Long non-coding RNAs (lncRNAs) are a heterogenous population of largely evolutionary preserved transcripts, over 200nt in length which play important roles in human health and disease. These versatile and numerous transcripts can possess RNA:Protein, RNA:RNA, and RNA:DNA binding potential. Some lncRNAs also encode short peptides which can have biological significance. Many, if not a plurality of known lncRNAs, are expressed in the brain, believed to help encode complex information and facilitate various neurologic processes. In fact, lncRNA expression appears to be positively correlated with organism and organ complexity in stark contrast to coding genes. Glioblastoma (GBM) is the most common primary brain tumor with a dismal prognosis. The 5-year survival rate for GBM is around 7% with a median survival of between 14-16mos. GBM standard of care has not changed much in past decades and there is a need for new therapeutic strategies. In this review we focus on six lncRNAs of significance to cancer highlighting their potential applications in GBM treatment. These lncRNAs are MALAT1/NEAT2, HOTAIR, ZFAS1, SOX2-OT, CASC19/PCAT2, and SAMMSON. Some of these lncRNAs are well characterized across numerous cancers, but most are poorly characterized in the context of GBM or glioma. Many lncRNAs, including those highlighted herein, have been implicated in therapy resistance including chemoresistance and radiation resistance. Some of these transcripts can be found in patient serum. lncRNAs then present potential as theragnostic, prognostic, and diagnostic potential for GBM and other cancers. They may also represent therapeutic opportunities directly and as concomitant therapies to address resistance mechanisms.

PURPOSE:Current radiotherapy (RT) in glioblastoma (GBM) is delivered as constant dose fractions (CDF), which do not account for intratumoral-heterogeneity and radio-selection in GBM. These factors contribute to differential treatment response complicating the therapeutic efficacy of this principle. Our study aims to investigate an alternative dosing strategy to overcome radio-resistance using a novel longitudinal radiation cytotoxicity assay. METHODS:Theoretical In-silico mathematical assumptions were combined with an in-vitro experimental strategy to investigate alternative radiation regimens. Patient-derived xenograft (PDX) brain tumor-initiating cells (BTICs) with differential radiation-sensitivities were tested individually with sham control and three regimens of the same nominal and average dose of 16 Gy (over four fractions), but with altered doses per fraction. Fractions were delivered conventionally (CDF: 4, 4, 4, 4 Gy), or as dynamic dose fractions (DDF) "ramped down" (RD: 7, 5, 3, 1 Gy), or DDF "ramped up" (RU: 1, 3, 5, 7 Gy), every 4 days. Interfraction-longitudinal data were collected by imaging cells every 5 days, and endpoint viability was taken on day 20. RESULTS:The proposed method of radiosensitivity assessment allows for longitudinal-interfraction investigation in addition to endpoint analysis. Delivering four-fraction doses in an RD manner proves to be most effective at overcoming acquired radiation resistance in BTICs (Relative cell viability: CDF vs. RD: P < 0.0001; Surviving fraction: CDF: vs. RD: P < 0.0001). CONCLUSIONS:Using in-silico cytotoxicity prediction modeling and an altered radiosensitivity assessment, we show DDF-RD is effective at inducing cytotoxicity in three BTIC lines with differential radiosensitivity.

