Jonathan B. Bell

A simple, low-cost staining method for rapid-throughput analysis of tumor spheroids

Tumor spheroids are becoming an important tool for the investigation of cancer stem cell (CSC) function in tumors; thus, low-cost and high-throughput methods for drug screening of tumor spheroids are needed. Using neurospheres as non-adherent three-dimensional (3-D) cultures, we developed a simple, low-cost acridine orange (AO)-based method that allows for rapid analysis of live neurospheres by fluorescence microscopy in a 96-well format. This assay measures the cross-section area of a spheroid, which corresponds to cell viability. Our novel method allows rapid screening of a panel of anti-proliferative drugs to assess inhibitory effects on the growth of cancer stem cells in 3-D cultures.

Targeting of glioblastoma cell lines and glioma stem cells by combined PIM kinase and PI3K-p110α inhibition

The PIM family of proteins encodes serine/threonine kinases with important roles in protein synthesis and cancer cell metabolism. In glioblastoma (GBM) cell lines, siRNA-mediated knockdown of PIM kinases or pharmacological inhibition of PIM kinases by SGI-1776 or AZD-1208 results in reduced phosphorylation of classic PIM effectors and also elements of the PI3K/mTOR pathway, suggesting interplay between PIM and mTOR signals in GBM cells. Combination of PIM kinase inhibitors with BYL-719, an inhibitor specific for the PI3K catalytic isoform p110α, results in enhanced antineoplastic effects in GBM cells. Additionally, pharmacologic inhibition of PIM kinases impairs growth of patient-derived glioma sphere cells, suggesting an important role for PIM kinases in cancer stem cell (CSC) function and survival. Such effects are further enhanced by concomitant inhibition of PIM kinase and p110α activities. Altogether these findings suggest that pharmacological PIM targeting in combination with PI3K inhibition may provide a unique therapeutic approach for the treatment of heterogeneous tumors containing populations of therapy-resistant CSCs in GBM.

MNK Inhibition Disrupts Mesenchymal Glioma Stem Cells and Prolongs Survival in a Mouse Model of Glioblastoma

Glioblastoma multiforme remains the deadliest malignant brain tumor, with glioma stem cells (GSC) contributing to treatment resistance and tumor recurrence. We have identified MAPK-interacting kinases (MNK) as potential targets for the GSC population in glioblastoma multiforme. Isoform-level subtyping using The Cancer Genome Atlas revealed that both MNK genes (MKNK1 and MKNK2) are upregulated in mesenchymal glioblastoma multiforme as compared with other subtypes. Expression of MKNK1 is associated with increased glioma grade and correlated with the mesenchymal GSC marker, CD44, and coexpression of MKNK1 and CD44 predicts poor survival in glioblastoma multiforme. In established and patient-derived cell lines, pharmacologic MNK inhibition using LY2801653 (merestinib) inhibited phosphorylation of the eukaryotic translation initiation factor 4E, a crucial effector for MNK-induced mRNA translation in cancer cells and a marker of transformation. Importantly, merestinib inhibited growth of GSCs grown as neurospheres as determined by extreme limiting dilution analysis. When the effects of merestinib were assessed in vivo using an intracranial xenograft mouse model, improved overall survival was observed in merestinib-treated mice. Taken together, these data provide strong preclinical evidence that pharmacologic MNK inhibition targets mesenchymal glioblastoma multiforme and its GSC population. Implications: These findings raise the possibility of MNK inhibition as a viable therapeutic approach to target the mesenchymal subtype of glioblastoma multiforme. Mol Cancer Res; 14(10); 984–93. ©2016 AACR.

Differential Response of Glioma Stem Cells to Arsenic Trioxide Therapy Is Regulated by MNK1 and mRNA Translation

Mesenchymal (MES) and proneural (PN) are two distinct glioma stem cell (GSC) populations that drive therapeutic resistance in glioblastoma (GBM). We screened a panel of 650 small molecules against patient-derived GBM cells to discover compounds targeting specific GBM subtypes. Arsenic trioxide (ATO), an FDA-approved drug that crosses the blood–brain barrier, was identified as a potent PN-specific compound in the initial screen and follow-up validation studies. Furthermore, MES and PN GSCs exhibited differential sensitivity to ATO. As ATO has been shown to activate the MAPK-interacting kinase 1 (MNK1)-eukaryotic translation initiation factor 4E (eIF4E) pathway and subsequent mRNA translation in a negative regulatory feedback manner, the mechanistic role of ATO resistance in MES GBM was explored. In GBM cells, ATO-activated translation initiation cellular events via the MNK1–eIF4E signaling axis. Furthermore, resistance to ATO in intracranial PDX tumors correlated with high eIF4E phosphorylation. Polysomal fractionation and microarray analysis of GBM cells were performed to identify ATOs effect on mRNA translation and enrichment of anti-apoptotic mRNAs in the ATO-induced translatome was found. Additionally, it was determined that MNK inhibition sensitized MES GSCs to ATO in neurosphere and apoptosis assays. Finally, examination of the effect of ATO on patients from a phase I/II clinical trial of ATO revealed that PN GBM patients responded better to ATO than other subtypes as demonstrated by longer overall and progression-free survival. Implications: These findings raise the possibility of a unique therapeutic approach for GBM, involving MNK1 targeting to sensitize MES GSCs to drugs like arsenic trioxide. Mol Cancer Res; 16(1); 32–46. ©2017 AACR.

HDL nanoparticles targeting sonic hedgehog subtype medulloblastoma

Medulloblastoma is the most common paediatric malignant brain cancer and there is a need for new targeted therapeutic approaches to more effectively treat these malignant tumours, which can be divided into four molecular subtypes. Here, we focus on targeting sonic hedgehog (SHH) subtype medulloblastoma, which accounts for approximately 25% of all cases. The SHH subtype relies upon cholesterol signalling for tumour growth and maintenance of tumour-initiating cancer stem cells (CSCs). To target cholesterol signalling, we employed biomimetic high-density lipoprotein nanoparticles (HDL NPs) which bind to the HDL receptor, scavenger receptor type B-1 (SCARB1), depriving cells of natural HDL and their cholesterol cargo. We demonstrate uptake of HDL NPs in SCARB1 expressing medulloblastoma cells and depletion of cholesterol levels in cancer cells. HDL NPs potently blocked proliferation of medulloblastoma cells, as well as hedgehog-driven Ewing sarcoma cells. Furthermore, HDL NPs disrupted colony formation in medulloblastoma and depleted CSC populations in medulloblastoma and Ewing sarcoma. Altogether, our findings provide proof of principle for the development of a novel targeted approach for the treatment of medulloblastoma using HDL NPs. These findings present HDL-mimetic nanoparticles as a promising therapy for sonic hedgehog (SHH) subtype medulloblastoma and possibly other hedgehog-driven cancers.

Abstract LB-009: Mnk targeting enhances vulnerability of medulloblastoma stem-like cancer cells to PI3K-p110alpha inhibition

Our recent work suggested that inhibition of mTORC1 activates Mnk in a PI3K-dependent manner, thereby providing a survival mechanism for medulloblastoma cells. The PI3K-Akt axis represents an important survival pathway that also confers therapy resistance to medulloblastoma stem cells, resulting in tumor recurrence. Here, we investigated a role for p110 isoforms of PI3K in medulloblastoma stem-like cancer cell biology and studied the potential of Mnk inhibition for sensitizing medulloblastoma stem-like cancer cells and orthotopic xenograft tumors to PI3K inhibition. We used medulloblastoma cell lines Daoy and D556 grown as conventional 2-D cultures or under stem-cell conditions as 3-D neurospheres to elucidate the roles of PI3K-Akt signaling in medulloblastoma proliferation, colony formation and stem cell function. We employed extreme limiting dilution analysis (ELDA) to ask whether concomitant Mnk inhibition enhances antineoplastic effects of PI3K inhibition on cancer stem cell growth. Additionally, in a preliminary intracereballar xenograft mouse study, we investigated the effects of pharmacologic PI3K and Mnk inhibition. We found that Akt activity greatly increased when 2-D cultures were converted into 3-D neurospheres. This Akt activation coincided with increased expression of CD133 and nestin, suggesting an important role for PI3K-Akt signaling in medulloblastoma stem cells. The p110a specific inhibitor BYL-719 blocked this Akt activation in neurospheres indicating this Akt activation is mediated by p110a. Consistently, of all class I PI3K catalytic isoforms (p110a, p110b, p110d and p110g) only knockdown of p110a disrupted stem cell frequencies in ELDA. We previously reported that mTORC1 inhibition engages Mnk signaling in a negative feedback manner to promote survival. Here we show that Mnk inhibition by CGP57380 sensitized medulloblastoma cells for pharmacologic inhibition and siRNA-mediated knockdown of p110a both in 2-D cancer cells and 3-D stem-like cancer cell cultures. After intracerebellar injection of medulloblastoma cells in nude mice, we found that combined targeting of PI3K-p110a and Mnk results in inhibition of tumor growth and increased survival. In summary, pharmacologic inhibition of PI3K-p110a by BYL-719 showed potent activity against medulloblastoma cells and stem-like cancer cells. Knockdown of p110a disrupted cancer stem cell frequency in ELDA and this effect was greatly enhanced by pharmacologic inhibition of Mnk. Finally, in an orthotopic mouse model we found that concomitant inhibition of p110a and Mnk prolonged survival and reduced tumor size. The striking effects of concomitant Mnk inhibition on stem-like cancer cells, neurospheres and tumors is particularly interesting as it suggests enhanced vulnerability of the therapy-resistant, tumor-initiating cancer stem cell population to p110a inhibition in medulloblastoma. Citation Format: Jonathan B. Bell, Frank Eckerdt, Quanhong Ma, Jessica R. Clymer, Stewart Goldman, Rintaro Hashizume, Leonidas C. Platanias. Mnk targeting enhances vulnerability of medulloblastoma stem-like cancer cells to PI3K-p110alpha inhibition. [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 LB-009.

