CD133 marks self-renewing cancer stem cells (CSCs) in a variety of solid tumors, and CD133+ tumor-initiating cells are known markers of chemo- and radio-resistance in multiple aggressive cancers, ...including glioblastoma (GBM), that may drive intra-tumoral heterogeneity. Here, we report three immunotherapeutic modalities based on a human anti-CD133 antibody fragment that targets a unique epitope present in glycosylated and non-glycosylated CD133 and studied their effects on targeting CD133+ cells in patient-derived models of GBM. We generated an immunoglobulin G (IgG) (RW03-IgG), a dual-antigen T cell engager (DATE), and a CD133-specific chimeric antigen receptor T cell (CAR-T): CART133. All three showed activity against patient-derived CD133+ GBM cells, and CART133 cells demonstrated superior efficacy in patient-derived GBM xenograft models without causing adverse effects on normal CD133+ hematopoietic stem cells in humanized CD34+ mice. Thus, CART133 cells may be a therapeutically tractable strategy to target CD133+ CSCs in human GBM or other treatment-resistant primary cancers.
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•Three immunotherapeutic modalities were developed to target CD133+ cells•Anti-CD133 DATEs and CAR-T cells are active in patient-derived glioblastoma (GBM) models•CD133-specific CAR-T (CART133) has enhanced activity in orthotopic GBM xenograft models•Intra-tumoral CART133 does not induce acute systemic toxicity in humanized mouse models
In this article, Singh and colleagues undertook a comparative evaluation of pre-clinical efficacy and safety of three immunotherapeutic modalities directed against CD133 braintumor-initiating cells. While all three modalities were efficacious in orthotopic GBM xenografts, CD133-specific CAR-T cells represented the most therapeutically tractable strategy against functionally important CD133+ GBM cells.
Glioblastoma (GBM) carries a dismal prognosis and inevitably relapses despite aggressive therapy. Many members of the Eph receptor tyrosine kinase (EphR) family are expressed by GBM stem cells (GSC), ...which have been implicated in resistance to GBM therapy. In this study, we identify several EphRs that mark a therapeutically targetable GSC population in treatment-refractory, recurrent GBM (rGBM). Using a highly specific EphR antibody panel and CyTOF (cytometry by time-of-flight), we characterized the expression of all 14 EphR in primary and recurrent patient-derived GSCs to identify putative rGBM-specific EphR. EPHA2 and EPHA3 coexpression marked a highly tumorigenic cell population in rGBM that was enriched in GSC marker expression. Knockdown of EPHA2 and EPHA3 together led to increased expression of differentiation marker GFAP and blocked clonogenic and tumorigenic potential, promoting significantly higher survival
Treatment of rGBM with a bispecific antibody against EPHA2/A3 reduced clonogenicity
and tumorigenic potential of xenografted recurrent GBM
via downregulation of AKT and ERK and increased cellular differentiation. In conclusion, we show that EPHA2 and EPHA3 together mark a GSC population in rGBM and that strategic cotargeting of EPHA2 and EPHA3 presents a novel and rational therapeutic approach for rGBM.
Treatment of rGBM with a novel bispecific antibody against EPHA2 and EPHA3 reduces tumor burden, paving the way for the development of therapeutic approaches against biologically relevant targets in rGBM.
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Mortality rates in patients diagnosed with central nervous system (CNS) tumors, originating in the brain or spinal cord, continue to remain high despite the advances in multimodal treatment regimens, ...including surgery, radiation, and chemotherapy. Recent success of adoptive cell transfer immunotherapy treatments using chimeric antigen receptor (CAR) engineered T cells against in chemotherapy resistant CD19 expressing B-cell lymphomas, has provided the foundation for investigating efficacy of CAR T immunotherapies in the context of brain tumor. Although significant efforts have been made in developing and translating the novel CAR T therapies for CNS tumors, including glioblastoma (GBM), researchers are yet to achieve a similar level of success as with liquid malignancies. In this review, we discuss strategies and considerations essential for developing robust preclinical models for the translation of T cell-based therapies for CNS tumors. Some of the key considerations include route of delivery, increasing persistence of T cells in tumor environment, remodeling of myeloid environment, establishing the window of treatment opportunity, harnessing endogenous immune system, designing multiple antigen targeting T cells, and rational combination of immunotherapy with the current standard of care. Although this review focuses primarily on CAR T therapies for GBM, similar strategies, and considerations are applicable to all CNS tumors in general.
