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Dual inhibition of CPT1A or TRAP1 and c-MET elicits growth reduction of PDXs. A and B, GBM12 PDX models were serially transplanted into mice. Following establishment of tumors, four treatment groups were randomly assigned. Thereafter, treatment was initiated with vehicle, crizotinib, etomoxir, or the combination of both by i.p. injections three times a week. On day 17, the experiment was terminated and a final measurement was taken, which was statistically evaluated (n ¼ 9; 2-3 tumors per animal; B). C and D, GBM12 PDX grafts were serially transplanted into mice. Following establishment of tumors, four treatment groups were randomly assigned. Thereafter, treatment was initiated with vehicle, gamitrinib (GTPP), crizotinib, or the combination of both by i.p. injections three times a week. On day 17, the experiment was terminated, and a final measurement was taken, which was statistically evaluated (n ¼ 8-9; 2-3 tumors per animal; D). E-G, Tumors from experiment C were harvested, fixed, and embedded in paraffin, followed by staining with standard hematoxylin and eosin (H&E) or were subjected to IHC for TUNEL or Ki67. H, Sections from the experiment in C were quantified for apoptotic bodies, number of TUNEL-positive cells, and number of mitosis per multiple high-power fields (n ¼ 4-7). Shown are mean and SD. Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ / ÃÃÃÃ , P < 0.001. Scale bar, 50 mm. I, U87 GBM cells were implanted in the right striatum of nude mice. Starting on day 5, four groups were formed: vehicle, etomoxir, crizotinib, etomoxir, and crizotinib. In total, nine treatments were administered, and animal survival is provided (Kaplan-Meier curve). The log-rank test was used to assess statistical significance (n ¼ 2-5 animals per group). Crizotinib and etomoxir versus etomoxir (P < 0.01), crizotinib and etomoxir versus crizotinib (P < 0.01), and crizotinib and etomoxir versus control (P < 0.01).
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The receptor kinase c-MET has emerged as a target for glioblastoma therapy. However, treatment resistance emerges inevitably. Here, we performed global metabolite screening with metabolite set enrichment coupled with transcriptome and gene set enrichment analysis and proteomic screening, and identified substantial reprogramming of tumor metabolism...
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... to synergistic reduction in proliferation of tumor cells ( Fig. 4A and B; Supplementary Fig. S6A-S6G), suggesting that oxidative phosphorylation operates as a prosurvival pathway in the context of MET inhibition. Given our earlier observation of an increase in polar and nonpolar metabolites related to FAO and an upregulation of transporters related to fatty acid transport, we hypothesized that MET regulates FAO and that MET inhibition will facilitate b-oxidation, which in turn will increase the OCR. ...
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... transcriptome analysis demonstrated that MET inhibition led to an increase of a master-regulator of oxidative metabolism, PGC1a, which was confirmed by real-time PCR analysis as well as on the protein level in several GBM model systems ( Fig. 4D-I; Supplementary Fig. S6C and S6G). In agreement, GSEA of chronically crizotinibtreated cells revealed an increase for the PPAR signaling pathway, which was accompanied by an activation of ERK and CREB signaling, which are known pathways to be upstream of PGC1a, suggesting their potential involvement in the transcriptional regulation of PGC1a through MET inhibition ...
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... palmitic acid, and OCR was measured (n ¼ 15-20). (Continued on the following page.) cancerres.aacrjournals.org Downloaded from treatment groups did not differ in tumor growth, we noted a substantial and statistical significant suppression in tumor growth in animals that received the combination treatment of crizotinib and etomoxir ( Fig. 6A and B). Next, we tested the drug combination of gamitrinib and crizotinib in vivo. To this end, we established GBM12 tumors in mice, formed treatment groups, and treated them with the indicated drug compounds. We noted a statistical significant reduction of tumor growth in the combination treatment with single treatments and controls (Fig. ...
