Enhanced expression of p21 promotes sensitivity of melanoma cells towards targeted therapies.

Metastatic melanoma patients benefit from the approved targeted BRAF inhibitor (BRAFi) therapy. Despite the great progress in the therapeutic approach to combat metastatic melanoma, fast emerging drug resistance in patients limits its long-term efficacy. In this study, we aimed to unravel the role of the p53 target gene CDKN1A/p21 in the response of melanoma cells towards BRAFi. We show that p53 activation increases BRAFi sensitivity in a synergistic manner exclusively in cells with a high expression of CDKN1A/p21. In a similar way, high expression of p21 was associated with a better response towards the mouse double minute 2 inhibitor (MDM2i) compared to those with low p21 expression. Indeed, p21 knockdown decreased the sensitivity towards both targeted therapies. The results indicate that the sensitivity of melanoma cells towards targeted therapies (BRAFi and MDM2i) is dependent on the p21 protein level in the cells. In addition to that, we found that p53 negatively regulates p73 expression; however, p73 seems not to have an influence on p53 expression. These findings offer new potential strategies for the treatment improvement of melanoma patients with high basal p21 levels with BRAFi by increasing treatment efficacy using combination therapies with p53 activating substances, which are able to further increase p21 expression levels. Furthermore, the data suggest that the expression and induction level of p21 could be used as a predictive biomarker in melanoma patients to forecast the outcome of a treatment with p53 activating substances and BRAFi. All in all, this manuscript shows the distinct role of p53 family members and its impact on melanoma therapy. In future, individualized treatment regimens based on p21 basal and induction levels could help melanoma patients with limited treatment options.

Targeting Rad51 as a strategy for the treatment of melanoma cells resistant to MAPK pathway inhibition.

Rad51 is an essential factor of the homologous recombination DNA repair pathway and therefore plays an important role in maintaining genomic stability. We show that RAD51 and other homologous recombination repair genes are overexpressed in metastatic melanoma cell lines and in melanoma patient samples, which correlates with reduced survival of melanoma patients. In addition, Rad51 expression in melanoma cells was regulated on a transcriptional level by the MAPK signaling pathway with Elk1 as the main downstream transcriptional effector. Most strikingly, melanoma cells which developed resistance towards MAPK inhibitors could be efficiently targeted by Rad51 inhibitors similar to their sensitive counterparts, leading to DNA damage, G2/M arrest and apoptosis. Furthermore, the treatment of MAPK inhibitor resistant cells with Rad51 inhibitors enhances the susceptibility of these cells for MAPK inhibitor treatment in vitro and in vivo. These data indicate that Rad51 plays a critical role in the survival of metastatic melanoma cells and is a promising target for the therapy of melanoma irrespective of its MAPK inhibitor resistance status.

Melanoma cells resistant towards MAPK inhibitors exhibit reduced TAp73 expression mediating enhanced sensitivity to platinum-based drugs.

The efficacy of targeted MAPK signalling pathway inhibitors (MAPKi) in metastatic melanoma therapy is limited by the development of resistance mechanisms that results in disease relapse. This situation still requires treatment alternatives for melanoma patients with acquired resistance to targeted therapy. We found that melanoma cells, which developed resistance towards MAPKi show an enhanced susceptibility to platinum-based drugs, such as cisplatin and carboplatin. We found that this enhanced susceptibility inversely correlates with the expression level of the p53 family member TAp73. We show that the lower expression of the TAp73 isoform in MAPKi-resistant melanoma cells enhances accumulation of DNA double-strand breaks upon cisplatin and carboplatin treatment by reducing the efficiency of nucleotide excision repair. These data suggest that a subgroup of melanoma patients with acquired resistance to MAPKi treatment and low TAp73 expression can benefit from chemotherapy with platinum-based drugs as a second-line therapy.

Human melanoma cells resistant to MAPK inhibitors can be effectively targeted by inhibition of the p90 ribosomal S6 kinase.

The clinical availability of small molecule inhibitors specifically targeting mutated BRAF marked a significant breakthrough in melanoma therapy. Despite a dramatic anti-tumour activity and improved patient survival, rapidly emerging resistance, however, greatly limits the clinical benefit. The majority of the already described resistance mechanisms involve a reactivation of the MAPK signalling pathway. The p90 ribosomal S6 kinase (RSK), a downstream effector of the MAPK signalling cascade, has been reported to enhance survival of melanoma cells in response to chemotherapy. Here, we can show that RSK activity is significantly increased in human melanoma cells with acquired resistance to the BRAFV600E/K inhibitor vemurafenib. Interestingly, inhibition of RSK signalling markedly impairs the viability of vemurafenib resistant melanoma cells and is effective both in two-dimensional and in three-dimensional culture systems, especially in a chronic, long-term application. The effect of RSK inhibition can be partly replicated by downregulation of the well-known RSK target, Y-box binding protein 1 (YB-1). Intriguingly, RSK inhibition also retains its efficacy in melanoma cells with combined resistance to vemurafenib and the MEK inhibitor trametinib. These data suggest that active RSK signalling might be an attractive novel therapeutic target in melanoma with acquired resistance to MAPK pathway inhibitors.

A Nexus Consisting of Beta-Catenin and Stat3 Attenuates BRAF Inhibitor Efficacy and Mediates Acquired Resistance to Vemurafenib.

Acquired resistance to second generation BRAF inhibitors (BRAFis), like vemurafenib is limiting the benefits of long term targeted therapy for patients with malignant melanomas that harbor BRAF V600 mutations. Since many resistance mechanisms have been described, most of them causing a hyperactivation of the MAPK- or PI3K/AKT signaling pathways, one potential strategy to overcome BRAFi resistance in melanoma cells would be to target important common signaling nodes. Known factors that cause secondary resistance include the overexpression of receptor tyrosine kinases (RTKs), alternative splicing of BRAF or the occurrence of novel mutations in MEK1 or NRAS. In this study we show that ß-catenin is stabilized and translocated to the nucleus in approximately half of the melanomas that were analyzed and which developed secondary resistance towards BRAFi. We further demonstrate that ß-catenin is involved in the mediation of resistance towards vemurafenib in vitro and in vivo. Unexpectedly, ß-catenin acts mainly independent of the TCF/LEF dependent canonical Wnt-signaling pathway in resistance development, which partly explains previous contradictory results about the role of ß-catenin in melanoma progression and therapy resistance. We further demonstrate that ß-catenin interacts with Stat3 after chronic vemurafenib treatment and both together cooperate in the acquisition and maintenance of resistance towards BRAFi.