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Vemurafenib resistance reprograms melanoma cells towards glutamine dependence.

Hernandez-Davies JE, Tran TQ, Reid MA, Rosales KR, Lowman XH, Pan M, Moriceau G, Yang Y, Wu J, Lo RS, Kong M - J Transl Med (2015)

Bottom Line: We demonstrated that MAPKi-acquired resistant cells uptook greater amounts of glutamine and have increased sensitivity to glutamine deprivation than their MAPKi-sensitive counterparts.In addition, it was found that both BPTES and L-DON were more effective at decreasing cell survival of MAPKi-resistant sub-lines than parental cell populations in vitro.When tested in vivo, we found that xenografts derived from resistant cells were more sensitive to BPTES or L-DON treatment than those derived from parental cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Beckman Research Institute of City of Hope Cancer Center, Duarte, CA, 91010, USA. jedavies@coh.org.

ABSTRACT

Background: (V600) BRAF mutations drive approximately 50% of metastatic melanoma which can be therapeutically targeted by BRAF inhibitors (BRAFi) and, based on resistance mechanisms, the combination of BRAF and MEK inhibitors (BRAFi + MEKi). Although the combination therapy has been shown to provide superior clinical benefits, acquired resistance is still prevalent and limits the overall survival benefits. Recent work has shown that oncogenic changes can lead to alterations in tumor cell metabolism rendering cells addicted to nutrients, such as the amino acid glutamine. Here, we evaluated whether melanoma cells with acquired resistance display glutamine dependence and whether glutamine metabolism can be a potential molecular target to treat resistant cells.

Methods: Isogenic BRAFi sensitive parental (V600) BRAF mutant melanoma cell lines and resistant (derived by chronic treatment with vemurafenib) sub-lines were used to assess differences in the glutamine uptake and sensitivity to glutamine deprivation. To evaluate a broader range of resistance mechanisms, isogenic pairs where the sub-lines were resistant to BRAFi + MEKi were also studied. Since resistant cells demonstrated increased sensitivity to glutamine deficiency, we used glutaminase inhibitors BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide] and L-L-DON (6-Diazo-5-oxo-L-norleucine) to treat MAPK pathway inhibitor (MAPKi) resistant cell populations both in vitro and in vivo.

Results: We demonstrated that MAPKi-acquired resistant cells uptook greater amounts of glutamine and have increased sensitivity to glutamine deprivation than their MAPKi-sensitive counterparts. In addition, it was found that both BPTES and L-DON were more effective at decreasing cell survival of MAPKi-resistant sub-lines than parental cell populations in vitro. We also showed that mutant NRAS was critical for glutamine addiction in mutant NRAS driven resistance. When tested in vivo, we found that xenografts derived from resistant cells were more sensitive to BPTES or L-DON treatment than those derived from parental cells.

Conclusion: Our study is a proof-of-concept for the potential of targeting glutamine metabolism as an alternative strategy to suppress acquired MAPKi-resistance in melanoma.

No MeSH data available.


Related in: MedlinePlus

Vemurafenib resistant cells uptake and use glutamine at a higher rate than vemurafenib sensitive cells. Melanoma M249 and M229 vemurafenib sensitive (parental) and single drug resistant (SDR) cell lines were plated to be 60% confluent and medium was changed after overnight incubation. Medium only was used as a control. Cells were then cultured for 24 h. Medium was then taken from growing cells and measurements were obtained of glutamine uptake, NH4+ production, glucose uptake, and lactate production using the Nova Bioprofiler 100 plus as described in materials and methods. Cells were counted and results are expressed as a glutamine uptake per cell (mmol/L) and NH4+ production per cell (mmol/L) and b glucose uptake per cell (mmol/L) and lactate production per cell (mmol/L) and are representative of the average (±standard deviation) of triplicate experiments.
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Fig1: Vemurafenib resistant cells uptake and use glutamine at a higher rate than vemurafenib sensitive cells. Melanoma M249 and M229 vemurafenib sensitive (parental) and single drug resistant (SDR) cell lines were plated to be 60% confluent and medium was changed after overnight incubation. Medium only was used as a control. Cells were then cultured for 24 h. Medium was then taken from growing cells and measurements were obtained of glutamine uptake, NH4+ production, glucose uptake, and lactate production using the Nova Bioprofiler 100 plus as described in materials and methods. Cells were counted and results are expressed as a glutamine uptake per cell (mmol/L) and NH4+ production per cell (mmol/L) and b glucose uptake per cell (mmol/L) and lactate production per cell (mmol/L) and are representative of the average (±standard deviation) of triplicate experiments.

Mentions: We first aimed to assess whether there were any differences in nutrient uptake between isogenic BRAFi sensitive parental V600BRAF mutant melanoma cell lines and single drug resistant (derived by chronic treatment with vemurafenib) sub-lines. We found that both M249 and M229 single drug resistant (SDR) cells had greater uptake of glutamine than their parental counterparts (Figure 1a). It is also known that glutaminase activity generates free ammonia, therefore, we also tested for NH4+ production [15]. We found that both M249 and M229 single drug resistant cells had greater amounts of ammonia production than parental cell populations, which is indicative of higher glutamine usage (Figure 1a). To exclude the possibility that the resistant cells display a general increase in metabolism, we also measured glucose uptake and lactate production in M249 and M229 parental and single drug resistant cells (Figure 1b). In contrast to the glutamine uptake, there were no significant differences in glucose uptake and lactate production between parental and single drug resistant cells (Figure 1b). These data suggest that single drug resistant cells may be more dependent on glutamine for growth and proliferation than vemurafenib-sensitive (parental) cell populations.Figure 1


Vemurafenib resistance reprograms melanoma cells towards glutamine dependence.

