Recurrent fusion of the v-myb avian myelobastosis viral oncogene homolog (MYB) and nuclear factor I/B (NFIB) generates the MYB-NFIB transcription factor, which has been detected in a high percentage of individuals with adenoid cystic carcinoma (ACC). To understand the functional role of this fusion protein in carcinogenesis, we generated a conditional mutant transgenic mouse that expresses MYB-NFIB along with p53 mutation in tissues that give rise to ACC: mammary tissue, salivary glands, or systemically in the whole body. Expression of the oncogene in mammary tissue resulted in hyperplastic glands that developed into adenocarcinoma in 27.3% of animals. Systemic expression of the MYB-NFIB fusion caused more rapid development of this breast phenotype, but mice died due to abnormal proliferation in the glomerular compartment of the kidney, which led to development of glomerulonephritis. These findings suggest the MYB-NFIB fusion is oncogenic and treatments targeting this transcription factor may lead to therapeutic responses in ACC patients.

The oncogene B-cell lymphoma 6 (BCL6) is associated with lymphomagenesis. Intriguingly, its expression is increased in preeclamptic placentas. Preeclampsia is one of the leading causes of maternal and perinatal mortality and morbidity. Preeclamptic placentas are characterized by various defects like deregulated differentiation and impaired fusion of trophoblasts. Its pathogenesis is however not totally understood. We show here that BCL6 is present throughout the cell fusion process in the fusogenic trophoblastic cell line BeWo. Suppression of BCL6 promotes trophoblast fusion, indicated by enhanced levels of fusion-related β-hCG, syncytin 1 and syncytin 2. Increased mRNA levels of these genes could also be observed in primary term cytotrophoblasts depleted of BCL6. Conversely, stable overexpression of BCL6 reduces the fusion capacity of BeWo cells. These data suggest that an accurately regulated expression of BCL6 is important for proper differentiation and successful syncytialization of trophoblasts. While deregulated BCL6 is linked to lymphomagenesis by blocking lymphocyte terminal differentiation, increased BCL6 in the placenta contributes to the development of preeclampsia by impairing trophoblast differentiation and fusion.

Because the emergence of drug resistance is a major limitation of current treatments for multiple myeloma (MM), it is necessary to continuously develop novel anticancer strategies. Here, using an inactivated Sendai virus (Hemagglutinating Virus of Japan; HVJ) envelope (HVJ-E), we discovered that increase of cytoplasmic Ca2+ by virus-cell fusion significantly induced apoptosis against human MM cells but not peripheral blood mononuclear cells from healthy donors. Interaction of F protein of HVJ-E with MM cells increased intracellular Ca2+ level of MMs by the induction of Ca2+ efflux from endoplasmic reticulum but not influx from extracellular region. The elevation of the Ca2+ cytoplasmic level induced SMAD1/5/8 phosphorylation and translocation into the nucleus, and SMAD1/5/8 and SMAD4 complex suppressed c-Myc transcription. Meanwhile, HVJ-E decreases S62 phosphorylation of c-Myc and promotes c-Myc protein degradation. Thus, HVJ-E-induced cell death of MM resulted from suppression of c-Myc by both destabilization of c-Myc protein and downregulation of c-Myc transcription. This study indicates that HVJ-E will be a promising tool for MM therapy.

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.

As an environmental poison, arsenic is responsible for many cancer deaths. Paradoxically, arsenic trioxide (ATO) presents also a powerful therapy used to treat refractory acute promyelocytic leukemia (APL) and is intensively investigated for treatment of other cancer types. Noteworthy, cancer therapy is frequently hampered by drug resistance, which is also often associated with enhancement of tumor aggressiveness. In this study, we analyzed ATO-selected cancer cells (A2780ATO) for the mechanisms underlying their enhanced tumorigenicity and aggressiveness. These cells were characterized by enhanced proliferation and spheroid growth as well as increased tumorigenicity of xenografts in SCID mice. Noteworthy, subsequent studies revealed that overexpression of Met receptor was the underlying oncogenic driver of these effects, as A2780ATO cells were characterized by collateral sensitivity against Met inhibitors. This finding was also confirmed by array comparative genomic hybridization (array CGH) and whole genome gene expression arrays, which revealed that Met overexpression by chronic ATO exposure was based on the transcriptional regulation via activation of AP-1. Finally, it was shown that treatment with the Met inhibitor crizotinib was also effective against A2780ATO cell xenografts in vivo, indicating that targeting of Met presents a promising strategy for the treatment of Met-overexpressing tumors after either arsenic exposure or failure to ATO treatment.

