Ryan E. Bash

Pharmacokinetics and efficacy of PEGylated liposomal doxorubicin in an intracranial model of breast cancer.

Introduction Breast cancer brain metastases (BCBM) are a challenging consequence of advanced BC. Nanoparticle agents, including liposomes, have shown enhanced delivery to solid tumors and brain. We compared pharmacokinetics (PK) and efficacy of PEGylated liposomal doxorubicin (PLD) with non-liposomal doxorubicin (NonL-doxo) in an intracranial model of BC. Methods Athymic mice were inoculated intracerebrally with MDA-MB-231-BR-luciferase-expressing cells. Tumor-bearing mice were administered PLD or NonL-doxo at 6mg/kg IV×1 and were euthanized prior to and 0.083, 1, 3, 6, 24, 72 and 96 h post-treatment. Samples were processed to measure sum total doxorubicin via HPLC. PLD and NonL-doxo were administered IV weekly as single agents (6 mg/kg) or in combination (4.5 mg/kg) with the PARP inhibitor, ABT-888, PO 25 mg/kg/day. Efficacy was assessed by survival and bioluminescence. Results Treatment with PLD resulted in approximately 1,500-fold higher plasma and 20-fold higher intracranial tumor sum total doxorubicin AUC compared with NonL-doxo. PLD was detected at 96 h; NonL-doxo was undetectable after 24 h in plasma and tumor. Median survival of PLD-treated animals was 32 days (d, [CI] 31–38), which was significantly longer than controls (26d [CI 25–28]; p = 0.0012) or NonL-doxo treatment (23.5d [CI 18–28], p = 0.0002). Combination treatment with PLD/ABT-888 yielded improved survival compared to NonL-doxo/ABT-888 (35d [CI 31–38] versus 29.5d [CI 25–34]; p = 0.006). Conclusions PLD provides both PK and efficacy advantage over NonL-doxo in the treatment of an in vivo model of BCBM. The results provide preclinical rationale to translate findings into early phase trials of PLD, with or without ABT-888, for patients with BCBM.

Evolutionary etiology of high-grade astrocytomas

Significance High-grade astrocytomas (HGAs), including glioblastomas (GBMs), are the most common human brain tumors, and they remain fatal with no effective treatment. The most prevalent form, primary GBM, presents clinically as advanced disease, thus providing no access to or understanding of early stages. We report a comprehensive study in the mouse that establishes causal relationships and an evolutionary etiology in HGA development. Events yielding disease, both engineered and spontaneous, indicate grade-specific roles culminating in the development of GBMs with characteristics of primary GBMs, including molecular alignment with the mesenchymal subclass, asymptomatic early disease, and rapid emergence of high-grade aggressive cancer. These genetically engineered models provide a path to basic understanding of disease etiology and a window into diagnostic and therapeutic discovery. Glioblastoma (GBM), the most common brain malignancy, remains fatal with no effective treatment. Analyses of common aberrations in GBM suggest major regulatory pathways associated with disease etiology. However, 90% of GBMs are diagnosed at an advanced stage (primary GBMs), providing no access to early disease stages for assessing disease progression events. As such, both understanding of disease mechanisms and the development of biomarkers and therapeutics for effective disease management are limited. Here, we describe an adult-inducible astrocyte-specific system in genetically engineered mice that queries causation in disease evolution of regulatory networks perturbed in human GBM. Events yielding disease, both engineered and spontaneous, indicate ordered grade-specific perturbations that yield high-grade astrocytomas (anaplastic astrocytomas and GBMs). Impaired retinoblastoma protein RB tumor suppression yields grade II histopathology. Additional activation of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) network drives progression to grade III disease, and further inactivation of phosphatase and tensin homolog (PTEN) yields GBM. Spontaneous missense mutation of tumor suppressor Trp53 arises subsequent to KRAS activation, but before grade III progression. The stochastic appearance of mutations identical to those observed in humans, particularly the same spectrum of p53 amino acid changes, supports the validity of engineered lesions and the ensuing interpretations of etiology. Absence of isocitrate dehydrogenase 1 (IDH1) mutation, asymptomatic low grade disease, and rapid emergence of GBM combined with a mesenchymal transcriptome signature reflect characteristics of primary GBM and provide insight into causal relationships.

Cooperativity between MAPK and PI3K signaling activation is required for glioblastoma pathogenesis

BACKGROUND Glioblastoma (GBM) genomes feature recurrent genetic alterations that dysregulate core intracellular signaling pathways, including the G1/S cell cycle checkpoint and the MAPK and PI3K effector arms of receptor tyrosine kinase (RTK) signaling. Elucidation of the phenotypic consequences of activated RTK effectors is required for the design of effective therapeutic and diagnostic strategies. METHODS Genetically defined, G1/S checkpoint-defective cortical murine astrocytes with constitutively active Kras and/or Pten deletion mutations were used to systematically investigate the individual and combined roles of these 2 RTK signaling effectors in phenotypic hallmarks of glioblastoma pathogenesis, including growth, migration, and invasion in vitro. A novel syngeneic orthotopic allograft model system was used to examine in vivo tumorigenesis. RESULTS Constitutively active Kras and/or Pten deletion mutations activated both MAPK and PI3K signaling. Their combination led to maximal growth, migration, and invasion of G1/S-defective astrocytes in vitro and produced progenitor-like transcriptomal profiles that mimic human proneural GBM. Activation of both RTK effector arms was required for in vivo tumorigenesis and produced highly invasive, proneural-like GBM. CONCLUSIONS These results suggest that cortical astrocytes can be transformed into GBM and that combined dysregulation of MAPK and PI3K signaling revert G1/S-defective astrocytes to a primitive gene expression state. This genetically-defined, immunocompetent model of proneural GBM will be useful for preclinical development of MAPK/PI3K-targeted, subtype-specific therapies.

