Eckel‐Passow, Jeanette E.

Abstract Signaling networks and the protein interactions that define them are of great interest, but techniques to assess these networks in unmanipulated primary human cells are limited. We have developed and applied a novel technological and analytical platform to measure proteins in shared complexes detected by exposed surface epitopes (PiSCES). Immunoprecipitation reactions are performed in multiplex using microsphere beads to measure 20+ proteins and 210+ distinct pair-wise protein associations that are important for T cell antigen receptor signaling. Jurkat T cells stimulated with superantigen generated an extensive PiSCES signature using this method and provided a dataset from which we optimized an analytical package. The system is currently being utilized to address the hypothesis that T cells isolated from skin lesions of autoimmune alopecia areta (AA) patients will have a unique PiSCES signature compared to those isolated from non-AA donors. To investigate this, we have isolated non-engineered primary cells from AA patient skin lesions and healthy controls, and have begun to identify differences in their PiSCES signatures, either directly after isolation or after stimulation with anti-CD3 and anti-CD28. We conclude that this novel PiSCES approach is capable of generating protein-protein interaction signatures that provide a unique view of TCR signalosome activity solicited from physiologic non-engineered primary human patient samples.

Abstract BACKGROUND The topologically associated domain (TAD) on 8q24 surrounding MYC is important for many types of cancer. The region is also important in the formation of IDH-mutant glioma; the risk allele rs55705857 being associated with the development of these tumors. The rs55705857 risk allele significantly increased the formation of tumors in an IDH-mutant mouse model. Cerebral organoids have been found to recapitulate the early development of the brain. We hypothesized that the rs55705857 risk allele may alter the phenotype of cerebral organoids. METHODS Isogenic induced pluripotent stem cells (iPSCs) were developed with (n=3) and without (n=2) the rs55705857 risk allele from the parental cell line PGP-1 (GM23338). These cells were cultured and differentiated into unpatterned cerebral organoids and observed throughout differentiation. Organoids were fixed, sectioned, and stained by H&E and immunofluorescence. RESULTS Organoids containing the risk allele were smaller throughout development, starting at the embryoid body stage and continuing up to over 75 days in culture, compared to two isogenic non-risk lines. At 4 weeks the risk allele containing organoids also contained a larger number of small rosette-like neuroepithelia, instead of the more continuous neuroepithelia observed in the non-risk allele organoids. At 75 days, the organoids with the risk allele maintained a larger proportion of Sox2 positive cells, which are enriched in the tight clusters of small cells with round nuclei. CONCLUSIONS The risk allele rs55705857 in the MYC TAD at 8q24 is altering the development of unpatterned cerebral organoids. This difference is visible as early as embryoid body development and persists through neuroepithelial development. Further studies of these differences may help our understanding of how rs55705857 accelerates IDH-mutant glioma development.

Abstract Missing MRI sequences are common in radiology due to differences in protocols, time constraints, and image artifacts. Missing sequences are a nuisance for subsequent algorithms, such as automated segmentation algorithms requiring multiple MRI sequences (e.g., HD-GLIO, which requires T2, T1, post-contrast T1 (T1c), and FLAIR). Accurate tumor segmentation is a fundamental step in extracting radiomics analysis to use, for example, to predict the IDH status of glioma or their overall survival. We validated a conditional (pix2pix) and super-resolution GAN (SOUP-GAN) for imputing MRI sequences on 285 adult glioma with complete MRI sequences available. We simulated missing T1 sequences and subsequently imputed them from T1c using pix2pix and from sagittal T1 using SOUP-GAN. Using HD-GLIO, we obtained the true segmentations from the complete MRI sequences. HD-GLIO was also used after replacing the original T1 sequence with the one imputed with pix2pix and SOUP-GAN. Thus, there were three sets of segmentations for each subject. We compared T2 and T1c radiomics features extracted with Pyradiomics using segmentations obtained with complete data with features extracted from segmentation obtained with the imputed T1 sequences using intraclass correlation coefficient (ICC) and Bland-Altman (BA) analysis. Median (interquartile range) ICC for shape features extracted from T2 and T1c were 0.82(0.76-0.92) using pix2pix and 0.89(0.86-0.94) using SOUP-GAN. Median ICC for first-order-statistics features were 0.90(0.83-0.93) for T2 and 0.90(0.88-0.92) for T1c using pix2pix, and 0.61(0.22-0.82) for T2 and 0.42(0.34-0.57) for T1c using SOUP-GAN. Median ICC for texture features were 0.86(0.82-0.87) for T2 and 0.87(0.79-0.90) for T1c using pix2pix, and 0.69(0.47-0.81) for T2 and 0.76(0.65-0.83) for T1c using SOUP-GAN. We show that T2 and T1c shape features (e.g., volumetric analysis) can be adequately extracted from segmentations obtained with an algorithm requiring multiple MRI sequences using T1 sequences imputed with GANs; however, the effect of imputation differed for first-order-statistics and texture features.