Abstract Background: Glioblastoma (GBM) has a median survival of <2 years and generally recurs within 6 months of treatment due to the development of chemo- and radiotherapy (RT) resistance. Tunneling nanotubes (TNTs) serve as intercellular conduits for establishing robust, tumor-promoting networks within the hypoxic tumor microenvironment. TNTs are provoked by hypoxia and can passage organelles like mitochondria, i.e., between astrocytes and stem-like brain tumor-initiating cells (BTICs). This is theorized to expand resistance properties to other cell types, as well as facilitate metabolic rescue in damaged cancer cells. We investigated the mitochondrial uptake (MU) abilities of RT-sensitive or RT-resistant BTICs from normal human astrocytes (NHAs) under hypoxic conditions. Methods: We used a Cytation5 Cell Imager to quantify MU by BTICs from NHAs in direct contact under normal (20%) or hypoxic oxygen tensions (5%). We obtained RT-sensitive (JX14P) patient-derived xenograft BTICs and generated a paired acquired-resistant (JX14P-RT) line. This was achieved by implanting primary tumors into flanks of athymic nude mice and serially treating with 6 fractions of 2Gy over 14 days for multiple passages until the median doubling time was halved. Cells were plated at a 1:1 ratio on Geltrex for 18h and exposed to 5% or 20% oxygen in serum-free media. NHAs were pre-labeled with a GFP-mitochondria tracker and BTICs were infected with a mCherry lentivirus. BTIC-MU was determined by quantifying double-positive cells in whole-well images. Viability was determined using CellTiterGlo, n = 4. Results: Time-lapse imaging revealed GFP-mitochondria transfer from NHAs to BTIC cells via TNTs stimulated by hypoxic conditions. We measured overall MU and cell viability in both BTIC lines. JX14P co-cultured with NHAs trended toward an increased MU (cell fraction) in hypoxia (Hyp) compared to Normoxia (Norm) (Norm = 32.61 ± 13, Hyp = 44.83 ± 5, P>0.167). JX14P exhibit higher cell viability (RLU) in hypoxia when mono- or co-cultured with NHAs (Mono: Norm = 10275 ± 901, Hyp = 12599 ± 579, P<0.0039, Co: Norm = 5415 ± 664, Hyp = 8341 ± 700, P<0.0001). JX14P-RT co-cultured with NHAs show a trend for more MU in hypoxia compared to Normoxia (Norm = 25.90 ± 12, Hyp = 38.12 ± 12, P>0.167). Compared to monoculture, JX14PRT exhibits higher cell viability in co-culture with NHAs under hypoxia (Mono: Norm = 9721 ± 255, Hyp = 10011 ± 1462, P>0.998, Co: Norm = 4928 ± 664, Hyp = 7805 ± 944, P<0.005). Conclusions: RT-sensitive or -resistant BTICs cocultured with NHAs exhibit increased cell viability under acute hypoxia compared to Normoxia with a trend toward increased MU. Results indicate a potential protective effect following direct interaction with NHAs under hypoxia. We are further exploring metabolic changes in each cell type following mitochondrial exchange. Citation Format: Lauren C. Nassour-Caswell, Nicholas J. Eustace, Christian T. Stackhouse, Hasan Alrefai, Patricia H. Hicks, Taylor L. Schanel, Joshua C. Anderson, Andee M. Beierle, Christopher D. Willey. Glioblastoma brain tumor-initiating cells are protected from hypoxia when co-cultured with normal human astrocytes revealing a potential role for mitochondrial transfer via tunneling nanotubes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1273.

