Antoinette Price

Inhibition of monocarboxylate transporter-4 depletes stem-like glioblastoma cells and inhibits HIF transcriptional response in a lactate-independent manner

Hypoxic regions are frequent in glioblastoma (GBM), the most common type of malignant adult brain tumor, and increased levels of tumor hypoxia have been associated with worse clinical outcomes. To unmask genes important in hypoxia, we treated GBM neurospheres in hypoxia and identified monocarboxylate transporter-4 (MCT4) as one of the most upregulated genes. To investigate the clinical importance of MCT4 in GBM, we examined clinical outcomes and found that MCT4 overexpression is associated with shorter patient survival. Consistent with this, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM, and MCT4 downregulation correlated with the less aggressive G-CIMP (Glioma CpG Methylator Phenotype) subset of GBM. Immunohistochemical analysis of tissue microarrays confirmed that MCT4 protein levels were increased in high-grade as compared with lower-grade astrocytomas, further suggesting that MCT4 is a clinically relevant target. To test the requirement for MCT4 in vitro, we transduced neurospheres with lentiviruses encoding short-hairpin RNAs (shRNAs) against MCT4, resulting in growth inhibition of 50–80% under hypoxia in two lines. MCT4 knockdown was associated with a decreased percentage of cells expressing the stem-cell marker CD133 and increased apoptotic fraction. We also found that flow-sorted CD133-positive cells had almost sixfold higher MCT4 levels than CD133-negative cells, suggesting that the stem-like population might have a greater requirement for MCT4. Most importantly, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo. Interestingly, whereas MCT4 is a well-characterized lactate exporter, we found that both intracellular and extracellular lactate levels did not change following MCT4 silencing, suggesting a novel lactate export-independent mechanism for growth inhibition in GBMs. To identify this potential mechanism, we performed microarray analysis on control and shMCT4-expressing neurospheres and found a dramatic reduction in the expression of multiple Hypoxia-Inducible Factor (HIF)-regulated genes following MCT4 knockdown. The overall reduction in HIF transcriptional response was further validated using a hypoxia response element (HRE)-dependent green-fluorescent protein (GFP) reporter line.

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

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

The TORC1/2 inhibitor TAK228 sensitizes atypical teratoid rhabdoid tumors to cisplatin-induced cytotoxicity

Background Atypical teratoid/rhabdoid tumors (AT/RTs) are deadly pediatric brain tumors driven by LIN28. Mammalian target of rapamycin (mTOR) is activated in many deadly, drug-resistant cancers and governs important cellular functions such as metabolism and survival. LIN28 regulates mTOR in normal cells. We therefore hypothesized that mTOR is activated downstream of LIN28 in AT/RT, and the brain-penetrating mTOR complex 1 and 2 (mTORC1/2) kinase inhibitor TAK228 would reduce AT/RT tumorigenicity. Methods Activation of mTOR in AT/RT was determined by measuring pS6 and pAKT (Ser473) by immunohistochemistry on tissue microarray of 18 primary AT/RT tumors. In vitro growth assays (BrdU and MTS), death assays (CC3, c-PARP by western blot), and survival curves of AT/RT orthotopic xenograft models were used to measure the efficacy of TAK228 alone and in combination with cisplatin. Results Lentiviral short hairpin RNA-mediated knockdown of LIN28A led to decreased mTOR activation. Primary human AT/RT had high levels of pS6 and pAKT (Ser473) in 21% and 87% of tumors by immunohistochemistry. TAK228 slowed cell growth, induced apoptosis in vitro, and nearly doubled median survival of orthotopic xenograft models of AT/RT. TAK228 combined with cisplatin synergistically slowed cell growth and enhanced cisplatin-induced apoptosis. Suppression of AKT sensitized cells to cisplatin-induced apoptosis and forced activation of AKT protected cells. Combined treatment with TAK228 and cisplatin significantly extended survival of orthotopic xenograft models of AT/RT compared with each drug alone. Conclusions TAK228 has efficacy in AT/RT as a single agent and synergizes with conventional chemotherapies by sensitizing tumors to cisplatin-induced apoptosis. These results suggest TAK228 may be an effective new treatment for AT/RT.

