NRAS mutant melanoma: Towards better therapies

in Cancer Treatment Reviews (2021), 99

Genetic alterations affecting RAS proteins are commonly found in human cancers. Roughly a fourth of melanoma patients carry activating NRAS mutations, rendering this malignancy particularly challenging to ... [more ▼]

Genetic alterations affecting RAS proteins are commonly found in human cancers. Roughly a fourth of melanoma patients carry activating NRAS mutations, rendering this malignancy particularly challenging to treat. Although the development of targeted as well as immunotherapies led to a substantial improvement in the overall survival of non-NRASmut melanoma patients (e.g. BRAFmut), patients with NRASmut melanomas have an overall poorer prognosis due to the high aggressiveness of RASmut tumors, lack of efficient targeted therapies or rapidly emerging resistance to existing treatments. Understanding how NRAS-driven melanomas develop therapy resistance by maintaining cell cycle progression and survival is crucial to develop more effective and specific treatments for this group of melanoma patients. In this review, we provide an updated summary of currently available therapeutic options for NRASmut melanoma patients with a focus on combined inhibition of MAPK signaling and CDK4/6-driven cell cycle progression and mechanisms of the inevitably developing resistance to these treatments. We conclude with an outlook on the most promising novel therapeutic approaches for melanoma patients with constitutively active NRAS. [less ▲]

From drug resistance mechanisms to microRNA function in melanoma

Doctoral thesis (2020)

Cutaneous melanoma is an aggressive skin cancer that emerges from the unrestrained proliferation of melanocytes, which are the pigment producing cells in the basal layer of the epidermis. Despite the fact ... [more ▼]

Cutaneous melanoma is an aggressive skin cancer that emerges from the unrestrained proliferation of melanocytes, which are the pigment producing cells in the basal layer of the epidermis. Despite the fact that it only accounts for approximately 5% of all skin cancers, melanoma is responsible for the vast majority of skin cancer-related deaths. As more than half of the patients with sporadic melanoma harbour activating mutations in the protein kinase BRAF, the development of small kinase inhibitors targeting mutated BRAF led to an increased overall survival of patients with metastatic melanoma. Despite the initially promising results, the rapidly emerging resistance to these targeted therapies remains a serious clinical issue. To investigate the mechanisms underlying resistance to targeted therapies, we used in vitro BRAF-mutant drug-sensitive and drug-resistant melanoma cell models that were generated in our laboratory. First, we performed a kinase inhibitor library screening with the aim to identify novel kinase inhibitor combinations to circumvent or delay BRAF inhibitor-induced resistance. We have characterised synergistic kinase inhibitors targeting the MAPK pathway and the cell cycle showing promising effects in BRAF-mutant drug-sensitive and -resistant cells, which could be used as an effective sequential or alternative treatment option for late-stage melanoma patients. Additionally, we investigated the impact of BRAF inhibitors at the transcriptional level by comparing miRNome and transcriptome changes in drug-sensitive and -resistant melanoma cells. We identified miRNAs (e.g. miR-509, miR-708) and genes (e.g. PCSK2, AXL) that were distinctly differentially expressed in resistant compared to sensitive cells. Subsequent co-expression analyses revealed a low MITF/AXL ratio in a subset of resistant cell lines, suggesting that miRNAs might be involved in the switch from one molecular phenotype to another, thus conferring tolerance to targeted therapies. Finally, we applied a method based on cross-linking ligation and sequencing of hybrids (qCLASH) to provide a comprehensive snapshot of the miRNA targetome in our BRAF-mutant melanoma cells. To our knowledge, we implemented for the first time a CLASH-based method to cancer cells, and identified over 8k direct and distinct miRNA-target interactions in melanoma cells, including many with non-predicted and non-canonical binding characteristics, thus expanding the pool of miRNA-target interactions. Taken together, these results provide new insights into complex and heterogeneous responses to BRAF inhibition, adding an additional level of complexity to drug-induced (post-) transcriptional network rewiring in melanoma. [less ▲]

Kinase inhibitor library screening identifies synergistic drug combinations effective in sensitive and resistant melanoma cells

in Journal of Experimental and Clinical Cancer Research (2019), 38(1),

Background: Melanoma is the most aggressive and deadly form of skin cancer with increasing case numbers worldwide. The development of inhibitors targeting mutated BRAF (found in around 60% of melanoma ... [more ▼]

Background: Melanoma is the most aggressive and deadly form of skin cancer with increasing case numbers worldwide. The development of inhibitors targeting mutated BRAF (found in around 60% of melanoma patients) has markedly improved overall survival of patients with late-stage tumors, even more so when combined with MEK inhibitors targeting the same signaling pathway. However, invariably patients become resistant to this targeted therapy resulting in rapid progression with treatment-refractory disease. The purpose of this study was the identification of new kinase inhibitors that do not lead to the development of resistance in combination with BRAF inhibitors (BRAFi), or that could be of clinical benefit as a 2nd line treatment for late-stage melanoma patients that have already developed resistance. Methods: We have screened a 274-compound kinase inhibitor library in 3 BRAF mutant melanoma cell lines (each one sensitive or made resistant to 2 distinct BRAFi). The screening results were validated by dose-response studies and confirmed the killing efficacies of many kinase inhibitors. Two different tools were applied to investigate and quantify potential synergistic effects of drug combinations: the Chou-Talalay method and the Synergyfinder application. In order to exclude that resistance to the new treatments might occur at later time points, synergistic combinations were administered to fluorescently labelled parental and resistant cells over a period of > 10 weeks. Results: Eight inhibitors targeting Wee1, Checkpoint kinase 1/2, Aurora kinase, MEK, Polo-like kinase, PI3K and Focal adhesion kinase killed melanoma cells synergistically when combined with a BRAFi. Additionally, combination of a Wee1 and Chk inhibitor showed synergistic killing effects not only on sensitive cell lines, but also on intrinsically BRAFi- and treatment induced-resistant melanoma cells. First in vivo studies confirmed these observations. Interestingly, continuous treatment with several of these drugs, alone or in combination, did not lead to emergence of resistance. Conclusions: Here, we have identified new, previously unexplored (in the framework of BRAFi resistance) inhibitors that have an effect not only on sensitive but also on BRAFi-resistant cells. These promising combinations together with the new immunotherapies could be an important step towards improved 1st and 2nd line treatments for late-stage melanoma patients. [less ▲]

Impact of BRAF kinase inhibitors on the miRNomes and transcriptomes of melanoma cells

in Biochimica et Biophysica Acta (2017)

Phosphorylation of the PDH complex precedes HIF-1-mediated effects and PDK1 upregulation during the first hours of hypoxic treatment in HCC cells

in Hypoxia (2016), 4