Hernandez-Davies JE, Tran TQ, Reid MA, Rosales KR, Lowman XH, Pan M, Moriceau G, Yang Y, Wu J, Lo RS, Kong M - J Transl Med (2015)

Vemurafenib resistant cells uptake and use glutamine at a higher rate than vemurafenib sensitive cells. Melanoma M249 and M229 vemurafenib sensitive (parental) and single drug resistant (SDR) cell lines were plated to be 60% confluent and medium was changed after overnight incubation. Medium only was used as a control. Cells were then cultured for 24 h. Medium was then taken from growing cells and measurements were obtained of glutamine uptake, NH4+ production, glucose uptake, and lactate production using the Nova Bioprofiler 100 plus as described in materials and methods. Cells were counted and results are expressed as a glutamine uptake per cell (mmol/L) and NH4+ production per cell (mmol/L) and b glucose uptake per cell (mmol/L) and lactate production per cell (mmol/L) and are representative of the average (±standard deviation) of triplicate experiments.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4490757&req=5

Fig1: Vemurafenib resistant cells uptake and use glutamine at a higher rate than vemurafenib sensitive cells. Melanoma M249 and M229 vemurafenib sensitive (parental) and single drug resistant (SDR) cell lines were plated to be 60% confluent and medium was changed after overnight incubation. Medium only was used as a control. Cells were then cultured for 24 h. Medium was then taken from growing cells and measurements were obtained of glutamine uptake, NH4+ production, glucose uptake, and lactate production using the Nova Bioprofiler 100 plus as described in materials and methods. Cells were counted and results are expressed as a glutamine uptake per cell (mmol/L) and NH4+ production per cell (mmol/L) and b glucose uptake per cell (mmol/L) and lactate production per cell (mmol/L) and are representative of the average (±standard deviation) of triplicate experiments.
Mentions: We first aimed to assess whether there were any differences in nutrient uptake between isogenic BRAFi sensitive parental V600BRAF mutant melanoma cell lines and single drug resistant (derived by chronic treatment with vemurafenib) sub-lines. We found that both M249 and M229 single drug resistant (SDR) cells had greater uptake of glutamine than their parental counterparts (Figure 1a). It is also known that glutaminase activity generates free ammonia, therefore, we also tested for NH4+ production [15]. We found that both M249 and M229 single drug resistant cells had greater amounts of ammonia production than parental cell populations, which is indicative of higher glutamine usage (Figure 1a). To exclude the possibility that the resistant cells display a general increase in metabolism, we also measured glucose uptake and lactate production in M249 and M229 parental and single drug resistant cells (Figure 1b). In contrast to the glutamine uptake, there were no significant differences in glucose uptake and lactate production between parental and single drug resistant cells (Figure 1b). These data suggest that single drug resistant cells may be more dependent on glutamine for growth and proliferation than vemurafenib-sensitive (parental) cell populations.Figure 1

Bottom Line: We demonstrated that MAPKi-acquired resistant cells uptook greater amounts of glutamine and have increased sensitivity to glutamine deprivation than their MAPKi-sensitive counterparts.In addition, it was found that both BPTES and L-DON were more effective at decreasing cell survival of MAPKi-resistant sub-lines than parental cell populations in vitro.When tested in vivo, we found that xenografts derived from resistant cells were more sensitive to BPTES or L-DON treatment than those derived from parental cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Beckman Research Institute of City of Hope Cancer Center, Duarte, CA, 91010, USA. jedavies@coh.org.

ABSTRACT

Background: (V600) BRAF mutations drive approximately 50% of metastatic melanoma which can be therapeutically targeted by BRAF inhibitors (BRAFi) and, based on resistance mechanisms, the combination of BRAF and MEK inhibitors (BRAFi + MEKi). Although the combination therapy has been shown to provide superior clinical benefits, acquired resistance is still prevalent and limits the overall survival benefits. Recent work has shown that oncogenic changes can lead to alterations in tumor cell metabolism rendering cells addicted to nutrients, such as the amino acid glutamine. Here, we evaluated whether melanoma cells with acquired resistance display glutamine dependence and whether glutamine metabolism can be a potential molecular target to treat resistant cells.

Methods: Isogenic BRAFi sensitive parental (V600) BRAF mutant melanoma cell lines and resistant (derived by chronic treatment with vemurafenib) sub-lines were used to assess differences in the glutamine uptake and sensitivity to glutamine deprivation. To evaluate a broader range of resistance mechanisms, isogenic pairs where the sub-lines were resistant to BRAFi + MEKi were also studied. Since resistant cells demonstrated increased sensitivity to glutamine deficiency, we used glutaminase inhibitors BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide] and L-L-DON (6-Diazo-5-oxo-L-norleucine) to treat MAPK pathway inhibitor (MAPKi) resistant cell populations both in vitro and in vivo.

Results: We demonstrated that MAPKi-acquired resistant cells uptook greater amounts of glutamine and have increased sensitivity to glutamine deprivation than their MAPKi-sensitive counterparts. In addition, it was found that both BPTES and L-DON were more effective at decreasing cell survival of MAPKi-resistant sub-lines than parental cell populations in vitro. We also showed that mutant NRAS was critical for glutamine addiction in mutant NRAS driven resistance. When tested in vivo, we found that xenografts derived from resistant cells were more sensitive to BPTES or L-DON treatment than those derived from parental cells.

Conclusion: Our study is a proof-of-concept for the potential of targeting glutamine metabolism as an alternative strategy to suppress acquired MAPKi-resistance in melanoma.

No MeSH data available.


Related in: MedlinePlus