The PTK7 and ROR2 Protein Receptors Interact in the Vertebrate WNT/Planar Cell Polarity (PCP) Pathway *

International audienceBackground: The planar cell polarity pathway plays important roles in morphogenetic processes. Results: PTK7 and ROR2 form a heterodimeric complex and bind to WNT5A, promoting JNK phosphorylation and regulating expression of paraxial protocadherin. Conclusion: PTK7 and ROR2 promote cell movement in mammalian cells and coordinate cell polarity during morphogenetic movements. Significance: We reveal new mechanisms of action of PTK7 in WNT/PCP signaling. The non-canonical WNT/planar cell polarity (WNT/PCP) pathway plays important roles in morphogenetic processes in vertebrates. Among WNT/PCP components, protein tyrosine kinase 7 (PTK7) is a tyrosine kinase receptor with poorly defined functions lacking catalytic activity. Here we show that PTK7 associates with receptor tyrosine kinase-like orphan receptor 2 (ROR2) to form a heterodimeric complex in mammalian cells. We demonstrate that PTK7 and ROR2 physically and functionally interact with the non-canonical WNT5A ligand, leading to JNK activation and cell movements. In the Xenopus embryo, Ptk7 functionally interacts with Ror2 to regulate protocadherin papc expression and morphogenesis. Furthermore , we show that Ptk7 is required for papc activation induced by Wnt5a. Interestingly, we find that Wnt5a stimulates the release of the tagged Ptk7 intracellular domain, which can translocate into the nucleus and activate papc expression. This study reveals novel molecular mechanisms of action of PTK7 in non-canonical WNT/PCP signaling that may promote cell and tissue movements

Ptk7-Deficient Mice Have Decreased Hematopoietic Stem Cell Pools as a Result of Deregulated Proliferation and Migration

International audienceHematopoietic stem cells (HSCs) located in adult bone marrow or fetal liver in mammals produce all cells from the blood system. Atthe top of the hierarchy are long-term HSCs endowed with lifelong self-renewal and differentiation properties. These features arecontrolled through key microenvironmental cues and regulatory pathways, such as Wnt signaling.We showed previously that PTK7,a tyrosine kinase receptor involved in planar cell polarity, plays a role in epithelial Wnt signaling; however, its function in hematopoiesishas remained unexplored. In this article, we show that PTK7 is expressed by hematopoietic stem and progenitor cells, withthe highest level of protein expression found on HSCs. Taking advantage of a Ptk7-deficient mouse strain, we demonstrate that loss ofPtk7 leads to a diminished pool of HSCs but does not affect in vitro or in vivo hematopoietic cell differentiation. This is correlatedwith increased quiescence and reduced homing abilities of Ptk7-deficient hematopoietic stem and progenitor cells, unraveling noveland unexpected functions for planar cell polarity pathways in HSC fate

