Publications by authors named "Ann C Mladek"

35 Publications

Protein kinase C and SRC signaling define reciprocally related subgroups of glioblastoma with distinct therapeutic vulnerabilities.

Cell Rep 2021 Nov;37(8):110054

Department of Cancer Biology, Mayo Clinic, Jacksonville, FL 32224, USA. Electronic address:

We report that atypical protein kinase Cι (PKCι) is an oncogenic driver of glioblastoma (GBM). Deletion or inhibition of PKCι significantly impairs tumor growth and prolongs survival in murine GBM models. GBM cells expressing elevated PKCι signaling are sensitive to PKCι inhibitors, whereas those expressing low PKCι signaling exhibit active SRC signaling and sensitivity to SRC inhibitors. Resistance to the PKCι inhibitor auranofin is associated with activated SRC signaling and response to a SRC inhibitor, whereas resistance to a SRC inhibitor is associated with activated PKCι signaling and sensitivity to auranofin. Interestingly, PKCι- and SRC-dependent cells often co-exist in individual GBM tumors, and treatment of GBM-bearing mice with combined auranofin and SRC inhibitor prolongs survival beyond either drug alone. Thus, we identify PKCι and SRC signaling as distinct therapeutic vulnerabilities that are directly translatable into an improved treatment for GBM.
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http://dx.doi.org/10.1016/j.celrep.2021.110054DOI Listing
November 2021

Multimodal platform for assessing drug distribution and response in clinical trials.

Neuro Oncol 2021 Aug 12. Epub 2021 Aug 12.

Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

Background: Response to targeted therapy varies between patients for largely unknown reasons. Here, we developed and applied an integrative platform using mass spectrometry imaging (MSI), phosphoproteomics, and multiplexed tissue imaging for mapping drug distribution, target engagement, and adaptive response to gain insights into heterogeneous response to therapy.

Methods: Patient derived xenograft (PDX) lines of glioblastoma were treated with adavosertib, a Wee-1 inhibitor, and tissue drug distribution was measured with MALDI MSI. Phosphoproteomics was measured in the same tumors to identify biomarkers of drug target engagement and cellular adaptive response. Multiplexed tissue imaging was performed on sister sections to evaluate spatial co-localization of drug and cellular response. The integrated platform was then applied on clinical specimens from glioblastoma patients enrolled in a Phase 1 clinical trial.

Results: PDX tumors exposed to different doses of adavosertib revealed intra and inter-tumoral heterogeneity of drug distribution and integration of the heterogeneous drug distribution with phosphoproteomics and multiplexed tissue imaging revealed new markers of molecular response to adavosertib. Analysis of paired clinical specimens from patients enrolled in the Phase 1 clinical trial informed the translational potential of the identified biomarkers in studying patient's response to adavosertib.

Conclusions: The multimodal platform identified a signature of drug efficacy and patient-specific adaptive responses applicable to pre-clinical and clinical drug development. The information generated by the approach may inform mechanisms of success and failure in future early phase clinical trials, providing information for optimizing clinical trial design and guiding future application into clinical practice.
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http://dx.doi.org/10.1093/neuonc/noab197DOI Listing
August 2021

Preclinical modeling in GBM PDX xenografts to guide clinical development of lisavanbulin - a novel tumor checkpoint controller targeting microtubules.

Neuro Oncol 2021 Jul 7. Epub 2021 Jul 7.

Department of Radiation Oncology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.

Background: Glioblastoma (GBM) is an incurable disease with few approved therapeutic interventions. Radiation therapy (RT) and temozolomide (TMZ) remain the standards of care. The efficacy and optimal deployment schedule of the orally bioavailable small-molecule tumor checkpoint controller lisavanbulin alone, and in combination with, standards of care were assessed using a panel of IDH-wildtype GBM patient-derived xenografts.

Methods: Mice bearing intracranial tumors received lisavanbulin +/- RT +/- TMZ and followed for survival. Lisavanbulin concentrations in plasma and brain were determined by liquid chromatography with tandem mass spectrometry, while flow cytometry was used for cell cycle analysis.

Results: Lisavanbulin monotherapy showed significant benefit (p<0.01) in 9 of 14 PDXs tested (median survival extension 9-84%) and brain-to-plasma ratios of 1.3 and 1.6 at 2- and 6-hours post-dose, respectively, validating previous data suggesting significant exposure in the brain. Prolonged lisavanbulin dosing from RT start until moribund was required for maximal benefit (GBM6: median survival lisavanbulin/RT 90 vs. RT alone 69 days, p=0.0001; GBM150: lisavanbulin/RT 143 days vs. RT alone 73 days, p=0.06). Similar observations were seen with RT/TMZ combinations (GBM39: RT/TMZ/lisavanbulin 502 days vs. RT/TMZ 249 days, p=0.0001; GBM26: RT/TMZ/lisavanbulin 172 days vs. RT/TMZ 121 days, p=0.04). Immunohistochemical analyses showed a significant increase in phospho-histone H3 with lisavanbulin treatment (p=0.01).

Conclusions: Lisavanbulin demonstrated excellent brain penetration, significant extension of survival alone or in RT or RT/TMZ combinations and was associated with mitotic arrest. These data provide a strong clinical rationale for testing lisavanbulin in combination with RT or RT/TMZ in GBM patients.
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http://dx.doi.org/10.1093/neuonc/noab162DOI Listing
July 2021

Heterogeneous delivery across the blood-brain barrier limits the efficacy of an EGFR-targeting antibody drug conjugate in glioblastoma.

Neuro Oncol 2021 May 29. Epub 2021 May 29.

Department of Radiation Oncology, Mayo Clinic, Rochester, MN.

Background: Antibody drug conjugates (ADCs) targeting the epidermal growth factor receptor (EGFR), such as depatuxizumab mafodotin (Depatux-M), is a promising therapeutic strategy for glioblastoma (GBM) but recent clinical trials did not demonstrate a survival benefit. Understanding the mechanisms of failure for this promising strategy is critically important.

Methods: PDX models were employed to study efficacy of systemic vs intracranial delivery of Depatux-M. Immunofluorescence and MALDI-MSI were performed to detect drug levels in the brain. EGFR levels and compensatory pathways were studied using quantitative flow cytometry, Western blots, RNAseq, FISH and phosphoproteomics.

Results: Systemic delivery of Depatux-M was highly effective in nine of 10 EGFR-amplified heterotopic PDXs with survival extending beyond one year in eight PDXs. Acquired resistance in two PDXs (GBM12 and GBM46) was driven by suppression of EGFR expression or emergence of a novel short-variant of EGFR lacking the epitope for the Depatux-M antibody. In contrast to the profound benefit observed in heterotopic tumors, only two of seven intrinsically sensitive PDXs were responsive to Depatux-M as intracranial tumors. Poor efficacy in orthotopic PDXs was associated with limited and heterogeneous distribution of Depatux-M into tumor tissues, and artificial disruption of the BBB or bypass of the BBB by direct intracranial injection of Depatux-M into orthotopic tumors markedly enhanced the efficacy of drug treatment.