Clonal evolution of cancer may be regulated by determinants of stemness, specifically self-renewal, and current therapies have not considered how genetic perturbations or properties of stemness ...affect such functional processes. Glioblastoma-initiating cells (GICs), identified by expression of the cell surface marker CD133, are shown to be chemoradioresistant. In the current study, we sought to elucidate the functional role of CD133 in self-renewal and identify compounds that can specifically target this CD133(+) treatment-refractory population.
Using gain/loss-of-function studies for CD133 we assessed the in vitro self-renewal and in vivo tumor formation capabilities of patient-derived glioblastoma cells. We generated a CD133 signature combined with an in silico screen to find compounds that target GICs. Self-renewal and proliferation assays on CD133-sorted samples were performed to identify the preferential action of hit compounds. In vivo efficacy of the lead compound pyrvinium was assessed in intracranial GIC xenografts and survival studies. Lastly, microarray analysis was performed on pyrvinium-treated GICs to discover core signaling events involved.
We discovered pyrvinium, a small-molecule inhibitor of GIC self-renewal in vitro and in vivo, in part through inhibition of Wnt/β-catenin signaling and other essential stem cell regulatory pathways. We provide a therapeutically tractable strategy to target self-renewing, chemoradioresistant, and functionally important CD133(+) stem cells that drive glioblastoma relapse and mortality.
Our study provides an integrated approach for the eradication of clonal populations responsible for cancer progression, and may apply to other aggressive and heterogeneous cancers.
Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor, representing 20% of newly diagnosed childhood central nervous system malignancies. Although advances in multimodal therapy ...yielded a 5-year survivorship of 80%, MB still accounts for the leading cause of childhood cancer mortality. In this work, we describe the epigenetic regulator BMI1 as a novel therapeutic target for the treatment of recurrent human Group 3 MB, a childhood brain tumor for which there is virtually no treatment option beyond palliation. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naive tumors will provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumor. Using a small molecule inhibitor against BMI1, PTC-028, we were able to demonstrate complete ablation of self-renewal of MB stem cells in vitro. When administered to mice xenografted with patient tumors, we observed significant reduction in tumor burden in both local and metastatic compartments and subsequent increased survival, without neurotoxicity. Strikingly, serial in vivo re-transplantation assays demonstrated a marked reduction in tumor initiation ability of recurrent MB cells upon re-transplantation of PTC-028-treated cells into secondary recipient mouse brains. As Group 3 MB is often metastatic and uniformly fatal at recurrence, with no current or planned trials of targeted therapy, an efficacious targeted agent would be rapidly transitioned to clinical trials.
PurposeGlioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of cancer stem cells (CSCs) ...drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here, we identify CD70 as a potential therapeutic target for recurrent GBM CSCs.Experimental designIn the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We use CD70 knockdown studies, subsequent RNAseq pathway analysis, and in vivo xenotransplantation to validate CD70’s role in GBM. Next, we developed and tested an anti-CD70 chimeric antigen receptor (CAR)-T therapy, which we validated in vitro and in vivo using our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples.ResultsCD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosis in vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations, notably putative M1 macrophages and CD4 T cells.ConclusionCD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable polytherapeutic avenue to co-target both GBM and its TIME.
Brain metastases (BM) result from the spread of primary tumors to the brain and are a leading cause of cancer mortality in adults. Secondary tissue colonization remains the main bottleneck in ...metastatic development, yet this "premetastatic" stage of the metastatic cascade, when primary tumor cells cross the blood-brain barrier and seed the brain before initiating a secondary tumor, remains poorly characterized. Current studies rely on specimens from fully developed macrometastases to identify therapeutic options in cancer treatment, overlooking the potentially more treatable "premetastatic" phase when colonizing cancer cells could be targeted before they initiate the secondary brain tumor. Here we use our established brain metastasis initiating cell (BMIC) models and gene expression analyses to characterize premetastasis in human lung-to-BM. Premetastatic BMIC engaged invasive and epithelial developmental mechanisms while simultaneously impeding proliferation and apoptosis. We identified the dopamine agonist apomorphine to be a potential premetastasis-targeting drug.
treatment with apomorphine prevented BM formation, potentially by targeting premetastasis-associated genes
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Low expression of these genes was associated with poor survival of patients with lung adenocarcinoma. These results illuminate the cellular and molecular dynamics of premetastasis, which is subclinical and currently impossible to identify or interrogate in human patients with BM. These data present several novel therapeutic targets and associated pathways to prevent BM initiation.
