Ewing Sarcoma Tumors Research Articles

Selected Articles from This Issue

Ewing sarcoma is largely driven by pathognomonic gene fusions involving EWSR1, particularly the EWS-FLI1 fusion, which are chiefly responsible for driving the transcriptional and metabolic rewiring associated with these cancers. However, previous work has also uncovered an oncogenic role for the scaffold protein menin in metabolic rewiring of Ewing sarcoma and other tumors, though the mechanism underlying this role is undefined. In this study, Jiménez and colleagues show that EWS-FLI1 and menin serve as upstream regulators of ATF4, a master regulator of the serine biosynthesis pathway. EWS-FLI1 directly binds the ATF4 promoter and both EWS-FLI1 and menin are integral for the induction of serine synthesis pathway downstream of ATF4, as inhibition of either gene resulted in loss of ATF4 expression and reduced activation of the serine biosynthesis gene program. Interestingly, menin inhibition also caused reduced ATF4 activity in B-cell acute lymphoblastic leukemia bearing MLL rearrangement, suggesting a broader role for menin in metabolic regulation. Taken together, the data uncover new mechanisms underlying the metabolic reprogramming observed in Ewing sarcoma and elucidate a pro-tumorigenic metabolic role for menin that may span a variety of different cancers.Prostate cancer is clinically managed by ablation of circulating testosterone and/or blockade of androgen receptor (AR) signaling, but invariably tumors progress to a castrate-resistant (CRPC) state. Numerous molecular drivers of castration resistance have been described, including hyperactivation of NF-κB signaling. However, strategies to overcome this resistance mechanism have proven elusive due to poor oral bioavailability of NF-κB pathway inhibitors. Here, Morel and colleagues demonstrate effective targeting of the NF-κB axis in CRPC in vivo using a novel orally bioavailable small molecule, dimethylaminoparthlide (DMAPT). Castration of mice bearing prostate tumors caused an increase in NF-κB signaling, leading to increased expression of AR and AR splice variants associated with a CRPC state. However, combination of castration with DMAPT prevented the induction of constitutively active AR variants. The authors show that this effect was specifically reliant on the effective inhibition of phosphorylated p65 NF-κB activity. Taken together, the data support the development of DMAPT as a therapeutic strategy to extend the efficacy of AR-targeted therapy by preventing or delaying the onset of castration resistance.Desmoplastic small round cell tumors (DSRCT) are rare, poorly differentiated cancers that are thought to arise from the peritoneum and which are marked by a pathognomonic EWSR1-WT1 gene fusion. The rarity of this tumor typ. has thus far precluded randomized trials and few laboratory models are available, highlighting an urgent need for additional insights and potential therapeutic targets. Here, Slotkin, Bowman, and their colleagues report genomic and transcriptomic data from a cohort of 78 DSRCT patients as well as a new bank of patient-derived xenografts for the study of DSRCT biology. The authors found that, aside from the pathognomonic fusion, DSRCT has a relatively stable genome with few recurrent mutations. Notably, however, FGFR4 was found to be highly expressed in DSRCT overall, with additional cases bearing FGFR4 amplification and/or activating mutations. These data provide new insights into the biology of DSRCT, including the role of FGFR4 as a potential therapeutic target, and present the first available bank of DSCRT laboratory models to the research community.c-Kit is a frequently activated proto-oncogene in acral and mucosal melanoma, but the c-Kit inhibitor dasatinib has limited efficacy in the clinic. In this study, Sabbah and colleagues perform pathway analysis to elucidate mechanisms of dasatinib resistance in melanoma. The authors report that dasatinib treatment achieves the goal of silencing the MAPK and PI3K/Akt pathways downstream of c-Kit, indicating an on-target effect. However, at pharmacological doses of dasatinib, the authors also observed activation of CREB transcriptional activity leading to induction of MITF and Bcl-2 expression via the CRTC3 pathway. Indeed, forced expression of MITF prevented any response to dasatinib in c-Kit mutant/amplified melanoma models, but inhibition of Bcl-2 using pharmacological agents restored sensitivity to dasatinib. Taken together, these data suggest that dasatinib treatment stimulates its own mechanism of resistance via induction of the pro-survival protein Bcl-2 and may also explain the modest benefits observed in dasatinib clinical trials.

Abstract 2163: Molecular characteristics of CDK4 and/or MDM2 amplification in Chinese soft tissue sarcoma (STS) patients

