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Dual function of fumarate hydratase in haematopoietic stem cell fates
Short Talk Presentations/Experimental Hematology 43 (2015) S45–S49
S49
2022 - AGE-ASSOCIATED CLONAL DOMINANCE OF MYELOID-BIASED HSC IS UNDERWRITTEN BY UNIQUE TRANSCRIPTIONAL AND EPIGENETIC ALTERATIONS Isabel Beerman1,2, Michael Ziller1, Amy Vandiver3, Raphael Gaudin2, Tomas Kirchhausen2, Andrew Feinberg3, Alexander Meissner1, and Derrick Rossi1,2 1 Harvard University, Boston, Massachusetts, USA; 2Boston Children’s Hospital, Boston, Massachusetts, USA; 3Johns Hopkins School of Medicine, Baltimore, Maryland, USA
2024 - DUAL FUNCTION OF FUMARATE HYDRATASE IN HAEMATOPOIETIC STEM CELL FATES Amelie V. Guitart1, Milica Vukovic1, Catarina Sepulveda1, Theano Panagopoulou1, Lewis Allen1, Patrick Pollard2, Nik Morton3, Andy Finch2, and Kamil Kranc1,2 1 MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom; 2Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom; 3Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom
Age-associated changes of the functional potential of hematopoietic stem cells (HSCs) are believed to underlie a variety of age-dependent blood cell pathophysiologies and contribute to the overall decline of the hematopoietic system. Modifications of the epigenetic landscape, DNA damage accumulation, and changes in cell polarity have all been identified as potential factors contributing to the diminished functional potential of aged HSCs. Additionally, changes in the HSC’s clonal composition, with a predominance of myeloid-biased clones in the aged compartment, have been implicated in the reduction of immune competence and elevated predisposition to myelogenous diseases in the elderly. To explore age-associated changes in the lineage-biased subsets, we analyzed genome-wide expression profiles of the CD150 subsets of HSCs isolated from young and aged mice and defined transcripts with expression patterns unique to each aging subset of hematopoietic stem cells. We also established the epigenetic profiles of the aging CD150 subsets and defined significant alterations in the DNA methylation patterns between the subsets and also during aging. The methylation and transcriptional profiles were then correlated to establish if epigenetic changes contributed to the altered expression in the HSC subsets. We also demonstrated that there is no significant difference in the DNA damage accumulation of the aged lineage-biased subsets when assaying for strand break accrual. Finally, using time-lapse microscopy on cells purified from the HSC reporter mouse (Fgd5ZsGreen-ZsGreen/+), we have examined the lineal hierarchy for the symmetric and asymmetric division preferences of the CD150 subsets. Taken together, we have defined the transcriptional and epigenetic profiles that underlie the age-associated dominance of the myeloid-biased HSCs.
Long-term hematopoietic stem cells (HSC) rely on glycolysis but switch to oxydative phosphorylation to comply with the energetic demands of self-renewal or differentiation. However, the tricarboxylic acid (TCA) cycle’s contribution to HSC metabolism remains unknown. In this study, we conditionally deleted fumarate hydratase (FH), one of the key enzymes of the TCA, which drives the hydration of fumarate into malate using a Cre-loxP in vivo approach. We demonstrate that Fh1 is indispensable to the haematopoietic system as its deletion is embryonic lethal due to a complete failure of the foetal liver hematopoietic function. Using a different mouse model, we validated these results in the adult. Metabolic analyses (combining LC/MS and SeaHorse approach) of Fh1-deficient HSCs and progenitor cells revealed that in addition to having a compromised TCA cycle, cellular concentration of fumarate is highly increased in these cell populations. To discriminate which of these two metabolic changes triggers the observed phenotypes we reintroduced the cytosolic isoform of Fh1 thereby restoring intracellular fumarate concentration while maintaining the TCA cycle impairment. This genetic addition is enough to rescue the embryonic lethality. Adult mice are viable and capable of ageing but present a myeloid lineage skewing. Serial transplantation revealed that HSCs harbouring a compromised TCA cycle are not able to maintain long-term self-renewal properties and have a very limited lymphopoietic potential. This work demonstrate an absolute requirement for Fh1 for embryonic and adult haematopoiesis. From the dual function of Fh1, we established that efficient clearance of fumarate is critical for hematopoietic function and that the TCA cycle is required for normal lymphopoiesis and for HSCs survival under stress condition.