These findings unveil molecular features of the premetastatic stage of lung-to-brain metastases and offer a potential therapeutic strategy to prevent brain metastases.
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Brain metastases (BM) are mainly treated palliatively with an expected survival of less than 12 months after diagnosis. In many solid tumors, the human neural stem cell marker glycoprotein CD133 is a ...marker of a tumor-initiating cell population that contributes to therapy resistance, relapse, and metastasis.
Here, we use a variant of our previously described CD133 binder to generate second-generation CD133-specific chimeric antigen receptor T cells (CAR-T) to demonstrate its specificity and efficacy against multiple patient-derived BM cell lines with variable CD133 antigen expression.
Using both lung- and colon-BM patient-derived xenograft models, we show that a CD133-targeting CAR-T cell therapy can evoke significant tumor reduction and survival advantage after a single dose, with complete remission observed in the colon-BM model.
In summary, these data suggest that CD133 plays a critical role in fueling the growth of BM, and immunotherapeutic targeting of this cell population is a feasible strategy to control the outgrowth of BM tumors that are otherwise limited to palliative care. See related commentary by Sloan et al., p. 477.
Glioblastoma (GBM) is a very aggressive and invasive tumor that relapses within nine months of diagnosis and remains incurable despite advances in multimodal therapy including surgical resection, ...chemotherapy and radiation. Poor patient outcome has been linked to both marker expression of brain tumor initiating cells (BTICs) and intratumoral heterogeneity (ITH), which have been associated with treatment resistance and tumor recurrence. ITH can be explained at the cellular level by the existence of multiple populations of cancer cells, including some which have acquired stemness properties like self-renewal, proliferation, and multilineage differentiation, also known as cancer stem cells (CSCs). In brain tumors, CSCs or BTICs, have been shown to be resistant to both chemotherapy and radiation treatment, allowing them to escape therapy and consequently allowing for tumor recurrence. As a result, therapies that focus on targeting the BTIC compartment within the bulk GBM tumor would provide better treatment and prognosis for patients. To profile ITH as it evolves through therapy delivery, we have developed a novel and dynamic BTIC patient-derived xenograft (PDX) model of human GBM recurrence, which allows for multimodal profiling of GBM BTICs at engraftment, after chemoradiotherapy delivery in a phase we have termed “minimal residual disease” (MRD), and at tumor recurrence. In this study, we present the profiling of the transcriptome and the cell-surface proteome at each of these stages, including validation of targets, novel and exclusive to recurrent treatment-refractory GBM, by CRISPR/Cas9 knockout and subsequent functional stem cell assays. Despite the fact that recurrent GBM is what ultimately leads to patient demise, it remains a largely unknown landscape. Virtually all of the current genomic, transcriptomic, and proteomic data is based on primary GBM. Hence, our study provides a unique therapeutic window into the often-overlooked elephant in the room: recurrent glioblastoma.
Abstract
No standardized treatment exists for patients with recurrent glioblastoma (GBM). Given the aggressive nature of the disease and difficulty in modeling tumor recurrence, minimal efforts have ...been made to design rational therapies against it. The roundabout guidance receptor 1 (ROBO1) protein is involved in axonal guidance during neurodevelopment and is aberrantly upregulated in glioma where it mediates glioma cell migration. Here, we present that ROBO1 is highly expressed on the surface of malignant and treatment-refractory brain tumor initiating cells (BTICs), prompting the development of an anti-ROBO1 CAR-T cell therapy. Using the binding region of a single-domain antibody targeting ROBO1, we developed second-generation anti-ROBO1 CAR-T cells specific and effective against ROBO1-expressing BTICs. Upon antigen exposure, anti-ROBO1 CAR-T cells upregulated markers of activation and degranulation. Additionally, treatment of orthotopic and patient-derived brain tumor xenograft models with anti-ROBO1 CAR-T cells resulted in reduced tumor burden and prolonged survival, demonstrating the therapy’s therapeutic potential for treating neoplastic brain malignancies.
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