Targeting neuroblastoma with a new inhibitor of the TAp73 interaction with MDM2 and mutant p53

TAp73 is a key tumour suppressor protein, regulating the transcription of unique and shared p53 target genes with crucial roles in tumorigenesis and therapeutic response. As such, in tumours with impaired p53 signalling, like neuroblastoma (NBL), TAp73 activation represents an encouraging strategy to suppress tumour growth and chemoresistance. In this work, we report a new TAp73-activating agent, the 1-carbaldehyde-3,4-dimethoxyxanthone (LEM2), with potent antitumour activity independent of p53 expression. LEM2 was able to release TAp73 from its interaction with both MDM2 and mutant p53 (mutp53), enhancing TAp73 transcriptional activity, cell cycle arrest, and apoptosis in p53-null and mutp53-expressing tumor cells. By cellular thermal shift assay (CETSA), LEM2 induced thermal stabilization of TAp73α but not of MDM2 or mutp53, suggesting the potential interaction of LEM2 with TAp73α. Interestingly, neither LEM2 alcohol 1-(hydroxymethyl)-3,4-dimethoxy-9H-xanthen-9-one (LEMred) or carboxylic acid 3,4-dimethoxy-9-oxo-9H-xanthene-1-carboxylic acid (LEMox) derivatives were able to inhibit the TAp73-MDM2 interaction supporting that LEM2 biological activity was due to the molecule itself and not to its potential derivatives. Consistently with an activation of the TAp73 pathway, LEM2 also displayed potent antitumour activity against patient-derived NBL cells, both alone and in combination of with doxorubicin and cisplatin. Collectively, besides its relevant contribution to the advance of TAp73 pharmacology, LEM2 may pave the way to improved therapeutic alternatives against NBL. Abstract


Targeting neuroblastoma with a new inhibitor of the TAp73 interaction with MDM2 and mutant p53
Characterization of antitumor activity TAp73 is a key tumour suppressor protein, regulating the transcription of unique and shared p53 target genes with crucial roles in tumorigenesis and therapeutic response. As such, in tumours with impaired p53 signalling, like neuroblastoma (NBL), TAp73 activation represents an encouraging strategy to suppress tumour growth and chemoresistance.
In this work, we report a new TAp73-activating agent, the 1-carbaldehyde-3,4dimethoxyxanthone (LEM2), with potent antitumour activity independent of p53 expression. LEM2 was able to release TAp73 from its interaction with both MDM2 and mutant p53 (mutp53), enhancing TAp73 transcriptional activity, cell cycle arrest, and apoptosis in p53-null and mutp53-expressing tumor cells. By cellular thermal shift assay (CETSA), LEM2 induced thermal stabilization of TAp73α but not of MDM2 or mutp53, suggesting the potential interaction of LEM2 with TAp73α. Interestingly, neither LEM2 alcohol 1-(hydroxymethyl)-3,4dimethoxy-9H-xanthen-9-one (LEMred) or carboxylic acid 3,4-dimethoxy-9-oxo-9H-xanthene-1-carboxylic acid (LEMox) derivatives were able to inhibit the TAp73-MDM2 interaction supporting that LEM2 biological activity was due to the molecule itself and not to its potential derivatives. Consistently with an activation of the TAp73 pathway, LEM2 also displayed potent antitumour activity against patient-derived NBL cells, both alone and in combination of with doxorubicin and cisplatin. Collectively, besides its relevant contribution to the advance of TAp73 pharmacology, LEM2 may pave the way to improved therapeutic alternatives against NBL.

Western blot
LEM2 was tested at 1µM for 16h or 24h. Immunoblots represent one of three independent experiments.

Sulforhodamine B assay
p73 protein levels. Immunoblots represent one of three independent experiments.
LEM2 was tested at 1µM for 16h or 24h. Immunoblots represent one of three independent experiments.
LEM2 was tested at 1 and 2µM on MDA-MB-468 cells for 24h. Immunoblots represent one of three independent experiments.

Potential application of LEM2 in NBL treatment
LEM2 was tested at 1 and 2µM on MDA-MB-468 cells for 24h. Immunoblots represent one of three independent experiments.

p73 is found inhibited in NBL cells by interaction with MDM2 and mutant p53
Lack of effective and non toxic strategies, specially in children with metastatic highrisk disease
• LEM2 growth inhibitory effect is TAp73dependent, and associated with cell cycle arrest, apoptosis, and induction of TAp73 target genes, but not with genotoxicity.
• LEM2 is an activator of TAp73 through disruption of its interaction with MDM2 and mutp53, and TAp73 stabilization.
• The potential application of LEM2 for NBL treatment was evidenced.