Inhibition of nonsense-mediated mRNA decay may improve stop codon read-through therapy for Duchenne muscular dystrophy.

Duchene muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are genetic neuromuscular disorders that affect skeletal and cardiac muscle resulting from mutations in the dystrophin gene (DMD), coding for dystrophin protein. Read-through therapies hold great promise for the treatment of genetic diseases harboring nonsense mutations, such as DMD/BMD, as they enable a complete translation of the affected mRNA. However, to date, most read-through drugs have not achieved a cure for patients. One possible explanation for the limitation of these therapies for DMD/BMD is that they rely on the presence of mutant dystrophin mRNAs. However, the mutant mRNAs containing premature termination codons are identified by the cellular surveillance mechanism, the nonsense-mediated mRNA decay (NMD) process, and are degraded. Here, we show that the combination of read-through drugs together with known NMD inhibitors have a synergistic effect on the levels of nonsense-containing mRNAs, among them the mutant dystrophin mRNA. This synergistic effect may enhance read-through therapies' efficacy and improve the current treatment for patients.

RBFOX2 modulates a metastatic signature of alternative splicing in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by aggressive local invasion and metastatic spread, leading to high lethality. Although driver gene mutations during PDA progression are conserved, no specific mutation is correlated with the dissemination of metastases1-3. Here we analysed RNA splicing data of a large cohort of primary and metastatic PDA tumours to identify differentially spliced events that correlate with PDA progression. De novo motif analysis of these events detected enrichment of motifs with high similarity to the RBFOX2 motif. Overexpression of RBFOX2 in a patient-derived xenograft (PDX) metastatic PDA cell line drastically reduced the metastatic potential of these cells in vitro and in vivo, whereas depletion of RBFOX2 in primary pancreatic tumour cell lines increased the metastatic potential of these cells. These findings support the role of RBFOX2 as a potent metastatic suppressor in PDA. RNA-sequencing and splicing analysis of RBFOX2 target genes revealed enrichment of genes in the RHO GTPase pathways, suggesting a role of RBFOX2 splicing activity in cytoskeletal organization and focal adhesion formation. Modulation of RBFOX2-regulated splicing events, such as via myosin phosphatase RHO-interacting protein (MPRIP), is associated with PDA metastases, altered cytoskeletal organization and the induction of focal adhesion formation. Our results implicate the splicing-regulatory function of RBFOX2 as a tumour suppressor in PDA and suggest a therapeutic approach for metastatic PDA.

Targeting splicing factors for cancer therapy.

Alternative splicing (AS) of mRNAs is an essential regulatory mechanism in eukaryotic gene expression. AS misregulation, caused by either dysregulation or mutation of splicing factors, has been shown to be involved in cancer development and progression, making splicing factors suitable targets for cancer therapy. In recent years, various types of pharmacological modulators, such as small molecules and oligonucleotides, targeting distinct components of the splicing machinery, have been under development to treat multiple disorders. Although these approaches have promise, targeting the core spliceosome components disrupts the early stages of spliceosome assembly and can lead to nonspecific and toxic effects. New research directions have been focused on targeting specific splicing factors for a more precise effect. In this Perspective, we will highlight several approaches for targeting splicing factors and their functions and suggest ways to improve their specificity.

S6K1 phosphorylates Cdk1 and MSH6 to regulate DNA repair.

The mTORC1 substrate, S6 Kinase 1 (S6K1), is involved in the regulation of cell growth, ribosome biogenesis, glucose homeostasis, and adipogenesis. Accumulating evidence has suggested a role for mTORC1 signaling in the DNA damage response. This is mostly based on the findings that mTORC1 inhibitors sensitized cells to DNA damage. However, a direct role of the mTORC1-S6K1 signaling pathway in DNA repair and the mechanism by which this signaling pathway regulates DNA repair is unknown. In this study, we discovered a novel role for S6K1 in regulating DNA repair through the coordinated regulation of the cell cycle, homologous recombination (HR) DNA repair (HRR) and mismatch DNA repair (MMR) mechanisms. Here, we show that S6K1 orchestrates DNA repair by phosphorylation of Cdk1 at serine 39, causing G2/M cell cycle arrest enabling homologous recombination and by phosphorylation of MSH6 at serine 309, enhancing MMR. Moreover, breast cancer cells harboring RPS6KB1 gene amplification show increased resistance to several DNA damaging agents and S6K1 expression is associated with poor survival of breast cancer patients treated with chemotherapy. Our findings reveal an unexpected function of S6K1 in the DNA repair pathway, serving as a tumorigenic barrier by safeguarding genomic stability.

