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Vascular tone is regulated by the relative contractile state of vascular smooth muscle cells (VSMCs). Several integrins directly modulate VSMC contraction by regulating calcium influx through L-type voltage-gated CaSVEP1 and integrin binding were confirmed by immunoprecipitation and cell binding assays. Human induced pluripotent stem cell-derived VSMCs were used in in vitro [CaWe confirmed the ligation of SVEP1 to integrin α9β1 and additionally found SVEP1 to directly bind to integrin α4β1. Inhibition of SVEP1, integrin α4β1 or α9β1 significantly enhanced [CaOur studies reveal a novel role for SVEP1 and the integrins α4β1 and α9β1 in reducing VSMC contractility. This could provide an explanation for the genetic associations with blood pressure risk at the SVEP1 and ITGA9 loci.

The endonuclease activity within the influenza virus cap-snatching process is a proven therapeutic target. The anti-influenza drug baloxavir is highly effective, but is associated with resistance mutations that threaten its clinical efficacy. The endonuclease resides within the N-terminal domain of the PA subunit (PAN) of the influenza RNA dependent RNA polymerase, and we report here complexes of PAN with RNA and DNA oligonucleotides to understand its specificity and the structural basis of baloxavir resistance mutations. The RNA and DNA oligonucleotides bind within the substrate binding groove of PAN in a similar fashion, explaining the ability of the enzyme to cleave both substrates. The individual nucleotides occupy adjacent conserved pockets that flank the two-metal active site. However, the 2′ OH of the RNA ribose moieties engage in additional interactions that appear to optimize the binding and cleavage efficiency for the natural substrate. The major baloxavir resistance mutation at position 38 is at the core of the substrate binding site, but structural studies and modeling suggest that it maintains the necessary virus fitness via compensating interactions with RNA. These studies will facilitate the development of new influenza therapeutics that spatially match the substrate and are less likely to elicit resistance mutations.

Aims Chronic heart failure (CHF) is a systemic syndrome with a poor prognosis and a need for novel therapies. We investigated whether whole blood transcriptomic profiling can provide new mechanistic insights into cardiovascular (CV) mortality in CHF. Methods and results Transcriptome profiles were generated at baseline from 944 CHF patients from the BIOSTAT-CHF study, of whom 626 survived and 318 died from a CV cause during a follow-up of 21 months. Multivariable analysis, including adjustment for cell count, identified 1153 genes (6.5%) that were differentially expressed between those that survived or died and strongly related to a validated clinical risk score for adverse prognosis. The differentially expressed genes mainly belonged to five non-redundant pathways: adaptive immune response, proteasome-mediated ubiquitin-dependent protein catabolic process, T-cell co-stimulation, positive regulation of T-cell proliferation, and erythrocyte development. These five pathways were selectively related (RV coefficients >0.20) with seven circulating protein biomarkers of CV mortality (fibroblast growth factor 23, soluble ST2, adrenomedullin, hepcidin, pentraxin-3, WAP 4-disulfide core domain 2, and interleukin-6) revealing an intricate relationship between immune and iron homeostasis. The pattern of survival-associated gene expression matched with 29 perturbagen-induced transcriptome signatures in the iLINCS drug-repurposing database, identifying drugs, approved for other clinical indications, that were able to reverse in vitro the molecular changes associated with adverse prognosis in CHF. Conclusion Systematic modelling of the whole blood protein-coding transcriptome defined molecular pathways that provide a link between clinical risk factors and adverse CV prognosis in CHF, identifying both established and new potential therapeutic targets.

The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSC–derived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3a–regulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.

Aims The present study aims to characterize the genetic risk architecture of bicuspid aortic valve (BAV) disease, the most common congenital heart defect. Methods and results We carried out a genome-wide association study (GWAS) including 2236 BAV patients and 11 604 controls. This led to the identification of a new risk locus for BAV on chromosome 3q29. The single nucleotide polymorphism rs2550262 was genome-wide significant BAV associated (P = 3.49 × 10−08) and was replicated in an independent case–control sample. The risk locus encodes a deleterious missense variant in MUC4 (p.Ala4821Ser), a gene that is involved in epithelial-to-mesenchymal transformation. Mechanistical studies in zebrafish revealed that loss of Muc4 led to a delay in cardiac valvular development suggesting that loss of MUC4 may also play a role in aortic valve malformation. The GWAS also confirmed previously reported BAV risk loci at PALMD (P = 3.97 × 10−16), GATA4 (P = 1.61 × 10−09), and TEX41 (P = 7.68 × 10−04). In addition, the genetic BAV architecture was examined beyond the single-marker level revealing that a substantial fraction of BAV heritability is polygenic and ∼20% of the observed heritability can be explained by our GWAS data. Furthermore, we used the largest human single-cell atlas for foetal gene expression and show that the transcriptome profile in endothelial cells is a major source contributing to BAV pathology. Conclusion Our study provides a deeper understanding of the genetic risk architecture of BAV formation on the single marker and polygenic level.

The discovery of genetic loci associated with complex diseases has outpaced the elucidation of mechanisms of disease pathogenesis. Here we conducted a genome-wide association study (GWAS) for coronary artery disease (CAD) comprising 181,522 cases among 1,165,690 participants of predominantly European ancestry. We detected 241 associations, including 30 new loci. Cross-ancestry meta-analysis with a Japanese GWAS yielded 38 additional new loci. We prioritized likely causal variants using functionally informed fine-mapping, yielding 42 associations with less than five variants in the 95% credible set. Similarity-based clustering suggested roles for early developmental processes, cell cycle signaling and vascular cell migration and proliferation in the pathogenesis of CAD. We prioritized 220 candidate causal genes, combining eight complementary approaches, including 123 supported by three or more approaches. Using CRISPR–Cas9, we experimentally validated the effect of an enhancer in MYO9B, which appears to mediate CAD risk by regulating vascular cell motility. Our analysis identifies and systematically characterizes >250 risk loci for CAD to inform experimental interrogation of putative causal mechanisms for CAD.

