Marianne Hütt-Cabezas

Functionally defined therapeutic targets in diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer. We performed a chemical screen in patient-derived DIPG cultures along with RNA-seq analyses and integrated computational modeling to identify potentially effective therapeutic strategies. The multi–histone deacetylase inhibitor panobinostat demonstrated therapeutic efficacy both in vitro and in DIPG orthotopic xenograft models. Combination testing of panobinostat and the histone demethylase inhibitor GSK-J4 revealed that the two had synergistic effects. Together, these data suggest a promising therapeutic strategy for DIPG.

Erratum: Functionally defined therapeutic targets in diffuse intrinsic pontine glioma(Nature Medicine (2015) 21 (555-559) DOI: 10.1038/nm.3855)

Catherine S Grasso, Yujie Tang, Nathalene Truffaux, Noah E Berlow, Lining Liu, Marie-Anne Debily, Michael J Quist, Lara E Davis, Elaine C Huang, Pamelyn J Woo, Anitha Ponnuswami, Spenser Chen, Tessa B Johung, Wenchao Sun, Mari Kogiso, Yuchen Du, Lin Qi, Yulun Huang, Marianne Hütt-Cabezas, Katherine E Warren, Ludivine Le Dret, Paul S Meltzer, Hua Mao, Martha Quezado, Dannis G van Vuurden, Jinu Abraham, Maryam Fouladi, Matthew N Svalina, Nicholas Wang, Cynthia Hawkins, Javad Nazarian, Marta M Alonso, Eric H Raabe, Esther Hulleman, Paul T Spellman, Xiao-Nan Li, Charles Keller, Ranadip Pal, Jacques Grill & Michelle Monje Nat. Med. 21, 555–559 (2015); doi:10.1038/nm.3855; published online 4 May 2015; corrected after print 15 June 2015

Activation of mTORC1/mTORC2 signaling in pediatric low-grade glioma and pilocytic astrocytoma reveals mTOR as a therapeutic target

BACKGROUND Previous studies support a role for mitogen-activated protein kinase pathway signaling, and more recently Akt/mammalian target of rapamycin (mTOR), in pediatric low-grade glioma (PLGG), including pilocytic astrocytoma (PA). Here we further evaluate the role of the mTORC1/mTORC2 pathway in order to better direct pharmacologic blockade in these common childhood tumors. METHODS We studied 177 PLGGs and PAs using immunohistochemistry and tested the effect of mTOR blockade on 2 PLGG cell lines (Res186 and Res259) in vitro. RESULTS Moderate (2+) to strong (3+) immunostaining was observed for pS6 in 107/177 (59%) PAs and other PLGGs, while p4EBP1 was observed in 35/115 (30%), pElF4G in 66/112 (59%), mTOR (total) in 53/113 (47%), RAPTOR (mTORC1 component) in 64/102 (63%), RICTOR (mTORC2 component) in 48/101 (48%), and pAkt (S473) in 63/103 (61%). Complete phosphatase and tensin homolog protein loss was identified in only 7/101 (7%) of cases. In PA of the optic pathways, compared with other anatomic sites, there was increased immunoreactivity for pS6, pElF4G, mTOR (total), RICTOR, and pAkt (P < .05). We also observed increased pS6 (P = .01), p4EBP1 (P = .029), and RICTOR (P = .05) in neurofibromatosis type 1 compared with sporadic tumors. Treatment of the PLGG cell lines Res186 (PA derived) and Res259 (diffuse astrocytoma derived) with the rapalog MK8669 (ridaforolimus) led to decreased mTOR pathway activation and growth. CONCLUSIONS These findings suggest that the mTOR pathway is active in PLGG but varies by clinicopathologic subtype. Additionally, our data suggest that mTORC2 is differentially active in optic pathway and neurofibromatosis type 1-associated gliomas. MTOR represents a potential therapeutic target in PLGG that merits further investigation.

Local delivery of cancer-cell glycolytic inhibitors in high-grade glioma

BACKGROUND 3-bromopyruvate (3-BrPA) and dichloroacetate (DCA) are inhibitors of cancer-cell specific aerobic glycolysis. Their application in glioma is limited by 3-BrPAs inability to cross the blood-brain-barrier and DCAs dose-limiting toxicity. The safety and efficacy of intracranial delivery of these compounds were assessed. METHODS Cytotoxicity of 3-BrPA and DCA were analyzed in U87, 9L, and F98 glioma cell lines. 3-BrPA and DCA were incorporated into biodegradable pCPP:SA wafers, and the maximally tolerated dose was determined in F344 rats. Efficacies of the intracranial 3-BrPA wafer and DCA wafer were assessed in a rodent allograft model of high-grade glioma, both as a monotherapy and in combination with temozolomide (TMZ) and radiation therapy (XRT). RESULTS 3-BrPA and DCA were found to have similar IC50 values across the 3 glioma cell lines. 5% 3-BrPA wafer-treated animals had significantly increased survival compared with controls (P = .0027). The median survival of rats with the 50% DCA wafer increased significantly compared with both the oral DCA group (P = .050) and the controls (P = .02). Rats implanted on day 0 with a 5% 3-BrPA wafer in combination with TMZ had significantly increased survival over either therapy alone. No statistical difference in survival was noted when the wafers were added to the combination therapy of TMZ and XRT, but the 5% 3-BrPA wafer given on day 0 in combination with TMZ and XRT resulted in long-term survivorship of 30%. CONCLUSION Intracranial delivery of 3-BrPA and DCA polymer was safe and significantly increased survival in an animal model of glioma, a potential novel therapeutic approach. The combination of intracranial 3-BrPA and TMZ provided a synergistic effect.

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.

The chromatin-modifying protein HMGA2 promotes atypical teratoid/rhabdoid cell tumorigenicity

Abstract Atypical teratoid/rhabdoid tumor (AT/RT) is an aggressive pediatric central nervous system tumor. The poor prognosis of AT/RT warrants identification of novel therapeutic targets and strategies. High-mobility Group AT-hook 2 (HMGA2) is a developmentally important chromatin-modifying protein that positively regulates tumor growth, self-renewal, and invasion in other cancer types. High-mobility group A2 was recently identified as being upregulated in AT/RT tissue, but the role of HMGA2 in brain tumors remains unknown. We used lentiviral short-hairpin RNA to suppress HMGA2 in AT/RT cell lines and found that loss of HMGA2 led to decreased cell growth, proliferation, and colony formation and increased apoptosis. We also found that suppression of HMGA2 negatively affected in vivo orthotopic xenograft tumor growth, more than doubling median survival of mice from 58 days to 153 days. Our results indicate a role for HMGA2 in AT/RT in vitro and in vivo and demonstrate that HMGA2 is a potential therapeutic target in these lethal pediatric tumors.

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