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Supplementary Materials and Methods

Figure S2, related to Figure 2. Comparison of glioma development induced in neonatal and adult G/tv-a and C/tv-a wt mice.

Table S1, related to Table 2. Mean AUC values were calculated from all dose-response curves generated for each group.

Figure S1, related to Figure 1. Double immunofluorescence stainings for GFP and chicken vimentin of neonatal G/tv-a and C/tv-a mouse brains infected with RCAS-eGFP.

Figure S3, related to Figure 4E. Representative photographs of double immunofluorescence stainings of GCs cultured in presence or absence of serum for 7 days.

Figure S4, related to Figure 4. Characterization of mouse neural stem cell cultures CCG/tv-a and CCC/tv-a.

The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors. Helleday and colleagues describe a nanomolar MTHFD2 inhibitor that causes replication stress and DNA damage accumulation in cancer cells via thymidine depletion, demonstrating a potential therapeutic strategy in AML tumors in vivo.

Supplementary methods for the article "Pharmacological targeting of MTHFD2 suppresses acute myeloid leukemia by inducing thymidine depletion and replication stress".

Glioblastoma is a devastating disease with an overall median survival of 8 months from diagnosis. The majority of patients die of a tumor relapse in close proximity of the resected primary tumor. Glioblastoma is intensely researched but most studies have been performed on tissues and cultures derived from the bulk tumor while it is the remaining edge cells that cause lethality. Investigations of edge cells are rare and few experimental models exist. Here we have established and analyzed a series of matched cell cultures derived from the tumor bulk and outer edge of six IDH wildtype glioblastoma patients with the purpose to understand glioblastoma edge cell biology. Tumor samples were resected guided by 5-ALA fluorescence using neuro-navigation and stringent procedures to not contaminate edge samples with bulk tumor cells. First bulk tumor samples were resected from 5-ALA fluorescent tissue. After removal of all fluorescent areas and careful irrigation of the cavity the edge sample was resected 1-2 cm outside of the fluorescent border in a non-eloquent area. Following dissociation the samples were used in sphere assays and for explantation. There was a significant difference in self-renewal across all patients between matched bulk and edge cultures, in line with results from sphere assays on acute cells, suggesting maintenance of glioblastoma cell properties in established cultures. Invasion analysis showed a reverse significant difference between matched bulk and edge cultures strengthening a general functional distinction between tumor bulk and edge cells across patients. To investigate the molecular basis of our findings we performed whole exome sequencing (WES) and combined single cell RNA- and ATAC-sequencing (10X Multiome). Analyses are ongoing but WES data does not support genetic causes for their differences while the 10X Multiome data indicate that epigenetic regulation may underlie the different properties of bulk and edge glioblastoma cells.

Grade IV astrocytoma/glioblastoma multiforme (GBM) is essentially incurable, partly due to its heterogenous nature, demonstrated even within the glioma-initiating cell (GIC) population. Increased therapy resistance of GICs is coupled to transition into a mesenchymal (MES) cell state. The GBM MES molecular signature displays a pronounced inflammatory character and its expression vary within and between tumors. Herein, we investigate how MES transition of GBM cells relates to inflammatory responses of normal astroglia. In response to CNS insults astrocytes enter a reactive cell state and participate in directing neuroinflammation and subsequent healing processes. We found that the MES signature show strong resemblance to gene programs induced in reactive astrocytes. Likewise, astrocyte reactivity gene signatures were enriched in therapy-resistant MES-like GIC clones. Variable expression of astrocyte reactivity related genes also largely defined intratumoral GBM cell heterogeneity at the single-cell level and strongly correlated with our previously defined therapy-resistance signature (based on linked molecular and functional characterization of GIC clones). In line with this, therapy-resistant MES-like GIC secreted immunoregulatory and tissue repair related proteins characteristic of astrocyte reactivity. Moreover, sensitive GIC clones could be made reactive through long-term exposure to the proinflammatory cytokine interleukin 1 beta (IL1β). IL1β induced a slow MES transition, increased therapy resistance, and a shift in DNA methylation profile towards that of resistant clones, which confirmed a slow reprogramming process. In summary, GICs enter through MES transition a reactive-astrocyte-like cell state, connected to therapy resistance. Thus, from a biological point of view, MES GICs would preferably be called 'reactive GICs'. The ability of GBM cells to mimic astroglial reactivity contextualizes the immunomodulatory and microenvironment reshaping abilities of GBM cells that generate a tumor-promoting milieu. This insight will be important to guide the development of future sensitizing therapies targeting treatment-resistant relapse-driving cell populations as well as enhancing the efficiency of immunotherapies in GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor which lacks efficient treatment and predictive biomarkers. Expression of the epithelial stem cell marker Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) has been described in GBM, but its functional role has not been conclusively elucidated. Here, we have investigated the role of LGR5 in a large repository of patient-derived GBM stem cell (GSC) cultures. The consequences of LGR5 overexpression or depletion have been analyzed using in vitro and in vivo methods, which showed that, among those with highest LGR5 expression (LGR5high ), there were two phenotypically distinct groups: one that was dependent on LGR5 for its malignant properties and another that was unaffected by changes in LGR5 expression. The LGR5-responding cultures could be identified by their significantly higher self-renewal capacity as measured by extreme limiting dilution assay (ELDA), and these LGR5high -ELDAhigh cultures were also significantly more malignant and invasive compared to the LGR5high -ELDAlow cultures. This showed that LGR5 expression alone would not be a strict marker of LGR5 responsiveness. In a search for additional biomarkers, we identified LPAR4, CCND2, and OLIG2 that were significantly upregulated in LGR5-responsive GSC cultures, and we found that OLIG2 together with LGR5 were predictive of GSC radiation and drug response. Overall, we show that LGR5 regulates the malignant phenotype in a subset of patient-derived GSC cultures, which supports its potential as a predictive GBM biomarker. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

