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Nature Protocols Jul 2012Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of...
Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. In addition, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pretreating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5-7 days to generate a completed ribosome profiling sequencing library. Sequencing and data analysis require a further 4-5 days.
Topics: Animals; Base Sequence; Gene Library; Harringtonines; Humans; Molecular Sequence Data; Peptide Chain Initiation, Translational; Protein Biosynthesis; RNA, Messenger; RNA, Ribosomal; Ribonucleases; Ribosomes; Saccharomyces cerevisiae; Sequence Analysis, RNA; Transcriptome
PubMed: 22836135
DOI: 10.1038/nprot.2012.086 -
Inhibition of the CDK2 and Cyclin A complex leads to autophagic degradation of CDK2 in cancer cells.Nature Communications May 2022Cyclin-dependent kinase 2 (CDK2) complex is significantly over-activated in many cancers. While it makes CDK2 an attractive target for cancer therapy, most inhibitors...
Cyclin-dependent kinase 2 (CDK2) complex is significantly over-activated in many cancers. While it makes CDK2 an attractive target for cancer therapy, most inhibitors against CDK2 are ATP competitors that are either nonspecific or highly toxic, and typically fail clinical trials. One alternative approach is to develop non-ATP competitive inhibitors; they disrupt interactions between CDK2 and either its partners or substrates, resulting in specific inhibition of CDK2 activities. In this report, we identify two potential druggable pockets located in the protein-protein interaction interface (PPI) between CDK2 and Cyclin A. To target the potential druggable pockets, we perform a LIVS in silico screening of a library containing 1925 FDA approved drugs. Using this approach, homoharringtonine (HHT) shows high affinity to the PPI and strongly disrupts the interaction between CDK2 and cyclins. Further, we demonstrate that HHT induces autophagic degradation of the CDK2 protein via tripartite motif 21 (Trim21) in cancer cells, which is confirmed in a leukemia mouse model and in human primary leukemia cells. These results thus identify an autophagic degradation mechanism of CDK2 protein and provide a potential avenue towards treating CDK2-dependent cancers.
Topics: Animals; Autophagy; CDC2-CDC28 Kinases; Cell Line, Tumor; Cyclin A; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cyclins; Homoharringtonine; Humans; Leukemia; Mice; Ribonucleoproteins
PubMed: 35595767
DOI: 10.1038/s41467-022-30264-0 -
Blood Feb 2022The majority of RUNX1 mutations in acute myeloid leukemia (AML) are missense or deletion-truncation and behave as loss-of-function mutations. Following standard therapy,...
The majority of RUNX1 mutations in acute myeloid leukemia (AML) are missense or deletion-truncation and behave as loss-of-function mutations. Following standard therapy, AML patients expressing mtRUNX1 exhibit inferior clinical outcome than those without mutant RUNX1. Studies presented here demonstrate that as compared with AML cells lacking mtRUNX1, their isogenic counterparts harboring mtRUNX1 display impaired ribosomal biogenesis and differentiation, as well as exhibit reduced levels of wild-type RUNX1, PU.1, and c-Myc. Compared with AML cells with only wild-type RUNX1, AML cells expressing mtRUNX1 were also more sensitive to the protein translation inhibitor homoharringtonine (omacetaxine) and BCL2 inhibitor venetoclax. Homoharringtonine treatment repressed enhancers and their BRD4 occupancy and was associated with reduced levels of c-Myc, c-Myb, MCL1, and Bcl-xL. Consistent with this, cotreatment with omacetaxine and venetoclax or BET inhibitor induced synergistic in vitro lethality in AML expressing mtRUNX1. Compared with each agent alone, cotreatment with omacetaxine and venetoclax or BET inhibitor also displayed improved in vivo anti-AML efficacy, associated with improved survival of immune-depleted mice engrafted with AML cells harboring mtRUNX1. These findings highlight superior efficacy of omacetaxine-based combination therapies for AML harboring mtRUNX1.
