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BRAF inhibition promotes BRAF mutant human melanoma cell survival under nutrient-deprived conditions through activation of mitochondrial metabolism
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EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
adhesion molecules in the superficial tumor, and with mesenchymal inductors, such as ETV5, and MAPK/ERK pathway at the invasive front. No conflict of interest. 480 BRAF inhibition promotes BRAF mutant human melanoma cell survival under nutrient-deprived conditions through activation of mitochondrial metabolism T. Delgado-Goni1 , S. Wantuch2 , P. Workman3 , R. Marais4 , M.O. Leach1 , M. Beloueche-Babari1 . 1 Institute of Cancer Research, Radiotherapy and Imaging, London, United Kingdom, 2 Institute of Cancer Research, Breast Cancer Research, London, United Kingdom, 3 Institute of Cancer Research, Cancer Therapeutics Unit, London, United Kingdom, 4 Cancer Research UK Manchester Institute, Molecular Oncology, Manchester, United Kingdom Background: BRAF-MEK1/2 signaling inhibitors have shown remarkable clinical activity in BRAF-mutant melanoma. However, responses are not durable suggesting activation of alternative tumour survival mechanisms that allow resistance to therapy. This work investigates the re-programming of glucose metabolism following BRAF inhibition as an adaptive response, and its significance for cell survival in a nutrient depleted environment. Material and Methods: WM266.4 (BRAFV600D) cells were treated with the BRAF inhibitor vemurafenib (2mM, 24 h) in standard culture media containing 100% 5mM [1-13C]glucose. Cell extracts were analyzed by 13C NMR spectroscopy to assess alterations in glucose metabolism. To assess the significance of these alterations for cell survival and for their dependence on glycolysis, glutamine and TCA metabolism, cell counts were obtained from WM266.4 and SKMEL28 (BRAFV600E) cells grown in different nutrientdeprived conditions, with or without vemurafenib, for 48 h: 1mM glucose, 1mM glucose without glutamine and 1mM glucose without glutamine and pyruvate. Results: Vemurafenib induced a reduction in [3-13C] extracellular lactate in treated WM266.4 cells with respect to controls (62.9±13.1%; P = 0.01). A significant decrease in the intracellular [3-13C]lactate levels after treatment (53.1±18.6%, P = 0.01), concomitant with a significant increase in [1-13C]glucose (up to 286.9±105.5%, P = 0.04), indicated decreased glycolysis and glucose utilization after internalization. A significant elevation in [2-13C] and [3-13C]glutamate (to 185.2±42.6% and 171.4±38.9% of controls, respectively, P < 0.05) and [2-13C]glutamine (135.1±14.1, P = 0.01) in treated cells indicated an increased pyruvate carboxylase (PC) flux, consistent with an increased [2-13C]/[4-13C] glutamate ratio (from 0.10±0.04 to 0.30±0.04 (P = 0.02). Accordingly, the enzyme activity of PC showed a significant increase (159±67%, P = 0.04) under BRAF inhibition. WM266.4 and SKMEL28 cells exhibited a significant reduction in cell counts in low glucose media that was emphasised when glutamine was removed. However, the number of viable cells was significantly higher in treated samples, indicating that vemurafenib reduces the dependency of these cells on glucose (p = 0.03 and p = 0.01 respectively) and glutamine (p < 0.01) for proliferation and survival. When pyruvate was removed, the growth advantage under treatment was abolished for both lines, consistent with the dependency of treated cells on mitochondrial metabolism, with PC flux requiring pyruvate availability. Conclusion: BRAF inhibition with vemurafenib in BRAF mutant human melanoma cells alters glucose utilization and activates mitochondrial metabolism with consequences that enable improved survival under nutrientdeprived conditions. This metabolic shift may enable survival and the emergence of resistant clones following treatment. No conflict of interest. 481 Single cell analysis of intratumour heterogeneity in childhood acute lymphoblastic leukaemia V. Turati1 , H. Mike2 , J. Herrero1 , A. John1 , S. Richardson1 , B. Gaal1 , L. Mark3 , S.E. Jacobsen4 , T. Enver1 . 