Abstract Background: Cyclin-dependent kinase 4 (CDK4) and mouse double minute 2 homolog (MDM2) amplification occur at high frequency in well-differentiated liposarcoma and dedifferentiated liposarcoma, which are generally considered to be diagnostic criteria for these tumors. Studies have also reported that CDK4 and/or MDM2 amplification can emerge in STS subtypes, such as Ewing's sarcoma (ES) and fibrosarcoma (FS). However, the clinical pathological and molecular characteristics of CDK4 and/or MDM2 amplification in STS are still unclear. We systematically depicted the clinical characteristics of MDM2 and CDK4 gene amplification in Chinese STS patients (pts), aiming to assist the precise diagnosis and treatment of STS. Methods: Totally 184 Chinese pts with different STS subtypes were included in this study. In terms of subtypes distribution, the cohort contained multiple STS subtypes including myofibroblastic sarcoma (MFS, n = 23), liposarcoma (LPS, n = 21), FS (n = 20), rhabdomyosarcoma (RMS, n = 19), liomyosarcoma (LMS, n = 12), undifferentiated pleomorphic sarcoma (n = 11), clear cell sarcoma (n = 10), synovial sarcoma (n = 8), ES (n = 7), hemangiosarcoma (n=7), other rare subtypes (n = 37) and unknown types (n = 9). The amplification status of CDK4 and MDM2 in the tumor tissues of these pts was reviewed by the DNA and RNA next generation sequencing method. Results: Totally 16.8% (31/184) of the Chinese STS pts harbored CDK4 and/or MDM2 amplification. Among the 31 pts, 17 were with co-amplification of CDK4 and MDM2, including 12 LPSs, two RMSs, one MFS, one LMS and one FS. Fourteen pts were with single amplification of MDM2 or CDK4, including LPS (n = 4), RMS (n = 3), MFS (n = 3), FS (n = 2), ES (n = 1), and malignant peripheral nerve sheath tumor (n = 1). In LPS, the ratios of co-amplification and single amplification of CDK4/MDM2 were 57.1% (12/21) and 19.0% (4/21) respectively. Other STS subtypes with high frequency of CDK4 and/or MDM2 amplification included RMS (26.3%, 5/19) and MFS (19.4%, 4/23). Meanwhile, our study revealed that co-amplification of CDK4 and MDM2 appeared to be a strong driver event in STS, which mutated exclusively with other driver genes, such as TP53, MYOD1, ATRX and PTEN. Pts with co-amplification of CDK4 and MDM2 were likely to benefit from the treatment of CDK4 and/or MDM2 inhibitors. However, more than half of the pts with single amplification of CDK4 or MDM2 were accompanied by mutations in other driven genes, such as FLI1-EWSR1 fusion, PAX3-FOXO1 fusion, MYOD1 p.L122R, TP53 p.R273S, and NRAS p.G13C. In these pts, single amplification of CDK4 or MDM2 may not be the main driver event, who may be less likely to benefit from CDK4 or MDM2 inhibitor monotherapy. Conclusion: Our study revealed the clinicopathologic and molecular features of CDK4 and/or MDM2 amplification in Chinese STS pts, which can guide the selection of personalized treatment strategies in STS pts. Citation Format: Gu Jin, Chunyang Wang, Xiaojuan Wang, Qifan He, Tonghui Ma. Molecular characteristics of CDK4 and/or MDM2 amplification in Chinese soft tissue sarcoma (STS) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2163.

Abstract 1546: Defining the immune microenvironment in Ewing's sarcoma to potentiate IL1RAP-targeted CAR-T immunotherapy

Abstract IL1RAP (Interleukin 1 receptor accessory protein) is differentially expressed in both Ewing sarcoma (EWS) and select hematologic malignancies (CML, AML). Using single domain antibody-like binders generated against IL1RAP to create chimeric antigen receptors (CARs) we demonstrated highly specific cytokine production and cytolytic activity against multiple EWS cell lines in vitro. Preliminary in vivo functionality of IL1RAP CAR-T indicates that additional approaches will be required to increase the efficacy of IL1RAP CAR-T for orthotopic EWS tumor models. To optimize CAR-T cell therapy for EWS, we used model cell lines to define tumor- and stromal-derived factors that must be overcome to obtain effective treatment. The human cell line TC71 (fusion gene positive) was administered both i.v. and i.m. to NSG mice, and subsequently treated with IL1RAP-specifc CAR-T cells. Tumor engraftment was not seen in the i.v. model, however the orthotopic i.m. model in NSG mice was successful, generating tumor and metastatic lesions surrounded by a collagen rich stromal element when CAR-T or control activated T cells were administered. This was not seen in control untreated tumors. Histochemical analysis of i.m. tumors demonstrated that T cells (CD3 positive) were confined to the tumor stromal boundary in proximity to cells of myeloid lineage (CD11b positive). We therefore analyzed cell surface and soluble factors produced by TC71 by flow cytometry, ELISA, and NanoString mRNA panels. TC71 was cultured alone or with interferon-gamma (IFNG), to model the presence of activated T cells and was found to constitutively express transcripts for both canonical and non-canonical chemokines: GPI, MIF, IL-32, TGFβ1; whereas CSF1, CXCL9, CXCL10 and CXCL11 were only induced by IFNG. MIF and TGF-β1 production were confirmed by ELISA. Flow cytometry revealed that numerous CAR-T targets (IL1RAP, CD276, EGFR and FGFR4) were expressed, as were the immune inhibitory molecules: PDL1, PDL2, Galectin-9, and CTLA-4. MHC class I and II and PDL1 were strongly induced by IFNG. We also observed transcripts for collagen related genes in TC71: COL16A1, 3A1, 5A3, LOXL1, P4HB and SERPINH1 both in vitro and in vivo. Our results indicate that tumor surface expression of immune inhibitory signals, PDL-1, PDL-2, CTLA4, as well as the soluble factors MIF, and TGF-b1, must be considered in armoring CAR-T for effective immune destruction of EWS, along with strategies designed to increase the transmigration of T cells through matrix proteins. Moreover, the chemokines produced by tumor alone, as well as those induced by IFNG, indicate a complex network for recruitment of local and bone marrow-derived stromal cells that create a physical as well as cellular barrier to adoptive immunotherapy which needs to be addressed for effective CAR-T therapy of this solid tumor. Citation Format: Rajesh Kumar, Peter Sullivan, Wei Li, Haifeng Zhang, Virginia Hoglund, Lingyang Wang, Yue Zhang, Poul H. Sorensen, Dimiter S. Dimitrov, Rimas J. Orentas. Defining the immune microenvironment in Ewing's sarcoma to potentiate IL1RAP-targeted CAR-T immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1546.

