Cellular vulnerabilities of glioblastoma

Glioblastoma (GB) is the most fatal and frequent malignant brain tumor, and it is driven by multiple oncogenic pathways. Despite intensive screening of genomic, transcriptomic, metabolic, and post-translational landscape of GB, targeted therapies have provided no improvements for the survival of GB patients. This incurability of GB is due to its infiltrative growth, intratumoral heterogeneity and intrinsic resistance towards treatment modalities which are driven by its subpopulations, such as glioblastoma stem cells (GSCs). Therefore, it is crucial to try to understand the mechanisms of GBs cellular resistance and potential vulnerabilities of GSCs. In this thesis we demonstrate alternative targets for GB therapy. Protein phosphatase 2A (PP2A) is inhibited in GB by non-genetic mechanisms, therefore, its therapeutic reactivation is possible. We described that small molecule reactivators of PP2A (SMAPs) efficiently cross the blood-brain barrier (BBB) and exhibit robust cytotoxicity towards heterogenous GB cell lines. Furthermore, we present specific kinases which inhibition induce synthetic lethality under PP2A reactivation. Collectively, these studies present SMAPs as a novel therapy for GB and propose an alternatives for multikinase inhibitors. In GB, nanoparticles have been researched for their potential to circumvent insufficient drug properties. However, opposed to traditional utilization of nanoparticles, we discovered an alternative use of them in GB. We demonstrated that mesoporous silica nanoparticles (MSNs) functionalized with polyethylenimine (PEI) induce cell death specifically in GSCs. The PEI-MSNs accumulated in the lysosomes of GSCs and caused lysosomal membrane permeabilization potentially through proton sponge effect. Furthermore, we determined that PEI-MSNs efficiently cross the BBB in mice. In summary, this thesis presents a novel therapy concepts for GB.Glioblastooman solutason haavoittuvuudet Glioblastooma (GB) on yleisin ja pahanlaatuisin aivosyöpä, jossa useat onkogeeniset signalointipolut ovat yliaktiivisia. Huolimatta genomiikan, transkriptomiikan, metabolomiikan ja translaation jälkeisten muutosten intensiivisestä seulonnasta GB:ssa, kohdennetut hoidot eivät ole tuottaneen lisäelinaikaa GB-potilaille. GB:n hoidon vaikeus johtuu sen infiltratiivisesta kasvusta, kasvaimen sisäisestä heterogeenisyydestä ja synnynnäisestä resistenssistä hoitoja vastaan. Syynä näihin on usein glioblastooman kantasolut. Tästä syystä, on erittäin tärkeää pyrkiä ymmärtämään GB:n solutason resistanssimekanismeja ja glioblastooman kantasolujen potentiaalisia heikkouksia. Tässä väitöskirjassa esitämme uusia kohteita GB:n hoitoon. GB:ssa proteiinifosfataasi 2A (PP2A) on estetty muilla tavoin kuin geneettisillä mekanismeilla. Tästä syystä sen terapeuttinen uudelleenaktivointi on mahdollista. Osoitimme tutkimuksissamme, että pienimolekyyliset PP2A aktivaattorit (SMAP) läpäisevät veri-aivoesteen ja ovat sytotoksisia GB:n heterogeenisiä solulinjoja kohtaan. Tämän lisäksi selvitimme, minkä kinaasien hiljentäminen altistaa GBsoluja entisestään PP2A:n aktivaatiolle. Yhteenvetona tutkimus esittää SMAP lääkkeet uutena terapiamuotona GB:n hoitoon ja ehdottaa vaihtoehtoja multikinaasiestäjille. Nanopartikkelitutkimus GB:aan liittyen on pääasiassa pyrkinyt parantamaan lääkkeiden ominaisuuksia. Me löysimme kuitenkin vaihtoehtoisen tavan käyttää nanopartikkeleita GB:ssa. Osoitimme, että mesohuokoiset piioksidi-nanopartikkelit, jotka on pinnoitettu polyetyyliemiinillä, aiheuttavat solukuoleman glioblastooman kantasoluissa. Kyseiset nanopartikkelit kerääntyivät glioblastooman kantasolujen lysosomeihin ja aiheuttivat sen membraanin tuhoutumisen ”proton sponge” efektin avulla. Kokonaisuudessaan väitöskirja esittää uusia heikkouksia glioblastooman kantasoluissa

Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells

Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially overcome kinase inhibitor resistance. However, in most cases the identification of the target kinases mediating therapeutic effects of multi-kinase inhibitors has been challenging. To tackle this important problem, we developed an actionable targets of multi-kinase inhibitors (AToMI) strategy and used it for characterization of glioblastoma target kinases of staurosporine derivatives displaying synergy with protein phosphatase 2A (PP2A) reactivation. AToMI consists of interchangeable modules combining drug-kinase interaction assay, siRNA high-throughput screening, bioinformatics analysis, and validation screening with more selective target kinase inhibitors. As a result, AToMI analysis revealed AKT and mitochondrial pyruvate dehydrogenase kinase PDK1 and PDK4 as kinase targets of staurosporine derivatives UCN-01, CEP-701, and K252a that synergized with PP2A activation across heterogeneous glioblastoma cells. Based on these proof-of-principle results, we propose that the application and further development of AToMI for clinically applicable multi-kinase inhibitors could provide significant benefits in overcoming the challenge of lack of knowledge of the target specificity of multi-kinase inhibitors.Peer reviewe

(2S, 4R)-4-[18F]Fluoroglutamine for In vivo PET Imaging of Glioma Xenografts in Mice: an Evaluation of Multiple Pharmacokinetic Models

Purpose: The glutamine analogue (2S, 4R)-4-[18F]fluoroglutamine ([18F]FGln) was investigated tofurther characterize its pharmacokinetics and acquire in vivo positron emission tomography (PET)images of separate orthotopic and subcutaneous glioma xenografts in mice.Procedures: [18F]FGln was synthesized at a high radiochemical purity as analyzed by high-performanceliquid chromatography. An orthotopic model was created by injecting luciferase-expressingpatient-derived BT3 glioma cells into the right hemisphere of BALB/cOlaHsd-Foxn1nu mousebrains (tumor growth monitored via in vivo bioluminescence), the subcutaneous model by injecting ratBT4C glioma cells into the flank and neck regions of Foxn1nu/nu mice. Dynamic PET images wereacquired after injecting 10–12 MBq of the tracer into mouse tail veins. Animals were sacrificed 63 minafter tracer injection, and ex vivo biodistributions were measured. Tumors and whole brains (with tumors)were cryosectioned, autoradiographed, and stained with hematoxylin-eosin. All images were analyzedwith CARIMAS software. Blood sampling of 6 Foxn1nu/nu and 6 C57BL/6J mice was performed after 9–14 MBq of tracer was injected at time points between 5 and 60 min then assayed for erythrocyte uptake,plasma protein binding, and plasma parent-fraction of radioactivity to correct PET image-derived whole-bloodradioactivity and apply the data to multiple pharmacokinetic models.Results: Orthotopic human glioma xenografts displayed PET image tumor-to-healthy brain region ratioof 3.6 and 4.8 while subcutaneously xenografted BT4C gliomas displayed (n = 12) a tumor-to-muscle(flank) ratio of 1.9 ± 0.7 (range 1.3–3.4). Using PET image-derived blood radioactivity corrected bypopulation-based stability analyses, tumor uptake pharmacokinetics fit Logan and Yokoi modeling forreversible uptake.Conclusions: The results reinforce that [18F]FGln has preferential uptake in glioma tissue versusthat of corresponding healthy tissue and fits well with reversible uptake models.</p

Monotherapy efficacy of blood-brain barrier permeable small molecule reactivators of protein phosphatase 2A in glioblastoma

