MEK Inhibition Suppresses Growth of Atypical Teratoid/Rhabdoid Tumors.

Atypical teratoid/rhabdoid (AT/RT) tumors are the most common malignant brain tumor of infancy and have a poor prognosis. We have previously identified very high expression of LIN28A and/or LIN28B in AT/RT tumors and showed that AT/RT have corresponding increased expression of the mitogen-activated protein (MAP) kinase pathway. Binimetinib is a novel inhibitor of mitogen-activated protein kinase (MAP2K1 or MEK), and is currently in pediatric phase II clinical trials for low-grade glioma. We hypothesized that binimetinib would inhibit growth of AT/RT cells by suppressing the MAP kinase pathway. Binimetinib inhibited AT/RT growth at nanomolar concentrations. Binimetinib decreased cell proliferation and induced apoptosis in AT/RT cells and significantly reduced AT/RT tumor growth in flank xenografts. Our data suggest that MAP kinase pathway inhibition could offer a potential avenue for treating these highly aggressive tumors.

MEK/MELK inhibition and blood-brain barrier deficiencies in atypical teratoid/rhabdoid tumors.

BACKGROUND: Atypical teratoid/rhabdoid tumors (AT/RT) are rare, but highly aggressive. These entities are of embryonal origin occurring in the central nervous system (CNS) of young children. Molecularly these tumors are driven by a single hallmark mutation, resulting in inactivation of SMARCB1 or SMARCA4. Additionally, activation of the MAPK signaling axis and preclinical antitumor efficacy of its inhibition have been described in AT/RT. METHODS: We established and validated a patient-derived neurosphere culture and xenograft model of sonic hedgehog (SHH) subtype AT/RT, at diagnosis and relapse from the same patient. We set out to study the vascular phenotype of these tumors to evaluate the integrity of the blood-brain barrier (BBB) in AT/RT. We also used the model to study combined mitogen-activated protein kinase kinase (MEK) and maternal embryonic leucine zipper kinase (MELK) inhibition as a therapeutic strategy for AT/RT. RESULTS: We found MELK to be highly overexpressed in both patient samples of AT/RT and our primary cultures and xenografts. We identified a potent antitumor efficacy of the MELK inhibitor OTSSP167, as well as strong synergy with the MEK inhibitor trametinib, against primary AT/RT neurospheres. Additionally, vascular phenotyping of AT/RT patient material and xenografts revealed significant BBB aberrancies in these tumors. Finally, we show in vivo efficacy of the non-BBB penetrable drugs OTSSP167 and trametinib in AT/RT xenografts, demonstrating the therapeutic implications of the observed BBB deficiencies and validating MEK/MELK inhibition as a potential treatment. CONCLUSION: Altogether, we developed a combination treatment strategy for AT/RT based on MEK/MELK inhibition and identify therapeutically exploitable BBB deficiencies in these tumors.

Genomic Alterations in Undifferentiated Malignant Tumors with Rhabdoid Phenotype and Loss of BRG1 Immunoexpression Identified by Fine Needle Aspirates.

OBJECTIVE: Evidence shows that the switch/sucrose nonfermenting chromatin remodeling complex plays a critical role in DNA repair, cancer progression and dedifferentiation. BRG1 is one of its key catalytic subunits. While the loss of BRG1 expression by immunocytochemistry has been identified in a subset of malignancies arising in various sites with undifferentiated/rhabdoid morphology and poor prognosis, the underlying basis for its loss is unclear. METHODS: A retrospective search was conducted in our cytopathology archive for undifferentiated malignant tumors with rhabdoid phenotype and BRG1 loss. Clinical information was obtained from electronic medical records. Next-generation sequencing was performed following macro-dissection of paraffin-embedded cellblock tissue. RESULTS: Three cases were identified; all presented with widely metastatic disease with no previously diagnosed primary malignancy, and subsequently died within 6 months of initial presentation. Cytologically, the aspirates showed dyshesive and undifferentiated cells with rhabdoid features. Extensive immunocytochemical workup demonstrated immunoreactivity with vimentin only and could not establish a specific lineage. BRG1 expression was absent, while INI1 expression was retained. Two cases harbored deleterious mutations in BRG1/SMARCA4. Pathogenic mutations in TP53 were identified in all tumors. CONCLUSIONS: BRG1 deficiency reflects underlying mutation in SMARCA4 gene in some but not all cases, suggesting that additional mechanisms may be causing BRG1 silencing. Pathogenic mutations in TP53 in all tumors are consistent with their highly aggressive nature. Recognizing the cytomorphology of this group of neoplasms and confirming their BRG1-deficient status by immunocytochemistry not only has prognostic implications, but may also impart potentially therapeutic value in the near future.

