The immunoregulatory effect of epigenetic therapy on myelodysplastic syndromes (MDS)

CD4+ T cells orchestrate immune responses and are actively engaged in shaping tumor immunity. Signal transducer and activator of transcription (STAT) signaling controls the epigenetic tuning of CD4+ T cell differentiation and polarization and perturbed STAT signaling networks in CD4+ T cells subvert antitumor immunity in malignancies. Azacitidine (AZA), the mainstay therapy for high-risk Myelodysplastic syndromes (HR-MDS), affects CD4+ T cell polarization and function, but whether this contributes to AZA efficacy is currently unknown. By employing functional proteomic, transcriptomic and mutational analyses in 73 HR-MDS patients undergoing AZA therapy, we demonstrate that responding patients exhibited a coordinated CD4+ T cell immune response and downregulated the inflammatory cytokine signaling pathways in CD4+ T cells after AZA, in contrast to non-responders who upregulated the same pathways. We further observed an AZA-mediated downregulation of intereukin-6—induced STAT3 phosphorylation in CD4+FOXP3- conventional CD4+ T cells (Tcons) that correlated independently with better response and survival, whereas it was also not associated with the mutation number and profile of the patients. The AZA-induced downregulation of IL-6/STAT3 axis in Tcons restored the STAT signaling architecture in CD4+ T cell subsets, while STAT signaling networks remained disorganized in patients who upregulated IL-6/STAT3 activity in Tcons. Given the pivotal role of CD4+ T cells in adaptive immunity, our findings suggest that the downregulation of IL-6/STAT3 pathway in Tcons potentially constitutes a previously unrecognized immune-mediated mechanism of action of AZA and set the scene for developing rational strategies of AZA combinations with IL-6/STAT3 axis inhibitors.Τα CD4+ T λεμφοκύτταρα οργανώνουν τις ανοσολογικές αποκρίσεις και παίζουν ενεργό ρόλο στην ανοσία εναντίον του όγκου. Τα STAT σηματοδοτικά μόρια ελέγχουν επιγενετικά την διαφοροποίηση και πόλωση των CD4+ Τ λεμφοκυττάρων, ενώ έκτοπη σηματοδότηση μέσω του STAT μονοπατιού στα CD4+ Τ κύτταρα υποσκάπτει την αντινεοπλασματική ανοσία στις κακοήθειες. Η αζακυτιδίνη (ΑΖΑ) που αποτελεί την κύρια θεραπεία για τα υψηλού κινδύνου Μυελοδυσπλαστικά σύνδρομα (ΥΚ-ΜΔΣ), επηρεάζει την πόλωση και λειτουργικότητα των CD4+ Τ λεμφοκυττάρων αλλά κατά πόσο αυτή η επιρροή συμβάλει στην αποτελεσματικότητα της ΑΖΑ παραμένει άγνωστο. Χρησιμοποιώντας λειτουργική πρωτεομική, μεταγραφική και μοριακή ανάλυση σε 73 ασθενείς με ΥΚ-ΜΔΣ υπό θεραπεία με ΑΖΑ, δείξαμε πως οι αποκρινόμενοι ασθενείς χαρακτηρίζονταν από οργανωμένη ανοσολογική απόκριση μεσολαβούμενη από CD4+ Τ λεμφοκύτταρα και υπορρυθμισμένα τα σηματοδοτικά μονοπάτια που σχετίζονται με την φλεγμονή στα CD4+ Τ λεμφοκύτταρα μετά την θεραπεία με ΑΖΑ, σε αντίθεση με τους μη αποκρινόμενους ασθενείς που είχαν υπερρυθμισμένα τα ίδια μονοπάτια. Επιπλέον, παρατηρήσαμε μια μεσολαβούμενη από την ΑΖΑ υπορρύθμιση της επαγόμενης από ιντερλευκίνη 6 (IL-6) φωσφορυλίωση του STAT3 στα CD4+FOXP3- T συμβατικά λεμφοκύτταρα (Tσυμ) που συσχετίστηκε με καλύτερη απόκριση και επιβίωση ανεξάρτητα από τα διαθέσιμα προγνωστικά συστήματα, ενώ δεν συσχετίσθηκε με τον αριθμό και το προφίλ των μεταλλάξεων στους ασθενείς. Η μεσολαβούμενη από την ΑΖΑ υπορρύθμιση του άξονα IL-6/STAT3 στα Τσυμ αποκατέστησε την STAT σηματοδοτική βιοϋπογραφή στους CD4+ Τ υποπληθυσμούς, ενώ παρέμεινε αποδιοργανωμένη στους ασθενείς που υπερρύθμισαν το αντίστοιχο μονοπάτι. Δεδομένου του κεντρικού ρόλου που διαδραματίζουν τα CD4+ T κύτταρα στην επίκτητη ανοσία, τα ευρήματα μας υποδηλώνουν ότι η υπορρύθμιση του άξονα IL-6/STAT3 στα Τσυμ πιθανότατα αποτελεί έναν μη χαρακτηρισμένο μεσολαβούμενο από το ανοσοποιητικό μηχανισμό δράσης της ΑΖΑ και θέτει το πλαίσιο ανάπτυξης νέων στρατηγικών συνδυασμού της ΑΖΑ με αναστολείς της οδού IL-6/STAT3

Isothiocyanate-induced Cell Cycle Arrest in a Novel In Vitro Exposure Protocol of Human Malignant Melanoma (A375) Cells

Background/Aim: Several studies have documented the effects of isothiocyanates (ITCs) on cancer prevention by inducing oxidative stress, activating apoptosis, affecting cell cycle regulation, etc. Previously, we have shown that ITCs, administered at low concentrations by the means of double-bolus are capable of potentiating cytotoxicity in human malignant melanoma (A375) cells by inducing apoptosis. The aim of the present study was to further investigate the effect of the treatment of A375 cells with ITCs on cell cycle progression and the levels of various cell cycle regulators. Materials and Methods: Cell cycle analysis was performed by means of flow cytometry whereas western immunoblotting was used to determine the expression levels of these protein regulators. Results: Our data showed an increase in the number of cells in the G2/M phase accompanied by a decrease in the G0/G1 phase, while several cell-cycle regulators were shown to be differentially expressed upon exposure to ITCs. Conclusion: ITCs induced cell cycle arrest in A375 cells

MRD Monitoring by Multiparametric Flow Cytometry in AML: Is It Time to Incorporate Immune Parameters?

Acute myeloid leukemia (AML) is a heterogeneous group of clonal myeloid disorders characterized by intrinsic molecular variability. Pretreatment cytogenetic and mutational profiles only partially inform prognosis in AML, whereas relapse is driven by residual leukemic clones and mere morphological evaluation is insensitive for relapse prediction. Measurable residual disease (MRD), an independent post-diagnostic prognosticator, has recently been introduced by the European Leukemia Net as a new outcome definition. However, MRD techniques are not yet standardized, thus precluding its use as a surrogate endpoint for survival in clinical trials and MRD-guided strategies in real-life clinical practice. AML resistance and relapse involve a complex interplay between clonal and immune cells, which facilitates the evasion of the leukemic clone and which is not taken into account when merely quantifying the residual leukemia. Multiparameter flow cytometry (MFC) offers the possibility of capturing an overall picture of the above interactions at the single cell level and can simultaneously assess the competence of anticancer immune response and the levels of residual clonal cells. In this review, we focus on the current status of MFC-based MRD in diverse AML treatment settings and introduce a novel perspective of combined immune and leukemia cell profiling for MRD assessment in AML

Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells.

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10(9) CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10(9) CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain