Sensitization to metabolic pathway perturbation. (A) Graphical representation of the proportion w... more Sensitization to metabolic pathway perturbation. (A) Graphical representation of the proportion which each metabolic category (Table S2) was represented in the top 10% sensitivity index scores. (B) The combined top ten pathways as defined by high-scoring sensitivity index genes for each TKI (INC80 or erlotinib). (PDF 801 kb)
Drug response and sensitivity index for siRNA treatment of triple-negative breast cancer (TNBC) c... more Drug response and sensitivity index for siRNA treatment of triple-negative breast cancer (TNBC) cell lines.. (XLSX 128 kb)
Quantitation of metabolic pool sizes, ratios for cell lines, and drug responses with statistical ... more Quantitation of metabolic pool sizes, ratios for cell lines, and drug responses with statistical values. Total ion count integrated over peak area for metabolite-specific mass ion validated by multiple precursor-product ion combinations. Mean, standard deviation, and ANOVA with studentâ s t test compares triple-negative breast cancer (TNBC) cell lines and treatment with INC280 or erlotinib vs control vehicle (DMSO). (XLSX 52 kb)
Genes and metabolic categories and pathways used in siRNA screen. Rate-limiting enzymes were coll... more Genes and metabolic categories and pathways used in siRNA screen. Rate-limiting enzymes were collated through KEGG annotation (http://www.genome.jp/kegg/), the Rate-Limiting Enzyme Regulation Database (http://rle.cbi.pku.edu.cn/home.cgi, [51]), and literature searches and categorized according to KEGG. (XLSX 25 kb)
RNAi screen, cell lines, and metabolic rates. (A) Schematic of RNAi screen (left) and transfectio... more RNAi screen, cell lines, and metabolic rates. (A) Schematic of RNAi screen (left) and transfection efficiency under screen conditions (right). (B) Characteristics of each cell line used in this study. (C) Schematic representing biological compartments and metabolic pathways assessed for metabolic rates. (PDF 957 kb)
Aberrant glutamatergic signaling has been implicated in multiple metabolic processes and several ... more Aberrant glutamatergic signaling has been implicated in multiple metabolic processes and several types of cancer. Our laboratory has previously illustrated the role of metabotropic glutamate receptor 1 (GRM1), a G-protein coupled receptor, in neoplastic transformation of melanocytes in vitro and spontaneous development of metastatic melanoma in vivo. Here, we demonstrate significant overexpression of glutaminase (GLS) in GRM1 expressing melanoma cells, resulting in excess production of GRM1’s natural ligand, glutamate, and the establishment of autocrine loops. GLS catalyzes the first step in glutamine metabolism–the conversion of glutamine to glutamate before entering the TCA cycle. Comparison of glutamate levels in circulating blood plasma between melanoma prone GRM1-transgenic and wild type mice showed elevated glutamate levels in melanoma prone GRM1-transgenic mice, suggesting that aberrant GRM1 expression also promotes an increase in circulating glutamate, to ensure constitutive activation of the GRM1 receptor and its downstream signaling effecters in vivo. We also discovered that manipulation of GRM1 expression by genetic means led to robust parallel changes in metabolite expression levels and intracellular glutamate concentrations by mass spectrometry analysis, as well as the endogenous levels of GLS in melanoma cells, suggesting a strong association between GRM1 and GLS. Inclusion of CB-839, a potent, selective, and orally bioavailable inhibitor of GLS, resulted in significant inhibition of GRM1+ melanoma cell proliferation compared to GRM1- ones. The sensitivity of GRM1+ melanoma cells to modulation of GLS points to the dependency of these cells to glutamate amounts via GLS activity. We also demonstrated that treatment with Riluzole, an inhibitor of glutamatergic signaling by decreasing the available ligand, glutamate, led to significantly reduced melanoma cell proliferation in vitro and tumor progression in vivo. We found that simultaneous targeting of glutamate production (CB-839) and release (Riluzole) in GRM1+ melanoma cells led to additive suppression of cell proliferation in vitro. Most importantly, disruption of GRM1 signaling through combined actions of CB-839 and Riluzole significantly suppressed tumor growth in two independent xenograft mouse models of melanoma, with no obvious symptoms of toxicity detected in these mice. Taken together, these results describe a novel pathway for GRM1-mediated tumorigenesis through rewiring of cellular metabolic networks. We are continuing to investigate the mechanisms and regulation of GLS in our experimental system, with the goal of developing a rational strategy for the treatment of GRM1-expressing cancers. Citation Format: Raj Shah, Simar Singh, Kevinn Eddy, Fabian Filipp, Suzie Chen. Concurrent inhibition of glutaminase (GLS) and metabotropic receptor 1 (GRM1) reduced glutamate bioavailability and led to a decrease in GRM1-expressing melanoma cell proliferation in vitro and tumor progression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1839.