Conclusions: Despite profound intrinsic sensitivity to Depatux-M, limited drug delivery into brain tumor may have been a key contributor to lack of efficacy in recently failed clinical trials.
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http://dx.doi.org/10.1093/neuonc/noab133DOI Listing
May 2021

Quantitative Analysis of Tyrosine Phosphorylation from FFPE Tissues Reveals Patient-Specific Signaling Networks.

Cancer Res 2021 Jul 20;81(14):3930-3941. Epub 2021 May 20.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.

Human tissue samples commonly preserved as formalin-fixed paraffin-embedded (FFPE) tissues after diagnostic or surgical procedures in the clinic represent an invaluable source of clinical specimens for in-depth characterization of signaling networks to assess therapeutic options. Tyrosine phosphorylation (pTyr) plays a fundamental role in cellular processes and is commonly dysregulated in cancer but has not been studied to date in FFPE samples. In addition, pTyr analysis that may otherwise inform therapeutic interventions for patients has been limited by the requirement for large amounts of frozen tissue. Here we describe a method for highly sensitive, quantitative analysis of pTyr signaling networks, with hundreds of sites quantified from one to two 10-μm sections of FFPE tissue specimens. A combination of optimized magnetic bead-based sample processing, optimized pTyr enrichment strategies, and tandem mass tag multiplexing enabled in-depth coverage of pTyr signaling networks from small amounts of input material. Phosphotyrosine profiles of flash-frozen and FFPE tissues derived from the same tumors suggested that FFPE tissues preserve pTyr signaling characteristics in patient-derived xenografts and archived clinical specimens. pTyr analysis of FFPE tissue sections from breast cancer tumors as well as lung cancer tumors highlighted patient-specific oncogenic driving kinases, indicating potential targeted therapies for each patient. These data suggest the capability for direct translational insight from pTyr analysis of small amounts of FFPE tumor tissue specimens. SIGNIFICANCE: This study reports a highly sensitive method utilizing FFPE tissues to identify dysregulated signaling networks in patient tumors, opening the door for direct translational insights from FFPE tumor tissue banks in hospitals.
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http://dx.doi.org/10.1158/0008-5472.CAN-21-0214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8286342PMC
July 2021

Efficacy of Tesevatinib in -Amplified Patient-Derived Xenograft Glioblastoma Models May Be Limited by Tissue Binding and Compensatory Signaling.

Mol Cancer Ther 2021 06 30;20(6):1009-1018. Epub 2021 Mar 30.

Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota.

Tesevatinib is a potent oral brain penetrant EGFR inhibitor currently being evaluated for glioblastoma therapy. Tesevatinib distribution was assessed in wild-type (WT) and triple knockout (TKO) FVB mice after dosing orally or via osmotic minipump; drug-tissue binding was assessed by rapid equilibrium dialysis. Two hours after tesevatinib dosing, brain concentrations in WT and TKO mice were 0.72 and 10.03 μg/g, respectively. Brain-to-plasma ratios (Kp) were 0.53 and 5.73, respectively. With intraperitoneal infusion, brain concentrations were 1.46 and 30.6 μg/g (Kp 1.16 and 25.10), respectively. The brain-to-plasma unbound drug concentration ratios were substantially lower (WT mice, 0.03-0.08; TKO mice, 0.40-1.75). Unbound drug concentrations in brains of WT mice were 0.78 to 1.59 ng/g. cytotoxicity and EGFR pathway signaling were evaluated using -amplified patient-derived glioblastoma xenograft models (GBM12, GBM6). pharmacodynamics and efficacy were assessed using athymic nude mice bearing either intracranial or flank tumors treated by oral gavage. Tesevatinib potently reduced cell viability [IC GBM12 = 11 nmol/L (5.5 ng/mL), GBM6 = 102 nmol/L] and suppressed EGFR signaling However, tesevatinib efficacy compared with vehicle in intracranial (GBM12, median survival: 23 vs. 18 days, = 0.003) and flank models (GBM12, median time to outcome: 41 vs. 33 days, = 0.007; GBM6, 44 vs. 33 days, = 0.007) was modest and associated with partial inhibition of EGFR signaling. Overall, tesevatinib efficacy in -amplified PDX GBM models is robust but relatively modest , despite a high brain-to-plasma ratio. This discrepancy may be explained by drug-tissue binding and compensatory signaling.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8172524PMC
June 2021

Salicylates enhance CRM1 inhibitor antitumor activity by induction of S-phase arrest and impairment of DNA-damage repair.

Blood 2021 01;137(4):513-523

Division of Hematology, Department of Internal Medicine.

Chromosome region maintenance protein 1 (CRM1) mediates protein export from the nucleus and is a new target for anticancer therapeutics. Broader application of KPT-330 (selinexor), a first-in-class CRM1 inhibitor recently approved for relapsed multiple myeloma and diffuse large B-cell lymphoma, have been limited by substantial toxicity. We discovered that salicylates markedly enhance the antitumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. Using salicylates in combination enables targeting of a range of blood cancers with a much lower dose of selinexor, thereby potentially mitigating prohibitive clinical adverse effects. Choline salicylate (CS) with low-dose KPT-330 (K+CS) had potent, broad activity across high-risk hematological malignancies and solid-organ cancers ex vivo and in vivo. The K+CS combination was not toxic to nonmalignant cells as compared with malignant cells and was safe without inducing toxicity to normal organs in mice. Mechanistically, compared with KPT-330 alone, K+CS suppresses the expression of CRM1, Rad51, and thymidylate synthase proteins, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, cell-cycle arrest in S-phase, and cell apoptosis. Moreover, the addition of poly (ADP-ribose) polymerase inhibitors further potentiates the K+CS antitumor effect. K+CS represents a new class of therapy for multiple types of blood cancers and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for cancer therapy in general.
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http://dx.doi.org/10.1182/blood.2020009013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845010PMC
January 2021

Genomic and Phenotypic Characterization of a Broad Panel of Patient-Derived Xenografts Reflects the Diversity of Glioblastoma.

Clin Cancer Res 2020 03 18;26(5):1094-1104. Epub 2019 Dec 18.

Mayo Clinic Arizona, Scottsdale, Arizona.

Purpose: Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization.

Experimental Design: PDXs were established from glioblastoma, IDH-wildtype ( = 93), glioblastoma, IDH-mutant ( = 2), diffuse midline glioma, H3 K27M-mutant ( = 1), and both primary ( = 60) and recurrent ( = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, = 83), RNA-sequencing ( = 68), and genome-wide methylation profiling ( = 76). WES data from 24 patient tumors was compared with derivative models.

Results: PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of , and , most at frequencies comparable with human glioblastoma. However, amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including amplification. However, in four patient-PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection.

Conclusions: Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-0909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056576PMC
March 2020

Molecular profiling of long-term IDH-wildtype glioblastoma survivors.

Neuro Oncol 2019 11;21(11):1458-1469

Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota.

Background: Glioblastoma (GBM) represents an aggressive cancer type with a median survival of only 14 months. With fewer than 5% of patients surviving 5 years, comprehensive profiling of these rare patients could elucidate prognostic biomarkers that may confer better patient outcomes. We utilized multiple molecular approaches to characterize the largest patient cohort of isocitrate dehydrogenase (IDH)-wildtype GBM long-term survivors (LTS) to date.

