Structure-Guided Discovery and Preclinical Assessment of Novel (Thiophen-3-yl)aminopyrimidine Derivatives as Potent ERK1/2 Inhibitors.

The RAS-RAF-MEK-ERK signaling cascade is abnormally activated in various tumors, playing a crucial role in mediating tumor progression. As the key component at the terminal stage of this cascade, ERK1/2 emerges as a potential antitumor target and offers a promising therapeutic strategy for tumors harboring BRAF or RAS mutations. Here, we identified 36c with a (thiophen-3-yl)aminopyrimidine scaffold as a potent ERK1/2 inhibitor through structure-guided optimization for hit 18. In preclinical studies, 36c showed powerful ERK1/2 inhibitory activities (ERK1/2 IC50 = 0.11/0.08 nM) and potent antitumor efficacy both in vitro and in vivo against triple-negative breast cancer and colorectal cancer models harboring BRAF and RAS mutations. 36c could directly inhibit ERK1/2, significantly block the phosphorylation expression of their downstream substrates p90RSK and c-Myc, and induce cell apoptosis and incomplete autophagy-related cell death. Taken together, this work provides a promising ERK1/2 lead compound for multiple tumor-treatment drug discovery.

Antitumor activity of extracellular signal-regulated kinases 1/2 inhibitor BVD-523 (ulixertinib) on thyroid cancer cells.

OBJECTIVE: This study aimed to investigate BVD-523 (ulixertinib), an adenosine triphosphate (ATP)-dependent extracellular signal-regulated kinases 1/2 inhibitor, for its antitumor potential in thyroid cancer. MATERIALS AND METHODS: Ten thyroid cancer cell lines known to carry mitogen-activated protein kinase (MAPK)-activated mutations, including v-Raf murine sarcoma viral oncogene homolog B (BRAF) and rat sarcoma virus (RAS) mutations, were examined. Cells were exposed to a 10-fold concentration gradient ranging from 0 to 3000 nM for 5 days. The half-inhibitory concentration was determined using the Cell Counting Kit-8 assay. Following BVD-523 treatment, cell cycle analysis was conducted using flow cytometry. In addition, the impact of BVD-523 on extracellular signal-regulated kinase (ERK)- dependent ribosomal S6 kinase (RSK) activation and the expression of cell cycle markers were assessed through western blot analysis. RESULTS: BVD-523 significantly inhibited thyroid cancer cell proliferation and induced G1/S cell cycle arrest dose-dependently. Notably, cell lines carrying MAPK mutations, especially those with the BRAF V600E mutation, exhibited heightened sensitivity to BVD-523's antitumor effects. Furthermore, BVD-523 suppressed cyclin D1 and phosphorylated retinoblastoma protein expression, and it robustly increased p27 levels in an RSK-independent manner. CONCLUSION: This study reveals the potent antitumor activity of BVD-523 against thyroid cancer cells bearing MAPK-activating mutations, offering promise for treating aggressive forms of thyroid cancer.

ML241 Antagonizes ERK 1/2 Activation and Inhibits Rotavirus Proliferation.

Rotavirus (RV) is the main pathogen that causes severe diarrhea in infants and children under 5 years of age. No specific antiviral therapies or licensed anti-rotavirus drugs are available. It is crucial to develop effective and low-toxicity anti-rotavirus small-molecule drugs that act on novel host targets. In this study, a new anti-rotavirus compound was selected by ELISA, and cell activity was detected from 453 small-molecule compounds. The anti-RV effects and underlying mechanisms of the screened compounds were explored. In vitro experimental results showed that the small-molecule compound ML241 has a good effect on inhibiting rotavirus proliferation and has low cytotoxicity during the virus adsorption, cell entry, and replication stages. In addition to its in vitro effects, ML241 also exerted anti-RV effects in a suckling mouse model. Transcriptome sequencing was performed after adding ML241 to cells infected with RV. The results showed that ML241 inhibited the phosphorylation of ERK1/2 in the MAPK signaling pathway, thereby inhibiting IκBα, activating the NF-κB signaling pathway, and playing an anti-RV role. These results provide an experimental basis for specific anti-RV small-molecule compounds or compound combinations, which is beneficial for the development of anti-RV drugs.

X-ray Screening of an Electrophilic Fragment Library and Application toward the Development of a Novel ERK 1/2 Covalent Inhibitor.

