Grant Funds Research on the Nrf2 Pathway in Ewing's Sarcoma
The Liddy Shriver Sarcoma Initiative is pleased to partner with the Brian Morden Foundation, the Wendy Walk and the Alan B. Slifka Foundation to fund a $100,000 grant for Ewing's sarcoma research at Johns Hopkins University. In the study, investigators aim to discover how a gene called Nrf2 helps make Ewing's sarcoma cells resistant to chemotherapy. Then they plan to interfere with the gene's function in the hopes of learning how to improve treatment response in patients.
Ewing's sarcoma is a rare and aggressive cancer that is usually diagnosed in children and young adults. Investigator David Loeb, MD, PhD explains the challenge the disease presents: "One of the biggest frustrations in taking care of kids with Ewing sarcoma is that these tumors are almost all very sensitive to chemotherapy in the beginning, but for too many kids, the tumor comes back. When it does, it is highly resistant and we have nothing curative to offer."
Shyam Biswal, PhD, the study's lead investigator, has been studying Nrf2 in lung cancers for about a decade, and it has emerged as an important driver of drug resistance in lung cancers. He believes that the same may be true of sarcomas: "We have some early hint that Nrf2 could be important for therapy resistance in sarcomas. Our studies with this grant will validate the target in sarcoma and test inhibitors of Nrf2. If these studies are positive, we can establish a solid foundation for developing a novel strategy to overcome therapeutic resistance in Ewing's sarcoma."
Dr. Loeb is hopeful about the potential of this research: "I think that if we are able to overcome some of the mechanisms of drug resistance in Ewing's sarcoma, our current treatment approaches will be more effective and cure rates will improve."
The Importance of Sarcoma-Specific Research
By Dr. David Loeb
Sarcomas are very different from carcinomas, especially at the genetic level. As more and more studies are published looking for mutations in cancers, it is becoming more and more clear that, unlike carcinomas, which tend to accumulate very large numbers of mutations, sarcomas have relatively few mutations in them. This means research focused on identifying these mutations will not likely have the same impact on sarcomas as on carcinomas, and different, non-mutational ways of changing gene expression must be identified.
This $100,000 grant is funded by the Liddy Shriver Sarcoma Initiative in partnership with the Brian Morden Foundation, the Wendy Walk and the Alan B. Slifka Foundation. Among other things, the Brian Morden Foundation funds Ewing's Sarcoma and childhood cancer research and supports patients, family, and staff of pediatric oncology units. The Wendy Walk continues to build on its five year history of providing hope, strength, and courage to those fighting these rare cancers while funding important international collaborative sarcoma research projects. The Alan B. Slifka Foundation is a private grant-making foundation that is dedicated to making a world safe for difference and healing. The Liddy Shriver Sarcoma Initiative greatly appreciates these very productive partnerships.
The Initiative also gratefully acknowledges the following support for this research: a donation from the Arlo and Susan Ellison family, donations from the friends of Max Ritvo, and donations made in memory of Michael Lio, Christi Campbell, and Nick Strub (the Nick Teddy Foundation).
ESUN Bulletin Copyright © January 2015 Liddy Shriver Sarcoma Initiative.
Developing Novel Therapeutic Intervention for Ewing Sarcoma
Ewing sarcoma is the second most common bone tumor in children, adolescents, and young adults. The introduction of systemic chemotherapy has dramatically improved the prognosis of patients presenting with localized disease, and current treatment regimens result in long term survival rates of approximately 75%.1 The vast majority of patients with Ewing sarcoma initially respond quite well to chemotherapy, and remission is common, even in patients presenting with widespread metastases. Unfortunately, relapse is common, and the prognosis for patients with metastatic disease remains poor, with cure rates in the 20% range.2 The discrepancy between good initial response to chemotherapy and poor patient survival suggests that the development of therapeutic resistance remains a primary roadblock to the cure of Ewing sarcoma.
