Sung-Hyeok Hong

Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model

Ewing sarcoma (ES) involves a tumor-specific chromosomal translocation that produces the EWS-FLI1 protein, which is required for the growth of ES cells both in vitro and in vivo. However, an EWS-FLI1-driven transgenic mouse model is not currently available. Here, we present data from six independent laboratories seeking an alternative approach to express EWS-FLI1 in different murine tissues. We used the Runx2, Col1a2.3, Col1a3.6, Prx1, CAG, Nse, NEFL, Dermo1, P0, Sox9 and Osterix promoters to target EWS-FLI1 or Cre expression. Additional approaches included the induction of an endogenous chromosomal translocation, in utero knock-in, and the injection of Cre-expressing adenovirus to induce EWS-FLI1 expression locally in multiple lineages. Most models resulted in embryonic lethality or developmental defects. EWS-FLI1-induced apoptosis, promoter leakiness, the lack of potential cofactors, and the difficulty of expressing EWS-FLI1 in specific sites were considered the primary reasons for the failed attempts to create a transgenic mouse model of ES.

YK-4-279 effectively antagonizes EWS-FLI1 induced leukemia in a transgenic mouse model

Ewing sarcoma is an aggressive tumor of bone and soft tissue affecting predominantly children and young adults. Tumor-specific chromosomal translocations create EWS-FLI1 and similar aberrant ETS fusion proteins that drive sarcoma development in patients. ETS family fusion proteins and over-expressed ETS proteins are also found in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Transgenic expression of EWS-FLI1 in mice promotes high penetrance erythroid leukemia with dense hepatic and splenic infiltrations. We identified a small molecule, YK-4-279, that directly binds to EWS-FLI1 and inhibits its oncogenic activity in Ewing sarcoma cell lines and xenograft mouse models. Herein, we tested in vivo therapeutic efficacy and potential side effects of YK-4-279 in the transgenic mouse model with EWS-FLI1 induced leukemia. A two-week course of treatment with YK-4-279 significantly reduced white blood cell count, nucleated erythroblasts in the peripheral blood, splenomegaly, and hepatomegaly of erythroleukemic mice. YK-4-279 inhibited EWS-FLI1 target gene expression in neoplastic cells. Treated animals showed significantly better overall survival compared to control mice that rapidly succumbed to leukemia. YK-4-279 treated mice did not show overt toxicity in liver, spleen, or bone marrow. In conclusion, this in vivo study highlights the efficacy of YK-4-279 to treat EWS-FLI1 expressing neoplasms and support its therapeutic potential for patients with Ewing sarcoma and other ETS-driven malignancies.

Neuropeptide Y receptor Y5 as an inducible pro-survival factor in neuroblastoma: implications for tumor chemoresistance

Neuroblastoma (NB) is a pediatric tumor of neural crest origin with heterogeneous phenotypes. Although low-stage tumors carry a favorable prognosis, >50% of high-risk NB relapses after treatment with a fatal outcome. Thus developing therapies targeting refractory NB remains an unsolved clinical problem. Brain-derived neurotrophic factor (BDNF) and its TrkB receptor are known to protect NB cells from chemotherapy-induced cell death, while neuropeptide Y (NPY), acting via its Y2 receptor (Y2R), is an autocrine proliferative and angiogenic factor crucial for maintaining NB tumor growth. Here we show that in NB cells, BDNF stimulates the synthesis of NPY and induces expression of another one of its receptors, Y5R. In human NB tissues, the expression of NPY and Y5R positively correlated with the expression of BDNF and TrkB. Functionally, BDNF triggered Y5R internalization in NB cells, whereas Y5R antagonist inhibited BDNF-induced p44/42 mitogen-activated protein kinase activation and its pro-survival activity. These observations suggested TrkB-Y5R transactivation that resulted in cross-talk between their signaling pathways. Additionally, NPY and Y5R were upregulated in a BDNF-independent manner in NB cells under pro-apoptotic conditions, such as serum deprivation and chemotherapy, as well as in cell lines and tissues derived from posttreatment NB tumors. Blocking Y5R in chemoresistant NB cells rich in this receptor sensitized them to chemotherapy-induced apoptosis and inhibited their growth in vivo by augmenting cell death. In summary, the NPY/Y5R axis is an inducible survival pathway activated in NB by BDNF or cellular stress. Upon such activation, Y5R augments the pro-survival effect of BDNF via its interactions with TrkB receptor and exerts an additional BDNF-independent anti-apoptotic effect, both of which contribute to NB chemoresistance. Therefore, the NPY/Y5R pathway may become a novel therapeutic target for patients with refractory NB, thus far an incurable form of this disease.