Background: \ud Spontaneous coronary artery dissection (SCAD) is an increasingly recognized cause of acute coronary syndromes (ACS) afflicting predominantly younger to middle-aged women. Observational studies have reported a high prevalence of extracoronary vascular anomalies, especially fibromuscular dysplasia (FMD) and a low prevalence of coincidental cases of atherosclerosis. PHACTR1/EDN1 is a genetic risk locus for several vascular diseases, including FMD and coronary artery disease, with the putative causal noncoding variant at the rs9349379 locus acting as a potential enhancer for the endothelin-1 (EDN1) gene.\ud \ud Objectives: \ud This study sought to test the association between the rs9349379 genotype and SCAD.\ud \ud Methods: \ud Results from case control studies from France, United Kingdom, United States, and Australia were analyzed to test the association with SCAD risk, including age at first event, pregnancy-associated SCAD (P-SCAD), and recurrent SCAD.\ud \ud Results: \ud The previously reported risk allele for FMD (rs9349379-A) was associated with a higher risk of SCAD in all studies. In a meta-analysis of 1,055 SCAD patients and 7,190 controls, the odds ratio (OR) was 1.67 (95% confidence interval [CI]: 1.50 to 1.86) per copy of rs9349379-A. In a subset of 491 SCAD patients, the OR estimate was found to be higher for the association with SCAD in patients without FMD (OR: 1.89; 95% CI: 1.53 to 2.33) than in SCAD cases with FMD (OR: 1.60; 95% CI: 1.28 to 1.99). There was no effect of genotype on age at first event, P-SCAD, or recurrence.\ud \ud Conclusions: \ud The first genetic risk factor for SCAD was identified in the largest study conducted to date for this condition. This genetic link may contribute to the clinical overlap between SCAD and FMD.

The recent identification of compounds that interact with the spliceosome (sudemycins, spliceostatin A, and meayamycin) indicates that these molecules modulate aberrant splicing via SF3B1 inhibition. Through whole transcriptome sequencing, we have demonstrated that treatment of Rh18 cells with sudemycin leads to exon skipping as the predominant aberrant splicing event. This was also observed following reanalysis of published RNA-seq data sets derived from HeLa cells after spliceostatin A exposure. These results are in contrast to previous reports that indicate that intron retention was the major consequence of SF3B1 inhibition. Analysis of the exon junctions up-regulated by these small molecules indicated that these sequences were absent in annotated human genes, suggesting that aberrant splicing events yielded novel RNA transcripts. Interestingly, the length of preferred downstream exons was significantly longer than the skipped exons, although there was no difference between the lengths of introns flanking skipped exons. The reading frame of the aberrantly skipped exons maintained a ratio of 2:1:1, close to that of the cassette exons (3:1:1) present in naturally occurring isoforms, suggesting negative selection by the nonsense-mediated decay (NMD) machinery for out-of-frame transcripts. Accordingly, genes involved in NMD and RNAs encoding proteins involved in the splicing process were enriched in both data sets. Our findings, therefore, further elucidate the mechanisms by which SF3B1 inhibition modulates pre-mRNA splicing.

The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.

[Image: see text] Improvements in the efficiency of the drug discovery process are needed in order to deliver life-saving medications to patients in a more cost-effective manner. While there are many reasons that the efficiency of this process has not gotten better, this Viewpoint proposes that the lack of integration of the three major disciplines (discovery, development, and clinical trials) plays a significant role in the ongoing high rate of failure in clinical trials for innovative drugs. Several specific proposals are made that may help to provide more integration, so that the gears of the human-driven drug discovery machine may mesh better in the future.

The reason why HIV cannot be cured by current therapy is because of viral persistence in resting T cells. One approach to permanent HIV remission that has received less attention is the so-called “block and lock” approach. The idea behind this approach is that the virus could be permanently disabled in patients if viral genome or surrounding chromatin could be altered to silence the virus, thus enabling patients to stop therapy. In this work, we have identified splicing factor 3B subunit 1 (SF3B1) as a potential target for this approach. SF3B1 interacts with the viral protein Tat, which is critical for viral transcription. Inhibition of SF3B1 prevents HIV transcription and reactivation from latency. Since there are preclinical inhibitors for this protein, our findings could pave the way to silence HIV transcription, potentially leading to prolonged or permanent remission., The main obstacle to an HIV cure is the transcriptionally inert proviruses that persist in resting CD4 T cells and other reservoirs. None of the current approaches has significantly reduced the size of the viral reservoir. Hence, alternative approaches, such as permanent blocking of viral transcription, to achieve a sustained remission, need urgent attention. To identify cellular factors that may be important for this approach, we sought for host targets that when altered could block HIV transcription and reactivation. Here, we identified splicing factor 3B subunit 1 (SF3B1) as a critical HIV dependency factor required for viral replication. SF3B1 is a splicing factor involved in directing chromatin and nascent gene transcripts to appropriate splice sites. Inhibitors of SF3B1 are currently in development for cancer and have been found to be nontoxic to normal cells compared to malignant cells. Knockdown of SF3B1 abrogated HIV replication in all cell types tested. SF3B1 interacted with viral protein Tat in vitro and in vivo. Genetic or pharmacologic inhibition of SF3B1 prevented Tat-mediated HIV transcription and RNA polymerase II association with the HIV promoter. In addition, an inhibitor of SF3B1 prevented HIV reactivation from latency irrespective of the latency-reversing agent used. The data show that SF3B1 is involved in viral transcription and reactivation from latency and may serve as a therapeutic target in the HIV cure efforts.

