8 S.Manenti: Cell signalling and cell cycle in myeloid malignancies
Team Research Project
Team 8 (S.Manenti): to understand cell cycle deregulations underlying myeloid malignancies
This part of our activity aims to characterize new regulations and new functions of CDC25A and CHK1. We identified new sites of phosphorylation on these proteins by mass spectrometry analysis, and we are currently investigating kinases involved in these modifications and establishing their importance for CHK1 and CDC25A functions.
We also investigate whether these proteins play a role during normal cell cycle or other important cellular processes, including apoptosis, differentiation, or autophagy. For instance, we recently identified a caspase-dependent cleavage of CDC25A generating a short form of the protein with increased activity and involved in the apoptotic process (Mazars et al, Cell Death Diff, 2009). In addition to CDC25A and CHK1, we also pay some attention to the mitotic kinase Plk1, that we recently found functionally linked with the mTOR signaling pathway (Renner et al, Blood, 2009).
Finally, since cell cycle regulation during normal haematopoietic differentiation is not extensively documented, we also investigate the importance of CDC25A, CHK1, and other cell cycle regulators, during this process. These studies are based on in vitro differentiation models starting from human or murine immature haematopoietic cells.
As far as possible, we translate our basic research data into haematological questions that we develop in oncogenic tyrosine kinase-driven myeloid malignancies models. This includes acute myeloid leukemia expressing the FLT3-ITD receptor, as well as myeloproliferative disease involving the JAK2V617F oncogene. For this more translational area of our research, we take advantage of close association with the Haematology Laboratory and Service of the IUCT hospital, in which several members of the team exert their professional hospital activity.
Cell cycle regulations in FLT3-ITD AML
We investigate how cell cycle proteins are regulated downstream of FLT3-ITD and what is their importance and their function for the proliferation and the resistance to therapeutic agents. We recently identified a STAT5-Pim signalling axis regulating CHK1 phosphorylation and functions in this model (Yuan et al, Leukemia, 2014; Yuan et al, Leukemia Research, 2014). We found that phosphorylation of CHK1 on Ser 280 by the Pim kinase is important for its function in DNA damage response, for resistance to therapeutic agents, as well as for proliferation of these cells. We are currently investigating how phosphorylation on this residue modifies the functional properties of CHK1, and how this kinase impacts the proliferation process of FLT3-ITD-positive cells. We are also interested in a more general characterization of CHK1 expression and activation status in AML, and its correlation with primary AML cells sensitivity to therapeutic agents and CHK1 inhibitors (Didier et al, Cancer Biol Ther, 2012).
In parallel, we recently identified the CDC25A phosphatase as an early target of signalling pathways in this model (Bertoli et al, submitted), and we are now further deciphering the functions of this protein as well as its different mechanisms of regulation in this cellular environment.
Cell cycle regulations in JAK2V617F myeloproliferative disease
Myeloproliferative disease like polycethemia vera or essential thrombocytemia are associated with an activating mutation (JAK2V617F) of the JAK2 tyrosine kinase which confers increased proliferation properties to the corresponding haematopoietic cells. We are interested in understanding cell cycle deregulations in this model. We identified a JAK/STAT/eIF2alpha pathway regulating CDC25A protein level, and we described the importance of this protein for the proliferation of these cells (Gautier et al, Blood, 2012). We are now further characterizing important players of cell proliferation downstream of JAK2V617F, and what are the molecular links between this oncogenic kinase and the translation intitiation factor eIF2alpha. We are also starting a study aimed to the characterization of CHK1 regulation and activity in this model.
