Jarek Maciaczyk

NOTCH pathway blockade depletes CD133-positive glioblastoma cells and inhibits growth of tumor neurospheres and xenografts

Cancer stem cells (CSCs) are thought to be critical for the engraftment and long‐term growth of many tumors, including glioblastoma (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. It has been shown that the NOTCH signaling pathway regulates normal stem cells in the brain, and that GBMs contain stem‐like cells with higher NOTCH activity. We therefore used low‐passage and established GBM‐derived neurosphere cultures to examine the overall requirement for NOTCH activity, and also examined the effects on tumor cells expressing stem cell markers. NOTCH blockade by γ‐secretase inhibitors (GSIs) reduced neurosphere growth and clonogenicity in vitro, whereas expression of an active form of NOTCH2 increased tumor growth. The putative CSC markers CD133, NESTIN, BMI1, and OLIG2 were reduced following NOTCH blockade. When equal numbers of viable cells pretreated with either vehicle (dimethyl sulfoxide) or GSI were injected subcutaneously into nude mice, the former always formed tumors, whereas the latter did not. In vivo delivery of GSI by implantation of drug‐impregnated polymer beads also effectively blocked tumor growth, and significantly prolonged survival, albeit in a relatively small cohort of animals. We found that NOTCH pathway inhibition appears to deplete stem‐like cancer cells through reduced proliferation and increased apoptosis associated with decreased AKT and STAT3 phosphorylation. In summary, we demonstrate that NOTCH pathway blockade depletes stem‐like cells in GBMs, suggesting that GSIs may be useful as chemotherapeutic reagents to target CSCs in malignant gliomas. STEM CELLS 2010;28:5–16

BRAF Activation Induces Transformation and Then Senescence in Human Neural Stem Cells: A Pilocytic Astrocytoma Model

Purpose: BRAF is frequently activated by gene fusion or point mutation in pilocytic astrocytoma, the most common pediatric brain tumor. We investigated the functional effect of constitutive BRAF activation in normal human neural stem and progenitor cells to determine its role in tumor induction in the brain. Experimental Design: The constitutively active BRAFV600E allele was introduced into human neurospheres, and its effects on MAPK (mitogen-activated protein kinase) signaling, proliferation, soft agarose colony formation, stem cell phenotype, and induction of cellular senescence were assayed. Immunohistochemistry was used to examine p16INK4a levels in pilocytic astrocytoma. Results: BRAFV600E expression initially strongly promoted colony formation but did not lead to significantly increased proliferation. BRAFV600E-expressing cells subsequently stopped proliferating and induced markers of oncogene-induced senescence including acidic β-galactosidase, PAI-1, and p16INK4a whereas controls did not. Onset of senescence was associated with decreased expression of neural stem cell markers including SOX2. Primary pilocytic astrocytoma cultures also showed induction of acidic β-galactosidase activity. Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16INK4a immunoreactivity in the majority of cases, but patients with tumors negative for p16INK4a had significantly shorter overall survival. Conclusions: BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation. Induction of senescence by BRAF may help explain the low-grade pathobiology of pilocytic astrocytoma, whereas worse clinical outcomes associated with tumors lacking p16INK4a expression could reflect failure to induce senescence or an escape from oncogene-induced senescence. Clin Cancer Res; 17(11); 3590–9. ©2011 AACR.

DNER, an epigenetically modulated gene, regulates glioblastoma-derived neurosphere cell differentiation and tumor propagation.

Neurospheres derived from glioblastoma (GBM) and other solid malignancies contain neoplastic stem‐like cells that efficiently propagate tumor growth and resist cytotoxic therapeutics. The primary objective of this study was to use histone‐modifying agents to elucidate mechanisms by which the phenotype and tumor‐promoting capacity of GBM‐derived neoplastic stem‐like cells are regulated. Using established GBM‐derived neurosphere lines and low passage primary GBM‐derived neurospheres, we show that histone deacetylase (HDAC) inhibitors inhibit growth, induce differentiation, and induce apoptosis of neoplastic neurosphere cells. A specific gene product induced by HDAC inhibition, Delta/Notch‐like epidermal growth factor‐related receptor (DNER), inhibited the growth of GBM‐derived neurospheres, induced their differentiation in vivo and in vitro, and inhibited their engraftment and growth as tumor xenografts. The differentiating and tumor suppressive effects of DNER, a noncanonical Notch ligand, contrast with the previously established tumor‐promoting effects of canonical Notch signaling in brain cancer stem‐like cells. Our findings are the first to implicate noncanonical Notch signaling in the regulation of neoplastic stem‐like cells and suggest novel neoplastic stem cell targeting treatment strategies for GBM and potentially other solid malignancies. STEM CELLS 2009;27:1473–1486

