Mikael Altun

Karolinska Institutet | KI · Department of Laboratory Medicine

PARP inhibitors (PARPi) predominantly targeting PARP1 and PARP2 have revolutionized cancer therapy by selectively killing cancer cells with defective DNA repair. However, achieving PARP1 or PARP2-selective inhibitors is difficult due to their close structural homology. Selectivity profiling is typically done with purified proteins, but these lack t...

Cellular target engagement technologies are reforming drug discovery by enabling quantification of intracellular drug binding; however, simultaneous assessment of drug-associated phenotypes has proven challenging. CeTEAM (cellular target engagement by accumulation of mutant) is a platform that can concomitantly evaluate drug-target interactions and...

Simple Summary Much of what we know about the loss of skeletal muscle strength and mass (sarcopenia) in old age comes from studies of small laboratory rodents. In humans, this condition is generally not noticeable until late middle age when it affects our mobility (clinical stage). As muscle wasting progresses with age, it poses a challenge to inde...

The facultative loss of muscle mass and function during aging (sarcopenia) poses a serious threat to our independence and health. Much of what we know about sarcopenia derive from studies on small rodents which serve as models for the human condition. Here we used outbred male rats to study the natural history of sarcopenia with the aim to compare...

Skeletal muscle adaptations to exercise have been associated with a range of health-related benefits, but cell type-specific adaptations within the muscle are incompletely understood. Here we use single-cell sequencing to determine the effects of exercise on cellular composition and cell type-specific processes in human skeletal muscle before and a...

Acute myeloid leukemia (AML) is an aggressive blood malignancy characterized by clonal accumulating of immature myeloid progenitors in the bone marrow and peripheral blood. Transcription factors are the most frequently mutated and dysregulated genes in AML and they have critical roles in AML pathogenesis and progression. In this study, we performed...

Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a gene...

Eukaryotic initiation factor 4A-III (eIF4A3), a core helicase component of the exon junction complex, is essential for splicing, mRNA trafficking, and nonsense-mediated decay processes emerging as targets in cancer therapy. Here, we unravel eIF4A3's tumor-promoting function by demonstrating its role in ribosome biogenesis (RiBi) and p53 (de)regulat...

Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a gene...

Background Autophagy is intensively studied in cancer, metabolic and neurodegenerative diseases, but little is known about its role in pathological conditions linked to altered neurotransmission. We examined the involvement of autophagy in levodopa (l‐dopa)‐induced dyskinesia, a frequent motor complication developed in response to standard dopamine...

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attrac...

The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we s...

The cell cycle is a highly conserved process involving the coordinated separation of a single cell into two daughter cells. To relate transcriptional regulation across the cell cycle with oscillatory changes in protein abundance and activity, we carried out a proteome- and phospho-proteome-wide mass spectrometry profiling. We compared protein dynam...

MTH1 prevents incorporation of oxidized deoxynucleoside triphosphates (e.g., 8-oxodGTP) into DNA. Earlier we demonstrated that MTH1 inhibitors TH588 and TH1579 possesses anti-cancer activity in various tumour types, while other structurally distinct MTH1 inhibitors failed to kill cancer cells or elevate the levels of oxidized nucleotides in DNA. In...

MTH1 prevents incorporation of oxidized deoxynucleoside triphosphates (e.g., 8-oxodGTP) into DNA. Earlier we demonstrated that MTH1 inhibitors TH588 and TH1579 possesses anti-cancer activity in various tumour types, while other structurally distinct MTH1 inhibitors failed to kill cancer cells or elevate the levels of oxidized nucleotides in DNA. In...

Aims To resolve timing and coordination of denervation atrophy and the re‐innervation recovery process to discern correlations indicative of common programs governing these processes Methods Female Sprague‐Dawley (SD) rats had a unilateral sciatic nerve crush. Based on longitudinal behavioural observations, the triceps surae muscle was analysed at...

The CC-genotype of the VDR polymorphism TaqI rs731236 has previously been associated with a higher risk of developing myopathy compared to TT-carriers. However, the mechanistic role of this polymorphism in skeletal muscle is not well defined. The effects of vitamin D on patients genotyped for the VDR polymorphism TaqI rs731236, comparing CC and TT-...

