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OBJECTIVE Medulloblastoma (MB) is the most common malignant pediatric brain tumor and accounts for approximately 20% of all pediatric CNS tumors. Current multimodal treatment is associated with a 70%–90% 5-year survival rate; however, the prognosis for patients with tumor dissemination and recurrent MB remains poor. The majority of survivors exhibit long-term neurocognitive complications; thus, more effective and less toxic treatments are critically needed. Tumor treating fields (TTFields) are low-intensity, alternating electric fields that disrupt cell division through physical interactions with key molecules during mitosis. Side effects from TTField therapy are minimal, making it an ideal candidate for MB treatment. METHODS To determine if TTFields can be an effective treatment for MB, the authors conducted an in vitro study treating multiple MB cell lines. Three MB molecular subgroups (SHH [sonic hedgehog], group 3, and group 4) were treated for 24, 48, and 72 hours at 100, 200, 300, and 400 kHz. Combinatorial studies were conducted with the small-molecule casein kinase 2 inhibitor CX-4945. RESULTS TTFields reduced MB cell growth with an optimal frequency of 300 kHz, and the most efficacious treatment time was 72 hours. Treatment with TTFields dysregulated actin polymerization and corresponded with a reduction in cell motility and invasion. TTFields also induced DNA damage (γH2AX, 53BP1) that correlated with an increase in apoptotic cells. The authors discovered that CX-4945 works synergistically with TTFields to reduce MB growth. In addition, combining CX-4945 and TTFields increased the cellular actin dysregulation, which correlated with a decrease in MB migration. CONCLUSIONS The findings of this study demonstrate that TTFields may be a novel and less toxic method to treat patients with MB.

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EGFRvIII is a tumor-specific variant of the epidermal growth factor receptor (EGFR). Although EGFRvIII is most commonly found in glioblastoma, its expression in other tumor types remains controversial. In this study, we investigated EGFRvIII expression and amplification in primary breast carcinoma. Our analyses confirmed the presence of EGFRvIII, but in the absence of amplification or rearrangement of the EGFR locus. Nested reverse transcriptase PCR and flow cytometry were used to detect a higher percentage of positive cases. EGFRvIII-positive cells showed increased expression of genes associated with self-renewal and epithelial–mesenchymal transition along with a higher percentage of stem-like cells. EGFRvIII also increased in vitro sphere formation and in vivo tumor formation. Mechanistically, EGFRvIII mediated its effects through the Wnt/β-catenin pathway, leading to increased β-catenin target gene expression. Inhibition of this pathway reversed the observed effects on cancer stem cell (CSC) phenotypes. Together, our findings show that EGFRvIII is expressed in primary breast tumors and contributes to CSC phenotypes in breast cancer cell lines through the Wnt pathway. These data suggest a novel function for EGFRvIII in breast tumorigenesis. Cancer Res; 72(10); 2657–71. ©2012 AACR.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Since surviving patients experience severe neurocognitive disabilities, better and more effective treatments are needed to enhance their quality of life. Casein kinase 2 (CK2) is known to regulate cell growth and survival in multiple cancers; however, the role of CK2 in MB is currently being studied. In this study, we verified the importance of CK2 in MB tumorigenesis and discovered that inhibition of CK2 using the small molecule inhibitor, CX-4945, can sensitize MB cells to a well-known and tolerated chemotherapeutic, temozolomide (TMZ). To study the role of CK2 in MB we modulated CK2 expression in multiple MB cells. Exogenous expression of CK2 enhanced cell growth and tumor growth in mice, while depletion or inhibition of CK2 expression decreased MB tumorigenesis. Treatment with CX-4945 reduced MB growth and increased apoptosis. We conducted a high-throughput screen where 4000 small molecule compounds were analyzed to identify compounds that increased the anti-tumorigenic properties of CX-4945. TMZ was found to work synergistically with CX-4945 to decrease cell survival and increase apoptosis in MB cells. O-6-methylguanine-DNA methyltransferase (MGMT) activity is directly correlated to TMZ sensitivity. We found that loss of CK2 activity reduced β-catenin expression, a known MGMT regulator, which in turn led to a decrease in MGMT expression and an increased sensitivity to TMZ. Our findings show that CK2 is important for MB maintenance and that treatment with CX-4945 can sensitize MB cells to TMZ treatment.