There is ample support for developmental regulation of glioblastoma stem cells (GSCs). To examine how cell lineage controls GSC function we have performed a cross-species epigenome analysis of mouse and human GSC cultures. We have analyzed and compared the chromatin-accessibility landscape of nine mouse GSC cultures of defined cell of origin and 60 patient-derived GSC cultures by assay for transposase-accessible chromatin using sequencing (ATAC-seq). This uncovered a variability of both mouse and human GSC cultures that was different from transcriptome analysis and better at predicting functional subgroups. In both species the chromatin accessibility-guided clusters were predominantly determined by distal regulatory element (DRE) regions, displayed contrasting sets of transcription factor binding motifs, and exhibited different functional and drug-response properties. Cross-species analysis of DRE regions in accessible chromatin revealed conserved epigenetic regulation of mouse and human GSCs. Human ATAC-seq data produced three distinct clusters with significant overlap to our previous mouse cell of origin- based stratification, where two of the clusters displayed significantly different patient survival. We conclude that epigenetic regulation of GSCs primarily is dictated by developmental origin which controls key GSC properties and affects therapeutic response.

We recently showed that the anti-helminthic compound mebendazole (MBZ) has immunomodulating activity in monocyte/macrophage models and induces ERK signalling. In the present study we investigated whether MBZ induced ERK activation is shared by other tubulin binding agents (TBAs) and if it is observable also in other human cell types. Curated gene signatures for a panel of TBAs in the LINCS Connectivity Map (CMap) database showed a unique strong negative correlation of MBZ with MEK/ERK inhibitors indicating ERK activation also in non-haematological cell lines. L1000 gene expression signatures for MBZ treated THP-1 monocytes also connected negatively to MEK inhibitors. MEK/ERK phosphoprotein activity testing of a number of TBAs showed that only MBZ increased the activity in both THP-1 monocytes and PMA differentiated macrophages. Distal effects on ERK phosphorylation of the substrate P90RSK and release of IL1B followed the same pattern. The effect of MBZ on MEK/ERK phosphorylation was inhibited by RAF/MEK/ERK inhibitors in THP-1 models, CD3/IL2 stimulated PBMCs and a MAPK reporter HEK-293 cell line. MBZ was also shown to increase ERK activity in CD4+ T-cells from lupus patients with known defective ERK signalling. Given these mechanistic features MBZ is suggested suitable for treatment of diseases characterized by defective ERK signalling, notably difficult to treat autoimmune diseases.

We and others have previously reported a correlation between high phosphodiesterase 3A (PDE3A) expression and selective sensitivity to phosphodiesterase (PDE) inhibitors. This indicates that PDE3A could serve both as a drug target and a biomarker of sensitivity to PDE3 inhibition. In this report, we explored publicly available mRNA gene expression data to identify cell lines with different PDE3A expression. Cell lines with high PDE3A expression showed marked in vitro sensitivity to PDE inhibitors zardaverine and quazinone, when compared with those having low PDE3A expression. Immunofluorescence and immunohistochemical stainings were in agreement with PDE3A mRNA expression, providing suitable alternatives for biomarker analysis of clinical tissue specimens. Moreover, we here demonstrate that tumor cells from patients with ovarian carcinoma show great variability in PDE3A protein expression and that level of PDE3A expression is correlated with sensitivity to PDE inhibition. Finally, we demonstrate that PDE3A is highly expressed in subsets of patient tumor cell samples from different solid cancer diagnoses and expressed at exceptional levels in gastrointestinal stromal tumor (GIST) specimens. Importantly, vulnerability to PDE3 inhibitors has recently been associated with co-expression of PDE3A and Schlafen family member 12 (SLFN12). We here demonstrate that high expression of PDE3A in clinical specimens, at least on the mRNA level, seems to be frequently associated with high SLFN12 expression. In conclusion, PDE3A seems to be both a promising biomarker and drug target for individualized drug treatment of various cancers.

There is ample support for developmental control of glioblastoma stem cells (GSCs), and a deeper knowledge of their epigenetic regulation could be central to more efficient glioblastoma (GBM) therapies. For this purpose, we analyzed the chromatin-accessibility landscape of nine mouse GSC cultures of defined cell of origin and 60 patient-derived GSC cultures by assay for transposase-accessible chromatin using sequencing (ATAC-seq). This uncovered an epigenetic variability of both mouse and human GSC cultures that differed from transcriptome clusters. Both mouse and human chromatin accessibility-guided clusters were predominantly determined by distal regulatory elements, displayed unique sets of transcription factor motif enrichment, and exhibited different functional and drug-response properties. Cross-species analysis of distal regulatory element regions in accessible chromatin of mouse and human cultures revealed conserved epigenetic regulation of GSCs.

