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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 glutaminase inhibitors. a M249 and M229 parental single drug resistant (SDR) cells were cultured in the presence of medium control (DMSO) or 10 μM BPTES for 48 h. SDR cell lines were cultured in combination of vemurafenib (1 μM) and BPTES. Surviving cell fraction percentage (survival %) compared to vehicle control (DMSO) was assessed using DIMSCAN technology (n = 6, ***p < 0.001, *p < 0.05). b M249 and M229 parental and single drug resistant cells were cultured in the presence of vehicle control (water) or 10 μM L-DON for 48 h. SDR cell lines received a combination of vemurafenib (1 μM) and L-DON. Surviving cell fraction percentage (survival %) compared to vehicle control (DMSO) was assessed using DIMSCAN technology (n = 6, ***p < 0.001, *p < 0.05). c M249 parental and double drug resistant (DDR) cells were cultured in the presence of medium control (DMSO) or 10 μM BPTES for 48 h. DDR cell lines were cultured in combination with vemurafenib (1 μM), selumetinib (1 μM), and BPTES. Surviving cell fraction percentage compared to vehicle control (DMSO) (survival %) was assessed using DIMSCAN technology (n = 6, ***p < 0.001). d M249 single (SDR) and double drug (DDR) resistant lines were cultured in the presence or absence of inhibitor (1 μM vemurafenib for SDR and 1 μM vemurafenib and 1 μM selumentib for DDR) and subsequently treated with 10 μM BPTES or deprived of glutamine (Gln) for 48 h. Surviving cell fraction percentage compared to vehicle control (DMSO) (survival %) was assessed using DIMSCAN technology.
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Fig3: Vemurafenib resistant cells are more sensitive to glutaminase inhibitors. a M249 and M229 parental single drug resistant (SDR) cells were cultured in the presence of medium control (DMSO) or 10 μM BPTES for 48 h. SDR cell lines were cultured in combination of vemurafenib (1 μM) and BPTES. Surviving cell fraction percentage (survival %) compared to vehicle control (DMSO) was assessed using DIMSCAN technology (n = 6, ***p < 0.001, *p < 0.05). b M249 and M229 parental and single drug resistant cells were cultured in the presence of vehicle control (water) or 10 μM L-DON for 48 h. SDR cell lines received a combination of vemurafenib (1 μM) and L-DON. Surviving cell fraction percentage (survival %) compared to vehicle control (DMSO) was assessed using DIMSCAN technology (n = 6, ***p < 0.001, *p < 0.05). c M249 parental and double drug resistant (DDR) cells were cultured in the presence of medium control (DMSO) or 10 μM BPTES for 48 h. DDR cell lines were cultured in combination with vemurafenib (1 μM), selumetinib (1 μM), and BPTES. Surviving cell fraction percentage compared to vehicle control (DMSO) (survival %) was assessed using DIMSCAN technology (n = 6, ***p < 0.001). d M249 single (SDR) and double drug (DDR) resistant lines were cultured in the presence or absence of inhibitor (1 μM vemurafenib for SDR and 1 μM vemurafenib and 1 μM selumentib for DDR) and subsequently treated with 10 μM BPTES or deprived of glutamine (Gln) for 48 h. Surviving cell fraction percentage compared to vehicle control (DMSO) (survival %) was assessed using DIMSCAN technology.

Mentions: To test whether we can target glutamine metabolism to treat resistant cells, we used glutaminase inhibitors, BPTES and L-DON to treat both parental and resistant cell populations. We then assessed cell survival upon treatment and observed whether resistant cell populations were in fact more sensitive to glutamine uptake. We found that, although both parental and single drug resistant populations were sensitive to glutaminase inhibitors BPTES and L-DON, single drug resistant cells in combination with vemurafenib were much more sensitive as demonstrated by reduced cell survival percentages (Figure 3a, b). To further examine whether double drug resistant cell lines were also sensitive to glutaminase inhibitors, we tested M249 double drug resistant cell line for survival after treatment with BPTES. We found that the double drug resistant line (similar to the single drug resistant line) was more sensitive to BPTES than parental counterparts (Figure 3c). To address whether this effect required BRAF inhibition or BRAF/MEK1 inhibition, we treated both M249 single and double drug resistant cell lines with BPTES and without glutamine in the presence or absence of BRAF/MEK inhibitors. We found that treatment of single or double drug resistant cell lines does not require the presence of the inhibitor to become sensitive to glutamine deprivation or BPTES treatment (Figure 3d). These results suggest that glutaminase inhibitors may be used as a strategy to target resistant cell populations.Figure 3


