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In this article, we discuss a class of MYC-interacting lncRNAs (long non-coding RNAs) that share the following criteria: They are direct transcriptional targets of MYC. Their expression is coordinated with the expression of MYC. They are required for sustained MYC-driven cell proliferation, and they are not essential for cell survival. We refer to these lncRNAs as "MYC facilitators" and discuss two representative members of this class of lncRNAs, SNHG17 (small nuclear RNA host gene) and LNROP (long non-coding regulator of POU2F2). We also present a general hypothesis on the role of lncRNAs in MYC-mediated transcriptional regulation.

Recent cryo-electron microscopic (cryo-EM) investigations have succeeded in the analysis of various structural conformations and functional states of PI3Kα, a dimer consisting of the catalytic subunit p110α and the regulatory subunit p85α of class IA of phosphoinositide 3-kinase. High resolution structures have been obtained of the unliganded and of BYL-719-bound PI3Kα. The latter provides information on excessively flexible domains of p85α that are then further analyzed with nanobodies and CXMS (chemical cross-linking, digestion and mass spectrometry). Analysis of p110α helical and kinase domain mutations reveals mutant-specific features that can be linked to the gain of function in enzymatic and signaling activities., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Peter K. Vogt reports a relationship with Umbra Therapeutics that includes: consulting or advisory., (Copyright © 2023 Elsevier B.V. All rights reserved.)

MYC controls most of the non-coding genome. Several long noncoding transcripts were originally identified in the human B cell line P496-3 and then shown to be required for MYC-driven proliferation of Burkitt lymphoma-derived RAMOS cells. In this study, we used RAMOS cells exclusively as a representative of the human B cell lineage. One of the MYC-controlled lncRNAs required for RAMOS cell proliferation is ENSG00000254887 which we will term LNROP (long non-coding regulator of POU2F2). In the genome, LNROP is located in close proximity of POU2F2, the gene encoding OCT2. OCT2 is a transcription factor with important roles in sustaining the proliferation of human B cells. Here we show that LNROP is a nuclear RNA and a direct target of MYC. Downregulation of LNROP attenuates the expression of OCT2. This effect of LNROP on the expression of OCT2 is unidirectional as downregulation of OCT2 does not alter the expression of LNROP. Our data suggest that LNROP is a cis-acting regulator of OCT2. To illustrate the downstream reach of LNROP, we chose a prominent target of OCT2, the tyrosine phosphatase SHP-1. Downregulation of OCT2 elevates the expression of SHP-1. Our data suggest the following path of interactions: LNROP enables the proliferation of B cells by positively and unidirectionally regulating the growth-stimulatory transcription factor OCT2. In actively proliferating B cells, OCT2 attenuates the expression and anti-proliferative activity of SHP-1., (© 2023. The Author(s).)

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that perform multiple and important cellular functions. The protein investigated here belongs to class IA of the PI3Ks; it is a dimer consisting of a catalytic subunit, p110α, and a regulatory subunit, p85α, and is referred to as PI3Kα. The catalytic subunit p110α is frequently mutated in cancer. The mutations induce a gain of function and constitute a driving force in cancer development. About 80% of these mutations lead to single-amino-acid substitutions in one of three sites of p110α: two in the helical domain of the protein (E542K and E545K) and one at the C-terminus of the kinase domain (H1047R). Here, we report the cryo-electron microscopy structures of these mutants in complex with the p110α-specific inhibitor BYL-719. The H1047R mutant rotates its sidechain to a new position and weakens the kα11 activation loop interaction, thereby reducing the inhibitory effect of p85α on p110α. E542K and E545K completely abolish the tight interaction between the helical domain of p110α and the N-terminal SH2 domain of p85α and lead to the disruption of all p85α binding and a dramatic increase in flexibility of the adaptor-binding domain (ABD) in p110α. Yet, the dimerization of PI3Kα is preserved through the ABD-p85α interaction. The local and global structural features induced by these mutations provide molecular insights into the activation of PI3Kα, deepen our understanding of the oncogenic mechanism of this important signaling molecule, and may facilitate the development of mutant-specific inhibitors.

Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα.

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases essential for growth and metabolism. Their aberrant activation is associated with many types of cancers. Here we used single-particle cryoelectron microscopy (cryo-EM) to determine three distinct conformations of full-length PI3Kα (p110α-p85α): the unliganded heterodimer PI3Kα, PI3Kα bound to the p110α-specific inhibitor BYL-719, and PI3Kα exposed to an activating phosphopeptide. The cryo-EM structures of unbound and of BYL-719-bound PI3Kα are in general accord with published crystal structures. Local deviations are presented and discussed. BYL-719 stabilizes the structure of PI3Kα, but three regions of low-resolution extra density remain and are provisionally assigned to the cSH2, BH, and SH3 domains of p85. One of the extra density regions is in contact with the kinase domain blocking access to the catalytic site. This conformational change indicates that the effects of BYL-719 on PI3Kα activity extend beyond competition with adenosine triphosphate (ATP). In unliganded PI3Kα, the DFG motif occurs in the "in" and "out" positions. In BYL-719-bound PI3Kα, only the DFG-in position, corresponding to the active conformation of the kinase, was observed. The phosphopeptide-bound structure of PI3Kα is composed of a stable core resolved at 3.8 Å. It contains all p110α domains except the adaptor-binding domain (ABD). The p85α domains, linked to the core through the ABD, are no longer resolved, implying that the phosphopeptide activates PI3Kα by fully releasing the niSH2 domain from binding to p110α. The structures presented here show the basal form of the full-length PI3Kα dimer and document conformational changes related to the activated and inhibited states., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

We report the discovery of a fluorescent small molecule probe. This probe exhibits an emission increase in the presence of the oncoprotein MYC that can be attenuated by a competing inhibitor. Hydrogen-deuterium exchange mass spectrometry analysis, rationalized by induced-fit docking, suggests it binds to the "coiled-coil" region of the leucine zipper domain. Point mutations of this site produced functional MYC constructs resistant to inhibition in an oncogenic transformation assay by compounds that displace the probe. Utilizing this probe, we have developed a high-throughput assay to identify MYC inhibitor scaffolds. Screening of a diversity library (N = 1408, 384-well) and a library of pharmacologically active compounds (N = 1280, 1536-well) yielded molecules with greater drug-like properties than the probe. One lead is a potent inhibitor of oncogenic transformation and is specific for MYC relative to resistant mutants and transformation-inducing oncogenes. This method is simple, inexpensive, and does not require protein modification, DNA binding, or the dimer partner MAX. This assay presents an opportunity for MYC inhibition researchers to discover unique scaffolds., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

MYC controls the transcription of large numbers of long noncoding RNAs (lncRNAs). Since MYC is a ubiquitous oncoprotein, some of these lncRNAs probably play a significant role in cancer. We applied CRISPR interference (CRISPRi) to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 noncoding loci that play positive roles in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RT-qPCR and in CRISPRi competition assays with individual GFP-expressing sgRNA constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID (SIN3-interacting domain) derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, SID is superior to the KRAB repressor domain, which serves routinely as a transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Tumor formation is generally linked to the acquisition of two or more driver genes that cause normal cells to progress from proliferation to abnormal expansion and malignancy. In order to understand genetic alterations involved in this process, we compared the transcriptomes of an isogenic set of breast epithelial cell lines that are non-transformed or contain a single or double knock-in (DKI) of PIK3CA (H1047R) or KRAS (G12V). Gene set enrichment analysis revealed that DKI cells were enriched over single mutant cells for genes that characterize a MYC target gene signature. This gene signature was mediated in part by the bromodomain-containing protein 9 (BRD9) that was found in the SWI-SNF chromatin-remodeling complex, bound to the MYC super-enhancer locus. Small molecule inhibition of BRD9 reduced MYC transcript levels. Critically, only DKI cells had the capacity for anchorage-independent growth in semi-solid medium, and CRISPR-Cas9 manipulations showed that PIK3CA and BRD9 expression were essential for this phenotype. In contrast, KRAS was necessary for DKI cell migration, and BRD9 overexpression induced the growth of KRAS single mutant cells in semi-solid medium. These results provide new insight into the earliest transforming events driven by oncoprotein cooperation and suggest BRD9 is an important mediator of mutant PIK3CA/KRAS-driven oncogenic transformation.