B-cell lymphoma 2 (BCL2) is an important apoptosis regulator during developmental and pathologica... more B-cell lymphoma 2 (BCL2) is an important apoptosis regulator during developmental and pathological states, and its overexpression is a key feature of several malignancies. Genomic data from The Cancer Genome Atlas (TCGA) reveals significant somatic copy number amplification, overexpression, and/or elevated protein activity of BCL2 in 50 % of diffuse large B-cell lymphoma (DLBC) patients. While its canonical role in mitochondria-directed apoptosis is well established, the effect of BCL2 on transcriptional and metabolic networks remains elusive. Using an established lymphocytic pro-B-cell line overexpressing BCL2, we identified dysregulated transcriptional and metabolic networks by transcriptomic profiling arrays. Elevated BCL2 levels affect transcription factor complexes and mitogenic programs of NF-κB/REL, HIF1A/ARNT, AP1, E2F, and STAT factors. Using stable isotope-assisted metabolic flux measurements we quantify that elevated BCL2 expression increases carbon utilization boosting cellular proliferation. Tumorigenic overexpression of BCL2 significantly increases glycolytic flux, glutaminolysis, and anaplerotic flux into the TCA cycle. At the same time, the mitochondrial acetyl-CoA pool is separated from the glycolytic one by inactivating the pyruvate dehydrogenase complex via transcriptional regulation of pyruvate dehydrogenase kinase (PDK3). As compensatory fuel, mitochondrial TCA cycle metabolism is supported by asparagine synthase (ASNS) and oxidative glutaminolysis creating targets for small molecule inhibition of glutaminase. Lymphoma cells overexpressing BCL2 contained more mitochondrial mass and were more sensitive to L-glutamine deprivation and glutaminase inhibition. Cells overexpressing a mutant BCL2 G145E, which is incapable of binding BH domain members, failed to increase proliferation, glycolysis, or glutaminolysis. Taken together, the oncogene BCL2 has the ability to ramp up a metabolic phenotype supporting proliferation independent of its anti-apoptotic role. The cellular model of BCL2 activation supports NF-κB-positive subtypes of DLBC and identifies metabolic bottlenecks with dependency on anaplerotic flux as an actionable BCL2 effector network in cancer.
Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy fo... more Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A...
Sensitization to metabolic pathway perturbation. (A) Graphical representation of the proportion w... more Sensitization to metabolic pathway perturbation. (A) Graphical representation of the proportion which each metabolic category (Table S2) was represented in the top 10% sensitivity index scores. (B) The combined top ten pathways as defined by high-scoring sensitivity index genes for each TKI (INC80 or erlotinib). (PDF 801 kb)
Drug response and sensitivity index for siRNA treatment of triple-negative breast cancer (TNBC) c... more Drug response and sensitivity index for siRNA treatment of triple-negative breast cancer (TNBC) cell lines.. (XLSX 128 kb)
Quantitation of metabolic pool sizes, ratios for cell lines, and drug responses with statistical ... more Quantitation of metabolic pool sizes, ratios for cell lines, and drug responses with statistical values. Total ion count integrated over peak area for metabolite-specific mass ion validated by multiple precursor-product ion combinations. Mean, standard deviation, and ANOVA with studentâ s t test compares triple-negative breast cancer (TNBC) cell lines and treatment with INC280 or erlotinib vs control vehicle (DMSO). (XLSX 52 kb)
Genes and metabolic categories and pathways used in siRNA screen. Rate-limiting enzymes were coll... more Genes and metabolic categories and pathways used in siRNA screen. Rate-limiting enzymes were collated through KEGG annotation (http://www.genome.jp/kegg/), the Rate-Limiting Enzyme Regulation Database (http://rle.cbi.pku.edu.cn/home.cgi, [51]), and literature searches and categorized according to KEGG. (XLSX 25 kb)
RNAi screen, cell lines, and metabolic rates. (A) Schematic of RNAi screen (left) and transfectio... more RNAi screen, cell lines, and metabolic rates. (A) Schematic of RNAi screen (left) and transfection efficiency under screen conditions (right). (B) Characteristics of each cell line used in this study. (C) Schematic representing biological compartments and metabolic pathways assessed for metabolic rates. (PDF 957 kb)
