Anti-leukemia effects of Omipalisib in Acute Myeloid Leukemia: inhibition of PI3K-AKT-mTOR signaling and suppression of Mitochondrial Biogenesis

doi:10.21203/rs.3.rs-2854129/v1

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Anti-leukemia effects of Omipalisib in Acute Myeloid Leukemia: inhibition of PI3K-AKT-mTOR signaling and suppression of Mitochondrial Biogenesis

https://doi.org/10.21203/rs.3.rs-2854129/v1

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published 11 Oct, 2023

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Omipalisib (GSK2126458), a potent dual PI3K/mTOR inhibitor, is reported to exhibit anti-tumor effect in several kinds of cancers. More than 50% of acute myeloid leukemia (AML) patients display a hyperactivation of PI3K/AKT/mTOR signaling. We investigated the anti-proliferative effect of omipalisib in AML cell lines with varied genetic backgrounds. The OCI-AML3 and THP-1 cell lines had a significant response to omipalisib, with IC₅₀ values of 17.45 nM and 8.93 nM, respectively. We integrated transcriptomic profile and metabolomic analyses, and followed by gene set enrichment analysis (GSEA) and metabolite enrichment analysis. Our findings showed that in addition to inhibiting PI3K/AKT/mTOR signaling and inducing cell cycle arrest at the G₀/G₁ phase, omipalisib also suppressed mitochondrial respiration and biogenesis. Furthermore, omipalisib downregulated several genes associated with serine, glycine, threonine, and glutathione metabolism, and decreased their protein and glutathione levels. In vivo experiments revealed that omipalisib significantly inhibited tumor growth and prolonged mouse survival without weight loss. Gedatolisib and dactolisib, another two PI3K/mTOR inhibitors, exerted similar effects without affecting mitochondria biogenesis. These results highlight the multifaceted anti-leukemic effect of omipalisib, revealing its potential as a novel therapeutic agent in AML treatment.

Biological sciences/Cancer/Cancer therapy/Targeted therapies

Biological sciences/Cancer/Cancer metabolism

acute myeloid leukemia

omipalisib

oxidative phosphorylation

mitochondria biogenesis

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by clonal expansion and impaired cell differentiation. Chromosomal alterations and mutations in leukemic cells are critical for their dysregulated survival and proliferation [1, 2]. Recently, owing to the understanding of the molecular landscape of AML through large-scale genomic analysis, the development of targeted therapies has greatly improved [3]. Although some new drugs, including FLT3 inhibitors (midostaurin and gilteritinib), IDH1/2 inhibitors (enasidenib and ivosidenib), and BCL2 inhibitor (venetoclax), have been approved by the FDA for the treatment of specific subgroups of AML [4], the development of effective and durable therapeutic strategies for other subgroups of AML remains unmet.

The phosphatidylinositol-3-kinase/AKT and the mammalian target of rapamycin (PI3K/AKT/mTOR) pathway plays a critical role in cell proliferation, differentiation, and survival [5, 6]. More than 50% of patients with AML display a hyperactivation of PI3K/AKT/mTOR signaling, accompanied by poor prognosis and chemotherapy resistance [7, 8]. Several somatic mutations, including mutations in receptor tyrosine kinases (FLT3 or KIT) and GTPases (NRAS), are associated with the dysregulated and constitutive activation of PI3K/AKT/mTOR signaling [9–11]. Considering the relevance of PI3K/AKT/mTOR signaling in leukemogenesis, targeting its key proteins has become an essential therapeutic strategy.

Several novel small-molecule drugs have been reported to target PI3K or mTOR in AML, such as PI3K δ/γ inhibitor duvelisib, PI3Kα inhibitor alpelisib, PI3K δ inhibitors idelalisib (CAL-101), pan-class I PI3K inhibitor buparlisib (BKM-120), as well as mTOR inhibitors temsirolimus (CCI-779) and everolimus (RAD001) in vitro and in vivo [12–16]. Dual PI3K/mTOR inhibitors, dactolisib (BEZ-235) and gedatolisib (PF-05212384), have been reported to suppress the proliferation of AML and reverse multidrug resistance in vitro [17, 18], but have not been successfully applied clinically in AML [19, 20]. Omipalisib (GSK2126458) is another potent dual PI3K/mTOR inhibitor [21], which has been shown to selectively inhibit the growth of various tumor cells [22–26]. In this study, we demonstrated the potent anti-leukemic effect of omipalisib in vitro and in vivo. Using transcriptomic analysis, metabolomics analysis, and subsequent experiments, we showed for the first time that omipalisib could inhibit glutathione metabolism by suppressing de novo serine synthesis and the folate/methionine cycle, inhibiting mitochondrial biogenesis, and suppressing mitochondrial respiration in myeloid leukemia cells.

