Jeffrey A. Toretsky

Downstream of mutant KRAS, the transcription regulator YAP is essential for neoplastic progression to pancreatic ductal adenocarcinoma.

Without the transcription regulator YAP, KRAS-mutant pancreatic neoplasia cannot progress into invasive cancer. Targeting YAP to Promote the Antitumor Response Pancreatic ductal adenocarcinoma is frequently associated with mutations in KRAS, but targeting RAS proteins is clinically challenging. From studies with genetically engineered mouse models and correlations in human samples, Zhang et al. determined that the protein YAP mediated the transition from pancreatic metaplasia to invasive adenocarcinoma by transcriptionally activating genes induced by KRAS signaling. Deleting Yap in Kras or Kras:Trp53 mutant mouse pancreatic epithelia prevented their proliferation, decreased the expression and secretion of KRAS-regulated proteins that mediate inflammation and migration, and promoted immune cell infiltration in the tumor microenvironment in mice. Because Yap deletion did not affect normal pancreatic development or function, the findings indicate that Yap may be a viable target for KRAS-mutant PDAC. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates and frequently carries oncogenic KRAS mutation. However, KRAS has thus far not been a viable therapeutic target. We found that the abundance of YAP mRNA, which encodes Yes-associated protein (YAP), a protein regulated by the Hippo pathway during tissue development and homeostasis, was increased in human PDAC tissue compared with that in normal pancreatic epithelia. In genetically engineered KrasG12D and KrasG12D:Trp53R172H mouse models, pancreas-specific deletion of Yap halted the progression of early neoplastic lesions to PDAC without affecting normal pancreatic development and endocrine function. Although Yap was dispensable for acinar to ductal metaplasia (ADM), an initial step in the progression to PDAC, Yap was critically required for the proliferation of mutant Kras or Kras:Trp53 neoplastic pancreatic ductal cells in culture and for their growth and progression to invasive PDAC in mice. Yap functioned as a critical transcriptional switch downstream of the oncogenic KRAS–mitogen-activated protein kinase (MAPK) pathway, promoting the expression of genes encoding secretory factors that cumulatively sustained neoplastic proliferation, a tumorigenic stromal response in the tumor microenvironment, and PDAC progression in Kras and Kras:Trp53 mutant pancreas tissue. Together, our findings identified Yap as a critical oncogenic KRAS effector and a promising therapeutic target for PDAC and possibly other types of KRAS-mutant cancers.

Assemblages: Functional units formed by cellular phase separation

The partitioning of intracellular space beyond membrane-bound organelles can be achieved with collections of proteins that are multivalent or contain low-complexity, intrinsically disordered regions. These proteins can undergo a physical phase change to form functional granules or other entities within the cytoplasm or nucleoplasm that collectively we term “assemblage.” Intrinsically disordered proteins (IDPs) play an important role in forming a subset of cellular assemblages by promoting phase separation. Recent work points to an involvement of assemblages in disease states, indicating that intrinsic disorder and phase transitions should be considered in the development of therapeutics.

YK-4-279 Inhibits ERG and ETV1 Mediated Prostate Cancer Cell Invasion

Background Genomic rearrangements involving the ETS family of transcription factors occur in 40–70% of prostate cancer cases. ERG and ETV1 are the most common ETS members observed in these genetic alterations. The high prevalence of these rearrangements and their biological significance represents a novel therapeutic target for the treatment of prostate cancer. Methods and Findings We recently reported the development of YK-4-279, a small molecule inhibitor of EWS-FLI1 oncoprotein in Ewings Sarcoma. Since ERG and ETV1 belong to the same class of ETS factors as FLI1, we tested the ability of YK-4-279 to inhibit biological functions of ERG and ETV1 proteins in prostate cancer. YK-4-279 inhibited ERG and ETV1 mediated transcriptional activity in a luciferase assay. YK-4-279 also decreased ERG and ETV1 downstream target mRNA and protein expression in ETV1-fusion positive LNCaP and ERG fusion positive VCaP cells. YK-4-279 reduced the motility of LNCaP cells in a scratch assay and the invasive phenotype of both LNCaP and VCaP cells in a HUVEC invasion assay. Fusion-negative PC3 cells were unresponsive to YK-4-279. SiRNA mediated ERG knockdown in VCaP cells resulted in a loss of drug responsiveness. Concurrently, transient ERG expression in PC-3 cells resulted in increased invasive potential, which was reduced by YK-4-279. Conclusion These data demonstrate that YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostate cancer cells leading to decreased motility and invasion. Therefore, YK-4-279 may have an impact on metastasis in prostate cancer and it may be further evaluated for its clinical applications in prostate cancer in addition to Ewings sarcoma.

Wnt/Frizzled signaling in Ewing sarcoma.

The Ewing sarcoma family of tumors (ESFT) is a set of neuroectodermal malignancies that typically presents in the second decade and has a poor prognosis due to metastatic disease. Wnt signaling has a critical role in the normal development of multiple neuroectodermal tissues and also contributes to the neoplastic properties of tumor cells of neuroectodermal origin.