1 UCL Cancer Institute, Cancer Biology, London, United Kingdom, 2 Institute of Child Health, Genetics & Genomic Medicine, London, United Kingdom, 3 Fluidigm Corporation, Single-cell Biology, San Francisco, USA, 4 University of Oxford, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom Background: Tumours are best viewed as a matrix of genetic and epigenetic heterogeneity which presents a rich substrate for selection and clonal evolution in response to therapy. Childhood Acute Lymphoblastic Leukaemia (cALL) is a paradigmatic disease for which static snapshots of mutational diversity at diagnosis revealed genetically distinct subclones arranged in complex branching architectures and identified genetic variegation in leukaemia propagating cells. The molecular mechanisms driving relapse of cALL are still, however, largely unknown and whether genetic or epigenetic factors can cause therapeutic resistance remains to be investigated. Material and Methods: To obtain a “real-time” longitudinal analysis of subclonal dynamics through treatment we established an in vivo mouse model that allows a patient tumour to be independently exposed to treatment multiple times. This has allowed us to track the fate of genetic subclones at single cell resolution by means of multicolour FISH with five mutation markers, enabling us to distinguish between deterministic and stochastic mechanisms of
selection during therapy. Single cell whole genome sequencing was adopted to extend the analysis’ resolution. Secondary transplantation and limiting dilution assays were used to assess the leukaemia initiating ability of resistant cells, and small cell number RNAseq (SMART-seq) was adopted to transcriptionally profile the latter. Results: Treatment of sensitive ALLs results in a striking reduction in leukaemic burden, but the overall extent of genetic diversity is unaffected, suggesting that resistance in ALL is largely independent of genetic variegation. Nevertheless, limiting dilution secondary transplantation assays suggest that exposure to treatment affects the functional properties of the tumour, enriching for cells with leukaemia initiating potential. Trascriptome analysis reveals that treatment-exposed cells express features characteristic of more primitive haematopoietic cells. Upon chemotherapy withdrawal cells revert to a treatment-na¨ıve signature consistent with the selection during treatment of a population of cells with stem-like properties. To further investigate the full extent of genetic heterogeneity and its relation to these transcriptional features of resistance, we are currently complementing our genetic studies with higher resolution single cell whole genome sequencing of selected specimens. Conclusions: Overall this analysis provides novel insight into the contribution of genetic and transcriptional/epigenetic heterogeneity to resistance to cytotoxic chemotherapy. In the case of cALL phenotypic heterogeneity appears to play a larger role then genetic diversity, particularly with regards to cell cycle state and developmental stage. No conflict of interest. 483 sept4/Arts regulates stem cell apoptosis and skin regeneration Y. Fuchs1 , S. Brown2 , T. Gorenc2 , J. Rodriguez2 , E. Fuchs3 , H. Steller2 . 1 Technion − Israel Institute of Technology, Lokey Center for Life Sciences and Engineering and The Faculty of Biology, Haifa, Israel, 2 Rockefeller University/HHMI, Strang Laboratory of Apoptosis and Cancer Biology, New York, USA, 3 Rockefeller University/HHMI, Laboratory of Mammalian Cell Biology and Development, New York, USA Adult stem cells are essential for tissue homeostasis and wound repair. Their proliferative capacity must be tightly regulated to prevent the emergence of unwanted and potentially dangerous cells, such as cancer cells. We found that mice deficient for the proapoptotic Sept4/ARTS gene have elevated numbers of hair follicle stem cells (HFSCs) that are protected against apoptosis. Sept4/ARTS−/− mice display dramatic improvement in wound healing and regeneration of hair follicles. These phenotypes depend on HFSCs, as indicated by lineage tracing. Inactivation of XIAP, a direct target of ARTS, abrogated these phenotypes and impaired wound healing. Our results indicate that apoptosis plays an important role in regulating stem cell-dependent regeneration and suggest that this pathway may be a target for regenerative medicine. Reference(s) Fuchs et al., Science 2013. No conflict of interest. 