There are several possible approaches to overcome therapeutic resistance, including chemotherapy dose escalation, use of molecularly targeted therapies in place of standard chemotherapeutics, and identification and inhibition of pathways involved in chemotherapy resistance. In Ewing sarcoma, dose escalation has failed to improve the survival of patients who present with metastatic disease, and toxicity has limited the ability to further escalate the treatment of patients who present with localized disease. Several laboratories are investigating molecularly targeted therapies for Ewing sarcoma, including small molecule inhibitors of the translocation oncogene, EWS-FLI1.3,4 The Biswal laboratory has taken the approach of identifying and targeting pathways that mediate a generalized resistance to chemotherapeutics, focusing on the transcription factor, nuclear factor erythroid-2 related factor-2 (Nrf2).5
In normal cells, in absence of stress, Nrf2 exists in an inactive state in the cytoplasm of most cells, bound to its inhibitor, Keap1 (Figure 1). Oxidative stress results in dissociation of Nrf2 from Keap1, translocation of Nrf2 to the nucleus, and increased expression of target genes involved in antioxidants, drug detoxification, drug efflux, and cell metabolism. This panel of target genes is central to chemotherapy resistance in a wide variety of tumor types. In lung and other cancers, Nrf2 is constitutively active due to either gain of function mutations in Nrf2 or loss of function mutations in Keap1.6-8 Constitutive activation of Nrf2 in these cancers is associated with resistance to both chemotherapy and radiotherapy and is also associated with more rapid tumor growth. These observations make Nrf2 an attractive target for interventions aimed at overcoming therapeutic resistance.
What is a transcription factor?
Transcription factors are proteins that enter the cell’s nucleus and turn on and off groups of genes with related functions. This allows cells to coordinate a response to signals from the environment. In this case, Nrf2 turns on and off a panel of genes associated with response to environmental stress. Cancer cells interpret chemotherapy as an environmental stress and turn on genes involved in detoxifying poisons in an effort to not be killed by the chemotherapy drugs. It is our expectation that inhibiting Nrf2 will increase the sensitivity of cancer cells, especially Ewing sarcoma cells, to chemotherapy.
In preliminary work, we investigated whether Nrf2 is expressed in sarcomas. Immunohistochemical analysis of a custom sarcoma tissue microarray revealed strong Nrf2 expression in Ewing sarcoma. We subsequently found high level Nrf2 expression, as well as expression of a number of known Nrf2 target genes, in a number of Ewing sarcoma cell lines. Since mutations of both Nrf2 and Keap1 have been reported in a variety of tumor types, we sequenced Nrf2 and Keap1 in Ewing sarcoma cell lines and were surprised not to find any mutations in either gene. To further investigate the mechanism by which Nrf2 is expressed in Ewing sarcoma, we transfected primary human mesenchymal stem cells with the EWS-FLI1 translocation oncogene and found that this led to upregulation of Nrf2 expression at both the mRNA and protein levels. Interestingly, we also found that Nrf2 binds to the promoter of EWSR1 (part of the EWS-FLI1 oncogene) and regulates its expression. Since it has been suggested that precise regulation of EWS-FLI1 expression is critical (too much EWS-FLI1 is toxic to mesenchymal cells, while loss of EWS-FLI1 is lethal to Ewing sarcoma cells), the mutual regulation of EWS-FLI1 and Nrf2 may play a critical role in Ewing sarcoma biology.
We next investigated the effect of inhibiting Nrf2 on Ewing sarcoma growth and resistance to chemotherapy. In separate work, in collaboration with the National Center for Advanced Translational Sciences (NCATS), the Biswal laboratory screened the Molecular Libraries Small Molecule Repository and identified potent, specific inhibitors of Nrf2 with favorable pharmacokinetic properties. We found that the small molecule Nrf2 inhibitors significantly augment the toxicity of doxorubicin towards several Ewing sarcoma cell lines in vitro. Similarly, we found that suppression of Nrf2 expression using an inducible shRNA both increased sensitivity of Ewing sarcoma cell lines to doxorubicin in vitro and delayed tumor growth in a mouse xenograft model, significantly prolonging the survival of mice.
Based on these findings, we conclude that EWS-FLI1 drives Nrf2 expression in Ewing sarcoma, and that inhibition of Nrf2, by either genetic means or using a small molecule inhibitor, increases sensitivity to chemotherapeutics and slows tumor growth. These findings allowed us to formulate the hypothesis that high Nrf2 signaling in Ewing sarcoma promotes tumor growth and confers drug resistance, and that Nrf2 inhibitors will be cytotoxic to Ewing sarcoma, and will also enhance sensitivity to standard chemotherapeutics, improving patient survival.