Purpose/Objective(s)Historically, radiotherapy fractions for Glioblastoma (GBM) are delivered in equal doses over a given number of days, though this regimen has not changed in the past century and does not accommodate for the development of radioresistance. Using mathematical oncological simulations, we propose that ramping up dose can overcome radioresistance in patient-derived xenograft (PDX) GBM cells more sufficiently than the same dose delivered in equal fractions.Materials/MethodsWe generated a radiation-sensitive (JX14P) and -resistant (JX14P-RT) PDX GBM cell line by implanting primary tumors into the flank of athymic nude mice. To achieve radioresistance, we treated these mice with 6 fractions of 2 Gy of radiation over 14 days. In silico simulations utilizing the radiosensitivity parameters based on the linear-quadratic model and JX14P and JX14P-RT doubling time, suggested a total regimen of 16 Gy, with an average of 4 Gy/fx, delivered in fractions of 1 Gy, 3 Gy, 5 Gy, or 7 Gy (Ramp-Up, "RU") would result in a smaller surviving fraction than the equivalent total dose of 4 Gy, 4 Gy, 4 Gy, 4 Gy (Equal Dosing, "ED") in JX14P and JX14PRT. The equivalent opposite regimen of 7 Gy, 5 Gy, 3 Gy, and 1 Gy (Ramp-Down, "RD") was also investigated. 5000 cells were plated in DMEM-F12 media with B27 supplement and 20 ng/ml EGF and FGF and irradiated every three days. Viability was determined using CellTiterGlo, nine days after the final dose of radiation. This experiment was done in triplicates. Statistical analysis of viability between the different regimens was assessed using a two-way ANOVA with a multiple comparisons Tukey test.ResultsOur preliminary results indicate that delivering a total dose of 16 Gy, alternating radiation fractionations every three days of 1 Gy, 3 Gy, 5 Gy, or 7 Gy, enhances cytotoxicity in both radiation-sensitive and radiation-resistant PDX models when compared to no treatment and fractions of equal dose (4 Gy) delivered in the same manner (Cell line vs luminescence, P<0.0001).ConclusionThis data suggests RU and RD dosing is superior to ED in the radiosensitive cell lines ED (P<0.0001). However, in the radioresistant cell lines, only RU was superior to ED (P<0.0001). We are also exploring the effects of this novel treatment regimen in our radiation-sensitive and -resistant PDX GBM cell lines when co-cultured with different starting proportions. This is the first known experiment where two regimens with the same average dose per fraction and total dose results in vastly different cytotoxicity.

Purpose/Objective(s) To molecularly characterize radiation-selected and control GBM patient-derived xenografts using targeted exome sequencing and RNA-seq for gene expression profiling to identify mechanisms of radiation resistance. Materials/Methods GBM is a devastating CNS malignancy, which in spite of chemotherapy and radiation, often recurs causing significant morbidity and mortality. While there exists numerous hypotheses concerning the treatment resistance of these tumors, the molecular nature of their resistance to therapy has not been fully characterized. To address this shortcoming, eight pairs of PDXs were created, with one of each pair being selected for radiation resistance and the other passaged in heterotopic mouse model as a control. Each tumor was molecularly characterized using targeted exome sequencing and RNA-seq for gene expression profiling. Previously validated bioinformatics tools (e.g., PatternCNV, bowtie2, SAMTools, wANNOVAR) in addition to custom scripts were used for analysis. Results At the genomic level, a recurrent locus of copy-number change in RT-selected pairs was identified at chromosome 12q, suggesting one possible mechanism of treatment resistance. At the tumor transcriptome level, the vast majority of genes that significantly change within one PDX pair are not conserved in all other pairs suggesting a diversity in adaptive response. Underlying patterns emerge, though, with genes involved in glycolysis, hypoxia response, and WNT pathway signaling being up-regulated in response to RT-selection. At the level of the tumor microenvironment, genes on the mitochondrial chromosome are disproportionately under-expressed in the mouse tissue with tumors that were RT-selected. Conclusion Our PDX lines generally maintain their classic genomic changes (e.g., EGFR mutation and structural alteration, and mutational profiles) after radiation selection, a finding qualitatively similar to previous studies in human primary and recurrent GBMs. A recurrent locus of amplification (chr12q) occurred suggesting importance in treatment resistance. The RNA-seq findings suggest that there are multiple pathways that undergo dysregulation in response to radiation. However, there is coherence in adaptation both within the tumor and in the tumor microenvironment. Additionally, the difference identified in the mouse transcriptome of the tumor-associated cells further underscores the importance of the tumor microenvironment and tumors' ability to selectively alter its composition. Future extensions of this work will include targeting the identified pathways with drugs known to cross the blood brain barrier.