Inhibition of DNA damage repair by the CDK4/6 inhibitor palbociclib delays irradiated intracranial atypical teratoid rhabdoid tumor and glioblastoma xenograft regrowth.

BACKGROUND: Radiation therapy is the most commonly used postsurgical treatment for primary malignant brain tumors. Consequently, investigating the efficacy of chemotherapeutics combined with radiation for treating malignant brain tumors is of high clinical relevance. In this study, we examined the cyclin-dependent kinase 4/6 inhibitor palbociclib, when used in combination with radiation for treating human atypical teratoid rhabdoid tumor (ATRT) as well as glioblastoma (GBM). METHODS: Evaluation of treatment antitumor activity in vitro was based upon results from cell proliferation assays, clonogenicity assays, flow cytometry, and immunocytochemistry for DNA double-strand break repair. Interpretation of treatment antitumor activity in vivo was based upon bioluminescence imaging, animal subject survival analysis, and staining of tumor sections for markers of proliferation and apoptosis. RESULTS: For each of the retinoblastoma protein (RB)-proficient tumor models examined (2 ATRTs and 2 GBMs), one or more of the combination therapy regimens significantly (P < .05) outperformed both monotherapies with respect to animal subject survival benefit. Among the combination therapy regimens, concurrent palbociclib and radiation treatment and palbociclib treatment following radiation consistently outperformed the sequence in which radiation followed palbociclib treatment. In vitro investigation revealed that the concurrent use of palbociclib with radiation, as well as palbociclib following radiation, inhibited DNA double-strand break repair and promoted increased tumor cell apoptosis. CONCLUSIONS: Our results support further investigation and possible clinical translation of palbociclib as an adjuvant to radiation therapy for patients with malignant brain tumors that retain RB expression.

Disrupting LIN28 in atypical teratoid rhabdoid tumors reveals the importance of the mitogen activated protein kinase pathway as a therapeutic target.

Atypical teratoid rhabdoid tumor (AT/RT) is among the most fatal of all pediatric brain tumors. Aside from loss of function mutations in the SMARCB1 (BAF47/INI1/SNF5) chromatin remodeling gene, little is known of other molecular drivers of AT/RT. LIN28A and LIN28B are stem cell factors that regulate thousands of RNAs and are expressed in aggressive cancers. We identified high-levels of LIN28A and LIN28B in AT/RT primary tumors and cell lines, with corresponding low levels of the LIN28-regulated microRNAs of the let-7 family. Knockdown of LIN28A by lentiviral shRNA in the AT/RT cell lines CHLA-06-ATRT and BT37 inhibited growth, cell proliferation and colony formation and induced apoptosis. Suppression of LIN28A in orthotopic xenograft models led to a more than doubling of median survival compared to empty vector controls (48 vs 115 days). LIN28A knockdown led to increased expression of let-7b and let-7g microRNAs and a down-regulation of KRAS mRNA. AT/RT primary tumors expressed increased mitogen activated protein (MAP) kinase pathway activity, and the MEK inhibitor selumetinib (AZD6244) decreased AT/RT growth and increased apoptosis. These data implicate LIN28/RAS/MAP kinase as key drivers of AT/RT tumorigenesis and indicate that targeting this pathway may be a therapeutic option in this aggressive pediatric malignancy.

Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2.

Inactivation of the switch/sucrose nonfermentable complex component SMARCB1 is extremely prevalent in pediatric malignant rhabdoid tumors (MRTs) or atypical teratoid rhabdoid tumors. This alteration is hypothesized to confer oncogenic dependency on EZH2 in these cancers. We report the discovery of a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity, (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide). The compound induces apoptosis and differentiation specifically in SMARCB1-deleted MRT cells. Treatment of xenograft-bearing mice with (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide) leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation. These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers.

Histone deacetylases expression in atypical teratoid rhabdoid tumors.

