Rewiring Of Cells Research Articles

The impact of autophagy during the development and survival of glioblastoma.

Glioblastoma is the most common and aggressive adult brain tumour, with poor median survival and limited treatment options. Following surgical resection and chemotherapy, recurrence of the disease is inevitable. Genomic studies have identified key drivers of glioblastoma development, including amplifications of receptor tyrosine kinases, which drive tumour growth. To improve treatment, it is crucial to understand survival response processes in glioblastoma that fuel cell proliferation and promote resistance to treatment. One such process is autophagy, a catabolic pathway that delivers cellular components sequestered into vesicles for lysosomal degradation. Autophagy plays an important role in maintaining cellular homeostasis and is upregulated during stress conditions, such as limited nutrient and oxygen availability, and in response to anti-cancer therapy. Autophagy can also regulate pro-growth signalling and metabolic rewiring of cancer cells in order to support tumour growth. In this review, we will discuss our current understanding of how autophagy is implicated in glioblastoma development and survival. When appropriate, we will refer to findings derived from the role of autophagy in other cancer models and predict the outcome of manipulating autophagy during glioblastoma treatment.

The Role of Aberrant Metabolism in Cancer: Insights Into the Interplay Between Cell Metabolic Reprogramming, Metabolic Syndrome, and Cancer

Metabolic syndrome (MetS) is characterized by hyperglycemia, hypertension, dyslipidemia and abdominal obesity. Patients with MetS or other metabolic disorders are more susceptible to cancer development and recurrence and have a worse long-term prognosis. Moreover, the metabolic reprogramming observed in cancer cells has also been described as one of the new hallmarks of cancer. Thus, aberrant metabolism has been proposed as an important risk factor for cancer. Chronic inflammation, reactive oxygen species (ROS), and oncogenic signaling pathways are considered as main potential triggers. Considering the strong association between metabolism and cancer, metabolism-modulating drugs, including metformin and statins, as well as adopting a healthy lifestyle, have been extensively investigated as strategies to combat cancer. Furthermore, strategies that interfere with the metabolic rewiring of cells may also have potent anti-cancer effects. In this article, we provide a comprehensive review of current knowledge on the relationship between aberrant metabolism and cancer and discuss the potential use of metabolism-targeting strategy for the treatment of cancer.

Multi-omics Reveal that c-Src Modulates the Mitochondrial Phosphotyrosine Proteome and Metabolism According to Nutrient Availability.

Src kinase family members, including c-Src, are involved in numerous signaling pathways and have been observed inside different cellular compartments. Notably, c-Src modulates carbohydrate and fatty acid metabolism and is involved in the metabolic rewiring of cancer cells. This kinase is found within mitochondria where it targets different proteins to impact on the organelle functions and overall metabolism. Surprisingly, no global metabolic characterization of Src has been performed although c-Src knock-out mice have been available for 30 years. Considering that c-Src is sensitive to various metabolites, c-Src might represent a crucial player in metabolic adjustments induced by nutrient stress. The aim of this work was to characterize the impact of c-Src on mitochondrial activity and overall metabolism using multi-omic characterization. Src+/+ and Src-/- mice were fed ad libitum or fasted during 24h and were then analyzed using multi-omics. We observed that deletion of c-Src is linked to lower phosphorylation of Y412-NDUFA8, inhibition of oxygen consumption and accumulation of metabolites involved in glycolysis, TCA cycle and amino acid metabolism in mice fed ad libitum. Finally, metabolomics and (phosphotyrosine) proteomics are differently impacted by Src according to nutrient availability. The findings presented here highlight that c-Src reduces mitochondrial metabolism and impacts the metabolic adjustment induced by nutrient stress.

Abstract PR09: KRas modulates pancreatic cancer cell metabolism and invasive potential through the lipase HSL