The recent identification of compounds that interact with the spliceosome (sudemycins, spliceostatin A, and meayamycin) indicates that these molecules modulate aberrant splicing via SF3B1 inhibition. Through whole transcriptome sequencing, we have demonstrated that treatment of Rh18 cells with sudemycin leads to exon skipping as the predominant aberrant splicing event. This was also observed following reanalysis of published RNA-seq data sets derived from HeLa cells after spliceostatin A exposure. These results are in contrast to previous reports that indicate that intron retention was the major consequence of SF3B1 inhibition. Analysis of the exon junctions up-regulated by these small molecules indicated that these sequences were absent in annotated human genes, suggesting that aberrant splicing events yielded novel RNA transcripts. Interestingly, the length of preferred downstream exons was significantly longer than the skipped exons, although there was no difference between the lengths of introns flanking skipped exons. The reading frame of the aberrantly skipped exons maintained a ratio of 2:1:1, close to that of the cassette exons (3:1:1) present in naturally occurring isoforms, suggesting negative selection by the nonsense-mediated decay (NMD) machinery for out-of-frame transcripts. Accordingly, genes involved in NMD and RNAs encoding proteins involved in the splicing process were enriched in both data sets. Our findings, therefore, further elucidate the mechanisms by which SF3B1 inhibition modulates pre-mRNA splicing.

Coronary artery disease (CAD) has substantial heritability and a polygenic architecture. However, the potential of genomic risk scores to help predict CAD outcomes has not been evaluated comprehensively, because available studies have involved limited genomic scope and limited sample sizes.This study sought to construct a genomic risk score for CAD and to estimate its potential as a screening tool for primary prevention.Using a meta-analytic approach to combine large-scale, genome-wide, and targeted genetic association data, we developed a new genomic risk score for CAD (metaGRS) consisting of 1.7 million genetic variants. We externally tested metaGRS, both by itself and in combination with available data on conventional risk factors, in 22,242 CAD cases and 460,387 noncases from the UK Biobank.The hazard ratio (HR) for CAD was 1.71 (95% confidence interval [CI]: 1.68 to 1.73) per SD increase in metaGRS, an association larger than any other externally tested genetic risk score previously published. The metaGRS stratified individuals into significantly different life course trajectories of CAD risk, with those in the top 20% of metaGRS distribution having an HR of 4.17 (95% CI: 3.97 to 4.38) compared with those in the bottom 20%. The corresponding HR was 2.83 (95% CI: 2.61 to 3.07) among individuals on lipid-lowering or antihypertensive medications. The metaGRS had a higher C-index (C = 0.623; 95% CI: 0.615 to 0.631) for incident CAD than any of 6 conventional factors (smoking, diabetes, hypertension, body mass index, self-reported high cholesterol, and family history). For men in the top 20% of metaGRS with2 conventional factors, 10% cumulative risk of CAD was reached by 48 years of age.The genomic score developed and evaluated here substantially advances the concept of using genomic information to stratify individuals with different trajectories of CAD risk and highlights the potential for genomic screening in early life to complement conventional risk prediction.

Schistosomiasis is a debilitating tropical disease caused by infection with parasitic blood flukes. Approximately 260 million people are infected worldwide, underscoring the clinical and socioeconomic impact of this chronic infection. Schistosomiasis is treated with the drug praziquantel (PZQ), which has proved the therapeutic mainstay for over three decades of clinical use. However, the molecular target(s) of PZQ remain undefined. Here we identify a molecular target for the antischistosomal eutomer — (R)-PZQ — which functions as a partial agonist of the human serotoninergic 5HT2B receptor. (R)-PZQ modulation of serotoninergic signaling occurs over a concentration range sufficient to regulate vascular tone of the mesenteric blood vessels where the adult parasites reside within their host. These data establish (R)-PZQ as a G-protein-coupled receptor ligand and suggest that the efficacy of this clinically important anthelmintic is supported by a broad, cross species polypharmacology with PZQ modulating signaling events in both host and parasite., Schistosomiasis is caused by infection with the flatworm Schistosoma, and praziquantel is the drug of choice for its treatment. Here, Chan and colleagues identify praziquantel as a ligand for the human serotoninergic 5-HT2B G-protein-coupled receptor, and reveal a function for praziquantel as a regulator of vascular tone in treated hosts.

Influenza is a seasonal and serious health threat, and the recent outbreak of H7N9 following the pandemic spread of H1N1 in 2009 has served to emphasize the importance of anti-influenza drug discovery. Zanamivir (Relenza#8482;) and oseltamivir (Tamiflu(®)) are two antiviral drugs currently recommended by the CDC for treating influenza. Both are examples of the successful application of structure-based drug design strategies. These strategies have combined computer- based approaches, such as docking- and pharmacophore-based virtual screening with X-ray crystallographic structural analyses. Docking is a routinely used computational method to identify potential hits from large compound libraries. This method has evolved from simple rigid docking approaches to flexible docking methods to handle receptor flexibility and to enhance hit rates in virtual screening. Virtual screening approaches can employ both ligand-based and structurebased pharmacophore models depending on the available information. The exponential growth in computing power has increasingly facilitated the application of computer-aided methods in drug discovery, and they now play significant roles in the search for novel therapeutics. An overview of these computational tools is presented in this review, and recent advances and challenges will be discussed. The focus of the review will be anti-influenza drug discovery and how advances in our understanding of viral biology have led to the discovery of novel influenza protein targets. Also discussed will be strategies to circumvent the problem of resistance emerging from rapid mutations that has seriously compromised the efficacy of current anti-influenza therapies.

Schistosomiasis is a highly prevalent neglected tropical disease caused by blood-dwelling helminths of the genus Schistosoma. Praziquantel (PZQ) is the only drug available widely for the treatment of this disease and is administered in racemic form, even though only the (R)-isomer has significant anthelmintic activity. Progress towards the development of a second generation of anthelmintics is hampered by a lack of understanding of the mechanism of action of PZQ. In this letter, we report an efficient protocol for the small-scale separation of enantiomers of 2(hydrolyzed PZQ) using supercritical fluid chromatography (SFC). The enantiopure 2 were then used to develop several molecular probes, which can potentially be used to help identify the protein target of PZQ and study its mode of action.

