Showing papers by "Sheila K. Singh published in 2022"

CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment

Abstract: Purpose Glioblastoma (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 design In 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. Results CD70 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. Conclusion CD70 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.

Discovery of HDAC6-Selective Inhibitor NN-390 with in Vitro Efficacy in Group 3 Medulloblastoma.

Abstract: Histone deacetylase 6 (HDAC6) has been targeted in clinical studies for anticancer effects due to its role in oncogenic transformation and metastasis. Through a second-generation structure-activity relationship (SAR) study, the design, and biological evaluation of the selective HDAC6 inhibitor NN-390 is reported. With nanomolar HDAC6 potency, >200-550-fold selectivity for HDAC6 in analogous HDAC isoform functional assays, potent intracellular target engagement, and robust cellular efficacy in cancer cell lines, NN-390 is the first HDAC6-selective inhibitor to show therapeutic potential in metastatic Group 3 medulloblastoma (MB), an aggressive pediatric brain tumor often associated with leptomeningeal metastases and therapy resistance. MB stem cells contribute to these patients' poor clinical outcomes. NN-390 selectively targets this cell population with a 44.3-fold therapeutic margin between patient-derived Group 3 MB cells in comparison to healthy neural stem cells. NN-390 demonstrated a 45-fold increased potency over HDAC6-selective clinical candidate citarinostat. In summary, HDAC6-selective molecules demonstrated in vitro therapeutic potential against Group 3 MB.

Dual Antigen T Cell Engagers Targeting CA9 as an Effective Immunotherapeutic Modality for Targeting CA9 in Solid Tumors

Abstract: Glioblastomas (GBM), the most common malignant primary adult brain tumors, are uniformly lethal and are in need of improved therapeutic modalities. GBM contain extensive regions of hypoxia and are enriched in therapy resistant brain tumor-initiating cells (BTICs). Carbonic anhydrase 9 (CA9) is a hypoxia-induced cell surface enzyme that plays an important role in maintenance of stem cell survival and therapeutic resistance. Here we demonstrate that CA9 is highly expressed in patient-derived BTICs. CA9+ GBM BTICs showed increased self-renewal and proliferative capacity. To target CA9, we developed dual antigen T cell engagers (DATEs) that were exquisitely specific for CA9-positive patient-derived clear cell Renal Cell Carcinoma (ccRCC) and GBM cells. Combined treatment of either ccRCC or GBM cells with the CA9 DATE and T cells resulted in T cell activation, increased release of pro-inflammatory cytokines and enhanced cytotoxicity in a CA9-dependent manner. Treatment of ccRCC and GBM patient-derived xenografts markedly reduced tumor burden and extended survival. These data suggest that the CA9 DATE could provide a novel therapeutic strategy for patients with solid tumors expressing CA9 to overcome treatment resistance.

Characterization of the minimal residual disease state reveals distinct evolutionary trajectories of human glioblastoma

Abstract: 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.

The Role of a Longitudinal, Multidisciplinary Clinic in Building a Unique Research Collaborative

Abstract: Multidisciplinary neuro-oncology clinics allow collaboration between various specialties and training levels. Building a tenable clinical research program based in the longitudinal dialogue and practice of collaborative clinicians and trainees can bridge clinical observations to research execution. However, forming a research team around a multidisciplinary clinic’s activities is constrained by a lack of literature or guidelines. As well, challenges in sustaining team logistics, communication, and productivity can persist without a standardized team framework. This perspective discusses the state of research teams in clinical oncology, and uses experiences from the McMaster Pediatric Brain Tumour Study Group to guide those seeking to form a research team based on the collective activities and observations of a multidisciplinary clinic.

Characterization of an RNA binding protein interactome reveals a context-specific post-transcriptional landscape of MYC-amplified medulloblastoma

Abstract: Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that the RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC-overexpressing mouse models and patient-derived xenografts. MSI1 inhibition abrogates tumor initiation and significantly prolongs survival in both models. We identify binding targets of MSI1 in normal neural and G3 MB stem cells and then cross referenced these data with unbiased large-scale screens at the transcriptomic, translatomic and proteomic levels to systematically dissect its functional role. Comparative integrative multi-omic analyses of these large datasets reveal cancer-selective MSI1-bound targets sharing multiple MYC associated pathways, providing a valuable resource for context-specific therapeutic targeting of G3 MB.