Topics: Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Core Binding Factor Alpha 2 Subunit; Drug Synergism; Homoharringtonine; Humans; Leukemia, Myeloid, Acute; Mutation; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-bcl-2; Sulfonamides
PubMed: 34601571
DOI: 10.1182/blood.2021013156 -
Journal of Hematology & Oncology Apr 2023Relapsed or refractory acute myeloid leukemia (R/R AML) has a dismal prognosis. The aim of this study was to investigate the activity and tolerability of venetoclax...
BACKGROUND
Relapsed or refractory acute myeloid leukemia (R/R AML) has a dismal prognosis. The aim of this study was to investigate the activity and tolerability of venetoclax combined with azacitidine plus homoharringtonine (VAH) regimen for R/R AML.
METHODS
This phase 2 trial was done at ten hospitals in China. Eligible patients were R/R AML (aged 18-65 years) with an Eastern Cooperative Oncology Group performance status of 0-2. Patients received venetoclax (100 mg on day 1, 200 mg on day 2, and 400 mg on days 3-14) and azacitidine (75 mg/m on days 1-7) and homoharringtonine (1 mg/m on days 1-7). The primary endpoint was composite complete remission rate [CRc, complete response (CR) plus complete response with incomplete blood count recovery (CRi)] after 2 cycles of treatment. The secondary endpoints include safety and survival.
RESULTS
Between May 27, 2020, and June 16, 2021, we enrolled 96 patients with R/R AML, including 37 primary refractory AML and 59 relapsed AML (16 relapsed after chemotherapy and 43 after allo-HSCT). The CRc rate was 70.8% (95% CI 60.8-79.2). In the patients with CRc, measurable residual disease (MRD)-negative was attained in 58.8% of CRc patients. Accordingly, overall response rate (ORR, CRc plus partial remission (PR)) was 78.1% (95% CI 68.6-85.4). At a median follow-up of 14.7 months (95% CI 6.6-22.8) for all patients, median overall survival (OS) was 22.1 months (95% CI 12.7-Not estimated), and event-free survival (EFS) was 14.3 months (95% CI 7.0-Not estimated). The 1-year OS was 61.5% (95% CI 51.0-70.4), and EFS was 51.0% (95% CI 40.7-60.5). The most common grade 3-4 adverse events were febrile neutropenia (37.4%), sepsis (11.4%), and pneumonia (21.9%).
CONCLUSIONS
VAH is a promising and well-tolerated regimen in R/R AML, with high CRc and encouraging survival. Further randomized studies are needed to be explored. Trial registration clinicaltrials.gov identifier: NCT04424147.
Topics: Humans; Azacitidine; Homoharringtonine; Leukemia, Myeloid, Acute; Bridged Bicyclo Compounds, Heterocyclic; Antineoplastic Combined Chemotherapy Protocols
PubMed: 37120593
DOI: 10.1186/s13045-023-01437-1 -
The Alkaloids. Chemistry and Biology 2017Cephalotaxus alkaloids represent a family of plant secondary metabolites known for 60 years. Significant activity against leukemia in mice was demonstrated for extracts... (Review)
Review
Cephalotaxus alkaloids represent a family of plant secondary metabolites known for 60 years. Significant activity against leukemia in mice was demonstrated for extracts of Cephalotaxus. Cephalotaxine (CET) (1), the major alkaloid of this series was isolated from Cephalotaxus drupacea species by Paudler in 1963. The subsequent discovery of promising antitumor activity among new Cephalotaxus derivatives reported by Chinese, Japanese, and American teams triggered extensive structure elucidation and biological studies in this family. The structural feature of this cephalotaxane family relies mainly on its tetracyclic alkaloid backbone, which comprises an azaspiranic 1-azaspiro[4.4]nonane unit (rings C and D) and a benzazepine ring system (rings A and B), which is linked by its C3 alcohol function to a chiral oxygenated side chain by a carboxylic function alpha to a tetrasubstituted carbon center. The botanical distribution of these alkaloids is limited to the Cephalotaxus genus (Cephalotaxaceae). The scope of biological activities of the Cephalotaxus alkaloids is mainly centered on the antileukemic activity of homoharringtonine (HHT) (2), which in particular demonstrated marked benefits in the treatment of orphan myeloid leukemia and was approved as soon as 2009 by European Medicine Agency and by US Food and Drug Administration in 2012. Its exact mechanism of action was partly elucidated and it was early recognized that HHT (2) inhibited protein synthesis at the level of the ribosome machinery. Interestingly, after a latency period of two decades, the topic of Cephalotaxus alkaloids reemerged as a prolific source of new natural structures. To date, more than 70 compounds have been identified and characterized. Synthetic studies also regained attention during the past two decades, and numerous methodologies were developed to access the first semisynthetic HHT (2) of high purity suitable for clinical studies, and then high grade enantiomerically pure CET (1), HHT (2), and analogs.