Papillary thyroid carcinoma (PTC) is the most frequent thyroid malignant neoplasia. Oncogene activation occurs in more than 70% of the cases. Indeed, about 40% of PTCs harbor mutations in BRAF gene, whereas RET rearrangements (RET/PTC oncogenes) are present in about 20% of cases. Finally, RAS mutations and TRK rearrangements account for about 5% each of these malignancies. We used RNA-Sequencing to identify fusion transcripts and mutations in cancer driver genes in a cohort of 18 PTC patients. Furthermore, we used targeted DNA sequencing to validate identified mutations. We extended the screening to 50 PTC patients and 30 healthy individuals. Using this approach we identified new missense mutations in CBL, NOTCH1, PIK3R4 and SMARCA4 genes. We found somatic mutations in DICER1, MET and VHL genes, previously found mutated in other tumors, but not described in PTC. We identified a new chimeric transcript generated by the fusion of WNK1 and B4GALNT3 genes, correlated with B4GALNT3 overexpression. Our data confirmed PTC genetic heterogeneity, revealing that gene expression correlates more with the mutation pattern than with tumor staging. Overall, this study provides new data about mutational landscape of this neoplasia, suggesting potential pharmacological adjuvant therapies against Notch signaling and chromatin remodeling enzymes.

Synovial sarcoma (SS) is a malignant soft tissue lesion and most commonly arises in young adults. Chromosomal translocation t(X;18)(p11;q11) results in the formation of SS18/SSX by gene fusion of the SS18 gene on chromosome 18 to either SSX1, SSX2, or SSX4 gene located on chromosome X, which is detected in more than 95% of SSs. Although multiple lines of evidence suggest that the SS18/SSX fusion is the oncogene in this tumor, the protein expression profiles associated with SS18/SSX have yet to be elucidated. In this study, we conducted proteomic studies using SS18/SSX knockdown in three SS cell lines to identify the regulated proteins associated with SS18/SSX in SS. Isobaric tags for relative and absolute quantitation (i-TRAQ) analyses identified approximate 1700-2,000 proteins regulated by the SS18/SSX fusion in each SS cell line. We also analyzed the three profiles to identify proteins that were similarly altered in all 3 cell lines and found 17 consistently upregulated and 18 consistently downregulated proteins, including TAGLN and ACTN4. In addition, network analyses identified several critical pathways including RUNX2 and SMARCA4. RUNX2 and SMARCA4 had the highest ranking in these identified pathways. In addition, we found that expression of TAGLN inhibited cell viability in SS cell lines. Our data suggest that the differentiation and cell growth of SS may be enhanced by the identified proteins induced by SS18/SSX. We believe that the findings obtained in the present functional analyses will help to improve our understanding of the relationship between SS18/SSX and malignant behavior in SS.

