DIPG Cell Lines Research Articles

Sequencing of ACVR1/ALK2 in patient derived DIPG cell lines

Sequencing of ACVR1/ALK2 in patient derived DIPG cell lines

Abstract A46: Heterotypic nucleosomes and PRC2 role in diffuse intrinsic pontine gliomas

Abstract Diffuse intrinsic pontine gliomas (DIPG) harbor a characteristic lysine-to-methionine mutation on histone H3 (H3K27M) that potently inhibits Polycomb Repressive Complex 2 (PRC2) enzymatic activity; however, the precise roles of H3K27M and PRC2 in tumorigenesis remain elusive. Here we perform epigenomic profiling of patient-derived H3K27M mutant DIPG cell lines and demonstrate that H3K27M resides in nucleosomes with H3K27acetylation (H3K27ac) and colocalizes with bromodomain proteins at actively transcribed genes. In contrast, PRC2 is excluded from H3K27M occupied regions but, strikingly, the PRC2 complex is required for maintaining oncogenic potential. Finally, we demonstrate that pharmacologic bromodomain inhibition suppresses tumor growth in vivo. Altogether our results indicate that H3K27M drives accumulation of H3K27ac and point to bromodomain proteins inhibition as a powerful clinical strategy against DIPG. Citation Format: Andrea Piunti. Heterotypic nucleosomes and PRC2 role in diffuse intrinsic pontine gliomas [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A46.

Combined use of CDK4/6 and mTOR inhibitors induce synergistic growth arrest of diffuse intrinsic pontine glioma cells via mutual downregulation of mTORC1 activity.

BackgroundDiffuse intrinsic pontine glioma (DIPG) is a lethal type of pediatric brain tumor that is resistant to conventional chemotherapies. Palbociclib is a putative novel DIPG treatment that restricts the proliferation of rapidly dividing cancer cells via selective inhibition of cyclin-dependent kinase (CDK) 4 and CDK6. However, implementing palbociclib as a monotherapy for DIPG is unfeasible, as CDK4/6 inhibitor resistance is commonplace and palbociclib does not readily cross the blood–brain barrier (BBB) or persist in the central nervous system. To inhibit the growth of DIPG cells, we aimed to use palbociclib in combination with the rapamycin analog temsirolimus, which is known to ameliorate resistance to CDK4/6 inhibitors and inhibit BBB efflux.Materials and methodsWe tested palbociclib and temsirolimus in three patient-derived DIPG cell lines. The expression profiles of key proteins in the CDK4/6 and mammalian target of rapamycin (mTOR) signaling pathways were assessed, respectively, to determine feasibility against DIPG. Moreover, we investigated effects on cell viability and examined in vivo drug toxicity.ResultsImmunoblot analyses revealed palbociclib and temsirolimus inhibited CDK4/6 and mTOR signaling through canonical perturbation of phosphorylation of the retinoblastoma (RB) and mTOR proteins, respectively; however, we observed noncanonical downregulation of mTOR by palbociclib. We demonstrated that palbociclib and temsirolimus inhibited cell proliferation in all three DIPG cell lines, acting synergistically in combination to further restrict cell growth. Flow cytometric analyses revealed both drugs caused G1 cell cycle arrest, and clonogenic assays showed irreversible effects on cell proliferation. Palbociclib did not elicit neurotoxicity in primary cultures of normal rat hippocampi or when infused into rat brains.ConclusionThese data illustrate the in vitro antiproliferative effects of CDK4/6 and mTOR inhibitors in DIPG cells. Direct infusion of palbociclib into the brain, in combination with systemic delivery of temsirolimus, represents a promising new approach to developing a much-needed treatment for DIPG.