Non-coding RNAs (ncRNAs) are emerging as potent regulators of biological process including human diseases such as cancer. Long non-coding RNAs (lncRNAs) are a dynamic and versatile class of ncRNAs for which there is extensive evidence of these transcripts modulating epigenetic programs to affect cellular phenotypes. Our initial studies using a model of glioblastoma (GBM) tumor recurrence has revealed a number of oncogenic lncRNAs associated with therapy resistance and/or tumor recurrence. We have validated the expression of these oncogenic lncRNAs in another cancer, pediatric rhabdomyosarcoma (RMS). There have been few effective therapeutic advances made in past decades for either RMS or GBM. Radiation therapy is commonly employed alongside surgery and standard chemotherapy, especially in the case of unresectable disease, advanced disease, or upon tumor recurrence. Tumors may inherently possess or acquire resistance to radiation therapy and other treatment modalities over the course of disease. Tumor therapy resistance and recurrence are mediated by complex epigenetic programs which lead to increased capacity for DNA damage repair or tolerance of double stranded breaks. LncRNAs may regulate mechanisms of tumor recurrence and therapy resistance across multiple cancer types. The functional roles of ZFAS1 modulating epithelial to mesenchymal transition in colon cancers and SAMMSON which drives aberrant mitochondrial function in melanoma have been described. CASC19/PCAT2, ZFAS1, DUXAP9, and SOX2-OT are overexpressed in multiple cancers with CASC19 having a significant negative association with GBM patient survival. In silico analysis identifies DNA-binding capacities harbored by these lncRNA transcripts, and their predicted binding sites are proximal to several cancer hallmark genes. We have verified the expression of isoforms of these lncRNA transcripts that contain DNA-binding domains through sequencing and qPCR. We hypothesize that these lncRNAs regulate transcriptional networks via recruitment of transcriptional or epigenetic machinery to genomic loci to modulate oncogenic programs and/or tumor suppressor pathways. As potent epigenetic regulators, lncRNAs present an attractive class of therapeutic targets that could complement standard treatments to reduce disease burden and improve patient survival. It is possible that acquired radiation resistance mediated by lncRNAs is conserved across multiple malignancies. We are developing chemically modified, novel antisense oligonucleotides to target these transcripts in GBM and RMS patient-derived xenografts. Using these systems, we hope to reveal novel associations with disease progression and oncogenic mechanisms that could be exploited through the direct targeting of lncRNAs. We hope our work will lead to the development of novel clinical agents to treat patients with RMS, GBM, and potentially other intractable cancers. Citation Format: Christian Tyler Stackhouse, Corinne Linardic, Malaika Kimmons, Samantha Weitzel. Therapeutic targeting of oncogenic long non-coding RNAs utilizing antisense oligonucleotides [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 5429.

Many subtypes of brain tumors are highly malignant and resistant to chemo- and radio- therapy. Tumor cells can shift their phenotype in response to treatments, the so-called adaptive resistance. Adaptive resistance mechanisms in malignant brain tumors are still poorly understood, and effective treatments have not yet been developed. To unveil such mechanisms, we have developed unique new experimental models to identify the adaptive resistance mechanisms to fractionated radiation in malignant brain tumors. We performed repeated irradiation (2-5Gy every 3-4 days, 3-6 weeks) on 6 human Glioma stem cells (GSCs), 2 mouse GSCs and 4 medulloblastomas (MB) cells in vitro and examined how tumor cells adapt to repeated irradiation. Brain tumor cells demonstrated dynamic adaptation to fractionated irradiation. They rapidly altered cell proliferation, intercellular adhesion, and stemness and acquired strong radioresistance. To identify genes responsible for radio-resistance, we performed RNA-seq analysis and CRISPR library screening using primary and radioresistant cells. We found that N-cadherin was upregulated in the majority of radioresistant GSCs. Stably transfecting N-cadherin in parental GSC rendered them 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 b-catenin at the cell surface, which suppressed Wnt/b-catenin proliferative signaling, and reduced neural differentiation. Moreover, N-cadherin increased Clusterin secretion, which protected GSCs against apoptosis after radiation treatment. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, which caused an EMT-like phenotype change in GSCs. The N-cadherin-mediated radioresistance phenotype could be reverted with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor. Adjuvant PPP combined with irradiation significantly extended the survival of orthotopically xenografted mice versus irradiation-only or drug-alone controls, supporting clinical translation. In conclusion, our data indicate that IGF1R inhibition can block the N-cadherin-mediated resistance pathway. Our study deepens our understanding of adaptive resistance during repeated irradiation in GBM, and validates the IGF1/N-cadherin/b-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability. These findings also confirmed that our radioresistant models effectively identify new adaptive resistance mechanisms in malignant brain tumors. (References: Osuka S, et. al., J Clin Invest. 2021;131(6):e136098) Citation Format: Satoru Osuka, Dan Zhu, Zhaobin Zhang, Chaoxi Li, Christian T. Stackhouse, Oltea Sampetrean, Jeffrey J. Olson, G. Yancey Gillespie, Hideyuki Saya, Christopher D. Willey, Erwin G. Van Meir. N-cadherin is a driver of adaptive radioresistance in malignant brain tumors [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 1430.