Cell culture and chemicals

Human leukemia cell lines, Kasumi-1, MV4-11, THP-1 (ATCC, Manassas, VA, USA), Molm-13, OCI-AML3 (DSMZ, Braunschweig, Germany), and SKNO-1 (JCRB, Osaka, Japan) were used in this study. Molm-13, MV4-11, and THP-1 cells were maintained in RPMI 1640 medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS). Kasumi-1 cells were maintained in RPMI 1640 medium supplemented with 20% FBS, SKNO-1 cells in RPMI 1640 medium supplemented with 10% FBS and 10 ng/ml GM-CSF (R&D systems, Minneapolis, MN, USA), and OCI-AML3 cells in MEMα medium (ThermoFisher Scientific) containing 20% FBS. All cell lines were cultured at 37°C with 5% CO₂. Omipalisib (GSK2126458) and gedatolisib (PF-05212384) were purchased from TargetMol (Boston, MA, USA). Dactolisib (NVP-BEZ235) was purchased from AbMole BioScience (Houston, TX, USA) All compounds were dissolved in DMSO.

Cell viability assay and flow cytometry

Following treatment with drugs for 72 h, cell viability was assessed by the Colorimetric CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) as previously described [27]. Each experiment was performed in triplicates.

For cell cycle analysis, following treatment with the drug for 24 h, the cells were fixed in 70% ethanol, and stained with propidium iodide (PI; BD Biosciences, Franklin Lakes, NJ, USA). To detect cell apoptosis, a Fluorescein Isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (BD Biosciences) was used. Following treatment with the drug for 72 h, the cells were stained with Annexin V-FITC and PI. All fluorescence intensities were measured using a CYTOFLEX^TM Flow Cytometer (Beckman Coulter Inc., Brea, CA, USA). Cell cycle distribution and apototic cells were analyzed using FlowJo 10.4 software

Western blot analysis

Following treatment with the drug for the indicated times, total cell lysates were separated on SDS-PAGEand then transferred to PVDF membranes (Millipore, Billerica, MO, USA). After blocking with BlockPRO™ 1 Min Protein-Free Blocking Buffer (Energenesis Biomedical Co., Taiwan), membranes were incubated with indicated primary antibodies (Table S1) in 4°C for overnight. Membranes were then washed with TBS and incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Cell Signaling Technology, Beverly, MA, USA) for 60 min at RT. Immunoblot signals were detected by chemiluminescence using Western Lightening Plus-ECL (Perkin Elmer, Waltham, MA, USA) and ChemiDoc™ XRS (Bio-Rad Laboratories).

RNA extraction, RNA-sequencing (RNA-seq), and quantitative RT-PCR

Total RNA was extracted using NucleoSpin® RNA extraction kit (Macherey-Nagel, Düren, Germany). RNA-seq was performed using an Illumina NovaSeq 6000 sequencer (Illumina, San Diego, CA) and subsequent bioinformatics analysis was performed as previously described [27]. Briefly, a total amount of 1μg RNA was used for library preparation. Library quality was assessed using an Agilent Bioanalyzer 2100 system with DNA High-Sensitivity Chips. Sequencing alignment of hg19 was performed using Rsubread (2.1.0), and differentially expressed gene (DEG) identification was conducted with EBSeq (1.26.0). These transcriptome files were posited in the Gene Expression Omnibus (GEO) database (ID: GSE224155). Pathway enrichment was then analyzed by gene set enrichment analysis (GSEA) (4.0.3) using the Molecular Signatures Database v7.0 (MSigDB, Broad Institute, Cambridge, MA, USA) and Metascape.

cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). The qRT-PCR was performed and analyzed on a QuantStudio 3 Real-Time PCR System (ThermoFisher Scientific) with 2x qPCRBIO SyGreen Blue Mix Lo-ROX (PCR Biosystems, London, UK). The primers used for specific gene detection are listed in Table S2.

Mitochondria analysis and mtDNA quantiﬁcation

Mitochondrial mass, ROS content, and mitochondrial membrane potential in cells were measured by CYTOFLEXTM Flow Cytometer (Beckman Coulter) using MitoTracker Green, MitoSOX kit, and TMRE (Invitrogen/ThermoFisher Scientific, Waltham, MA, USA), respectively, according to the manufacturer’s instructions.

To determine the mtDNA copy number, total DNA was isolated from cell lines or tissues using DNeasy kits (Qiagen, Venlo, Netherlands). Nuclear and mitochondrial DNA content was analyzed using qPCR. The primers used for qPCR are listed in Table S2.

Metabolomics Analysis

Cell pellets were extracted in methanol using Geno Grinder 2010 (SPEX, Pittsburgh, CA, USA). After extraction, the supernatant was collected and evaporated in a centrifuge vaporizer (EYELA, Tokyo, Japan). The residue was reconstituted in 50% methanol, centrifuged, and was filtered with a 0.2 μm Minisart RC 4 filter (Sartorius, Goettingen, Germany) for HPLC-QTOF-MS analysis.

