A chemical screen identifies the chemotherapeutic drug topotecan as a specific inhibitor of the B-MYB/MYCN axis in neuroblastoma

Francesco Sottile, Ilaria Gnemmi, Sandra Cantilena, Walter C D'Acunto, Arturo Sala, Francesco Sottile, Ilaria Gnemmi, Sandra Cantilena, Walter C D'Acunto, Arturo Sala

Abstract

The transcription factor MycN is the prototypical neuroblastoma oncogene and a potential therapeutic target. However, its strong expression caused by gene amplification in about 30% of neuroblastoma patients is a considerable obstacle to the development of therapeutic approaches aiming at eliminating its tumourigenic activity. We have previously reported that B-Myb is essentially required for transcription of the MYCN amplicon and have also shown that B-MYB and MYCN are engaged in a feed forward loop promoting the survival/proliferation of neuroblastoma cells. We postulated that pharmacological strategies breaking the B-MYB/MYCN axis should result in clinically desirable effects. Thus, we implemented a high throughput chemical screen, using a curated library of ~1500 compounds from the National Cancer Institute, whose endpoint was the identification of small molecules that inhibited B-Myb. At the end of the screening, we found that the compounds pinafide, ellipticine and camptothecin inhibited B-Myb transcriptional activity in luciferase assays. One of the compounds, the topoisomerase-1 inhibitor camptothecin, is of considerable clinical interest since its derivatives topotecan and irinotecan are currently used as first and second line treatment agents for various types of cancer, including neuroblastoma. We found that neuroblastoma cells with amplification of MYCN are more sensitive than MYCN negative cells to camptothecin and topotecan killing. Campothecin and topotecan caused selective down-regulation of B-Myb and MycN expression in neuroblastoma cells. Notably, forced overexpression of B-Myb could antagonize the killing effect of topotecan and camptothecin, demonstrating that the transcription factor is a key target of the drugs. These results suggest that camptothecin and its analogues should be more effective in patients whose tumours feature amplification of MYCN and/or overexpression of B-MYB.

Figures

Figure 1. Chemical screen
Figure 1. Chemical screen
Each dot represents relative luciferase activity (RLU) expressed as percentages relative to the value obtained in cells treated with the vehicle DMSO (indicated by the black diamond and ctrl), which was set as 100%. The highlighted compound codes 50N E8, 50N G9, and 63 F6 indicate ellipticine, camptothecin and pinafide, respectively.
Figure 2. Validation of compounds identified in…
Figure 2. Validation of compounds identified in the primary screen
GIMEN (MYCN non-amplified neuroblastoma cells) were transiently co-transfected with the MYB-responsive promoter pGL2-mim1 and the pcDna-B-MYB/empty vectors or with the p53 responsive p21luc promoter and pCMVp53/empty vectors. A renilla luciferase plasmid was added to the transfection mix for normalisation. Cells were treated with the compounds ellipticine, pinafide and camptothecin at the indicated concentrations for 24h. Error bars indicate standard deviations and the asterisk indicates statistically significant differences (Student's T test p ≤ 0,05) between the activities of cells treated with the compound relative to untreated cells (indicated by ctrl).
Figure 3. MTS proliferation assay in neuroblastoma…
Figure 3. MTS proliferation assay in neuroblastoma cell lines exposed to ellipticine and pinafide
Proliferation of MYCN amplified (panel A) or MYCN non amplified (panel B) cells in the presence of increasing concentrations of Ellipticine and Pinafide, as indicated. Note that cell viability is only reduced by high concentrations (250-500nM) of pinafide, in a MYCN-independent manner. Neuroblastoma cell lines were generally resistant to the killing effect of ellipticine. Error bars indicate standard deviations.
Figure 4. MTS proliferation assay in neuroblastoma…
Figure 4. MTS proliferation assay in neuroblastoma cell lines exposed to camptothecin (CPT) or topotecan (TPT)
A) MYCN-amplified NB cells; B) MYCN non-amplified NB cells. Error bars indicate standard deviations and statistical significance was verified by Student's T test (*= p<0,05 **=p<0,01 ***=p<0,001 of 48hrs 10v20v250nM vs. CTR; 72h 10v20v250nM vs. CTR )
Figure 5. Effects of camptothecin (CPT) and…
Figure 5. Effects of camptothecin (CPT) and topotecan (TPT) on the B-MYB/MYCN axis
Western blot analysis showing the expression of B-Myb and MycN in the indicated neuroblastoma cell lines treated with CPT or TPT (20nM) for 8-16-24-32hrs. Expression of the housekeeping gene GAPDH was used as loading control.
Figure 6. Effects of camptothecin (CPT) and…
Figure 6. Effects of camptothecin (CPT) and topotecan (TPT) on cell cycle proteins and PARP cleavage
Western blot analysis showing the expression of Cyclin A and cyclin B in the indicated neuroblastoma cell lines treated with CPT or TPT (20nM) for 8-16-24-32hrs. Cleavage of PARP is indicated by the appearance of a 89KDa fragment. The housekeeping gene GAPDH was used as loading control.
Figure 7. B-MYB rescues the killing effect…
Figure 7. B-MYB rescues the killing effect of camptothecin (CPT) and topotecan (TPT) in MYCN amplified cells
MTS proliferation assay showing the effect of CPT and TPT in LAN-5 cell clones transfected with pCMV-empty or pCMV-B-MYB plasmids. Bars indicate densitometric values relative to vehicle treated controls. Standard deviations is indicated by the error bars, statistical significance was assessed using the Student's T-test (*= p<0,05; **=p<0,01; ***=p<0,001; o= p<0,05 oo=p<0,01; ooo=p<0,001;)

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