Citations

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.

Abstract Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in astrocytic glioma and are frequently coupled with TP53 and ATRX mutations. Collectively, these alterations cause genomic instability leading to high basal DNA double strand breaks (DSBs). Understanding how IDH/TP53/ATRX mutant cells process endogenous DSBs may help exploit inhibitors of DNA damage response (DDR) for the treatment of patients with IDH mutant gliomas. Through systematic effort to uncover the mechanisms involved in repair of endogenous DSBs in IDH1/2 mutant GBMs, we have discovered that high basal phosphorylated DNA-PK (p-DNA-PK) was characteristic of an IDH1/TP53/ATRX mutant GBM164 patient derived xenograft (PDX) but not in another IDH1 mutant GBM196 PDX. Immunofluorescence (IF) studies in patient specimen from which GBM164 was derived showed that p-DNA-PK co-localized with g-H2AX, 53BP1 or H4K20me2 (but not p-RPA) the known surrogates of DSBs. In contrast, p-DNA-PK was absent in the patient specimen from which GBM196 was derived, which otherwise had equally intense g-H2AX immunostaining colocalized with p-RPA. An independent IF study involving 11 IDH1 wild-type (WT) and 11 IDH1 mutant GBM patient samples, the p-DNA-PK was observed in 3 (27%) of 11 IDH1 mutant samples while IDH1 WT tumors were negative for p-DNA-PK. A telomere specific fluorescence in situ hybridization (Tel-FISH) confirmed elevated alternative lengthening of telomere (ALT) activity in GBM196 (but not in GBM164) indicative of HR proficiency. Consistently, HR related genes, including BRCA1 and MRE11A, were found upregulated in ALT-positive GBM196 as compared to those in GBM164. Interestingly, ALT+ GBM196 cells were highly vulnerable to inhibitors of ATM and ATR pathways. In conclusion, IDH1/TP53/ATRX mutant gliomas can be subdivided into HR-mediated ALT-positive group, which repairs the endogenous DSBs by HR (e.g. GBM196) and an ALT-negative/p-DNA-PK group, which repairs DSBs by c-NHEJ (e.g. GBM164) and this subdivision can be developed as a prescient biomarker of sensitivity to DDR inhibitors.

Abstract Choosing effective chemotherapies for intravenous delivery to brain tumors is challenging, especially given the protective nature of the blood brain barrier (BBB). Connecting drug distribution to non-invasive, pre-surgical magnetic resonance imaging (MRI) could allow for predictive insight into drug distribution. In a previous study, we found that T2Gd images were predictive of a low BBB penetrant drug (Cefazolin), and FLAIR images were predictive of a high BBB penetrant drug (Levetiracetam). While these results are promising, we further seek to explore how advanced MRI sequences might inform image-based models of drug distribution. Prior to surgery, we acquired advanced dynamic contrast enhanced (DCE) and diffusion weighted imaging (DWI) MRI sequences for eight brain tumor patients (7 gliomas and 1 metastatic adenocarcinoma) in addition to the anatomic MRIs. All resulting quantitative maps and acquired images were co-registered. Prior to incision, patients received injections of cefazolin and levetiracetam. Next, multiple blood samples and biopsies were collected during surgery. Biopsies and plasma samples were analyzed for drug concentration using liquid chromatography mass spectrometry (LCMS), and biopsy drug levels were reported as Brain-Plasma Ratio (BPR). Mean image intensity was extracted from a 15x15 voxel window surrounding the biopsy location. We performed linear regression analyses to determine which combination of images were predictive of BPR. We found that considering quantitative imaging improved our initial ability to predict BPR for both drugs. For cefazolin, the third diffusion tensor eigenvalue (L3) map was significantly correlated with BPR (p< 0.001, R2= 0.36). For levetiracetam, the best model consisted of a combination of images and maps with the L3 map and the isotropic diffusion map (P) being the most influential (p= 0.001, R2= 0.63). Advanced MRI-based modeling is a promising tool for forecasting drug distribution in brain tumors and could be of great importance for understanding efficacy and selecting therapeutic strategies.

Abstract Glioblastoma (GBM) remains the most common adult brain cancer, with a dismal average patient survival of less than two years. No new treatments have been approved for GBM since the introduction of the alkylating agent temozolomide in 2005. Even then, temozolomide treatment only increases the average survival of GBM patients by a few months. Thus, novel therapeutic options are direly needed. The aurora kinases A and B are targetable and overexpressed in GBM, and their expression is highly correlated with patient survival outcomes. Our lab has found that small molecule aurora kinase inhibition reduces GBM tumor growth in vitro and in vivo, however, eventually tumors still grow. Computational analysis integrating compound transcriptional response signatures from the LINCS L1000 dataset with the single-cell RNA-sequencing data of patient GBM tumors resected at the University of Miami predicts that aurora inhibition targets a subset of cells present within any GBM tumor. Results of in vivo single-cell perturbation experiments with the aurora kinase inhibitor alisertib coincide with our predictions and reveal a cellular transcriptional phenotype resistant to aurora kinase inhibition, characterized by a mesenchymal expression program. We find that small molecules that are predicted to target different cell populations from alisertib, including this resistant mesenchymal population, synergize with alisertib to kill GBM cells. As a whole, we have identified the cellular population resistant to aurora kinase inhibition and have developed an analytical framework that identifies synergistic small molecule combinations by identifying compounds that target transcriptionally distinct cellular populations within GBM tumors.

Abstract Purpose: The epigenetic mechanisms involved in transcriptional regulation leading to malignant phenotype in gliomas remains poorly understood. Topoisomerase IIB (TOP2B), an enzyme that decoils and releases torsional forces in DNA, is overexpressed in a subset of gliomas. Therefore, we investigated its role in epigenetic regulation in these tumors. Experimental Design: To investigate the role of TOP2B in epigenetic regulation in gliomas, we performed paired chromatin immunoprecipitation sequencing for TOP2B and RNA-sequencing analysis of glioma cell lines with and without TOP2B inhibition and in human glioma specimens. These experiments were complemented with assay for transposase-accessible chromatin using sequencing, gene silencing, and mouse xenograft experiments to investigate the function of TOP2B and its role in glioma phenotypes. Results: We discovered that TOP2B modulates transcription of multiple oncogenes in human gliomas. TOP2B regulated transcription only at sites where it was enzymatically active, but not at all native binding sites. In particular, TOP2B activity localized in enhancers, promoters, and introns of PDGFRA and MYC, facilitating their expression. TOP2B levels and genomic localization was associated with PDGFRA and MYC expression across glioma specimens, which was not seen in nontumoral human brain tissue. In vivo, TOP2B knockdown of human glioma intracranial implants prolonged survival and downregulated PDGFRA. Conclusions: Our results indicate that TOP2B activity exerts a pleiotropic role in transcriptional regulation of oncogenes in a subset of gliomas promoting a proliferative phenotype.

Abstract 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 Wee1 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 the 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 preclinical 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.