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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.
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.
Myelodysplastic syndrome (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 might 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 a summary of some of the latest findings about the function, the prognostic impact and potential treatment strategies of iron in patients with MDS and AML.
Lead-optimization strategies for compounds targeting c-Myc G-quadruplex (G4) DNA are being pursued to develop anticancer drugs. Here, we investigate the structure-activity- relationship (SAR) of a newly synthesized series of molecules based on the pyrrolidine-substituted 5-nitro indole scaffold to target G4 DNA. Our synthesized series allows modulation of flexible elements with a structurally preserved scaffold. Biological and biophysical analyses illustrate that substituted 5-nitroindole scaffolds bind to the c-Myc promoter G-quadruplex. These compounds downregulate c-Myc expression and induce cell-cycle arrest in the sub-G1/G1 phase in cancer cells. They further increase the concentration of intracellular reactive oxygen species. NMR spectra show that three of the newly synthesized compounds interact with the terminal G-quartets (5′- and 3′-ends) in a 2 : 1 stoichiometry.
Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.
In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein–protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.
We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.
Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene BCR-ABL1T315I differ from BCR-ABL1 native (BCR-ABL1) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin β3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1T315I+ tumor burden and significantly prolonged survival of mice with BCR-ABL1T315I+ CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin β3/ILK-mediated signaling pathway in BCR-ABL1T315I+ cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting.
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.
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.
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.
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.