Jung Kwon Lee

Legumain (Lgmn) is a virulence factor found in the protozoan parasites Blastocystis and Trichomonas , which affect both humans and animals. However, its specific targets and cytotoxic mechanisms on host cells are not well understood. Recent findings show that Lgmn cleaves PAR2 at the N ₃₀ -R ₃₁ residue, a site also targeted by L-asparaginase, a vital treatment for acute lymphoblastic leukemia (ALL), a severe hematologic cancer that poses high risks to children. This emphasizes the urgent need for more effective therapeutic strategies. Here, we demonstrate that Lgmn induces ER Ca ²⁺ release via the µ-OR1-G _αi and PAR2-G _αq pathways. In PAR2-knockdown ALL cells, the stimulation of adenylate cyclase (AC) with forskolin or treatment with 8-CPT-cAMP effectively inhibits Lgmn-induced µ-OR1-mediated ER Ca ²⁺ release, indicating that Lgmn’s stimulation of µ-OR1 results in the downregulation of AC and a subsequent decrease in cAMP levels. Additionally, the PKA-specific inhibitor 14–22 amide alone triggers ER Ca ²⁺ release, and subsequent treatment with Lgmn does not enhance this effect, suggesting that PKA inhibition plays a role and that the Lgmn-µ-OR1-AC-cAMP axis can be bypassed in µ-OR1-mediated ER Ca ²⁺ release. Furthermore, we observed a corresponding reduction in the phosphorylation of PLCβ3 at Ser1105 and BAD at Ser118, both of which are regulated by PKA. The Lgmn-induced ER Ca ²⁺ release ultimately leads to apoptosis in ALL cells, which can be reversed by blocking ER Ca ²⁺ release. Our results thus provide novel insights into the specific targets of Lgmn secreted from the protozoa and demonstrate how this virulence factor induces cytotoxic effects on host cells, paving the way for innovative therapeutic strategies for patients with ALL.

L-asparaginase is an indispensable chemotherapeutic drug for patients with acute lymphoblastic leukemia (aLL), a life-threatening lymphoid neoplasm and the prime cause of cancer death among children. Previously, we reported that L-asparaginase kills aLL cells via an excessive rise in [Ca²⁺]_i due to IP3R-mediated ER Ca²⁺ release followed by stimulation of the intrinsic apoptotic pathway (Blood, 133, 2222-2232). We also demonstrated that L-asparaginase triggers ER Ca²⁺ release by targeting the G-protein-coupled receptor (GPCR), protease-activated receptor 2 (PAR2) (Cell Death & Discovery, 10:366). However, how L-asparaginase stimulates PAR2 remains unknown. Here, we show that elastase, which can disarm trypsin-mediated PAR2 activation by cleaving a S₆₇-V₆₈ residue downstream of the tethered ligand (TL) and removing it from PAR2, abrogates L-asparaginase-induced ER Ca²⁺ release, indicating that L-asparaginase targets the TL-containing PAR2 N-terminal extracellular domain to induce ER Ca²⁺ release. Inactive forms (T₁₁₁V/K₁₈₄T or D₁₁₂T/K₁₈₄T) of L-asparaginase do not induce ER Ca²⁺ release in μ-opioid receptor 1 (µ-OR1)-knockdown aLL cells, suggesting that L-asparaginase action on PAR2 requires its enzymatic activity. Time-lapse confocal microscopy of cells expressing mRFP-hPAR2-eYFP and nanoluciferase (Nluc) reporter release assays of cells expressing Nluc-hPAR2-eYFP showed that L-asparaginase cleaves PAR2 at the N-terminal extracellular I₂₆-G₇₁ domain. Cleavage assay of a PAR2 N-terminal peptide by L-asparaginase and subsequent LC-MS/MS analysis show that L-asparaginase is a PAR2 protease that cleaves N₃₀-R₃₁ and R₃₁-S₃₂ residues, unmasking the PAR2 TL. Thus, our findings reveal for the first time the molecular mechanism through which L-asparaginase activates PAR2, leading to perturbation of intracellular Ca²⁺ homeostasis and aLL cell apoptosis.