Extracellular small RNAs (sRNAs), including microRNAs (miRNAs), are promising biomarkers for diseases such as Duchenne muscular dystrophy (DMD), although their biological relevance is largely unknown. To investigate the relationship between intracellular and extracellular sRNA levels on a global scale, we performed sRNA sequencing in four muscle types and serum from wild-type, dystrophic mdx, and mdx mice in which dystrophin protein expression was restored by exon skipping. Differentially abundant sRNAs were identified in serum (mapping to miRNA, small nuclear RNA [snRNA], and PIWI-interacting RNA [piRNA] loci). One novel candidate biomarker, miR-483, was increased in both mdx serum and muscle, and also elevated in DMD patient sera. Dystrophin restoration induced global shifts in miRNA (including miR-483) and snRNA-fragment abundance toward wild-type levels. Specific serum piRNA-like sRNAs also responded to exon skipping therapy. Absolute miRNA expression in muscle was positively correlated with abundance in the circulation, although multiple highly expressed miRNAs in muscle were not elevated in mdx serum, suggesting that both passive and selective release mechanisms contribute to serum miRNA levels. In conclusion, this study has revealed new insights into the sRNA biology of dystrophin deficiency and identified novel DMD biomarkers., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: The main regulatory subunits of Class IA phosphatidylinositol 3-kinase (PI3K), p85α and p85β, initiate diverse cellular activities independent of binding to the catalytic subunit p110. Several of these signaling processes directly or indirectly contribute to a regulation of PI3K and could become targets for therapeutic efforts. Areas covered: This review will highlight two general areas of p85 activity: (1) direct interaction with regulatory proteins and with determinants of the cytoskeleton, and (2) a genetic analysis by deletion and domain switches identifying new functions for p85 domains. Expert Opinion: Isoform-specific activities of regulatory subunits have long been at the periphery of the PI3K field. Our understanding of these unique functions of the regulatory subunits is fragmentary and raises many important questions. At this time, there is insufficient information to translate this knowledge into the clinic, but some tempting targets have emerged that could move the field forward with the help of novel technologies in drug design and identification.

MYC is a key transcriptional regulator involved in cellular proliferation and has established roles in transcriptional elongation and initiation, microRNA regulation, apoptosis, and pluripotency. Despite this prevalence, functional chemical probes of MYC function at the protein level have been limited. Previously, we discovered 5a, that binds to MYC with potency and specificity, downregulates the transcriptional activities of MYC and shows efficacy in vivo. However, this scaffold posed intrinsic pharmacokinetic liabilities, namely, poor solubility that precluded biophysical interrogation. Here, we developed a screening platform based on field-effect transistor analysis (Bio-FET), surface plasmon resonance (SPR), and a microtumor formation assay to analyze a series of new compounds aimed at improving these properties. This blind SAR campaign has produced a new lead compound of significantly increased in vivo stability and solubility for a 40-fold increase in exposure. This probe represents a significant advancement that will not only enable biophysical characterization of this interaction and further SAR, but also contribute to advances in understanding of MYC biology., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Our understanding of isoform-specific activities of phosphatidylinositol 3-kinase (PI3K) is still rudimentary, and yet, deep knowledge of these non-redundant functions in the PI3K family is essential for effective and safe control of PI3K in disease. The two major isoforms of the regulatory subunits of PI3K are p85α and p85β, encoded by the genes PIK3R1 and PIK3R2, respectively. These isoforms show distinct functional differences that affect and control cellular PI3K activity and signaling [1-4]. In this study, we have further explored the differences between p85α and p85β by genetic truncations and substitutions. We have discovered unexpected activities of the mutant proteins that reflect regulatory functions of distinct p85 domains. These results can be summarized as follows: Deletion of the SH3 domain increases oncogenic and PI3K signaling activity. Deletion of the combined SH3-RhoGAP domains abolishes these activities. In p85β, deletion of the cSH2 domain reduces oncogenic and signaling activities. In p85α, such a deletion has an activating effect. The deletions of the combined cSH2 and iSH2 domains and also the deletion of the cSH2, iSH2 and nSH2 domains yield results that go in the same direction, generally activating in p85α and reducing activity in p85β. The contrasting functions of the cSH2 domains are verified by domain exchanges with the cSH2 domain of p85β exerting an activating effect and the cSH2 domain of p85α an inactivating effect, even in the heterologous isoform. In the cell systems studied, protein stability was not correlated with oncogenic and signaling activity. These observations significantly expand our knowledge of the isoform-specific activities of p85α and p85β and of the functional significance of specific domains for regulating the catalytic subunits of class IA PI3K., Competing Interests: CONFLICTS OF INTEREST The authors have declared that no conflict of interest exists.

