Research Article: Acadesine Kills Chronic Myelogenous Leukemia (CML) Cells through PKC-Dependent Induction of Autophagic Cell Death

Date Published: November 18, 2009

Publisher: Public Library of Science

Author(s): Guillaume Robert, Issam Ben Sahra, Alexandre Puissant, Pascal Colosetti, Nathalie Belhacene, Pierre Gounon, Paul Hofman, Fréderic Bost, Jill-Patrice Cassuto, Patrick Auberger, Georg Häcker.

Abstract: CML is an hematopoietic stem cell disease characterized by the t(9;22) (q34;q11) translocation encoding the oncoprotein p210BCR-ABL. The effect of acadesine (AICAR, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) a compound with known antileukemic effect on B cell chronic lymphoblastic leukemia (B-CLL) was investigated in different CML cell lines. Acadesine triggered loss of cell metabolism in K562, LAMA-84 and JURL-MK1 and was also effective in killing imatinib-resistant K562 cells and Ba/F3 cells carrying the T315I-BCR-ABL mutation. The anti-leukemic effect of acadesine did not involve apoptosis but required rather induction of autophagic cell death. AMPK knock-down by Sh-RNA failed to prevent the effect of acadesine, indicating an AMPK-independent mechanism. The effect of acadesine was abrogated by GF109203X and Ro-32-0432, both inhibitor of classical and new PKCs and accordingly, acadesine triggered relocation and activation of several PKC isoforms in K562 cells. In addition, this compound exhibited a potent anti-leukemic effect in clonogenic assays of CML cells in methyl cellulose and in a xenograft model of K562 cells in nude mice. In conclusion, our work identifies an original and unexpected mechanism by which acadesine triggers autophagic cell death through PKC activation. Therefore, in addition to its promising effects in B-CLL, acadesine might also be beneficial for Imatinib-resistant CML patients.

Partial Text: CML is a myeloproliferative syndrome linked to a hematopoietic stem cell disorder leading to increased production of granulocytes at all stages of differentiation [1]. CML patients carry the t(9;22) (q34;q11) translocation [1], which is responsible for the expression of p210 BCR-ABL, a constitutively active tyrosine kinase [2]. The role of BCR-ABL in the pathogenesis of CML is well documented [3], [4]. Of note, BCR-ABL mediates several survival pathways including STAT5/Bcl-xL, Ras/Raf/MEK/Erk-1/2, PI3K/Akt and NF-kB, that collectively confer proliferative advantages and resistance to apoptosis [5], [6], [7], [8]. Imatinib (Gleevec) which targets the ATP-binding site of different tyrosine kinases including BCR-ABL [9], [10], selectively induces growth arrest and apoptosis of BCR-ABL positive leukemia cells with minimal effect on normal hematopoietic progenitors [11], [12], [13].

Acadesine has currently used in phaseI/II for the treatment of B-CLL [31]. In the present study we evaluated the potential anti-leukemic effect and the mechanism of action of acadesine on CML cell lines both in vitro and in vivo. The data presented herein demonstrate that acadesine exerts a potent anti-leukemic effect on different CML cell lines. Although acadesine does activate the AMPK pathway in CML cells, several lines of evidence indicate that this kinase is not involved in the anti-leukemic effect of acadesine. Firstly, AMPK knock-down by specific sh-RNA or si-RNA did not interfere with acadesine-mediated growth arrest and inhibition of cell metabolism. Secondly, the PKC inhibitors GFX and R0- efficiently inhibited the antileukemic effect of acadesine, but failed to affect AMPK phosphorylation in response to this AMPK activator (not shown). Importantly, acadesine activated several PKC isoforms, including α, β and γ in K562 cells. Altogether, our findings support the involvement of PKC but not AMPK in the anti-leukemic properties of acadesine. In several models of leukemia, acadesine has been reported to trigger AMPK activation, an effect accompanied by an important inhibition of their proliferation potential. This effect has often been associated with caspase activation and induction of apoptosis [32], [33]. Interestingly, this is clearly not the case in the present study, since we were unable to detect any increase in caspase activity upon acadesine stimulation of different CML cell lines. Accordingly, acadesine also failed to induce apoptosis in the different CML cell lines used in the present study. Thus, we conclude, that the anti-leukemic effect of acadesine did not involve induction of apoptosis in several CML cell lines.



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