484 Tumor progression marker for breast cancer − survivin gene (BIRC5) Y. Shlyakhtunou1 . 1 Vitebsk State Order of Peoples’ Friendship Medical University, Oncology, Vitebsk, Belarus Breast cancer is a leader in the structure of morbidity and mortality of the female population from malignant tumors. Distant metastases are the main cause of death of patients, a substrate for the development of which are circulating tumor cells (CTCs). However, only one-search ethics cells is not sufficient to form a complete picture of the nature and course of the tumor process in individual patients. Determination of the expression of tumor-genes responsible for the different processes of tumor progression allows a more complete picture. Such genes include the gene survivin (BIRC5) family of inhibitors of apoptosis (IAP). Objective: To study the expression of the gene survivin in the tumor tissue of breast carcinoma, as well as CTCs in the peripheral blood of patients with breast cancer. Material and Methods: Using real-time PCR was investigated expression of the gene survivin in 16 samples of primary invasive ductal carcinoma of the breast, three samples of benign tumors − fibroadenoma of the breast, as well as 26 samples of peripheral blood of patients with breast cancer at various stages of tumor and stage specific treatment, and 3 healthy people are controlled. After homogenization of frozen tumor samples was extracted RNA, followed by cDNA synthesis and determination of expression of survivin. Were isolated from peripheral blood using CTCs microspheres carrying antibodies to EpCAM, after several stages passed as tumor RNA extraction followed by standard stages real-time PCR. Results: In primary breast carcinoma was determined by high expression of survivin gene in all 16 samples with the average value (M±m) 1.40±0.49 (min 1.21; max 3.41). The highest figures were determined expression in tumors of medium and high grade (G II−III) with lymphovenous invasion
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
adhesion molecules in the superficial tumor, and with mesenchymal inductors, such as ETV5, and MAPK/ERK pathway at the invasive front. No conflict of interest. 480 BRAF inhibition promotes BRAF mutant human melanoma cell survival under nutrient-deprived conditions through activation of mitochondrial metabolism T. Delgado-Goni1 , S. Wantuch2 , P. Workman3 , R. Marais4 , M.O. Leach1 , M. Beloueche-Babari1 . 1 Institute of Cancer Research, Radiotherapy and Imaging, London, United Kingdom, 2 Institute of Cancer Research, Breast Cancer Research, London, United Kingdom, 3 Institute of Cancer Research, Cancer Therapeutics Unit, London, United Kingdom, 4 Cancer Research UK Manchester Institute, Molecular Oncology, Manchester, United Kingdom Background: BRAF-MEK1/2 signaling inhibitors have shown remarkable clinical activity in BRAF-mutant melanoma. However, responses are not durable suggesting activation of alternative tumour survival mechanisms that allow resistance to therapy. This work investigates the re-programming of glucose metabolism following BRAF inhibition as an adaptive response, and its significance for cell survival in a nutrient depleted environment. Material and Methods: WM266.4 (BRAFV600D) cells were treated with the BRAF inhibitor vemurafenib (2mM, 24 h) in standard culture media containing 100% 5mM [1-13C]glucose. Cell extracts were analyzed by 13C NMR spectroscopy to assess alterations in glucose metabolism. To assess the significance of these alterations for cell survival and for their dependence on glycolysis, glutamine and TCA metabolism, cell counts were obtained from WM266.4 and SKMEL28 (BRAFV600E) cells grown in different nutrientdeprived conditions, with or without vemurafenib, for 48 h: 1mM glucose, 1mM glucose without glutamine and 1mM glucose without glutamine and pyruvate. Results: Vemurafenib induced a reduction in [3-13C] extracellular lactate in treated WM266.4 