Kranc, Kamil

Forrester, Lesley

Panagopoulou, Theoni Ioanna

Medical Research Council (MRC)

2017-09-22T14:33:57Z

2017-09-22T14:33:57Z

2017-07-08

The finely tuned regulation of haematopoiesis is crucial in order to maintain life-long haematopoiesis. The disruption of the balance among cell fates, can lead to malignant transformation. It has become increasingly evident that the metabolic regulation haematopoietic stem cells is critical for stem cell fate decisions. Haematopoietic stem cells reside in a hypoxic microenvironment within the bone marrow and are thought to mainly utilize glycolysis rather than oxidative phosphorylation in order to maintain their pool. However recent evidence suggests that oxidative phosphorylation is critical for quiescent HSCs and in several cases, for leukaemic stem cells (LSCs). One of the key parts of mitochondrial respiration is the tricarboxylic cycle (TCA), providing co-factors for its efficient activity. The TCA functions by catalysing the oxidation of pyruvate via key enzymatic activities. A key component of the TCA cycle is fumarate hydratase (Fh1) which catalyses the hydration of fumarate into malate within the mitochondria, but also catalyses the same reaction in the cytoplasm. FH is a tumour suppressor in human lyomeioma and renal kidney cancer (HLRCC). Previous work conducted by our team has shown that Fh1 is essential for foetal and adult haematopoiesis, as Fh1 deletion within the haematopoietic system is embryonic lethal. Furthermore, conditional deletion of Fh1 in donor cells of the Mx1-Cre system that were injected in lethally irradiated recipients, resulted in the complete reduction of their chimerism in the peripheral blood of recipient mice. Mechanistically, these phenotypes were mostly associated with supra-physiological levels of fumarate as a result of Fh1 deletion. Interestingly, by employing mice that ubiquitously express the human cytosolic isoform of FH (FHCyt, which lacks the mitochondrial targeting sequence and therefore is excluded from the mitochondria), we rescued the embryonic lethality that Fh1 causes, and reduced the levels of fumarate. Importantly, although FHCyt expression restored fumarate-associated lethality, it did not restore the mitochondrial defects, allowing us to study the importance of genetically intact TCA in the context of haematopoiesis. Here I investigated the impact that a genetic truncation of the TCA cycle (as a result of the lack of the mitochondrial isoform of Fh1) has on leukaemic transformation and on aged haematopoiesis. Fh1fl/fl; FHCyt; Vav-iCre mice of approximately 60 weeks old displayed and expansion in the pool of early stem and progenitor compartment (Lin- Sca-1+ c-Kit+), as well as in the early progenitors HPC-1 (LSK CD48+ CD150-) and HPC-2 (LSK CD48+ CD150+). Furthermore, the mice exhibited a drastic depletion of B cells (CD19+ B220+) and an expansion in the frequency of the myeloid compartment (Mac-1+ Gr1+). In order to assess the importance of the TCA cycle in malignant transformation, I isolated stem and progenitor cells from Fh1fl/fl; FHCyt; Vav-iCre (and control (Fh1fl/fl; FHCyt Vav-iCre negative or Fh1fl/fl Vav-iCre negative)) E 14.5 day old embryos and infected them with retroviruses expressing Meis1 and Hoxa9, and generated pre-leukaemic cells (pre-LCs). Genetically intact TCA was required for the efficient generation of leukaemia-initiating cells (LICs), as injection of pre-LCs lacking mitochondrial Fh1 into sub-lethally irradiated recipient mice, resulted in 76 % of leukaemia-free mice while injection of control pre-LCs resulted in 25 % of leukaemia-free mice. However, the genetic perturbation of the TCA did not exert and effect on the long-term self-renewal capacity of LICs. Inducible deletion of mitochondrial Fh1 in established LICs of the Mx1-Cre background using poly (I:C) did not affect their ability to generate AML in primary and secondary recipient mice. These data indicate that genetically intact TCA is required for the efficient generation of LICs in vivo but is dispensable for their long-term self-renewal capacity, highlighting the metabolic rewiring that occurs at different stages of leukaemic transformation. In an effort to understand whether, similarly to HLRCC, Fh1 plays a tumour-suppressive role in malignant haematopoiesis, I isolated LSK cells from the foetal liver of E 14.5 old embryos lacking both isoforms of Fh1. Fh1fl/fl; Vav-iCre cells transduced with Meis1/Hoxa9 or MLL-AF9, MLL-ENL, AML-ETO (chromosomal translocations involved in AML development) -expressing retroviruses, failed to generate colonies in methylcellulose, indicating that stem and progenitor cells require Fh1 to undergo in vitro transformation by these oncogenes. Furthermore, acute deletion of Fh1 (via the use of lentivirally-expressed Cre) in pre-LCs generated using the Meis1/Hoxa9 retroviruses, rendered them unable to generate colonies in methylcellulose, indicating that Fh1 is required for the self-renewal capacity of pre- LCs in vitro. Similarly, when LICs (Fh1fl/fl; Vav-iCre negative) isolated from primary recipient mice were infected with Cre to induce deletion of Fh1, they were unable to generate colonies indicating that Fh1 is required for the self-renewal capacity of LICs in vitro. Finally, in order to identify whether Fh1 is important for LIC self-renewal in vivo I generated Fh1fl/fl; Mx1-Cre pre-LCs by infecting stem and progenitor cells of E 14.5 embryos with Meis1/Hoxa9 retroviruses, and injected them into sub-lethally irradiated mice. After the mice developed AML, I induced the deletion of Fh1, by injecting the mice with poly (I:C). Interestingly, the percentage of LICs in the peripheral blood of recipient mice was drastically decreased, leaving recipient mice leukaemia-free for the remaining time they were monitored. Surprisingly however, approximately 50 % of the recipient mice exhibited a drastic increase in LIC chimerism after two weeks post poly (I:C). Assessment of LICs isolated from recipient mice indicated that Fh1 was fully deleted. These data indicate that while in some cases Fh1 is required for LIC self-renewal in vivo, in other cases it is dispensable. Therefore, the tumour-suppressive roles of Fh1 are likely tissue-specific and do not extend to haematopoietic cells. Overall, this study agrees with published work supporting the notion that intact mitochondrial respiration is important (in varying degrees), in both the contexts of normal and malignant haematopoiesis.

