Modulation of RNA splicing as a potential treatment for cancer

Close to 90% of human genes are transcribed into pre-mRNA that undergoes alternative splicing, producing multiple mRNAs and proteins from single genes. This process is largely responsible for human proteome diversity, and about half of genetic disease-causing mutations affect splicing. Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense therapeutics that modify gene expression by directing pre-mRNA splice site usage. Bauman et al. investigated an SSO that up-regulated the expression of an anti-cancer splice variant while simultaneously eliminating an over-expressed cancer-causing splice variant. This was accomplished by targeting pre-mRNA of the apoptotic regulator Bcl-x, which is alternatively spliced to express anti- and pro-apoptotic splice variants Bcl-xL and Bcl-xS, respectively. High expression of Bcl-xL is a hallmark of many cancers and is considered a general mechanism used by cancer cells to evade apoptosis. Redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS by SSO induced apoptotic and chemosensitizing effects in various cancer cell lines. Importantly, the paper shows that delivery of Bcl-x SSO using a lipid nanoparticle redirected Bcl-x splicing and reduced tumor burden in melanoma lung metastases. This was the first demonstration of SSO efficacy in tumors in vivo. SSOs are not limited to be solely potential anti-cancer drugs.?SSOs were first applied to repair aberrant splicing in thalassemia, a genetic disease, they have been used to create novel proteins (e.g., ?7TNFR1), and they have recently progressed to clinical trials for patients with Duchenne muscular dystrophy.     

Bi-specific splice-switching PMO oligonucleotides conjugated via a single peptide active in a mouse model of Duchenne muscular dystrophy

The potential for therapeutic application of splice-switching oligonucleotides (SSOs) to modulate pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo oligonucleotide uptake efficacy. Biological activities can be significantly enhanced through the use of synthetically conjugated cationic cell penetrating peptides (CPPs). Studies to date have focused on the delivery of a single SSO conjugated to a CPP, but here we describe the conjugation of two phosphorodiamidate morpholino oligonucleotide (PMO) SSOs to a single CPP for simultaneous delivery and pre-mRNA targeting of two separate genes, exon 23 of the Dmd gene and exon 5 of the Acvr2b gene, in a mouse model of Duchenne muscular dystrophy. Conjugations of PMOs to a single CPP were carried out through an amide bond in one case and through a triazole linkage (‘click chemistry’) in the other. The most active bi-specific CPP–PMOs demonstrated comparable exon skipping levels for both pre-mRNA targets when compared to individual CPP–PMO conjugates both in cell culture and in vivo in the mdx mouse model. Thus, two SSOs with different target sequences conjugated to a single CPP are biologically effective and potentially suitable for future therapeutic exploitation.     

Novel splicing factor RBM25 modulates Bcl-x pre-mRNA 5' splice site selection

RBM25 has been shown to associate with splicing cofactors SRm160/300 and assembled splicing complexes, but little is known about its splicing regulation. Here, we characterize the functional role of RBM25 in alternative pre-mRNA splicing. Increased RBM25 expression correlated with increased apoptosis and specifically affected the expression of Bcl-x isoforms. RBM25 stimulated proapoptotic Bcl-x_S 5′ splice site (5′ ss) selection in a dose-dependent manner, whereas its depletion caused the accumulation of antiapoptotic Bcl-x_L. Furthermore, RBM25 specifically bound to Bcl-x RNA through a CGGGCA sequence located within exon 2. Mutation in this element abolished the ability of RBM25 to enhance Bcl-x_S 5′ ss selection, leading to decreased Bcl-x_S isoform expression. Binding of RBM25 was shown to promote the recruitment of the U1 small nuclear ribonucleoprotein particle (snRNP) to the weak 5′ ss; however, it was not required when a strong consensus 5′ ss was present. In support of a role for RBM25 in modulating the selection of a 5′ ss, we demonstrated that RBM25 associated selectively with the human homolog of yeast U1 snRNP-associated factor hLuc7A. These data suggest a novel mode for Bcl-x_S 5′ ss activation in which binding of RBM25 with exonic element CGGGCA may stabilize the pre-mRNA-U1 snRNP through interactions with hLuc7A.     

