Isabella Taylor

The transcriptional modulator HMGA2 promotes stemness and tumorigenicity in glioblastoma.

Glioblastoma (GBM) contains a population of stem-like cells that promote tumor invasion and resistance to therapy. Identifying and targeting stem cell factors in GBM may lead to the development of more effective therapies. High Mobility Group AT-hook 2 (HMGA2) is a transcriptional modulator that mediates motility and self-renewal in normal and cancer stem cells. We identified increased expression of HMGA2 in the majority of primary human GBM tumors and cell lines compared to normal brain. Additionally, HMGA2 expression was increased in CD133+ GBM neurosphere cells compared to CD133- cells. Targeting HMGA2 with lentiviral short hairpin RNA (shRNA) led to decreased GBM stemness, invasion, and tumorigenicity. Ectopic expression of HMGA2 in GBM cell lines promoted stemness, invasion, and tumorigenicity. Our data suggests that targeting HMGA2 in GBM may be therapeutically beneficial.

Disrupting NOTCH Slows Diffuse Intrinsic Pontine Glioma Growth, Enhances Radiation Sensitivity, and Shows Combinatorial Efficacy With Bromodomain Inhibition.

Abstract NOTCH regulates stem cells during normal development and stemlike cells in cancer, but the roles of NOTCH in the lethal pediatric brain tumor diffuse intrinsic pontine glioma (DIPG) remain unknown. Because DIPGs express stem cell factors such as SOX2 and MYCN, we hypothesized that NOTCH activity would be critical for DIPG growth. We determined that primary DIPGs expressed high levels of NOTCH receptors, ligands, and downstream effectors. Treatment of the DIPG cell lines JHH-DIPG1 and SF7761 with the &ggr;-secretase inhibitor MRK003 suppressed the level of the NOTCH effectors HES1, HES4, and HES5; inhibited DIPG growth by 75%; and caused a 3-fold induction of apoptosis. Short hairpin RNAs targeting the canonical NOTCH pathway caused similar effects. Pretreatment of DIPG cells with MRK003 suppressed clonogenic growth by more than 90% and enhanced the efficacy of radiation therapy. The high level of MYCN in DIPG led us to test sequential therapy with the bromodomain inhibitor JQ1 and MRK003, and we found that JQ1 and MRK003 inhibited DIPG growth and induced apoptosis. Together, these results suggest that dual targeting of NOTCH and MYCN in DIPG may be an effective therapeutic strategy in DIPG and that adding a &ggr;-secretase inhibitor during radiation therapy may be efficacious initially or during reirradiation.

Notch signaling activation in pediatric low-grade astrocytoma.

Abstract Pilocytic astrocytoma (PA) is the most common primary brain tumor in children; various signaling pathways have been implicated in its biology. The Notch signaling pathway has been found to play a role in the development, stem cell biology, and pathogenesis of several cancers, but its role in PA has not been investigated. We studied alterations in Notch signaling components in tumor tissue from 18 patients with PA and 4 with other low-grade astrocytomas to identify much needed therapeutic targets. We found that Notch pathway members were overexpressed at the mRNA (NOTCH1, NOTCH2, HEY1, HEY2) and protein (HES1) levels in PAs at various anatomic sites compared with non-neoplastic brain samples. These changes were not associated with specific BRAF alterations. Inhibiting the Notch pathway in the pediatric low-grade astrocytoma cell lines Res186 and Res259 using either RNA interference or a &ggr;-secretase inhibitor resulted in variable, but significant, reduction in cell growth and migration. This study suggests a potential role for Notch signaling in pediatric low-grade astrocytoma tumorigenesis and that Notch signaling may be a viable pathway therapeutic target.

Abstract 3549: C-MYC sensitizes GBM with primitive features to glutamine metabolism disruption

