1. ¹INSERM, Unit 'Apoptosis, Cancer and Immunity', Villejuif, France
2. ²Institut Gustave Roussy, Villejuif, France
3. ³Faculté Paris Sud-Université Paris XI, Villejuif, France
4. ⁴INSERM U542, Hôpital Paul Brousse, Université Paris XI, Villejuif, France
5. ⁵Service d'Hématologie Clinique, Hôpital Avicenne, AP-HP, Université Paris XIII, Bobigny, France
6. ⁶CNRS, Institut André Lwoff, Villejuif, France

Correspondence: Dr G Kroemer, CNRS-FRE 2939, Institut Gustave Roussy, PR1, 39 rue Camille Desmoulins, F-94805 Villejuif, France. E-mail: kroemer@igr.fr

Received 24 May 2006; Revised 11 October 2006; Accepted 3 November 2006; Published online 8 January 2007.

Abstract

CD34⁺ bone marrow blasts from high-risk myelodysplastic syndrome (MDS) patients as well as MDS patient-derived cell lines (P39 and MOLM13) constitutively activate the nuclear factor-B (NF-B) pathway and undergo apoptosis when NF-B is inhibited. Here, we show that the combination of conventional chemotherapeutic agents (daunorubicin, mitoxantrone, 5-azacytidine or camptothecin) with the NF-B inhibitor BAY11-7082 did not yield a synergistic cytotoxicity. In contrast, BAY11-7082 (which targets the NF-B-activating I-B kinase (IKK) complex) or knockdown of essential components of the NF-B system (such as the IKK1 and IKK2 subunits of the IKK complex and the p65 subunit of NF-B), by small interfering RNAs sensitized MDS cell lines to starvation-induced apoptosis. The combination of BAY11-7082 and nutrient depletion synergistically killed the acute myeloid leukemia (AML) cell line U937 as well as primary CD34⁺ bone marrow blasts from AML and high-risk MDS patients. The synergistic killing by BAY11-7082, combined with nutrient depletion, led to cell death accompanied by all hallmarks of apoptosis, including an early loss of the mitochondrial transmembrane potential, the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, activation of caspase-3, phosphatidylserine exposure on the plasma membrane surface and nuclear chromatin condensation. Transmission electron microscopy revealed the presence of numerous autophagic vacuoles in the cytoplasm before cells underwent nuclear apoptosis. Nonetheless, cell death was neither inhibited by the pan-caspase inhibitor z-VAD-fmk nor by knockdown of AIF or of essential components of the autophagy pathway (ATG5, ATG6/Beclin-1, ATG10, ATG12). In contrast, external supply of glucose, insulin or insulin-like growth factor-I could retard the cell death induced by BAY11-7082 combined with starvation. These results suggest that in MDS cells, NF-B inhibition can precipitate a bioenergetic crisis that leads to an autophagic stress response followed by apoptotic cell death.