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.

Despite the success in treating newly diagnosed pediatric acute lymphoblastic leukemia (aLL), the long-term cure rate for the 20% of children who relapse is poor, making relapsed aLL the primary cause of cancer death in children. By unbiased genome-wide retroviral RNAi screening and knockdown studies, we previously discovered opioid receptor mu 1 (OPRM1) as a new aLL cell resistance biomarker for the aLL chemotherapeutic drug, l-asparaginase, i.e., OPRM1 loss triggers l-asparaginase resistance. Indeed, aLL cell OPRM1 level is inversely proportional to l-asparaginase IC50: the lower the OPRM1 level, the higher the l-asparaginase IC50, indicating that aLL cells expressing reduced OPRM1 levels show resistance to l-asparaginase. In the current study, we utilized OPRM1-expressing and -knockdown aLL cells as well as relapsed patient aLL cells to identify candidate targeted therapy for l-asparaginase-resistant aLL. In OPRM1-expressing cells, l-asparaginase induces apoptosis via a cascade of events that include OPRM1-mediated decline in [cAMP]_i, downregulation of PKA-mediated BAD S₁₁₈ phosphorylation that can be reversed by 8-CPT-cAMP, cyt C release from the mitochondria, and subsequent caspase activation and PARP1 cleavage. The critical role of PKA inhibition due to a decrease in [cAMP]_i in this apoptotic process is evident in the killing of OPRM1-knockdown and low OPRM1-expressing relapsed patient aLL cells by the PKA inhibitors, H89 and 14–22 amide. These findings demonstrate for the first time that PKA can be targeted to kill aLL cells resistant to l-asparaginase due to OPRM1 loss, and that H89 and 14–22 amide may be utilized to destroy l-asparaginase-resistant patient aLL cells.

L-Asparaginase (l-ASNase) is a strategic component of treatment protocols for acute lymphoblastic leukemia (ALL). It causes asparagine deficit, resulting in protein synthesis inhibition and subsequent leukemic cell death and ALL remission. However, patients often relapse because of the development of resistance, but the underlying mechanism of ALL cell resistance to l-asparaginase remains unknown. Through unbiased genome-wide RNA interference screening, we identified huntingtin associated protein 1 (HAP1) as an ALL biomarker for l-asparaginase resistance. Knocking down HAP1 induces l-asparaginase resistance. HAP1 interacts with huntingtin and the intracellular Ca2+ channel, inositol 1,4,5-triphosphate receptor to form a ternary complex that mediates endoplasmic reticulum (ER) Ca2+ release upon stimulation with inositol 1,4,5-triphosphate3. Loss of HAP1 prevents the formation of the ternary complex and thus l-asparaginase-mediated ER Ca2+ release. HAP1 loss also inhibits external Ca2+ entry, blocking an excessive rise in [Ca2+]i, and reduces activation of the Ca2+-dependent calpain-1, Bid, and caspase-3 and caspase-12, leading to reduced number of apoptotic cells. These findings indicate that HAP1 loss prevents l-asparaginase–induced apoptosis through downregulation of the Ca2+-mediated calpain-1-Bid-caspase-3/12 apoptotic pathway. Treatment with BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)] reverses the l-asparaginase apoptotic effect in control cells, supporting a link between l-asparaginase-induced [Ca2+]i increase and apoptotic cell death. Consistent with these findings, ALL patient leukemic cells with lower HAP1 levels showed resistance to l-asparaginase, indicating the clinical relevance of HAP1 loss in the development of l-asparaginase resistance, and pointing to HAP1 as a functional l-asparaginase resistance biomarker that may be used for the design of effective treatment of l-asparaginase-resistant ALL.