Haploidentical Hematopoietic Cell Transplantation Followed By Donor Natural Killer Cell Infusion and Sirolimus Maintenance Can Successfully Treat Patients with High-Risk Ewing Sarcoma

Background: Approximately 30-40% of patients with newly diagnosed localized Ewing Sarcoma (EWS), and 60-80% with disseminated EWS, will eventually relapse. Patients with late relapse have a 5 year overall survival (OS) of 30%, while patients with early relapse (<2 years from initial diagnosis) have a more dismal 5 year OS of <10% (Stahl Ped Blood Cancer 2011). Unfortunately, conventional treatments including chemotherapy, autologous transplantation, surgery, and radiation therapy often lead to additional morbidities without substantial gains in disease remissions or survival. Allogeneic hematopoietic cell transplantation (HCT) using human leukocyte antigen (HLA)-matched related donors has shown some success for patients with high-risk EWS (Baird BBMT 2012). Additionally, EWS cells are exquisitely sensitive to natural killer (NK) cell cytotoxic activity (Cho CCR 2010), and many EWS tumors have upregulated the mammalian target of rapamycin (mTOR) pathway making them susceptible to anti-mTOR agents (Wan et al The Oncologist 2007). With growing literature supporting the safety and efficacy of HLA-haploidentical HCT with post-transplant cyclophosphamide (CY) for hematological malignancies, we developed a novel treatment regimen adding a single infusion of donor NK cells on day +7 after HLA-haploidentical HCT, followed by sirolimus as part of a GVHD prophylaxis strategy. We hypothesized that this multi-faceted immunotherapy approach, in combination with an mTOR-inhibitor post-transplant, would be well-tolerated and efficacious in patients with relapsed EWS.Methods: Patients received reduced-intensity conditioning using fludarabine (150 mg/m2), CY (29 mg/kg) and 3 Gy total body irradiation, followed by infusion of HLA-haploidentical related marrow (n=4) or peripheral blood stem cells (n=2). Post-transplant immune suppression consisted of CY (50 mg/kg, days +3 and +4), tacrolimus (beginning day +5, and transitioned to sirolimus from day +28 to 6 months), and mycophenolate mofetil (MMF) (day +9 to +84). MMF initiation was delayed until day +9 to decrease concerns of inhibiting NK activity. For NK cell therapy, non-mobilized peripheral blood mononuclear cells were collected from the same haplo donors on day +6 using apheresis. NK cells were selected on day +7 using the Miltenyi CliniMACS system (CD3 depletion followed by CD56 selection) and were administered as a single, fresh infusion that same day without prior culturing, expansion, or exogenous cytokine exposure.Results: Four pediatric and 2 adult patients with median age of 18.5 (10.5-34.0) years having relapsed EWS were enrolled on this Phase II study. All patients had stable or undetectable gross disease at HCT. Patients had 1 (n=3), 2 (n=2), or 5 (n=1) relapses prior to HCT. Median time from EWS diagnosis to first relapse was 22.5 (15-56) months. Two patients had prior autologous HCTs. Median HCT-CI score at time of transplant was 4 (2-5), demonstrating increased risk for transplant-related mortality (TRM). Patients received a median CD34 dose of 5.24 (1.3-8.3) x 106/kg. NK cell products had a median log T cell depletion of 6.25 (5.18-6.42), median NK recovery of 54% (47-71%), and median NK purity of 91% (78-96%). Excellent viability (>99%) was seen in all NK products. Donor NK infusions were well-tolerated without fevers or other adverse reactions. Sustained full donor chimerism (>95% CD3) was achieved in all 6 patients. One patient developed stage 1 (grade II) gut graft-versus-host disease (GVHD) at day +52 which was treated with budesonide. To date, no patients have developed chronic GVHD nor have died from TRM. With a median follow-up of 1.3 years (range, 180 days - 2.7 years), 1 year overall survival is estimated at 75% (see figure). Three patients have relapsed or progressed at 187, 284, and 455 days after HCT, of which 1 patient (who had 5 prior relapses), died of progressive disease at day +350. The other 2 patients have received salvage treatments. They remain alive with full-donor chimerism and low-level/absent disease at 1.7 and 2.7 years post-HCT.Summary: This dual immunotherapy approach, using HLA-haploidentical HCT with donor NK cell infusion, followed by sirolimus maintenance, was well-tolerated by all patients. While longer follow-up is needed, preliminary results of this ongoing novel approach demonstrate feasibility in EWS patients, with better than expected 1 year OS and promising disease control. [Display omitted] DisclosuresThakar:Miltenyi Biotec: Other: funding of partial costs - disposable agents. Hari:Janssen: Honoraria; Sanofi: Honoraria, Research Funding; Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy; Takeda Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Spectrum: Consultancy, Research Funding; Amgen: Consultancy, Honoraria, Research Funding.