Heterogeneous Nuclear Ribonucleoprotein K Represses the Production of Pro-apoptotic Bcl-xS Splice Isoform

The Bcl-x pre-mRNA is alternatively spliced to produce the anti-apoptotic Bcl-x_L and the pro-apoptotic Bcl-x_S isoforms. By performing deletion mutagenesis on a human Bcl-x minigene, we have identified a novel exonic element that controls the use of the 5′ splice site of Bcl-x_S. The proximal portion of this element acts as a repressor and is located downstream of an enhancer. Further mutational analysis provided a detailed topological map of the regulatory activities revealing a sharp transition between enhancer and repressor sequences. Portions of the enhancer can function when transplanted in another alternative splicing unit. Chromatography and immunoprecipitation assays indicate that the silencer element interacts with heterogeneous ribonucleoprotein particle (hnRNP) K, consistent with the presence of putative high affinity sites for this protein. Finally, down-regulation of hnRNP K by RNA interference enhanced splicing to Bcl-x_S, an effect seen only when the sequences bound by hnRNP K are present. Our results therefore document a clear role for hnRNP K in preventing the production of the pro-apoptotic Bcl-x_S splice isoform.Alternative splicing is a major mechanism used to augment the number of proteins encoded by the genome. It is estimated that as many as 97% of multiple exon pre-mRNAs undergo alternative splicing (1, 2). Disruption of alternative splicing by mutating important regulatory sequences or by altering the expression or activity of proteins controlling splice site selection has been linked with different diseases, including cancer (3–7). Apoptosis is an important and complex cellular program involved in development and differentiation in higher organisms (8, 9). However, its aberrant control often contributes to cancer development and the resistance of cancer cells to drug therapy (10–13).Genes implicated in the apoptotic pathway are alternatively spliced often to produce protein isoforms with distinct or even antagonistic activities (14, 15). A good example is the apoptotic regulator Bcl-x, which is alternatively spliced to produce two major isoforms, the anti-apoptotic Bcl-x_L protein and the shorter pro-apoptotic Bcl-x_S isoform (16). This alternative splicing decision involves a competition between two 5′ splice sites; the use of the downstream site creates Bcl-x_L, and the use of the upstream one produces Bcl-x_S (Fig. 1A). Bcl-x_L is always the predominant form in cancer cells, and overexpressing it can confer resistance to chemotherapeutic agents (17–22). On the other hand, overexpression of the pro-apoptotic Bcl-x_S isoform enhances sensitivity to the topoisomerase inhibitor etoposide and to taxol in a breast cancer cell line, while triggering apoptosis in melanoma cell lines (23, 24). Using antisense technologies to improve the production of the Bcl-x_S splice variant can also induce apoptosis in cancer cells (25–27).Open in a separate windowFIGURE 1.A, alternative splicing of Bcl-x produces two major isoforms, Bcl-x_L and Bcl-x_S. B, regulation of Bcl-x alternative splicing. The enhancer elements are shown as white boxes, and the repressors are black. The pointed and flat arrows indicate positive and negative regulation, respectively. Protein kinase C inhibition relieves repression caused by the SB1 element on the Bcl-x_S splice site (36). The repressor elements CRCE1, recognized by SAP155, and CRCE2 mediate the production of Bcl-x_S by ceramide as when induced by gemcitabine in A549 cells (38, 39). hnRNP F/H binds to the B2G element to enhance the production of the Bcl-x_S isoform (41). RBM25, through an element located upstream of the Bcl-x_S splice site, can also augment its use (44). A large intronic region (IRE) mediates the Bcl-x_L increase caused by interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and 12-O-tetradecanoylphorbol-13-acetate (TPA) (35). Finally, the B3 region also enhances Bcl-x_L formation through the binding of SRp30c to AM2 and ML2 and the U1 snRNP to two cryptic 5′ splice sites (42).Alternative splicing is regulated by different proteins bound to sequence elements near splice sites. A variety of mechanisms is used to