High-grade glioma (HGG) is a group of primary malignant brain tumors with dismal prognosis. Whereas adult HGG has been studied extensively, childhood HGG, a relatively rare disease, is less well-characterized. Here, we present two novel platelet-derived growth factor (PDGF)-driven mouse models of pediatric supratentorial HGG. Tumors developed from two different cells of origin reminiscent of neural stem cells (NSC) or oligodendrocyte precursor cells (OPC). Cross-species transcriptomics showed that both models are closely related to human pediatric HGG as compared with adult HGG. Furthermore, an NSC-like cell-of-origin enhanced tumor incidence, malignancy, and the ability of mouse glioma cells (GC) to be cultured under stem cell conditions as compared with an OPC-like cell. Functional analyses of cultured GC from these tumors showed that cells of NSC-like origin were more tumorigenic, had a higher rate of self-renewal and proliferation, and were more sensitive to a panel of cancer drugs compared with GC of a more differentiated origin. These two mouse models relevant to human pediatric supratentorial HGG propose an important role of the cell-of-origin for clinicopathologic features of this disease. Cancer Res; 77(3); 802–12. ©2016 AACR.

The identity of the glioblastoma (GBM) cell of origin and its contributions to disease progression and treatment response remain largely unknown. We have analyzed how the phenotypic state of the initially transformed cell affects mouse GBM development and essential GBM cell (GC) properties. We find that GBM induced in neural stem-cell-like glial fibrillary acidic protein (GFAP)-expressing cells in the subventricular zone of adult mice shows accelerated tumor development and produces more malignant GCs (mGC1GFAP) that are less resistant to cancer drugs, compared with those originating from more differentiated nestin- (mGC2NES) or 2,′3′-cyclic nucleotide 3′-phosphodiesterase (mGC3CNP)-expressing cells. Transcriptome analysis of mouse GCs identified a 196 mouse cell origin (MCO) gene signature that was used to partition 61 patient-derived GC lines. Human GC lines that clustered with the mGC1GFAP cells were also significantly more self-renewing, tumorigenic, and sensitive to cancer drugs compared with those that clustered with mouse GCs of more differentiated origin.

Background Pharmacological treatment of complex diseases using more than two drugs is commonplace in the clinic due to better efficacy, decreased toxicity and reduced risk for developing resistance. However, many of these higher-order treatments have not undergone any detailed preceding in vitro evaluation that could support their therapeutic potential and reveal disease related insights. Despite the increased medical need for discovery and development of higher-order drug combinations, very few reports from systematic large-scale studies along this direction exist. A major reason is lack of computational tools that enable automated design and analysis of exhaustive drug combination experiments, where all possible subsets among a panel of pre-selected drugs have to be evaluated. Results Motivated by this, we developed COMBImage2, a parallel computational framework for higher-order drug combination analysis. COMBImage2 goes far beyond its predecessor COMBImage in many different ways. In particular, it offers automated 384-well plate design, as well as quality control that involves resampling statistics and inter-plate analyses. Moreover, it is equipped with a generic matched filter based object counting method that is currently designed for apoptotic-like cells. Furthermore, apart from higher-order synergy analyses, COMBImage2 introduces a novel data mining approach for identifying interesting temporal response patterns and disentangling higher- from lower- and single-drug effects. COMBImage2 was employed in the context of a small pilot study focused on the CUSP9v4 protocol, which is currently used in the clinic for treatment of recurrent glioblastoma. For the first time, all 246 possible combinations of order 4 or lower of the 9 single drugs consisting the CUSP9v4 cocktail, were evaluated on an in vitro clonal culture of glioma initiating cells. Conclusions COMBImage2 is able to automatically design and robustly analyze exhaustive and in general higher-order drug combination experiments. Such a versatile video microscopy oriented framework is likely to enable, guide and accelerate systematic large-scale drug combination studies not only for cancer but also other diseases. Electronic supplementary material The online version of this article (10.1186/s12859-019-2908-0) contains supplementary material, which is available to authorized users.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor which lacks efficient treatment and predictive biomarkers. Expression of the epithelial stem cell marker Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) has been described in GBM, but its functional role has not been conclusively elucidated. Here, we have investigated the role of LGR5 in a large repository of patient-derived GBM stem cell (GSC) cultures. The consequences of LGR5 overexpression or depletion have been analyzed using in vitro and in vivo methods, which showed that, among those with highest LGR5 expression (LGR5

