Figure - available via license: Creative Commons Attribution 2.5 Generic
Content may be subject to copyright.
(A) A panel of ESFT and non-ESFT cells were treated with a dose range of small molecule. Cell viability was measured by WST after 72 hours of treatment. One representative graph from a cytotoxicity assay is shown. Graphs show IC50 values for (B) ESFT and (C) non-ESFT cells (**, p < 0.05, using a two-tailed Student's t-test). (D) ESFT and non-ESFT cells were treated with 10μM small molecule for 18 hours. Graph shows fold caspase-3 activity of treated cell lysates to control cell lysates. (E) A4573 cells were assayed for caspase-3 activation with increasing concentrations of YK-4-279 and (S)-YK-4-279 for 18 hours. For all panels, black bars represent YK-4-279, blue bars represent (S)-YK-4-279, and red bars represent (R)-YK-4-279.
Source publication
Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only located within the tumor. However, there are currently no agents targeted toward transcription factors, which are often considered to be 'undruggable.' A considerable body of evidence is accruing that refutes this claim based upon the intrinsic disorder...
Similar publications
The small arginine-rich protein protamine condenses complete genomic DNA into the sperm head. Here, we applied its high RNA binding capacity for spontaneous electrostatic assembly of therapeutic nanoparticles decorated with tumour-cell-specific antibodies for efficiently targeting siRNA. Fluorescence microscopy and DLS measurements of these nanocar...
Citations
... Targeting EWS-FLI1 directly remains challenging; however, pharmacological targeting of the EWS-FLI1 protein complex, for example using the YK-4-279 and its analog TK-216, is a potentially promising strategy. YK-4-279 blocks the interaction of EWS-FLI1 with helicase A [161,162] or DDX5 [94] and reverses the transcriptional activation of EWS-FLI1. YK-4-279 also leads to an isoform shift from ARID1A-L to ARID1A-S and prevents the interaction between EWS-FLI1 and ARID1A-L [104]. ...
Simple Summary
Ewing sarcoma (EwS) is a highly aggressive and metastatic cancer in children and adolescents. Canonical therapy mainly comprises the combination of intensive chemotherapy, radiation, and local surgery, which give rise to acute and chronic adverse effects. Drugs targeting EwS without side effects are in urgent demand. Genetically, EwS is characterized by chromosomal translocations with a low mutation burden. As a result, the chimeric protein EWS-ETS, mainly EWS-FLI1(85%), is critical for the malignancy of EwS. EWS-FLI1 directly binds to GGAA microsatellites in enhancers and promotors of the target genes and recruits multiple transcription factors or epigenetic regulators to reprogramme the epigenome. Direct targeting EWS-FLI1 is difficult due to the disordered structure, we mainly review the current knowledge of EWS-FLI1 property, the EWS-FLI1 protein complex, and the downstream pathways, we also summarize the targeted therapy of EwS by taking advantage of the EWS-FLI1 protein complex and the immunotherapy of the genes activated by EWS-FLI1.
Abstract
Ewing sarcoma (EwS) is a rare and predominantly pediatric malignancy of bone and soft tissue in children and adolescents. Although international collaborations have greatly improved the prognosis of most EwS, the occurrence of macrometastases or relapse remains challenging. The prototypic oncogene EWS-FLI1 acts as an aberrant transcription factor that drives the cellular transformation of EwS. In addition to its involvement in RNA splicing and the DNA damage response, this chimeric protein directly binds to GGAA repeats, thereby modifying the transcriptional profile of EwS. Direct pharmacological targeting of EWS-FLI1 is difficult because of its intrinsically disordered structure. However, targeting the EWS-FLI1 protein complex or downstream pathways provides additional therapeutic options. This review describes the EWS-FLI1 protein partners and downstream pathways, as well as the related target therapies for the treatment of EwS.
... 127 Trabectedin, mithramycin, TK216, and LSD1 inhibitors can affect EWSR-FLI1 epigenetic reprogramming. [127][128][129][130][131][132][133] Table 4 compiles selected trials with a focus on ES or osteosarcoma. RNA interference, protein degraders, and immunotherapy directed to the novel epitope are also potential strategies to target the oncoprotein. ...