achieve regulation. Some splicing factors act by recruiting or inhibiting the binding of different components of the spliceosome. Others may change the conformation of the pre-mRNA to mask a splice site or to bring a pair of splice sites into closer proximity (28, 29).Although individual factors can have a strong and specific effect on splicing decisions, alternative splicing often relies on a combination of factors to determine the appropriate levels of isoforms. The implication of multiple proteins likely provides additional levels of regulation that helps attuned splicing control to a variety of stresses, environmental cues, and growth conditions. In several cases, the interaction of regulatory factors can be antagonistic. For example, in the Drosophila male-specific-lethal-2 (msl-2) pre-mRNA, recruitment of SXL to a uridine-rich region interferes with the binding of TIA-1 that is necessary for efficient U1 snRNP² recruitment at the 5′ splice site (30). On the same pre-mRNA, SXL also diminishes U2AF recognition of the polypyrimidine tract at the 3′ splice site. TIA proteins bound to a U-rich element on the avian myosin phosphatase targeting subunit-1 (MYPT1) pre-mRNA repress the binding of PTB (31). PTB can also reduce the recruitment of ETR-3 to intronic elements near exon 5 of cardiac troponin T (32). In neurons, the binding of PTB to the introns surrounding the N1 exon of c-src is antagonized by nPTB protein, promoting exon inclusion. On the hnRNP A1 pre-mRNA, PTB diminishes the binding of SRp30c to the intronic CE9 element, reducing the inhibition by this protein on the use of the downstream 3′ splice site (33). SC35 and hnRNP A1 have partially overlapping binding sites on the human immunodeficiency virus 1 (HIV-1) tat exon 2. Preferential binding of SC35 enhances the inclusion of the exon, whereas hnRNP A1, by reducing SC35 binding, increases exclusion (34). Thus, the competition provided by an overlapping or a closely abutting pair of enhancer/ silencer represents a simple and frequent mechanism of splicing control.The regulation of Bcl-x alternative splicing has received some attention in recent years leading to the discovery of several cis-acting elements and a few trans-acting control factors (Fig. 1B). Intronic regions downstream from the Bcl-x_L 5′ splice site have been implicated as mediating signals from cytokines such as interleukin-6 and granulocyte-macrophage colony-stimulating factor (35). In addition, we have reported that an element located 187 nt upstream of the Bcl-x_S splice site mediates a protein kinase C-dependent signal that represses splicing to the Bcl-x_S donor site (36). On the other hand, ceramide enhances the use of the Bcl-x_S 5′ splice site by lifting the repression mediated by two other elements (37, 38). The activity of one of these apparently involves SAP155 (39). The RNA-binding protein Sam68, under the control of the tyrosine kinase Fyn, can also increase the production of Bcl-x_S in cooperation with hnRNP A1 (40), and this effect is inhibited by overexpression of ASF/SF2. The Bcl-x sequences bound by the above factors remain to be identified. We also uncovered enhancer elements for Bcl-x_S and Bcl-x_L. hnRNP F and H bind downstream of the Bcl-x_S 5′ splice site to stimulate splicing to that site (41). Enhancement of Bcl-x_L is conferred by SRp30c, which binds upstream of the 5′ splice site to antagonize the repressor activity of pseudo 5′ splice sites (42). Recently, the SR protein SC35 was shown to increase the production of Bcl-x_S (43). Finally, the binding of RBM25 to a sequence element upstream of the Bcl-x_S 5′ splice site stimulated its use, possibly by recruiting U1 snRNP through its interaction with the U1-associated protein hLuc7A (44). Thus, the region located between the two competing 5′ splice sites of Bcl-x is densely populated by splicing control elements.In this study, we have pursued our characterization of Bcl-x splicing control by examining the contribution of sequences directly upstream of the Bcl-x_S donor site. Our mutational approach identified a region containing flanking enhancer and silencer activities. The activity of the repressor portion is mediated by hnRNP K, which makes this protein an anti-apoptotic regulator.     

