cPLA2α inhibition leads to RIP1 kinase-dependent cell death in acute leukemia cells

cPLA2α inhibition leads to RIP1 kinase-dependent cell death in acute leukemia cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article cPLA 2 α inhibition leads to RIP1 kinase-dependent cell death in acute leukemia cells Felicity Ashcroft, Nur Mahammad, Astrid Feuerherm, Pegah Abdollahi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6922842/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Receptor-interacting protein kinase (RIP1) sits at the interface between inflammation and cell fate decisions. Through its kinase activity RIP1 is key to initiating necroptosis, which is promising alternative to treat cancers, particularly those resistant to pro-apoptotic drugs. We demonstrate that by inhibiting the proinflammatory type II cytosolic phospholipase, cPLA 2 α , we can initiate RIP1 kinase-dependent cell death via necroptosis in T-cell acute lymphoblastic leukemia (T-ALL) cells. When we block cPLA 2 α activity using the thiazolyl ketone AVX420, we find a marked reduction in the viability of two T-ALL cell lines, CCRF-CEM and Jurkat. This is associated with an increase in the phosphorylation of both RIP3 and MLKL, indicating the cells are undergoing necroptosis. We also see increased levels of apoptotic markers and lipid peroxidation, indicating that cells also undergo apoptosis and ferroptosis. Pre-treatment with the RIP1 kinase inhibitor, necrostatin-1, completely blocks cell death from AVX420 treatment, whereas pan-caspase inhibition and lipid-ROS scavengers are only partially effective. The inhibition of cPLA 2 α leads to a rapid accumulation of mitochondrial-associated reactive oxygen species (mt-ROS), which is blocked either by necrostatin-1 or total ROS scavengers, but not by pan-caspase inhibition or liproxstatin-1. The increase in mt-ROS precedes the loss of mitochondrial membrane potential and accumulation of lipid-ROS, indicating apoptosis and ferroptosis are responses to unresolved oxidative stress in the necroptotic cells. Our findings suggest that in T-ALL cells, cPLA 2 α inhibition activates RIP1 kinase. This leads to necroptosis and oxidative stress-dependent cell death. cPLA 2 α could therefore be a relevant therapeutic target for treating refractory or relapsed disease where resistance to apoptosis is inherent. Biological sciences/Cancer Health sciences/Medical research/Drug development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Necroptosis is one of the more recently described mechanisms of programmed cell death and is executed by the oligomerization of mixed lineage kinase domain-like protein (MLKL) to form the necrosome, leading to cell rupture and inflammation ( 1 , 2 ). Necroptosis can be induced by the activation of death receptors, e.g., the TNFα-receptor, or in response to stress induced by heat or altered pH as recently ( 3 ). The master regulator of necroptosis is receptor interacting protein kinase 1 (RIP1). Through post translational modifications, RIP1 integrates death receptor activation with survival signaling through NF-κB to determine cell fate. Physiologically, necroptosis appears important for eliminating infected cells, particularly when viral infections impair apoptosis, and can help promote innate immune responses through release of inflammatory mediators. Excessive necroptosis has been linked with some chronic inflammatory conditions and has the potential both to increase tumour progression and play a role as a potential therapeutic strategy. Cytosolic phospholipase A2 α (cPLA 2 α) is a member of the PLA 2 superfamily that cleave fatty acids from the sn-2 position of membrane phospholipids. Six major groups are classified by their localization, regulatory mechanisms, and specificity ( 4 ) where cPLA 2 α is the best studied member of the type IV group. Inactive cPLA 2 α is found in the cytosol, and its membrane translocation and activation are regulated by increased Ca 2+ concentration, phospholipid binding, and phosphorylation. It exhibits a strong selectivity for phospholipids with the highly reactive polyunsaturated fatty acid arachidonic acid (AA) at the sn-2 position ( 4 ) and both of the major cleavage products (free AA, and lysophosphatidylcholine (LPC)) are metabolized to potent lipid signaling molecules that are coupled to the activity of NF-κB and with established roles in physiological and pathological inflammation and cancer ( 5 – 7 ). T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of T-cell cancer that originates in the thymus. It predominantly affects young individuals, where chemotherapy has a high curative potential ( 8 , 9 ). In adults, however, higher rates of primary refractory disease and relapse contribute to a 5-year survival of around 30–40%, highlighting the limited treatment options. We recently demonstrated that cell lines originating from T-cell acute leukemias were highly sensitive to treatment with the thiazolyl ketone inhibitor of cPLA 2 α, AVX420, and further demonstrated that this was a result of an accumulation of ROS in these cells ( 10 ). Here, we investigate why T-ALL cells are sensitive to cPLA 2 α inhibition and provide evidence that loss of cPLA 2 α causes a rapid and RIP1-dependent accumulation of mitochondrial-associated ROS (mt-ROS) leading to necroptosis, as well as apoptosis, and ferroptosis dependent on the cellular context. Materials and Methods 2.1 Materials Cell culture media RPMI 1640 (#R0883), Gentamycin (#G1397), Propidium Iodide (#P4170), Ribonuclease A (#R6513), Dimethyl sulfoxide (DMSO) (#2650), Valinomycin (#V0627), Ferrostatin-1 (#SML0583), Cu-ATSM (#SML0769), Liproxstatin-1 (#SML1414), 1S,3R-RSL 3 (#SML2234), ML210 (#SML0521), Necrostatin-1 (#480065), and N-Acetyl-L-Cysteine (#A7250) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against cleaved PARP-1 (CST #5625), cleaved caspase-3 (CST #9664), β-Actin (CST #4970), Phospho-MLKL (Ser358) (CST#91689), and Phospho-RIP3 (Ser227) (CST#93654) were obtained from Cell Signaling Technology (Danvers, MA, USA). Doxorubicin (#15007) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Fetal Bovine Serum (FBS) (#10270106) was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Z-VAD-FMK (Pan caspase -inhibitor) (#HY-16658B) and SM-164 (#HY-15989) were purchased from MedChemExpress (Monmouth Junction, United States). BV-6 ((#inh-bv-6) was purchased from Invivogen (San Diego, California, United States). L-glutamine (#17-605E) was purchased from Lonza Pharma & Biotech (Hochbergerstrasse, Basel, Switzerland). AVX420 ((Methyl 2-(2-(4-heptyloxy)-phenoxy)-acetyl)thiazole-4-carboxylate) was synthesized in the laboratory of organic chemistry at the University of Athens by George Kokotos. AVX420 was stored at -80ºC in DMSO. 2.2 Methods 2.2.1 Maintenance and plating of T-ALL cells The human acute T-cell lymphoblastic leukemia cell lines CCRF-CEM and Jurkat E6.1 were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and grown in RPMI 1640 medium, supplemented with L-glutamine (2 mM), Gentamycin (0.1 mg/ml), and 10% FBS (complete medium) at 37ºC with 5% CO 2 . The cells were confirmed to be Mycoplasma -free. The cells were harvested by centrifugation and counted using the TC20 automated cell counter from BIO-RAD (Hercules, California, USA) using 0.4% Trypan blue from NanoEnTek (Waltham, MA, USA) to check viability. The cells were seeded in complete medium at a density of 200,000 cells/ml in either 96 or 24-well plates. On the same day, the cells were treated and incubated at 5% CO 2 and 37ºC for the indicated time. 2.2.2 Cell viability and PI exclusion assays Cell viability was assessed using the CellTiter-Glo® (CTG) assay. Cells were seeded in 96-well plates, and the assay was performed according to the manufacturer’s instructions. Luminescence was measured using a Cytation 5 Cell Imaging Multimode Reader (BioTek Instruments, Winooski, VT, USA). To assess cell membrane integrity and distinguish live from dead cells, a propidium iodide (PI) exclusion assay was used. Cells were plated in 24-well plates, treated as indicated, and incubated with 1.4 µg/mL PI for 5 minutes at room temperature. Data were acquired using a CytoFLEX flow cytometer (Beckman Coulter, USA) with a filter set optimized for PI detection (excitation: 488 nm; emission: 585/42 nm). 2.2.3 Cell Cycle Analysis Cells were plated in 24-well plates, treated as indicated, then harvested and washed with PBS. For fixation, ice-cold 70% ethanol was added dropwise while vortexing to prevent clumping, followed by incubation at 4°C for 30 minutes. Fixed cells were centrifuged, washed with PBS, and treated with 50 µL of RNase A (50 µg/mL) for 20 minutes at room temperature. DNA was stained with PI (2.5 µg/mL) and incubated on ice for 15 minutes in the dark. Samples were analyzed using a NovoCyte flow cytometer (ACEA Biosciences, Agilent Technologies, USA) with a PI-optimized filter set (excitation: 488 nm, emission: 585/40 nm). Flow cytometry data were used to assess cell cycle distribution. 2.2.4 Annexin V-FITC assay The annexin V-fluorescein isothiocyanate (FITC) kit from Tau Technologies (Albuquerque, NM, USA) was used to measure phosphatidylserine (PS) externalization. Cells were plated in 24-well plates. following the protocol we have described previously ( 11 ). Data was acquired through either an LSRII flow cytometer from BD Biosciences (San Jose, CA, USA) or a CytoFLEX flow cytometer from Beckman Coulter (California, USA). Subsequent data analysis was performed using Novoexpress Software v1.5.0 from Agilent Technologies (Santa Clara, USA). 2.2.5 Caspase-3/7 Activity Assay Caspase activity was measured using a Caspase-3/7 Glo assay kit (Promega Corporation, Madison, WI, USA) following the protocol described in our previous study ( 11 ). This assay produces a luminescent substrate with a four-peptide sequence, which, after cleavage by caspase-3/7, generates a light signal. The resulting luminescence was recorded with the Cytation 5 cell imaging multimode reader (Biotek Instruments, Winooski, VT, USA). 