PURPOSE: Atypical teratoid rhabdoid tumors (ATRTs) are rare, highly malignant central nervous system tumors that occur during infancy and early childhood. Their poor outcome and resistance to conventional chemotherapies and radiotherapy, urges the development of new therapies. Recent studies have evaluated the effects of histone deacetylase inhibitors (HDACi) as a new potential treatment for ATRTs. However, most HDACi act unselectively against all, or at least several, histone deacetylase (HDAC) family members. We hypothesized that specific HDAC family members are deregulated in ATRT and therefore a more selective class of HDACi would be beneficial to patients with ATRT. METHODS: To test our hypothesis, we evaluated the expression level of different HDAC family members in ATRTs. Eight ATRTs were compared to six medulloblastoma samples in regards to the level of expression of the 18 HDAC family members as determined by microarray gene expression profiling. RESULTS: HDAC1 was the only member of the HDAC family to be significantly differentially expressed in ATRTs (FC = 4.728; p value = 0.00003). CONCLUSIONS: A class of HDACi specifically targeting HDAC1 may allow for the desired therapeutic benefits with fewer side effects for children with ATRT.

Targeting Aurora Kinase A enhances radiation sensitivity of atypical teratoid rhabdoid tumor cells.

Atypical teratoid/rhabdoid tumors (ATRT) are rare, highly malignant, embryonal CNS tumors with a poor prognosis. Therapy relies on highly toxic chemotherapy and radiotherapy. To improve outcomes and decrease morbidity, more targeted therapy is required. Gene expression analysis revealed elevated expression of multiple kinases in ATRT tissues. Aurora Kinase A was one of the candidate kinases. The objective of this study was to evaluate the impact of Aurora Kinase A inhibition in ATRT cell lines. Our analysis revealed that inhibition of Aurora Kinase A induces cell death in ATRT cells and the small molecule inhibitor MLN 8237 sensitizes these cells to radiation. Furthermore, inhibition of Aurora Kinase A resulted in decreased activity of pro-proliferative signaling pathways. These data indicate that inhibition of Aurora Kinase A is a promising small molecule target for ATRT therapy.

Aurora A is a repressed effector target of the chromatin remodeling protein INI1/hSNF5 required for rhabdoid tumor cell survival.

Rhabdoid tumors (RT) are aggressive pediatric malignancies with poor prognosis. INI1/hSNF5 is a component of the chromatin remodeling SWI/SNF complex and a tumor suppressor deleted in RT. Previous microarray studies indicated that reintroduction of INI1/hSNF5 into RT cells leads to repression of a high degree of mitotic genes including Aurora Kinase A (Aurora A, STK6). Here, we found that INI1/SNF5 represses Aurora A transcription in a cell-type-specific manner. INI1-mediated repression was observed in RT and normal cells but not in non-RT cell lines. Chromatin immunoprecipitation (ChIP) assay indicated that INI1/hSNF5 associates with Aurora A promoter in RT and normal cells but not in non-RT cells. Real-time PCR and immunohistochemical analyses of primary human and mouse RTs harboring mutations in INI1/hSNF5 gene indicated that Aurora A was overexpressed/derepressed in these tumor cells, confirming that INI1/hSNF5 represses Aurora A in vivo. Knockdown of Aurora A impaired cell growth, induced mitotic arrest and aberrant nuclear division leading to decreased survival, and increased cell death and caspase 3/7-mediated apoptosis in RT cells (but not in normal cells). These results indicated that Aurora A is a direct downstream target of INI1/hSNF5-mediated repression in RT cells and that loss of INI1/hSNF5 leads to aberrant overexpression of Aurora A in these tumors, which is required for their survival. We propose that a high degree of Aurora A expression may play a role in aggressive behavior of RTs and that targeting expression or activity of this gene is a novel therapeutic strategy for these tumors.

Immunohistochemical analyses of alpha1 and alpha3 Na+/K+-ATPase subunit expression in medulloblastomas.

BACKGROUND: The levels of expression of the α1 and α3 subunits of the Na(+)/K(+)-ATPase (the NaK sodium pump) in medulloblastomas are unclear. PATIENTS AND METHODS: This study investigated the expression of the NaK subunits using immunohistochemical methods in 29 medulloblastomas including 23 classic, three large-cell/anaplastic and three nodular/desmoplastic medulloblastomas, as well as in three atypical teratoid/rhabdoid tumors (AT/RTs). RESULTS: There was overexpression of the α1 or α3 NaK subunits in more than half of the medulloblastomas and atypical AT/RTs, with about one-third of these tumours displaying overexpression of both subunits. CONCLUSION: These preliminary data suggest that targeting these subunits in AT/RTs and medulloblastomas that overexpress these proteins may lead to therapeutic benefit. These findings warrant confirmation in larger numbers of patients than those used in this study. Moreover, it should be determined whether inhibition of the α1/α3 NaK subunits can be integrated into the risk stratification schemes already in use for medulloblastoma patients.

The tyrosine kinase c-Abl promotes proliferation and is expressed in atypical teratoid and malignant rhabdoid tumors.

BACKGROUND: Atypical teratoid/rhaboid tumors (AT/RTs) and extracranial malignant rhabdoid tumors are highly malignant neoplasms with a dismal prognosis. These tumors predominantly affect infants and targeted, adjuvant treatment approaches would be highly desirable. METHODS: In the current study, the authors investigated the expression and functional role of tyrosine kinases in 2 malignant rhabdoid tumor cell lines (A204 and G401) and in a series of 5 AT/RTs and 18 malignant rhabdoid tumors (13 rhabdoid tumors of the kidney and 5 extrarenal rhabdoid tumors). RESULTS: Both cell lines consistently expressed the tyrosine kinase c-Abl, which promoted proliferation as assessed by small interfering RNA knockdown. Blockage of c-Abl using the tyrosine kinase inhibitor imatinib resulted in reduced cellular growth in both cell lines. Furthermore, c-Abl was expressed in all rhabdoid tumors, whereas expression of platelet-derived growth factor receptor subtypes alpha and beta was infrequent and c-Kit expression was absent. CONCLUSIONS: The current data pointed toward a role for c-Abl in the biology of malignant rhabdoid tumors and provided a rationale for the investigation of tyrosine kinase inhibitors that target c-Abl for the treatment of these aggressive tumors.

Rhabdoid tumor growth is inhibited by flavopiridol.

PURPOSE: Rhabdoid tumors are aggressive and incurable pediatric malignancies. INI1/hSNF5, a tumor suppressor biallelically deleted/inactivated in rhabdoid tumors, directly represses cyclin D1. Rhabdoid tumors and cells are exquisitely dependent on cyclin D1 for genesis and survival, suggesting that targeting the cyclin/cyclin-dependent kinase (cdk) axis may be an effective therapeutic strategy for these tumors. Because cdk inhibitors have not been used for preclinical or clinical testing on rhabdoid tumors, we investigated the effect of flavopiridol, a pan-cdk inhibitor with promising clinical activity, on rhabdoid tumors. EXPERIMENTAL DESIGN: The effect of flavopiridol on rhabdoid cells was tested in vitro using survival, cell cycle, and apoptosis assays. Its effect was assessed in vivo using xenografted rhabdoid tumor models. Immunoblot and immunohistochemical analysis was used to assess the effect of flavopiridol on cyclin D1 and p21 expression in vitro and in vivo, respectively. RESULTS: Nanomolar concentrations of flavopiridol inhibited rhabdoid cell growth (IC(50) approximately 200 nmol/L), induced G(1) and G(2) arrest, and apoptosis in vitro in a concentration-dependent manner. These effects were correlated with the down-modulation of cyclin D1, up-regulation of p21, and induction of caspase 3/7 activities. Flavopiridol (at 7.5 mg/kg) significantly inhibited the growth of xenografted rhabdoid tumors, and its effect was correlated with the induction of p21 and down-modulation of cyclin D1. CONCLUSIONS: Flavopiridol is effective in inducing cell cycle arrest and cytotoxicity in rhabdoid tumors. Its effects are correlated with the down-regulation of cyclin D1 and the up-regulation of p21. Flavopiridol is potentially a novel chemotherapeutic agent for rhabdoid tumors.

Etodolac, a selective cyclooxygenase-2 inhibitor, induces apoptosis by activating caspases in human malignant rhabdoid tumor cells (FRTK-1).

