Widespread hypertranscription in aggressive human cancers.

Cancers are often defined by the dysregulation of specific transcriptional programs; however, the importance of global transcriptional changes is less understood. Hypertranscription is the genome-wide increase in RNA output. Hypertranscription's prevalence, underlying drivers, and prognostic significance are undefined in primary human cancer. This is due, in part, to limitations of expression profiling methods, which assume equal RNA output between samples. Here, we developed a computational method to directly measure hypertranscription in 7494 human tumors, spanning 31 cancer types. Hypertranscription is ubiquitous across cancer, especially in aggressive disease. It defines patient subgroups with worse survival, even within well-established subtypes. Our data suggest that loss of transcriptional suppression underpins the hypertranscriptional phenotype. Single-cell analysis reveals hypertranscriptional clones, which dominate transcript production regardless of their size. Last, patients with hypertranscribed mutations have improved response to immune checkpoint therapy. Our results provide fundamental insights into gene dysregulation across human cancers and may prove useful in identifying patients who would benefit from novel therapies.

Comprehensive profiling of mRNA splicing indicates that GC content signals altered cassette exon inclusion in Ewing sarcoma.

Ewing sarcoma (EwS) is a small round blue cell tumor and is the second most frequent pediatric bone cancer. 85% of EwS tumors express the fusion oncoprotein EWS-FLI1, the product of a t(11;22) reciprocal translocation. Prior work has indicated that transcription regulation alone does not fully describe the oncogenic capacity of EWS-FLI1, nor does it provide an effective means to stratify patient tumors. Research using EwS cell lines and patient samples has suggested that EWS-FLI1 also disrupts mRNA biogenesis. In this work we both describe the underlying characteristics of mRNA that are aberrantly spliced in EwS tumor samples as well as catalogue mRNA splicing events across other pediatric tumor types. Here, we also use short- and long-read sequencing to identify cis-factors that contribute to splicing profiles we observe in Ewing sarcoma. Our analysis suggests that GC content upstream of cassette exons is a defining factor of mRNA splicing in EwS. We also describe specific splicing events that discriminate EwS tumor samples from the assumed cell of origin, human mesenchymal stem cells derived from bone marrow (hMSC-BM). Finally, we identify specific splicing factors PCBP2, RBMX, and SRSF9 by motif enrichment and confirm findings from tumor samples in EwS cell lines.

Development of an Ewing sarcoma cell line with resistance to EWS­FLI1 inhibitor YK­4­279.

Despite Ewing sarcoma (ES) being the second most common pediatric malignancy of bone and soft tissue, few novel therapeutic approaches have been introduced over the past few decades. ES contains a pathognomonic chromosomal translocation that leads to a fusion protein between EWSR1 and an ets family member, most often FLI1. EWS­FLI1 is the most common type of fusion protein and is a well­vetted therapeutic target. A small molecule inhibitor of EWS­FLI1, YK­4­279 (YK) was developed with the intention to serve as a targeted therapy option for patients with ES. The present study investigated resistance mechanisms by developing an ES cell line specifically resistant to YK. The ES cell line A4573 was treated with YK to create resistant cells by long term continuous exposure. The results revealed that resistance in A4573 was robust and sustainable, with a >27­fold increase in IC50 lasting up to 16 weeks in the absence of the compound. Resistant ES cells were still sensitive to standard of care drugs, including doxorubicin, vincristine and etoposide, which may be valuable in future combination treatments in the clinic. Resistant ES cells revealed an increased expression of CD99. RNA sequencing and qPCR validation of resistant ES cells confirmed an increased expression of ANO1, BRSK2 and IGSF21, and a reduced expression of COL24A1, PRSS23 and RAB38 genes. A functional association between these genes and mechanism of resistance remains to be investigated. The present study created a cell line to investigate YK resistance.

EWS-FLI1 modulated alternative splicing of ARID1A reveals novel oncogenic function through the BAF complex.

