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1.
Nature ; 548(7668): 471-475, 2017 08 24.
Article in English | MEDLINE | ID: mdl-28813415

ABSTRACT

Cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are required for the initiation and progression of various malignancies. Pharmacological inhibitors of CDK4/6 have shown significant activity against several solid tumours. Their primary mechanism of action is thought to be the inhibition of phosphorylation of the retinoblastoma tumour suppressor, inducing G1 cell cycle arrest in tumour cells. Here we use mouse models of breast carcinoma and other solid tumours to show that selective CDK4/6 inhibitors not only induce tumour cell cycle arrest, but also promote anti-tumour immunity. We confirm this phenomenon through transcriptomic analysis of serial biopsies from a clinical trial of CDK4/6 inhibitor treatment for breast cancer. The enhanced anti-tumour immune response has two underpinnings. First, CDK4/6 inhibitors activate tumour cell expression of endogenous retroviral elements, thus increasing intracellular levels of double-stranded RNA. This in turn stimulates production of type III interferons and hence enhances tumour antigen presentation. Second, CDK4/6 inhibitors markedly suppress the proliferation of regulatory T cells. Mechanistically, the effects of CDK4/6 inhibitors both on tumour cells and on regulatory T cells are associated with reduced activity of the E2F target, DNA methyltransferase 1. Ultimately, these events promote cytotoxic T-cell-mediated clearance of tumour cells, which is further enhanced by the addition of immune checkpoint blockade. Our findings indicate that CDK4/6 inhibitors increase tumour immunogenicity and provide a rationale for new combination regimens comprising CDK4/6 inhibitors and immunotherapies as anti-cancer treatment.


Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/immunology , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Animals , Antigen Presentation/drug effects , Antigen Presentation/immunology , Biological Mimicry/drug effects , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Female , Humans , Interferons/metabolism , Mice , Phosphorylation/drug effects , RNA, Double-Stranded/genetics , Repressor Proteins/biosynthesis , Signal Transduction/drug effects , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , Transcriptome , Viruses/drug effects , Viruses/genetics , Viruses/immunology
2.
Proc Natl Acad Sci U S A ; 115(40): E9325-E9332, 2018 10 02.
Article in English | MEDLINE | ID: mdl-30224479

ABSTRACT

The PI3K-Akt-mTOR signaling pathway is a master regulator of RNA translation. Pharmacological inhibition of this pathway preferentially and coordinately suppresses, in a 4EBP1/2-dependent manner, translation of mRNAs encoding ribosomal proteins. However, it is unclear whether mechanistic target of rapamycin (mTOR)-4EBP1/2 is the exclusive translation regulator of this group of genes, and furthermore, systematic searches for novel translation modulators have been immensely challenging because of difficulties in scaling existing RNA translation profiling assays. Here, we developed a rapid and highly scalable approach for gene-specific quantitation of RNA translation, termed Targeted Profiling of RNA Translation (TPRT). We applied this technique in a chemical screen for translation modulators, and identified numerous preclinical and clinical therapeutic compounds, with diverse nominal targets, that preferentially suppress translation of ribosomal proteins. Surprisingly, some of these compounds act in a manner that bypasses canonical regulation by mTOR-4EBP1/2. Instead, these compounds exert their translation effects in a manner that is dependent on GCN2-eIF2α, a central signaling axis within the integrated stress response. Furthermore, we were also able to identify metabolic perturbations that also suppress ribosomal protein translation in an mTOR-independent manner. Together, we describe a translation assay that is directly applicable to large-scale RNA translation studies, and that enabled us to identify a noncanonical, mTOR-independent mode for translation regulation of ribosomal proteins.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carrier Proteins/metabolism , Multiprotein Complexes/metabolism , Protein Biosynthesis , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Ribosomal Proteins/biosynthesis , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Carrier Proteins/genetics , Cell Line, Transformed , Cell Line, Tumor , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Humans , Multiprotein Complexes/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Ribosomal Proteins/genetics , TOR Serine-Threonine Kinases/genetics
3.
J Am Chem Soc ; 137(19): 6112-5, 2015 May 20.
Article in English | MEDLINE | ID: mdl-25924099

ABSTRACT

The simultaneous intracellular delivery of multiple types of payloads, such as hydrophobic drugs and nucleic acids, typically requires complex carrier systems. Herein, we demonstrate a self-deliverable form of nucleic acid-drug nanostructure that is composed almost entirely of payload molecules. Upon light activation, the nanostructure sheds the nucleic acid shell, while the core, which consists of prodrug molecules, disintegrates via an irreversible self-immolative process, releasing free drug molecules and small molecule fragments. We demonstrate that the nanostructures exhibit enhanced stability against DNase I compared with free DNA, and that the model drug (camptothecin) released exhibits similar efficacy as free, unmodified drugs toward cancer cells.