Fragment-based drug discovery (FBDD) has become an established method for the identification of efficient starting points for drug discovery programs. In recent years, electrophilic fragment screening has garnered increased attention from both academia and industry to identify novel covalent hits for tool compound or drug development against challenging drug targets. Herein, we describe the design and characterization of an acrylamide-focused electrophilic fragment library and screening campaign against extracellular signal-regulated kinase 2 (ERK2) using high-throughput protein crystallography as the primary hit-finding technology. Several fragments were found to have covalently modified the adenosine triphosphate (ATP) binding pocket Cys166 residue. From these hits, 22, a covalent ATP-competitive inhibitor with improved potency (ERK2 IC50 = 7.8 µM), was developed.

Identification of a novel target site for ATP-independent ERK2 inhibitors.

Extracellular signal-regulated kinase 2 (ERK2) controls vital physiological processes involving proliferation and differentiation and is a drug target molecule for many diseases such as cancers. In silico screening focusing on an allosteric site that plays a crucial role in substrate anchoring conferred an ERK2 inhibitor (compound 1). However, a competitive binding assay indicated that compound 1 did not bind to the allosteric site. Here, the crystal structure of ERK2 in complex with compound 1 revealed a novel binding site. This finding demonstrates the feasibility of developing new types of ERK2 inhibitors.

Swine acute diarrhea syndrome coronavirus replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered enteric coronavirus. We have previously shown that the caspase-dependent FASL-mediated and mitochondrion-mediated apoptotic pathways play a central role in SADS-CoV-induced apoptosis, which facilitates viral replication. However, the roles of intracellular signaling pathways in SADS-CoV-mediated cell apoptosis and the relative advantages that such pathways confer on the host or virus remain largely unknown. In this study, we show that SADS-CoV induces the activation of ERK during infection, irrespective of viral biosynthesis. The knockdown or chemical inhibition of ERK1/2 significantly suppressed viral protein expression and viral progeny production. The inhibition of ERK activation also circumvented SADS-CoV-induced apoptosis. Taken together, these data suggest that ERK activation is important for SADS-CoV replication, and contributes to the virus-mediated changes in host cells. Our findings demonstrate the takeover of a particular host signaling mechanism by SADS-CoV and identify a potential approach to inhibiting viral spread.

USP19 exacerbates lipogenesis and colorectal carcinogenesis by stabilizing ME1.

Lipogenesis plays a critical role in colorectal carcinogenesis, but precisely how remains unclear. Here, we show that ERK2 phosphorylates ME1 at T103, thereby inhibiting its polyubiquitination and proteasomal degradation and enhancing its interaction with USP19. USP19 antagonizes RNF1-mediated ME1 degradation by deubiquitination, which in turn promotes lipid metabolism and NADPH production and suppresses ROS. Meanwhile, ROS dramatically increases PD-L1 mRNA levels through accelerating expression of the transcription factor NRF2. Increased lipid metabolism is correlated with ERK2 activity and colorectal carcinogenesis in human patients. Therefore, the combination of ERK2 inhibitor and anti-PD-L1 antibody significantly inhibits spontaneous and chemically induced colorectal carcinogenesis. Collectively, the USP19-ME1 axis plays a vital role in colorectal carcinogenesis and may also provide a potential therapeutic target.

Carbon dioxide inhibits COVID-19-type proinflammatory responses through extracellular signal-regulated kinases 1 and 2, novel carbon dioxide sensors.

Mitogen-activated protein kinase (MAPK) signalling pathways are crucial for developmental processes, oncogenesis, and inflammation, including the production of proinflammatory cytokines caused by reactive oxygen species and upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. There are no drugs that can effectively prevent excessive inflammatory responses in endothelial cells in the lungs, heart, brain, and kidneys, which are considered the main causes of severe coronavirus disease 2019 (COVID-19). In this work, we demonstrate that human MAPKs, i.e. extracellular signal-regulated kinases 1 and 2 (ERK1/2), are CO2 sensors and CO2 is an efficient anti-inflammatory compound that exerts its effects through inactivating ERK1/2 in cultured endothelial cells when the CO2 concentration is elevated. CO2 is a potent inhibitor of cellular proinflammatory responses caused by H2O2 or the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. ERK1/2 activated by the combined action of RBD and cytokines crucial for the development of severe COVID-19, i.e. interferon-gamma (IFNγ) and tumour necrosis factor-α (TNFα), are more effectively inactivated by CO2 than by dexamethasone or acetylsalicylic acid in human bronchial epithelial cells. Previously, many preclinical and clinical studies showed that the transient application of 5-8% CO2 is safe and effective in the treatment of many diseases. Therefore, our research indicates that CO2 may be used for the treatment of COVID-19 as well as the modification of hundreds of cellular pathways.

Lithium chloride inhibits the migration and invasion of osteosarcoma cells by blocking nuclear translocation of phospho-Erk.