2.2.6 Preparation of Plates for ROS and Mitochondrial Membrane Potential Detection Experiments After treatment with the compound and controls, cells were washed with PBS and resuspended in DPBS with 1% BSA and dye. They were incubated with Deep Red, BODIPY™ 581/591 C11, and MitoSOX™ (1 h), or JC-1 (15 min) at 37°C, 5% CO₂, following manufacturer’s instructions. The acquired data were analyzed using Novoexpress Software v1.5.0 from Agilent Technologies (Santa Clara, USA). 2.2.6.1 ROS Detection with Deep Red Dye The level of reactive oxygen species (ROS) was assessed using the Cellular ROS Assay Kit (Cat. No. ab186029; Abcam, Cambridge, UK). This kit utilizes a deep red dye that can permeate cell membranes and emit deep red fluorescence upon interacting with ROS. For the assay, the 200X concentrated deep red dye reagent was diluted in DPBS (Dulbecco's Phosphate Buffered Saline) to achieve a 1X final concentration in solution, which was then added to the samples. Data was acquired using a CytoFLEX flow cytometer (Beckman Coulter, California, USA) with a filter set optimized for the deep red dye detection (excitation at 640 nm and emission at 675/25 nm). 2.2.6.2 Lipid ROS Detection with BODIPY™ 581/591 C11 Lipid-derived ROS was identified using the lipid peroxidation sensor BODIPY™ 581/591 C11 (Cat. No. D3861, Thermo Fisher Scientific, Waltham, MA, USA). In the procedure, 1 mM BODIPY solution was diluted in DPBS to obtain a 2 µM final concentration, which was subsequently added to the samples. Data was analyzed using a CytoFLEX flow cytometer (Beckman Coulter, California, USA) with filter sets optimized for BODIPY detection (excitation at 488 nm and emission at 525/40 nm). 2.2.6.3 Mitochondrial ROS Detection with MitoSOX ™ The MitoSOX red mitochondrial superoxide indicator for live-cell imaging (Cat. No. M36008, Thermo Fisher Scientific) was used to evaluate the production of mitochondrial reactive oxygen species (ROS). MitoSOX can permeate live cells and selectively target mitochondria. The solution was prepared by diluting the 1mM MitoSOX reagent in DPBS to achieve a final concentration of 0.5 µM. Data acquisition was executed using the CytoFLEX flow cytometer from Beckman Coulter (California, USA), with a 488 nm excitation laser and a 585/42 nm bandpass emission filter set for detecting MitoSOX Red fluorescence. 2.2.6.4 Mitochondrial Membrane Potential Sensor with JC-1 The mitochondrial membrane potential was assessed using JC-1 (Cat. No. T3168, Thermo Fisher Scientific). JC-1 is a membrane-permeant dye that specifically targets mitochondria and is commonly employed to assess mitochondrial health. The solution was prepared by diluting a 5 mg/ml stock of JC-1 in DPBS to a final concentration of 5 µg/ml and added to the samples. Data collection was carried out with an LSRII flow cytometer from BD Biosciences (San Jose, CA, USA), using a 488 nm excitation laser and the following filter sets: a 530/30 nm bandpass filter for detecting the green monomer form (low membrane potential) and a 585/42 nm bandpass filter for detecting the red J-aggregate form (high membrane potential). 2.2.7 Western Blot Protein extraction and Western blotting were performed as described in our previous study ( 11 ). Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and measured with a Cytation 5 Cell Imaging Multimode Reader (BioTek Instruments, Winooski, VT, USA). Western blotting was then carried out according to our previous protocol ( 11 ) and imaged using a Syngene G:BOX Chemi XX6 gel documentation system (Frederick, USA). Image analysis was conducted using Image Studio software (LI-COR Biosciences, Lincoln, NE, USA) and ImageJ 1.43q (NIH, USA). 2.2.8 Statistical Analysis GraphPad Prism 9.3.1 (San Diego, CA, USA) was utilized for statistical analysis and generating figures. For statistical evaluations, one-way ANOVA was employed, and Dunnett's posthoc analysis was used to correct for multiple comparisons when comparing treated samples to the untreated control. Differences were considered significant if the p-value was less than 0.05. Results 3.1 Loss of T-ALL cell viability by inhibition of cPLA 2 α . We tested the effect of the cPLA 2 α inhibitor AVX420 on the viability of two T-ALL cell models, CCRF-CEM and Jurkat. Using CTG assays, we found a dose-dependent inhibition of viability in both cell lines. Half-maximal inhibitory concentration (IC50s) were calculated and found to be 0.51 and 0.52 µM in CCRF-CEM and Jurkat cells respectively after a 24-hour treatment. AVX420 was slightly more effective in the CCRF-CEM cells versus Jurkat after 72 hours, with an IC50 of 0.28 versus 0.4 µM (Fig. S1 A ). In contrast to the chemotherapy agent doxorubicin, which caused pronounced arrest of cells in G2/M phase of the cell cycle, we found no discernable effect of AVX420 on cell cycle distribution, and cell death was therefore considered to be the primary reason for reduced ATP production observed using CTG assays ( Fig. 1 , Fig. S1 B) . 3.2 cPLA 2 α inhibition initiates multiple regulated cell death pathways in T-ALL To identify the cause of cell death, we first looked at whether the cells were undergoing apoptosis, as previously observed in multiple myeloma cells ( 11 ). Apoptosis can be triggered by the presence of extracellular death signals or the absence of survival signals, and its execution is dependent on the coordinated activation of caspase enzymes ( 12 ). To identify apoptosis, we measured externalized phosphatidylserine (PS) by Annexin V-FITC staining using flow cytometry and activity of effector caspases with the caspase 3/7 glo assay using a plate-based assay. We found a dose-dependent increase in the proportion of early apoptotic cells (Annexin V positive / PI negative) and an increase in the activity of effector caspases in response to AVX420 in both CCRF-CEM and Jurkat cells (Fig. 2 A, B). We confirmed that caspase-3 and PARP-1 proteins were cleaved in response to AVX420 in CCRF-CEM cells using Western blotting (Fig. 2 C, Fig. S2A ). To explore whether apoptosis is the major cause of cell death, we preincubated the cells with the pan-caspase inhibitor Z-Val-Ala-Asp-Fluoro Methyl Ketone (Z-VAD) before treatment with AVX420. As expected, Z-VAD preincubation completely blocked both the effector caspase activity and cleavage of caspase 3 and PARP proteins, however, PS externalization and cell death were only partially blocked, particularly in CCRF-CEM cells, indicating a significant proportion of cell death was caspase-independent (Fig. 2 D, Fig. S2B, C). We then investigated the involvement of two additional RCD pathways, necroptosis and ferroptosis. Necroptosis is executed by the mixed lineage kinase domain-like protein (MLKL) upon activation by receptor-interacting protein (RIP) kinases RIP1 and RIP3 ( 1 , 2 ) in response to tumor necrosis factor TNFα-receptor signaling ( 13 , 14 ). Ferroptosis, is distinguished by the accumulation of iron and lipid peroxidation, which leads to membrane rupture. It can be caused by redox imbalance in the cells, ( 15 , 16 ) and free radical scavengers such as ferrostatin-1, liproxstatin-1, and exogenous glutathione (GSH) are potent inhibitors of ferroptotic cell death ( 17 ). To determine whether necroptosis was activated in response to cPLA 2 α inhibition, we measured the phosphorylation of two key proteins in the necroptosis cascade, RIP3 and MLKL. As a positive control, necroptosis was induced with a combination of TNF-α, Smac mimetic SM-164, and Z-VAD-FMK, the combination denoted “TSZ” [40]. cPLA 2 α inhibition led to a dose-dependent increase in the phosphorylation of both RIP3 and MLKL, which was partially blocked by pre-treatment with the RIP1 inhibitor necrostatin-1 (Fig. 3 A, Fig. S3A, Fig. S2C ). To determine whether ferroptosis was induced in response to cPLA 2 α inhibition, we treated cells with AVX420 and measured the levels of lipid peroxides (lipid-ROS) using flow cytometric analysis of Bodipy 581/591 C11 staining. The glutathione peroxidase 4 (GPX4) inhibitor RSL-3 was used to induce ferroptosis as a positive control ( 18 ). cPLA 2 α inhibition caused a dose-dependent increase in lipid-ROS, suggesting ferroptosis was also underway (Fig. 3 B) and we concluded that apoptosis, necroptosis and ferroptosis all occur in response to cPLA 2 α inhibition in T-ALL cells. 3.4 RIP1 inhibition protects cells from loss of cPLA 2 α activity. To investigate the relative importance of ferroptosis and necroptosis to cell death resulting from cPLA 2 α inhibition, we treated cells with either known inhibitors of ferroptosis (ferrostatin-1, liproxstatin-1, or CuATSM) or the RIP1 inhibitor necrostatin-1 before addition of AVX420, and measured cell viability after 24 hours using CTG assays. As positive controls, the GPX4 degradation agent ML210 ( 19 ) and necroptosis inducers TBZ (TNF-α, Smac mimetic BV-6, and Z-VAD-FMK) [41] were used in addition to RSL3 and TSZ to induce ferroptosis and necroptosis, respectively. Pretreatment with ferroptosis inhibitors restored CCRF-CEM cell viability to > 80% when used in combination with either RSL-3 or ML210 compared to approximately 60% when used in combination with AVX420. Necrostatin-1 on the other hand was more effective against AVX420, restoring viability to approximately 80% versus only 40% when used in combination with TSZ or TBZ ( Fig. 4 A ) . Combining cell death inhibitors did not give additional protection in viability assays and combining ferroptosis inhibitors with necrostatin-1 was detrimental compared to using necrostatin-1 alone (Fig. S2B) . When we tested the same inhibitors using propidium iodide exclusion assay to measure cell death, necrostatin-1 provided complete resistance to the effects of AVX420 in both cell lines, while as we observed previously, Z-VAD provided only partial resistance ( Fig. 4 B ) . The ferroptosis inhibitor liproxstatin had contrasting effects in