Lee Jung Kwon, Kamran Hamza, Lee Ki-Young
Department of Cell Biology & Anatomy, Arnie Charbonneau Cancer and Alberta Children's Hospital Research Institutes, University of Calgary, Calgary, AB, Canada.
Cell Death Discov. 2024 Aug 15;10(1):366. doi: 10.1038/s41420-024-02142-9.
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 [Ca] due to IP3R-mediated ER Ca 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 Ca release remain elusive. Here, we show that L-asparaginase targets µ-OR1 and PAR2 and induces IP3R-mediated ER Ca 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 and G, 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 Ca release, suggesting that activation of µ-OR1 negatively regulates AC and cAMP. In addition, the PKA inhibitor 14-22 amide (myr) alone evokes ER Ca release, and subsequent L-asparaginase treatment does not induce further ER Ca release, indicating the involvement of PKA inhibition in L-asparaginase-induced µ-OR1-mediated ER Ca 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 Ca release, and (b) BAD Ser118 phosphorylation, which leads to caspase activation and apoptosis. Thus, our findings offer new insights into the Ca-mediated mechanisms behind L-asparaginase-induced aLL cell apoptosis and suggest that PKA may be targeted for therapeutic intervention for aLL.
L-天冬酰胺酶是急性淋巴细胞白血病（ALL）的一种标准治疗选择，ALL是一种血液系统癌症，在儿童癌症患者中致死率最高。此前，我们证明L-天冬酰胺酶通过IP3R介导的内质网钙释放导致[Ca]致命性升高，随后激活钙蛋白酶-1-Bid-半胱天冬酶-3/12，从而杀死ALL细胞（《血液》，第133卷，2222 - 2232页）。然而，触发IP3R介导的内质网钙释放的L-天冬酰胺酶上游靶点仍不清楚。在此，我们表明L-天冬酰胺酶靶向μ-阿片受体1（µ-OR1）和蛋白酶激活受体2（PAR2），并在ALL细胞中诱导IP3R介导的内质网钙释放。在此过程中，µ-OR1起主要作用，而PAR2起次要作用。利用PAR2和µ-OR1基因敲低的细胞，我们证明L-天冬酰胺酶对µ-OR1和PAR2的刺激分别通过G蛋白和G蛋白传递其信号。在PAR2基因敲低的细胞中，用福斯可林刺激腺苷酸环化酶或用8-环磷酸腺苷（8-CPT-cAMP）处理可减少L-天冬酰胺酶诱导的µ-OR1介导的内质网钙释放，这表明µ-OR1的激活对腺苷酸环化酶和环磷酸腺苷起负调节作用。此外，蛋白激酶A（PKA）抑制剂14 - 22酰胺（myr）单独即可引发内质网钙释放，随后用L-天冬酰胺酶处理不会诱导进一步的内质网钙释放，这表明PKA抑制参与了L-天冬酰胺酶诱导的µ-OR1介导的内质网钙释放，该过程可绕过L-天冬酰胺酶-µ-OR1-腺苷酸环化酶-环磷酸腺苷循环。这与以下情况相符：（a）依赖PKA的抑制性磷脂酶Cβ3（PLCβ3）丝氨酸1105磷酸化减少，这促使PLCβ3激活和内质网钙释放；（b）BAD丝氨酸118磷酸化减少，这导致半胱天冬酶激活和细胞凋亡。因此，我们的研究结果为L-天冬酰胺酶诱导ALL细胞凋亡背后的钙介导机制提供了新的见解，并表明PKA可能成为ALL治疗干预的靶点。