Metabolomic profiling were conducted by the Metabolomics Core Laboratory at the Center of Genomic Medicine at National Taiwan University. All the extracts were analyzed using an Agilent 1290 UHPLC system coupled with a 6540-QTOF (Agilent Technologies, Santa Clara, CA, USA) coupled with an Acquity HSS T3 column (100 × 2.1 mm, 1.8 μm; Waters, Milford, MA, USA). The mobile phase comprised solvent A (0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The gradient elution program was as follows: 0-1.5 min:2% solvent B; 1.5-9 min: linear gradient from 2% to 50% solvent B; 9-14 min: linear gradient from 50% to 95% solvent B; and finally, 14-17 min: 95% solvent B. The ﬂow rate was 300 μl/min. A Jet Stream electrospray ionization source with a capillary voltage of 4.0 kV was used in positive mode for sample ionization. The MS parameters were set as follows: gas temperature, 325 °C; gas ﬂow, 5 L/min; nebulizer pressure, 40 psi; sheath gas temperature, 325 °C; and sheath gas ﬂow, 10 L/min. The scan range was set at 50-1700 m/z. Metabolite intensities were analyzed using z-transformation. Analysis of the targeted metabolomics dataset was performed by using MetaboAnalyst 5.0.

Cellular bioenergetic analysis

Oxygen consumption rate (OCR) measurements were performed using a Seahorse an XFe24 extracellular flux Analyzer (Agilent Technologies/Seahorse Bioscience, Billerica, MA, USA) according to the manufacturer’s instructions. Cells were pretreated with drug for 24 h and seeded in a 24-well cell culture microplate precoated with Cell-Tak® Cell and Tissue Adhesive (Corning, Corning, NY). The Cell Mito Stress Test Kit (Agilent Technologies/Seahorse Bioscience) was used to measure mitochondrial respiration. After baseline measurements, oligomycin A, FCCP, rotenone, and antimycin A were sequentially applied to the microplate. The results were analyzed using the wave 2.4 (Agilent Technologies/Seahorse Bioscience).

In vivo efficacy of omipalisib in a murine model

A proper amount of OCI-AML3 cells were subcutaneously inoculated into CAnN.Cg-Foxn1^nu/CrlNarl mice (National Laboratory of Animal Breeding and Research Center, Taipei, Taiwan). When the tumor size reached 100 mm³, the mice were randomly divided into three groups, one receiving oral omipalisib with 0.2 mg/kg (n = 10), the other one receiving oral omipalisib of 1 mg/kg (n = 11) and one vehicle group (n = 11). Treatments were administered for two days, followed by a 1-day rest, for a total of seven cycles.

All the mice were regularly monitored for body weight and tumor growth. Tumor volumes were calculated using the following formula: (length × width²) × 0.5 in millimeters. The mice were euthanized for further analysis when the tumor volume reached 1500 mm³. The xenograft experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the College of Medicine, National Taiwan University (#20180457, 30 Jan 2019), and conformed to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health.

Statistical analysis

Statistically significant differences were evaluated using a two-sided Student’s t-test. Error bars indicate means ± S.D. or means ± S.E.M. of at least three independent triplicate experiments. The IC₅₀ values were determined using the AAT Bioquest IC₅₀ calculator Principal component analysis (PCA) was performed by using ClustVis. P values of less than 0.05 were considered statistically significant.

Omipalisib potently suppressed AML cell proliferation

We investigated the activity of the PI3K/AKT/mTOR signaling pathway in various leukemia cell lines. Reverse phase protein array (RPPA) analysis of leukemia cell lines from the Cancer Cell Line Encyclopedia (CCLE) database indicated that OCI-AML3, THP-1, NB4, MV4-11, and MOLM-13 cells had enhanced PI3K/AKT/mTOR signaling (Fig. S1A). Furthermore, Genomics of Drug Sensitivity in Cancer (GDSC) data analysis indicated that AML cells are more sensitive to omipalisib, a PI3K/mTOR dual inhibitor, than other solid tumor cells (Fig. S1B). Consequently, we determined the cytotoxic effects of omipalisib on a panel of leukemia cell lines, including OCI-AML3, THP-1, SKNO-1, Kasumi-1, MV4-11, and MOLM-13. We found that all AML cell lines except SKNO-1 were sensitive to omipalisib, with an IC₅₀ in the sub-nanomolar range (Fig. 1A). In contrast, the cytotoxic effects of gedatolisib, another PI3K/mTOR dual inhibitor, were inconsistent in various AML cell lines (Fig. 1B). OCI-AML3 and THP-1 cells were more sensitive to omipalisib and less sensitive to gedatolisib, suggesting that omipalisib may have additional anti-leukemic mechanisms. Peripheral blood mononuclear cells (PBMCs) from normal controls showed a relatively higher IC₅₀ (211.71 nM for omipalisib (Fig. S1C)), suggesting its clinical applicability.

Immunoblotting experiments revealed that, as expected, the phosphorylation of AKT and mTOR (Fig. 1C) and the downstream molecules S6K and 4E-BP1 (Fig. 1D) were significantly abrogated in OCI-AML3 and THP-1 cells after 4 h of omipalisib treatment. The cell cycle analysis revealed that omipalisib significantly induced G₀/G₁ cell cycle arrest (Fig. 1E, F). Additionally, immunoblotting indicated that the cyclin-dependent kinase (CDK) inhibitor, p27, was upregulated (Fig. 1G, H) in OCI-AML3 and THP-1 after 24 h of treatment. These data indicated that omipalisib inhibited the PI3K/AKT/mTOR signaling pathway and subsequently suppressed AML proliferation.