RBM10 regulates alternative splicing

RBM10, originally called S1-1, is a nuclear RNA-binding protein with domains characteristic of RNA processing proteins. It has been reported that RBM10 constitutes spliceosome complexes and that RBM5, a close homologue of RBM10, regulates alternative splicing of apoptosis-related genes, Fas and cFLIP. In this study, we examined whether RBM10 has a regulatory function in splicing similar to RBM5, and determined that it indeed regulates alternative splicing of Fas and Bcl-x genes. RBM10 promotes exon skipping of Fas pre-mRNA as well as selection of an internal 5′-splice site in Bcl-x pre-mRNA. We propose a consensus RBM10-binding sequence at 5′-splice sites of target exons and a mechanistic model of RBM10 action in the alternative splicing.     

Group VIA Phospholipase A2 (iPLA2β) Modulates Bcl-x 5′-Splice Site Selection and Suppresses Anti-apoptotic Bcl-x(L) in β-Cells

Diabetes is a consequence of reduced β-cell function and mass, due to β-cell apoptosis. Endoplasmic reticulum (ER) stress is induced during β-cell apoptosis due to various stimuli, and our work indicates that group VIA phospholipase A₂β (iPLA₂β) participates in this process. Delineation of underlying mechanism(s) reveals that ER stress reduces the anti-apoptotic Bcl-x(L) protein in INS-1 cells. The Bcl-x pre-mRNA undergoes alternative pre-mRNA splicing to generate Bcl-x(L) or Bcl-x(S) mature mRNA. We show that both thapsigargin-induced and spontaneous ER stress are associated with reductions in the ratio of Bcl-x(L)/Bcl-x(S) mRNA in INS-1 and islet β-cells. However, chemical inactivation or knockdown of iPLA₂β augments the Bcl-x(L)/Bcl-x(S) ratio. Furthermore, the ratio is lower in islets from islet-specific RIP-iPLA₂β transgenic mice, whereas islets from global iPLA₂β^−/− mice exhibit the opposite phenotype. In view of our earlier reports that iPLA₂β induces ceramide accumulation through neutral sphingomyelinase 2 and that ceramides shift the Bcl-x 5′-splice site (5′SS) selection in favor of Bcl-x(S), we investigated the potential link between Bcl-x splicing and the iPLA₂β/ceramide axis. Exogenous C₆-ceramide did not alter Bcl-x 5′SS selection in INS-1 cells, and neutral sphingomyelinase 2 inactivation only partially prevented the ER stress-induced shift in Bcl-x splicing. In contrast, 5(S)-hydroxytetraenoic acid augmented the ratio of Bcl-x(L)/Bcl-x(S) by 15.5-fold. Taken together, these data indicate that β-cell apoptosis is, in part, attributable to the modulation of 5′SS selection in the Bcl-x pre-mRNA by bioactive lipids modulated by iPLA₂β.     

Synthesis of 2′-O-(N-methylcarbamoylethyl) 5-methyl-2-thiouridine and its application to splice-switching oligonucleotides

The effect of 2′-O-(N-methylcarbamoyl)ethyl (MCE) modification on splice-switching oligonucleotides (SSO) was systematically evaluated. The incorporation of five MCE nucleotides at the 5′-termini of SSOs effectively improved the splice switching effect. In addition, the incorporation of 2′-O-(N-methylcarbamoylethyl)-5-methyl-2-thiouridine (s²T_MCE), a duplex-stabilizing nucleotide with an MCE modification, into SSOs further improved splice switching. These SSOs may be useful for the treatment of genetic diseases associated with splicing errors.     

