An in vivo model of glioblastoma radiation resistance identifies long noncoding RNAs and targetable kinases.

Key molecular regulators of acquired radiation resistance in recurrent glioblastoma (GBM) are largely unknown, with a dearth of accurate preclinical models. To address this, we generated 8 GBM patient-derived xenograft (PDX) models of acquired radiation therapy-selected (RTS) resistance compared with same-patient, treatment-naive (radiation-sensitive, unselected; RTU) PDXs. These likely unique models mimic the longitudinal evolution of patient recurrent tumors following serial radiation therapy. Indeed, while whole-exome sequencing showed retention of major genomic alterations in the RTS lines, we did detect a chromosome 12q14 amplification that was associated with clinical GBM recurrence in 2 RTS models. A potentially novel bioinformatics pipeline was applied to analyze phenotypic, transcriptomic, and kinomic alterations, which identified long noncoding RNAs (lncRNAs) and targetable, PDX-specific kinases. We observed differential transcriptional enrichment of DNA damage repair pathways in our RTS models, which correlated with several lncRNAs. Global kinomic profiling separated RTU and RTS models, but pairwise analyses indicated that there are multiple molecular routes to acquired radiation resistance. RTS model-specific kinases were identified and targeted with clinically relevant small molecule inhibitors. This cohort of in vivo RTS patient-derived models will enable future preclinical therapeutic testing to help overcome the treatment resistance seen in patients with GBM.

Myristoylated alanine-rich C-kinase substrate effector domain phosphorylation regulates the growth and radiation sensitization of glioblastoma.

Glioblastoma harbors frequent alterations in receptor tyrosine kinases, phosphatidylinositol­3 kinase (PI3K) and phosphatase and tensin homolog (PTEN) that dysregulate phospholipid signaling driven tumor proliferation and therapeutic resistance. Myristoylated alanine­rich C­kinase substrate (MARCKS) is a 32 kDa intrinsically unstructured protein containing a polybasic (+13) effector domain (ED), which regulates its electrostatic sequestration of phospholipid phosphatidylinositol (4,5)­bisphosphate (PIP2), and its binding to phosphatidylserine, calcium/calmodulin, filamentous actin, while also serving as a nuclear localization sequence. MARCKS ED is phosphorylated by protein kinase C (PKC) and Rho­associated protein kinase (ROCK) kinases; however, the impact of MARCKS on glioblastoma growth and radiation sensitivity remains undetermined. In the present study, using a tetracycline­inducible system in PTEN­null U87 cells, we demonstrate that MARCKS overexpression suppresses growth and enhances radiation sensitivity in vivo. A new image cytometer, Xcyto10, was utilized to quantify differences in MARCKS ED phosphorylation on localization and its association with filamentous actin. The overexpression of the non­phosphorylatable ED mutant exerted growth­suppressive and radiation­sensitizing effects, while the pseudo­phosphorylated ED mutant exhibited an enhanced colony formation and clonogenic survival ability. The identification of MARCKS protein­protein interactions using co­immunoprecipitation coupled with tandem mass spectrometry revealed novel MARCKS­associated proteins, including importin­ß and ku70. On the whole, the findings of this study suggest that the determination of the MARCKS ED phosphorylation status is essential to understanding the impact of MARCKS on cancer progression.

Enhanced Sensitivity of Patient-Derived Pediatric High-Grade Brain Tumor Xenografts to Oncolytic HSV-1 Virotherapy Correlates with Nectin-1 Expression.

Pediatric high-grade brain tumors and adult glioblastoma are associated with significant morbidity and mortality. Oncolytic herpes simplex virus-1 (oHSV) is a promising approach to target brain tumors; oHSV G207 and M032 (encodes human interleukin-12) are currently in phase I clinical trials in children with malignant supratentorial brain tumors and adults with glioblastoma, respectively. We sought to compare the sensitivity of patient-derived pediatric malignant brain tumor and adult glioblastoma xenografts to these clinically-relevant oHSV. In so doing we found that pediatric brain tumors were more sensitive to the viruses and expressed significantly more nectin-1 (CD111) than adult glioblastoma. Pediatric embryonal and glial tumors were 74-fold and 14-fold more sensitive to M002 and 16-fold and 6-fold more sensitive to G207 than adult glioblastoma, respectively. Of note, pediatric embryonal tumors were more sensitive than glial tumors. Differences in sensitivity may be due in part to nectin-1 expression, which predicted responses to the viruses. Treatment with oHSV resulted in prolonged survival in both pediatric and adult intracranial patient-dervied tumor xenograft models. Our results suggest that pediatric brain tumors are ideal targets for oHSV and that brain tumor expression of nectin-1 may be a useful biomarker to predict patient response to oHSV.