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... ( Fig. 6A and B). Next, we tested the drug combination of gamitrinib and crizotinib in vivo. To this end, we established GBM12 tumors in mice, formed treatment groups, and treated them with the indicated drug compounds. We noted a statistical significant reduction of tumor growth in the combination treatment with single treatments and controls (Fig. 6C and D). The same experiments as performed in the GBM12 model were subsequently conducted in GBM14. Akin to the GBM12 model, we found that both combination treatments (crizotinib and gamitrinib; crizotinib and etomoxir) led to a more pronounced reduction in tumor growth than each single treatment or control ( Supplementary Fig. S11A and ...
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... confirm how the combination treatment (crizotinib and gamitrinib) reduced tumor growth, we performed histologic analysis of tumors with hematoxylin and eosin, TUNEL, and Ki67 staining (Fig. 6E-H). Through TUNEL staining, we found that overall there is more cell death in tumor slides from the combination treatment. The proliferation index (Ki67) was reduced in the combination treatment as well. These findings suggest both a reduced proliferation of tumor cells and an enhanced induction of cell death, which provides an ...
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... and after completion of a treatment regimen of either vehicle, single (crizotinib, etomoxir), or the combination treatment, we noted a small, but significant survival extension of animals receiving the combination treatment, suggesting that this treatment concept may have potential following optimization of delivery or inhibitor modification (Fig. ...
Citations
... Another inhibitor, crizotinib, which targets MET kinase (112,113), induces the metabolic reprogramming of GBM cells. This reprogramming, characterized by heightened oxidative phosphorylation and fatty acid oxidation, is also mediated by upregulated PGC1a expression and facilitated by increased CREB phosphorylation after Crizotinib exposure (14,114). The mTORC1 pathway, crucial for cell growth and proliferation in GBM (115), is often activated by epidermal growth factor receptor (EGFR). ...
PGC1α, a central player in mitochondrial biology, holds a complex role in the metabolic shifts seen in cancer cells. While its dysregulation is common across major cancers, its impact varies. In some cases, downregulation promotes aerobic glycolysis and progression, whereas in others, overexpression escalates respiration and aggression. PGC1α’s interactions with distinct signaling pathways and transcription factors further diversify its roles, often in a tissue-specific manner. Understanding these multifaceted functions could unlock innovative therapeutic strategies. However, challenges exist in managing the metabolic adaptability of cancer cells and refining PGC1α-targeted approaches. This review aims to collate and present the current knowledge on the expression patterns, regulators, binding partners, and roles of PGC1α in diverse cancers. We examined PGC1α’s tissue-specific functions and elucidated its dual nature as both a potential tumor suppressor and an oncogenic collaborator. In cancers where PGC1α is tumor-suppressive, reinstating its levels could halt cell proliferation and invasion, and make the cells more receptive to chemotherapy. In cancers where the opposite is true, halting PGC1α’s upregulation can be beneficial as it promotes oxidative phosphorylation, allows cancer cells to adapt to stress, and promotes a more aggressive cancer phenotype. Thus, to target PGC1α effectively, understanding its nuanced role in each cancer subtype is indispensable. This can pave the way for significant strides in the field of oncology.
... Non-exclusive expression in the tumour cells, with immune cells including macrophages and T cell and oligodendrocytes also expressing these genes was our findings in the SHM analysis, epigenetic-mediated downregulation of RPL and RPS genes is confirmed also with the ChromHMM approach, mostly mediated by the loss of the 4 activating chromatin states and the gain of poised gene body state(Fig.S9a and table S3). Signalling pathways related to the receptor tyrosine kinase MET49 and ECM organisation 50 are also found enriched by genes epigenetically downregulated in GIC as compared to iNSC (Fig.S9a) as well as deregulation of keratinisation, previously described in a glioblastoma context 51 . Importantly, we identified an enrichment for cellular response to hypoxia(Fig.S9a), ...
Analysis of chromatin remodelling in neoplastic stem cells as compared to ontogenetically related neural stem cells, reveals multifactorial epigenetic regulation of signalling pathways known to contribute to glioblastoma development. It also identifies novel epigenetically regulated druggable target genes on a patient-specific level, including SMOX and GABBR2 which could be further developed for future translational approaches to more effectively treat this neoplasm.
