Investigating the Role of H3.3K27M during Cellular Transformation and Differentiation.

Diffuse Intrinsic Pontine Glioma (DIPG) is the most fatal brain tumour in childhood and current therapeutic interventions are failing. There is an urgent need for better understanding of DIPG pathogenesis and novel therapeutic approaches. Discovery of histone 3 variant mutations in DIPG has enabled the development of more faithful disease models and highlighted a role for histone epigenetics in gliomagenesis. The H3.3K27M mutant occurs in 65% of DIPGs and is the focus of this thesis. We have previously demonstrated that H3.3K27M transforms immortalized normal human astrocytes (iNHAs). We expand on that initial finding by testing the ability of these cells to form colonies at high confluency (HC). We find that H3.3K27M iNHAs execute a cell-autonomous program promoting cellular invasiveness, EMT and ECM reorganization through transcriptional dysregulation at HC. Importantly, the transcriptome of H3.3K27M iNHAs significantly overlaps with the astrocytic cell program recently described in DIPG. We identify the cell surface protein FPR3 as a putative H3.3K27M target likely upregulated to support EMT. We speculate that downregulation of cell-cell and cell-matrix adhesion proteins by H3.3K27M is central to colony formation and demonstrate that protocadherin 7 (PCDH7) is reduced in our model as in human glioblastoma. This study lays the necessary groundwork for further exploration of these novel putative H3.3K27M targets. Since our lab and others have determined that H3.3K27M alone is not a potent driver of transformation, we investigated its impact on cell state. We utilized the thoroughly characterized C2C12 myoblast differentiation model. The mechanism underlying H3.3K27M-induced transcriptional activation centres on dysregulation of the PRC2 complex and loss of the repressive H3K27me3 mark. Consistent with this, our investigation of the impact of H3.3K27M in differentiating myoblasts revealed massive H3K27me3 loss throughout differentiation and precocious differentiation through activation of the master muscle regulator factors (MRFs) MyoD and myogenin followed by transient cell cycle exit. Importantly, differentiating H3.3K27M myoblasts did not completely downregulate cell cycle factors cyclin B1 and cyclin D1 likely stalling complete terminal differentiate. This work has important implications for our understanding of how cellular decision-making is impacted by H3.3K27M and hints that mutant expression may disrupt proper cell cycle regulation.Ph.D

Study of the biodistribution of fluorescein in glioma infiltrated mouse brain and histopathologic correlation of intraoperative findings in high-grade gliomas resected under fluorescein fluorescence-guidance

OBJECT Intravenous fluorescein sodium has been used during resection of high-grade gliomas to help the surgeon visualize tumor margins. Several studies have reported improved rates of gross-total resection (GTR) using high doses of fluorescein sodium under white light. The recent introduction of a fluorescein-specific camera that allows for high-quality intraoperative imaging and use of very low dose fluorescein has drawn new attention to this fluorophore. However, the ability of fluorescein to specifically stain glioma cells is not yet well understood. METHODS The authors designed an in vitro model to assess fluorescein uptake in normal human astrocytes and U251 malignant glioma cells. An in vivo experiment was also subsequently designed to study fluorescein uptake by intracranial U87 malignant glioma xenografts in male nonobese diabetic/severe combined immunodeficient mice. A genetically induced mouse glioma model was used to adjust for the possible confounding effect of an inflammatory response in the xenograft model. To assess the intraoperative application of this technology, the authors prospectively enrolled 12 patients who underwent fluorescein-guided resection of their high-grade gliomas using low-dose intravenous fluorescein and a microscope-integrated fluorescence module. Intraoperative fluorescent and nonfluorescent specimens at the tumor margins were randomly analyzed for histopathological correlation. RESULTS The in vitro and in vivo models suggest that fluorescein demarcation of glioma-invaded brain is the result of distribution of fluorescein into the extracellular space, most likely as a result of an abnormal blood-brain barrier. Glioblastoma tumor cell–specific uptake of fluorescein was not observed, and tumor cells appeared to mostly exclude fluorescein. For the 12 patients who underwent resection of their high-grade gliomas, the histopathological analysis of the resected specimens at the tumor margin confirmed the intraoperative fluorescent findings. Fluorescein fluorescence was highly specific (up to 90.9%) while its sensitivity was 82.2%. False negatives occurred due to lack of fluorescence in areas of diffuse, low-density cellular infiltration. Margins of contrast enhancement based on intraoperative MRI–guided StealthStation neuronavigation correlated well with fluorescent tumor margins. GTR of the contrast-enhancing area as guided by the fluorescent signal was achieved in 100% of cases based on postoperative MRI. CONCLUSIONS Fluorescein sodium does not appear to selectively accumulate in astrocytoma cells but in extracellular tumor cell-rich locations, suggesting that fluorescein is a marker for areas of compromised blood-brain barrier within high-grade astrocytoma. Fluorescein fluorescence appears to correlate intraoperatively with the areas of MR enhancement, thus representing a practical tool to help the surgeon achieve GTR of the enhancing tumor regions

Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations

Diffuse intrinsic pontine glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children, with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and to the selection of therapies on the basis of assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic makeup of this brain cancer, with nearly 80% found to harbor a p.Lys27Met histone H3.3 or p.Lys27Met histone H3.1 alteration. However, DIPGs are still thought of as one disease, with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs, we integrated whole-genome sequencing with methylation, expression and copy number profiling, discovering that DIPGs comprise three molecularly distinct subgroups (H3-K27M, silent and MYCN) and uncovering a new recurrent activating mutation affecting the activin receptor gene ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of the downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer

EXPERIMENTAL THERAPEUTICS AND PHARMACOLOGY

The multifunctional protein - tissue inhibitor of metalloproteinases-1 (TIMP-1) - has been associated with poor prognosis in several types of cancers including glioblastomas. Glioblastomas are the most common and malignant primary brain tumor known for being highly invasive and resistant to therapy. New treatment strategies are continuously being explored and currently vascular endothelial growth factor (VEGF) inhibitors administered in combination with Irinotecan is the most promising second line treatment. TIMP-1 has been associated with decreased response to chemotherapy in breast and colorectal cancer and especially the family of topoisomerase (TOP) inhibitors, such as Irinotecan, has been suggested to be affected by TIMP-1. In the present study, we investigated whether a high TIMP-1 expression in glioblastoma cells played a role in TOP inhibitor resistance. We established two TIMP-1 over-expressing cell lines and evaluated the sensitivity towards the TOP1 inhibitor SN-38 and the TOP2 inhibitor Epirubicin using a viability and a cytotoxicity assay. In addition, we investigated the invasive features of the cells in a brain slice culture model as well as in an orthotopic xenograft model. The results showed that TIMP-1 over-expressing U87MG cell line sub-clones were significantly more resistant than the controls when exposed to SN-38 and Epirubicin. The same tendency was seen for the TIMP-1 over-expressing A172 sub-clones. No significant differences in invasion patterns were observed for TIMP-1 over-expressing sub-clones when compared to controls. In conclusion, the present study suggests that TIMP-1 over-expression reduces the effect of TOP inhibitors in the glioblastoma cell line U87MG. There was no significant effect of TIMP-1 over-expression on tumor cell invasion. The association found between TIMP-1 cellular levels and the effect of TOP inhibitors needs to be validated in clinical patient material. Pediatric supratentorial high-grade astrocytomas (pHGAs) and diffuse intrinsic pontine gliomas (DIPG) are devastating pediatric malignancies for which no effective therapies exist. Poly-(ADP-Ribose)-Polymerase (PARP) protein expression is found in ∼60% of DIPGs suggesting PARP may be a potential therapeutic target. PARP1/2 were characterized by Western-blotting in normal human astrocytes (NHA), pHGA cell lines (SJG2, SF-188), DIPG cell lines (DIPG-M, DIPG58), and one murine brainstem glioma cell line (mBSG). Cell viability in response to different dosages of Olaparib, Veliparib, or Niraparib was determined using the MTT Assay. PARP activity, apoptosis, and DNA damage was determined by Western blotting against PAR, cleaved PARP, and phosphorylated yH2AX, respectively. Cell cycle phases were analyzed using FACS and western blot for p21. Western blotting demonstrated that, compared with NHAs, PARP1 were highly expressed in SJG2, DIPG-M, and DIPG-58 cells. PARP2 expression was only detected in SJG2 cells. All PARP inhibitors reduced PARP activity as indicated by reduced PAR levels. Olaparib reduced SJG2, mBSG, DIPG58 and DIPGM cell viability at concentrations of 5 or 10uM uM (P < 0.05), Whereas Niraparib induced cytotoxicity at concentrations of 2uM and above (P < 0.05). Olaparib and Niraparib induced DNA damage and apoptosis in SJG2 at doses of 5, 10uM and 2, 5, 10uM, respectively. Niraparib induced G2 arrest in mBSG demonstrated by FACS and increased levels of p21 (P < 0.05). Our data provides in vitro evidence that PARP inhibition may be an effective therapeutic avenue for treatment of pHGA and DIPG. Furthermore while all PARP inhibitors suppress PARP activity not all PARP inhibitors reduce cell viability. Thus not all PARP inhibitors can be expected to be equally efficacious in a clinical trial setting. Toca 511 (vocimagene amiretrorepvec), an amphotropic retroviral replicating vector (RRV), can successfully and safely deliver a functional, optimized yeast cytosine deaminase (CD) gene to tumors in orthotopic glioma models. Within infected cells, CD converts 5-fluorocytosine (5-FC) to the anti-cancer drug 5-FU. The combination of Toca 511 with oral extended release 5-FC (Toca FC), is currently in clinical trials for recurrent High Grade Glioma (HGG, NCT01156584 and NCT01470794). Temozolomide (TMZ), in combination with radiation