Developing Implantable Scaffolds to Enhance Neural Stem Cell Therapy for Post-Operative Glioblastoma.

Pre-clinical and clinical studies have shown that engineered tumoricidal neural stem cells (tNSCs) are a promising treatment strategy for the aggressive brain cancer glioblastoma (GBM). Yet, stabilizing human tNSCs within the surgical cavity following GBM resection is a significant challenge. As a critical step toward advancing engineered human NSC therapy for GBM, we used a preclinical variant of the clinically utilized NSC line HB1.F3.CD and mouse models of human GBM resection/recurrence to identify a polymeric scaffold capable of maximizing the transplant, persistence, and tumor kill of NSC therapy for post-surgical GBM. Using kinetic bioluminescence imaging, we found that tNSCs delivered into the mouse surgical cavity wall by direct injection persisted only 3 days. We found that delivery of tNSCs into the cavity on nanofibrous electrospun poly-l-lactic acid scaffolds extended tNSC persistence to 8 days. Modifications to fiber surface coating, diameter, and morphology of the scaffold failed to significantly extend tNSC persistence in the cavity. In contrast, tNSCs delivered into the post-operative cavity on gelatin matrices (GEMs) persisted 8-fold longer as compared to direct injection. GEMs remained permissive to tumor-tropic homing, as tNSCs migrated off the scaffolds and into invasive tumor foci both in vitro and in vivo. To mirror envisioned human brain tumor trials, we engineered tNSCs to express the prodrug/enzyme thymidine kinase (tNSCstk) and transplanted the therapeutic cells in the post-operative cavity of mice bearing resected orthotopic patient-derived GBM xenografts. Following administration of the prodrug ganciclovir, residual tumor volumes in mice receiving GEM/tNSCs were reduced by 10-fold at day 35, and median survival was extended from 31 to 46 days. Taken together, these data begin to define design parameters for effective scaffold/tNSC composites and suggest a new approach to maximizing the efficacy of tNSC therapy in human patient trials.

Generation and Profiling of Tumor-Homing Induced Neural Stem Cells from the Skin of Cancer Patients.

The conversion of human fibroblasts into personalized induced neural stem cells (iNSCs) that actively seek out tumors and deliver cytotoxic agents is a highly promising approach for treating various types of cancer. However, the ability to generate iNSCs from the skin of cancer patients has not been explored. Here, we take an important step toward clinical application by generating iNSCs from skin biopsies of human patients undergoing treatment for the aggressive brain cancer, glioblastoma (GBM). We then utilized a panel of functional and genomic studies to investigate the efficacy and tumor-homing capacity of these patient-derived cells, as well as genomic analysis, to characterize the impact of interpatient variability on this personalized cell therapy. From the skin-tissue biopsies, we established fibroblasts and transdifferentiated the cells into iNSCs. Genomic and functional testing revealed marked variability in growth rates, therapeutic agent production, and gene expression during fibroblast-to-iNSC conversion among patient lines. In vivo testing showed patient-derived iNSCs home to tumors, yet rates and expression of homing-related pathways varied among patients. With the use of surgical-resection mouse models of invasive human cluster of differentiation 133+ (CD133+) GBM cells and serial kinetic imaging, we found that "high-performing" patient-derived iNSC lines reduced the volume of GBM cells 60-fold and extended survival from 28 to 45 days. Treatment with "low-performing" patient lines had minimal effect on tumor growth, but the anti-tumor effect could be rescued by increasing the intracavity dose. Together, these data show, for the first time, that tumor-homing iNSCs can be generated from the skin of cancer patients and efficaciously suppress tumor growth. We also begin to define genetic markers that could be used to identify cells that will contain the most effective attributes for tumor homing and kill in human patients, including high gene expression of the semaphorin-3B (SEMA3B), which is known to be involved in neuronal cell migration. These studies should serve as an important guide toward clinical GBM therapy, where the personalized nature of optimized iNSC therapy has the potential to avoid transplant rejection and maximize treatment durability.