Mebendazole (MBZ), a drug commonly used for helminitic infections, has recently gained substantial attention as a repositioning candidate for cancer treatment. However, the mechanism of action behind its anticancer activity remains unclear. To address this problem, we took advantage of the curated MBZ-induced gene expression signatures in the LINCS Connectivity Map (CMap) database. The analysis revealed strong negative correlation with MEK/ERK1/2 inhibitors. Moreover, several of the most upregulated genes in response to MBZ exposure were related to monocyte/macrophage activation. The MBZ-induced gene expression signature in the promyeloblastic HL-60 cell line was strongly enriched in genes involved in monocyte/macrophage pro-inflammatory (M1) activation. This was subsequently validated using MBZ-treated THP-1 monocytoid cells that demonstrated gene expression, surface markers and cytokine release characteristic of the M1 phenotype. At high concentrations MBZ substantially induced the release of IL-1β and this was further potentiated by lipopolysaccharide (LPS). At low MBZ concentrations, cotreatment with LPS was required for MBZ-stimulated IL-1β secretion to occur. Furthermore, we show that the activation of protein kinase C, ERK1/2 and NF-kappaB were required for MBZ-induced IL-1β release. MBZ-induced IL-1β release was found to be dependent on NLRP3 inflammasome activation and to involve TLR8 stimulation. Finally, MBZ induced tumor-suppressive effects in a coculture model with differentiated THP-1 macrophages and HT29 colon cancer cells. In summary, we report that MBZ induced a pro-inflammatory (M1) phenotype of monocytoid cells, which may, at least partly, explain MBZ's anticancer activity observed in animal tumor models and in the clinic.

Proximity ligation assay (PLA) is a unique method in which single-stranded oligonucleotides are conjugated to affinity binders of proteins, followed by amplification of the signal by DNA polymerization and hybridization of complementary oligonucleotides labeled with fluorogenic or chromogenic readout. Here, a brief overview of the field of protein analysis describes the background and the initial development of the technique for the detection of protein–protein interactions via the proximity probes mentioned. In this context, PLA can constrain the general problem of cross-reactivity in protein detection by affinity binders, by ensuring that only cognate pairs of proximity probes result in a signal. Thereafter, this chapter deals mainly with derivatives methods and their applications, with a particular interest in improved specificity, application to various biological materials, and multiplexing. The method has been applied in situ and in solution, adapted for the detection of posttranslational modifications such as phosphorylation and interactions between proteins and specific DNA sequences, and multiplexed to a certain extent, which illustrates its versatility. A technique free from enzymatic reaction, the hybridization chain reaction, can be considered a cost-effective alternative particularly suitable to molecular diagnostics. Finally, we explore further development toward higher-level multiplexing and sensitivity. At this point it is not clear what level can be achieved by PLA, but the assay is compatible with a wide range of readout, including separate real-time amplification reactions and novel microfluidic read-out platforms.

AKN-028 is a novel tyrosine kinase inhibitor with preclinical activity in acute myeloid leukemia (AML), presently undergoing investigation in a phase I/II study. It is a potent inhibitor of the FMS-like kinase 3 (FLT3) but shows in vitro activity in a wide range of AML samples. In the present study, we have characterized the effects of AKN-028 on AML cells in more detail. AKN-028 induced a dose-dependent G0/1 arrest in AML cell line MV4-11. Treatment with AKN-028 caused significantly altered gene expression in all AML cell types tested (430 downregulated, 280 upregulated transcripts). Subsequent gene set enrichment analysis revealed enrichment of genes associated with the proto-oncogene and cell cycle regulator c-Myc among the downregulated genes in both AKN-028 and midostaurin treated cells. Kinase activity profiling in AML cell lines and primary AML samples showed that tyrosine kinase activity, but not serine/threonine kinase activity, was inhibited by AKN-028 in a dose dependent manner in all samples tested, reaching approximately the same level of kinase activity. Cells sensitive to AKN-028 showed a higher overall tyrosine kinase activity than more resistant ones, whereas serine/threonine kinase activity was similar for all primary AML samples.In summary, AKN-028 induces cell cycle arrest in AML cells, downregulates Myc-associated genes and affect several signaling pathways. AML cells with high global tyrosine kinase activity seem to be more sensitive to the cytotoxic effect of AKN-028 in vitro.

LSD1 has emerged as a potential therapeutic target for a number of cancer types (e.g. AML, SCLC, colorectal, breast, liver, prostate, glioblastoma, Ewing sarcoma), as well as sickle cell anaemia and Alzheimer’s disease. Irreversible LSD1 catalytic inhibitors have shown limited clinical efficacy in AML and SCLC, while solid tumours are largely unaddressed. This is in contrast to LSD1 knockdown with impact on a wide range of cancers, indicating that LSD1 functions other than enzymatic should be targeted. We have developed novel small molecules that modulate LSD1 via an allosteric site - without inhibiting its enzymatic function - inducing a 60% reduction of nuclear LSD1 levels. The sensitivity profile in a cancer cell line panel is unique and dissimilar to >300 diverse reference compounds. In vitro efficacy is observed in glioma-initiating clones that are highly resistant to standard-of-care temozolimide as well as catalytic LSD1 inhibitors. Efficacy in the sub-µM range is observed with other solid tumour models, e.g. prostate cancer. The compounds exhibit synergy (Bliss independence >40%) with HDAC inhibitors as evaluated by viability in cellular cancer models, including lung, liver and glioblastoma. Pharmacokinetic studies show good blood-brain-barrier penetration and oral availability of the allosteric LSD1 modulator BEA-17. A repeat dose of 25 mg/kg was well tolerated by NOD SCID mice, leading to µM level accumulation in the brain. Results from orthotopic glioblastoma PDX models, and in vivo hollow-fiber models of other solid tumours will be presented, as well as mechanistic insights from biophysical assays and gene expression analysis. Citation Format: Wei B. Emond, Matthis Geitmann, Malin Jarvius, Konrad Koehler, Per Källblad, Mia Niklasson, Vendela Parrow, Rajiv Sawant, Maria Sjöberg, Johan Winquist, Anna Segerman, Ulf Bremberg. LSD1 modulation by allosteric ligands [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3843.