Deficiency for the Cysteine Protease Cathepsin L Impairs Myc-Induced Tumorigenesis in a Mouse Model of Pancreatic Neuroendocrine Cancer

Motivated by the recent implication of cysteine protease cathepsin L as a potential target for anti-cancer drug development, we used a conditional MycER^TAM;Bcl-x_L model of pancreatic neuroendocrine tumorigenesis (PNET) to assess the role of cathepsin L in Myc-induced tumor progression. By employing a cysteine cathepsin activity probe in vivo and in vitro, we first established that cathepsin activity increases during the initial stages of MycER^TAM;Bcl-x_L tumor development. Among the cathepsin family members investigated, only cathepsin L was predominately produced by beta-tumor cells in neoplastic pancreata and, consistent with this, cathepsin L mRNA expression was rapidly upregulated following Myc activation in the beta cell compartment. By contrast, cathepsins B, S and C were highly enriched in tumor-infiltrating leukocytes. Genetic deletion of cathepsin L had no discernible effect on the initiation of neoplastic growth or concordant angiogenesis. However, the tumors that developed in the cathepsin L-deficient background were markedly reduced in size relative to their typical wild-type counterparts, indicative of a role for cathepsin L in enabling expansive tumor growth. Thus, genetic blockade of cathepsin L activity is inferred to retard Myc-driven tumor growth, encouraging the potential utility of pharmacological inhibitors of cysteine cathepsins in treating late stage tumors.     

Enhanced delivery of peptide-morpholino oligonucleotides with a small molecule to correct splicing defects in the lung

Pulmonary diseases offer many targets for oligonucleotide therapeutics. However, effective delivery of oligonucleotides to the lung is challenging. For example, splicing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) affect a significant cohort of Cystic Fibrosis (CF) patients. These individuals could potentially benefit from treatment with splice switching oligonucleotides (SSOs) that can modulate splicing of CFTR and restore its activity. However, previous studies in cell culture used oligonucleotide transfection methods that cannot be safely translated in vivo. In this report, we demonstrate effective correction of a splicing mutation in the lung of a mouse model using SSOs. Moreover, we also demonstrate effective correction of a CFTR splicing mutation in a pre-clinical CF patient-derived cell model. We utilized a highly effective delivery strategy for oligonucleotides by combining peptide-morpholino (PPMO) SSOs with small molecules termed OECs. PPMOs distribute broadly into the lung and other tissues while OECs potentiate the effects of oligonucleotides by releasing them from endosomal entrapment. The combined PPMO plus OEC approach proved to be effective both in CF patient cells and in vivo in the mouse lung and thus may offer a path to the development of novel therapeutics for splicing mutations in CF and other lung diseases.     

Functional Consequences for Apoptosis by Transcription Elongation Regulator 1 (TCERG1)-Mediated Bcl-x and Fas/CD95 Alternative Splicing

Here, we present evidence for a specific role of the splicing-related factor TCERG1 in regulating apoptosis in live cells by modulating the alternative splicing of the apoptotic genes Bcl-x and Fas. We show that TCERG1 modulates Bcl-x alternative splicing during apoptosis and its activity in Bcl-x alternative splicing correlates with the induction of apoptosis, as determined by assessing dead cells, sub-G1-phase cells, annexin-V binding, cell viability, and cleavage of caspase-3 and PARP-1. Furthermore, the effect of TCERG1 on apoptosis involved changes in mitochondrial membrane permeabilization. We also found that depletion of TCERG1 reduces the expression of the activated form of the pro-apoptotic mitochondrial membrane protein Bak, which remains inactive by heterodimerizing with Bcl-x_L, preventing the initial step of cytochrome c release in Bak-mediated mitochondrial apoptosis. In addition, we provide evidence that TCERG1 also participates in the death receptor-mediated apoptosis pathway. Interestingly, TCERG1 also modulates Fas/CD95 alternative splicing. We propose that TCERG1 sensitizes a cell to apoptotic agents, thus promoting apoptosis by regulating the alternative splicing of both the Bcl-x and Fas/CD95 genes. Our findings may provide a new link between the control of alternative splicing and the molecular events leading to apoptosis.     

