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.

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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.

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. Roundabout guidance receptor 1 (ROBO1) protein is involved in axonal guidance during neurodevelopment and aberrant ROBO1 signaling axis is associated with higher tumorigenicity in GBM. ROBO1 was highly expressed on the surface of malignant and treatment-refractory brain tumor initiating cells (BTICs) in rGBM, brain metastasis (BM) and medulloblastoma (MB). Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition. We therefore developed and validated second-generation CAR-T cells against ROBO1, which demonstrated upregulation of activation markers, enhanced cytokine release, markedly increased proliferation, and induction of potent and specific tumor cell death compared to untransduced cells in all the three brain cancers. These findings were further validated in vivo in rGBM, MB and BM models, where ROBO1 CAR-T cells showed significant reduction in tumor burden and increase in survival of treated mice. We conclude that functional reprogramming drives tumorigenicity and dependence on a multi-targetable PTP4A2-ROBO1 signaling axis at GBM recurrence.