A synthetic lethal screen identifies HDAC4 as a potential target in MELK overexpressing cancers

Abstract Maternal embryonic leucine zipper kinase (MELK) is frequently overexpressed in cancer, but the role of MELK in cancer is still poorly understood. MELK was shown to have roles in many cancer-associated processes including tumor growth, chemotherapy resistance, and tumor recurrence. To determine whether the frequent overexpression of MELK can be exploited in therapy, we performed a high-throughput screen using a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential when MELK is overexpressed. We identified two such genes: LAG2 and HDA3. LAG2 encodes an inhibitor of the Skp, Cullin, F-box containing (SCF) ubiquitin-ligase complex, while HDA3 encodes a subunit of the HDA1 histone deacetylase complex. We find that one of these synthetic lethal interactions is conserved in mammalian cells, as inhibition of a human homolog of HDA3 (Histone Deacetylase 4, HDAC4) is synthetically toxic in MELK overexpression cells. Altogether, our work identified a novel potential drug target for tumors that overexpress MELK.

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A synthetic lethal screen identifies HDAC4 as a potential target in MELK overexpressing cancers

10.1101/2021.07.16.452653 ◽

2021 ◽

Author(s):

Floris Foijer ◽

Lin Zhou ◽

Fernando R Rosas Bringas ◽

Bjorn Bakker ◽

Judith E Simon ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Mammalian Cells ◽

Leucine Zipper ◽

Chemotherapy Resistance ◽

Synthetic Lethal ◽

Ubiquitin Ligase Complex ◽

Synthetic Lethal Interactions ◽

A Subunit ◽

Scf Ubiquitin Ligase

Maternal embryonic leucine zipper kinase (MELK) is frequently overexpressed in cancer, but the role of MELK in cancer is still poorly understood. MELK was shown to have roles in many cancer-associated processes including tumor growth, chemotherapy resistance, and tumor recurrence. To determine whether the frequent overexpression of MELK can be exploited in therapy, we performed a high-throughput screen using a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential when MELK is overexpressed. We identified two such genes: LAG2 and HDA3. LAG2 encodes an inhibitor of the SCF ubiquitin-ligase complex, while HDA3 encodes a subunit of the HDA1 histone deacetylase complex. We find that one of these synthetic lethal interactions is conserved in mammalian cells, as inhibition of a human homolog of HDA3 (HDAC4) is synthetically toxic in MELK overexpression cells. Altogether, our work might provide a new angle of how to exploit MELK overexpression in cancers and might thus lead to novel intervention strategies.

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Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination

Science ◽

10.1126/science.aap7607 ◽

2018 ◽

Vol 362 (6419) ◽

pp. 1177-1182 ◽

Cited By ~ 52

Author(s):

M. Steklov ◽

S. Pandolfi ◽

M. F. Baietti ◽

A. Batiuk ◽

P. Carai ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Human Disease ◽

Mammalian Cells ◽

Leucine Zipper ◽

Ras Signaling ◽

Ras Proteins ◽

Ubiquitin Ligase Complex ◽

Cullin 3 ◽

Guanosine Triphosphatase

The leucine zipper–like transcriptional regulator 1 (LZTR1) protein, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, is implicated in human disease, yet its mechanism of action remains unknown. We found that Lztr1 haploinsufficiency in mice recapitulates Noonan syndrome phenotypes, whereas LZTR1 loss in Schwann cells drives dedifferentiation and proliferation. By trapping LZTR1 complexes from intact mammalian cells, we identified the guanosine triphosphatase RAS as a substrate for the LZTR1-CUL3 complex. Ubiquitome analysis showed that loss of Lztr1 abrogated Ras ubiquitination at lysine-170. LZTR1-mediated ubiquitination inhibited RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations disrupted either LZTR1-CUL3 complex formation or its interaction with RAS proteins. RAS regulation by LZTR1-mediated ubiquitination provides an explanation for the role of LZTR1 in human disease.