Identification of mutation-specific targeted therapies is a critical challenge in precision medicine. Synthetic lethality provides the basis for an approach to identify new therapeutic targets for mutations that are difficult to target directly, since in synthetic lethal (SL) interactions, an alteration in one gene leads to dependency on a second gene. Neither alteration by itself is essential for survival, but together these alterations lead to cancer cell death. We previously developed a new computational method, Mining Synthetic Lethals (MiSL), that can infer relationships from primary human tumor genomic and transcriptomic data and enable the identification of SL interactions in the context of native human tumors. The underlying assumption of MiSL is that SL partners of a mutation will be selectively amplified or never deleted in primary tumor samples harboring the mutation. Here, we used MiSL to identify novel SL partners of KRAS mutations in lung cancer. KRAS activating mutations are highly prevalent in lung cancer and aside from early clinical data on a novel covalent inhibitor for KRAS G12C mutations, there is no reported treatment to date for patients with KRAS mutations. We applied MiSL to identify SL interactions with KRAS G12 mutations in non-small cell lung cancer (NSCLC) and identified 220 SL partner genes. Some of these genes were foreseeable dependencies, such as MAPK, growth factor and inflammatory signaling associated genes; while others were novel SL candidates and included genes associated with splicing. Since we have access to a splicing-modulating drug in preclinical development, sudemycin D6 (SD6) which targets SF3B1, a spliceosome subunit, we focused on testing the SF3B1 interactors identified by MiSL, SLU7 and SRRM2. To enable validation studies, we first verified KRAS growth dependency in a panel of NSCLC cell lines using siRNA knock-down (KD). Next, we tested siRNA KD of predicted MiSL candidates on growth of KRAS-dependent (H358 and H441 – KRAS mutant) and KRAS-independent (H2228 – KRAS WT) cell lines. We confirmed that the KD of SLU7 expression reduced viability of KRAS mutant (75 to 85% reduced) but not KRAS wild-type (WT) cells lines. Similar differential viability was also observed with siRNA KD of SF3B1. Pharmacological inhibition of SF3B1 using SD6 also demonstrated that KRAS mutant cell lines were more sensitive to SD6 compared to a KRAS WT line (>5-30x). Finally, we validated these findings in an in vivo tumor xenograft model by treating KRAS WT and KRAS mutant tumors with SD6 and showed that the KRAS mutant tumors were significantly more inhibited by SD6 than KRAS WT tumors. Studies are ongoing to further confirm the SL interaction in lung cancer cell lines that are isogenic except for the KRAS mutation. In conclusion, SL predictions based on primary KRAS G12 mutant tumors indicated that splicing modulation is a vulnerability of KRAS tumors. Finally, synthetic lethal targets identified by primary tumor data mining can provide new therapeutic targets for KRAS specific mutant cancers, which can lead to personalized treatment options for NSCLC cancer patients. Citation Format: Claire E Repellin, Puja Patel, Yihui Shi, Helena Gong, Leena Shewade, Thomas Webb, Lidia Sambucetti, Subarna Sinha. Primary tumor data analysis reveals novel synthetic lethal dependencies between KRAS mutation and the spliceosome [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C018. doi:10.1158/1535-7163.TARG-19-C018

Identification of predictive biomarkers for targeted drugs is an active research area in precision oncology. Usually, a genetic alteration in the drug target is used to identify responders, which can limit the use of the drug in other responsive cancers. Alternatively, cell line-based drug sensitivity data are used to build machine-learning models to identify predictive biomarkers. These methods require large amounts of data and the black-box models developed are often hard to interpret. Synthetic lethality provides an alternate approach for predictive biomarker identification of targeted drugs. In synthetic lethal (SL) interactions, a defect in one gene leads to dependency on a second gene. Neither defect by itself is essential for survival, but together they lead to cell death. Thus, the inhibition of the drug target in the presence of a cancer-specific genetic alteration would result in cancer cell death. The genetic alterations can be used as biomarkers for the drug. We developed a novel computational pipeline for predictive biomarker identification based on our tool, Mining Synthetic Lethals (MiSL), that mines SL interactions from pan-cancer primary tumor data (such as TCGA). Since MiSL utilizes primary human tumor data, it enables the identification of SL interactions in the native context of human tumors and is more likely to find relationships relevant to in vivo tumor biology than shRNA/CRISPR screens. To discover biomarkers of a targeted drug, we used MiSL to identify genetic alterations that are SL with targets of the drug in a specific cancer type. As proof of principle, we applied our method to identify biomarkers of sudemycin-D6 (SD6), a potent splicing modulator, in non-small cell lung cancer (NSCLC). First, we identified putative drug targets of SD6. Since SD6 is known to inhibit SF3B1, any gene belonging to the same protein complex as SF3B1 was termed a SD6 drug target. Using these SD6 drug targets, our MiSL-based pipeline identified KRAS mutations, present in ~30% of NSCLC patients, as a SD6 biomarker. To confirm our predictions, we treated a panel of KRAS-mutant-and-dependent NSCLC lines (H358, H23 and H441) and a KRAS-WT NSCLC line (H2228) with SD6 and measured cell viability. All KRAS-mutant lines tested were significantly more sensitive to SD6 (> 5-30X lower IC50) than the KRAS-WT line. Furthermore, the KRAS-mutant lines were also more sensitive (5-20X lower IC50), compared to the KRAS-WT line, to a splicing modulator of a different class – a kinase inhibitor that phosphorylates components of the spliceosome. In vivo studies are underway to further validate our in vitro findings. This study identifies KRAS mutation as a novel predictive biomarker for splicing modulators in NSCLC, which could lead to new therapeutic options for KRAS-mutated NSCLC. Furthermore, our work demonstrates the feasibility of a synthetic lethality-based pipeline to predict biomarkers for targeted drugs. Citation Format: Claire E. Repellin, Puja Patel, Yihui Shi, Helena Gong, Thomas R. Webb, Lidia Sambucetti, Subarna Sinha. Synthetic lethality-based predictive biomarker identification of splicing modulators in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4015.