Glioblastoma (GBM) is among the most lethal of known human cancers, with a median survival of less than 15 months. The highly infiltrative nature and genetic heterogeneity of GBM renders treatment difficult. Therefore, better and more targeted therapies are needed for patients with GBM. There is a new WHO subset of GBM that contains primitive neuronal components (GBM-PNC). These tumors can arise from a histologically classic GBM, and often the GBM-PNC portions of the tumor contain C-MYC or N-MYC amplifications. High MYC expression is known to alter cellular metabolism, increasing reliance on glutamine, which may create opportunities for therapeutic intervention. We hypothesized that depriving GBM-PNC cells of glutamine using metabolic inhibitors would suppress growth and tumorigenicity. To create genetically appropriate GBM-PNC models, we derived cortex (CTX) human neural stem cells and transformed them through lentiviral expression of mutant p53, constitutively-active AKT and hTERT. Transformed neurospheres were then lentivirally transduced with either C-MYC or BMI1. These models formed aggressive tumors in mice and recapitulated the histological features of GBM with expression of NESTIN, GFAP, and MAP2. When treated with the glutamine metabolic inhibitors DON or Acivicin, transformed neurospheres that expressed C-MYC had decreased cellular proliferation (BrdU incorporation, P Citation Format: Brad Andrew Poore, Isabella Taylor, Jeffrey Rubens, Allison Hanaford, Micah Maxwell, Charles Eberhart, Eric Raabe. C-MYC sensitizes GBM with primitive features to glutamine metabolism disruption [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3549. doi:10.1158/1538-7445.AM2017-3549

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 4222: Targeting HMGA2 suppresses GBM stemness, invasion and tumorigenicity

Glioblastoma Multiforme (GBM) causes the majority of brain tumor-related deaths in the United States. The high invasiveness of GBM prevents complete surgical resection, allowing tumor recurrence. The lack of curative treatment highlights a dire need to develop specific targeted therapeutics. Identifying and targeting novel molecular markers in glioma stem-like cells (GSCs) thought to be critical for tumor invasion and recurrence could lead to development of better therapies. HMGA2, a non-histone transcriptional modulator, is a regulator of normal and cancer stem cells. The significance of HMGA2 as a potential therapeutic target in GBM has not been investigated. We found increased HMGA2 expression in a subset of primary human GBM tumors, in multiple patient-derived GBM neurosphere and adherent cell lines, and in the mesenchymal subgroup of GBM in The Cancer Genome Atlas (TCGA) database. Lentiviral short hairpin RNA (shRNA)-lowered HMGA2 protein levels and significantly reduced invasion and clonogenicity of GBM cell lines in transwell invasion and soft agar colony formation assays. Pharmacological inhibition of HMGA proteins using the DNA minor-groove binding drug Netropsin significantly inhibited growth of the U-87 MG GBM cell line (P Citation Format: Harpreet Kaur, Marianne Hutt-Cabezas, Isabella Taylor, Laura Asnaghi, Fausto Rodriguez, Brent A. Orr, Charles G. Eberhart, Eric H. Raabe. Targeting HMGA2 suppresses GBM stemness, invasion and tumorigenicity. [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 4222. doi:10.1158/1538-7445.AM2015-4222

Abstract 3099: Dual targeting of mTOR and Notch disrupts growth and promotes apoptosis in diffuse intrinsic pontine glioma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Diffuse intrinsic pontine gliomass (DIPGss) account for 10% of pediatric brain tumors and have a universally poor prognosis. Our laboratory has shown that targeting the NOTCH pathway in glioblastoma (GBM) with gamma secretase inhibitors (GSI), inhibits the growth and viability of tumor neurospheres. Primary DIPG as well as DIPG cell lines express have very high levels of NOTCH activity as measured by western blot and qPCR assays. AKT amplification is observed in primary DIPG, and TORC1/TORC2 activation occurs in DIPG cell lines as measured by western blot. The mTOR and NOTCH pathways are known to interact and alter cancer stem cell growth, stemness, and metabolism. We hypothesized that dual targeting of NOTCH and mTOR in DIPG would lead to decreased proliferation and increased apoptosis compared to targeting either pathway in isolation. In established DIPG cell lines, we observed that GSI treatment decreases NOTCH activity in a dose dependent manner as evidenced by decreased expression of NOTCH targets expression of Hes1 and Hey1 via as measured by western blot and qPCR assays. We also observed a dose-dependent reduction in DIPG proliferation and growth after gamma-secretase inhibition as measured by BrdU (Day 7, DMSO to 5 uM MRK003 had p value <0.05) and MTT assays (Day 5 and Day 7, DMSO to 2 uM and 5 uM MRK003 p value <0.05). However, inhibition of growth and increased apoptosis were not complete. We observed a similar decrease in proliferation and increase in apoptosis using the dual TORC1/TORC2 inhibitor PPT242. Combination therapy with MRK003 and PP242 led to significantly increased apoptosis compared to either treatment alone (cell cycle Sub-G1: DMSO 13.2% vs 1000nM PP242-5uM M003 28.9%). This data suggest that dual targeting of the mTOR and NOTCH pathways with GSI will decrease DIPG cell growth and proliferation, increase cell death, and reduce tumorigenicity. Citation Format: Isabella C. Taylor, Marianne Hutt-Cabezas, Melanie Weingart, Kathy Warren, Howard Chang, Javad Nazarian, Charles G. Eberhart, Eric H. Raabe. Dual targeting of mTOR and Notch disrupts growth and promotes apoptosis in diffuse intrinsic pontine glioma. [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 3099. doi:10.1158/1538-7445.AM2014-3099

