Ki-Young 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.

This study introduces a rapid, cost-effective, and efficient method for in ovo xenografting of patient-derived acute lymphoblastic leukemia (ALL) cells, encompassing both B-cell and T-cell lineages. Using fertilized chicken embryos, we injected patient-derived B-ALL and T-ALL cells into the vasculature of embryos 11 days post-fertilization (11dpf). Remarkably, four days following the injection, the engrafted human leukemia cells exhibited significant survival, proliferation, and vascular colonization within the developing chicken embryo. By 15dpf, we detected a notable increase in CD10/CD19+ B-ALL and CD4/CD8+ T-ALL cells in blood samples from the embryo's vasculature, confirming successful engraftment. This system facilitates efficient and reproducible assessment of leukemia cell behavior in a living organism without the considerable costs and ethical constraints associated with other animal models. This rapid approach provides a high-throughput and biologically relevant in vivo platform for evaluating potential therapeutic compounds. The in ovo patient-derived xenograft (PDX)-ALL system presented here offers a promising tool for preclinical drug screening, mechanistic studies, and potentially for personalized medicine approaches in leukemia research.

This study introduces a rapid, cost-effective, and efficient method for in ovo xenografting of patient-derived acute lymphoblastic leukemia (ALL) cells, encompassing both B-cell and T-cell lineages. Using fertilized chicken embryos, we injected patient-derived B-ALL and T-ALL cells into the vasculature of embryos 11 days post-fertilization (11dpf). Remarkably, four days following the injection, the engrafted human leukemia cells exhibited significant survival, proliferation, and vascular colonization within the developing chicken embryo. By 15dpf, we detected a notable increase in CD10/CD19+ B-ALL and CD4/CD8+ T-ALL cells in blood samples from the embryo's vasculature, confirming successful engraftment. This system facilitates efficient and reproducible assessment of leukemia cell behavior in a living organism without the considerable costs and ethical constraints associated with other animal models. This rapid approach provides a high-throughput and biologically relevant in vivo platform for evaluating potential therapeutic compounds. The in ovo patient-derived xenograft (PDX)-ALL system presented here offers a promising tool for preclinical drug screening, mechanistic studies, and potentially for personalized medicine approaches in leukemia research.

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.

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 μ-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_αi, but not G_βϒ, indicating that the process is dependent on G_αi. 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_αi-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 PLCβ3 Ser1105 phosphorylation that leads to PLCβ3 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_αi-dependent downregulation of the AC-cAMP-PKA-PLCβ3/BAD pathway. The fact that 14–22 amide (myr) alone effectively kills ALL cells suggests that PKA may be targeted for ALL therapy.

Editorial: Biomarkers and therapeutic strategies in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (aLL) is a malignant cancer in the blood and bone marrow characterized by rapid expansion of lymphoblasts. It is a common pediatric cancer and the principal basis of cancer death in children. Previously, we reported that L-asparaginase, a key component of acute lymphoblastic leukemia chemotherapy, causes IP3R-mediated ER Ca²⁺ release, which contributes to a fatal rise in [Ca²⁺]_cyt, eliciting aLL cell apoptosis via upregulation of the Ca²⁺-regulated caspase pathway (Blood, 133, 2222–2232). However, the cellular events leading to the rise in [Ca²⁺]_cyt following L-asparaginase-induced ER Ca²⁺ release remain obscure. Here, we show that in acute lymphoblastic leukemia cells, L-asparaginase causes mitochondrial permeability transition pore (mPTP) formation that is dependent on IP3R-mediated ER Ca²⁺ release. This is substantiated by the lack of L-asparaginase-induced ER Ca²⁺ release and loss of mitochondrial permeability transition pore formation in cells depleted of HAP1, a key component of the functional IP3R/HAP1/Htt ER Ca²⁺ channel. L-asparaginase induces ER Ca²⁺ transfer into mitochondria, which evokes an increase in reactive oxygen species (ROS) level. L-asparaginase-induced rise in mitochondrial Ca²⁺ and reactive oxygen species production cause mitochondrial permeability transition pore formation that then leads to an increase in [Ca²⁺]_cyt. Such rise in [Ca²⁺]_cyt is inhibited by Ruthenium red (RuR), an inhibitor of the mitochondrial calcium uniporter (MCU) that is required for mitochondrial Ca²⁺ uptake, and cyclosporine A (CsA), an mitochondrial permeability transition pore inhibitor. Blocking ER-mitochondria Ca²⁺ transfer, mitochondrial ROS production, and/or mitochondrial permeability transition pore formation inhibit L-asparaginase-induced apoptosis. Taken together, these findings fill in the gaps in our understanding of the Ca²⁺-mediated mechanisms behind L-asparaginase-induced apoptosis in acute lymphoblastic leukemia cells.

Loss of cyclin-dependent kinase 5 (Cdk5) in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) increases ER–mitochondria tethering and ER Ca²⁺ transfer to the mitochondria, subsequently increasing mitochondrial Ca²⁺ concentration ([Ca²⁺]_mt). This suggests a role for Cdk5 in regulating intracellular Ca²⁺ dynamics, but how Cdk5 is involved in this process remains to be explored. Using ex vivo primary mouse embryonic fibroblasts (MEFs) isolated from Cdk5^−/− mouse embryos, we show here that loss of Cdk5 causes an increase in cytosolic Ca²⁺concentration ([Ca²⁺]_cyt), which is not due to reduced internal Ca²⁺ store capacity or increased Ca²⁺ influx from the extracellular milieu. Instead, by stimulation with ATP that mediates release of Ca²⁺ from internal stores, we determined that the rise in [Ca²⁺]_cyt in Cdk5^−/− MEFs is due to increased inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca²⁺ release from internal stores. Cdk5 interacts with the IP3R1 Ca²⁺ channel and phosphorylates it at Ser₄₂₁. Such phosphorylation controls IP3R1-mediated Ca²⁺ release as loss of Cdk5, and thus, loss of IP3R1 Ser₄₂₁ phosphorylation triggers an increase in IP3R1-mediated Ca²⁺ release in Cdk5^−/− MEFs, resulting in elevated [Ca²⁺]_cyt. Elevated [Ca²⁺]_cyt in these cells further induces the production of reactive oxygen species (ROS), which upregulates the levels of Nrf2 and its targets, Prx1 and Prx2. Cdk5^−/− MEFs, which have elevated [Ca²⁺]_cyt, proliferate at a faster rate compared to wt, and Cdk5^−/− embryos have increased body weight and size compared to their wt littermates. Taken together, we show that altered IP3R1-mediated Ca²⁺ dynamics due to Cdk5 loss correspond to accelerated cell proliferation that correlates with increased body weight and size in Cdk5^−/− embryos.