... TRAP1 is a major chaperone of the respiratory complex of the electron transport chain, therefore interfering with TRAP1 disintegrates oxidative phosphorylation (131). Besides, TRAP1 is upregulated in Glioblastoma multiforme (GBM) compared with normal brain cells. ...
Mitochondrial heat shock protein 90 (mtHsp90), including Tumor necrosis factor receptor-associated protein 1 (TRAP1) and Hsp90 translocated from cytoplasm, modulating cellular metabolism and signaling pathways by altering the conformation, activity, and stability of numerous client proteins, and is highly expressed in tumors. mtHsp90 inhibition results in the destabilization and eventual degradation of its client proteins, leading to interference with various tumor-related pathways and efficient control of cancer cell development. Among these compounds, gamitrinib, a specific mtHsp90 inhibitor, has demonstrated its safety and efficacy in several preclinical investigations and is currently undergoing evaluation in clinical trials. This review aims to provide a comprehensive overview of the present knowledge pertaining to mtHsp90, encompassing its structure and function. Moreover, our main emphasis is on the development of mtHsp90 inhibitors for various cancer therapies, to present a thorough overview of the recent pre-clinical and clinical advancements in this field.
... For the drug treatment experiments, 10 days after the initial tumour implantation, the mice were injected intracranially with IgG (3 µg), anti-MET404 monoclonal antibody (3 µg), onartuzumab (3 µg) or anti-MET404 combined with onartuzumab (3 µg each) through a screw guide (Protech International) every 3 days. Crizotinib (MCE, HY-50878) was successively resolved in dimethyl sulfoxide (DMSO), PEG-300, Tween-80 and saline as recommended by the manufacturer, and administered to the mice via oral gavage (50 mg/kg) every other day, as previously reported 56 . The control mice were administered with the solvent. ...
Activated by its single ligand, hepatocyte growth factor (HGF), the receptor tyrosine kinase MET is pivotal in promoting glioblastoma (GBM) stem cell self-renewal, invasiveness and tumorigenicity. Nevertheless, HGF/MET-targeted therapy has shown limited clinical benefits in GBM patients, suggesting hidden mechanisms of MET signalling in GBM. Here, we show that circular MET RNA (circMET) encodes a 404-amino-acid MET variant (MET404) facilitated by the N ⁶ -methyladenosine (m ⁶ A) reader YTHDF2. Genetic ablation of circMET inhibits MET404 expression in mice and attenuates MET signalling. Conversely, MET404 knock-in (KI) plus P53 knock-out (KO) in mouse astrocytes initiates GBM tumorigenesis and shortens the overall survival. MET404 directly interacts with the MET β subunit and forms a constitutively activated MET receptor whose activity does not require HGF stimulation. High MET404 expression predicts poor prognosis in GBM patients, indicating its clinical relevance. Targeting MET404 through a neutralizing antibody or genetic ablation reduces GBM tumorigenicity in vitro and in vivo, and combinatorial benefits are obtained with the addition of a traditional MET inhibitor. Overall, we identify a MET variant that promotes GBM tumorigenicity, offering a potential therapeutic strategy for GBM patients, especially those with MET hyperactivation.
... Recent research from our group provided evidence that CPI-613 interferes with GBM growth in vitro and in vivo [1]. Similarly, several combination therapies, involving etomoxir, have shown preclinical efficacy [14,16]. ...
... While certain off-target effects might not be entirely excluded, it appears that succinate dehydrogenase is one of the key targets affected by gamitrinib [29,30]. Consistently, several studies have shown that gamitrinib potently suppresses the oxygen consumption rate of various tumor cells, including GBM [16,31]. Due to its profound effect on metabolism, gamitrinib elicited a mitochondrial unfolded stress response with up-regulation of CEBP/beta and CHOP that in turn suppressed NF-κB activity in GBM cells. ...
