Reprogramming Of Cellular Metabolism Research Articles

Abstract LB125: The role of succinate dehydrogenase in ovarian cancer metabolism

Abstract The reprogramming of cellular metabolism is an important hallmark of cancer allowing tumor cells to survive and proliferate. Recent data revealed that subsets of ovarian cancer with invasive and metastatic phenotype are highly metabolically active with increased mitochondrial respiration. To better understand the metabolic profiles of ovarian cancer, we utilized patient-derived xenografts (PDXs) derived from high-grade serous ovarian carcinoma (HGSOC), and performed comprehensive analysis of metabolic pathways by Reverse Phase Protein Array (RPPA). The RPPA analysis revealed PDX subsets with high or low mitochondrial activity and metabolism, and we identified succinate dehydrogenase (SDHA) to be a top differentially expressed metabolic protein between those PDX subsets. SDHA is a mitochondrial enzyme that participates in the TCA cycle and the electron transport chain, which are both essential for cellular metabolism. Analysis of the TCGA data demonstrated that SDHA gene alterations are highly prevalent in HGSOC patients (19% cases). Despite these findings, the impact of SDHA amplification on cancer biology is poorly understood. To assess the effect of SDHA overexpression on energy metabolism, we used SDHA-overexpressing ovarian cancer cell lines and measured cellular respiration by Seahorse. The results revealed that the overexpression of SDHA leads to a significant increase in basal and maximal respiration shifting cellular metabolism towards increased oxidative phosphorylation in mitochondria. Further, we determined if SDHA overexpression redirects cellular metabolism in response to deprivation of selected nutrients. In the conditions with low glucose or glutamine, SDHA overexpressing cells significantly increased basal respiration when compared with controls. In galactose medium that disrupts glycolysis, the SDHA lost its ability to induce cellular respiration, which indicates that SDHA relays on glycolysis to increase mitochondrial respiration. Together, our data indicate that SDHA requires glycolysis to fuel TCA cycle and and support cellular respiration. Next, we performed in vitro drug screening and found that SDHA overexpressing cells are highly sensitive to glycolysis inhibitor Shikonin. Our pilot in vivo data showed that Shikonin significantly reduces growth of SDHA-amplified tumors. In summary, our studies indicate that SDHA upregulation plays a role in reprogramming of cellular metabolism and pathogenesis of ovarian cancer. Moreover, the unique metabolic state of ovarian cancer associated with SDHA amplification could be successfully targetable offering a potential new treatment strategy for ovarian cancer patients. Citation Format: Magdalena Cybula, Maria Rostworowska, Lin Wang, Summer Wang, Ana Luiza Drumond-Bock, Magdalena Bieniasz. The role of succinate dehydrogenase in ovarian cancer metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB125.

TRAP1 Chaperones the Metabolic Switch in Cancer.

Mitochondrial function is dependent on molecular chaperones, primarily due to their necessity in the formation of respiratory complexes and clearance of misfolded proteins. Heat shock proteins (Hsps) are a subset of molecular chaperones that function in all subcellular compartments, both constitutively and in response to stress. The Hsp90 chaperone TNF-receptor-associated protein-1 (TRAP1) is primarily localized to the mitochondria and controls both cellular metabolic reprogramming and mitochondrial apoptosis. TRAP1 upregulation facilitates the growth and progression of many cancers by promoting glycolytic metabolism and antagonizing the mitochondrial permeability transition that precedes multiple cell death pathways. TRAP1 attenuation induces apoptosis in cellular models of cancer, identifying TRAP1 as a potential therapeutic target in cancer. Similar to cytosolic Hsp90 proteins, TRAP1 is also subject to post-translational modifications (PTM) that regulate its function and mediate its impact on downstream effectors, or ‘clients’. However, few effectors have been identified to date. Here, we will discuss the consequence of TRAP1 deregulation in cancer and the impact of post-translational modification on the known functions of TRAP1.

Regulation of Treg Cell Metabolism and Function in Non-Lymphoid Tissues.

