TP53 mutant MDM2-amplified cell lines selected for resistance to MDM2-p53 binding antagonists retain sensitivity to ionizing radiation.

Non-genotoxic reactivation of the p53 pathway by MDM2-p53 binding antagonists is an attractive treatment strategy for wild-type TP53 cancers. To determine how resistance to MDM2/p53 binding antagonists might develop, SJSA-1 and NGP cells were exposed to growth inhibitory concentrations of chemically distinct MDM2 inhibitors, Nutlin-3 and MI-63, and clonal resistant cell lines generated. The p53 mediated responses of parental and resistant cell lines were compared. In contrast to the parental cell lines, p53 activation by Nutlin-3, MI-63 or ionizing radiation was not observed in either the SJSA-1 or the NGP derived cell lines. An identical TP53 mutation was subsequently identified in both of the SJSA-1 resistant lines, whilst one out of three identified mutations was common to both NGP derived lines. Mutation specific PCR revealed these mutations were present in parental SJSA-1 and NGP cell populations at a low frequency. Despite cross-resistance to a broad panel of MDM2/p53 binding antagonists, these MDM2-amplified and TP53 mutant cell lines remained sensitive to ionizing radiation (IR). These results indicate that MDM2/p53 binding antagonists will select for p53 mutations present in tumours at a low frequency at diagnosis, leading to resistance, but such tumours may nevertheless remain responsive to alternative therapies, including IR.

MDM2-p53 protein-protein interaction inhibitors: a-ring substituted isoindolinones.

Structure-activity relationships for the MDM2-p53 inhibitory activity of a series of A-ring substituted 2-N-benzyl-3-(4-chlorophenyl)-3-(1-(hydroxymethyl)cyclopropyl)methoxy)isoindolinones have been investigated, giving rise to compounds with improved potency over their unsubstituted counterparts. Isoindolinone A-ring substitution with a 4-chloro group for the 4-nitrobenzyl, 4-bromobenzyl and 4-cyanobenzyl derivatives (10a-c) and substitution with a 6-tert-butyl group for the 4-nitrobenzyl derivative (10j) were found to confer additional potency. Resolution of the enantiomers of 10a showed that potent MDM2-p53 activity resided in the (-)-enantiomer ((-)-10a; IC(50)=44 ± 6 nM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compounds 10a and (-)-10a increase p53 protein levels, activate p53-dependent MDM2 and p21 transcription in MDM2 amplified cells, and show improved selectivity for growth inhibition in wild type p53 cell lines over the parent compound.

Isoindolinone inhibitors of the murine double minute 2 (MDM2)-p53 protein-protein interaction: structure-activity studies leading to improved potency.

Inhibition of the MDM2-p53 interaction has been shown to produce an antitumor effect, especially in MDM2 amplified tumors. The isoindolinone scaffold has proved to be versatile for the discovery of MDM2-p53 antagonists. Optimization of previously reported inhibitors, for example, NU8231 (7) and NU8165 (49), was guided by MDM2 NMR titrations, which indicated key areas of the binding interaction to be explored. Variation of the 2-N-benzyl and 3-alkoxy substituents resulted in the identification of 3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (74) as a potent MDM2-p53 inhibitor (IC(50) = 0.23 ± 0.01 µM). Resolution of the enantiomers of 74 showed that potent MDM2-p53 activity primarily resided with the (+)-R-enantiomer (74a; IC(50) = 0.17 ± 0.02 µM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compound 74a activates p53, MDM2, and p21 transcription in MDM2 amplified cells and shows moderate selectivity for wild-type p53 cell lines in growth inhibition assays.

Different mechanisms are involved in apoptosis induced by melanoma gangliosides on human monocyte-derived dendritic cells.

Tumor escape is linked to multiple mechanisms, notably the liberation, by tumor cells, of soluble factors that inhibit the function of dendritic cells (DC). We have shown that melanoma gangliosides impair DC differentiation and induce their apoptosis. The present study was aimed to give insight into the mechanisms involved. DC apoptosis was independent of the catabolism of gangliosides since lactosylceramide did not induce cell death. Apoptosis induced by GM3 and GD3 gangliosides was not blocked by inhibitors of de novo ceramide biosynthesis, whereas the acid sphingomyelinase inhibitor desipramine only prevented apoptosis induced by GM3. Furthermore, our results suggest that DC apoptosis was triggered via caspase activation, and it was ROS dependent with GD3 ganglioside, suggesting that GM3 and GD3 induced apoptosis through different mechanisms.

Immunosuppressive networks in the tumour environment and their effect in dendritic cells.

The failure of the immune system to provide protection against tumour cells is an important immunological problem. It is now evident that inadequate function of the host immune system is one of the main mechanisms by which tumours escape from immune control, as well as an important factor that limits the success of cancer immunotherapy. In recent years, it has become increasingly clear that defects in dendritic cells have a crucial role in non-responsiveness to tumours. This article focuses on the functional consequences and recently described mechanisms of the dendritic-cell defects in cancer.

Dendritic cells dysfunction in tumour environment.

Several studies indicate that most tumours are immunogenic and they rarely succeed to induce an efficient immune response. Many mechanisms have been involved in the tumour escape from host immune surveillance. The tumour microenvironment has emerged as an important component contributing to dendritic cells (DCs) dysfunction. There is evidence that DCs play a key role in the induction of tumour-specific immune responses, especially via cross-priming through MHC-class I antigens presentation. In this review we will discuss the potential role of the tumour microenvironment in DCs dysfunction.

Melanoma-derived gangliosides impair migratory and antigen-presenting function of human epidermal Langerhans cells and induce their apoptosis.

Gangliosides are ubiquitous, membrane-associated, glycosphingolipids, the composition and production of which is altered in many tumour cells. They have been shown to inhibit the in vitro generation and differentiation of dendritic cells (DCs) from progenitors, but their effect on human tissue-residing DCs is yet to be investigated. In the present study, we analysed the effect of GM3 and GD3 gangliosides purified from human melanoma tumours on the phenotypic and functional maturation of human epidermal Langerhans cells (LCs), the first immune barrier against the tumour cells. We showed that both gangliosides impaired spontaneous LC maturation induced by a short in vitro culture, as assessed by significant down-regulation of co-stimulation (CD40, CD54, CD80, CD86) and maturation markers (CD83, CCR7), which correlated to an impaired ability of the cells to mount allogeneic T cell proliferation. Furthermore, the ganglioside-treated cells displayed less ability to migrate towards CCL19/macrophage inflammatory protein 3 beta, the chemokine that specifically binds CCR7 and mediates LC migration to lymph nodes. Lastly, we showed that both GM3 and GD3 gangliosides enhance LC spontaneous apoptosis. Globally, these in vitro results might explain, at least in part, the altered number and distribution of LCs in melanoma-bearing patients. They underscore a new mechanism for gangliosides to impede the host immune response by inducing LC dysfunction in the tumour microenvironment.