Reoperation in adult patients with recurrent glioblastoma: A matched cohort analysis

Abstract: Abstract Background Despite maximal safe cytoreductive surgery and postoperative adjuvant therapies, glioblastoma (GBM) inevitably recurs and leads to deterioration of neurological status and eventual death. There is no consensus regarding the benefit of repeat resection for enhancing survival or quality of life in patients with recurrent GBM. We aimed to examine if reoperation for GBM recurrence incurs a survival benefit as well as examine its complication profile. Methods We performed a single-center retrospective chart review on all adult patients who underwent resection of supratentorial GBM between January 1, 2008 and December 1, 2013 at our center. Patients with repeat resection were manually matched for age, sex, tumor location, and Karnofsky Performance Status (KPS) with patients who underwent single resection to compare overall survival (OS), and postoperative morbidity. Results Of 237 patients operated with GBM, 204 underwent single resection and 33 were selected for repeat surgical resections. In a matched analysis there was no difference in the OS between groups (17.8 ± 17.6 months vs 17 ± 13.5 months, P = .221). In addition, repeat surgical resection had a higher rate of postoperative neurological complications compared to the initial surgery. Conclusions When compared with matched patients who underwent a single surgical resection, patients undergoing repeat surgical resection did not show significant increase in OS and may have incurred more neurological complications related to the repeat resection. Further studies are required to assess which patients would benefit from repeat surgical resection and optimize timing of the repeat resection in selected patients.

Exth-81. itga5 is a novel immunotherapeutic target against treatment refractory medulloblastoma

Abstract: Medulloblastoma (MB) is the most common type of malignant pediatric brain cancer. Current standard of care (SOC) involves maximal safe resection and neuraxis radiotherapy and chemotherapy in individuals older than 3 years. To date, these cytotoxic SOC combined with craniospinal irradiation led to devastating neurocognitive and developmental deficits impacting quality of life for pediatric patients. The biological heterogeneity of MB is highlighted by the existence of four distinct molecular subgroups (WNT, SHH, Group 3, and Group 4). Group 3 and Group 4 have the poorest patient outcomes because of their aggressive, metastatic nature, and so often remain treatment refractory to SOC. Group 3 has a poor prognosis due to its high incidence of leptomeningeal spread and an overall survival rate of less than 50%. The cytotoxic nature and lack of response in specific subtypes to SOC underscores the urgent need for developing and translating novel treatment options including immunotherapies. In our earlier work, we have developed a therapy-adapted patient derived xenograft (PDX) model of the Group 3 MB as the tumor cells undergoes therapy in vitro and in vivo. N-glycocapture surfaceome profiling of the MB cells through this PDX model identified Integrin α5 (ITGA5) as one of the most differentially expressed targets found at recurrence when compared to engraftment and untreated timepoints. Through shRNA knockdown and small molecule inhibition, we identify ITGA5 expression marks a MB cell subpopulation with increased self-renewal ability both in vitro and in vivo. Access to recurrent MB (rMB) post-therapy allowed us to investigate the changes in the surfaceome of MB cells using proteomics profiling to identify promising rMB-specific targets for rational development of novel immunotherapies.

852 Investigating the functional role of GPNMB in glioblastoma and the tumor immune microenvironment and its targeted elimination using CAR-Ts

Abstract: Background Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Due to GBM displaying extreme heterogeneity and immune suppression, prognosis remains dismal. Glycoprotein nonmetastatic melanoma protein B (GPNMB) has previously been identified as a clinically rele-vant target in GBM while being absent in normal brain tissues and shown to be active in the tumor immune microenvironment. 1,2 Chimeric Antigen Receptor T-cells (CAR-Ts) fail clinical trials because multiple antigens will be required to eliminate all GBM subpopulations. 3,4 We previously proved CD133 to be an effective CAR-T target in GBM models. 5 Methods Immunohistochemistry was performed on patient derived xenograft (PDX) brains and tissue microarrays of 16 patient matched primary/recurrent GBMs as well as 23 normal organ tissues. Whole cell proteomics was performed on 43 matched primary/recurrent GBM samples. Flow cytometry measured surface expression levels of CD133 and GPNMB to confirm CAR-T accessibility. CRISPR/Cas9 was used to eliminate expression in GBM lines to measure proliferation and mouse survival times. GPNMB knockout clones were gener-ated in GL261 and engrafted in immunocompetent mice to examine single cell transcriptomes using sciRNAseq at end-point. A second-generation CAR-T was developed to target GPNMB-expressing populations, and efficacy was interrogated