CCRF-CEM and Jurkat cells, providing partial protection from cell death in CCRF-CEM cells, but not in Jurkat. Cu-ATSM, a different ferroptosis inhibitor, provided some protection against AVX420- induced death in Jurkat cells, and this effect was additive in combination with Z-VAD ( Fig. 4 B ) . In addition to inhibition of ferroptosis, CuATSM can protect damaged mitochondria by delivering Cu 2+ ( 20 ), which could explain the discrepancy in these results. So far, we have demonstrated that cPLA 2 α inhibition initiates apoptosis, necroptosis, and ferroptosis which together contribute to the loss of cell viability in T-ALL cells. Necrostatin-1 blocked all AVX420-dependent cell death in both cell types implying that RIP1 may be mechanistically upstream of all three cell death pathways in T-ALL. 3.5 cPLA 2 α inhibition causes RIP1-dependent mitochondrial ROS accumulation Our previous findings indicate that unresolved oxidative stress, which could be rescued with ROS scavengers, underlies the cellular response to AVX420 in cancer ( 10 ). Mitochondria play a central role in integrating metabolic and cell death signaling. To explore the role of mitochondria in the response to cPLA 2 α inhibition, we measured the levels of mitochondrial ROS using MitoSOX, a mitochondrial dye that fluoresces when oxidized by superoxide, and JC-1, a cationic dye that indirectly indicates mitochondrial membrane potential (MMP). Treatment with AVX420 caused a clear, dose-dependent increase in the levels of mt-ROS after 4 hours in both cell lines (Fig. 5 A) but had no effect on the MMP (Fig. 5 B). Valinomycin, a potassium ionophore and known mitochondrial respiration uncoupler ( 21 ), on the other hand, caused a highly significant loss of MMP (Fig. 5 B). Performing a time course analysis following treatment with 0.5 µM AVX420 showed that the increased levels of both mt-ROS and lipid-ROS occurred rapidly, within 1 hour, and were found in close to 80% of cells 12 hours after treatment. Accumulation of ROS preceded effects on mitochondrial membrane potential, which began 8 to 12 hours after treatment and mirrored the reduction in viable cells ( Fig. 5 C ) . In addition to its central role in the control of cell fate decisions in response to TNFα signaling, RIP1 can associate with mitochondria, and both activate and be activated by mt-ROS ( 22 , 23 ). To test whether the observed mt-ROS accumulation was dependent on the activity of RIP1 we preincubated cells with necrostatin-1 and measured mt-ROS after treatment with AVX420. Necrostatin-1 was as effective as universal ROS scavengers GSH and NAC at preventing mt-ROS accumulation whereas both liproxstatin-1, and Z-VAD were completely ineffective ( Fig. 6 A ) . The increases in lipid-ROS and total ROS on the other hand were effectively blocked by liproxstatin-1, as well as necrostatin-1 and the universal ROS scavengers but not by Z-VAD (Fig. 6 B and Fig. S6A ). In CCRF-CEM cells, liproxstatin-1 and necrostatin-1 both prevented mitochondrial uncoupling, while in Jurkat, a significant protective effect was only seen using necrostatin-1 ( Fig. 6 C ). In both cases, Z-VAD had no protective effect. The two cell lines showed contrasting responses to liproxstatin. In both cases liproxstatin prevented lipid-ROS accumulation but in Jurkat this did not prevent loss of mitochondrial membrane potential and cell death, suggesting that apoptosis and necroptosis dominate in Jurkat cells, but in CCRF-CEM ferroptosis is an important RCD pathway. Discussion Superoxide anions produced during oxidative metabolism in mitochondria are the primary source of intracellular ROS and drive signaling events that regulate cell growth and survival ( 24 ). ROS-dependent signaling is typically pro-tumorigenic and can contribute to the initiation and progression of the cancer ( 25 , 26 ). Both the higher ROS levels seen in some cancers, and increased antioxidant response signaling e.g. by Nrf-2 ( 26 ) are vulnerabilities that can be targeted therapeutically. Proliferative signaling can, for example, be inhibited by lowering ROS levels using antioxidants, or in contrast, programmed cell death can be triggered by inhibiting the cell’s antioxidant response system, leading to oxidative stress-induced regulated cell death ( 27 – 32 ). Here, we found that inhibition of cPLA 2 α increased mt-ROS and induced necroptosis, apoptosis, and ferroptosis in T-ALL cells, implying cPLA 2 α may be important in the cell’s antioxidant response system. In neurons, LOX-dependent AA metabolism can amplify ROS levels ( 33 – 36 ) and in this context, cPLA 2 α inhibition can suppress ROS and restrict inflammatory tissue damage ( 37 , 38 ). In contrast, we find that in leukemia cell lines, cPLA 2 α inhibition increases ROS, which is consistent with studies in U937 leukemia cells where cPLA 2 α was shown to be cytoprotective against ROS induced by peroxynitrate treatment ( 39 ). Other phospholipase A2 enzymes protect cells from oxidative stress by removing oxidized lipid species from membrane phospholipids. Lp-PLA2, for example, protects against ferroptosis in gastric cancer cells by catalyzing the metabolism of oxidized phosphatidylethanolamine ( 40 ) and iPLA2γ removes oxidized phospholipids and participates in lipid remodeling in mitochondria ( 41 ). A similar mechanism could be considered, assuming cPLA2α localizes to mitochondria, where its highly oxidizable substrates are present in abundance ( 42 , 43 ). Mitochondrial localization of cPLA 2 α has been reported in nerve tissue ( 44 , 45 ) however, unlike iPLA2γ, cPLA 2 α does not have a known mitochondrial localization sequence, and the mechanism for mitochondrial localization in astrocytes was dependent on its interaction with mitochondrial antiviral signaling protein (MAVS) ( 45 ). Critically, we found that the RIP1 inhibitor necrostatin-1, as well as affording the T-ALL cell lines protection from all AVX420-induced programmed cell death, prevented the increase in mt-ROS. RIP1 is an important molecular switch between cell survival and cell death in inflammatory conditions ( 46 ) and it is the kinase activity of the protein that drives the formation of complexes that promote apoptosis and necroptosis. Phosphorylation at Ser25 by IKKα/β is key to keeping the kinase activity of RIP1 in check ( 47 ). Given the established role of cPLA 2 α in activation of NF-κB and IKKα/β ( 5 , 6 , 48 , 49 ), we suggest that inhibiting cPLA 2 α could activate RIP1 by suppressing inhibitory phosphorylation, and that RIP1 activity initiates ROS accumulation as demonstrated in murine embryonic fibroblasts ( 50 ). We showed previously that exogenous addition of arachidonic acid could not rescue AVX420-induced ROS formation or cell death ( 10 ), therefore LPC-derived signaling molecules would be likely candidates in maintaining NF-κB/IKKα/β activity in this case. Alternatively, mt-ROS itself is a key regulator of the autophosphorylation of RIP1 at Ser 161 which drives RIP3 recruitment to the necrosome ( 51 ). If cPLA 2 α inhibition were the primary driver of increased mt-ROS, this could trigger a positive feedback loop involving RIP1/RIP3 activation, explaining the RIP1-dependent phenotype. These alternate models are summarized in Fig. 7 . Further investigations into the links between cPLA 2 α, NFκB, and RIP1 signaling will be key to delineating these processes. Exploiting the connections between oxidative stress and programmed cell death is an emerging tool in the fight against cancer ( 52 ). High innate level of ROS and/or defective antioxidant response mechanisms can make certain leukemias, and aggressive breast and lung cancers particularly sensitive to ROS inducers ( 53 – 55 ). Inhibitors of cPLA 2 α could have significant therapeutic value in these cancers, particularly in acute lymphocytic leukemia, where novel targeted therapeutics with better tolerability are sorely needed. Declarations Acknowledgments: A special thanks to Samah Elsaadi for his valuable guidance, insights, and encouragement throughout this study. Additionally, we would like to acknowledge that Figure 7 was created with the help of BioRender.com. Author Contributions: Conceptualization, B.J., A.J.F., F.J.A., M.B., and N.M.; methodology, N.M.; software, N.M.; validation, N.M., F.J.A., A.J.F., M.B. P.A., and B.J.; formal analysis, N.M.; investigation, N.M.; resources, B.J., M.B., and A.J.F.; data curation, N.M., A.J.F., and F.J.A.; manuscript preparation, N.M., F.J.A; manuscript review and editing, F.J.A., A.J.F., M.B., P.A. and B.J.; visualization, N.M., F.J.A. and A.J.F.; supervision, B.J., M.B., A.J.F. and F.J.A.; project administration, B.J. and A.J.F.; funding acquisition, B.J. and A.J.F. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by The Research Council of Norway grant no. 311392 and Coegin Pharma. Conflicts of Interest: B.J., A.J.F. and F.J.A hold shares in Coegin Pharma. N.M. and F.J.A. work for Coegin Pharma. All other authors report no conflicts. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6922842","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":475061272,"identity":"8f61b13b-67ff-4ced-8f90-201647c3cea4","order_by":0,"name":"Felicity Ashcroft","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYBAC9gbmhgMMBmA2G4jgBwrh18JzgBGkxQCuRbIHJERIC5BC1iKRQEALe2PjgR8FfxIb2I8/e/Bzj42EveTb448L2xjy+HFp4TnYcLDHwCCxgSfH3LDnWZoEj3ReYvPMNoZiSRx22UskNhxmAGlhyGGTZjhwuI5HOsewmbeNIXHDARy2yD+EauF//gykRYJH8gwBLRKMUC0SCWYQLRI8BLTwJIL8YmzcJvHGTLLnANAvZ3IMZ/Ock0icicMvPOyHD3/48UdOtp8//ZnEjwM2EuztZww+85TZJPbjCDEYcGxDE5AgoAEYcgRVjIJRMApGwcgFAG1hWJIXp5k+AAAAAElFTkSuQmCC","orcid":"","institution":"Norwegian University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Felicity","middleName":"","lastName":"Ashcroft","suffix":""},{"id":475061273,"identity":"0ac5a3f8-4791-455d-9182-a4af803c3814","order_by":1,"name":"Nur Mahammad","email":"","orcid":"","institution":"Norwegian University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Nur","middleName":"","lastName":"Mahammad","suffix":""},{"id":475061274,"identity":"8df0ce29-40c5-4be0-96b5-a0debedd83d6","order_by":2,"name":"Astrid Feuerherm","email":"","orcid":"","institution":"Norwegian University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Astrid","middleName":"","lastName":"Feuerherm","suffix":""},{"id":475061275,"identity":"0554ac7a-3194-47e3-a58c-e5ccc37e3dd1","order_by":3,"name":"Pegah Abdollahi","email":"","orcid":"","institution":"Oslo University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pegah","middleName":"","lastName":"Abdollahi","suffix":""},{"id":475061276,"identity":"4fc995aa-2892-4993-944c-68225f60dfd8","order_by":4,"name":"Magne Børset","email":"","orcid":"https://orcid.org/0000-0001-5179-2835","institution":"Norwegian University of Science and Technology, Trondheim, Norway","correspondingAuthor":false,"prefix":"","firstName":"Magne","middleName":"","lastName":"Børset","suffix":""},{"id":475061277,"identity":"e65f55dc-953c-49bb-869b-e61a4b14ecbd","order_by":5,"name":"Berit Johansen","email":"","orcid":"","institution":"Norwegian University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Berit","middleName":"","lastName":"Johansen","suffix":""}],"badges":[],"createdAt":"2025-06-18 12:06:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6922842/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6922842/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86924087,"identity":"d5d7a7a6-c98f-4437-84fc-335e1651033a","added_by":"auto","created_at":"2025-07-17 08:13:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":135848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecPLA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eα\u003c/strong\u003e \u003cstrong\u003einhibition results in decreased viability in T-ALL cells. A.\u003c/strong\u003e Dose-dependent induction of cell death in CCRF and Jurkat cells exposed to AVX420 at the indicated concentrations for 24 hours. The percentage of live and dead cells was assessed by propidium iodide exclusion using flow cytometry. \u003cstrong\u003eB.\u003c/strong\u003e Cell cycle analysis in CCRF and Jurkat cells after treatment with AVX420 or doxorubicin for 24 hours. The percentage of cells in G1, G2, and S was calculated based on propidium iodide staining of fixed cells measured with flow cytometry. Data are the mean ± standard deviation of at least three independent experiments. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, compared to vehicle treated control.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6922842/v1/fe215172b890eee6f547413f.png"},{"id":86924382,"identity":"42145adb-0e77-4bab-a1d3-a4972e9d2e4d","added_by":"auto","created_at":"2025-07-17 08:21:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":194616,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAVX420 induces caspase-dependent cell death in T-ALL cells.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Flow cytometric analysis of annexin V and propidium iodide staining of CCRF-CEM and Jurkat cells treated with indicated concentrations of AVX420 or doxorubicin for 24 hours. The proportions of live, apoptotic, and dead cells in each sample were estimated by quadrant analysis of Annexin V versus PI staining. \u003cstrong\u003eB. \u003c/strong\u003eCaspase 3/7 activity determined by caspase glow assay in CCRF-CEM and Jurkat treated with indicated concentrations of AVX420 or doxorubicin for 24 hours in the presence or absence of Z-VAD. \u003cstrong\u003eC.\u003c/strong\u003e Representative Western blot showing the levels of cleaved caspase-3, β-actin, and PARP-1 in CCRF-CEM cell lysates having been treated with the indicated concentrations of AVX420 or doxorubicin for 24 hours in the presence or absence of Z-VAD. Additional Western blots along with densitometric analysis are available in supplemental information \u003cstrong\u003eFig S2A\u003c/strong\u003e. \u003cstrong\u003eD. \u003c/strong\u003e\u0026nbsp;Viability determined by propidium iodide exclusion in CCRF-CEM and Jurkat cells, where cells were pretreated with Z-VAD-FMK for 30 minutes, followed by treatment with AVX420 or doxorubicin at the indicated doses for 24 hours. Representative dot plots are shown in the Figure as well. Graphical data are presented as the mean + standard deviation of at least three independent experiments. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, compared to vehicle-treated control. #p\u0026lt;0.01, ##p\u0026lt;0.0001 when only viable populations were compared between single dose and combination in Figure D. +p\u0026lt;0.05, ++p\u0026lt;0.01, +++p\u0026lt;0.001, ++++p\u0026lt;0.0001 when only apoptotic populations were compared between single dose and combination in Figure D.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6922842/v1/25dc56c7b4eea789ed92f881.png"},{"id":86924090,"identity":"09b98419-5ecc-48cd-abfe-16f6b56763bf","added_by":"auto","created_at":"2025-07-17 08:13:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":118941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAVX420 induces necroptotic and ferroptotic cell death in T-ALL cells.\u003c/strong\u003e A\u003cstrong\u003e. \u003c/strong\u003eRepresentative Western blot and densitometric analysis showing P-RIP3, P-MLKL, and beta-actin in CCRF-CEM cell lysates treated with the indicated concentrations of AVX420 and TSZ in the presence and absence of Necrostatin-1 for 24 hours. \u003cstrong\u003eB.\u003c/strong\u003e Flow cytometric analysis of Bodipy 581/591 C11 staining in CCRF-CEM and Jurkat cells treated with indicated concentrations of AVX420 for 4 hours. Representative dot plots showing the gating strategy are presented in \u003cstrong\u003eFig. S3B.\u003c/strong\u003e The cell viability was verified by propidium iodide exclusion and shown in \u003cstrong\u003eFig. S3C\u003c/strong\u003e. Graphical data are presented as the mean + standard deviation of at least three independent experiments. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, compared to vehicle-treated control.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6922842/v1/ffded58c3a23fced216d6a85.png"},{"id":86924092,"identity":"b490bdd4-19ed-456b-8245-98f872eeae7c","added_by":"auto","created_at":"2025-07-17 08:13:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":112642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApoptosis, ferroptosis, and necroptosis inhibitors prevent cell death induced by inhibition of cPLA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eα. A.\u003c/strong\u003e CCRF-CEM cells were pretreated with ferrostatin-1, liproxstatin-1, CuATSM for 30 minutes, followed by treatment with AVX420 and the ferroptosis inducers RSL-3 or ML210 and necroptosis inducers TSZ or TBZ for 24 hours. Cell viability was assessed using the CTG viability assay. \u003cstrong\u003eB. \u003c/strong\u003eCCRF-CEM and Jurkat cells were pretreated with apoptosis, ferroptosis, and necroptosis inhibitors, followed by exposure to AVX420 and corresponding positive control agents for 24 hours. The data shown in this figure represent the most relevant doses from these experiments. Detailed flow cytometry analysis of cells stained with propidium iodide, including results from additional dosing conditions, is provided in Supplementary \u003cstrong\u003eFigure S4.\u003c/strong\u003e Data is presented as the mean + standard deviation, and each measurement was repeated at least three times independently. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, compared to respective single doses.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6922842/v1/f9f614e65711dd53267c0c99.png"},{"id":86924381,"identity":"2102b6aa-83c0-4c81-ae2d-1244cb9406b6","added_by":"auto","created_at":"2025-07-17 08:21:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":103010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAVX420 induces mitochondrial ROS in T-ALL cells.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e CCRF-CEM and Jurkat cells were treated with varying doses of AVX420 for 4 hours. Following this, the presence of mitochondrial ROS was ascertained with the aid of MitoSOX staining and examined using flow cytometry. Supplemental \u003cstrong\u003eFig. S5A \u003c/strong\u003eshows dot plots with the gating strategy\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eB.\u003c/strong\u003e CCRF-CEM and Jurkat cells were exposed to AVX420 in a dose range of 0.25 µM to 4 µM. Following incubation periods of 4 and 24 hours, mitochondrial membrane rupture was assessed using flow cytometry after JC-1 staining. Supplemental \u003cstrong\u003eFig. S5B \u003c/strong\u003eshows dot plots with the gating strategy\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eC.\u003c/strong\u003e CCRF-CEM cells were treated with 0.5 µM AVX420 from 1 hour to 24 hours. Subsequent assessments of lipid ROS, mitochondrial ROS, mitochondrial membrane potential, and cellular viability were conducted using a flow cytometer. For these evaluations, the cells were stained with Bodipy 581/591 C11, MitoSOX, JC-1, and propidium iodide, respectively. Data is presented as the mean + standard deviation, and each measurement was repeated at least three times independently. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, compared to untreated control.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6922842/v1/919cfc8224a684d1a5df1bbd.png"},{"id":86924093,"identity":"ca1e0d4b-aac6-45e0-9262-14fe379b451c","added_by":"auto","created_at":"2025-07-17 08:13:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":168842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRIP1 inhibitor prevents ROS induction initiated by cPLA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eα\u003c/strong\u003e \u003cstrong\u003einhibition in T-ALL cells. \u003c/strong\u003eZ-VAD, Lip-1, CuAT, Nec-1, GSH, and NAC were used at single concentrations of 50 µM, 5 µM, 2.5 µM, 50 µM, 2.5 mM, and 2.5 mM respectively.