Constitutive activation of GNAQ/11 is the initiative oncogenic event in uveal melanoma(UM).Direct targeting GNAQ/11 has yet to be proven feasible as they are vital for a plethora of cellular functions.In search of gen...Constitutive activation of GNAQ/11 is the initiative oncogenic event in uveal melanoma(UM).Direct targeting GNAQ/11 has yet to be proven feasible as they are vital for a plethora of cellular functions.In search of genetic vulnerability for UM,we found that inhibition of euchromatic histone lysine methyltransferase 2(EHMT2)expression or activity significantly reduced the proliferation and migration capacity of cancer cells.Notably,elevated expression of EHMT2 had been validated in UM samples.Furthermore,Kaplan-Meier survival analysis indicated high EHMT2 protein level was related to poor recurrence-free survival and a more advanced T stage.Chromatin immunoprecipitation sequencing analysis and the following mechanistic investigation showed that ARHGAP29 was a downstream target of EHMT2.Its transcription was suppressed by EHMT2 in a methyltransferasedependent pattern in GNAQ/11-mutant UM cells,leading to elevated RhoA activity.Rescuing constitutively active RhoA in UM cells lacking EHMT2 restored oncogenic phenotypes.Simultaneously blocking EHMT2 and GNAQ/11 signaling in vitro and in vivo showed a synergistic effect on UM growth,suggesting the driver role of these two key molecules.In summary,our study shows evidence for an epigenetic program of EHMT2 regulation that influences UM progression and indicates inhibiting EHMT2 and MEK/ERK simultaneously as a therapeutic strategy in GNAQ/11-mutant UM.展开更多
Importance:Postzygotic mutations in the GNAQ/GNA11 genes,which encode the G-protein nucleotide binding protein alpha subunits,have been identified in patients with phakomatosis pigmentovascularis(PPV).However,little i...Importance:Postzygotic mutations in the GNAQ/GNA11 genes,which encode the G-protein nucleotide binding protein alpha subunits,have been identified in patients with phakomatosis pigmentovascularis(PPV).However,little is known about the Chinese population.Objective:To identify pathogenic mutations in pediatric patients with PPV within the Chinese population.Methods:We performed whole-exome sequencing(WES)using skin lesion tissues from pediatric patients diagnosed with PPV.Additionally,ultradeep-targeted sequencing was conducted to validate the somatic mutations.A genotype-phenotype correlation was analyzed by integrating data from previous reports with the findings of the present study.Results:Thirteen patients were enrolled,all diagnosed with the cesioflammea type of PPV,except for one patient with an unclassifiable type.We identified somatic GNA11 c.547C>T(p.R183C)variant in seven patients and GNAQ c.548G>A(p.R183Q)in four patients,with low allelic fractions ranging from 2.1%to 8.6%through ultradeep sequencing.Besides,a GNAQ c.548G>A(p.R183Q)variant was detected through targeted sequencing in one of two patients who did not exhibit detectable variants via WES.The genotype-phenotype correlation analysis,involving 15 patients with a GNA11 variant and 10 with a GNAQ variant,revealed that facial capillary malformation(87%vs.50%,P=0.075)and ocular melanocytosis(80%vs.40%,P=0.087)appeared to be more frequent in patients with GNA11 mutation compared to those with GNAQ mutations.All four patients diagnosed with cesiomarmorata type or overlapping cesioflammea and cesiomarmorata type PPV carried the GNA11 variant.Interpretation:Our study demonstrated that the majority of PPV patients in the Chinese population carried a postzygotic variant of GNAQ/GNA11,thus further confirming the pathogenic role of GNAQ/GNA11 mosaicism in the development of PPV cesioflammea type.展开更多