Methods: Retrospective analysis was performed on 49 archived formalin-fixed paraffin embedded tumor specimens from patients diagnosed with GBM at the Mayo Clinic between December 1995 and September 2013. These patient samples were subdivided into 2 groups based on survival (12 LTS, 37 short-term survivors [STS]) and subsequently examined by mutation sequencing, copy number analysis, methylation profiling, and gene expression.

Results: Of the 49 patients analyzed in this study, LTS were younger at diagnosis (P = 0.016), more likely to be female (P = 0.048), and MGMT promoter methylated (UniD, P = 0.01). IDH-wildtype STS and LTS demonstrated classic GBM mutations and copy number changes. Pathway analysis of differentially expressed genes showed LTS enrichment for sphingomyelin metabolism, which has been linked to decreased GBM growth, invasion, and angiogenesis. STS were enriched for DNA repair and cell cycle control networks.

Conclusions: While our findings largely report remarkable similarity between these LTS and more typical STS, unique attributes were observed in regard to altered gene expression and pathway enrichment. These attributes may be valuable prognostic markers and are worth further examination. Importantly, this study also underscores the limitations of existing biomarkers and classification methods in predicting patient prognosis.
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http://dx.doi.org/10.1093/neuonc/noz129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827834PMC
November 2019

Xenograft-based, platform-independent gene signatures to predict response to alkylating chemotherapy, radiation, and combination therapy for glioblastoma.

Neuro Oncol 2019 09;21(9):1141-1149

Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan.

Background: Predictive molecular biomarkers to select optimal treatment for patients with glioblastoma and other cancers are lacking. New strategies are needed when large randomized trials with correlative molecular data are not feasible.

Methods: Gene signatures (GS) were developed from 31 orthotopic glioblastoma patient-derived xenografts (PDXs), treated with standard therapies, to predict benefit from radiotherapy (RT-GS), temozolomide (Chemo-GS), or the combination (ChemoRT-GS). Independent validation was performed in a heterogeneously treated clinical cohort of 502 glioblastoma patients with overall survival as the primary endpoint. Multivariate Cox analysis was used to adjust for confounding variables and evaluate interactions between signatures and treatment.

Results: PDX models recapitulated the clinical heterogeneity of glioblastoma patients. RT-GS, Chemo-GS, and ChemoRT-GS were correlated with benefit from treatment in the PDX models. In independent clinical validation, higher RT-GS scores were associated with increased survival only in patients receiving RT (P = 0.0031, hazard ratio [HR] = 0.78 [0.66-0.92]), higher Chemo-GS scores were associated with increased survival only in patients receiving chemotherapy (P < 0.0001, HR = 0.66 [0.55-0.8]), and higher ChemoRT-GS scores were associated with increased survival only in patients receiving ChemoRT (P = 0.0001, HR = 0.54 [0.4-0.74]). RT-GS and ChemoRT-GS had significant interactions with treatment on multivariate analysis (P = 0.0009 and 0.02, respectively), indicating that they are bona fide predictive biomarkers.

Conclusions: Using a novel PDX-driven methodology, we developed and validated 3 platform-independent molecular signatures that predict benefit from standard of care therapies for glioblastoma. These signatures may be useful to personalize glioblastoma treatment in the clinic and this approach may be a generalizable method to identify predictive biomarkers without resource-intensive randomized trials.
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http://dx.doi.org/10.1093/neuonc/noz090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736132PMC
September 2019

Automatic 3D Nonlinear Registration of Mass Spectrometry Imaging and Magnetic Resonance Imaging Data.

Anal Chem 2019 05 22;91(9):6206-6216. Epub 2019 Apr 22.

Department of Neurosurgery, Brigham and Women's Hospital , Harvard Medical School , Boston , Massachusetts 02115 , United States.

Multimodal integration between mass spectrometry imaging (MSI) and radiology-established modalities such as magnetic resonance imaging (MRI) would allow the investigations of key questions in complex biological systems such as the central nervous system. Such integration would provide complementary multiscale data to bridge the gap between molecular and anatomical phenotypes, potentially revealing new insights into molecular mechanisms underlying anatomical pathologies presented on MRI. Automatic coregistration between 3D MSI/MRI is a computationally challenging process due to dimensional complexity, MSI data sparsity, lack of direct spatial-correspondences, and nonlinear tissue deformation. Here, we present a new computational approach based on stochastic neighbor embedding to nonlinearly align 3D MSI to MRI data, identify and reconstruct biologically relevant molecular patterns in 3D, and fuse the MSI datacube to the MRI space. We demonstrate our method using multimodal high-spectral resolution matrix-assisted laser desorption ionization (MALDI) 9.4 T MSI and 7 T in vivo MRI data, acquired from a patient-derived, xenograft mouse brain model of glioblastoma following administration of the EGFR inhibitor drug of Erlotinib. Results show the distribution of some identified molecular ions of the EGFR inhibitor erlotinib, a phosphatidylcholine lipid, and cholesterol, which were reconstructed in 3D and mapped to the MRI space. The registration quality was evaluated on two normal mouse brains using the Dice coefficient for the regions of brainstem, hippocampus, and cortex. The method is generic and can therefore be applied to hyperspectral images from different mass spectrometers and integrated with other established in vivo imaging modalities such as computed tomography (CT) and positron emission tomography (PET).
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http://dx.doi.org/10.1021/acs.analchem.9b00854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691508PMC
May 2019

PARP Inhibitors for Sensitization of Alkylation Chemotherapy in Glioblastoma: Impact of Blood-Brain Barrier and Molecular Heterogeneity.

Front Oncol 2018 22;8:670. Epub 2019 Jan 22.

Departments of Radiation Oncology, Mayo Clinic, Rochester, MN, United States.

Prognosis of patients with glioblastoma (GBM) remains dismal despite maximal surgical resection followed by aggressive chemo-radiation therapy. Almost every GBM, regardless of genotype, relapses as aggressive recurrent disease. Sensitization of GBM cells to chemo-radiation is expected to extend survival of patients with GBM by enhancing treatment efficacy. The PARP family of enzymes has a pleiotropic role in DNA repair and metabolism and has emerged as an attractive target for sensitization of cancer cells to genotoxic therapies. However, despite promising results from a number of preclinical studies, progress of clinical trials involving PARP inhibitors (PARPI) has been slower in GBM as compared to other malignancies. Preclinical studies have uncovered limitations of PARPI-mediated targeting of base excision repair, considered to be the likely mechanism of sensitization for temozolomide (TMZ)-resistant GBM. Nevertheless, PARPI remain a promising sensitizing approach for at least a subset of GBM tumors that are inherently sensitive to TMZ. Our PDX preclinical trial has helped delineate promoter hyper-methylation as a biomarker of the PARPI veliparib-mediated sensitization. In clinical trials, promoter hyper-methylation now is being studied as a potential predictive biomarker not only for response to TMZ therapy alone, but also PARPI-mediated sensitization of TMZ therapy. Besides the combination approach being investigated, IDH1/2 mutant gliomas associated with 2-hydroxygluterate (2HG)-mediated homologous recombination (HR) defect may potentially benefit from PARPI monotherapy. In this article, we discuss existing results and provide additional data in support of potential alternative mechanisms of sensitization that would help identify potential biomarkers for PARPI-based therapeutic approaches to GBM.
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http://dx.doi.org/10.3389/fonc.2018.00670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349736PMC
January 2019

Brain Distribution and Active Efflux of Three panRAF Inhibitors: Considerations in the Treatment of Melanoma Brain Metastases.