Splicing factor SF3B1 is frequently mutated in chronic lymphocytic leukemia (CLL) patients and has been suggested as a potential therapeutic target. In this study, we demonstrated that SF3B1 modulator sudemycin D6 (SD6) effectively inhibits cell growth and induces apoptosis in CLL cells. RNA sequencing analysis revealed significant increases in global intron retention in SD6-treated CLL cells. Pathway analysis of the genes associated with increased intron retention suggested that B-cell receptor (BCR) and PI3K signaling pathways were among the most important pathways being affected by SD6. The increases in intron retention were inversely correlated with decreases in mRNA and protein levels of the affected BCR/PI3K pathway molecules including BLNK, BTK, AKT, PLCγ2, and PI3Kδ. SD6 treatment also induced a time-dependent exon-skipping event in MCL-1 mRNA and resulted in significant down-regulation of another anti-apoptotic gene TRAF1, thus collectively contributing to the SD6-induced apoptosis. Furthermore, SD6 treatment can overcome the pro-survival and pro-growth signals and synergize with established CLL therapies ibrutinib, idelalisib, and venetoclax to induce apoptosis in primary CLL cells co-cultured with bone marrow stromal cells and T-cell-derived cytokines. Finally, in vivo treatment with SD6 at 10mg/kg/day for 7 days significantly inhibited the growth of xenograft tumors that were established by subcutaneous inoculation of 5×106 MEC1 CLL cells into NOD mice. Collectively, these results provide a strong rationale for the future clinical development of spliceosome modulators and potential combination therapies for the treatment of CLL. Citation Format: Qimei Han, Jianbo Wang, Austin Y. Shull, Fang Shi, Libin Deng, Jeong-Hyeon Choi, Eun-Jeong Park, Shuo Tu, Lirong Pei, Farrukh T. Awan, Roni Bollag, Locke J. Bryan, Hong-bo Xin, Chandraiah Lagisetti, Thomas R. Webb, Victor Jin, Huidong Shi. Modulation of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5230.

Somatic mutations in spliceosome genes are detectable in ∼50% of patients with myelodysplastic syndromes (MDS). We hypothesize that cells harbouring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome. We focus on mutant U2AF1 and utilize sudemycin compounds that modulate pre-mRNA splicing. We find that haematopoietic cells expressing mutant U2AF1(S34F), including primary patient cells, have an increased sensitivity to in vitro sudemycin treatment relative to controls. In vivo sudemycin treatment of U2AF1(S34F) transgenic mice alters splicing and reverts haematopoietic progenitor cell expansion induced by mutant U2AF1 expression. The splicing effects of sudemycin and U2AF1(S34F) can be cumulative in cells exposed to both perturbations—drug and mutation—compared with cells exposed to either alone. These cumulative effects may result in downstream phenotypic consequences in sudemycin-treated mutant cells. Taken together, these data suggest a potential for treating haematological cancers harbouring U2AF1 mutations with pre-mRNA splicing modulators like sudemycins., Spliceosome mutations occur in approximately 50% of patients with myelodysplastic syndromes. Here, the authors show that tumour cells harbouring the S34F mutation in the U2AF spliceosome gene is sensitive to compounds that further perturb the spliceosome.

KRAS is the most frequently mutated oncogene in human cancer, but its therapeutic targeting remains challenging. Here, we report a synthetic lethal screen with a library of deubiquitinases and identify USP39, which encodes an essential splicing factor, as a critical gene for the viability of KRAS-dependent cells. We show that splicing fidelity inhibitors decrease preferentially the proliferation rate of KRAS-active cells. Moreover, depletion of DHX38, encoding an USP39-interacting splicing factor, also reduces the viability of these cells. In agreement with these results, USP39 depletion caused a significant reduction in pre-mRNA splicing efficiency, as demonstrated through RNA-seq experiments. Furthermore, we show that USP39 is up-regulated in lung and colon carcinomas and its expression correlates with KRAS levels and poor clinical outcome. Accordingly, our work provides critical information for the development of splicing-directed antitumor treatments and supports the potential of USP39-targeting strategies as the basis of new anticancer therapies.

The identification of potent spliceosome modulators that demonstrate antitumor activity indicates that this complex may be a target for drug development. Several natural products have been demonstrated to bind to the SF3b1 subunit of this macromolecule and these agents modulate alternative RNA splicing. In this article we describe their biological properties, discuss the validity of the spliceosome as a therapeutic target, and propose that alteration of alternative splicing represents a viable approach for inducing tumor-selective cytotoxicity.

The splicing of pre-mRNA is a critical process in normal cells and is deregulated in cancer. Compounds that modulate this process have recently been shown to target a specific vulnerability in tumors. We have developed a novel cell-based assay that specifically activates luciferase in cells exposed to SF3B1 targeted compounds, such as sudemycin D6. This assay was used to screen a combined collection of approved drugs and bioactive compounds. This screening approach identified several active hits, the most potent of which were CGP-74514A and aminopurvalanol A, both have been reported to be cyclin-dependent kinases (CDKs) inhibitors. We found that these compounds, and their analogs, show significant cdc2-like kinase (CLK) inhibition and clear structure-activity relationships (SAR) at CLKs. We prepared a set of analogs and were able to ‘dial out’ the CDK activity and simultaneously developed CLK inhibitors with low nanomolar activity. Thus, we have demonstrated the utility of our exon-skipping assay and identified new molecules that exhibit potency and selectivity for CLK, as well as some structurally related dual CLK/CDK inhibitors.

KRAS is the most frequently mutated oncogene in human cancer, but its therapeutic targeting remains challenging. Here, we report a synthetic lethal screen with a library of deubiquitinases and identify USP39, which encodes an essential splicing factor, as a critical gene for the viability of KRAS-dependent cells. We show that splicing fidelity inhibitors decrease preferentially the proliferation rate of KRAS-active cells. Moreover, depletion of DHX38, encoding an USP39-interacting splicing factor, also reduces the viability of these cells. In agreement with these results, USP39 depletion caused a significant reduction in pre-mRNA splicing efficiency, as demonstrated through RNA-seq experiments. Furthermore, we show that USP39 is up-regulated in lung and colon carcinomas and its expression correlates with KRAS levels and poor clinical outcome. Accordingly, our work provides critical information for the development of splicing-directed antitumor treatments and supports the potential of USP39-targeting strategies as the basis of new anticancer therapies.