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of only 9%, due in part to its robust metastatic capability. Metastatic invasion is a particularly energy-intensive cellular process; thus, novel therapeutic strategies could include targeting the energy source for tumor cell invasion and migration. Excess lipids, present due to increased fatty acid synthesis or obesity, could provide a fuel source for cancer progression and metastasis. Neutral lipids are stored in organelles called lipid droplets (LDs). How LDs contribute to PDAC tumor cell invasion and metastasis remains unknown. We hypothesize that PDAC cells can store excess lipid to drive metastatic invasion. Indeed, introduction of excess exogenous fatty acids augments LD production and accumulation in PDAC cells and leads to enhanced PDAC cell motility and invasion in vitro. LDs are metabolized by the concerted action of lipases. Importantly, we have found that treatment of tumor cells with lipase inhibitors blocks tumor cell invasion, indicating that utilization of stored lipids via lipase-mediated LD breakdown is required for invasive migration. Hormone sensitive lipase (HSL) is a lipolytic enzyme, best characterized in adipose tissue, that is critical to the breakdown and utilization of LDs in vitro and in vivo. We have made the surprising observation that oncogenic KRas downregulates expression of HSL in pancreatic cancer cells, which shifts the cells towards lipid storage. In PDAC, mutational activation of the oncogene KRas occurs in 95% of tumors and contributes to metabolic rewiring of tumor cells. Our data suggest that the metabolic shift induced by mutant KRas is comprised of decreased LD utilization via downregulation of HSL. Indeed, re-expression of HSL shifts tumor cell metabolism towards elevated oxidative phosphorylation. Importantly, KRas-mutant cells still require the catalytic activity of HSL for tumor cell invasion, even though HSL expression is downregulated. Using redox ratio imaging, we have found that migratory cells selectively utilize oxidative phosphorylation during the process of migration to metabolize stored lipid droplets and fuel invasive migration. Finally, depletion of LD stores via overexpression of HSL blocks tumor cell invasion in vitro and metastasis in mice, even in the context of oncogenic KRas. These data indicate that regulation of HSL is important for both lipid storage and maintaining cancer cell invasiveness. Therefore, we propose a novel mechanism by which oncogenic KRas mediates HSL activity in a tunable manner, thereby altering the metabolism of PDAC cells and leading to increased metastatic ability. This abstract is also being presented as Poster B47. Citation Format: Cody Rozeveld, Ryan Schulze, Lizhi Zhang, Gina L Razidlo. KRas modulates pancreatic cancer cell metabolism and invasive potential through the lipase HSL [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr PR09.

Aconitase 2 inhibits the proliferation of MCF-7 cells promoting mitochondrial oxidative metabolism and ROS/FoxO1-mediated autophagic response

BackgroundDeregulation of the tricarboxylic acid cycle (TCA) due to mutations in specific enzymes or defective aerobic metabolism is associated with tumour growth. Aconitase 2 (ACO2) participates in the TCA cycle by converting citrate to isocitrate, but no evident demonstrations of its involvement in cancer metabolism have been provided so far.MethodsBiochemical assays coupled with molecular biology, in silico, and cellular tools were applied to circumstantiate the impact of ACO2 in the breast cancer cell line MCF-7 metabolism. Fluorescence lifetime imaging microscopy (FLIM) of NADH was used to corroborate the changes in bioenergetics.ResultsWe showed that ACO2 levels are decreased in breast cancer cell lines and human tumour biopsies. We generated ACO2- overexpressing MCF-7 cells and employed comparative analyses to identify metabolic adaptations. We found that increased ACO2 expression impairs cell proliferation and commits cells to redirect pyruvate to mitochondria, which weakens Warburg-like bioenergetic features. We also demonstrated that the enhancement of oxidative metabolism was supported by mitochondrial biogenesis and FoxO1-mediated autophagy/mitophagy that sustains the increased ROS burst.ConclusionsThis work identifies ACO2 as a relevant gene in cancer metabolic rewiring of MCF-7 cells, promoting a different utilisation of pyruvate and revealing the potential metabolic vulnerability of ACO2-associated malignancies.

The Folate Cycle Enzyme MTHFR Is a Critical Regulator of Cell Response to MYC-Targeting Therapies