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive tumor presenting in the kidney and soft tissue in childhood. However, effective treatment for MRT has not been established. We investigated the antitumor effect of etodolac, a selective cyclooxygenase-2 inhibitor, on MRT cells in vitro using the MRT cell line FRTK-1. Etodolac induced apoptosis of FRTK-1 cells through activation of caspase-8, -9 and -3. Moreover, several caspase inhibitors completely or partially inhibited etodolac-induced apoptosis. Our data indicated that etodolac had an antitumor effect on MRT cells and holds promise as a novel therapeutic strategy for MRT.

Establishment and characterization of a new cell line, FRTK-1, derived from human malignant rhabdoid tumor of the kidney, with overexpression of epidermal growth factor receptor and cyclooxygenase-2.

A new human malignant rhabdoid tumor (MRT) cell line (designated FRTK-1) was established from MRT of the kidney of an 18-month-old boy. The cell line is maintained for over 24 months with more than 100 passages. FRTK-1 cells in vitro showed 2 different growth patterns, adherent and non-adherent patterns. The FRTK-1 cells showed the same morphological and immunophenotypical characteristics as primary tumor cells of kidney. Cytogenetic and molecular analyses revealed a non-sense mutation in the hSNF5/INI1 gene and loss of expression of hSNF5/INI1 gene product protein. Epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) were expressed in the FRTK-1 cells. Until now, there has been no report of MRT cell lines with expression of COX-2. Therefore, FRTK-1 cell line might be useful for investigating biological behavior and developing new molecular targeting antitumor drugs for MRT with expression of EGFR or COX-2.

Characterization and inhibition of fatty acid synthase in pediatric tumor cell lines.

The current study characterizes the lipogenic enzyme fatty acid synthase (FAS; EC 2.3.1.85) in pediatric tumor cell lines of neural or neural crest origin [medulloblastoma (Daoy), malignant rhabdoid tumor of kidney (SM II), retinoblastoma (Y79), and neuroblastoma (SK-N-SH)]. Constitutive FAS content and activity in these lines were compared to human fibroblast cell line Hs27. Hs27 exhibits low levels of FAS and recapitulates enzyme status in normal human tissues under most physiological conditions. Western analysis detected significantly larger amounts of FAS protein in Y79 and SK-N-SH than Daoy, SM II and Hs27. Incorporation of radiolabeled malonyl-CoA into total cellular lipid revealed that enzyme activity correlated with amount. Increased FAS content and activity in Y79 and SK-N-SH relative to the other cell lines and Hs27, in particular, implied enzyme activation in retinoblastoma and neuroblastoma lineages. The enzyme also showed evidence of hormonal regulation, as dexamethasone induced FAS protein in Daoy and SK-N-SH. However, hormonal induction of FAS protein levels did not correlate with activity levels, which led us to speculate phosphorylation as a means of regulating the enzyme's activity. Finally, the FAS inhibitor cerulenin was investigated for its ability to suppress tumor cell growth. After four days of propagation, short-term treatment of cell lines with drug produced mean IC50s less than 10.5 micrograms/ml (i.e., 5.6 +/- 1.9 for SM II; 9.3 +/- 1.5 for Daoy; 10.2 +/- 0.2 for SK-N-SH; and 10.4 +/- 2.6 for Y79). Annexin V assays revealed that cerulenin initiated apoptosis. The antineoplastic properties of cerulenin documented here are consistent with prior studies showing its cytotoxic effects upon other types of cancer cells and illustrate the potential utility of FAS inhibition as a novel chemotherapeutic approach.

Detection of telomerase activity in oral lesions.

Telomerase is a ribonucleoprotein complex intimately associated with cell immortalization and neoplastic transformation. In almost all types of cancer this enzyme is reactivated and stabilizes telomere length. It may be necessary for continuous cell proliferation. In this study we used a non-radioactive polymerase chain reaction assay to analyze telomerase activity in various tissue specimens taken from the oral cavity. Four of 4 (100%) squamous cell carcinoma cell lines, 28 of 29 (96%) malignant tumors, 10 of 28 (36%) benign lesions, and none of the 14 (0%) oral control tissues possessed telomerase activity. Moreover, 4 of 15 (27%) oral rinses and 3 of 3 (100%) samples of ascites and pleural effusion taken from patients with oral malignancy were telomerase positive. These findings indicate that the evaluation of telomerase activity in tissue and body fluid specimens may provide information useful in the diagnosis of oral malignancy.