MDM2 Inhibitor Nutlin-3a Triggers Autophagic Cell Death In Addition to Apoptosis In Leukemia Cell Lines with Wild-Type p53

Nutlin-3a is a small molecule inhibitor of MDM2 and has been shown to induce apoptosis and cell cycle arrest in various cancer models in a p53 dependent manner. Autophagy is a programmed cell death that can occur concurrently with apoptosis or in its absence. There is significant debate whether autophagy is a protective mechanism or a bona fide mechanism of cell death. While autophagy can function as tumor cell defense mechanism against cellular stress induced death, mutation/loss of alleles of certain genes regulating autophagy have been associated with development of cancer (e.g. Beclin-1 in breast cancer [Nature, 1999, 402: 672–676]). Multiple proteins involved in autophagy are transcriptional targets of p53 but Nutlin-3a has not been evaluated for its role in inducing autophagy. Here we present data suggesting that low dose Nutlin-3a induces autophagy in addition to apoptosis in leukemia cell lines in a p53 dependent manner.

OCI-AML-3 cells (p53-WT) treated with Nutlin-3a (2.5 and 5.0μM for 48, 72 and 96 hrs) were stained with mono-dansyl-cadaverine (MDC), a dye that accumulates in acidic autophagic vacuoles. OCI-AML-3 cells showed increasing staining with MDC in a dose and time dependent fashion by both flow cytometry (54%, 57% and 51% MDC positive after treatment with Nutlin-3a 5.0μM for 48, 72 and 96 hrs) and by confocal microscopy. Nutlin-3a treated cells also were positive for Annexin-V (flow cytometry 22%, 26% and 36% at 48, 72 and 96 hrs time points), and some of the cells were double-positive for Annexin-V and MDC (9.2%, 5% and 7% at 48, 72 and 96 hrs) suggesting that both apoptosis and autophagy can occur simultaneously. Autophagy induction was confirmed by Transmission Electron Microscopy (TEM). Large, multiple autophagic vacuoles were observed in OCI-AML-3 cells treated with Nutlin-3a. OCI-AML-3 cells with stable p53 knockdown by shRNA or HL-60 cells (p53-null) did not show increased MDC staining by flow cytometry (both cell lines) or autophagic vacuoles by TEM (HL-60) after similar treatment. Western blot analysis showed increases in LC3-II and in conjugation of Atg5/12, early and late autophagy markers respectively, in OCI-AML-3 cells after treatment with Nutlin-3a. Increased expression of the autophagy markers (LC3-II and Atg 5/12 conjugate) were also seen by Western blot analysis in the ALL cell lines REH and NALM-6 (both p53-WT) after treatment with Nutlin-3a. Western blot and/or RT-PCR analysis showed upregulation of other p53 related proteins involved in autophagy e.g. DRAM, AMPK-β, LKB1, pLKB1 in OCI-AML-3 cells treated with Nutlin-3a. As mTOR/Akt pathway inhibits autophagy, analysis of mTOR targets showed downregulation of the total and phospho-ribosomal-S6-protein levels, whereas there was no change in total or phospho-4-EBP-1 levels. Knockdown of Beclin-1 (ATG6), one of the proteins required for initiation of the formation of autophagic vacuoles, caused reduction in autophagic vacuoles (MDC staining by confocal microscopy) in OCI-AML-3 and REH cells without affecting apoptosis induction (Annexin V by flow cytometry). Pharmacologic inhibition of late autophagy by Bafilomycin (10nM for 2 hours) reduced MDC staining in OCI-AML-3 cells treated with Nutlin-3a for 48 hrs (32% without and 9% with Bafilomycin) while having limited inhibition of apoptosis (Annexin V positive 42% without and 33% with Bafilomycin).

Nutlin-3a induces autophagy in leukemia cells by a p53 dependent manner. We also demonstrate that autophagy could go hand-in-hand with apoptosis and in a fraction of cells both processes may occur concomitantly. Inhibition of autophagy does not necessarily enhance apoptosis.

Andreeff:Roche: Research Funding. Borthakur:ASCO: Research Funding.