cPLA2α inhibition leads to RIP1 kinase-dependent cell death in acute leukemia cells

Receptor-interacting protein kinase (RIP1) sits at the interface between inflammation and cell fate decisions. Through its kinase activity RIP1 is key to initiating necroptosis, which is promising alternative to treat cancers, particularly those resistant to pro-apoptotic drugs. We demonstrate that by inhibiting the proinflammatory type II cytosolic phospholipase, cPLA ₂ α , we can initiate RIP1 kinase-dependent cell death via necroptosis in T-cell acute lymphoblastic leukemia (T-ALL) cells. When we block cPLA ₂ α activity using the thiazolyl ketone AVX420, we find a marked reduction in the viability of two T-ALL cell lines, CCRF-CEM and Jurkat. This is associated with an increase in the phosphorylation of both RIP3 and MLKL, indicating the cells are undergoing necroptosis. We also see increased levels of apoptotic markers and lipid peroxidation, indicating that cells also undergo apoptosis and ferroptosis. Pre-treatment with the RIP1 kinase inhibitor, necrostatin-1, completely blocks cell death from AVX420 treatment, whereas pan-caspase inhibition and lipid-ROS scavengers are only partially effective. The inhibition of cPLA ₂ α leads to a rapid accumulation of mitochondrial-associated reactive oxygen species (mt-ROS), which is blocked either by necrostatin-1 or total ROS scavengers, but not by pan-caspase inhibition or liproxstatin-1. The increase in mt-ROS precedes the loss of mitochondrial membrane potential and accumulation of lipid-ROS, indicating apoptosis and ferroptosis are responses to unresolved oxidative stress in the necroptotic cells. Our findings suggest that in T-ALL cells, cPLA ₂ α inhibition activates RIP1 kinase. This leads to necroptosis and oxidative stress-dependent cell death. cPLA ₂ α could therefore be a relevant therapeutic target for treating refractory or relapsed disease where resistance to apoptosis is inherent.