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.

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.

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, 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 increased cell-cell adhesion, slower proliferation, an elevation of stemness properties 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 b-catenin at the cell surface, which suppressed Wnt/ b-catenin proliferative signaling, reduced neural differentiation, and protected against apoptosis through Clusterin secretion. Restoration of wild type N-cadherin, but not mutant N-cad lacking b-catenin binding region, led to induce radioresistance in N-cadherin knockout GSCs, indicating the importance of the binding between N-cadherin and b-catenin. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, and the radiation resistance phenotype could be reverted with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor, supporting clinical translation. Moreover, the elevation of N-cad and Clusterin are related to prognosis of GBM in the TCGA dataset. In conclusion, our data indicate that IGF1R inhibitor can block the N-cadherin-mediated resistance pathway. Our study deepens our understanding of adaptive radioresistance during repeated irradiation in GBM, and validates the IGF1/N-cadherin/b-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability.

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.

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, we generated radioresistant human and mouse GSCs by exposing them to repeated cycles of irradiation. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by increased cell-cell adhesion, slower proliferation, an elevation of stemness properties and N-cadherin expression. Stably transfecting N-cadherin in parental GSC rendered them 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 b-catenin at the cell surface, which suppressed Wnt/b-catenin proliferative signaling, and reduced neural differentiation. Transfection of wild type N-cadherin, but not mutant N-cadherin lacking the b-catenin binding region, restored radioresistance in N-cadherin knockout GSCs, indicating the importance of the binding between N-cadherin and b-catenin. Moreover, N-cadherin increased Clusterin secretion, which protected GSCs against apoptosis after radiation treatment. N-cadherin knockout decreased Clusterin secretion and sensitized the cells to radiation therapy. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, which induced an EMT-like phenotype change in GSCs. The N-cadherin-mediated radioresistance phenotype could be reverted with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor. Adjuvant PPP combined with irradiation significantly extended the survival of orthotopically xenografted mice versus irradiation-only or drug alone controls, supporting clinical translation. Moreover, elevated N-cadherin and Clusterin mRNA expression is related to prognosis of GBM in the TCGA dataset. In conclusion, our data indicate that IGF1R inhibition can block the N-cadherin-mediated resistance pathway. Our study deepens our understanding of adaptive radioresistance during repeated irradiation in GBM, and validates the IGF1/N-cadherin/β-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability. Citation Format: Satoru Osuka, Dan Zhu, Zhaobin Zhang, Chaoxi Li, Christian T. Stackhouse, Oltea Sampetrean, Jeffrey J. Olson, G. Yancey Gillespie, Hideyuki Saya, Christopher D. Willey, Erwin G. Van Meir. IGF1/N-cadherin/b-catenin/Clusterin signaling axis can mediates adaptive radioresistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3078.

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 β-catenin at the cell surface, which suppressed Wnt/β-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.

