An in vivo model of glioblastoma radiation resistance identifies long noncoding RNAs and targetable kinases.

Key molecular regulators of acquired radiation resistance in recurrent glioblastoma (GBM) are largely unknown, with a dearth of accurate preclinical models. To address this, we generated 8 GBM patient-derived xenograft (PDX) models of acquired radiation therapy-selected (RTS) resistance compared with same-patient, treatment-naive (radiation-sensitive, unselected; RTU) PDXs. These likely unique models mimic the longitudinal evolution of patient recurrent tumors following serial radiation therapy. Indeed, while whole-exome sequencing showed retention of major genomic alterations in the RTS lines, we did detect a chromosome 12q14 amplification that was associated with clinical GBM recurrence in 2 RTS models. A potentially novel bioinformatics pipeline was applied to analyze phenotypic, transcriptomic, and kinomic alterations, which identified long noncoding RNAs (lncRNAs) and targetable, PDX-specific kinases. We observed differential transcriptional enrichment of DNA damage repair pathways in our RTS models, which correlated with several lncRNAs. Global kinomic profiling separated RTU and RTS models, but pairwise analyses indicated that there are multiple molecular routes to acquired radiation resistance. RTS model-specific kinases were identified and targeted with clinically relevant small molecule inhibitors. This cohort of in vivo RTS patient-derived models will enable future preclinical therapeutic testing to help overcome the treatment resistance seen in patients with GBM.

N-cadherin upregulation mediates adaptive radioresistance in glioblastoma.

Glioblastoma (GBM) is composed of heterogeneous tumor cell populations, including those with stem cell properties, termed glioma stem cells (GSCs). GSCs are innately less radiation sensitive than the tumor bulk and are believed to drive GBM formation and recurrence after repeated irradiation. However, it is unclear how GSCs adapt to escape the toxicity of repeated irradiation used in clinical practice. To identify important mediators of adaptive radioresistance in GBM, we generated radioresistant human and mouse GSCs by exposing them to repeat cycles of irradiation. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by a reduction in cell proliferation and an increase in cell-cell adhesion and N-cadherin expression. Increasing N-cadherin expression rendered parental GSCs radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs lost their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of ß-catenin at the cell surface, which suppressed Wnt/ß-catenin proliferative signaling, reduced neural differentiation, and protected against apoptosis through Clusterin secretion. N-cadherin upregulation was induced by radiation-induced IGF1 secretion, and the radiation resistance phenotype could be reverted with picropodophyllin, a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor, supporting clinical translation.

The impact of provider training on patient education for pediatric early food introduction.

BACKGROUND: Pediatric food allergies (FAs) present significant health and economic problems. Currently, there are no cures for FAs. Recent studies suggest that early introduction (EI), between 4 and 6 months of age, of commonly allergenic foods (CAFs) may reduce the risk of developing FAs. This contradicts the current standard of care, food avoidance. LOCAL PROBLEM: A federally qualified health center saw 894 patients aged 0-24 months during a 12-month period with only 18.9% receiving nutrition education. New dietary recommendations to prevent FA were not in place. METHODS: A retrospective chart review was used to evaluate use of an order set with patient education on EI to CAFs in the electronic medical record (EMR). INTERVENTIONS: Providers attended training on EI to CAFs and use of the EMR order set. Data were collected on the use of the order set over a 3-month period. RESULTS: Provider training significantly improved knowledge of FA as well as EI guidelines. After 3 months of implementation, 25.95% of eligible encounters contained the EI order set; 52% of patients received the order set during the measurement period. In the impact population, patients 4-12 months of age, 74.55% of patients received the order set. CONCLUSIONS: Evidence-based clinical content in EMR order sets coupled with provider training ensure clinical decision support in identifying, monitoring, and optimizing quality care standards.

Cancer Explant Models.

