Abstract 3360: Myristoylated alanine-rich C-kinase substrate (MARCKS) as a target for pathological tunneling nanotubes in glioblastoma

Abstract Background: Glioblastoma (GBM) is a lethal brain malignancy with a median survival of <2 years due to relapse in 100% of cases. Tunneling nanotubes (TNTs) facilitate the development of treatment resistance by establishing tumor-promoting networks between GBM and surrounding cells. Only having emerged in 2004, many key players remain to be identified which is why we propose that MARCKS regulates TNT directionality through phosphorylation of its effector domain (ED). Methods: We quantified TNT sinuosity, numbers, branching points, and average length in (3) PTEN-null doxycycline-inducible U87-MARCKS-mutant models in serum-free conditions. Mutants overexpress MARCKS as wild-type (U87-WT), non-phosphorylated (U87-NP), or pseudo-phosphorylated (U87-PP). TNTs were characterized using immunofluorescence, confocal imaging, ICY manual TNT plugin, and negative staining. We also investigated a cytotoxic peptide derived from MARCKS-ED, termed MED2, on PTEN-null brain tumor-initiating cell (BTIC) TNTs. Statistical analysis was conducted with one or two-way ANOVA and multiple comparisons. Results: The most striking difference between all mutants is the loss of rigidity and directionality in U87-NP TNTs. Negative staining revealed U87-NP TNTs have deformed bodies with clubbed tips compared to ordered filaments in U87-WT and U87-PP. 4h MED2 treatment in non-mutated BTICs results in cytotoxicity as well as truncated TNTs that phenotypically resemble U87-NP. Conclusions: This is the first report supporting MARCKS to be a novel regulator of TNTs through ED phosphorylation. Future directions will investigate the functionality of TNTs in U87 mutants to determine if the lack of MARCKS phosphorylation renders TNTs nonfunctional. Defining MARCKS' role in GBM TNTs not only advances the field of cellular communication but also offers a new perspective on GBM treatment. Quantitative TNT Characterization Sinuosity TNT Number Branch Points Average Length MARCKS/F-actin Correlation U87-WT 1.00 +/- 0.00 29.0 +/- 2.94 17.0 +/- 2.94 9.80 +/- 1.74 R2=0.9245 U87-NP 1.21 +/- 0.10 11.5 +/- 1.29 9.00 +/- 0.82 9.90 +/- 1.79 R2=0.5497 U87-PP 1.03 +/- 0.02 34.3 +/- 11.2 8.50 +/- 3.41 7.90 +/- 1.05 R2=0.7249 P-value WT vs. NP: P=0.0026 WT vs. PP: P=0.7804 NP vs. PP: P=0.0070 WT vs. NP: P=0.0126 WT vs. PP: P=0.5344 NP vs. PP: P=0.0025 WT vs. NP: P=0.0053 WT vs. PP: P=0.0036 NP vs. PP: P=0.9616 WT vs. NP: P=0.9975 WT vs. PP: P=0.0363 NP vs. PP: P=0.0312 U87-WT: P<0.0001 U87-NP: P<0.0001 U87-PP: P<0.0001 BTIC + No treatment 1.00 +/- 0.00 13.5 +/- 1.29 8.00 +/- 1.83 23.2 +/- 10.3 Not determined BTIC + 3μM MED2 1.48 +/- 0.33 13.3 +/- 3.5 7.50 +/- 1.92 15.0 +/- 11.7 Not determined P-value NT vs. MED2: P=0.0198 NT vs. MED2: P>0.9999 NT vs. MED2: P>0.9999 NT vs. MED2 P=0.2003 Not determined Citation Format: Lauren C. Nassour-Caswell, Nicholas J. Eustace, Shane T. Rich-New, Taylor L. Schanel, Hasan Alrefai, Andee M. Beierle, Manoj Kumar, Patricia H. Hicks, Johsua C. Anderson, Christopher D. Willey. Myristoylated alanine-rich C-kinase substrate (MARCKS) as a target for pathological tunneling nanotubes in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3360.