Engineered Mesenchymal Stem Cell/Nanomedicine Spheroid as an Active Drug Delivery Platform for Combinational Glioblastoma Therapy.

Mesenchymal stem cell (MSC) has been increasingly applied to cancer therapy because of its tumor-tropic capability. However, short retention at target tissue and limited payload option hinder the progress of MSC-based cancer therapy. Herein, we proposed a hybrid spheroid/nanomedicine system, comprising MSC spheroid entrapping drug-loaded nanocomposite, to address these limitations. Spheroid formulation enhanced MSC's tumor tropism and facilitated loading of different types of therapeutic payloads. This system acted as an active drug delivery platform seeking and specifically targeting glioblastoma cells. It enabled effective delivery of combinational protein and chemotherapeutic drugs by engineered MSC and nanocomposite, respectively. In an in vivo migration model, the hybrid spheroid showed higher nanocomposite retention in the tumor tissue compared with the single MSC approach, leading to enhanced tumor inhibition in a heterotopic glioblastoma murine model. Taken together, this system integrates the merits of cell- and nanoparticle- mediated drug delivery with the tumor-homing characteristics of MSC to advance targeted combinational cancer therapy.

Sustained Delivery of Doxorubicin via Acetalated Dextran Scaffold Prevents Glioblastoma Recurrence after Surgical Resection.

The primary cause of mortality for glioblastoma (GBM) is local tumor recurrence following standard-of-care therapies, including surgical resection. With most tumors recurring near the site of surgical resection, local delivery of chemotherapy at the time of surgery is a promising strategy. Herein drug-loaded polymer scaffolds with two distinct degradation profiles were fabricated to investigate the effect of local drug delivery rate on GBM recurrence following surgical resection. The novel biopolymer, acetalated dextran (Ace-DEX), was compared with commercially available polyester, poly(l-lactide) (PLA). Steady-state doxorubicin (DXR) release from Ace-DEX scaffolds was found to be faster when compared with scaffolds composed of PLA, in vitro. This increased drug release rate translated to improved therapeutic outcomes in a novel surgical model of orthotopic glioblastoma resection and recurrence. Mice treated with DXR-loaded Ace-DEX scaffolds (Ace-DEX/10DXR) resulted in 57% long-term survival out to study completion at 120 days compared with 20% survival following treatment with DXR-loaded PLA scaffolds (PLA/10DXR). Additionally, all mice treated with PLA/10DXR scaffolds exhibited disease progression by day 38, as defined by a 5-fold growth in tumor bioluminescent signal. In contrast, 57% of mice treated with Ace-DEX/10DXR scaffolds displayed a reduction in tumor burden, with 43% exhibiting complete remission. These results underscore the importance of polymer choice and drug release rate when evaluating local drug delivery strategies to improve prognosis for GBM patients undergoing tumor resection.

Neural stem cell therapy for cancer.

Cancers of the brain remain one of the greatest medical challenges. Traditional surgery and chemo-radiation therapy are unable to eradicate diffuse cancer cells and tumor recurrence is nearly inevitable. In contrast to traditional regenerative medicine applications, engineered neural stem cells (NSCs) are emerging as a promising new therapeutic strategy for cancer therapy. The tumor-homing properties allow NSCs to access both primary and invasive tumor foci, creating a novel delivery platform. NSCs engineered with a wide array of cytotoxic agents have been found to significantly reduce tumor volumes and markedly extend survival in preclinical models. With the recent launch of new clinical trials, the potential to successfully manage cancer in human patients with cytotoxic NSC therapy is moving closer to becoming a reality.

Engineering toxin-resistant therapeutic stem cells to treat brain tumors.