http://hdl.handle.net/1842/23584

en

The University of Edinburgh

Guitart A, Panagopoulou T, Vukovic M, Sepulveda C, Sas Z, Gonzalez MV, Allen L, Armesilla-Diaz A,Edwards-Hicks J, Wills J, Easterbrook A, Corman D, Pollard P, Morton N, Carter R, Finch A, Kranc KR. Fumarate hydratase is a critical metabolic regulator of haematopoietic stem cell functions. Journal of Experimental Medicine.

Vukovic M, Subramani C, Sepulveda C, Guitart A, Allen L, Panagopoulou T, Holyoake T, Ratcliffe PJ, Kranc KR. Adult haematopoietic stem cells lacking Hif-1α self-renew normally. Blood. 2016; 127, 2841-2846.

Vukovic M, Guitart AV, Sepulveda C, Villacreces A, O'Duibhir E, Panagopoulou T, Ivens A, Menendez-Gonzalez J, Iglesias JM, Allen L, Glykofrydis F, Subramani C, Armesilla-Diaz A, Post AE, Schaak K, Gezer D, So CW, Holyoake TL, Wood A, O'Carroll D, Ratcliffe PJ, Kranc KR. Hif-1α and Hif-2α synergize to suppress AML development but are dispensable for disease maintenance. J Exp Med. 2015 Dec14;212(13):2223-34.

fumarate hydratase

acute myeloid leukaemia

Role of the metabolic enzyme fumarate hydratase in aged haematopoiesis and malignant transformation

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy