Triggering mitochondrial apoptosis using 'mito-priming' technique

Triggering mitochondrial apoptosis using 'mito-priming' technique

Apoptosis often requires mitochondrial outer membrane permeabilization, a process targeted by Bcl-2-binding BH3 mimetics. Researchers describe and apply 'mito-priming', a method that allows triggering mitochondrial apoptosis in a synchronous manner, facilitating the investigation of mitochondrial apoptosis and its regulation by Bcl-2 proteins.

Following most apoptotic stimuli, mitochondrial outer membrane permeabilization (MOMP) is essential for cell death. MOMP leads to the release of mitochondrial intermembrane space proteins such as cytochrome c that activate caspase proteases causing rapid cell death.

However, even in the absence of caspase activity, MOMP typically kills cells and therefore represents a point-of-no-return. Given this pivotal role in dictating life and death, MOMP is highly regulated, primarily by pro- and anti-apoptotic members of the Bcl-2 protein family.

Evasion from apoptosis is considered a hallmark of cancer. Paradoxically, while apoptotic inhibition promotes cancer, tumor cells often display increased apoptotic sensitivity relative to normal tissue. Underlying this increased sensitivity, are altered levels of pro- and anti-apoptotic Bcl-2 proteins. Due to the pro-apoptotic stresses that cancer cells encounter, anti-apoptotic Bcl-2 function is required for cell survival to counteract pro-apoptotic BH3-only protein function. Cancer cells in this state are termed ‘primed-to-die’ and are sensitive to apoptosis-inducing therapies. Importantly, targeted anti-cancer therapies called BH3 mimetics have recently been developed to exploit this Bcl-2 dependency. In a manner similar to BH3-only proteins, BH3 mimetics bind to and inhibit anti-apoptotic Bcl-2 function.

Due to the wide-ranging roles of apoptosis in health and disease, the regulation of MOMP by Bcl-2 proteins has been intensively studied. Nevertheless, methods to investigate mitochondrial apoptosis are complicated by commonly used treatments, such as staurosporine, that induce MOMP over many hours, in an asynchronous manner and often with off-target, non-MOMP-dependent effects.

Current methods to ‘cleanly’ induce mitochondrial apoptosis include ER/tamoxifen or doxycycline-based induction of BH3-only protein activity. However, these approaches remain far from ideal due to various factors that include low potency, lack of general applicability, extended time of induction and, in some cases, direct effects of the chemical inducer on mitochondrial function.

Circumventing these problems, authors wanted to develop a technique that would rapidly and synchronously induce apoptosis over a cellular population in an effective manner. Ideally, such an approach would be applicable to any cell of choice. A second criterion was that any technique should also permit investigation of Bcl-2-mediated regulation of MOMP, for example allowing investigation of BH3-only protein specificity for the executioner proteins BAX or BAK.

This method, called mito-priming, uses co-expression of pro- and anti-apoptotic Bcl-2 proteins to engineer Bcl-2 addiction. On addition of Bcl-2 targeting BH3 mimetics, mito-primed cells undergo apoptosis in a rapid and synchronous manner.

Using this method authors have comprehensively surveyed the efficacy of BH3 mimetic compounds, identifying potent and specific MCL-1 inhibitors. Furthermore, by combining different pro- and anti-apoptotic Bcl-2 pairings together with CRISPR/Cas9-based genome editing, they find that tBID and PUMA can preferentially kill in a BAK-dependent manner.

In summary, mito-priming represents a facile and robust means to trigger mitochondrial apoptosis.