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1.
Nat Chem Biol ; 16(11): 1208-1217, 2020 11.
Article in English | MEDLINE | ID: mdl-32958952

ABSTRACT

The immunomodulatory drug (IMiD) thalidomide and its derivatives lenalidomide and pomalidomide are therapeutic agents used in the treatment of multiple myeloma. Although pomalidomide offers considerable clinical benefits to patients with lenalidomide-resistant multiple myeloma, the molecular mechanisms underlying its superior efficacy remain unclear. Here we show that ARID2, a component of the polybromo-associated BAF (PBAF) chromatin-remodeling complex, is a pomalidomide-induced neosubstrate of CRL4CRBN. BRD7, another subunit of PBAF, is critical for pomalidomide-induced ARID2 degradation. ARID2 is involved in transcriptional regulation of pomalidomide target genes including MYC. Pomalidomide is more effective than lenalidomide in degrading ARID2 and is capable of inhibiting MYC expression and proliferation in lenalidomide-resistant cell lines. Notably, ARID2 expression is associated with a poor prognosis and is higher in chemoresistant minimal residual disease (MRD) populations, and in patients with relapsed/refractory multiple myeloma. These findings suggest that ARID2 is a promising target for overcoming lenalidomide resistance in patients with multiple myeloma.


Subject(s)
Antineoplastic Agents/pharmacology , Multiple Myeloma/metabolism , Thalidomide/analogs & derivatives , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Antineoplastic Agents/metabolism , Cell Line, Tumor , Chromosomal Proteins, Non-Histone/metabolism , Drug Resistance, Neoplasm/drug effects , Drug Screening Assays, Antitumor , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , Humans , Lenalidomide/pharmacology , Multiple Myeloma/drug therapy , Mutation , Protein Binding , Proteolysis/drug effects , RNA, Messenger , RNA, Small Interfering , Thalidomide/metabolism , Thalidomide/pharmacology , Time Factors , Transcription Factors/genetics , Ubiquitination
2.
Sci Rep ; 10(1): 4012, 2020 03 04.
Article in English | MEDLINE | ID: mdl-32132601

ABSTRACT

Pomalidomide, a derivative of thalidomide, is an effective treatment for multiple myeloma. The drug exerts its effects through CRBN, a component of the E3 ubiquitin ligase complex CRL4CRBN. To search for novel factors involved in the anti-cancer activity of pomalidomide, we performed a genome-wide shRNA library screen and identified 445 genes as those affecting pomalidomide sensitivity. Genes encoding components of the ubiquitin-proteasome pathway, such as subunits of the CRL4CRBN complex, the COP9 signalosome, and the 26S proteasome, were among the pomalidomide-affecting genes. Karyopherin beta 1 (KPNB1) was identified as a novel pomalidomide-affecting gene. KPNB1 was required for the nuclear import of CRBN and for the CRBN-directed, pomalidomide-dependent degradation of a clinically relevant substrate, the transcription factor Aiolos. By contrast, the cytoplasmic translation factor GSPT1 was degraded following treatment with the thalidomide derivative CC-885 only when CRBN was present in the cytoplasm, indicating that subcellular distribution of CRBN is critical for the efficacy of thalidomide-based medications.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Multiple Myeloma/metabolism , Neoplasm Proteins/metabolism , Thalidomide/analogs & derivatives , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Cell Line, Tumor , Genome-Wide Association Study , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Neoplasm Proteins/genetics , Phenylurea Compounds/pharmacology , Thalidomide/pharmacology , Ubiquitin-Protein Ligases/genetics
4.
Mol Cell Biol ; 35(20): 3459-70, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26217014

ABSTRACT

Restores TBP function 1 (Rtf1) is generally considered to be a subunit of the Paf1 complex (PAF1C), a multifunctional protein complex involved in histone modification and transcriptional or posttranscriptional regulation. Rtf1, however, is not stably associated with the PAF1C in most species except Saccharomyces cerevisiae, and its biochemical functions are not well understood. Here, we show that human Rtf1 is a transcription elongation factor that may function independently of the PAF1C. Rtf1 requires "Rtf1 coactivator" activity, which is most likely unrelated to the PAF1C or DSIF, for transcriptional activation in vitro. A mutational study revealed that the Plus3 domain of human Rtf1 is critical for its coactivator-dependent function. Transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation studies in HeLa cells showed that Rtf1 and the PAF1C play distinct roles in regulating the expression of a subset of genes. Moreover, contrary to the finding in S. cerevisiae, the PAF1C was apparently recruited to the genes examined in an Rtf1-independent manner. The present study establishes a role for human Rtf1 as a transcription elongation factor and highlights the similarities and differences between the S. cerevisiae and human Rtf1 proteins.


Subject(s)
Nuclear Proteins/physiology , Transcription Factors/physiology , Gene Ontology , HeLa Cells , Humans , Transcription Elongation, Genetic , Transcriptional Activation , Ubiquitination
5.
Mol Pharmacol ; 84(6): 824-33, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24043703

ABSTRACT

Salicylic acid is a classic nonsteroidal anti-inflammatory drug. Although salicylic acid also induces mitochondrial injury, the mechanism of its antimitochondrial activity is not well understood. In this study, by using a one-step affinity purification scheme with salicylic acid-immobilized beads, ferrochelatase (FECH), a homodimeric enzyme involved in heme biosynthesis in mitochondria, was identified as a new molecular target of salicylic acid. Moreover, the cocrystal structure of the FECH-salicylic acid complex was determined. Structural and biochemical studies showed that salicylic acid binds to the dimer interface of FECH in two possible orientations and inhibits its enzymatic activity. Mutational analysis confirmed that Trp301 and Leu311, hydrophobic amino acid residues located at the dimer interface, are directly involved in salicylic acid binding. On a gel filtration column, salicylic acid caused a shift in the elution profile of FECH, indicating that its conformational change is induced by salicylic acid binding. In cultured human cells, salicylic acid treatment or FECH knockdown inhibited heme synthesis, whereas salicylic acid did not exert its inhibitory effect in FECH knockdown cells. Concordantly, salicylic acid treatment or FECH knockdown inhibited heme synthesis in zebrafish embryos. Strikingly, the salicylic acid-induced effect in zebrafish was partially rescued by FECH overexpression. Taken together, these findings illustrate that FECH is responsible for salicylic acid-induced inhibition of heme synthesis, which may contribute to its antimitochondrial and anti-inflammatory function. This study establishes a novel aspect of the complex pharmacological effects of salicylic acid.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Ferrochelatase/antagonists & inhibitors , Heme/antagonists & inhibitors , Mitochondria/drug effects , Salicylic Acid/adverse effects , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Cell Line, Tumor , Crystallography, X-Ray , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Escherichia coli Proteins/chemistry , Ferrochelatase/biosynthesis , Ferrochelatase/chemistry , Heme/biosynthesis , Humans , Mitochondria/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Protein Multimerization , Protoporphyrins/metabolism , Salicylic Acid/chemistry , Zebrafish
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