Metabolic rewiring of neoplastic cells engenders metabolic liabilities that can be exploited to design innovative therapeutic strategies, including those to increase the therapeutic index of existing anticancer therapies. We hypothesized that metabolic perturbation may substantially influence cell response to therapies targeting major oncogenes which are involved in active hijacking of neoplastic cell metabolism. In that regard, MYC represents a paradigmatic oncogene as this transcription factor is deregulated in more than 50% of human cancers and reprograms many aspect of cell metabolism. MYC expression is controlled by clusters of super-enhancer genomic regions densely occupied by transcription factors and chromatin regulators ― including BET bromodomain proteins, and CDK7 and CDK9 kinases. Two cohorts of patients with Acute Myeloid Leukemia, AML (TCGA-LAML, n=198 and GSE14468, n=526) were queried with multiple gene sets in order to reveal a core of metabolic gene signatures, which are connected to the folate cycle, and whose activation was poorly correlated with an active MYC transcriptional program in AML. According to these data, we established that folate cycle disruption upon folic acid starvation consistently enhanced resistance to MYC targeting by BET or CDK7 inhibitors (JQ1, OTX015, THZ1) as well as BRD4-directed shRNAs in a large panel of human AML cell lines harboring a wide variety of genetic alterations (n=7), in MLL-translocated primary patient samples with AML (n=4), and in animals injected with MLL-AF9-positive leukemic cells. Using an shRNA-based screening approach against enzymes from the folate cycle, we revealed that the knockdown of the rate-limiting enzyme in the folate cycle, 5,10-methylenetetrahydroflate reductase (MTHFR), significantly increased resistance to OTX015 in AML cell lines (n=4) and in animals transplanted with Mthfr-depleted blasts. Previous reports have identified and extensively studied two common genetic variants in the MTHFR gene, C677T and A1298C, encoding two MTHFR enzyme variants with reduced activity in about 10% of Caucasians. We introduced in KG1a cells these two non-synonymous single nucleotide polymorphisms in MTHFR using CRISPR-Cas9, thereby generating isogenic cell lines exhibiting all combinations of variants. Although the clones which are heterozygous for any of the two variants had similar sensitivity to OTX015 as wild-type clones, 677 TT and 1298 CC homozygous KG1a clones were significantly more resistant to OTX015 than their wild-type counterpart, an effect that was alleviated by exogenous overexpression of wild-type MTHFR or supplementation of cells with the end-product metabolite synthesized by MTHFR, 5-CH3 THF. Consistent with these data, MLL-translocated patients displaying a homozygous and compound heterozygous MTHFR genotype for any of the two variants (n=8) responded significantly less to OTX015 than those with wild-type homozygous and heterozygous MTHFR genotypes (n=8). Finally, we established that the loss of a single copy of Mthfr which phenocopies in mice a partial impairment in MTHFR activity caused by non-synonymous single nucleotide polymorphisms on MTHFR, was sufficient to attenuate sensitivity to JQ1 of MLL-AF9-driven leukemias. Using metabolomics profiling, we pointed out that a major effect of folate cycle disturbance in AML cells is the intracellular accumulation of S-adenosyl-homocysteine, SAH, which is the downstream effector of MTHFR knockdown triggering BET inhibitor resistance. Given that SAH is a potent inhibitor of SAM-dependent methylation reactions, we determined that folate cycle impairment decreases H3K27 and H3K9 methyltransferase activities and subsequent methylation of H3K27 and H3K9 histone marks across the whole genome of AML cells. By combining ChIP- and RNA-sequencing approaches, we demonstrated that decreased methylation levels of H3K27 and H3K9 histone marks upon folate cycle alteration combined with BET inhibition activates SPI1 and IRF / Interferon signaling transcriptional programs. SPI1 knockdown significantly reduced the resistance to OTX015 of AML cells whose MTHFR expression was suppressed or MLL-AF9-transformed Mthfr knockout primary murine cells. Our data provide a rationale for screening MTHFR polymorphisms and the folate cycle status to exclude patients least likely and nominate those most likely to benefit from MYC-targeting therapies. Disclosures Dombret: CELGENE: Consultancy, Honoraria; AGIOS: Honoraria; Institut de Recherches Internationales Servier (IRIS): Research Funding. Stegmaier:Rigel Pharmaceuticals: Consultancy; Novartis: Research Funding.

The Warburg Effect Is Associated With Tumor Aggressiveness in Testicular Germ Cell Tumors.