Autophagy in Acute Myeloid Leukemia
Over the past few years, autophagy becomes a precious therapeutic target for treating cancers. Indeed, autophagy, which literally translated means “eat yourself”, is more than a lysosomal degradation pathway involving the elimination of protein aggregates and unwanted cellular components. Autophagy is a survival mechanism that ensures energy and cellular homeostasis by recycling the degraded material. It is well established that autophagy is involved in maintaining cancer state, metastasis and resistance to cancer therapy. However, whether autophagy acts as a tumour suppressor or tumour growth promoter is still a matter of debate. Therefore, depending on the context either promoting or inhibiting autophagy could be a mean to treat cancers. This apparent controversy is particularly true in haematological malignancies. Indeed on one hand several anticancer treatments induce autophagy helping the leukemic cells to recover, and on another hand, autophagy could promote the degradation of specific oncogenes (e.g. PML-RARA, BCR-ABL) favouring then leukemic cell death. Our lab research is focused on the contribution of autophagy in drug resistance particularly in acute myeloid leukemia (AML). We investigate whether the level of autophagy could have a prognosis value and could be linked to specific mutations (e.g. FLT-ITD). In parallel, we want to better understand whether autophagy controls signalling output and oncogenic functions and how it impacts on response to anti cancer treatments, to eventually address if autophagy modulation could to be used as a therapeutic tool in AML.
Stéphane Manenti's team is part of Labex TOUCAN & PHUC CAPTOR
- Cell cycle : the cell division cycle is a succession of phases during which a cell divides into two daughter cells genetically identical. The cell duplicates its content during the interphase (composed of the G1, S and G2 phases), and then divides into two cells during mitosis. Among critical regulators of cell cycle progression are the cyclin-dependent kinase complexes (cyclin-CDKs) which are hetero-dimers of a regulatory cyclin and a cyclin-dependent kinase (CDK ; the catalytic subunits). CDKs can phosphorylate specific substrates, thereby promoting cell cycle progression. Cyclin-CDKs are tightly regulated at multiple levels, including by CDC25 phophatases (see below).
- CDC25A : a dual specificity phosphatase removing phosphate groups from Thr14 ant Tyr15 residues of cyclin dependent kinases, leading to catalytic activation of these kinases during the different phases of the cell cycle. In mammals, 3 CDC25 phosphatases (A, B and C) are identified. CDC25A activates different CDKs all along the cell cycle, while CDC25B and C are more restrained to G2 and M phases. CDC25A is often over-expressed in different cancers, an its knock-out in mice is lethal at early stage of differentiation.
- CHK1 : a ser/thr kinase acting as a central actor of the DNA damage response, by phosphorylating different cell cycle related substrates and inducing by this way cell cycle arrest called checkpoints.
- Haematopoiesis : includes all the differentiation and proliferation events allowing renewing of all blood cells. In humans, this complex process takes place in the bone marrow. Schematically, haematopoietic stem cells (HSC) present in specific compartments of the bone marrow are submitted to extra-cellular stimuli leading to differentiation into progenitors of the different haematopoietic lineages (myeloid and lymphoid), and ultimately, to all the blood cellular components. Approximately 1011–1012 new blood cells are produced daily in order to maintain steady state levels in the peripheral circulation
- Acute myeloid leukemia/myeloproliferative disease : many myeloid malignancies linked to haematopoietic cells defects have been described. Acute myeloid leukemia are characterized by increased proliferation and resistance of leukemic cells, together with a block in differentiation that can occur at different stages of the haematopoietic process. This leads to accumulation in the bone marrow of undifferentiated leukemic cells called « blasts ». Myeloproliferative disease are also characterized by excessive proliferation and resistance of haematopoietic cells, but since there are no defects in haematopoietic differentiation, they lead to an excess of differentiated myeloid cells in the bone marrow and in the blood. Mutation of cytoplasmic tyrosine kinases are often at the origin of these pathologies, as for instance BCR-ABL in the case of chronic myeloid leukemia (CML), or JAK2V617F in the case of polycythemia vera, primary myelofibrosis, or essential thrombocythemia.
- Autophagy : a catabolic process in which cytoplasm bulk, proteins and organelles are sequestered in autophagosomal vesicles and delivered to the lysosomes for degradation. This highly conserved mechanism controls the turnover of stable and defective proteins and thereby secures cellular homeostasis. However autophagy is not only a sink, as degraded material (amino acid, lipids etc) are recycled and re-used by the cell. Then, autophagy is a sensor of the metabolic state and allows cells to adapt to poor growth environments. Autophagy protects cells from death during periods of nutrient deprivation or environmental stress. Hence, autophagy is considered an essential survival mechanism.