Damage to the DNA in our cells can cause harmful changes that, if unchecked, can lead to the development of cancer. To help prevent this, cellular mechanisms are in place to repair defects in the DNA. A particular process, known as the mTORC1-S6K1 pathway is suspected to be important for repair because when this pathway is blocked, cells become more sensitive to DNA damage. It is still unknown how the various proteins involved in the mTORC1-S6K1 pathway contribute to repairing DNA. One of these proteins, S6K1, is an enzyme involved in coordinating cell growth and survival. The tumor cells in some forms of breast cancer produce more of this protein than normal, suggesting that S6K1 benefits these cells' survival. However, it is unclear exactly how the enzyme does this. Amar-Schwartz, Ben-Hur, Jbara et al. studied the role of S6K1 using genetically manipulated mouse cells and human cancer cells. These experiments showed that the protein interacts with two other proteins involved in DNA repair and activates them, regulating two different repair mechanisms and protecting cells against damage. These results might explain why some breast cancer tumors are resistant to radiotherapy and chemotherapy treatments, which aim to kill tumor cells by damaging their DNA. If this is the case, these findings could help clinicians choose more effective treatment options for people with cancers that produce additional S6K1. In the future, drugs that block the activity of the enzyme could make cancer cells more susceptible to chemotherapy.

Single allele loss-of-function mutations select and sculpt conditional cooperative networks in breast cancer.

The most common events in breast cancer (BC) involve chromosome arm losses and gains. Here we describe identification of 1089 gene-centric common insertion sites (gCIS) from transposon-based screens in 8 mouse models of BC. Some gCIS are driver-specific, others driver non-specific, and still others associated with tumor histology. Processes affected by driver-specific and histology-specific mutations include well-known cancer pathways. Driver non-specific gCIS target the Mediator complex, Ca++ signaling, Cyclin D turnover, RNA-metabolism among other processes. Most gCIS show single allele disruption and many map to genomic regions showing high-frequency hemizygous loss in human BC. Two gCIS, Nf1 and Trps1, show synthetic haploinsufficient tumor suppressor activity. Many gCIS act on the same pathway responsible for tumor initiation, thereby selecting and sculpting just enough and just right signaling. These data highlight ~1000 genes with predicted conditional haploinsufficient tumor suppressor function and the potential to promote chromosome arm loss in BC.

Splice-switching as cancer therapy.

In light of recent advances in RNA splicing modulation as therapy for specific genetic diseases, there is great optimism that this approach can be applied to treatment of cancer as well. Dysregulation of alternative RNA splicing is a common aberration detected in many cancers and thus, provides an attractive target for therapeutics. Here, we present and compare two promising approaches that are currently being investigated to manipulate alternative splicing and their potential use in therapy. The first strategy makes use of splice-switching oligonucleotides, whereas the second strategy uses CRISPR (clustered regularly interspaced short palindromic repeat Cas (CRISPR-associated) technology. We will discuss both the challenges and limitations of these technologies and progress being made to implement splice-switching as a potential cancer therapy.

Alternative Splicing of the Inhibitory Immune Checkpoint Receptor SLAMF6 Generates a Dominant Positive Form, Boosting T-cell Effector Functions.

SLAMF6 is a homotypic receptor of the Ig-superfamily associated with progenitor-exhausted T cells. Here we show that in humans, SLAMF6 has three splice isoforms involving its V-domain. Although the canonical receptor inhibited T-cell activation through SAP recruitment, the short isoform SLAMF6Δ17-65 had a strong agonistic effect. The costimulatory action depended on protein phosphatase SHP1 and led to a cytotoxic molecular profile mediated by the expression of TBX21 and RUNX3. Patients treated with immune checkpoint blockade showed a shift toward SLAMF6Δ17-65 in peripheral blood T cells. We developed splice-switching antisense oligonucleotides (ASO) designed to target the relevant SLAMF6 splice junction. Our ASOs enhanced SLAMF6Δ17-65 expression in human tumor-infiltrating lymphocytes and improved their capacity to inhibit human melanoma in mice. The yin-yang relationship of SLAMF6 splice isoforms may represent a balancing mechanism that could be exploited to improve cancer immunotherapy.

