Evgeny Kulesskiy

Heparan sulfate proteoglycan syndecan-3 is a novel receptor for GDNF, neurturin, and artemin.

Syndecan-3 may act alone or as a coreceptor with RET to promote cell spreading, neurite outgrowth, and migration of cortical neurons by GNDF, NRTN, and ARTN.

N-syndecan deficiency impairs neural migration in brain

N-syndecan (syndecan-3) is a transmembrane proteoglycan that is abundantly expressed in the major axonal pathways and in the migratory routes of the developing brain. When ligated by heparin-binding (HB) growth-associated molecule (GAM; pleiotrophin), N-syndecan mediates cortactin–Src kinase-dependent neurite outgrowth. However, the functional role of N-syndecan in brain development remains unexplored. In this study, we show that N-syndecan deficiency perturbs the laminar structure of the cerebral cortex as a result of impaired radial migration. In addition, neural migration in the rostral migratory stream is impaired in the N-syndecan–null mice. We suggest that the migration defect depends on impaired HB-GAM–induced Src kinase activation and haptotactic migration. Furthermore, we show that N-syndecan interacts with EGF receptor (EGFR) at the plasma membrane and is required in EGFR-induced neuronal migration.

Quantitative scoring of differential drug sensitivity for individually optimized anticancer therapies

We developed a systematic algorithmic solution for quantitative drug sensitivity scoring (DSS), based on continuous modeling and integration of multiple dose-response relationships in high-throughput compound testing studies. Mathematical model estimation and continuous interpolation makes the scoring approach robust against sources of technical variability and widely applicable to various experimental settings, both in cancer cell line models and primary patient-derived cells. Here, we demonstrate its improved performance over other response parameters especially in a leukemia patient case study, where differential DSS between patient and control cells enabled identification of both cancer-selective drugs and drug-sensitive patient sub-groups, as well as dynamic monitoring of the response patterns and oncogenic driver signals during cancer progression and relapse in individual patient cells ex vivo. An open-source and easily extendable implementation of the DSS calculation is made freely available to support its tailored application to translating drug sensitivity testing results into clinically actionable treatment options.

Antifungal Application of Nonantifungal Drugs

ABSTRACT Candida species are the cause of 60% of all mycoses in immunosuppressed individuals, leading to ∼150,000 deaths annually due to systemic infections, whereas the current antifungal therapies either have toxic side effects or are insufficiently efficient. We performed a screening of two compound libraries, the Enzo and the Institute for Molecular Medicine Finland (FIMM) oncology collection library, for anti-Candida activity based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. From a total of 844 drugs, 26 agents showed activity against Candida albicans. Of those, 12 were standard antifungal drugs (SADs) and 7 were off-target drugs previously reported to be active against Candida spp. The remaining 7 off-target drugs, amonafide, tosedostat, megestrol acetate, melengestrol acetate, stanozolol, trifluperidol, and haloperidol, were identified with this screen. The anti-Candida activities of the new agents were investigated by three individual assays using optical density, ATP levels, and microscopy. The antifungal activities of these drugs were comparable to those of the SADs found in the screen. The aminopeptidase inhibitor tosedostat, which is currently in a clinical trial phase for anticancer therapy, displayed a broad antifungal activity against different Candida spp., including Candida glabrata. Thus, this screen reveals agents that were previously unknown to be anti-Candida agents, which allows for the design of novel therapies against invasive candidiasis.

Transmembrane Prostatic Acid Phosphatase (TMPAP) Interacts with Snapin and Deficient Mice Develop Prostate Adenocarcinoma

The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP), a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930s. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP) and a transmembrane type-I (TMPAP). The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP−/−) with C57BL/6J background. The PAP−/− mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells) and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma.

Phosphoproteomics to Characterize Host Response During Influenza A Virus Infection of Human Macrophages