Ion therapy guideline (Version 2020)

Ion therapy guideline (Version 2020)

A review of Leila Rose Foundation support for families affected by rare childhood cancer in Australia over the past decade.

BackgroundThe Leila Rose Foundation (“the Foundation”) was established in April 2011, to address financial toxicity as well as the gaps in knowledge and support for families affected by a rare childhood cancer diagnosis in Australia.AimThe aim of this brief report is to analyze the diagnostic trends surrounding the rare cancer diagnoses for patients referred to the Foundation over the past decade and to present case studies evaluating the role of the Foundation's Family Support Coordinator in providing tailored, individualized support for families.MethodsEligibility for family support is restricted to children ≤ 14 years of age at diagnosis with a cancer that has an incidence less than 5% of all childhood cancers in Australia as reflected by national registry data. The analysis of diagnostic trends in this report, was based upon a systematic review of enrolment records. The role of the Family Support Coordinator is presented in four different case studies.ResultsAs at 1 November 2020, the Foundation has supported 197 families affected by rare childhood cancer. Financial support of $825,000 has been provided directly to these families. Enrollment records demonstrate that 35 patients representing 18% of all enrollments have had a unique diagnosis that has not been recorded for any other enrolled patient highlighting that these diagnoses are very rare. The most frequent diagnoses have included Medulloblastoma, Ewing's Sarcoma and Wilm's Tumor (20, 19, 19 patients respectively). The Family Support Coordinator role has provided individualized support for families which has been greatly appreciated based upon ad hoc family feedback.ConclusionsChallenges remain in terms of improving outcomes for families affected by rare childhood cancer. The Foundation is committed to leaving no stone unturned and delivering its unique support services to families in order to reduce the burden caused by a rare childhood cancer diagnosis both now and in the future.

Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma

Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10mg/kg once a day, intraperitoneal administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with invivo efficacy reported to date.

Targeting EYA3 in Ewing Sarcoma Retards Tumor Growth and Angiogenesis.

EWSR1/FLI1, the most common fusion gene in Ewing sarcoma, upregulates expression of the Eyes Absent 3 (EYA3) transactivator-phosphatase protein. The purpose of this study was to investigate molecular and cellular mechanisms through which EYA3 might promote Ewing sarcoma tumor growth and to determine whether the EYA3 tyrosine phosphatase activity represents a viable therapeutic target. We used genetic and pharmacologic modulation of EYA3 in cell line-based xenografts to examine how loss of EYA3 tyrosine phosphatase activity affects tumor growth and angiogenesis. Molecular mechanisms were evaluated in vivo and in vitro through analyses of tumor tissue and multicellular tumor spheroids. Our results show that both loss of EYA3 in Ewing sarcoma cells and pharmacologic inhibition of the EYA3 tyrosine phosphatase activity inhibit tumor growth and tumor angiogenesis. EYA3 regulates levels of VEGFA in Ewing tumors, as well as promoting DNA damage repair and survival of Ewing sarcoma tumor cells. Target engagement is demonstrated in tumor tissue through elevated levels of the EYA3 substrate H2AX-pY142 upon loss of EYA3 or with Benzarone treatment. The efficacy of EYA3 tyrosine phosphatase inhibition in attenuating tumor growth and angiogenesis is corroborated in an Ewing sarcoma patient-derived tumor xenograft. Together, the results presented here validate EYA3 as a target for the development of novel Ewing sarcoma therapeutic strategies, and set the stage for evaluating the efficacy of combining the antiangiogenic and anti-cell survival effects of EYA3 inhibition with cytotoxic chemotherapy.

The Clinical Characteristics and Prediction Nomograms for Primary Spine Malignancies.

BackgroundPrimary spine malignancies (PSMs) are relatively rare in bone tumors. Due to their rarity, the clinical characteristics and prognostic factors are still ambiguous. In this study, we aim to identify the clinical features and proposed prediction nomograms for patients with PSMs.MethodsPatients diagnosed with PSMs including chordoma, osteosarcoma, chondrosarcoma, Ewing sarcoma, and malignant giant cell tumor of bone (GCTB) between 1975 and 2016 were selected from the Surveillance, Epidemiology, and End Results (SEER) database. The patient and tumor characteristics were described based on clinical information. The significant prognostic factors of overall survival (OS) and cancer-specific survival (CSS) were identified by the univariate and multivariate Cox analysis. Then, the nomograms for OS and CSS were established based on the selected predictors and their accuracy was explored by the Cox–Snell residual plot, area under the curve (AUC) of receiver operator characteristic (ROC) and calibration curve.ResultsThe clinical information of 1,096 patients with PSMs was selected from the SEER database between 1975 and 2016. A total of 395 patients were identified with full survival and treatment data between 2004 and 2016. Chordoma is the commonest tumor with 400 cases, along 172 cases with osteosarcoma, 240 cases with chondrosarcoma, 262 cases with Ewing sarcoma and 22 cases with malignant GCTB. The univariate and multivariate analyses revealed that older age (Age > 60), distant metastasis, chemotherapy, and Surgery were independent predictors for OS and/or CSS. Based on these results, the nomograms were established with a better applicability (AUC for CSS: 0.784; AUC for OS: 0.780).ConclusionsThis study provides the statistics evidence for the clinical characteristics and predictors for patients with PSMs based on a large size population. Additionally, precise prediction nomograms were also established with a well-applicability.

