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Identification of drug-resistance genes for the development of effective combined therapies for melanoma

Final Report Summary - MELANOMA DRUG RESIST (Identification of drug-resistance genes for the development of effective combined therapies for melanoma)

Malignant melanoma causes 9000 deaths each year in the USA alone, more than one every hour. It represents the common tumor whose incidence has increased the most in the last 30 years, thus resulting in a lifetime risk of developing malignant melanoma of 1/55 in men and 1/56 for women in 2010 in UK, and an estimated incidence of 200,000 new cases each year worldwide. Melanomas are tumours that arise from the pigmented cells of the skin: the melanocytes. Due to their physiological functions, melanocytes have inborn features that make them really aggressive once they transform into a tumour. During an embryo’s development they originate from the neural crest, which later becomes the spinal cord, and migrate throughout the body to reach their natural location. This intrinsic migratory activity is reactivated in melanoma tumors, thus making them highly likely to spread into metastases and, consequently, to be lethal. Additionally, as melanocytes are born to shield us from the mutagenic UV radiation that hits us from the sun every day, they can easily bear the accumulation of mutations, DNA lesions that drive the tumors themselves and increase their aggressiveness. These features make malignant melanoma a very aggressive disease and the first cause of death for skin cancer.
Indeed, when melanoma is diagnosed early, surgical removal is generally curative. Metastatic melanoma, however, has a poor prognosis and is refractory to chemotherapy, with survival from stage IV melanoma ranging from 8 to 18 months after diagnosis. While recent progress has been made with therapies that potentiate the immune response in melanoma patients, these modalities are still curative only in a fraction of the patients. Therefore, the development of novel and more effective therapeutic regimens is an unmet need.

Aims and experimental outline
The aim of this project was to identify new therapeutic approaches for the treatment of melanoma. In details, the 2 specific aims were: 1): To identify genes that confer
resistance/sensitivity to clinically relevant targeted therapies for melanoma. 2): To exploit the newly
developed knowledge on drug-resistance genes to design novel and more effective personalized
combination therapies. We decided to focus on a subtype of the disease, defined as BRAF/NRAS wild type melanomas, that represent about 30% of human tumors, since for those patients no targeted therapies are currently available. In order to achieve these aims, we assembled a collection of 21 BRAF/NRAS wild type melanoma cell lines and deeply characterized them for mutations, copy number variations, gene and microRNA expression (outlined in Figure 1).
We then designed a high-throughput drug screening to measure the sensitivity of our cell line collection to a combination of 180 drugs that are clinically relevant. In details, we screened 60 library drugs (tested at different concentrations to generate the survival curve) that cover the inhibition of the pathways most frequently deregulated in cancer, and we combined them with 3 anchor drugs (used at 2 different concentration) that are in clinical use. We choose to screen drug combinations instead of single drugs in order to maximize the chance of identifying a treatment that is highly effective and less prone to the occurrence of resistance, a phenomenon very frequent with single targeted drugs that limit the clinical benefits in patients. The rationale of the screening was that the identification of drug combinations that are synergistic and strongly active in multiple cell lines represents an approach to prioritize those combinations for further preclinical development and eventually clinical translation.

The characterization of the genomic landscape of our collection of BRAF/NRAS wild type melanomas allowed us to compare them with BRAF/NRAS wild type human tumors (from TCGA repository) and showed that our model is representative of the lesion occurring in human tumors (see Figure 1). By the high-throughput drug screening we determined the pattern of drug sensitivity and drug combination synergy across the whole collection of cell lines (see Figure 2). We measured the sensitivity to each drug or drug combination as Area Under the Curve, where on the Y axis we represent the viability of each cell-line compared to vehicle treated controls, and on the X axis the concentration of the library drug that we used. Then we used the value of the delta AUC (calculated as depicted in Figure 2B) to measure the synergy of the drug combination. As expected, the combination of the library drug with the different anchor drug achieved a stronger killing effect in than the library drug alone (Figure 1A). We then analysed ~7000 survival curved and observed that the synergy (delta AUC>2, Figure 2C) is a very rare event and occurs in about 1% of cases. Additionally, most of the drug combination resulted to be synergistic in only 1 or 2 cell lines (Figure 2D). Therefore we prioritize those drug combinations recurrent in >2 cell lines for further investigation and attempted a systematic validation by 2 independent viability assays. By this approach, we identified a top synergistic drug combination that was confirmed to be effective in 23% of the cell lines by the 2 independent assays (Figure 3A-B).
We the attempted to screen for synergy to the drug combination in 3 additional cell line collections. Overall, we confirmed the drugs synergy firstly using an independent collection of BRAF/NRAS wild type melanoma cell lines (n=7), then a collection of BRAF/NRAS wild type patient derived xenotransplant cultures (n=3), and finally a collection of BRAFV600E melanoma cell lines (n=9) (Figure 3D). These results robustly confirmed the synergy between the 2 drugs in a relevant fraction of melanoma cell lines from 4 different cell line collections.
Finally, we attempted to confirm the synergy of our drug combination in vivo. We utilized a patient derived xenotransplant mouse model and treated 4 cohorts of 5 mice each (each mouse with 2 tumors) with vechicle, drug1, drug2 and drug1 plus drug 2 combination. The drug combination resulted to be well tolerated in vivo. Remarkably, we observed a significant shrinkage of the tumors treated with the drug combination, while the single drug alone just delayed tumor growth. These results confirm in vivo the synergy between the 2 drugs. (Figure 4).

Conclusion and impact
We have assembled and deeply characterized for genomic lesion, gene and microRNA expression, sensitivity to single drug and drug combination a collection of 21 BRAF/NRAS wild type melanoma cell lines that is representative of the human tumor counterpart. Our collection represents the deepest characterized collection of cell lines for this subtype of melanoma. These data represents a useful resource of hypothesis testing that will be made publicly available upon publication of our manuscript.
By our screening we identified a synergistic drug combination that is effective in killing a significant fraction of BRAF/NRAS wild type melanomas. We are completing our study to try to identify the mechanism of drug synergy and markers of drug synergy. Nonetheless, our robust validation by 2 assays in 4 independent collections of cell lines and the confirmation of the synergy in vivo indicate that the newly identified drug combination has a strong clinical translation potential. Overall, we have identified a new drug combination that can have a significant impact on the treatment of patients affected by BRAF/NRAS wild type melanomas.

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