Small, noncoding RNAs are short untranslated RNA molecules, some of which have been associated with cancer development. Recently we showed that a class of small RNAs generated during the maturation process of tRNAs (tRNA-derived small RNAs, hereafter "tsRNAs") is dysregulated in cancer. Specifically, we uncovered tsRNA signatures in chronic lymphocytic leukemia and lung cancer and demonstrated that the ts-4521/3676 cluster (now called " ts-101 " and " ts-53 ," respectively), ts-46 , and ts-47 are down-regulated in these malignancies. Furthermore, we showed that tsRNAs are similar to Piwi-interacting RNAs (piRNAs) and demonstrated that ts-101 and ts-53 can associate with PiwiL2, a protein involved in the silencing of transposons. In this study, we extended our investigation on tsRNA signatures to samples collected from patients with colon, breast, or ovarian cancer and cell lines harboring specific oncogenic mutations and representing different stages of cancer progression. We detected tsRNA signatures in all patient samples and determined that tsRNA expression is altered upon oncogene activation and during cancer staging. In addition, we generated a knocked-out cell model for ts-101 and ts-46 in HEK-293 cells and found significant differences in gene-expression patterns, with activation of genes involved in cell survival and down-regulation of genes involved in apoptosis and chromatin structure. Finally, we overexpressed ts-46 and ts-47 in two lung cancer cell lines and performed a clonogenic assay to examine their role in cell proliferation. We observed a strong inhibition of colony formation in cells overexpressing these tsRNAs compared with untreated cells, confirming that tsRNAs affect cell growth and survival., Competing Interests: The authors declare no conflict of interest.

Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling., (Copyright © 2016 Elsevier Inc. All rights reserved.)

HIV-1 provirus integration results in a persistent latently infected reservoir that is recalcitrant to combined antiretroviral therapy (cART) with lifelong treatment being the only option. The "shock and kill" strategy aims to eradicate latent HIV by reactivating proviral gene expression in the context of cART treatment. Gene-specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising single guide RNAs (sgRNAs) with a nuclease-deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the long terminal repeat promoter of HIV-1 and identified a "hotspot" for activation within the viral enhancer sequence. Activating sgRNAs transcriptionally modulated the latent proviral genome across multiple different in vitro latency cell models including T cells comprising a clonally integrated mCherry-IRES-Tat (LChIT) latency system. We detected consistent and effective activation of latent virus mediated by activator sgRNAs, whereas latency reversal agents produced variable activation responses. Transcriptomic analysis revealed dCas9-VP64/sgRNAs to be highly specific, while the well-characterized chemical activator TNFα induced widespread gene dysregulation. CRISPR-mediated gene activation represents a novel system which provides enhanced efficiency and specificity in a targeted latency reactivation strategy and represents a promising approach to a "functional cure" of HIV/AIDS.

Nuclear run-on (NRO) is a method that measures transcriptional activity via the quantification of biochemically labeled nascent RNA molecules derived from nuclear isolates. Widespread use of this technique has been limited because of its technical difficulty relative to steady-state total mRNA analyses. Here we describe a detailed protocol for the quantification of transcriptional activity in human cell cultures. Nuclei are first isolated and NRO transcription is performed in the presence of bromouridine. Labeled nascent transcripts are purified by immunoprecipitation, and transcript levels are determined by reverse-transcription quantitative PCR (RT-qPCR). Data are then analyzed using standard techniques described elsewhere. This method is rapid (the protocol can be completed in 2 d) and cost-effective, exhibits negligible detection of background noise from unlabeled transcripts, requires no radioactive materials and can be performed from as few as 500,000 nuclei. It also takes advantage of the high sensitivity, specificity and dynamic range of RT-qPCR.

A point mutation in PIK3CA, the gene encoding the α isoform of class I phosphatidylinositol 3-kinase, induces extensive remodeling of the transcriptome and proteome, resulting in a gene signature that specifically resembles that of the basal subtype, but not other types, of breast cancer.

We have compared the proteome, transcriptome, and metabolome of two cell lines: the human breast epithelial line MCF-10A and its mutant descendant MCF-10A-H1047R. These cell lines are derived from the same parental stock and differ by a single amino acid substitution (H1047R) caused by a single nucleotide change in one allele of the PIK3CA gene, which encodes the catalytic subunit p110α of PI3K (phosphatidylinositol 3-kinase). They are considered isogenic. The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF-10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse, with examples in structural protein levels, the DNA repair machinery, and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer.

Using RNA-seq (RNA sequencing) of ribosome-depleted RNA, we have identified 1,273 lncRNAs (long non-coding RNAs) in P493-6 human B-cells. Of these, 534 are either up- or downregulated in response to MYC overexpression. An increase in MYC occupancy near their TSS (transcription start sites) was observed for MYC-responsive lncRNAs suggesting these are direct MYC targets. MYC binds to the same TSS across several cell lines, but the number of TSS bound depends on cellular MYC levels and increases with higher MYC concentrations. Despite this concordance in promoter binding, a majority of expressed lncRNAs are specific for one cell line, suggesting a determinant role of additional, possibly differentiation-specific factors in the activity of MYC-bound lncRNA promoters. A significant fraction of the lncRNA transcripts lack polyadenylation. The RNA-seq data were confirmed on eight selected lncRNAs by NRO (nuclear run-on) and RT-qPCR (quantitative reverse transcription PCR).

Expression of the regulatory subunit p85β of PI3K induces oncogenic transformation of primary avian fibroblasts. The transformed cells proliferate at an increased rate compared with nontransformed controls and show elevated levels of PI3K signaling. The oncogenic activity of p85β requires an active PI3K-TOR signaling cascade and is mediated by the p110α and p110β isoforms of the PI3K catalytic subunit. The data suggest that p85β is a less effective inhibitor of the PI3K catalytic subunit than p85α and that this reduced level of p110 inhibition accounts for the oncogenic activity of p85β.

The oncogenic bHLH-LZ transcription factor Myc forms binary complexes with its binding partner Max. These and other bHLH-LZ-based protein-protein interactions (PPI) in the Myc-Max network are essential for the physiological and oncogenic activities of Myc. We have generated a genetically determined and highly specific protein-fragment complementation assay based on Renilla luciferase to analyze the dynamic interplay of bHLH-LZ transcription factors Myc, Max, and Mxd1 in vivo. We also applied this PPI reporter to quantify alterations of nuclear Myc-Max complexes in response to mutational events, competitive binding by the transcriptional repressor Mxd1, or perturbations by small-molecule Myc inhibitors, including recently identified potent PPI inhibitors from a Kröhnke pyridine library. We show that the specificity of Myc-Max PPI reduction by the pyridine inhibitors directly correlates with their efficient and highly specific potential to interfere with the proliferation of human and avian tumor cells displaying deregulated Myc expression. In a direct comparison with known Myc inhibitors using human and avian cell systems, the pyridine compounds reveal a unique inhibitory potential even at sub-micromolar concentrations combined with remarkable specificity for the inhibition of Myc-driven tumor cell proliferation. Furthermore, we show in direct comparisons using defined avian cell systems that different Max PPI profiles for the variant members of the Myc protein family (c-Myc, v-Myc, N-Myc, L-Myc) correlate with their diverse oncogenic potential and their variable sensitivity to the novel pyridine inhibitors.