... In turn, loss of NF-kb function sensitized GBM cells to death-receptor mediated apoptosis (extrinsic) [25]. Several preclinical studies have shown efficacy in GBM model systems [16,31]. It should be noted that gamitrinib synergized with BH3-mimetic to kill GBM cells in vitro and in an orthotopic patient-derived xenograft model of GBM in mice [23]. ...
While glycolysis is abundant in malignancies, mitochondrial metabolism is significant as well. Mitochondria harbor the enzymes relevant for cellular respiration, which is a critical pathway for both regeneration of reduction equivalents and energy production in the form of ATP. The oxidation of NADH2 and FADH2 are fundamental since NAD and FAD are the key components of the TCA-cycle that is critical to entertain biosynthesis in cancer cells. The TCA-cycle itself is predominantly fueled through carbons from glucose, glutamine, fatty acids and lactate. Targeting mitochondrial energy metabolism appears feasible through several drug compounds that activate the CLPP protein or interfere with NADH-dehydrogenase, pyruvate-dehydrogenase, enzymes of the TCA-cycle and mitochondrial matrix chaperones. While these compounds have demonstrated anti-cancer effects in vivo, recent research suggests which patients most likely benefit from such treatments. Here, we provide a brief overview of the status quo of targeting mitochondrial energy metabolism in glioblastoma and highlight a novel combination therapy.
... Cellular mesenchymal-epithelial transition factor (c-Met), an oncogenic transmembrane receptor tyrosine kinase (RTK), plays an essential function in invasive growth during embryo development and liver regeneration [1,2]. c-Met signaling pathway has been implicated as a critical regulator for maintaining intracellular redox homeostasis and oxidative stress [3]. ...
... (3-(5-Fluoropyrimidin-2-yl)phenyl)methanol (12) White solid, yield 55% (1.12 g) from compound 10 (10 mmol, 1.32 g). 1 ...
... 3-(1-(3-(5-Fluoropyrimidin-2-yl)benzyl)-6-oxo-1,6-dihydropyridazin-3-yl)benzonitrile (15) Yellow solid, yield 68% (1.30 g) from compound 12 (5 mmol, 1.02 g). 1 ...
Cellular mesenchymal–epithelial transition factor (c-Met), an oncogenic transmembrane receptor tyrosine kinase (RTK), plays an essential role in cell proliferation during embryo development and liver regeneration. Thioredoxin reductase (TrxR) is overexpressed and constitutively active in most tumors closely related to cancer recurrence. Multi-target-directed ligands (MTDLs) strategy provides a logical approach to drug combinations and would adequately address the pathological complexity of cancer. In this work, we designed and synthesized a series of selenium-containing tepotinib derivatives by means of selenium-based bioisosteric modifications and evaluated their antiproliferative activity. Most of these selenium-containing hybrids exhibited potent dual inhibitory activity toward c-Met and TrxR. Among them, compound 8b was the most active, with an IC50 value of 10 nM against MHCC97H cells. Studies on the mechanism of action revealed that compound 8b triggered cell cycle arrest at the G1 phase and caused ROS accumulations by targeting TrxR, and these effects eventually led to cell apoptosis. These findings strongly suggest that compound 8b serves as a dual inhibitor of c-Met and TrxR, warranting further exploitation for cancer therapy.
... BRAF inhibitors reduce tumor glycolysis while inducing mitochondrial oxidative phosphorylation (OXPHOS) by triggering the expression of a mitochondrial biogenesis gene signature, thereby conferring resistance of BRAF V600E -mutant melanoma to BRAF inhibitors 22,23 . MET inhibitors enhance mitochondrial OXPHOS and fatty acid oxidation (FAO) in glioblastoma, resulting in acylcarnitine accumulation and reduced therapeutic efficacy 24 . This evidence that drugresistant tumor cells rely more on mitochondrial OXPHOS and less on glycolysis challenges the contention that tumor cells are usually characterized by the Warburg effect, that is, the production of ATP mostly from glycolysis and not oxidative phosphorylation, even under conditions of high oxygen availability. ...