Connections between epigenetic reprogramming and transcription or splicing create novel mechanistic networks that can be targeted with tailored therapies. Multiple subunits of the chromatin remodeling BAF complex, including ARID1A, play a role in oncogenesis, either as tumor suppressors or oncogenes. Recent work demonstrated that EWS-FLI1, the oncogenic driver of Ewing sarcoma (ES), plays a role in chromatin regulation through interactions with the BAF complex. However, the specific BAF subunits that interact with EWS-FLI1 and the precise role of the BAF complex in ES oncogenesis remain unknown. In addition to regulating transcription, EWS-FLI1 also alters the splicing of many mRNA isoforms, but the role of splicing modulation in ES oncogenesis is not well understood. We have identified a direct connection between the EWS-FLI1 protein and ARID1A isoform protein variant ARID1A-L. We demonstrate here that ARID1A-L is critical for ES maintenance and supports oncogenic transformation. We further report a novel feed-forward cycle in which EWS-FLI1 leads to preferential splicing of ARID1A-L, promoting ES growth, and ARID1A-L reciprocally promotes EWS-FLI1 protein stability. Dissecting this interaction may lead to improved cancer-specific drug targeting.

The ETS Inhibitors YK-4-279 and TK-216 Are Novel Antilymphoma Agents.

PURPOSE: Transcription factors are commonly deregulated in cancer, and they have been widely considered as difficult to target due to their nonenzymatic mechanism of action. Altered expression levels of members of the ETS-transcription factors are often observed in many different tumors, including lymphomas. Here, we characterized two small molecules, YK-4-279 and its clinical derivative, TK-216, targeting ETS factors via blocking the protein-protein interaction with RNA helicases, for their antilymphoma activity. EXPERIMENTAL DESIGN: The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination; validation experiments on in vivo models; and transcriptome and coimmunoprecipitation experiments. RESULTS: YK-4-279 and TK-216 demonstrated an antitumor activity across several lymphoma cell lines, which we validated in vivo. We observed synergistic activity when YK-4-279 and TK-216 were combined with the BCL2 inhibitor venetoclax and with the immunomodulatory drug lenalidomide. YK-4-279 and TK-216 interfere with protein interactions of ETS family members SPIB, in activated B-cell-like type diffuse large B-cell lymphomas, and SPI1, in germinal center B-cell-type diffuse large B-cell lymphomas. CONCLUSIONS: The ETS inhibitor YK-4-279 and its clinical derivative TK-216 represent a new class of agents with in vitro and in vivo antitumor activity in lymphomas. Although their detailed mechanism of action needs to be fully defined, in DLBCL they might act by targeting subtype-specific essential transcription factors.

Inhibition of the oncogenic fusion protein EWS-FLI1 causes G2-M cell cycle arrest and enhanced vincristine sensitivity in Ewing's sarcoma.

Ewing's sarcoma (ES) is a rare and highly malignant cancer that grows in the bones or surrounding tissues mostly affecting adolescents and young adults. A chimeric fusion between the RNA binding protein EWS and the ETS family transcription factor FLI1 (EWS-FLI1), which is generated from a chromosomal translocation, is implicated in driving most ES cases by modulation of transcription and alternative splicing. The small-molecule YK-4-279 inhibits EWS-FLI1 function and induces apoptosis in ES cells. We aimed to identify both the underlying mechanism of the drug and potential combination therapies that might enhance its antitumor activity. We tested 69 anticancer drugs in combination with YK-4-279 and found that vinca alkaloids exhibited synergy with YK-4-279 in five ES cell lines. The combination of YK-4-279 and vincristine reduced tumor burden and increased survival in mice bearing ES xenografts. We determined that independent drug-induced events converged to cause this synergistic therapeutic effect. YK-4-279 rapidly induced G2-M arrest, increased the abundance of cyclin B1, and decreased EWS-FLI1-mediated generation of microtubule-associated proteins, which rendered cells more susceptible to microtubule depolymerization by vincristine. YK-4-279 reduced the expression of the EWS-FLI1 target gene encoding the ubiquitin ligase UBE2C, which, in part, contributed to the increase in cyclin B1. YK-4-279 also increased the abundance of proapoptotic isoforms of MCL1 and BCL2, presumably through inhibition of alternative splicing by EWS-FLI1, thus promoting cell death in response to vincristine. Thus, a combination of vincristine and YK-4-279 might be therapeutically effective in ES patients.

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

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.

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

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.

Single enantiomer of YK-4-279 demonstrates specificity in targeting the oncogene EWS-FLI1.