Testicular Germ Cell Tumors (TGCTs) are a rare group of neoplasms and the most common solid malignancy arising in young male adults. Despite the good response of these tumors to platinum-based chemotherapy, some patients are refractory to treatment and present poor clinical outcomes. During carcinogenesis and tumor development, cancer cells reprogram energy metabolism toward a hyper-glycolytic phenotype, an emerging hallmark of cancer. This phenomenon, known as the Warburg effect or aerobic glycolysis, involves overexpression of metabolism-related proteins, like glucose and monocarboxylate transporters, pH regulators and intracellular glycolytic enzymes. The metabolic profile of TGCTs is very little explored and, recently, this metabolic rewiring of cancer cells has been associated with aggressive clinicopathological characteristics of these tumors. The overexpression of monocarboxylate transporter 4 (MCT4) in TGCTs has been pointed out as a poor prognostic factor, as well as a promising therapeutic target. As a result, the main aim of the present study was to evaluate the prognostic value of key metabolism-related proteins in TGCTs. The immunohistochemical expressions of CD44 (as a monocarboxylate transporter chaperone), glucose transporter 1 (GLUT1), carbonic anhydrase IX (CAIX), hexokinase II (HKII) and lactate dehydrogenase V (LDHV) were evaluated in a series of 148 adult male patients with TGCTs and associated with clinicopathological parameters. In addition, paired normal tissues were also evaluated. The sample included 75 seminoma and 73 non-seminoma tumors. GLUT1 and CD44 expression was significantly increased in malignant samples when compared to paired normal samples. Conversely, HKII and LDHV expressions were significantly decreased in malignant samples. Concerning the clinicopathological values, CAIX expression was significantly associated with disease recurrence, while HKII expression was significantly associated with aggressive characteristics of TGCTs, including higher staging and non-seminoma histology. In conclusion, this study brings new insights on the metabolic characteristics of TGCTs, showing alterations in the expression of proteins related with the Warburg effect, as well as associations of the hyper-glycolytic and acid-resistant phenotype with aggressive clinicopathological parameters.

Autocrine STAT3 activation in HPV positive cervical cancer through a virus-driven Rac1-NFκB-IL-6 signalling axis.

Persistent human papillomavirus (HPV) infection is the leading cause of cervical cancer. Although the fundamental link between HPV infection and oncogenesis is established, the specific mechanisms of virus-mediated transformation are not fully understood. We previously demonstrated that the HPV encoded E6 protein increases the activity of the proto-oncogenic transcription factor STAT3 in primary human keratinocytes; however, the molecular basis for STAT3 activation in cervical cancer remains unclear. Here, we show that STAT3 phosphorylation in HPV positive cervical cancer cells is mediated primarily via autocrine activation by the pro-inflammatory cytokine Interleukin 6 (IL-6). Antibody-mediated blockade of IL-6 signalling in HPV positive cells inhibits STAT3 phosphorylation, whereas both recombinant IL-6 and conditioned media from HPV positive cells leads to increased STAT3 phosphorylation within HPV negative cervical cancer cells. Interestingly, we demonstrate that activation of the transcription factor NFκB, involving the small GTPase Rac1, is required for IL-6 production and subsequent STAT3 activation. Our data provides new insights into the molecular re-wiring of cancer cells by HPV E6. We reveal that activation of an IL-6 signalling axis drives the autocrine and paracrine phosphorylation of STAT3 within HPV positive cervical cancers cells and that activation of this pathway is essential for cervical cancer cell proliferation and survival. Greater understanding of this pathway provides a potential opportunity for the use of existing clinically approved drugs for the treatment of HPV-mediated cervical cancer.

Can Metabolic Pathways Be Therapeutic Targets in Rheumatoid Arthritis?

The metabolic rewiring of tumor cells and immune cells has been viewed as a promising source of novel drug targets. Many of the molecular pathways implicated in rheumatoid arthritis (RA) directly modify synovium metabolism and transform the resident cells, such as the fibroblast-like synoviocytes (FLS), and the synovial tissue macrophages (STM), toward an overproduction of enzymes, which degrade cartilage and bone, and cytokines, which promote immune cell infiltration. Recent studies have shown metabolic changes in stromal and immune cells from RA patients. Metabolic disruption in the synovium provide the opportunity to use in vivo metabolism-based imaging techniques for patient stratification and to monitor treatment response. In addition, these metabolic changes may be therapeutically targetable. Thus, resetting metabolism of the synovial membrane offers additional opportunities for disease modulation and restoration of homeostasis in RA. In fact, rheumatologists already use the antimetabolite methotrexate, a chemotherapy agent, for the treatment of patients with inflammatory arthritis. Metabolic targets that do not compromise systemic homeostasis or corresponding metabolic functions in normal cells could increase the drug armamentarium in rheumatic diseases for combination therapy independent of systemic immunosuppression. This article summarizes what is known about metabolism in synovial tissue cells and highlights chemotherapies that target metabolism as potential future therapeutic strategies for RA.

Mitophagy in Cancer: A Tale of Adaptation.