Regulator T cells (Tregs) play pivotal roles in maintaining immune tolerance and regulating immune responses against pathogens and tumors. Reprogramming of cellular metabolism has been determined as a crucial process that connects microenvironmental cues and signaling networks to influence homeostasis and function of tissue Tregs. In adaptation to a variety of non-lymphoid tissues, Tregs coordinate local immune signals and signaling networks to rewire cellular metabolic programs to sustain their suppressive function. Altered Treg metabolism in turn shapes Treg activation and function. In light of the advanced understanding of immunometabolism, manipulation of systemic metabolites has been emerging as an attractive strategy aiming to modulate metabolism and function of tissue Tregs and improve the treatment of immune-related diseases. In this review, we summarize key immune signals and metabolic programs involved in the regulation of tissue Tregs, review the mechanisms underlying the differentiation and function of Tregs in various non-lymphoid tissues, and discuss therapeutic intervention of metabolic modulators of tissue Tregs for the treatment of autoimmune diseases and cancer.

Connective Tissue Growth Factor in Idiopathic Pulmonary Fibrosis: Breaking the Bridge.

CTGF is upregulated in patients with idiopathic pulmonary fibrosis (IPF), characterized by the deposition of a pathological extracellular matrix (ECM). Additionally, many omics studies confirmed that aberrant cellular senescence-associated mitochondria dysfunction and metabolic reprogramming had been identified in different IPF lung cells (alveolar epithelial cells, alveolar endothelial cells, fibroblasts, and macrophages). Here, we reviewed the role of the CTGF in IPF lung cells to mediate anomalous senescence-related metabolic mechanisms that support the fibrotic environment in IPF.

DDR1 promotes LoVo cell proliferation by regulating energy metabolism.

Cellular energy metabolism dysregulation is associated with colorectal cancer (CRC) development and progression. Discoidin domain receptor 1a (DDR1a), one of the five DDR1 isoforms, is closely related to cell proliferation, invasion, and apoptosis in various tumors. Whether it participates in cellular metabolic reprogramming and regulates CRC initiation and progression remains unclear. In this study, we compared the expression of DDR1 in CRC tissues and adjacent tissues from 126 postoperative CRC samples. Moreover, lentivirus-mediated DDR1a overexpression and knockdown were performed in LoVo cells, and cell viability and proliferation were determined by CCK-8 and BrdU assays, respectively. Oxygen consumption rate, extracellular acidification rate, and lactate production were used to determine the effect of DDR1a on metabolic reprogramming. Clinically, CRC patients with high DDR1 expression had poor differentiation and were at an advanced TNM stage. DDR1a promoted LoVo cell proliferation, mitochondrial function, and extracellular acidification. Moreover, DDR1a knockdown inhibited intracellular lactic acid production in LoVo cells, while a pyruvate kinase inhibitor (diamide, 200 μM) significantly reversed this progression. Taken together, our results reveal that DDR1 plays a crucial role in maintaining intracellular environment homeostasis through metabolic reprogramming.

The Double-Edged Sword of SIRT3 in Cancer and Its Therapeutic Applications.

Reprogramming of cellular energy metabolism is considered an emerging feature of cancer. Mitochondrial metabolism plays a crucial role in cancer cell proliferation, survival, and metastasis. As a major mitochondrial NAD+-dependent deacetylase, sirtuin3 (SIRT3) deacetylates and regulates the enzymes involved in regulating mitochondrial energy metabolism, including fatty acid oxidation, the Krebs cycle, and the respiratory chain to maintain metabolic homeostasis. In this article, we review the multiple roles of SIRT3 in various cancers, and systematically summarize the recent advances in the discovery of its activators and inhibitors. The roles of SIRT3 vary in different cancers and have cell- and tumor-type specificity. SIRT3 plays a unique function by mediating interactions between mitochondria and intracellular signaling. The critical functions of SIRT3 have renewed interest in the development of small molecule modulators that regulate its activity. Delineation of the underlying mechanism of SIRT3 as a critical regulator of cell metabolism and further characterization of the mitochondrial substrates of SIRT3 will deepen our understanding of the role of SIRT3 in tumorigenesis and progression and may provide novel therapeutic strategies for cancer targeting SIRT3.