Pseudomonas exotoxin (PE) potently blocks protein synthesis by catalyzing the inactivation of elongation factor-2 (EF-2). Targeted PE-cytotoxins have been used as antitumor agents, although their effective clinical translation in solid tumors has been confounded by off-target delivery, systemic toxicity, and short chemotherapeutic half-life. To overcome these limitations, we have created toxin-resistant stem cells by modifying endogenous EF-2, and engineered them to secrete PE-cytotoxins that target specifically expressed (interleukin-13 receptor subunit alpha-2) or overexpressed (epidermal growth factor receptor) in glioblastomas (GBM). Molecular analysis correlated efficacy of PE-targeted cytotoxins with levels of cognate receptor expression, and optical imaging was applied to simultaneously track the kinetics of protein synthesis inhibition and GBM cell viability in vivo. The release of IL13-PE from biodegradable synthetic extracellular matrix (sECM) encapsulated stem cells in a clinically relevant GBM resection model led to increased long-term survival of mice compared to IL13-PE protein infusion. Moreover, multiple patient-derived GBM lines responded to treatment, underscoring its clinical relevance. In sum, integrating stem cell-based engineering, multimodal imaging, and delivery of PE-cytotoxins in a clinically relevant GBM model represents a novel strategy and a potential advancement in GBM therapy.

Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells.

Exosomes have recently come into focus as "natural nanoparticles" for use as drug delivery vehicles. Our objective was to assess the feasibility of an exosome-based drug delivery platform for a potent chemotherapeutic agent, paclitaxel (PTX), to treat MDR cancer. Herein, we developed different methods of loading exosomes released by macrophages with PTX (exoPTX), and characterized their size, stability, drug release, and in vitro antitumor efficacy. Reformation of the exosomal membrane upon sonication resulted in high loading efficiency and sustained drug release. Importantly, incorporation of PTX into exosomes increased cytotoxicity more than 50 times in drug resistant MDCKMDR1 (Pgp+) cells. Next, our studies demonstrated a nearly complete co-localization of airway-delivered exosomes with cancer cells in a model of murine Lewis lung carcinoma pulmonary metastases, and a potent anticancer effect in this mouse model. We conclude that exoPTX holds significant potential for the delivery of various chemotherapeutics to treat drug resistant cancers. FROM THE CLINICAL EDITOR: Exosomes are membrane-derived natural vesicles of ~40 - 200 nm size. They have been under extensive research as novel drug delivery vehicles. In this article, the authors developed exosome-based system to carry formulation of PTX and showed efficacy in the treatment of multi-drug resistant cancer cells. This novel system may be further developed to carry other chemotherapeutic agents in the future.

Local administration of irinotecan using an implantable drug delivery device stops high-grade glioma tumor recurrence in a glioblastoma tumor model.

The treatment for Glioblastoma is limited due to the presence of the blood brain barrier, which restricts the entry of chemotherapeutic drugs into the brain. Local delivery into the tumor resection margin has the potential to improve efficacy of chemotherapy. We developed a safe and clinically translatable irinotecan implant for local delivery to increase its efficacy while minimizing systemic side effects. Irinotecan-loaded implants were manufactured using hot melt extrusion, gamma sterilized at 25 kGy, and characterized for their irinotecan content, release, and drug diffusion. Their therapeutic efficacy was evaluated in a patient-derived xenograft mouse resection model of glioblastoma. Their safety and translatability were evaluated using histological analysis of brain tissue and serum chemistry analysis. Implants containing 30% and 40% w/w irinotecan were manufactured without plasticizer. The 30% and 40% implants showed moderate local toxicity up to 2- and 6-day post-implantation. Histopathology of the implantation site showed signs of necrosis at days 45 and 14 for the 30% and 40% implants. Hematological analysis and clinical chemistry showed no signs of serious systemic toxicity for either implant. The 30% implants had an 80% survival at day 148, with no sign of tumor recurrence. Gamma sterilization and 12-month storage had no impact on the integrity of the 30% implants. This study demonstrates that the 30% implants are a promising novel treatment for glioblastoma that could be quickly translated into the clinic.

Auto-loaded TRAIL-exosomes derived from induced neural stem cells for brain cancer therapy.

