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Heatmap of dysregulated BMIT pathways

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 in vivo. Treatment of rGBM with a bispecific antibody against EPHA2/A3 reduced clonogenicity in vitro and tumorigenic potential of xenografted recurrent GBM in vivo 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.Significance: 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. Cancer Res; 78(17); 5023–37. ©2018 AACR.

Supplementary figures of additional heatmaps generated from the RNA sequencing analyses, snapshots of dysregulated BMIT genes within KEGG and GO datasets, and futher Apomorphine analyses.

Protein expression of EphA2, EphA3, CD133, CD15, Bmi1, Sox2, FoxG1 and ITGA6 in GBM.

Supplementary information depicting n numbers of in vivo experiments, primer sequences, and top drug hits.

Patient demographics.

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. In vivo treatment with apomorphine prevented BM formation, potentially by targeting premetastasis-associated genes KIF16B, SEPW1, and TESK2. 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.Significance: These findings unveil molecular features of the premetastatic stage of lung-to-brain metastases and offer a potential therapeutic strategy to prevent brain metastases. Cancer Res; 78(17); 5124–34. ©2018 AACR.

INTRO: Patients with brain metastases (BM) face a 90% mortality rate within one year of their diagnosis and they lack targeted therapeutic options, particularly preventative or interceptional ones. METHODS: The Singh lab has generated a large in-house biobank of patient-derived BM cell lines that are established from patient-derived BM from primary lung and breast cancers and melanoma. We use these BM cell lines to generate murine orthotopic xenograft models of BM and interrogate the biological processes that lead to BM. These models have successfully recapitulated all the stages of their respective metastatic cascades and allowed characterization of a “premetastatic” population of BM cells that have just seeded the brains of mice before forming mature, clinically detectable tumors. Pre-metastatic cell populations are impossible to detect in human patients but present a therapeutic window wherein metastasizing cells can be targeted and eradicated before establishing clinically detectable and difficult to treat brain tumors. RESULTS: Targeting premetastatic BM cells is a feasible interceptional strategy to block BM, but druggable targets are still very limited. Here, we applied RNA sequencing of premetastatic BM cells to reveal a unique deregulated transcriptomic profile that is specific to premetastatic cells regardless of primary tumor origin. Subsequent Connectivity Map analysis revealed compounds that we biologically characterized in vitro for selective anti-BMIC phenotypes. This effort led us to identify a tool compound that exhibits anti-BM activity in vitro, while remaining ineffective against normal brain cell controls. Follow up preclinical studies showed that treatment with this tool compound reduces the tumor burden of mice compared to placebo, while providing a significant survival advantage. Mass spectrometry-based metabolomics and CRISPR knock-out studies directly validated our tool compound’s target, Target X, as a targetable therapeutic vulnerability in BM, where pharmacological and genetic perturbation of Target X attenuates BM cell proliferation both in vitro and in vivo. We have now begun a large-scale medicinal chemistry campaign to develop a novel, brain penetrant Target X-inhibitor with a drug-like pre-clinical profile validated by our in vivo experimental models. This advanced drug candidate will be ready for later stage preclinical development and subsequent clinical development. CONCLUSION: This potential first-in-class anti-metastatic therapy may provide an alternative interceptional treatment strategy for patients experiencing BM that are otherwise limited to palliation. Our work provides a new model for target discovery and validation to develop more effective preventative therapeutic strategies for patients with metastatic disease. Citation Format: Agata M. Kieliszek, Daniel Mobilio, Blessing I. Bassey-Archibong, Jarrod Johnson, Nikoo Aghaei, William Gwynne, Dillon McKenna, Minomi K. Subapanditha, Chitra Venugopal, Jakob Magolan, Sheila K. Singh. Uncovering a new therapeutic vulnerability for preventing brain metastases [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 2474.

Recurrence of solid tumors renders patients vulnerable to a distinctly advanced, highly treatment-refractory disease state that has an increased mutational burden and novel oncogenic drivers not detected at initial diagnosis. Improving outcomes for recurrent cancers requires a better understanding of cancer cell populations that expand from the post-therapy, minimal residual disease (MRD) state. We profiled barcoded tumor stem cell populations through therapy at tumor initiation/engraftment, MRD and recurrence in our therapy-adapted, patient-derived xenograft models of glioblastoma (GBM). Tumors showed distinct patterns of recurrence in which clonal populations exhibited either an a priori, pre-existing fitness advantage, or a priori equipotency fitness acquired through therapy. Characterization of the MRD state by single-cell and bulk RNA sequencing revealed a tumor-intrinsic immunomodulatory signature with strong prognostic significance at the transcriptomic level and in proteomic analysis of cerebrospinal fluid (CSF) collected from GBM patients at all stages of disease. Our results provide insight into the innate and therapy-driven dynamics of human GBM, and the prognostic value of interrogating the MRD state in solid cancers.

Description, Gene expression profiling of medulloblastoma cells undergoing therapy identifies BPIFB4 as a novel target for recurrent disease., Medulloblastoma (MB) remains a leading cause of cancer-related mortality among children. The paucity of MB samples collected at relapse has hindered the functional understanding of molecular mechanisms driving therapy failure. New models capable of accurately recapitulating tumor progression in response to conventional therapeutic interventions are urgently needed. In this study, we developed a therapy-adapted PDX MB model that has a distinct advantage of generating human MB recurrence. The comparative gene expression analysis of MB cells collected throughout therapy led to identification of genes specifically up-regulated after therapy, including one previously undescribed in the setting of brain tumors, bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4). Subsequent functional validation resulted in a markedly diminished in vitro proliferation, self-renewal, and longevity of MB cells, translating into extended survival and reduced tumor burden in vivo. Targeting endothelial nitric oxide synthase, a downstream substrate of BPIFB4, impeded growth of several patient-derived MB lines at low nanomolar concentrations.

Differentiation is a central key capability of stem cells. Their ability to be multipotent and undergo self-renewal are key identifying features of stem cells. A differentiation assay allows for study of one of the essential features of stem cells, the ability to differentiate into all of the cell types of its lineage, in order to ensure that the cells cultured and utilized in key experiments indeed have stem cell properties. Neural stem cells when plated in differentiation media, differentiate into all three neural lineages: Neurons, Astrocytes, and Oligodendrocytes. Brain tumor initiating cells (BTICs) are cells present in brain tumors that possess stem cell properties and are able to self-renew and differentiate into neural lineages. In the current chapter, we discuss protocols involved in immunofluorescence staining and identification of differentiated cells from BTIC populations.

As a useful biotechnology, flow cytometry has revolutionized the field of cell analysis through its dynamic system that employs fluidics, optics, and electronics. It was first used to analyze DNA, but is often used to determine biomarker expression, as well as to characterize and sort cells, in accordance with various parameters. A common application of flow cytometry is the identification and isolation of a distinct cancer cell population, known as cancer stem cells (CSCs). Various biomarkers have been used to elucidate this proportion of cells within the brain, termed brain tumor initiating cells (BTICs). Here, we discuss methodology to prepare BTICs for flow cytometric analysis that includes the expression of markers.