2023 - TARGETING PRE-LEUKEMIC STEM CELLS IN T-ACUTE LYMPHOBLASTIC LEUKEMIA Bastien Gerby, Guillaume Laflamme, Benjamin Kwok, Philippe Roux, Josee Hebert, Guy Sauvageau, and Trang Hoang Institute of Research in Immunology and Cancer, Montreal, Quebec, Canada Current chemotherapy of pediatric T cell acute lymphoblastic leukemia (T-ALL) efficiently reduces the tumor mass and induces long-term remission. Nonetheless, preleukemic stem cells (pre-LSCs) that were not active during treatment are spared and can eventually evolve to malignancy causing relapse. Moreover, current treatment is associated with undesirable consequences including reproductive, obesity and muscoluskeletal problems and remains ineffective in adolescent and adult T-ALL. Using transgenic mouse models expressing the SCL and LMO1 oncogenic transcription factors in the thymus, we recently showed that these oncogenes reprogram normal pro-T cells into aberrantly self-renewing pre-LSCs1. Based on the evidence that pre-LSCs in AML are resistant to current induction therapy2,3, we designed a strategy to identify drugs that inhibit the cell viability of pre-LSCs maintained by co-culture with stromal cells expressing the NOTCH1 ligand DL4 to mimick the thymic microenvironement. We developed a robust high throughput (HTS) phenotypic screen of a library of 3581 chemical compounds. This screening confirmed chemotherapeutic agents commonly used in the treatment of T-ALL and revealed new compounds. Surprisingly for relatively quiescent cells, we identified microtubule-targeting drugs (MTDs) as the most robust and consistently active family of inhibitors, suggesting new and unexpected function of MTDs. Indeed, we report that the MTD UM0119979 abrogates pre-LSCs viability and self-renewal activity by inhibiting SCL function. Moreover, the UM0119979 exhibits a potent antileukemic effect on primary human T-ALL blasts in vitro and in vivo without affecting normal HSCs functions. Together, our findings demonstrate the feasibility of targeting pre-LSCs and identify a new drug for consideration in T-ALL treatment.
S49
2022 - AGE-ASSOCIATED CLONAL DOMINANCE OF MYELOID-BIASED HSC IS UNDERWRITTEN BY UNIQUE TRANSCRIPTIONAL AND EPIGENETIC ALTERATIONS Isabel Beerman1,2, Michael Ziller1, Amy Vandiver3, Raphael Gaudin2, Tomas Kirchhausen2, Andrew Feinberg3, Alexander Meissner1, and Derrick Rossi1,2 1 Harvard University, Boston, Massachusetts, USA; 2Boston Children’s Hospital, Boston, Massachusetts, USA; 3Johns Hopkins School of Medicine, Baltimore, Maryland, USA
2024 - DUAL FUNCTION OF FUMARATE HYDRATASE IN HAEMATOPOIETIC STEM CELL FATES Amelie V. Guitart1, Milica Vukovic1, Catarina Sepulveda1, Theano Panagopoulou1, Lewis Allen1, Patrick Pollard2, Nik Morton3, Andy Finch2, and Kamil Kranc1,2 1 MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom; 2Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom; 3Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom
Age-associated changes of the functional potential of hematopoietic stem cells (HSCs) are believed to underlie a variety of age-dependent blood cell pathophysiologies and contribute to the overall decline of the hematopoietic system. Modifications of the epigenetic landscape, DNA damage accumulation, and changes in cell polarity have all been identified as potential factors contributing to the diminished functional potential of aged HSCs. Additionally, changes in the HSC’s clonal composition, with a predominance of myeloid-biased clones in the aged compartment, have been implicated in the reduction of immune competence and elevated predisposition to myelogenous diseases in the elderly. To explore age-associated changes in the lineage-biased subsets, we analyzed genome-wide expression profiles of the CD150 subsets of HSCs isolated from young and aged mice and defined transcripts with expression patterns unique to each aging subset of hematopoietic stem cells. We also established the epigenetic profiles of the aging CD150 subsets and defined significant alterations in the DNA methylation patterns between the subsets and also during aging. The methylation and transcriptional profiles were then correlated to establish if epigenetic changes contributed to the altered expression in the HSC subsets. We also demonstrated that there is no significant difference in the DNA damage accumulation of the aged lineage-biased subsets when assaying for strand break accrual. Finally, using time-lapse microscopy on cells purified from the HSC reporter mouse (Fgd5ZsGreen-ZsGreen/+), we have examined the lineal hierarchy for the symmetric and asymmetric division preferences of the CD150 subsets. Taken together, we have defined the transcriptional and epigenetic profiles that underlie the age-associated dominance of the myeloid-biased HSCs.