Lithium chloride (LiCl) is an important mood-stabilizing therapeutic agent for bipolar disorders, which has also been shown to inhibit cancer cell metastasis. Investigations of LiCl-induced signaling have focused mainly on extracellular signal regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3 (GSK-3). However, little is known about the differences in cellular activities resulting from specific signaling via each of these pathways. In this study, we investigated the difference in responses between the Wnt/ß-catenin and ERK pathways by LiCl or epidermal growth factor (EGF) treatment of osteosarcoma cells. In particular, we analyzed the mechanisms responsible for differences in cell mobility and cell proliferation when pERK or ß-catenin is activated. In osteosarcoma cells treated with LiCl or EGF, active ß-catenin and p-ERK protein levels were significantly increased compared to those in the control group. However, in wound healing and transwell invasion assays, U2OS and SaOS2 cell migration was significantly reduced by LiCl treatment but increased by EGF treatment. In addition, the proliferation of U2OS cells was reduced by LiCl treatment but increased by EGF treatment. Using immunofluorescence microscopy, we observed nuclear accumulation of phosphorylated ERK (pERK) with EGF treatment, but pERK was restricted to the perinuclear area with LiCl treatment. These results were confirmed using immunoblot assays after subcellular fractionation. Together, these data suggest that LiCl interferes with the translocation of pERK from the cytoplasm to the nucleus.

Standardized Extract of Asparagus officinalis Stem Attenuates SARS-CoV-2 Spike Protein-Induced IL-6 and IL-1ß Production by Suppressing p44/42 MAPK and Akt Phosphorylation in Murine Primary Macrophages.

Excessive host inflammation following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with severity and mortality in coronavirus disease 2019 (COVID-19). We recently reported that the SARS-CoV-2 spike protein S1 subunit (S1) induces pro-inflammatory responses by activating toll-like receptor 4 (TLR4) signaling in macrophages. A standardized extract of Asparagus officinalis stem (EAS) is a unique functional food that elicits anti-photoaging effects by suppressing pro-inflammatory signaling in hydrogen peroxide and ultraviolet B-exposed skin fibroblasts. To elucidate its potential in preventing excessive inflammation in COVID-19, we examined the effects of EAS on pro-inflammatory responses in S1-stimulated macrophages. Murine peritoneal exudate macrophages were co-treated with EAS and S1. Concentrations and mRNA levels of pro-inflammatory cytokines were assessed using enzyme-linked immunosorbent assay and reverse transcription and real-time polymerase chain reaction, respectively. Expression and phosphorylation levels of signaling proteins were analyzed using western blotting and fluorescence immunomicroscopy. EAS significantly attenuated S1-induced secretion of interleukin (IL)-6 in a concentration-dependent manner without reducing cell viability. EAS also markedly suppressed the S1-induced transcription of IL-6 and IL-1ß. However, among the TLR4 signaling proteins, EAS did not affect the degradation of inhibitor κBα, nuclear translocation of nuclear factor-κB p65 subunit, and phosphorylation of c-Jun N-terminal kinase p54 subunit after S1 exposure. In contrast, EAS significantly suppressed S1-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and Akt. Attenuation of S1-induced transcription of IL-6 and IL-1ß by the MAPK kinase inhibitor U0126 was greater than that by the Akt inhibitor perifosine, and the effects were potentiated by simultaneous treatment with both inhibitors. These results suggest that EAS attenuates S1-induced IL-6 and IL-1ß production by suppressing p44/42 MAPK and Akt signaling in macrophages. Therefore, EAS may be beneficial in regulating excessive inflammation in patients with COVID-19.

Role of MAPK ERK1/2 and p38 in the Regulation of Secretory Functions of Different Populations of Neuroglia in Ethanol-Induced Neurodegeneration.

We studied the participation of ERK1/2 and p38 in secretion of neurotrophic growth factors by various types of neuroglia under conditions of in vitro and in vivo modeled ethanol-induced neurodegeneration. The inhibitory role of these protein kinases in the production of neurotrophins by intact astrocytes and the absence of their participation in the regulation of functions of oligodendrocytes and microglial cells were shown. Under conditions of ethanol neurotoxicity, the role of ERK1/2 and p38 in the production of growth factors by glial elements was significantly changed. Neurodegeneration modeled in vitro led to inversion of the role of both protein kinases in the secretion of neurotrophins by astroglia and inhibition of the cytokine-synthesizing function of oligodendrocytes and microglial cells by ERK1/2 and p38. In mice receiving ethanol per os for a long time (as well as in cells in vitro exposed to ethanol), mitogen-activated kinases stimulated the function of astrocytes and inhibited the production of growth factors by microglial cells. At the same time, chronic alcoholization was accompanied by the appearance of the stimulating role of ERK1/2 and p38 in the implementation of the secretory function by oligodendrocytes.

