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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 melanoma tumors are sensitive to glutaminase inhibitor treatment in vivo. a Nod Scid Gamma (NSG) mice were injected with 5 × 106 of M249 parental or single drug resistant cells subcutaneously on the right flank. When tumor size reached an average of 100 mm3 tumor cell volume, treatment with 15 mg/kg of BPTES or vehicle control (DMSO) was provided every other day through intraperitoneal injection. Measurements were taken for tumor length and width. Tumor volume (mm3) was calculated by multiplying (length × width × width)/2. Graph represents mean tumor volume ± SD. b Nude mice were injected with 2 × 106 of M249 resistant cells subcutaneously on right flank. When tumor size reached an average of 100 mm3 tumor cell volume, treatment with 20 mg/kg of L-DON or vehicle control (water) was provided twice a week through intraperitoneal injection. Graph represents mean tumor volume ± SD.
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Fig5: Vemurafenib resistant melanoma tumors are sensitive to glutaminase inhibitor treatment in vivo. a Nod Scid Gamma (NSG) mice were injected with 5 × 106 of M249 parental or single drug resistant cells subcutaneously on the right flank. When tumor size reached an average of 100 mm3 tumor cell volume, treatment with 15 mg/kg of BPTES or vehicle control (DMSO) was provided every other day through intraperitoneal injection. Measurements were taken for tumor length and width. Tumor volume (mm3) was calculated by multiplying (length × width × width)/2. Graph represents mean tumor volume ± SD. b Nude mice were injected with 2 × 106 of M249 resistant cells subcutaneously on right flank. When tumor size reached an average of 100 mm3 tumor cell volume, treatment with 20 mg/kg of L-DON or vehicle control (water) was provided twice a week through intraperitoneal injection. Graph represents mean tumor volume ± SD.

Mentions: Since in vitro data demonstrated the potential of glutaminase inhibitors to treat resistant cell lines, we asked whether these inhibitors would also be effective in vivo. To do this, xenograft experiments with NSG mice were used to inject mice with both M249 parental (vemurafenib sensitive) and M249 single drug resistant cell lines and subsequently treat with glutaminase inhibitor BPTES. As shown in Figure 5a, BPTES dramatically suppressed tumor growth in mice injected with M249 single drug resistant cells compared to mice injected with the parental (sensitive) cells. As the BPTES treatment displayed minimal effect on tumor growth from parental cells, we next focused to validate whether targeting glutamine metabolism is efficient to inhibit resistant tumor growth using a different glutaminase inhibitor, L-DON. Similarly, we found that L-DON effectively blocked vemurafenib resistant tumor growth (Figure 5b).Figure 5


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 melanoma tumors are sensitive to glutaminase inhibitor treatment in vivo. a Nod Scid Gamma (NSG) mice were injected with 5 × 106 of M249 parental or single drug resistant cells subcutaneously on the right flank. When tumor size reached an average of 100 mm3 tumor cell volume, treatment with 15 mg/kg of BPTES or vehicle control (DMSO) was provided every other day through intraperitoneal injection. Measurements were taken for tumor length and width. Tumor volume (mm3) was calculated by multiplying (length × width × width)/2. Graph represents mean tumor volume ± SD. b Nude mice were injected with 2 × 106 of M249 resistant cells subcutaneously on right flank. When tumor size reached an average of 100 mm3 tumor cell volume, treatment with 20 mg/kg of L-DON or vehicle control (water) was provided twice a week through intraperitoneal injection. Graph represents mean tumor volume ± SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4490757&req=5

Fig5: Vemurafenib resistant melanoma tumors are sensitive to glutaminase inhibitor treatment in vivo. a Nod Scid Gamma (NSG) mice were injected with 5 × 106 of M249 parental or single drug resistant cells subcutaneously on the right flank. When tumor size reached an average of 100 mm3 tumor cell volume, treatment with 15 mg/kg of BPTES or vehicle control (DMSO) was provided every other day through intraperitoneal injection. Measurements were taken for tumor length and width. Tumor volume (mm3) was calculated by multiplying (length × width × width)/2. Graph represents mean tumor volume ± SD. b Nude mice were injected with 2 × 106 of M249 resistant cells subcutaneously on right flank. When tumor size reached an average of 100 mm3 tumor cell volume, treatment with 20 mg/kg of L-DON or vehicle control (water) was provided twice a week through intraperitoneal injection. Graph represents mean tumor volume ± SD.
Mentions: Since in vitro data demonstrated the potential of glutaminase inhibitors to treat resistant cell lines, we asked whether these inhibitors would also be effective in vivo. To do this, xenograft experiments with NSG mice were used to inject mice with both M249 parental (vemurafenib sensitive) and M249 single drug resistant cell lines and subsequently treat with glutaminase inhibitor BPTES. As shown in Figure 5a, BPTES dramatically suppressed tumor growth in mice injected with M249 single drug resistant cells compared to mice injected with the parental (sensitive) cells. As the BPTES treatment displayed minimal effect on tumor growth from parental cells, we next focused to validate whether targeting glutamine metabolism is efficient to inhibit resistant tumor growth using a different glutaminase inhibitor, L-DON. Similarly, we found that L-DON effectively blocked vemurafenib resistant tumor growth (Figure 5b).Figure 5

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