Sarkaria, Jann N.

BRAF-targeted kinase inhibitors (KIs) are used to treat malignancies including BRAF-mutant non–small cell lung cancer, colorectal cancer, anaplastic thyroid cancer, and, most prominently, melanoma. However, KI selection criteria in patients remain unclear, as are pharmacokinetic/pharmacodynamic (PK/PD) mechanisms that may limit context-dependent efficacy and differentiate related drugs. To address this issue, we imaged mouse models of BRAF-mutant cancers, fluorescent KI tracers, and unlabeled drug to calibrate in silico spatial PK/PD models. Results indicated that drug lipophilicity, plasma clearance, faster target dissociation, and, in particular, high albumin binding could limit dabrafenib action in visceral metastases compared to other KIs. This correlated with retrospective clinical observations. Computational modeling identified a timed strategy for combining dabrafenib and encorafenib to better sustain BRAF inhibition, which showed enhanced efficacy in mice. This study thus offers principles of spatial drug action that may help guide drug development, KI selection, and combination.

High-grade gliomas (HGG) are devastating diseases in children and adults. In the pediatric population, diffuse midline gliomas (DMG) harboring H3K27 alterations are the most aggressive primary malignant brain tumors. With no effective therapies available, children typically succumb to disease within one year of diagnosis. In adults, glioblastoma (GBM) remains largely intractable, with a median survival of approximately 14 months despite standard clinical care of radiation and temozolomide. Therefore, effective therapies for these tumors remain one of the most urgent and unmet needs in modern medicine. Interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is a cell-surface transmembrane protein upregulated in many HGGs, including DMG and adult GBM, posing a potentially promising therapeutic target for these tumors. In this study, we investigated the pharmacological effects of GB-13 (also known as IL13.E13K-PE4E), a novel peptide–toxin conjugate that contains a targeting moiety designed to bind IL-13Rα2 with high specificity and a point-mutant cytotoxic domain derived from Pseudomonas exotoxin A. Glioma cell lines demonstrated a spectrum of IL-13Rα2 expression at both the transcript and protein level. Anti-tumor effects of GB-13 strongly correlated with IL-13Rα2 expression and were reflected in apoptosis induction and decreased cell proliferation in vitro. Direct intratumoral administration of GB-13 via convection-enhanced delivery (CED) significantly decreased tumor burden and resulted in prolonged survival in IL-13Rα2-upregulated orthotopic xenograft models of HGG. In summary, administration of GB-13 demonstrated a promising pharmacological response in HGG models both in vitro and in vivo in a manner strongly associated with IL-13Rα2 expression, underscoring the potential of this IL-13Rα2-targeted therapy in a subset of HGG with increased IL-13Rα2 levels.

Abstract Background RBBP4 activates transcription by histone acetylation, but the partner histone acetyltransferases are unknown. Thus, we investigated the hypothesis that RBBP4 interacts with p300 in a complex in glioblastoma (GBM). Methods shRNA silencing of RBBP4 or p300 and RNAseq was used to identify genes co-regulated by RBBP4 and p300 in GBM43 patient-derived xenograft (PDX). RBBP4/p300 complex was demonstrated using proximity ligation assay (PLA) and ChIPseq delineated histone H3 acetylation and RBBP4/p300 complex binding in promoters/enhancers. Temozolomide (TMZ)-induced DNA double strand breaks (DSBs) were evaluated by γ-H2AX and proliferation by CyQuant and live cell monitoring assays. In vivo efficacy was based on survival of mice with orthotopic tumors. Results shRBBP4 and shp300 downregulated 4768 genes among which 1485 (31%) were commonly downregulated by both shRNAs, while upregulated genes were 2484, including 863 (35%) common genes. The pro-survival genes were the top-ranked among the downregulated genes, including C-MYC. RBBP4/p300 complex was demonstrated in the nucleus, and shRBBP4 or shp300 significantly sensitized GBM cells to TMZ compared to the control shNT in vitro (P < .05). Moreover, TMZ significantly prolonged the survival of mice bearing GBM22-shRBBP4 orthotopic tumors compared with control shNT tumors (median shNT survival 52 days vs. median shRBBP4 319 days; P = .001). CREB-binding protein (CBP)/p300 inhibitor CPI-1612 suppressed H3K27Ac and RBBP4/p300 complex target proteins, including C-MYC, and synergistically sensitized TMZ in vitro. Pharmacodynamic evaluation confirmed brain penetration by CPI-1612 supporting further investigation to evaluate efficacy to sensitize TMZ. Conclusions RBBP4/p300 complex is present in GBM cells and is a potential therapeutic target.

