Investigational New Drugs

, Volume 30, Issue 5, pp 1830–1840 | Cite as

The cytotoxic activity of Aplidin in chronic lymphocytic leukemia (CLL) is mediated by a direct effect on leukemic cells and an indirect effect on monocyte-derived cells

  • Pablo E. Morande
  • Samanta R. Zanetti
  • Mercedes Borge
  • Paula Nannini
  • Carolina Jancic
  • Raimundo F. Bezares
  • Alicia Bitsmans
  • Miguel González
  • Andrea L. Rodríguez
  • Carlos M. Galmarini
  • Romina Gamberale
  • Mirta Giordano
PRECLINICAL STUDIES

Summary

Aplidin is a novel cyclic depsipeptide, currently in Phase II/III clinical trials for solid and hematologic malignancies. The aim of this study was to evaluate the effect of Aplidin in chronic lymphocytic leukemia (CLL), the most common leukemia in the adult. Although there have been considerable advances in the treatment of CLL over the last decade, drug resistance and immunosuppression limit the use of current therapy and warrant the development of novel agents. Here we report that Aplidin induced a dose- and time-dependent cytotoxicity on peripheral blood mononuclear cells (PBMC) from CLL patients. Interestingly, Aplidin effect was markedly higher on monocytes compared to T lymphocytes, NK cells or the malignant B-cell clone. Hence, we next evaluated Aplidin activity on nurse-like cells (NLC) which represent a cell subset differentiated from monocytes that favors leukemic cell progression through pro-survival signals. NLC were highly sensitive to Aplidin and, more importantly, their death indirectly decreased neoplasic clone viability. The mechanisms of Aplidin-induced cell death in monocytic cells involved activation of caspase-3 and subsequent PARP fragmentation, indicative of death via apoptosis. Aplidin also showed synergistic activity when combined with fludarabine or cyclophosphamide. Taken together, our results show that Aplidin affects the viability of leukemic cells in two different ways: inducing a direct effect on the malignant B-CLL clone; and indirectly, by modifying the microenvironment that allows tumor growth.

Keywords

Aplidin Plitidepsin Tumor microenvironment Chronic lymphocytic leukemia Monocytes Myeloid cells 

Notes

Acknowledgements

The authors would like to thank all patients and donors for their participation in this study; Dr Analía Trevani for assistance with fluorescence microscopy; Ms Beatriz Loria and Ms Mabel Horvat for technical assistance. This work was supported by grants from Agencia Nacional de Promoción Científica (Argentina), CONICET and Fundación Florencio Fiorini.

Conflict of interest

C.M. Galmarini: employment, PharmaMar. The other authors reported no potential conflicts of interest.

Supplementary material

10637_2011_9740_MOESM1_ESM.jpg (351 kb)
Fig. 1S Sensitivity of CLL cells to the combination of Aplidin with fludarabine or 4-OH-cyclophosphamide. PBMC isolated from CLL patients were incubated with Aplidin at 10, 50 or 100 nM, alone or in combination with the indicated concentrations of fludarabine (Fluda) or 4-OH-cyclophosphamide (4-OH-C). Percentages of cell death were calculated by 7-AAD dye incorporation and flow cytometry analysis. Shown are the curves obtained when performing cultures for 24 h (panel A, n = 5) or 48 h (panel B, n = 6) (JPEG 351 kb)
10637_2011_9740_MOESM2_ESM.jpg (508 kb)
Fig. 2S Characterization of nurse-like cells (NLC) from CLL samples. PBMC from CLL patients were cultured for 10–15 days on 24- or 48- well flat-bottomed microtiter to allow the differentiation of NLC. a The outgrowth of NLC was assessed by phase-contrast microscopy. Black bars = 10 μm. b NLC were differentiated in chambered coverglass slides and labeled with phycoerythrin-anti-CD14 antibody and TO-PRO®-3 stain as a nuclear counterstain. Displayed is a representative fluorescent micrography showing CD14 expression on membrane NLC (red) and nuclei (blue) White bar = 10 μm. c Differentiation of NLC was also assessed by flow cytometry analysis. A representative dot plot of the side-angle (SSC) and forward-angle (FSC) of lymphocytes and NLC is shown (JPEG 507 kb)
10637_2011_9740_MOESM3_ESM.jpg (158 kb)
Fig. 3S Monocytes from healthy donors are highly sensitive to Aplidin-induced cell death. a PBMC from 3 healthy donors (3 × 106/ml) were cultured for 24 h with different doses of Aplidin or 0.01% DMSO as vehicle (Control). Cells were washed and labeled with anti-CD14 fluorescent antibody to determine the percentage of monocytes by flow cytometry. Shown are representative dot plots of FSC versus CD14 expression under control or Aplidin (100 nM) conditions. b Purified human monocytes were incubated with Aplidin at 10, 50 or 100 nM for 48 h. Percentages of cell death as revealed by 7-AAD dye is shown (n = 3) (JPEG 157 kb)

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Pablo E. Morande
    • 1
  • Samanta R. Zanetti
    • 1
  • Mercedes Borge
    • 1
  • Paula Nannini
    • 1
  • Carolina Jancic
    • 1
  • Raimundo F. Bezares
    • 3
  • Alicia Bitsmans
    • 4
  • Miguel González
    • 1
  • Andrea L. Rodríguez
    • 1
  • Carlos M. Galmarini
    • 5
  • Romina Gamberale
    • 1
    • 2
  • Mirta Giordano
    • 1
    • 2
    • 6
  1. 1.Laboratory of Immunology, IIHEMANational Academy of MedicineBuenos AiresArgentina
  2. 2.Department of Microbiology, Parasitology and Immunology, School of MedicineUniversity of Buenos AiresBuenos AiresArgentina
  3. 3.Department of HematologyHospital Teodoro AlvarezBuenos AiresArgentina
  4. 4.Department of HematologyHospital Ramos MejíaBuenos AiresArgentina
  5. 5.Cell Biology DepartmentPharmaMar SAUMadridSpain
  6. 6.Laboratorio de Inmunología OncológicaIIHEMA, Academia Nacional de MedicinaBuenos AiresArgentina

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