In a fluorescence polarization screen for the MYC-MAX interaction, we have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. The Kd of KJ-Pyr-9 for MYC in vitro is 6.5 ± 1.0 nM, as determined by backscattering interferometry; KJ-Pyr-9 also interferes with MYC-MAX complex formation in the cell, as shown in a protein fragment complementation assay. KJ-Pyr-9 specifically inhibits MYC-induced oncogenic transformation in cell culture; it has no or only weak effects on the oncogenic activity of several unrelated oncoproteins. KJ-Pyr-9 preferentially interferes with the proliferation of MYC-overexpressing human and avian cells and specifically reduces the MYC-driven transcriptional signature. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells.

MicroRNA deregulation is frequent in human colorectal cancers (CRCs), but little is known as to whether it represents a bystander event or actually drives tumor progression in vivo. We show that miR-135b overexpression is triggered in mice and humans by APC loss, PTEN/PI3K pathway deregulation, and SRC overexpression and promotes tumor transformation and progression. We show that miR-135b upregulation is common in sporadic and inflammatory bowel disease-associated human CRCs and correlates with tumor stage and poor clinical outcome. Inhibition of miR-135b in CRC mouse models reduces tumor growth by controlling genes involved in proliferation, invasion, and apoptosis. We identify miR-135b as a key downsteam effector of oncogenic pathways and a potential target for CRC treatment., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Addition of short (6 to 16 amino acids) peptide sequences to the N-terminus of p110α induces a gain of function. Such sequences include the common Flag, His, and VSV tags as well as random sequences. An N-terminal myristylation signal generally believed to activate p110α by providing a constitutive membrane address is also activating, if myristylation is mutationally abolished. The gain of function seen with N-terminally tagged (NTT) p110α constructs extends to signaling, oncogenic transformation and stimulation of cell growth. The activating effect of N-terminal tags requires a functional Ras-binding domain in p110α. Mutations in that domain (T208D and K227A) abolish the gains of function in oncogenicity and signaling. The dominant negative mutant of Ras, RasN17, interferes with transformation induced by NTT p110α. In contrast, binding to p85 activity is not required for cellular transformation and enhanced signaling by NTT p110α.

Cells transformed by the p110α-H1047R mutant of PI3K show increased tyrosine phosphorylation of Stat3. This activation of Stat3 is important for the transformation process, because a dominant-negative mutant of Stat3 interferes with PI3K-induced oncogenesis. GDC-0941, a specific inhibitor of PI3K reduces the level of Stat3 phosphorylation. The effect of PI3K on Stat3 appears to be mediated by a member of the Tec kinase family. The Tec kinase inhibitor LFM-A13 blocks Stat3 phosphorylation in H1047R-transformed cells. The Janus kinase inhibitor AG490 and the Src kinase inhibitor Src-1, as well as rapamycin, have no effect on Stat3 phosphorylation in H1047R-transformed cells. The H1047R-transformed cells also release a factor that induces Stat3 phosphorylation in normal cells with possible effects on the cellular microenvironment. In some human tumor cell lines, the enhanced phosphorylation of Stat3 is inhibited by both PI3K and by Tec kinase inhibitors, suggesting that the link between PI3K and Stat3 is significant in human cancer.

Akt (cellular homolog of murine thymoma virus akt8 oncogene) is an essential component of the PI3K (phosphatidylinositol 3-kinase) pathway. Its activity is stimulated by receptor tyrosine kinases and G-protein coupled receptors and plays a critical role in the regulation of cell proliferation, differentiation and apoptosis. A gain of function in Akt can lead to uncontrolled cell proliferation and resistance to apoptosis, both hallmarks of oncogenic transformation. In this communication, we have investigated the phosphorylation at the Akt residues T308, S473 and T450 and their roles in oncogenic transformation and signaling. We find that T450 phosphorylation has only a minimal part in these activities. In contrast, the phosphorylation of T308 and of S473 fulfills essential, distinct, and non-overlapping functions that we define with inactivating and with phosphomimetic mutations of these sites.

