Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of six amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together, these data allow us to develop a general model for activation of TpoR and explain the critical role of the JM W515 residue in the regulation of the activity of the receptor

Thrombopoietin receptor : regulation, dimerization, oncogenic activation and bimodal signaling

Philadelphia negative myeloproliferative neoplasms include polycythemia vera, essential thrombocythemia and primary myelofibrosis. Those MPNs are clonal diseases for which the phenotype is almost always driven by exon-specific somatic mutations in three genes: JAK2 exon 14, MPL exon 10 and CALR exon 9. MPL/TpoR is a homodimeric receptor that is a central player for developing a full JAK2 V617F or CALR mutant disease in mice. However, the receptor itself can suffer from pathological activating and dimerizing mutations within exon 10, namely W515X and S505N. This thesis addresses TpoR physiologic and oncogenic activation, dimerization and orientation dependent signaling including dimeric interfaces and transmembrane domain tilt angle. This thesis explains also transmembrane and juxtamembrane regulation mechanisms that are sometimes human specific and that prevent activation of a receptor otherwise primed for activation.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

Thrombopoietin receptor : regulation, dimerization, oncogenic activation and bimodal signaling

Philadelphia negative myeloproliferative neoplasms include polycythemia vera, essential thrombocythemia and primary myelofibrosis. Those MPNs are clonal diseases for which the phenotype is almost always driven by exon-specific somatic mutations in three genes: JAK2 exon 14, MPL exon 10 and CALR exon 9. MPL/TpoR is a homodimeric receptor that is a central player for developing a full JAK2 V617F or CALR mutant disease in mice. However, the receptor itself can suffer from pathological activating and dimerizing mutations within exon 10, namely W515X and S505N. This thesis addresses TpoR physiologic and oncogenic activation, dimerization and orientation dependent signaling including dimeric interfaces and transmembrane domain tilt angle. This thesis explains also transmembrane and juxtamembrane regulation mechanisms that are sometimes human specific and that prevent activation of a receptor otherwise primed for activation.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

The Thrombopoietin Receptor: Structural Basis of Traffic and Activation by Ligand, Mutations, Agonists, and Mutated Calreticulin.

A well-functioning hematopoietic system requires a certain robustness and flexibility to maintain appropriate quantities of functional mature blood cells, such as red blood cells and platelets. This review focuses on the cytokine receptor that plays a significant role in thrombopoiesis: the receptor for thrombopoietin (TPO-R; also known as MPL). Here, we survey the work to date to understand how this receptor functions at a molecular level throughout its lifecycle, from traffic to the cell surface, dimerization and binding cognate cytokine via its extracellular domain, through to its subsequent activation of associated Janus kinases and initiation of downstream signaling pathways, as well as the regulation of these processes. Atomic level resolution structures of TPO-R have remained elusive. The identification of disease-causing mutations in the receptor has, however, offered some insight into structure and function relationships, as has artificial means of receptor activation, through TPO mimetics, transmembrane-targeting receptor agonists, and engineering in dimerization domains. More recently, a novel activation mechanism was identified whereby mutated forms of calreticulin form complexes with TPO-R via its extracellular N-glycosylated domain. Such complexes traffic pathologically in the cell and persistently activate JAK2, downstream signal transducers and activators of transcription (STATs), and other pathways. This pathologic TPO-R activation is associated with a large fraction of human myeloproliferative neoplasms

Une anémie de Biermer se dissimulant sous les traits d’une anémie normocytaire

L’anémie mégaloblastique par carence en vitamine B12 ou en folate est bien connue. Son diagnostic est aisément posé par l’association d’une carence en vitamine et d’une macrocytose globulaire. Toutefois, diverses circonstances peuvent masquer la macrocytose et, donc, compliquer le diagnostic. Nous rapportons le cas d’une patiente se présentant pour une anémie hémolytique normocytaire qui s’avère être après mise au point, une anémie de Biermer associée à une alpha-thalassémie mineure.[A pernicious anemia masking itself under the guise of a normocytic anemia] Megaloblastic anemia due to vitamin B12 or folate deficiency is a well-known clinical entity. Diagnosis is easily made on the basis of vitamin deficiency associated with macrocytosis. Several conditions can, however, hide the conventional macrocytosis, rendering the diagnosis less straightforward. We report the case of a 45-year-old woman presenting with relatively well-tolerated severe anemia (7g/dL) and mild thrombocytopenia. The mean corpuscular volume (MCV) was normal (86,9 fL) with low reticulocytosis. Biology revealed signs of hemolysis and a low vitamin B12 level combined with anti-intrinsinc factor antibodies, which corroborated the diagnosis of pernicious anemia. To exclude a bicytopenia of central origine, a bone marrow aspiration was performed, which confirmed the megloblastosis. the normal MCV prompted us to search for a cause that could mask the classic macrocytosis observed in megaloblastic anemias. Ferritin and C-reactive protein levels were in the normal range, as was hemoglobin electrophoresis. We finally detected an α+thalassemia with 3,7 kb deletion while using molecular biology techniques. The patient recovered within a few weeks after being given intramuscular vitamin B12. This case of normocytic hemolytic anemia that turned out to be a pernicious anemia associated with an alpha-thalassemia minor illustrates that normal MCV does not suffice to rule out vitamin B12 deficiency. The major reasons for normocytic anemia under these circumstances are its combination with microcytic anemia, mostly due to iron deficiency and thalassemia, or its coexistence with macrocytic erythrocytes comprising erythrocyte fragment

Can the 72-hour rule based on "Blast/Abn Lymph" flag on Sysmex XN-10 optimize the workflow in hematology laboratory?

Despite the continuing improvement of automated blood cell counters, confirmation by blood smear examination remains the gold standard in case of anomalies. With a constant goal of standardisation, different experts committees (e.g. the French-speaking cellular hematology group (Groupe francophone d'hématologie cellulaire, GFHC and the ISLH International society for laboratory hematology) recently published criteria for microscopic analysis of blood smears. Cornet et al. evaluated the application of those criteria and propose to suppress any review for 72 hours when a "Blast/Abn lymph" flag is triggered for a sample with no abnormal cell on the microscopic review. The aims of our study were to retrospectively evaluate whether this 72-hour rule adequately operates and whether it is possible to extend the arbitrary 72-hour timeframe to 96h and 144h. To achieve this goal, 40,688 blood samples were collected from three French-speaking hospitals. 1,548 samples presented an isolated "Blast/Abn lymph" flag. Only 221 samples presented the application of the 72-hour rule at least once for our study period. We were able to extend this rule to 144 hours for 10 samples of them. All blood smears for which the rule was applied were verified and there was no abnormal cell on smears at 72 and 144 hours. In conclusion, the 72-hour rule derived from the GFHC's criteria is secure and reduces the slide review rate and thus the production costs and the turnaround time of hemogram results. Further investigations could confirm that its extension to 144 hours is also adequate

Diagnostic testing in myeloid malignancies by next-generation sequencing: recommendations from the Commission Personalised Medicine

Molecular diagnostics have an increasing impact on diagnosis, risk stratification and targeted treatment in haemato-oncology. In the framework of a pilot study for the implementation of next-generation sequencing in the Belgian healthcare system, the Commission of Personalised Medicine was founded to give professional and evidence-based advice on the molecular analysis in haemato-oncology. This paper describes its recommendations for NGS analysis in myeloid malignancies. In addition, the minimally required set of genes that must be analysed is defined and algorithms for molecular workflow in myeloid malignancies are proposed