Targeting cancer stem-like cells in glioblastoma and colorectal cancer through metabolic pathways

Cancer stem‐like cells (CSCs) are thought to be the main cause of tumor occurrence, progression and therapeutic resistance. Strong research efforts in the last decade have led to the development of several tailored approaches to target CSCs with some very promising clinical trials underway; however, until now no anti‐CSC therapy has been approved for clinical use. Given the recent improvement in our understanding of how onco‐proteins can manipulate cellular metabolic networks to promote tumorigenesis, cancer metabolism research may well lead to innovative strategies to identify novel regulators and downstream mediators of CSC maintenance. Interfering with distinct stages of CSC‐associated metabolics may elucidate novel, more efficient strategies to target this highly malignant cell population. Here recent discoveries regarding the metabolic properties attributed to CSCs in glioblastoma (GBM) and malignant colorectal cancer (CRC) were summarized. The association between stem cell markers, the response to hypoxia and other environmental stresses including therapeutic insults as well as developmentally conserved signaling pathways with alterations in cellular bioenergetic networks were also discussed. The recent developments in metabolic imaging to identify CSCs were also summarized. This summary should comprehensively update basic and clinical scientists on the metabolic traits of CSCs in GBM and malignant CRC.

Pharmacologic Wnt Inhibition Reduces Proliferation, Survival, and Clonogenicity of Glioblastoma Cells

Abstract Wingless (Wnt) signaling is an important pathway in gliomagenesis and in the growth of stem-like glioma cells. Using immunohistochemistry to assess the translocation of &bgr;-catenin protein, we identified intranuclear staining suggesting Wnt pathway activation in 8 of 43 surgical samples (19%) from adult patients with glioblastoma and in 9 of 30 surgical samples (30%) from pediatric patients with glioblastoma. Wnt activity, evidenced by nuclear &bgr;-catenin in our cohort and high expression of its target AXIN2 (axis inhibitor protein 2) in published glioma datasets, was associated with shorter patient survival, although this was not statistically significant. We determined the effects of the porcupine inhibitor LGK974 on 3 glioblastoma cell lines with elevated AXIN2 and found that it reduced Wnt pathway activity by 50% or more, as assessed by T-cell factor luciferase reporters. Wnt inhibition led to suppression of growth, proliferation in cultures, and modest induction of cell death. LGK974 reduced NANOG messenger RNA levels and the fraction of cells expressing the stem cell marker CD133 in neurosphere cultures, induced glial differentiation, and suppressed clonogenicity. These data indicate that LGK974 is a promising new agent that can inhibit the canonical Wnt pathway in vitro, slow tumor growth, and deplete stem-like clonogenic cells, thereby providing further support for targeting Wnt in patients with glioblastoma.

DiSCoVERing Innovative Therapies for Rare Tumors: Combining Genetically Accurate Disease Models with In Silico Analysis to Identify Novel Therapeutic Targets.

Purpose: We used human stem and progenitor cells to develop a genetically accurate novel model of MYC-driven Group 3 medulloblastoma. We also developed a new informatics method, Disease-model Signature versus Compound-Variety Enriched Response (“DiSCoVER”), to identify novel therapeutics that target this specific disease subtype. Experimental Design: Human neural stem and progenitor cells derived from the cerebellar anlage were transduced with oncogenic elements associated with aggressive medulloblastoma. An in silico analysis method for screening drug sensitivity databases (DiSCoVER) was used in multiple drug sensitivity datasets. We validated the top hits from this analysis in vitro and in vivo. Results: Human neural stem and progenitor cells transformed with c-MYC, dominant-negative p53, constitutively active AKT and hTERT formed tumors in mice that recapitulated Group 3 medulloblastoma in terms of pathology and expression profile. DiSCoVER analysis predicted that aggressive MYC-driven Group 3 medulloblastoma would be sensitive to cyclin-dependent kinase (CDK) inhibitors. The CDK 4/6 inhibitor palbociclib decreased proliferation, increased apoptosis, and significantly extended the survival of mice with orthotopic medulloblastoma xenografts. Conclusions: We present a new method to generate genetically accurate models of rare tumors, and a companion computational methodology to find therapeutic interventions that target them. We validated our human neural stem cell model of MYC-driven Group 3 medulloblastoma and showed that CDK 4/6 inhibitors are active against this subgroup. Our results suggest that palbociclib is a potential effective treatment for poor prognosis MYC-driven Group 3 medulloblastoma tumors in carefully selected patients. Clin Cancer Res; 22(15); 3903–14. ©2016 AACR.