therapy, is the most commonly used first-line chemotherapy treatment for patients with glioblastoma, the most common and aggressive form of primary brain cancer. A separate study (Takahashi et al., this meeting) addresses the potential radiation synergy with Toca 511/5-FC treatment. A subset of patients with certain genetic alterations does not respond well to TMZ treatment and the overall median survival for patients who respond remains poor, suggesting combinatorial approaches may be necessary to significantly improve patient outcomes. To determine whether Toca 511 and 5-FC therapy is compatible with TMZ, we examined the effect of TMZ in combination with Toca 511 and 5-FC in TMZ-sensitive and resistant glioma lines both in vitro and in vivo. We show that in vitro TMZ delays but does not prevent RRV spread, nor interfere with Toca 511 and 5-FC mediated cell killing in glioma tumor cells, and in vivo there is no significant hematologic effect from the combination of 5-FC and the clinically relevant dose of TMZ. A synergistic long-term survival advantage is observed in mice bearing an orthotopic TMZ-sensitive glioma tumor after Toca 511 administration followed by co-administration of TMZ in combination with 5-FC. These results provide support for the investigation of this novel combination treatment strategy for patients with newly diagnosed glioblastoma. BACKGROUND: LAZ is a 21-aminosteroid that has radioprotective effects against radiation-induced lipid peroxidation. Also antiproliferative effects have been reported against glioblastoma cell lines. DESIGN/METHODS: LAZ PEGylated liposomes (Lipo G) were developed at the University of Houston.. Glioblastoma cell line U87-expressing firefly luciferase reporter gene (100,000 cells in 2 µL) was injected intracranially in each SCID mouse. There were 4 treatment groups (n = 8-9, each): brain model (M) without treatment (control), radiation 2Gy weekly (M + R), Lipo G at 5 mg/kg dose intraperitoneally twice per week (M + L) and radiation with Lipo G (M + R + L). Treatment lasted three weeks. Tumor size was monitored using bioluminescence imaging (BLI), in each mouse. Mice were sacrificed after 3 weeks. Brain was harvested. Lipid peroxidation of brain tissues was quantified by measuring malondialdehyde (MDA) as a surrogate biomarker. Survival was evaluated using Kaplan Meier analysis at P= 0.05. RESULTS: BLI intensity was 4002.03 ± 1737.67, 2034 ± 737.72, 1387.36 ± 684.53 and 2498.89 ± 2521.32 % for M, M + R, M + L and M + R + L, respectively. Tumor size of the M + L group was reduced by 65% compared to control. There was no significant difference in tumor size of radiated groups compared to control group. MDA brain concentration in M + L and M + R + L groups was significantly less than in M + R group (8.27 ± 0.78 and 10.37 ± 3.30 µM/gm vs. 23.09± 3.79 µM/gm). The survival mean was 22.67, 25.33, 25.22 and 27.13 days for M, M + R, M + R + L and M + L groups, respectively. Mean survival of LAZ treated groups (M + L and M + R + L) was significantly longer than that of the control group. CONCLUSIONS: LAZ liposomal formulations reduced tumor growth by 65%. LAZ also protected brain tissue from radiation-induced lipid peroxidation by reducing MDA concentration by 50%. These provocative data warrant further investigation of LAZ as a radiation protectant and chemotherapeutic agent. Patients with malignant brain tumors have a median survival of approximately one year following diagnosis, regardless of currently available treatments which include surgery followed by radiation and chemotherapy. Improvement in the survival of brain cancer patients requires the design of new therapeutic modalities that take advantage of common phenotypes. One such phenotype is the metabolic dysregulation that is a hallmark of cancer cells. It has therefore been postulated that one approach to treating brain tumors may be by metabolic alteration such as that which occurs through the use of the ketogenic diet (KD). The KD is high-fat, low-carbohydrate diet that induces ketosis and has been utilized for the non-pharmacologic treatment of refractory epilepsy. We and others have shown that this diet enhances survival and potentiates standard therapy in mouse models of malignant gliomas, yet the anti-tumor mechanisms are not fully understood. It has been previously shown that caloric restriction, which induces ketosis, reduces microvessel density in mouse and human brain tumor models, suggesting an anti-angiogenic effect. We now report that in animals fed KetoCal® (KC)(4:1 fat:protein/carbohydrates) ad libitum, peritumoral edema is significantly reduced early in tumor progression when compared to those fed a standard rodent diet. Gene expression profiling demonstrated that KC decreases the expression of the gene encoding vascular endothelial growth factor B (VEGFB) and angiopoetin 1 receptor (TEK). Furthermore, protein analysis showed a reduction of platelet endothelial cell adhesion molecule 1 (PECAM1/CD31) in tumors from animals maintained on KC. Taken together our data suggests that KC alters the angiogenic processes involved in malignant progression of gliomas. A greater understanding of the effects of the ketogenic diet as an adjuvant therapy will allow for a more rational approach to its clinical use