Medulloblastoma (MB) is the most common malignant brain tumor in children and accounts for approximately 20% of all pediatric central nervous system tumors. Current multimodal treatment has led to a 70-90% five-year overall survival rate, however, the prognosis for patients with tumor dissemination and recurrent MB remains poor. Additionally, the majority of survivors exhibit long-term neurocognitive and neuroendocrine complications because of the cytotoxic drugs and high dose radiation. Consequently, more effective and less toxic treatments are necessary in order to raise the quality of life for these young patients. Tumor Treating Fields (TTFields) are an exciting new therapeutic opportunity for MB. TTFields are low intensity, alternating electric fields that disrupt cell division through physical interactions with key molecules during mitosis. The side effects from TTFields treatments are incredibly low, making it an ideal candidate for MB therapy. Currently TTFields were found to increase the survival of adult brain tumor patients by dysregulating the cytoskeletal network leading to mitotic arrest as well as other varied multi-modal mechanisms of action. To determine if TTFields can be an effective treatment for MB, we conducted an in vitro study treating multiple MB cell lines with TTFields. We discovered that TTFields treatment reduced cell growth in three different MB molecular subgroups (SHH, Group 3, and Group 4) with an optimal frequency of 200-300 kHz. We also observed that TTFields treatment dysregulated actin polymerization that corresponded with a reduction in cell motility and invasion. TTFields treatment also increased DNA damage (γH2AX, 53BP1) that correlated with an increase in apoptotic cells. To expand on these initial findings, we conducted a combinatorial study with the small molecule inhibitor, CX-4945. CX-4945 is an inhibitor to casein kinase 2 and is currently being used in a phase I clinical trial to treat recurrent MB (NCT03904862). We discovered that CX-4945 enhanced the growth reduction of TTFields treatment. In addition, combining CX-4945 and TTFields increased the number of cells with dysregulated actin which correlated with a decrease in MB migration and invasion. Our findings demonstrate that TTFields may be a novel and less toxic method to treat MB patients. Citation Format: Ryan T. Nitta, Gordon Li. Tumor treating fields induce DNA damage and apoptosis in medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1833.

Medulloblastoma (MB) is the most common malignant brain tumor in children and accounts for approximately 20% of all pediatric central nervous system tumors. Current multimodal treatment has led to a 70-90% five-year overall survival rate, however, the prognosis for patients with tumor dissemination and recurrent MB remains poor. Additionally, the majority of survivors exhibit long-term neurocognitive and neuroendocrine complications because of the cytotoxic drugs and high dose radiation. Consequently, more effective and less toxic treatments are necessary in order to raise the quality of life for these young patients. Tumor Treating Fields (TTFields) are an exciting new therapeutic opportunity for MB. TTFields are low intensity, alternating electric fields that disrupt cell division through physical interactions with key molecules during mitosis. The side effects from TTFields treatments are incredibly low, making it an ideal candidate for MB therapy. Currently TTFields were found to increase the survival of adult brain tumor patients by dysregulating the cytoskeletal network leading to mitotic arrest and as well as other varied multi-modal mechanisms of action. To determine if TTFields can be an effective treatment for MB, we conducted an in vitro study treating multiple MB cell lines with TTFields. We discovered that TTFields treatment reduced MB cell growth (Daoy, Med1-MB, D425, and D283) with an optimal frequency of 200-250 kHz. We also observed that TTFields treatment dysregulated actin polymerization that corresponded with a reduction in cell motility and invasion. TTFields treatment also caused DNA damage (γH2AX, 53BP1) that correlated with an increase in apoptotic cells. To expand on these initial findings, we conducted a combinatorial study with the small molecule inhibitor, CX-4945. CX-4945 is an inhibitor to casein kinase 2 and is currently being used in a phase I clinical trial to treat recurrent MB (NCT03904862). We discovered that CX-4945 enhanced the growth reduction of TTFields treatment. In addition, combining CX-4945 and TTFields increased the number of cells with dysregulated actin which correlated with a decrease in MB migration and invasion. Our findings demonstrate that TTFields may be a novel and less toxic method to treat MB patients. Citation Format: Ryan T. Nitta, Gordon Li. Tumor treating fields induce DNA damage and apoptosis in medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3049.

Among high-grade brain tumors, glioblastoma is particularly difficult to treat, in part due to its highly infiltrative nature which contributes to the malignant phenotype and high mortality in patients. In order to better understand the signaling pathways underlying glioblastoma invasion, we performed the first large-scale CRISPR-Cas9 loss of function screen specifically designed to identify genes that facilitate cell invasion. We tested 4,574 genes predicted to be involved in trafficking and motility. Using a transwell invasion assay, we discovered 33 genes essential for invasion. Of the 11 genes we selected for secondary testing using a wound healing assay, 6 demonstrated a significant decrease in migration. The strongest regulator of invasion was mitogen-activated protein kinase 4 (MAP4K4). Targeting of MAP4K4 with single guide RNAs or a MAP4K4 inhibitor reduced migration and invasion in vitro. This effect was consistent across three additional patient derived glioblastoma cell lines. Analysis of epithelial-mesenchymal transition markers in U138 cells with lack or inhibition of MAP4K4 demonstrated protein expression consistent with a non-invasive state. Importantly, MAP4K4 inhibition limited migration in a subset of human glioma organotypic slice cultures. Our results identify MAP4K4 as a novel potential therapeutic target to limit glioblastoma invasion.

The original version of this Article omitted Suzana A. Kahn, Siddhartha S. Mitra & Samuel H. Cheshier as jointly supervising authors. This has now been corrected in both the PDF and HTML versions of the Article.