Overcoming the challenges of understanding and treating cancer requires reliable patient-derived models of cancer (PDMCs). For decades, cancer research and therapeutic development relied primarily on cancer cell lines because of their prevalence, reproducibility, and simplicity to maintain. However, findings from research conducted in cell lines are rarely recapitulated in vivo and seldom directly translatable to patients. The tumor microenvironment (TME), tumor-stromal interactions, and associations with host immune cells produce profound changes in tumor phenotype and complexity not captured in traditional monolayer cell culture. In this chapter, we present various cancer explant models and discuss their applicability based on specific research aims. We discuss the appropriateness of these models for basic science questions, drug screening/development, and for personalized, precision medicine. We also consider logistical factors such as resource cost, technical difficulty, and accessibility. We finish this chapter with a practical guide intended to help the reader select the cancer explant model system(s) that best address their research aims.

Exploring the Roles of lncRNAs in GBM Pathophysiology and Their Therapeutic Potential.

Glioblastoma (GBM) remains the most devastating primary central nervous system malignancy with a median survival of around 15 months. The past decades of research have not yielded significant advancements in the treatment of GBM. In that same time, a novel class of molecules, long non-coding RNAs (lncRNAs), has been found to play a multitude of roles in cancer and normal biology. The increased accessibility of next generation sequencing technologies and the advent of lncRNA-specific microarrays have facilitated the study of lncRNA etiology. Molecular and computational methods can be applied to predict lncRNA function. LncRNAs can serve as molecular decoys, scaffolds, super-enhancers, or repressors. These molecules can serve as phenotypic switches for GBM cells at the expression and/or epigenetic levels. LncRNAs can affect stemness/differentiation, proliferation, invasion, survival, DNA damage response, and chromatin dynamics. Aberrant expression of these transcripts may facilitate therapy resistance, leading to tumor recurrence. LncRNAs could serve as novel theragnostic or prognostic biomarkers in GBM and other cancers. RNA-based therapeutics may also be employed to target lncRNAs as a novel route of treatment for primary or recurrent GBM. In this review, we explore the roles of lncRNAs in GBM pathophysiology and posit their novel therapeutic potential for GBM.

A Novel Assay for Profiling GBM Cancer Model Heterogeneity and Drug Screening.

Accurate patient-derived models of cancer are needed for profiling the disease and for testing therapeutics. These models must not only be accurate, but also suitable for high-throughput screening and analysis. Here we compare two derivative cancer models, microtumors and spheroids, to the gold standard model of patient-derived orthotopic xenografts (PDX) in glioblastoma multiforme (GBM). To compare these models, we constructed a custom NanoString panel of 350 genes relevant to GBM biology. This custom assay includes 16 GBM-specific gene signatures including a novel GBM subtyping signature. We profiled 11 GBM-PDX with matched orthotopic cells, derived microtumors, and derived spheroids using the custom NanoString assay. In parallel, these derivative models underwent drug sensitivity screening. We found that expression of certain genes were dependent on the cancer model while others were model-independent. These model-independent genes can be used in profiling tumor-specific biology and in gauging therapeutic response. It remains to be seen whether or not cancer model-specific genes may be directly or indirectly, through changes to tumor microenvironment, manipulated to improve the concordance of in vitro derivative models with in vivo models yielding better prediction of therapeutic response.

Extra-coding RNAs regulate neuronal DNA methylation dynamics.

Epigenetic mechanisms such as DNA methylation are essential regulators of the function and information storage capacity of neurons. DNA methylation is highly dynamic in the developing and adult brain, and is actively regulated by neuronal activity and behavioural experiences. However, it is presently unclear how methylation status at individual genes is targeted for modification. Here, we report that extra-coding RNAs (ecRNAs) interact with DNA methyltransferases and regulate neuronal DNA methylation. Expression of ecRNA species is associated with gene promoter hypomethylation, is altered by neuronal activity, and is overrepresented at genes involved in neuronal function. Knockdown of the Fos ecRNA locus results in gene hypermethylation and mRNA silencing, and hippocampal expression of Fos ecRNA is required for long-term fear memory formation in rats. These results suggest that ecRNAs are fundamental regulators of DNA methylation patterns in neuronal systems, and reveal a promising avenue for therapeutic targeting in neuropsychiatric disease states.