ORIGINAL PAPER Kinetics of ERK1/2 activation determine sensitivity of acute myeloid leukaemia cells to the induction of apoptosis by the novel small molecule ingenol 3-angelate (PEP005) Peter Hampson • Keqing Wang • Lisa Milverton • Elisabeth Ersvaer • Oystein Bruserud • Janet M. Lord Published online: 14 May 2010 Ó Springer Science+Business Media, LLC 2010 Abstract The novel small molecule ingenol 3-angelate (PEP005) has been shown previously to induce apoptosis in leukaemic cell lines and primary AML cells, an effect that requires the expression of protein kinase C-delta (PKCd). Here we have investigated signalling events downstream of PKCd that determine sensitivity of AML cells to PEP005. We show that activation of ERK1/2 MAP kinase occurred in both sensitive and resistant cells and that induction of apoptosis required sustained signalling through the ERK1/2 pathway. Inhibition of ERK1/2 signalling using the MEK inhibitor PD98059 inhibited PEP005-induced apoptosis and activation of ERK1/2 was shown to occur downstream of PKC activation. The data show that PEP005-induced apoptosis is both PKC and ERK1/2 dependent and indicate that chronic activation of ERK1/2 in leukaemic cells delivers a pro-apoptotic rather than a proliferative or sur- vival signal. Keywords Acute myeloid leukaemia Á ERK1/2 Á PKC Á Apoptosis Introduction Ingenol 3-angelate, otherwise known as PEP005, is a hydrophobic diterpene ester isolated from the sap of the plant Euphorbia peplus. Recent studies have shown that PEP005 has potent anti-neoplastic effects against cancer cell lines both in vitro and in vivo, and the compound has now entered phase II clinical trials for the treatment of non- melanoma skin cancer and actinic keratoses [1]. PEP005 has been shown to be a potent activator of the protein kinase C (PKC) family of isoenzymes, activating cPKCs and nPKCs in the low nanomolar range [2]. Work from our own group subsequently showed that PEP005 was able to induce apoptosis in a number of leukaemic cell lines, an effect that was dependent upon the expression and activa- tion of PKCd [3]. The downstream signalling events leading to apoptosis were not investigated in that study though in vitro studies in melanoma cell lines have dem- onstrated that PEP005 can induce PKC-dependent hyper- activation of ERK1/2 and subsequently cause cell-cycle arrest and senescence [4]. Moreover, other studies have shown that activation of PKCd can lead to ERK1/2 phos- phorylation [5, 6], suggesting another potential down- stream mechanism of action for PEP005. Mitogen-activated protein kinase (MAPK) is a family of serine/threonine protein kinases which comprises extra- cellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinases. These MAPKs act as transducers of extracellular signalling and play an important role in a number of cellular processes such as proliferation, differentiation and apoptosis [7]. ERK1/2 is generally activated by cytokines or growth factors and mediates mitogenic and anti-apoptotic signals [7]. On the other hand, stress stimuli activated p38 and JNK are pri- marily involved in regulating cell apoptosis and differen- tiation [8]. Dysregulation of MAPK signalling pathways, specifically ERK1/2 pathways, have been implicated in the pathogenesis of malignant diseases. Indeed, in almost one-third of human cancers the ERK1/2 pathway is P. Hampson (&) Á K. Wang Á L. Milverton Á J. M. Lord MRC Centre for Immune Regulation, School of Immunity and Infection, Birmingham University Medical School, Birmingham B15 2TT, UK e-mail: p.hampson@bham.ac.uk E. Ersvaer Á O. Bruserud Section for Haematology, Department of Medicine, Haukeland University Hospital, 5021 Bergen, Norway 123 Apoptosis (2010) 15:946–955 DOI 10.1007/s10495-010-0507-7