J Pharmacol Exp Ther 2019 03 8;368(3):446-461. Epub 2019 Jan 8.

Brain Barriers Research Center, Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota (G.G., M.K., A.S.M., K.E.P., W.F.E.); and Radiation Oncology, Mayo Clinic, Rochester, Minnesota (A.C.M., J.N.S.)

Targeted inhibition of RAF and MEK by molecularly targeted agents has been employed as a strategy to block aberrant mitogen-activated protein kinase (MAPK) signaling in melanoma. While the use of BRAF and MEK inhibitors, either as a single agent or in combination, improved efficacy in BRAF-mutant melanoma, initial responses are often followed by relapse due to acquired resistance. Moreover, some BRAF inhibitors are associated with paradoxical activation of the MAPK pathway, causing the development of secondary malignancies. The use of panRAF inhibitors, i.e., those that target all isoforms of RAF, may overcome paradoxical activation and resistance. The purpose of this study was to perform a quantitative assessment and evaluation of the influence of efflux mechanisms at the blood-brain barrier (BBB), in particular, Abcb1/P-glycoprotein (P-gp) and Abcg2/breast cancer resistance protein (Bcrp), on the brain distribution of three panRAF inhibitors: CCT196969 [1-(3-(-butyl)-1-phenyl-1-pyrazol-5-yl)-3-(2-fluoro-4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)phenyl)urea], LY3009120 1-(3,3-Dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido(2,3-d)pyrimidin-6-yl)phenyl)urea, and MLN2480 [4-pyrimidinecarboxamide, 6-amino-5-chloro--[(1)-1-[5-[[[5-chloro-4-(trifluoromethyl)-2-pyridinyl]amino]carbonyl]-2-thiazolyl]ethyl]-]. In vitro studies using transfected Madin-Darby canine kidney II cells indicate that only LY3009120 and MLN2480 are substrates of Bcrp, and none of the three inhibitors are substrates of P-gp. The three panRAF inhibitors show high nonspecific binding in brain and plasma. In vivo studies in mice show that the brain distribution of CCT196969, LY3009120, and MLN2480 is limited, and is enhanced in transgenic mice lacking P-gp and Bcrp. While MLN2480 has a higher brain distribution, LY3009120 exhibits superior in vitro efficacy in patient-derived melanoma cell lines. The delivery of a drug to the site of action residing behind a functionally intact BBB, along with drug potency against the target, collectively play a critical role in determining in vivo efficacy outcomes.
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http://dx.doi.org/10.1124/jpet.118.253708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374543PMC
March 2019

Integrated mapping of pharmacokinetics and pharmacodynamics in a patient-derived xenograft model of glioblastoma.

Nat Commun 2018 11 21;9(1):4904. Epub 2018 Nov 21.

Department of Radiology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 02115, USA.

Therapeutic options for the treatment of glioblastoma remain inadequate despite concerted research efforts in drug development. Therapeutic failure can result from poor permeability of the blood-brain barrier, heterogeneous drug distribution, and development of resistance. Elucidation of relationships among such parameters could enable the development of predictive models of drug response in patients and inform drug development. Complementary analyses were applied to a glioblastoma patient-derived xenograft model in order to quantitatively map distribution and resulting cellular response to the EGFR inhibitor erlotinib. Mass spectrometry images of erlotinib were registered to histology and magnetic resonance images in order to correlate drug distribution with tumor characteristics. Phosphoproteomics and immunohistochemistry were used to assess protein signaling in response to drug, and integrated with transcriptional response using mRNA sequencing. This comprehensive dataset provides simultaneous insight into pharmacokinetics and pharmacodynamics and indicates that erlotinib delivery to intracranial tumors is insufficient to inhibit EGFR tyrosine kinase signaling.
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http://dx.doi.org/10.1038/s41467-018-07334-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249307PMC
November 2018

Efficacy of the MDM2 Inhibitor SAR405838 in Glioblastoma Is Limited by Poor Distribution Across the Blood-Brain Barrier.

Mol Cancer Ther 2018 09 3;17(9):1893-1901. Epub 2018 Jul 3.

Mayo Clinic, Rochester, Minnesota.

Controversy exists surrounding whether heterogeneous disruption of the blood-brain barrier (BBB), as seen in glioblastoma (GBM), leads to adequate drug delivery sufficient for efficacy in GBM. This question is especially important when using potent, targeted agents that have a poor penetration across an intact BBB. Efficacy of the murine double minute-2 (MDM2) inhibitor SAR405838 was tested in patient-derived xenograft (PDX) models of GBM. efficacy of SAR405838 was evaluated in PDX models with varying MDM2 expression and those with high (GBM108) and low (GBM102) expression were evaluated for flank and orthotopic efficacy. BBB permeability, evaluated using TexasRed-3 kDa dextran, was significantly increased in GBM108 through VEGFA overexpression. Drug delivery, MRI, and orthotopic survival were compared between BBB-intact (GBM108-vector) and BBB-disrupted (GBM108-VEGFA) models. MDM2-amplified PDX lines with high MDM2 expression were sensitive to SAR405838 in comparison with MDM2 control lines in both and heterotopic models. In contrast with profound efficacy observed in flank xenografts, SAR405838 was ineffective in orthotopic tumors. Although both GBM108-vector and GBM108-VEGFA readily imaged on MRI following gadolinium contrast administration, GBM108-VEGFA tumors had a significantly enhanced drug and gadolinium accumulation, as determined by MALDI-MSI. Enhanced drug delivery in GBM108-VEGFA translated into a marked improvement in orthotopic efficacy. This study clearly shows that limited drug distribution across a partially intact BBB may limit the efficacy of targeted agents in GBM. Brain penetration of targeted agents is a critical consideration in any precision medicine strategy for GBM. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125211PMC
September 2018

Restricted Delivery of Talazoparib Across the Blood-Brain Barrier Limits the Sensitizing Effects of PARP Inhibition on Temozolomide Therapy in Glioblastoma.

Mol Cancer Ther 2017 Dec 25;16(12):2735-2746. Epub 2017 Sep 25.

Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota.