Trophoblast lineage specific expression of the alternative splicing factor RBFOX2 suggests a role in placental development.

INTRODUCTION: RBFOX2, an RNA-binding protein, controls tissue-specific alternative splicing of exons in diverse processes of development. The progenitor cytotrophoblast of the human placenta differentiates into either the syncytiotrophoblast, formed via cell fusion, or the invasive extravillous trophoblast lineage. The placenta affords a singular system where a role for RBFOX2 in both cell invasion and cell fusion may be studied. We investigated a role for RBFOX2 in trophoblast cell differentiation, as a foundation for investigations of RBFOX2 in embryo implantation and placental development. METHODS: Immunohistochemistry of RBFOX2 was performed on placental tissue sections from three trimesters of pregnancy and from pathological pregnancies. Primary trophoblast cell culture and immunofluorescence were employed to determine RBFOX2 expression upon cell fusion. Knockdown of RBFOX2 expression was performed with ßhCG and syncytin-1 as molecular indicators of fusion. RESULTS: In both normal and pathological placentas, RBFOX2 expression was confined to the cytotrophoblast and the extravillous trophoblast, but absent from the syncytiotrophoblast. Additionally, we showed that primary trophoblasts that spontaneously fused in cell culture downregulated RBFOX2 expression. In functional experiments, knockdown expression of RBFOX2 significantly upregulated ßhCG, while the upregulation of syncytin-1 did not reach statistical significance. DISCUSSION: RBFOX2, by conferring mRNA diversity, may act as a regulator switch in trophoblast differentiation to either the fusion or invasive pathways. By studying alternative splicing we further our understanding of placental development, yielding possible insights into preeclampsia, where expression of antiangiogenic isoforms produced through alternative splicing play a critical role in disease development and severity.

Pharmacological induction of selective endoplasmic reticulum retention as a strategy for cancer therapy.

The integrated stress response (ISR) converges on eIF2α phosphorylation to regulate protein synthesis. ISR is activated by several stress conditions, including endoplasmic reticulum (ER) stress, executed by protein kinase R-like endoplasmic reticulum kinase (PERK). We report that ER stress combined with ISR inhibition causes an impaired maturation of several tyrosine kinase receptors (RTKs), consistent with a partial block of their trafficking from the ER to the Golgi. Other proteins mature or are secreted normally, indicating selective retention in the ER (sERr). sERr is relieved upon protein synthesis attenuation and is accompanied by the generation of large mixed disulfide bonded complexes, including ERp44. sERr was pharmacologically recapitulated by combining the HIV-protease inhibitor nelfinavir with ISRIB, an experimental drug that inhibits ISR. Nelfinavir/ISRIB combination is highly effective to inhibit the growth of RTK-addicted cell lines and hepatocellular (HCC) cells in vitro and in vivo. Thus, pharmacological sERr can be utilized as a modality for cancer treatment.

Dissecting the role of crosstalk between glioblastoma subpopulations in tumor cell spreading.

Glioblastoma (GBM) is a highly infiltrative brain cancer, which is thus difficult to operate. GBM cells frequently harbor Epidermal Growth Factor Receptor amplification (EGFRwt) and/or activating mutation (EGFRvIII), generating at least two different cellular subpopulations within the tumor. We examined the relationship between the diffusive architectures of GBM tumors and the paracrine interactions between those subpopulations. Our aim was to shed light on what drives GBM cells to reach large cell-cell distances, and whether this characteristic can be manipulated. We established a methodology that quantifies the infiltration abilities of cancer cells through computation of cell-cell separation distance distributions in 3D. We found that aggressive EGFRvIII cells modulate the migration and infiltrative properties of EGFRwt cells. EGFRvIII cells secrete HGF and IL6, leading to enhanced activity of Src protein in EGFRwt cells, and rendering EGFRwt cells higher velocity and augmented ability to spread. Src inhibitor, dasatinib, at low non-toxic concentrations, reduced the infiltrative properties of EGFRvIII/EGFRwt neurospheres. Furthermore, dasatinib treatment induced compact multicellular microstructure packing of EGFRvIII/EGFRwt cells, impairing their ability to spread. Prevention of cellular infiltration or induction of compact microstructures may assist the detection of GBM tumors and tumor remnants in the brains and improve their surgical removal.

