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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 are more sensitive to glutamine deprivation. a Melanoma M249 and b M229 single drug resistant (SDR) cells were cultured in the presence of medium with or without glutamine (Gln) or glucose (Gluc). After 24 h representative images and surviving fractions (survival %) compared to control cells in complete medium are shown using DIMSCAN, a microcomputer fluorescence-based cytotoxicity assay (n = 6, **p < 0.01). Western blot analysis was also used to assess levels of cleaved PARP as an indicator of apoptosis in both c M249 and d M229 parental and SDR cell lines. e Apoptosis was also assessed using flow cytometry of M249 parental and resistance cells stained with AnnexinV and DAPI. M249 parental and SDR cells were cultured in the presence of medium with and without glutamine for 24 h. Representative dot blots and percentages of AnnexinV positive cells are shown. f Melanoma double drug resistant (DDR) cell line M249 DDR5 was cultured in the presence of medium with and without glutamine (Gln) or glucose (Gluc). After 24 h representative images and surviving fractions (survival %) compared to control cells in complete medium are shown using DIMSCAN, a microcomputer fluorescence-based cytotoxicity assay (n = 6, ***p < 0.001). g Apoptosis was also assessed using flow cytometry of M249 parental and DDR cells stained with AnnexinV and DAPI. M249 parental and DDR cells were cultured in the presence of medium with and without glutamine for 24 h. Representative dot blots and percentages of AnnexinV positive cells are shown.
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Fig2: Vemurafenib resistant cells are more sensitive to glutamine deprivation. a Melanoma M249 and b M229 single drug resistant (SDR) cells were cultured in the presence of medium with or without glutamine (Gln) or glucose (Gluc). After 24 h representative images and surviving fractions (survival %) compared to control cells in complete medium are shown using DIMSCAN, a microcomputer fluorescence-based cytotoxicity assay (n = 6, **p < 0.01). Western blot analysis was also used to assess levels of cleaved PARP as an indicator of apoptosis in both c M249 and d M229 parental and SDR cell lines. e Apoptosis was also assessed using flow cytometry of M249 parental and resistance cells stained with AnnexinV and DAPI. M249 parental and SDR cells were cultured in the presence of medium with and without glutamine for 24 h. Representative dot blots and percentages of AnnexinV positive cells are shown. f Melanoma double drug resistant (DDR) cell line M249 DDR5 was cultured in the presence of medium with and without glutamine (Gln) or glucose (Gluc). After 24 h representative images and surviving fractions (survival %) compared to control cells in complete medium are shown using DIMSCAN, a microcomputer fluorescence-based cytotoxicity assay (n = 6, ***p < 0.001). g Apoptosis was also assessed using flow cytometry of M249 parental and DDR cells stained with AnnexinV and DAPI. M249 parental and DDR cells were cultured in the presence of medium with and without glutamine for 24 h. Representative dot blots and percentages of AnnexinV positive cells are shown.

Mentions: In order to test this, we aimed to deprive M249 and M229 single drug resistant cells of glutamine and glucose to observe the effects on cell survival. We found that both M249 and M229 single drug resistant (SDR) populations were more sensitive to glutamine deprivation than glucose deprivation (Figure 2a, b). In addition, both parental and single drug resistant cells were deprived of glutamine and tested for apoptosis by measuring PARP cleavage using Western blot analysis. M249 and M229 single drug resistant cells both had higher levels of PARP cleavage upon glutamine deprivation than parental counterparts indicating increased levels of apoptosis (Figure 2c, d). In correlation with the Western blot results, there were more apoptotic cells upon glutamine deprivation in the M249 single drug resistant line than the parental line measured by Annexin V staining (Figure 2e). To further evaluate a broader range of resistance mechanisms, melanoma isogenic sub-lines with acquired vemurafenib and MEK inhibitor double drug resistance (DDR) were also used. We tested the double drug resistant (DDR) M249 cell line containing both BRAF amplication and MEK1 mutations for sensitivity to glutamine. Similar to the single drug resistant lines, the M249 double drug resistant line was found to be more sensitive to glutamine deprivation than glucose deprivation (Figure 2f). In addition, we tested for apoptosis via Annexin V staining upon glutamine deprivation and found that M249 double drug resistant cells were also more sensitive to glutamine deprivation than parental counterparts (Figure 2g). These results highly suggest that resistant cells are more dependent on glutamine for their survival, and that targeting glutamine metabolism with inhibitors aimed at blocking glutamine usage may be a way to specifically kill the vemurafenib resistant cells.Figure 2