Alterations in cellular metabolome after pharmacological inhibition of Notch in glioblastoma cells

Notch signaling can promote tumorigenesis in the nervous system and plays important roles in stem‐like cancer cells. However, little is known about how Notch inhibition might alter tumor metabolism, particularly in lesions arising in the brain. The gamma‐secretase inhibitor MRK003 was used to treat glioblastoma neurospheres, and they were subdivided into sensitive and insensitive groups in terms of canonical Notch target response. Global metabolomes were then examined using proton magnetic resonance spectroscopy, and changes in intracellular concentration of various metabolites identified which correlate with Notch inhibition. Reductions in glutamate were verified by oxidation‐based colorimetric assays. Interestingly, the alkylating chemotherapeutic agent temozolomide, the mTOR‐inhibitor MLN0128, and the WNT inhibitor LGK974 did not reduce glutamate levels, suggesting that changes to this metabolite might reflect specific downstream effects of Notch blockade in gliomas rather than general sequelae of tumor growth inhibition. Global and targeted expression analyses revealed that multiple genes important in glutamate homeostasis, including glutaminase, are dysregulated after Notch inhibition. Treatment with an allosteric inhibitor of glutaminase, compound 968, could slow glioblastoma growth, and Notch inhibition may act at least in part by regulating glutaminase and glutamate.

The effect of neurosphere culture conditions on the cellular metabolism of glioma cells

Malignant gliomas, with an average survival time of 16-19 months after initial diagnosis, account for one of the most lethal tumours overall. Current standards in patient care provide only unsatisfying strategies in diagnostic and treatment for high-grade gliomas. Here we describe metabolic phenomena in the choline and glycine network associated with stem cell culture conditions in the classical glioma cell line U87. Using high-resolution proton magnetic resonance spectroscopy of cell culture metabolic extracts we compare the metabolic composition of U87 chronically propagated as adherent culture in medium supplemented with serum to serum-free neurosphere growth. We found that the switch to neurosphere growth, besides the increase of cells expressing the putative glioma stem cell marker CD133, modulated a number of intracellular metabolites including choline, creatine, glycine, and myo-inositol that have been previously reported as potential diagnostic markers in various tumours. These findings highlight the critical influence of culture conditions on glioma cell metabolism, and therefore particular caution should be drawn to the use of in vitro system research in order to investigate cancer metabolism.

Abstract 3454: LIN28 is expressed in glioblastomas and promotes KRAS-mediated transformation of human neural stem cells

The pluripotency factor LIN28 is activated in many cancers arising outside the central nervous system, but its role in brain tumors is poorly understood. LIN28 inhibits the maturation of let-7, which in turn represses KRAS, c-myc, HMGA2 and other oncogenic factors, many of which are known to be activated in glioblastoma (GBM). Because malignant gliomas are thought to arise from normal human neural stem cells (hNSC), we hypothesized that the stem cell factor LIN28 plays a role in the development of high grade gliomas. Using immunohistochemistry, we detected LIN28 protein in a subset of gliomas, with higher expression in GBMs than in lower grade tumors. To examine if LIN28 would facilitate transformation of hNSC, we infected these cells with lentivirus encoding LIN28 together with dominant negative R248W p53 (DN-p53), constitutively active KRAS (CA-KRAS) and hTERT (hNSC-LIN28/DN-p53/CA-KRAS/hTERT), which are all commonly dysregulated in GBM. As controls, hNSC were also infected with DN-p53/CA-KRAS/hTERT, LIN28/DN-p53/hTERT, DN-p53/hTERT, or with GFP. The lentivirus transduced hNSCs continued to express the stem cell markers CD133, SOX2, Nestin and OLIG2, and retained the potential to differentiate along neuronal and glial lineages. The LIN28/DN-p53/CA-KRAS/hTERT expressing cells showed increased proliferation by BrdU assay compared to normal hNSCs. To test their tumorigenecity in vivo, hNSC-LIN28/DN-p53/CA-KRAS/hTERT were injected into the brains of immunocompromised mice. Eight weeks after injection, 66.7% (12 of 18) of the mice developed invasive brain tumors resembling GBMs which were positive for Nestin and GFAP, and negative for synaptophysin. In contrast, control injections including hNSC-DN-p53/hTERT (5 mice), hNSC-LIN28/DN-p53/hTERT (5 mice) and hNSC-GFP (10 mice) did not generate tumors over 6 months. Interestingly, hNSC-DN-p53/CA-KRAS/hTERT cultures proliferated very poorly, suggesting that both LIN28 and activated KRAS may play key roles in transformation. In support of this possibility, we found using quantitative RT-PCR and western blotting that hNSC-LIN28/DN-p53/CA-KRAS/hTERT expressed higher levels of KRAS and phospho-MAPK than hNSC-DN-p53/CA-KRAS/hTERT lacking LIN28, suggesting this stem cell factor is required to facilitate MAPK activation and complete transformation of hNSCs. To further confirm the role of LIN28 in the tumorigenicity of GBMs, a tumor-derived neurosphere line (JHH-GBM14) was infected with LIN28 (GBM14-LIN28) or GFP control (GBM14-GFP) and injected as orthotopic xenografts into immunocompromised mice. In addition to forming brain tumors larger than GBM14-GFP controls, GBM14-LIN28 tumors significantly invaded into normal tissues including the corpus callosum and the contralateral brain. Our data show that LIN28 is expressed in human GBMs and that LIN28 can facilitate tumorigenicity in orthotopic GBM mouse models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3454. doi:10.1158/1538-7445.AM2011-3454

