The MYC-Regulated RNA-Binding Proteins hnRNPC and LARP1 Are Drivers of Multiple Myeloma Cell Growth and Disease Progression and Negatively Predict Patient Survival.

Multiple myeloma (MM) is a malignant plasma cell disorder in which the MYC oncogene is frequently dysregulated. Due to its central role, MYC has been proposed as a drug target; however, the development of a clinically applicable molecule modulating MYC activity remains an unmet challenge. Consequently, an alternative is the development of therapeutic options targeting proteins located downstream of MYC. Therefore, we aimed to identify undescribed MYC-target proteins in MM cells using Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and mass spectrometry. We revealed a cluster of proteins associated with the regulation of translation initiation. Herein, the RNA-binding proteins Heterogeneous Nuclear Ribonucleoprotein C (hnRNPC) and La Ribonucleoprotein 1 (LARP1) were predominantly downregulated upon MYC depletion. CRISPR-mediated knockout of either hnRNPC or LARP1 in conjunction with redundant LARP family proteins resulted in a proliferative disadvantage for MM cells. Moreover, high expression levels of these proteins correlate with high MYC expression and with poor survival and disease progression in MM patients. In conclusion, our study provides valuable insights into MYC's role in translation initiation by identifying hnRNPC and LARP1 as proliferation drivers of MM cells and as both predictive factors for survival and disease progression in MM patients.

PDP1 is a key metabolic gatekeeper and modulator of drug resistance in FLT3-ITD-positive acute myeloid leukemia.

High metabolic flexibility is pivotal for the persistence and therapy resistance of acute myeloid leukemia (AML). In 20-30% of AML patients, activating mutations of FLT3, specifically FLT3-ITD, are key therapeutic targets. Here, we investigated the influence of FLT3-ITD on AML metabolism. Nuclear Magnetic Resonance (NMR) profiling showed enhanced reshuffling of pyruvate towards the tricarboxylic acid (TCA) cycle, suggesting an increased activity of the pyruvate dehydrogenase complex (PDC). Consistently, FLT3-ITD-positive cells expressed high levels of PDP1, an activator of the PDC. Combining endogenous tagging of PDP1 with genome-wide CRISPR screens revealed that FLT3-ITD induces PDP1 expression through the RAS signaling axis. PDP1 knockdown resulted in reduced cellular respiration thereby impairing the proliferation of only FLT3-ITD cells. These cells continued to depend on PDP1, even in hypoxic conditions, and unlike FLT3-ITD-negative cells, they exhibited a rapid, PDP1-dependent revival of their respiratory capacity during reoxygenation. Moreover, we show that PDP1 modifies the response to FLT3 inhibition. Upon incubation with the FLT3 tyrosine kinase inhibitor quizartinib (AC220), PDP1 persisted or was upregulated, resulting in a further shift of glucose/pyruvate metabolism towards the TCA cycle. Overexpression of PDP1 enhanced, while PDP1 depletion diminished AC220 resistance in cell lines and peripheral blasts from an AC220-resistant AML patient in vivo. In conclusion, FLT3-ITD assures the expression of PDP1, a pivotal metabolic regulator that enhances oxidative glucose metabolism and drug resistance. Hence, PDP1 emerges as a potentially targetable vulnerability in the management of AML.

Selection-free endogenous tagging of cell lines by bicistronic co-expression of the surface antigen NGFR.

