Reprofiling using a zebrafish melanoma model reveals drugs cooperating with targeted therapeutics

Abstract

Phenotype-guided re-profiling of approved drug molecules presents an accelerated route to developing anticancer therapeutics by bypassing the target-identification bottleneck of target-based approaches and by sampling drugs already in the clinic. Further, combinations incorporating targeted therapies can be screened for both efficacy and toxicity. Previously we have developed an oncogenic-RAS-driven zebrafish melanoma model that we now describe display melanocyte hyperplasia while still embryos. Having devised a rapid method for quantifying melanocyte burden, we show that this phenotype can be chemically suppressed by incubating V12RAS transgenic embryos with potent and selective small molecule inhibitors of either MEK or PI3K/mTOR. Moreover, we demonstrate that combining MEK inhibitors (MEKi) with dual PI3K/mTOR inhibitors (PI3K/mTORi) resulted in a super-additive suppression of melanocyte hyperplasia. The robustness and simplicity of our novel screening assay inspired us to perform a modest screen of FDA approved compounds for their ability to potentiate MEKi PD184352 or PI3K/mTORi NVPBEZ235 suppression of V12RAS-driven melanocyte hyperplasia. Through this route, we confirmed Rapamycin as a compound that could synergize with MEKi and even more so with PI3K/mTORi to suppress melanoma development, including suppressing the growth of cultured human melanoma cells. Further, we discovered two additional compounds—Disulfiram and Tanshinone—that also co-operate with MEKi to suppress the growth of transformed zebrafish melanocytes and showed activity toward cultured human melanoma cells. In conclusion, we provide proof-of-concept that our phenotype-guided screen could be used to identify compounds that affect melanoma development and prompt further evaluation of Disulfiram and Tanshinone as possible partners for combination therapy.

Zebrafish as a Model Organism for the Development of Drugs for Skin Cancer

“…The compounds, drugs or small molecules, can be added directly to the water environment of the zebrafish [ 3 ]. Moreover, given the high genetic and physiological similarities with humans, zebrafish can be used as a useful and cost-effective vehicle for high-throughput screening (HTS) [ 2 , 4 , 5 , 6 ].…”

“…On the contrary, in the next stages of tumor progression, melanoma cells invade the subcutaneous tissues (vertical growth phase (VGP) melanoma) and eventually progress toward the metastatic phase. At this stage, few therapeutic options are available, and melanoma frequently relapses and becomes incurable [ 6 , 24 ].…”

“…Hence, using the phenotype-guided drug discovery, it is possible to identify an effective compound by looking at phenotype alteration in the whole organism, independently of the specific target. Therefore, this approach improves the development of new drugs and, at the same time, allows the identification of the pathways that are implicated in the development or progression of the specific disease [ 6 ]. Two examples of the use of zebrafish for phenotype-based drug discovery are given by the screening of chemical molecules as suppressors of two specific genetic mutations: (1) gridlock and (2) Crumbs homolog ( crb ) mutations [ 45 ].…”

Zebrafish (Danio rerio) as a Model Organism

“…In later stages of tumor progression, the melanoma cells invade the subcutaneous tissues due to vertical growth phase (VGP) of melanoma and eventually lead toward the metastatic phase. At this stage, very limited therapeutic options are available, and melanoma frequently deteriorates and becomes untreatable [73,74].…”

Selective Inhibition of Kras Signaling by Combination of Low Dose Rapamycin and Paclitaxel in Vivo

“…Похожие результаты получены и на трансгенной мо-дели меланомы зебрафиш tg(mitfa:V12HRAS;mitfa:GFP), развитие которой обусловлено гиперэкспрессией му-тантной формы гена HRAS в меланоцитах. В этом слу-чае ингибирование каскадов MEK/ERK и PI3K/AKT приводило к торможению гиперплазии меланоцитов и снижению скорости роста опухолей [17].…”