Abstract 5453: YK-4-279 inhibits ETS-positive prostate cancer cell metastasis in a mouse xenograft model.

Chromosomal translocations involving the ETS family of transcription factors are found in a majority of prostate cancers, including the most clinically aggressive forms. These translocations produce a chimeric gene, which fuses the promoter region of an androgen responsive gene to the coding region of ETS factors, most frequently ETV1 or ERG. Over-expression of ETS factors in prostate cancer cells results in a more invasive phenotype. The high prevalence of these rearrangements, and their biological significance represents a novel therapeutic target for the treatment of prostate cancer. We recently demonstrated that the small molecule YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostate cancer cells leading to decreased motility and invasion in-vitro. Here we present our findings in an in-vivo mouse xenograft model. SCID-beige mice were subcutaneously injected with LNCaP and PC-3 xenografts. Mice were treated with YK-4-279 and its effect on primary tumor size and lung metastasis was observed. YK-4-279 treatment resulted in reduced metastasis of the tumor from primary site to lungs. YK-4-279 also affected expression of ETV1 target genes such as MMP7 and MMP13. ETS fusion-negative PC-3 xenografts were unresponsive to YK-4-279. Our results demonstrate that YK-4-279 decreases tumor growth and inhibits metastasis in fusion-positive prostate cancer cell xenografts. Therefore, YK-4-279 may have an impact on preventing metastasis in prostate cancer patients, which is a leading cause of death. Citation Format: Said Rahim, Sarah Justvig, Sung-Hyeok Hong, Yali Kong, Milton L. Brown, Colm Morrissey, Jeffrey A. Toretsky, Aykut Uren. YK-4-279 inhibits ETS-positive prostate cancer cell metastasis in a mouse xenograft model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5453. doi:10.1158/1538-7445.AM2013-5453

Abstract B13: Perineural invasion in Ewing sarcoma—a novel mechanism and new therapeutic opportunities

Tumor dissemination and relapse are the major problems in Ewing sarcoma (ES) treatment, yet the mechanisms driving these processes are unknown. To elucidate the routes of ES metastatic spread, we used an orthotopic xenograft model. ES cells were injected into the gastrocnemius muscles of SCID/beige mice. Once the primary tumors reached the desired volume, they were excised by limb amputation. Subsequently, tumor dissemination was monitored by MRI and confirmed by histopathologic analysis. Interestingly, aside from typical hematogenous metastases, such as bone and lung lesions, we have also observed frequent perineural tumor dissemination manifested by the presence of migratory ES cells along the nerves adjacent to the primary tumors. This phenomenon was associated with formation of recurrent tumors at the amputation sites, as well as pelvic tumors with spine involvement. Interestingly, the level of perineural invasion (PNI) was dependent on the expression of neuropeptide Y (NPY) in ES cells. NPY is a neuronal protein released from peripheral sympathetic neurons, but also highly expressed in ES cells along with its receptors. The xenografts derived from ES cell lines not releasing endogenous NPY (TC71, TC32) exhibited frequent PNI in tumor-bearing limbs, as well as a high number of recurrent tumors at the surgery site and spine metastases (70% and 100% of mice with evidence of PNI for TC71 and TC32 xenografts, respectively). This phenomenon was less common in ES xenografts derived from NPY-rich SK-ES1 cells (17% of mice with signs of PNI). In line with these observations, NPY knockdown in SK-ES1 xenografts drastically accelerated formation of spinal tumors (60% of mice). Notably, in 40% of mice bearing SK-ES1/NPY shRNA xenografts the spinal tumors developed before the primary tumor growth was detectable at the site of ES cell injection. Thus, our in vivo experiments suggested that a lack of endogenous NPY in ES cells expressing high levels of its receptors triggers chemotactic effects of this peptide released from neighboring peripheral nerves, facilitating PNI. Indeed, in a transwell migration assay, NPY exerted significant chemotactic activity in SK-ES1/NPY shRNA cells, but not in the original SK-ES1 cell line. An even more profound chemotactic effect specific to the SK-ES1/NPY shRNA cells was observed with NPY-rich conditioned media obtained from neuroblastoma cells, which can serve as a model of peripheral sympathetic neurons. Further studies are required to determine which NPY receptors are responsible for its chemotactic properties. If the presence of perineural tumor growth is confirmed in human tumors, factors responsible for PNI in ES, e.g., NPY receptors, may become targets for novel therapies preventing disease dissemination and recurrence. Citation Format: Susana Galli, Sung-Hyeok Hong, Jason U. Tilan, Mina Adnani, Shiya Zhu, Yassi Fallah, Yi-Chien Lee, Olga Rodriguez, Chris Albanese, Ewa Izycka-Swieszewska, Joanna Kitlinska. Perineural invasion in Ewing sarcoma—a novel mechanism and new therapeutic opportunities [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B13.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