Medulloblastoma is the most common malignant brain tumor of childhood. Group 3 medulloblastoma, the most aggressive molecular subtype, frequently disseminates through the leptomeningeal cerebral spinal fluid (CSF) spaces in the brain and spinal cord. The mechanism of dissemination through the CSF remains poorly understood, and the molecular pathways involved in medulloblastoma metastasis and self-renewal are largely unknown. Here we show that NOTCH1 signaling pathway regulates both the initiation of metastasis and the self-renewal of medulloblastoma. We identify a mechanism in which NOTCH1 activates BMI1 through the activation of TWIST1. NOTCH1 expression and activity are directly related to medulloblastoma metastasis and decreased survival rate of tumor-bearing mice. Finally, medulloblastoma-bearing mice intrathecally treated with anti-NRR1, a NOTCH1 blocking antibody, present lower frequency of spinal metastasis and higher survival rate. These findings identify NOTCH1 as a pivotal driver of Group 3 medulloblastoma metastasis and self-renewal, supporting the development of therapies targeting this pathway., Group 3 medulloblastoma is an aggressive pediatric brain tumour that disseminates through the leptomeningeal cerebral spinal fluid. Here, the authors show that in Group 3 medulloblastoma NOTCH1 activates BMI1 through the activation of TWIST1, driving metastasis and self-renewal, and in mouse models a NOTCH1 blocking antibody decreased spinal metastases.

A major limitation of targeted cancer therapy is the rapid emergence of drug resistance, which often arises through mutations at or downstream of the drug target or through intrinsic resistance of subpopulations of tumor cells. Medulloblastoma (MB), the most common pediatric brain tumor, is no exception, and MBs that are driven by sonic hedgehog (SHH) signaling are particularly aggressive and drug-resistant. To find new drug targets and therapeutics for MB that may be less susceptible to common resistance mechanisms, we used a developmental phosphoproteomics approach in murine granule neuron precursors (GNPs), the developmental cell of origin of MB. The protein kinase CK2 emerged as a driver of hundreds of phosphorylation events during the proliferative, MB-like stage of GNP growth, including the phosphorylation of three of the eight proteins commonly amplified in MB. CK2 was critical to the stabilization and activity of the transcription factor GLI2, a late downstream effector in SHH signaling. CK2 inhibitors decreased the viability of primary SHH-type MB patient cells in culture and blocked the growth of murine MB tumors that were resistant to currently available Hh inhibitors, thereby extending the survival of tumor-bearing mice. Because of structural interactions, one CK2 inhibitor (CX-4945) inhibited both wild-type and mutant CK2, indicating that this drug may avoid at least one common mode of acquired resistance. These findings suggest that CK2 inhibitors may be effective for treating patients with MB and show how phosphoproteomics may be used to gain insight into developmental biology and pathology.

The original version of this Article contained an error in the spelling of the author David Solow-Cordero, which was incorrectly given as David Solow-Codero. This has now been corrected in both the PDF and HTML versions of the Article.

Published online; International audience; A variety of drugs targeting monoamine receptors are routinely used in human pharmacology. We assessed the effect of these drugs on the viability of tumor-initiating cells isolated from patients with glioblastoma. Among the drugs targeting monoamine receptors, we identified prazosin, an a1-and a2B-adrenergic receptor antagonist, as the most potent inducer of patient-derived glioblastoma-initiating cell death. Prazosin triggered apoptosis of glioblastoma-initiating cells and of their differentiated progeny, inhibited glioblastoma growth in orthotopic xenografts of patient-derived glioblastoma-initiating cells, and increased survival of glioblastoma-bearing mice. We found that prazosin acted in glioblastoma-initiating cells independently from adrenergic receptors. Its off-target activity occurred via a PKCd-dependent inhibition of the AKT pathway, which resulted in caspase-3 activation. Blockade of PKCd activation prevented all molecular changes observed in prazosin-treated glioblastoma-initiating cells, as well as prazosin-induced apoptosis. Based on these data, we conclude that prazosin, an FDA-approved drug for the control of hyperten-sion, inhibits glioblastoma growth through a PKCd-dependent mechanism. These findings open up promising prospects for the use of prazosin as an adjuvant therapy for glioblastoma patients.

The purpose of this article is to update the neurosurgical field on current molecular markers important to glioblastoma biology, treatment, and prognosis. The highlighted biologic markers in this article include epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and O6-methylguanine-DNA methyltransferase (MGMT).