Abstract Glioblastoma (GBM) is composed of a variety of tumor cell populations including those with stem cell properties, known as glioma stem cells (GSCs). GSCs are innately less sensitive to radiation than the tumor bulk and are believed to drive GBM formation and recurrence following repeated irradiation. However, it is unclear how GSCs adapt to avoid the toxicity of repeated irradiation used in clinical practice. We established radioresistant human and mouse GSCs by exposing them to repeated rounds of irradiation in order to uncover critical mediators of adaptive radioresistance. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by increased cell-cell adhesion, slower proliferation, an elevation of stemness properties 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 reduced their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of β-catenin at the cell surface, which decreased Wnt/ β-catenin proliferative signaling, reduced neural differentiation, and protected against apoptosis through Clusterin secretion. Restoration of wild type N-cadherin, but not mutant N-cad lacking β-catenin binding region, led to increased radioresistance in N-cadherin knockout GSCs, indicating the importance of the binding between N-cadherin and β-catenin. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, and the radiation resistance phenotype can be reversed with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor, supporting clinical translation. Moreover, the elevation of N-cad and Clusterin are related to prognosis of GBM in the TCGA dataset. In conclusion, our data indicate that IGF1R inhibitor can block the N-cadherin-mediated resistance pathway. Our research provides a deeper understanding of adaptive radioresistance after repeated irradiation, and validates the IGF1/N-cadherin/β-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability.

Glioblastoma multiforme (GBM) almost invariably recurs and tumors exhibit resistance to conventional therapies. One mechanism of resistance is enhanced DNA damage response (DDR) to alkylating chemotherapy or radiation therapy (RT). We have generated 8 novel GBM patient-derived xenograft (PDX) models of tumor recurrence following serial in vivo irradiation (6 x 2Gy fractions over 2 weeks for 6+ rounds). RNA sequencing has revealed enrichment of a number of DDR pathways in the RT selected (RTS) PDX. Differential enrichment across the RTS PDX suggests multiple molecular routes to acquired RT resistance. We have also identified differential enrichment of molecular signatures for cell cycle progression, stemness, and chromatin remodeling all suggesting decreased proliferation, increased stemness, and more compacted chromatin states associated with RTS PDX. We have identified altered kinase signaling in RTS PDX that may suggest targetable signaling pathways using small molecule inhibitors. Integrated ‘–omics’ analysis has identified SRC family kinases and altered expression of collagens related to the RTS profile. Long non-coding RNAs (lncRNA) have the potential to regulate molecular phenotypes through nucleic acid binding. We have identified 269 lncRNAs significantly differentially expressed in the RTS condition. We have determined that a number of these transcripts have DNA binding potential in gene regulatory regions proximal to kinase, DDR, cell cycle, stemness, and chromatin remodeling genes. Analysis of lncRNAs and genes proximal to their binding sites has revealed regulatory networks potentially governing cell fate and phenotype. We have observed complex correlations of some of these transcripts, such as ZFAS1, which has a positive correlation with expression of stemness-promoting genes and simultaneous inverse correlation with cell cycle genes. This suggests ZFAS1 could be a phenotypic switch between a RT-sensitive proliferating cell and a RT-resistant non-proliferating stem-like cell. LncRNAs may represent a novel therapeutic target for the treatment of therapy resistant, recurrent GBM.

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.