Abstract 4850: The minor groove binding agent quinacrine inhibits growth and increases apoptotic death in diffuse intrinsic pontine glioma tumor cells

Abstract Diffuse intrinsic pontine glioma (DIPG) is an invasive, incurable and aggressive pediatric brain tumor found in the brainstem. Improved understanding of the biology of DIPG tumors is urgently needed to develop novel treatments. Our previous studies have shown that DIPG tumors and cell lines express high levels of the DNA-binding stem cell factor high mobility group AT-hook 2 (HMGA2).. Targeting HMGA2 using lentiviral shRNA decreased DIPG cell invasion, proliferation and increased apoptosis in our studies. We hypothesized that inhibiting HMGA2 using DNA minor groove binding drugs like quinacrine would decrease DIPG proliferation and increase apoptotic cell death. We used three DIPG cell lines (JHHDIPG1, JHHDIPG16A, and SUDIPG13) to test the effect of quinacrine. Quinacrine has traditionally been used as an anti-malarial drug and is known to strongly bind to DNA. We used BrdU incorporation as a measure of proliferation and expression of cleaved caspase-3 (CC-3) as a measure of apoptosis. Treatment of DIPG cells with quinacrine showed a dose dependent increase in cell death (CC-3 expression) from 3uM to 20uM, with the most effective doses being from 3uM to 5uM. In all three DIPG cell lines, treatment with 1uM to 5uM quinacrine showed a significant reduction in cell proliferation (BrdU) from 3uM to 5uM compared to vehicle-treated cells (P<0.0001). Additionally, treatment of DIPG cell lines with 1uM to 5uM quinacrine showed a significant increase in apoptotic cell death (CC-3 expression) compared to vehicle control cells from 3uM to 5uM (P<0.001). Our data suggests that minor groove binding agents like quinacrine could be effective therapies for this lethal brain tumor. Our future studies are aimed at testing the efficacy of quinacrine in inhibiting DIPG tumorigenicity and elucidating the mechanism of action. Citation Format: Huizi Guo, Harpreet Kaur, Sepehr Akhtarkhavari, Charles G. Eberhart, Eric H. Raabe. The minor groove binding agent quinacrine inhibits growth and increases apoptotic death in diffuse intrinsic pontine glioma tumor cells [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 4850.

Abstract 3182: A human neural stem cell glial brain tumor model identifies the relative contribution of different oncogenic elements to malignant transformation

Abstract Diffuse intrinsic pontine glioma (DIPG) is an invasive malignancy of the brainstem that accounts for greater than 80% of pediatric brainstem gliomas. In the past 40 years, there have been no significant advances in DIPG treatments and survival, so it remains a leading cause of death from pediatric brain tumors. Nearly 80% of DIPG harbor a point mutation in H3F3A or HIST1H3B, and the presence of this H3.3K27M mutation is inversely correlated with reduced OS, suggesting that epigenetic dysregulation is a key driver to cause the pathogenesis of DIPG. However, it is unclear how the H3.3K27M mutation and the other common alterations in DIPG contribute to tumorigenicity in human neural stem cells. To address the relative contributions of different oncogenic elements to DIPG, we sought to add common DIPG elements in a stepwise fashion to human neural stem cells derived from the developing hindbrain. We chose oncogenic elements that are known to present in DIPG, including the stem cell factor BMI1, mutant (R248W) p53, H3.3K27M, constitutively active AKT (representing the activation of mTOR detected in the vast majority of DIPG) and hTERT. We found that the combination of the stem cell factor BMI1 along with mutant (R248W) p53 and hTERT could immortalize neural stem cells, but was insufficient to form orthotopic xenograft tumors when placed in the pons of immunodeficient mice. Addition of activated AKT led to aggressive tumor formation, with a glial phenotype as evidenced by robust GFAP expression and absence of synaptophysin expression. All mice bearing these four genetic alterations succumbed to their tumors within 100 days of implantation. We found that introduction of H3.3K27M mutation reduces the level of tri-methylation of H3.3KK27 to that seen in patient-derived DIPG cell lines. Furthermore, we found that H3.3KK27 mutation introduction increases expression of LIN28B, a stem cell factor, which is also found in patient-derived DIPGs. Our previous study showing that LIN28A, another family member of LIN28 proteins, regulates invasion and tumorigenicity in adult high-grade gliomas suggests that H3.3K27M mutation might facilitate invasiveness of DIPG through LIN28B and its downstream effectors HMGA2, SNAI1, and SLUG. In summary, we have developed a human hindbrain neural stem cell DIPG model that has both accurate cell of origin and genetic defects including H3.3K27M mutation. Our models allow for assessment of the relative contribution to transformation of each genetic element in a stepwise fashion in the likely cell of origin of this deadly tumor. Citation Format: Youngran Park, Ping An, Dacheng Ding, Charles G. Eberhart, Eric H. Raabe. A human neural stem cell glial brain tumor model identifies the relative contribution of different oncogenic elements to malignant transformation [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 3182.