Altered gene expression in conjunctival squamous cell carcinoma.

Conjunctival squamous cell carcinoma is a malignancy of the ocular surface. The molecular drivers responsible for the development and progression of this disease are not well understood. We therefore compared the transcriptional profiles of eight snap-frozen conjunctival squamous cell carcinomas and one in situ lesion with normal conjunctival specimens in order to identify diagnostic markers or therapeutic targets. RNA was analyzed using oligonucleotide microarrays, and a wide range of transcripts with altered expression identified, including many dysregulated in carcinomas arising at other sites. Among the upregulated genes, we observed more than 30-fold induction of the matrix metalloproteinases, MMP-9 and MMP-11, as well as a prominent increase in the mRNA level of a calcium-binding protein important for the intracellular calcium signaling, S100A2, which was induced over 20-fold in the tumor cohort. Clusterin was the most downregulated gene, with an approximately 180-fold reduction in the mRNA expression. These alterations were all confirmed by qPCR in the samples used for initial microarray analysis. In addition, immunohistochemical analysis confirmed the overexpression of MMP-11 and S100A2, as well as reductions in clusterin, in several independent in situ carcinomas of conjunctiva. These data identify a number of alterations, including upregulation of MMP-9, MMP-11, and S100A2, as well as downregulation of clusterin, associated with epithelial tumorigenesis in the ocular surface.

mTORC1 inhibition in pediatric low-grade glioma depletes glutathione and therapeutically synergizes with carboplatin

BACKGROUND Pediatric low-grade glioma (pLGG) often initially responds to front-line therapies such as carboplatin, but more than 50% of treated tumors eventually progress and require additional therapy. With the discovery that pLGG often contains mammalian target of rapamycin (mTOR) activation, new treatment modalities and combinations are now possible for patients. The purpose of this study was to determine if carboplatin is synergistic with the mTOR complex 1 inhibitor everolimus in pLGG. METHODS We treated 4 pLGG cell lines and 1 patient-derived xenograft line representing various pLGG genotypes, including neurofibromatosis type 1 loss, proto-oncogene B-Raf (BRAF)-KIAA1549 fusion, and BRAFV600E mutation, with carboplatin and/or everolimus and performed assays for growth, cell proliferation, and cell death. Immunohistochemistry as well as in vivo and in vitro metabolomics studies were also performed. RESULTS Carboplatin synergized with everolimus in all of our 4 pLGG cell lines (combination index <1 at Fa 0.5). Combination therapy was superior at inhibiting tumor growth in vivo. Combination treatment increased levels of apoptosis as well as gamma-H2AX phosphorylation compared with either agent alone. Everolimus treatment suppressed the conversion of glutamine and glutamate into glutathione both in vitro and in vivo. Exogenous glutathione reversed the effects of carboplatin and everolimus. CONCLUSIONS The combination of carboplatin and everolimus was effective at inducing cell death and slowing tumor growth in pLGG models. Everolimus decreased the amount of available glutathione inside the cell, preventing the detoxification of carboplatin and inducing increased DNA damage and apoptosis.

Abstract 2102: Targeting mTOR downstream of LIN28 in atypical teratoid rhabdoid tumors promotes apoptosis and suppresses tumorigenicity