Ritanserin, a potent serotonin 2A receptor antagonist, represses MEK/ERK signalling pathway to restore PAX6 production and function in aniridia-like cellular model.

Aniridia is a panocular inherited rare eye disease linked to heterozygous mutations on the PAX6 gene, which fail to properly produce sufficient protein essential for normal eye development and function. Most of the patients suffer from aniridia-related keratopathy, a progressive opacification of the cornea. There is no effective treatment for this blinding disease. Here we screen for small compounds and identified Ritanserin, a serotonin 2A receptor antagonist, that can rescue PAX6 haploinsufficiency of mutant limbal cells, defective cell migration and PAX6-target gene expression. We further demonstrated that Ritanserin activates PAX6 production through the selective inactivation of the MEK/ERK signaling pathway. Our data strongly suggest that repurposing this therapeutic molecule could be effective in preventing or treating existing blindness by restoring corneal transparency.

Structure based designing of thiazolidinone-pyrimidine derivatives as ERK2 inhibitors: Synthesis and in vitro evaluation.

Breast cancer has been associated with an overexpression of various molecular targets; accordingly, various target-specific chemotherapeutic agents have been developed. Inhibition of ERK2, a member of MAPK pathway, is an important target involved in the treatment of both oestrogen receptor-positive and triple-negative breast cancer. Thus, in continuation of our previous work on the ERK2 target, we here report novel inhibitors of this kinase. Out of three lead molecules reported in our previous study, we selected the thiazolidinone-pyrimidine scaffold for further development of small molecule inhibitors of ERK2. Analogues of the lead molecule were docked in the target kinase, followed by molecular dynamic simulations and MM-GBSA calculations. Analogues maintaining key interactions with amino acid residues in the ATP-binding domain of ERK2 were selected and duly synthesized. In vitro biochemical evaluation of these molecules against ERK2 kinase disclosed that two molecules possess significant kinase inhibitory potential with IC50 values ≤ 0.5 µM.

Dual targeting of JAK2 and ERK interferes with the myeloproliferative neoplasm clone and enhances therapeutic efficacy.

Myeloproliferative neoplasms (MPN) show dysregulated JAK2 signaling. JAK2 inhibitors provide clinical benefits, but compensatory activation of MAPK pathway signaling impedes efficacy. We hypothesized that dual targeting of JAK2 and ERK1/2 could enhance clone control and therapeutic efficacy. We employed genetic and pharmacologic targeting of ERK1/2 in Jak2V617F MPN mice, cells and patient clinical isolates. Competitive transplantations of Jak2V617F vs. wild-type bone marrow (BM) showed that ERK1/2 deficiency in hematopoiesis mitigated MPN features and reduced the Jak2V617F clone in blood and hematopoietic progenitor compartments. ERK1/2 ablation combined with JAK2 inhibition suppressed MAPK transcriptional programs, normalized cytoses and promoted clone control suggesting dual JAK2/ERK1/2 targeting as enhanced corrective approach. Combined pharmacologic JAK2/ERK1/2 inhibition with ruxolitinib and ERK inhibitors reduced proliferation of Jak2V617F cells and corrected erythrocytosis and splenomegaly of Jak2V617F MPN mice. Longer-term treatment was able to induce clone reductions. BM fibrosis was significantly decreased in MPLW515L-driven MPN to an extent not seen with JAK2 inhibitor monotherapy. Colony formation from JAK2V617F patients' CD34+ blood and BM was dose-dependently inhibited by combined JAK2/ERK1/2 inhibition in PV, ET, and MF subsets. Overall, we observed that dual targeting of JAK2 and ERK1/2 was able to enhance therapeutic efficacy suggesting a novel treatment approach for MPN.

Discovery of ASTX029, A Clinical Candidate Which Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (15) through structure-guided optimization of our previously published isoindolinone lead (7). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.

Escin Sodium Improves the Prognosis of Acute Pancreatitis via Promoting Cell Apoptosis by Suppression of the ERK/STAT3 Signaling Pathway.