Dddr-03. development of novel anti-brain metastasis inhibitors

Abstract: The current standard of care for (surgery and radiation) for brain metastases (BM) is inadequate as BM have a 90% mortality rate within one year of diagnosis, posing a large unmet clinical need. The Singh lab has generated a large in-house biobank of patient-derived BM cell lines that are established from BM patient tumor samples. 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 BM cascades and additionally captured 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 represent a therapeutic window wherein metastasizing cells can be targeted and eradicated before establishing clinically detectable and difficult to treat brain tumors. RNA sequencing of pre-metastatic BM cells revealed a unique deregulated transcriptomic profile that is specific to pre-metastatic cells despite the tumor of origin. Connectivity Map analysis was applied to the gene expression signatures of pre-metastatic BM cells to identity a compound (Drug A) which selectively inhibits BM cell proliferation but is not blood-brain barrier (BBB) penetrant and has not been previously considered in the context of brain metastasis. We synthesized a BBB-penetrant analogue of Drug A and found, using our patient-derived xenograft (PDX) models, that it increased survival benefit relative to both placebo and Drug A. Beginning with this promising scaffold, we will conduct structure-activity hypothesis-driven medicinal chemistry campaigns to optimize this scaffold for brain permeation while maintaining selective anti-BM activity. Development of novel small molecules that target premetastatic BM cells could slow or prevent the formation of BM and dramatically improve the prognosis of at-risk cancer patients.

Abstract 564: Targeting axonal guidance with anti-ROBO1 CAR T cells: A new therapeutic strategy for malignant brain cancer

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. Citation Format: Sheila Kumari Singh, Chirayu R. Chokshi, Benjamin Brakel, Martin A. Rossotti, Chitra Venugopal, Sabra Salim, Kevin Henry. Targeting axonal guidance with anti-ROBO1 CAR T cells: A new therapeutic strategy for malignant brain cancer [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 564.

Functional discovery of targetable dependencies in recurrent glioblastoma

Abstract: Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. Here, we explore the functional drivers of post-treatment recurrent GBM. By conducting genome-wide CRISPR-Cas9 screens in patient-derived GBM models, we uncover distinct genetic dependencies in recurrent tumor cells that were absent in their patient-matched primary predecessors, accompanied by increased mutational burden and differential transcript and protein expression. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Mechanistically, genetic perturbation or small molecule inhibition of PTP4A2 represses axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1), exploiting a functional dependency on ROBO signaling. Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition. We conclude that functional reprogramming drives tumorigenicity and dependence on a multi-targetable PTP4A2-ROBO1 signaling axis at GBM recurrence. and sensitivity to TMZ in primary pre-treatment tumor cells 6-9 . However, these studies do not examine changes at post-treatment tumor recurrence, and thus cannot explain treatment failure in ~70% of GBM patients 10 . Here, we conduct a genome-scale comparison between patient-matched pre- and post-treatment GBM cells at the functional, transcriptomic, and proteomic levels. We uncover a therapeutic vulnerability for protein tyrosine phosphatase 4A2 (PTP4A2) at tumor recurrence, and introduce a modulatory role for PTP4A2 on axonal guidance proteins. Normalization for AAVS1, PTP4A2 (�rst gRNA) PTP4A2 gRNA). DEGs extracted using treat() (limma) and log 2 (fold change)>1 and antibody binding to these proteins was assessed by indirect titration ELISA. The a�nities and kinetics of the interactions between monomeric single domain antibodies and human ROBO1 (25°C, pH 7.4) were determined using surface plasmon resonance. ROBO1 and other proteins were immobilized on a sensor chip CM5 (GE Healthcare) by amine coupling and antibodies were �owed over the antigen surfaces on a Biacore T200 instrument (GE Healthcare). Data from multi-cycle kinetic analysis were �t to a 1:1 binding model.

Abstract 976: Interrogating the pre-metastastic gene signature to block brain metastases