We report the synthesis of a pyrimidinone library that targets anaplastic lymphoma kinase (ALK), an oncogenic receptor tyrosine kinase. This library was generated in three steps from a versatile commercially available starting material. Some compounds within this library showed single digit micromolar inhibition of ALK in vitro, while showing minimal inhibition of other homologous insulin receptor family kinases including the human insulin receptor kinase (IRK), at the highest concentrations investigated. We also present initial ALK structure–activity relationships for this library.

Two unrelated bacterial natural products, FR901464 and pladienolide B, have previously been shown to have significant antitumor activity in vivo. These compounds target the SF3b subunit of the spliceosome, with a derivative of pladienolide (E7107) entering clinical trials for cancer. However, due to the structural complexity of these molecules, their research and development has been significantly constrained. We have generated a set of novel analogues (Sudemycins) that possess the pharmacophore that is common to FR901464 and pladienolide, via a flexible enantioselective route, which allows for the production of gram quantities of drug. These compounds demonstrate cytotoxicity toward human tumor cell lines in culture and exhibit antitumor activity in a xenograft model. Here, we present evidence that Sudemycins are potent modulators of alternative splicing in human cells, both of endogenous genes and from minigene constructs. Furthermore, levels of alternative splicing are increased in tumor cells relative to normal cells, and these modifications can be observed in human tumor xenografts in vivo following exposure of animals to the drug. In addition, the change in the splicing pattern observed with the Sudemycins are similar to that observed with Spliceostatin A, a molecule known to interact with the SF3b subunit of the spliceosome. Hence, we conclude that Sudemycins can regulate the production of alternatively spliced RNA transcripts and these alterations are more prevalent in tumors, as compared to normal cells, following drug exposure. These studies suggest that modulation of alternative splicing may play a role in the antitumor activity of this class of agents.

Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the Western countries but is relatively rare in East Asia. Mutations in myeloid differentiation primary response gene 88 (MYD88) occur in 2.0-4.4% of Caucasian patients with CLL. However, Asian subjects showed a higher MYD88 mutation frequency of 8% and MYD88 mutations were associated with unfavorable prognosis in Asian CLL patients with mutated IGHV gene. To further explore the molecular and pathological mechanisms of the MYD88 L265P mutation in CLL, we performed a pilot study to investigate the transcriptome-wide differential expression between CLL patients with and without MYD88 L265P mutation. Methods: mRNA sequencing was performed on a cohort of 15 Chinese CLL patients with MYD88 L265P mutation (n=5) or wild-type MYD88 gene (n=10). Differential expression and pathway enrichment analyses were performed based on FPKM values after processing the raw sequencing reads. The alternative splicing events were analyzed using JuncBASE and the differences in splicing patterns were compared between the two groups. The MYD88 L265P and wild-type genes were overexpressed in the MEC-1 cell line using lentiviral vectors, and stable cell lines were established after puromycin selection. Real time quantitative PCR was used to determine the mRNA expression of spliceosome pathway genes. The protein expression of TLR pathway was assessed with Western Blot. The PrestoBlue™ assay was used for determining IC-50 of spliceosome inhibitor-treated CLL cell lines, and flow cytometry was utilized for apoptosis and cell death analyses. Results: mRNA sequencing analysis revealed that 4231 genes showed significant differences in mRNA expression between MYD88 mutant and wild-type CLL patients. Of these genes, 3033 were up regulated and 1198 were down regulated in patients with MYD88 L265P mutation. Pathway analysis demonstrated that tumor necrosis factor (TNF) signaling, cell cycle, spliceosome, ribosome, p53 signaling, and NF-κB signaling pathways were significantly enriched among differentially expressed genes. Interestingly, about two thirds (19 out of 30) of differentially expressed genes in the spliceosome pathway were down regulated in CLL samples with MYD88 mutation. Quantitative RT-PCR confirmed the down regulation of spliceosome genes including SF3A1, SF3A2, SF3B4, SF3B5 and PUF60 in MYD88 mutated samples in an independent CLL patient cohort. Analysis of alternative splicing events revealed a total of 180 genes with inter-group splicing differences, among which differential splicing events were most common in the genes involved in immune system regulation, i. e. CD22, CD79B, CD200 and IKZF1 etc. The MEC1 cells stably overexpressing MYD88 L265P demonstrated decreases in RelA and TRAF6 expression as compared to wild type MYD88-overexpressing cells, which was in agreement with RNA-seq analysis results in CLL patient samples. In addition, we found that overexpression of MYD88 L265P mutant resulted in decreased mRNA expression of several spliceosome genes such as SF3A1, SF3A2, SF3B4, SF3B5 and PUF60 in the stable cell line. Furthermore, MYD88 L265P increased the sensitivity of MEC1 cells to spliceosome inhibitor sudemycin D6 (SD6) by 3 fold, while reducing their sensitivity to Ibrutinib by two fold. Conclusion: We discovered the differential mRNA expression landscapes related to MYD88 mutation status in CLL and their contributions to the progression of CLL through multiple pathways. Of particular interest was the down regulation of spliceosome pathway genes. In a CLL cell line model, we found that, compared with the MYD88 wild-type CLL cell line, CLL cells overexpressing the mutant MYD88 were more sensitive to spliceosome inhibitor SD6, suggesting that inhibiting spliceosome function may be a potential therapeutic target for CLL patients with mutant MYD88 gene. In summary, our findings provide novel insights for further understanding of the molecular and pathological mechanisms of MYD88 mutation in CLL. Disclosures Lagisetti: St. Jude Children's Research Hospital: Patents & Royalties: Chandraiah Lagisetti is listed as inventors on patents assigned to St. Jude Children's Research Hospital covering sudemycin D6 and was employed by SRI Biosciences at the time these experiments were carried out. Webb:St. Jude Children's Research Hospital: Patents & Royalties: Thomas R. Webb is listed as inventors on patents assigned to St. Jude Children's Research Hospital covering sudemycin D6 and was employed by SRI Biosciences at the time these experiments were carried out..