Efficacy of Carboplatin Alone and in Combination with ABT888 in Intracranial Murine Models of BRCA-Mutated and BRCA–Wild-Type Triple-Negative Breast Cancer

Patients with breast cancer brain metastases have extremely limited survival and no approved systemic therapeutics. Triple-negative breast cancer (TNBC) commonly metastasizes to the brain and predicts poor prognosis. TNBC frequently harbors BRCA mutations translating to platinum sensitivity potentially augmented by additional suppression of DNA repair mechanisms through PARP inhibition. We evaluated brain penetrance and efficacy of carboplatin ± the PARP inhibitor ABT888, and investigated gene-expression changes in murine intracranial TNBC models stratified by BRCA and molecular subtype status. Athymic mice were inoculated intracerebrally with BRCA-mutant: SUM149 (basal), MDA-MB-436 (claudin-low); or BRCA–wild-type (wt): MDA-MB-468 (basal), MDA-MB-231BR (claudin-low). TNBC cells were treated with PBS control [intraperitoneal (IP), weekly], carboplatin (50 mg/kg/wk, IP), ABT888 (25 mg/kg/d, oral gavage), or their combination. DNA damage (γ-H2AX), apoptosis (cleaved caspase-3, cC3), and gene expression were measured in intracranial tumors. Carboplatin ± ABT888 significantly improved survival in BRCA-mutant intracranial models compared with control, but did not improve survival in BRCA-wt intracranial models. Carboplatin + ABT888 revealed a modest survival advantage versus carboplatin in BRCA-mutant models. ABT888 yielded a marginal survival benefit in the MDA-MB-436, but not in the SUM149 model. BRCA-mutant SUM149 expression of γ-H2AX and cC3 proteins was elevated in all treatment groups compared with control, whereas BRCA-wt MDA-MB-468 cC3 expression did not increase with treatment. Carboplatin treatment induced common gene-expression changes in BRCA-mutant models. Carboplatin ± ABT888 penetrates the brain and improves survival in BRCA-mutant intracranial TNBC models with corresponding DNA damage and gene-expression changes. Combination therapy represents a potential promising treatment strategy for patients with TNBC brain metastases warranting further clinical investigation. Mol Cancer Ther; 14(4); 920–30. ©2015 AACR.

Attachment to roots and virulence of a chvB mutant of Agrobacterium tumefaciens are temperature sensitive.

Agrobacterium tumefaciens chvB mutants are unable to produce beta-1,2 glucan. They are nonattaching and avirulent and show reduced motility at room temperature. At lower temperatures (16 degrees C), chvB mutants became virulent on Bryophyllum daigremontiana and Lycopersicon esculentum and were able to attach to L. esculentum, Arabidopsis thaliana, Daucus carota, and Tagetes erecta roots. The mutant bacteria also recovered wild-type motility at lower temperatures. Two other nonattaching mutants of A. tumefaciens, AttR and AtrA, were unaffected by the lowered temperature, remaining nonattaching and avirulent.

Core pathway mutations induce de-differentiation of murine astrocytes into glioblastoma stem cells that are sensitive to radiation but resistant to temozolomide

BACKGROUND Glioma stem cells (GSCs) from human glioblastomas (GBMs) are resistant to radiation and chemotherapy and may drive recurrence. Treatment efficacy may depend on GSCs, expression of DNA repair enzymes such as methylguanine methyltransferase (MGMT), or transcriptome subtype. METHODS To model genetic alterations in human GBM core signaling pathways, we induced Rb knockout, Kras activation, and Pten deletion mutations in cortical murine astrocytes. Neurosphere culture, differentiation, and orthotopic transplantation assays were used to assess whether these mutations induced de-differentiation into GSCs. Genome-wide chromatin landscape alterations and expression profiles were examined by formaldehyde-assisted isolation of regulatory elements (FAIRE) seq and RNA-seq. Radiation and temozolomide efficacy were examined in vitro and in an allograft model in vivo. Effects of radiation on transcriptome subtype were examined by microarray expression profiling. RESULTS Cultured triple mutant astrocytes gained unlimited self-renewal and multilineage differentiation capacity. These cells harbored significantly altered chromatin landscapes that were associated with downregulation of astrocyte- and upregulation of stem cell-associated genes, particularly the Hoxa locus of embryonic transcription factors. Triple-mutant astrocytes formed serially transplantable glioblastoma allografts that were sensitive to radiation but expressed MGMT and were resistant to temozolomide. Radiation induced a shift in transcriptome subtype of GBM allografts from proneural to mesenchymal. CONCLUSION A defined set of core signaling pathway mutations induces de-differentiation of cortical murine astrocytes into GSCs with altered chromatin landscapes and transcriptomes. This non-germline genetically engineered mouse model mimics human proneural GBM on histopathological, molecular, and treatment response levels. It may be useful for dissecting the mechanisms of treatment resistance and developing more effective therapies.