Radical radiotherapy for paediatric solid tumour metastases: An overview of current European protocols and outcomes of a SIOPE multicenter survey.

About 20% of children with solid tumours (ST) present with distant metastases (DM). Evidence regarding the use of radical radiotherapy of these DM is sparseand open for personal interpretation. The aim of this survey was to review European protocols and to map current practice regarding the irradiation of DM across SIOPE-affiliated countries. Radiotherapy guidelines for metastatic sites (bone, brain, distant lymph nodes, lung and liver) in eight European protocols for rhabdomyosarcoma, non-rhabdomyosarcoma soft-tissue sarcoma, Ewing sarcoma, neuroblastomaand renal tumours were reviewed. SIOPE centres irradiating ≥50 children annually were invited to participate in an online survey. Radiotherapy to at least one metastatic site was recommended in all protocols, except for high-risk neuroblastoma. Per protocol, dose prescription varied per site, and information on delineation and treatment planning/delivery was generally missing. Between July and September 2019, 20/27 centres completed the survey. Around 14% of patients were deemed to have DM from ST at diagnosis, of which half were treated with curative intent. A clear cut-off for a maximum number of DM was not used in half of the centres. Regardless of the tumour type and site, conventional radiotherapy regimens were most commonly used to treat DM. When stereotactic radiotherapy was used, a wide range of fractionation regimens were applied. Current radiotherapy guidelines for DM do not allow a consistent approach in a multicentre setting. Prospective (randomised) trials are needed to define the role of radical irradiation of DM from paediatric ST.

P21-activated kinases as viable therapeutic targets for the treatment of high-risk Ewing sarcoma.

Ewing sarcoma (ES) is the second most common bone tumor in children and young adults. Unfortunately, there have been minimal recent advancements in improving patient outcomes, especially in metastatic and recurrent diseases. In this study, we investigated the biological role of p21-activated kinases (PAKs) in ES, and the ability to therapeutically target them in high-risk disease. Via informatics analysis, we established the inverse association of PAK1 and PAK4 expression with clinical stage and outcome in ES patients. Through expression knockdown and small-molecule inhibition of PAKs, utilizing FRAX-597, KPT-9274, and PF-3758309 in multiple ES cell lines and patient-derived xenograft models, we further explored the role of PAKs in ES tumor growth and metastatic capabilities. In vitro studies in several ES cell lines indicated that diminishing PAK1 and PAK4 expression reduces tumor cell viability, migratory, and invasive properties. In vivo studies using PAK4 inhibitors, KPT-9274 and PF-3758309 demonstrated significant inhibition of primary and metastatic tumor formation, while transcriptomic analysis of PAK4-inhibitor-treated tumors identified concomitant suppression of Notch, β-catenin, and hypoxia-mediated signatures. In addition, the analysis showed enrichment of anti-tumor immune regulatory mechanisms, including interferon (IFN)-ɣ and IFN-α responses. Altogether, our molecular and pre-clinical studies are the first to establish a critical role for PAKs in ES development and progression, and consequently as viable therapeutic targets for the treatment of high-risk ES in the near future.

Abstract PO-041: Genome-wide CRISPR/Cas9 screen reveals mitochondrial gene mutation as a driver for drug resistance in Ewing sarcoma