P-glycoprotein (Pgp) is an ATP-dependent efflux pump involved in transport of xenobiotics from cells that, when overexpressed, can mediate multidrug resistance in mammalian cells. Pgp may be a candidate target for new anthelmintics, as it plays critical roles in normal cell physiology, in removal of drugs from cells, and potentially in the development of drug resistance. Schistosomes are parasitic flatworms that cause schistosomiasis, which affects hundreds of millions of people worldwide. Here, we express SMDR2, a Pgp homologue from Schistosoma mansoni (Platyhelminthes), in Chinese hamster ovary (CHO) cells and use fluorescence-based assays to examine the functional and pharmacological properties of this transporter. Membrane vesicles from stably transfected CHO cells expressing recombinant SMDR2 show significant increases in rhodamine transport and ATP hydrolysis compared with those from control cells or cells transfected with empty vector. SMDR2-mediated transport is inhibited by the Pgp modulators verapamil (IC50=12.1 μM) and nifedipine, and also by praziquantel, the current drug of choice against schisotosomiasis (IC50=17.4 μM). Efflux measurements of a fluorescent analog of praziquantel indicate that it is also a substrate for SMDR2. The interaction of praziquantel with SMDR2 may offer new strategies for potentiating the action of praziquantel and possibly overcoming drug resistance.—Kasinathan, R. S., Goronga, T., Messerli, S. M., Webb, T. R., Greenberg, R. M. Modulation of a Schistosoma mansoni multidrug transporter by the antischistosomal drug praziquantel.

As an effective strategy of the drug discovery for peptide-binding GPCRs based on the natural ligands, beta-turn peptidomimetic compound library with benzodiazepine skeleton was constructed using solid and solution phase parallel synthesis with four different scaffolds containing Phe, Lys, Ser and Glu, respectively. The usefulness of 162 library compounds was evaluated by the cell based screening at melanocortin 4 receptor in CHO-k1 cells, to find hit compounds showing agonistic effect at the receptor. The screening of library afforded three hit compounds including the most effective analog, (S)-3-benzyl-7-(4-fluorobenzyloxy)-4-(4-methoxyphenethyl)-4,5-dihydro-1H-benzo[e][1,4]diazepin-2(3H)-one, 13aiE, of which EC50 was determined as 13 μM.

We report the design and synthesis of an insulin receptor kinase family-targeted inhibitor template using the inhibitor conformation observed in an IGF1R/inhibitor co-crystal complex by application of a novel molecular design approach that we have recently published. The synthesis of the template involves a one pot Opatz cyclization reaction that provides a versatile indole ester in good yields. We also developed the required chemistry to elaborate this template with additional substituents and have used this chemistry to prepare some initial compounds that show selective inhibition of anaplastic lymphoma kinase (ALK).

Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.

We report the design and highly enantioselective synthesis of a potent analogue of the spliceosome inhibitor FR901464, based on a non-natural product scaffold. The design of this compound was facilitated by a pharmacophore hypothesis that assumed key interaction types that are common to FR901464 and an otherwise unrelated natural product (pladienolide). The synthesis allows for the preparation of numerous novel analogues. We present results on the in vitro activity for this compound against several tumor cell lines.

Kevlar and Nomex are high-performance polymers which have wide varieties of applications in daily life. Recently, they have been proposed to be biocidal materials when reacted with household bleach (sodium hypochlorite solution) because they contain amide moieties which can be chlorinated to generate biocidal N-halamine functional groups. Although Nomex can be chlorinated without any significant decomposition, Kevlar decomposes under the same chlorination conditions. In this study, two mimics for each of the polymers were synthesized to simulate the carboxylate and diaminophenylene components of the materials. It was found that the p-diaminophenylene component of the Kevlar mimic is oxidized to a quinone-type structure upon treatment with hypochlorous acid, which then decomposes. However, such a mechanism for the Nomex mimic is not possible. In this paper, based upon these observations, a plausible answer will be provided to the title question.

The spliceosome has recently emerged as a new target for cancer chemotherapy and novel antitumor spliceosome targeted agents are under development. Here, we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small-molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel-based assay for the measurement of drug exposure in human leukocytes. The first assay utilizes a highly specific bioluminescent splicing reporter, based on the skipping of exons 4-11 of a Luc-MDM2 construct, which specifically yields active luciferase when treated with small-molecule spliceosome modulators. We demonstrate that this reporter can be used to monitor alternative splicing in whole cells in vitro. We describe here that cell lines carrying the reporter can be used in vivo for the efficient pharmacodynamic analysis of agents during drug optimization and development. We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression. The second assay relies on the treatment of freshly drawn human blood with SD6 ex vivo treatment. Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments. The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

The optimization of screening hits on a promising new target for therapy of certain cancers involving anaplastic lymphoma kinase (ALK) inspired the development of this efficient solid-phase chemistry. A series of novel pyridones have been recently discovered as inhibitors of ALK, which led to the design of focused libraries around the pyridone scaffold. A stepwise process involving iterative template modification based on both medicinal chemistry insights and computational ranking of virtual libraries was employed in the design. The unique solid-phase chemistry has addressed the need for rapid optimization of this "early lead" series. Herein the methodology and scope of the chemistry, as well as its application for library synthesis, are discussed.