Intrinsic astrocyte heterogeneity influences tumor growth in glioma mouse models.

The influence of cellular origin on glioma pathogenesis remains elusive. We previously showed that mutations inactivating Rb and Pten and activating Kras transform astrocytes and induce tumorigenesis throughout the adult mouse brain. However, it remained unclear whether astrocyte subpopulations were susceptible to these mutations. We therefore used genetic lineage tracing and fate mapping in adult conditional, inducible genetically engineered mice to monitor transformation of glial fibrillary acidic protein (GFAP) and glutamate aspartate transporter (GLAST) astrocytes and immunofluorescence to monitor cellular composition of the tumor microenvironment over time. Because considerable regional heterogeneity exists among astrocytes, we also examined the influence of brain region on tumor growth. GFAP astrocyte transformation induced uniformly rapid, regionally independent tumor growth, but transformation of GLAST astrocytes induced slowly growing tumors with significant regional bias. Transformed GLAST astrocytes had reduced proliferative response in culture and in vivo and malignant progression was delayed in these tumors. Recruited glial cells, including proliferating astrocytes, oligodendrocyte progenitors and microglia, were the majority of GLAST, but not GFAP astrocyte‐derived tumors and their abundance dynamically changed over time. These results suggest that intrinsic astrocyte heterogeneity, and perhaps regional brain microenvironment, significantly contributes to glioma pathogenesis.

Combination therapy with potent PI3K and MAPK inhibitors overcomes adaptive kinome resistance to single agents in preclinical models of glioblastoma

Background Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Prognosis remains poor despite multimodal therapy. Developing alternative treatments is essential. Drugs targeting kinases within the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) effectors of receptor tyrosine kinase (RTK) signaling represent promising candidates. Methods We previously developed a non-germline genetically engineered mouse model of GBM in which PI3K and MAPK are activated via Pten deletion and KrasG12D in immortalized astrocytes. Using this model, we examined the influence of drug potency on target inhibition, alternate pathway activation, efficacy, and synergism of single agent and combination therapy with inhibitors of these 2 pathways. Efficacy was then examined in GBM patient-derived xenografts (PDX) in vitro and in vivo. Results PI3K and mitogen-activated protein kinase kinase (MEK) inhibitor potency was directly associated with target inhibition, alternate RTK effector activation, and efficacy in mutant murine astrocytes in vitro. The kinomes of GBM PDX and tumor samples were heterogeneous, with a subset of the latter harboring MAPK hyperactivation. Dual PI3K/MEK inhibitor treatment overcame alternate effector activation, was synergistic in vitro, and was more effective than single agent therapy in subcutaneous murine allografts. However, efficacy in orthotopic allografts was minimal. This was likely due to dose-limiting toxicity and incomplete target inhibition. Conclusion Drug potency influences PI3K/MEK inhibitor-induced target inhibition, adaptive kinome reprogramming, efficacy, and synergy. Our findings suggest that combination therapies with highly potent, brain-penetrant kinase inhibitors will be required to improve patient outcomes.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

MerTK as a therapeutic target in glioblastoma.

Background Glioma-associated macrophages and microglia (GAMs) are components of the glioblastoma (GBM) microenvironment that express MerTK, a receptor tyrosine kinase that triggers efferocytosis and can suppress innate immune responses. The aim of the study was to define MerTK as a therapeutic target using an orally bioavailable inhibitor, UNC2025. Methods We examined MerTK expression in tumor cells and macrophages in matched patient GBM samples by double-label immunohistochemistry. UNC2025-induced MerTK inhibition was studied in vitro and in vivo. Results MerTK/CD68+ macrophages increased in recurrent tumors while MerTK/glial fibrillary acidic protein-positive tumor cells did not. Pharmacokinetic studies showed high tumor exposures of UNC2025 in a syngeneic orthotopic allograft mouse GBM model. The same model mice were randomized to receive vehicle, daily UNC2025, fractionated external beam radiotherapy (XRT), or UNC2025/XRT. Although median survival (21, 22, 35, and 35 days, respectively) was equivalent with or without UNC2025, bioluminescence imaging (BLI) showed significant growth delay with XRT/UNC2025 treatment and complete responses in 19%. The responders remained alive for 60 days and showed regression to 1%-10% of pretreatment BLI tumor burden; 5 of 6 were tumor free by histology. In contrast, only 2% of 98 GBM mice of the same model treated with XRT survived 50 days and none survived 60 days. UNC2025 also reduced CD206+ macrophages in mouse tumor samples. Conclusions These results suggest that MerTK inhibition combined with XRT has a therapeutic effect in a subset of GBM. Further mechanistic studies are warranted.