Endogenous protein tagging, in contrast to exogenous overexpression of tagged proteins, allows to characterize specific protein functions under defined physiological or pathophysiological conditions without the influence of non-physiological protein levels. The development of generic and homology-independent tagging strategies, exploiting the CRISPR/spCas9 gene editing system in combination with generic tag donor plasmids, allows targeted and precise gene modification in mammalian cells for almost any desirable gene. So far, fluorescent tags or antibiotic resistance cassettes coupled to the endogenous fusion protein expression have been applied to isolate correctly modified clones. However, both can be challenging, especially when endogenously controlled expression of the tagged protein is weak or regulated by cellular signals. Here, we expand the strategy to selection-free endogenous tagging by exploiting exogenous co-expression of surface antigens. These endogenously regulated, but still easily accessible surface antigens allow simple identification and isolation of clones harboring correctly tagged alleles via common sorting procedures (e.g. FACS/MACS). Using metabolically controlled interaction studies of the endogenously tagged mTORC1-regulating GATOR2 complex protein WDR59, we show that endogenous GFP-labeling does not affect complex association of fusion proteins and downstream signaling via mTORC1. In addition, exogenous co-expression of the NGFR surface antigen does not influence conditional protein-protein interactions.•A method for selection-free, site-specific, homology-independent endogenous genetic tagging.•Production of fusion genes for protein visualization in living cells or determination of protein-protein-interactions.•Expression of a fusion protein mirroring physiological expression in its natural genetic context.

The proteogenomic subtypes of acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive blood cancer with a poor prognosis. We report a comprehensive proteogenomic analysis of bone marrow biopsies from 252 uniformly treated AML patients to elucidate the molecular pathophysiology of AML in order to inform future diagnostic and therapeutic approaches. In addition to in-depth quantitative proteomics, our analysis includes cytogenetic profiling and DNA/RNA sequencing. We identify five proteomic AML subtypes, each reflecting specific biological features spanning genomic boundaries. Two of these proteomic subtypes correlate with patient outcome, but none is exclusively associated with specific genomic aberrations. Remarkably, one subtype (Mito-AML), which is captured only in the proteome, is characterized by high expression of mitochondrial proteins and confers poor outcome, with reduced remission rate and shorter overall survival on treatment with intensive induction chemotherapy. Functional analyses reveal that Mito-AML is metabolically wired toward stronger complex I-dependent respiration and is more responsive to treatment with the BCL2 inhibitor venetoclax.

Identification of the Cysteine Protease Legumain as a Potential Chronic Hypoxia-Specific Multiple Myeloma Target Gene.

Multiple myeloma (MM) is the second most common hematologic malignancy, which is characterized by clonal proliferation of neoplastic plasma cells in the bone marrow. This microenvironment is characterized by low oxygen levels (1-6% O2), known as hypoxia. For MM cells, hypoxia is a physiologic feature that has been described to promote an aggressive phenotype and to confer drug resistance. However, studies on hypoxia are scarce and show little conformity. Here, we analyzed the mRNA expression of previously determined hypoxia markers to define the temporal adaptation of MM cells to chronic hypoxia. Subsequent analyses of the global proteome in MM cells and the stromal cell line HS-5 revealed hypoxia-dependent regulation of proteins, which directly or indirectly upregulate glycolysis. In addition, chronic hypoxia led to MM-specific regulation of nine distinct proteins. One of these proteins is the cysteine protease legumain (LGMN), the depletion of which led to a significant growth disadvantage of MM cell lines that is enhanced under hypoxia. Thus, herein, we report a methodologic strategy to examine MM cells under physiologic hypoxic conditions in vitro and to decipher and study previously masked hypoxia-specific therapeutic targets such as the cysteine protease LGMN.

Genome-scale integration of transcriptome and metabolome unveils squalene synthase and dihydrofolate reductase as targets against AML cells resistant to chemotherapy.

