PKA inhibition is a central step in D,L-methadone-induced ER Ca²⁺ release and subsequent apoptosis in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is a hematologic cancer that mostly affects children. It accounts for over a quarter of ALL pediatric cancers, causing most of the cancer death among children. Previously, we demonstrated that D,L-methadone causes ALL cell apoptosis via mu-opioid receptor 1 (OPRM1)-triggered ER Ca2+ release and decrease in Ca2+ efflux, elevating [Ca2+](i). However, the precise mechanism by which D,L-methadone induces ER Ca2+ release remains to be defined. Here, we show that in ALL cells, D,L-methadone-induced ER Ca2+ release is blocked by inhibition of G(alpha i) but not G(beta Upsilon), indicating that the process is dependent on G(alpha i). Activation of adenylyl cyclase (AC) with forskolin or treatment with 8-CPT-cAMP blocks D,L-methadone-induced ER Ca2+ release, indicating that the latter results from G(alpha i)-dependent downregulation of AC and cAMP. The 14-22 amide (myr) PKA inhibitor alone elicits ER Ca2+ release, and subsequent treatment with D,L-methadone does not cause additional ER Ca2+ release, indicating that PKA inhibition is a key step in D,L-methadone-induced ER Ca2+ release and can bypass the D,L-methadone-OPRM1-AC-cAMP step. This is consistent with the decrease in PKA-dependent (i) inhibitory PLC beta 3 Ser1105 phosphorylation that leads to PLC beta 3 activation and ER Ca2+ release, and (ii) BAD Ser118 phosphorylation, which together ultimately result in caspase activation and apoptosis. Thus, our findings indicate that D,L-methadone-induced ER Ca2+ release and subsequent apoptosis in ALL cells is mediated by G(alpha i)-dependent downregulation of the AC-cAMP-PKA-PLC beta 3/BAD pathway. The fact that 14-22 amide (myr) alone effectively kills ALL cells suggests that PKA may be targeted for ALL therapy.