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MYC Oncogene Contributions to Release of Cell Cycle Brakes

Genes ◽

10.3390/genes10030244 ◽

2019 ◽

Vol 10 (3) ◽

pp. 244 ◽

Cited By ~ 26

Author(s):

Lucía García-Gutiérrez ◽

María Dolores Delgado ◽

Javier León

Keyword(s):

Cell Cycle ◽

Transcriptional Level ◽

Synthetic Lethal ◽

Myc Oncogene ◽

Synthetic Lethal Interactions ◽

A Cell ◽

Functional Inactivation ◽

P27 Gene ◽

Cell Cycle Inhibitors

Promotion of the cell cycle is a major oncogenic mechanism of the oncogene c-MYC (MYC). MYC promotes the cell cycle by not only activating or inducing cyclins and CDKs but also through the downregulation or the impairment of the activity of a set of proteins that act as cell-cycle brakes. This review is focused on the role of MYC as a cell-cycle brake releaser i.e., how MYC stimulates the cell cycle mainly through the functional inactivation of cell cycle inhibitors. MYC antagonizes the activities and/or the expression levels of p15, ARF, p21, and p27. The mechanism involved differs for each protein. p15 (encoded by CDKN2B) and p21 (CDKN1A) are repressed by MYC at the transcriptional level. In contrast, MYC activates ARF, which contributes to the apoptosis induced by high MYC levels. At least in some cells types, MYC inhibits the transcription of the p27 gene (CDKN1B) but also enhances p27’s degradation through the upregulation of components of ubiquitin ligases complexes. The effect of MYC on cell-cycle brakes also opens the possibility of antitumoral therapies based on synthetic lethal interactions involving MYC and CDKs, for which a series of inhibitors are being developed and tested in clinical trials

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Targeting Glutathione Metabolism: Partner in Crime in Anticancer Therapy

Nutrients ◽

10.3390/nu11081926 ◽

2019 ◽

Vol 11 (8) ◽

pp. 1926 ◽

Cited By ~ 19

Author(s):

Enrico Desideri ◽

Fabio Ciccarone ◽

Maria Rosa Ciriolo

Keyword(s):

Therapeutic Strategy ◽

State Level ◽

Anticancer Therapy ◽

Chemotherapeutic Agents ◽

Glutathione Metabolism ◽

Low Molecular Weight ◽

Steady State Level ◽

Synthetic Lethal ◽

Synthetic Lethal Interactions

Glutathione (GSH) is the predominant low-molecular-weight antioxidant with a ubiquitous distribution inside the cell. The steady-state level of cellular GSH is dependent on the balance between synthesis, hydrolysis, recycling of glutathione disulphide (GSSG) as well as cellular extrusion of reduced, oxidized, or conjugated-forms. The augmented oxidative stress typical of cancer cells is accompanied by an increase of glutathione levels that confers them growth advantage and resistance to a number of chemotherapeutic agents. Targeting glutathione metabolism has been widely investigated for cancer treatment although GSH depletion as single therapeutic strategy has resulted largely ineffective if compared with combinatorial approaches. In this review, we circumstantiate the role of glutathione in tumour development and progression focusing on how interfering with different steps of glutathione metabolism can be exploited for therapeutic purposes. A dedicated section on synthetic lethal interactions with GSH modulators will highlight the promising option of harnessing glutathione metabolism for patient-directed therapy in cancer.

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Both N- and C-terminal domains of RelB are required for full transactivation: role of the N-terminal leucine zipper-like motif

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1572-1582.1993 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1572-1582

Author(s):

P Dobrzanski ◽

R P Ryseck ◽

R Bravo

Keyword(s):

Transcriptional Activation ◽

Mammalian Cells ◽

Transcriptional Activity ◽

Leucine Zipper ◽

Wild Type ◽

Nf Kappa B ◽

Hybrid Molecules ◽

In Cis ◽

Rel Homology Domain

RelB, a member of the Rel family of transcription factors, can stimulate promoter activity in the presence of p50-NF-kappa B or p50B/p49-NF-kappa B in mammalian cells. Transcriptional activation analysis reveals that the N and C termini of RelB are required for full transactivation in the presence of p50-NF-kappa B. RelB/p50-NF-kappa B hybrid molecules containing the Rel homology domain of p50-NF-kappa B and the N and C termini of RelB have high transcriptional activity compared with wild-type p50-NF-kappa B. The N and C termini of RelB cooperate in transactivation in cis or trans configuration. Alterations in the structure of the leucine zipper-like motif present in the N terminus of RelB significantly decrease the transcriptional capacity of RelB and of different RelB/p50-NF-kappa B hybrid molecules.