The Secreted Metabolome of Hela Cells under Effect of Crotamine, a Cell-Penetrating Peptide from a Rattlesnake Using NMR-Based Metabolomics Analyses

Sequestering and reprogramming of cellular metabolism represents one of the principal hallmarks of several cells. Antimicrobial peptides have been shown to exhibit selective anticancer activities. In this study, the secreted metabolome of HeLa cells under action of the antimicrobial peptide Crotamine from the venom of the South American rattlesnake Crotalus durissus terrificus was evaluated. Crotamine has been shown to be selective for highly proliferating cells and is able to extend the in vivo lifespan. The present study using a cell line of cervical cancer, HeLa cells, provide insights into how Crotamine acts in cell metabolism. NMR spectroscopy was used to identify and quantify relative metabolite levels, which are associated with Crotamine uptake. Statistical analysis reveals that Crotamine dramatically affects metabolites related to glycolysis, metabolism and biosynthesis of amino acids and pyruvate metabolism. The developed machine learning model is found to be robust by ROC curve analysis, suggesting that the metabolic state of HeLa cells treated with Crotamine is different from the control samples. To account for metabolite levels, it is suggested that Crotamine would have to act on glycolysis, which, in turn, affects several other metabolic pathways, such as, glutathione metabolism, TCA cycle and pyruvate metabolism. The observed metabolic changes shed light into the mode of Crotamine function.

The interaction between end-metabolites and immune escape

Abstract Emerging data from metabolites-relating trails in cancers demonstrate that a common mechanism of resistance to many novel classes of immune therapeutics is the emergence of immune escape due to the reprogramming of cellular metabolism. Among them, current work about end-metabolites mostly focuses on the intersection between lactate acid, adenosine, reactive oxygen species (ROS), and tumour immune escape. In this article, we aim to review the evidence to date for the dynamic interplay between the three end-metabolites and tumour immune escape for potential approaches to overcome obstacles in the efficacy and durability of immune cancer therapies. We have organized known end-metabolites-associated immune escape mechanisms into three hallmarks: (1) decreased immunogenicity of cancer cells which constitutes defective antigen presentation and the attenuated expression of costimulatory molecules on tumour cells, (2) immunosuppressive microenvironment with aberrant angiogenesis inhibits the differentiation, maturation, and immune deviation of immune cells while drives the activation of immunosuppressive cells by immune-suppressive mediators (cytokines and other factors), (3) immune tolerance retained by inhibitory molecules and depletion of immune cells.

Pyruvate Dehydrogenase Kinase Inhibition by Dichloroacetate in Melanoma Cells Unveils Metabolic Vulnerabilities.

Melanoma is characterized by high glucose uptake, partially mediated through elevated pyruvate dehydrogenase kinase (PDK), making PDK a potential treatment target in melanoma. We aimed to reduce glucose uptake in melanoma cell lines through PDK inhibitors dichloroacetate (DCA) and AZD7545 and through PDK knockdown, to inhibit cell growth and potentially unveil metabolic co-vulnerabilities resulting from PDK inhibition. MeWo cells were most sensitive to DCA, while SK-MEL-2 was the least sensitive, with IC50 values ranging from 13.3 to 27.0 mM. DCA strongly reduced PDH phosphorylation and increased the oxygen consumption rate:extracellular acidification rate (OCR:ECAR) ratio up to 6-fold. Knockdown of single PDK isoforms had similar effects on PDH phosphorylation and OCR:ECAR ratio as DCA but did not influence sensitivity to DCA. Growth inhibition by DCA was synergistic with the glutaminase inhibitor CB-839 (2- to 5-fold sensitization) and with diclofenac, known to inhibit monocarboxylate transporters (MCTs) (3- to 8-fold sensitization). CB-839 did not affect the OCR:ECAR response to DCA, whereas diclofenac strongly inhibited ECAR and further increased the OCR:ECAR ratio. We conclude that in melanoma cell lines, DCA reduces proliferation through reprogramming of cellular metabolism and synergizes with other metabolically targeted drugs.

A non-dividing cell population with high pyruvate dehydrogenase kinase activity regulates metabolic heterogeneity and tumorigenesis in the intestine

Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.

SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas.

SUMMARYInherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation.

Signal Transduction during Metabolic and Inflammatory Reprogramming in Pulmonary Vascular Remodeling.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by (mal)adaptive remodeling of the pulmonary vasculature, which is associated with inflammation, fibrosis, thrombosis, and neovascularization. Vascular remodeling in PAH is associated with cellular metabolic and inflammatory reprogramming that induce profound endothelial and smooth muscle cell phenotypic changes. Multiple signaling pathways and regulatory loops act on metabolic and inflammatory mediators which influence cellular behavior and trigger pulmonary vascular remodeling in vivo. This review discusses the role of bioenergetic and inflammatory impairments in PAH development.