AbstractBrain tumours have a poor prognosis and lack effective treatments. The blood–brain barrier (BBB) represents a major hurdle to drug delivery to brain tumours. In some locations in the tumour, the BBB may be disrupted to form the blood–brain tumour barrier (BBTB). This leaky BBTB enables diagnosis of brain tumours by contrast enhanced magnetic resonance imaging; however, this disruption is heterogeneous throughout the tumour. Thus, relying on the disrupted BBTB for achieving effective drug concentrations in brain tumours has met with little clinical success. Because of this, it would be beneficial to design drugs and drug delivery strategies to overcome the ‘normal’ BBB to effectively treat the brain tumours. In this review, we discuss the role of BBB/BBTB in brain tumour diagnosis and treatment highlighting the heterogeneity of the BBTB. We also discuss various strategies to improve drug delivery across the BBB/BBTB to treat both primary and metastatic brain tumours. Recognizing that the BBB represents a critical determinant of drug efficacy in central nervous system tumours will allow a more rapid translation from basic science to clinical application. A more complete understanding of the factors, such as BBB–limited drug delivery, that have hindered progress in treating both primary and metastatic brain tumours, is necessary to develop more effective therapies.

Abstract Motivation Mass spectrometry imaging (MSI) provides rich biochemical information in a label-free manner and therefore holds promise to substantially impact current practice in disease diagnosis. However, the complex nature of MSI data poses computational challenges in its analysis. The complexity of the data arises from its large size, high-dimensionality and spectral nonlinearity. Preprocessing, including peak picking, has been used to reduce raw data complexity; however, peak picking is sensitive to parameter selection that, perhaps prematurely, shapes the downstream analysis for tissue classification and ensuing biological interpretation. Results We propose a deep learning model, massNet, that provides the desired qualities of scalability, nonlinearity and speed in MSI data analysis. This deep learning model was used, without prior preprocessing and peak picking, to classify MSI data from a mouse brain harboring a patient-derived tumor. The massNet architecture established automatically learning of predictive features, and automated methods were incorporated to identify peaks with potential for tumor delineation. The model’s performance was assessed using cross-validation, and the results demonstrate higher accuracy and a substantial gain in speed compared to the established classical machine learning method, support vector machine. Availability and implementation https://github.com/wabdelmoula/massNet. The data underlying this article are available in the NIH Common Fund’s National Metabolomics Data Repository (NMDR) Metabolomics Workbench under project id (PR001292) with http://dx.doi.org/10.21228/M8Q70T. Supplementary information Supplementary data are available at Bioinformatics online.

Abstract INTRODUCTION Glioblastoma (GBM) is a diffusely invasive primary brain tumor with significant spread of tumor cells to the periphery of visible image abnormality. Enhancement of Gadolinium (Gd) contrast agent on magnetic resonance imaging (MRI) has historically been considered a confirmation of local breakdown of the blood brain barrier (BBB) and sufficient drug delivery to the bulk of tumors. In this work, we used GBM-derived xenograft (PDX) models to compare drug delivery in GBM brain for high and low BBB-permeable drugs. MATERIALS AND METHODS Five patient-derived orthotopic xenograft models from two GBM cell lines (GBM39 and GBM12) were co-dosed with erlotinib and osimertinib, two drugs with low and high BBB-permeability, respectively. T1Gd and T2-weighted MRIs were acquired from all animals prior to model sacrifice. Tumors were manually segmented on denoised and standardized MRIs and intensity patterns were captured using first and second order statistical features in the moving 3x3 kernel. We compared drug levels found in Matrix Assisted Laser Desorption Ionization (MALDI) in T1Gd enhancement, T2 enhancement, and normal brain. We also performed linear regression modeling to predict drug levels using MRI features. Model performance was measured using root mean squared error (RMSE). RESULTS Our analysis showed correlations between imaging features and MALDI drug levels. Osimertinib had a uniform distribution across the brain for all animals and all cell lines, consistent with our expectation for a high BBB-penetrant drug. Erlotinib showed the highest drug levels in T2 for GBM39 and in T1Gd for GBM12. Regression models showed promising results for predicting Erlotinib with a low RMSE of 0.037. CONCLUSION Our preliminary results suggest MRI can be predictive of drug levels for low-BBB penetrant drugs. Understanding the relationship between MRIs and drug distribution in diffuse tumors can be beneficial to developing effective treatment.

Abstract High grade gliomas are devastating diseases. In the pediatric population, diffuse midline gliomas with the H3K27M mutation (H3K27M DMG) are the most aggressive primary malignant brain tumors. With no effective therapies available, children typically succumb to disease within one year of diagnosis. In adults, glioblastoma (GBM) remains a death sentence despite standard clinical care. Therefore, effective therapies for these tumors remain one of the most urgent and unmet needs in modern medicine. Interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is a cell-surface transmembrane protein upregulated in H3K27M DMG and GBM versus normal brain tissue, posing a potentially promising therapeutic target for both tumors. In this study, we investigated the pharmacological effects of a novel peptide-toxin conjugate, IL13.E13K-PE4E (also known as GB-13), that contains a targeting moiety designed to bind IL-13Rα2 with high specificity and a point-mutant cytotoxic domain derived from Pseudomonas exotoxin A. We demonstrated that IL13.E13K-PE4E was a potent killer of cultured H3K27M DMG and GBM cells in vitro. Intratumoral administration of IL13.E13K-PE4E via convection-enhanced delivery (CED) decreased tumor burden and prolonged survival in both H3K27M DMG and GBM murine xenograft models. Furthermore, we observed enhanced drug tissue retention and volume of distribution after CED, suggesting IL13.E13K-PE4E is capable of covering the target area and remaining at the site of infusion long enough to impart therapeutic effects. In summary, administration of IL13.E13K-PE4E demonstrated a potent pharmacological response in H3K27M DMG and GBM models both in vitro and in vivo in a manner strongly associated with IL13Rα2 expression, underscoring the potential of IL13Rα2 targeted therapy in a subset of these tumors.

Abstract While dose escalation of radiotherapy (DERT) has failed to improve overall survival (OS) or progression-free survival (PFS) for glioblastoma in previous studies, a recent phase II clinical trial utilizing 18F-DOPA-PET-directed DERT demonstrated improved PFS in MGMT-unmethylated patients and OS in MGMT-methylated patients compared to historical controls. This planned secondary analysis sought to determine 1) how 18F-DOPA-PET changes RT volumes beyond standard MRI-planning, 2) which patients benefit most and least from this protocol, 3) which are mostly likely to experience clinically significant radionecrosis after DERT, and 4) patterns of failure after DERT. For 69 evaluable patients, median MRI-defined, PET-defined, and combined low-dose gross tumor volumes (GTV51) were 54 cc (range 9-248), 23 cc (0.4-179), and 62 cc (10-260), respectively. Median MRI-defined, PET-defined, and combined high-dose GTVs (GTV76) were 32 cc (range 4-136), 6 cc (0.1-138), and 34 cc (4-162), respectively. 18F-DOPA-PET resulted in a median volumetric expansion of 13% (0-243%) and 5% (0-217%) from MRI-defined low-dose and high-dose GTV’s, respectively. Central failures ( >95% of recurrence tumor volume) occurred within the 76 Gy, 60 Gy, and 51 Gy isodose lines in 32 (46%), 60 (87%), and 64 (93%) patients, respectively. Recursive partitioning analysis stratified patients by OS and PFS. Patients with 18F-DOPA-PET-defined GTV76 > 7.8cc, MRI-defined GTV76 > 42.7cc, and MGMT promotor-unmethylated tumors had the shortest OS, while patients with smaller PET and MRI-defined tumors had the longest OS (median 10.4 vs. 64.6 months, p< 0.001). Similarly, PFS was worst in patients with 18F-DOPA-PET-defined GTV76 > 2.17 cc who had biopsy only (median PFS 3.2 months, p< 0.001). Patients with 18F-DOPA-PET-defined GTV51 > 50 cc had the highest risk of grade 3+ radionecrosis (p< 0.001). In conclusion, larger 18F-DOPA-PET and MRI-defined tumor volumes were associated with worse outcomes, and 18F-DOPA-PET-directed DERT appears to reduce risk of central recurrence in high-dose volumes.