Abstract 2464: Disrupting the epigenetic modifier HMGA2 in DIPG and GBM inhibits tumor invasion, growth and tumorigenicity

Abstract Diffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are lethal, incurable brain tumors with poor prognosis. Identifying and targeting novel molecular markers regulating tumor invasion and growth can lead to development of better therapies. HMGA2, a minor-groove DNA-binding protein, is a transcriptional modulator in normal and cancer stem cells. We hypothesized that HMGA2 contributes to pediatric high grade glioma invasion and tumorigenicity through its ability to alter the transcription of large numbers of genes. We found increased expression of HMGA2 in a subset of GBM and DIPG tumors by western blotting and immunohistochemistry. We confirmed this result in multiple patient-derived tumor cell lines, including 3 DIPG neurosphere cell lines. Lentiviral short hairpin RNA (shRNA)-mediated reduction of HMGA2 in GBM cells significantly reduced invasion in transwell assay and colony formation in soft agar assay (shHMGA2 vs. shControl, P<0.01). Similarly, shRNA mediated suppression of HMGA2 in DIPG cell lines reduced proliferation as measured by BrdU incorporation, reduced invasion by transwell assay and increased apoptosis by cleaved caspase-3 (CC-3) immunofluorescence (shHMGA2 vs. shControl, P<0.01). Pharmacological inhibition of HMGA proteins using the DNA minor-groove binding drug Netropsin significantly inhibited growth (BrdU assay) and increased apoptosis (CC-3 immunofluorescence) of DIPG cell lines (P<0.01). Mice bearing orthotopic GBM xenograft cell lines transduced with HMGA2 shRNA lived longer (median survival = 108 days) compared to control shRNA (67.5 days, P<0.0001 by log-rank analysis). Our results suggest an oncogenic role of HMGA2 in promoting DIPG and GBM invasion and growth, identifying HMGA2 as a potential therapeutic target in these devastating tumors. Netropsin or other minor groove binding agents may be effective therapeutics for pediatric high-grade gliomas. Citation Format: Harpreet Kaur, Marianne Hütt-Cabezas, Isabella Taylor, Melanie Weingart, Fausto Rodriguez, Charles G. Eberhart, Eric H. Raabe. Disrupting the epigenetic modifier HMGA2 in DIPG and GBM inhibits tumor invasion, growth and 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 2464.

Abstract IA02: Human ES cells for modeling brain cancer

Abstract Diffuse Intrinsic Pontine Gliomas (DIPG) are a highly aggressive tumor of the brainstem occurring primarily in children. The outlook of patients with this disease is particularly dire due to the tumor's inaccessible location and its lethal course over one or two years. Recent sequencing data identified, for the first time in human cancer, heterozygous mutations in the histone H3 that create a lysine to methionine amino acid substitution. Using a human ES-based platform, we demonstrate the exquisitely cell-context specific oncogenic role of the H3K27M mutation. The model recapitulated the histological features and in vivo progression of the human tumor, as well as its transcriptomal and epigenetic profile. Genome wide analyses indicate a resetting of the transformed precursors to a developmentally more primitive state, with evidence of major modifications of histone marks at master regulatory genes. Drug screens in the ES model identified a compound that targets menin, a protein with multiple partners the mixed leukemia lineage protein MLL. The compounds have been validated in patient derived DIPG cell lines and are undergoing intense preclinical assessments. The ES model, coupled with more recent gene editing technology, provides a very powerful platform for the dissection of the requirements of neoplastic transformation in specific tumors, the specification of cells of origin, as well insight into the downstream effectors of individual and combined mutations. Human pluripotent stem cells have an excellent potential as platform for modeling cancer. Work to extend these findings to additional mutations and tumor types is ongoing. Ref. Funato et al. Science. 2014 Dec 19;346 (6216) Citation Format: Viviane Tabar. Human ES cells for modeling brain cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA02.

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

Abstract 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 5049: Targeting the Notch and mTOR pathways in diffuse intrinsic pontine glioma.