Transcriptomic analysis revealed that omipalisib downregulated amino acid metabolisms and mitochondrial biogenesis in AML

We performed RNA sequencing (RNA-seq) to compare omipalisib- and DMSO-treated OCI-AML3 cells after exposure to the drug for 24 h. We identified a total of 2056 differentially expressed genes (DEGs), of which 811 were downregulated and 1245 were upregulated (p < 0.05 and -1 ≥ log₂FC ≥ 1) post omipalisib treatment for 24 h (Fig. 2A). Interestingly, the most suppressed genes after omipalisib treatment were those associated with the cell cycle, DNA metabolic processes, PI3K/AKT/mTOR signaling, the MYC pathway, and E2F pathway (Fig. 2B). Gene set enrichment analysis (GSEA) further showed marked suppression of the KEGG pathway gene sets, including “Gly, Ser and Thr metabolism”, “Cys and Met metabolism”, “Carbon metabolism”, and “Oxidative phosphorylation” following omipalisib treatment (Fig. 2C). Furthermore, we found that gene sets that contain genes encoding components of mitochondrial biogenesis and the respiratory electron transporter chain were significantly suppressed in the omipalisib-treated group (Fig. 2D). Based on the results of these transcriptomic analyses, we proposed that omipalisib not only suppresses cell proliferation through PI3K/AKT/mTOR signaling inhibition but also reprograms cellular metabolisms and impairs mitochondrial biogenesis.

Omipalisib reduced oxidative phosphorylation and impaired mitochondrial biogenesis in AML

To examine the effects of omipalisib on mitochondrial biogenesis and oxidative phosphorylation, a Seahorse XF Cell Mito Stress Assay was performed. We found a significant decrease in basal OCR, maximal respiration, spare-respiratory capacity, and proton leak-driven OCR in omipalisib-treated OCI-AML3 cells (Fig. 3A). Similar results were observed in THP-1 cells (Fig. S2A). Flow cytometry analysis revealed that omipalisib decreased mitochondrial membrane potential and mitochondrial mass but increase mitochondrial ROS in OCI-AML3 cells (Fig. 3B). In addition, qRT-PCR results demonstrated that omipalisib significantly decreased the mtDNA content (Fig. 3C) and inhibited the expression of genes associated with mitochondrial biosynthesis, including PPARGC1B, TFAM, and NUDFS6 (Fig. 3D). Immunoblot analysis indicated that the protein levels of PGC1β were significantly decreased in OCI-AML3 cells after 72 h omipalisib treatment (Fig. 3E). These results indicated that omipalisib suppressed mitochondrial respiration and inhibited mitochondrial biogenesis in myeloid leukemia cells.

AML cells exhibit metabolic changes in response to omipalisib

To determine the role of metabolic reprogramming in response to omipalisib treatment, we performed a metabolomic analysis of OCI-AML3 cells after omipalisib treatment for 24 h. In total, 82 metabolites were identified in the targeted metabolomic analysis. PCA demonstrated a clear separation between the omipalisib- and the DMSO-treated group (Fig. 4A). Of the 82 metabolites, 22 significantly increased or decreased (p-value ≤ 0.05) after omipalisib treatment compared to the DMSO controls (Fig. 4B). Metabolomics analyses indicated that omipalisib treatment significantly decreased the intracellular levels of L-valine, L-alanine, L-tryptophan, L-phenylalanine, L-tyrosine, L-histidine, and pyruvate (Fig. 4C and Table S3). Metabolite set enrichment analysis (MSEA) revealed that these metabolites are involved in the biosynthesis and degradation of various amino acids (Fig. 4D and Table S4). These results indicate that omipalisib suppressed amino acid metabolism in OCI-AML3 cells.

Omipalisib inhibited glutathione metabolism through suppressing de novo serine synthesis and folate/methionine cycle in AML

Joint-Pathway Analysis (MetaboAnalyst 5.0) which combined transcriptomic and metabolomic data revealed that “glutathione metabolism” (p-value = 0.000278, impact = 0.36548) and “glycine, serine, and threonine metabolism” (p-value = 0.004847, impact = 0.25803) were the most significantly downregulated pathways in the omipalisib-treated group (Fig. 5A and Table S5). Subsequently, qRT-PCR analysis confirmed that the expression of several genes related to de novo serine synthesis pathway, regulators of glycine biosynthesis, glutathione synthesis, and glycolysis was significantly decreased in omipalisib-treated OCI-AML3 cells (Fig. 5B). Similar results were obtained in THP-1 cells (Fig. S2B). In concordance with the transcriptomic alternations, the metabolomic profiling data also showed that the cellular levels of glutathione and pyruvate were significantly decreased (Fig. 5C). Immunoblotting analysis indicated that the expression of PGHDH and PSAT1 was significantly decreased in OCI-AML3 cells after 24 h of omipalisib treatment (Fig. 5D). In summary, we propose that omipalisib inhibits glycolysis, de novo serine synthesis, the folate/methionine cycle, and glutathione, resulting in decreased glutathione and pyruvate levels (Fig. 5E).