Long-term hematopoietic stem cells (HSC) rely on glycolysis but switch to oxydative phosphorylation to comply with the energetic demands of self-renewal or differentiation. However, the tricarboxylic acid (TCA) cycle’s contribution to HSC metabolism remains unknown. In this study, we conditionally deleted fumarate hydratase (FH), one of the key enzymes of the TCA, which drives the hydration of fumarate into malate using a Cre-loxP in vivo approach. We demonstrate that Fh1 is indispensable to the haematopoietic system as its deletion is embryonic lethal due to a complete failure of the foetal liver hematopoietic function. Using a different mouse model, we validated these results in the adult. Metabolic analyses (combining LC/MS and SeaHorse approach) of Fh1-deficient HSCs and progenitor cells revealed that in addition to having a compromised TCA cycle, cellular concentration of fumarate is highly increased in these cell populations. To discriminate which of these two metabolic changes triggers the observed phenotypes we reintroduced the cytosolic isoform of Fh1 thereby restoring intracellular fumarate concentration while maintaining the TCA cycle impairment. This genetic addition is enough to rescue the embryonic lethality. Adult mice are viable and capable of ageing but present a myeloid lineage skewing. Serial transplantation revealed that HSCs harbouring a compromised TCA cycle are not able to maintain long-term self-renewal properties and have a very limited lymphopoietic potential. This work demonstrate an absolute requirement for Fh1 for embryonic and adult haematopoiesis. From the dual function of Fh1, we established that efficient clearance of fumarate is critical for hematopoietic function and that the TCA cycle is required for normal lymphopoiesis and for HSCs survival under stress condition.
2023 - TARGETING PRE-LEUKEMIC STEM CELLS IN T-ACUTE LYMPHOBLASTIC LEUKEMIA Bastien Gerby, Guillaume Laflamme, Benjamin Kwok, Philippe Roux, Josee Hebert, Guy Sauvageau, and Trang Hoang Institute of Research in Immunology and Cancer, Montreal, Quebec, Canada Current chemotherapy of pediatric T cell acute lymphoblastic leukemia (T-ALL) efficiently reduces the tumor mass and induces long-term remission. Nonetheless, preleukemic stem cells (pre-LSCs) that were not active during treatment are spared and can eventually evolve to malignancy causing relapse. Moreover, current treatment is associated with undesirable consequences including reproductive, obesity and muscoluskeletal problems and remains ineffective in adolescent and adult T-ALL. Using transgenic mouse models expressing the SCL and LMO1 oncogenic transcription factors in the thymus, we recently showed that these oncogenes reprogram normal pro-T cells into aberrantly self-renewing pre-LSCs1. Based on the evidence that pre-LSCs in AML are resistant to current induction therapy2,3, we designed a strategy to identify drugs that inhibit the cell viability of pre-LSCs maintained by co-culture with stromal cells expressing the NOTCH1 ligand DL4 to mimick the thymic microenvironement. We developed a robust high throughput (HTS) phenotypic screen of a library of 3581 chemical compounds. This screening confirmed chemotherapeutic agents commonly used in the treatment of T-ALL and revealed new compounds. Surprisingly for relatively quiescent cells, we identified microtubule-targeting drugs (MTDs) as the most robust and consistently active family of inhibitors, suggesting new and unexpected function of MTDs. Indeed, we report that the MTD UM0119979 abrogates pre-LSCs viability and self-renewal activity by inhibiting SCL function. Moreover, the UM0119979 exhibits a potent antileukemic effect on primary human T-ALL blasts in vitro and in vivo without affecting normal HSCs functions. Together, our findings demonstrate the feasibility of targeting pre-LSCs and identify a new drug for consideration in T-ALL treatment.