... Notably, OXPHOS activation was a determinant of KRAS-mutant NSCLC resistance to MEK inhibition, as targeted inhibition of OXPHOS significantly augmented trametinib sensitivity in both primary and acquired resistance tumor models in vitro and in vivo. Our findings and those of recently published studies 21,24 , support the notion that the mechanisms underlying metabolic plasticity may represent a more attractive therapeutic target for circumventing drug resistance. ...
MEK is a canonical effector of mutant KRAS; however, MEK inhibitors fail to yield satisfactory clinical outcomes in KRAS-mutant cancers. Here, we identified mitochondrial oxidative phosphorylation (OXPHOS) induction as a profound metabolic alteration to confer KRAS-mutant non-small cell lung cancer (NSCLC) resistance to the clinical MEK inhibitor trametinib. Metabolic flux analysis demonstrated that pyruvate metabolism and fatty acid oxidation were markedly enhanced and coordinately powered the OXPHOS system in resistant cells after trametinib treatment, satisfying their energy demand and protecting them from apoptosis. As molecular events in this process, the pyruvate dehydrogenase complex (PDHc) and carnitine palmitoyl transferase IA (CPTIA), two rate-limiting enzymes that control the metabolic flux of pyruvate and palmitic acid to mitochondrial respiration were activated through phosphorylation and transcriptional regulation. Importantly, the co-administration of trametinib and IACS-010759, a clinical mitochondrial complex I inhibitor that blocks OXPHOS, significantly impeded tumor growth and prolonged mouse survival. Overall, our findings reveal that MEKi therapy creates a metabolic vulnerability in the mitochondria and further develop an effective combinatorial strategy to circumvent MEKi resistance in KRAS-driven NSCLC.
... Indeed, when we inhibit oxidative phosphorylation by targeting IDH2, resistance is impaired, providing more direct evidence that this metabolic switch is important for cancer development. These findings are consistent with studies in other cancer types where PPARGC1A-driven programmes play an important role in drug resistance in melanomas [50] and glioblastoma [51]. In both cases, PPARGC1A-dependent metabolic reprogramming is elicited following inhibition of either a RTK (MET) or a downstream pathway component (BRaf), demonstrating a common response to RTK pathway inhibition in rewiring metabolic programmes in OAC and these cancers. ...
Oesophageal adenocarcinoma (OAC) patients show poor survival rates and there are few targeted molecular therapies available. However, components of the receptor tyrosine kinase (RTK) driven pathways are commonly mutated in OAC, typified by high frequency amplifications of the RTK ERBB2. ERBB2 can be therapeutically targeted, but this has limited clinical benefit due to the acquisition of drug resistance. Here we examined how OAC cells adapt to ERBB2 inhibition as they transition to a drug resistant state. ERBB2 inhibition triggers widespread remodelling of the accessible chromatin landscape and the underlying gene regulatory networks. The transcriptional regulators HNF4A and PPARGC1A play a key role in this network rewiring. Initially, inhibition of cell cycle associated gene expression programmes is observed, with compensatory increases in the programmes driving changes in metabolic activity. Both PPARGC1A and HNF4A are required for the acquisition of resistance to ERBB2 inhibition and PPARGC1A is instrumental in promoting a switch to dependency on oxidative phosphorylation. Our work therefore reveals the molecular pathways that support the acquisition of a resistant state and points to potential new therapeutic strategies to combat cellular adaptation and ensuing drug resistance.
... A further example of an increase of OXPHOS and fatty acid oxidation is represented by inhibition of the MET kinase (MET proto-oncogene, receptor tyrosine kinase). The MET gene is amplified in a fraction of GBMs and therefore this protein constitutes a viable target [77]. However, as in other contexts, interference with MET was not durable in GBM, raising the question of how resistance could occur so rapidly. ...
... One possible explanation may be metabolic reprogramming following treatment with the MET inhibitor, crizotinib (Table 1). Indeed, crizotinib treatment led to an up-regulation of acyl-carnitines and a transcriptional signature of fatty acid oxidation [77]. Carbon tracing experiments and extracellular flux analysis confirmed the increase of oxidative energy metabolism fueled in part by palmitic acid [77]. ...