Poly ADP-ribose polymerase (PARP) inhibitors, including talazoparib, potentiate temozolomide efficacy in multiple tumor types; however, talazoparib-mediated sensitization has not been evaluated in orthotopic glioblastoma (GBM) models. This study evaluates talazoparib ± temozolomide in clinically relevant GBM models. Talazoparib at 1-3 nmol/L sensitized T98G, U251, and GBM12 cells to temozolomide, and enhanced DNA damage signaling and G-M arrest cyclical therapy with talazoparib (0.15 mg/kg twice daily) combined with low-dose temozolomide (5 mg/kg daily) was well tolerated. This talazoparib/temozolomide regimen prolonged tumor stasis more than temozolomide alone in heterotopic GBM12 xenografts [median time to endpoint: 76 days versus 50 days temozolomide ( = 0.005), 11 days placebo ( < 0.001)]. However, talazoparib/temozolomide did not accentuate survival beyond that of temozolomide alone in corresponding orthotopic xenografts [median survival 37 vs. 30 days with temozolomide ( = 0.93), 14 days with placebo, < 0.001]. Average brain and plasma talazoparib concentrations at 2 hours after a single dose (0.15 mg/kg) were 0.49 ± 0.07 ng/g and 25.5±4.1 ng/mL, respectively. The brain/plasma distribution of talazoparib in Bcrp versus wild-type (WT) mice did not differ, whereas the brain/plasma ratio in Mdr1a/b mice was higher than WT mice (0.23 vs. 0.02, < 0.001). Consistent with the brain distribution, overexpression of MDR1 decreased talazoparib accumulation in MDCKII cells. These results indicate that talazoparib has significant MDR1 efflux liability that may restrict delivery across the blood-brain barrier, and this may explain the loss of talazoparib-mediated temozolomide sensitization in orthotopic versus heterotopic GBM xenografts. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716902PMC
December 2017

Heterogeneous Binding and Central Nervous System Distribution of the Multitargeted Kinase Inhibitor Ponatinib Restrict Orthotopic Efficacy in a Patient-Derived Xenograft Model of Glioblastoma.

J Pharmacol Exp Ther 2017 11 28;363(2):136-147. Epub 2017 Aug 28.

Brain Barriers Research Center, Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota (J.K.L., M.K., K.E.P., S.Z., W.F.E.) and Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota (S.K.G., K.K.B., B.L.C., A.C.M., D.J.M., J.N.S.)

This study investigated how differences in drug distribution and free fraction at different tumor and tissue sites influence the efficacy of the multikinase inhibitor ponatinib in a patient-derived xenograft model of glioblastoma (GBM). Efficacy studies in GBM6 flank (heterotopic) and intracranial (orthotopic) models showed that ponatinib is effective in the flank but not in the intracranial model, despite a relatively high brain-to-plasma ratio. In vitro binding studies indicated that flank tumor had a higher free (unbound) drug fraction than normal brain. The total and free drug concentrations, along with the tissue-to-plasma ratio (Kp) and its unbound derivative (Kp,uu), were consistently higher in the flank tumor than the normal brain at 1 and 6 hours after a single dose in GBM6 flank xenografts. In the orthotopic xenografts, the intracranial tumor core displayed higher Kp and Kp,uu values compared with the brain-around-tumor (BAT). The free fractions and the total drug concentrations, hence free drug concentrations, were consistently higher in the core than in the BAT at 1 and 6 hours postdose. The delivery disadvantages in the brain and BAT were further evidenced by the low total drug concentrations in these areas that did not consistently exceed the in vitro cytotoxic concentration (IC). Taken together, the regional differences in free drug exposure across the intracranial tumor may be responsible for compromising efficacy of ponatinib in orthotopic GBM6.
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http://dx.doi.org/10.1124/jpet.117.243477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625285PMC
November 2017

Inhibiting DNA-PK radiosensitizes human osteosarcoma cells.

Biochem Biophys Res Commun 2017 04 12;486(2):307-313. Epub 2017 Mar 12.

Department of Orthopedic Surgery, Mayo Clinic School of Graduate Medical Education, Mayo Clinic, Rochester, MN 55902, USA; Department of Biomedical Engineering and Physiology, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Rochester, MN 55902, USA.

Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PK), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PK in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PK inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PK was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PK inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma.
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http://dx.doi.org/10.1016/j.bbrc.2017.03.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489116PMC
April 2017

Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins.

Cell Rep 2016 Mar 10;14(11):2587-98. Epub 2016 Mar 10.

Department of Radiation Oncology, Mayo Clinic, Rochester, MN 55905, USA.

Here we provide evidence that RBBP4 modulates temozolomide (TMZ) sensitivity through coordinate regulation of two key DNA repair genes critical for recovery from TMZ-induced DNA damage: methylguanine-DNA-methyltransferase (MGMT) and RAD51. Disruption of RBBP4 enhanced TMZ sensitivity, induced synthetic lethality to PARP inhibition, and increased DNA damage signaling in response to TMZ. Moreover, RBBP4 silencing enhanced TMZ-induced H2AX phosphorylation and apoptosis in GBM cells. Intriguingly, RBBP4 knockdown suppressed the expression of MGMT, RAD51, and other genes in association with decreased promoter H3K9 acetylation (H3K9Ac) and increased H3K9 tri-methylation (H3K9me3). Consistent with these data, RBBP4 interacts with CBP/p300 to form a chromatin-modifying complex that binds within the promoter of MGMT, RAD51, and perhaps other genes. Globally, RBBP4 positively and negatively regulates genes involved in critical cellular functions including tumorigenesis. The RBBP4/CBP/p300 complex may provide an interesting target for developing therapy-sensitizing strategies for GBM and other tumors.
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http://dx.doi.org/10.1016/j.celrep.2016.02.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4805508PMC
March 2016

Delineation of MGMT Hypermethylation as a Biomarker for Veliparib-Mediated Temozolomide-Sensitizing Therapy of Glioblastoma.

J Natl Cancer Inst 2016 May 27;108(5). Epub 2015 Nov 27.

Department of Radiation Oncology (SKG, BLC, ACM, KKB, JLP, MAS, LC, GJK, JNS), Division of Medical Oncology (SHK), Molecular Pharmacology and Experimental Therapeutics (FBA, JMR), Division of Biomedical Statistics and Informatics (PAD, JEEP, KVB), and Laboratory Medicine and Pathology (GS, RBJ), Mayo Clinic, Rochester MN; The University of Texas, MD Anderson Cancer Center, Houston, TX (RGV, EPS).

Background: Sensitizing effects of poly-ADP-ribose polymerase inhibitors have been studied in several preclinical models, but a clear understanding of predictive biomarkers is lacking. In this study, in vivo efficacy of veliparib combined with temozolomide (TMZ) was evaluated in a large panel of glioblastoma multiforme (GBM) patient-derived xenografts (PDX) and potential biomarkers were analyzed.

Methods: The efficacy of TMZ alone vs TMZ/veliparib was compared in a panel of 28 GBM PDX lines grown as orthotopic xenografts (8-10 mice per group); all tests of statistical significance were two-sided. DNA damage was analyzed by γH2AX immunostaining and promoter methylation of DNA repair gene O6-methylguanine-DNA-methyltransferase (MGMT) by Clinical Laboratory Improvement Amendments-approved methylation-specific polymerase chain reaction.

Results: The combination of TMZ/veliparib statistically significantly extended survival of GBM models (P < .05 by log-rank) compared with TMZ alone in five of 20 MGMT-hypermethylated lines (average extension in median survival = 87 days, range = 20-150 days), while the combination was ineffective in six MGMT-unmethylated lines. In the MGMT promoter-hypermethylated GBM12 line (median survival with TMZ+veliparib = 189 days, 95% confidence interval [CI] = 59 to 289 days, vs TMZ alone = 98 days, 95% CI = 49 to 210 days, P = .04), the profound TMZ-sensitizing effect of veliparib was lost when MGMT was overexpressed (median survival with TMZ+veliparib = 36 days, 95% CI = 28 to 38 days, vs TMZ alone = 35 days, 95% CI = 32 to 37 days, P = .87), and a similar association was observed in two nearly isogenic GBM28 sublines with an intact vs deleted MGMT locus. In comparing DNA damage signaling after dosing with veliparib/TMZ or TMZ alone, increased phosphorylation of damage-responsive proteins (KAP1, Chk1, Chk2, and H2AX) was observed only in MGMT promoter-hypermethylated lines.

Conclusion: Veliparib statistically significantly enhances (P < .001) the efficacy of TMZ in tumors with MGMT promoter hypermethylation. Based on these data, MGMT promoter hypermethylation is being used as an eligibility criterion for A071102 (NCT02152982), the phase II/III clinical trial evaluating TMZ/veliparib combination in patients with GBM.
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http://dx.doi.org/10.1093/jnci/djv369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862419PMC
May 2016

Evaluation of novel imidazotetrazine analogues designed to overcome temozolomide resistance and glioblastoma regrowth.

Mol Cancer Ther 2015 Jan 28;14(1):111-9. Epub 2014 Oct 28.

Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota.

The cellular responses to two new temozolomide (TMZ) analogues, DP68 and DP86, acting against glioblastoma multiforme (GBM) cell lines and primary culture models are reported. Dose-response analysis of cultured GBM cells revealed that DP68 is more potent than DP86 and TMZ and that DP68 was effective even in cell lines resistant to TMZ. On the basis of a serial neurosphere assay, DP68 inhibits repopulation of these cultures at low concentrations. The efficacy of these compounds was independent of MGMT and MMR functions. DP68-induced interstrand DNA cross-links were demonstrated with H2O2-treated cells. Furthermore, DP68 induced a distinct cell-cycle arrest with accumulation of cells in S phase that is not observed for TMZ. Consistent with this biologic response, DP68 induces a strong DNA damage response, including phosphorylation of ATM, Chk1 and Chk2 kinases, KAP1, and histone variant H2AX. Suppression of FANCD2 expression or ATR expression/kinase activity enhanced antiglioblastoma effects of DP68. Initial pharmacokinetic analysis revealed rapid elimination of these drugs from serum. Collectively, these data demonstrate that DP68 is a novel and potent antiglioblastoma compound that circumvents TMZ resistance, likely as a result of its independence from MGMT and mismatch repair and its capacity to cross-link strands of DNA.
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http://dx.doi.org/10.1158/1535-7163.MCT-14-0113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297195PMC
January 2015

Discordant in vitro and in vivo chemopotentiating effects of the PARP inhibitor veliparib in temozolomide-sensitive versus -resistant glioblastoma multiforme xenografts.

Clin Cancer Res 2014 Jul 16;20(14):3730-41. Epub 2014 May 16.

Authors' Affiliations: Departments of Radiation Oncology, and

Purpose: Effective sensitizing strategies potentially can extend the benefit of temozolomide (TMZ) therapy in patients with glioblastoma (GBM). We previously demonstrated that robust TMZ-sensitizing effects of the [poly (ADP-ribose) polymerase] (PARP) inhibitor veliparib (ABT-888) are restricted to TMZ-sensitive GBM xenografts. The focus of this study is to provide an understanding for the differential sensitization in paired TMZ-sensitive and -resistant GBM models.

Experimental Design: The impact of veliparib on TMZ-induced cytotoxicity and DNA damage was evaluated in vitro and in vivo in models of acquired TMZ resistance (GBM12TMZ-mgmt(High), GBM12TMZ-mgmt(Low), and U251TMZ), inherent TMZ resistance (T98G), and TMZ-sensitive (U251 and GBM12). In vivo drug efficacy, pharmacokinetics, and pharmacodynamics were analyzed using clinically relevant dosing regimens.

Results: Veliparib enhanced TMZ cytotoxicity and DNA-damage signaling in all GBM models in vitro with more pronounced effects in TMZ-resistant lines at 3 to 10 μmol/L veliparib. In vivo, combined TMZ/veliparib, compared with TMZ alone, significantly delayed tumor growth and enhanced DNA-damage signaling and γH2AX levels in the sensitive GBM12 xenograft line but not in the resistant GBM12TMZ lines. The pharmacokinetic profile of veliparib was similar for GBM12 and GBM12TMZ tumors with Cmax (∼1.5 μmol/L) in tissue significantly lower than concentrations associated with optimal in vitro sensitizing effects for resistant tumors. In contrast, robust suppression of PARP-1 expression by shRNA significantly increased TMZ sensitivity of U251TMZ in vitro and in vivo.

Conclusions: In vitro cytotoxicity assays do not adequately model the therapeutic index of PARP inhibitors, as concentrations of veliparib and TMZ required to sensitize TMZ-resistant cancer cells in vivo cannot be achieved using a tolerable dosing regimen.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-3446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4111895PMC
July 2014

Efficacy of protracted temozolomide dosing is limited in MGMT unmethylated GBM xenograft models.

Neuro Oncol 2013 Jun 10;15(6):735-46. Epub 2013 Mar 10.

Department of Radiation Oncology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA.

Background: Temozolomide (TMZ) is important chemotherapy for glioblastoma multiforme (GBM), but the optimal dosing schedule is unclear.

Methods: The efficacies of different clinically relevant dosing regimens were compared in a panel of 7 primary GBM xenografts in an intracranial therapy evaluation model.

Results: Protracted TMZ therapy (TMZ daily M-F, 3 wk every 4) provided superior survival to a placebo-treated group in 1 of 4 O(6)-DNA methylguanine-methyltransferase (MGMT) promoter hypermethylated lines (GBM12) and none of the 3 MGMT unmethylated lines, while standard therapy (TMZ daily M-F, 1 wk every 4) provided superior survival to the placebo-treated group in 2 of 3 MGMT unmethylated lines (GBM14 and GBM43) and none of the methylated lines. In comparing GBM12, GBM14, and GBM43 intracranial specimens, both GBM14 and GBM43 mice treated with protracted TMZ had a significant elevation in MGMT levels compared with placebo. Similarly, high MGMT was found in a second model of acquired TMZ resistance in GBM14 flank xenografts, and resistance was reversed in vitro by treatment with the MGMT inhibitor O(6)-benzylguanine, demonstrating a mechanistic link between MGMT overexpression and TMZ resistance in this line. Additionally, in an analysis of gene expression data, comparison of parental and TMZ-resistant GBM14 demonstrated enrichment of functional ontologies for cell cycle control within the S, G2, and M phases of the cell cycle and DNA damage checkpoints.

Conclusions: Across the 7 tumor models studied, there was no consistent difference between protracted and standard TMZ regimens. The efficacy of protracted TMZ regimens may be limited in a subset of MGMT unmethylated tumors by induction of MGMT expression.
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http://dx.doi.org/10.1093/neuonc/not010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661094PMC
June 2013

ATM inhibitor KU-55933 increases the TMZ responsiveness of only inherently TMZ sensitive GBM cells.