Bone sarcomas are rare heterogeneous tumors that affect patients of all ages including children, adolescent young adults, and older adults. They include many aggressive subtypes and patient groups with poor outcomes, poor access to clinical trials, and lack of defined standard therapeutic strategies. Conventional chondrosarcoma remains a surgical disease, with no defined role for cytotoxic therapy and no approved targeted systemic therapies. Here, we discuss promising novel targets and strategies undergoing evaluation in clinical trials. Multiagent chemotherapy has greatly improved outcomes for patients with Ewing sarcoma (ES) and osteosarcoma, but management of those with high-risk or recurrent disease remains challenging and controversial. We describe the impact of international collaborative trials, such as the rEECur study, that aim to define optimal treatment strategies for those with recurrent, refractory ES, and evidence for high-dose chemotherapy with stem-cell support. We also discuss current and emerging strategies for other small round cell sarcomas, such as CIC-rearranged, BCOR-rearranged tumors, and the evaluation of emerging novel therapeutics and trial designs that may offer a new paradigm to improve survival in these aggressive tumors with notoriously bad (to the bone) outcomes.
... Recently, however, YK-4-279 and its derivative TK-216 have been reported to inhibit the protein-protein interaction between EWS-FLI1 and RNA helicase A, resulting in the cell growth inhibition of Ewing's sarcoma. 8,9 DAX1, also known as NR0B1, is an orphan member of the nuclear receptor superfamily. 10 DAX1 has a unique structure: its C-terminal region contains a ligand-binding domain (LBD), as do other nuclear receptors, whereas the N-terminal region has no DNA-binding domain but contains 3.5 repeats of the LXXLL/LXXML motif, which plays important roles in binding to transcriptional coactivators or other proteins. ...
Objective:
EWS-FLI1 is the most common oncogenic fusion protein in Ewing's sarcoma family tumors (ESFTs). DAX1, an orphan member of the nuclear receptor superfamily, is up-regulated by EWS-FLI1 and plays a key role in the transformed phenotype of ESFTs.
Methods:
To discover a functional inhibitor of DAX1 and EWS-FLI1, we screened small-molecular inhibitors using a DAX1 reporter assay system.
Results:
K-234 and its derivatives, which were dihydroorotate dehydrogenase (DHODH) inhibitors, showed inhibitory effects in the reporter assay. K-234 inhibited the growth of Ewing's sarcoma with various fusion types, and K-234 derivatives altered the expression of EWS-FLI1-regulated genes. The DAX1 expression had no effect on the growth inhibitory effect of the K-234 derivatives, while DHODH overexpression or uridine treatment attenuated their inhibitory effects, suggesting that inhibition by K-234 derivatives occurs through DHODH inhibition. An in vivo study showed that a K-234 derivative clearly inhibited tumor growth in an Ewing's sarcoma xenograft mouse model.
Conclusion:
Taken together, the present results suggest that DHODH inhibitors can inhibit the function of DAX1/EWS-FLI1 in ESFTs and might be a therapeutic agent with potent anti-tumor activity for Ewing's sarcoma patients.
... Fortis column (C18, 250 mm × 4.6 mm, 5 µm) was used for analysis with ammonium acetate (20 mM): acetonitrile: methanol (10:45:45%v/v) as mobile phase at 1 mL/min ow rate. The wavelength of detection was at 484 nm [15]. ...
... The EWS-FLI1 fusion protein driving ES oncogenesis, it has proven to be a promising target for drug development [43]. Agents that molecularly-targeted the EWS-FLI1 interaction with RNA helicase A (RHA) which helps in the formation of a protein-protein transcriptional complexes, are in the preclinical stage of development (eg, small-molecule YK-4-279 and peptide E9 or ESAP1) [15,[44][45][46]. Other agents identi ed include chemotherapeutic drugs with proven clinical e caciousness (eg, doxorubicin and etoposide). ...