Combinatorial Drug Testing in 3D Microtumors Derived from GBM Patient-Derived Xenografts Reveals Cytotoxic Synergy in Pharmacokinomics-informed Pathway Interactions.

Glioblastoma multiforme (GBM), the most common form of primary malignant brain cancer in adults, is a devastating disease for which effective treatment has remained elusive for over 75 years. One reason for the minimal progress during this time is the lack of accurate preclinical models to represent the patient's tumor's in vivo environment, causing a disconnect in drug therapy effectiveness between the laboratory and clinic. While patient-derived xenografts (PDX's or xenolines) are excellent human tumor representations, they are not amenable to high throughput testing. Therefore, we developed a miniaturized xenoline system (microtumors) for drug testing. Nineteen GBM xenolines were profiled for global kinase (kinomic) activity revealing actionable kinase targets associated with intracranial tumor growth rate. Kinase inhibitors for these targets (WP1066, selumetinib, crizotinib, and cediranib) were selected for single and combination therapy using a fully human-derived three-dimensional (3D) microtumor model of GBM xenoline cells embedded in HuBiogel for subsequent molecular and phenotype assays. GBM microtumors closely resembled orthotopically-implanted tumors based on immunohistochemical analysis and displayed kinomic and morphological diversity. Drug response testing could be reproducibly performed in a 96-well format identifying several synergistic combinations. Our findings indicate that 3D microtumors can provide a suitable high-throughput model for combination drug testing.

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Pediatric medulloblastoma xenografts including molecular subgroup 3 and CD133+ and CD15+ cells are sensitive to killing by oncolytic herpes simplex viruses.

BACKGROUND: Childhood medulloblastoma is associated with significant morbidity and mortality that is compounded by neurotoxicity for the developing brain caused by current therapies, including surgery, craniospinal radiation, and chemotherapy. Innate therapeutic resistance of some aggressive pediatric medulloblastoma has been attributed to a subpopulation of cells, termed cancer-initiating cells or cancer stemlike cells (CSCs), marked by the surface protein CD133 or CD15. Brain tumors characteristically contain areas of pathophysiologic hypoxia, which has been shown to drive the CSC phenotype leading to heightened invasiveness, angiogenesis, and metastasis. Novel therapies that target medulloblastoma CSCs are needed to improve outcomes and decrease toxicity. We hypothesized that oncolytic engineered herpes simplex virus (oHSV) therapy could effectively infect and kill pediatric medulloblastoma cells, including CSCs marked by CD133 or CD15. METHODS: Using 4 human pediatric medulloblastoma xenografts, including 3 molecular subgroup 3 tumors, which portend worse patient outcomes, we determined the expression of CD133, CD15, and the primary HSV-1 entry molecule nectin-1 (CD111) by fluorescence activated cell sorting (FACS) analysis. Infectability and cytotoxicity of clinically relevant oHSVs (G207 and M002) were determined in vitro and in vivo by FACS, immunofluorescent staining, cytotoxicity assays, and murine survival studies. RESULTS: We demonstrate that hypoxia increased the CD133+ cell fraction, while having the opposite effect on CD15 expression. We established that all 4 xenografts, including the CSCs, expressed CD111 and were highly sensitive to killing by G207 or M002. CONCLUSIONS: Pediatric medulloblastoma, including Group 3 tumors, may be an excellent target for oHSV virotherapy, and a clinical trial in medulloblastoma is warranted.

ROCK Inhibition Facilitates In Vitro Expansion of Glioblastoma Stem-Like Cells.

Due to their stem-like characteristics and their resistance to existing chemo- and radiation therapies, there is a growing appreciation that cancer stem cells (CSCs) are the root cause behind cancer metastasis and recurrence. However, these cells represent a small subpopulation of cancer cells and are difficult to propagate in vitro. Glioblastoma is an extremely deadly form of brain cancer that is hypothesized to have a subpopulation of CSCs called glioblastoma stem cells (GSCs; also called brain tumor initiating cells, BTICs). We propose the use of selective Rho-kinase (ROCK) inhibitors, Y-27632 and fasudil, to promote GSC/BTIC-like cell survival and propagation in vitro. ROCK inhibitors have been implicated in suppressing apoptosis, and it was hypothesized that they would increase the number of GSC/BTIC-like cells grown in vitro and improve cloning efficiencies. Indeed, our data demonstrate that transient and continuous supplementation of non-toxic concentrations of Y-27632 and fasudil inhibited apoptosis, enhanced the cells' ability to form spheres, and increased stem cell marker expressing GSC/BTIC-like cell subpopulation. Our data indicated that pharmacological and genetic (siRNA) inhibitions of the ROCK pathway facilitates in vitro expansion of GSC/BTIC-like cells. Thus, ROCK pathway inhibition shows promise for future optimization of CSC culture media.

