LNX1 Modulates Notch1 Signaling to Promote Expansion of the Glioma Stem Cell Population during Temozolomide Therapy in Glioblastoma

SIMPLE SUMMARY: Glioblastoma is the most common adult malignant brain tumor. It is an aggressive tumor that returns even after surgical removal and temozolomide-based chemotherapy and radiation. Our goal was to understand what genes are altered by temozolomide and how those genes may contribute to tumor return. Our work shows that one of the genes altered is LNX1, which increases the expression of Notch1, a gene important for glioblastoma progression. We further showed that the elevation of LNX1 and Notch1 results in an increase in the tumor stem cell population, a subpopulation of cells thought to help propagate a more aggressive tumor. Finally, we showed that forced reduction in LNX1 expression results in increased survival of animals implanted with glioblastoma. Together, these results suggest that LNX1 may be a novel therapeutic target that would allow modulation of Notch1 activity and the stem cell population, potentially resulting in increased patient survival. ABSTRACT: Glioblastoma (GBM) is the most common primary brain malignancy in adults, with a 100% recurrence rate and 21-month median survival. Our lab and others have shown that GBM contains a subpopulation of glioma stem cells (GSCs) that expand during chemotherapy and may contribute to therapeutic resistance and recurrence in GBM. To investigate the mechanism behind this expansion, we applied gene set expression analysis (GSEA) to patient-derived xenograft (PDX) cells in response to temozolomide (TMZ), the most commonly used chemotherapy against GBM. Results showed significant enrichment of cancer stem cell and cell cycle pathways (False Discovery Rate (FDR) < 0.25). The ligand of numb protein 1 (LNX1), a known regulator of Notch signaling by targeting negative regulator Numb, is strongly upregulated after TMZ therapy (p < 0.0001) and is negatively correlated with survival of GBM patients. LNX1 is also upregulated after TMZ therapy in multiple PDX lines with concomitant downregulations in Numb and upregulations in intracellular Notch1 (NICD). Overexpression of LNX1 results in Notch1 signaling activation and increased GSC populations. In contrast, knocking down LNX1 reverses these changes, causing a significant downregulation of NICD, reduction in stemness after TMZ therapy, and resulting in more prolonged median survival in a mouse model. Based on this, we propose that during anti-GBM chemotherapy, LNX1-regulated Notch1 signaling promotes stemness and contributes to therapeutic resistance.