Transdifferentiation (TD), a somatic cell reprogramming process that eliminates pluripotent intermediates, creates cells that are ideal for personalized anti-cancer therapy. Here, we provide the first evidence that extracellular vesicles (EVs) from TD-derived induced neural stem cells (Exo-iNSCs) are an efficacious treatment strategy for brain cancer. We found that genetically engineered iNSCs generated EVs loaded with the tumoricidal gene product TRAIL at nearly twice the rate of their parental fibroblasts, and TRAIL produced by iNSCs was naturally loaded into the lumen of EVs and arrayed across their outer membrane (Exo-iNSC-TRAIL). Uptake studies in ex vivo organotypic brain slice cultures showed that Exo-iNSC-TRAIL selectively accumulates within tumor foci, and co-culture assays demonstrated that Exo-iNSC-TRAIL killed metastatic and primary brain cancer cells more effectively than free TRAIL. In an orthotopic mouse model of brain cancer, Exo-iNSC-TRAIL reduced breast-to-brain tumor xenografts by approximately 3000-fold compared to treatment with free TRAIL, with all Exo-iNSC-TRAIL treated animals surviving through 90 days post-treatment. In additional in vivo testing against aggressive U87 and invasive GBM8 glioblastoma tumors, Exo-iNSC-TRAIL also induced a statistically significant increase in survival. These studies establish a novel, easily generated, stable, tumor-targeted EV to efficaciously treat multiple forms of brain cancer.

Correction: Specific transfection of inflamed brain by macrophages: A new therapeutic strategy for neurodegenerative diseases.

[This corrects the article DOI: 10.1371/journal.pone.0061852.].

Auto-loaded TRAIL-exosomes derived from induced neural stem cells for brain cancer therapy.

Transdifferentiation (TD), a somatic cell reprogramming process that eliminates pluripotent intermediates, creates cells that are ideal for personalized anti-cancer therapy. Here, we provide the first evidence that extracellular vesicles (EVs) from TD-derived induced neural stem cells (Exo-iNSCs) are an efficacious treatment strategy for brain cancer. We found that genetically engineered iNSCs generated EVs loaded with the tumoricidal gene product TRAIL at nearly twice the rate as their parental fibroblasts, and the TRAIL produced by iNSCs were naturally loaded into the lumen of EVs and arrayed across their outer membrane (Exo-iNSC-TRAIL). Uptake studies in ex vivo organotypic brain slice cultures showed Exo-iNSC-TRAIL selectively accumulates within tumor foci, and co-culture assays showed that Exo-iNSC-TRAIL killed metastatic and primary brain cancer cells more effectively than free TRAIL. In an orthotopic mouse model of brain cancer, Exo-iNSC-TRAIL reduced breast-to-brain tumor xenografts around 3000-fold greater than treatment with free TRAIL, with all Exo-iNSC-TRAIL treated animals surviving through 90 days post-treatment. In additional in vivo testing against aggressive U87 and invasive GBM8 glioblastoma tumors, Exo-iNSC-TRAIL also induced a statistically significant increase in survival. These studies establish a new easily generated, stable, tumor-targeted EV to efficaciously treat multiple forms of brain cancer.

Trans-lesion synthesis and mismatch repair pathway crosstalk defines chemoresistance and hypermutation mechanisms in glioblastoma.

Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides an aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O6mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.

Real-time multi-modality imaging of glioblastoma tumor resection and recurrence.

The lack of relevant pre-clinical animal models incorporating the clinical scenario of Glioblastoma multiforme (GBM) resection and recurrence has contributed significantly to the inability to successfully treat GBM. A multi-modality imaging approach that allows real-time assessment of tumor resection during surgery and non-invasive detection of post-operative tumor volumes is urgently needed. In this study, we report the development and implementation of an optical imaging and magnetic resonance imaging (MRI) approach to guide GBM resection during surgery and track tumor recurrence at multiple resolutions in mice. Intra-operative fluorescence-guided surgery allowed real-time monitoring of intracranial tumor removal and led to greater than 90 % removal of established intracranial human GBM. The fluorescent signal clearly delineated tumor margins, residual tumor, and correlated closely with the clinically utilized fluorescence surgical marker 5-aminolevulinic acid/porphyrin. Post-operative non-invasive optical imaging and MRI confirmed near-complete tumor removal, which was further validated by immunohistochemistry (IHC). Longitudinal non-invasive imaging and IHC showed rapid recurrence of multi-focal tumors that exhibited a faster growth rate and altered blood-vessel density compared to non-resected tumors. Surgical tumor resection significantly extended long-term survival, however mice ultimately succumbed to the recurrent GBM. This multi-modality imaging approach to GBM resection and recurrence in mice should provide an important platform for investigating multiple aspects of GBM and ultimately evaluating novel therapeutics.

