Srikanth R. Ambati

Pre-clinical efficacy of PU-H71, a novel HSP90 inhibitor, alone and in combination with bortezomib in Ewing sarcoma

Ewing sarcoma is characterized by multiple deregulated pathways that mediate cell survival and proliferation. Heat shock protein 90 (HSP90) is a critical component of the multi‐chaperone complexes that regulate the disposition and activity of a large number of proteins involved in cell‐signaling systems. We tested the efficacy of PU‐H71, a novel HSP90 inhibitor in Ewing sarcoma cell lines, primary samples, benign mesenchymal stromal cells and hematopoietic stem cells. We performed cell cycle analysis, clonogenic assay, immunoblot analysis and reverse phase protein array in Ewing cell lines and in vivo experiments in NSG and nude mice using the A673 cell line. We noted a significant therapeutic window in the activity of PU‐H71 against Ewing cell lines and benign cells. PU‐H71 treatment resulted in G2/M phase arrest. Exposure to PU‐H71 resulted in depletion of critical proteins including AKT, pERK, RAF‐1, c‐MYC, c‐KIT, IGF1R, hTERT and EWS‐FLI1 in Ewing cell lines. Our results indicated that Ewing sarcoma tumor growth and the metastatic burden were significantly reduced in the mice injected with PU‐H71 compared to the control mice. We also investigated the effects of bortezomib, a proteasome inhibitor, alone and in combination with PU‐H71 in Ewing sarcoma. Combination index (CI)‐Fa plots and normalized isobolograms indicated synergism between PU‐H71 and bortezomib. Ewing sarcoma xenografts were significantly inhibited when mice were treated with the combination compared to vehicle or either drug alone. This provides a strong rationale for clinical evaluation of PU‐H71 alone and in combination with bortezomib in Ewing sarcoma.

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

Ewing sarcoma is a primitive round cell sarcoma with a peak incidence in adolescence that is driven by a chimeric oncogene created from the fusion of the EWSR1 gene with a member of the ETS family of genes. Patients with metastatic and recurrent disease have dismal outcomes and need better therapeutic options. We screened a library of 309,989 chemical compounds for growth inhibition of Ewing sarcoma cells to provide the basis for the development of novel therapies and to discover vulnerable pathways that might broaden our understanding of the pathobiology of this aggressive sarcoma. This screening campaign identified a class of benzyl-4-piperidone compounds that selectively inhibit the growth of Ewing sarcoma cell lines by inducing apoptosis. These agents disrupt 19S proteasome function through inhibition of the deubiquitinating enzymes USP14 and UCHL5. Functional genomic data from a genome-wide shRNA screen in Ewing sarcoma cells also identified the proteasome as a node of vulnerability in Ewing sarcoma cells, providing orthologous confirmation of the chemical screen findings. Furthermore, shRNA-mediated silencing of USP14 or UCHL5 in Ewing sarcoma cells produced significant growth inhibition. Finally, treatment of a xenograft mouse model of Ewing sarcoma with VLX1570, a benzyl-4-piperidone compound derivative currently in clinical trials for relapsed multiple myeloma, significantly inhibited in vivo tumor growth. Overall, our results offer a preclinical proof of concept for the use of 19S proteasome inhibitors as a novel therapeutic strategy for Ewing sarcoma. Cancer Res; 76(15); 4525-34. ©2016 AACR.

High affinity and covalent-binding microtubule stabilizing agents show activity in chemotherapy-resistant acute myeloid leukemia cells

Treatment failure in acute myeloid leukemia (AML) is frequently due to the persistence of a cell population resistant to chemotherapy through different mechanisms, in which drug efflux via ATP-binding cassette (ABC) proteins, specifically P-glycoprotein, is one of the most recognized. However, disappointing results from clinical trials employing inhibitors for these transporters have demonstrated the need to adopt different strategies. We hypothesized that microtubule targeting compounds presenting high affinity or covalent binding could overcome the effect of ABC transporters. We therefore evaluated the activity of the high-affinity paclitaxel analog CTX-40 as well as the covalent binder zampanolide (ZMP) in AML cells. Both molecules were active in chemosensitive as well as in chemoresistant cell lines overexpressing P-glycoprotein. Moreover, ZMP or CTX-40 in combination with daunorubicin showed synergistic killing without increased in vitro hematopoietic toxicity. In a primary AML sample, we further demonstrated that ZMP and CTX-40 are active in progenitor and differentiated leukemia cell populations. In sum, our data indicate that high affinity and covalent-binding anti-microtubule agents are active in AML cells otherwise chemotherapy resistant.

