Claire Racaud-Sultan

Expression of β-catenin by acute myeloid leukemia cells predicts enhanced clonogenic capacities and poor prognosis

Activation of the Wnt/β-catenin pathway has recently been shown to be crucial to the establishment of leukemic stem cells in chronic myeloid leukemia. We sought to determine whether β-catenin was correlated to clonogenic capacity also in the acute myeloid leukemia (AML) setting. Eighty-two patients were retrospectively evaluated for β-catenin expression by Western blot. β-Catenin was expressed (although at various protein levels) in 61% of patients, and was undetectable in the remaining cases. In our cohort, β-catenin expression was correlated with the clonogenic proliferation of AML-colony forming cells (AML-CFC or CFU-L) in methylcellulose in the presence of 5637-conditioned medium, and more strikingly with self-renewing of leukemic cells, as assessed in vitro by a re-plating assay. In survival analyses, β-catenin appeared as a new independent prognostic factor predicting poor event-free survival and shortened overall survival (both with P<0.05). Furthermore, variations in β-catenin protein levels were dependent on post-transcriptional mechanisms involving the Wnt/β-catenin pathway only in leukemic cells. Indeed, β-catenin negative leukemic cells were found to increase β-catenin in response to Wnt3a agonist in contrast to normal counterparts. Altogether, our data pave the way to the evaluation of Wnt pathway inhibition as a new rationale for eradicating the clonogenic pool of AML cells.

A crosstalk between the Wnt and the adhesion-dependent signaling pathways governs the chemosensitivity of acute myeloid leukemia

Relapses following chemotherapy are a major hindrance to patients’ survival in acute myeloid leukemia (AML). To investigate the role of the hematopoietic niche in the chemoresistance of leukemic cells, we examined two pathways: one mediated by adhesion molecules/integrins, and the other by soluble factors of the morphogen Wnt pathway. In our study, both the adhesion of leukemic blasts to fibronectin and the addition of Wnt antagonists induced, independently, resistance of AML cells to daunorubicin in a cell survival assay. Using pharmacological inhibitors and siRNA, we showed that both resistance pathways required the activity of the glycogen synthase kinase 3β (GSK3β). Moreover, the AML cell protection downstream of GSK3β was mediated by NF-κB. A link between the adhesion and the Wnt pathway was found, as adhesion of U937 on human osteoblasts, a component of the hematopoietic niche, triggered the secretion of the Wnt antagonist sFRP-1 and supported resistance to daunorubicin. The osteoblast-conditioned medium could also confer chemoresistance to U937 cells cultured in suspension, and this cell protective effect was abrogated after depletion of sFRP-1. In the context of this potential double in vivo resistance, modulators of the common signal GSK3β and of its target NF-κB could represent important novel therapeutic tools.

Hammada scoparia flavonoids and rutin kill adherent and chemoresistant leukemic cells

In search for compounds able to reduce cell adhesion-mediated drug resistance (CAM-DR), we studied effects of Hammada scoparia extracts on leukemic cells adherent or in suspension. We show that H. scoparia flavonoidic fraction and its compound rutin induce apoptosis specifically in adherent leukemic cells and abolish CAM-DR. Importantly, rutin inhibited survival of adherent leukemic progenitors (CD34(+)38(-)123(+)) but spared normal progenitors (CD34(+)38(-)). The pro-apoptotic effects of rutin were correlated with a decrease of active GSK3β and inhibitors of GSK3β reproduced rutin-induced cytotoxicity. This study uncovers the potential of H. scoparia flavonoids and rutin to overcome CAM-DR in acute myeloid leukemia.

Focal adhesion kinase splice variants maintain primitive acute myeloid leukemia cells through altered Wnt signaling.

Focal adhesion kinase (FAK) activity contributes to many advanced cancer phenotypes, but little is known about its role in human acute myeloid leukemia (AML). Here, we show that FAK splice variants are abnormally expressed in the primitive leukemic cells of poor prognosis AML patients. In the CD34+38−123+ long‐term culture‐initiating cell‐enriched leukemic cells of these patients, FAK upregulates expression of Frizzled‐4 and phosphorylates Pyk2 to enable the required association of Pyk2 with the Wnt5a/Frizzled‐4/LRP5 endocytosis complex and downstream activation of β‐catenin, thereby replacing the Wnt3a‐controlled canonical pathway used by normal hematopoietic stem cells. Transduction of primitive normal human hematopoietic cells with FAK splice variants induces a marked increase in their clonogenic activity and signaling via the Wnt5a‐controlled canonical pathway. Targeting FAK or β‐catenin efficiently eradicates primitive leukemic cells in vitro suggesting that FAK could be a useful therapeutic target for improved treatment of poor prognosis AML cases. STEM CELLS2012;30:1597–1610