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA LIN28 is a somatic cell reprogramming and stem cell factor that binds to and regulates RNA involved in growth, invasion and metabolic genes. We have previously shown that LIN28 is upregulated in atypical teratoid rhabdoid tumors (AT/RT) and contributes to the aggressive nature of these tumors. One of the canonical downstream targets of LIN28 is the mTOR pathway. We hypothesized that AT/RT tumorgenicity is dependent on mTOR signaling downstream of LIN28 and inhibition of this pathway would disrupt tumor growth. We found that primary human AT/RT samples have high expression of the mTOR pathway as determined by immunohistochemistry staining for P-S6 and P-AKT Ser473 (21% of tumors with 2+ P-S6 staining; 87% with 2+ p-AKT staining). The dual TORC1/2 inhibitor MLN0128 has good brain penetration and is currently being tested in phase I clinical trials. Treatment of AT/RT cell lines with MLN0128 inhibits TORC1/2 targets in vitro and suppresses cell proliferation at 100nM concentration in multiple AT/RT cell lines (MTS assay for BT12 p<0.005 vs DMSO control; BT37 p = 0.006 vs DMSO control; and CHLA-06 p<0.005 vs DMSO control by t-test). MLN0128 also slows cell proliferation via BrdU assay (BT12 p<0.005 vs DMSO control; BT37 p<0.005 100nM vs DMSO control; CHLA-06 p<0.005 100nM vs DMSO control by t-test) and induces apoptosis measured by Western Blot for cleaved PARP and cleaved caspase 3 assay (BT12 p<0.005 100nM vs DMSO; BT37 p<0.005 100nM vs DMSO; CHLA-06 p = 0.007 100nM vs DMSO by t-test). MLN0128 induces apoptosis synergistically with cisplatin, which is the backbone of conventional AT/RT therapy (CC3 assay BT37 p<0.005 vs DMSO by t-test) and slows cell growth (MTS assay BT37 p<0.005 vs DMSO control by t-test). MLN0128 treatment of AT/RT xenograft mouse models extends overall survival from a median of 23 days to 39 days (Log-rank test p = 0.002). Targeting the mTOR pathway with MLN0128 leads to potent in vitro and in vivo activity against AT/RT and can be combined with conventional chemotherapy to provide an important survival benefit. MLN0128 may be a candidate for future clinical trials to treat this deadly tumor. Citation Format: Jeffrey Rubens, Antoinette Price, Brent Orr, Charles Eberhart, Eric Raabe. Targeting mTOR downstream of LIN28 in atypical teratoid rhabdoid tumors promotes apoptosis and suppresses tumorigenicity. [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 2102.

Abstract 504: Glutamine metabolic inhibitors suppress growth and tumorigenicity in MYC and MYCN-driven pediatric malignancies

The MYC family of oncogenes is associated with aggressive pediatric cancers such as medulloblastoma and neuroblastoma. These transcription factors promote oncogenesis in part by altering cellular metabolism. In multiple types of cancer, high-level MYC expression drives increased glutamine metabolism. We hypothesized that MYC-driven medulloblastoma and MYCN amplified neuroblastoma would be sensitive to the glutamine metabolic inhibitors 6-diazo-5-oxo-l-norleucine (DON) and Acivicin. These drugs have previously be used in pediatric clinical trials, but never extensively clinically tested in MYC-driven malignancies. The high MYC medulloblastoma cell lines D425Med and D283Med experienced an 82% decrease in growth as measured by MTS assay when treated with Acivicin or DON (p Citation Format: Allison Hanaford, Catherine Guerra, Charles Eberhart, Eric Raabe, Antoinette Price. Glutamine metabolic inhibitors suppress growth and tumorigenicity in MYC and MYCN-driven pediatric malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 504. doi:10.1158/1538-7445.AM2015-504

Abstract 3272: Targeting LIN28 and the RAS/MAP kinase pathway in atypical teratoid rhabdoid tumors