Glioblastoma is a fatal disease in which most targeted therapies have clinically failed. However, pharmacological reactivation of tumour suppressors has not been thoroughly studied as yet as a glioblastoma therapeutic strategy. Tumour suppressor protein phosphatase 2A is inhibited by non-genetic mechanisms in glioblastoma, and thus, it would be potentially amendable for therapeutic reactivation. Here, we demonstrate that small molecule activators of protein phosphatase 2A, NZ-8-061 and DBK-1154, effectively cross the in vitro model of blood-brain barrier, and in vivo partition to mouse brain tissue after oral dosing. In vitro, small molecule activators of protein phosphatase 2A exhibit robust cell-killing activity against five established glioblastoma cell lines, and nine patient-derived primary glioma cell lines. Collectively, these cell lines have heterogeneous genetic background, kinase inhibitor resistance profile and stemness properties; and they represent different clinical glioblastoma subtypes. Moreover, small molecule activators of protein phosphatase 2A were found to be superior to a range of kinase inhibitors in their capacity to kill patient-derived primary glioma cells. Oral dosing of either of the small molecule activators of protein phosphatase 2A significantly reduced growth of infiltrative intracranial glioblastoma tumours. DBK-1154, with both higher degree of brain/blood distribution, and more potent in vitro activity against all tested glioblastoma cell lines, also significantly increased survival of mice bearing orthotopic glioblastoma xenografts. In summary, this report presents a proof-of-principle data for blood-brain barrier-permeable tumour suppressor reactivation therapy for glioblastoma cells of heterogenous molecular background. These results also provide the first indications that protein phosphatase 2A reactivation might be able to challenge the current paradigm in glioblastoma therapies which has been strongly focused on targeting specific genetically altered cancer drivers with highly specific inhibitors. Based on demonstrated role for protein phosphatase 2A inhibition in glioblastoma cell drug resistance, small molecule activators of protein phosphatase 2A may prove to be beneficial in future glioblastoma combination therapies.Peer reviewe

Circumventing drug treatment? Intrinsic lethal effects of polyethyleneimine (PEI)-functionalized nanoparticles on glioblastoma cells cultured in stem cell conditions

Glioblastoma (GB) is the most frequent malignant tumor originating from the central nervous system. Despite breakthroughs in treatment modalities for other cancer types, GB remains largely irremediable due to the high degree of intratumoral heterogeneity, infiltrative growth, and intrinsic resistance towards multiple treatments. A sub-population of GB cells, glioblastoma stem cells (GSCs), act as a reservoir of cancer-initiating cells and consequently, constitute a significant challenge for successful therapy. In this study, we discovered that PEI surface-functionalized mesoporous silica nanoparticles (PEI-MSNs), without any anti-cancer drug, very potently kill multiple GSC lines cultured in stem cell conditions. Very importantly, PEI-MSNs did not affect the survival of established GB cells, nor other types of cancer cells cultured in serum-containing medium, even at 25 times higher doses. PEI-MSNs did not induce any signs of apoptosis or autophagy. Instead, as a potential explanation for their lethality under stem cell culture conditions, we demonstrate that the internalized PEI-MSNs accumulated inside lysosomes, subsequently causing a rupture of the lysosomal membranes. We also demonstrate blood–brain-barrier (BBB) permeability of the PEI-MSNs in vitro and in vivo. Taking together the recent indications for the vulnerability of GSCs for lysosomal targeting and the lethality of the PEI-MSNs on GSCs cultured under stem cell culture conditions, the results enforce in vivo testing of the therapeutic impact of PEI-functionalized nanoparticles in faithful preclinical GB models.</p

Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells

Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially overcome kinase inhibitor resistance. However, in most cases the identification of the target kinases mediating therapeutic effects of multi-kinase inhibitors has been challenging. To tackle this important problem, we developed an actionable targets of multi-kinase inhibitors (AToMI) strategy and used it for characterization of glioblastoma target kinases of staurosporine derivatives displaying synergy with protein phosphatase 2A (PP2A) reactivation. AToMI consists of interchangeable modules combining drug-kinase interaction assay, siRNA high-throughput screening, bioinformatics analysis, and validation screening with more selective target kinase inhibitors. As a result, AToMI analysis revealed AKT and mitochondrial pyruvate dehydrogenase kinase PDK1 and PDK4 as kinase targets of staurosporine derivatives UCN-01, CEP-701, and K252a that synergized with PP2A activation across heterogeneous glioblastoma cells. Based on these proof-of-principle results, we propose that the application and further development of AToMI for clinically applicable multi-kinase inhibitors could provide significant benefits in overcoming the challenge of lack of knowledge of the target specificity of multi-kinase inhibitors