Abstract: BACKGROUND: The incidence of brain metastases (BM) is tenfold higher than that of primary brain tumors. BM predominantly originate from primary lung, breast, and melanoma tumors with a 90% mortality rate within one year of diagnosis, posing a large unmet clinical need to identify novel therapies against BM. The goal of this work is to uncover the molecular factors that drive the formation of BM and investigate whether we can slow down and ultimately block BM formation. METHODS: The Singh lab has generated a large in-house biobank of patient-derived BM cell lines that are established from BM patient tumor samples. 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 BM cascades and additionally captured a “pre-metastatic” 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 represent a therapeutic window wherein metastasizing cells can be targeted and eradicated before establishing clinically detectable and difficult to treat brain tumors. RESULTS: RNA sequencing of pre-metastatic BM cells revealed a unique deregulated transcriptomic profile that is specific to pre-metastatic cells despite the tumor of origin. Subsequent Connectivity Map analysis revealed compounds that we biologically characterized in vitro for selective anti-BMIC phenotypes. This effort led to a lead compound that exhibits anti-BM activity in vitro, while remaining ineffective against normal brain cell controls. Preliminary in vivo work has shown that following both orthotopic and intracardiac injection of BM cells, treatment with this lead compound reduces the tumor burden compared to mice being treated by a vehicle control, while providing a significant survival advantage. Ongoing mechanistic investigations aim to delineate the protein target of this compound in the context of the observed selective anti-BM phenotype. CONCLUSION: Identification of novel small molecules that target premetastatic BM cells could slow or prevent the formation of BM and dramatically improve the prognosis of at-risk cancer patients. Citation Format: Agata Kieliszek, Blessing Bassey-Archibong, Chitra Venugopal, Sheila Singh. Interrogating the pre-metastastic gene signature to block brain metastases [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 976.

Abstract 3999: HLA-G, SPAG9 and STAT3 signalling: An alliance that promotes early-stage brain metastases

Abstract: Brain metastases (BM) are the most common brain tumours in adults and a prominent cause of cancer-related mortality globally. Leading sources of BM are cancers of the lung, breast and melanoma, which together account for approximately 80% of all BM. Unfortunately, current clinical modalities for BM including surgery, radiation therapy and chemotherapy still offer limited efficacy and median survival times of 4 - 12 months in treated patients, emphasizing the need for more effective therapeutic strategies and generally a better understanding of the disease. We recently identified the presence of stem-like cells termed “brain metastasis-initiating cells” or BMICs in patient-derived BM from lung, breast and melanoma cancers that are able to recapitulate the complete brain metastatic cascade in pre-clinical models of BM. Through these models, we serendipitously captured lung, breast and melanoma BMICs at the “pre-metastatic” stage of BM - a stage where circulating metastatic cells have seeded the brain, but not yet formed full-blown (macro-metastatic) brain lesions. Transcriptomic analysis of pre-metastatic and macro-metastatic lung, breast and melanoma BMICs revealed a unique genetic profile in pre-metastatic BMICs that was distinct from their macro-metastatic counterparts. Further analysis identified several genes commonly up-regulated in all pre-metastatic BMIC cohorts irrespective of their primary tumour of origin. Intriguingly, we found that inhibition of the non-classical human leukocyte class I antigen-G or HLA-G gene (one of the top up-regulated genes in the pre-metastatic cohorts), reduced the ability of BMICs to form mature brain lesions. Correspondingly, HLA-G over-expression increased the capacity of BMICs to establish secondary brain tumours. Mechanistically, we discovered that over-expressing HLA-G levels in BMICs (to simulate the high levels that occurs in pre-metastatic BMICs), increased the activation of STAT3 signalling and this was mediated in part via a novel HLA-G binding partner - SPAG9. Our work thus uncovered a potential cooperative role between HLA-G, SPAG9 and STAT3 signalling during the early stages of BM. Indeed, attenuation of SPAG9 protein levels or STAT3 signalling in HLA-G over-expressing BMICs using CRISPR knockout and a STAT3 inhibitor respectively obstructed the ability of high HLA-G levels to promote mature brain lesions. This is the first study to reveal a role for an HLA-G-SPAG9-STAT3 axis in BM and highlights the potential of targeting this axis to inhibit BM, which will markedly extend patient survival. Citation Format: Blessing I. Bassey-Archibong, Chirayu R. Chokshi, Nikoo Aghaei, Agata Kieliszek, Nazanin Tatari, Dillon McKenna, Mohini Singh, Minomi Subapanditha, Arun Parmar, Neil Savage, Yu Lu, Chitra Venugopal, Sheila Singh. HLA-G, SPAG9 and STAT3 signalling: An alliance that promotes early-stage brain metastases [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 3999.