Topics: Animals; Antineoplastic Agents, Phytogenic; Harringtonines; Humans
PubMed: 28838429
DOI: 10.1016/bs.alkal.2017.07.001 -
Cell Nov 2011The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. Here we present a suite of techniques, based on ribosome...
The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. Here we present a suite of techniques, based on ribosome profiling (the deep sequencing of ribosome-protected mRNA fragments), to provide genome-wide maps of protein synthesis as well as a pulse-chase strategy for determining rates of translation elongation. We exploit the propensity of harringtonine to cause ribosomes to accumulate at sites of translation initiation together with a machine learning algorithm to define protein products systematically. Analysis of translation in mouse embryonic stem cells reveals thousands of strong pause sites and unannotated translation products. These include amino-terminal extensions and truncations and upstream open reading frames with regulatory potential, initiated at both AUG and non-AUG codons, whose translation changes after differentiation. We also define a class of short, polycistronic ribosome-associated coding RNAs (sprcRNAs) that encode small proteins. Our studies reveal an unanticipated complexity to mammalian proteomes.
Topics: Algorithms; Animals; Artificial Intelligence; Embryoid Bodies; Embryonic Stem Cells; Genomics; Harringtonines; High-Throughput Nucleotide Sequencing; Kinetics; Mice; Open Reading Frames; Peptide Chain Initiation, Translational; Protein Biosynthesis; RNA; Ribosomes; Sequence Analysis, RNA
PubMed: 22056041
DOI: 10.1016/j.cell.2011.10.002 -
Viruses Jun 2020As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases...
As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here, we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified the most potent sera from recovered patients for the treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19.
Topics: Amodiaquine; Animals; Antiviral Agents; Betacoronavirus; COVID-19; Caco-2 Cells; Cell Line, Tumor; Chlorocebus aethiops; Coronavirus Infections; Drug Therapy, Combination; Emetine; HEK293 Cells; HT29 Cells; Homoharringtonine; Humans; Immune Sera; Immunization, Passive; Indoles; Nelfinavir; Neutralization Tests; Pandemics; Pneumonia, Viral; Pyrans; Pyrroles; SARS-CoV-2; Vero Cells; COVID-19 Serotherapy
PubMed: 32545799
DOI: 10.3390/v12060642 -
Clinical Lymphoma, Myeloma & Leukemia Oct 2021To evaluate the efficacy and safety of homoharringtonine (HHT) in acute myeloid leukemia (AML). (Meta-Analysis)
Meta-Analysis Review
BACKGROUND
To evaluate the efficacy and safety of homoharringtonine (HHT) in acute myeloid leukemia (AML).
METHODS
PubMed, Cochrane Library, Embase, China National Knowledge of Infrastructure, and Wanfang data were systematically searched until October 31, 2020, for AML treatment with and without HHT. Fixed- and random-effect models were used to pool main outcomes, and between-study heterogeneity was assessed.
RESULTS
A total of 37 articles (2846 patients) fitting our criterion were included. The pooled overall response rate for the patients treated with HHT was 82% (CI, 77.9%-85.6%; I = 73.5%), and the complete response rate was 63.4% (CI, 58.8%-68%; I = 67.3%). Our study showed that patients treated with HHT have more overall response and complete response benefits and less cardiotoxicity and relapse rate. Subgroup analysis showed that patients with AML treated with HHT have significant overall response benefits in patients younger than 60 (odds ratio [OR], 1.63; CI, 1.33-2; I = 1.7%; P < .001), the newly diagnosed (OR, 1.59; CI, 1.15-2.21; I = 34.7%; P = .006), and relapsed/refractory patients (OR, 2.13; CI, 1.38-3.29; I = 32.3%; P = .001). Better complete remission benefits were observed in patients younger than 60 (OR, 1.32; CI, 1.1-1.59; I = 7%; P = .004), the newly diagnosed (OR, 1.32; CI, 1.08-1.62; I = 33.5%; P = .006), and relapsed/refractory patients (OR, 1.81; CI, 1.19-2.77; P = .006). For elderly patients, HHT treatment reduced relapse risk by 76.6% (OR, 0.23; CI, 0.09-0.63; I = 0%; P = .004).