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Atypical teratoid rhabdoid tumor (AT/RT) is a highly malignant central nervous system neoplasm that primarily affects very young children and has a very poor prognosis. Aside from presumed founder loss of function mutations in the SMARCB1 (BAF47/INI1/SNF5) chromatin remodeling gene, little is known of molecular drivers of AT/RT. LIN28A and LIN28B are stem cell factors that regulate thousands of RNAs and are expressed in aggressive cancers. One of the canonical downstream targets of LIN28 is the RAS/MEK/ERK pathway. Due to the increased stem cell factor expression in AT/RT, we hypothesized that LIN28 contributes to tumorigenesis in these neoplasms through the regulation of multiple pro-growth, stemness, and metabolic pathways. We identified increased levels of LIN28A in AT/RT primary tumors compared to normal brain using immunohistochemistry (P = 0.026 by Mann-Whitney test). We detected LIN28A or LIN28B in 100% of AT/RT cell lines by western blot or qPCR. 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. Colonies formed by BT37 and CHLA-06 cells with LIN28A knockdown were reduced by between 50 and 90 percent (BT37 p = 0.0002 sh800 vs pLKO, CHLA-06 p = 0.009 sh802 vs pLKO). A Cleaved caspase 3 (CC3) assay for apoptosis showed that LIN28A knockdown in BT37 and CHLA-06 cells led to a 4 to 6 fold increase in the percentage of cells expressing CC3 compared to controls measured by immunofluorescence (BT37 p = 0.0005 sh800 vs pLKO; CHLA-06 p = 0.004 sh802 vs pLKO). 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 - p = 0.007 by Log-rank test) showing that LIN28A is critical to AT/RT cell line tumorigenesis. We found lower KRAS expression in LIN28A knockdown cell lines compared to pLKO control using qPCR. We also found high expression levels of phospho-ERK in AT/RT primary tumors by immunohistochemistry. Increased ERK signaling correlated with LIN28A expression in AT/RT (P = 0.008, r = 0.57 by Spearman correlation). Trametinib is a MEK1 and MEK2 inhibitor that has been developed through phase III clinical trials in adult melanoma. Trametinib inhibited the KRAS pathway in CHLA-06-ATRT and BT37 AT/RT cell lines as measured by suppression of p-ERK by Western blot. Treatment with Trametinib for 5 days suppressed the growth of BT37 and CHLA-06 cells (MTS assay for BT37 p = 0.003 for 1 nM and p = 0.002 for 10 nM vs DMSO control; for CHLA-06 p<0.001 for 1 nM and 10nM vs DMSO control by t-test). These data implicate LIN28/RAS/MAP kinase as key drivers of AT/RT tumorigenesis and indicate that targeting this pathway may serve as a novel therapeutic option in this aggressive pediatric malignancy. Citation Format: Jeffrey Rubens, Melanie Weingart, Antoinette Price, Marianne Hutt-Cabezas, Isabella Taylor, Sariah Allen, Brent Orr, Charles Eberhart, Eric Raabe. Targeting LIN28 and the RAS/MAP kinase pathway in atypical teratoid rhabdoid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3272. doi:10.1158/1538-7445.AM2015-3272

Abstract 494: MYC, dominant negative p53 and AKT transform human neural stem cells into primitive neuro-ectodermal tumors sensitive to glutaminase inhibitors