Abstract Ewing sarcoma is a pediatric bone cancer caused by a chromosomal translocation event that generates the pathognomonic fusion protein, EWS/FLI, which acts as aberrant transcription factor. EWS/FLI is responsible for activating and repressing important genes to drive oncogenic transformation and tumorigenesis. EWS/FLI is notoriously difficult to target with small molecule inhibitors based on its lack of enzymatic activity and intrinsically disordered EWS domain, thus rational drug design is presently unfeasible. Other potential therapeutic targets are interacting partners of EWS/FLI, including epigenetic modulators, such as lysine specific demethylase-1 (LSD1). Inhibition of LSD1 by a reversible, non-competitive inhibitor, SP-2509, has been shown to cause extensive changes to gene expression and induce apoptosis in Ewing sarcoma cells. A Phase I clinical trial (NCT03600649) is currently ongoing to study the effect an SP-2509 analog, Seclidemstat (SP-2577), on patients with recurrent or refractory Ewing sarcoma tumors. Thus, understanding potential drug resistance mechanisms is vital to ensure maximal efficacy of Seclidemstat treatment for patients in the present, and in future clinical trials with this targeted therapeutic drug. Preliminary investigation using a genome-wide CRISPR/Cas9 knock-out (GeCKo) screen has revealed that the loss of genes important to mitochondrial function, specifically in the electron transport chain (ETC), play a distinct role in SP-2509 drug-resistance in Ewing sarcoma cells. We validated the GeCKo screen using cell viability assays in an Ewing sarcoma cell line (A673) via knockout (KO) of mitochondrial genes that were identified by the GeCKo screen. Consistent with the screen, we found select mitochondrial genes led to an increase in IC50 values (i.e. drug-resistance) to SP-2509, compared to WT A673 cells. We found that drug resistance could be enhanced even greater by generating monoclonal cell populations containing complete mitochondrial gene KO. We performed RNA-sequencing and metabolomics analyses to examine SP-2509 response differences between WT and our generated drug resistance cell lines. We similarly determined that treating A673 cells with mitochondrial ETC inhibitors recapitulated drug resistance. Currently, the mechanism that links mitochondrial dysfunction in Ewing sarcoma cells and resistance to the epigenetic drug SP-2509 is incompletely understood. However, our ongoing studies aim to uncover this link and ultimately provide consequential information that will help patients overcome Ewing sarcoma. Citation Format: Jack P. Tokarsky, Jesse Crow, Lillian Guenther, Emily Theisen, Kimberly Stegmaier, Stephen Lessnick. Genome-wide CRISPR/Cas9 screen reveals mitochondrial gene mutation as a driver for drug resistance in Ewing sarcoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-041.

Eltrombopag inhibits the proliferation of Ewing sarcoma cells via iron chelation and impaired DNA replication

BackgroundThe treatment of Ewing sarcoma, an aggressive bone and soft tissue sarcoma, is associated with suboptimal outcomes and significant side-effects. Consequently, there is an urgent need to identify novel therapies that will improve outcomes for children and adults with Ewing sarcoma tumors while also decreasing treatment-related toxicities.MethodsWe analyzed data from the PRISM drug repurposing screen, which tested the activity of 4518 drugs across 578 cancer cell lines, to identify drugs that selectively inhibit the growth of Ewing sarcoma cell lines. We then tested the effects of a top hit from the screen on cell proliferation, cell cycle progression, and activation of the DNA damage pathway using Ewing sarcoma cell lines. We also used a CRISPR/Cas9 gene knockout approach to investigate the role of Schlafen 11 (SLFN11), a restriction factor for DNA replication stress that is overexpressed in Ewing sarcoma tumors, in mediating the sensitivity of Ewing sarcoma cells to the drug.ResultsWe found that eltrombopag, an FDA-approved thrombopoietin-receptor agonist (TPO-RA) that is currently being evaluated as a treatment for chemotherapy-induced thrombocytopenia, inhibits the growth of Ewing sarcoma cell lines in vitro in proliferation and colony formation assays. However, from a mechanistic standpoint, the thrombopoietin receptor is not expressed in Ewing sarcoma cells and we show that eltrombopag impairs DNA replication and causes DNA damage in Ewing sarcoma cells by chelating iron, a known “off-target” effect of the drug. We also found that the sensitivity of Ewing sarcoma cells to eltrombopag is mediated, in part, by SLFN11, which regulates the cellular response to DNA replication stress.ConclusionsEwing sarcoma cell lines are sensitive to eltrombopag and this drug could improve outcomes for patients with Ewing sarcoma tumors by both targeting the tumor, via chelation of iron and inhibition of DNA replication, and reducing chemotherapy-induced thrombocytopenia, via stimulation of the thrombopoietin receptor.

A novel HDGF-ALCAM axis promotes the metastasis of Ewing sarcoma via regulating the GTPases signaling pathway.

Ewing sarcoma (ES) is a type of highly aggressive pediatric tumor in bones and soft tissues and its metastatic spread remains the most powerful predictor of poor outcome. We previously identified that the transcription factor hepatoma-derived growth factor (HDGF) promotes ES tumorigenesis. However, the mechanisms underlying ES metastasis remain unclear. Here, we show that HDGF drives ES metastasis in vitro and in vivo, and HDGF reduces metastasis-free survival (MFS) in two independent large cohorts of human ES patients. Integrative analyses of HDGF ChIP-seq and gene expression profiling in ES cells reveal that HDGF regulates multiple metastasis-associated genes, among which activated leukocyte cell adhesion molecule (ALCAM) emerges as a major HDGF target and a novel metastasis-suppressor in ES. HDGF down-regulates ALCAM, induces expression and activation of the downstream effectors Rho-GTPase Rac1 and Cdc42, and promotes actin cytoskeleton remodeling and cell-matrix adhesion. In addition, repression of ALCAM and activation of Rac1 and Cdc42 are required for the pro-metastatic functions of HDGF in vitro. Moreover, analyses in murine models with ES tumor orthotopic implantation and experimental metastasis, as well as in human ES samples, demonstrate the associations among HDGF, ALCAM, and GTPases expression levels. Furthermore, high HDGF/low ALCAM expression define a subgroup of patients harboring the worst MFS. These findings suggest that the HDGF/ALCAM/GTPases axis represents a promising therapeutic target for limiting ES metastasis.

The translational repressor 4E-BP1 regulates RRM2 levels and functions as a tumor suppressor in Ewing sarcoma tumors.