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A Subunit of the Anaphase-Promoting Complex Is a Centromere-Associated Protein in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.18.1.468 ◽

1998 ◽

Vol 18 (1) ◽

pp. 468-476 ◽

Cited By ~ 39

Author(s):

Pia-Marie Jörgensen ◽

Eva Brundell ◽

Maria Starborg ◽

Christer Höög

Keyword(s):

Ubiquitin Ligase ◽

Mammalian Cells ◽

Chinese Hamster ◽

Separation Process ◽

Soluble Form ◽

Anaphase Promoting Complex ◽

Mitotic Chromosomes ◽

Sister Chromatids ◽

Ubiquitin Ligase Complex ◽

A Subunit

ABSTRACT Sister chromatids in early mitotic cells are held together mainly by interactions between centromeres. The separation of sister chromatids at the transition between the metaphase and the anaphase stages of mitosis depends on the anaphase-promoting complex (APC), a 20S ubiquitin-ligase complex that targets proteins for destruction. A subunit of the APC, called APC-α in Xenopus (and whose homologs are APC-1, Cut4, BIME, and Tsg24), has recently been identified and shown to be required for entry into anaphase. We now show that the mammalian APC-α homolog, Tsg24, is a centromere-associated protein. While this protein is detected only during the prophase to the anaphase stages of mitosis in Chinese hamster cells, it is constitutively associated with the centromeres in murine cells. We show that there are two forms of this protein in mammalian cells, a soluble form associated with other components of the APC and a centromere-bound form. We also show that both the Tsg24 protein and the Cdc27 protein, another APC component, are bound to isolated mitotic chromosomes. These results therefore support a model in which the APC by ubiquitination of a centromere protein regulates the sister chromatid separation process.

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Inducing toxicity by introducing a leucine-zipper-like motif in frog antimicrobial peptide, magainin 2

Biochemical Journal ◽

10.1042/bj20110056 ◽

2011 ◽

Vol 436 (3) ◽

pp. 609-620 ◽

Cited By ~ 35

Author(s):

Brijesh Kumar Pandey ◽

Saurabh Srivastava ◽

Manish Singh ◽

Jimut Kanti Ghosh

Keyword(s):

Antimicrobial Peptides ◽

Mammalian Cells ◽

Leucine Zipper ◽

Lipid Vesicles ◽

Aqueous Environment ◽

Structural Element ◽

Human Red Blood Cells ◽

Naturally Occurring ◽

Hydrophobic Amino Acids

Cytotoxicity, a major obstacle in therapeutic application of antimicrobial peptides, is controlled by leucine-zipper-like sequences in melittin and other naturally occurring antimicrobial peptides. Magainin 2 shows significantly lower cytotoxicity than many naturally occurring antimicrobial peptides and lacks this structural element. To investigate the consequences of introducing a leucine zipper sequence in magainin 2, a novel analogue (Mag-mut) was designed by rearranging only the positions of its hydrophobic amino acids to include this structural element. Both magainin 2 and Mag-mut showed appreciable similarities in their secondary structures in the presence of negatively charged lipid vesicles, in localizing and permeabilizing the selected bacteria and exhibiting bactericidal activities. However, Mag-mut bound and localized strongly on to the mammalian cells tested and exhibited significantly higher cytotoxicity than magainin 2. Only Mag-mut, but not magainin 2, permeabilized human red blood cells and zwitterionic lipid vesicles. In contrast with magainin 2, Mag-mut self-assembled in an aqueous environment and bound co-operatively on to zwitterionic lipid vesicles. The peptides formed pores of different sizes on to a selected mammalian cell. The results of the present study indicate an important role of the leucine zipper sequence in the cytotoxicity of Mag-mut and demonstrate that its introduction into a non-toxic peptide, without altering the amino acid composition, can render cytotoxicity.

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Hemozoin produced by mammals confers heme tolerance

eLife ◽

10.7554/elife.49503 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 10

Author(s):

Rini H Pek ◽

Xiaojing Yuan ◽

Nicole Rietzschel ◽

Jianbing Zhang ◽

Laurie Jackson ◽

...

Keyword(s):

Mammalian Cells ◽

Blood Feeding ◽

Degradation Pathway ◽

Synthetic Lethal ◽

Heme Transport ◽

Synthetic Lethal Interactions ◽

Free Heme ◽

Perinatal Lethality ◽

Erythroid Maturation ◽

High Concentrations

Free heme is cytotoxic as exemplified by hemolytic diseases and genetic deficiencies in heme recycling and detoxifying pathways. Thus, intracellular accumulation of heme has not been observed in mammalian cells to date. Here we show that mice deficient for the heme transporter SLC48A1 (also known as HRG1) accumulate over ten-fold excess heme in reticuloendothelial macrophage lysosomes that are 10 to 100 times larger than normal. Macrophages tolerate these high concentrations of heme by crystallizing them into hemozoin, which heretofore has only been found in blood-feeding organisms. SLC48A1 deficiency results in impaired erythroid maturation and an inability to systemically respond to iron deficiency. Complete heme tolerance requires a fully-operational heme degradation pathway as haplo insufficiency of HMOX1 combined with SLC48A1 inactivation causes perinatal lethality demonstrating synthetic lethal interactions between heme transport and degradation. Our studies establish the formation of hemozoin by mammals as a previously unsuspected heme tolerance pathway.

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Both N- and C-terminal domains of RelB are required for full transactivation: role of the N-terminal leucine zipper-like motif.

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1572 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1572-1582 ◽

Cited By ~ 53

Author(s):

P Dobrzanski ◽

R P Ryseck ◽

R Bravo

Keyword(s):

Transcriptional Activation ◽

Mammalian Cells ◽

Transcriptional Activity ◽

Leucine Zipper ◽

Wild Type ◽

Nf Kappa B ◽

Hybrid Molecules ◽

In Cis ◽

Rel Homology Domain

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Improper Coordination of BamA and BamD Results in Bam Complex Jamming by a Lipoprotein Substrate

mBio ◽

10.1128/mbio.00660-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 9

Author(s):

Muralidhar Tata ◽

Anna Konovalova

Keyword(s):

Genetic Analysis ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Synthetic Lethal ◽

Bam Complex ◽

Synthetic Lethal Interactions ◽

Bona Fide ◽

Redundant Functions ◽

Kinetics Of

ABSTRACT The β-barrel assembly machinery, the Bam complex, is central to the biogenesis of integral outer membrane proteins (OMPs) as well as OMP-dependent surface-exposed lipoproteins, such as regulator of capsule synthesis protein F (RcsF). Previous genetic analysis established the model that nonessential components BamE and BamB have overlapping, redundant functions to enhance the kinetics of the highly conserved BamA/BamD core. Here we report that BamE plays a specialized nonredundant role in the Bam complex required for surface exposure of RcsF. We show that the lack of bamE, but not bamB, completely abolishes assembly of RcsF/OMP complexes and establish that the inability to assemble RcsF/OMP complexes is a molecular reason underlying all synthetic lethal interactions of ΔbamE. Our genetic analysis and biochemical cross-linking suggest that RcsF accumulates on BamA when BamA cannot engage with BamD because of its limited availability or the incompatible conformation. The role of BamE is to promote proper coordination of RcsF-bound BamA with BamD to complete OMP assembly around RcsF. We show that in the absence of BamE, RcsF is stalled on BamA, thus blocking its function, and we identify the lipoprotein RcsF as a bona fide jamming substrate of the Bam complex. IMPORTANCE The β-barrel assembly machinery, the Bam complex, consists of five components, BamA to -E, among which BamA and BamD are highly conserved and essential. The nonessential components are believed to play redundant roles simply by improving the rate of β-barrel folding. Here we show that BamE contributes a specific and nonoverlapping function to the Bam complex. BamE coordinates BamA and BamD to form a complex between the lipoprotein RcsF and its partner outer membrane β-barrel protein, allowing RcsF to reach the cell surface. In the absence of BamE, RcsF accumulates on BamA, thus blocking the activity of the Bam complex. As the Bam complex is a major antibiotic target in Gram-negative bacteria, the discovery that a lipoprotein can act as a jamming substrate may open the door for development of novel Bam complex inhibitors.

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