Abstract Glioblastoma (GBM) is a highly malignant type of adult brain tumor with a poor prognosis. Temozolomide (TMZ), a DNA alkylating agent, has been widely used as an effective first-line chemotherapeutic agent for the treatment of GBM patients. The efficacy of TMZ in GBM depends on the absence of the DNA repair protein MGMT which reverses the DNA damage induced by TMZ. The MGMT promoter is hypermethylated in about 45% of GBMs, resulting in lack of MGMT expression and increased responsiveness to TMZ. TMZ is less effective in MGMT unmethylated GBMs. We propose that EGFR inhibition downregulates MGMT and sensitizes glioma cells to TMZ and a combination of pretreatment with erlotinib followed by TMZ could be a useful therapeutic approach in MGMT expressing GBMs. As our experimental model, we used multiple MGMT unmethylated lines from the Mayo Clinic patient derived xenografts (PDXs) panel. Our data demonstrate that exposure of cells to erlotinib for 48h results in downregulation of MGMT at the mRNA and protein level. Additionally, EGFR inhibition activates the AP-1 transcription factor, and overexpression of AP-1 components Fos and Jun results in decreased MGMT expression in TMZ resistant PDXs, suggesting that AP-1 acts as a transcriptional repressor of MGMT. We further identified that the mice implanted with TMZ resistant PDXs pretreated with afatinib followed by TMZ treatment survived longer compared to those treated with TMZ alone. Thus, the use of EGFR inhibition may enhance the sensitivity of MGMT unmethylated GBMs to TMZ.

Abstract Glioblastoma (GBM), the most common primary brain tumor in adults, remains uniformly fatal due to the lack of effective targeted therapies for this aggressive malignancy. Genomic amplification of epidermal growth factor receptor (EGFR) occurs in 40-60% of primary GBM. Half of all EGFR-amplified cases of GBM also harbor EGFRvIII, a constitutively active, truncated variant of EGFR. The incidence of EGFR alterations in GBM makes inhibition of EGFR/EGFRvIII an attractive therapeutic approach. Depatuxizumab mafodotin (ABT-414) is an antibody-drug conjugate comprised of ABT-806, a mAB against EGFR, and a cytotoxic payload (monomethyl auristatin F). ABT-414 therapy initially showed promising results as an EGFRvIII therapy in vivo and in vitro; however, ABT414 failed to provide a survival benefit in phase I/II clinical trials, potentially due to therapeutic resistance. In this study, we seek to investigate the mechanisms of resistance to ABT-414 by performing whole exome, transcriptome and single cell RNA seq on ABT-414 resistant GBM PDX tumors. Our data showed an enrichment of mutations unique to the ABT-414 resistant tumors, including a point mutation (S466I) in TEK/TIE2 transmembrane angiopoietin receptor. In vitro expression of TEK S466I in GBM cells showed an increase in activation of ERK and STAT3 and increased TEK/TIE2 immunoprecipitation with EGFR compared to the wild-type TEK receptor. Furthermore, gene ontology analysis reveals that ABT-414-treated flank tumors exhibit increased activation of extracellular matrix organization and CNS developmental processes compared to flank tumors in the control treatment groups. ABT-414-treated tumors also demonstrate increased expression of inhibitor of differentiation (ID)1 and ID3, associated with stem-like phenotype in glioblastoma. Taken together, our data indicate that resistance to ABT-414 is mediated by both de novo mutations not detected in the parent tumor and adaptive dysregulation of pathways which may lead to dedifferentiation and therapeutic resistance.