Abstract 2443: Ewing sarcoma progression associates with increasing chromosomal instability: A role for neuropeptide Y and its Y5 receptor

Ewing sarcoma (ES) is a tumor driven by EWS-ETS fusion proteins. Yet, the same fusions are present in localized and metastatic tumors that carry strikingly different prognoses. Despite low levels of genomic instability in primary ES tumors, the presence of complex karyotypes is one of a few adverse prognostic factors, implicating an acquired chromosomal instability (CIN) in ES progression. As transcriptional targets of EWS-ETS, neuropeptide Y (NPY) and its Y5 receptor (Y5R) are highly expressed in ES and further activated by hypoxia. We have found that overexpression of Y5R leads to defects in cytokinesis, followed by formation of polyploid cells, chromosome loss and CIN. Thus, the goal of our study was to determine whether CIN that is driven by hypoxia-induced activation of NPY/Y5R axis promotes ES metastases. ES cells were injected into gastrocnemius muscles of SCID/beige mice. Hypoxia in the resulting primary tumors was created by 72h ligation of the femoral artery. Then, the tumors were excised and mice were monitored for metastases. Tissues and cells derived from primary tumors and metastases were subjected to cytogenetic analyses. ES metastasis was associated with progressive genomic changes in tumor cells. Cells derived from primary tumors exhibited increases in nuclear sizes and ploidy, as compared to the original cells. Tumor hypoxia exacerbated this effect. This initial increase in ploidy was followed by a decrease in nuclear size, increase in mitotic errors and reduced chromosome numbers in cells from metastatic tissues, suggesting that ES progression associates with increased CIN and is triggered by cell polyploidization. This notion was confirmed by increased DNA copy number alterations in tissues from ES metastases observed in xenografts derived from 2 different cell lines and a clinical case of matched primary tumor and metastasis tissue (array-CGH). In SK-ES1 xenografts, these alterations involved gains in the locus of Y5R. Consequently, FISH identified an SK-ES1 clone with 3 copies of the Y5R gene. The percent of cells with Y5R gene amplification increased with the degree of SK-ES1 progression, with 16-24% cells in the original SK-ES1 cell line, 40-60% in primary tumors and 86-100% in metastases. This was associated with an increase in Y5R expression in metastatic tissues. Thus, the metastasis in SK-ES1 xenografts associated with a selection of the clone with amplified Y5R. SK-ES1 cells subjected to hypoxia in vitro presented with similar increases in nuclear sizes and enrichment in the clone with amplified Y5R (48%), as was observed in primary tumors. Y5R activation in normoxic SK-ES1 cells mimicked this effect. Our findings support the role for acquired CIN in ES progression and metastasis and implicate the hypoxia-induced activation of the NPY/Y5R axis as its potential trigger. Thus, Y5R antagonist may serve as an adjuvant treatment to prevent ES CIN and progression. Citation Format: Akanksha Mahajan, Sung-Hyeok Hong, Jason U. Tilan, Susana Galli, Congyi Lu, Jasmine Rodgers, Anju Duttargi, Rachel Acree, Luciane R. Cavalli, Joanna B. Kitlinska. Ewing sarcoma progression associates with increasing chromosomal instability: A role for neuropeptide Y and its Y5 receptor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2443.