More than 85%of patients with uveal melanoma(UM)carry a GNAQ or GNA11 mutation at a hotspot codon(Q209)that encodes G proteinαsubunit q/11 polypeptides(Gα_(q/11)).GNAQ/11 relies on palmitoylation for membrane associ...More than 85%of patients with uveal melanoma(UM)carry a GNAQ or GNA11 mutation at a hotspot codon(Q209)that encodes G proteinαsubunit q/11 polypeptides(Gα_(q/11)).GNAQ/11 relies on palmitoylation for membrane association and signal transduction.Despite the palmitoylation of GNAQ/11 was discovered long before,its implication in UM remains unclear.Here,results of palmitoylation-targeted mutagenesis and chemical interference approaches revealed that the loss of GNAQ/11 palmitoylation substantially affected tumor cell proliferation and survival in UM cells.Palmitoylation inhibition through the mutation of palmitoylation sites suppressed GNAQ/11^(Q209L)-induced malignant transformation in NIH3T3 cells.Importantly,the palmitoylation-deficient oncogenic GNAQ/11 failed to rescue the cell death initiated by the knock down of endogenous GNAQ/11 oncogenes in UM cells,which are much more dependent on Gα_(q/11) signaling for cell survival and proliferation than other melanoma cells without GNAQ/11 mutations.Furthermore,the palmitoylation inhibitor,2-bromopalmitate,also specifically disrupted Gα_(q/11) downstream signaling by interfering with the MAPK pathway and BCL2 survival pathway in GNAQ/11-mutant UM cells and showed a notable synergistic effect when applied in combination with the BCL2 inhibitor,ABT-199,in vitro.The findings validate that GNAQ/11 palmitoylation plays a critical role in UM and may serve as a promising therapeutic target for GNAQ/11-driven UM.展开更多
Uveal melanoma is the most common intraocular cancer in the adult eye.R183 and Q209 were found to be mutational hotspots in exon 4 and exon 5 of GNAQ and GNA11 in Caucasians.However,only a few studies have reported so...Uveal melanoma is the most common intraocular cancer in the adult eye.R183 and Q209 were found to be mutational hotspots in exon 4 and exon 5 of GNAQ and GNA11 in Caucasians.However,only a few studies have reported somaticmutations in GNAQ or GNA11 in uveal melanoma in Chinese.We extracted somatic DNA from paraffin-embedded biopsies of 63 Chinese uveal melanoma samples and sequenced the entire coding regions of exons 4 and 5 in GNAQ and GNA11.The results showed that 33%of Chinese uveal melanoma samples carried Q209 mutations while none had R183 mutation in GNAQ or GNA11.In addition,seven novel missense somatic mutations in GNAQ(Y192C,F194L,P170S,D236N,L232F,V230A,and M227I)and four novel missense somatic mutations in GNA11(R166C,I200T,S225F,and V206M)were found in our study.The high mutation frequency of Q209 and the novel missense mutations detected in this study suggest that GNAQ and GNA11 are common targets for somatic mutations in Chinese uveal melanoma.展开更多
基金supported by the Science and Technology Commission of Shanghai(20DZ2270800,China)the National Natural Science Foundation of China(grants 82073889)+1 种基金the Innovative Research Team of High-level Local Universities in Shanghai(SHSMU-ZDCX20210900,China)China Postdoctoral Science Foundation(2022M722120,China)and Shanghai Sailing Program(22YF1422800,China)。
文摘Constitutive activation of GNAQ/11 is the initiative oncogenic event in uveal melanoma(UM).Direct targeting GNAQ/11 has yet to be proven feasible as they are vital for a plethora of cellular functions.In search of genetic vulnerability for UM,we found that inhibition of euchromatic histone lysine methyltransferase 2(EHMT2)expression or activity significantly reduced the proliferation and migration capacity of cancer cells.Notably,elevated expression of EHMT2 had been validated in UM samples.Furthermore,Kaplan-Meier survival analysis indicated high EHMT2 protein level was related to poor recurrence-free survival and a more advanced T stage.Chromatin immunoprecipitation sequencing analysis and the following mechanistic investigation showed that ARHGAP29 was a downstream target of EHMT2.Its transcription was suppressed by EHMT2 in a methyltransferasedependent pattern in GNAQ/11-mutant UM cells,leading to elevated RhoA activity.Rescuing constitutively active RhoA in UM cells lacking EHMT2 restored oncogenic phenotypes.Simultaneously blocking EHMT2 and GNAQ/11 signaling in vitro and in vivo showed a synergistic effect on UM growth,suggesting the driver role of these two key molecules.In summary,our study shows evidence for an epigenetic program of EHMT2 regulation that influences UM progression and indicates inhibiting EHMT2 and MEK/ERK simultaneously as a therapeutic strategy in GNAQ/11-mutant UM.
