Final Report Summary - DECIDE (Decision-making within cells and differentiation entity therapies)
The overarching aims of DECIDE have been to: 1) contribute towards a better understanding of haematopoiesis and 2) generate new analogues of 1 alpha, 25-dihydroxyvitamin D3 (1,25D) that have a reduced calcaemic action and are potent anti-cancer agents (termed an1,25D). Outcomes from the work by the Fellows have moved forward both of these areas.
For decades the pathways of development for various blood cell types have been depicted as a hierarchical scheme. The apex is the multipotent haematopoietic stem cell (HSC) which first chooses between either the myeloid or lymphoid developmental options. Cells then become more restricted in their lineage potentials in a stepwise manner through oligo-potent haematopoietic progenitors (HPC). This classical view has been challenged by many models including our pairwise model. In this model, lineage choices are viewed as a continuum, there aren’t underlying branch points and the ordering of invariant relationships between each of the cell lineages is based on the sets of incomplete potentials demonstrated for various HPC. In recent years, two notions that substantially change the way we view haematopoiesis have been of interest to investigators. The first is that HSCs immediately adopt a single fate without transiting a series of intermediate HPC, as supported by the notion that HSCs are either multipotent or uni-potent. Second is the viewpoint that some haematopoietic cytokines instruct lineage fate in addition to providing signals for cell survival and proliferation.
Do HSC and HPC have a uni-lineage signature? Early Progenitors with Lymphoid and Myeloid potentials (EPLM) are viewed as one population of cells. RNA sequencing of single EPLM has revealed at least 3 groups of cells, with a B cell, dendritic or myeloid signature. Single cell PCR studies have revealed HSC that express the mRNAs for the receptors for Flt3 ligand (Flt3L) or erythropoietin or macrophage colony-stimulating factor. Flt3L and interleukin 7 (IL-7) are viewed as instructive for B cell development. Instead, work has shown that Flt3L provides a proliferative signal and IL-7 a signal for survival to B cell committed progenitors. These cytokines have been used, with stem cell factor, to grow bone marrow and foetal liver pro-B cells in vitro. Studies of a Flt3L transgenic mouse support the notion that Flt3L can divert cells away from the megakaryocyte and erythroid pathways towards myeloid and lymphoid development. But, we have been unable to observe an instructive action of Flt3L on single HSCs when cultured in a high amount of Flt3L.
Mesenchymal stromal cells are important for haematopoiesis as revealed by ablation of FAP+ve stromal cells in the bone marrow of mice disrupts the integrity of haematopoiesis resulting in cytopenia. Experiments to investigate the interactions of MSC with developing HSC and HPC have been conducted almost entirely under normoxia (20% oxygen). But, HSC reside in a hypoxic environment and it has been shown that this changes the nature of the secretome of MSC. Of importance to bone marrow transplantation is that MSCs cells have been shown to be highly radio-resistant, this radio-resistance is increased in hypoxic conditions and Hif-1alpha, which is induced by hypoxia, plays an important role in the DNA damage response to radiation.
The findings from DECIDE support our continuum model of the availability of lineages options to HSC. Whilst a lineage signature is evident in some HSC and in their progeny, as for EPLM, other closely related fates are retained as clandestine options. Steady state haematopoiesis is often studied and clandestine options may be important when the system is stressed and there is an increased demand for a particular cell type. The extent to which cytokines, as presented by MSC and their secretome, play an instructive role during haematopoiesis requires further investigation.
Work in WP2 has led to the synthesis of new an1,25D that have a substantially reduced calcaemic action and that are potent anticancer agents. This is important because the long term limitation for the use of 1,25D in cancer therapy has been the achievement of an effective therapeutic dose because of its calcaemic action. The new an1,25Ds mean that a more effective dose of 1,25D is likely to be achievable and current cancer trials of 1,25D should benefit. The new an1,25D arose from reviewing structure and biological activities of existing an1,25Ds. Beneficial modifications were identified and new chemistry was required to synthesise even newer an1,25Ds. Two of the new analogues are substantially less calcaemic than 1,25D, as tested in mice, and they are a log fold more potent than 1,25D against acute myeloid leukaemia cell lines. They also potently decreased the in vitro cloning potential of the human colon cancer cell line HT29. The new an1,25D bind effectively to the receptor for 1,25D. However, an important finding is that the enhanced anticancer action of an1,25Ds does not correlate with their binding affinity or transactivation potency for the vitamin D receptor (VDR). Hence, the precise mode of action of the new an1,25D remains unclear. The influence of an1,25Ds on the conformation of VDR may mediate their selectivity or a balance between VDR-driven transcription events and rapid signals or an alternative receptor.
All-trans retinoic acid (ATRA) combined with arsenic trioxide have provided a cure for over 95% of patients with acute promyelocytic leukaemia. Attention was focussed on whether ATRA can prolong the survival of children with high-risk neuroblastoma (NB). ATRA together with depletion of polyamines (via an analogue verlindamycin) has been shown to enhance the differentiation of NB cells and be more effective than the use of either of the agents alone.