Longitudinal monitoring of cardiac siderosis using cardiovascular magnetic resonance T2* in patients with thalassemia major on various chelation regimens: a 6-year study.

Cardiovascular magnetic resonance (CMR) and hepatic magnetic resonance imaging (MRI) have become reliable noninvasive tools to monitor iron excess in thalassemia major (TM) patients. However, long‐term studies are lacking. We reviewed CMR and hepatic MRI T2* imaging on 54 TM patients who had three or more annual measurements. They were managed on various chelation regimens. Patients were grouped according to their degree of cardiac siderosis: severe (T2*, <10 msec), mild to moderate (T2* = 10–20 msec), and no cardiac siderosis (T2*, >20 msec). We looked at the change in cardiac T2*, liver iron concentration (LIC) and left ventricular ejection fraction (LVEF) at years 3 and 5. In patients with severe cardiac siderosis, cardiac T2* (mean ± SD) improved from 6.9 ± 1.6 at baseline to 13.6 ± 10.0 by year 5, mean ΔT2* = 6.7 (P = 0.04). Change in cardiac T2* at year 3 was not significant in the severe group. Patients with mild to moderate cardiac siderosis had mean cardiac T2* of 14.6 ± 2.9 at baseline which improved to 26.3 ± 9.5 by year 3, mean ΔT2* = 11.7 (P = 0.01). At baseline, median LICs (mg/g dry weight) in patients with severe, mild–moderate, and no cardiac siderosis were 3.6, 2.8, and 3.3, whereas LVEFs (mean ± SD) (%) were 56.3 ± 10.1, 60 ± 5, and 66 ± 7.6, respectively. No significant correlation was noted between Δ cardiac T2* and Δ LIC, Δ cardiac T2*, and Δ LVEF at years 3 and 5. Throughout the observation period, patients with no cardiac siderosis maintained their cardiac T2* above 20 msec. The majority of patients with cardiac siderosis improve cardiac T2* over time with optimal chelation. Am. J. Hematol. 88:652–656, 2013.

BO-1055, a novel DNA cross-linking agent with remarkable low myelotoxicity shows potent activity in sarcoma models

DNA damaging agents cause rapid shrinkage of tumors and form the basis of chemotherapy for sarcomas despite significant toxicities. Drugs having superior efficacy and wider therapeutic windows are needed to improve patient outcomes. We used cell proliferation and apoptosis assays in sarcoma cell lines and benign cells; γ-H2AX expression, comet assay, immunoblot analyses and drug combination studies in vitro and in patient derived xenograft (PDX) models. BO-1055 caused apoptosis and cell death in a concentration and time dependent manner in sarcoma cell lines. BO-1055 had potent activity (submicromolar IC50) against Ewing sarcoma and rhabdomyosarcoma, intermediate activity in DSRCT (IC50 = 2-3μM) and very weak activity in osteosarcoma (IC50 >10μM) cell lines. BO-1055 exhibited a wide therapeutic window compared to other DNA damaging drugs. BO-1055 induced more DNA double strand breaks and γH2AX expression in cancer cells compared to benign cells. BO-1055 showed inhibition of tumor growth in A673 xenografts and caused tumor regression in cyclophosphamide resistant patient-derived Ewing sarcoma xenografts and A204 xenografts. Combination of BO-1055 and irinotecan demonstrated synergism in Ewing sarcoma PDX models. Potent activity on sarcoma cells and its relative lack of toxicity presents a strong rationale for further development of BO-1055 as a therapeutic agent.

Plasma DNA-Based Molecular Diagnosis, Prognostication, and Monitoring of Patients With EWSR1 Fusion-Positive Sarcomas

Purpose Ewing Sarcoma (ES) and Desmoplastic Small Round Cell Tumors (DSRCT) are aggressive sarcomas molecularly characterized by EWSR1 gene fusions. As pathognomonic genomic events in these respective tumor types, EWSR1 fusions represent robust potential biomarkers for disease monitoring. Patients and Methods To investigate the feasibility of identifying EWSR1 fusions in plasma derived cell-free DNA (cfDNA) from ES and DSRCT patients, we evaluated two complementary approaches in samples from 17 patients with radiographic evidence of disease. The first approach involved identification of patient-specific genomic EWSR1 fusion breakpoints in formalin-fixed, paraffin-embedded tumor DNA using a broad, hybridization capture-based next generation sequencing (NGS) panel, followed by design of patient-specific droplet digital PCR (ddPCR) assays for plasma cfDNA interrogation . The second approach employed a disease-tailored targeted hybridization capture-based NGS panel applied directly to cfDNA which included EWSR1 as well as several other genes with potential prognostic utility. Results EWSR1 fusions were identified in 11/11 (100%) ES and 5/6 (83%) DSRCT samples by ddPCR, while 10/11 (91%) and 4/6 (67%) were identified by NGS. The ddPCR approach had higher sensitivity, ranging between 0.009-0.018% sensitivity. However, the hybrid capture-based NGS assay identified the precise fusion breakpoints in the majority of cfDNA samples, as well as mutations in TP53 and STAG2, two other recurrent, clinically significant alterations in ES, all without prior knowledge of the tumor sequencing results. Conclusion These results provide a compelling rationale for an integrated approach utilizing both NGS and ddPCR for plasma cfDNA-based biomarker evaluations in prospective cooperative group studies.

Intracardiac Low-grade Sarcoma Following Treatment for Ewing Sarcoma

A 16-year-old male was diagnosed with Ewing sarcoma of the ribcage with pulmonary metastases. Six months after completion of scheduled therapy, he was found to have a new intracardiac mass, presumed recurrent Ewing sarcoma. EWSR1 fusion was not detected by droplet digital polymerase chain reaction from blood plasma. After no improvement with salvage chemotherapy, he underwent surgical resection that identified a low-grade spindle cell sarcoma. Despite the near-synchronous presentation of 2 unrelated sarcomas, extensive genomic analyses did not reveal any unifying somatic or germline mutations nor any apparent cancer predisposition. This case also highlights the potential role of utilizing plasma cell-free DNA for diagnosing tumors in locations where biopsy confers high morbidity.

A single-center experience with undifferentiated embryonal sarcoma of the liver

Undifferentiated embryonal sarcoma of the liver (UESL) is a rare aggressive mesenchymal pediatric tumor. Previously, reported outcomes have been very poor. Here, we report a single‐center experience of five patients with UESL treated with upfront gross total resection and adjuvant chemotherapy. We have a median follow‐up of 8 years with a range from 5 to 19 years with 100% event‐free survival.

Abstract 1625: Ureidomustine, a novel DNA-crosslinking agent shows activity in sarcoma preclinical models and lacks toxicity in normal tissues