基金Beijing Natural Science Foundation:Grant/Award Number:7222058The Open Project of Henan Clinical Research Center of Childhood Diseases:Grant/Award Number:YJZX202209+2 种基金National Regional Medical Center Opening Project:Grant/Award Number:NRMC0101Investigator Initiated Project:Grant/Award Number:[2022]-E-028-YBCH Young Investigator Program(BCHYIP):Grant/Award Number:3-1-014-01-36。
文摘Importance:Postzygotic mutations in the GNAQ/GNA11 genes,which encode the G-protein nucleotide binding protein alpha subunits,have been identified in patients with phakomatosis pigmentovascularis(PPV).However,little is known about the Chinese population.Objective:To identify pathogenic mutations in pediatric patients with PPV within the Chinese population.Methods:We performed whole-exome sequencing(WES)using skin lesion tissues from pediatric patients diagnosed with PPV.Additionally,ultradeep-targeted sequencing was conducted to validate the somatic mutations.A genotype-phenotype correlation was analyzed by integrating data from previous reports with the findings of the present study.Results:Thirteen patients were enrolled,all diagnosed with the cesioflammea type of PPV,except for one patient with an unclassifiable type.We identified somatic GNA11 c.547C>T(p.R183C)variant in seven patients and GNAQ c.548G>A(p.R183Q)in four patients,with low allelic fractions ranging from 2.1%to 8.6%through ultradeep sequencing.Besides,a GNAQ c.548G>A(p.R183Q)variant was detected through targeted sequencing in one of two patients who did not exhibit detectable variants via WES.The genotype-phenotype correlation analysis,involving 15 patients with a GNA11 variant and 10 with a GNAQ variant,revealed that facial capillary malformation(87%vs.50%,P=0.075)and ocular melanocytosis(80%vs.40%,P=0.087)appeared to be more frequent in patients with GNA11 mutation compared to those with GNAQ mutations.All four patients diagnosed with cesiomarmorata type or overlapping cesioflammea and cesiomarmorata type PPV carried the GNA11 variant.Interpretation:Our study demonstrated that the majority of PPV patients in the Chinese population carried a postzygotic variant of GNAQ/GNA11,thus further confirming the pathogenic role of GNAQ/GNA11 mosaicism in the development of PPV cesioflammea type.
基金This work was supported by the Key Project of National Natural Science Foundation of China(No.81530006 to Ruibao Ren)Shanghai Collaborative Innovation Program on Regenerative Medicine and Stem Cell Research(No.2019CXJQ01 to Ruibao Ren)+2 种基金National Natural ScienceFoundation of China(No.81870112 to Ruibao Ren,No.81770171 to Bo Jiao,and No.81970134 to Ping Liu)Samuel Waxman Cancer Research Foundation(to Ruibao Ren)the Innovative Research Team of High-level Local Universities in Shanghai.
文摘More than 85%of patients with uveal melanoma(UM)carry a GNAQ or GNA11 mutation at a hotspot codon(Q209)that encodes G proteinαsubunit q/11 polypeptides(Gα_(q/11)).GNAQ/11 relies on palmitoylation for membrane association and signal transduction.Despite the palmitoylation of GNAQ/11 was discovered long before,its implication in UM remains unclear.Here,results of palmitoylation-targeted mutagenesis and chemical interference approaches revealed that the loss of GNAQ/11 palmitoylation substantially affected tumor cell proliferation and survival in UM cells.Palmitoylation inhibition through the mutation of palmitoylation sites suppressed GNAQ/11^(Q209L)-induced malignant transformation in NIH3T3 cells.Importantly,the palmitoylation-deficient oncogenic GNAQ/11 failed to rescue the cell death initiated by the knock down of endogenous GNAQ/11 oncogenes in UM cells,which are much more dependent on Gα_(q/11) signaling for cell survival and proliferation than other melanoma cells without GNAQ/11 mutations.Furthermore,the palmitoylation inhibitor,2-bromopalmitate,also specifically disrupted Gα_(q/11) downstream signaling by interfering with the MAPK pathway and BCL2 survival pathway in GNAQ/11-mutant UM cells and showed a notable synergistic effect when applied in combination with the BCL2 inhibitor,ABT-199,in vitro.The findings validate that GNAQ/11 palmitoylation plays a critical role in UM and may serve as a promising therapeutic target for GNAQ/11-driven UM.
基金This work was supported by State Key Laboratory of Ophthalmology(Zhongshan Ophthalmic Center,Sun Yat-Sen University),“100 talents plan”from Sun Yatsen University,the Open Research Funds of the State Key Laboratory of Ophthalmology(2017KF05)Funds of the Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science(2017B030314025)+1 种基金Medical Research Fund of Guangdong Province(No.A2018337)Guangzhou Institute of Pediatrics/GuangzhouWomen and Children’s Medical Center(No.IP-2018-002).
文摘Uveal melanoma is the most common intraocular cancer in the adult eye.R183 and Q209 were found to be mutational hotspots in exon 4 and exon 5 of GNAQ and GNA11 in Caucasians.However,only a few studies have reported somaticmutations in GNAQ or GNA11 in uveal melanoma in Chinese.We extracted somatic DNA from paraffin-embedded biopsies of 63 Chinese uveal melanoma samples and sequenced the entire coding regions of exons 4 and 5 in GNAQ and GNA11.The results showed that 33%of Chinese uveal melanoma samples carried Q209 mutations while none had R183 mutation in GNAQ or GNA11.In addition,seven novel missense somatic mutations in GNAQ(Y192C,F194L,P170S,D236N,L232F,V230A,and M227I)and four novel missense somatic mutations in GNA11(R166C,I200T,S225F,and V206M)were found in our study.The high mutation frequency of Q209 and the novel missense mutations detected in this study suggest that GNAQ and GNA11 are common targets for somatic mutations in Chinese uveal melanoma.