J Neurooncol 2012 Dec 3;110(3):349-57. Epub 2012 Oct 3.

Mayo Clinic, Guggenheim 6-01B, 200 1st Street SW, Rochester, MN 55905, USA.

Ataxia telangiectasia mutated (ATM) kinase is critical in sensing and repairing DNA double-stranded breaks (DSBs) such as those induced by temozolomide (TMZ). ATM deficiency increases TMZ sensitivity, which suggests that ATM inhibitors may be effective TMZ sensitizing agents. In this study, the TMZ sensitizing effects of 2 ATM specific inhibitors were studied in established and xenograft-derived glioblastoma (GBM) lines that are inherently sensitive to TMZ and derivative TMZ-resistant lines. In parental U251 and U87 glioma lines, the addition of KU-55933 to TMZ significantly increased cell killing compared to TMZ alone [U251 survival: 0.004 ± 0.0015 vs. 0.08 ± 0.01 (p < 0.001), respectively, and U87 survival: 0.02 ± 0.005 vs. 0.04 ± 0.002 (p < 0.001), respectively] and also elevated the fraction of cells arrested in G2/M [U251 G2/M fraction: 61.8 ± 1.1 % vs. 35 ± 0.8 % (p < 0.001), respectively, and U87 G2/M fraction 25 ± 0.2 % vs.18.6 ± 0.4 % (p < 0.001), respectively]. In contrast, KU-55933 did not sensitize the resistant lines to TMZ, and neither TMZ alone or combined with KU-55933 induced a G2/M arrest. While KU-55933 did not enhance TMZ induced Chk1/Chk2 activation, it increased TMZ-induced residual γ-H2AX foci in the parental cells but not in the TMZ resistant cells. Similar sensitization was observed with either KU-55933 or CP-466722 combined with TMZ in GBM12 xenograft line but not in GBM12TMZ, which is resistant to TMZ due to MGMT overexpression. These findings are consistent with a model where ATM inhibition suppresses the repair of TMZ-induced DSBs in inherently TMZ-sensitive tumor lines, which suggests an ATM inhibitor potentially could be deployed with an improvement in the therapeutic window when combined with TMZ.
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http://dx.doi.org/10.1007/s11060-012-0979-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3535329PMC
December 2012

p16-Cdk4-Rb axis controls sensitivity to a cyclin-dependent kinase inhibitor PD0332991 in glioblastoma xenograft cells.

Neuro Oncol 2012 Jul 18;14(7):870-81. Epub 2012 Jun 18.

Department of Radiation Oncology, Mayo Clinic, Rochester, MN 55905, USA.

Deregulation of the p16(INK4a)-Cdk4/6-Rb pathway is commonly detected in patients with glioblastoma multiforme (GBM) and is a rational therapeutic target. Here, we characterized the p16(INK4a)-Cdk4/6-Rb pathway in the Mayo panel of GBM xenografts, established from primary tissue samples from patients with GBM, and evaluated their response to PD0332991, a specific inhibitor of Cdk4/6. All GBM xenograft lines evaluated in this study had disruptions in the p16(INK4a)-Cdk4/6-Rb pathway. In vitro evaluation using short-term explant cultures from selected GBM xenograft lines showed that PD0332991 effectively arrested cell cycle in G1-phase and inhibited cell proliferation dose-dependently in lines deleted for CDKN2A/B-p16(INK4a) and either single-copy deletion of CDK4 (GBM22), high-level CDK6 amplification (GBM34), or deletion of CDKN2C/p18(INK4c) (GBM43). In contrast, 2 GBM lines with p16(INK4a) expression and either CDK4 amplification (GBM5) or RB mutation (GBM28) were completely resistant to PD0332991. Additional xenograft lines were screened, and GBM63 was identified to have p16(INK4a) expression and CDK4 amplification. Similar to the results with GBM5, GBM63 was resistant to PD0332991 treatment. In an orthotopic survival model, treatment of GBM6 xenografts (CDKN2A/B-deleted and CDK4 wild-type) with PD0332991 significantly suppressed tumor cell proliferation and prolonged survival. Collectively, these data support the concept that GBM tumors lacking p16(INK4a) expression and with nonamplified CDK4 and wild-type RB status may be more susceptible to Cdk4/6 inhibition using PD0332991.
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http://dx.doi.org/10.1093/neuonc/nos114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3379801PMC
July 2012

Inhibition of histone deacetylation potentiates the evolution of acquired temozolomide resistance linked to MGMT upregulation in glioblastoma xenografts.

Clin Cancer Res 2012 Aug 6;18(15):4070-9. Epub 2012 Jun 6.

Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota 55905, USA.

Purpose: The therapeutic benefit of temozolomide in glioblastoma multiforme (GBM) is limited by resistance. The goal of this study was to elucidate mechanisms of temozolomide resistance in GBM.

Experimental Design: We developed an in vivo GBM model of temozolomide resistance and used paired parental and temozolomide-resistant tumors to define the mechanisms underlying the development of resistance and the influence of histone deacetylation (HDAC) inhibition.

Results: Analysis of paired parental and resistant lines showed upregulation of O6-methylguanine-DNA methyltransferase (MGMT) expression in 3 of the 5 resistant xenografts. While no significant change was detected in MGMT promoter methylation between parental and derivative-resistant samples, chromatin immunoprecipitation showed an association between MGMT upregulation and elevated acetylation of lysine 9 of histone H3 (H3K9-ac) and decreased dimethylation (H3K9-me2) in GBM12 and GBM14. In contrast, temozolomide resistance development in GBM22 was not linked to MGMT expression, and both parental and resistant lines had low H3K9-ac and high H3K9-me2 within the MGMT promoter. In the GBM12TMZ-resistant line, MGMT reexpression was accompanied by increased recruitment of SP1, C-JUN, NF-κB, and p300 within the MGMT promoter. Interestingly, combined treatment of GBM12 flank xenografts with temozolomide and the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) favored the evolution of temozolomide resistance by MGMT overexpression as compared with treatment with temozolomide alone.

Conclusion: This study shows, for the first time, a unique mechanism of temozolomide resistance development driven by chromatin-mediated MGMT upregulation and highlights the potential for epigenetically directed therapies to influence the mechanisms of resistance development in GBM.
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http://dx.doi.org/10.1158/1078-0432.CCR-12-0560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3716364PMC
August 2012

Radiosensitizing effects of temozolomide observed in vivo only in a subset of O6-methylguanine-DNA methyltransferase methylated glioblastoma multiforme xenografts.

Int J Radiat Oncol Biol Phys 2009 Sep;75(1):212-9

Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota 55905, USA.

Purpose: Concurrent temozolomide (TMZ) and radiation therapy (RT) followed by adjuvant TMZ is standard treatment for patients with glioblastoma multiforme (GBM), although the relative contribution of concurrent versus adjuvant TMZ is unknown. In this study, the efficacy of TMZ/RT was tested with a panel of 20 primary GBM xenografts.