Rôle du récepteur PTK7 dans l'hématopoièse murine

La tumorigenèse est un processus complexe provoqué par l'accumulation d'altérations génétiques contribuant à la transformation progressive d'une cellule normale en une cellule cancéreuse. Les travaux menés en hématologie ont permis d'identifier de nombreux mécanismes de signalisation impliqués dans des processus cellulaires clés comme la prolifération, la différenciation et la survie cellulaire, particulièrement au niveau des cellules souches hématopoïétiques (CSH). Parmi ces mécanismes, ceux déclenchés par les récepteurs à activité tyrosine kinase comme c-KIT ou FLT-3 ont été largement décrits au cours de ces dernières années. PTK7 est un récepteur tyrosine kinase de la famille des pseudokinases impliqué dans le développement embryonnaire qui joue un rôle important dans la polarité planaire. Le gène humain initialement cloné à partir de cellules de cancer du côlon code pour une protéine surexprimée dans de nombreuses tumeurs malignes. Nous avons confirmé sa surexpression dans les leucémies aiguës myéloïdes humaines et démontré que cet évènement représente un facteur indépendant de mauvais pronostic, en modulant la réponse aux agents cytotoxiques. Afin de mieux comprendre le rôle de PTK7 dans l'hématopoïèse physiologique, mon projet a consisté à générer une souris déficiente pour cette protéine et à étudier sa fonction dans la biologie des CSH. J'ai montré que les cellules déficientes pour PTK7 présentent un défaut de domiciliation vers la moelle osseuse. Ce travail contribue à mettre en évidence le rôle des protéines de la polarité dans le système hématopoïétique et, plus particulièrement, dans la biologie des CSH.Tumorigenesis is a multiple step process resulting from accumulation of genetic alterations leading to progressive transformation of normal cells into tumoral cells. Many signaling pathways have been described as key processes implicated in cell proliferation, cell differentiation and cell survival, in particular in mature hematotopoietic cells and hematopoietic stem cells (HSCs). Among these signaling pathways, those controlled by tyrosine kinase receptors such as c-KIT or FLT-3 have been extensively described during the last two decades.PTK7 is a pseudokinase receptor of the tyrosine kinase receptor family involved in embryonic development and described for its role in planar cell polarity. The human gene has been initially cloned from colon carcinoma cells and is frequently overexpressed in solid tumors. We described PTK7 overexpression in acute myeloid leukemia and demonstrated that it represents an independent poor prognosis factor acting as a modulator of the chemotherapeutic response.To better understand the physiological role of PTK7 in the hematopoietic system, my project consisted in the generation of a PTK7 deficient mouse model, and in the study of its function, in particular in HSC biology. My work demonstrated that PTK7 deficient HSCs have a general homing defect and poorly colonize hematopoietic organs including the bone marrow. This work contributes to a better understanding of PTK7 functions and, more generally, sheds light on the role of cell polarity proteins in the biology of HSCs

Etude du récepteur PTK7 dans l'hématopoïèse normale et pathologique (mise en place d'un modèle murin)

AIX-MARSEILLE2-BU Pharmacie (130552105) / SudocSudocFranceF

PTK7: a cell polarity receptor with multiple facets.

International audiencePTK7 is a tyrosine kinase receptor implicated in planar cell polarity, a process with multiple implications at the cellular and organism levels. Loss of function of PTK7 leads to profound morphogenetic defects in the mouse, such as neural tube defects, misorientation of stereocilia in the inner ear, and impaired polarized cell movements. The planar cell polarity pathway is classically assigned to a non-canonical Wnt pathway, which does not rely on b-catenin transcriptional activity. We recently revealed that PTK7 is implicated in b-catenin-dependent developmental processes in mammalian and Xenopus systems. Based on data recently obtained by our group as well as others, we discuss how PTK7 could be involved in canonical and non-canonical Wnt pathways, and which implications are expected from these data in physiology and physiopathology

Acute erythroid leukemias have a distinct molecular hierarchy from non-erythroid acute myeloid leukemias.

International audienc

Persistence of PNH Clones over Time: Insights from the Mid-Term Analysis of the French Nation-Wide Multicenter Prospective Observational Study