The neuregulin 1 (NRG1) fusion is a recently identified novel driver oncogene in invasive mucinous adenocarcinoma of the lung (IMA). After identification of a case of SLC3A2-NRG1 in a patient with IMA, we verified this fusion gene in a cohort of 59 patients with IMA. Targeted cancer panel sequencing and RT-PCR identified the possible coexistence of other driver oncogenes. Among 59 IMAs, we found 16 NRG1 fusions (13 SLC3A2-NRG1 and 3 CD74-NRG1). Of 16 patients with NRG1 fusions, concurrent KRAS codon 12 mutations were found in 10 cases. We also found concurrent NRAS Q61L mutation and EML4-ALK fusion in additional two cases with NRG1 fusions. When comparing overall survival (OS) according to the presence of NRG1 fusions showed that patients harboring NRG1 fusions had significantly inferior OS than those without NRG1 fusions (hazard ratio = 0.286; 95% confidence interval, .094 to .865). Ectopic expression of the SLC3A2-NRG1 fusion in lung cancer cells increased cell migration, proliferation and tumor growth in vitro and in xenograft models, suggesting oncogenic function for the fusion protein. We found that the SLC3A2-NRG1 fusion promoted ERBB2-ERBB3 phosphorylation and heteroduplex formation and activated the downstream PI3K/AKT/mTOR pathway through paracrine signaling. These findings suggested that the SLC3A2-NRG1 fusion was a driver in IMA with an important prognostic impact. SLC3A2-NRG1 should be considered a therapeutic target for patients with IMA.

While many solid tumors are defined by the presence of a particular oncogene, the role that this oncogene plays in driving transformation through the acquisition of aneuploidy and overcoming growth arrest are often not known. Further, although aneuploidy is present in many solid tumors, it is not clear whether it is the cause or effect of malignant transformation. The childhood sarcoma, Alveolar Rhabdomyosarcoma (ARMS), is primarily defined by the t(2;13)(q35;q14) translocation, creating the PAX3-FOXO1 fusion protein. It is unclear what role PAX3-FOXO1 plays in the initial stages of tumor development through the acquisition and persistence of aneuploidy. In this study we demonstrate that PAX3-FOXO1 serves as a driver mutation to initiate a cascade of mRNA and miRNA changes that ultimately reprogram proliferating myoblasts to induce the formation of ARMS. We present evidence that cells containing PAX3-FOXO1 have changes in the expression of mRNA and miRNA essential for maintaining proper chromosome number and structure thereby promoting aneuploidy. Further, we demonstrate that the presence of PAX3-FOXO1 alters the expression of growth factor related mRNA and miRNA, thereby overriding aneuploid-dependent growth arrest. Finally, we present evidence that phosphorylation of PAX3-FOXO1 contributes to these changes. This is one of the first studies describing how an oncogene and post-translational modifications drive the development of a tumor through the acquisition and persistence of aneuploidy. This mechanism has implications for other solid tumors where large-scale genomics studies may elucidate how global alterations contribute to tumor phenotypes allowing the development of much needed multi-faceted tumor-specific therapeutic regimens.

The differentiation status of tumors is used as a prognostic indicator, with tumors comprised of less differentiated cells exhibiting higher levels of aggressiveness that correlate with a poor prognosis. Although oncogenes contribute to blocking differentiation, it is not clear how they globally alter miRNA expression during differentiation to achieve this result. The pediatric sarcoma Alveolar Rhabdomyosarcoma, which is primarily characterized by the expression of the PAX3-FOXO1 oncogenic fusion protein, consists of undifferentiated muscle cells. However, it is unclear what role PAX3-FOXO1 plays in promoting the undifferentiated state. We demonstrate that expression of PAX3-FOXO1 globally alters the expression of over 80 individual miRNA during early myogenic differentiation, resulting in three primary effects: 1) inhibition of the expression of 51 miRNA essential for promoting myogenesis, 2) promoting the aberrant expression of 43 miRNA not normally expressed during myogenesis, and 3) altering the expression pattern of 39 additional miRNA. Combined, these changes are predicted to have an overall negative effect on myogenic differentiation. This is one of the first studies describing how an oncogene globally alters miRNA expression to block differentiation and has clinical implications for the development of much needed multi-faceted tumor-specific therapeutic regimens.

