Publications by authors named "Jonathan R Hart"

33 Publications

Synthetic fluorescent MYC probe: Inhibitor binding site elucidation and development of a high-throughput screening assay.

Bioorg Med Chem 2021 Jul 6;42:116246. Epub 2021 Jun 6.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bmc.2021.116246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8279689PMC
July 2021

An MXD1-derived repressor peptide identifies noncoding mediators of MYC-driven cell proliferation.

Proc Natl Acad Sci U S A 2020 03 10;117(12):6571-6579. Epub 2020 Mar 10.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037;

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.
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http://dx.doi.org/10.1073/pnas.1921786117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104257PMC
March 2020

Cooperates with to Promote MYC Activity and Tumorigenesis via the Bromodomain Protein BRD9.

Cancers (Basel) 2019 Oct 24;11(11). Epub 2019 Oct 24.

Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

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.
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http://dx.doi.org/10.3390/cancers11111634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896067PMC
October 2019

Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD.

Mol Ther Nucleic Acids 2018 Dec 17;13:1-15. Epub 2018 Aug 17.

Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK; Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.omtn.2018.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140421PMC
December 2018

Isoform-specific activities of the regulatory subunits of phosphatidylinositol 3-kinases - potentially novel therapeutic targets.

Expert Opin Ther Targets 2018 10 24;22(10):869-877. Epub 2018 Sep 24.

a Department of Molecular Medicine , The Scripps Research Institute , La Jolla , CA , USA.

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.
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http://dx.doi.org/10.1080/14728222.2018.1522302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197473PMC
October 2018

Synthetic molecules for disruption of the MYC protein-protein interface.

Bioorg Med Chem 2018 08 11;26(14):4234-4239. Epub 2018 Jul 11.

Department of Chemistry, BCC-582, 10550 N Torrey Pines Road, The Scripps Research Institute, La Jolla, CA 92037, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bmc.2018.07.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214403PMC
August 2018

Domain analysis reveals striking functional differences between the regulatory subunits of phosphatidylinositol 3-kinase (PI3K), p85α and p85β.

Oncotarget 2017 Aug 3;8(34):55863-55876. Epub 2017 Aug 3.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, San Diego, CA, USA.

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.
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http://dx.doi.org/10.18632/oncotarget.19866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593529PMC
August 2017

tsRNA signatures in cancer.

Proc Natl Acad Sci U S A 2017 07 10;114(30):8071-8076. Epub 2017 Jul 10.

Department of Cancer Biology and Medical Genetics, The Ohio State University Comprehensive Cancer Center, Columbus, OH 43210;

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 cluster (now called "" and "," respectively), , and are down-regulated in these malignancies. Furthermore, we showed that tsRNAs are similar to Piwi-interacting RNAs (piRNAs) and demonstrated that and 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 and 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 and 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.
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http://dx.doi.org/10.1073/pnas.1706908114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544330PMC
July 2017

A butterfly effect in cancer.

Mol Cell Oncol 2016 Jan 14;3(1):e1029063. Epub 2015 Apr 14.

The Scripps Research Institute, Chemical Physiology ; La Jolla, CA USA.

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.
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http://dx.doi.org/10.1080/23723556.2015.1029063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4845179PMC
January 2016

A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling.

Cell 2016 Apr;165(3):643-55

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA; Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2016.03.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006944PMC
April 2016

MINCR is not a MYC-induced lncRNA.

Proc Natl Acad Sci U S A 2016 Feb 25;113(5):E496-7. Epub 2016 Jan 25.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037;

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http://dx.doi.org/10.1073/pnas.1519903113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747730PMC
February 2016

Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex.

Mol Ther 2016 Mar 19;24(3):488-98. Epub 2015 Nov 19.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.

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.
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http://dx.doi.org/10.1038/mt.2015.202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786915PMC
March 2016

Stopping MYC in its tracks.

Aging (Albany NY) 2015 Jul;7(7):463-4

Institute of Biochemistry and Center for Molecular Biosciences (CMBI), University of Innsbruck, Austria.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4543033PMC
http://dx.doi.org/10.18632/aging.100780DOI Listing
July 2015

A brave new MYC-amplified world.

Aging (Albany NY) 2015 Jul;7(7):459-60

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4543031PMC
http://dx.doi.org/10.18632/aging.100777DOI Listing
July 2015

Quantification of nascent transcription by bromouridine immunocapture nuclear run-on RT-qPCR.

Nat Protoc 2015 Aug 16;10(8):1198-211. Epub 2015 Jul 16.

1] Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA. [2] School of Biotechnology and Biomedical Sciences, University of New South Wales, Sydney, New South Wales, Australia.

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.
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http://dx.doi.org/10.1038/nprot.2015.076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4790731PMC
August 2015

MYC regulates the non-coding transcriptome.