61st Annual Meeting and Exposition of the American-Society-of-Hematology (ASH), Orlando, FL, DEC 07-10, 2019International audienceIntroduction: Since the publication of the International Guidelines (Borowitz, 2010; Illingworth, 2018), no study has assessed the long-term evolution of paroxysmal nocturnal hemoglobinuria (PNH) clones using high-resolution flow cytometry. The sole evaluation, performed by Sugimori et al, using a 2-color flow cytometry test, showed the disappearance of PNH clones in 24% of patients with bone marrow (BM) failure over 5 years (Sugimori, 2009). A diagnostic practice harmonization using high-resolution flow cytometry has spread in France since 2013 through an on-going inter-laboratory comparison program (Debliquis, 2015). Thus, our HPNAFC group has been able to initiate a French nation-wide multicenter prospective observational study.Objective: We aimed to assess the evolution of PNH clones over a long term period using mostly high sensitivity test, which is required for minor clone assessment, with validated flow cytometry data.Methods: All patients of any age with a PNH clone or GPI-deficient cells ≥0.01%, newly- or previously-diagnosed, detected in France from February 29th 2016, could be included in this Observatory, provided that the center had validated a PNH flow cytometry quality control. For each patient, the baseline assessment was always considered as the initial PNH clone detection, even if it occurred before the initiation of the Observatory. Thus, this strategy allowed the collection of cases with long-term follow-up. Referent cytometrist of each center included patients in the e-CRF available on the HPNAFC website providing clinical and biological information as well as flow cytometry raw data files. This study was approved by the national research ethics board.Results: As of July 15th 2019, 48 participating flow cytometry laboratories across France have enrolled 356 patients with a PNH clone or GPI-deficient cells ≥ 0.01%. All cases have been carefully reviewed by the 2 principal investigators, who both thoroughly re-examined flow cytometry data and the e-CRF filling that led to the update of roughly one third of the submitted files. This enabled the validation of 200 patients at diagnosis, the remaining 156 being ongoing. One hundred and three of the 200 validated patients displayed at least one follow-up point (more than 3 months apart from the diagnosis) with a clone size determined at diagnosis (see flow chart figure 1A). For 8/103 patients, exchanges with centers are still ongoing. Thus, we were able to assess the evolution of PNH clones of 95 patients with 2 [range: 1-8] follow-up points over a period of 4.1 [0.3-14.2] years, corresponding to 200 validated follow-up points. The patient median age at diagnosis was 40 years old [10-85] with 3 pediatric cases (<18y) and a M/F sex ratio of 0.86. Diagnoses were made between 2003 and 2018 with clinical information available in 97% of cases: 19 patients (20%) had hemolytic anemia and most patients (n=73, 77%) displayed BM failure including aplastic anemia (n=62), myelodysplastic syndrome (n=7) and unexplained cytopenia(s) (n=4). No case of thrombosis was included. All patients with hemolytic anemia showed an increasing clone size over time including the two who were not treated with eculizumab (Figure 1B; median size at diagnosis on neutrophils: 81.3% vs median size over 5 years: 96.3%). The median clone size at diagnosis for patients with BM failure was 1.5% on neutrophils with a very wide range [0.01-97.87], almost half of them being less than 1%. When comparing the diagnosis point with the latest follow-up point, PNH clone size increased in 37 patients, decreased in 16 of them and remained stable in 20 cases (Figure 1C, D). Nine of the 37 patients reached a PNH clone size above 50% and 4 of them received a treatment by eculizumab in a median delay of 5 years [1.5-6.0]. Interestingly, no patient showed spontaneous disappearance of PNH clones, pending the use of a high-resolution flow cytometry test. The only five patients with undetectable PNH clones (Figure 1D, red lines) were those who underwent BM transplantation.Conclusion: This multicenter study based on robust flow cytometry analysis showed no disappearance of PNH clones, including minor ones, over a long period of time, regardless of the clinical manifestations, except for patients who underwent BM transplantation. Moreover, PNH clone size increased in half of patients with BM failure, justifying a long term PNH clone size monitoring, even in these patients

Protein tyrosine kinase 7 has a conserved role in Wnt/β-catenin canonical signalling.

International audienceThe receptor protein tyrosine kinase 7 (PTK7) was recently shown to participate in noncanonical Wnt/planar cell polarity signalling during mouse and frog embryonic development. In this study, we report that PTK7 interacts with β-catenin in a yeast two-hybrid assay and mammalian cells. PTK7-deficient cells exhibit weakened β-catenin/T-cell factor transcriptional activity on Wnt3a stimulation. Furthermore, Xenopus PTK7 is required for the formation of Spemann's organizer and for Siamois promoter activation, events that require β-catenin transcriptional activity. Using epistatic assays, we demonstrate that PTK7 functions upstream from glycogen synthase kinase 3. Taken together, our data reveal a new and conserved role for PTK7 in the Wnt canonical signalling pathway