Chemical Library Screening Identifies Novel Inhibitors of Cyclin D2 (CCND2) Transactivation That Selectively Induce Apoptosis in Multiple Myeloma Cells.
Bibliographic record
Abstract
Abstract Multiple myeloma tumors universally target one of the three human cyclin D genes (CCND1, CCND2 or CCND3) for dysregulation (Bergsagel et al., 2005, Blood, 106:296). Using a lentivirus expressing CCND2 RNAi we first tested the effects of selective cyclin D2 knock down on My5 and H929 myeloma cell lines and found G0/G1 phase arrest, increased apoptosis and significant selective disadvantage in transfected cells. By comparison, knockout mouse models indicate that most somatic tissues can develop in the total absence of cyclin D1, D2 and D3 (Kozar et al., 2004, Cell,118:477). Targeted inhibition of specific cyclin D expression is therefore a rational therapeutic strategy in myeloma. To identify novel pharmaceutical inhibitors of CCND2 transactivation we developed an assay employing NIH 3T3 cells stably co-expressing the CCND2 transactivator c-Maf and the cyclin D2 promoter driving firefly luciferase (luc) and screened the Lopac (n=1280), Prestwick (n=1120) and Spectrum (n=2000) libraries of drugs and natural compounds. In a parallel MTS assay, the effect of each compound on 3T3 viability was determined, allowing exclusion of compounds that caused secondary suppression of CCND2 due to non-specific cytotoxicity. From the screen we identified 10 c-Maf independent putative CCND2 inhibitors. These included monensin, patulin, β-lapachone, camptothecin, dihydrogambogic acid, gentian violet, thapsigargin, brefeldin A, pristimerin and kinetin riboside. Three of the 10 compounds (gentian violet, thapsigargin and patulin) were not studied further due to toxicity cited in the literature. Subsequent validation studies using selected compounds in human myeloma cell lines (HMCL) confirmed successful suppression of both cyclin D2 and D1 proteins. Each of these compounds was then shown to be cytotoxic to a genetically diverse and standardized panel of 14 HMCL in MTT assays: monensin (10–760 nM), camptothecin (5–700nM), dihydrogambogic acid (250–800 nM), pristimerin (150–500 nM) and kinetin riboside (2.5–20μM). Cell cycle analysis confirmed induction of G0/G1 phase arrest for most compounds, consistent with cyclin D inhibition. However, camptothecin and b-lapachone induced S-phase arrest, suggesting secondary suppression of cyclin D by virtue of S-phase activity. Unsorted myeloma patient bone marrow samples demonstrated selective activity for pristimerin, dihydrogambogic acid and kinetin riboside against CD138+ myeloma cells compared with non malignant hematopoietic cells; by contrast monensin showed almost equal toxicity for normal cells. The triterpenoid, pristimerin, showed potent anti-myeloma activity and was examined in greater detail. Studies confirm that pristimerin rapidly inhibits cyclin D1, D2 and D3 expression (<6 hours) at nanomolar concentrations and induces apoptosis of primary myeloma cells characterized by caspase 9 cleavage and Annexin V binding. While pristimerin is cytotoxic to HMCL and patient myeloma cells at 0.1–0.15 mg/L, toxicity studies in vivo indicate that the drug is tolerated in mice at 2.5 mg/kg i.p. daily. In vivo activity against a xenograft model is currently being determined. Overall this targeted chemical biology screen has identified several compounds, including the triterpenoid, pristimerin, that are being further characterized for promising preclinical anti-myeloma activity.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".