Bsci-22 determining the effect of novel small molecule drugs against the migration of brain metastasis initiating cells (bmics)

Abstract: Abstract BACKGROUND Brain metastases are secondary tumors that predominantly arise from the spread of lung, skin, and breast cancers. The current standard of care for brain metastases is complete surgical resection, with a median survival of four months. Therefore, there is a dire need to discover new therapies that effectively target brain metastases. To do this, we have identified anti-brain metastasis drugs that specifically target brain metastasis initiating cells (BMICs), a cancer stem cell population that is thought to escape standard therapies and has the ability to leave their primary tumor, seed the brain, and form a secondary brain tumor. Since the migration of the BMICs is essential to the development of brain metastases in patients, the main goal of this study was to determine the effect our anti-brain metastasis drugs have against the migration of lung, skin, and breast BMICs. METHODS This migration assay utilizes a bi-well silicone structure which effectively establishes a ‘wound’ healing-like migration assay. BMICs are plated in optimized equal concentrations in each silicone bi-well structure to successfully form two cellular mono-layers that are separated by a middle silicone wall. Once cells adhere to the plate the silicone structure is removed and the area between the two cell populations is imaged over time with an in vitro imaging system. RESULTS This optimized assay has been used to screen our anti-brain metastasis drugs against the migration of lung, breast, and skin BMICs. Thus far our drugs have been tested against lung and skin BMICs which resulted in a significant decrease in BMIC migration. SIGNIFICANCE Since brain metastasis arises from the migration of cancer cells to a secondary organ, it is crucial to discover the effect of anti-brain metastasis drugs on BMIC migration prior to the initiation of preclinical animal trials.

Bsci-13 development of novel anti-brain metastasis inhibitors

Abstract: The current standard of care (surgery and radiation) for brain metastases (BM) is inadequate as BM have a 90% mortality rate within one year of diagnosis, posing a large unmet clinical need. The Singh lab has generated a large in-house biobank of patient-derived BM cell lines that are established from BM patient tumor samples. 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 BM cascades and additionally captured a “pre-metastatic” 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 represent a therapeutic window wherein metastasizing cells can be targeted and eradicated before establishing clinically detectable and difficult to treat brain tumors. RNA sequencing of pre-metastatic BM cells revealed a unique deregulated transcriptomic profile that is specific to pre-metastatic cells despite the tumor of origin. Connectivity Map analysis was applied to the gene expression signatures of pre-metastatic BM cells to identity a compound (Drug A) which selectively inhibits BM cell proliferation but is not blood-brain barrier (BBB) penetrant and has not been previously considered in the context of brain metastasis. We synthesized a BBB-penetrant analogue of Drug A and found, using our patient-derived xenograft (PDX) models, that it increased survival benefit relative to both placebo and Drug A. Beginning with this promising scaffold, we will conduct structure-activity hypothesis-driven medicinal chemistry campaigns to optimize this scaffold for brain permeation while maintaining selective anti-BM activity. Development of novel small molecules that target pre-metastatic BM cells could slow or prevent the formation of BM and dramatically improve the prognosis of at-risk cancer patients.

Epco-07. non-genomic determinants of tumor cell phenotypes in adult glioblastoma

Abstract: Non-genomic determinates of oncogenic cellular phenotypes is an emerging concept that expanded our model of tumor hallmarks. We hypothesized that oncogenic programs in adult glioblastoma (GBM) such as angiogenesis, proliferation, DNA repair, epithelial to mesenchymal transition and quiescent states are achieved independently of mutational background or clonal linage. We therefore explored the assortations of large-scale chromosomal rearrangements and canonical driver mutations with oncogenic programs in single cell RNA sequencing (scRNA-seq) of over 3000 tumor cells in 4 adult GBM using open data. We find recurring patterns where tumor cells from diverse mutational background and clonal linage converge upon oncogenic tumor phenotypes. We validate this observation in 9 tumors comprising over 16,000 cells and in xenograft animal models of GBM pre- and post temozolamide treatment. We finally explore the epigenetic associations of oncogenic phenotypes via computational label transfer from scRNA-seq to chromatic accessibility data from single cell ATAC sequencing (scATAC-seq). We find open genomic regions associated with canonical regulators, such as the highly oncogenic mesenchymal phenotype driven by TWIST1. Our results suggest a paradigm shift towards non-genetic determinants of oncogenic phenotypes in GBM complementing the conventional concept of cancer clonal evolution.