Bcl-xL deamidation is regulated by multiple ion transporters and is intramolecularly catalyzed

In susceptible tumor cells, DNA-damaging antineoplastic agents induce an increase in intracellular pH during the premitochondrial stage of apoptosis. The rate of nonenzymatic deamidation of two asparagines in the anti-apoptotic protein Bcl-x_L is accelerated by this increase in pH. Deamidation of these asparagines is a signal for the degradation of Bcl-x_L, which is a component of the apoptotic response to DNA damage. It has previously been shown that the increase in pH is mediated by the ion transporter Na⁺/H⁺ exchanger 1 in some cells. Here we demonstrate that one or more additional ion transporters also have a role in the regulation of Bcl-x_L deamidation in at least some tumor cell lines and fibroblasts. As a second, independent finding, we report that there are histidines in close proximity to the Bcl-x_L deamidation sites that are highly conserved in land-dwelling species and we present evidence that deamidation of human Bcl-x_L is intramolecularly catalyzed in a manner that is dependent upon these histidines. Further, we present evidence that these histidines act as a pH-sensitive switch that enhances the effect of the increase in pH on the rate of Bcl-x_L deamidation. The conservation of such histidines implies that human Bcl-x_L is in essence “designed” to be deamidated, which provides further evidence that deamidation serves as a bona fide regulatory post-translational modification of Bcl-x_L.     

Survival motor neuron protein reduction deregulates autophagy in spinal cord motoneurons in vitro

Spinal muscular atrophy (SMA) is a genetic disorder characterized by degeneration of spinal cord motoneurons (MNs), resulting in muscular atrophy and weakness. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene and decreased SMN protein. SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoproteins and pre-mRNA splicing requirements. SMN reduction causes neurite degeneration and cell death without classical apoptotic features, but the direct events leading to SMN degeneration in SMA are still unknown. Autophagy is a conserved lysosomal protein degradation pathway whose precise roles in neurodegenerative diseases remain largely unknown. In particular, it is unclear whether autophagosome accumulation is protective or destructive, but the accumulation of autophagosomes in the neuritic beadings observed in several neurite degeneration models suggests a close relationship between the autophagic process and neurite collapse. In the present work, we describe an increase in the levels of the autophagy markers including autophagosomes, Beclin1 and light chain (LC)3-II proteins in cultured mouse spinal cord MNs from two SMA cellular models, suggesting an upregulation of the autophagy process in Smn (murine survival motor neuron protein)-reduced MNs. Overexpression of Bcl-x_L counteracts LC3-II increase, contributing to the hypothesis that the protective role of Bcl-x_L observed in some SMA models may be mediated by its role in autophagy inhibition. Our in vitro experimental data indicate an upregulation in the autophagy process and autophagosome accumulation in the pathogenesis of SMA, thus providing a valuable clue in understanding the mechanisms of axonal degeneration and a possible therapeutic target in the treatment of SMA.     

Mutations in U5 snRNA loop 1 influence the splicing of different genes in vivo

The U5 snRNA loop 1 is characterized by the conserved sequence G₁C₂C₃U₄U₅U₆Y₇A₈Y₉ and is essential for the alignment of exons during the second step of pre-mRNA splicing in Saccharo myces cerevisiae. Despite this sequence conservation the size, rather than sequence, of loop 1 is critical for exon alignment in vitro. To determine the in vivo requirements for U5 loop 1 a library of loop 1 sequences was transformed into a yeast strain where the endogenous U5 gene was deleted. Comparison of viable mutations in loop 1 revealed that position 6 was invariant and positions 5 and 7 displayed some sequence conservation. These data indicate positions 5, 6 and 7 in loop 1 are important for U5 function in vivo. A screen for mutations that suppress the temperature-sensitive phenotype of three loop 1 mutants produced eight intragenic suppressors all containing alterations in loop 1. Further analysis of these temperature-sensitive mutants revealed that each displayed distinct cell cycle arrest phenotypes and pre-mRNA splicing inhibition patterns. The cell cycle arrest is likely attributed to inefficient splicing of α-tubulin pre-mRNA in one mutant and actin pre-mRNA in another. These results suggest that various mutations in loop 1 may affect the splicing of different pre-mRNAs in vivo.     