The development of resistance to chemotherapeutic agents, such as Doxorubicin (DOX) and cytarabine (AraC), is one of the greatest challenges to the successful treatment of Acute Myeloid Leukemia (AML). Such acquisition is often underlined by a metabolic reprogramming that can provide a therapeutic opportunity, as it can lead to the emergence of vulnerabilities and dependencies to be exploited as targets against the resistant cells. In this regard, genome-scale metabolic models (GSMMs) have emerged as powerful tools to integrate multiple layers of data to build cancer-specific models and identify putative metabolic vulnerabilities. Here, we use genome-scale metabolic modelling to reconstruct a GSMM of the THP1 AML cell line and two derivative cell lines, one with acquired resistance to AraC and the second with acquired resistance to DOX. We also explore how, adding to the transcriptomic layer, the metabolomic layer enhances the selectivity of the resulting condition specific reconstructions. The resulting models enabled us to identify and experimentally validate that drug-resistant THP1 cells are sensitive to the FDA-approved antifolate methotrexate. Moreover, we discovered and validated that the resistant cell lines could be selectively targeted by inhibiting squalene synthase, providing a new and promising strategy to directly inhibit cholesterol synthesis in AML drug resistant cells.

Amino acid sensory complex proteins in mTORC1 and macroautophagy regulation.

Autophagy is the highly conserved catabolic process, which enables the survival of a cell under unfavorable environmental conditions. In a constantly changing environment, cells must be capable of dynamically oscillating between anabolism and catabolism in order to maintain cellular homeostasis. In this context, the activity of the mechanistic Target Of Rapamycin Complex 1 (mTORC1) is of major importance. As a central signaling node, it directly controls the process of macroautophagy and thus cellular metabolism. Thereby, the control of mTORC1 is equally crucial as the regulation of cellular homeostasis itself, whereby particular importance is attributed to amino acid sensory proteins. In this review, we describe the recent findings of macroautophagy and mTORC1 regulation by upstream amino acid stimuli in different subcellular localizations. We highlight in detail which proteins of the sensor complexes play a specific role in this regulation and point out additional non-canonical functions, e.g. in the regulation of macroautophagy, which have received little attention so far.

Metabolic Plasticity Is an Essential Requirement of Acquired Tyrosine Kinase Inhibitor Resistance in Chronic Myeloid Leukemia.

Tyrosine kinase inhibitors (TKIs) are currently the standard chemotherapeutic agents for the treatment of chronic myeloid leukemia (CML). However, due to TKI resistance acquisition in CML patients, identification of new vulnerabilities is urgently required for a sustained response to therapy. In this study, we have investigated metabolic reprogramming induced by TKIs independent of BCR-ABL1 alterations. Proteomics and metabolomics profiling of imatinib-resistant CML cells (ImaR) was performed. KU812 ImaR cells enhanced pentose phosphate pathway, glycogen synthesis, serine-glycine-one-carbon metabolism, proline synthesis and mitochondrial respiration compared with their respective syngeneic parental counterparts. Moreover, the fact that only 36% of the main carbon sources were utilized for mitochondrial respiration pointed to glycerol-phosphate shuttle as mainly contributors to mitochondrial respiration. In conclusion, CML cells that acquire TKIs resistance present a severe metabolic reprogramming associated with an increase in metabolic plasticity needed to overcome TKI-induced cell death. Moreover, this study unveils that KU812 Parental and ImaR cells viability can be targeted with metabolic inhibitors paving the way to propose novel and promising therapeutic opportunities to overcome TKI resistance in CML.

The Clinical Significance of Iron Overload and Iron Metabolism in Myelodysplastic Syndrome and Acute Myeloid Leukemia.

Myelodysplasticsyndrome (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell diseases leading to an insufficient formation of functional blood cells. Disease-immanent factors as insufficient erythropoiesis and treatment-related factors as recurrent treatment with red blood cell transfusions frequently lead to systemic iron overload in MDS and AML patients. In addition, alterations of function and expression of proteins associated with iron metabolism are increasingly recognized to be pathogenetic factors and potential vulnerabilities of these diseases. Iron is known to be involved in multiple intracellular and extracellular processes. It is essential for cell metabolism as well as for cell proliferation and closely linked to the formation of reactive oxygen species. Therefore, iron can influence the course of clonal myeloid disorders, the leukemic environment and the occurrence as well as the defense of infections. Imbalances of iron homeostasis may induce cell death of normal but also of malignant cells. New potential treatment strategies utilizing the importance of the iron homeostasis include iron chelation, modulation of proteins involved in iron metabolism, induction of leukemic cell death via ferroptosis and exploitation of iron proteins for the delivery of antileukemic drugs. Here, we provide an overview of some of the latest findings about the function, the prognostic impact and potential treatment strategies of iron in patients with MDS and AML.