\u003cem\u003e \u003c/em\u003e\u003cstrong\u003eA. \u003c/strong\u003eCCRF-CEM and Jurkat cells were pretreated with ferrostatin-1, liproxstatin-1, CuATSM, necrostatin-1, GSH, and NAC for 30 minutes, followed by treatment with AVX420 for 4 hours. Mitochondrial ROS was detected using flow cytometry after MitoSOX staining. Supplemental \u003cstrong\u003eFig. S6B \u003c/strong\u003eshows dot plots with the gating strategy\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eB.\u003c/strong\u003e CCRF-CEM and Jurkat cells were pretreated with ferrostatin-1, liproxstatin-1, CuATSM, necrostatin-1, GSH, and NAC for 30 minutes, followed by treatment with AVX420 and RSL-3 for 4 hours. Lipid ROS was detected using flow cytometry after BODIPY™ 581/591 C11 staining. Supplemental \u003cstrong\u003eFig. S6C \u003c/strong\u003eshows dot plots with the gating strategy\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eC.\u003c/strong\u003e CCRF-CEM and Jurkat cells were pretreated with ferrostatin-1, liproxstatin-1, CuATSM, necrostatin-1, GSH, and NAC for 30 minutes, followed by treatment with AVX420, Staurosporine and Valinomycin. Following a 24-hour incubation, cells were stained with JC-1, and the level of mitochondrial membrane rupture was evaluated using flow cytometry. Supplemental \u003cstrong\u003eFig. S6D \u003c/strong\u003eshows dot plots with the gating strategy\u003cstrong\u003e.\u003c/strong\u003e \u0026nbsp;Z-VAD, Z-VAD-FMK; Lip-1, Liproxstatin-1; CuAT, CuATSM; Nec-1, Necrostatin-1; GSH, Glutathione; NAC, N-Acetyl Cysteine. Data are presented as the mean + standard deviation, and each measurement was repeated at least three times independently. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, compared to respective single doses.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6922842/v1/35c6dd9e5824776b9f37ffe0.png"},{"id":86925338,"identity":"284234de-72ab-4643-9c8c-e7e7953eb826","added_by":"auto","created_at":"2025-07-17 08:29:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":73010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel for a cPLA₂α-NFκB-RIP1 signaling axis that inhibits programmed cell death.\u003c/strong\u003e Our data supports a model whereby in T-ALL cells, activation of NF-κB by cPLA\u003csub\u003e2\u003c/sub\u003eα can suppress RIP1 kinase activity. Loss of cPLA\u003csub\u003e2\u003c/sub\u003eα activity triggers RIP1 kinase-dependent mt-ROS accumulation and programmed cell death (filled arrows). \u0026nbsp;Alternatively, cPLA\u003csub\u003e2\u003c/sub\u003eα may itself protect against lipid peroxidation, and inhibition leads to mt-ROS accumulation sensed by RIP1 and amplified by RIP3 activation (dashed arrows). In each case, loss of mitochondrial redox homeostasis amplifies lipid-ROS formation and initiates programmed cell death. Inhibitors are shown italicized in grey.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6922842/v1/70d98236716e4fb01d051bb4.png"},{"id":86925785,"identity":"a2d0184f-25f4-4562-8ad0-9f1450e1513a","added_by":"auto","created_at":"2025-07-17 08:37:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1620356,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6922842/v1/70b3b9ac-4eae-4b33-9b9c-06e974aa1459.pdf"},{"id":86924096,"identity":"f46e306e-c618-4d06-9fba-86573bb4ae84","added_by":"auto","created_at":"2025-07-17 08:13:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6624636,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6922842/v1/d9eb08ef37fd1130e48679eb.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"\u003cp\u003ecPLA\u003csub\u003e2\u003c/sub\u003eα inhibition leads to RIP1 kinase-dependent cell death in acute leukemia cells\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNecroptosis is one of the more recently described mechanisms of programmed cell death and is executed by the oligomerization of mixed lineage kinase domain-like protein (MLKL) to form the necrosome, leading to cell rupture and inflammation (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Necroptosis can be induced by the activation of death receptors, e.g., the TNFα-receptor, or in response to stress induced by heat or altered pH as recently (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The master regulator of necroptosis is receptor interacting protein kinase 1 (RIP1). Through post translational modifications, RIP1 integrates death receptor activation with survival signaling through NF-κB to determine cell fate. Physiologically, necroptosis appears important for eliminating infected cells, particularly when viral infections impair apoptosis, and can help promote innate immune responses through release of inflammatory mediators. Excessive necroptosis has been linked with some chronic inflammatory conditions and has the potential both to increase tumour progression and play a role as a potential therapeutic strategy.\u003c/p\u003e\u003cp\u003eCytosolic phospholipase A2 α (cPLA\u003csub\u003e2\u003c/sub\u003eα) is a member of the PLA\u003csub\u003e2\u003c/sub\u003e superfamily that cleave fatty acids from the sn-2 position of membrane phospholipids. Six major groups are classified by their localization, regulatory mechanisms, and specificity (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) where cPLA\u003csub\u003e2\u003c/sub\u003eα is the best studied member of the type IV group. Inactive cPLA\u003csub\u003e2\u003c/sub\u003eα is found in the cytosol, and its membrane translocation and activation are regulated by increased Ca\u003csup\u003e2+\u003c/sup\u003e concentration, phospholipid binding, and phosphorylation. It exhibits a strong selectivity for phospholipids with the highly reactive polyunsaturated fatty acid arachidonic acid (AA) at the sn-2 position (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) and both of the major cleavage products (free AA, and lysophosphatidylcholine (LPC)) are metabolized to potent lipid signaling molecules that are coupled to the activity of NF-κB and with established roles in physiological and pathological inflammation and cancer (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of T-cell cancer that originates in the thymus. It predominantly affects young individuals, where chemotherapy has a high curative potential (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). In adults, however, higher rates of primary refractory disease and relapse contribute to a 5-year survival of around 30\u0026ndash;40%, highlighting the limited treatment options. We recently demonstrated that cell lines originating from T-cell acute leukemias were highly sensitive to treatment with the thiazolyl ketone inhibitor of cPLA\u003csub\u003e2\u003c/sub\u003eα, AVX420, and further demonstrated that this was a result of an accumulation of ROS in these cells (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Here, we investigate why T-ALL cells are sensitive to cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition and provide evidence that loss of cPLA\u003csub\u003e2\u003c/sub\u003eα causes a rapid and RIP1-dependent accumulation of mitochondrial-associated ROS (mt-ROS) leading to necroptosis, as well as apoptosis, and ferroptosis dependent on the cellular context.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCell culture media RPMI 1640 (#R0883), Gentamycin (#G1397), Propidium Iodide (#P4170), Ribonuclease A (#R6513), Dimethyl sulfoxide (DMSO) (#2650), Valinomycin (#V0627), Ferrostatin-1 (#SML0583), Cu-ATSM (#SML0769), Liproxstatin-1 (#SML1414), 1S,3R-RSL 3 (#SML2234), ML210 (#SML0521), Necrostatin-1 (#480065), and N-Acetyl-L-Cysteine (#A7250) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against cleaved PARP-1 (CST #5625), cleaved caspase-3 (CST #9664), β-Actin (CST #4970), Phospho-MLKL (Ser358) (CST#91689), and Phospho-RIP3 (Ser227) (CST#93654) were obtained from Cell Signaling Technology (Danvers, MA, USA). Doxorubicin (#15007) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Fetal Bovine Serum (FBS) (#10270106) was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Z-VAD-FMK (Pan caspase -inhibitor) (#HY-16658B) and SM-164 (#HY-15989) were purchased from MedChemExpress (Monmouth Junction, United States). BV-6 ((#inh-bv-6) was purchased from Invivogen (San Diego, California, United States). L-glutamine (#17-605E) was purchased from Lonza Pharma \u0026amp; Biotech (Hochbergerstrasse, Basel, Switzerland). AVX420 ((Methyl 2-(2-(4-heptyloxy)-phenoxy)-acetyl)thiazole-4-carboxylate) was synthesized in the laboratory of organic chemistry at the University of Athens by George Kokotos. AVX420 was stored at -80\u0026ordm;C in DMSO.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Methods\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1 Maintenance and plating of T-ALL cells\u003c/h2\u003e\u003cp\u003eThe human acute T-cell lymphoblastic leukemia cell lines CCRF-CEM and Jurkat E6.1 were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and grown in RPMI 1640 medium, supplemented with L-glutamine (2 mM), Gentamycin (0.1 mg/ml), and 10% FBS (complete medium) at 37\u0026ordm;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells were confirmed to be \u003cem\u003eMycoplasma\u003c/em\u003e-free.\u003c/p\u003e\u003cp\u003eThe cells were harvested by centrifugation and counted using the TC20 automated cell counter from BIO-RAD (Hercules, California, USA) using 0.4% Trypan blue from NanoEnTek (Waltham, MA, USA) to check viability. The cells were seeded in complete medium at a density of 200,000 cells/ml in either 96 or 24-well plates. On the same day, the cells were treated and incubated at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37\u0026ordm;C for the indicated time.