Emerging Roles of the Tumor Suppressor p53 in Metabolism.

Metabolism plays critical roles in maintaining the homeostasis of cells. Metabolic abnormalities are often considered as one of the main driving forces for cancer progression, providing energy and substrates of biosynthesis to support neoplastic proliferation effectively. The tumor suppressor p53 is well known for its roles in inducing cell cycle arrest, apoptosis, senescence and ferroptosis. Recently, emerging evidence has shown that p53 is also actively involved in the reprogramming of cellular metabolism. In this review, we focus on recent advances in our understanding of the interplay between p53 and metabolism of glucose, fatty acid as well as amino acid, and discuss how the deregulation of p53 in these processes could lead to cancer.

NFE2L1-mediated proteasome function protects from ferroptosis

ObjectiveFerroptosis continues to emerge as a novel modality of cell death with important therapeutic implications for a variety of diseases, most notably cancer and degenerative diseases. While susceptibility, initiation, and execution of ferroptosis have been linked to reprogramming of cellular lipid metabolism, imbalances in iron-redox homeostasis, and aberrant mitochondrial respiration, the detailed mechanisms of ferroptosis are still insufficiently well understood. Methods and resultsHere we show that diminished proteasome function is a new mechanistic feature of ferroptosis. The transcription factor nuclear factor erythroid-2, like-1 (NFE2L1) protects from ferroptosis by sustaining proteasomal activity. In cellular systems, loss of NFE2L1 reduced cellular viability after the induction of both chemically and genetically induced ferroptosis, which was linked to the regulation of proteasomal activity under these conditions. Importantly, this was reproduced in a Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD) patient-derived cell line carrying mutated glutathione peroxidase-4 (GPX4), a critical regulator of ferroptosis. Also, reduced proteasomal activity was associated with ferroptosis in Gpx4-deficient mice. In a mouse model for genetic Nfe2l1 deficiency, we observed brown adipose tissue (BAT) involution, hyperubiquitination of ferroptosis regulators, including the GPX4 pathway, and other hallmarks of ferroptosis. ConclusionOur data highlight the relevance of the NFE2L1-proteasome pathway in ferroptosis. Manipulation of NFE2L1 activity might enhance ferroptosis-inducing cancer therapies as well as protect from aberrant ferroptosis in neurodegeneration, general metabolism, and beyond.

Cellular stress responses and metabolic reprogramming in cancer progression and dormancy.

Recurrent disease after prolonged cancer dormancy is a major cause of cancer associated mortality, yet many of the mechanisms that are engaged to initiate dormancy as well as later recurrence remain incompletely understood. It is known that cancer cells initiate adaptation mechanisms to adapt tightly regulated cellular processes to non-optimal growth environments; Recent investigations have begun to elucidate the contribution of these mechanisms to malignant progression, with intriguing studies now defining cellular stress as a key contributor to the development and maintenance of cancer dormancy. This review will focus on our current understanding of stress responses facilitating malignant cell adaptation and metabolic reprogramming to establish cancer dormancy.

Understanding the molecular mechanisms of macrophage polarization and metabolic reprogramming in endometriosis: A narrative review.

BackgroundEndometriosis is an estrogen‐dependent disease and causes pelvic pain and infertility. The limits of current pharmacotherapy in women who desire to become pregnant prompt the development of various targeted molecules for more effective treatment. A review article focused on the unique aspect of cellular metabolic reprogramming of endometriotic cells has been reported. The cellular metabolic pathways are reprogrammed to adapt to a variety of environmental stresses (e.g., nutrient starvation or glucose deprivation, hypoxic stress, excessive reactive oxygen species generation, and other environmental factors). This review aims to summarize macrophage polarization and metabolic reprogramming in endometriosis.MethodsA literature search was performed between January 2000 and March 2022 in the PubMed and Google Scholar databases using a combination of specific terms.ResultsMacrophage cellular metabolism has a marked influence on its phenotype and function. Preclinical studies showed that metabolic conversion toward glycolysis or oxidative phosphorylation drives macrophage polarization to M1 or M2 phenotype, respectively. Such cellular metabolic rewiring can offer new therapeutic opportunities.ConclusionA better understanding of metabolic reprogramming biology in endometriosis‐associated macrophages is essential in considering novel therapeutic approach for endometriosis. However, there are currently no detailed studies on therapeutic strategies targeting the cellular metabolic properties of endometriosis‐associated macrophages.