Philadelphia chromosome-positive (Ph+) Acute Lymphoblastic Leukemia (ALL) accounts for 25-30% of adult ALL and its incidence increases with age in adults >40 years old. Irrespective of age, the ABL1 fusion genes are markers of poor prognosis and amplification of the NUP214-ABL1 oncogene can be detected mainly in patients with T-ALL. T cell malignancies harboring the ABL1 fusion genes are sensitive to many cytotoxic agents, but up to date complete remissions have not been achieved. The PI3K/Akt/mTOR signaling pathway is often activated in leukemias and plays a crucial role in leukemogenesis.We analyzed the effects of three BCR-ABL1 tyrosine kinase inhibitors (TKIs), alone and in combination with a panel of selective PI3K/Akt/mTOR inhibitors, on three NUP214-ABL1 positive T-ALL cell lines that also displayed PI3K/Akt/mTOR activation. Cells were sensitive to anti BCR-ABL1 TKIs Imatinib, Nilotinib and GZD824, that specifically targeted the ABL1 fusion protein, but not the PI3K/Akt/mTOR axis. Four drugs against the PI3K/Akt/mTOR cascade, GSK690693, NVP-BGT226, ZSTK474 and Torin-2, showed marked cytotoxic effects on T-leukemic cells, without affecting the NUP214-ABL1 kinase and related pathway. Dephosphorylation of pAkt and pS6 showed the cytotoxicity of these compounds. Either single or combined administration of drugs against the different targets displayed inhibition of cellular viability associated with a concentration-dependent induction of apoptosis, cell cycle arrest in G0/G1 phase and autophagy, having the combined treatments a significant synergistic cytotoxic effect. Co-targeting NUP214-ABL1 fusion gene and PI3K/Akt/mTOR signaling pathway could represent a new and effective pharmacological strategy to improve the outcome in NUP214-ABL1 positive T-ALL.

Ewing sarcoma (ES) is an aggressive tumor defined by EWSR1 gene fusions that behave as an oncogene. Here we demonstrate that RING1B is highly expressed in primary ES tumors, and its expression is independent of the fusion oncogene. RING1B-depleted ES cells display an expression profile enriched in genes functionally involved in hematological development but RING1B depletion does not induce cellular differentiation. In ES cells, RING1B directly binds the SCN8A sodium channel promoter and its depletion results in enhanced Nav1.6 expression and function. The signaling pathway most significantly modulated by RING1B is NF-κB. RING1B depletion results in enhanced p105/p50 expression, which sensitizes ES cells to apoptosis by FGFR/SHP2/STAT3 blockade. Reduced NaV1.6 function protects ES cells from apoptotic cell death by maintaining low NF-κB levels. Our findings identify RING1B as a trait of the cell-of-origin and provide a potential targetable vulnerability.

A subset of lung cancers is dependent on the anaplastic lymphoma kinase (ALK) oncogene for survival, a mechanism that is exploited by the use of the ALK inhibitor crizotinib. Despite exceptional initial tumor responses to ALK inhibition by crizotinib, durable clinical response is limited and the emergence of drug resistance occurs. Furthermore, intrinsic resistance is frequently observed, where patients fail to respond initially to ALK-inhibitor therapy. These events demonstrate the underlying complexity of a molecularly-defined oncogene-driven cancer and highlights the need to identify compensating survival pathways. Using a loss-of-function whole genome short-hairpin (shRNA) screen, we identified MYCBP as a determinant of response to crizotinib, implicating the MYC signaling axis in resistance to crizotinib-treated ALK+ NSCLC. Further analysis reveals that ALK regulates transcriptional expression of MYC and activates c-MYC transactivation of c-MYC target genes. Inhibition of MYC by RNAi or small molecules sensitizes ALK+ cells to crizotinib. Taken together, our findings demonstrate a dual oncogene mechanism, where ALK positively regulates the MYC signaling axis, providing an additional oncogene target whose inhibition may prevent or overcome resistance.

RhoE is a small GTPase involved in the regulation of actin cytoskeleton dynamics, cell cycle and apoptosis. The role of RhoE in cancer is currently controversial, with reports of both oncogenic and tumor-suppressive functions for RhoE. Using RhoE-deficient mice, we show here that the absence of RhoE blunts contact-inhibition of growth by inhibiting p27Kip1 nuclear translocation and cooperates in oncogenic transformation of mouse primary fibroblasts. Heterozygous RhoE+/gt mice are more susceptible to chemically induced skin tumors and RhoE knock-down results in increased metastatic potential of cancer cells. These results indicate that RhoE plays a role in suppressing tumor initiation and progression.

