Apoptosis is a distinct genetic and biochemical pathway essential to all multicellular organisms and critical for the crafting of multiple distinct lineages. An intact death pathway is required for the maintenance of normal tissue homeostasis, a careful balance between proliferation and cell death. In addition, as shown by our transgenic and knockout models, genetic aberrations in the death pathway can be a primary pathogenic event in disease.
We noted that BCL-2 inhibits apoptosis in the genesis of lymphoma. We and others cloned the t(14;18) chromosomal breakpoint of human follicular lymphoma and demonstrated that this fusion increases expression of BCL-2. Transgenic mice bearing a Bcl-2–Ig minigene recapitulating the chromosomal translocation provided a definitive link to cancer, as they developed follicular hyperplasia due to extended cell survival that progressed to high-grade lymphomas. We localized BCL-2 to mitochondria and established that the survival effect resulted from the blocking of apoptosis by BCL-2. Thus BCL-2 plays a primary role in oncogenesis by inhibiting apoptosis and is the first member of a new category of oncogenes—regulators of cell death.
We extended the BCL-2 family by identifying conserved homologs, including BAX, the first partner protein that promotes apoptosis. The susceptibility to cell death is determined by the competing interests of pro- versus anti-apoptotic BCL-2 members, which serve as a major control point in the pathway. BCL-2 members possess conserved BCL-2 homolog domains (BH1–4) required for dimerization and regulation of apoptosis. The anti-apoptotic members are more highly conserved, and each possesses a pocket that binds BH3 domains from selected pro-apoptotic members. Pro-apoptotic members can be further subdivided into the more fully conserved “multidomain and “BH3-only subsets. We noted that multidomain BAX and BAK constitute an obligate gateway to the intrinsic pathway of cell death, operating at both the mitochondria and endoplasmic reticulum (ER). Pro-apoptotic BAX and BAK are activated in response to death stimuli, undergoing homo-oligomerization that results in the permeabilization of the outer mitochondrial membrane and the release of cytochrome c and caspase activation. BAX and BAK also operate at the endoplasmic reticulum to control Ca2+ levels. Selected death stimuli can be classified as ER or mitochondrial dependent (Figure 1).
We further integrated the pathway by identifying BH3-only pro-apoptotic molecules BID and BAD, which through their post-translational modification interconnect extracellular death and survival cues with the core apoptotic pathway. A pro-apoptotic cascade exists in which BH3-only members function as upstream death ligands that induce allosteric activation of multidomain BAX and BAK. In contrast, anti-apoptotic molecules such as BCL-2, BCL-XL, and MCL-1 serve a principal role of sequestering BH3-only molecules in stable complexes, preventing activation of BAX and BAK (Figure 2). Recently, we marshaled evidence for a two-class model for BH3 domains: BID-like domains activate BAX and BAK, while BAD-like domains sensitize cells by occupying the pocket of anti-apoptotic members. We are pursuing BH3 mimetics that can initiate apoptosis at definable points in the genetic pathway and serve as prototype therapeutics (Movie 1 and Movie 2).
Our genetic gain- and loss-of-function mouse models indicate the contribution of disordered apoptosis to cancer, immunodeficiency, autoimmunity, infertility, and degenerative disease. Our mice deficient for BID or BAD also develop malignancies. This demonstrates that even the upstream BH3-only molecules are required to maintain cellular homeostasis and to suppress tumorigenesis in select cell types.
We asked whether a greater rationale exists for the localization of BCL-2 members to intracellular organelles, especially the mitochondria. We observed that BCL-2 proteins reside in macromolecular complexes that connect mitochondrial physiology and the core apoptotic pathway. A mitochondrial metabolic channel, VDAC2, keeps the potentially lethal BAK molecule inactive (Figure 3). A combination of proteomics, genetics, and physiology revealed the BH3-only molecule BAD resides in a functional holoenzyme complex in liver mitochondria with WAVE-1, PKA (protein kinase A), PP1 (protein phosphatase 1), and glucokinase. BAD serves an unanticipated role in integrating glycolysis and apoptosis, two major pathways critical for cell survival (Figure 4).
Finally, we successfully generated modified BH3 α helices that bound with increased affinity to multidomain BCL-2 member pockets. Using hydrocarbon stapling, a novel hydrocarbon cross-linking strategy, we generated BH3 peptides called "stabilized alpha-helix of BCL-2 domains" (SAHBs), which proved to be helical, protease resistant, and cell permeable (Figure 5). A SAHB of the BH3 domain from BID specifically activated the apoptotic pathway to kill leukemia cells and effectively inhibited the growth of human leukemia xenografts in vivo.
Overall, we are pursuing a combination of genetics, biochemistry, and structural biology to define the mammalian apoptotic pathway, integrating it from death signals to final effector mechanisms.