Abstract 60: Functional mapping reveals widespread remodelling and unrecognized pathway dependencies in recurrent glioblastoma

Abstract: Glioblastoma is a highly fatal brain cancer. The underlying functional drivers of treatment resistance and disease recurrence are unclear. By applying a genome-wide CRISPR-Cas9 library to patient-derived glioblastoma stem cell models, we systematically map genetic dependencies in patient-matched pre-treatment primary and post-treatment recurrent tumor cells. These insights reveal a large-scale remodelling of genetic dependency profiles at disease recurrence, arming recurrent tumor cells with newly-acquired genetic drivers and further loss of tumor suppressors. These analyses support parallel tumor-intrinsic mechanisms of treatment resistance which rely on acquisition of immunosuppressive capacity, including a defective mismatch repair pathway, ablation of PTEN activity, and a novel combination of de novo mutations in SWI/SNF components. We map a multilayered genetic and functional response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel driver of self-renewal, proliferation and tumorigenicity at glioblastoma recurrence. Mechanistically, genetic perturbation and a small molecule inhibitor of PTP4A2 results in greater survival and reduced tumor growth in patient-derived models of recurrent glioblastoma. Citation Format: Chirayu R. Chokshi, Kevin Brown, Chitra Venugopal, Jason Moffat, Sheila K. Singh. Functional mapping reveals widespread remodelling and unrecognized pathway dependencies in recurrent glioblastoma [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 60.

Modl-05. discovery of dynamic minimal residual disease states in adult glioblastoma using single cell technology

Abstract: Persister states are proposed oncogenic substrates of disease recurrence in cancer. Recent concepts suggest persisters may occur in dynamic states of the cell cycle (such as cycling and non-cycling states). We therefore investigated biological programs at the post-treatment minimal residual disease (MRD) state following standard chemoradiotherapy in patient-derived xenograft models of GBM. Our analysis of single cell RNA sequencing (scRNA-seq) data from 2704 tumor cells (929 cells post treatment, 1775 matched controls) yielded a cellular profile for non-cycling and cycling persister states. We validated these programs in 3 independent scRNA-seq datasets of human glioblastoma specimens consisting of over 16,000 cells from various genetic backgrounds, including an internal two patient-matched primary and recurrent GBM pairs with over 13,000 cells. Finally, we determined that clones identified based on large scale chromosomal rearrangements converge on previously identified persister states including the dynamic states discovered in our study. Our results provide new evidence towards dynamic persister states in glioblastoma, further analysis of these dynamic states is critical to targeting this incurable disease.

Syst-15 targeting axonal guidance with anti-robo1 car t cells: a new therapeutic strategy for malignant brain cancer

Abstract: No standardized treatments exist for patients with treatment-refractory brain metastasis, glioblastoma and other recurrent brain tumours. Given the aggressive nature of these diseases and difficulty in modelling tumour recurrence, minimal efforts have been made to design rational therapies against them. Neurodevelopmental pathways are often highjacked and go awry in the progression of these cancers. The roundabout guidance receptor 1 (ROBO1) protein is involved in axonal guidance during neurodevelopment, and we have shown that aberrant ROBO signalling promotes invasiveness and tumour growth in glioblastoma. Likewise, this signalling may contribute to the metastasis and growth of metastatic brain tumours, making the ROBO1-expressing tumour cell population an attractive and functionally relevant therapeutic target. Here, we present that ROBO1 is highly expressed on the surface of malignant and treatment-refractory brain tumour 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 malignant brain cancers, upregulating markers of activation and degranulation upon exposure to ROBO1-expressing BTICs. Additionally, orthotopic patient-derived xenograft models of malignant brain tumours treated with anti-ROBO1 CAR-T cells had a reduced tumour burden and prolonged survival, demonstrating therapeutic potential for treating brain malignancies.

The Road to CAR T-Cell Therapies for Pediatric CNS Tumors: Obstacles and New Avenues

Abstract: Pediatric central nervous system (CNS) tumors are the most common solid tumors diagnosed in children and are the leading cause of pediatric cancer-related death. Those who do survive are faced with the long-term adverse effects of the current standard of care treatments of chemotherapy, radiation, and surgery. There is a pressing need for novel therapeutic strategies to treat pediatric CNS tumors more effectively while reducing toxicity – one of these novel modalities is chimeric antigen receptor (CAR) T-cell therapy. Currently approved for use in several hematological malignancies, there are promising pre-clinical and early clinical data that suggest CAR-T cells could transform the treatment of pediatric CNS tumors. There are, however, several challenges that must be overcome to develop safe and effective CAR T-cell therapies for CNS tumors. Herein, we detail these challenges, focusing on those unique to pediatric patients including antigen selection, tumor immunogenicity and toxicity. We also discuss our perspective on future avenues for CAR T-cell therapies and potential combinatorial treatment approaches.