Subject(s)
Antineoplastic Agents, Phytogenic/administration & dosage , Camptothecin/administration & dosage , DNA/administration & dosage , Delayed-Action Preparations/chemistry , Nanostructures/chemistry , Prodrugs/administration & dosage , Humans , Light , Models, Molecular
4.
Curr Protoc Mol Biol ; 125(1): e71, 2019 01.
Article in English | MEDLINE | ID: mdl-30346115

ABSTRACT

This unit describes a reverse transcription-quantitative PCR (RT-qPCR)-based method for gene-targeted measurement of RNA translation levels. The method includes washing and lysing cells with a buffer containing cycloheximide to enrich ribosomal accumulation at translation initiation sites (TIS), followed by enzymatic treatment to generate ribosomal footprints, reverse transcription targeted towards TIS of specific transcripts of interest to generate complementary DNA (cDNA), and qPCR to measure the abundance of these footprints. This method enables time- and cost-effective assessment of changes in translation levels across focused panels of genes and across numerous samples. © 2018 by John Wiley & Sons, Inc.


Subject(s)
Gene Expression Profiling/methods , RNA/genetics , Real-Time Polymerase Chain Reaction/methods , Transcription, Genetic , Animals , DNA, Complementary/genetics , DNA, Complementary/metabolism , Gene Expression Regulation , Humans , Protein Biosynthesis , Proteins/genetics , Proteins/metabolism , RNA/isolation & purification , RNA/metabolism , Reverse Transcription
5.
iScience ; 9: 149-160, 2018 Nov 30.
Article in English | MEDLINE | ID: mdl-30391850

ABSTRACT

The role of maternal and embryonic leucine zipper kinase (MELK) in cancer cell proliferation has been contentious, with recent studies arriving at disparate conclusions. We investigated the in vitro dependency of cancer cells on MELK under a range of assay conditions. Abrogation of MELK expression has little effect under common culture conditions, in which cells are seeded at high densities and reach confluence in 3-5 days. However, MELK dependency becomes clearly apparent in clonogenic growth assays using either RNAi or CRISPR technologies to modulate MELK expression. This dependency is in sharp contrast to that of essential genes, such as those encoding classic mitotic kinases, but is similar to that of other oncogenes including MYC and KRAS. Our study provides an example demonstrating some of the challenges encountered in cancer target validation, and reveals how subtle, but important, technical variations can ultimately lead to divergent outcomes and conclusions.

6.
Cell Rep ; 25(11): 2972-2980.e5, 2018 12 11.
Article in English | MEDLINE | ID: mdl-30540933

ABSTRACT

PARP inhibitors have shown promising clinical activities for patients with BRCA mutations and are changing the landscape of ovarian cancer treatment. However, the therapeutic mechanisms of action for PARP inhibition in the interaction of tumors with the tumor microenvironment and the host immune system remain unclear. We find that PARP inhibition by olaparib triggers robust local and systemic antitumor immunity involving both adaptive and innate immune responses through a STING-dependent antitumor immune response in mice bearing Brca1-deficient ovarian tumors. This effect is further augmented when olaparib is combined with PD-1 blockade. Our findings thus provide a molecular mechanism underlying antitumor activity by PARP inhibition and lay a foundation to improve therapeutic outcome for cancer patients.


Subject(s)
BRCA1 Protein/deficiency , Immunity , Membrane Proteins/metabolism , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/immunology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Animals , BRCA1 Protein/metabolism , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Female , HEK293 Cells , Humans , Immunity/drug effects , Mice, Inbred C57BL , Ovarian Neoplasms/pathology , Phthalazines/pharmacology , Phthalazines/therapeutic use , Piperazines/pharmacology , Piperazines/therapeutic use , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Programmed Cell Death 1 Receptor/metabolism , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/metabolism , Treatment Outcome
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