2-APB and CBD-Mediated Targeting of Charged Cytotoxic Compounds Into Tumor Cells Suggests the Involvement of TRPV2 Channels.

Targeted delivery of therapeutic compounds to particular cell types such that they only affect the target cells is of great clinical importance since it can minimize undesired side effects. For example, typical chemotherapeutic treatments used in the treatment of neoplastic disorders are cytotoxic not only to cancer cells but also to most normal cells when exposed to a critical concentration of the compound. As such, many chemotherapeutics exhibit severe side effects, often prohibiting their effective use in the treatment of cancer. Here, we describe a new means for facilitated delivery of a clinically used chemotherapy compound' doxorubicin, into hepatocellular carcinoma cell line (BNL1 ME). We demonstrate that these cells express a large pore, cation non-selective transient receptor potential (TRP) channel V2. We utilized this channel to shuttle doxorubicin into BNL1 ME cells. We show that co-application of either cannabidiol (CBD) or 2-APB, the activators of TRPV2 channels, together with doxorubicin leads to significantly higher accumulation of doxorubicin in BNL1 ME cells than in BNL1 ME cells that were exposed to doxorubicin alone. Moreover, we demonstrate that sub-effective doses of doxorubicin when co-applied with either 2-APB or CBD lead to a significant decrease in the number of living BNL1 ME cell and BNL1 ME cell colonies in comparison to application of doxorubicin alone. Finally, we demonstrate that the doxorubicin-mediated cell death is significantly more potent, requiring an order of magnitude lower dose, when co-applied with CBD than with 2-APB. We suggest that CBD may have a dual effect in promoting doxorubicin-mediated cell death by facilitating the entry of doxorubicin via TRPV2 channels and preventing its clearance from the cells by inhibiting P-glycoprotein ATPase transporter. Collectively, these results provide a foundation for the use of large pore cation-non selective channels as "natural" drug delivery systems for targeting specific cell types.

Intra-Tumoral Metabolic Zonation and Resultant Phenotypic Diversification Are Dictated by Blood Vessel Proximity.

Differential exposure of tumor cells to blood-borne and angiocrine factors results in diverse metabolic microenvironments conducive for non-genetic tumor cell diversification. Here, we harnessed a methodology for retrospective sorting of fully functional, stroma-free cancer cells solely on the basis of their relative distance from blood vessels (BVs) to unveil the whole spectrum of genes, metabolites, and biological traits impacted by BV proximity. In both grafted mouse tumors and natural human glioblastoma (GBM), mTOR activity was confined to few cell layers from the nearest perfused vessel. Cancer cells within this perivascular tier are distinguished by intense anabolic metabolism and defy the Warburg principle through exercising extensive oxidative phosphorylation. Functional traits acquired by perivascular cancer cells, namely, enhanced tumorigenicity, superior migratory or invasive capabilities, and, unexpectedly, exceptional chemo- and radioresistance, are all mTOR dependent. Taken together, the study revealed a previously unappreciated graded metabolic zonation directly impacting the acquisition of multiple aggressive tumor traits.

Specific inhibition of splicing factor activity by decoy RNA oligonucleotides.

Alternative splicing, a fundamental step in gene expression, is deregulated in many diseases. Splicing factors (SFs), which regulate this process, are up- or down regulated or mutated in several diseases including cancer. To date, there are no inhibitors that directly inhibit the activity of SFs. We designed decoy oligonucleotides, composed of several repeats of a RNA motif, which is recognized by a single SF. Here we show that decoy oligonucleotides targeting splicing factors RBFOX1/2, SRSF1 and PTBP1, can specifically bind to their respective SFs and inhibit their splicing and biological activities both in vitro and in vivo. These decoy oligonucleotides present an approach to specifically downregulate SF activity in conditions where SFs are either up-regulated or hyperactive.

