BH3 profiling can determine dependence on antiapoptotic Bcl-2 family members and can predict response to chemotherapeutic agents.

A18 Bcl-2 family member proteins regulate the mitochondrial pathway of apoptosis. These family members can be divided into anti-apoptotic proteins and pro-apoptotic proteins. Pro-apoptotic proteins can be further divided into multi-domain (Bax and Bak) and BH3 only (including Bid, Bim, Bad, Noxa,etc.). All Bcl-2 family proteins have at least one BH domain which share sequence homology and are involved in protein interactions. Anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-XL, Mcl-1) function by binding and sequestering pro-apoptotic proteins. Anti-apoptotic proteins show preferential binding to certain pro-apoptotic BH3-only proteins, as determined by fluorescent polarization. For example, Bcl-2 can bind to Bad, whereas Mcl-1 can bind to Noxa.
 BH3 profiling is a technique that utilizes this distinct binding code in order to determine whether cells are dependent on anti-apoptotic proteins for survival. Peptides that match the BH3 domain sequence of Bcl-2 family proteins are used. When added to isolated mitochondria these BH3 peptides can induce release of cytochrome c, which provides a functional read out for the measurement of dependence on anti-apoptotic proteins. BH3 profiling can distinguish between Bcl-2 and Mcl-1 dependence in primary leukemia from genetically designed mouse models and cell lines, as well as leukemia samples from human patients. In addition, we show that Bcl-2 and Mcl-1 are primed with the pro-apoptotic BH3-only proteins Bim and Puma and not Bax or Bak. BH3 profiling can also identify three classes of apoptotic blocks, which are (A) loss of pro-apoptotic BH3 only protein, (B) loss of pro-apoptotic Bax and Bak, and (C) overexpression of anti-apoptotic proteins (Bcl-2, Bcl-XL, Mcl-1). Class A and B blocks would be resistant to chemotherapeutic agents, while a Class C block would be sensitive to chemotherapeutic agents. Cells that are Bcl-2 dependent according to BH3 profiling would be sensitive to the Bcl-2 antagonist ABT-737.