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase ( IDH) –mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1 R132H -driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

Abstract Background EGFR targeting antibody-drug conjugates (ADCs) are highly effective against EGFR-amplified tumors, but poor distribution across the blood–brain barrier (BBB) limits their efficacy in glioblastoma (GBM) when administered systemically. We studied whether convection-enhanced delivery (CED) can be used to safely infuse ADCs into orthotopic patient-derived xenograft (PDX) models of EGFRvIII mutant GBM. Methods The efficacy of the EGFR-targeted ADCs depatuxizumab mafodotin (Depatux-M) and Serclutamab talirine (Ser-T) was evaluated in vitro and in vivo. CED was performed in nontumor and tumor-bearing mice. Immunostaining was used to evaluate ADC distribution, pharmacodynamic effects, and normal cell toxicity. Results Dose-finding studies in orthotopic GBM6 identified single infusion of 2 μg Ser-T and 60 μg Depatux-M as safe and effective associated with extended survival prolongation (>300 days and 95 days, respectively). However, with serial infusions every 21 days, four Ser-T doses controlled tumor growth but was associated with lethal toxicity approximately 7 days after the final infusion. Limiting dosing to two infusions in GBM108 provided profound median survival extension of over 200 days. In contrast, four Depatux-M CED doses were well tolerated and significantly extended survival in both GBM6 (158 days) and GBM108 (310 days). In a toxicity analysis, Ser-T resulted in a profound loss in NeuN+ cells and markedly elevated GFAP staining, while Depatux-M was associated only with modest elevation in GFAP staining. Conclusion CED of Depatux-M is well tolerated and results in extended survival in orthotopic GBM PDXs. In contrast, CED of Ser-T was associated with a much narrower therapeutic window.

Abstract The Mayo Clinic collection of patient-derived xenograft (PDXs) has been developed from the direct implantation of glioblastoma (GBM) samples into immunodeficient mice. Over 105 models have been extensively characterized with regards to histology, invasion, flank and orthotopic growth rates, and molecular profiling. Evaluation of orthotopic tumors demonstrated infiltration of tumor cells into the brain parenchymal in 90% of models with contralateral hemisphere involvement in 64%. Extensive molecular profiling confirmed the PDX collection captures the genetic heterogeneity of GBM, with the majority of typical molecular alterations seen at a frequency similar to the TCGA including gain of chromosome 7 and loss of chromosome 10. TERT promoter mutations (86%) and homozygous deletion of CDKN2A (70%) were the most frequent. Additional alterations in the p53 pathway included TP53 mutations (36%) and MDM2/4 amplification (10%/2%). IDH mutations were found in three models while MGMT promoter methylation was observed in 45% of PDXs. The available molecular sequencing results are viewable on cBioPortal. These genetically diverse models are extremely useful for biomarker discovery and efficacy evaluations. Using an innovative 1x1x1 PDX preclinical trial design, we are establishing a workflow to evaluate control and drug treatment in an individual animal across PDX lines in the flank. As our first use of this strategy, a small molecule MDM2 inhibitor was evaluated in 17 PDX lines with variable MDM2 and TP53 statuses. Tumors harboring MDM2 amplification without TP53 mutation (n=5) showed profound response, with 4 PDXs showing a complete response that lasted >150 days. Response in TP53 wild-type tumors lacking MDM2 amplification was variable, with a 1.9 to 4.8 fold longer time to endpoint (mean 3; median 2.7). A more traditionally powered intracranial efficacy study performed with one MDM2 amplified (GBM108) and two non-amplified (GBM14, GBM10) PDX lines confirmed these findings of reduced and more variable response to therapy in MDM2 non-amplified TP53 wild-type tumors. Future studies will aim to examine novel biomarkers indicative of therapeutic response to better understand molecular features associated with MDM2 inhibitor efficacy. With over 20 years of experience, our group has extensive expertise in development of clinically relevant study designs focused on examining efficacy, tolerability, and pharmacokinetic/pharmacodynamic assessments. Our diverse collection of clinically relevant GBM PDX models provides a highly sought-after resource which has been utilized by the pharmaceutical community with 58 working relationships to date (26 contracts, 32 collaborations). Through these collaborations, we aim to expand the use of our highly characterized GBM PDX panel for biomarker discovery using similar strategies. Citation Format: Danielle M. Burgenske, Ann C. Mladek, Shulan Tian, Mark A. Schroeder, Zeng Hu, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Rachael Vaubel, Jann N. Sarkaria. Evaluation of novel therapeutics using the Mayo Clinic GBM Patient-Derived Xenograft (PDX) collection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4001.