Abstract 2949: YK-4-279 is a small molecule inhibitor of ETV1 and inhibits metastasis in a mouse model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The erythroblastosis virus E26 transforming sequences (ETS) family of transcription factors consists of a highly conserved group of genes that play important roles in cellular proliferation, differentiation, migration and invasion. Chromosomal translocations fusing ETS factors to promoters of androgen responsive genes have been found in a majority of prostate cancers, including the most clinically aggressive forms. ERG and ETV1 are the most commonly translocated ETS proteins. Over-expression of these proteins in prostate cancer cells results in a more invasive phenotype. The high prevalence of these rearrangements, and their biological significance represents a novel therapeutic target for the treatment of prostate cancer. We recently demonstrated that the small molecule YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostate cancer cells leading to decreased motility and invasion in-vitro. Here, we present our findings in an in-vivo mouse xenograft model. SCID-beige mice were subcutaneously implanted with fusion-positive LNCaP and fusion-negative PC-3 tumors. Animals were treated with YK-4-279 and its effect on tumor size, lung metastasis and survival were observed. YK-4-279 treatment resulted in decreased tumor size in the LNCaP cohort only. A reduction in tumor metastasis to the lungs was observed in compound treated LNCaP animals with comparable tumor sizes. YK-4-279 also increased survival in LNCaP mice. Expression of ETV1 target genes MMP7, FKBP10 and GLYATL2 was reduced as well. ETS fusion-negative PC-3 xenografts were unresponsive to the compound. YK-4-279 is a chiral molecule that exists as a racemic mixture of R and S enantiomers. As part of this study, we also established that (S) -YK-4-279 is the active enantiomer in prostate cancer cells. (S) -YK-4-279 binds to ETV1 with comparable kinetics as the racemic mixture and inhibits ETV1 activity. (R) -YK-4-279 does not demonstrate ETV1 binding or inhibition. Our results demonstrate that YK-4-279 is a potent inhibitor of ETV1 and should be further evaluated for its clinical applications in prostate cancer. Citation Format: Said Rahim, Sarah Justvig, Sung-Hyeok Hong, Perrer Tosso, Haydar Celik, Yasemin Sayedigar-Kont, Milton Brown, Colm Morrissey, Jeffrey Toretsky, Aykut Uren. YK-4-279 is a small molecule inhibitor of ETV1 and inhibits metastasis in a mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2949. doi:10.1158/1538-7445.AM2014-2949

Abstract LB-197: YK-4-279 is effective in treating EWS-FLI1 induced myeloid/erythroid leukemia in a transgenic mouse model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ewing sarcoma is an aggressive neoplasm affecting bone and soft tissue occurring almost exclusively in children and young adults. It is characterized by a distinct chromosomal translocation involving EWS and FLI1 genes making targeted therapy feasible since the resulting oncoprotein, EWS-FLI1, is exclusively expressed in the cancerous cells. The fusion protein has been shown to confer its tumorigenic phenotype through aberrant transcriptional activity, splicing, and protein-protein interactions. Previous work from our lab has identified a small molecule inhibitor, YK-4-279 that directly binds to EWS-FLI1 and inhibits its oncogenic activity in Ewing Sarcoma cell lines. Uncertainty about the cell of origin for Ewing sarcoma contributes to the lack of in vivo models for the disease, which has hindered preclinical study efforts. Torchia et al. (Mol. and Cell. Bio. 2007. 27(22): 7918-7934) has generated a transgenic mouse model where expression of EWS-FLI1 was targeted to the bone marrow, spleen, and liver by crossing Rosa26-loxP-stop-loxP-EWS-FLI1 mice with mice that express cre recombinase under Mx1-promoter, which responds to pIpC. Expression of EWS-FLI1 in these tissues induce a rapid expansion of primitive myeloid progenitors leading to rapid leukemia development. We used this mouse model to assess the efficacy of YK-4-279 in disrupting oncogenic activity of EWS-FLI1 in vivo. The mice were injected with 1mg of pIpC at an age of one month to induce leukemia. Once an increase in white blood cell count is observed following pIpC injection, the mice were randomly assigned to a treatment or control groups. Treatment of the transgenic mice daily, five times a week, intraperitoneally with 75mg/kg YK-4-279 vs. vehicle led to improved overall survival. Median survival for control group was 10.5 days while for treatment group was 24 days (p=0.045). Mice treated with 75mg/kg YK-4-279 showed significant reduction of disease burden within two weeks as monitored by weekly White Blood Cell count (DMSO vs. 75mg/kg YK Week 1: p=0.023 and Week 2: p=0.004). Mice treated with 150mg/kg YK-4-279 showed even more reduction of disease burden where one week after treatment WBC for DMSO group increased on average by 6495 cells/ul of blood while for YK-4-279 group it decreased by 1278 cells/ul of blood. Two weeks after the treatment started, the WBC for DMSO group increased even more by an average of 17,500 cells/ul of blood while the disease was stabilized for 150mg/kg YK-4-279 treated group (DMSO vs. 150mg/kg YK-4-279 Week 1: p-value<0.0001 and Week 2: p-value<0.002). Additionally, we observed reduced proliferation of blast cells in the peripheral blood by differential counting in YK-4-279 treated group vs. those treated with vehicle alone. Furthermore, we show that following two weeks treatment, YK-4-279 inhibits genes that are up-regulated in response to EWS-FLI1 activation in EF/+ Mx1cre+ mice. In conclusion, findings from this in vivo study highlight the effect of YK-4-279 in treating EWS-FLI1 induced leukemia and suggest its potential for early clinical trials in humans. Citation Format: Tsion Z. Minas, Jenny Han, Sung-Hyeok Hong, Jeffrey Toretsky, Aykut Uren. YK-4-279 is effective in treating EWS-FLI1 induced myeloid/erythroid leukemia in a transgenic mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-197. doi:10.1158/1538-7445.AM2014-LB-197

