35 research outputs found
Transcriptional transactivation by selected short random peptides attached to lexA-GFP fusion proteins
BACKGROUND: Transcriptional transactivation is a process with remarkable tolerance for sequence diversity and structural geometry. In studies of the features that constitute transactivating functions, acidity has remained one of the most common characteristics observed among native activation domains and activator peptides. RESULTS: We performed a deliberate search of random peptide libraries for peptides capable of conferring transcriptional transactivation on the lexA DNA binding domain. Two libraries, one composed of C-terminal fusions, the other of peptide insertions within the green fluorescent protein structure, were used. We show that (i) peptide sequences other than C-terminal fusions can confer transactivation; (ii) though acidic activator peptides are more common, charge neutral and basic peptides can function as activators; and (iii) peptides as short as 11 amino acids behave in a modular fashion. CONCLUSIONS: These results support the recruitment model of transcriptional activation and, combined with other studies, suggest the possibility of using activator peptides in a variety of applications, including drug development work
A Comprehensive Patient-Derived Xenograft Collection Representing the Heterogeneity of Melanoma
Therapy of advanced melanoma is changing dramatically. Following mutational and biological subclassification of this heterogeneous cancer, several targeted and immune therapies were approved and increased survival significantly. To facilitate further advancements through pre-clinical in vivo modeling, we have established 459 patient-derived xenografts (PDX) and live tissue samples from 384 patients representing the full spectrum of clinical, therapeutic, mutational, and biological heterogeneity of melanoma. PDX have been characterized using targeted sequencing and protein arrays and are clinically annotated. This exhaustive live tissue resource includes PDX from 57 samples resistant to targeted therapy, 61 samples from responders and non-responders to immune checkpoint blockade, and 31 samples from brain metastasis. Uveal, mucosal, and acral subtypes are represented as well. We show examples of pre-clinical trials that highlight how the PDX collection can be used to develop and optimize precision therapies, biomarkers of response, and the targeting of rare genetic subgroups
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mRNA decay in Saccharomyces cerevisiae.
mRNA decay is an important step in the control of gene expression. To study mRNA degradation I have exploited the genetic, biochemical, and molecular tools available in Saccharomyces cerevisiae. These studies provided insight into the signals within individual transcripts which specify their half-lives, the various mechanisms by which mRNAs are degraded, and the trans-acting factors which both perform and control nucleolytic events. I identified a 65 nucleotide segment from the coding region of the unstable MATÉ‘l mRNA which was capable of targeting both the MATÉ‘l and stable PGKI transcripts for rapid degradation. This "instability element" was divided into two parts, one located in the first 33, and the second in the latter 32, nucleotides. The first part could be functionally replaced by different mRNA sequences containing rare codons, and while unable to promote mRNA decay by itself, enhanced degradation mediated by the second part. I determined that the MATÉ‘l Instability Element (MIE) targets mRNAs for rapid degradation by increasing the rates of two nucleolytic steps in a pathway of mRNA decay common to several stable and unstable yeast transcripts. The initial step in this pathway is shortening of the poly(A) tail of an mRNA. Subsequently, mRNAs are decapped, after which the transcript body is degraded in a 5' to 3' exonucleolytic manner. The MIE promotes decay of the MATÉ‘l mRNA through an increase in its decapping rate. In contrast, PGKI mRNA decay was stimulated through an increase in its rate of deadenylation. The observation that the poly(A) tail must be removed prior to mRNA decapping suggests that the poly(A) tail inhibits decapping. I determined that the major poly(A)binding protein (Pablp) is required for the inhibition of decapping mediated by the poly(A) tail. Pablp is also required for normal deadenylation rates. Pablp therefore affects mRNA decapping and deadenylation, the two rate determining steps in a common pathway of mRNA decay. Determining how Pablp, and additional trans-acting factors, exert influence over both decapping and deadenylation will provide a greater understanding of the basis of differential rates of mRNA degradation
The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity
The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available1. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines, this collection allowed identification of genetic, lineage, and gene-expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Together, our results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of ‘personalized’ therapeutic regimen
Pre-clinical Selectivity Profile of the CDK4/6 Inhibitor Ribociclib Compared With That of Palbociclib and Abemaciclib
A hallmark of cancer is unchecked cell division. Retinoblastoma protein (Rb) is a human tumor suppressor that guards the entry point into S phase by binding E2F transcription factors and keeping them inactive. Many growth promoting stimuli increase the expression of D-cyclins, which bind to and activate CDK4/6 kinases. The D-cyclin bound CDK4/6 holoenzymes phosphorylate Rb resulting in the release of E2F, which in turn activates genes required for S phase entry and DNA replication. Numerous oncogenic aberrations converge at the CDK4/6-Rb pathway, providing a strong rationale for developing CDK4/6 inhibitors as cancer therapeutics.
Ribociclib (LEE011) is a selective CDK4/6 inhibitor that has received FDA Breakthrough designation and Priority Review for treatment of hormone receptor positive breast cancer in combination with letrozole and is being tested in additional clinical trials. Here, we describe the pre-clinical selectivity profile of ribociclib in biochemical and cellular assay. Ribociclib inhibits both CDK4 - cyclin D1 and CDK6 - cyclin D3 kinase activity with nanomolar IC50s in biochemical assays. To comprehensively address selectivity of ribociclib in direct comparison with two other clinical CDK4/6 inhibitors, palbociclib and abemaciclib, we made use of the KinomeScan assay platform (DiscoverX) consisting of >450 kinase active site-directed competition binding assays. Based on the binding constants (Kd) for CDK4 and CDK6, we adjusted the test concentrations in the kinase selectivity panel to account for higher potency of abemaciclib. Data indicated that both ribociclib and palbociclib have high selectivity for CDK4 (CDK6 was not covered in the panel) with very few distinct additional binding events detected. In contrast, abemaciclib is a much more promiscuous kinase inhibitor.