Sex differences in the GSK3β-mediated survival of adherent leukemic progenitors

Therapeutic resistance of acute myeloid leukemia stem cells, enriched in the CD34+38−123+ progenitor population, is supported by extrinsic factors such as the bone marrow niche. Here, we report that when adherent onto fibronectin or osteoblast components, CD34+38−123+ progenitors survive through an integrin-dependent activation of glycogen synthase kinase 3β (GSK3β) by serine 9-dephosphorylation. Strikingly, GSK3β-mediated survival was restricted to leukemic progenitors from female patients. GSK3β inhibition restored sensitivity to etoposide, and impaired the clonogenic capacities of adherent leukemic progenitors from female patients. In leukemic progenitors from female but not male patients, the scaffolding protein RACK1, activated downstream of α5β1-integrin engagement, was specifically upregulated and controlled GSK3β activation through the phosphatase protein phosphatase 2A (PP2A). In a mirrored manner, survival of adherent progenitors (CD34+38−) from male but not female healthy donors was partially dependent on this pathway. We conclude that the GSK3β-dependent survival pathway might be sex-specific in normal immature population and flip-flopped upon leukemogenesis. Taken together, our results strengthen GSK3β as a promising target for leukemic stem cell therapy and reveal gender differences as a new parameter in anti-leukemia therapy.

Anti-inflammatory and anticancer effects of flavonol glycosides from Diplotaxis harra through GSK3β regulation in intestinal cells

Abstract Context and objective: Diplotaxis harra (Forssk.) Boiss. (Brassicaceae) is traditionally used as an antidiabetic, anti-inflammatory or anticancer agent. In these pathologies, the glycogen synthase kinase 3 β (GSK3β) is overactivated and represents an interesting therapeutic target. Several flavonoids can inhibit GSK3β and the purpose of this study was to search for the compounds in Diplotaxis harra which are able to modulate GSK3β. Materials and methods: Methanol extracts from D. harra flowers were prepared and the bio-guided fractionation of their active compounds was performed using inflammatory [protease-activated receptor 2 (PAR2)-stimulated IEC6 cells] and cancer (human Caco-2 cell line) intestinal cells. 50–100 μg/mL of fractions or compounds purified by HPLC were incubated with cells whose inhibited form of GSK3β (Pser9 GSK3β) and survival were analyzed by Western blot at 1 h and colorimetric assay at 24 h, respectively. LC-UV-MS profiles and MS-MS spectra were used for the characterization of extracts and flavonoids-enriched fractions, and the identification of pure flavonoids was achieved by MS and NMR analysis. Results: The methanol extract from D. harra flowers and its flavonoid-enriched fraction inhibit GSK3β in PAR2-stimulated IEC6 cells. GSK3β inhibition by the flavonoid-enriched D. harra fraction was dependent on PKC activation. The flavonoid-enriched D. harra fraction and its purified compound isorhamnetin-3,7-di-O-glucoside induced a 20% decrease of PAR2-stimulated IEC6 and Caco-2 cell survival. Importantly, normal cells (non-stimulated IEC6 cells) were spared by these treatments. Conclusion: This work indicates that flavonoids from D. harra display cytotoxic activity against inflammatory and cancer intestinal cells which could depend on GSK3β inhibition.

Thrombin modifies growth, proliferation and apoptosis of human colon organoids: a protease‐activated receptor 1‐ and protease‐activated receptor 4‐dependent mechanism

Thrombin is massively released upon tissue damage associated with bleeding or chronic inflammation. The effects of this thrombin on tissue regrowth and repair has been scarcely addressed and only in cancer cell lines. Hence, the purpose of the present study was to determine thrombins pharmacological effects on human intestinal epithelium growth, proliferation and apoptosis, using three‐dimensional cultures of human colon organoids.

SFRP CO-03 – Culture et caractérisation de colonosphères humains

Objectifs Les cellules souches intestinales (CSI) permettent le developpement de mini-intestins in vitro . Notre but etait de cultiver et caracteriser des colonospheres humains, monocouche de cellules epitheliales organisee en 3D. Materiels et methodes A partir de resection colique, les cryptes sont re-suspendues dans du Matrigel puis ensemencees. Un milieu complet est ajoute pour reconstituer le microenvironnement des CSI, remplace par un milieu de differenciation pendant 5 jours. Les colonospheres ont ete fixes a J8 pour analyse immunohistochimique. Resultats principaux Les colonospheres ont un pole apical (actine +) oriente vers une lumiere centrale et un pole baso-lateral (β-catenine +). Il y a une zone apoptotique centrale (caspase 3 active +) et des zones de proliferation (Ki-67 +) ; des cellules immatures (CD44 et CD24 +) et des cellules differenciees : Goblet (mucine 2 +), enteroendocrines (chromogranine A +) et colonocytes (cytokeratine 20 +). Apres differenciation, nous retrouvons une diminution de cellules immatures et l’expression d’occludine (jonctions serrees), dans des zones distinctes. Conclusions Ce modele est bien etabli dans nos mains, avec des perspectives de recherche sur les maladies inflammatoires chroniques de l’intestin.