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 are more sensitive to glutamine deprivation. a Melanoma M249 and b M229 single drug resistant (SDR) cells were cultured in the presence of medium with or without glutamine (Gln) or glucose (Gluc). After 24 h representative images and surviving fractions (survival %) compared to control cells in complete medium are shown using DIMSCAN, a microcomputer fluorescence-based cytotoxicity assay (n = 6, **p < 0.01). Western blot analysis was also used to assess levels of cleaved PARP as an indicator of apoptosis in both c M249 and d M229 parental and SDR cell lines. e Apoptosis was also assessed using flow cytometry of M249 parental and resistance cells stained with AnnexinV and DAPI. M249 parental and SDR cells were cultured in the presence of medium with and without glutamine for 24 h. Representative dot blots and percentages of AnnexinV positive cells are shown. f Melanoma double drug resistant (DDR) cell line M249 DDR5 was cultured in the presence of medium with and without glutamine (Gln) or glucose (Gluc). After 24 h representative images and surviving fractions (survival %) compared to control cells in complete medium are shown using DIMSCAN, a microcomputer fluorescence-based cytotoxicity assay (n = 6, ***p < 0.001). g Apoptosis was also assessed using flow cytometry of M249 parental and DDR cells stained with AnnexinV and DAPI. M249 parental and DDR cells were cultured in the presence of medium with and without glutamine for 24 h. Representative dot blots and percentages of AnnexinV positive cells are shown.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4490757&req=5

Fig2: Vemurafenib resistant cells are more sensitive to glutamine deprivation. a Melanoma M249 and b M229 single drug resistant (SDR) cells were cultured in the presence of medium with or without glutamine (Gln) or glucose (Gluc). After 24 h representative images and surviving fractions (survival %) compared to control cells in complete medium are shown using DIMSCAN, a microcomputer fluorescence-based cytotoxicity assay (n = 6, **p < 0.01). Western blot analysis was also used to assess levels of cleaved PARP as an indicator of apoptosis in both c M249 and d M229 parental and SDR cell lines. e Apoptosis was also assessed using flow cytometry of M249 parental and resistance cells stained with AnnexinV and DAPI. M249 parental and SDR cells were cultured in the presence of medium with and without glutamine for 24 h. Representative dot blots and percentages of AnnexinV positive cells are shown. f Melanoma double drug resistant (DDR) cell line M249 DDR5 was cultured in the presence of medium with and without glutamine (Gln) or glucose (Gluc). After 24 h representative images and surviving fractions (survival %) compared to control cells in complete medium are shown using DIMSCAN, a microcomputer fluorescence-based cytotoxicity assay (n = 6, ***p < 0.001). g Apoptosis was also assessed using flow cytometry of M249 parental and DDR cells stained with AnnexinV and DAPI. M249 parental and DDR cells were cultured in the presence of medium with and without glutamine for 24 h. Representative dot blots and percentages of AnnexinV positive cells are shown.
Mentions: In order to test this, we aimed to deprive M249 and M229 single drug resistant cells of glutamine and glucose to observe the effects on cell survival. We found that both M249 and M229 single drug resistant (SDR) populations were more sensitive to glutamine deprivation than glucose deprivation (Figure 2a, b). In addition, both parental and single drug resistant cells were deprived of glutamine and tested for apoptosis by measuring PARP cleavage using Western blot analysis. M249 and M229 single drug resistant cells both had higher levels of PARP cleavage upon glutamine deprivation than parental counterparts indicating increased levels of apoptosis (Figure 2c, d). In correlation with the Western blot results, there were more apoptotic cells upon glutamine deprivation in the M249 single drug resistant line than the parental line measured by Annexin V staining (Figure 2e). To further evaluate a broader range of resistance mechanisms, melanoma isogenic sub-lines with acquired vemurafenib and MEK inhibitor double drug resistance (DDR) were also used. We tested the double drug resistant (DDR) M249 cell line containing both BRAF amplication and MEK1 mutations for sensitivity to glutamine. Similar to the single drug resistant lines, the M249 double drug resistant line was found to be more sensitive to glutamine deprivation than glucose deprivation (Figure 2f). In addition, we tested for apoptosis via Annexin V staining upon glutamine deprivation and found that M249 double drug resistant cells were also more sensitive to glutamine deprivation than parental counterparts (Figure 2g). These results highly suggest that resistant cells are more dependent on glutamine for their survival, and that targeting glutamine metabolism with inhibitors aimed at blocking glutamine usage may be a way to specifically kill the vemurafenib resistant cells.Figure 2

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