The beta-turn has been implicated as an important conformation for biological recognition of peptides or proteins. We adapted the concept of general Calpha atom positioning from the cluster analysis and recombination of each ideal beta-turn conformation pattern by Garland and Dean (J. Comput.-Aided Mol. Des. 1999, 13, 469) as one strategy of designing non-peptide beta-turn scaffolds. Herein, the Calpha positions of tetrahydro-1,4-benzodiazepin-2-one scaffold were analyzed after the calculation of the low-energy conformer using a semiempirical protocol. Three points of corresponding Calpha carbons for diverse substitutions in the scaffold were designated, and an efficient solid-phase synthesis of the peptidomimetic library was developed. The scaffold itself was synthesized in solution phase starting from 5-hydroxy-2-nitrobenzaldehyde and loaded to the 4-formyl-3,5-dimethoxyphenoxy (PL-FDMP) resin with high efficiency of reductive amination. Various building blocks for the derivatization of the 7-hydroxyl and N-1 amide nitrogen could be introduced via selective alkylation. Cleavage, parallel column chromatography, and NMR analysis of 62 final compounds confirmed the feasibility of this peptidomimetic library synthesis.

The coordination chemistry of silver(I) with the nitrogen-bridged ligands (C6H5)2PN(R)P(C6H5)2 [R = H (dppa); R = CH3 (dppma)] has been investigated by 31P NMR and electrospray mass spectrometry (ESMS). Species observed by 31P NMR include Ag2(μ-dppa)2+, Ag2(μ-dppa)22+, Ag2(μ-dppa)32+, Ag2(μ-dppma)2+, Ag2(μ-dppma)22+, and Ag(η2-dppma)2+. Species observed by ESMS at low cone voltages were Ag2(dppa)22+, Ag2(dppa)32+, Ag2(dppma)22+, and Ag(dppma)2+. (C6H5)2PN(CH3)P(C6H5)2 showed a strong tendency to chelate, while (C6H5)2PN(H)P(C6H5)2 preferred to bridge. Differences in the bridging versus chelating behavior of the ligands are assigned to the Thorpe−Ingold effect, where the methyl group on nitrogen sterically interacts with the phenyl groups on phosphorus. The crystal structure of the three-coordinate dinuclear silver(I) complex {Ag2[(C6H5)2PN(H)P(C6H5)2]3}(BF4)2 has been determined. Bond distances include Ag−Ag = 2.812(1) A, Ag(1)−P(av) = 2.492(3) A, and Ag(2)−P(av) = 2.509(3) A. The compound crystallizes in...

The splicing of pre-mRNA is a critical process in normal cells and is deregulated in cancer. Compounds that modulate this process have recently been shown to target a specific vulnerability in tumors. We have developed a novel cell-based assay that specifically activates luciferase in cells exposed to SF3B1 targeted compounds, such as sudemycin D6. This assay was used to screen a combined collection of approved drugs and bioactive compounds. This screening approach identified several active hits, the most potent of which were CGP-74514A and aminopurvalanol A, both have been reported to be cyclin-dependent kinases (CDKs) inhibitors. We found that these compounds, and their analogs, show significant cdc2-like kinase (CLK) inhibition and clear structure-activity relationships (SAR) at CLKs. We prepared a set of analogs and were able to ‘dial out’ the CDK activity and simultaneously developed CLK inhibitors with low nanomolar activity. Thus, we have demonstrated the utility of our exon-skipping assay and identified new molecules that exhibit potency and selectivity for CLK, as well as some structurally related dual CLK/CDK inhibitors. Citation Format: Yihui Shi, Jaehyeon Park, Chandraiah Lagisetti, Wei Zhou, Lidia C. Sambucetti, Thomas R. Webb. A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor scaffold [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4193. doi:10.1158/1538-7445.AM2017-4193

Background Reprogramming cellular gene transcription sustains HTLV-1 viral persistence that ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL). We hypothesized that besides these quantitative transcriptional effects, HTLV-1 qualitatively modifies the pattern of cellular gene expression. Results Exon expression analysis shows that patients’ untransformed and malignant HTLV-1+ CD4+ T-cells exhibit multiple alternate exon usage (AEU) events. These affect either transcriptionally modified or unmodified genes, culminate in ATLL, and unveil new functional pathways involved in cancer and cell cycle. Unsupervised hierarchical clustering of array data permitted to isolate exon expression patterns of 3977 exons that discriminate uninfected, infected, and transformed CD4+ T-cells. Furthermore, untransformed infected CD4+ clones and ATLL samples shared 486 exon modifications distributed in 320 genes, thereby indicating a role of AEUs in HTLV-1 leukemogenesis. Exposing cells to splicing modulators revealed that Sudemycin E reduces cell viability of HTLV-1 transformed cells without affecting primary control CD4+ cells and HTLV-1 negative cell lines, suggesting that the huge excess of AEU might provide news targets for treating ATLL. Conclusions Taken together, these data reveal that HTLV-1 significantly modifies the structure of cellular transcripts and unmask new putative leukemogenic pathways and possible therapeutic targets. Electronic supplementary material The online version of this article (doi:10.1186/s12977-014-0119-3) contains supplementary material, which is available to authorized users.

MYC (also known as c-MYC) overexpression or hyperactivation is one of the most common drivers of human cancer. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. MYC is a transcription factor, and many of its pro-tumorigenic functions have been attributed to its ability to regulate gene expression programs. Notably, oncogenic MYC activation has also been shown to increase total RNA and protein production in many tissue and disease contexts. While such increases in RNA and protein production may endow cancer cells with pro-tumour hallmarks, this increase in synthesis may also generate new or heightened burden on MYC-driven cancer cells to process these macromolecules properly. Here we discover that the spliceosome is a new target of oncogenic stress in MYC-driven cancers. We identify BUD31 as a MYC-synthetic lethal gene in human mammary epithelial cells, and demonstrate that BUD31 is a component of the core spliceosome required for its assembly and catalytic activity. Core spliceosomal factors (such as SF3B1 and U2AF1) associated with BUD31 are also required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces an increase in total precursor messenger RNA synthesis, suggesting an increased burden on the core spliceosome to process pre-mRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYC-hyperactivated cells leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of many essential cell processes. Notably, genetic or pharmacological inhibition of the spliceosome in vivo impairs survival, tumorigenicity and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing, and that components of the spliceosome may be therapeutic entry points for aggressive MYC-driven cancers.