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase family and have been implicated in tumorigenesis. One isoform in particular, JNK2α, has been shown to be frequently activated in primary brain tumors, to enhance several tumorigenic phenotypes and to increase tumor formation in mice. As JNK is frequently activated in non-small cell lung carcinoma (NSCLC), we investigated the role of the JNK2α isoform in NSCLC formation by examining its expression in primary tumors and by modulating its expression in cultured cell lines. We discovered that 60% of the tested primary NSCLC tumors had three-fold higher JNK2 protein and two- to three-fold higher JNK2α mRNA expression than normal lung control tissue. To determine the importance of JNK2α in NSCLC progression, we reduced JNK2α expression in multiple NSCLC cell lines using short hairpin RNA. Cell lines deficient in JNK2α had decreased cellular growth and anchorage-independent growth, and the tumors were four-fold smaller in mass. To elucidate the mechanism by which JNK2α induces NSCLC growth, we analyzed the JNK substrate, signal transducer and activator of transcription 3 (STAT3). Our data demonstrates for the first time that JNK2α can regulate the transcriptional activity of STAT3 by phosphorylating the Ser727 residue, thereby regulating the expression of oncogenic genes, such as c-Myc. Furthermore, reintroduction of JNK2α2 or STAT3 restored the tumorigenicity of the NSCLC cells, demonstrating that JNK2α is important for NSCLC progression. Our studies reveal a novel mechanism in which phosphorylation of STAT3 is mediated by a constitutively active JNK2 isoform, JNK2α.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor, accounting for about 20% of all cases. While surgery, craniospinal irradiation, and chemotherapy have resulted in a cure rate of 70%, the surviving patients are afflicted with neurocognitive impairment, endocrine dysfunction, and a severe decrease in quality of life. Consequently, better and more effective treatments are needed to treat these young patients. Casein kinase 2 (CK2) is an intriguing therapeutic target for MB because it is dysregulated in all the MB molecular subgroups and patients with high CK2 expression have a significantly worse prognosis. To elucidate the role of CK2 in MB we transduced multiple MB cell lines with CK2 isoforms. We discovered that a CK2 isoform, CK2a, increased tumorigenesis in both the Sonic Hedgehog and Group 3 MB subgroups, while knocking down expression ameliorated MB growth. Through these transduced cell lines we also determined that CK2 can regulate MB tumorigenesis through b-catenin and potentially GLI1. Moreover, mice orthotopically injected with MB cells over-expressing CK2a or CK2b displayed a reduced survival compared to control. We extended our analysis to a CK2 inhibitor, CX-4945, which is currently undergoing phase I/II clinical trials for toxicity and safety. Treatment with CX-4945 reduced MB cell growth and also reduced expression of MGMT, a well-known regulator of temozolomide (TMZ) efficacy. Our findings were corroborated when we screened 4,000 FDA approved compounds to identify molecules that work synergistically with CX-4945. TMZ was one of the major compounds that were identified in the screen, suggesting that CX-4945 treatment can sensitize MB cells to TMZ by dysregulating MGMT. Combinatorial treatment with CX-4945 and TMZ had a synergistic effect on reducing MB tumor growth. Together, our findings suggest that CK2 is a novel therapeutic target for MB and that combining CX-4945 and TMZ can lead to a promising new MB therapy. Citation Format: Ryan Nitta, Sara Matilda Bolin, Ginikachi Nwagbo, Teresa Purzner, Suzana Kahn, Yoon-Jae Cho, Gordon Li. Casein kinase 2 is a major regulator of medulloblastoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-322.

Background Glioblastoma (GBM) is the most malignant primary brain tumor in adults, with a median survival time of one and a half years. Traditional treatments, including radiation, chemotherapy, and surgery, are not curative, making it imperative to find more effective treatments for this lethal disease. γ-Glutamyl transferase (GGT) is a family of enzymes that was shown to control crucial redox-sensitive functions and to regulate the balance between proliferation and apoptosis. GGT7 is a novel GGT family member that is highly expressed in brain and was previously shown to have decreased expression in gliomas. Since other members of the GGT family were found to be altered in a variety of cancers, we hypothesized that GGT7 could regulate GBM growth and formation. Methods To determine if GGT7 is involved in GBM tumorigenesis, we modulated GGT7 expression in two GBM cell lines (U87-MG and U138) and monitored changes in tumorigenicity in vitro and in vivo. Results We demonstrated for the first time that GBM patients with low GGT7 expression had a worse prognosis and that 87% (7/8) of primary GBM tissue samples showed a 2-fold decrease in GGT7 expression compared to normal brain samples. Exogenous expression of GGT7 resulted in a 2- to 3-fold reduction in proliferation and anchorage-independent growth under minimal growth conditions (1% serum). Decreasing GGT7 expression using either short interfering RNA or short hairpin RNA consistently increased proliferation 1.5- to 2-fold. In addition, intracranial injections of U87-MG cells with reduced GGT7 expression increased tumor growth in mice approximately 2-fold, and decreased mouse survival. To elucidate the mechanism by which GGT7 regulates GBM growth, we analyzed reactive oxygen species (ROS) levels in GBM cells with modulated GGT7 expression. We found that enhanced GGT7 expression reduced ROS levels by 11-33%. Conclusion Our study demonstrates that GGT7 is a novel player in GBM growth and that GGT7 can play a critical role in tumorigenesis by regulating anti-oxidative damage. Loss of GGT7 may increase the cellular ROS levels, inducing GBM occurrence and growth. Our findings suggest that GGT7 can be a promising biomarker and a potential therapeutic target for GBM. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1232-y) contains supplementary material, which is available to authorized users.