The t(12;21) translocation generating the ETV6/RUNX1 fusion gene represents the most frequent chromosomal rearrangement in childhood leukemia. Presence of ETV6/RUNX1 alone is usually not sufficient for leukemia onset, and additional genetic alterations have to occur in ETV6/RUNX1-positive cells to cause transformation. We have previously generated an ETV6/RUNX1 transgenic mouse model where the expression of the fusion gene is restricted to CD19-positive B cells. Since BCL2 family members have been proposed to play a role in leukemogenesis, we investigated combined effects of ETV6/RUNX1 with exogenous expression of the antiapoptotic protein BCL2 by crossing ETV6/RUNX1 transgenic animals with Vav-BCL2 transgenic mice. Strikingly, co-expression of ETV6/RUNX1 and BCL2 resulted in significantly shorter disease latency in mice, indicating oncogene cooperativity. This was associated with faster development of follicular B cell lymphoma and exacerbated immune complex glomerulonephritis. ETV6/RUNX1-BCL2 double transgenic animals displayed increased B cell numbers and immunoglobulin titers compared to Vav-BCL2 transgenic mice. This led to pronounced deposition of immune complexes in glomeruli followed by accelerated development of immune complex glomerulonephritis. Thus, our study reveals a previously unrecognized synergism between ETV6/RUNX1 and BCL2 impacting on malignant disease and autoimmunity.

The genomic features and arising mechanisms of coding cancer somatic gene fusions (CSGFs) largely remain elusive. In this study, we show the gene origin stratification pattern of CSGF partners that fusion partners in human cancers are significantly enriched for genes with the gene age ofEuteleostomes and with the gene family age of Bilateria. GC skew (a measurement of G, C nucleotide content bias, (G-C)/(G+C)) is a useful measurement to indicate the DNA leading strand, lagging strand, replication origin, and replication terminal and DNA-RNA R-loop formation. We find that GC skew bias at the 5 prime (5') but not the 3 prime (3') partners of CSGFs, coincident with the polarity feature of gene expression breadth that the 5' partners are more ubiquitous while the 3' fusion partners are more tissue specific in general. We reveal distinct length and composition distributions of 5' and 3' of CSGFs, including sequence features corresponded to the 5' untranslated regions (UTRs), 3' UTRs, and the N-terminal sequences of the encoded proteins. Oncogenic somatic gene fusions are most enriched for the 5' and 3' genes' somatic amplification alongside a substantial proportion of other types of combinations. At the function level, 5' partners of CSGFs appear more likely to be tumour suppressor genes while many 3' partners appear to be proto-oncogene. Such distinct polarities of CSGFs at the evolutionary, structural, genomic and functional levels indicate the heterogeneous arsing mechanisms of CSGFs including R-loops and suggest potential novel targeted therapeutics specific to CSGF functional categories.

The Smyd2 protein (Set- and Mynd domain containing protein 2) is a methyl-transferase that can modify both histones and cytoplasmic proteins. Smyd2 is over-expressed in several cancer types and was shown to be limiting for tumor development in the pancreas. However, genetic evidence for a role of Smyd2 in other cancers or in mouse development was missing to date. Using germ line-deleted mouse strains, we now show that Smyd2 and the related protein Smyd3 are dispensable for normal development. Ablation of Smyd2 did not affect hematopoiesis, but retarded the development of leukemia promoted by MLL-AF9, a fusion oncogene associated with acute myeloid leukemia (AML) in humans. Smyd2-deleted leukemic cells showed a competitive disadvantage relative to wild-type cells, either in vitro or in vivo. The Smyd2 gene was directly activated by the oncogenic transcription factor Myc in either MLL9-AF9-induced leukemias, Myc-induced lymphomas, or fibroblasts. However, unlike leukemias, the development of lymphomas was not dependent upon Smyd2. Our data indicate that Smyd2 has a critical role downstream of Myc in AML.