Abstract Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brain tumor which is known to have frequent alteration of mTOR signaling. Analysis of DIPG tissue obtained through the Mid-Atlantic DIPG Consortium (MADC) also shows high level activation of Notch pathway effectors HES4 and HES5. We have established JHH DIPG1, a human DIPG xenograft and neurosphere cell line from a rapid autopsy specimen obtained through the MADC. Notch 1 and 3 and Notch downstream effectors, Hes1, Hes5 and Hey1, are highly expressed in JHH DIPG1 and a second DIPG line, SU DIPG1. These DIPG cell lines also express high level phospho-S6 and phospho-AKT 473, indicating activation of TORC1 and TORC2. Notch and mTOR pathway signaling is equal to or exceeding that observed in glioblastoma neurosphere cell lines. Notch targeting using a gamma-secretase inhibitor leads to a dose-dependent reduction in cell proliferation of up to 30% (p=0.05) as well as inhibition of phospho-AKT 473, suggesting that inhibition of the NOTCH pathway targets TORC2 complexes. BrdU incorporation also showed that treatment of DIPG cell lines with a TORC1 inhibitor also has up to a 60% attenuation of cell proliferation (p<0.005) in treated DIPG cells compared to non-treated cells and a dose-dependent reduction in phospho-S6, with concomitant upregulation of phospho-AKT 473 indicating TORC2 activation. Dual TORC1 and TORC2 inhibition by combining a rapalog and a gamma-secretase inhibitor may prove to be synergistic in DIPG. Simultaneous treatment with a rapalog and gamma-secretase inhibition suppresses TORC1 and TORC2, as measured by western blot. We are currently investigating the possible mechanism of action of these drugs in DIPG. Our laboratory is investigating additional metabolic inhibitors that can be used with TORC1 and TORC2 inhibitors to further improve treatment options for DIPG. We are currently exploring the role of the Notch and mTOR pathways in DIPG in pre-clinical studies in mice, with the goal of moving targeted inhibitors into clinical trials for these currently highly lethal tumors. Citation Format: Marianne Hutt, Wendy Goldstein, Javad Nazarian, Antoinette Price, Kah Jing Lim, Katherine Warren, Howard Chang, Charles Eberhart, Eric Raabe. Targeting the Notch and mTOR pathways in diffuse intrinsic pontine glioma. [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 5049. doi:10.1158/1538-7445.AM2013-5049

Abstract 2480: High-level activation of the Notch pathway in diffuse intrinsic pontine glioma

Abstract Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brain tumor. Due to its critical location within the brainstem, and its characteristic MRI signature, biopsy is rarely performed. Since many patients die at home in hospice care, autopsy specimens have traditionally been rare. Because of this difficulty in obtaining tissue, three institutions (Johns Hopkins, Children's National Medical Center, and the National Institutes of Health - Pediatric Oncology Branch) have formed the Mid-Atlantic DIPG Consortium (MADC), to collect and disseminate rapid-autopsy material from patients with DIPG. Using a fresh tissue specimen obtained from a rapid autopsy and disseminated through the MADC, we have established a human DIPG xenograft and neurosphere cell line, JHH-DIPG1. In vitro, these cells are GFAP positive and express human neural stem cell markers often observed in glioblastoma, including human specific SOX2 and NESTIN. They also show marked invasive properties in vitro as measured by transwell invasion assay. In vivo, JHH-DIPG1 forms tumor xenografts in approximately six months in immunocompromised mice. Tumor cells diffusely infiltrate distant brain structures, recapitulating the invasive phenotype of DIPG. Due to their high level expression of neural stem cell markers, and the well-known roles of the Notch pathway in stem cell biology and aggressive brain tumors, we examined the expression of members of the Notch pathway in DIPG. Notch downstream effectors, specifically Hes1, Hes5 and Hey1, are highly expressed in JHH-DIPG1, at levels equal to or exceeding those observed in neurosphere cell lines derived from adult supratentorial glioblastoma. The JHH-DIPG1 tumor cell line represents one of only a handful of pediatric DIPG cell lines extant, and the high level of Notch pathway activation in these cells suggests that blockade of the Notch pathway using gamma secretase inhibitors and shRNAs against the canonical Notch effector CBF may be efficacious in DIPG. Our laboratory is actively investigating the role of the Notch pathway in DIPG using this and other DIPG cell lines, with the goal of moving Notch inhibition into clinical trials for these currently highly lethal tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2480. doi:1538-7445.AM2012-2480