Cancer cell lines grown as two-dimensional (2D) cultures have been an essential model for studying cancer biology and anticancer drug discovery. However, 2D cancer cell cultures have major limitations, as they do not closely mimic the heterogeneity and tissue context of in vivo tumors. Developing three-dimensional (3D) cell cultures, such as multicellular tumor spheroids, has the potential to address some of these limitations. Here, we combined a high-throughput gene expression profiling method with a tumor spheroid-based drug-screening assay to identify context-dependent treatment responses. As a proof of concept, we examined drug responses of quiescent cancer cells to oxidative phosphorylation (OXPHOS) inhibitors. Use of multicellular tumor spheroids led to discovery that the mevalonate pathway is upregulated in quiescent cells during OXPHOS inhibition, and that OXPHOS inhibitors and mevalonate pathway inhibitors were synergistically toxic to quiescent spheroids. This work illustrates how 3D cellular models yield functional and mechanistic insights not accessible via 2D cultures.

Choroid plexus tumors are intraventricular neoplasms predominantly affecting young children. In contrast to choroid plexus papillomas, choroid plexus carcinomas progress frequently, necessitating the development of adjuvant treatment concepts. Platelet derived growth factor (PDGF) signaling has been shown to support growth in a variety of tumors. The finding of PDGF receptor expression in choroid plexus tumors prompted us to elucidate PDGF receptor activation state using a novel method, in situ proximity ligation assay, on formalin-fixed, paraffin-embedded, archival samples of 19 choroid plexus tumors. As assessed by in situ proximity ligation assay, the proportion of phosphorylated PDGF receptor alpha was low in choroid plexus papillomas and choroid plexus carcinomas, whereas phosphorylated PDGF receptor beta was found to be significantly higher in choroid plexus carcinomas. In the immortalized choroid plexus epithelial cell line Z310 expressing PDGF receptor beta, PDGF-BB exhibited a time- and dose-dependent proliferative response, which was significantly attenuated by imatinib (gleevec). In conclusion, our findings suggest that PDGF receptor beta is functionally involved in the biology of choroid plexus tumors and may represent a molecular target for therapy. In addition, this study demonstrates the feasibility and usefulness of in situ proximity ligation assay for monitoring receptor tyrosine kinase activation in formalin-fixed, paraffin-embedded, archival tissues.

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor beta (PDGFRbeta) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRbeta in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRbeta in porcine aortic endothelial cells transfected with the beta-receptor, but not in cells transfected with the alpha-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRbeta. We furthermore visualized tyrosine phosphorylated PDGFRbeta in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.

CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.

Intratumoral heterogeneity is a hallmark of glioblastoma multiforme and thought to negatively affect treatment efficacy. Here, we establish libraries of glioma-initiating cell (GIC) clones from patient samples and find extensive molecular and phenotypic variability among clones, including a range of responses to radiation and drugs. This widespread variability was observed as a continuum of multitherapy resistance phenotypes linked to a proneural-mesenchymal shift in the transcriptome. Multitherapy resistance was associated with a semi-stable cell state that was characterized by an altered DNA methylation pattern at promoter regions of mesenchymal master regulators and enhancers. The gradient of cell states within the GIC compartment constitutes a distinct form of heterogeneity. Our findings may open an avenue toward the development of new therapeutic rationales designed to reverse resistant cell states.

To find drugs suitable for repositioning for use against leukemia, samples from patients with chronic lymphocytic, acute myeloid and lymphocytic leukemias as well as peripheral blood mononuclear cells (PBMC) were tested in response to 1266 compounds from the LOPAC1280 library (Sigma). Twenty-five compounds were defined as hits with activity in all leukemia subgroups (M drug concentration. Only one of these compounds, quinacrine, showed low activity in normal PBMCs and was therefore selected for further preclinical evaluation. Mining the NCI-60 and the NextBio databases demonstrated leukemia sensitivity and the ability of quinacrine to reverse myeloid leukemia gene expression. Mechanistic exploration was performed using the NextBio bioinformatic software using gene expression analysis of drug exposed acute myeloid leukemia cultures (HL-60) in the database. Analysis of gene enrichment and drug correlations revealed strong connections to ribosomal biogenesis nucleoli and translation initiation. The highest drug–drug correlation was to ellipticine, a known RNA polymerase I inhibitor. These results were validated by additional gene expression analysis performed in-house. Quinacrine induced early inhibition of protein synthesis supporting these predictions. The results suggest that quinacrine have repositioning potential for treatment of acute myeloid leukemia by targeting of ribosomal biogenesis.

Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes-oligonucleotides attached to antibodies against the two target proteins-guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-gamma (IFN-gamma) signaling and low-molecular-weight inhibitors.

Glioblastoma Multiforme (GBM) is the most frequent adult primary malignant brain tumor that remains incurable despite aggressive treatment. The cell of origin (COO) for GBM is unknown but assumed to be a glial stem or progenitor cell. GBM harbours hierarchical tumor cells called glioma stem cells (GSCs) that maintain tumor growth, drive tumor progression and cause tumor relapse due to their increased resistance to therapy. We have analyzed the significance of cellular origin for GBM development and GSC properties by comparing mouse GBMs and GSCs derived thereof induced in neural stem cells (NSCs), glial-restricted precursor cells (GPCs) or oligodendrocyte precursor cells (OPCs) by identical mutations. There were striking differences in GBM development and the phenotypes of GSCs and their response to drugs owing to the COO. Global gene expression analysis of mouse GSC lines displayed a clear separation due to COO and differential gene expression analysis identified a COO gene signature of 175 genes. Cross-species bioinformatics analyses were performed. First we analyzed the human cancer genome atlas (TCGA) GBM tissue samples and the mouse GSC expression data for a collection of TCGA GBM subtype signature genes. This showed that we could model both Proneural and Mesenchymal GBMs in mice by merely switching the COO. Next, we used the mouse COO gene signature to stratify a large number of newly established human glioma stem cell lines. This produced two groups of human GSCs; the NSC origin group and the progenitor cell (PC) origin group in which the mouse GPC- and OPC-derived genes were combined. Importantly, patient survival was significantly different between the NSC and PC COO groups with a better prognosis for the PC group patients. Thus, the cell of origin is essential for GBM biology and needs to be considered for more accurate patient stratification, target identification and drug discovery.

The overall aim of this study was to find ways to sensitize treatment resistant glioma-initiating cell (GIC) clones to conventional therapy. Heterogeneity is substantial in glioblastoma multiforme (GBM) and through establishment of clonal GIC cultures from patient biopsies we have demonstrated a wide variety in the responses to drugs and radiation. A multitherapy resistance phenotype was linked to proneural-mesenchymal transition (PMT) in the transcriptome. The variety in therapy response was observed as a continuum of phenotypes. The distribution of phenotypes resembled a normal distribution and multitherapy resistance was associated with low DNA methylation grade in promoter regions of mesenchymal master regulators (FOSL2, RUNX1). Our data thus implied that the transition is bi-directional and epigenetically regulated (Segerman et al, Cell Reports - accepted in principle). To investigate if spontaneous changes in drug and radiation response occur, we have derived subclones from a resistant clone. Both subclones with higher and lower therapy resistance than the parental clone were generated. Also molecularly the subclones largely reconstituted the original clonal variation. PMT shows similarities to epithelial-mesenchymal transition (EMT), which is induced by extrinsic factors. We therefore specifically analyzed the gene expression data for signaling receptors differentially expressed in resistant vs. sensitive clones as well as cognate ligands. To estimate the importance of a particular signaling pathway, expression of co-receptors and ligands was taken into account. We found several cases of coherent upregulation of receptor and ligand indicative of autocrine loops. Regarding pathways that appeared to be overactive in resistant vs. sensitive clones, there was a prominent overlap with EMT. It was also apparent that several pathways were activated concomitantly. We are currently focusing on identifying combinations of drugs (and antibodies) that sensitize resistant clones to conventional treatment through modulation of cell signaling patterns. In the initial screen temozolomide (TMZ) response is used as an indicator of achieved sensitization. The strategy is to iteratively combine primarily antagonists of signaling receptors connected to resistance. We are also exploring the effect of stimulating pathways with apparently higher activity in sensitive clones (e.g. addition of ligands).The concept of sensitizing glioma and other types of cancer cells by targeting the mesenchymal character through usage of e.g. signaling receptor inhibitors is not new and has shown promise. Indeed, our preliminary data look encouraging. In conclusion, our data show that multitherapy resistance is connected to a plastic cell-state. Also, receptors and ligands that are differentially expressed in resistant and sensitive clones engage pathways regulating EMT. Citation Format: Mia Niklasson, Malin Jarvius, Caroline Haglund, Efthymia Chantzi, Tobias Bergström, Frida Nyberg, Annika Hermansson, Mårten Fryknäs, Mats Gustafsson, Bo Segerman, Rolf Larsson, Bengt Westermark, Anna Segerman. Targeting of a mesenchymal profile in order to sensitize multitherapy resistant glioblastoma clones [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4175. doi:10.1158/1538-7445.AM2017-4175

