Abstract 2: Phosphorylation of BNIP3 is a switch between life and death in cancer cells
Authors: Frazier William , Liu Katherine , Dimitry Julie , Capoccia Benjamin , Manning Pamela , Karr Robert 
Published: August 2015

Antibodies vs CD47 (CD47mAbs) that block the CD47-SIRPalpha interaction promote the phagocytosis of cancer cells and have efficacy in several tumor models. A select few CD47mAbs also directly kill cancer cells by lowering cellular cAMP levels leading to mitochondrial damage and cell death. These CD47mAbs are thus referred to as “dual-function” mAbs. Activation of protein kinase A prevents CD47mAb-mediated death suggesting that phosphorylation of one or more target proteins in the cancer cell can block the death mechanism. BNIP3, a member of the BH3-only family, is induced by hypoxia and oncogenes and is necessary for induction of cell death by dual-function CD47mAbs. BNIP3 can activate autophagy, a pro-survival function, and can also induce cell death by damaging mitochondria. The role of BNIP3 in cancer is controversial and context-dependent with some cancers over-expressing BNIP3 compared to low levels of expression in normal tissue, while other cancers cannot tolerate BNIP3 expression and silence the gene, often by methylation of the BNIP3 promoter. This suggests that cancers that tolerate BNIP3 expression employ an as yet unknown mechanism to protect themselves from its toxic effects. The C-terminal transmembrane (TM) domain of BNIP3 (residues 164-184) penetrates the outer mitochondrial membrane allowing the extreme C-terminal ten residue tail of BNIP3 (RRLTTSTSTF, residues 185-194) to extend across the intermembrane space to bind OPA1 on the inner mitochondrial membrane, a key step in the death mechanism. We used a phosphosite-specific antibody to detect phosphorylation of BNIP3 immunoprecipitated from Jurkat leukemia cells at residue T188 which resides in a canonical protein kinase A site (RRLT, amino acids 185-188). Using mass spectrometry of 6His-tagged BNIP3 isolated from HEK293 cells treated with 8BrcAMP, we identified as many as 4 additional phosphorylated sites in the C-terminal tail sequence (residues 189-194, TSTSTF). We generated phosphomimetic (S/T to D) and unphosphorylated (S/T to A or N) mutations at these residues and expressed the mutant BNIP3 proteins in 293 cells. All of the BNIP3 mutants associated with mitochondria but only the phosphomimetic mutants prevented BNIP3-induced mitochondrial damage and cell death. In contrast, mutation of the phosphorylated S/T residues to unphosphorylated residues resulted in rapid and extensive cell death. Importantly, phosphomimetic C-terminal BNIP3 residues blocked cell death without preventing autophagy, providing evidence that the two roles of BNIP3 can be regulated independently. We replicated these results in one lung cancer and three breast cancer cell lines. These findings suggest that phosphorylation at the C-terminus of BNIP3 is a switch that determines the pro-survival vs pro-death effects of BNIP3. Dual-function CD47mAbs may act by dephosphorylating BNIP3 expressed in tumor cells thus unleashing its killing potential.

Citation Format: William Frazier, Katherine Liu, Julie Dimitry, Benjamin Capoccia, Pamela Manning, Robert Karr. Phosphorylation of BNIP3 is a switch between life and death in cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2. doi:10.1158/1538-7445.AM2015-2