Current approaches in enhancing TRAIL therapies in glioblastoma.

Glioblastoma (GBM) is the most prevalent, aggressive, primary brain cancer in adults and continues to pose major medical challenges due in part to its high rate of recurrence. Extensive research is underway to discover new therapies that target GBM cells and prevent the inevitable recurrence in patients. The pro-apoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted attention as an ideal anticancer agent due to its ability to selectively kill cancer cells with minimal toxicity in normal cells. Although initial clinical evaluations of TRAIL therapies in several cancers were promising, later stages of clinical trial results indicated that TRAIL and TRAIL-based therapies failed to demonstrate robust efficacies due to poor pharmacokinetics, resulting in insufficient concentrations of TRAIL at the therapeutic site. However, recent studies have developed novel ways to prolong TRAIL bioavailability at the tumor site and efficiently deliver TRAIL and TRAIL-based therapies using cellular and nanoparticle vehicles as drug loading cargos. Additionally, novel techniques have been developed to address monotherapy resistance, including modulating biomarkers associated with TRAIL resistance in GBM cells. This review highlights the promising work to overcome the challenges of TRAIL-based therapies with the aim to facilitate improved TRAIL efficacy against GBM.

Cell-based therapies for glioblastoma: Promising tools against tumor heterogeneity.

Glioblastoma (GBM) is a highly aggressive tumor with a devastating impact on quality-of-life and abysmal survivorship. Patients have very limited effective treatment options. The successes of targeted small molecule drugs and immune checkpoint inhibitors seen in various solid tumors have not translated to GBM, despite significant advances in our understanding of its molecular, immune, and microenvironment landscapes. These discoveries, however, have unveiled GBM's incredible heterogeneity and its role in treatment failure and survival. Novel cellular therapy technologies are finding successes in oncology and harbor characteristics that make them uniquely suited to overcome challenges posed by GBM, such as increased resistance to tumor heterogeneity, modularity, localized delivery, and safety. Considering these advantages, we compiled this review article on cellular therapies for GBM, focusing on cellular immunotherapies and stem cell-based therapies, to evaluate their utility. We categorize them based on their specificity, review their preclinical and clinical data, and extract valuable insights to help guide future cellular therapy development.

Magnetic Control of Protein Expression via Magneto-mechanical Actuation of ND-PEGylated Iron Oxide Nanocubes for Cell Therapy.