Glioblastoma (GBM), the most common primary brain malignancy, has a very poor prognosis despite decades of research. Current treatment with maximal safe resection, concurrent and adjuvant temozolomide (TMZ), and radiation treatment (XRT) with tumor-treating fields have provided marginal improvement in outcomes, yet median survival is only 15 months. Our group believes that inadequate preclinical models have hampered translational efforts for GBM. We posit that patient-derived xenografts (PDX) are superior to traditional immortalized glioma lines and can be an important tool for exploring mechanisms of therapy resistance. We screened a large GBM PDX panel to identify those with inherent XRT sensitivity, then produced the largest cohort of acquired XRT resistant PDX lines through serial in vivo XRT selection. PDX were profiled at the transcriptome and kinome levels. GBM PDX were propagated in athymic nude mice in vivo and screened for XRT sensitivity. Acquired XRT resistance was produced through serial flank XRT of sensitive tumors with serial passaging. GBM PDX were transcriptionally profiled by RNA-Seq and global kinase activity (kinomic) profiling using a microarray processor. Informatics tools include Differential Gene Expression; Differential Gene Correlation Analysis; BEDtools; Weighted Correlation Network Analysis; Fast Exponential Monte Carlo; Pathway, Annotated-list, and Gene-signature Electronic Repository; Biomedical Entity Expansion, Ranking, and Exploration; GeneOntology and GeneTerrain visualization; and using the long non-coding RNA (lncRNA) tools Triplexator and ASSA. 20 PDX were screened for baseline XRT sensitivity by measuring tumor doubling time (TDT) following flank XRT (2 Gy x 6 over 2 weeks) and PDX were deemed XRT sensitive if TDT>20 days. Of the 10 inherently XRT sensitive PDX, 8 lines were rendered XRT resistant through serial flank irradiation (2 Gy x 6 over 2 weeks) repeated during up to 6 in vivo passages. Median parental line TDT was 35 days versus 5 days for the XRT resistant lines (p<0.0001, Gehan-Breslow-Wilcoxon test). Differential RNA-Seq revealed that acquired XRT resistance was associated with a number of altered lncRNA transcripts that were mapped to corresponding gene expression targets with high correlation. Interestingly, many of the lncRNA transcripts were decreased in the XRT resistant setting. Similarly, acquired XRT resistance promoted a global reduction in kinomic activity as compared to parental and inherently resistant tumors, though JAK, FGFR, and Ephrin family kinases were increased. Interactive bioinformatic visualization tools allow for exploration of these data sets. This robust cohort of inherent and acquired XRT resistant GBM PDX provide a suitable preclinical model for novel therapeutic testing.

Recurrence of therapy resistant Glioblastoma multiforme (GBM) is responsible for patient mortality. Not all patients qualify for surgical resection or for chemotherapy, but radiation is almost a universally tolerated therapy. We are modeling acquired radiation resistance using 8 radiation-sensitive GBM patient-derived xenolines (PDX) made resistant by six irradiation series (6x2Gy each) in vivo. In 4 resistance-induced PDX, MGMT (O6-methylguanine–DNA methyltransferase) protein expression increased over original isogenic PDX. Paradoxically, temozolomide screening of orthotopic radiation-resistant PDX with increased MGMT expression revealed increased, not decreased chemo-sensitivity. This suggests an unanticipated mechanism associating acquired radiation resistance and alkylating chemotherapy sensitivity. RNA-seq data of long non-coding RNAs (lncRNAs) has revealed associations with patient overall survival and age at diagnosis. Out of 24,076 lncRNAs, 5 are significantly differentially expressed with regard to patient overall survival and age at diagnosis. The functions of lnc-ZNF117-1, lnc-DCUN1D4-1, and LINC01397 are unknown, but they are enriched in brain over other tissues. Tissue specific expression and function are unknown for lnc-TBL1XR1-5, but it is highly expressed in HepG2 (hepatocellular carcinoma) as well as in non-functioning pituitary adenomas (NFPAs). The lnc-CDH17-1 transcript is also highly expressed in NFPAs. All 5 of these lncRNAs have complex secondary and tertiary structures, but their physiologic or pathologic functions are unknown. LncRNAs most likely contribute to acquired resistance through epigenetic regulation of transcription and chromatin state. Deep sequencing of total RNA isolated from intracranial radiation–sensitive/–resistant PDX to relate transcriptional programs and therapy resistance is underway. These data will be paired with kinomic profiling of matched tumors to elucidate basal signaling modality changes between radiation–sensitive/–resistant tumors. Analysis of differentially expressed lncRNA will be used to predict lncRNA structure/function, elucidating druggable mechanisms mediated by lncRNAs.

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.