Dynamics of Circulating γδ T Cell Activity in an Immunocompetent Mouse Model of High-Grade Glioma.

Human γδ T cells are potent effectors against glioma cell lines in vitro and in human/mouse xenograft models of glioblastoma, however, this effect has not been investigated in an immunocompetent mouse model. In this report, we established GL261 intracranial gliomas in syngeneic WT C57BL/6 mice and measured circulating γδ T cell count, phenotype, Vγ/Vδ repertoire, tumor histopathology, NKG2D ligands expression, and T cell invasion at day 10-12 post-injection and at end stage. Circulating γδ T cells transiently increased and upregulated Annexin V expression at post-tumor day 10-12 followed by a dramatic decline in γδ T cell count at end stage. T cell receptor repertoire showed no changes in Vγ1, Vγ4, Vγ7 or Vδ1 subsets from controls at post-tumor day 10-12 or at end stage except for an end-stage increase in the Vδ4 population. Approximately 12% of γδ T cells produced IFN-γ. IL-17 and IL-4 producing γδ T cells were not detected. Tumor progression was the same in TCRδ-/- C57BL/6 mice as that observed in WT mice, suggesting that γδ T cells exerted neither a regulatory nor a sustainable cytotoxic effect on the tumor. WT mice that received an intracranial injection of γδ T cells 15m following tumor placement showed evidence of local tumor growth inhibition but this was insufficient to confer a survival advantage over untreated controls. Taken together, our findings suggest that an early nonspecific proliferation of γδ T cells followed by their depletion occurs in mice implanted with syngeneic GL261 gliomas. The mechanism by which γδ T cell expansion occurs remains a subject for further investigation of the mechanisms responsible for this immune response in the setting of high-grade glioma.

The role of Src family kinases in growth and migration of glioma stem cells.

Src family kinases (SFKs) are highly expressed and active in clinical glioblastoma multiforme (GBM) specimens. SFKs inhibitors have been demonstrated to inhibit proliferation and migration of glioma cells. However, the role of SFKs in glioma stem cells (GSCs), which are important for treatment resistance and recurrence, has not been reported. Here, we examined the expression pattern of individual members of SFKs and their functional role in CD133⁺ GSCs in comparison to primary glioma cells. We found that Fyn, c-Src and Yes were robustly expressed in GSCs while Lck was absent. Knockdown of c-Src, Yes or treatment with the SFK inhibitor dasatinib inhibited the migration of GSCs, but had no impact on their growth or self-renewal. These results suggest that SFKs represent an effective target for GSC migration but not for their growth.

Therapeutic potential of AZD1480 for the treatment of human glioblastoma.

Aberrant activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway has been implicated in glioblastoma (GBM) progression. To develop a therapeutic strategy to inhibit STAT-3 signaling, we have evaluated the effects of AZD1480, a pharmacologic inhibitor of JAK1 and JAK2. In this study, the in vitro efficacy of AZD1480 was tested in human and murine glioma cell lines. AZD1480 treatment effectively blocks constitutive and stimulus-induced JAK1, JAK2, and STAT-3 phosphorylation in both human and murine glioma cells, and leads to a decrease in cell proliferation and induction of apoptosis. Furthermore, we used human xenograft GBM samples as models for the study of JAK/STAT-3 signaling in vivo, because human GBM samples propagated as xenografts in nude mice retain both the hallmark genetic alterations and the invasive phenotype seen in vivo. In these xenograft tumors, JAK2 and STAT-3 are constitutively active, but levels vary among tumors, which is consistent with the heterogeneity of GBMs. AZD1480 inhibits constitutive and stimulus-induced phosphorylation of JAK2 and STAT-3 in these GBM xenograft tumors in vitro, downstream gene expression, and inhibits cell proliferation. Furthermore, AZD1480 suppresses STAT-3 activation in the glioma-initiating cell population in GBM tumors. In vivo, AZD1480 inhibits the growth of subcutaneous tumors and increases survival of mice bearing intracranial GBM tumors by inhibiting STAT-3 activity, indicating that pharmacologic inhibition of the JAK/STAT-3 pathway by AZD1480 should be considered for study in the treatment of patients with GBM tumors.

