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Patients with head and neck malignancies commonly develop metastatic disease, yet rarely do these carcinomas metastasize to the brain. Stereotactic radiosurgery (SRS) is routinely employed to treat brain metastases (BM). This study was undertaken to examine the efficacy of SRS for BM from primary head and neck carcinomas. From 2000 to 2016, a total of 19 patients with 38 lesions were retrospectively identified. All patients presented with a primary head and neck malignancy and subsequently developed metastatic disease to the brain treated with SRS at our institution. Actuarial rates for overall survival (OS), local control (LC) and distant brain metastases (DBM) were calculated using Kaplan-Meier estimates. Median follow up was 6.8 months and median survival was 15.8 months. Eleven lesions received post-operative SRS to a surgical cavity and 27 lesions received definitive SRS to a metastasis. The median dose prescribed was 18 Gy. One-year actuarial rate for LC was 77.3% (95% confidence interval [CI] 44-92%) while 1 year and 2 year rates of OS were 52.9% (CI 28-73%) and 31.7% (CI 11-55%) respectively. The median time to develop DBM was 8.4 months. Three patients (16%) underwent repeat SRS following development of new BM and three patients (16%) underwent salvage whole brain radiotherapy (WBRT). SRS may be utilized in the treatment of patients with primary head and neck malignancies metastasized to the brain with high efficacy. Patients with well-controlled systemic disease and good performance status may benefit the most from definitive SRS while avoiding WBRT.

Despite recent advances in the treatment of medulloblastoma, patients in high-risk categories still face very poor outcomes. Evidence indicates that a subpopulation of cancer stem cells contributes to therapy resistance and tumour relapse in these patients. To prevent resistance and relapse, the development of treatment strategies tailored to target subgroup specific signalling circuits in high-risk medulloblastomas might be similarly important as targeting the cancer stem cell population. We have previously demonstrated potent antineoplastic effects for the PI3Kα selective inhibitor alpelisib in medulloblastoma. Here, we performed studies aimed to enhance the anti-medulloblastoma effects of alpelisib by simultaneous catalytic targeting of the mTOR kinase. Pharmacological mTOR inhibition potently enhanced the suppressive effects of alpelisib on cancer cell proliferation, colony formation and apoptosis and additionally blocked sphere-forming ability of medulloblastoma stem-like cancer cells in vitro. We identified the HH effector GLI1 as a target for dual PI3Kα and mTOR inhibition in SHH-type medulloblastoma and confirmed these results in HH-driven Ewing sarcoma cells. Importantly, pharmacologic mTOR inhibition greatly enhanced the inhibitory effects of alpelisib on medulloblastoma tumour growth in vivo. In summary, these findings highlight a key role for PI3K/mTOR signalling in GLI1 regulation in HH-driven cancers and suggest that combined PI3Kα/mTOR inhibition may be particularly interesting for the development of effective treatment strategies in high-risk medulloblastomas.

Medulloblastoma is a highly malignant pediatric brain tumor associated with poor outcome. Developing treatments that target the cancer stem cell (CSC) population in medulloblastoma are important to prevent tumor relapse and induce long-lasting clinical responses. We utilized medulloblastoma neurospheres that display CSC characteristics and found activation of the PI3K/AKT pathway in sphere-forming cells. Of all class IA PI3Ks, only the PI3Kα isoform was required for sphere formation by medulloblastoma cells. Knockdown of p110α, but not p110β or p110δ, significantly disrupted cancer stem cell frequencies as determined by extreme limiting dilution analysis (ELDA), indicating an essential role for the PI3Kα catalytic isoform in medulloblastoma CSCs. Importantly, pharmacologic inhibition of the MAPK-interacting kinase (MNK) enhanced the antineoplastic effects of targeted PI3Kα inhibition in medulloblastoma. This indicates that MNK signaling promotes survival in medulloblastoma, suggesting dual PI3Kα and MNK inhibition may provide a novel approach to target and eliminate medulloblastoma CSCs. We also observed a significant reduction in tumor formation in subcutaneous and intracranial mouse xenograft models, which further suggests that this combinatorial approach may represent an efficient therapeutic strategy for medulloblastoma. Implications: These findings raise the possibility of a unique therapeutic approach for medulloblastoma, involving MNK targeting to sensitize medulloblastoma CSCs to PI3Kα inhibition.

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.

// Asneha Iqbal 1, 2 , Frank Eckerdt 1 , Jonathan Bell 1, 3 , Ichiro Nakano 4 , Francis J. Giles 1, 3 , Shi-Yuan Cheng 1 , Rishi R. Lulla 1, 2 , Stewart Goldman 1, 2 , Leonidas C. Platanias 1, 3, 5 1 Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL, USA 2 Division of Hematology/Oncology/Stem Cell Transplantation, Ann and Robert H. Lurie Children’s Hospital of Chicago, Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 3 Division of Hematology/Oncology, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA 4 Department of Neurological Surgery and James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA 5 Department of Medicine, Jesse Brown VA Medical Center, Chicago, IL, USA Correspondence to: Leonidas C. Platanias, e-mail: l-platanias@northwestern.edu Keywords: glioblastoma, PIM kinase, mTOR signaling, p110α Received: February 04, 2016 Accepted: April 05, 2016 Published: April 21, 2016 ABSTRACT 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.

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.

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.

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 ATO's 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.

The mTOR pathway controls mRNA translation of mitogenic proteins and is a central regulator of metabolism in malignant cells. Development of malignant cell resistance is a limiting factor to the effects of mTOR inhibitors, but the mechanisms accounting for such resistance are not well understood. We provide evidence that mTORC1 inhibition by rapamycin results in engagement of a negative feedback regulatory loop in malignant medulloblastoma cells, involving phosphorylation of the eukaryotic translation-initiation factor eIF4E. This eIF4E phosphorylation is Mnk2- mediated, but Mnk1-independent, and acts as a survival mechanism for medulloblastoma cells. Pharmacological targeting of Mnk1/2 or siRNA-mediated knockdown of Mnk2 sensitizes medulloblastoma cells to mTOR inhibition and promotes suppression of malignant cell proliferation and anchorage-independent growth. Altogether, these findings provide evidence for the existence of a Mnk2-controlled feedback loop in medulloblastoma cells that accounts for resistance to mTOR inhibitors, and raise the potential for combination treatments of mTOR and Mnk inhibitors for the treatment of medulloblastoma.