Cancer-specific mutations in the iSH2 (inter-SH2) and nSH2 (N-terminal SH2) domains of p85alpha, the regulatory subunit of phosphatidylinositide 3-kinase (PI3K), show gain of function. They induce oncogenic cellular transformation, stimulate cellular proliferation, and enhance PI3K signaling. Quantitative determinations of oncogenic activity reveal large differences between individual mutants of p85alpha. The mutant proteins are still able to bind to the catalytic subunits p110alpha and p110beta. Studies with isoform-specific inhibitors of p110 suggest that expression of p85 mutants in fibroblasts leads exclusively to an activation of p110alpha, and p110alpha is the sole mediator of p85 mutant-induced oncogenic transformation. The characteristics of the p85 mutants are in agreement with the hypothesis that the mutations weaken an inhibitory interaction between p85alpha and p110alpha while preserving the stabilizing interaction between p85alpha iSH2 and the adapter-binding domain of p110alpha.

The catalytic and regulatory subunits of class I phosphoinositide 3-kinase (PI3K) have oncogenic potential. The catalytic subunit p110α and the regulatory subunit p85 undergo cancer-specific gain-of-function mutations that lead to enhanced enzymatic activity, ability to signal constitutively, and oncogenicity. The β, γ, and δ isoforms of p110 are cell-transforming as overexpressed wild-type proteins. Class I PI3Ks have the unique ability to generate phosphoinositide 3,4,5 trisphosphate (PIP(3)). Class II and class III PI3Ks lack this ability. Genetic and cell biological evidence suggests that PIP(3) is essential for PI3K-mediated oncogenicity, explaining why class II and class III enzymes have not been linked to cancer. Mutational analysis reveals the existence of at least two distinct molecular mechanisms for the gain of function seen with cancer-specific mutations in p110α; one causing independence from upstream receptor tyrosine kinases, the other inducing independence from Ras. An essential component of the oncogenic signal that is initiated by PI3K is the TOR (target of rapamycin) kinase. TOR is an integrator of growth and of metabolic inputs. In complex with the raptor protein (TORC1), it controls cap-dependent translation, and this function is essential for PI3K-initiated oncogenesis.

In glioblastomas, an Akt-independent, PTEN (phosphatase and tensin homolog deleted on chromosome ten)-regulated signaling pathway links EGFR (epidermal growth factor receptor) to the phosphorylation of TOR (target of rapamycin) and of the ribosomal protein S6 and to the control of cell replication. Although PKCalpha (protein kinase Calpha) has been identified as an essential component, the detailed wiring of this previously unexplored noncanonical pathway remains to be worked out.

Research on PI 3-kinase (PI3K) is undergoing significant shifts in emphasis. Questions that have been dormant for some time are coming to the forefront, such as the relationship of PTEN to PI3K and the role of AKT in PI3K-driven oncogenesis. Two non-alpha isoforms of Class I PI3K are now established as important determinants in cancer: p110beta and p110delta. The oncogenic activities of p110beta include a non-catalytic function, a finding that will have immediate consequences for drug development.

Signaling by class I phosphatidylinositol 3-kinase (PI3K) controls cell growth, replication, motility, and metabolism. The PI3K pathway commonly shows gain of function in cancer. Two small GTPases, Rheb (Ras homolog enriched in brain) and Ras (rat sarcoma viral oncogene), play important roles in PI3K signaling. Rheb activates the TOR (target of rapamycin) kinase in a GTP-dependent manner; it links TOR to upstream signaling components, including the tuberous sclerosis complex (TSC) and Akt (homolog of the Akt8 murine lymphoma viral oncoprotein). Constitutively active, GTP-bound Rheb is oncogenic in cell culture, and activity that requires farnesylation. Ras activates PI3K by recruitment to the plasma membrane and possibly by inducing a conformational change in the catalytic subunit p110 of PI3K. In return, Ras signaling through the MAP kinase (MAPK) pathway is activated by PIP(3), the product of PI3K. Loss of Ras function can interfere with PI3K signaling. Various lines of evidence suggest complementary roles for PI3K and MAPK signaling in oncogenesis.