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 of transcription factors. Our previous studies show that RNA Helicase A (RHA) enhances the oncogenesis of EWS-FLI1, a putative intrinsically disordered protein. Interruption of this protein-protein complex by small molecule inhibitors validates this interaction as a unique therapeutic target. Single enantiomer activity from a chiral compound has been recognized as strong evidence for specificity in a small molecule-protein interaction. Our compound, YK-4-279, has a chiral center and can be separated into two enantiomers by chiral HPLC. We show that there is a significant difference in activity between the two enantiomers. (S)-YK-4-279 is able to disrupt binding between EWS-FLI1 and RHA in an immunoprecipitation assay and blocks the transcriptional activity of EWS-FLI1, while (R)-YK-4-279 cannot. Enantiospecific effects are also established in cytotoxicity assays and caspase assays, where up to a log-fold difference is seen between (S)-YK-4-279 and the racemic YK-4-279. Our findings indicate that only one enantiomer of our small molecule is able to specifically target a protein-protein interaction. This work is significant for its identification of a single enantiomer effect upon a protein interaction suggesting that small molecule targeting of intrinsically disordered proteins can be specific. Furthermore, proving YK-4-279 has only one functional enantiomer will be helpful in moving this compound towards clinical trials.

Schizosaccharomyces pombe Dss1p is a DNA damage checkpoint protein that recruits Rad24p, Cdc25p, and Rae1p to DNA double-strand breaks.

Schizosaccharomyces pombe Dss1p and its homologs function in multiple cellular processes including recombinational repair of DNA and nuclear export of messenger RNA. We found that Tap-tagged Rad24p, a member of the 14-3-3 class of proteins, co-purified Dss1p along with mitotic activator Cdc25p, messenger RNA export/cell cycle factor Rae1p, 19 S proteasomal factors, and recombination protein Rhp51p (a Rad51p homolog). Using chromatin immunoprecipitation, we found that Dss1p recruited Rad24p and Rae1p to the double-strand break (DSB) sites. Furthermore, Cdc25p also recruited to the DSB site, and its recruitment was dependent on Dss1p, Rad24p, and the protein kinase Chk1p. Following DSB, all nuclear Cdc25p was found to be chromatin-associated. We found that Dss1p and Rae1p have a DNA damage checkpoint function, and upon treatment with UV light Deltadss1 cells entered mitosis prematurely with indistinguishable timing from Deltarad24 cells. Taken together, these results suggest that Dss1p plays a critical role in linking repair and checkpoint factors to damaged DNA sites by specifically recruiting Rad24p and Cdc25p to the DSBs. We suggest that the sequestration of Cdc25p to DNA damage sites could provide a mechanism for S. pombe cells to arrest at G(2)/M boundary in response to DNA damage.

The nuclear export signal of splicing factor Uap56p interacts with nuclear pore-associated protein Rae1p for mRNA export in Schizosaccharomyces pombe.

Mammalian UAP56 or its homolog Sub2p in Saccharomyces cerevisiae are members of the ATP-dependent RNA helicase family and are required for splicing and nuclear export of mRNA. Previously we showed that in Schizosaccharomyces pombe Uap56p is critical for mRNA export. It links the mRNA adapter Mlo3p, a homolog of Yra1p in S. cerevisiae or Aly in mammals, to nuclear pore-associated mRNA export factor Rae1p. In this study we show that, in contrast to S. cerevisiae, Uap56p in S. pombe is not required for pre-mRNA splicing. The putative RNA helicase function of Uap56p is not required for mRNA export. However, the RNA-binding motif of Uap56p is critical for nuclear export of mRNA. Within Uap56p we identified nuclear import and export signals that may allow it to shuttle between the nucleus and the cytoplasm. We found that Uap56p interacts with Rae1p directly via its nuclear export signal, and this interaction is critical for the nuclear export activity of Uap56p as well as for exporting mRNA. RNA binding and the ability to shuttle between the nucleus and cytoplasm are important features of mRNA export carriers such as HIV-Rev. Our results suggest that Uap56p could function similarly as an export carrier of mRNA in S. pombe.

Combined efficacy of tamoxifen and coenzyme Q10 on the status of lipid peroxidation and antioxidants in DMBA induced breast cancer.

