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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

Knock down of NRAS in vemurafenib resistant cells reduces sensitivity to glutaminase inhibitor. Melanoma M249 resistant cells were infected with shNRAS or shControl lentiviral particles and selected for puromycin resistance after 48 h. a Quantitative PCR was used to measure relative mRNA expression of NRAS in shControl and shNRAS infected cells. b shNRAS or shControl cells were cultured in the presence of 10 μM BPTES for 48 h or with vehicle control (DMSO). Surviving cell fraction percentage (survival %) compared to vehicle control (DMSO) was determined using DIMSCAN analysis (n = 6, *p < 0.05).
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Fig4: Knock down of NRAS in vemurafenib resistant cells reduces sensitivity to glutaminase inhibitor. Melanoma M249 resistant cells were infected with shNRAS or shControl lentiviral particles and selected for puromycin resistance after 48 h. a Quantitative PCR was used to measure relative mRNA expression of NRAS in shControl and shNRAS infected cells. b shNRAS or shControl cells were cultured in the presence of 10 μM BPTES for 48 h or with vehicle control (DMSO). Surviving cell fraction percentage (survival %) compared to vehicle control (DMSO) was determined using DIMSCAN analysis (n = 6, *p < 0.05).

Mentions: To determine whether resistance acquired NRAS mutations had a role in the transformation of M249 resistant cells to glutamine dependence, cells with stable knockdown of NRAS and scrambled shRNA controls were prepared using short hairpin RNA (shRNA). First, we assessed whether the shRNA was successful at reducing levels of NRAS in the cell. Indeed, relative mRNA expression of NRAS was reduced in shNRAS knockdown cells well below control levels (Figure 4a). Control and shNRAS knock down cells were subsequently treated with BPTES and assessed for cell survival without the presence of BRAF inhibitor vemurafenib in the medium. We found that knocking down NRAS allowed cells to become less sensitized to the glutaminase inhibitor BPTES when compared to control cell lines (Figure 4b). These results suggest that resistance acquired mutations in NRAS contribute to reprogramming of resistant cells to glutamine dependence.Figure 4


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)

Knock down of NRAS in vemurafenib resistant cells reduces sensitivity to glutaminase inhibitor. Melanoma M249 resistant cells were infected with shNRAS or shControl lentiviral particles and selected for puromycin resistance after 48 h. a Quantitative PCR was used to measure relative mRNA expression of NRAS in shControl and shNRAS infected cells. b shNRAS or shControl cells were cultured in the presence of 10 μM BPTES for 48 h or with vehicle control (DMSO). Surviving cell fraction percentage (survival %) compared to vehicle control (DMSO) was determined using DIMSCAN analysis (n = 6, *p < 0.05).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Knock down of NRAS in vemurafenib resistant cells reduces sensitivity to glutaminase inhibitor. Melanoma M249 resistant cells were infected with shNRAS or shControl lentiviral particles and selected for puromycin resistance after 48 h. a Quantitative PCR was used to measure relative mRNA expression of NRAS in shControl and shNRAS infected cells. b shNRAS or shControl cells were cultured in the presence of 10 μM BPTES for 48 h or with vehicle control (DMSO). Surviving cell fraction percentage (survival %) compared to vehicle control (DMSO) was determined using DIMSCAN analysis (n = 6, *p < 0.05).
Mentions: To determine whether resistance acquired NRAS mutations had a role in the transformation of M249 resistant cells to glutamine dependence, cells with stable knockdown of NRAS and scrambled shRNA controls were prepared using short hairpin RNA (shRNA). First, we assessed whether the shRNA was successful at reducing levels of NRAS in the cell. Indeed, relative mRNA expression of NRAS was reduced in shNRAS knockdown cells well below control levels (Figure 4a). Control and shNRAS knock down cells were subsequently treated with BPTES and assessed for cell survival without the presence of BRAF inhibitor vemurafenib in the medium. We found that knocking down NRAS allowed cells to become less sensitized to the glutaminase inhibitor BPTES when compared to control cell lines (Figure 4b). These results suggest that resistance acquired mutations in NRAS contribute to reprogramming of resistant cells to glutamine dependence.Figure 4

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