Abstract Knowledge about inherited and acquired genetics of adult diffuse glioma has expanded significantly over the past decade. Genomewide association studies (GWAS) stratified by histologic subtype identified six germline variants that were associated specifically with glioblastoma (GBM) and 12 that were associated with lower grade glioma. A GWAS performed using the 2016 WHO criteria, stratifying patients by IDH mutation and 1p/19q codeletion (as well as TERT promoter mutation), discovered that many of the known variants are associated with specific WHO glioma subtypes. In addition, the GWAS stratified by molecular group identified two additional novel regions: variants in D2HGDH that were associated with tumors that had an IDH mutation and a variant near FAM20C that was associated with tumors that had both IDH mutation and 1p/19q codeletion. The results of these germline associations have been used to calculate polygenic risk scores, from which to estimate relative and absolute risk of overall glioma and risk of specific glioma subtypes. We will review the concept of polygenic risk models and their potential clinical utility, as well as discuss the published adult diffuse glioma polygenic risk models. To date, these prior genetic studies have been done on European populations. Using the published glioma polygenic risk model, we show that the genetic associations published to date do not generalize across genetic ancestries, demonstrating that genetic studies need to be done on more diverse populations.

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.

Abstract Abnormalities on brain MRI may be identified in ~15% of individuals aged 50-66. It can be difficult to discriminate glioma, CNS lymphoma, Inflammatory Demyelinating Disease (CNSIDD) and solitary metastasis, and misdiagnosis may expose patients to unnecessary anxiety, surgery, or radiotherapy. CNS lymphoma requires only biopsy, solitary metastasis may be resected and radiated or radiated empirically, and high-grade glioma requires maximal safe resection followed by chemoradiation. CNSIDD should only be biopsied when diagnostic uncertainty requires it, and resection and radiotherapy are unnecessary, introducing unwarranted morbidity. Polygenic risk models can identify patients at the highest risk of developing glioma; we hypothesized that these polygenic models could help with differential diagnosis of indeterminate brain lesions. We also hypothesized that race would be an important contributing factor in the models. In the initial discovery and validation European (EUR) cohorts the mean probability of glioma for IDHmut non-codeleted glioma was 0.55 and 0.52, respectively, and in healthy controls was 0.19 and 0.21, respectively. To further evaluate sensitivity, we analyzed additional genotype data from 867 gliomas (764 EUR, 54 AFR, 24 AMR, 15 EAS, 10 SAS) from The Cancer Genome Atlas (TCGA). Across 764 EUR IDHmut non-codeleted glioma, the mean probability 0.53, whereas across 54 AFR and 24 AMR the mean was 0.22 and 0.32, respectively. To evaluate specificity, we analyzed 3200 TCGA patients with primary tumor types that commonly metastasize to the brain (2676 EUR, 365 AFR, 46 AMR, 95 EAS, 18 SAS), and 236 AFR healthy controls. For patients with non-CNS primary tumors, the mean probability ranged from 0.09-0.18 for EUR and 0.04-0.07 for AFR. For AFR healthy controls, the mean was 0.05. Overall, race is a significant factor for polygenic risk models. Further work entails evaluating polygenic risk models in IDHwt glioma, CNS lymphoma and CNSIDD cohorts as well as in additional races.

Abstract BACKGROUND Determination of the causation of germline single nucleotide polymorphisms (SNPs) located in non-coding regions of the genome is challenging. The genomic region of 8q24 has been identified as important in many kinds of cancer, linked to a topologically associated domain (TAD) encompassing MYC; this TAD contains a GWAS SNP (rs55705857) associated with IDH-mutant glioma. METHODS Germline genotyping data from 622 IDH-mutant glioma and 668 controls were used to fine map the rs55705857 locus by detailed haplotype analysis. Chromatin immunoprecipitation sequencing (ChIP-seq) of histone markers H3K4me1, H3K4me3, H3K27ac and H3K36me3 was performed on normal brain samples (n=8) and human glioma samples (n=11 IDH-wt and 52 IDH-mut). RNAseq from 9 normal and 83 brain tumors (n=26 IDH-wt and 55 IDH-mut) were used to assess differential gene expression. RESULTS Fine-mapping identified rs55705857 SNP as the most likely causative allele (OR=8.69; p<0.001) within 8q24 for the development of IDH-mutant glioma. At rs55705857, both H3K27ac and H3K4me1 in IDH-mutant vs IDH-wt tumors were increased 3.05- and 1.58-fold, respectively (DiffBind; p=5.81×10-7 and p=2.31×10-3). ChromHMM analysis of the marks indicated that promoter and enhancer functions were significantly increased, and the activity broadened at rs55705857 in IDH-mut gliomas compared to IDH-wt tumors and normal brain samples. This enhancement correlated with significant increased MYC expression in IDH-mut gliomas (p=3.1×10-13), as well as alterations of Myc signaling targets. Publicly available ATACseq, ChIPseq and long-range DNA interaction data demonstrated that the rs55705857 locus is open and interacts with the MYC promoter. CONCLUSIONS Fine-mapping of the 8q24 locus provided strong evidence that rs55705857 is the causative 8q24 locus associated with IDH-mut glioma. Functional experiments suggest that IDH mutation facilitates rs55705857 interaction with MYC to alter downstream MYC targets.