Gedatolisib and dactolisib exerted similar effects on glutathione metabolism and oxidative phosphorylation in AML

Dactolisib (BEZ-235) and gedatolisib (PF-05212384), two other dual PI3K/mTOR inhibitors, have been reported to suppress proliferation and migration and reverse multidrug resistance in AML [17, 18]. These two inhibitors suppressed the expression of several genes associated with de novo serine synthesis, the folate/methionine cycle, glutathione synthesis, and glycolysis (Fig. S3A, B). Gedatolisib suppressed basal OCR, proton leak, and maximal respiration in OCI-AML3 cells (Fig. S3C). Moreover, gedatolisib increased mitochondrial ROS and decreased mitochondrial membrane potential but it did not suppress mitochondrial mass and mtDNA content in OCI-AML3 cells (Fig. S3D, E). In addition, gedatolisib and dactolisib did not suppress the expression of mitochondrial biosynthesis-related genes (Fig. S3F). These results indicated that gedatolisib and dactolisib could only suppress glutathione metabolism and mitochondrial respiration rather than mitochondrial biogenesis.

Omipalisib suppressed the growth of subcutaneous OCI-AML3 xenograft tumors

To evaluate the anti-leukemic efficacy of omipalisib in vivo, OCI-AML3 xenografted nude mice were treated with vehicle (DMSO) or omipalisib (0.2 mg/kg or 1 mg/kg). Omipalisib was well-tolerated by the mice, and there were no side effects on body weight during the dosing period (Fig. 6A). In line with the in vitro results, mice treated with 0.2 or 1 mg/kg omipalisib showed retarded OCI-AML3 tumor growth (Fig. 6B, C). Compared with the vehicle group, oral administration of 0.2 or 1 mg/kg omipalisib significantly prolonged the survival of mice bearing OCI-AML3 tumors (Fig. 6D).

This study demonstrates the potent anti-leukemic effect of omipalisib in vitro and in vivo. While its effects on other cancers [22–26] have been studied, its impact on leukemia was previously unknown. Our results show that omipalisib effectively inhibits PI3K/AKT/mTOR signaling pathway, triggers cell cycle arrest, and regulates metabolic pathways in AML cells. This is the first study to investigate the anti-malignant activity of omipalisib and its detailed mechanisms in AML by integrating transcriptome, metabolome, and functional assays.

Our analysis of metabolomic and transcriptomic data revealed that omipalisib suppressed de novo serine synthesis, the folate/methionine cycle, and glutathione metabolism, which are dysregulated in various cancers [28–31], including FLT3-ITD-driven AML [32]. These metabolic pathways are critical for protein, nucleotide and lipid synthesis, generation of glutathione and NADPH, and methylation reactions [33, 34]. Omipalisib and other PI3K/mTOR dual inhibitors significantly suppressed the expression of genes involved in these metabolic pathways, including PHGDH, PSAT1, PSPH, SHMT1/2, and MTHFD1/2. Dysregulation of serine metabolism through the mTORC1-ATF4 axis has also been reported in FLT3-ITD AML [32], highlighting the potential of serine metabolism as treatment target in AML.

Notably, we found that omipalisib, but not gedatolisib or dactolisib, suppressed oxidative phosphorylation and disrupted mitochondrial biogenesis. We demonstrated that omipalisib significantly inhibited mitochondrial respiration rate, decreased membrane potential, and increased mitochondrial ROS. Furthermore, omipalisib strongly decreased mitochondrial mass, mtDNA, and the expression of several genes related to mitochondrial biogenesis, as well as their protein levels. Additionally, AML cells have higher mitochondrial biogenesis than normal hematopoietic cells [35, 36], and AML progression requires increased mitochondrial biogenesis and oxidative phosphorylation [37, 38]. Therefore, mitochondrial metabolism and biogenesis are potential therapeutic targets for AML treatment.

Peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1 (PGC1) is a family of transcriptional coactivators that promotes mitochondrial biogenesis, oxidative phosphorylation, and fatty acid oxidation in various cancers [39–41]. Abnormal expression of PGC1α (PPARGC1A) or PGC1β (PPARGC1B) has been implicated in tumorigenesis in several cancers [42–50]. DepMap analysis showed that PPARGC1B expression was essential for OCI-AML3 cell survival (Fig. S4A). Analysis of TCGA LAML [51] and OHSU AML [52] transcriptomic databases revealed higher expression of PPARGC1B than that of PPARGC1A in both cohorts (Fig. S4B), with high PPARGC1B expression correlating with poorer overall survival in AML patients (Fig. S4C). Omipalisib may target PGC1β, but further studies are required to confirm this hypothesis.