Mutations in the LMNA gene, which encodes all A-type lamins, including lamin A and lamin C, cause a variety of tissue-specific degenerative diseases termed laminopathies. Little is known about the pathogenesis of these disorders. Previous studies have indicated that A-type lamins interact with the retinoblastoma protein (pRB). Here we probe the functional consequences of this association and further examine links between nuclear structure and cell cycle control. Since pRB is required for cell cycle arrest by p16(ink4a), we tested the responsiveness of multiple lamin A/C-depleted cell lines to overexpression of this CDK inhibitor and tumor suppressor. We find that the loss of A-type lamin expression results in marked destabilization of pRB. This reduction in pRB renders cells resistant to p16(ink4a)-mediated G(1) arrest. Reintroduction of lamin A, lamin C, or pRB restores p16(ink4a)-responsiveness to Lmna(-/-) cells. An array of lamin A mutants, representing a variety of pathologies as well as lamin A processing mutants, was introduced into Lmna(-/-) cells. Of these, a mutant associated with mandibuloacral dysplasia (MAD R527H), as well as two lamin A processing mutants, but not other disease-associated mutants, failed to restore p16(ink4a) responsiveness. Although our findings do not rule out links between altered pRB function and laminopathies, they fail to support such an assertion. These findings do link lamin A/C to the functional activation of a critical tumor suppressor pathway and further the possibility that somatic mutations in LMNA contribute to tumor progression.

The retinoblastoma protein (pRB) is a critical regulator of cell proliferation and differentiation and an important tumor suppressor. In the G 1 phase of the cell cycle, pRB localizes to perinucleolar sites associated with lamin A/C intranuclear foci. Here, we examine pRB function in cells lacking lamin A/C, finding that pRB levels are dramatically decreased and that the remaining pRB is mislocalized. We demonstrate that A-type lamins protect pRB from proteasomal degradation. Both pRB levels and localization are restored upon reintroduction of lamin A. Lmna -/- cells resemble Rb -/- cells, exhibiting altered cell-cycle properties and reduced capacity to undergo cell-cycle arrest in response to DNA damage. These findings establish a functional link between a core nuclear structural component and an important cell-cycle regulator. They further raise the possibility that altered pRB function may be a contributing factor in dystrophic syndromes arising from LMNA mutation.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. While surgery, craniospinal irradiation, and chemotherapy have resulted in a cure rate of 70–75%, the surviving patients are afflicted with neurocognitive impairment, endocrine dysfunction, and a severe decrease in quality of life. Consequently, better and more effective treatments are needed to treat these young patients. Casein kinase 2 (CK2) is an intriguing therapeutic target for MB because it is dysregulated in all the MB molecular subgroups and patients with high CK2 expression have a significantly worse prognosis. To elucidate the role of CK2 in MB we transduced multiple MB cell lines with CK2 isoforms. We discovered that a CK2 isoform, CK2alpha, increased MB tumorigenesis in both the SHH and Grade 3 molecular subgroups, while knocking down its expression ameliorated MB growth. We also found that CK2 can regulate MB tumorigenesis through beta-catenin and potentially GLI1. We extended our analysis to a CK2 inhibitor, CX-4945, which is currently undergoing phase I/II clinical trials. Treatment with CX-4945 reduced MB cell growth and tumor growth in mice that were intracranially injected with MB cells. Consistent with previous research we found that CX-4945 specifically inhibited CK2 and has the ability to cross the blood-brain-barrier. Treatment with CX-4945 also reduced expression of MGMT, a well-known regulator of temozolomide (TMZ) efficacy. Combinatorial treatments with CX-4945 and TMZ had a synergistic effect on MB tumor reduction. Our findings were corroborated when we screened 4,000 FDA approved compounds to identify molecules that work synergistically with CX-4945. TMZ was one of the major compounds that were identified in the screen suggesting that CX-4945 treatment can sensitize MB cells to TMZ by dysregulating MGMT. Together, our findings suggest that CK2 is a novel MB therapeutic target and combining CX-4945 and TMZ can lead to a promising new MB therapy.

Glioblastoma (GBM) is the most common and fatal primary brain tumor in humans and it is essential that new and better therapies are developed to treat this disease. Previous research suggests that casein kinase 2 (CK2), may be a promising therapeutic target for GBMs. CK2 has enhanced expression or activity in numerous cancers, including GBM and it has been demonstrated that inhibitors of CK2 regressed tumor growth in GBM xenograft mouse models. Our studies demonstrate that the CK2 subunit, CK2α, is overexpressed in and plays an important role in regulating brain tumor initiating cells (BTIC) in GBM. Initial studies showed that two GBM cell lines (U87-MG and U138) transduced with CK2α had enhanced proliferation and anchorage-independent growth. Inhibition of CK2α using siRNA or small molecule inhibitors (TBBz, CX-4945) reduced cell growth and decreased tumor size and increased the survival rate in GBM xenograft mouse models. We also verified that inhibition of CK2α decreased the activity of a well-known GBM initiating cell regulator, β-catenin. Loss of CK2α decreased two β-catenin-regulated genes that are involved in GBM initiating cell growth, OCT4 and NANOG. To determine the importance of CK2α in GBM stem cell maintenance, we reduced CK2α activity in primary GBM samples and tumor spheres derived from GBM patients. We discovered that loss of CK2α activity reduced the sphere forming capacity of BTIC and decreased numerous GBM stem cell markers including CD133, CD90, CD49f, and A2B5. Our study suggests that CK2α is involved in GBM tumorigenesis by maintaining BTIC through the regulation of β-catenin.