To improve the specificity of the current diagnosis and treatment of Ewing's sarcoma (ES), efforts are underway to develop theranostic targeting probes. In particular, tumor specific markers and microenvironment represent potential targets. In addition, tumor exosomes are considered as possible nanocarriers for drugs to achieve tumor targeting and therapy. Herein, the development of CS2-YK@Exo , by packaging the pH-responsive ES-targeting near-infrared probe CS2-YK into ES exosomes is presented. The engineered exosome can serve as shuttle vehicles for drug uptake by tumor cells, and the pH-activatable characteristics of CS2-YK@Exo in acidic tumor environments can realize a precise image-guided drug delivery for the treatment of ES. The in vivo and in vitro studies verified the targeting, specificity, efficacy, and safety of the probe. Subcutaneous ES tumor mouse models, orthotopic models, and lung metastasis models are established to evaluate the real-time target imaging capabilities of the probe. This study demonstrates the potential of CS2-YK@Exo as a delivery nanosystem for tumor-targeted imaging and release of therapeutics to achieve real-time monitoring of therapy.
... Four of the tested compounds showed a significant effect and were either able to reduce tumor size (topoisomerase I inhibitor irinotecan (100 μM); BCL-X L inhibitor A-1331852 (10 μM)) or to slow down tumor growth compared to the respective control group (MCL-1 inhibitor S63845 (10 μM); YK-4-279 (10 μM), initially reported to block the interaction between EWS:FLI1 and RNA helicase A, but more recently identified as a microtubule destabilizing agent [30][31][32][33]) (Fig. 3). All other tested compounds did not show any significant effect in our in vivo model (Fig. S1). ...
Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited.
Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model.
In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.
... TWIST1 plays a role in ES metastasis, as its suppression reduces metastasis but does not affect the development of the original tumor [93]. HULC's sponging effect on miR-186-5p was established in the evaluation of the small molecule YK-4-279, which reduces the transcriptional activity of EWS-FLI1 [94,95]. Treatment of ES cell lines with YK-4-279 downregulated HULC, decreasing its sponging effect on miR-186-5p thereby increasing TWIST1 transcript binding with miR-186-5p and reducing TWIST1 protein levels [92]. ...
Ewing’s sarcoma (ES) is a pediatric sarcoma caused by a chromosomal translocation. Unlike in most cancers, the genomes of ES patients are very stable. The translocation product of the EWS-FLI1 fusion is most often the predominant genetic driver of oncogenesis, and it is pertinent to explore the role of epigenetic alterations in the onset and progression of ES. Several types of noncoding RNAs, primarily microRNAs and long noncoding RNAs, are key epigenetic regulators that have been shown to play critical roles in various cancers. The functions of these epigenetic regulators are just beginning to be appreciated in ES. Here, we performed a comprehensive literature review to identify these noncoding RNAs. We identified clinically relevant tumor suppressor microRNAs, tumor promoter microRNAs and long noncoding RNAs. We then explored the known interplay between different classes of noncoding RNAs and described the currently unmet need for expanding the noncoding RNA repertoire of ES. We concluded the review with a discussion of epigenetic regulation of ES via regulatory noncoding RNAs. These noncoding RNAs provide new avenues of exploration to develop better therapeutics and identify novel biomarkers.
... With this idea in mind, several groups have attempted to develop drugs able to interfere with the transcriptional activity of EWSR1-FLI1 fusion proteins. Among these drugs are trabectedin and its analogs [41,42], Mithramycin [43,44], and YK-4-279 and derivatives [45,46]. Although these drugs have reached clinical testing, the results obtained have been, at the moment, discouraging because of the elevated toxicity in some cases [47] and for the intrinsic difficulty in targeting transcription factors with pharmacological approaches [48], not to mention that pharmacological inhibition of EWSR1-FLI1 could require the administration of a drug for prolonged periods to achieve a durable response, as occurs in the case of tyrosine kinase inhibitors (for example, imatibib) [49]. ...