In the past years, we have learnt that tumors co-evolve with their microenvironment, and that the active interaction between cancer cells and stromal cells plays a pivotal role in cancer initiation, progression and treatment response. Among the players involved, the pathways regulating mitochondrial functions have been shown to be crucial for both cancer and stromal cells. This is perhaps not surprising, considering that mitochondria in both cancerous and non-cancerous cells are decisive for vital metabolic and bioenergetic functions and to elicit cell death. The central part played by mitochondria also implies the existence of stringent mitochondrial quality control mechanisms, where a specialized autophagy pathway (mitophagy) ensures the selective removal of damaged or dysfunctional mitochondria. Although the molecular underpinnings of mitophagy regulation in mammalian cells remain incomplete, it is becoming clear that mitophagy pathways are intricately linked to the metabolic rewiring of cancer cells to support the high bioenergetic demand of the tumor. In this review, after a brief introduction of the main mitophagy regulators operating in mammalian cells, we discuss emerging cell autonomous roles of mitochondria quality control in cancer onset and progression. We also discuss the relevance of mitophagy in the cellular crosstalk with the tumor microenvironment and in anti-cancer therapy responses.

Metabolic rewiring of stem cells and differentiated cells in cancer: the hypothetical consequences of a GABA deficiency in endocrine pancreas

Metabolic rewiring of stem cells and differentiated cells in cancer: the hypothetical consequences of a GABA deficiency in endocrine pancreas

Metabolism and Epigenetic Interplay in Cancer: Regulation and Putative Therapeutic Targets.

Alterations in the epigenome and metabolism affect molecular rewiring of cancer cells facilitating cancer development and progression. Modulation of histone and DNA modification enzymes occurs owing to metabolic reprogramming driven by oncogenes and expression of metabolism-associated genes is, in turn, epigenetically regulated, promoting the well-known metabolic reprogramming of cancer cells and, consequently, altering the metabolome. Thus, several malignant traits are supported by the interplay between metabolomics and epigenetics, promoting neoplastic transformation. In this review we emphasize the importance of tumour metabolites in the activity of most chromatin-modifying enzymes and implication in neoplastic transformation. Furthermore, candidate targets deriving from metabolism of cancer cells and altered epigenetic factors is emphasized, focusing on compounds that counteract the epigenomic-metabolic interplay in cancer.

Metabolic Regulation of Innate Immunity to Fungal Infection.

In recent years, the renewed interest in immune cell metabolism has driven the emergence of a research field aimed at studying the role of metabolic processes during innate and adaptive immune responses. Although the specific requirements of myeloid cells after the canonical lipopolysaccharide/TLR4 stimulation have been extensively addressed, recent evidence suggests that this model may not represent a universally accurate metabolic blueprint. Instead, different microbial stimuli, pathogens, or tissue microenvironments trigger specific and complex metabolic rewiring of myeloid cells. This chapter aims to provide an overview of the metabolic heterogeneity in activated myeloid cells during fungal disease. Directions for future research in dissecting the uniqueness of metabolic signatures during fungal infection are suggested to ultimately provide new tailored diagnostic and therapeutic interventions.

Vancomycin-Dependent Response in Live Drug-Resistant Bacteria by Metabolic Labeling.

The surge in drug-resistant bacterial infections threatens to overburden healthcare systems worldwide. Bacterial cell walls are essential to bacteria, thus making them unique targets for the development of antibiotics. We describe a cellular reporter to directly monitor the phenotypic switch in drug-resistant bacteria with temporal resolution. Vancomycin-resistant enterococci (VRE) escape the bactericidal action of vancomycin by chemically modifying their cell-wall precursors. A synthetic cell-wall analogue was developed to hijack the biosynthetic rewiring of drug-resistant cells in response to antibiotics. Our study provides the first in vivo VanX reporter agent that responds to cell-wall alteration in drug-resistant bacteria. Cellular reporters that reveal mechanisms related to antibiotic resistance can potentially have a significant impact on the fundamental understanding of cellular adaption to antibiotics.

Nuclear mTOR acts as a transcriptional integrator of the androgen signaling pathway in prostate cancer

Androgen receptor (AR) signaling reprograms cellular metabolism to support prostate cancer (PCa) growth and survival. Another key regulator of cellular metabolism is mTOR, a kinase found in diverse protein complexes and cellular localizations, including the nucleus. However, whether nuclear mTOR plays a role in PCa progression and participates in direct transcriptional cross-talk with the AR is unknown. Here, via the intersection of gene expression, genomic, and metabolic studies, we reveal the existence of a nuclear mTOR-AR transcriptional axis integral to the metabolic rewiring of PCa cells. Androgens reprogram mTOR-chromatin associations in an AR-dependent manner in which activation of mTOR-dependent metabolic gene networks is essential for androgen-induced aerobic glycolysis and mitochondrial respiration. In models of castration-resistant PCa cells, mTOR was capable of transcriptionally regulating metabolic gene programs in the absence of androgens, highlighting a potential novel castration resistance mechanism to sustain cell metabolism even without a functional AR. Remarkably, we demonstrate that increased mTOR nuclear localization is indicative of poor prognosis in patients, with the highest levels detected in castration-resistant PCa tumors and metastases. Identification of a functional mTOR targeted multigene signature robustly discriminates between normal prostate tissues, primary tumors, and hormone refractory metastatic samples but is also predictive of cancer recurrence. This study thus underscores a paradigm shift from AR to nuclear mTOR as being the master transcriptional regulator of metabolism in PCa.