An increasing amount of experimental and epidemiological evidence implicates the involvement of oxygen derived radicals in the pathogenesis of cancer development. It is well known that chemical carcinogenesis is multistage process. Free radicals arefound to be involved in both initiation and promotion of multistage carcinogenesis. Tamoxifen (TAM) is a potent antioxidant and a non-steroidal antiestrogen drug most used in the chemotherapy and chemoprevention of breast cancer. Besides its anticarcinogenic potential, it also produces some adverse toxic side effects, while taken for a long time. In order to minimise the side effects and to improve the antioxidant efficacy of tamoxifen, coenzyme Q10 (CoQ10) was added. Hence the present study was designed to investigate the combined efficacy of TAM along with CoQ10 in 7, 12 dimethyl benz(a)anthracene (DMBA) induced peroxidative damage in rat mammary carcinoma. The experimental setup comprised of one control and five experimental groups and it was carried out in adult female Sprague-Dawley rats. Mammary carcinoma was induced by oral administration of DMBA (25 mg kg(-1) body wt) and the treatment was started by the oral administration of TAM (10 mg kg(-1) body wt day(-1)) and CoQ10 (40 mg kg(-1) body wt day(-1)) dissolved in olive oil and continued for 28 days. Rats induced with DMBA showed a decline in the thiol capacity of the cell accompanied by high malondialdehyde content levels along with lowered activities of antioxidant status (superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione). In contrast, glutathione metabolising enzymes (glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione-S-transferase) were increased significantly in chemically induced carcinoma bearing rats. Administration of TAM along with CoQ10 restored the activities to a significant level thereby preventing cancer cell proliferation. This study highlights the increased antioxidant enzyme activities in relation to the susceptibility of cells to carcinogenic agents and the response of tumour cells to the chemotherapeutic agents.

Energy-modulating vitamins--a new combinatorial therapy prevents cancer cachexia in rat mammary carcinoma.

Mitochondria are the major intracellular organelles producing ATP molecules via the electron transport chain. Cancer cells have a deviant energy metabolism, and a high rate of glycolysis is related to a high degree of dedifferentiation and proliferation. The overall net ATP production is diminished with cancer, which ultimately leads to cancer cachexia. The present study was designed to investigate the altered energy metabolism in cancer cells and to enhance ATP production in the normal host cell metabolism by enhancing the activities of mitochondrial enzymes, using energy-modulating vitamins, and thus prevent cancer cachexia. Female Sprague-Dawley rats were selected for the experimental study. Mammary carcinoma was induced by the oral administration of 7,12-dimethylbenz[a]anthracene (25 mg/kg body weight), and treatment was started by the oral administration of the energy-modulating vitamins riboflavin (45 mg/kg body weight per d), niacin (100 mg/kg body weight per d) and coenzyme Q10 (40 mg/kg body weight per d) for 28 d. Mitochondria were isolated from the mammary gland and liver of all four groups, and the Krebs cycle and oxidative phosphorylation enzymes were assayed. In mammary carcinoma-bearing animals, the activities of the Krebs cycle and oxidative phosphorylation enzymes were significantly decreased. These activities were restored to a greater extent in animals treated with energy-modulating vitamins. From these experimental results, one may hypothesize that the combination therapy of energy-modulating vitamins could be of major therapeutic value in breast cancer.

Augmented efficacy of tamoxifen in rat breast tumorigenesis when gavaged along with riboflavin, niacin, and CoQ10: effects on lipid peroxidation and antioxidants in mitochondria.