Methods And Materials: Mice with intracranial xenografts were treated with TMZ, RT, TMZ/RT, or placebo. Survival ratio for a given treatment/line was defined as the ratio of median survival for treatment vs. placebo.

Results: The median survival ratio was significantly higher for O6-methylguanine-DNA methyltransferase (MGMT) methylated tumors versus unmethylated tumors following treatment with TMZ (median survival ratio, 3.6 vs. 1.5, respectively; p = 0.008) or TMZ/RT (5.7 vs. 2.3, respectively; p = 0.001) but not RT alone (1.7 vs. 1.6; p = 0.47). In an analysis of variance, MGMT methylation status and p53 mutation status were significantly associated with treatment response. When we analyzed the additional survival benefit conferred specifically by combined therapy, only a subset (5 of 11) of MGMT methylated tumors derived substantial additional benefit from combined therapy, while none of the MGMT unmethylated tumors did. Consistent with a true radiosensitizing effect of TMZ, sequential treatment in which RT (week 1) was followed by TMZ (week 2) proved significantly less effective than TMZ followed by RT or concurrent TMZ/RT (survival ratios of 4.0, 9.6 and 12.9, respectively; p < 0.0001).

Conclusions: Concurrent treatment with TMZ and RT provides significant survival benefit only in a subset of MGMT methylated tumors and provides superior antitumor activity relative to sequential administration of RT and TMZ.
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http://dx.doi.org/10.1016/j.ijrobp.2009.04.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773462PMC
September 2009

Effective sensitization of temozolomide by ABT-888 is lost with development of temozolomide resistance in glioblastoma xenograft lines.

Mol Cancer Ther 2009 Feb 27;8(2):407-14. Epub 2009 Jan 27.

Department of Neurologic Surgery, Mayo Clinic, Rochester, MN 55905, USA.

Resistance to temozolomide and radiotherapy is a major problem for patients with glioblastoma but may be overcome using the poly(ADP-ribose) polymerase inhibitor ABT-888. Using two primary glioblastoma xenografts, the efficacy of ABT-888 combined with radiotherapy and/or temozolomide was evaluated. Treatment with ABT-888 combined with temozolomide resulted in significant survival prolongation (GBM12: 55.1%, P = 0.005; GBM22: 54.4%, P = 0.043). ABT-888 had no effect with radiotherapy alone but significantly enhanced survival in GBM12 when combined with concurrent radiotherapy/temozolomide. With multicycle therapy, ABT-888 further extended the survival benefit of temozolomide in the inherently sensitive GBM12 and GBM22 xenograft lines. However, after in vivo selection for temozolomide resistance, the derivative GBM12TMZ and GBM22TMZ lines were no longer sensitized by ABT-888 in combination with temozolomide, and a similar lack of efficacy was observed in two other temozolomide-resistant tumor lines. Thus, the sensitizing effects of ABT-888 were limited to tumor lines that have not been previously exposed to temozolomide, and these results suggest that patients with newly diagnosed glioblastoma may be more likely to respond to combined temozolomide/poly(ADP-ribose) polymerase inhibitor therapy than patients with recurrent disease.
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http://dx.doi.org/10.1158/1535-7163.MCT-08-0854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2692390PMC
February 2009

Evaluation of MGMT promoter methylation status and correlation with temozolomide response in orthotopic glioblastoma xenograft model.

J Neurooncol 2009 Mar 15;92(1):23-31. Epub 2008 Nov 15.

Department of Radiation Oncology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA.

CpG methylation within the O6-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with enhanced survival of glioblastoma multiforme (GBM) patients treated with temozolomide (TMZ). Although MGMT promoter is methylated in approximately 50% of GBM, several studies have reported a lack of correlation between MGMT methylation and protein expression levels and consequently inaccurate discrimination of TMZ sensitive and resistant patients. To understand the limitations of currently used assays, TMZ responsiveness of 13 GBM xenograft lines was correlated with MGMT protein expression and MGMT promoter methylation determined by (1) standard methylation-specific polymerase chain reaction (MS-PCR), (2) quantitative MS-PCR (qMS-PCR), and (3) bisulfite sequencing. For each xenograft line, mice with established intracranial xenografts were treated with vehicle control or TMZ (66 mg/kgx5 days), and TMZ response was defined as relative prolongation in median survival for TMZ-treated versus control-treated mice. The relative survival benefit with TMZ was inversely related to MGMT protein expression (r=-0.75; P=0.003) and directly correlated with qMS-PCR (r=0.72; P=0.006). There was a direct correlation between MGMT methylation signal by qMS-PCR and the number of methylated CpG sites within the region amplified by MS-PCR (r=0.78, P=0.002). However, bisulfite sequencing revealed heterogeneity in the extent of CpG methylation in those tumors with a robust qMS-PCR signal. Three of the 4 GBM lines with a qMS-PCR signal greater than 10% had at least 1 unmethylated CpG site, while only one line was fully methylated at all 12 CpG sites. These data highlight one potential limitation of the evaluation of MGMT methylation by MS-PCR assay and suggest that more detailed evaluation of methylation at individual CpG sites relative to TMZ response may be worth pursuing.
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http://dx.doi.org/10.1007/s11060-008-9737-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2790867PMC
March 2009

PTEN loss does not predict for response to RAD001 (Everolimus) in a glioblastoma orthotopic xenograft test panel.

Clin Cancer Res 2008 Jun;14(12):3993-4001

Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota, USA.

Purpose: Hyperactivation of the phosphatidylinositol 3-kinase/Akt signaling through disruption of PTEN function is common in glioblastoma multiforme, and these genetic changes are predicted to enhance sensitivity to mammalian target of rapamycin (mTOR) inhibitors such as RAD001 (everolimus).

Experimental Design: To test whether PTEN loss could be used as a predictive marker for mTOR inhibitor sensitivity, the response of 17 serially transplantable glioblastoma multiforme xenografts was evaluated in an orthotopic therapy evaluation model. Of these 17 xenograft lines, 7 have either genomic deletion or mutation of PTEN.

Results: Consistent with activation of Akt signaling, there was a good correlation between loss of PTEN function and elevated levels of Akt phosphorylation. However, of the 7 lines with disrupted PTEN function, only 1 tumor line (GBM10) was significantly sensitive to RAD001 therapy (25% prolongation in median survival), whereas 1 of 10 xenograft lines with wild-type PTEN was significantly sensitive to RAD001 (GS22; 34% prolongation in survival). Relative to placebo, 5 days of RAD001 treatment was associated with a marked 66% reduction in the MIB1 proliferation index in the sensitive GBM10 line (deleted PTEN) compared with a 25% and 7% reduction in MIB1 labeling index in the insensitive GBM14 (mutant PTEN) and GBM15 (wild-type PTEN) lines, respectively. Consistent with a cytostatic antitumor effect, bioluminescent imaging of luciferase-transduced intracranial GBM10 xenografts showed slowed tumor growth without significant tumor regression during RAD001 therapy.

Conclusion: These data suggest that loss of PTEN function is insufficient to adequately predict responsiveness to mTOR inhibitors in glioblastoma multiforme.
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http://dx.doi.org/10.1158/1078-0432.CCR-07-4152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3972037PMC
June 2008
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