Preclinical evaluation of ex vivo expanded/activated γδ T cells for immunotherapy of glioblastoma multiforme.

We have previously shown that expanded/activated γδ T cells from healthy donors are cytotoxic to GBM cell lines and primary GBM explants. In this report, we examined the therapeutic effect of intracranial infusion of expanded/activated γδ T cells on human minimal and established U251 tumor xenografts in athymic nude mice. Immunohistochemistry was used to determine the presence of NKG2D ligands on cell lines and tumors, and blocking studies were used to determine the effect of these ligands on γδ T cell recognition. Expanded/activated γδ T cells were prepared by 18-day culture in RPMI, human serum (HS), anti-CD2, IL-12, IFN-γ, and OKT-3. Anti-GBM activity of the cell product was assessed using in vitro cytotoxicity assays against the GBM cell line U251MG in suspension and in adherent culture. Ex vivo expanded/activated γδ T cells were of the effector/memory phenotype, expressed Th1 cytokines, and effectively killed U251 cells in vitro. Xenografts were prepared using a U251 cell line following transfection with a firefly luciferase gene to monitor tumor progression. Mice treated with γδ T cells showed slower progression of both new and established GBM xenografts versus mice that received vehicle only as determined by photon emission over time. Median survival was improved in all γδ T cell treated groups between 32 and 50 days by Kaplan-Meier analysis. U251 cells expressed ULBP-2 and ULBP-3, although blocking of these reduced in vitro cytotoxicity of γδ T cells to U251MG by only 33 and 25%, respectively. These studies show that expanded/activated γδ T cells can mediate killing of new or established GBM xenografts, reduce tumor progression, and constitute a potentially effective novel immunotherapeutic strategy against GBM.

Engineered herpes simplex viruses efficiently infect and kill CD133+ human glioma xenograft cells that express CD111.

Oncolytic herpes simplex viruses (HSV) hold promise for therapy of glioblastoma multiforme (GBM) resistant to traditional therapies. We examined the ability of genetically engineered HSV to infect and kill cells that express CD133, a putative marker of glioma progenitor cells (GPC), to determine if GPC have an inherent therapeutic resistance to HSV. Expression of CD133 and CD111 (nectin-1), the major entry molecule for HSV, was variable in six human glioma xenografts, at initial disaggregation and after tissue culture. Importantly, both CD133+ and CD133- populations of glioma cells expressed CD111 in similar relative proportions in five xenografts, and CD133+ and CD133- glioma cell subpopulations were equally sensitive to killing in vitro by graded dilutions of wild-type HSV-1(F) or several different gamma(1)34.5-deleted viruses. GPC did not display an inherent resistance to HSV. While CD111 expression was an important factor for determining sensitivity of glioma cells to HSV oncolysis, it was not the only factor. Our findings support the notion that HSV will not be able to effectively enter, infect, and kill cells in tumors that have low CD111 expression (<20%). However, virotherapy with HSV may be very effective against CD111+ GPC resistant to traditional therapies.

Increased efficacy of an interleukin-12-secreting herpes simplex virus in a syngeneic intracranial murine glioma model.

Long-term survivors of glioblastoma multiforme, the most common form of primary intracranial malignancy in adults, are extremely rare. Experimental animal models that more closely resemble human disease are essential for the identification of effective novel therapies. We report here an extensive analysis of the 4C8 glioma model to assess its suitability for evaluating novel type 1 herpes simplex virus (HSV-1) therapies of malignant glioma. We first determined that expression of major histocompatibility complex I and II and of alphavbeta3 in the 4C8 model was comparable to that seen in human glioma cells. Next, using a panel of Delta(gamma1)34.5 HSVs, we demonstrated that, in vitro, 4C8 cells were as sensitive as human glioma cells to both infection and lysis and that the 4C8 cells supported the production of foreign gene products. Replication competence of HSV was demonstrated in vitro. Finally, 4C8 intracranial gliomas were established in immunologically competent syngeneic B6D2F1 mice, treated by intratumoral injection of selected engineered HSVs, including the interleukin-12-expressing virus, M002. Survival data from these studies demonstrated that 4C8 cells in vivo are sensitive to both direct oncolysis and HSV-mediated interleukin-12 expression. Fluorescence-activated cell sorting analyses of immune-related infiltrating cells supported the concept that survival was prolonged in part because of antitumor actions of these cells. We conclude that the 4C8/B6D2F1 syngeneic glioma model is suitable for preclinical evaluation of HSV-based therapies and that M002 is a superior virus for the treatment of murine glioma in this model.