Non-Surgical Treatment for Racemic Neurocysticercosis with Compression of the Optic Nerve There are very few reports in the literature to guide treatment for patients with racemic neurocysticercosis with direct compression of the optic nerves leading to vision loss. Although the current approach to a patient with sudden neurologic decline with a large mass lesion is surgical decompression, we present a case of a patient who improved with antiparasitics and steroids alone. A 32-year-old Hispanic male presented with a several-month history of headache and new onset left eye vision loss.

Casein kinase 2 (CK2) is an oncogenic protein kinase which contributes to tumor development, proliferation, and suppression of apoptosis in multiple cancer types. The mechanism by which CK2 expression and activity leads to tumorigenesis in glioblastoma (GBM), a stage IV primary brain tumor, is being studied. Recent studies demonstrate that CK2 plays an important role in GBM formation and growth through the inhibition of tumor suppressors and activation of oncogenes. In addition, intriguing new reports indicate that CK2 may regulate GBM formation in a novel manner; CK2 may play a critical role in cancer stem cell (CSC) maintenance. Since glial CSCs have the ability to self-renew and initiate tumor growth, new treatments which target these CSCs are needed to treat this fatal disease. Inhibition of CK2 is potentially a novel method to inhibit GBM growth and reoccurrence by targeting the glial CSCs. A new, orally available, selective CK2 inhibitor, CX-4945 has had promising results when tested in cancer cell lines, in vivo xenograft models, and human clinical trials. The development of CK2 targeted inhibitors, starting with CX-4945, may lead to a new class of more effective cancer therapies.

EGFRvIII is a tumor-specific variant of the epidermal growth factor receptor (EGFR). Although EGFRvIII is most commonly found in glioblastoma, its expression in other tumor types remains controversial. In this study, we investigated EGFRvIII expression and amplification in primary breast carcinoma. Our analyses confirmed the presence of EGFRvIII, but in the absence of amplification or rearrangement of the EGFR locus. Nested reverse transcriptase PCR and flow cytometry were used to detect a higher percentage of positive cases. EGFRvIII-positive cells showed increased expression of genes associated with self-renewal and epithelial–mesenchymal transition along with a higher percentage of stem-like cells. EGFRvIII also increased in vitro sphere formation and in vivo tumor formation. Mechanistically, EGFRvIII mediated its effects through the Wnt/β-catenin pathway, leading to increased β-catenin target gene expression. Inhibition of this pathway reversed the observed effects on cancer stem cell (CSC) phenotypes. Together, our findings show that EGFRvIII is expressed in primary breast tumors and contributes to CSC phenotypes in breast cancer cell lines through the Wnt pathway. These data suggest a novel function for EGFRvIII in breast tumorigenesis. Cancer Res; 72(10); 2657–71. ©2012 AACR.

Despite numerous clinical trials over the past 2 decades, the overall survival for children diagnosed with diffuse intrinsic pontine glioma (DIPG) remains 9–10 months. Radiation therapy is the only treatment with proven effect and novel therapies are needed. Epidermal growth factor receptor variant III (EGFRvIII) is the most common variant of the epidermal growth factor receptor and is expressed in many tumor types but is rarely found in normal tissue. A peptide vaccine targeting EGFRvIII is currently undergoing investigation in phase 3 clinical trials for the treatment of newly diagnosed glioblastoma (GBM), the tumor in which this variant receptor was first discovered. In this study, we evaluated EGFRvIII expression in pediatric DIPG samples using immunohistochemistry with a double affinity purified antibody raised against the EGFRvIII peptide. Staining of pediatric DIPG histological samples revealed expression in 4 of 9 cases and the pattern of staining was consistent with what has been seen in EGFRvIII transfected cells as well as GBMs from adult trials. In addition, analysis of tumor samples collected immediately post mortem and of DIPG cells in culture by RT-PCR, western blot analysis, and flow cytometry confirmed EGFRvIII expression. We were therefore able to detect EGFRvIII expression in 6 of 11 DIPG cases. These data suggest that EGFRvIII warrants investigation as a target for these deadly pediatric tumors.