... Indeed, crizotinib treatment led to an up-regulation of acyl-carnitines and a transcriptional signature of fatty acid oxidation [77]. Carbon tracing experiments and extracellular flux analysis confirmed the increase of oxidative energy metabolism fueled in part by palmitic acid [77]. In the context of crizotinib, it was noted that glucose was critical to provide carbons to the TCA cycle such that citrate was substantially more labeled by glucose carbons following crizotinib exposure. ...
Glioblastoma WHO IV (GBM), the most common primary brain tumor in adults, is a heterogenous malignancy that displays a reprogrammed metabolism with various fuel sources at its disposal. Tumor cells primarily appear to consume glucose to entertain their anabolic and catabolic metabolism. While less effective for energy production, aerobic glycolysis (Warburg effect) is an effective means to drive biosynthesis of critical molecules required for relentless growth and resistance to cell death. Targeting the Warburg effect may be an effective venue for cancer treatment. However, past and recent evidence highlight that this approach may be limited in scope because GBM cells possess metabolic plasticity that allows them to harness other substrates, which include but are not limited to, fatty acids, amino acids, lactate, and acetate. Here, we review recent key findings in the literature that highlight that GBM cells substantially reprogram their metabolism upon therapy. These studies suggest that blocking glycolysis will yield a concomitant reactivation of oxidative energy pathways and most dominantly beta-oxidation of fatty acids.
... Kang and colleagues (14) first described the efficacy of Gamitrinib in a GBM cell line U87MG. Subsequent studies reported the therapeutic efficacy of Gamitrinib in other GBM cell lines, including T98G, LN229, U251MG, and A172 (9,10,20,21). Unfortunately, studies that systematically investigate the effect of Gamitrinib are lacking and the antitumor mechanisms in gliomas are still mostly unknown. ...
... Gamitrinib was first described in 2009 and has shown the antitumor activity in preclinical models for multiple cancers, including prostate cancer, melanoma, lung cancer, colon cancer, breast cancer, and GBM (9,10,(16)(17)(18)21). Among these studies, Chae and colleagues (9) investigated the antitumor activity in breast, prostate, lung, and brain tumors, and demonstrated that mitochondrial, but not cytosolic, HSP90s is a global integrator of tumor bioenergetics, autophagy, and interorganelle stress-response signaling (10). ...
Purpose:
To investigate the anti-tumor activity of a mitochondrial-localized HSP90 inhibitor, Gamitrinib, in multiple glioma models, and to elucidate the anti-tumor mechanisms of Gamitrinib in gliomas.
Experimental design:
A broad panel of primary and temozolomide (TMZ)-resistant human glioma cell lines were screened by cell viability assays, flow cytometry and crystal violet assays to investigate the therapeutic efficacy of Gamitrinib. Seahorse assays were used to measure the mitochondrial respiration of glioma cells. Integrated analyses of RNA sequencing (RNAseq) and reverse phase protein array (RPPA) data were performed to reveal the potential anti-tumor mechanisms of Gamitrinib. Neurospheres, patient-derived organoids (PDOs), cell line-derived xenografts (CDX) and patient-derived xenografts (PDX) models were generated to further evaluate the therapeutic efficacy of Gamitrinib.
Results:
Gamitrinib inhibited cell proliferation and induced cell apoptosis and death in 17 primary glioma cells, 6 TMZ-resistant glioma cells, 4 neurospheres and 3 PDOs. Importantly, Gamitrinib significantly delayed the tumor growth and improved survival of mice in both CDX and PDX models in which tumors were either subcutaneously or intracranially implanted. Integrated computational analyses of RNAseq and RPPA data revealed that Gamitrinib exhibited its anti-tumor activity via (1) suppressing mitochondrial biogenesis, OXPHOS and cell cycle progression and (2) activating the energy-sensing AMP-activated kinase, DNA damage and stress response.
Conclusions:
These preclinical findings established the therapeutic role of Gamitrinib in gliomas and revealed the inhibition of mitochondrial biogenesis and tumor bioenergetics as the primary anti-tumor mechanisms in gliomas.