Next, we sought to determine the relative potencies of the three inhibitors against CDK4 vs CDK6 in cellular assays. When testing different routinely used readouts of cellular viability, we found that assays that measure metabolic activity (such as CTG) tend to underestimate the effects of CDK4/6 inhibition. Therefore, assays that either directly or indirectly assessed cell number were used in these studies. We first identified cancer cell lines primarily dependent on either CDK4 or CDK6 as judged by combined RNA expression analysis and shRNA/CRISPR-based functional assays. When determining IC50 values for these cell lines with the three CDK4/6 inhibitors, we found that both ribociclib and abemaciclib demonstrated greater activity in CDK4-relative to CDK6-dependent cells, whereas palbociclib was similarly active in both cells types.
In conclusion, the high degree of selectivity of ribociclib suggests that off-target kinase inhibition is an unlikely complication in patients. Moreover, the apparent preference for CDK4 over CDK6 could be an advantage in certain cancer types that are primarily dependent on CDK4
Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitors In Breast Cancer: Current Status and Future Development.
8Evidence suggests that the epidermal growth factor receptor (EGFR) and its ligands are involved in the pathogenesis of different human carcinomas, including breast cancer. Results of phase II clinical trials of EGFR tyrosine kinase inhibitors (TKIs) have shown that these compounds have little activity in breast cancer patients when used as single agents. The potential pitfalls of these clinical trials, and the molecular mechanisms that might be involved in regulating the sensitivity/resistance of breast cancer cells to EGFR TKIs are discussed in this brief article. In particular, preclinical findings clearly demonstrate that breast cancer cells are able to activate different mechanisms to escape the anti-tumor effects of drugs directed against growth factor-driven pathways. Therefore, it is conceivable that significant blockade of tumor growth might be obtained only through contemporary blockade of different growth promoting pathways, at least in advanced disease. In addition, preclinical and clinical findings support the use of EGFR TKIs in specific subgroups of breast cancer patients, such as estrogen receptor positive (ER+), tamoxifen resistant patients. In this regard, we describe potential future applications of these compounds in combination with other agents in the treatment of breast carcinoma.nonenoneNORMANNO N; DE LUCA A; MONICA R. MAIELLO; MANCINO M; DANTONIO M; MACALUSO M; CAPONIGRO A; A. GIORDANONormanno, N; DE LUCA, A; MONICA R., Maiello; Mancino, M; Dantonio, M; Macaluso, M; Caponigro, A; Giordano, Antoni
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in breast cancer: Current status and future development
Evidence suggests that the epidermal growth factor receptor (EGFR) and its ligands are involved in the pathogenesis of different human carcinomas, including breast cancer. Results of phase II clinical trials of EGFR tyrosine kinase inhibitors (TKIs) have shown that these compounds have little activity in breast cancer patients when used as single agents. The potential pitfalls of these clinical trials, and the molecular mechanisms that might be involved in regulating the sensitivity/resistance of breast cancer cells to EGFR TKIs are discussed in this brief article. In particular, preclinical findings clearly demonstrate that breast cancer cells are able to activate different mechanisms to escape the anti-tumor effects of drugs directed against growth factor-driven pathways. Therefore, it is conceivable that significant blockade of tumor growth might be obtained only through contemporary blockade of different growth promoting pathways, at least in advanced disease. In addition, preclinical and clinical findings support the use of EGFR TKIs in specific subgroups of breast cancer patients, such as estrogen receptor positive (ER+), tamoxifen resistant patients. In this regard, we describe potential future applications of these compounds in combination with other agents in the treatment of breast carcinoma
Down-regulation of class II phosphoinositide 3-kinase alpha expression below a critical threshold induces apoptotic cell death.
Members of the phosphoinositide 3-kinase (PI3K) family collectively control multiple cellular responses, including proliferation, growth, chemotaxis, and survival. These diverse effects can partly be attributed to the broad range of downstream effectors being regulated by the products of these lipid kinases, the 3'-phosphoinositides. However, an additional layer of complexity is introduced by the existence of multiple PI3K enzyme isoforms. Much has been learned over the last years on the roles of the classes I and III PI3K members in cellular signaling, but little is known about the isoform-specific tasks done by the class II PI3Ks (C2alpha, beta, and gamma). In this study, we used quantitative reverse transcription-PCR and RNA interference in mammalian cells to gain further insight into the function of these lesser studied PI3K enzymes. We find that PI3K-C2alpha, but not PI3K-C2beta, has an important role in controlling cell survival and by using a panel of RNA interference reagents, we were able to determine a critical threshold of PI3K-C2alpha mRNA levels, below which the apoptotic program is switched on, via the intrinsic cell death pathway. In addition, knockdown of PI3K-C2alpha to levels that by themselves do not induce apoptosis sensitize cells to the anticancer agent Taxol (paclitaxel). Lastly, we report that lowering the levels of PI3K-C2alpha in a number of cancer cell lines reduces their proliferation and cell viability, arguing that PI3K inhibitors targeting not only the class Ialpha isoform but also class IIalpha may contribute to an effective anticancer strategy