Ureidomustine (BO-1055) is a water-soluble N- mustard derivative that has antitumor activity against a number of human tumors including prostate, colon cancers and gliomas. In this study, we investigated its pre-clinical activity in sarcoma models. We tested the activity of BO-1055 on early passages from Ewing sarcoma patient samples and patient-derived xenografts, preclinical models of rhabdomyosarcoma, desmoplastic small round cell tumor (DSRCT) and osteosarcoma. We used cell proliferation, viability, clonogenicity and cell death assays, and immunoblotting for differential expression of proteins in cells that are sensitive and resistant to BO-1055. We studied the toxicity of BO-1055 on cardiac myocytes, hematopoietic stem and progenitor cells (HSPCs), epithelial, mesenchymal and endothelial cells, and performed animal toxicity experiments in mice. Efficacy of BO-1055 was tested in NSG mice bearing A673 xenografts (Ewing sarcoma) treated at doses of 10mg/kg, 20mg/kg and 30mg/kg, Q2D×5, via i.v. injection after the tumors grew to 250-500 mm3. We also investigated the efficacy of this agent in NSG mice bearing A204 xenografts and Ewing sarcoma patient-derived xenografts that were resistant to cyclophosphamide. The antitumor effects of BO-1055 in combination with standard chemotherapeutic agents were also evaluated. Ureidomustine exhibited significant cytotoxicity against the in vitro growth of Ewing sarcoma and rhabdomyosarcoma with sub μM IC50 values and was more cytotoxic than conventional DNA damaging agents. BO-1055 had less cytotoxicity against a panel of normal HSPCs. BO-1055 had moderate activity (IC50 = 2-4 μM) on DSRCT cells and had only weak activity (IC50>10μM) against osteosarcoma cell lines. Notably, this agent has no cross-resistance to Taxol and Vinblastine. Growth arrest in G2/M phase was noted at 24h and maximal apoptosis and cell death at 48h and 72h after exposure of A673 cells. Apoptosis was due to the induction of caspase 3 and 7 activity in a dose- and time- dependent manner. Cardiotoxicity was evaluated by binding of BO-1055 to hERG, which was significantly less than that of Astemizole indicating that BO-1055 has less propensity for QTc prolongation. BO-1055 significantly suppressed Ewing sarcoma patient-derived xenografts that were resistant to cyclophosphamide. Remarkably, this agent induced complete suppression of tumors in nude mice bearing A204 xenografts, without significant weight loss. In two-drug combination studies, BO-1055 exhibited synergism with etoposide, SN-38 (active metabolite of irinotecan), doxorubicin and PU-H71 (HSP-90 inhibitor). Because of its broad therapeutic window, lack of cross-resistance, better safety profile and synergistic cytotoxicity with standard drugs, BO-1055 has high potential for clinical application for treatment of Ewing sarcoma and rhabdomyosarcoma patients. Citation Format: Srikanth R. Ambati, Shieh JaeHung, Benet Pera, Elissa W.P. Wong, Eloisi Caldas Lopes, Elizabeth Peguero, Tsann-Long Su, Malcolm A.S. Moore. Ureidomustine, a novel DNA-crosslinking agent shows activity in sarcoma preclinical models and lacks toxicity in normal tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1625. doi:10.1158/1538-7445.AM2015-1625

Abstract PR09: High-throughput chemical screening identifies a novel class of proteasome inhibitors with significant activity against Ewing sarcoma

To identify novel small molecules with potential therapeutic activity against Ewing sarcoma (ES) and to define critical biological pathways in ES, we performed a high-throughput chemical screen, followed by functional, biochemical and genetic studies of 2 compounds of interest. We screened a library of 309,989 compounds for agents with selective activity against a panel of 5 ES cell lines. We identified 23 agents with significant activity (IC50 To further characterize these compounds, we synthesized additional analogs for in vitro and in vivo studies. One analog (compound E) was 10-fold more potent than compounds P and W in growth inhibition assays, and induced apoptosis and cell cycle arrest in multiple ES cell lines. Treatment of mice with ES xenograft tumors resulted in tumor growth regression, with significantly more efficacy than mice treated with bortezomib. A compound structurally very similar to compound E (b-AP15) has recently been demonstrated to inhibit the deubiquitinating enzymes USP14 and UCHL5 (D9Arcy P et al, Nat Med 2011). These enzymes associate with the 19S proteasome, and promote 20S proteasomal function. In a previous genome wide loss-of-function shRNA screen against ES cell line CHP100, we identified both USP14 and UCHL5 as hits, and have now validated these findings in multiple additional ES cell lines. To further investigate compound E as an alternative proteasome inhibitor, we studied its effect in a colon cancer cell line (HCT-116) with an established resistance mutation (PSMB5 M104V) to bortezomib. While the cell line demonstrated > 10-fold resistance to bortezomib as compared to the wild-type cell line, there was no significant difference in susceptibility to compounds E and P. We have identified a class of proteasome inhibitors with significant activity against in vitro and in vivo models of ES. Previous in vitro studies of 20S proteasome inhibitors, such as bortezomib, have demonstrated efficacy in ES and other solid tumor types. However in vivo studies and clinical trials of bortezomib in patients with solid tumors have yielded only modest effects. Alternative proteasome inhibitors may therefore be promising candidates for future therapy against ES as well as other solid tumor types. This abstract is also presented as Poster A36. Citation Format: Neerav Shukla, Romel Somwar, Srikanth Ambati, Roger S. Smith, Melinda Merchant, Rachel Kobos, Ouathek Ouerfelli, Hakim Djaballah, Marc Ladanyi. High-throughput chemical screening identifies a novel class of proteasome inhibitors with significant activity against Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR09.