Mismatch repair (MMR) is critical to maintaining the integrity of the genome, and deficiencies in MMR are correlated with cancerous transformations. Bulky rhodium intercalators target DNA base mismatches with high specificity. Here we describe the application of bulky rhodium intercalators to inhibit cellular proliferation differentially in MMR-deficient cells compared with cells that are MMR-proficient. Preferential inhibition by the rhodium complexes associated with MMR deficiency is seen both in a human colon cancer cell line and in normal mouse fibroblast cells; the inhibition of cellular proliferation depends strictly on the MMR deficiency of the cell. Furthermore, our assay of cellular proliferation is found to correlate with DNA mismatch targeting by the bulky metallointercalators. It is the Delta-isomer that is active both in targeting base mismatches and in inhibiting DNA synthesis. Additionally, the rhodium intercalators promote strand cleavage at the mismatch site with photoactivation, and we observe that the cellular response is enhanced with photoactivation. Targeting DNA mismatches may therefore provide a cell-selective strategy for chemotherapeutic design.

Single-nucleotide polymorphisms are the largest source of genetic variation in humans. We report a method for the discovery of single-nucleotide polymorphisms within genomic DNA. Pooled genomic samples are amplified, denatured, and annealed to generate mismatches at polymorphic DNA sites. Upon photoactivation, these DNA mismatches are then cleaved site-specifically by using a small molecular probe, a bulky metallointercalator, Rhchrysi or Rhphzi. Fluorescent labeling of the cleaved products and separation by capillary electrophoresis permits rapid identification with single-base resolution of the single-nucleotide polymorphism site. This method is remarkably sensitive and minor allele frequencies as low as 5% can be readily detected.

Here we report the synthesis of luminescent ruthenium complexes that bind DNA base pair mismatches. [Ru(bpy)2(tpqp)]Cl2 (tpqp = 7,8,13,14-tetrahydro-6-phenylquino[8,7-k][1,8]phenanthroline), [Ru(bpy)2(pqp)]Cl2 (pqp = 6-phenylquino[8,7-k][1,8]phenanthroline), and [Ru(bpy)2(tactp)]Cl2 [tactp = 4,5,9,18-tetraazachryseno[9,10-b]triphenylene] have been synthesized, and their spectroscopic properties in the absence and presence of DNA have been examined. While [Ru(bpy)2(pqp)]2+ shows no detectable luminescence, [Ru(bpy)2(tpqp)]2+ is luminescent in the absence and presence of DNA with an excited-state lifetime of 10 ns and a quantum yield of 0.002. Although no increase in emission intensity is associated with binding to mismatch-containing DNA, luminescence quenching experiments and measurements of steady-state fluorescence polarization provide evidence for preferential binding to oligonucleotides containing a CC mismatch. Furthermore, by marking the site of binding through singlet oxygen sensitized damage, the complex has been shown to target a CC mismatch site directly with a specific binding affinity, Kb = 4 x 10(6) M(-1). [Ru(bpy)2(tactp)]2+, an analogue of [Ru(bpy)2(dppz)]2+ containing a bulky intercalating ligand, is luminescent in aqueous solution at micromolar concentrations and exhibits a 12-fold enhancement in luminescence in the presence of DNA. The complex, however, tends to aggregate in aqueous solution; we find a dimerization constant of 9.8 x 10(5) M(-1). Again, by singlet oxygen sensitization it is apparent that [Ru(bpy)2(tactp)]2+ binds preferentially to a CC mismatch; using a DNase I footprinting assay, a binding constant to a CC mismatch of 8 x 10(5) M(-1) is found. Hence results with these novel luminescent complexes support the concept of using a structurally demanding ligand to obtain selectivity in targeting single base mismatches in DNA. The challenge is coupling the differential binding we can obtain to differential luminescence., (Copyright 2004 American Chemical Society)

A rhodium(III) complex, rac-[Rh(bpy)(2)phzi](3+) (bpy, 2,2'-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)(2)(chrysi)](3+) [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 x 10(7) M(-1), respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design.