Oncotarget 2014 Dec;5(24):12543-54

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA.

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).
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4350361PMC
http://dx.doi.org/10.18632/oncotarget.3033DOI Listing
December 2014

The butterfly effect in cancer: a single base mutation can remodel the cell.

Proc Natl Acad Sci U S A 2015 Jan 12;112(4):1131-6. Epub 2015 Jan 12.

Departments of Molecular and Experimental Medicine and

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.
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http://dx.doi.org/10.1073/pnas.1424012112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4313835PMC
January 2015

Oncogenic activity of the regulatory subunit p85β of phosphatidylinositol 3-kinase (PI3K).

Proc Natl Acad Sci U S A 2014 Nov 10;111(47):16826-9. Epub 2014 Nov 10.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037

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.
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http://dx.doi.org/10.1073/pnas.1420281111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4250105PMC
November 2014

In vivo quantification and perturbation of Myc-Max interactions and the impact on oncogenic potential.

Oncotarget 2014 Oct;5(19):8869-78

Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4253403PMC
http://dx.doi.org/10.18632/oncotarget.2588DOI Listing
October 2014

Inhibitor of MYC identified in a Kröhnke pyridine library.

Proc Natl Acad Sci U S A 2014 Aug 11;111(34):12556-61. Epub 2014 Aug 11.

Chemistry, The Scripps Research Institute, La Jolla, CA 92037; and

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.
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http://dx.doi.org/10.1073/pnas.1319488111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151726PMC
August 2014

MicroRNA-135b promotes cancer progression by acting as a downstream effector of oncogenic pathways in colon cancer.

Cancer Cell 2014 Apr;25(4):469-83

Cancer Research UK Beatson Institute, Glasgow G61 1BD, UK.

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.
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http://dx.doi.org/10.1016/j.ccr.2014.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3995091PMC
April 2014

Addition of N-terminal peptide sequences activates the oncogenic and signaling potentials of the catalytic subunit p110α of phosphoinositide-3-kinase.

Cell Cycle 2011 Nov 1;10(21):3731-9. Epub 2011 Nov 1.

The Scripps Research Institute, Department of Molecular and Experimental Medicine, La Jolla, CA, USA.

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.
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http://dx.doi.org/10.4161/cc.10.21.17920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266008PMC
November 2011

Essential role of Stat3 in PI3K-induced oncogenic transformation.

Proc Natl Acad Sci U S A 2011 Aug 25;108(32):13247-52. Epub 2011 Jul 25.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.

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.
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http://dx.doi.org/10.1073/pnas.1110486108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3156164PMC
August 2011

Phosphorylation of AKT: a mutational analysis.

Oncotarget 2011 Jun;2(6):467-76

The Scripps Research Institute, Molecular and Experimental Medicine, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3139455PMC
http://dx.doi.org/10.18632/oncotarget.293DOI Listing
June 2011

Cancer-derived mutations in the regulatory subunit p85alpha of phosphoinositide 3-kinase function through the catalytic subunit p110alpha.

Proc Natl Acad Sci U S A 2010 Aug 16;107(35):15547-52. Epub 2010 Aug 16.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.

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.
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http://dx.doi.org/10.1073/pnas.1009652107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2932613PMC
August 2010

Phosphatidylinositol 3-kinase: the oncoprotein.

Curr Top Microbiol Immunol 2010 ;347:79-104

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.

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.
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http://dx.doi.org/10.1007/82_2010_80DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2955792PMC
January 2011

Akt demoted in glioblastoma.

Sci Signal 2009 Apr 21;2(67):pe26. Epub 2009 Apr 21.

The Scripps Research Institute, Department of Molecular and Experimental Medicine, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

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.
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http://dx.doi.org/10.1126/scisignal.267pe26DOI Listing
April 2009

PI 3-kinase and cancer: changing accents.

Curr Opin Genet Dev 2009 Feb;19(1):12-7

Department of Molecular and Experimental Medicine, Division of Oncovirology, The Scripps Research Institute, La Jolla, CA 92037, USA.

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.
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http://dx.doi.org/10.1016/j.gde.2008.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2746202PMC
February 2009

Biochemical and biological characterization of tumor-associated mutations of p110alpha.

Methods Enzymol 2008 ;438:291-305

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.

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.
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http://dx.doi.org/10.1016/S0076-6879(07)38020-8DOI Listing
June 2008

DNA mismatch-specific targeting and hypersensitivity of mismatch-repair-deficient cells to bulky rhodium(III) intercalators.

Proc Natl Acad Sci U S A 2006 Oct 9;103(42):15359-63. Epub 2006 Oct 9.

Division of Chemistry and Chemical Engineering, California Institute of Technology Pasadena, CA 91125, USA.

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.
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http://dx.doi.org/10.1073/pnas.0607576103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1622828PMC
October 2006
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