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 glutaminase inhibitors. a M249 and M229 parental single drug resistant (SDR) cells were cultured in the presence of medium control (DMSO) or 10 μM BPTES for 48 h. SDR cell lines were cultured in combination of vemurafenib (1 μM) and BPTES. Surviving cell fraction percentage (survival %) compared to vehicle control (DMSO) was assessed using DIMSCAN technology (n = 6, ***p < 0.001, *p < 0.05). b M249 and M229 parental and single drug resistant cells were cultured in the presence of vehicle control (water) or 10 μM L-DON for 48 h. SDR cell lines received a combination of vemurafenib (1 μM) and L-DON. Surviving cell fraction percentage (survival %) compared to vehicle control (DMSO) was assessed using DIMSCAN technology (n = 6, ***p < 0.001, *p < 0.05). c M249 parental and double drug resistant (DDR) cells were cultured in the presence of medium control (DMSO) or 10 μM BPTES for 48 h. DDR cell lines were cultured in combination with vemurafenib (1 μM), selumetinib (1 μM), and BPTES. Surviving cell fraction percentage compared to vehicle control (DMSO) (survival %) was assessed using DIMSCAN technology (n = 6, ***p < 0.001). d M249 single (SDR) and double drug (DDR) resistant lines were cultured in the presence or absence of inhibitor (1 μM vemurafenib for SDR and 1 μM vemurafenib and 1 μM selumentib for DDR) and subsequently treated with 10 μM BPTES or deprived of glutamine (Gln) for 48 h. Surviving cell fraction percentage compared to vehicle control (DMSO) (survival %) was assessed using DIMSCAN technology.
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Related In: Results  -  Collection

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Fig3: Vemurafenib resistant cells are more sensitive to glutaminase inhibitors. a M249 and M229 parental single drug resistant (SDR) cells were cultured in the presence of medium control (DMSO) or 10 μM BPTES for 48 h. SDR cell lines were cultured in combination of vemurafenib (1 μM) and BPTES. Surviving cell fraction percentage (survival %) compared to vehicle control (DMSO) was assessed using DIMSCAN technology (n = 6, ***p < 0.001, *p < 0.05). b M249 and M229 parental and single drug resistant cells were cultured in the presence of vehicle control (water) or 10 μM L-DON for 48 h. SDR cell lines received a combination of vemurafenib (1 μM) and L-DON. Surviving cell fraction percentage (survival %) compared to vehicle control (DMSO) was assessed using DIMSCAN technology (n = 6, ***p < 0.001, *p < 0.05). c M249 parental and double drug resistant (DDR) cells were cultured in the presence of medium control (DMSO) or 10 μM BPTES for 48 h. DDR cell lines were cultured in combination with vemurafenib (1 μM), selumetinib (1 μM), and BPTES. Surviving cell fraction percentage compared to vehicle control (DMSO) (survival %) was assessed using DIMSCAN technology (n = 6, ***p < 0.001). d M249 single (SDR) and double drug (DDR) resistant lines were cultured in the presence or absence of inhibitor (1 μM vemurafenib for SDR and 1 μM vemurafenib and 1 μM selumentib for DDR) and subsequently treated with 10 μM BPTES or deprived of glutamine (Gln) for 48 h. Surviving cell fraction percentage compared to vehicle control (DMSO) (survival %) was assessed using DIMSCAN technology.
Mentions: To test whether we can target glutamine metabolism to treat resistant cells, we used glutaminase inhibitors, BPTES and L-DON to treat both parental and resistant cell populations. We then assessed cell survival upon treatment and observed whether resistant cell populations were in fact more sensitive to glutamine uptake. We found that, although both parental and single drug resistant populations were sensitive to glutaminase inhibitors BPTES and L-DON, single drug resistant cells in combination with vemurafenib were much more sensitive as demonstrated by reduced cell survival percentages (Figure 3a, b). To further examine whether double drug resistant cell lines were also sensitive to glutaminase inhibitors, we tested M249 double drug resistant cell line for survival after treatment with BPTES. We found that the double drug resistant line (similar to the single drug resistant line) was more sensitive to BPTES than parental counterparts (Figure 3c). To address whether this effect required BRAF inhibition or BRAF/MEK1 inhibition, we treated both M249 single and double drug resistant cell lines with BPTES and without glutamine in the presence or absence of BRAF/MEK inhibitors. We found that treatment of single or double drug resistant cell lines does not require the presence of the inhibitor to become sensitive to glutamine deprivation or BPTES treatment (Figure 3d). These results suggest that glutaminase inhibitors may be used as a strategy to target resistant cell populations.Figure 3

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