The Role of Nrf2 in Ewing Sarcoma
Our preliminary data suggest that the gene that drives 85% of Ewing sarcoma, EWS-FLI1, which is a transcription factor (see previous sidebar), and turn on Nrf2. In addition, blocking Nrf2 can not only make Ewing sarcoma cells more sensitive to chemotherapy drugs, at least in the lab, but can also make tumors grow more slowly in laboratory mice. This makes us think that Nrf2 may play two important roles in Ewing sarcoma: 1) promoting tumor growth and 2) promoting resistance to chemotherapy drugs. Thus, an inhibitor of Nrf2 may make Ewing sarcoma cells more sensitive to chemotherapy, but it also might be toxic to sarcoma cells all by itself.
Purpose of Investigation
The purpose of this investigation is two-fold. We will investigate the biology of Nrf2 in Ewing sarcoma, and we will also determine whether small molecule Nrf2 inhibitors have the potential to improve the treatment of patients with Ewing sarcoma, either by directly killing tumor cells or by overcoming the therapeutic resistance that limits the ability of standard chemotherapy drugs to eradicate the disease.
Specific Aim 1: To identify the downstream transcriptional targets of Nrf2 in Ewing sarcoma cells which lead to therapeutic resistance. The transcriptional program of Nrf2 in carcinomas has been well described, but the panel of Nrf2 target genes in sarcomas may be different, reflecting a distinct developmental lineage (mesenchymal origin, as opposed to the epithelial origin of carcinomas). In fact, our preliminary work has demonstrated that some Nrf2 target genes previously identified in carcinomas (such as genes involved in glutathione biosynthesis and recycling) are also targets in Ewing sarcoma, but we have also identified several novel target genes not previously reported in lung cancer. We will transfect a panel of 8 Ewing sarcoma cell lines with doxycycline-inducible Nrf2 shRNA. To control for “off target effects” we will use 2 independent shRNAs and a nontargeting control. We will perform RNA sequencing before and after induction of the shRNA. Ingenuity pathway analysis will allow us to decipher the network of Nrf2-dependent pathways in Ewing sarcoma, and this information will influence how Nrf2 inhibitors will be combined with other cytotoxic agents in future preclinical work, as well as in clinical trials. In addition, these results will increase our understanding of the contribution of Nrf2 to Ewing sarcoma biology and help guide the design of experiments described under Specific Aims 2 and 3. In silico analysis of regulatory regions of genes which are regulated by Nrf2 will identify those which might be direct Nrf2 targets. Chromatin immunoprecipitation and promoter/reporter assays will be performed to validate key genes as directly regulated by Nrf2.
Specific Aim 2: To determine if attenuation of Nrf2 signaling by specific lead small molecule inhibitors can block Ewing sarcoma tumor growth. We will test small molecule inhibitors of Nrf2, identified in ongoing work with NCATS. We will evaluate these inhibitors in three complementary ways:
- Gene expression analysis. We will treat Ewing sarcoma cells with Nrf2 inhibitors, or with an inactive analog as control, in vitro and evaluate expression of known Nrf2 target genes, as well as Nrf2 target genes identified under Specific Aim 1.
- Growth in soft agar. Ewing sarcoma cell lines will be treated with small molecule Nrf2 inhibitors, or an inactive analog control, and anchorage independent growth on soft agar will be evaluated.
- Xenograft assay. Ewing sarcoma cell lines will be implanted subcutaneously into the flank of immune deficient mice. After verifying tumor growth, animals will be randomized to receive either a small molecule Nrf2 inhibitor or an inactive control. Serial measurements of tumor size will determine effect of the drug on tumor growth. Expression of Nrf2 target genes (both previously known genes and those identified under Specific Aim 1) will be evaluated in treated and untreated tumors to validate target inhibition. Results of experiments using subcutaneous tumor implantation will be validated in an orthotopic xenograft model (tumor cells implanted in the tibia).
Specific Aim 3: To determine if abrogation of Nrf2 signaling can enhance the cytotoxicity of chemotherapy drugs toward Ewing sarcoma cells in vitro and in vivo. Because treatment resistance is a prominent problem in Ewing sarcoma, and because Nrf2 regulates the expression of a number of genes involved in resistance to chemotherapeutics, we will determine whether Nrf2 inhibitors enhance the cytotoxicity of chemotherapy drugs toward Ewing sarcoma cells in vitro and in vivo.
- In vitro. Ewing sarcoma cell lines will be treated in vitro with a panel of chemotherapy drugs (focusing on those with known clinical utility, including doxorubicin, etoposide, cyclophosphamide, ifosfamide, topotecan, irinotecan, vincristine, and temozolomide, but informed by results of Specific Aim 1) and either one of the Nrf2 inhibitors or an inactive analog as a critical control. A variety of drug concentrations will be used, MTT assays will be performed to quantify cytotoxicity, and results will be analyzed using CompuSyn software to evaluate synergy.