Monolayer, two-dimensional (2D) cell cultures have been a predominant in vitro model in anticancer drug discovery and high-throughput screening (HTS). However, 2D cultures of cancer cells lack numerous properties of in vivo tumors, such as tissue-like structure, cell-cell interactions and nutrient/oxygen gradients. Thus, in recent years there has been an increased interest in 3D cell cultures, such as multicellular tumors spheroids (MCTS), to address some of these limitations. Recently, we and others have applied MCTS for HTS and identified oxidative phosphorylation (OXPHOS) as a selective vulnerability of quiescent cancer cells persisting in hypoxic and nutrient-deprived milieu. However, prolonged continuous exposure to OXPHOS inhibitors is necessary for the cytotoxic effect. Thus, there is a need to identify processes that could be co-targeted for enhanced anticancer activity. Here, we present two distinct HTS approaches to identify combination partner molecules for OXPHOS inhibitors. Since we were interested in targeting non-dividing nutrient-deprived cancer cells, we used quiescent MCTS (Q-MCTS), as an in vitro model. Cells in Q-MCTS experience glucose concentrations and pH similar to those observed in deep tumor parenchyma in vivo. In our first screening approach, we have applied high-throughput gene-expression profiling to study drug effects in MCTS at a large scale. Using L1000 Gene Expression Profiling method, we generated a dataset of over 1000 drug-induced gene-expression profiles and found that co-targeting of OXPHOS and the mevalonate pathway results in selective synergistic toxicity in quiescent cancer cells. In the other approach, we screened a library of 1650 biologically active compounds, with or without addition of the FDA-approved anthelmintic agent nitazoxanide (an OXPHOS inhibitor with high drug repurposing potential). After the screen, we selected molecules that demonstrated pronounced synergy when combined with nitazoxanide, but not when used alone. Then, we validated the hits in an extensive dose-response combination experiments in Q-MCTS and chose 14 compounds that demonstrated strong synergistic interaction with nitazoxanide at broad range of concentrations. These included antifungal agents, kinase inhibitors and others. In summary, we here report on novel approaches, utilizing 3D cell cultures, to identify drug combinations targeting quiescent cancer cells. By high-throughput gene-expression profiling and large-scale combinatorial drug screens, we were able to identify drug combinations preferentially toxic to quiescent cells. This work also demonstrates how 3D cell cultures yield functional insights that are not accessible through standard 2D cultures. Citation Format: Wojciech Senkowski, Madiha Nazir, Malin Jarvius, Jenny Rubin, Johan Lengqvist, Mats G. Gustafsson, Peter Nygren, Kim Kultima, Rolf Larsson, Mårten Fryknäs. High-throughput drug combination screening in tumor spheroids identifies context-dependent synthetic lethalities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4990. doi:10.1158/1538-7445.AM2017-4990

Inhibition of mitochondrial oxidative phosphorylation (OXPHOS) has recently emerged as a promising strategy for treatment of therapy-resistant cancer cells. These cells often reside within hypoxic tumor regions, where nutrient concentrations are low. Recently, OXPHOS inhibitors have been demonstrated to be toxic to quiescent, nutrient-deprived cells in multicellular tumor spheroids. Such spheroids, formed without medium exchange over the culture period, can serve as an appropriate model to mimic quiescent in vivo tumor regions. These spheroids exhibit low cell proliferation and comprise necrotic cores, contrary to commonly used spheroids cultured with frequent medium change. We here aimed to characterize how quiescent cells respond to OXPHOS inhibition and thereby identify processes that could be co-targeted for enhanced toxicity. We treated HCT116 colon cancer cell line, grown as monolayer cultures and spheroids, with a range of OXPHOS inhibitors (n = 10, including FDA-approved drugs, e.g. nitazoxanide) and other compounds (n = 14) at escalating doses and in 4 biological replicates. Then, we obtained global gene expression profiles (n = 1149, including 144 vehicle controls) of all treatment conditions using L1000 Gene Expression Profiling method. We found that upon exposure to OXPHOS inhibitors cells grown as nutrient-deprived spheroids significantly and in dose-dependent manner upregulate expression of genes involved in biosynthesis of cholesterol. This response was not observed for spheroids cultured with medium change or monolayer cell cultures. Thus, we were interested if simultaneous exposure to OXPHOS inhibitors and statins, inhibitors of mevalonate (cholesterol precursor) synthesis, would result in enhanced cytotoxic effects in quiescent, metabolically stressed cells. We here demonstrate that combination of OXPHOS inhibitors and statins results in pronounced synergistic cytotoxicity in metabolically stressed spheroids. This effect was observed for various classes of OXPHOS inhibitors and different types of statins, indicating that the observed synergy was not a result of off-target effects. This notion was further strengthened by the finding that mevalonate largely abrogated the synergistic effects. In conclusion, we here report that statins enhance the toxic effects of OXPHOS inhibitors in quiescent, metabolically stressed cells. Our results can serve as a foundation for further studies on targeting therapy-resistant and nutrient-deprived cancer cells by inhibition of OXPHOS. We also demonstrate, for the first time, that the L1000 Gene Expression Profiling can be used to study 3D cell cultures. Importantly, our findings underscore the importance of using a relevant cellular model for target discovery endeavors. Citation Format: Wojciech Senkowski, Malin Jarvius, Kim Kultima, Jenny Rubin, Mats Gustafsson, Peter Nygren, Rolf Larsson, Mårten Fryknäs. Mitochondrial inhibitors and statins: a lethal combination for metabolically stressed cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 213.