Beta-Catenin Accelerates Human Papilloma Virus Type-16 Mediated Cervical Carcinogenesis in Transgenic Mice

Human papilloma virus (HPV) is the principal etiological agent of cervical cancer in women, and its DNA is present in virtually all of these tumors. However, exposure to the high-risk HPV types alone is insufficient for tumor development. Identifying specific collaborating factors that will lead to cervical cancer remains an unanswered question, especially because millions of women are exposed to HPV. Our earlier work using an in vitro model indicated that activation of the canonical Wnt pathway in HPV-positive epithelial cells was sufficient to induce anchorage independent growth. We therefore hypothesized that constitutive activation of this pathway might function as the “second hit.” To address this possibility, we developed two double-transgenic (DT) mouse models, K14-E7/ΔN87βcat and K14-HPV16/ΔN87βcat that express either the proteins encoded by the E7 oncogene or the HPV16 early region along with constitutively active β-catenin, which was expressed by linking it to the keratin-14 (K14) promoter. We initiated tumor formation by treating all groups with estrogen for six months. Invasive cervical cancer was observed in 11% of the K14-ΔN87βcat mice, expressing activated β-catenin and in 50% of the animals expressing the HPV16 E7 oncogene. In double-transgenic mice, coexpression of β-catenin and HPV16 E7 induced invasive cervical cancer at about 7 months in 94% of the cases. We did not observe cervical cancer in any group unless the mice were treated with estrogen. In the second model, K14-HPV16 mice suffered cervical dysplasias, but this phenotype was not augmented in HPV16/ΔN87βcat mice. In summary, the phenotypes of the K14-E7/ΔN87βcat mice support the hypothesis that activation of the Wnt/β-catenin pathway in HPV-associated premalignant lesions plays a functional role in accelerating cervical carcinogenesis.

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing.

Significance Alternative splicing of RNA allows a limited number of coding regions in the human genome to produce proteins with diverse functionality. Alternative splicing has also been implicated as an oncogenic process. Identifying aspects of cancer cells that differentiate them from noncancer cells remains an ongoing challenge, and our research suggests that alternatively spliced mRNA and subsequent protein isoforms will provide new anticancer targets. We determined that the key oncoprotein of Ewing sarcoma (ES), EWS-FLI1, regulates alternative splicing in multiple cell line models. These experiments establish oncogenic aspects of splicing that are specific to cancer cells and thereby illuminate potentially oncogenic splicing shifts as well as provide a useful stratification mechanism for ES patients. The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron–exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4–279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4–279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.

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.

RNA helicase A activity is inhibited by oncogenic transcription factor EWS-FLI1

RNA helicases impact RNA structure and metabolism from transcription through translation, in part through protein interactions with transcription factors. However, there is limited knowledge on the role of transcription factor influence upon helicase activity. RNA helicase A (RHA) is a DExH-box RNA helicase that plays multiple roles in cellular biology, some functions requiring its activity as a helicase while others as a protein scaffold. The oncogenic transcription factor EWS-FLI1 requires RHA to enable Ewing sarcoma (ES) oncogenesis and growth; a small molecule, YK-4-279 disrupts this complex in cells. Our current study investigates the effect of EWS-FLI1 upon RHA helicase activity. We found that EWS-FLI1 reduces RHA helicase activity in a dose-dependent manner without affecting intrinsic ATPase activity; however, the RHA kinetics indicated a complex model. Using separated enantiomers, only (S)-YK-4-279 reverses the EWS-FLI1 inhibition of RHA helicase activity. We report a novel RNA binding property of EWS-FLI1 leading us to discover that YK-4-279 inhibition of RHA binding to EWS-FLI1 altered the RNA binding profile of both proteins. We conclude that EWS-FLI1 modulates RHA helicase activity causing changes in overall transcriptome processing. These findings could lead to both enhanced understanding of oncogenesis and provide targets for therapy.

Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level.

ABSTRACT Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC–MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5′ untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane.

Systemic levels of neuropeptide Y and dipeptidyl peptidase activity in patients with Ewing sarcoma--associations with tumor phenotype and survival.

Ewing sarcoma (ES) is driven by fusion of the Ewing sarcoma breakpoint region 1 gene (EWSR1) with an E26 transformation‐specific (ETS) transcription factor (EWS‐ETS), most often the Friend leukemia integration 1 transcription factor (FLI1). Neuropeptide Y (NPY) is an EWS‐FLI1 transcriptional target; it is highly expressed in ES and exerts opposing effects, ranging from ES cell death to angiogenesis and cancer stem cell propagation. The functions of NPY are regulated by dipeptidyl peptidase IV (DPPIV), a hypoxia‐inducible enzyme that cleaves the peptide and activates its growth‐promoting actions. The objective of this study was to determine the clinically relevant functions of NPY by identifying the associations between patients ES phenotype and their NPY concentrations and DPP activity.