Ewing sarcoma is an aggressive bone cancer affecting children and young adults. The main molecular hallmark of Ewing sarcoma are chromosomal translocations that produce chimeric oncogenic transcription factors, the most frequent of which is the aberrant transcription factor EWSR1–FLI1. Because this is the principal oncogenic driver of Ewing sarcoma, its inactivation should be the best therapeutic strategy to block tumor growth. In this study, we genetically inactivated EWSR1–FLI1 using CRISPR-Cas9 technology in order to cause permanent gene inactivation. We found that gene editing at the exon 9 of FLI1 was able to block cell proliferation drastically and induce senescence massively in the well-studied Ewing sarcoma cell line A673. In comparison with an extensively used cellular model of EWSR1–FLI1 knockdown (A673/TR/shEF), genetic inactivation was more effective, particularly in its capability to block cell proliferation. In summary, genetic inactivation of EWSR1–FLI1 in A673 Ewing sarcoma cells blocks cell proliferation and induces a senescence phenotype that could be exploited therapeutically. Although efficient and specific in vivo CRISPR-Cas9 editing still presents many challenges today, our data suggest that complete inactivation of EWSR1–FLI1 at the cell level should be considered a therapeutic approach to develop in the future.
... The BCL-XL inhibitor A-1331852 (10 µM) reduced tumor size by -5% (control group 65% increase) ( Fig. 3D). YK-4-279, an inhibitor reported to block the interaction between EWS-FLI1 and RNA helicase A decreased tumor growth to 62% (control group 134% increase) (Fig. 3E) 20,21 . ...
Ewing sarcoma is a pediatric bone and soft tissue cancer for which new therapies to improve disease outcome and to reduce adverse effects of current standard treatments are urgently needed. To identify new and effective drugs, phenotypic drug screening has proven to be a powerful method and a cancer model ideally suited for this approach is the larval zebrafish xenograft system. Complementing mouse xenografts, zebrafish offer high-througput screening possibilities in an intact complex vertebrate organism. Here, we generated Ewing sarcoma xenografts in zebrafish larvae and established a workflow for automated imaging of xenografts, tumor cell recognition within transplanted zebrafish and quantitative tumor size analysis over consecutive days by high-content imaging. The increased throughput of our in vivo screening setup allowed us to identify combination therapies effective against Ewing sarcoma cells. Especially, combined inhibition of MCL-1 and BCL-XL, two anti-apoptotic proteins, was highly efficient at eradicating tumor cells in our zebrafish xenograft assays with two Ewing sarcoma cell lines and with patient-derived cells. Transcriptional analysis across Ewing sarcoma cell lines and tumors revealed that MCL-1 and BCL2L1, coding for BCL-XL, are the most abundantly expressed anti-apoptotic genes, suggesting that combined MCL-1/BCL-XL inhibition might be a broadly applicable strategy for Ewing sarcoma treatment.
... The small-molecule drug YK-4-279 mimics the effects of DN-ETS for cell proliferation, survival, invasion, and ETS function Specific targeted therapy against ETS factors may hold promise as a melanoma treatment. Recently, the small-molecule inhibitor YK-4-279 has been utilized to efficiently target the translocation product EWS-FLI1 in Ewing sarcoma (11,22). This compound also efficiently inhibits other EWS translocation products with ETS family members (ERG, ETV1) specifically through the ETS domain (12). ...
The failure of once promising target-specific therapeutic strategies often arises from redundancies in gene expression pathways. Even with new melanoma treatments, many patients are not responsive or develop resistance, leading to disease progression in terms of growth and metastasis. We previously discovered that the transcription factors ETS1 and PAX3 drive melanoma growth and metastasis by promoting the expression of the MET receptor. Here, we find that there are multiple ETS family members expressed in melanoma and that these factors have redundant functions. The small molecule YK-4-279, initially developed to target the ETS gene–containing translocation product EWS-FLI1, significantly inhibited cellular growth, invasion, and ETS factor function in melanoma cell lines and a clinically relevant transgenic mouse model, BrafCA;Tyr-CreERT2;Ptenf/f. One of the antitumor effects of YK-4-279 in melanoma is achieved via interference of multiple ETS family members with PAX3 and the expression of the PAX3-ETS downstream gene MET. Expression of exogenous MET provided partial rescue of the effects of YK-4-279, further supporting that MET loss is a significant contributor to the antitumor effects of the drug. This is the first study identifying multiple overlapping functions of the ETS family promoting melanoma. In addition, targeting all factors, rather than individual members, demonstrated impactful deleterious consequences in melanoma progression. Given that multiple ETS factors are known to have oncogenic functions in other malignancies, these findings have a high therapeutic impact.