DNA binding as an anti-inflammatory Mice that lack the gene encoding 8-oxoguanine DNA glycosylase 1 (OGG1) show resistance to inflammation. This enzyme binds to sites of oxidative DNA damage and initiates DNA base excision repair. Visnes et al. developed a small-molecule drug that acts as a potent and selective active-site inhibitor that stops OGG1...

Nucleosome assembly proteins (NAPs) are histone chaperones with an important role in chromatin structure and epigenetic regulation of gene expression. We find that high gene expression levels of mouse Nap1l3 are restricted to haematopoietic stem cells (HSCs) in mice. Importantly, with shRNA or CRISPR-Cas9 mediated loss of function of mouse Nap1l3 a...

Epigenetic alterations contribute to leukemogenesis in childhood acute myeloid leukemia and therefore are of interest for potential therapeutic strategies. Here, we performed large-scale RNA interference screens using small hairpin RNAs in acute myeloid leukemia cells and non-transformed bone marrow cells to identify leukemia-specific dependencies....

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Novel therapies are undergoing clinical trials, for example, the Hsp90 inhibitor, XL888, in combination with BRAF inhibitors for the treatment of therapy-resistant melanomas. Unfortunately, our data show that this combination elicits a heterogeneous response in a panel of melanoma cell lines including PDX-derived models. We sought to understand the...

The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in...

Non-synchronized Fucci based cell phase sorting and RNA sequencing to identify cycling transcripts. (A) Using high-throughput imaging analysis, U2OS Fucci cells were stained and imaged for DAPI, EdU incorporation and Fucci status. Results are first shown separately (top and bottom left), then classified according to DNA status according to DAPI and...

(A) Protein expression levels of PAX6 in HeLa-Fucci cells analyzed by fluorescent imaging correlating immunostaining of PAX6 to cell cycle phase determined by DNA content (DAPI), represented as boxplots. (B) Examples of receptors and associated proteins significantly oscillating in HeLa and U2OS cells at FDR≤0.001. (EPS)

Examples of relative distribution of transcripts in cell cycle phases. (A) Live HeLa-Fucci cells were sorted in 3 cell cycle populations based on expression of Fucci markers. (B) Post-sort validation was done by flow cytometry analysis of expression of Fucci markers as well as of DNA content (staining by propidium iodide, PI). (C, D) Analysis of tr...

(A) A comparison between HeLa-Fucci cell cycle transcriptome and the Whitfield et al. data set [19] indicates number of shared transcripts. (B) Distribution plots of the θ value for HeLa-Fucci versus the full hit-list of the Seed Match Category reported by [18]. (C) STRING analysis (using the web interphase available at http://string-db.org) of TFs...

A schematic illustration of a network incorporating FGF, Notch and WNT signaling oscillates over the cell cycle. (EPS)

Molecular clock synchronization with the cell cycle. (A) Plot of the θ-value for core circadian genes in U2OS-Fucci cells (p-value≤0.001). (B) Venn diagram between cell cycle oscillating transcripts in U2OS-Fucci (FDR≤0.001), HeLa-Fucci cells (FDR≤0.001) and published circadian clock transcriptome in non-proliferating liver cells [55]. (EPS)

MiFlowCyt—Hela Fucci and U2OS Fucci sortings. (PDF)

RNA sequencing and TriComp data. (CSV)

GO cell cycle term summaries. (XLSX)

Transcription factor results. (XLSX)

GO term enrichment of developmental transcription factors. (XLSX)

Ubiquitin-specific protease 7 is a validated anticancer target; thus, selective USP7 inhibitors are of great interest. In this issue of Cell Chemical Biology, Lamberto et al. (2017) and Pozhidaeva et al. (2017) report important insights into the structural inhibitor-enzyme interplay, lighting the way toward the development of selective inhibitors.

hMTH1 is an 8-oxodGTPase that prevents mis-incorporation of free oxidized nucleotides into genomic DNA. Base excision and mismatch repair pathways also restrict the accumulation of oxidized lesions in DNA by removing the mis-inserted 8-oxo-7,8-dihydro-2'-deoxyguanosines (8-oxodGs). In this study, we aimed to investigate the interplay between hMYH D...