Ex Vivo Experiments Show That IDH1/2-Mutant Inhibitors Can be Safely Used As Adjuvants to Regular Chemotherapy in IDH1/2-Mutated Acute Myeloid Leukemia

Background: Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2MT) occur in up to 20% of MDS, MDS/MPN overlap and (s)AML. These mutations induce a metabolic rewiring of cancer cells that may impose metabolic vulnerabilities which can be pharmacologically targeted. In carcinoma cells, IDH1MT sensitize cells to oxidative stress induced by irradiation and treatment with electron transport chain complex I inhibitors, such as phenformin and metformin. Conversely, IDH1MT inhibitors protect IDH1MT cells against these treatment modalities. As a consequence, IDH1MT inhibitors and cytotoxic drugs should not be used together in patients with IDH1MT carcinoma and this has clinical relevance since IDH1/2MT inhibitors are already in phase I clinical trials. The underlying mechanism is that IDH1MT dramatically reduce the cellular production capacity of NADPH, an important antioxidant, and IDH1MT inhibitors restore the NADPH production capacity. Of note, IDH1 and IDH2 are the most important providers of NADPH in most cells.Methods: Primary IDH1/2MT, IDH1/2WT and TET2MT AML cells (n=15) were cultured and treated with the IDH1MT inhibitor AGI-5198, the IDH2MT inhibitor AGI-6780, irradiation, phenformin, metformin, daunorubicin or cytarabine. Cell numbers were counted 5 days after therapy exposure. In addition, cells were adhered to microscopy slides and maximal IDH1/2 and G6PD activity was determined using quantitative enzyme cytochemistry. ROS levels were fluorometrically measured using flow cytometry.Results: We observed that IDH1/2MT do not sensitize primary AML cells to irradiation, phenformin, metformin, daunorubicin or cytarabine and IDH1/2MT inhibitors had no effect on therapy sensitivity in primary AML cells. Because it is argued that inactivating TET2MT function downstream of IDH1/2MT, TET2MT AML cells served as a negative control and showed no sensitization neither.Although IDH1/2MT greatly reduce the IDH1/2-mediated NADPH production capacity in AML cells (P < 0.0001), IDH1/2 are not important NADPH providers in myeloid cells and the contribution of glucose-6-phosphate dehydrogenase (G6PD) to the total NADPH production pool is much larger (maximal G6PD activity is ~4 times higher than maximal IDH1/2 activity, Fig. 1). This contrasts the situation in glioblastoma, where IDH1/2 provide 65% of all NADPH. Large-scale mRNA-sequencing in AML and glio(blasto)ma datasets revealed no differences in IDH1/2 and G6PD expression between types of cancer, suggesting that post-translational modifications perform key roles in regulating the maximal activity of these enzymes (Fig. 2). To investigate the implications of these results on cellular redox potentials, we performed ROS measurements after treatment with irradiation and observed that IDH1/2MT do not increase ROS in AML cells, whereas IDH1/2MT do increase ROS in glioma cells.Discussion: Increased sensitivity to oxidative stress is not a metabolic vulnerability in IDH1/2MT AML and this is likely due to the fact that maximal NADPH production capacity is affected to only a small extent in IDH1/2MT AML. Therapies that increase oxidative stress, such as irradiation and metformin, counteract with IDH1/2MT inhibitors in glio(blasto)ma cells because there, IDH1/2MT have profound implications on cellular NADPH production capacities and ROS levels. Our results suggest that IDH1/2MT do not induce sensitivity to cytotoxic drugs in AML cells and thatIDH1/2MT inhibitors can be safely used adjuvantly to the AML standard of care daunorubicin and cytarabine, or whole-body irradiation in the context of bone marrow transplantations. These data are crucial for phase II and III clinical trial designs ofIDH1/2MT inhibitors.G6PD (a, c, e, g) and IDH1 activity (b, d, f, h) staining of IDH1R132 non-mutated (a, b, e, f) and mutated (c, d, g, h) primary AML cells (a-d) and glioblastoma (e-h) cryostat sections. The amount of blue color (nitro BT-formazan) directly reflects IDH1/2 and G6PD activity (production of NADPH). [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Whole-Exome Sequencing Identifies Germline IDH2 and IDH3 mutations That Predispose to Myeloid Neoplasms