The FIP1-like-1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRα) fusion oncogene is the driver factor in a subset of patients with hypereosinophilic syndrome (HES)/chronic eosinophilic leukemia (CEL). Most FIP1L1-PDGFRα-positive patients respond well to the tyrosine kinase inhibitor (TKI) imatinib. Resistance to imatinib in HES/CEL has been described mainly due to the T674I mutation in FIP1L1-PDGFRα, which is homologous to the imatinib-resistant T315I mutation in BCR-ABL. Development of novel TKIs is imperative to overcome resistance to imatinib. We synthesized S116836, a novel TKI. In this study, we evaluated the antitumor activity of S116836 in FIP1L1-PDGFRα-expressing cells. The results showed that S116836 potently inhibited PDGFRα and its downstream signaling molecules such as STAT3, AKT, and Erk1/2. S116836 effectively inhibited the growth of the WT and T674I FIP1L1-PDGFRα-expressing neoplastic cells in vitro and in nude mouse xenografts. Moreover, S116836 induced intrinsic pathway of apoptosis as well as the death receptor pathway, coincided with up-regulation of the proapoptotic BH3-only protein Bim-EL through the Erk1/2 pathway. In conclusion, S116836 is active against WT and T674I FIP1L1-PDGFRα-expressing cells, and may be a prospective agent for the treatment of HES/CEL.

Ewing sarcoma (EwS) is an aggressive pediatric bone cancer in need of more effective therapies than currently available. Most research into novel targeted therapeutic approaches is focused on the fusion oncogene EWSR1-FLI1, which is the genetic hallmark of this disease. In this study, a broad range of 3,325 experimental compounds, among them FDA approved drugs and natural products, were screened for their effect on EwS cell viability depending on EWS-FLI1 expression. In a network-based approach we integrated the results from drug perturbation screens and RNA sequencing, comparing EWS-FLI1-high (normal expression) with EWS-FLI1-low (knockdown) conditions, revealing novel interactions between compounds and EWS-FLI1 associated biological processes. The top candidate list of druggable EWS-FLI1 targets included genes involved in translation, histone modification, microtubule structure, topoisomerase activity as well as apoptosis regulation. We confirmed our in silico results using viability and apoptosis assays, underlining the applicability of our integrative and systemic approach. We identified differential sensitivities of Ewing sarcoma cells to BCL-2 family inhibitors dependent on the EWS-FLI1 regulome including altered MCL-1 expression and subcellular localization. This study facilitates the selection of effective targeted approaches for future combinatorial therapies of patients suffering from Ewing sarcoma.

The human 5-lipoxygenase (5-LO), encoded by the ALOX5 gene, is the key enzyme in the formation of pro-inflammatory leukotrienes. ALOX5 gene transcription is strongly stimulated by calcitriol (1α, 25-dihydroxyvitamin D3) and TGFβ (transforming growth factor-β). Here, we investigated the influence of MLL (activator of transcript initiation), AF4 (activator of transcriptional elongation) as well as of the leukemogenic fusion proteins MLL-AF4 (ectopic activator of transcript initiation) and AF4-MLL (ectopic activator of transcriptional elongation) on calcitriol/TGFβ-dependent 5-LO transcript elongation. We present evidence that the AF4 complex directly interacts with the vitamin D receptor (VDR) and promotes calcitriol-dependent ALOX5 transcript elongation. Activation of transcript elongation was strongly enhanced by the AF4-MLL fusion protein but was sensitive to Flavopiridol. By contrast, MLL-AF4 displayed no effect on transcriptional elongation. Furthermore, HDAC class I inhibitors inhibited the ectopic effects caused by AF4-MLL on transcriptional elongation, suggesting that HDAC class I inhibitors are potential therapeutics for the treatment of t(4;11)(q21;q23) leukemia.