- In vivo. Promising drug combinations identified in vitro will be tested in vivo. Ewing sarcoma cell lines will be implanted subcutaneously in the flank of immune deficient mice. After established tumors are palpable, mice will be divided into four cohorts: vehicle control, Nrf2 inhibitor, chemotherapy drug, or the combination. Mice will be treated for 4 weeks, during which time tumor growth rate will be monitored. After 4 weeks, animals will be euthanized, tumor weight determined, and tumors will be evaluated histologically, and gene expression analysis will be performed to verify inhibition of Nrf2 in animals treated with Nrf2 inhibitor. Selected, highly promising drug combinations will be further tested in an orthotopic tumor model of Ewing sarcoma (pretibial implantation, with amputation after tumor growth to allow for assessment of an effect on metastatic disease).
Ewing sarcoma is very sensitive to chemotherapy, but despite achieving a radiographic complete remission, up to 30% of patients who present with localized disease, and almost all patients who present with metastases will ultimately suffer a relapse and succumb to their disease. Thus, treatment resistance is a major barrier to cure for patients with Ewing sarcoma. We have demonstrated that Nrf2, a transcription factor that regulates the expression of a large number of genes that mediate resistance to chemotherapy, is expressed at high levels in Ewing sarcoma. By identifying Nrf2 target genes, and using this information to design in vitro and in vivo experiments to test the ability of Nrf2 inhibitors to either directly kill Ewing sarcoma cells or to augment their sensitivity to chemotherapeutics, we are taking the first step towards introducing these agents into clinical use. In collaboration with NCATS, we are designing and validating small molecule Nrf2 inhibitors for clinical use, and the experiments funded by this grant will guide the introduction of these agents to the treatment of Ewing sarcoma patients. We expect these drugs to have substantial anti-cancer activity on their own, in addition to increasing response to established drugs, and the proposed preclinical studies will provide the support needed to justify early phase clinical trials on a Nrf2 inhibitor in patients with high risk Ewing sarcoma. The ability to overcome chemotherapy resistance with small molecule Nrf2 inhibitors will dramatically enhance the effectiveness of current treatments and improve the survival of patients not cured by current treatment methods.
1. Womer RB, West DC, Krailo MD, Dickman PS, Pawel BR, et al. (2012) Randomized controlled trial of interval-compressed chemotherapy for the treatment of localized Ewing sarcoma: a report from the Children's Oncology Group. J Clin Oncol 30: 4148-4154.
2. Bernstein ML, Devidas M, Lafreniere D, Souid AK, Meyers PA, et al. (2006) Intensive therapy with growth factor support for patients with Ewing tumor metastatic at diagnosis: Pediatric Oncology Group/Children's Cancer Group Phase II Study 9457--a report from the Children's Oncology Group. J Clin Oncol 24: 152-159.
3. Erkizan HV, Kong Y, Merchant M, Schlottmann S, Barber-Rotenberg JS, et al. (2009) A small molecule blocking oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits growth of Ewing's sarcoma. Nat Med 15: 750-756.
4. Grohar PJ, Woldemichael GM, Griffin LB, Mendoza A, Chen QR, et al. (2011) Identification of an inhibitor of the EWS-FLI1 oncogenic transcription factor by high-throughput screening. J Natl Cancer Inst 103: 962-978.
5. Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, et al. (2006) Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS Med 3: e420.
6. Zhang P, Singh A, Yegnasubramanian S, Esopi D, Kombairaju P, et al. (2010) Loss of Kelch-like ECH-associated protein 1 function in prostate cancer cells causes chemoresistance and radioresistance and promotes tumor growth. Mol Cancer Ther 9: 336-346.
7. Mitsuishi Y, Taguchi K, Kawatani Y, Shibata T, Nukiwa T, et al. (2012) Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming. Cancer Cell 22: 66-79.
8. Shibata T, Kokubu A, Saito S, Narisawa-Saito M, Sasaki H, et al. (2011) NRF2 mutation confers malignant potential and resistance to chemoradiation therapy in advanced esophageal squamous cancer. Neoplasia 13: 864-873.
ESUN Bulletin Copyright © January 2015 Liddy Shriver Sarcoma Initiative.