Background: Somatic mutations in isocitrate dehydrogenases 1 and 2 (IDH1/2MT) occur in up to 20% of certain types of myeloid neoplasms, chiefly MDS, MDS/MPN overlap and AML. These mutations induce a metabolic rewiring of cancer cells that affects alpha-ketoglutarate (aKG)-dependent dioxygenases such as TET2 DNA demethylases and Jumanji histone demethylases, which leads to global DNA and chromatin hypermethylation and leukemogenesis. In addition to somatic IDH1/2MT, germline IDH1/2MT occur in the context of Ollier and Maffucci syndromes which predispose to cartilaginous tumors and D 2HG aciduria, which predisposes to brain tumors. We hypothesized that germline mutations in IDH1/2 or the functionally related IDH3 may predispose to myeloid neoplasms.Methods: From 409 patients with MDS, MPN or AML, tumor samples from the myeloid compartment and germline samples from CD3+ lymphocytes were subjected to whole-exome sequencing. Myeloid cells were adhered to microscopy slides and maximal IDH3, IDH1/2 and, glucose-6-phosphate dehydrogenase (G6PD) glutamate dehydrogenase (GDH) activity was determined using quantitative enzyme cytochemistry.Results: In 409 patients with myeloid diseases, we found 8 patients with germline variants in IDH2 or IDH3. Combined, these variants occur more frequently in these 409 patients with myeloid neoplasms than in the general population (OR = 4.05, P =.0024), suggesting that germline IDH2/3MT predispose for myeloid diseases (Table 1). Of note, germline IDH2/3MT were completely mutually exclusive with somatic TET2MT, suggesting overlapping functions. Notably, all but one of these variants were indicated by two independent software programs to be deleterious for the enzymatic activity. To validate this, we determined maximal IDH3 activity in myeloid cells derived from a 47-year old AML patient with a germline IDH3MT (c.G626A, p.G209E) (and family history of leukemia) and her sister with IDH3WT. Maximal IDH3 activity was downregulated in the IDH3MT sample, while maximal IDH1/2, G6PD and GDH activity were unchanged (Fig. 1).1 patient had a family history of MDS and 3 patients had a family history of colon, prostrate, gastric, lung and head and neck carcinoma in multiple family members. One patient had 4 family members affected with different cancers. The high age of the affected patients suggest that although germline IDH2/3MT may predispose to myeloid neoplasms, the development of disease occurs slowly.Discussion: Whereas somatic IDH2MT are common in MDS and other myeloid neoplasms, somatic IDH3MT are not frequently observed, nor in myeloid neoplasms, nor in other types of cancer that are regularly affected by IDH1/2MT. We show that deleterious germline IDH2/3MT may predispose to myeloid neoplasms and we postulate that this may occur via decreases in IDH2/3 activity. IDH3G209E is deleterious for IDH3 enzymatic function and may decrease intracellular aKG levels. This will restrict the function of aKG-dependent dioxygenases such as TET2 and Jumonji and mimic somatic IDH1/2MT and somatic inactivating TET2MT. This suggests that IDH1/2/3 are key enzymes and aKG is a central metabolite in maintaining normal function in myeloid cells. Given the family histories of 5/8 of the affected patients, screenings for germline IDH2/3 variants may reveal novel recurring IDH2/3MT that relate to various types of cancer.Representative photomicrographs of primary IDH3G209E MDS and IDH3WT myeloid cells after staining IDH3 activity in the presence of various isocitrate concentrations, IDH2 activity (1 mM isocitrate) and GDH activity (5 mM glutamate) using quantitative enzyme cytochemistry. The conversion of colorless tetrazolium salt to the purple formazan directly reflects enzyme activity thus darker cells have a higher maximal activity of the investigated enzyme.Table 1Clinical characteristics from 6 patients with germline IDH2/3MT for which clinical information was available.Patient IDAgeDiagnosisGenderOS (months)VariantSNP nrOccurrence in general population150RCMDF23IDH2T495Mrs1180539400,004848266CMML-1M46IDH3AR360Crs1163749960,004161349RAEB-1F16IDH3BR359Wrs3776821520,000154470RCMDM67IDH2T495Mrs1180539400,004848576MDS-UF17IDH3BR334Wrs3746135880,00008675sAMLM5IDH2R261Hrs1181017770,00177 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract A171: Targeting tumor metabolism by novel EGFR inhibitor in head and neck cancer