Aberrant glutamatergic signaling has been implicated in multiple metabolic processes and several ... more Aberrant glutamatergic signaling has been implicated in multiple metabolic processes and several types of cancer. Our laboratory has previously illustrated the role of metabotropic glutamate receptor 1 (GRM1), a G-protein coupled receptor, in neoplastic transformation of melanocytes in vitro and spontaneous development of metastatic melanoma in vivo. Here, we demonstrate significant overexpression of glutaminase (GLS) in GRM1 expressing melanoma cells, resulting in excess production of GRM1’s natural ligand, glutamate, and the establishment of autocrine loops. GLS catalyzes the first step in glutamine metabolism–the conversion of glutamine to glutamate before entering the TCA cycle. Comparison of glutamate levels in circulating blood plasma between melanoma prone GRM1-transgenic and wild type mice showed elevated glutamate levels in melanoma prone GRM1-transgenic mice, suggesting that aberrant GRM1 expression also promotes an increase in circulating glutamate, to ensure constitutive activation of the GRM1 receptor and its downstream signaling effecters in vivo. We also discovered that manipulation of GRM1 expression by genetic means led to robust parallel changes in metabolite expression levels and intracellular glutamate concentrations by mass spectrometry analysis, as well as the endogenous levels of GLS in melanoma cells, suggesting a strong association between GRM1 and GLS. Inclusion of CB-839, a potent, selective, and orally bioavailable inhibitor of GLS, resulted in significant inhibition of GRM1+ melanoma cell proliferation compared to GRM1- ones. The sensitivity of GRM1+ melanoma cells to modulation of GLS points to the dependency of these cells to glutamate amounts via GLS activity. We also demonstrated that treatment with Riluzole, an inhibitor of glutamatergic signaling by decreasing the available ligand, glutamate, led to significantly reduced melanoma cell proliferation in vitro and tumor progression in vivo. We found that simultaneous targeting of glutamate production (CB-839) and release (Riluzole) in GRM1+ melanoma cells led to additive suppression of cell proliferation in vitro. Most importantly, disruption of GRM1 signaling through combined actions of CB-839 and Riluzole significantly suppressed tumor growth in two independent xenograft mouse models of melanoma, with no obvious symptoms of toxicity detected in these mice. Taken together, these results describe a novel pathway for GRM1-mediated tumorigenesis through rewiring of cellular metabolic networks. We are continuing to investigate the mechanisms and regulation of GLS in our experimental system, with the goal of developing a rational strategy for the treatment of GRM1-expressing cancers. Citation Format: Raj Shah, Simar Singh, Kevinn Eddy, Fabian Filipp, Suzie Chen. Concurrent inhibition of glutaminase (GLS) and metabotropic receptor 1 (GRM1) reduced glutamate bioavailability and led to a decrease in GRM1-expressing melanoma cell proliferation in vitro and tumor progression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1839.
B-cell lymphoma 2 (BCL2) is an important apoptosis regulator during developmental and pathologica... more B-cell lymphoma 2 (BCL2) is an important apoptosis regulator during developmental and pathological states, and its overexpression is a key feature of several malignancies. Genomic data from The Cancer Genome Atlas (TCGA) reveals significant somatic copy number amplification, overexpression, and/or elevated protein activity of BCL2 in 50 % of diffuse large B-cell lymphoma (DLBC) patients. While its canonical role in mitochondria-directed apoptosis is well established, the effect of BCL2 on transcriptional and metabolic networks remains elusive. Using an established lymphocytic pro-B-cell line overexpressing BCL2, we identified dysregulated transcriptional and metabolic networks by transcriptomic profiling arrays. Elevated BCL2 levels affect transcription factor complexes and mitogenic programs of NF-κB/REL, HIF1A/ARNT, AP1, E2F, and STAT factors. Using stable isotope-assisted metabolic flux measurements we quantify that elevated BCL2 expression increases carbon utilization boosting cellular proliferation. Tumorigenic overexpression of BCL2 significantly increases glycolytic flux, glutaminolysis, and anaplerotic flux into the TCA cycle. At the same time, the mitochondrial acetyl-CoA pool is separated from the glycolytic one by inactivating the pyruvate dehydrogenase complex via transcriptional regulation of pyruvate dehydrogenase kinase (PDK3). As compensatory fuel, mitochondrial TCA cycle metabolism is supported by asparagine synthase (ASNS) and oxidative glutaminolysis creating targets for small molecule inhibition of glutaminase. Lymphoma cells overexpressing BCL2 contained more mitochondrial mass and were more sensitive to L-glutamine deprivation and glutaminase inhibition. Cells overexpressing a mutant BCL2 G145E, which is incapable of binding BH domain members, failed to increase proliferation, glycolysis, or glutaminolysis. Taken together, the oncogene BCL2 has the ability to ramp up a metabolic phenotype supporting proliferation independent of its anti-apoptotic role. The cellular model of BCL2 activation supports NF-κB-positive subtypes of DLBC and identifies metabolic bottlenecks with dependency on anaplerotic flux as an actionable BCL2 effector network in cancer.
Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy fo... more Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A...
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