in-ovo-xenografting-patient-derived-acute-lymphoblastic-leukemia-all

<em>In Ovo</em> Xenografting of Patient-Derived Acute Lymphoblastic Leukemia (ALL) Cells (PDX-ALL)

This study introduces a rapid, cost-effective, and efficient method for in ovo xenografting of patient-derived acute lymphoblastic leukemia (ALL) cells, ...

University of Calgary

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 () as an ALL biomarker for l-asparaginase resistance. Knocking down HAP1 induces l-asparaginase resistance. HAP1 interacts with huntingtin and the intracellular Ca channel, inositol 1,4,5-triphosphate receptor to form a ternary complex that mediates endoplasmic reticulum (ER) Ca release upon stimulation with inositol 1,4,5-triphosphate Loss of HAP1 prevents the formation of the ternary complex and thus l-asparaginase-mediated ER Ca release. HAP1 loss also inhibits external Ca entry, blocking an excessive rise in [Ca], and reduces activation of the Ca-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 Ca-mediated calpain-1-Bid-caspase-3/12 apoptotic pathway. Treatment with BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-','-tetraacetic acid tetrakis(acetoxymethyl ester)] reverses the l-asparaginase apoptotic effect in control cells, supporting a link between l-asparaginase-induced [Ca] 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  as a functional l-asparaginase resistance biomarker that may be used for the design of effective treatment of l-asparaginase-resistant ALL.

HAP1 loss confers l-asparaginase resistance in ALL by downregulating the calpain-1-Bid-caspase-3/12 pathway.

Various methodologies have been employed for the therapeutic interpolation of the progressive brain disorder Alzheimer's disease. Thus, β-secretase inhibition is significant to prevent disease progression in the early stages.

Neuroprotective Effect of β-secretase Inhibitory Peptide from Pacific Hake (Merluccius productus) Fish Protein Hydrolysate.

The search continues for improved therapy for acute lymphoblastic leukemia (aLL), the most common malignancy in children. Recently, D,L-methadone was put forth as sensitizer for aLL chemotherapy. However, the specific target of D,L-methadone in leukemic cells and the mechanism by which it induces leukemic cell apoptosis remain to be defined. Here, we demonstrate that D,L-methadone induces leukemic cell apoptosis through activation of the mu1 subtype of opioid receptors (OPRM1). D,L-Methadone evokes IP3R-mediated ER Ca release that is inhibited by OPRM1 loss. In addition, the rate of Ca extrusion following D,L-methadone treatment is reduced, but is accelerated by loss of OPRM1. These D,L-methadone effects cause a lethal rise in [Ca] that is again inhibited by OPRM1 loss, which then prevents D,L-methadone-induced apoptosis that is associated with activation of calpain-1, truncation of Bid, cytochrome C release, and proteolysis of caspase-3/12. Chelating intracellular Ca with BAPTA-AM reverses D,L-methadone-induced apoptosis, establishing a link between the rise in [Ca] and D,L-methadone-induced apoptosis. Altogether, our findings point to OPRM1 as a specific target of D,L-methadone in leukemic cells, and that OPRM1 activation by D,L-methadone disrupts IP3R-mediated ER Ca release and rate of Ca efflux, causing a rise in [Ca] that upregulates the calpain-1-Bid-cytochrome C-caspase-3/12 apoptotic pathway.

D,L-Methadone causes leukemic cell apoptosis via an OPRM1-triggered increase in IP3R-mediated ER Ca release and decrease in Ca efflux, elevating [Ca].

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 Ca release, which contributes to a fatal rise in [Ca], eliciting aLL cell apoptosis  upregulation of the Ca-regulated caspase pathway (Blood, 133, 2222-2232). However, the cellular events leading to the rise in [Ca] following L-asparaginase-induced ER Ca 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 Ca release. This is substantiated by the lack of L-asparaginase-induced ER Ca release and loss of mitochondrial permeability transition pore formation in cells depleted of HAP1, a key component of the functional IP3R/HAP1/Htt ER Ca channel. L-asparaginase induces ER Ca transfer into mitochondria, which evokes an increase in reactive oxygen species (ROS) level. L-asparaginase-induced rise in mitochondrial Ca and reactive oxygen species production cause mitochondrial permeability transition pore formation that then leads to an increase in [Ca]. Such rise in [Ca] is inhibited by Ruthenium red (RuR), an inhibitor of the mitochondrial calcium uniporter (MCU) that is required for mitochondrial Ca uptake, and cyclosporine A (CsA), an mitochondrial permeability transition pore inhibitor. Blocking ER-mitochondria Ca 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 Ca-mediated mechanisms behind L-asparaginase-induced apoptosis in acute lymphoblastic leukemia cells.