Abstract Cellular metabolism is substantially altered during tumor progression, and associated with poor prognosis and therapeutic resistance of human cancers. Therefore, the metabolic rewiring of cancer cells has been viewed as a promising source for novel drug targets, and being actively pursued in the development of selective antineoplastic agents. EGFR targeted agents currently approved or under investigation for head and neck squamous cell carcinoma (HNSCC) include EGFR monoclonal antibodies or EGFR tyrosine kinase inhibitors (EGFR-TKI). However, HNSCC patients failing to these agents treatment, due to the development of acquired tumor resistance, will have a compromised survival. We have identified a novel EGFR-TKI, BPR3K007S0. Based on the EC50 in enzyme-based assay of wild-type EGFR &amp; EGFR mutant L858R protein, BPR3K007S0 is more effective than that of clinical used EGFR-TKI gefitinib (Iressa). We further evaluated the drug effects on EGFR phosphorylation in HNSCC cells, and found that BPR3K007S0 inhibited phosphorylation of EGFR in a time-dependent manner. Further study demonstrated that BPR3K007S0 decreased HNSCC cell proliferation and colony formation by causing G1-phase arrest, which was strongly associated with a marked decrease of the level of cyclin E and cdk2 with concomitant induction of p21 and p27. Notably, BPR3K007S0 showed much potent effect toward cell growth in vitro and in vivo than that of gefitinib in HNSCC cells, implicating that the anticancer effect of BPR3K007S0 is not limited to EGFR inhibition. More importantly, we found that BPR3K007S0 significantly suppressed the expression of a wide range of metabolic genes. Among them, the levels of c-Myc and hexokinase 2 were significantly decreased in a time-dependent manner by treatment with BPR3K007S0. Gene manipulation study demonstrated that knockdown of c-Myc leads to a decreased level of hexokinase 2. In this system, silence of EGFR did not affect c-Myc expression, suggesting that c-Myc may not be the EGFR downstream target in used HNSCC cells. Taken together, these results revealed that BPR3K007S0 has added benefits by targeting of EGFR and cancer metabolism, and are likely to be superior to gefitinib in HNSCC models. Thus, development of multi-kinase and metabolic inhibitors holds promises for overcoming treatment resistance of HNSCC. (Grant support: DOH102-TD-M-111-102001) Citation Format: Ching-Chuan Kuo, Hsing-Pang Hsieh, Hsing-Jien Kung. Targeting tumor metabolism by novel EGFR inhibitor in head and neck cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A171.

Metabolic exchanges within tumor microenvironment

Tumor progression toward malignancy often requires a metabolic rewiring of cancer cells to meet changes in metabolic demand to forefront nutrient and oxygen withdrawal, together with strong anabolic requests to match high proliferation rate. Tumor microenvironment highly contributes to metabolic rewiring of cancer cells, fostering complete nutrient exploitation, favoring OXPHOS of lipids and glutamine at the expense of glycolysis and enhancing exchanges via extracellular microvesicles or exosomes of proteins, lipids and small RNAs among tumor and stromal cells. Noteworthy, the same molecular drivers of metabolic reprogramming within tumor and stroma are also able to elicit motility, survival and self-renewal on cancer cells, thereby sustaining successful escaping strategies to circumvent the hostile hypoxic, acidic and inflammatory environment. This review highlights the emerging role of nutrients and vesicle-mediated exchanges among tumor and stromal cells, defining their molecular pathways and offering new perspectives to develop treatments targeting this complex metabolic rewiring.

A novel pyruvate kinase M2 activator compound that suppresses lung cancer cell viability under hypoxia.

Pyruvate kinase M2 isoform (PKM2), a rate-limiting enzyme in the final step of glycolysis, is known to be associated with the metabolic rewiring of cancer cells, and considered an important cancer therapeutic target. Herein, we report a novel PKM2 activator, PA-12, which was identified via the molecular docking-based virtual screening. We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 μM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium. In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia. We also verified that the effects of PA-12 were dependent on PKM2 expression in cancer cells, demonstrating the specificity of PA-12 for PKM2 protein. Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.