CONCLUSIONS
HHT can be a reliable choice with less cardiotoxicity for patients with AML, especially for the newly diagnosed or patients younger than 60. For elderly intolerant patients, the use of HHT can reduce relapse.
Topics: Antineoplastic Agents, Phytogenic; Homoharringtonine; Humans; Leukemia, Myeloid, Acute
PubMed: 34301487
DOI: 10.1016/j.clml.2021.06.002 -
Biochemical Pharmacology Jul 2023Fusion with host cell membrane is the main mechanism of infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we propose that a new strategy...
Fusion with host cell membrane is the main mechanism of infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we propose that a new strategy to screen small-molecule antagonists blocking SARS-CoV-2 membrane fusion. Using cell membrane chromatography (CMC), we found that harringtonine (HT) simultaneously targeted SARS-CoV-2 S protein and host cell surface TMPRSS2 expressed by the host cell, and subsequently confirmed that HT can inhibit membrane fusion. HT effectively blocked SARS-CoV-2 original strain entry with the IC of 0.217 μM, while the IC in delta variant decreased to 0.101 μM, the IC in Omicron BA.1 variant was 0.042 μM. Due to high transmissibility and immune escape, Omicron subvariant BA.5 has become the dominant strain of the SARS-CoV-2 virus and led to escalating COVID-19 cases, however, against BA.5, HT showed a surprising effectiveness. The IC in Omicron BA.5 was even lower than 0.0019 μM. The above results revealed the effect of HT on Omicron is very significant. In summary, we characterize HT as a small-molecule antagonist by direct targeting on the Spike protein and TMPRSS2.
Topics: Humans; COVID-19; SARS-CoV-2; Harringtonines
PubMed: 37211174
DOI: 10.1016/j.bcp.2023.115617 -
Molecular Oncology Jul 2023Bromodomain-containing protein 4 (BRD4) inhibitors have been clinically developed to treat acute myeloid leukemia (AML), but their application is limited by the...
Bromodomain-containing protein 4 (BRD4) inhibitors have been clinically developed to treat acute myeloid leukemia (AML), but their application is limited by the possibility of drug resistance, which is reportedly associated with the activation of the WNT/β-catenin pathway. Meanwhile, homoharringtonine (HHT), a classic antileukemia drug, possibly inhibits the WNT/β-catenin pathway. In this study, we attempted to combine a novel BRD4 inhibitor (ACC010) and HHT to explore their synergistic lethal effects in treating AML. Here, we found that co-treatment with ACC010 and HHT synergistically inhibited cell proliferation, induced apoptosis, and arrested the cell cycle in FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)-positive AML cells in vitro, and significantly inhibiting AML progression in vivo. Mechanistically, ACC010 and HHT cooperatively downregulated MYC and inhibited FLT3 activation. Further, when HHT was added, ACC010-resistant cells demonstrated a good synergy. We also extended our study to the mouse BaF3 cell line with FLT3-inhibitor-resistant FLT3-ITD/tyrosine kinase domain mutations and AML cells without FLT3-ITD. Collectively, our results suggested that the combination treatment of ACC010 and HHT might be a promising strategy for AML patients, especially those carrying FLT3-ITD.
Topics: Animals; Mice; Apoptosis; beta Catenin; Cell Line, Tumor; fms-Like Tyrosine Kinase 3; Homoharringtonine; Leukemia, Myeloid, Acute; Mutation; Nuclear Proteins; Protein Kinase Inhibitors; Transcription Factors; Humans
PubMed: 36567628
DOI: 10.1002/1878-0261.13368