatment of multiple myeloma and relapsed mantle cell lymphoma. In vitro cell viability assays performed on Ewing sarcoma cell lines have demonstrated drug susceptibility with IC₅₀ levels as low as 20 nmol/L (31), but subsequent xenograft studies performed by the Pediatric Preclinical Testing Program identified limited activity in Ewing sarcoma or any other pediatric solid tumor type (32). Furthermore, in the COG ADVL0916 study, which evaluated the use of bortezomib in combination with escalating doses of vorinostat in 23 children with solid tumors, including 2 patients with Ewing sarcoma, no responses were observed (33). More generally, numerous phase II studies have failed to demonstrate meaningful activity of bortezomib in adult solid tumors (34–42). The poor activity of bortezomib in most solid tumor patients has been attributed to poor tumor penetration of the drug (43). Clinical dosing schedules for bortezomib in multiple myeloma achieve only 70% proteasome inhibition in blood cells with complete recovery between doses, suggesting that the pharmacodynamic profile in solid tumors may be even less favorable (44).

Novel inhibitors of the 20S proteasome with potentially advantageous pharmacologic properties are being investigated in solid tumors. Carfilzomib is a 20S inhibitor, which, unlike bortezomib, irreversibly inhibits the chymotrypsin-like activity of the 20S proteasome (45). A recent phase I/II study for adult patients with advanced solid tumors was conducted in which patients were treated twice weekly on consecutive days for 3 weeks per 28-day cycle. Among the 65 patients treated in the phase II portion of this trial, none achieved a partial response or better (46). Although other studies evaluating the efficacy of carfilzomib in solid tumors are ongoing, these disappointing results highlight the need for the development and evaluation of alternative proteasome inhibition strategies.

Thus, while in vitro data suggest that proteasome function may be an effective therapeutic target in some solid tumors, clinical experience with 20S inhibitors in patients with solid tumors has been disappointing, suggesting that alternate strategies for proteasome inhibition should be pursued.

Here, we have provided strong preclinical evidence that benzyl-4-piperidone compounds are highly active in Ewing sarcoma. Two analogues were identified as lead compounds from our high-throughput chemical screen on the basis of stringent requirements for both potency and selectivity. We have demonstrated that treatment of Ewing sarcoma cell lines with this family of compounds leads to accumulation of polyubiquitinated proteins, without inhibition of the 20S proteasome, thereby acting at a site within the ubiquitin–proteasome system distinct from that targeted by proteasome inhibitors currently employed in clinical practice. Our findings are supported by recent publications that have identified the 19S proteasome component as the target for this group of compounds (25, 47). We also demonstrate that this class of compounds act synergistically with bortezomib and can overcome bortezomib resistance mutations (data not shown). This is an important observation for the potential evaluation of dual 19S and 20S inhibition in future trials for both liquid and solid tumor types. Finally, we have also established the susceptibility of Ewing sarcoma to proteasomal disruption through an unbiased functional genomics screen and through specific knockdown experiments targeting the DUBs USP14 and UCHL5. Our data supports USP14 as a primary target given its identification as a lead hit on our genome-wide shRNA screen as well as preferential b-AP15 inhibitory activity against USP14 seen in our in vitro binding studies.

We have identified a highly active novel class of proteasome inhibitors from a screen of an exceptionally broad library of chemicals against Ewing sarcoma and have validated these functionally and preclinically. Importantly, a clinical compound is now available for this novel class of proteasome inhibitors; VLX1570 was recently developed in an effort to improve on the chemical properties of b-AP15 for use in a clinical setting. This novel derivative was generated by substituting the piperidine ring in b-AP15 with an azepane ring. VLX1570 has a superior clinical profile compared with b-AP15 with significantly improved solubility as well as an increase in biologic activity (29). A phase I/II trial assessing the safety and efficacy of VLX1570 in patients with relapsed/refractory multiple myeloma is ongoing (NCT02372240). Our findings now provide a compelling rationale for a clinical trial evaluating VLX1570 as a novel therapeutic strategy in patients with relapsed/refractory Ewing sarcoma.

M. Merchant is employed at AstraZeneca as Medical Science Director. P. D'Arcy has ownership interest (including patents) in Vivolux. S. Linder has ownership interest as a shareholders of Vivolux. No potential conflicts of interest were disclosed by the other authors.

Conception and design: N. Shukla, R. Somwar, R.S. Smith, M. Merchant, H. Djaballah, M. Ladanyi

Development of methodology: N. Shukla, R. Somwar, P. D'Arcy, C. Antczak, G. Yang, C.K.Y. Ng, I. Khodos, O. Ouerfelli, H. Djaballah

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N. Shukla, R. Somwar, R.S. Smith, S. Ambati, S. Munoz, M. Merchant, C. Antczak, D. Shum, C. Radu, G. Yang, I. Khodos, E. de Stanchina, J.S. Reis-Filho, O. Ouerfelli, S. Linder

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N. Shukla, R. Somwar, R.S. Smith, S. Ambati, S. Munoz, R. Kobos, C. Antczak, B. Binder, C. Radu, G. Yang, B.S. Taylor, C.K.Y. Ng, B. Weigelt, J.S. Reis-Filho, H. Djaballah, M. Ladanyi

Writing, review, and/or revision of the manuscript: N. Shukla, R. Somwar, R.S. Smith, S. Ambati, M. Merchant, C. Antczak, G. Yang, C.K.Y. Ng, B. Weigelt, J.S. Reis-Filho, O. Ouerfelli, S. Linder, M. Ladanyi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. Somwar, S. Munoz, X. Wang, I. Khodos, H. Djaballah

Study supervision: N. Shukla, R. Somwar, S. Linder, M. Ladanyi

Other (performed experiments): P. D'Arcy

Other (synthesized all molecules and wrote the experiments): G. Yang

Other (designed, written and drawn all chemical structures and data): O. Ouerfelli

The authors thank Joseph Olechnowicz for editorial assistance.

This study was funded by the Ewing's Research Foundation, the Sarcoma Foundation of America, the Hyundai Hope on Wheels Program, the Ewing Sarcoma Research Fund, Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research, and the Experimental Therapeutics Center of Memorial Sloan Kettering Cancer Center. Research infrastructure support was provided by the National Cancer Institute under grant no. P30CA008748.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.