Glioblastoma multiforme (GBM) remains in the group of incurable malignancies, with a median survival of 15 months. Intratumoral heterogeneity is a key factor driving relapse by providing the basis for escape of therapy-resistant cells. While the heterogeneous genomic and transcriptomic landscape of cancers is currently thoroughly characterized, phenotypic heterogeneity within tumors is less well understood. The overall goal in this study was to identify pre-existing treatment resistant clones with disease relapse potential and biomarkers linked to resistance vs sensitivity. To enable functional studies of isolated tumor clones we adopted an in vitro subcloning strategy. An adherent glioma neural stem cell culturing protocol was used to enrich for cells with stem-like properties and to permit efficient phenotypic screening. We established six libraries of clonal cell cultures from fresh GBM surgical specimens, corresponding to five different patient tumors. In all 708 clonal cultures were expanded. These were considered representatives of the self-renewing compartment within the tumor cell population. We found a remarkable variation in drug and radiation response within the clone libraries. A striking observation was that clones resistant to one drug, also tended to be resistant to most of the drugs we used, regardless of the drug's mechanism of action. This indicates that resistance in large parts is mediated by a general mechanism. Most clones carried genetic aberrations that were unique or found only in a few clones but it was primarily the transcriptome data that demonstrated a clear link to the phenotypic data. A continuous gradient between multi-resistance and sensitivity was found to be connected to a gradual transition between a mesenchymal (MES), in resistant clones, and a more proneural (PN) and proliferative character in sensitive clones. The continuous distributions and lack of discrete groups in clonal response phenotypes and in linked signatures points to a general cell-state related resistance mechanism. We also found that resistant clones had a lower methylation level in promoters of known master regulators of MES subtype associated genes. This indicates that the phenotypic heterogeneity is driven by fluctuations in the epigenetic status. Taken together, our findings imply that intratumoral heterogeneity in GBM includes general clonal resistance mechanisms among glioma- initiating cells driven by epigenetic mechanisms. This evokes hope that new therapeutic approaches involving epigenetic reprogramming can be applied to sensitize cells toward conventional treatment. Citation Format: Anna Segerman, Maria Niklasson, Caroline Haglund, Tobias Bergstrom, Malin Jarvius, Yuan Xie, Demet Caglayan Simov, Annika Hermansson, Marianne Kastemar, Zeinab Naimaie-Ali, Malin Berglund, Ann Westermark, Magnus Sundstrom, Goran Hesselager, Lene Uhrbom, Mats Gustafsson, Rolf Larsson, Marten Fryknas, Bo Segerman, Bengt Westermark. GBM exhibits phenotypic microheterogeneity and harbors pre-existing multi- resistant clones with a mesenchymal transition signature. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2415.

Platelet-derived growth factor receptor (PDGFR) signaling has been implicated in the pathogenesis of glioblastomas and represents a target for the tyrosine kinase inhibitor imatinib. To examine the prognostic or predictive role of PDGFRs in recurrent glioblastomas, expression was examined in tumor samples of 101 patients of CSTI571BDE40, a randomized trial comparing hydroxyurea monotherapy and a combination of hydroxyurea and imatinib. Furthermore, PDGFRα phosphorylation was investigated using in situ proximity ligation assay. PDGFRα protein was expressed in 33% of tumors and was associated with male sex, young age, presence of R132H mutated isocitrate dehydrogenase 1 protein and short median survival (142 vs. 187 days, p = 0.028). Tumor PDGFRα phosphorylation was also associated with short survival (p = 0.030). The subset of patients with PDGFRα positive glioblastoma did not have longer survival on treatment with hydroxyurea and imatinib compared with hydroxyurea monotherapy. In conclusion, both PDGFRα protein expression and phosphorylation status had a prognostic role in recurrent glioblastomas but did not define a group that showed benefit from the combination therapy consisting of hydroxyurea and imatinib.

An essential skill for every researcher is to learn how to select and apply the most appropriate methods for the questions they are trying to answer. With the extensive variety of methods available, it is increasingly important to scrutinize the advantages and disadvantages of these techniques prior to making a decision on which to use. In this article, we describe an approach to evaluate methods by reducing them into subcomponents. This is exemplified by a brief description of some commonly used proteomics methods. The same approach can also be used in method development by rearranging subcomponents in order to create new methods, as demonstrated with the development of proximity ligation assays (PLAs). PLA is a method as designed in our laboratory for detection of proteins, protein-protein interactions and post-translational modifications. Fundamentally, protein-recognition events are converted into detectable DNA molecules. The technique uses protein-DNA conjugates as binders for the targets of interest. Binding of two or more conjugates to the target results in assembly of an assay-specific DNA molecule. Subsequent amplification of the DNA molecule generates a signal that can be detected using PCR, for detection of minute amounts of proteins in serum, or standard fluorescence microscopy for detection of protein-protein interactions in tissue sections. Lastly, we apply the approach of recombining subcomponents to develop a few novel hypothetical methods hoping this might stimulate the readers to utilize this approach themselves.

Protein diagnostics by proximity ligation : Combining multiple recognition and DNA amplification for improved protein analysis