Adenosiini 2A-reseptoreiden PET-kuvantaminen neuroinflamaatiossa [11C]TMSX-merkkiaineella

Siirretty Doriast

Circumventing Drug Treatment? : Intrinsic Lethal Effects of Polyethyleneimine (PEI)-Functionalized Nanoparticles on Glioblastoma Cells Cultured in Stem Cell Conditions

Glioblastoma (GB) is the most frequent malignant tumor originating from the centralnervous system. Despite breakthroughs in treatment modalities for other cancer types, GB remainslargely irremediable due to the high degree of intratumoral heterogeneity, infiltrative growth, andintrinsic resistance towards multiple treatments. A sub-population of GB cells, glioblastoma stem cells(GSCs), act as a reservoir of cancer-initiating cells and consequently, constitute a significant challengefor successful therapy. In this study, we discovered that PEI surface-functionalized mesoporoussilica nanoparticles (PEI-MSNs), without any anti-cancer drug, very potently kill multiple GSClines cultured in stem cell conditions. Very importantly, PEI-MSNs did not affect the survival ofestablished GB cells, nor other types of cancer cells cultured in serum-containing medium, even at25 times higher doses. PEI-MSNs did not induce any signs of apoptosis or autophagy. Instead, asa potential explanation for their lethality under stem cell culture conditions, we demonstrate thatthe internalized PEI-MSNs accumulated inside lysosomes, subsequently causing a rupture of thelysosomal membranes. We also demonstrate blood–brain-barrier (BBB) permeability of the PEI-MSNs in vitroandin vivo. Taking together the recent indications for the vulnerability of GSCs for lysosomaltargeting and the lethality of the PEI-MSNs on GSCs cultured under stem cell culture conditions,the results enforcein vivotesting of the therapeutic impact of PEI-functionalized nanoparticles infaithful preclinical GB models.Peer reviewe

Monotherapy efficacy of blood-brain barrier permeable small molecule reactivators of protein phosphatase 2A in glioblastoma

Contains fulltext : 226259.pdf (publisher's version ) (Open Access

PP2A-based triple-strike therapy overcomes mitochondrial apoptosis resistance in brain cancer cells

Mitochondrial glycolysis and hyperactivity of the phosphatidylinositol 3-kinase-protein kinase B (AKT) pathway are hallmarks of malignant brain tumors. However, kinase inhibitors targeting AKT (AKTi) or the glycolysis master regulator pyruvate dehydrogenase kinase (PDKi) have failed to provide clinical benefits for brain tumor patients. Here, we demonstrate that heterogeneous glioblastoma (GB) and medulloblastoma (MB) cell lines display only cytostatic responses to combined AKT and PDK targeting. Biochemically, the combined AKT and PDK inhibition resulted in the shutdown of both target pathways and priming to mitochondrial apoptosis but failed to induce apoptosis. In contrast, all tested brain tumor cell models were sensitive to a triplet therapy, in which AKT and PDK inhibition was combined with the pharmacological reactivation of protein phosphatase 2A (PP2A) by NZ-8-061 (also known as DT-061), DBK-1154, and DBK-1160. We also provide proof-of-principle evidence for in vivo efficacy in the intracranial GB and MB models by the brain-penetrant triplet therapy (AKTi + PDKi + PP2A reactivator). Mechanistically, PP2A reactivation converted the cytostatic AKTi + PDKi response to cytotoxic apoptosis, through PP2A-elicited shutdown of compensatory mitochondrial oxidative phosphorylation and by increased proton leakage. These results encourage the development of triple-strike strategies targeting mitochondrial metabolism to overcome therapy tolerance in brain tumors.Peer reviewe