Real-Time NMR Spectroscopy for Studying Metabolism.

Current metabolomics approaches utilize cellular metabolite extracts, are destructive, and require high cell numbers. We introduce here an approach that enables the monitoring of cellular metabolism at lower cell numbers by observing the consumption/production of different metabolites over several kinetic data points of up to 48 hours. Our approach does not influence cellular viability, as we optimized the cellular matrix in comparison to other materials used in a variety of in-cell NMR spectroscopy experiments. We are able to monitor real-time metabolism of primary patient cells, which are extremely sensitive to external stress. Measurements are set up in an interleaved manner with short acquisition times (approximately 7 minutes per sample), which allows the monitoring of up to 15 patient samples simultaneously. Further, we implemented our approach for performing tracer-based assays. Our approach will be important not only in the metabolomics fields, but also in individualized diagnostics.

Metabolic Plasticity of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is one of the most common and life-threatening leukemias. A highly diverse and flexible metabolism contributes to the aggressiveness of the disease that is still difficult to treat. By using different sources of nutrients for energy and biomass supply, AML cells gain metabolic plasticity and rapidly outcompete normal hematopoietic cells. This review aims to decipher the diverse metabolic strategies and the underlying oncogenic and environmental changes that sustain continuous growth, mediate redox homeostasis and induce drug resistance in AML. We revisit Warburg's hypothesis and illustrate the role of glucose as a provider of cellular building blocks rather than as a supplier of the tricarboxylic acid (TCA) cycle for energy production. We discuss how the diversity of fuels for the TCA cycle, including glutamine and fatty acids, contributes to the metabolic plasticity of the disease and highlight the roles of amino acids and lipids in AML metabolism. Furthermore, we point out the potential of the different metabolic effectors to be used as novel therapeutic targets.

Correction: LSD1 inhibition by tranylcypromine derivatives interferes with GFI1-mediated repression of PU.1 target genes and induces differentiation in AML.

The original version of this Article contained an error in the Acknowledgements section.

CRISPR/Cas9-mediated generic protein tagging in mammalian cells.

Systematic protein localization and protein-protein interaction studies to characterize specific protein functions are most effectively performed using tag-based assays. Ideally, protein tags are introduced into a gene of interest by homologous recombination to ensure expression from endogenous control elements. However, inefficient homologous recombination makes this approach difficult in mammalian cells. Although gene targeting efficiency by homologous recombination increased dramatically with the development of designer endonuclease systems such as CRISPR/Cas9 capable of inducing DNA double-strand breaks with unprecedented accuracy, the strategies still require synthesis or cloning of homology templates for every single gene. Recent developments have shown that endogenous protein tagging can be achieved efficiently in a homology independent manner. Hence, combinations between CRISPR/Cas9 and generic tag-donor plasmids have been used successfully for targeted gene modifications in mammalian cells. Here, we developed a tool kit comprising a CRISPR/Cas9 expression vector with several EGFP encoding plasmids that should enable tagging of almost every protein expressed in mammalian cells. By performing protein-protein interaction and subcellular localization studies of mTORC1 signal transduction pathway-related proteins expressed in HEK293T cells, we show that tagged proteins faithfully reflect the behavior of their native counterparts under physiological conditions.

Chronic Hypoxia Enhances ß-Oxidation-Dependent Electron Transport via Electron Transferring Flavoproteins.