Ribonucleotide reductase (RNR), which is a heterodimeric tetramer composed of RRM1 and RRM2 subunits, is the rate-limiting enzyme in the synthesis of deoxyribonucleoside triphosphates (dNTPs) and essential for both DNA replication and the repair of DNA damage. The activity of RNR is coordinated with the cell cycle and regulated by fluctuations in the level of the RRM2 subunit. Multiple cancer types, including Ewing sarcoma tumors, are sensitive to inhibitors of RNR or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. Here, we show that the expression of the RRM2 protein is dependent on active protein synthesis and that 4E-BP1, a repressor of cap-dependent protein translation, specifically regulates the level of the RRM2 protein. Furthermore, inhibition of mTORC1/2, but not mTORC1, activates 4E-BP1, inhibits protein synthesis, and reduces the level of the RRM2 protein in multiple sarcoma cell lines. This effect of mTORC1/2 inhibitors on protein synthesis and RRM2 levels was rescued in cell lines with the CRISPR/Cas9-mediated knockout of 4E-BP1. In addition, the inducible expression of a mutant 4E-BP1 protein that cannot be phosphorylated by mTOR blocked protein synthesis and inhibited the growth of Ewing sarcoma cells in vitro and in vivo in a xenograft. Overall, these results provide insight into the multifaceted regulation of RRM2 protein levels and identify a regulatory link between protein translation and DNA replication.

Chimeric Antigen Receptor-modified T cells targeting EphA2 for the immunotherapy of paediatric bone tumours

Chimeric Antigen Receptor (CAR) T-cell therapy, as an approved treatment option for patients with B cell malignancies, demonstrates that genetic modification of autologous immune cells is an effective anti-cancer regimen. Erythropoietin-producing Hepatocellular receptor tyrosine kinase class A2 (EphA2) is a tumour associated antigen expressed on a range of sarcomas, including paediatric osteosarcoma (OS) and Ewing sarcoma (ES). We tested human EphA2 directed CAR T cells for their capacity to target and kill human OS and ES tumour cells using in vitro and in vivo assays, demonstrating that EphA2 CAR T cells have potent anti-tumour efficacy in vitro and can eliminate established OS and ES tumours in vivo in a dose and delivery route dependent manner. Next, in an aggressive metastatic OS model we demonstrated that systemically infused EphA2 CAR T cells can traffic to and eradicate tumour deposits in murine livers and lungs. These results support further pre-clinical evaluation of EphA2 CAR T cells to inform the design of early phase clinical trial protocols to test the feasibility and safety of this immune cell therapy in paediatric bone sarcoma patients.

Pediatric sarcomas display a variable EpCAM expression in a histology-dependent manner

EpCAM is a transmembrane glycoprotein typically overexpressed in cancer of epithelial origin and mainly involved in the epithelial-to–mesenchymal transition (EMT) of tumor cells that spread and disseminate. Strategies for the targeting and capture of EpCAM-expressing tumor cells are showing promise in cancers prone to metastatize, both as diagnostic tools and potential therapies. Sarcomas are among the most aggressive tumors in children, with a common mesenchymal origin that comprises both soft tissue sarcomas (STS) and bone sarcomas. The aim of this study was to assess EpCAM expression in pediatric sarcomas and correlate its expression with disease progression. To do so, we analyzed a set of cell lines and primary tumor tissues from rhabdomyosarcoma (RMS), Ewing sarcoma (ES), synovial sarcoma (SS) and desmoplastic small round cell tumor (DSRCT) STS, or osteosarcoma (OS) bone cancer. We demonstrated that EpCAM was variably expressed in pediatric sarcomas, with DSRCT, a rare, aggressive and almost fatal tumor type, characterized by the highest EpCAM expression levels. Interestingly, although EpCAM expression was lower in RMS tumors, high levels at diagnosis correlated with reduced patients' overall survival (p < 0.05). Indeed, membrane-bound EpCAM was detected in circulating sarcoma tumor cells, revealing its potential to be used as dissemination biomarker in this type of childhood cancers. This reinforces the concept that pediatric sarcomas do express both epithelial and mesenchymal markers and reside in an intermediate condition that most likely contributes to their aggressive phenotype and low survival rate.

Abstract 6594: Targeting Ewing sarcoma and osteosarcoma with anti-MCAM chimeric antigen receptor modified NK cells

