SMAC Mimetic Induced Cell Death: Mode of Action and Overcoming Resistance
Cancers are characterized by uncontrolled growth and proliferation. One of the key regulators that act to prevent tumor development is programmed cell death, or apoptosis. Defects in the ability of tumors to undergo apoptosis are as important in cancer progression as the loss of signaling controls that limit growth and proliferation. Hence, a fundamental approach, that to date has not been fully exploited, is to attempt to reestablish proper apoptotic signaling allowing cells that have lost normal regulatory controls to essentially commit suicide.
Among key regulators of cell death is the inhibitor of apoptosis (IAP) family of proteins that act to suppress activation of enzymes, the caspases, responsible for carrying out the cell death program. An endogenous protein called second mitochondrial activator of caspases (smac) is released from the mitochondria upon genotoxic stress, such as DNA damage, and binds to IAPs, un-inhibiting caspases, allowing apoptosis to occur. In many cancers this process is defective, with the observation that IAPs are often over expressed and that cancer cells become resistant to genotoxic stress and do not release smac from the mitochondria. The nature of the interaction between smac and IAPs presents itself as an ideal target. A four amino acid motif of smac binds to and displaces caspases from the IAPs. As a means to bypass the need to induce genotoxic stress, a small molecule mimetic of the four amino acid motif was synthesized and shown to be able to synergize with pro-apoptotic stimuli to induce apoptosis, and was also shown to have single agent efficacy. My research has aimed to identify the mechanism of why some cell lines are sensitive to single agent smac mimetic. I identified autocrine TNF production, both basal and smac mimetic induced, as a key feature of cells able to respond to single agent treatment. Additionally, I was able to identify key components responsible for cell death to occur by conducting a limited, targeted siRNA knockdown screen of TNF signaling components to identify receptor interacting protein kinase I (RIPK1) as a key component involved in caspase-8 activation. Furthermore, I was also interested in understanding why a majority of cells do not respond to smac mimetic, either as a single agent or in combination with TNF. I determined at least two mechanisms whereby this was achieved. Firstly, one of the key mechanisms of smac mimetic action is to induce the degradation of cellular IAP1 (cIAP1) and cIAP2. This degradation is key for the proper formation of an active RIPK1- caspases 8 complex. Some cells are highly sensitive to TNF induced up-regulation of cIAP2, which becomes refractive to degradation following the initial smac mimetic treatment, owing to loss of cIAP1. The return of cIAP2 blocks formation of the RIPK1-caspase 8 complex and the presence of cIAP2 accounts for resistance in some cell lines. Secondly, there is another class of cells that do not express cIAP2, basally or in response to TNF, that are nonetheless resistant. These cells are defective in responding to TNF and are thus unable to properly recruit RIPK1 to the TNF receptor. These cells are also defecting in nuclear factor-kappaB (NF-?B) signaling and possess, in relative terms, far less RIPK1 than do sensitive cells and simply do not have enough RIPK1 to incorporate into the death inducing complex. As a last goal, I wanted to determine if it was possible to force cells that are resistant to become sensitive. Given the key role that TNF plays in smac mimetic sensitivity, it seemed like a good bet that interfering with signaling downstream of the receptor might allow events at the receptor to still occur, but block downstream pro-survival events from happening. Utilizing both siRNAs and chemical inhibition of NF-?B I was able to sensitize previously resistant cells to smac mimetic and TNF treatment. Additionally, I was able to demonstrate that targeting parallel pathways that regulate cIAP2 also sensitized cells. Specifically, targeting protein kinase B (AKT) and targeting the epidermal growth factor receptor with erlotinib (Tarceva) were both highly effective. These results will hopefully expand the therapeutic use of smac mimetic as well as other established chemotherapeutic. Such combinatorial therapy offers the hope of limiting the toxicity of current therapies and expanding the pool of patients that might be responsive.