The RNA-Binding Protein QKI Suppresses Cancer-Associated Aberrant Splicing

Lung cancer is the leading cause of cancer-related death worldwide. Aberrant splicing has been implicated in lung tumorigenesis. However, the functional links between splicing regulation and lung cancer are not well understood. Here we identify the RNA-binding protein QKI as a key regulator of alternative splicing in lung cancer. We show that QKI is frequently down-regulated in lung cancer, and its down-regulation is significantly associated with a poorer prognosis. QKI-5 inhibits the proliferation and transformation of lung cancer cells both in vitro and in vivo. Our results demonstrate that QKI-5 regulates the alternative splicing of NUMB via binding to two RNA elements in its pre-mRNA, which in turn suppresses cell proliferation and prevents the activation of the Notch signaling pathway. We further show that QKI-5 inhibits splicing by selectively competing with a core splicing factor SF1 for binding to the branchpoint sequence. Taken together, our data reveal QKI as a critical regulator of splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated regulation of the Notch signaling pathway.     

Human IAP-Like Protein Regulates Programmed Cell Death Downstream of Bcl-xL and Cytochrome c

The gene encoding human IAP-like protein (hILP) is one of several mammalian genes with sequence homology to the baculovirus inhibitor-of-apoptosis protein (iap) genes. Here we show that hILP can block apoptosis induced by a variety of extracellular stimuli, including UV light, chemotoxic drugs, and activation of the tumor necrosis factor and Fas receptors. hILP also protected against cell death induced by members of the caspase family, cysteine proteases which are thought to be the principal effectors of apoptosis. hILP and Bcl-x_L were compared for their ability to affect several steps in the apoptotic pathway. Redistribution of cytochrome c from mitochondria, an early event in apoptosis, was not blocked by overexpression of hILP but was inhibited by Bcl-x_L. In contrast, hILP, but not Bcl-x_L, inhibited apoptosis induced by microinjection of cytochrome c. These data suggest that while Bcl-x_L may control mitochondrial integrity, hILP can function downstream of mitochondrial events to inhibit apoptosis.     

Towards the next generation of dual Bcl-2/Bcl-xL inhibitors

Structural modifications of the left-hand side of compound 1 were identified which retained or improved potent binding to Bcl-2 and Bcl-x_L in in vitro biochemical assays and had strong activity in an RS4;11 apoptotic cellular assay. For example, sulfoxide diastereomer 13 maintained good binding affinity and comparable cellular potency to 1 while improving aqueous solubility. The corresponding diastereomer (14) was significantly less potent in the cell, and docking studies suggest that this is due to a stereochemical preference for the R_S versus S_S sulfoxide. Appending a dimethylaminoethoxy side chain (27) adjacent to the benzylic position of the biphenyl moiety of 1 improved cellular activity by approximately three-fold, and this activity was corroborated in cell lines overexpressing Bcl-2 and Bcl-x_L.     

T Cell Factor-1 Controls the Lifetime of CD4+ CD8+ Thymocytes In Vivo and Distal T Cell Receptor α-Chain Rearrangement Required for NKT Cell Development

Natural killer T (NKT) cells are a component of innate and adaptive immune systems implicated in immune, autoimmune responses and in the control of obesity and cancer. NKT cells develop from common CD4+ CD8+ double positive (DP) thymocyte precursors after the rearrangement and expression of T cell receptor (TCR) Vα14-Jα18 gene. Temporal regulation and late appearance of Vα14-Jα18 rearrangement in immature DP thymocytes has been demonstrated. However, the precise control of lifetime of DP thymocytes in vivo that enables distal rearrangements remains incompletely defined. Here we demonstrate that T cell factor (TCF)-1, encoded by the Tcf7 gene, is critical for the extended lifetime of DP thymocytes. TCF-1-deficient DP thymocytes fail to undergo TCR Vα14-Jα18 rearrangement and produce significantly fewer NKT cells. Ectopic expression of Bcl-x_L permits Vα14-Jα18 rearrangement and rescues NKT cell development. We report that TCF-1 regulates expression of RORγt, which regulates DP thymocyte survival by controlling expression of Bcl-x_L. We posit that TCF-1 along with its cofactors controls the lifetime of DP thymocytes in vivo.