Abstract Ewing sarcoma and osteosarcoma are the two most common pediatric malignant bone tumors. Patients who present with metastatic disease or relapse have a dismal average 5-year survival of only 20%. Novel therapeutic approaches are desperately needed. The melanoma cell adhesion molecule (MCAM/CD146/MUC18) is highly expressed in sarcoma and metastatic pediatric tumors. MCAM depletion inhibits Ewing sarcoma cell migration in vitro and tumor metastasis in vivo. High MCAM levels in patient tumors are associated with poor survival in Ewing sarcoma. MCAM therefore constitutes a unique and novel target for immunotherapy against MCAM+ sarcomas. We previously demonstrated that natural killer (NK) cell engineered to express chimeric antigen receptor (CAR) against CD20 significantly reduced tumor burden, prevented tumor dissemination, and extended survival of treated mice compared to mock NK treatment in humanized Burkitt lymphoma xenograft mouse model. We hypothesize that anti-MCAM CAR NK cell will enhance the inherent NK-sarcoma cytotoxicity by better targeting sarcoma cells. Here we develop an anti-MCAM CAR NK cell and investigate its efficacy in promoting NK cell cytotoxicity against Ewing sarcoma and osteosarcoma. MCAM expression in sarcoma cell lines and tumor samples were validated by flow cytometry and western blotting. Peripheral blood mononuclear cells (PBMCs) are expanded into NK cells ex vivo using K562-mbIL21-41BBL artificial antigen presenting cells (aAPC). Anti-MCAM CAR NK cells are generated by non-viral electroporation of anti-MCAM CAR mRNA into expanded NK cells. Anti-MCAM CAR NK cytotoxicity is evaluated in vitro by luciferase based cytotoxicity assay. We detected various levels of MCAM expression in Ewing sarcoma and osteosarcoma cell lines and tumors. In addition, we noticed an interesting patchy expression pattern of MCAM in the Ewing sarcoma tumor sample. Importantly, we found a significantly increased cytotoxicity of anti-MCAM CAR NK cells compared to mock NK cells (59.9±10 vs 13.3±3.7 at E:T ratio of 5:1, p=0.006; and 65.6±7 vs 13.7±3.4 at 10:1, p=0.004) against Ewing sarcoma TC71 cells after co-culturing for 4 hours. A similar result was observed at the time point of 48 hours (91.5±8 vs 59±0.5 at E:T of 5:1, p=0.018). Anti-MCAM CAR NK cells also showed significantly enhanced cytotoxicity against U2OS osteosarcoma cells compared to mock NK cells (32.6±23.2 vs 0.5±19.5 at E:T of 0.5:1, p=0.004; 47.1±27.4 vs 10.2±24 at E:T of 1:1, p=0.001) at the time point of 48 hours. In summary, these findings demonstrated enhanced efficacy of anti-MCAM CAR NK cells compared to mock NK cells against Ewing sarcoma and osteosarcoma cells and support a further preclinical investigation whether targeting MCAM by CAR NK will limit sarcoma growth and/or metastasis and increase survival in vivo. Citation Format: Aliza Gardenswartz, Wen Luo, Jeremy M. Rosenblum, Janet Ayello, Mitchell S. Cairo. Targeting Ewing sarcoma and osteosarcoma with anti-MCAM chimeric antigen receptor modified NK cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6594.

CDK9 as a Valuable Target in Cancer: From Natural Compounds Inhibitors to Current Treatment in Pediatric Soft Tissue Sarcomas.

Cyclin-Dependent Kinases (CDKs) are well-known reliable targets for cancer treatment being often deregulated. Among them, since the transcription-associated CDK9 represents the sentry of cell transcriptional homeostasis, it can be a valuable target for managing cancers in which the transcriptional machinery is dysregulated by tumor-driver oncogenes. Here we give an overview of some natural compounds identified as CDK inhibitors with reported activity also against CDK9, that were taken as a model for the development of highly active synthetic anti-CDK9 agents. After, we summarize the data on CDK9 inhibition in a group of rare pediatric solid tumors such as rhabdomyosarcoma, Ewing’s sarcoma, synovial sarcoma and malignant rhabdoid tumors (soft tissue sarcomas), highlighting the more recent results in this field. Finally, we discuss the perspective and challenge of CDK9 modulation in cancer.

Abstract 5714: Targeting serine metabolism impairs tumor growth in preclinical models of Ewing sarcoma

Abstract Despite a growing body of knowledge about the genomic landscape of Ewing sarcoma (ES), translation of basic discoveries into targeted therapies and significant clinical gains has remained elusive. Recent insights have revealed that the oncogenic transcription factor EWS-FLI1 can impact ES cellular metabolism, raising the possibility of targeting metabolic dependencies as a novel therapeutic strategy. Here, we show that 3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme in de novo serine synthesis, is highly expressed in human ES cells and that EWS-FLI1 regulates PHGDH expression and de novo serine synthesis. PHGDH knockdown resulted in decreased ES cell proliferation, especially under serine limitation, and significantly inhibited xenograft tumorigenesis in preclinical orthotopic models of ES. Additionally, the PHGDH inhibitor NCT-503 caused a dose-dependent decrease in cellular proliferation. Moreover, we report a novel drug combination in which nicotinamide phosphoribosyltransferase (NAMPT) inhibition, which blocks production of the PHGDH substrate NAD+, synergized with NCT-503 to abolish ES cellular proliferation and tumor growth. Furthermore, we show that serine deprivation inhibited ES cell proliferation and tumorigenesis, indicating that ES cells depend on exogenous serine in addition to de novo serine synthesis. Our findings suggest that serine metabolism is critical for ES tumorigenesis, and that targeting serine metabolism should be further investigated as a potential therapeutic strategy for ES. Citation Format: Sameer Issaq, Arnulfo Mendoza, Tracy Rosales, Christine Heske, Craig Thomas, Ralph DeBerardinis, Lee Helman. Targeting serine metabolism impairs tumor growth in preclinical models of Ewing sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5714.