Caveolin-1 Regulates Cellular Metabolism: A Potential Therapeutic Target in Kidney Disease

The kidney is an energy-consuming organ, and cellular metabolism plays an indispensable role in kidney-related diseases. Caveolin-1 (Cav-1), a multifunctional membrane protein, is the main component of caveolae on the plasma membrane. Caveolae are represented by tiny invaginations that are abundant on the plasma membrane and that serve as a platform to regulate cellular endocytosis, stress responses, and signal transduction. However, caveolae have received increasing attention as a metabolic platform that mediates the endocytosis of albumin, cholesterol, and glucose, participates in cellular metabolic reprogramming and is involved in the progression of kidney disease. It is worth noting that caveolae mainly depend on Cav-1 to perform the abovementioned cellular functions. Furthermore, the mechanism by which Cav-1 regulates cellular metabolism and participates in the pathophysiology of kidney diseases has not been completely elucidated. In this review, we introduce the structure and function of Cav-1 and its functions in regulating cellular metabolism, autophagy, and oxidative stress, focusing on the relationship between Cav-1 in cellular metabolism and kidney disease; in addition, Cav-1 that serves as a potential therapeutic target for treatment of kidney disease is also described.

Targeting immunometabolism in host-directed therapies to fungal disease.

Fungal infections affect over a billion people and are responsible for more than 1.5 million deaths each year. Despite progress in diagnostic and therapeutic approaches, the management of severe fungal infections remains a challenge. Recently, the reprogramming of cellular metabolism has emerged as a central mechanism through which the effector functions of immune cells are supported to promote antifungal activity. An improved understanding of the immunometabolic signatures that orchestrate antifungal immunity, together with the dissection of the mechanisms that underlie heterogeneity in individual immune responses, may therefore unveil new targets amenable to adjunctive host-directed therapies. In this review, we highlight recent advances in the metabolic regulation of host-fungus interactions and antifungal immune responses, and outline targetable pathways and mechanisms with promising therapeutic potential.

CSIG-02. NRF2-GUIDED GLUTATHIONE SYNTHESIS IS AN ESSENTIAL METABOLIC PATHWAY IN IN IDH1-MUTATED GLIOMA

Abstract BACKGROUND Isocitrate dehydrogenase (IDH) mutations are common genetic abnormalities in WHO Grade II/III glioma, which result in the reprogramming of cellular metabolism and redox homeostasis. Many lines of evidence showed that IDH mutations are critical for glioma formation, whereas the therapeutic options for IDH-mutated cancers remain limited. METHODS In the present study, we used the patient-derived glioma cell lines to investigate the role of nuclear factor erythroid 2-related factor 2 (NRF2) governed glutathione de novo synthesis. Further, we evaluated the therapeutic value of NRF2 inhibitors in IDH1-mutated cells and preclinical orthotopic models. RESULTS The neomorphic activity of mutant IDH reprogrammed the metabolic pathways involving enzyme cofactors such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). The depletion of NAD(P) in IDH1-mutated cells resulted in elevated oxidative stress and constitutive activation of NRF2-governed cytoprotective pathways through the decoupling of NRF2 from its E3 ligase Kelch-like ECH-associated protein 1 (Keap1). Activation of NRF2 enhanced glutathione synthesis by enhancing the gene transcription of GCLC, GCLM, and SLC7A11, which are the critical for glutathione de novo synthesis. Further, evidence from both in vitro assays and patient cohort indicated that NRF2 governed glutathione synthesis is important for maintaining the redox homeostasis and cell survival, especially in IDH1-mutated glioma. Finally, Blockade of the NRF2/glutathione metabolic pathway exhibited synergistic cytotoxicity with the metabolic stress in IDH1-mutated cells, which results in overwhelming oxidative damage, as well as a substantial reduction in tumor cell proliferation and xenograft expansion. CONCLUSION In this study, we highlighted that NRF2 plays critical roles in the disease progression of IDH1-mutated glioma by prompting glutathione synthesis. Targeting NRF2 governed glutathione metabolism could serve as a valuable synthetic lethality approach for IDH1-mutated malignancies.