The two new an1,25D are promising candidates for development as anticancer agents. They still need to be tested in animal models of cancer and refined to render resistance to degradation and improve pharmacokinetics, lipophilicity and solubility. The beneficial modifications identified so far can also be extended to generate even more potent an1,25Ds with no calcaemic action.
The Fellows were trained in cell biology technologies, pharmaceuticals and business and generic skills at 4 Schools, held in Birmingham, Basel, Wroclaw and Galway. In Birmingham, the Fellows received training in management and project governance, research ethics, haematopoiesis, drug manufacturing practice and presentation skills. In Basel, Fellows received further training in presentation skills and scientists from Novartis provided excellent training in pharmaceuticals. The 3rd School provided training in protein science technologies, cancer stem cells and writing skills. The 4th School provide training in cell therapeutics and an1,25D as a therapeutic modality for AML and to ESRs to help them to brand themselves in regard to job seeking.
As to dissemination, thirty three papers have been published to date, 6 papers are soon to be submitted and the book entitled “Diversity, Versatility and Leukaemia”, by Brown and Sanchez-Garcia, appeared in 2016. An important paper is that describing the roles of Flt3 and IL-7 (doi: 10.1073/pnas.1613316113). The issue of The Journal of Steroid Biochemistry and Molecular Biology that was the Proceedings of the 18th Vitamin D Workshop contained a number of papers on new an1,25Ds and their activities. A commemorative issue of the International Journal of Molecular Sciences (IJMS), in honour of Prof. Milan Uskokovic and entitled “Role of vitamin D in cancer therapeutics", also contained a series of research articles on new an1,25Ds. An issue of the IJMS entitled “The biology and treatment of acute myeloid leukaemia” is being edited by Marcinkowska and Brown. The Fellows presented posters and/or gave talks at 33 conferences, attending at total of 42 meetings. There have been 12 public outreach events/activities, including the British, Lower Silesia, Poland, Big Bang, Think Corner and Fun Palace Science festivals and Curing Perfect (http://curingperfect.com). To date, 47,492 individuals worldwide have visited our website, 24 newsletters have been distributed to the 87 subscribers and our Twitter account has 69 followers.
Ten Fellows have been awarded their PhD, one is writing her thesis and one is on a 4 year program. Lilly von Münchow, Sharmin Nadkarni and Narasimha Rao were awarded suma cum laudae and Llucia Albertí Servera a PhD magna cum laudae, and Ciaran Mooney after two years work.
More information about the DECIDE project can be found at www.birmingham.ac.uk/DECIDE or by emailing the Co-ordinator Geoffrey Brown at g.brown@bham.ac.uk
For decades the pathways of development for various blood cell types have been depicted as a hierarchical scheme. The apex is the multipotent haematopoietic stem cell (HSC) which first chooses between either the myeloid or lymphoid developmental options. Cells then become more restricted in their lineage potentials in a stepwise manner through oligo-potent haematopoietic progenitors (HPC). This classical view has been challenged by many models including our pairwise model. In this model, lineage choices are viewed as a continuum, there aren’t underlying branch points and the ordering of invariant relationships between each of the cell lineages is based on the sets of incomplete potentials demonstrated for various HPC. In recent years, two notions that substantially change the way we view haematopoiesis have been of interest to investigators. The first is that HSCs immediately adopt a single fate without transiting a series of intermediate HPC, as supported by the notion that HSCs are either multipotent or uni-potent. Second is the viewpoint that some haematopoietic cytokines instruct lineage fate in addition to providing signals for cell survival and proliferation.
Do HSC and HPC have a uni-lineage signature? Early Progenitors with Lymphoid and Myeloid potentials (EPLM) are viewed as one population of cells. RNA sequencing of single EPLM has revealed at least 3 groups of cells, with a B cell, dendritic or myeloid signature. Single cell PCR studies have revealed HSC that express the mRNAs for the receptors for Flt3 ligand (Flt3L) or erythropoietin or macrophage colony-stimulating factor. Flt3L and interleukin 7 (IL-7) are viewed as instructive for B cell development. Instead, work has shown that Flt3L provides a proliferative signal and IL-7 a signal for survival to B cell committed progenitors. These cytokines have been used, with stem cell factor, to grow bone marrow and foetal liver pro-B cells in vitro. Studies of a Flt3L transgenic mouse support the notion that Flt3L can divert cells away from the megakaryocyte and erythroid pathways towards myeloid and lymphoid development. But, we have been unable to observe an instructive action of Flt3L on single HSCs when cultured in a high amount of Flt3L.
Mesenchymal stromal cells are important for haematopoiesis as revealed by ablation of FAP+ve stromal cells in the bone marrow of mice disrupts the integrity of haematopoiesis resulting in cytopenia. Experiments to investigate the interactions of MSC with developing HSC and HPC have been conducted almost entirely under normoxia (20% oxygen). But, HSC reside in a hypoxic environment and it has been shown that this changes the nature of the secretome of MSC. Of importance to bone marrow transplantation is that MSCs cells have been shown to be highly radio-resistant, this radio-resistance is increased in hypoxic conditions and Hif-1alpha, which is induced by hypoxia, plays an important role in the DNA damage response to radiation.