Primitive neuroectodermal tumors (PNET) are highly aggressive tumors that can arise in different regions of the brain. A subset of PNET have genomic amplification of MYC, and the MYC-regulator LIN28A is known to define a group of PNET. Both MYC and LIN28 regulate metabolism in normal and malignant cells. We hypothesized that a combination of dominant negative R248Wp53, MYC, AKT and hTERT would transform human neural stem cells into PNET-like tumors. We used lentiviral vectors to infect human neural stem cells derived from hindbrain and cortex with the same packages of oncogenic elements. Hindbrain neural stem cells transduced with MYC/R248Wp53/AKT/hTERT proliferated at an increased rate compared to control cells as measured by BrdU incorporation (Hindbrain pBABE vs. Hindbrain MYC/R248Wp53/AKT/hTERT: 4% vs. 50% BrdU positive, t-test p = 0.001). Similar results were observed in cortex derived human neural stem cells transduced with MYC/R248Wp53/AKT/hTERT (Cortex pBABE vs Cortex MYC/R248Wp53/AKT/hTERT: 5% vs. 24% BrdU positive, t-test p = 0.027). Both hindbrain and cortex derived human neural stem cells transformed with MYC /R248Wp53/hTERT, and MYC/R248Wp53/hTERT/AKT formed aggressive orthotopic xenograft tumors with histologic characteristics of PNET. The latency of tumor formation decreased with increasing addition of oncogenes. We detected increased glutaminase (GLS) in our MYC-driven human neural stem cell models compared to SV40 immortalized neural stem cells, and hypothesized that GLS inhibitors would inhibit the growth of MYC-driven and LIN28 driven brain tumors. The glutaminase inhibitors DON (6-Diazo-5-oxo-L-norleucine) and acivicin suppressed the growth of our MYC-driven neural stem cell models of PNET as well as the PNET cell line PFSK (p = 0.003) as measured by BrdU and MTT growth assay. Cell cycle analysis of MYC-transduced human neural stem cell models showed increased cell death as measured by sub G1 population after treatment with DON and acivicin. This data shows that glutaminase inhibition may be a useful therapeutic modality in PNET. Citation Format: Isabella Taylor, Sama Ahsan, Antoinette Price, Charles Eberhart, Eric H. Raabe. MYC, dominant negative p53 and AKT transform human neural stem cells into primitive neuro-ectodermal tumors sensitive to glutaminase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 494. doi:10.1158/1538-7445.AM2015-494

Abstract 2930: Silencing MCT4 inhibits GBM growth, HIF response, and CD133-positive fraction in a lactate-independent fashion.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Glioblastomas (GBM) contain a hypoxic core surrounded by proliferative cells. Our group and others have shown that GBM stem cells favor a hypoxic microenvironment, and it is believed that many reside in the tumor core. Conventional therapies target the tumor bulk, but may spare stem cells in the hypoxic niche. To patch this therapeutic loophole, we sought to target the GBM stem cell microenvironment by identifying genes that are important for survival in hypoxia. We identified monocarboxylate transporter 4 (MCT4) as one of the most upregulated genes in our GBM neurosphere lines in response to hypoxia. Clinically, GBM patients with a two-fold or more upregulation of MCT4 have a significantly shorter survival (p = 0.036) than patients with intermediate expression. Consistent with this data, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM (p<0.0001). Using immunohistochemical analysis, we also found that MCT4 protein levels are increased in high-grade as compared to lower grade astrocytomas (p<0.0001), confirming the clinical importance of MCT4. To test the requirement for MCT4 under hypoxia in vitro, we silenced MCT4 in neurospheres using lentiviruses encoding short hairpin RNA (shRNA) specific for MCT4. Cell growth was inhibited in hypoxia by ∼60% in HSR-GBM1 and ∼70% in JHH-GBM10 in these neurosphere lines following MCT4 knockdown. Interestingly, while MCT4 was expressed at lower levels in normoxia, silencing it in 21% oxygen also significantly inhibited growth. CD133-positive stem-like cells expressed a significantly higher amount of MCT4 compared to CD133-negative cells, and decreased proliferation in vitro following MCT4 silencing was associated with reduced CD133-positive stem-like cells and increased apoptosis. This suggests that MCT4 is critical for the survival of CD133-positive stem-like cells under hypoxia, and its inhibition targets the stem cell pool in the hypoxic niche in tumors. Importantly, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo (p=0.009). Interestingly, while multiple earlier studies had identified and characterized MCT4 as a lactate exporter, we found that both extracellular and intracellular lactate levels did not change following MCT4 silencing. Instead, MCT4 inhibition led to downregulation of the HIF (Hypoxia-Inducible Factor) response, reducing HIF target gene expression and pointing to a potential novel lactate-independent function of MCT4 and a unique mechanism for growth inhibition in malignant gliomas. Citation Format: Kah Suan Lim, Kah Jing Lim, Brent A. Orr, Antoinette C. Price, Charles G. Eberhart, Eli E. Bar. Silencing MCT4 inhibits GBM growth, HIF response, and CD133-positive fraction in a lactate-independent fashion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2930. doi:10.1158/1538-7445.AM2013-2930