Presenter:
Alexander Colón
Glioblastoma is the most aggressive brain tumor, notorious for its therapeutic resistance, immunosuppressed tumor microenvironment (TME), and paucity of active tumor-infiltrating lymphocytes (TILs) capable of triggering an antitumoral response. Immune checkpoint blockers (ICB) targeting suppressive molecules such as programmed cell death protein 1 (PD- 1) and lymphocyte activation gene 3 (LAG-3) have shown efficacy in melanoma treatment. In preclinical studies, combination anti-PD-1 and anti-LAG-3 treatment has been shown to enhance immune activation and increase TILs in the TME. It is hypothesized that this co- inhibition is also associated with phenotypic changes observed in T cells, shifting to a natural killer (NK) / natural killer T (NKT) cell signature. NK/NKT cells interact with other cells in an HLA-independent manner, so the T cell adoption of a NK-like phenotype could improve immune activation and cancer cell killing. To investigate T cell reprogramming, we expanded T cells from peripheral blood mononuclear cells (PBMCs) of a glioblastoma patient (under a Duke IRB-approved protocol) using anti-CD3/anti-CD28 stimulation with anti-PD1 (nivolumab) or anti-PD1 + anti-Lag3 (relatlimab) over a 14-day period. We aimed to characterize the phenotypic expression of T cell and NK cell signatures at different timepoints throughout treatment based on growth kinetics, spectral flow cytometry, and bulk RNA sequencing on days 0, 3, 7, 10, and 14. Data from these in vitro studies will help demonstrate if this shift occurs simultaneously with T cell activation and to what degree phenotypic changes are observed. This pre-clinical study is conducted in parallel with our upcoming clinical trial of PD-1/LAG-3 co-inhibition in newly diagnosed glioblastoma (NCT06816927). Mechanistic studies will complement analyses of human biospecimens to better understand the effects of nivolumab and relatlimab on T cell activation, proliferation, exhaustion, and memory formation, as well as generate new insights into the role of NK/NKT cells. LCP1 Loss Reduces IL-6-Induced STAT3 Nuclear Translocation in CSA
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Presenter: Alexander Colón
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Background: Chondrosarcoma (CSA) is the second most common malignancy of bone with limited treatment options for metastatic disease beyond surgical resection. Our lab has demonstrated that the actin-binding protein, LCP1 (lymphocyte cytosolic protein 1), is associated with poor prognosis and metastatic behavior in CSA; however, the mechanisms by which it promotes tumor progression remain unclear. Confocal microscopy demonstrated the role of LCP1 in cytoskeletal organization in CSA cells. Additionally, a biotinylation and proteomics screen revealed interactions between LCP1 and key signaling proteins, including STAT3 and the upstream IL-6 receptor subunit IL6ST, suggesting the potential role of LCP1 in oncogenic cell signaling and transcriptional regulation. Surprisingly, LCP1 knock-out in our CSA cells resulted in increased cytoplasmic phosphorylated STAT3. Given the role of LCP1 in cytoskeletal functions, we hypothesized that LCP1 is required for STAT3 nuclear translocation and transcriptional activity in response to IL-6 stimulation. Methods: To test this hypothesis, we utilized the CSA cell line sw1353 and compared nuclear STAT3 quantities in LCP1 wildtype and knockdown conditions, in response to IL-6 stimulation. Nuclear STAT3 was quantified using an enzyme-linked immunosorbent assay (ELISA) after harvesting nuclear and cytoplasmic protein fractionations. To analyze these results, we performed a one and two-way ANOVA and Tukey's multiple comparison test. Results: Our results showed that IL-6 stimulation led to increased nuclear STAT3 compared to all non-stimulated groups (p=<0.0001). Importantly, LCP1 knockdown demonstrated a decrease in nuclear STAT3 compared to non-knockdown samples in all groups. (p= <0.0016). Conclusions: These findings support our hypothesis that LCP1 plays a critical role in mediating STAT3 nuclear transport during IL-6 signaling. As STAT3 is a known oncogenic transcriptional activator, this opens potential for LCP1 as a promising therapeutic target for disrupting metastatic signaling pathways in CSA.
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Duke University / 2025
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Presenter: Alexander Colón