In this study, we elucidated the cytotoxicity and metabolic alterations caused by omipalisib in AML, both in vitro and in vivo. The combination of metabolomic profiling and transcriptomic analyses, followed by various precise experiments, uncovered the molecular mechanisms of omipalisib, including the inhibition of serine and glutathione biosynthesis and impairment of oxidative phosphorylation and mitochondrial biogenesis in AML. Recently, a randomized, placebo-controlled study demonstrated acceptable tolerability of omipalisib in idiopathic pulmonary fibrosis [53]. Participants receiving 2.0 mg omipalisib twice daily achieved a maximum observed concentration (C_max) of 170 nM, which is well above the effective dose used in this study. To date, omipalisib has been demonstrated to be an effective therapeutic strategy in combination with CDK4/6 and autophagy inhibitors for the treatment of various cancer types [23, 24]. Omipalisib has the advantages of sensitizing cancer cells to radiotherapy and chemotherapy [22, 54]. Therefore, combined treatment strategy using omipalisib is an effective future protocol for AML.

Acknowledgements

This study was supported by research grants from the Taiwan Health Foundation, and the Ministry of Science and Technology, Taiwan (MOST 111-2320-B-002-058 and MOST 108-2320-B-002-050-MY3, respectively). We acknowledge the technical supports of the Metabolomics Core Laboratory at the Center of Genomic Medicine, National Taiwan University, and the Immune Research Core of the Department of Medical Research at the National Taiwan University Hospital.

Author Contributions

Conceptualization and Supervision: L.-I. L.; Methodology: C.-Y. T, Y.-H.F., J.-W. L.; Bioinformatics analysis: C.-Y. T, Y.-H.F.; Animal experiments: C.-Y. T, D.-L. O; Data curation: C.-Y. T, D.-L. O; Funding acquisition: L.-I. L. and H.-A. H.; Writing – original draft: C.-Y. T.; Writing – review & editing: L.-I. L. and J.-W. L.

Competing interests

The authors declare that they have no conflict of interest.