Engineered cells used as smart vehicles for delivery of secreted therapeutic proteins enable effective treatment of cancer and certain degenerative, autoimmune, and genetic disorders. However, current cell-based therapies use mostly invasive tools for tracking proteins and do not allow for controlled secretion of therapeutic proteins, which could result in unconstrained killing of surrounding healthy tissues or ineffective killing of host cancer cells. Regulating the expression of therapeutic proteins after success of therapy remains elusive. In this study, a noninvasive therapeutic approach mediated by magneto-mechanical actuation (MMA) was developed to remotely regulate the expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein, which is secreted by transduced cells. Stem cells, macrophages, and breast cancer cells were transduced with a lentiviral vector encoding the SGpL2TR protein. SGpL2TR comprises TRAIL and GpLuc domains optimized for cell-based applications. Our approach relies on the remote actuation of cubic-shape highly magnetic field responsive superparamagnetic iron oxide nanoparticles (SPIONs) coated with nitrodopamine PEG (ND-PEG), which are internalized within the cells. Cubic ND-PEG-SPIONs actuated by superlow frequency alternating current magnetic fields can translate magnetic forces into mechanical motion and in turn spur mechanosensitive cellular responses. Cubic ND-PEG-SPIONs were artificially designed to effectively operate at low magnetic field strengths (<100 mT) retaining approximately 60% of their saturation magnetization. Compared to other cells, stems cells were more sensitive to the interaction with actuated cubic ND-PEG-SPIONs, which clustered near the endoplasmic reticulum (ER). Luciferase, ELISA, and RT-qPCR analyses revealed a marked TRAIL downregulation (secretion levels were depleted down to 30%) when intracellular particles at 0.100 mg/mL Fe were actuated by magnetic fields (65 mT and 50 Hz for 30 min). Western blot studies indicated actuated, intracellular cubic ND-PEG-SPIONs can cause mild ER stress at short periods (up to 3 h) of postmagnetic field treatment thus leading to the unfolded protein response. We observed that the interaction of TRAIL polypeptides with ND-PEG can also contribute to this response. To prove the applicability of our approach, we used glioblastoma cells, which were exposed to TRAIL secreted from stem cells. We demonstrated that in the absence of MMA treatment, TRAIL essentially killed glioblastoma cells indiscriminately, but when treated with MMA, we were able to control the cell killing rate by adjusting the magnetic doses. This approach can expand the capabilities of stem cells to serve as smart vehicles for delivery of therapeutic proteins in a controlled manner without using interfering and expensive drugs, while retaining their potential to regenerate damaged tissue after treatment. This approach brings forth new alternatives to regulate protein expression noninvasively for cell therapy and other cancer therapies.

Longitudinal 3-D Visualization of Microvascular Disruption and Perfusion Changes in Mice During the Evolution of Glioblastoma Using Super-Resolution Ultrasound.

Glioblastoma is an aggressive brain cancer with a very poor prognosis in which less than 6% of patients survive more than five-year post-diagnosis. The outcome of this disease for many patients may be improved by early detection. This could provide clinicians with the information needed to take early action for treatment. In this work, we present the utilization of a non-invasive, fully volumetric ultrasonic imaging method to assess microvascular change during the evolution of glioblastoma in mice. Volumetric ultrasound localization microscopy (ULM) was used to observe statistically significant ( ) reduction in the appearance of functional vasculature over the course of three weeks. We also demonstrate evidence suggesting the reduction of vascular flow for vessels peripheral to the tumor. With an 82.5% consistency rate in acquiring high-quality vascular images, we demonstrate the possibility of volumetric ULM as a longitudinal method for microvascular characterization of neurological disease.

Utilizing induced neural stem cell-based delivery of a cytokine cocktail to enhance chimeric antigen receptor-modified T-cell therapy for brain cancer.

Chimeric antigen receptor (CAR)-modified T-cell therapy has shown enormous clinical promise against blood cancers, yet efficacy against solid tumors remains a challenge. Here, we investigated the potential of a new combination cell therapy, where tumor-homing induced neural stem cells (iNSCs) are used to enhance CAR-T-cell therapy and achieve efficacious suppression of brain tumors. Using in vitro and in vivo migration assays, we found iNSC-secreted RANTES/IL-15 increased CAR-T-cell migration sixfold and expansion threefold, resulting in greater antitumor activity in a glioblastoma (GBM) tumor model. Furthermore, multimodal imaging showed iNSC delivery of RANTES/IL-15 in combination with intravenous administration of CAR-T cells reduced established orthotopic GBM xenografts 2538-fold within the first week, followed by durable tumor remission through 60 days post-treatment. By contrast, CAR-T-cell therapy alone only partially controlled tumor growth, with a median survival of only 19 days. Together, these studies demonstrate the potential of combined cell therapy platforms to improve the efficacy of CAR-T-cell therapy for brain tumors.

Trans-Lesion Synthesis and Mismatch Repair Pathway Crosstalk Defines Chemoresistance and Hypermutation Mechanisms in Glioblastoma.

Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides a new aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O6mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define novel molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.

Trans-Lesion Synthesis and Mismatch Repair Pathway Crosstalk Defines Chemoresistance and Hypermutation Mechanisms in Glioblastoma.

Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides a new aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O6mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define novel molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.