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2 Cell viability and PI exclusion assays\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCell viability was assessed using the CellTiter-Glo\u0026reg; (CTG) assay. Cells were seeded in 96-well plates, and the assay was performed according to the manufacturer\u0026rsquo;s instructions. Luminescence was measured using a Cytation 5 Cell Imaging Multimode Reader (BioTek Instruments, Winooski, VT, USA). To assess cell membrane integrity and distinguish live from dead cells, a propidium iodide (PI) exclusion assay was used. Cells were plated in 24-well plates, treated as indicated, and incubated with 1.4 \u0026micro;g/mL PI for 5 minutes at room temperature. Data were acquired using a CytoFLEX flow cytometer (Beckman Coulter, USA) with a filter set optimized for PI detection (excitation: 488 nm; emission: 585/42 nm).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3 Cell Cycle Analysis\u003c/h2\u003e\u003cp\u003eCells were plated in 24-well plates, treated as indicated, then harvested and washed with PBS. For fixation, ice-cold 70% ethanol was added dropwise while vortexing to prevent clumping, followed by incubation at 4\u0026deg;C for 30 minutes. Fixed cells were centrifuged, washed with PBS, and treated with 50 \u0026micro;L of RNase A (50 \u0026micro;g/mL) for 20 minutes at room temperature. DNA was stained with PI (2.5 \u0026micro;g/mL) and incubated on ice for 15 minutes in the dark. Samples were analyzed using a NovoCyte flow cytometer (ACEA Biosciences, Agilent Technologies, USA) with a PI-optimized filter set (excitation: 488 nm, emission: 585/40 nm). Flow cytometry data were used to assess cell cycle distribution.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4 Annexin V-FITC assay\u003c/h2\u003e\u003cp\u003eThe annexin V-fluorescein isothiocyanate (FITC) kit from Tau Technologies (Albuquerque, NM, USA) was used to measure phosphatidylserine (PS) externalization. Cells were plated in 24-well plates. following the protocol we have described previously (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Data was acquired through either an LSRII flow cytometer from BD Biosciences (San Jose, CA, USA) or a CytoFLEX flow cytometer from Beckman Coulter (California, USA). Subsequent data analysis was performed using Novoexpress Software v1.5.0 from Agilent Technologies (Santa Clara, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.2.5 Caspase-3/7 Activity Assay\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCaspase activity was measured using a Caspase-3/7 Glo assay kit (Promega Corporation, Madison, WI, USA) following the protocol described in our previous study (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). This assay produces a luminescent substrate with a four-peptide sequence, which, after cleavage by caspase-3/7, generates a light signal. The resulting luminescence was recorded with the Cytation 5 cell imaging multimode reader (Biotek Instruments, Winooski, VT, USA).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.2.6 Preparation of Plates for ROS and Mitochondrial Membrane Potential Detection Experiments\u003c/h2\u003e\u003cp\u003eAfter treatment with the compound and controls, cells were washed with PBS and resuspended in DPBS with 1% BSA and dye. They were incubated with Deep Red, BODIPY\u0026trade; 581/591 C11, and MitoSOX\u0026trade; (1 h), or JC-1 (15 min) at 37\u0026deg;C, 5% CO₂, following manufacturer\u0026rsquo;s instructions. The acquired data were analyzed using Novoexpress Software v1.5.0 from Agilent Technologies (Santa Clara, USA).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section4\"\u003e\u003ch2\u003e2.2.6.1 ROS Detection with Deep Red Dye\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe level of reactive oxygen species (ROS) was assessed using the Cellular ROS Assay Kit (Cat. No. ab186029; Abcam, Cambridge, UK). This kit utilizes a deep red dye that can permeate cell membranes and emit deep red fluorescence upon interacting with ROS. For the assay, the 200X concentrated deep red dye reagent was diluted in DPBS (Dulbecco's Phosphate Buffered Saline) to achieve a 1X final concentration in solution, which was then added to the samples. Data was acquired using a CytoFLEX flow cytometer (Beckman Coulter, California, USA) with a filter set optimized for the deep red dye detection (excitation at 640 nm and emission at 675/25 nm).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section4\"\u003e\u003ch2\u003e2.2.6.2 Lipid ROS Detection with BODIPY\u0026trade; 581/591 C11\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eLipid-derived ROS was identified using the lipid peroxidation sensor BODIPY\u0026trade; 581/591 C11 (Cat. No. D3861, Thermo Fisher Scientific, Waltham, MA, USA). In the procedure, 1 mM BODIPY solution was diluted in DPBS to obtain a 2 \u0026micro;M final concentration, which was subsequently added to the samples. Data was analyzed using a CytoFLEX flow cytometer (Beckman Coulter, California, USA) with filter sets optimized for BODIPY detection (excitation at 488 nm and emission at 525/40 nm).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section4\"\u003e\u003ch2\u003e2.2.6.3 Mitochondrial ROS Detection with MitoSOX\u003csup\u003e\u0026trade;\u003c/sup\u003e\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe MitoSOX red mitochondrial superoxide indicator for live-cell imaging (Cat. No. M36008, Thermo Fisher Scientific) was used to evaluate the production of mitochondrial reactive oxygen species (ROS). MitoSOX can permeate live cells and selectively target mitochondria. The solution was prepared by diluting the 1mM MitoSOX reagent in DPBS to achieve a final concentration of 0.5 \u0026micro;M. Data acquisition was executed using the CytoFLEX flow cytometer from Beckman Coulter (California, USA), with a 488 nm excitation laser and a 585/42 nm bandpass emission filter set for detecting MitoSOX Red fluorescence.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section4\"\u003e\u003ch2\u003e2.2.6.4 Mitochondrial Membrane Potential Sensor with JC-1\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe mitochondrial membrane potential was assessed using JC-1 (Cat. No. T3168, Thermo Fisher Scientific). JC-1 is a membrane-permeant dye that specifically targets mitochondria and is commonly employed to assess mitochondrial health. The solution was prepared by diluting a 5 mg/ml stock of JC-1 in DPBS to a final concentration of 5 \u0026micro;g/ml and added to the samples. Data collection was carried out with an LSRII flow cytometer from BD Biosciences (San Jose, CA, USA), using a 488 nm excitation laser and the following filter sets: a 530/30 nm bandpass filter for detecting the green monomer form (low membrane potential) and a 585/42 nm bandpass filter for detecting the red J-aggregate form (high membrane potential).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.2.7 Western Blot\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eProtein extraction and Western blotting were performed as described in our previous study (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Protein concentrations were determined using the Pierce\u0026trade; BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and measured with a Cytation 5 Cell Imaging Multimode Reader (BioTek Instruments, Winooski, VT, USA). Western blotting was then carried out according to our previous protocol (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) and imaged using a Syngene G:BOX Chemi XX6 gel documentation system (Frederick, USA). Image analysis was conducted using Image Studio software (LI-COR Biosciences, Lincoln, NE, USA) and ImageJ 1.43q (NIH, USA).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.2.8 Statistical Analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eGraphPad Prism 9.3.1 (San Diego, CA, USA) was utilized for statistical analysis and generating figures. For statistical evaluations, one-way ANOVA was employed, and Dunnett's posthoc analysis was used to correct for multiple comparisons when comparing treated samples to the untreated control. Differences were considered significant if the p-value was less than 0.05.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Loss of T-ALL cell viability by inhibition of cPLA\u003csub\u003e2\u003c/sub\u003eα .\u003c/h2\u003e\u003cp\u003eWe tested the effect of the cPLA\u003csub\u003e2\u003c/sub\u003eα inhibitor AVX420 on the viability of two T-ALL cell models, CCRF-CEM and Jurkat. Using CTG assays, we found a dose-dependent inhibition of viability in both cell lines. Half-maximal inhibitory concentration (IC50s) were calculated and found to be 0.51 and 0.52 \u0026micro;M in CCRF-CEM and Jurkat cells respectively after a 24-hour treatment. AVX420 was slightly more effective in the CCRF-CEM cells versus Jurkat after 72 hours, with an IC50 of 0.28 versus 0.4 \u0026micro;M \u003cb\u003e(Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e). In contrast to the chemotherapy agent doxorubicin, which caused pronounced arrest of cells in G2/M phase of the cell cycle, we found no discernable effect of AVX420 on cell cycle distribution, and cell death was therefore considered to be the primary reason for reduced ATP production observed using CTG assays \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.2 cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition initiates multiple regulated cell death pathways in T-ALL\u003c/h2\u003e\u003cp\u003eTo identify the cause of cell death, we first looked at whether the cells were undergoing apoptosis, as previously observed in multiple myeloma cells (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Apoptosis can be triggered by the presence of extracellular death signals or the absence of survival signals, and its execution is dependent on the coordinated activation of caspase enzymes (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). To identify apoptosis, we measured externalized phosphatidylserine (PS) by Annexin V-FITC staining using flow cytometry and activity of effector caspases with the caspase 3/7 glo assay using a plate-based assay. We found a dose-dependent increase in the proportion of early apoptotic cells (Annexin V positive / PI negative) and an increase in the activity of effector caspases in response to AVX420 in both CCRF-CEM and Jurkat cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). We confirmed that caspase-3 and PARP-1 proteins were cleaved in response to AVX420 in CCRF-CEM cells using Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cb\u003eFig. S2A\u003c/b\u003e). To explore whether apoptosis is the major cause of cell death, we preincubated the cells with the pan-caspase inhibitor Z-Val-Ala-Asp-Fluoro Methyl Ketone (Z-VAD) before treatment with AVX420. As expected, Z-VAD preincubation completely blocked both the effector caspase activity and cleavage of caspase 3 and PARP proteins, however, PS externalization and cell death were only partially blocked, particularly in CCRF-CEM cells, indicating a significant proportion of cell death was caspase-independent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cb\u003eFig. S2B, C).\u003c/b\u003e We then investigated the involvement of two additional RCD pathways, necroptosis and ferroptosis. Necroptosis is executed by the mixed lineage kinase domain-like protein (MLKL) upon activation by receptor-interacting protein (RIP) kinases RIP1 and RIP3 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) in response to tumor necrosis factor TNFα-receptor signaling (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Ferroptosis, is distinguished by the accumulation of iron and lipid peroxidation, which leads to membrane rupture. It can be caused by redox imbalance in the cells, (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) and free radical scavengers such as ferrostatin-1, liproxstatin-1, and exogenous glutathione (GSH) are potent inhibitors of ferroptotic cell death (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine whether necroptosis was activated in response to cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition, we measured the phosphorylation of two key proteins in the necroptosis cascade, RIP3 and MLKL. As a positive control, necroptosis was induced with a combination of TNF-α, Smac mimetic SM-164, and Z-VAD-FMK, the combination denoted \u0026ldquo;TSZ\u0026rdquo; [40]. cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition led to a dose-dependent increase in the phosphorylation of both RIP3 and MLKL, which was partially blocked by pre-treatment with the RIP1 inhibitor necrostatin-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cb\u003eFig. S3A, Fig. S2C\u003c/b\u003e). To determine whether ferroptosis was induced in response to cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition, we treated cells with AVX420 and measured the levels of lipid peroxides (lipid-ROS) using flow cytometric analysis of Bodipy 581/591 C11 staining. The glutathione peroxidase 4 (GPX4) inhibitor RSL-3 was used to induce ferroptosis as a positive control (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition caused a dose-dependent increase in lipid-ROS, suggesting ferroptosis was also underway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and we concluded that apoptosis, necroptosis and ferroptosis all occur in response to cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition in T-ALL cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4 RIP1 inhibition protects cells from loss of cPLA\u003csub\u003e2\u003c/sub\u003eα activity.\u003c/h2\u003e\u003cp\u003eTo investigate the relative importance of ferroptosis and necroptosis to cell death resulting from cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition, we treated cells with either known inhibitors of ferroptosis (ferrostatin-1, liproxstatin-1, or CuATSM) or the RIP1 inhibitor necrostatin-1 before addition of AVX420, and measured cell viability after 24 hours using CTG assays. As positive controls, the GPX4 degradation agent ML210 (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) and necroptosis inducers TBZ (TNF-α, Smac mimetic BV-6, and Z-VAD-FMK) [41] were used in addition to RSL3 and TSZ to induce ferroptosis and necroptosis, respectively. Pretreatment with ferroptosis inhibitors restored CCRF-CEM cell viability to \u0026gt;\u0026thinsp;80% when used in combination with either RSL-3 or ML210 compared to approximately 60% when used in combination with AVX420. Necrostatin-1 on the other hand was more effective against AVX420, restoring viability to approximately 80% versus only 40% when used in combination with TSZ or TBZ \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Combining cell death inhibitors did not give additional protection in viability assays and combining ferroptosis inhibitors with necrostatin-1 was detrimental compared to using necrostatin-1 alone \u003cb\u003e(Fig. S2B)\u003c/b\u003e. When we tested the same inhibitors using propidium iodide exclusion assay to measure cell death, necrostatin-1 provided complete resistance to the effects of AVX420 in both cell lines, while as we observed previously, Z-VAD provided only partial resistance \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The ferroptosis inhibitor liproxstatin had contrasting effects in CCRF-CEM and Jurkat cells, providing partial protection from cell death in CCRF-CEM cells, but not in Jurkat. Cu-ATSM, a different ferroptosis inhibitor, provided some protection against AVX420- induced death in Jurkat cells, and this effect was additive in combination with Z-VAD \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. In addition to inhibition of ferroptosis, CuATSM can protect damaged mitochondria by delivering Cu\u003csup\u003e2+\u003c/sup\u003e (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), which could explain the discrepancy in these results. So far, we have demonstrated that cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition initiates apoptosis, necroptosis, and ferroptosis which together contribute to the loss of cell viability in T-ALL cells. Necrostatin-1 blocked all AVX420-dependent cell death in both cell types implying that RIP1 may be mechanistically upstream of all three cell death pathways in T-ALL.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.5 cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition causes RIP1-dependent mitochondrial ROS accumulation\u003c/h2\u003e\u003cp\u003eOur previous findings indicate that unresolved oxidative stress, which could be rescued with ROS scavengers, underlies the cellular response to AVX420 in cancer (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Mitochondria play a central role in integrating metabolic and cell death signaling. To explore the role of mitochondria in the response to cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition, we measured the levels of mitochondrial ROS using MitoSOX, a mitochondrial dye that fluoresces when oxidized by superoxide, and JC-1, a cationic dye that indirectly indicates mitochondrial membrane potential (MMP). Treatment with AVX420 caused a clear, dose-dependent increase in the levels of mt-ROS after 4 hours in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) but had no effect on the MMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Valinomycin, a potassium ionophore and known mitochondrial respiration uncoupler (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), on the other hand, caused a highly significant loss of MMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Performing a time course analysis following treatment with 0.5 \u0026micro;M AVX420 showed that the increased levels of both mt-ROS and lipid-ROS occurred rapidly, within 1 hour, and were found in close to 80% of cells 12 hours after treatment. Accumulation of ROS preceded effects on mitochondrial membrane potential, which began 8 to 12 hours after treatment and mirrored the reduction in viable cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn addition to its central role in the control of cell fate decisions in response to TNFα signaling, RIP1 can associate with mitochondria, and both activate and be activated by mt-ROS (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). To test whether the observed mt-ROS accumulation was dependent on the activity of RIP1 we preincubated cells with necrostatin-1 and measured mt-ROS after treatment with AVX420. Necrostatin-1 was as effective as universal ROS scavengers GSH and NAC at preventing mt-ROS accumulation whereas both liproxstatin-1, and Z-VAD were completely ineffective \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The increases in lipid-ROS and total ROS on the other hand were effectively blocked by liproxstatin-1, as well as necrostatin-1 and the universal ROS scavengers but not by Z-VAD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB \u003cb\u003eand Fig. S6A\u003c/b\u003e). In CCRF-CEM cells, liproxstatin-1 and necrostatin-1 both prevented mitochondrial uncoupling, while in Jurkat, a significant protective effect was only seen using necrostatin-1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cb\u003e).\u003c/b\u003e In both cases, Z-VAD had no protective effect. The two cell lines showed contrasting responses to liproxstatin. In both cases liproxstatin prevented lipid-ROS accumulation but in Jurkat this did not prevent loss of mitochondrial membrane potential and cell death, suggesting that apoptosis and necroptosis dominate in Jurkat cells, but in CCRF-CEM ferroptosis is an important RCD pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSuperoxide anions produced during oxidative metabolism in mitochondria are the primary source of intracellular ROS and drive signaling events that regulate cell growth and survival (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). ROS-dependent signaling is typically pro-tumorigenic and can contribute to the initiation and progression of the cancer (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Both the higher ROS levels seen in some cancers, and increased antioxidant response signaling e.g. by Nrf-2 (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) are vulnerabilities that can be targeted therapeutically. Proliferative signaling can, for example, be inhibited by lowering ROS levels using antioxidants, or in contrast, programmed cell death can be triggered by inhibiting the cell\u0026rsquo;s antioxidant response system, leading to oxidative stress-induced regulated cell death (\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHere, we found that inhibition of cPLA\u003csub\u003e2\u003c/sub\u003eα increased mt-ROS and induced necroptosis, apoptosis, and ferroptosis in T-ALL cells, implying cPLA\u003csub\u003e2\u003c/sub\u003eα may be important in the cell\u0026rsquo;s antioxidant response system. In neurons, LOX-dependent AA metabolism can amplify ROS levels (\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) and in this context, cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition can suppress ROS and restrict inflammatory tissue damage (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In contrast, we find that in leukemia cell lines, cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition increases ROS, which is consistent with studies in U937 leukemia cells where cPLA\u003csub\u003e2\u003c/sub\u003eα was shown to be cytoprotective against ROS induced by peroxynitrate treatment (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Other phospholipase A2 enzymes protect cells from oxidative stress by removing oxidized lipid species from membrane phospholipids. Lp-PLA2, for example, protects against ferroptosis in gastric cancer cells by catalyzing the metabolism of oxidized phosphatidylethanolamine (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) and iPLA2γ removes oxidized phospholipids and participates in lipid remodeling in mitochondria (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). A similar mechanism could be considered, assuming cPLA2α localizes to mitochondria, where its highly oxidizable substrates are present in abundance (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Mitochondrial localization of cPLA\u003csub\u003e2\u003c/sub\u003eα has been reported in nerve tissue (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e) however, unlike iPLA2γ, cPLA\u003csub\u003e2\u003c/sub\u003eα does not have a known mitochondrial localization sequence, and the mechanism for mitochondrial localization in astrocytes was dependent on its interaction with mitochondrial antiviral signaling protein (MAVS) (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCritically, we found that the RIP1 inhibitor necrostatin-1, as well as affording the T-ALL cell lines protection from all AVX420-induced programmed cell death, prevented the increase in mt-ROS. RIP1 is an important molecular switch between cell survival and cell death in inflammatory conditions (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) and it is the kinase activity of the protein that drives the formation of complexes that promote apoptosis and necroptosis. Phosphorylation at Ser25 by IKKα/β is key to keeping the kinase activity of RIP1 in check (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Given the established role of cPLA\u003csub\u003e2\u003c/sub\u003eα in activation of NF-κB and IKKα/β (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e), we suggest that inhibiting cPLA\u003csub\u003e2\u003c/sub\u003eα could activate RIP1 by suppressing inhibitory phosphorylation, and that RIP1 activity initiates ROS accumulation as demonstrated in murine embryonic fibroblasts (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). We showed previously that exogenous addition of arachidonic acid could not rescue AVX420-induced ROS formation or cell death (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), therefore LPC-derived signaling molecules would be likely candidates in maintaining NF-κB/IKKα/β activity in this case. Alternatively, mt-ROS itself is a key regulator of the autophosphorylation of RIP1 at Ser 161 which drives RIP3 recruitment to the necrosome (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). If cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition were the primary driver of increased mt-ROS, this could trigger a positive feedback loop involving RIP1/RIP3 activation, explaining the RIP1-dependent phenotype. These alternate models are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Further investigations into the links between cPLA\u003csub\u003e2\u003c/sub\u003eα, NFκB, and RIP1 signaling will be key to delineating these processes.\u003c/p\u003e\u003cp\u003eExploiting the connections between oxidative stress and programmed cell death is an emerging tool in the fight against cancer (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). High innate level of ROS and/or defective antioxidant response mechanisms can make certain leukemias, and aggressive breast and lung cancers particularly sensitive to ROS inducers (\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Inhibitors of cPLA\u003csub\u003e2\u003c/sub\u003eα could have significant therapeutic value in these cancers, particularly in acute lymphocytic leukemia, where novel targeted therapeutics with better tolerability are sorely needed.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA special thanks to Samah Elsaadi for his valuable guidance, insights, and encouragement throughout this study. Additionally, we would like to acknowledge that Figure 7 was created with the help of BioRender.com.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, B.J., A.J.F., F.J.A., M.B., and N.M.; methodology, N.M.; software, N.M.; validation, N.M., F.J.A., A.J.F., M.B. P.A., and B.J.; formal analysis, N.M.; investigation, N.M.; resources, B.J., M.B., and A.J.F.; data curation, N.M., A.J.F., and F.J.A.; manuscript preparation, N.M., F.J.A; manuscript review and editing, F.J.A., A.J.F., M.B., P.A. and B.J.; visualization, N.M., F.J.A. and A.J.F.; supervision, B.J., M.B., A.J.F. and F.J.A.; project administration, B.J. and A.J.F.; funding acquisition, B.J. and A.J.F. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was supported by The Research Council of Norway grant no. 311392 and Coegin Pharma.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e B.J., A.J.F. and F.J.A hold shares in Coegin Pharma. N.M. and F.J.A. work for Coegin Pharma. All other authors report no conflicts. The funders were not involved in the study design, data collection, analysis, manuscript composition, or the decision to publish the findings.\u003cbr\u003e \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell. 2009;137(6):1112-23.\u003c/li\u003e\n \u003cli\u003eHe S, Wang L, Miao L, Wang T, Du F, Zhao L, et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell. 2009;137(6):1100-11.\u003c/li\u003e\n \u003cli\u003eYe K, Chen Z, Xu Y. The double-edged functions of necroptosis. Cell Death Dis. 2023;14(2):163.\u003c/li\u003e\n \u003cli\u003eDennis EA, Cao J, Hsu YH, Magrioti V, Kokotos G. Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention. 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Blood. 2013;122(19):3322-30.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6922842/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6922842/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eReceptor-interacting protein kinase (RIP1) sits at the interface between inflammation and cell fate decisions. Through its kinase activity RIP1 is key to initiating necroptosis, which is promising alternative to treat cancers, particularly those resistant to pro-apoptotic drugs. We demonstrate that by inhibiting the proinflammatory type II cytosolic phospholipase, cPLA\u003csub\u003e2\u003c/sub\u003eα , we can initiate RIP1 kinase-dependent cell death via necroptosis in T-cell acute lymphoblastic leukemia (T-ALL) cells. When we block cPLA\u003csub\u003e2\u003c/sub\u003eα activity using the thiazolyl ketone AVX420, we find a marked reduction in the viability of two T-ALL cell lines, CCRF-CEM and Jurkat. This is associated with an increase in the phosphorylation of both RIP3 and MLKL, indicating the cells are undergoing necroptosis. We also see increased levels of apoptotic markers and lipid peroxidation, indicating that cells also undergo apoptosis and ferroptosis. Pre-treatment with the RIP1 kinase inhibitor, necrostatin-1, completely blocks cell death from AVX420 treatment, whereas pan-caspase inhibition and lipid-ROS scavengers are only partially effective. The inhibition of cPLA\u003csub\u003e2\u003c/sub\u003eα leads to a rapid accumulation of mitochondrial-associated reactive oxygen species (mt-ROS), which is blocked either by necrostatin-1 or total ROS scavengers, but not by pan-caspase inhibition or liproxstatin-1. The increase in mt-ROS precedes the loss of mitochondrial membrane potential and accumulation of lipid-ROS, indicating apoptosis and ferroptosis are responses to unresolved oxidative stress in the necroptotic cells. Our findings suggest that in T-ALL cells, cPLA\u003csub\u003e2\u003c/sub\u003eα inhibition activates RIP1 kinase. This leads to necroptosis and oxidative stress-dependent cell death. cPLA\u003csub\u003e2\u003c/sub\u003eα could therefore be a relevant therapeutic target for treating refractory or relapsed disease where resistance to apoptosis is inherent.\u003c/p\u003e","manuscriptTitle":"cPLA2α inhibition leads to RIP1 kinase-dependent cell death in acute leukemia cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 08:13:36","doi":"10.21203/rs.3.rs-6922842/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b150487e-3dcd-4041-a534-42e7bb054e9e","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":51673655,"name":"Biological sciences/Cancer"},{"id":51673656,"name":"Health sciences/Medical research/Drug development"}],"tags":[],"updatedAt":"2025-07-17T08:13:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-17 08:13:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6922842","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6922842","identity":"rs-6922842","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}