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MOLECULAR EVIDENCE THAT ATOVAQUONE IS A NEW STAT3-INHIBITOR COUNTERBALANCING LEUKEMIC CELL PRO-SURVIVAL IN LARGE GRANULAR LYMPHOCYTE LEUKEMIA

PF367

Teramo, A.1, 2; Barilà, G.1, 2; Calabretto, G.1, 2; Vicenzetto, C.1, 2; Gasparini, V. R.1, 2; Leoncin, M.1; Facco, M.1, 2; Semenzato, G.1, 2; Zambello, R.1, 2

doi: 10.1097/01.HS9.0000559680.60909.38
Poster Session I: Chronic lymphocytic leukemia and related disorders - Biology & translational research
Free

1Department of Medicine, Hematology and Clinical Immunology Branch, University of Padua

2Veneto Institute of Molecular Medicine (VIMM), Padua, Italy

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Background:

Large Granular Lymphocyte Leukemia (LGLL) is a rare heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGLs) with cytotoxic activity. The hallmark of leukemic LGLs is a constitutive activation of STAT3 leading to enhanced triggering of many pro-survival genes. Moreover, 30-40% of patients carry mutations in STAT3 gene bringing to the constitutive activation of the malignant clone.

No specific treatment is now available for the patients affected by LGLL and the in-use therapy is based on low doses of immunosuppressive drugs, i.e. Methotrexate (MTX), Cyclophosphamide (CTX) and Cyclosporin A (CyA). Anyway, these therapies show limited efficacy, induce many side effects with the Overall Response Rate (ORR) being around 50% and a high rate of relapse in the responding patients.

The JAK/STAT signaling pathway, which is central to LGL leukemia pathogenesis, includes several players that provide the basis for new targeted treatments. This offers the rationale for using Atovaquone (ATQ, Mepron™), a recently FDA-approved anti-microbial agent characterized by a low adverse effect profile that has been recently demonstrated (Xiang et al., 2016) to work as a powerful STAT3 inhibitor with anti-cancer efficacy in both animal models and human cell lines.

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Aims:

Our study focuses on in vitro tests to evaluate the effects of ATQ as inhibitor of STAT3 phosphorylation and of LGLs survival, with the goal to develop a targeted approach to LGLL, likely devoid of adverse effects.

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Methods:

Patients' Peripheral Blood Mononuclear Cells (PBMC) were purified to perform cultures to test serial drug concentrations. Using in vitro cells culture, the inhibition of STAT3 protein phosphorylation and the expression of its target proteins, MCL1 and BCL2, were evaluated by Western Blot assay (WB). In addition, the cytotoxicity and the apoptotic selectivity of ATQ was investigated by Annexin V test; a staining with anti-CD57, CD56 or CD16 was used to identify leukemic LGLs from PBMCs. Finally, by RT-PCR, STAT3 activity was analyzed along its transcriptional levels and target genes.

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Results:

Through in vitro experiments we initially tested the best concentration inhibiting cell viability and STAT3 activation on patients' PBMCs. We observed that ATQ was efficiently able to inhibit STAT3 phosphorylation at a concentration of 25 μM. The results of the apoptosis analysis showed that ATQ has a dose and time dependent cytotoxic effect. The IC50 was reached at a dose of 25 μM after 24 h of culture. ATQ demonstrated a selective cytotoxicity against leukemic LGLs (reaching 3.9 ± 0.7 fold increase of apoptosis vs untreated condition after 48 h of culture) sparing non-leukemic cells (ATQ treated apoptosis vs untreated in non-LGL PBMC = 1.18 ± 0.3 fold change). By comparing the in vitro cell effect of ATQ with that of the commonly used therapies, ATQ showed a cytotoxic effect consistent with CTX and CyA, but higher than MTX (p < 0.01). Concerning anti-apoptotic factors under the STAT3 control, after 6 h ATQ treatment the expression of MCL1 and BCL2 showed only minimal changes, thereby suggesting that different mechanisms of action take place.

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Summary/Conclusion:

These in vitro data indicate that ATQ has a promising action on the inhibition of the JAK/STAT pro-survival pathway in leukemic LGLs, resulting in a selective cytotoxic effect against LGLs through MCL1- and BCL2-independent mechanisms.

Copyright © 2019 The Authors. Published by Wolters Kluwer Health Inc., on behalf of the European Hematology Association.