ATR and CHK1 maintain cancer cell survival under replication stress and inhibitors of both kinases are currently undergoing clinical trials. As ATR activity is increased after CHK1 inhibition, we hypothesized that this may indicate an increased reliance on ATR for survival. Indeed, we observe that replication stress induced by the CHK1 inhibitor AZ...

Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more s...

Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incor...

Posttranslational modification with ubiquitin (Ub) controls many cellular processes, and aberrant ubiquitination can contribute to cancer, immunopathology, and neurodegeneration. The versatility arises from the ability of Ub to form polymer chains with eight distinct linkages via lysine side chains and the N terminus. In this study, we engineered D...

Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resista...

Background: Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. One area where such activity-based probes have been applied is the ubiquitin-proteasome pathway, which is emerging as an important therapeutic target. A family of reagents has been developed that specifically label...

Novel ubiquitin-based active site probes including a fluorescent tag have been developed and evaluated. A new, functionalizable electrophilic trap is utilized allowing for late stage diversification of the probe. Attachment of fluorescent dyes allowed direct detection of endogenous deubiquitinating enzyme (DUB) activities in cell extracts by in-gel...

A proper cellular adaptation to low oxygen levels is essential for processes such as development, growth, metabolism, and angiogenesis. The response to decrease in oxygen supply, referred to as hypoxia, is also involved in numerous human diseases including cancer, inflammatory conditions, and vascular disease. The hypoxia-inducible factor 1-α (HIF-...

Converting lead compounds into drug candidates is a crucial step in drug development, requiring early assessment of potency, selectivity, and off-target effects. We have utilized activity-based chemical proteomics to determine the potency and selectivity of deubiquitylating enzyme (DUB) inhibitors in cell culture models. Importantly, we characteriz...

ABSTRACT: A large quantitative study was carried out to compare the proteome of respiratory syncytial virus (RSV) infected versus uninfected cells in order to determine novel pathways regulated during viral infection. RSV infected and mock-infected HEp2 cells were lysed and proteins separated by preparative isoelectric focussing using offgel fracti...

2-oxoglutarate (2-OG)-dependent oxygenases have diverse roles in human biology. The inhibition of several 2-OG oxygenases is being targeted for therapeutic intervention, including for cancer, anemia, and ischemic diseases. We report a small-molecule probe for 2-OG oxygenases that employs a hydroxyquinoline template coupled to a photoactivable cross...

Photoactivated cross-linking of peptides to proteins is a useful strategy for identifying enzyme-substrate and protein-protein interactions in cell lysates as demonstrated by studies on the human hypoxia inducible factor system.

Among the hallmarks of aged organisms are an accumulation of misfolded proteins and a reduction in skeletal muscle mass ("sarcopenia"). We have examined the effects of aging and dietary restriction (which retards many age-related changes) on components of the ubiquitin proteasome system (UPS) in muscle. The hindlimb muscles of aged (30 months old)...

Microbial pathogens exploit the ubiquitin system to facilitate infection and manipulate the immune responses of the host. In this study, susceptibility to Yersinia enterocolitica and Yersinia pseudotuberculosis invasion was found to be increased upon overexpression of the deubiquitinating enzyme otubain 1 (OTUB1), a member of the ovarian tumour dom...

Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmic-reticulum-associated degradation (ERAD). Although the involvement of membrane-associated ubiquitin-conjugating enzymes and ligases in these processes is well documented, their regulation by ubiquitin deconjugases is less well understood. By screening...

OTUB (otubain) 1 is a human deubiquitinating enzyme that is implicated in mediating lymphocyte antigen responsiveness, but whose molecular function is generally not well defined. A structural analysis of OTUB1 shows differences in accessibility to the active site and in surface properties of the substrate-binding regions when compared with its clos...

Several disturbances occurring during aging of humans and rodents alike stem from changes in sensory and motor functions. Using a battery of behavioral tests we have studied alterations in performance with advancing age in female and male rats of some frequently used strains. In parallel, we collected survival and body weight data. The median survi...

Motor disturbances and wasting of skeletal muscles (sarcopenia) causes significant impairment of daily life activities and is a major underlying cause for hospitalization in senescence. Herein we review data and present new findings on aging-specific changes in motoneurons, skeletal muscle and the interplay between motoneurons and target muscle fib...