Hypoxia poses a stress to cells and decreases mitochondrial respiration, in part by electron transport chain (ETC) complex reorganization. While metabolism under acute hypoxia is well characterized, alterations under chronic hypoxia largely remain unexplored. We followed oxygen consumption rates in THP-1 monocytes during acute (16 h) and chronic (72 h) hypoxia, compared to normoxia, to analyze the electron flows associated with glycolysis, glutamine, and fatty acid oxidation. Oxygen consumption under acute hypoxia predominantly demanded pyruvate, while under chronic hypoxia, fatty acid- and glutamine-oxidation dominated. Chronic hypoxia also elevated electron-transferring flavoproteins (ETF), and the knockdown of ETF⁻ubiquinone oxidoreductase lowered mitochondrial respiration under chronic hypoxia. Metabolomics revealed an increase in citrate under chronic hypoxia, which implied glutamine processing to α-ketoglutarate and citrate. Expression regulation of enzymes involved in this metabolic shunting corroborated this assumption. Moreover, the expression of acetyl-CoA carboxylase 1 increased, thus pointing to fatty acid synthesis under chronic hypoxia. Cells lacking complex I, which experienced a markedly impaired respiration under normoxia, also shifted their metabolism to fatty acid-dependent synthesis and usage. Taken together, we provide evidence that chronic hypoxia fuels the ETC via ETFs, increasing fatty acid production and consumption via the glutamine-citrate-fatty acid axis.

LSD1 inhibition by tranylcypromine derivatives interferes with GFI1-mediated repression of PU.1 target genes and induces differentiation in AML.

LSD1 has emerged as a promising epigenetic target in the treatment of acute myeloid leukemia (AML). We used two murine AML models based on retroviral overexpression of Hoxa9/Meis1 (H9M) or MN1 to study LSD1 loss of function in AML. The conditional knockout of Lsd1 resulted in differentiation with both granulocytic and monocytic features and increased ATRA sensitivity and extended the survival of mice with H9M-driven AML. The conditional knockout led to an increased expression of multiple genes regulated by the important myeloid transcription factors GFI1 and PU.1. These include the transcription factors GFI1B and IRF8. We also compared the effect of different irreversible and reversible inhibitors of LSD1 in AML and could show that only tranylcypromine derivatives were capable of inducing a differentiation response. We employed a conditional knock-in model of inactive, mutant LSD1 to study the effect of only interfering with LSD1 enzymatic activity. While this was sufficient to initiate differentiation, it did not result in a survival benefit in mice. Hence, we believe that targeting both enzymatic and scaffolding functions of LSD1 is required to efficiently treat AML. This finding as well as the identified biomarkers may be relevant for the treatment of AML patients with LSD1 inhibitors.

Polarization of Human Macrophages by Interleukin-4 Does Not Require ATP-Citrate Lyase.

Macrophages exposed to the Th2 cytokines interleukin (IL) IL-4 and IL-13 exhibit a distinct transcriptional response, commonly referred to as M2 polarization. Recently, IL-4-induced polarization of murine bone marrow-derived macrophages (BMDMs) has been linked to acetyl-CoA levels through the activity of the cytosolic acetyl-CoA-generating enzyme ATP-citrate lyase (ACLY). Here, we studied how ACLY regulated IL-4-stimulated gene expression in human monocyte-derived macrophages (MDMs). Although multiple ACLY inhibitors attenuated IL-4-induced target gene expression, this effect could not be recapitulated by silencing ACLY expression. Furthermore, ACLY inhibition failed to alter cellular acetyl-CoA levels and histone acetylation. We generated ACLY knockout human THP-1 macrophages using CRISPR/Cas9 technology. While these cells exhibited reduced histone acetylation levels, IL-4-induced gene expression remained intact. Strikingly, ACLY inhibitors still suppressed induction of target genes by IL-4 in ACLY knockout cells, suggesting off-target effects of these drugs. Our findings suggest that ACLY may not be the major regulator of nucleocytoplasmic acetyl-CoA and IL-4-induced polarization in human macrophages. Furthermore, caution should be warranted in interpreting the impact of pharmacological inhibition of ACLY on gene expression.