Abstract 2607: BMI1 and dominant negative p53 cooperate to suppress AKT-mediated oncogene-induced senescence and promote transformation in human neural stem cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Purpose: BMI-1 is required for the maintenance of normal brain stem cells and is associated with aggressive brain tumors such as glioblastoma multiforme (GBM). BMI1 is a polycomb group protein that can modify chromatin to silence the pro-senescence tumor suppressor p16INK4a. The PI3K/AKT pathway is also associated with clinically aggressive brain tumors. Interestingly, persistent activation of AKT can cause oncogene-induced senescence. Dominant negative R248W p53 mutation blocks induction of the tumor suppressor p21CIP1 and is found in aggressive brain tumors such as GBM. Telomere maintenance through telomerase enzyme activity is also found in aggressive brain tumors. We investigated if constitutively active AKT, dominant negative p53, BMI1, and hTERT can cooperate to bring about the transformation of human neural stem and progenitor cells, and in particular if BMI1 can suppress AKT induced senescence. Experimental Design: We used lentivirus to introduce singly and in combination BMI1, R248W p53, hTERT and constitutively active AKT into human cortex derived stem and progenitor cells. We assayed the effects of one, two, three or four oncogenes on the proliferation and induction of cellular senescence. We measured proliferation using bromodeoxyurine (BrdU) and senescence by western blotting for p16INK4a and p21 CIP1. Results: Increased expression of BMI1 alone did not have a significant effect on human neural stem cell proliferation, but did lead to the suppression of p16INK4a. AKT alone transduced cells showed no increased proliferation compared to control cells, but did show upregulation of p16INK4a. AKT alone expressing cells stopped growing after several passages in culture, in association with increased p16 INK4a expression. Cells transduced with BMI1 and AKT initially grew at a similar rate to control cells, but after several passages, similar to AKT alone transduced cells, BMI1/AKT cells stopped proliferating and expressed p21CIP1and p16INK4a. Control cells continued to proliferate for multiple additional passages. Cells transduced with R248W p53/hTERT/BMI1/AKT proliferated at an increased rate compared to control cells (17% BrdU positive for pBABE control cells compared to 37% for R248W p53/hTERT/BMI1/AKT p=0.0003, t-test). These cells did not undergo senescence, did not induce p16INK4a or p21CIP1 and continued proliferating more than control or single oncogene transduced cells long after other cells had senesced. Human neural stem cells transduced with R248W p53/hTERT/BMI1/AKT formed aggressive orthotopic xenograft glial tumors, while control cells did not form tumors. Conclusions: BMI1 alone cannot suppress AKT induced senescence in human cortex derived neural stem cells. But BMI1 combined with R248W p53 and hTERT caused suppression of AKT induced senescence, and resulting cells showed signs of oncogenic transformation Citation Format: Dacheng Ding, Ulf Kahlert, Jarek Maciaczyk, Guido Nikkhah, Charles Eberhart, Eric Raabe. BMI1 and dominant negative p53 cooperate to suppress AKT-mediated oncogene-induced senescence and promote transformation in human neural stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2607. doi:10.1158/1538-7445.AM2013-2607