Requirement for ER-mitochondria Ca transfer, ROS production and mPTP formation in L-asparaginase-induced apoptosis of acute lymphoblastic leukemia 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 Ca release and decrease in Ca efflux, elevating [Ca]. However, the precise mechanism by which D,L-methadone induces ER Ca release remains to be defined. Here, we show that in ALL cells, D,L-methadone-induced ER Ca release is blocked by inhibition of G, but not G, indicating that the process is dependent on G. Activation of adenylyl cyclase (AC) with forskolin or treatment with 8-CPT-cAMP blocks D,L-methadone-induced ER Ca release, indicating that the latter results from G-dependent downregulation of AC and cAMP. The 14-22 amide (myr) PKA inhibitor alone elicits ER Ca release, and subsequent treatment with D,L-methadone does not cause additional ER Ca release, indicating that PKA inhibition is a key step in D,L-methadone-induced ER Ca 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 Ca 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 Ca release and subsequent apoptosis in ALL cells is mediated by G-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.

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

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], downregulation of PKA-mediated BAD S 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] 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.

PKA inhibition kills L-asparaginase-resistant leukemic cells from relapsed acute lymphoblastic leukemia patients.

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-asparaginase induces IP3R-mediated ER Ca release by targeting µ-OR1 and PAR2 and kills acute lymphoblastic leukemia cells.

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 [Ca] due to IP3R-mediated ER Ca release followed by stimulation of the intrinsic apoptotic pathway (Blood, 133, 2222-2232). We also demonstrated that L-asparaginase triggers ER Ca 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 S-V residue downstream of the tethered ligand (TL) and removing it from PAR2, abrogates L-asparaginase-induced ER Ca release, indicating that L-asparaginase targets the TL-containing PAR2 N-terminal extracellular domain to induce ER Ca release. Inactive forms (TV/KT or DT/KT) of L-asparaginase do not induce ER Ca 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 I-G 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 N-R and R-S 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 Ca homeostasis and aLL cell apoptosis.

Article	Year
HAP1 loss confers l-asparaginase resistance in ALL by downregulating the calpain-1-Bid-caspase-3/12 pathway. Blood\| PubMed ID: 30819925	2019
Neuroprotective Effect of β-secretase Inhibitory Peptide from Pacific Hake (Merluccius productus) Fish Protein Hydrolysate. Current Alzheimer research\| PubMed ID: 31724512	2019
D,L-Methadone causes leukemic cell apoptosis via an OPRM1-triggered increase in IP3R-mediated ER Ca release and decrease in Ca efflux, elevating [Ca]. Scientific reports\| PubMed ID: 33441856	2021
Requirement for ER-mitochondria Ca transfer, ROS production and mPTP formation in L-asparaginase-induced apoptosis of acute lymphoblastic leukemia cells. Frontiers in cell and developmental biology\| PubMed ID: 36895789	2023
PKA inhibition is a central step in D,L-methadone-induced ER Ca release and subsequent apoptosis in acute lymphoblastic leukemia. Frontiers in cell and developmental biology\| PubMed ID: 38721527	2024
PKA inhibition kills L-asparaginase-resistant leukemic cells from relapsed acute lymphoblastic leukemia patients. Cell death discovery\| PubMed ID: 38802344	2024
L-asparaginase induces IP3R-mediated ER Ca release by targeting µ-OR1 and PAR2 and kills acute lymphoblastic leukemia cells. Cell death discovery\| PubMed ID: 39147734	2024
L-asparaginase is a PAR2 N-terminal protease that unmasks the PAR2 tethered ligand. Cell death discovery\| PubMed ID: 40195325	2025

Jung Kwon Lee

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