IGF1R-IRS1/2 Pharmacological Inhibitors Act By Distinct Cellular and Molecular Mechanisms and Reveals Vulnerabilities for Treatment of Acute Myeloid Leukemia

Background: Preclinical rationale for targeting the insulin-like growth factor 1 (IGF1R)-Insulin Receptor Substrates 1 and 2 (IRS1/2) signaling in acute myeloid leukemia (AML), particularly in cells harboring the FLT3-ITD mutation, has been recently provided [Blood (2018) 132 Supp: 1512 and [Blood (2019) 134 Supp: 2689]. However, little is known about the non-canonical molecular mechanisms regulated by IGF1R-IRS1/2 signaling and pharmacological inhibition of this pathway in AML. Aims: To depict distinctive non-explicit molecular effects of linsitinib (IGF1R tyrosine kinase inhibitor) and NT157 (IGF1R-IRS1/2 allosteric inhibitor) treatment in FLT3-ITD-mutated AML cells. Material and methods: The MOLM-13 (homozygous) and MV4-11 (heterozygous) FLT3-ITD-mutated AML cell lines were treated with linsitinib (10 µM) or NT157 (1 µM) for 24 hours and used for label-free proteomic quantification analysis (n=3). Raw MS/MS data were processed using the SORCERER system and proteins were identified with built-in Andromeda search engine based on the human Uniprot proteome database. False discovery rate cutoffs were set to 1% on peptide, protein, and site decoy level, only allowing high quality identification to pass. Expression values were normalized across experimental conditions by quantile normalization based on the Limma-Voom pipeline, and then systematically compared similarities and differences in protein expression across experimental conditions by applying the Benjamin-Hochberg correction for multiple comparisons. To depict pathways associated to IGF1R, IRS1 and IRS2 gene expression related to processes identified by the proteomic data, we performed a gene-set enrichment analysis (GSEA) using the curated genesets for oncogenic events and molecular functions (MSigDB, Broad Institute) from RNA-seq data of the Cancer Genome Atlas AML cohort (n=173). Results: Considering a ≥ 2-fold change difference in both directions, linsitinib treatment downregulated 6 and 18 and upregulated 13 and 116 proteins in MOLM-13 and MV4-11 cells, respectively. Likewise, NT157 downregulated 12 and 126 and upregulated 204 and 297 proteins. When compared directly, linsitinib reduced expression of 11 and 35 and increased expression of 110 and 70 proteins in MOLM-13 and MV4-11 cells, respectively. Gene ontology identified that linsitinib resulted in upregulation of 7 molecular functions, while the NT157 ensued the upregulation of 18 and downregulation of 17 molecular functions pathways in a consistently manner between all comparisons. Of note, linsitinib activates post-transcriptional regulatory mechanisms, RNA metabolism (RNA binding P=1.15E-12; RNA processing P=8.64E-7) and reduced the protein and macromolecule metabolism (cellular protein metabolism P=3.86E-6). NT157 affected several of mitochondrial functions (increasing proton transmembrane transport activity P=1.55E-12, reducing oxidoreductase activity P=9.11E-10, and oxidative phosphorylation P=5.19E-8). Altogether, these data highlighted that NT157 profounder cytotoxic effect is a result of reprogramming of cellular energetics metabolism, and that linsitinib altered transcription and translation processes, probably as a result of autophagy, a mechanism originally described by our group [Blood (2017) 130 Supp: 3966]. GSEA analysis revealed that high IGF1R expression is positively enriched with RPS14 signature (Normalized Enriched Score [NES]=2.23; FDR-q<0.001), a ribosomal protein related to pathophysiology of myeloid neoplasms related to chromosome 5q deletion. Both IRS1 and IRS2 transcriptional signatures were associated with cellular growth signaling, such as AKT (NES=1.86; FDR-q= 0.006) and MYC (NES=1.67; FDR-q= 0.005), mitochondrial function [mitochondrial gene expression (NES=1.71; FDR-q= 0.001)]. Conclusion: Our proteomic data shed light on new and non-explicit mechanisms related to IGF1R-IRS1/2 inhibitors. Linsitinib modulates molecular processes related to RNA transcription and translation, while NT157 profoundly affect the cellular energetics, and, at least in part, explain the differential pre-clinical efficiency. Moreover, allosteric pharmacological inhibition of IGF1R-IRS1/2 pathway seems a more promising strategy than the tyrosine kinase inhibition, especially for AML subgroup more dependent of mitochondrial metabolism, such as AML with FLT3 mutation. DisclosuresNo relevant conflicts of interest to declare.