cells with respect to controls (62.9±13.1%; P = 0.01). A significant decrease in the intracellular [3-13C]lactate levels after treatment (53.1±18.6%, P = 0.01), concomitant with a significant increase in [1-13C]glucose (up to 286.9±105.5%, P = 0.04), indicated decreased glycolysis and glucose utilization after internalization. A significant elevation in [2-13C] and [3-13C]glutamate (to 185.2±42.6% and 171.4±38.9% of controls, respectively, P < 0.05) and [2-13C]glutamine (135.1±14.1, P = 0.01) in treated cells indicated an increased pyruvate carboxylase (PC) flux, consistent with an increased [2-13C]/[4-13C] glutamate ratio (from 0.10±0.04 to 0.30±0.04 (P = 0.02). Accordingly, the enzyme activity of PC showed a significant increase (159±67%, P = 0.04) under BRAF inhibition. WM266.4 and SKMEL28 cells exhibited a significant reduction in cell counts in low glucose media that was emphasised when glutamine was removed. However, the number of viable cells was significantly higher in treated samples, indicating that vemurafenib reduces the dependency of these cells on glucose (p = 0.03 and p = 0.01 respectively) and glutamine (p < 0.01) for proliferation and survival. When pyruvate was removed, the growth advantage under treatment was abolished for both lines, consistent with the dependency of treated cells on mitochondrial metabolism, with PC flux requiring pyruvate availability. Conclusion: BRAF inhibition with vemurafenib in BRAF mutant human melanoma cells alters glucose utilization and activates mitochondrial metabolism with consequences that enable improved survival under nutrientdeprived conditions. This metabolic shift may enable survival and the emergence of resistant clones following treatment. No conflict of interest. 481 Single cell analysis of intratumour heterogeneity in childhood acute lymphoblastic leukaemia V. Turati1 , H. Mike2 , J. Herrero1 , A. John1 , S. Richardson1 , B. Gaal1 , L. Mark3 , S.E. Jacobsen4 , T. Enver1 . 1 UCL Cancer Institute, Cancer Biology, London, United Kingdom, 2 Institute of Child Health, Genetics & Genomic Medicine, London, United Kingdom, 3 Fluidigm Corporation, Single-cell Biology, San Francisco, USA, 4 University of Oxford, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom Background: Tumours are best viewed as a matrix of genetic and epigenetic heterogeneity which presents a rich substrate for selection and clonal evolution in response to therapy. Childhood Acute Lymphoblastic Leukaemia (cALL) is a paradigmatic disease for which static snapshots of mutational diversity at diagnosis revealed genetically distinct subclones arranged in complex branching architectures and identified genetic variegation in leukaemia propagating cells. The molecular mechanisms driving relapse of cALL are still, however, largely unknown and whether genetic or epigenetic factors can cause therapeutic resistance remains to be investigated. Material and Methods: To obtain a “real-time” longitudinal analysis of subclonal dynamics through treatment we established an in vivo mouse model that allows a patient tumour to be independently exposed to treatment multiple times. This has allowed us to track the fate of genetic subclones at single cell resolution by means of multicolour FISH with five mutation markers, enabling us to distinguish between deterministic and stochastic mechanisms of
selection during therapy. Single cell whole genome sequencing was adopted to extend the analysis’ resolution. Secondary transplantation and limiting dilution assays were used to assess the leukaemia initiating ability of resistant cells, and small cell number RNAseq (SMART-seq) was adopted to transcriptionally profile the latter. Results: Treatment of sensitive ALLs results in a striking reduction in leukaemic burden, but the overall extent of genetic diversity is unaffected, suggesting that resistance in ALL is largely independent of genetic variegation. Nevertheless, limiting dilution secondary transplantation assays suggest that exposure to treatment affects the functional properties of the tumour, enriching for cells with leukaemia initiating potential. Trascriptome analysis reveals that treatment-exposed cells express features