Abstract 4949: The role of symmetric cell division in post-therapy glioma-initiating cell expansion

Abstract

Abstract The purpose of this study is to elucidate the mechanism by which LNX1 regulates Notch1 to increase symmetric self-renewal of glioma-initiating cells (GICs) and thereby to expand the GIC population in glioblastoma (GBM) during therapy. GBM is an aggressive primary malignancy of the brain with an almost 100% recurrence rate. Our lab and others have shown that GBM contains a subpopulation of GICs that may drive the therapeutic resistance capabilities of recurrent GBM. We have shown that the GIC population expands following administration of temozolomide (TMZ), the most commonly used drug for antiglioma chemotherapy. While it has been suggested that this expansion relies solely on Darwinian selection, we and others have demonstrated that cellular plasticity-driven expansion of therapeutically resistant GICs post-therapy may play a role in disease recurrence. GICs follow the normal stem cell pattern of cell division. They can divide in three ways: (1) asymmetric division to form one GIC and one differentiated cancer cell, (2) symmetric self-renewal to form two daughter GICs, or (3) symmetric differentiation to form two cancer cells. Here, we used ImageStream flow cytometry analysis to show that symmetric self-renewal, where one GIC produces two daughter GICs, occurs at a rate of 24% in GBM cells treated with vehicle as compared to 54% following TMZ therapy (p<0.0001). Gene set expression analysis (GSEA) on GBM patient-derived xenograft (PDX) cells after treatment with vehicle or TMZ showed a statistically significant enrichment of 29 genes involved in polarized cell division after TMZ (FDRq<0.25). LNX1, a Notch1 regulator known to regulate symmetric cell division, was found to be enriched 8.7-fold in multiple GBM PDX lines (p<0.0001). Furthermore, Western blotting of multiple GBM PDX cell lines showed that Notch1 antagonist Numb is significantly downregulated and that Notch1 and Notch1 downstream markers (MAML1, Hes1, and p21) are significantly upregulated after therapy. Knocking down LNX1 reverses these changes, causing a significant downregulation of Notch1 downstream marker Hes-1 (p < .001). Furthermore, knocking down LNX1 reduced the frequency of GICs after TMZ therapy. The stem cell frequency in control cells was 1/128 as compared to 1/54 after therapy (p<0.001). In comparison, LNX1 knockdown cells had a stem cell frequency of 1/84 as compared to 1/92 after TMZ therapy (p=0.84). Finally, based on The Cancer Genome Atlas data, LNX1 mRNA expression is negatively correlated with overall survival in patients with GBM (p=0.015). Taken together, we propose that therapeutic stress-induced LNX1 expression can alter the Notch pathway, which subsequently allows symmetric self-renewal to predominate in GBM and therefore provides a potentially valuable therapeutic target to prevent GBM recurrence. Citation Format: Shivani Baisiwala, Robert Hall, Louisa Warnke, Anne Christensen, Jack Shireman, Cheol H. Park, Atique U. Ahmed. The role of symmetric cell division in post-therapy glioma-initiating cell expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4949.