Assi SA, Imperato MR, Coleman DJL, Pickin A, Potluri S, Ptasinska A, et al. Subtype-specific regulatory network rewiring in acute myeloid leukemia. Nature Genetics. 2019;51:151-62.
Jahn N, Terzer T, Sträng E, Dolnik A, Cocciardi S, Panina E, et al. Genomic heterogeneity in core-binding factor acute myeloid leukemia and its clinical implication. Blood Advances. 2020;4(24):6342-52.
Carter JL, Hege K, Yang J, Kalpage HA, Su Y, Edwards H, et al. Targeting multiple signaling pathways: the new approach to acute myeloid leukemia therapy. Signal Transduction and Targeted Therapy. 2020;5(1):288.
Daver N, Wei AH, Pollyea DA, Fathi AT, Vyas P, DiNardo CD. New directions for emerging therapies in acute myeloid leukemia: the next chapter. Blood Cancer Journal. 2020; 10: 107.
Manning BD, Toker A. AKT/PKB Signaling: Navigating the Network. Cell. 2017;169(3):381-405.
Saxton RA, Sabatini DM. mTOR Signaling in growth, metabolism, and fisease. Cell. 2017;168(6):960-76.
Sophie P, Nicolas C, Jérôme T, Valérie B, Pascale C-L, Lise W, et al. Role of the PI3K/AKT and mTOR signaling pathways in acute myeloid leukemia. Haematologica. 2010;95(5):819-28.
Bertacchini J, Guida M, Accordi B, Mediani L, Martelli AM, Barozzi P, et al. Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis. Leukemia. 2014;28(11):2197-205.
Brandts CH, Sargin B, Rode M, Biermann C, Lindtner B, Schwäble J, et al. Constitutive activation of Akt by Flt3 internal tandem duplications is necessary for increased survival, proliferation, and myeloid transformation. Cancer Research. 2005;65(21):9643-50.
Malaise M, Steinbach D, Corbacioglu S. Clinical implications of c-Kit mutations in acute myelogenous leukemia. Current Hematologic Malignancy Reports. 2009;4(2):77-82.
Wang S, Wu Z, Li T, Li Y, Wang W, Hao Q, et al. Mutational spectrum and prognosis in NRAS-mutated acute myeloid leukemia. Scientific Reports. 2020;10(1):12152.
Pillinger G, Loughran NV, Piddock RE, Shafat MS, Zaitseva L, Abdul-Aziz A, et al. Targeting PI3Kδ and PI3Kγ signalling disrupts human AML survival and bone marrow stromal cell mediated protection. Oncotarget. 2016;7(26)39784-95.
Colamonici M, Blyth G, Saleiro D, Szilard A, Bliss-Moreau M, Giles FJ, et al. Dual targeting of acute myeloid leukemia progenitors by catalytic mTOR inhibition and blockade of the p110α subunit of PI3 kinase. Oncotarget. 2015;6(10):8062-70.
Nguyen LXT, Sesay A, Mitchell BS. Effect of CAL-101, a PI3Kδ inhibitor, on ribosomal RNA synthesis and cell proliferation in acute myeloid leukemia cells. Blood Cancer J. 2014;4(7):e228.
Allegretti M, Ricciardi MR, Licchetta R, Mirabilii S, Orecchioni S, Reggiani F, et al. The pan-class I phosphatidyl-inositol-3 kinase inhibitor NVP-BKM120 demonstrates anti-leukemic activity in acute myeloid leukemia. Scientific Reports. 2015;5(1):18137.
Zeng Z, Sarbassov DD, Samudio IJ, Yee KWL, Munsell MF, Ellen Jackson C, et al. Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML. Blood. 2006;109(8):3509-12.
Deng L, Jiang L, Lin X-h, Tseng K-F, Liu Y, Zhang X, et al. The PI3K/mTOR dual inhibitor BEZ235 suppresses proliferation and migration and reverses multidrug resistance in acute myeloid leukemia. Acta Pharmacologica Sinica. 2017;38(3):382-91.
Lindblad O, Cordero E, Puissant A, Macaulay L, Ramos A, Kabir NN, et al. Aberrant activation of the PI3K/mTOR pathway promotes resistance to sorafenib in AML. Oncogene. 2016;35(39):5119-31.
Lang F, Wunderle L, Badura S, Schleyer E, Brüggemann M, Serve H, et al. A phase I study of a dual PI3-kinase/mTOR inhibitor BEZ235 in adult patients with relapsed or refractory acute leukemia. BMC Pharmacology and Toxicology. 2020;21(1):70.
Vargaftig J, Farhat H, Ades L, Briaux A, Benoist C, Turbiez I, et al. Phase 2 Trial of Single Agent Gedatolisib (PF-05212384), a dual PI3K/mTOR Inhibitor, for adverse prognosis and relapse/refractory AML: clinical and transcriptomic results. Blood. 2018;132(Supplement 1):5233.
Knight SD, Adams ND, Burgess JL, Chaudhari AM, Darcy MG, Donatelli CA, et al. Discovery of GSK2126458, a highly potent inhibitor of PI3K and the mammalian target of rapamycin. ACS Medicinal Chemistry Letters. 2010;1(1):39-43.
Liu T, Sun Q, Li Q, Yang H, Zhang Y, Wang R, et al. Dual PI3K/mTOR Inhibitors, GSK2126458 and PKI-587, suppress tumor progression and increase radiosensitivity in nasopharyngeal carcinoma. Molecular Cancer Therapeutics. 2015;14(2):429.
Wong K, Di Cristofano F, Ranieri M, De Martino D, Di Cristofano A. PI3K/mTOR inhibition potentiates and extends palbociclib activity in anaplastic thyroid cancer. Endocrine-Related Cancer. 2019;26(4):425-36.
Bernard M, Cardin GB, Cahuzac M, Ayad T, Bissada E, Guertin L, et al. Dual inhibition of autophagy and PI3K/AKT/MTOR pathway as a therapeutic strategy in head and neck squamous cell carcinoma. Cancers. 2020;12(9): 2371.
McKinnon T, Venier R, Yohe M, Sindiri S, Gryder B, Shern JF, et al. Functional screening of FGER4-driven tumorigenesis identifies PI3K/mTOR inhibition as a therapeutic strategy in rhabdomyosarcoma. Oncogene. 2018; 37(20):2630-44.
Feng Y, Jiang Y, Hao F. GSK2126458 has the potential to inhibit the proliferation of pancreatic cancer uncovered by bioinformatics analysis and pharmacological experiments. Journal of Translational Medicine. 2021;19(1):373.
Fu YH, Tseng CY, Lu JW, Lu WH, Lan PQ, Chen CY, et al. Deciphering the role of pyrvinium pamoate in the generation of integrated stress response and modulation of mitochondrial function in myeloid leukemia cells through transcriptome analysis. Biomedicines. 2021;9(12)1869.