Flotillins Regulate Focal Adhesions by Interacting with α-Actinin and by Influencing the Activation of Focal Adhesion Kinase.

Cell-matrix adhesion and cell migration are physiologically important processes that also play a major role in cancer spreading. In cultured cells, matrix adhesion depends on integrin-containing contacts such as focal adhesions. Flotillin-1 and flotillin-2 are frequently overexpressed in cancers and are associated with poor survival. Our previous studies have revealed a role for flotillin-2 in cell-matrix adhesion and in the regulation of the actin cytoskeleton. We here show that flotillins are important for cell migration in a wound healing assay and influence the morphology and dynamics of focal adhesions. Furthermore, anchorage-independent growth in soft agar is enhanced by flotillins. In the absence of flotillins, especially flotillin-2, phosphorylation of focal adhesion kinase and extracellularly regulated kinase is diminished. Flotillins interact with α-actinin, a major regulator of focal adhesion dynamics. These findings are important for understanding the molecular mechanisms of how flotillin overexpression in cancers may affect cell migration and, especially, enhance metastasis formation.

High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing.

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)-a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

Cytochrome P450 enzymes but not NADPH oxidases are the source of the NADPH-dependent lucigenin chemiluminescence in membrane assays.

Measuring NADPH oxidase (Nox)-derived reactive oxygen species (ROS) in living tissues and cells is a constant challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous studies have used NADPH-stimulated assays in membrane fractions which have been suggested to reflect Nox activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse (Nox1-Nox2-Nox4-/-) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells overexpressing Nox enzymes could not be recapitulated in NADPH-stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases. Using a combination of native protein electrophoresis, NADPH-stimulated assays and mass spectrometry, mitochondrial proteins and cytochrome P450 were identified as possible source of the assay signal. Cells lacking functional mitochondrial complexes, however, displayed a normal activity in NADPH-stimulated membrane assays suggesting that mitochondrial oxidoreductases are unlikely sources of the signal. Microsomes overexpressing P450 reductase, cytochromes b5 and P450 generated a NADPH-dependent signal in assays utilizing lucigenin, L-012 and dihydroethidium (DHE). Knockout of the cytochrome P450 reductase by CRISPR/Cas9 technology (POR-/-) in HEK293 cells overexpressing Nox4 or Nox5 did not interfere with ROS production in intact cells. However, POR-/- abolished the signal in NADPH-stimulated assays using membrane fractions from the very same cells. Moreover, membranes of rat smooth muscle cells treated with angiotensin II showed an increased NADPH-dependent signal with lucigenin which was abolished by the knockout of POR but not by knockout of p22phox. IN CONCLUSION: the cytochrome P450 system accounts for the majority of the signal of Nox activity chemiluminescence based assays.

Ltbp4 regulates Pdgfrß expression via TGFß-dependent modulation of Nrf2 transcription factor function.

Latent transforming growth factor beta binding protein 4 (LTBP4) belongs to the fibrillin/LTBP family of proteins and plays an important role as a structural component of extracellular matrix (ECM) and local regulator of TGFß signaling. We have previously reported that Ltbp4S knock out mice (Ltbp4S-/-) develop centrilobular emphysema reminiscent of late stage COPD, which could be partially rescued by inactivating the antioxidant protein Sestrin 2 (Sesn2). More recent studies showed that Sesn2 knock out mice upregulate Pdgfrß-controlled alveolar maintenance programs that protect against cigarette smoke induced pulmonary emphysema. Based on this, we hypothesized that the emphysema of Ltbp4S-/- mice is primarily caused by defective Pdgfrß signaling. Here we show that LTBP4 induces Pdgfrß signaling by inhibiting the antioxidant Nrf2/Keap1 pathway in a TGFß-dependent manner. Overall, our data identified Ltbp4 as a major player in lung remodeling and injury repair.