Abstract 5044: MYC drives formation of primative neuro-ectodermal tumors in human neural stem cells derived from multiple brain regions.

Primitive neuroectodermal tumors (PNET) are thought to arise from neural stem cells. They are highly aggressive tumors, and they can arise in different regions of the brain. A subset of PNET has genomic amplification of MYC, and the MYC-regulator LIN28A is known to define a group of PNET. The AKT/PTEN pathway is commonly altered in aggressive brain tumors. hTERT is expressed in the majority of PNET. Deletion or mutation of p53 is also a common event in PNET. We hypothesized that a combination of dominant negative R248Wp53, c-myc, AKT and hTERT would transform human neural stem cells into a model for PNET. We used lentiviral vectors to infect human neural stem cells derived from hindbrain and cortex with the same packages of oncogenic elements. After antibiotic selection, we performed western blotting and qPCR to verify the presence of MYC and constitutively active AKT. Increased TP53 expression on western blot was used as a proxy for inactivation of the p53 pathway by dominant negative R248W p53. Increased hTERT expression was verified by qPCR. 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). We then tested the tumor forming ability of cortex and hindbrain derived human neural stem cells, by performing orthotopic intracranial injections on immunocompromised mice. No tumors formed from human neural stem cells infected with one or two oncogenic elements. However, 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. These PNET-like tumors were synaptophysin positive, GFAP negative, had a high proliferative index, and showed signs of leptomeningal dissemination. Taken together, these experiments show that human neural stem cells from different brain regions, when transduced with the same package of oncogenes, form PNET-like orthotopic xenograft tumors. These data suggest that MYC9s transforming power may trump loco-regional differences in neural stem and progenitor cells. Citation Format: Isabella Taylor, Ulf Kahlert, Jarek Maciaczyk, Guido Nikkhah, Charles Eberhart, Eric H. Raabe. MYC drives formation of primative neuro-ectodermal tumors in human neural stem cells derived from multiple brain regions. [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 5044. doi:10.1158/1538-7445.AM2013-5044

Abstract 5051: MYC-driven models of primitive neuroectodermal tumor are sensitive to inhibitors of glutamine metabolism.

Brain tumors are a diverse group of cancers in which alterations in growth regulation pathways drive tumorigenicity. Histologically similar tumors may have significant differences in underlying molecular pathways and this in turn may have significant implications for the development of targeted treatment. The MYC family of proteins promotes proliferation and an aggressive phenotype in diverse cancers. Primitive Neuroectodermal Tumors (PNETs) are high grade tumors that can arise throughout the neuro-axis. One subset of PNETs is MYC amplified, and some PNETs are known to express LIN28A, a key regulator of MYC. MYC expression in tumors leads to increased reliance on glutamine metabolism (i.e. the Warburg effect). We hypothesized that MYC-driven PNET tumors would up regulate glutamine metabolism and that glutaminase inhibitors would selectively target MYC-driven tumors. We tested this hypothesis using the MYC-positive PNET cell line PFSK as well as MYC-transformed human neural stem cells. The glutamine methabolic inhibitors DON (6-Diazo-5-oxo-L-norleucine) and acivicin were used to disrupt glutamine metabolism in these cells. Using Tetrazolium dye reduction (MTT assay) and Bromodeoxyuridine assays (BrdU) as indicators for cell growth and replication, we found that PFSK growth is inhibited by acivicin at 10 micromolar concentration (MTT activity reduced by 88%, p Citation Format: Sama F. Ahsan, Allison Hanaford, Melanie Weingart, Isabella Taylor, Charles Eberhart, Eric Raabe. MYC-driven models of primitive neuroectodermal tumor are sensitive to inhibitors of glutamine metabolism. [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 5051. doi:10.1158/1538-7445.AM2013-5051