Abstract B044: Dual inhibition of CRK and CRKL as a strategy to block glioblastoma invasion

Abstract Glioblastoma is the most common and most aggressive primary malignant brain tumor with a dismal prognosis, mainly due to its highly invasive nature. Diffuse infiltration of glioblastoma cells into surrounding brain tissues severely limits the effectiveness of surgical resection and focal radiation, resulting in nearly inevitable recurrence. Therefore, therapeutic interventions that suppress glioblastoma motility and block invasion could serve as an additional layer of therapy to improve the efficacy of current treatments. CT10 regulator of kinase (CRK) and CRK-like (CRKL) have been reported to play crucial roles in tumor cell migration and invasion in many cancers. We previously identified CRK and CRKL as central mediators of glioblastoma cell motility and proliferation. Transient gene knockdown of both CRK and CRKL completely abolished glioblastoma cell migration and invasion, and partially inhibited glioblastoma cell proliferation. On the other hand, overexpression of CRK or CRKL enhanced glioblastoma migration and invasion. These findings led us to hypothesize that CRK and CRKL are critical drivers of glioblastoma infiltration in vivo and that inhibiting these proteins could improve treatment outcomes. Using an orthotopic xenograft mouse model, we demonstrate that glioblastoma cells with stable CRK and CRKL double knockdown exhibit delayed tumor formation and prolonged survival in immunodeficient mice, confirming the functional importance of CRK and CRKL in vivo. We are studying how CRK/CRKL knockdown affects glioblastoma growth and invasion in the mouse brain. Since the lack of CRK and CRKL inhibitors made it challenging to validate CRK and CRKL as therapeutic targets further, we developed novel peptide inhibitors of CRK and CRKL designed to bind the SH2 domains of CRK and CRKL. A peptide sequence was derived from the binding motifs of p130Cas, a well-known CRK/CRKL-binding protein. The peptide was optimized through a molecular modeling-based study of its structure-function relationship. The optimized peptide effectively disrupted the CRK/CRKL-mediated protein-protein interactions in vitro. To enable delivery into tumor cells, the CRK/CRKL-antagonist peptide was conjugated to a cholesterol-polylysine-trimaleimide nanosponge. Treatment of glioblastoma cells in culture with the nanosponge-coupled peptide resulted in efficient uptake by glioblastoma cells and robust inhibition of glioblastoma cell migration. Ongoing work will evaluate the in vivo efficacy of the nanosponge-coupled CRK/CRKL-antagonist peptide in orthotopic glioblastoma models to determine whether it recapitulates the anti-cancer effect of genetic double knockdown. These findings establish CRK and CRKL as promising therapeutic targets in glioblastoma. Our study introduces CRK/CRKL-antagonist peptides as a potential new class of anti-cancer therapeutics to enhance current glioblastoma treatment strategies. Citation Format: Taeju Peter. Park, Piyanka Hettiarachchi, Anil Ahsan, David K. Johnson, Anuradha Roy, Justin T. Douglas, Obdulia Covarrubias-Zambrano, Stefan Bossmann. Dual inhibition of CRK and CRKL as a strategy to block glioblastoma invasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr B044.