Reactive oxygen species (ROS) play a major role in causing mitochondrial changes linked to cancer and metastasis. Uptake of antioxidants by tissue to reduce the ROS production could be instrumental in controlling cancer. Tamoxifen (TAM), a nonsteroidal anti-estrogen drug most used in the chemotherapy and chemoprevention of breast cancer. Riboflavin, niacin and coenzyme Q10 (CoQ10) are proved to be potent antioxidants and protective agents against many diseases including cancer. The objective of this research is to determine the therapeutic efficacy of combinatorial therapy on mammary carcinoma bearing rats in terms of the mitochondrial lipid peroxidation and antioxidant status especially MnSOD. Female albino rats of Sprague-Dawley strain were selected for the investigation. Mammary carcinoma was induced with 7,12-dimethyl benz(a)anthracene (DMBA: 25 mg), and the treatment was started by the oral administration of TAM (10 mg/kg body weight/day) along with riboflavin (45 mg/kg body weight/day), niacin (100 mg/kg body weight/day) and CoQ10 (40 mg/kg body weight/day) for 28 days. The levels of lipid peroxides, activities of enzymic and non-enzymic antioxidants were measured in the mitochondria isolated from the mammary gland and liver of control and experimental rats. Rats treated with DMBA showed an increase in mitochondrial lipid peroxidation (mammary gland 52.3%; liver 25.1%) accompanied by high malondialdehyde levels along with lowered activities of mitochondrial enzymic antioxidants [superoxide dismutase (mammary gland 19.9%; liver 24.8%), catalase (mammary gland 50%; liver 19.7%), glutathione peroxidase (mammary gland 47.8%; liver 31.1%)] and non-enzymic antioxidants [reduced glutathione (mammary gland 14.3%; liver 13.3%), Vitamin C (mammary gland 6.49%; liver 21.4%) and E (mammary gland 20.3%; liver 22.2%)]. Administration of combinatorial therapy restored lipid peroxide level and the activities of enzymic and non-enzymic antioxidants to near normalcy. In addition, antitumour activity was also found to be enhanced which is evident from the increased expression of tumour suppressor gene MnSOD thereby preventing cancer cell proliferation. These results suggested that TAM treatment is the most effective during co-administration of riboflavin, niacin and CoQ10 in terms of mitochondrial antioxidant and antitumour activity.

Therapeutic effect of tamoxifen and energy-modulating vitamins on carbohydrate-metabolizing enzymes in breast cancer.

BACKGROUND: Cancer cells have an abnormal energetic metabolism. One of the earliest discovered hallmarks of cancer had its roots in bioenergetics, as many tumours were found in the 1920s to exhibit a high glycolytic phenotype. An animal with cancer shows significant and progressive energy loss from the host (i.e. noncancerous) tissues, which could occur by the establishment of a systemic energy-depriving cycle involving the interaction of tumour glycolysis and host gluconeogenesis. Tamoxifen (TAM) is a nonsteroidal antioestrogen that is widely used in adjuvant therapy for all stages of breast carcinoma. To improve the therapeutic efficacy of TAM and to expand its usage in the treatment of breast cancer, it is necessary to establish an energy-enhancing programme. In order to provide sufficient energy and to prevent cancer cachexia, TAM can be supplemented with energy-modulating vitamins (EMV). In this investigation the augmentation of the efficacy of TAM by the effects of EMV supplementation on carbohydrate-metabolizing enzymes, the mitochondrial Krebs cycle and respiratory enzymes was evaluated in the mammary gland of carcinoma-bearing rats. METHODS: Female albino Sprague-Dawley rats were selected for the investigation. The experimental set-up included one control and four experimental groups. Mammary carcinoma was induced with 7,12- dimethyl benz(a)anthracene (25 mg), and TAM was administered orally (10 mg/kg body weight per day) along with EMV which comprised riboflavin (45 mg/kg per day), niacin (100 mg/kg per day) and coenzyme Q(10) (40 mg/kg per day). RESULTS: Measurements were made on tumour tissue and surrounding normal tissue in all experimental groups. Tumour tissue showed significant (P<0.05) increases in the glycolytic enzymes hexokinase, phosphoglucoisomerase and aldolase, and significant decreases in the gluconeogenic enzymes glucose-6-phosphatase and fructose-1,6-biphosphatase. In contrast, the surrounding tissue showed significant decreases in glycolytic enzymes and significant increases in gluconeogenic enzymes. The activities of the mitochondrial Krebs cycle enzymes isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, and respiratory chain enzymes NADH dehydrogenase and cytochrome c oxidase were significantly reduced in both tumour and surrounding tissue of the mammary carcinoma-bearing rats. These biochemical disturbances were effectively counteracted by supplementation with EMV, which restored the activities of all these enzyme to their respective control levels. CONCLUSION: Combination therapy of TAM with EMV not only alters carbohydrate metabolism but can also prevent body weight loss by enhancing the host energy metabolism.