The findings from DECIDE support our continuum model of the availability of lineages options to HSC. Whilst a lineage signature is evident in some HSC and in their progeny, as for EPLM, other closely related fates are retained as clandestine options. Steady state haematopoiesis is often studied and clandestine options may be important when the system is stressed and there is an increased demand for a particular cell type. The extent to which cytokines, as presented by MSC and their secretome, play an instructive role during haematopoiesis requires further investigation.
Work in WP2 has led to the synthesis of new an1,25D that have a substantially reduced calcaemic action and that are potent anticancer agents. This is important because the long term limitation for the use of 1,25D in cancer therapy has been the achievement of an effective therapeutic dose because of its calcaemic action. The new an1,25Ds mean that a more effective dose of 1,25D is likely to be achievable and current cancer trials of 1,25D should benefit. The new an1,25D arose from reviewing structure and biological activities of existing an1,25Ds. Beneficial modifications were identified and new chemistry was required to synthesise even newer an1,25Ds. Two of the new analogues are substantially less calcaemic than 1,25D, as tested in mice, and they are a log fold more potent than 1,25D against acute myeloid leukaemia cell lines. They also potently decreased the in vitro cloning potential of the human colon cancer cell line HT29. The new an1,25D bind effectively to the receptor for 1,25D. However, an important finding is that the enhanced anticancer action of an1,25Ds does not correlate with their binding affinity or transactivation potency for the vitamin D receptor (VDR). Hence, the precise mode of action of the new an1,25D remains unclear. The influence of an1,25Ds on the conformation of VDR may mediate their selectivity or a balance between VDR-driven transcription events and rapid signals or an alternative receptor.
All-trans retinoic acid (ATRA) combined with arsenic trioxide have provided a cure for over 95% of patients with acute promyelocytic leukaemia. Attention was focussed on whether ATRA can prolong the survival of children with high-risk neuroblastoma (NB). ATRA together with depletion of polyamines (via an analogue verlindamycin) has been shown to enhance the differentiation of NB cells and be more effective than the use of either of the agents alone.
The two new an1,25D are promising candidates for development as anticancer agents. They still need to be tested in animal models of cancer and refined to render resistance to degradation and improve pharmacokinetics, lipophilicity and solubility. The beneficial modifications identified so far can also be extended to generate even more potent an1,25Ds with no calcaemic action.
The Fellows were trained in cell biology technologies, pharmaceuticals and business and generic skills at 4 Schools, held in Birmingham, Basel, Wroclaw and Galway. In Birmingham, the Fellows received training in management and project governance, research ethics, haematopoiesis, drug manufacturing practice and presentation skills. In Basel, Fellows received further training in presentation skills and scientists from Novartis provided excellent training in pharmaceuticals. The 3rd School provided training in protein science technologies, cancer stem cells and writing skills. The 4th School provide training in cell therapeutics and an1,25D as a therapeutic modality for AML and to ESRs to help them to brand themselves in regard to job seeking.
As to dissemination, thirty three papers have been published to date, 6 papers are soon to be submitted and the book entitled “Diversity, Versatility and Leukaemia”, by Brown and Sanchez-Garcia, appeared in 2016. An important paper is that describing the roles of Flt3 and IL-7 (doi: 10.1073/pnas.1613316113). The issue of The Journal of Steroid Biochemistry and Molecular Biology that was the Proceedings of the 18th Vitamin D Workshop contained a number of papers on new an1,25Ds and their activities. A commemorative issue of the International Journal of Molecular Sciences (IJMS), in honour of Prof. Milan Uskokovic and entitled “Role of vitamin D in cancer therapeutics", also contained a series of research articles on new an1,25Ds. An issue of the IJMS entitled “The biology and treatment of acute myeloid leukaemia” is being edited by Marcinkowska and Brown. The Fellows presented posters and/or gave talks at 33 conferences, attending at total of 42 meetings. There have been 12 public outreach events/activities, including the British, Lower Silesia, Poland, Big Bang, Think Corner and Fun Palace Science festivals and Curing Perfect (http://curingperfect.com). To date, 47,492 individuals worldwide have visited our website, 24 newsletters have been distributed to the 87 subscribers and our Twitter account has 69 followers.
Ten Fellows have been awarded their PhD, one is writing her thesis and one is on a 4 year program. Lilly von Münchow, Sharmin Nadkarni and Narasimha Rao were awarded suma cum laudae and Llucia Albertí Servera a PhD magna cum laudae, and Ciaran Mooney after two years work.
More information about the DECIDE project can be found at www.birmingham.ac.uk/DECIDE or by emailing the Co-ordinator Geoffrey Brown at g.brown@bham.ac.uk