Cancer stem cells (CSCs) represent a small subpopulation of a tumor that can self-renew, differentiate, and contribute to metastasis and tumor recurrence. Their unique molecular machinery allows for their resistance to most conventional cancer therapies. Hence, depletion of CSCs in cancer therapy remains a high unmet medical need. Telomerase expression, which plays a central role in replicative immortality as one of the hallmarks of cancer, is present in all CSCs studied to date. Inhibition of telomerase represents an attractive therapeutic approach to target CSCs. Imetelstat is a potent oligonucleotide inhibitor of telomerase that is currently in Phase II clinical trials against multiple cancer indications. Several recent studies demonstrate that imetelstat inhibits telomerase in CSCs and leads to depletion of CSCs in vitro and in vivo. Interestingly, multiple pathways seem to be involved in the depletion of CSCs by imetelstat, depending on the cell type studied. The molecular mechanisms of imetelstat’s effect on CSCs are currently under investigation.

The c-Jun N-terminal kinases (JNK) are important regulators of cell growth, proliferation, and apoptosis. JNKs are typically activated by a sequence of events that include phosphorylation of its T-P-Y motif by an upstream kinase, followed by homodimerization and translocation to the nucleus. Constitutive activation of JNK has been found in a variety of cancers including non-small cell lung carcinomas, gliomas, and mantle cell lymphoma. In vitro studies show that constitutive activation of JNK induces a transformed phenotype in fibroblasts and enhances tumorigenicity in a variety of cell lines. Interestingly, a subset of JNK isoforms was recently found to autoactivate rendering the proteins constitutively active. These constitutively active JNK proteins were found to play a pivotal role in activating transcription factors that increase cellular growth and tumor formation in mice. In this chapter, we describe techniques and methods that have been successfully used to study the three components of JNK activation. Use of these techniques may lead to a better understanding of the components of JNK pathways and how JNK is activated in cancer cells.

c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) family and are important regulators of cell growth, proliferation, and apoptosis. Typically, a sequential series of events are necessary for MAPK activation: phosphorylation, dimerization, and then subsequent translocation to the nucleus. Interestingly, a constitutively active JNK isoform, JNK2alpha2, possesses the ability to autophosphorylate and has been implicated in several human tumors, including glioblastoma multiforme. Because overexpression of JNK2alpha2 enhances several tumorigenic phenotypes, including cell growth and tumor formation in mice, we studied the mechanisms of JNK2alpha2 autophosphorylation and autoactivation. We find that JNK2alpha2 dimerization in vitro and in vivo occurs independently of its autophosphorylation but is dependent on nine amino acids, known as the alpha-region. Alanine scanning mutagenesis of the alpha-region reveals that five specific mutants (L218A, K220A, G221A, I224A, and F225A) prevent JNK2alpha2 dimerization rendering JNK2alpha2 inactive and incapable of stimulating tumor formation. Previous studies coupled with additional mutagenesis of neighboring isoleucines and leucines (I208A, I214A, I231A, and I238A) suggest that a leucine zipper may play an important role in JNK2alpha2 homodimerization. We also show that a kinase-inactive JNK2alpha2 mutant can interact with and inhibit wild type JNK2alpha2 autophosphorylation, suggesting that JNK2alpha2 undergoes trans-autophosphorylation. Together, our results demonstrate that JNK2alpha2 differs from other MAPK proteins in two major ways; its autoactivation/autophosphorylation is dependent on dimerization, and dimerization most likely precedes autophosphorylation. In addition, we show that dimerization is essential for JNK2alpha2 activity and that prevention of dimerization may decrease JNK2alpha2 induced tumorigenic phenotypes.

Medulloblastoma (MB) is a malignant brain tumor that originates in the cerebellum in children and spreads via the cerebrospinal fluid to the leptomeningeal spaces of the brain and of the spinal cord. MB is stratified into four distinct groups, according to genetic and clinical features, and patients from group 3 (also known as c-Myc-amplified group) have the highest risk of developing metastatic disease and, consequently, a poor prognosis. Treatment protocols involve surgery, craniospinal radiation, and high-dose chemotherapy, which frequently cause disabling neurotoxic effects in long-term survivors. We used MB cell lines and primary cells isolated from patients from the c-Myc-amplified group to develop a spontaneous spinal metastasis orthotopic xenograft model as a tool to understand the cellular determinants of leptomeningeal and spinal dissemination. Human cells isolated from the primary site tumors expressed 10 fold higher NICD1 (Notch1 Intracellular Domain), the active form of Notch1, than cells isolated from spinal metastatic sites (as quantified by western blot and imunohistochemistry), suggesting the downregulation of canonical Notch1 signaling pathway in MB metastasis. Moreover, flow cytometry analyses revealed that a higher percentage of cells isolated from metastatic sites expressed full-length surface Notch1 as compared to the primary site tumor. These differences cannot be explained by enrichment in the stem cell population at primary tumor sites, as we observed that MB cells isolated from primary tumors and metastatic sites exhibit equivalent self-renewal potential and express equivalent levels of CD133 and CD15. Our goal is to understand the role of notch in regulating medulloblastoma metastasis. Citation Format: Suzana A. Kahn, Sharareh Gholamin, Michael Zhang, Ryan Nitta, Irving Weissman, Siddhartha Mitra, Samuel Cheshier. Involvement of Notch1 signaling pathway in medulloblastoma metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4041. doi:10.1158/1538-7445.AM2014-4041