Abstract B26: MAPK-interacting kinase inhibition sensitizes glioblastoma and glioma stem cells to arsenic trioxide

Glioblastoma (GBM) is the deadliest primary brain tumor with a median survival of around one year. Arsenic trioxide (ATO) is an emerging therapy for the treatment of GBM and other malignant brain tumors. The cytotoxic effects of ATO are mainly mediated by the production of reactive oxygen species and induction of cell death pathways. However, glioma stem cells in heterogeneous GBM tumors impart resistance by activation of survival pathways, thereby preventing therapeutic responses to cytotoxic agents such as ATO. We have previously shown that ATO responses in leukemia are antagonized by the MAPK-interacting kinases (MNKs), which activate protein translation and survival pathways including the eukaryotic translation initiation factor 4E (eIF4E) in response to ATO treatment. Yet, the role of MNK signaling in GBM and glioma stem cells and the potential of using MNK inhibitors to sensitize GBM to ATO has not been explored. In this study, we sought to determine the mechanisms by which MNK signaling regulates arsenic trioxide responses in GBM and glioma stem cells. GBM cell lines were treated with ATO in the presence or absence of MNK inhibitors or siRNA against MNK isoforms. Western blots of treated samples were analyzed with antibodies against phosphorylated eIF4E, the key downstream effector of the MNKs. Following treatment with ATO and MNK inhibitors, proliferation rate and apoptosis were determined by WST-1 assay and Annexin V-FITC/PI staining. GBM cell lines were grown under stem cell conditions and subjected to qPCR and flow cytometry to monitor CD44 expression and aldehyde dehydrogenase (ALDH) activity, both markers of stemness. Patient-derived glioma stem cell lines displaying mesenchymal-like phenotype were treated with ATO and MNK inhibitors and analyzed by neurosphere formation assay. Treatment of GBM cell lines with ATO resulted in MNK activation and induction of eIF4E phosphorylation in a MNK1-depedent manner. Furthermore, MNK inhibition sensitized GBM cells to the anti-proliferative and pro-apoptotic effects of ATO. Knockdown of MNK1 in GBM cell lines grown under stem cell conditions decreased neurosphere formation. Finally, pharmacological MNK inhibition sensitized mesenchymal-like glioma stem cells to ATO. Our results suggest ATO in combination with MNK inhibition might represent a new approach for the treatment of GBM, in particular the therapy-resistant glioma stem cell subpopulation. Citation Format: Jonathan B. Bell, Frank Eckerdt, Ahmet Dirim Arslan, Asneha Iqbal, Angel A. Alvarez, Shi-Yuan Cheng, Ichiro Nakano, Leonidas C. Platanias. MAPK-interacting kinase inhibition sensitizes glioblastoma and glioma stem cells to arsenic trioxide. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B26.