Long Noncoding RNA MALAT1 Regulates Cancer Glucose Metabolism by Enhancing mTOR-Mediated Translation of TCF7L2.

Reprogrammed glucose metabolism of enhanced aerobic glycolysis (or the Warburg effect) is known as a hallmark of cancer. The roles of long noncoding RNAs (lncRNA) in regulating cancer metabolism at the level of both glycolysis and gluconeogenesis are mostly unknown. We previously showed that lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) acts as a proto-oncogene in hepatocellular carcinoma (HCC). Here, we investigated the role of MALAT1 in regulating cancer glucose metabolism. MALAT1 upregulated the expression of glycolytic genes and downregulated gluconeogenic enzymes by enhancing the translation of the metabolic transcription factor TCF7L2. MALAT1-enhanced TCF7L2 translation was mediated by upregulation of SRSF1 and activation of the mTORC1-4EBP1 axis. Pharmacological or genetic inhibition of mTOR and Raptor or expression of a hypophosphorylated mutant version of eIF4E-binding protein (4EBP1) resulted in decreased expression of TCF7L2. MALAT1 expression regulated TCF7L2 mRNA association with heavy polysomes, probably through the TCF7L2 5'-untranslated region (UTR), as determined by polysome fractionation and 5'UTR-reporter assays. Knockdown of TCF7L2 in MALAT1-overexpressing cells and HCC cell lines affected their metabolism and abolished their tumorigenic potential, suggesting that the effects of MALAT1 on glucose metabolism are essential for its oncogenic activity. Taken together, our findings suggest that MALAT1 contributes to HCC development and tumor progression by reprogramming tumor glucose metabolism. SIGNIFICANCE: These findings show that lncRNA MALAT1 contributes to HCC development by regulating cancer glucose metabolism, enhancing glycolysis, and inhibiting gluconeogenesis via elevated translation of the transcription factor TCF7L2.

Modulation of MKNK2 alternative splicing by splice-switching oligonucleotides as a novel approach for glioblastoma treatment.

The gene encoding the kinase Mnk2 (MKNK2) is alternatively spliced to produce two isoforms-Mnk2a and Mnk2b. We previously showed that Mnk2a is downregulated in several types of cancer and acts as a tumor suppressor by activation of the p38-MAPK stress pathway, inducing apoptosis. Moreover, Mnk2a overexpression suppressed Ras-induced transformation in culture and in vivo. In contrast, the Mnk2b isoform acts as a pro-oncogenic factor. In this study, we designed modified-RNA antisense oligonucleotides and screened for those that specifically induce a strong switch in alternative splicing of the MKNK2 gene (splice switching oligonucleotides or SSOs), elevating the tumor suppressive isoform Mnk2a at the expense of the pro-oncogenic isoform Mnk2b. Induction of Mnk2a by SSOs in glioblastoma cells activated the p38-MAPK pathway, inhibited the oncogenic properties of the cells, re-sensitized the cells to chemotherapy and inhibited glioblastoma development in vivo. Moreover, inhibition of p38-MAPK partially rescued glioblastoma cells suggesting that most of the anti-oncogenic activity of the SSO is mediated by activation of this pathway. These results suggest that manipulation of MKNK2 alternative splicing by SSOs is a novel approach to inhibit glioblastoma tumorigenesis.

TRPM2 Mediates Neutrophil Killing of Disseminated Tumor Cells.

Neutrophils play a critical role in cancer, with both protumor and antitumor neutrophil subpopulations reported. The antitumor neutrophil subpopulation has the capacity to kill tumor cells and limit metastatic spread, yet not all tumor cells are equally susceptible to neutrophil cytotoxicity. Because cells that evade neutrophils have greater chances of forming metastases, we explored the mechanism neutrophils use to kill tumor cells. Neutrophil cytotoxicity was previously shown to be mediated by secretion of H2O2 We report here that neutrophil cytotoxicity is Ca2+ dependent and is mediated by TRPM2, a ubiquitously expressed H2O2-dependent Ca2+ channel. Perturbing TRPM2 expression limited tumor cell proliferation, leading to attenuated tumor growth. Concomitantly, cells expressing reduced levels of TRPM2 were protected from neutrophil cytotoxicity and seeded more efficiently in the premetastatic lung.Significance: These findings identify the mechanism utilized by neutrophils to kill disseminated tumor cells and to limit metastatic spread. Cancer Res; 78(10); 2680-90. ©2018 AACR.