Significance
These findings identify YK-4-279 as a promising therapeutic agent against melanoma by targeting multiple ETS family members and blocking their ability to act as transcription factors.
... The small molecular drug YK-4-279 has been proposed to block the interaction of RNA Helicase A (RHA) with the EWS/ETS fusion protein, crucial for the oncogenic transcriptional activity [8,9]. Efficacy against ES as well as ETS fusion-positive prostate cancer and SPIB-or SPI1-driven B-cell lymphoma models has been demonstrated in vitro and in vivo [10][11][12][13][14]. Currently, a derivative of YK-4-279 called TK216 is being tested in patients with relapsed and refractory ES in a clinical phase 1 study (NCT02657005). ...
... At the molecular level, YK-4-279 blocks the interaction of an oncogenic EWS/ETS fusion protein with its cofactor RHA. However, YK-4-279 does not only exert anti-cancer activity in ES [8,10,11,24], but also in prostate cancer [12,13,32,33], lymphoma [14], neuroblastoma [17,31] and glioma [10,15]. Ongoing research uncovered that YK-4-279 can also interfere with the mitotic machinery, leading to a pronounced mitotic arrest, and displays synergistic effects with vincristine [34]. ...
... At the molecular level, YK-4-279 blocks the interaction of an oncogenic EWS/ETS fusion protein with its cofactor RHA. However, YK-4-279 does not only exert anti-cancer activity in ES [8,10,11,24], but also in prostate cancer [12,13,32,33], lymphoma [14], neuroblastoma [17,31] and glioma [10,15]. Ongoing research uncovered that YK-4-279 can also interfere with the mitotic machinery, leading to a pronounced mitotic arrest, and displays synergistic effects with vincristine [34]. ...
Simple Summary
ETS transcription factors are potent oncogenic drivers in several cancer types and represent promising therapeutic targets. However, molecular factors influencing response to ETS factor inhibition are widely unknown so far. Here, we uncover that sensitivity of cancer cells against ETS factor blockade by the small molecule inhibitor YK-4-279 is strongly promoted by p53 loss in a MAPK-driven background. Induction of a parthanatos-like cell death based on a deregulated MAPK/ETS1/p53/PARP1 signal axis is identified as underlying molecular mechanism. Hence, this study suggests a novel and biomarker-driven therapeutic strategy for p53-deleted tumours, generally known for their profound therapy resistance.
Abstract
The small-molecule E26 transformation-specific (ETS) factor inhibitor YK-4-279 was developed for therapy of ETS/EWS fusion-driven Ewing’s sarcoma. Here we aimed to identify molecular factors underlying YK-4-279 responsiveness in ETS fusion-negative cancers. Cell viability screenings that deletion of P53 induced hypersensitization against YK-4-279 especially in the BRAFV600E-mutated colon cancer model RKO. This effect was comparably minor in the BRAF wild-type HCT116 colon cancer model. Out of all ETS transcription factor family members, especially ETS1 overexpression at mRNA and protein level was induced by deletion of P53 specifically under BRAF-mutated conditions. Exposure to YK-4-279 reverted ETS1 upregulation induced by P53 knock-out in RKO cells. Despite upregulation of p53 by YK-4-279 itself in RKOp53 wild-type cells, YK-4-279-mediated hyperphosphorylation of histone histone H2A.x was distinctly more pronounced in the P53 knock-out background. YK-4-279-induced cell death in RKOp53-knock-out cells involved hyperPARylation of PARP1, translocation of the apoptosis-inducible factor AIF into nuclei, and induction of mitochondrial membrane depolarization, all hallmarks of parthanatos. Accordingly, pharmacological PARP as well as BRAFV600E inhibition showed antagonistic activity with YK-4-279 especially in the P53 knock-out background. Taken together, we identified ETS factor inhibition as a promising strategy for the treatment of notoriously therapy-resistant p53-null solid tumours with activating MAPK mutations.