Abstract 3105: Neuropeptide Y Y5 receptor in neuroblastoma chemoresistance

Neuroblastoma (NB) is a pediatric tumor with heterogeneous phenotypes. While low stage tumors carry favorable prognosis, over 50% of high risk NB relapses after treatment with fatal outcome. Thus, developing therapies targeting this refractory form of NB remains an unsolved clinical problem. Neuropeptide Y (NPY) is a sympathetic neurotransmitter released from NB cells. High systemic levels of NPY are associated with poor clinical outcome of the disease, which is in agreement with its proliferative effect in NB cells and angiogenic properties. While all of the above functions of NPY are mediated mainly by its Y2 receptor (Y2R), predominantly expressed in NB and endothelial cells, some NB cell lines additionally express NPY Y5R. The goal of our study was to elucidate functions of Y5R/NPY pathway in NB. We have shown that, in contrast to the constitutively expressed Y2R, expression of Y5R was induced in pro-apoptotic conditions, such as serum deprivation, hypoxia or lack of attachment. Under such cellular stress, blocking Y5R by its selective antagonist or siRNA augmented NB cell death, suggesting pro-survival activity of Y5R/NPY axis. This effect was associated with a decrease in activity of p44/42 MAPK, a known mediator of NPY neuroprotective actions. This anti-apoptotic activity of Y5R contributed to chemoresistance of NB cells. Expression of Y5R and NPY was significantly increased in NB cells treated in vitro with chemotherapy. This effect was more pronounced in cells derived from relapsing tumors of patients that were previously treated with chemotherapy, suggesting pre-activation of the pathways inducing Y5R/NPY expression in these cells. Additionally, these refractory NB cell lines had elevated basal levels of Y5R and NPY expression, as compared to corresponding cell lines derived from the same patients at diagnosis. In line with this observation, 100% of surviving NB cells in tissues derived from chemotherapy-treated NB tumors was highly positive for Y5R, while in non-treated tumors only single, isolated Y5R-positive cells were observed. Blocking Y5R in chemoresistant NB cells rich in this receptor sensitized them to chemotherapy-induced apoptosis, as shown by a decrease in the number of viable cells and increase in caspase 3/7 activity. Consequently, Y5R significantly inhibited growth of NB xenografts derived from chemoresistant NB cells, which was associated with a 4-fold increase in cell death, while no significant changes in the levels of NB cell proliferation and tumor vascularization was observed. In summary, Y5R/NPY axis is an inducible pro-survival pathway activated in NB under cellular stress. This Y5R-mediated anti-apoptotic effect contributes to NB chemoresistance, implicating this receptor as a novel therapeutic target for patients with refractory NB, thus far lacking adequate treatment. Citation Format: Emily Trinh, Magdalena Czarnecka, Sung-Hyeok Hong, Congyi Lu, Samantha Martin, Susana Galli, Ewa Izycka-Swieszewska, Anna Kuan-Celarier, David Christian, Meredith Horton, Jason U. Tilan, Joanna B. Kitlinska. Neuropeptide Y Y5 receptor in neuroblastoma chemoresistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3105. doi:10.1158/1538-7445.AM2014-3105