Acute pancreatitis (AP), an inflammatory disorder of the pancreas, can cause systemic inflammatory responses. Escin Sodium (ES), a natural mixture of triterpene saponins extracted from the dry ripe fruit of Fructus Aesculi or horse chestnut crude, has been demonstrated to have antiedematous, anti-inflammatory, and antiexudative effects. We here aim to investigate the effects of ES pretreatment on AP in vivo and in vitro and explore its potential molecular mechanism. In the present study, we demonstrated that ES pretreatment could apparently decrease amylase and lipase, downregulate inflammatory cytokines, and attenuate pancreatic damage. Additionally, the increased expression of apoptotic-related proteins and the results of flow cytometry demonstrated the effects of ES on promoting apoptosis in acinar cells. Moreover, ES could enhance mitochondrial membrane potential (MMP, ΔΨm) and reactive oxygen species (ROS) level and reduce intracellular calcium concentration, which are closely related to mitochondrial-mediated death. The effect of ES pretreatment on acinar cell apoptosis was furtherly confirmed by the regulatory pathway of the ERK/STAT3 axis. These results suggest that ES attenuates the severity of AP by enhancing cell apoptosis via suppressing the ERK/STAT3 signaling pathway. These findings provide evidence for ES which is treated as a novel and potent therapeutic for the treatment of AP.

Diminished PLK2 Induces Cardiac Fibrosis and Promotes Atrial Fibrillation.

[Figure: see text].

Mesenchymal stem cell-derived exosomal miR-21a-5p promotes M2 macrophage polarization and reduces macrophage infiltration to attenuate atherosclerosis.

Atherosclerosis (AS) is the main pathological basis for ischemic cardiovascular and cerebrovascular diseases. Mesenchymal stem cell (MSC)-derived exosomes have the potential to alleviate AS, while the underlying mechanism remains unclear. Here, we aimed to investigate the mechanism of MSC-derived exosomes in AS. The AS mouse model was prepared by feeding ApoE-/- mice with high-fat diet. AS mice were administered with MSC-derived exosomes, and the atherosclerotic plaque area was analyzed by Oil Red O staining. Mouse RAW264.7 macrophages were incubated with MSC-derived exosomes. The macrophage infiltration, macrophage proportion, and cell migration were estimated by immunohistochemistry, flow cytometry, or Transwell assay. The relationship between miR-21a-5p and kruppel-like factor 6 (KLF6) or extracellular signal-regulated protein kinases 2 (ERK2) was verified by luciferase reporter assay. We found that MSC-derived exosomes promoted M2 polarization of macrophages and reduced plaque area and macrophage infiltration in AS mice. miR-21a-5p overexpression caused an increase of M2 macrophages in RAW264.7 cells and led to a decrease in migration of RAW264.7 cells. Moreover, both KLF6 and ERK2 are the targets of miR-21a-5p. MSC-derived exosomes containing miR-21a-5p promoted M2 polarization of RAW264.7 cells by suppressing KLF6 expression. MSC-derived exosomes containing miR-21a-5p inhibited migration of RAW264.7 cells through inhibiting the ERK1/2 signaling pathway. In conclusion, MSC-derived exosomes containing miR-21a-5p promote macrophage polarization and reduce macrophage infiltration by targeting KLF6 and ERK1/2 signaling pathways, thereby attenuating the development of AS. Thus, MSC-derived exosomes may be a promising treatment for AS.

Betulin, a Newly Characterized Compound in Acacia auriculiformis Bark, Is a Multi-Target Protein Kinase Inhibitor.

The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/ß (GSK-3 α/ß), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.

Combined intermittent fasting and ERK inhibition enhance the anti-tumor effects of chemotherapy via the GSK3ß-SIRT7 axis.

Dietary interventions such as intermittent fasting (IF) have emerged as an attractive strategy for cancer therapies; therefore, understanding the underlying molecular mechanisms is pivotal. Here, we find SIRT7 decline markedly attenuates the anti-tumor effect of IF. Mechanistically, AMP-activated protein kinase (AMPK) phosphorylating SIRT7 at T263 triggers further phosphorylation at T255/S259 by glycogen synthase kinase 3ß (GSK3ß), which stabilizes SIRT7 by decoupling E3 ligase UBR5. SIRT7 hyperphosphorylation achieves anti-tumor activity by disrupting the SKP2-SCF E3 ligase, thus preventing SKP2-mediated K63-linked AKT polyubiquitination and subsequent activation. In contrast, GSK3ß-SIRT7 axis is inhibited by EGF/ERK2 signaling, with ERK2 inactivating GSK3ß, thus accelerating SIRT7 degradation. Unfavorably, glucose deprivation or chemotherapy hijacks the GSK3ß-SIRT7 axis via ERK2, thus activating AKT and ensuring survival. Notably, Trametinib, an FDA-approved MEK inhibitor, enhances the efficacy of combination therapy with doxorubicin and IF. Overall, we have revealed the GSK3ß-SIRT7 axis that must be fine-tuned in the face of the energetic and oncogenic stresses in malignancy.