Recapitulating the clinical scenario of BRCA-associated pancreatic cancer in pre-clinical models.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. BRCA-associated PDAC comprises a clinically relevant subtype. A portion of these patients are highly susceptible to DNA damaging therapeutics, however, responses are heterogeneous and clinical resistance evolves. We have developed unique patient-derived xenograft (PDX) models from metastatic lesions of germline BRCA-mutated patients obtained at distinct time points; before treatment and at progression. Thus, closely mimicking clinical scenarios, to further investigate treatment naïve and resistant patients. DNA was isolated from six BRCA-mutated PDXs and classified by whole-genome sequencing to stable-genome or homologous recombination deficient (HRD)-genome. The sensitivity to DNA-damaging agents was evaluated in vivo in three BRCA-associated PDAC PDXs models: (1) HRD-genome naïve to treatments; (2) stable-genome naïve to treatment; (3) HRD-genome resistant to treatment. Correlation between disease course at tissue acquisition and response to PARP inhibitor (PARPi)/platinum was demonstrated in PDXs in vivo. Only the HRD-genome PDX, naïve to treatment, was sensitive to PARP inhibitor/cisplatin treatments. Our results demonstrate heterogeneous responses to DNA damaging agents/PARPi in BRCA-associated PDX thus reflecting the wide clinical spectrum. An HRD-genome PDX generated from a naïve to treatment biopsy was sensitive to platinum/PARPi whereas no benefit was observed in treating a HRD-genome PDXs generated from a patient that had acquired resistance nor stable-genome PDXs.

The role of alternative splicing in cancer drug resistance.

One of the major challenges in cancer treatment today is that many patients develop resistance to the therapeutic agents, resulting in treatment failure. Alternative splicing can significantly alter the coding region of drug targets. Here, we highlight several reports that provide key examples of alternative splicing events that occur in various cancers and play a role in resistance to cancer therapy. These examples present prime targets for future study and development of splicing modulation therapy. Modulation of alternative splicing has recently been approved as treatment for several diseases, although not yet for cancer. We propose that a similar approach may be successfully adapted to combat cancer therapy resistance, in cases where alternative splicing is known to be the mechanism that contributes to the resistance.

CLIP6-PNA-Peptide Conjugates: Non-Endosomal Delivery of Splice Switching Oligonucleotides.

Efficient delivery of oligonucleotides still remains a challenge in the field of oligonucleotide based therapy. Peptide nucleic acid (PNA), a DNA analogue that is typically synthesized by solid phase peptide chemistry, has been conjugated to a variety of cell penetrating peptides (CPP) as a means of improving its cellular uptake. These CPPs typically deliver their cargoes into cells by an endosomal-dependent mechanism resulting in lower bioavailability of the cargo. Herein, we designed and synthesized PNA-peptide conjugates as splice switching oligonucleotides (SSO) targeting the Mnk2 gene, a therapeutic target in cancer. In humans, the MKNK2 gene, is alternatively spliced, generating isoforms with opposite biological activities: Mnk2a and Mnk2b. It was found that the Mnk2a isoform is down-regulated in breast, lung, brain, and colon tumors and is a tumor suppressor, whereas MnK2b is oncogenic. We have designed and synthesized PNAs that were conjugated to either of the following peptides: a nuclear localization sequence (NLS) or a cytosol localizing internalization peptide (CLIP6). CLIP6-PNA demonstrates effective cellular uptake and exclusively employs a nonendosomal mechanism to cross the cellular membranes of glioblastoma cells (U87). Simple incubation of PNA-peptide conjugates in human glioblastoma cells up-regulates the Mnk2a isoform leading to cancer cell death.