Pollari S, Käkönen S-M, Edgren H, Wolf M, Kohonen P, Sara H, et al. Enhanced serine production by bone metastatic breast cancer cells stimulates osteoclastogenesis. Breast Cancer Research and Treatment. 2011;125(2):421-30.
Mullarky E, Mattaini KR, Vander Heiden MG, Cantley LC, Locasale JW. PHGDH amplification and altered glucose metabolism in human melanoma. Pigment Cell & Melanoma Research. 2011;24(6):1112-5.
Liu J, Guo S, Li Q, Yang L, Xia Z, Zhang L, et al. Phosphoglycerate dehydrogenase induces glioma cells proliferation and invasion by stabilizing forkhead box M1. Journal of Neuro-Oncology. 2013;111(3):245-55.
Sun WY, Kim HM, Jung W-H, Koo JS. Expression of serine/glycine metabolism-related proteins is different according to the thyroid cancer subtype. Journal of Translational Medicine. 2016;14(1):168.
Bjelosevic S, Gruber E, Newbold A, Shembrey C, Devlin JR, Hogg SJ, et al. Serine biosynthesis is a metabolic vulnerability in FLT3-ITD–driven acute myeloid leukemia. Cancer Discovery. 2021;11(6):1582.
Locasale JW. Serine, glycine and one-carbon units: cancer metabolism in full circle. Nature Reviews Cancer. 2013;13(8):572-83.
Yang M, Vousden KH. Serine and one-carbon metabolism in cancer. Nature Reviews Cancer. 2016;16(10):650-62.
Škrtić M, Sriskanthadevan S, Jhas B, Gebbia M, Wang X, Wang Z, et al. Inhibition of mitochondrial translation as a therapeutic strategy for human acute myeloid leukemia. Cancer Cell. 2011;20(5):674-88.
Basak NP, Banerjee S. Mitochondrial dependency in progression of acute myeloid leukemia. Mitochondrion. 2015;21:41-8.
Schimmer AD, Škrtić M. Therapeutic potential of mitochondrial translation inhibition for treatment of acute myeloid leukemia. Expert Review of Hematology. 2012;5(2):117-9.
Farge T, Saland E, de Toni F, Aroua N, Hosseini M, Perry R, et al. Chemotherapy-resistant human acute myeloid leukemia cells are not enriched for leukemic stem cells but require oxidative metabolism. Cancer Discovery. 2017;7(7):716.
Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM. A Cold-Inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell. 1998;92(6):829-39.
Lin J, Puigserver P, Donovan J, Tarr P, Spiegelman BM. Peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β), a novel PGC-1-related transcription coactivator associated with host cell factor. Journal of Biological Chemistry. 2002;277(3):1645-8.
Andersson U, Scarpulla Richard C. PGC-1-Related Coactivator, a novel, serum-inducible coactivator of nuclear respiratory factor 1-dependent transcription in mammalian cells. Molecular and Cellular Biology. 2001;21(11):3738-49.
Shiota M, Yokomizo A, Tada Y, Inokuchi J, Tatsugami K, Kuroiwa K, et al. Peroxisome proliferator-activated receptor γ coactivator-1α interacts with the androgen receptor (AR) and promotes prostate cancer cell growth by activating the AR. Molecular Endocrinology. 2010;24(1):114-27.
McGuirk S, Gravel S-P, Deblois G, Papadopoli DJ, Faubert B, Wegner A, et al. PGC-1α supports glutamine metabolism in breast cancer. Cancer & Metabolism. 2013;1(1):22.
Vazquez F, Lim J-H, Chim H, Bhalla K, Girnun G, Pierce K, et al. PGC1α expression defines a subset of human melanoma tumors with increased mitochondrial capacity and resistance to oxidative stress. Cancer Cell. 2013;23(3):287-301.
LaGory Edward L, Wu C, Taniguchi Cullen M, Ding C-Kuang C, Chi J-T, von Eyben R, et al. Suppression of PGC-1α is critical for reprogramming oxidative metabolism in renal cell carcinoma. Cell Reports. 2015;12(1):116-27.
Sancho P, Burgos-Ramos E, Tavera A, Bou Kheir T, Jagust P, Schoenhals M, et al. MYC/PGC-1α balance determines the metabolic phenotype and plasticity of pancreatic cancer stem cells. Cell Metabolism. 2015;22(4):590-605.
Fisher Kurt W, Das B, Kim Hyun S, Clymer Beth K, Gehring D, Smith Deandra R, et al. AMPK promotes aberrant PGC1β expression to support human colon tumor cell survival. Molecular and Cellular Biology. 2015;35(22):3866-79.
Zhang H, Li L, Chen Q, Li M, Feng J, Sun Y, et al. PGC1β regulates multiple myeloma tumor growth through LDHA-mediated glycolytic metabolism. Molecular Oncology. 2018;12(9):1579-95.
Chen X, Lv Y, Sun Y, Zhang H, Xie W, Zhong L, et al. PGC1β regulates breast tumor growth and metastasis by SREBP1-mediated HKDC1 expression. Frontiers in Oncology. 2019;9:290.
Ghilardi C, Moreira-Barbosa C, Brunelli L, Ostano P, Panini N, Lupi M, et al. PGC1α/β expression predicts therapeutic response to oxidative phosphorylation inhibition in ovarian cancer. Cancer Research. 2022;82(7):1423-34.
Chang K, Creighton CJ, Davis C, Donehower L, Drummond J, Wheeler D, et al. The Cancer Genome Atlas Pan-Cancer analysis project. Nature Genetics. 2013;45(10):1113-20.
Tyner JW, Tognon CE, Bottomly D, Wilmot B, Kurtz SE, Savage SL, et al. Functional genomic landscape of acute myeloid leukaemia. Nature. 2018;562(7728):526-31.
Lukey PT, Harrison SA, Yang S, Man Y, Holman BF, Rashidnasab A, et al. A randomised, placebo-controlled study of omipalisib (PI3K/mTOR) in idiopathic pulmonary fibrosis. European Respiratory Journal. 2019;53(3):1801992.
Du J, Chen F, Yu J, Jiang L, Zhou M. The PI3K/mTOR Inhibitor omipalisib suppresses nonhomologous end joining and sensitizes cancer cells to radio- and chemotherapy. Molecular Cancer Research. 2021;19(11):1889-99.

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Anti-leukemia effects of Omipalisib in Acute Myeloid Leukemia: inhibition of PI3K-AKT-mTOR signaling and suppression of Mitochondrial Biogenesis

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