L-asparaginase induces IP3R-mediated ER Ca2+ release by targeting µ-OR1 and PAR2 and kills acute lymphoblastic leukemia cells

L-asparaginase is a standard therapeutic option for acute lymphoblastic leukemia (aLL), a hematologic cancer that claims the most lives of pediatric cancer patients. Previously, we demonstrated that L-asparaginase kills aLL cells via a lethal rise in [Ca²⁺]_i due to IP3R-mediated ER Ca²⁺ release followed by calpain-1-Bid-caspase-3/12 activation (Blood, 133, 2222-2232). However, upstream targets of L-asparaginase that trigger IP3R-mediated ER Ca²⁺ release remain elusive. Here, we show that L-asparaginase targets µ-OR1 and PAR2 and induces IP3R-mediated ER Ca²⁺ release in aLL cells. In doing so, µ-OR1 plays a major role while PAR2 plays a minor role. Utilizing PAR2- and µ-OR1-knockdown cells, we demonstrate that L-asparaginase stimulation of µ-OR1 and PAR2 relays its signal via G_αi and G_αq, respectively. In PAR2-knockdown cells, stimulation of adenylate cyclase with forskolin or treatment with 8-CPT-cAMP reduces L-asparaginase-induced µ-OR1-mediated ER Ca²⁺ release, suggesting that activation of µ-OR1 negatively regulates AC and cAMP. In addition, the PKA inhibitor 14-22 amide (myr) alone evokes ER Ca²⁺ release, and subsequent L-asparaginase treatment does not induce further ER Ca²⁺ release, indicating the involvement of PKA inhibition in L-asparaginase-induced µ-OR1-mediated ER Ca²⁺ release, which can bypass the L-asparaginase-µ-OR1-AC-cAMP loop. This coincides with (a) the decreases in PKA-dependent inhibitory PLCβ3 Ser1105 phosphorylation, which prompts PLCβ3 activation and ER Ca²⁺ release, and (b) BAD Ser118 phosphorylation, which leads to caspase activation and apoptosis. Thus, our findings offer new insights into the Ca²⁺-mediated mechanisms behind L-asparaginase-induced aLL cell apoptosis and suggest that PKA may be targeted for therapeutic intervention for aLL.