Influenza A viruses cause infections in the human respiratory tract and give rise to annual seasonal outbreaks, as well as more rarely dreaded pandemics. Influenza A viruses become quickly resistant to the virus-directed antiviral treatments, which are the current main treatment options. A promising alternative approach is to target host cell factors that are exploited by influenza viruses. To this end, we characterized the phosphoproteome of influenza A virus infected primary human macrophages to elucidate the intracellular signaling pathways and critical host factors activated upon influenza infection. We identified 1675 phosphoproteins, 4004 phosphopeptides and 4146 nonredundant phosphosites. The phosphorylation of 1113 proteins (66%) was regulated upon infection, highlighting the importance of such global phosphoproteomic profiling in primary cells. Notably, 285 of the identified phosphorylation sites have not been previously described in publicly available phosphorylation databases, despite many published large-scale phosphoproteome studies using human and mouse cell lines. Systematic bioinformatics analysis of the phosphoproteome data indicated that the phosphorylation of proteins involved in the ubiquitin/proteasome pathway (such as TRIM22 and TRIM25) and antiviral responses (such as MAVS) changed in infected macrophages. Proteins known to play roles in small GTPase–, mitogen-activated protein kinase–, and cyclin-dependent kinase- signaling were also regulated by phosphorylation upon infection. In particular, the influenza infection had a major influence on the phosphorylation profiles of a large number of cyclin-dependent kinase substrates. Functional studies using cyclin-dependent kinase inhibitors showed that the cyclin-dependent kinase activity is required for efficient viral replication and for activation of the host antiviral responses. In addition, we show that cyclin-dependent kinase inhibitors protect IAV-infected mice from death. In conclusion, we provide the first comprehensive phosphoproteome characterization of influenza A virus infection in primary human macrophages, and provide evidence that cyclin-dependent kinases represent potential therapeutic targets for more effective treatment of influenza infections.

HB-GAM (pleiotrophin) reverses inhibition of neural regeneration by the CNS extracellular matrix

Chondroitin sulfate (CS) glycosaminoglycans inhibit regeneration in the adult central nervous system (CNS). We report here that HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin), a CS-binding protein expressed at high levels in the developing CNS, reverses the role of the CS chains in neurite growth of CNS neurons in vitro from inhibition to activation. The CS-bound HB-GAM promotes neurite growth through binding to the cell surface proteoglycan glypican-2; furthermore, HB-GAM abrogates the CS ligand binding to the inhibitory receptor PTPσ (protein tyrosine phosphatase sigma). Our in vivo studies using two-photon imaging of CNS injuries support the in vitro studies and show that HB-GAM increases dendrite regeneration in the adult cerebral cortex and axonal regeneration in the adult spinal cord. Our findings may enable the development of novel therapies for CNS injuries.

Precision Cancer Medicine in the Acoustic Dispensing Era Ex Vivo Primary Cell Drug Sensitivity Testing

Cancer therapy is increasingly becoming individualized, but there are also big gaps between the molecular knowledge of individual cancers we can generate today and what can be applied in the clinic. In an attempt to bridge this knowledge gap between cancer genetic and molecular profiling and clinically useful information, an individualized systems medicine program has been established at the Institute for Molecular Medicine Finland (FIMM), University of Helsinki, and the Helsinki University Hospital. Central to this program is drug sensitivity and resistance testing (DSRT), in which responses of primary cancer cells to a comprehensive clinical oncology and signal transduction drug collection are monitored. The drug sensitivity information is used with molecular profiling to establish hypotheses on individual cancer-selective targeting drug combinations and their predictive biomarkers, which can be explored in the clinic. Here, we describe how acoustic droplet ejection is enabling DSRT in our cancer individualized systems medicine program to (1) generate consistent but configurable assay-ready plates and determine how this affects data quality, (2) flexibly prepare drug combination testing plates, (3) dispense reagents and cells to the assay plates, and (4) perform ultra-miniaturized follow-up assays on the cells from DSRT plates.

Methods for High-throughput Drug Combination Screening and Synergy Scoring

Gene products or pathways that are aberrantly activated in cancer but not in normal tissue hold great promises for being effective and safe anticancer therapeutic targets. Many targeted drugs have entered clinical trials but so far showed limited efficacy mostly due to variability in treatment responses and often rapidly emerging resistance. Toward more effective treatment options, we will need multi-targeted drugs or drug combinations, which selectively inhibit the viability and growth of cancer cells and block distinct escape mechanisms for the cells to become resistant. Functional profiling of drug combinations requires careful experimental design and robust data analysis approaches. At the Institute for Molecular Medicine Finland (FIMM), we have developed an experimental-computational pipeline for high-throughput screening of drug combination effects in cancer cells. The integration of automated screening techniques with advanced synergy scoring tools allows for efficient and reliable detection of synergistic drug interactions within a specific window of concentrations, hence accelerating the identification of potential drug combinations for further confirmatory studies.

Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins

Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.