characteristic of more primitive haematopoietic cells. Upon chemotherapy withdrawal cells revert to a treatment-na¨ıve signature consistent with the selection during treatment of a population of cells with stem-like properties. To further investigate the full extent of genetic heterogeneity and its relation to these transcriptional features of resistance, we are currently complementing our genetic studies with higher resolution single cell whole genome sequencing of selected specimens. Conclusions: Overall this analysis provides novel insight into the contribution of genetic and transcriptional/epigenetic heterogeneity to resistance to cytotoxic chemotherapy. In the case of cALL phenotypic heterogeneity appears to play a larger role then genetic diversity, particularly with regards to cell cycle state and developmental stage. No conflict of interest. 483 sept4/Arts regulates stem cell apoptosis and skin regeneration Y. Fuchs1 , S. Brown2 , T. Gorenc2 , J. Rodriguez2 , E. Fuchs3 , H. Steller2 . 1 Technion − Israel Institute of Technology, Lokey Center for Life Sciences and Engineering and The Faculty of Biology, Haifa, Israel, 2 Rockefeller University/HHMI, Strang Laboratory of Apoptosis and Cancer Biology, New York, USA, 3 Rockefeller University/HHMI, Laboratory of Mammalian Cell Biology and Development, New York, USA Adult stem cells are essential for tissue homeostasis and wound repair. Their proliferative capacity must be tightly regulated to prevent the emergence of unwanted and potentially dangerous cells, such as cancer cells. We found that mice deficient for the proapoptotic Sept4/ARTS gene have elevated numbers of hair follicle stem cells (HFSCs) that are protected against apoptosis. Sept4/ARTS−/− mice display dramatic improvement in wound healing and regeneration of hair follicles. These phenotypes depend on HFSCs, as indicated by lineage tracing. Inactivation of XIAP, a direct target of ARTS, abrogated these phenotypes and impaired wound healing. Our results indicate that apoptosis plays an important role in regulating stem cell-dependent regeneration and suggest that this pathway may be a target for regenerative medicine. Reference(s) Fuchs et al., Science 2013. No conflict of interest. 484 Tumor progression marker for breast cancer − survivin gene (BIRC5) Y. Shlyakhtunou1 . 1 Vitebsk State Order of Peoples’ Friendship Medical University, Oncology, Vitebsk, Belarus Breast cancer is a leader in the structure of morbidity and mortality of the female population from malignant tumors. Distant metastases are the main cause of death of patients, a substrate for the development of which are circulating tumor cells (CTCs). However, only one-search ethics cells is not sufficient to form a complete picture of the nature and course of the tumor process in individual patients. Determination of the expression of tumor-genes responsible for the different processes of tumor progression allows a more complete picture. Such genes include the gene survivin (BIRC5) family of inhibitors of apoptosis (IAP). Objective: To study the expression of the gene survivin in the tumor tissue of breast carcinoma, as well as CTCs in the peripheral blood of patients with breast cancer. Material and Methods: Using real-time PCR was investigated expression of the gene survivin in 16 samples of primary invasive ductal carcinoma of the breast, three samples of benign tumors − fibroadenoma of the breast, as well as 26 samples of peripheral blood of patients with breast cancer at various stages of tumor and stage specific treatment, and 3 healthy people are controlled. After homogenization of frozen tumor samples was extracted RNA, followed by cDNA synthesis and determination of expression of survivin. Were isolated from peripheral blood using CTCs microspheres carrying antibodies to EpCAM, after several stages passed as tumor RNA extraction followed by standard stages real-time PCR. Results: In primary breast carcinoma was determined by high expression of survivin gene in all 16 samples with the average value (M±m) 1.40±0.49 (min 1.21; max 3.41). The highest figures were determined expression in tumors of medium and high grade (G II−III) with lymphovenous invasion