Background: Activation of telomerase is essential for the indefinite replication potential of most cancer cells. Inhibition of telomerase is expected to lead to loss of telomere maintenance resulting in cell cycle arrest and/or cancer cell death, making telomerase inhibition an attractive anti-cancer approach. Cancer stem cells (CSCs) are rare cells in tumors implicated in cancer initiation and potentiation, as well as persistence or recurrence after standard treatment. Unlike normal tissue counterparts, all CSCs tested to date have upregulated telomerase activity. Recent identification of several non-canonical roles of telomerase components may expand the functionality of telomerase-inhibiting drugs. Imetelstat, a potent telomerase inhibitor currently in Phase II clinical trials, has been shown to reduce proliferative potential in multiple cancer models. Several recent studies have demonstrated that imetelstat depletes CSCs in various tumor types. The relatively rapid onset of CSC depletion could probably indicate a mechanism independent of telomere shortening. To understand the contributions of non-canonical pathways in the response of cancer cells to imetelstat treatment, we analyzed modulations of key signaling pathways in imetelstat-treated glioblastoma multiforme cancer cell lines (U87-MG and U118) and their CSC and bulk subsets. Results: Imetelstat inhibited telomerase activity, proliferative capacity and colony-forming potential in the U87 and U118 cell lines. Imetelstat treatment (2 weeks at 3uM) resulted in decreased expression of casein kinase 2 (CK2) subunits alpha and beta, and a reduction in phosphorylation of downstream CK2 substrates such as the DNA repair protein XRCC1. In addition, imetelstat reduced the transcriptional activity of beta-catenin, which is regulated by members of the CK2 family, in U87-MG cells. Cyclin D1, which is regulated by beta-catenin activity, was found to be down regulated by imetelstat treatment. These pathway modulations were not observed in T98G, a cell line resistant to imetelstat-mediated proliferation effects. Reducing CK2-alpha expression had an additive effect on cell growth inhibition in combination with imetelstat in U87-MG cells. Knock-down of the CK2-alpha, but not CK2-beta subunits, with siRNAs rapidly reduced the numbers of CSCs in U87-MG and U118 cell lines. Concomitant with the down-regulation of CK2-alpha signaling, imetelstat treatment depleted the number of CSCs in U78-MG and U118 cell lines. Preliminary data indicate a more marked reduction of CK2-alpha levels in the CSC subset of U87-MG cells compared to the bulk cells upon short term imetelstat treatment (1 week at 3uM). These in-vitro exposure concentrations of imetelstat are comparable to those attainable intratumorally in a xenograft mouse model. Conclusions: Our results suggest that CK2-alpha signaling and dysregulation of its downstream targets may play a key role in survival of CSCs as well as bulk tumor cells in U87 and U118 glioblastoma cell lines. These novel insights may help identify drugs that can act synergistically with imetelstat in treating cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A56.

Background EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM), breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community. Results In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAbDMvIII, specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC) and immunofluorescence (IF) and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 107 M-1 as determined by enzyme-linked immunosorbent assay (ELISA). Conclusion This recombinant antibody thus holds great potential to be used as a research reagent and diagnostic tool in research laboratories and clinics because of its high quality, easy viability and unique versatility. This antibody is also a strong candidate to be investigated for further in vivo therapeutic studies.

c-Jun N-terminal kinases (JNKs) are members of the mitogen activated protein kinase (MAPK) family and have been implicated in tumorigenesis. One isoform in particular, JNK2alpha, has been shown to be frequently activated in primary brain tumors, to enhance several tumorigenic phenotypes, and to increase tumor formation in mice. It was reported that there is frequent activation of JNK in non-small cell lung carcinoma (NSCLC). In this study, we investigated the role of the JNK2alpha isoform in NSCLC formation by examining its expression in primary tumors and by modulating its expression in cultured cell lines. We discovered 70% of the tested primary NSCLC tumors had 2 to 3-fold higher JNK2alpha protein and mRNA expression compared to normal lung tissue indicating a possible role of JNK2alpha in NSCLC tumorigenesis. To determine the importance of JNK2alpha in NSCLC progression, we reduced JNK2α in multiple NSCLC cell lines using short hairpin RNA. Cell lines deficient in JNK2alpha had decreased cellular growth and anchorage-independent growth, and the tumors were 4-fold smaller in mass. To elucidate the mechanism by which JNK2alpha induces NSCLC growth, we analyzed the JNK substrate, STAT3. Our data show that JNK2alpha can regulate the transcriptional activity of STAT3 by phosphorylating the Ser727 residue of STAT3 thereby regulating expression of oncogenic genes, such as c-Myc protein. Our studies revealed a novel mechanism in which phosphorylation of STAT3 is mediated by a constitutively active JNK2 isoform, JNK2alpha. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 310.